BIOMETRIC AND BIOCHEMICAL STUDIES

ON HOT PEPPER

By

Qumer Iqbal

M.Sc. (Hons.) Agriculture

A thesis submitted in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

IN

HORTICULTURE

INSTITUTE OF HORTICULTURAL SCIENCES

UNIVERSITY OF AGRICULTURE, FAISALABAD

(PAKISTAN)

2009

From the beginning it was clear to me…

this is not about me or what I can do,

rather this is about what ALLAH wants me to do and

how He will enable me to do it.

Dedicated to

My Beloved Parents &

Loving Sister Rukhsana Iqbal (Late)

i

AACCKKNNOOWWLLEEDDGGEEMMEENNTTSS

All praises and thanks are for Almighty Allah, who is the entire source of all knowledge

and kinds of wisdom endowed to mankind. His bounteous blessing that flourished my

thoughts and fulfilled my ambitions and my modest efforts in the form of this write up

and made this material contribution towards the deep ocean of scientific knowledge

already existing and which is a permanent source of benefit for his humanity and

creature.

I present my humble gratitude from the deep sense of heart to the Holy Prophet

Muhammad (Peace Be Upon Him) that without him the life would have been worthless.

All praises be to the Holy Prophet Muhammad (Peace Be Upon him), the city of

knowledge, the illuminating torch and the rescuer of humanity from going astray, whose

teachings enlightened my heart and flourished my thoughts.

This thesis owes its existence to the help, support, and inspiration of many people. In the

first place, I would like to express my sincere appreciation and gratitude to my supervisor

Professor Dr. Muhammad Amjad (PhD Salford, UK) for his support and

encouragement during the more than four years of this research work. He has provided

me an optimum working environment at the Institute, where a lack of resources is

something unimaginable due to his managerial skills and foresight. His uncompromising

quest for excellence significantly shapes everyone at the institute. I am extremely

honored to be one of his students and I wish to boost the knowledge and confidence he

has given me.

I offer my sincere thanks to Dr. Muhammad Rafique Asi, Senior Scientist, Nuclear

Institute for Agriculture and Biology (NIAB), Faisalabad, co-supervisor, for his inspiring

guidance, keen interest and unsuited help during my study period. I am also indebted to

Dr. Muhammad Asif Ali and Dr. Riaz Ahmad who have not only been a source of

enthusiasm and encouragement over many years, but have also agreed to serve on my

supervisory committee. They have provided me the necessary knowledge in making my

proposal and for inculcating in me the excitement of doing research work.

I am also thankful to all the teaching staff in the Institute of Horticultural Sciences,

University of Agriculture, Faisalabad; my lab mates Khurrum Ziaf, Muzammil

ii

Jehangir, Javed Saqi, Aamir Nawaz, Tanveer Ahmad and in particular Dr. Aamir

Shakeel who have set the pace of implementation with mind-boggling ingenuity and

speed. I am very grateful to them for creating the cooperative spirit and the excellent

working atmosphere to make a unique setting for intellectual explorations.

I feel myself helpless in searching proper words to pay back and infect no one can ever

be able to do some for his father who provided me all what I needed. I am also proud of

my mother who inspired and encouraged me, always remembered me in her prayers. I

present my special thanks from core of my heart, to my brother (Zafar Iqbal), bhabi,

loving sisters and cute Wishah Fatima (my niece) who are a constant reminder that there

are people who still inspire my world.

I am also thankful to Higher Education Commission, Government of Pakistan, for

providing all financial support for this study.

(QUMER IQBAL)

iii

TTAABBLLEE OOFF CCOONNTTEENNTTSS Title Page ACKNOWLEDGEMENTS i

TABLE OF CONTENTS iii LIST OF TABLES vii

LIST OF FIGURES ix

ABSTRACT xi Chapter 1. INTRODUCTION 1 Chapter 2. REVIEW OF LITERATURE 6 2.1. Use of plastic mulches in vegetable production 7 2.1.1 Effect of plastic mulches on soil temperature 7 2.1.2. Effect of plastic mulches on soil moisture 9 2.1.3. Effect of plastic mulches on weed control 10 2.1.4. Effect of plastic mulches on plant growth and yield 10 2.1.5. Mulch type/color (black and clear plastic mulch) 12 2.1.6. Effect of mulch type/color on insects/pests and diseases 13 2.2. Hot pepper as a source of antioxidants 14 2.2.1. Capsaicinoids 15 22..22..22.. Carotenoids 18 2.2.3. Ascorbic acid 20 2.2.4. Phenolic compounds 21 2.3. Aflatoxin contamination as a serious concern 22 2.3.1. Chemistry of aflatoxin 23 2.3.2. Aflatoxins and health 25 2.3.3. Occurrence 26 2.3.4. Aflatoxins and storage 29 2.4. Aflatoxins, microbial load and irradiation 31 Chapter 3. MATERIALS AND METHODS 36 3.1. Effect of plastic mulches on growth and yield of hot peppers 36 3.1.1. Hot pepper hybrids 36 3.1.2. Plastic mulch 36 3.1.3. Layout 36 3.1.4. Methods 37 3.1.5. Soil temperature 37 3.1.6. Plant height 37 3.1.7. Stem diameter 37 3.1.8. Root fresh weight 38 3.1.9. Root dry weight 38 3.1.10. Root length 38 3.1.11. Leaf area plant-1 38 3.1.12. Days taken to first flowering 38

iv

3.1.13. Fresh fruit yield plant-1 38 3.1.14. Dry fruit yield plant-1 38 3.1.15. Harvest index 38 3.1.16. Fruit length 38 3.1.17. Fruit diameter 39 3.1.18. Determination of nutrient elements 39 i. Leaf sampling 39 ii. Sample preparation 39 iii. Total nitrogen 39 iv. Digestion procedure 39 v. Distillation 40 vi. Quantification of nitrogen 40 vii. Estimation phosphorus and potassium 40 viii. Digestion procedure 40 ix. Phosphorus estimation 41 x. Potassium estimation 41 3.2. Quantification of antioxidant constituents in hot peppers harvested at different stages

41

3.2.1. Stage designations 41 3.2.2. Determination of capsaicin and dihydrocapsaicin 42 i. Capsaicinoid standards 42 ii. Extraction of capsaicinoid 42 iii.Liquid chromatographic analysis 42 iv. Quantification of capsaicinoids 43 3.2.3. Capsaicin to dihydrocapsaicin ratio 43 3.2.4. Total capsaicinoids 43 3.2.5. Total carotenoids 43 3.2.6. Ascorbic acid 43 3.2.7. Total phenolics 44 3.3. Evaluation of hot peppers for aflatoxin contamination, microbial load and antioxidant quality during storage

44

3.3.1. Packaging and storage of samples 44 3.3.2. Moisture contents 44 3.3.3. Mycological studies 45 i. Sterilization of glass ware 45 ii. Media preparation 45 iii. Sabouraud agar (SA) composition 45 iv. Aspergillus flavus parasiticus agar (AFPA) ccomposition 45 v. Preparation of serial dilution 45 vi. Total fungal counts 46 vii. Aspergillus counts 46 3.3.4. Determination of aflatoxins 46 i. Aflatoxin standards 46

v

ii. Sample extraction and clean-up 46 iii. Liquid chromatographic analysis 47 iv. Quantification of aflatoxins 47 3.3.5. Antioxidants determination 47 3.4. Effect of gamma-radiation on microbial load and antioxidant quality of

aflatoxin contaminated samples during storage 47

3.5: Statistical analysis 48 Chapter 4. RESULTS AND DISCUSSION 49 4.1. Experiment I. Effect of plastic mulches on growth and yield of hot peppers 49 4.1.1: Soil temperature 49 4.1.2. Plant height at first harvest 51 4.1.3. Stem diameter 51 4.1.4. Root fresh weight 53 4.1.5. Root dry weight 53 4.1.6. Root length 55 4.1.7. Leaf area plant-1 57 4.1.8. Days taken to first flowering 57 4.1.9. Fresh fruit yield plant-1 62 4.1.10. Dry fruit yield plant-1 64

4.1.11. Harvest index 64 4.1.12. Fruit length 67 4.1.13. Fruit diameter 69 4.1.14. Leaf nitrogen content at fruit set 69 4.1.15. Leaf phosphorus contents at fruit set 73 4.1.16. Leaf potash content at fruit set 73 4.1.17. Conclusion 76 4.2. Experiment II. Quantification of antioxidant constituents in hot peppers

harvested at different stages 77

4.2.1. Capsaicin 77 4.2.2. Dihydrocapsaicin 81 4.2.3. Capsaicin to dihydrocapsaicin ratio 83 4.2.4. Total capsaicinoids 86 4.2.5. Total carotenoids 86 4.2.6. Ascorbic acid 91 4.2.7. Total phenolic contents 93 4.2.8. Conclusion 95 4.3. Experiment III. Evaluation of hot peppers for aflatoxin contamination,

microbial load and antioxidant quality during storage 96

4.3.1. Moisture contents 96 4.3.2. Total aflatoxins 96 4.3.3. Total fungal counts 103 4.3.4. Aspergillus flavus/parasiticus counts 105 4.3.5. Capsaicin 108

vi

4.3.6. Dihydrocapsaicin 110 4.3.7. Total carotenoids 113 4.3.8. Ascorbic acid 115 4.3.9. Total phenolic contents 118 4.3.10. Conclusion 120 4.4. Experiment IV. Effect of gamma-radiation on microbial load and antioxidant

quality of aflatoxin contaminated samples during storage 121

4.4.1. Moisture contents 121 4.4.2. Total aflatoxins 121 4.4.3. Total fungal counts 125 4.4.4. Aspergillus flavus/parasiticus counts 125 4.4.5. Capsaicin 129 4.4.6. Dihydrocapsaicin 132 4.4.7. Total carotenoids 132 4.4.8. Ascorbic acid 136 4.4.9. Total phenolic contents 136 4.4.10. Conclusion 139 Chapter 5. SUMMARY 140 REFERENCES 146

vii

LLIISSTT OOFF TTAABBLLEESS

Table Title Page 4.1.1 Effect of hybrids and plastic mulch on plant height (cm) in hot pepper. 52 4.1.2 Effect of hybrids and plastic mulch on stem diameter (mm) in hot pepper. 54 4.1.3 Effect of hybrids and plastic mulch on root fresh weight (g) in hot

pepper. 54 4.1.4 Effect of hybrids and plastic mulch on root dry weight (g) in hot pepper. 56 4.1.5 Effect of hybrids and plastic mulch on root length (cm) in hot pepper. 56 4.1.6 Effect of hybrids and plastic mulch on leaf area plant-1(cm2) in hot

pepper. 58 4.1.7 Effect of hybrids and plastic mulch on days taken to first flowering in

hot pepper. 60 4.1.8 Effect of hybrids and plastic mulch on fresh fruit yield plant-1 (kg) in hot

pepper. 63 4.1.9 Effect of hybrids and plastic mulch on dry fruit yield plant-1 (kg) in hot

pepper. 65 4.1.10 Effect of hybrids and plastic mulch on harvest index (%) in hot pepper. 66 4.1.11 Effect of hybrids and plastic mulch on fruit length (cm) in hot pepper. 68 4.1.12 Effect of hybrids and plastic mulch on fruit diameter (mm) in hot pepper. 70 4.1.13 Effect of hybrids and plastic mulch on leaf nitrogen (%) at fruit set in hot

pepper. 72 4.1.14 Effect of hybrids and plastic mulch on leaf phosphorus (%) at fruit set in

hot pepper. 74 4.1.15 Effect of hybrids and plastic mulch on leaf potash (%) at fruit set in hot

pepper. 74 4.2.1 Mean square values from analysis of variance for capsaicin and

dihydrocapsaicin of hot pepper hybrids harvested at different stages. 78 4.2.2 Mean square values from analysis of variance for capsaicin to

dihydrocapsaicin ratio and total capsaicinoids of hot pepper hybrids harvested at different stages. 84

4.2.3 Mean square values from analysis of variance for, total carotenoids, ascorbic acid and total phenolics content of hot pepper hybrids harvested at different stages. 88

4.3.1 Mean square values from analysis of variance for moisture contents (%age) of hot pepper hybrids under different storage conditions. 97

4.3.2. Mean square values from analysis of variance for total aflatoxins and total fungal counts of hot pepper hybrids under different storage conditions. 99

4.3.3 Mean square values from analysis of variance for Aspergillus flavus/parasiticus counts and capsaicin contents of hot pepper hybrids under different storage conditions. 106

4.3.4 Mean square values from analysis of variance for dihydrocapsaicin and total carotenoid contents of hot pepper hybrids under different storage conditions. 111

viii

4.3.5 Mean square values from analysis of variance for ascorbic acid and total phenolic contents of hot pepper hybrids under different storage conditions. 116

4.4.1 Mean square values from analysis of variance for moisture contents (%age) and total aflatoxins in hot pepper hybrids as affected by gamma radiation and storage 122

4.4.2 Mean square values from analysis of variance for total fungal counts and Aspergillus counts in hot pepper hybrids as affected by gamma radiation and storage 126

4.4.3 Mean square values from analysis of variance for capsaicin and dihydrocapsaicin concentration in hot pepper hybrids as affected by gamma radiation and storage 130

4.4.4 Mean square values from analysis of variance for total carotenoids, ascorbic acid and total phenolic contents in hot pepper hybrids as affected by gamma radiation and storage 134

ix

LLIISSTT OOFF FFIIGGUURREESS

Figure Title Page 2.1 Chemical structures of capsaicin (A) and dihydrocapsaicin (B). 16 2.2 Chemical structures of aflatoxins 24

4.1.1 Average soil temperature at a depth of 10cm after transplanting over three months for 15-day periods during the year 2005-06 and 2006-07 at 12.00-1.00 solar hour. 50

4.1.2 Interactive effect of hybrid x plastic mulch on plant height (cm) during the year 2006-07. 52

4.1.3 Interactive effect of hybrid x plastic mulch on leaf area per plant (cm2) during the year 2005-06 (A) and 2006-07 (B). 59

4.1.4 Interactive effect of hybrid x plastic mulch on days taken to first flowering during the year 2005-06 (A) and 2006-07 (B). 61

4.1.5 Interactive effect of hybrid x plastic mulch on fresh fruit yield per plant (kg) during the year 2006-07. 63

4.1.6 Interactive effect of hybrid x plastic mulch on dry fruit yield per plant (kg) during the year 2006-07. 65

4.1.7 Interactive effect of hybrid x plastic mulch on fruit length (cm) during the year 2006-07. 68

4.1.8 Interactive effect of hybrid x plastic mulch on fruit diameter (mm) during the year 2005-06 (A) and 2006-07 (B). 71

4.1.9 Interactive effect of hybrid x plastic mulch on leaf nitrogen content (%) during the year 2005-06. 72

4.1.10 Interactive effect of hybrid x plastic mulch on leaf potash contents during the year 2005-06 (A) and 2006-07 (B). 75

4.2.1 Chromatographs of capsaicinoid compounds. A-Standard peaks and B-Peaks of sample 79

4.2.2 Pattern of capsaicin mg 100g-1 distribution in hot peppers harvested at different stages. 80

4.2.3 Pattern of dihydrocapsaicin distribution mg 100g-1 in hot peppers harvested at different stages. 82

4.2.4 Pattern of capsaicin to dihydrocapsaicin ratio in hot peppers harvested at different stages. 85

4.2.5 Pattern of total capsaicinoids distribution in hot peppers harvested at different stages. 87

4.2.6 Pattern of total carotenoids mg 100g-1 distribution in hot peppers harvested at different stages. 89

4.2.7 Pattern of ascorbic acid mg 100g-1 distribution in hot peppers harvested at different stages. 92

4.2.8 Pattern of total phenolic contents mg 100g-1 distribution in hot peppers harvested at different stages. 94

4.3.1 Moisture (%) in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene 98

x

Bag and JB: Jute Bag) during five month storage. 4.2.2 Chromatographs of aflatoxins. A-Standard peaks and B-Peaks of sample 100 4.3.3 Aflatoxin contamination in hot peppers viz. Sky Red (A), Maha (B) and

Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 101

4.3.4 Total fungal counts in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 104

4.3.5 Aspergillus flavus/parasiticus counts in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 107

4.3.6 Capsaicin concentration in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 109

4.3.7 Dihydrocapsaicin concentration in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 112

4.3.8 Total carotenoid content in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 114

4.3.9 Ascorbic acid content in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 117

4.3.10 Total phenolic content in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. 119

4.4.1 Effect of gamma radiation on moisture contents in hot peppers during three months storage. 123

4.4.2 Effect of gamma radiation on total aflatoxins in hot peppers during three months storage. 124

4.4.3 Effect of gamma radiation on total fungal counts in hot peppers during three months storage. 127

4.4.4 Effect of gamma radiation on Aspergillus flavus/parasiticus counts in hot peppers during storage. 128

4.4.5 Effect of gamma radiation on capsaicin concentration in hot peppers during three months storage. 131

4.4.6 Effect of gamma radiation on dihydrocapsaicin concentration in hot peppers three months storage. 133

4.4.7 Effect of gamma radiation on total carotenoids concentration in hot peppers during three months storage. 135

4.4.8 Effect of gamma radiation on ascorbic acid concentration in hot peppers during three months storage. 137

4.4.9 Effect of gamma radiation on ascorbic acid concentration in hot peppers during three months storage. 138

xi

ABSTRACT

Studies were conducted to evaluate the impact of plastic mulches viz. black, clear and

bare soil in the modification of plant growing environments on three hot pepper hybrids

namely Sky Red, Maha and Wonder King in poly/plastic tunnels during the year 2005-06

and 2006-07. Hot pepper hybrids and plastic mulches had significant effect on plant

growth and yield attributes. By using clear plastic mulch intensive weed proliferation was

problematic issue; however under black plastic mulch almost complete weed suppression

was achieved which results in increased fruit yield than hot peppers grown under clear

plastic mulch and bare soil (control).

The pattern of antioxidant accumulation was envisaged in hot peppers harvested at

different stages; immature green, mature green, color break, red ripe and dried fruit.

Capsaicinoids had significant distribution in mature green stage while progressive

accretion of carotenoids and ascorbic acid was observed at dried and red ripe stage of all

hybrids, respectively. However, the pattern of total phenolic contents biosynthesis was

found significant at immature green stage in Sky Red where as in Maha at color break

stage and in case of Wonder King at red ripe stage.

Aflatoxin contamination in hot pepper hybrids was investigated under various

temperatures (20, 25 and 30°C) and packaging regimes (polyethylene and jute bags)

during five months storage period. Aflatoxin detection under these conditions had lower

levels than the existing regulatory limits ascribed by European Commission (EC No.

1881/2006) that is 10µg kg-1 for total aflatoxins. Aflatoxin contamination and microbial

load was increased significantly with the increase in temperature and storage duration

which was heavily infested when samples packed in jute bags and stored at 25 and 30°C

respectively. Storage duration and temperature regimes had inverse relation on

antioxidant quality of hot pepper ecotypes as well.

Further attempts were made to decontaminate aflatoxin contaminated samples of hot

pepper hybrids (from previous study) subjected to gamma radiation (2, 4 and 6 kGy) and

its effect on antioxidant stability was again assessed after three month storage. Higher the

irradiation dose, lower the concentration of carotenoids and ascorbic acid ascertained in

hot pepper ecotypes; however, capsaicinoids and polyphenols rendered greater stability at

higher irradiation dose during storage. Irradiated samples of hot peppers had 7%

xii

reduction in aflatoxin contamination as compared to non-irradiated (control). Total fungal

population had inverse relation with increasing radiation dose and complete inhibition

was observed when irradiated at 6 kGy and no further fungal proliferation was seen

during three months storage at ambient conditions.

1

CChhaapptteerr 11

IINNTTRROODDUUCCTTIIOONN

Pepper, chilli, chile, or chili belongs to the Solanaceae family genus Capsicum and is

closely related to tomato, eggplant, potato and tobacco. The genus Capsicum represents a

diverse plant group and includes twenty seven species; five domesticated and twenty two

un-domesticated (Bosland, 1993). Capsicum annuum, C. frutescens, C. chinense, C.

baccatum and C. pubescens are considered domesticated species of peppers. The use and

uses of the numerous cultivars within the five domesticated species have grown

exponentially. Despite their vast trait differences, virtually all commercially cultivated

Capsicum cultivars in the world belong to C. annuum. Peppers are quite diverse and may

be classified by the trade according to the end use. Peppers grown for their characteristics

hot flavor are of genus Capsicum, C. annuum L. var. annuum principally and C.

frutescens L. to a lesser extent. A second pepper type valued for its brilliant deep red

color is paprika, evoking none or only negligible pungency. Both types have a distinct

aroma, valuable in certain formulations. A third pepper type classified according to end

use is C. annuum L. var. annuum, the large sized fleshy bell pepper used as fresh

vegetable and valued for its aroma, color and crisp texture, but evokes no pungency

(Govindarajan and Sathyanarayana, 1991).

Hot pepper is an important agricultural crop, not only because of its economic

importance, but also due to nutritional and medicinal value of its fruits. These are the

excellent source of natural colors and antioxidant compounds (Howard et al., 2000). A

wide spectrum of antioxidant vitamins, carotenoids, capsaicinoids and phenolic

compounds are present in hot pepper fruits. The intake of these compounds in food is an

important health-protecting factor by prevention of widespread human diseases. As

consumption continues to increase, hot peppers could provide important amounts of

nutritional antioxidants to the human diet. Levels of these antioxidants can vary with

genotype, stage of harvest/maturity and plant part consumed as well as storage and

processing conditions (Daood et al., 1996 and Marin et al., 2004) but maturation affects

synthesis of these compounds which influence hot pepper quality e.g. differences in hot

2

pepper color, shape and capsaicin level changes continuously during maturation.

Important nutrients like ascorbic acid and provitamin A increased from green stage to the

red stage (Howard et al., 1994 and Sidonia et al., 2005). Therefore, stage of harvest is an

important factor affecting the content of these antioxidants in hot pepper fruits. Fixing the

harvesting stage for high antioxidant contents would be helpful in deciding maximum

retention of antioxidant compounds important for improved human health and nutrition.

Thus it becomes pertinent to study these variations at different harvest stages in hot

pepper cultivars to select the best one for health benefits.

The National Master Agriculture Research Plan 1996-2005 for Pakistan identified hot

pepper as a crop requiring research to increase and stabilize yield and quality (PARC,

1996). Acreage under hot peppers is increasing particularly in Punjab due to a shift in

production trend from cotton based farming to non-traditional crop production which in

turn is due to a decline in income from cotton crop. In Pakistan, dry chilli peppers are

cultivated on an area of 66 thousand hectares with 130 thousand tonnes of production

while in China dry chilli pepper are cultivated on an area of 40 thousand hectares with

production of 250 thousand tonnes. In Pakistan, yield per hectare is 1.96 tonnes as

compared to 6.25 tonnes in other dry chilli pepper producing countries like China (FAO,

2007); the yield gap is too high. Major factors limiting pepper production are lack of high

yielding and disease resistant varieties, poor cultural practices and uncertain weather

conditions.

Hot pepper is a summer vegetable grown in southern Punjab and Sindh, Pakistan. The

normal planting time in Punjab starts from mid February and fruit pickings continues up

to August. In the month of July and August, the spell of rains starts which is

characteristic feature of monsoon season. In this season, splash of rains deteriorates the

fruit quality because of high humidity which accelerates the rottening and dried fruit

becomes more susceptible for aflatoxin contamination. The presence of aflatoxin is a

serious concern for dry chilli pepper exporters from Pakistan.

During the last decade, the area under protected cultivation (poly/plastic tunnels) of

vegetables like hot peppers, tomatoes and cucumbers in plastic tunnels is increasing

steadily. Hot pepper is one of the potential crop to be grown in poly/plastic tunnels. The

climate of Punjab is suitable for simple unheated poly/plastic tunnels for indoor vegetable

3

production which is well suited for the purposes of over-bridging the gap in vegetable

markets during cool months or to extend the season to be earlier in the market than the

produce of the open field. Similarly, hot peppers for spice production gave advantage to

be harvested and dried before the onset of rainy season. In poly/plastic tunnels, plastic

mulches have been used in many regions of the world for commercial production of

vegetables in order to maximize water use efficiency by the plant, to reduce weeds and to

get early and quality produce. But in Pakistan, it is a new technique and area is growing

up year after year. So far no research information is available for local farming

community on hot pepper production in poly/plastic tunnels using plastic mulch.

Mulching with plant residues and synthetic material is a well-established technique for

increasing the profitability of many horticultural crops (Gimenez et al., 2002). The

practice consists in placing mulch over the soil surface to create a favorable soil-water-

plant relation. Mulches create a microenvironment by retaining soil moisture and by

changing root-zone temperatures and the quantity and quality of light reflected back to

the plants. This change in reflected light alters plant growth and development. Plastic

mulches affect plant microclimate by modifying the soil energy balance and restricting

soil water evaporation, thereby affecting plant growth and yield. Vegetable crops grown

under plastic mulches have shown earlier (7 to 14 days) and increased yields (2 to 3

times) over bare soil (Lamont, 1993 and Ibarra-Jimenez et al., 2004).

Hot pepper production in Pakistan not only fulfills domestic needs but also helps in

earning foreign exchange. Pakistan earned Rs. 192.32 million during 2004-05 by

exporting red chilli pepper to Middle East, USA and other countries (Amjad et al., 2007).

But in recent years export of dried red chilli peppers from Pakistan has declined after the

detection of aflatoxin by European Union (EU) food authorities (Russell and Paterson,

2007). Therefore, the presence of aflatoxin is a serious concern for dry chilli exporters

from Pakistan. Aflatoxin contamination can occur in the field, during crop production,

but also during storage if conditions are favorable. The filamentous fungi that are major

producers of aflatoxin are Aspergillus flavus and Aspergillus parasiticus. Dry chilli

pepper is one of the favorite spice in Pakistan and is very sensitive to aflatoxin

contamination depending on atmospheric temperature, humidity, drying and processing

conditions (Cokosoyler, 1999). They are usually sun dried in open fields and stored under

4

poor conditions which might results in aflatoxin contamination. Contamination with A.

flavus and subsequent production of aflatoxin during storage is considered as one of the

most serious safety problems of peppers in Pakistan. Development of storage fungi in a

post-harvest commodity can also be influenced by length of time in storage. The longer

the storage time, the greater the possibility of building up environmental conditions

conducive to aflatoxigenic moulds proliferation and subsequent mycotoxin production.

Moreover, since temperature and relative humidity are important factors for aflatoxin

production, it is of great interest to evaluate the effect of these parameters on aflatoxin

production during storage. The permissible limit to export dry chilli for aflatoxin B1 is 5

ppb and total aflatoxin 10 ppb as per regulatory limits in the European Commission

(Russell and Paterson, 2007).

With increasing awareness and knowledge about aflatoxins, a lot of efforts have been

made to eliminate aflatoxin completely or to reduce their contents in the foods and

feedstuffs to significantly lower levels because they are potent source of health hazards to

both man and farm animals. Spices such as red peppers are frequently exposed to insects

and microorganisms during cultivation and storage which may be potential contamination

sources in foods even when added in small amounts. A major concern of food processors

is to assure that the microbial load of ingredients and processing aids does not contribute

to spoilage of food and does not diminish its microbial safety. The microbial numbers can

be reduced by the use of chemicals, such as ethylene oxide and methyl bromide but these

chemicals now have been considered as dangerous to humans and/or the environment.

Many studies have shown that irradiation is a safe process; therefore in 1994 WHO

declared that irradiation of food is safe from nutritional and toxicological point of view

(Dwyer et al., 2003). Food irradiation is the process of exposing food to a carefully

controlled amount of energy in the form of high-speed particles or rays. Interest in the

irradiation process is increasing because of persistently high food losses from infestation,

contamination and spoilage; mounting concerns over food-borne diseases and growing

international trade in food products that must meet strict import standards of quality and

quarantine (Sadecka, 2007). Despite the considerable potential benefits offered by the

application of irradiation in the processing of food products, there has been significant

consumer resistance to the process which has effectively constrained the introduction of

5

the technology (Henson, 1995). Dried foods, such as red peppers are less sensitive to

irradiation than hydrated ones and their irradiation has been authorized at a maximum

dose of 10 kGy and 30 kGy in Korea and United States respectively (Olson, 1998). These

irradiation doses further need to be optimized to be effective against a particular strain of

aflatoxin as well as to be safe to maintain the hot pepper quality. Keeping in view the

needs of hot pepper producers, processors and exporters, this study was planned to

provide some of the necessary information for the safe production, handling and storage

of hot pepper in order to limit microbial load and aflatoxin contamination and thereby

helping to maintain a viable export trade.

The specific objectives of present study were:

1. To assess the impact of plastic mulches on hot pepper productivity

2. To quantify the accumulation of antioxidant constituents in hot peppers harvested

at different stages

3. To measure the extent of aflatoxin contamination, microbial load and antioxidant

quality of whole peppers under different storage conditions

4. To assess the effect of gamma-radiation on microbial load and antioxidant quality

of aflatoxin contaminated whole peppers during storage

6

CChhaapptteerr 22

RREEVVIIEEWW

OOFF

LLIITTEERRAATTUURREE

The popularity of hot peppers for spice, vegetable and other uses increases every year. In

this regard, peppers are principal component of curry and chilli powder, it can be used to

make pepper sauce, red pepper and paprika. It can also be used as dried or fresh whole

peppers to make canned and pickled peppers. Hot peppers are also used in the medical

field with pungency being an important pharmacological property. These are also

extremely good sources of many essential nutrients and are richer sources of vitamin A

and C. Another major use of pepper is as a coloring agent in food industry to color a wide

variety of processed foods.

A lot of research work has been carried out on various aspects of hot peppers however;

the literature available pertaining to the present study has been reviewed under the

following headings:

2.1: Use of plastic mulches in vegetable production

2.1.1 Effect of plastic mulches on soil temperature

2.1.2 Effect of plastic mulches on soil moisture

2.1.3 Effect of plastic mulches on weed control

2.1.4 Effect of plastic mulches on plant growth and yield

2.1.5 Mulch type/color (black and clear plastic mulch)

2.1.6 Effect of mulch type/color on insect/pest and disease

2.2: Hot peppers as a source of antioxidants

2.2.1 Capsaicinoids

2.2.2 Carotenoids

2.2.3 Ascorbic acid

2.2.4 Phenolic compounds

7

2.3: Aflatoxin contamination as a concern

2.3.1 Chemistry of aflatoxins

2.3.2 Aflatoxins and health

2.3.3 Occurrence

2.3.4 Aflatoxins and storage

2.4: Aflatoxins, microbial load and gamma radiation

2.1: Use of plastic mulches in vegetable production

Since the beginning of civilization, the man has developed technologies to increase the

efficiency of food production. The use of plastic mulch in commercial vegetable

production is one of these traditional techniques that have been used since 1950’s. A

favorable soil-water-plant relation is created by placing mulch over the soil surface. The

microclimate surrounding the plant and soil is significantly affected by mulch i.e. the

thermodynamic environment, the moisture, the erosion, the physical soil structure, the

incidence of pests and diseases, crop growth and yield.

In order to maximize water use efficiency by the plant and to improve the quality of

produce, the use of mulch has become an important cultural practice in many regions of

the world for the commercial production of vegetable crops. In Pakistan, use of inorganic

mulches is a new technique and area under plastic mulch is slowly growing up year after

year. The type of mulching material is not fully documented, especially for hot peppers

cultivation in poly/plastic tunnels. Mulching with plant residues and synthetic material is

a well-established technique for increasing the profitability of many horticultural crops

(Gimenez et al., 2002). Mulches create a microenvironment by retaining soil moisture

and changing root-zone temperatures and the quantity and quality of light reflected back

to the plants which alter plant growth and development (Csizinszky et al., 1995). Plastic

mulches affect plant microclimate by modifying the soil energy balance and restricting

soil water evaporation, thereby affecting plant growth and its yield (Tarara, 2000 and

Voorhees et al., 1981).

2.1.1: Effect of plastic mulches on soil temperature

Different forms of plastic mulch are available varying from woven plastic to smooth

plastic and embossed plastic films. Now-a-days 100% compostable and biodegradable

mulches are also available in advanced countries and these are more environment

8

friendly. In addition to the surface structure, the color and thickness of the mulch creates

lot of variations which have an effect on the plant microclimate and in particular the soil

temperature. Soil temperatures can be increased in the field by applying plastic mulches.

Darrow (1966) asserted that soil heat loss can be hindered by plastic coverings leading to

a sustained increase in soil temperature. Himelrick et al. (1993) found that soil

temperatures were warmest with clear plastic mulch followed in order of decreasing

temperatures by black-on-white, black, white on-black and bare ground. Soil temperature

is increased by 5 to 10°C by the application of plastic mulches when compared to bare

soil (Elmer and Ferrandino, 1991). This well documented temperature rise is often used

as an explanation for increased production of crops grown on plastic mulch (Grubinger et

al., 1993 and Davis, 1994). Plastic mulches modify the soil temperature regime according

to their optical properties (Ham et al., 1993). Changes in root zone temperature can affect

the uptake and translocation of essential nutrients, therefore influencing root and shoot

growth (Tindall et al., 1990). Increased soil temperatures also affect the crop in other

ways. Extreme solar heating of the soi1 can lead to improve plant health by controlling

soil-borne pathogens (Katan et al., 1976).

Each of the different colored mulches used in the production of crops causes different

temperature effects. Black plastic film is the most common form of mulch and has been

shown to cause a significant temperature rise in soils (Wein et al., 1993). Clear plastic

mulch is often used for soil sterilization (solarisation) - the plastic film is fixed over wet

soil to trap solar heat which kills weeds and soil pathogens. Clear plastic is believed to

achieve higher soil temperatures than black plastic. This happens because much of the

incident radiation is absorbed by colored films (Argall and Stewart, 1990) and does not

pass through to the soil. Ham et al. (1993) showed that the placement of the mulch was

important to raise temperature. Results indicated that clear plastic heated soil less than

black plastic, if it was placed tightly across the soil with good contact between the soil

surface and the mulch. They also suggested that if clear plastic mulches placed loosely

over the soil an insulating air layer develops which results in the soil heat storage and

reducing heat loss. Locher et al. (2005) revealed that use of dark colored mulch is the

safest solution because even in case of high air temperature and solar radiation, the soil

does not warm to a harmful degree. They observed that in case of light colored mulches

9

(clear, violet, light green) the soil temperature increased 2.5-2.9°C higher than in case of

the un-mulched control. They also mentioned in their studies that dark colored mulches

(black, dark green, red) increased soil temperature 1.4-2.1°C compared to the un-mulched

(control). Overall studies indicated that higher yields of sweet peppers were achieved

from mulched treatments due to higher soil temperatures than the un-mulched treatment.

2.1.2: Effect of plastic mulches on soil moisture

Water is essential for growth and development. It is also a major cost in agricultural

systems. The success of many agricultural forms relies on conservative and efficient use

of water. Moisture retention is undoubtedly the most common reason for which mulch is

applied to soil. Mulch is used to protect the soil from direct exposure to the sun which

would evaporate moisture from the soil surface and cause drying of the soil profile. The

protective interface established by the mulch stops raindrop splash by absorbing the

impact energy of the rain, hence reducing soil surface crust formation. The mulch also

slows soil surface runoff allowing a longer infiltration time. These features result in

improved water infiltration rates and higher soil moisture. An auxiliary benefit of mulch

reducing soil splash is the decreased need for additional cleaning prior to processing of

the herb foliage (Barker, 1990). Organic and inorganic mulches have been shown to

improve the moisture retention of soil. This extended water holding ability enables plants

to survive during low rainfall periods.

The use of plastic mulch can be improved if under-mulch irrigation is used in

combination with soil moisture monitoring. The influence of rainfall events are not as

great when plastic mulch is used, necessitating active irrigation management. Under-

mulch, irrigation of vegetable crops has been shown to improve crop yields more than

overhead irrigation systems (Clough et al., 1990). Mulch enables the soil moisture levels

to maintain for longer periods. In some cases while providing improved moisture

conditions within the soil, the mulch changes the plants microclimate so that it uses more

water (Clark and Moore, 1991 and Zajicek and Heilman, 1991), thus negating the initial

benefit. Plastic mulch conserved 47.08% of water and increased yield by 47.67% in

tomato when compared to un-mulched control (Friake et al., 1990). Palada et al. (2003)

concluded that plastic mulching resulted in 33 to 52% more efficient use of irrigation

water in bell pepper compared to bare soil.

10

2.1.3: Effect of plastic mulches on weed control

Weed control in crops is a difficult, time consuming and expensive task. Plastic mulches

have the potential to alter soil temperature, crop water use, improve crop quality and in

some cases reduce weed competition, thereby improving crop development and

increasing yields (Lamont, 2005 and Ngouaajio and Ernest, 2005). Black plastic mulch is

both effective at warming the soil and reducing weed competition. Clear plastic mulch

provides greater soil warming, but it does not reduce the weed competition (Lamont,

2005). Dark colored mulches lay across the soil and around the crop reduce the amount of

light reaching the soil and thus inhibit weed germination and smother emerging weeds.

Mulching for weed control can take a number of forms: inorganic or organic mulches can

be applied and left in situ to control the weeds; living mulches can be grown to choke out

weeds before planting the mulches are either killed with chemicals or complete their life

cycle before the growing season of the herb. Solarisation uses an inorganic mulch and

solar energy to disinfect the soil, the mulch being removed prior to planting. Similarly

100% weed control was observed in cassava peel with black plastic mulch as compared

to bare soil (Aniekwe et al., 2004).

2.1.4: Effect of plastic mulches on plant growth and yield

Organic or inorganic soil mulches influence the crop in a number of ways. Plastic

mulches can offer a barrier against weeds, moisture loss, nutrient loss, erosion, insect and

disease injury while encouraging plant establishment and an earlier crop of potentially

higher quality (Mugalla et al., 1996). The combined effects of soil temperature, soil

moisture and weed suppression not only work to improve crop growth but they also

facilitate hand picking and lead to higher yield and increased fruit size (Scheerens and

Brenneman, 1994). Increase in soil temperature by application of plastic mulch caused a

significant reduction in pathogen levels, at lower cost than un-mulched, fumigated soi1

fields and at lower phytotoxicity levels (Abdul-Baki et al., 1996). The effect of plastic

mulch and its color improve soil structure, crop growth and its development. Growth,

yield and nutrient uptake are affected by plastic mulch and initial nitrogen levels in the

soil (Wein and Minotti, 1988). Karp et al. (2006) reported that mulching treatment

significantly influenced nutrient content of leaves and chlorophyll contents (381 SPAD

units) were significantly lower in control plants compared with plants grown on different

11

mulches (498 and 542 SPAD units). Chilli plants grown on plastic mulch had

significantly higher N and K contents in leaf tissues at early fruiting stage when

compared with bare soil (Hassan et al., 1995). Plastic mulches increased crop growth

(3.2–4.0 cm), dry root mass (12.2–50.1%), nitrogen fixing activity (3.3–12.8%), leaf

chlorophyll content (41–78%) more reproductive buds (63.3–94.1%) and starts flowering

9 days earlier in groundnut than un-mulched control (Hu et al., 1995).

Plastic mulches absorbed incident radiation that can be readily transmitted to the soil

surface and the air is relatively immobile near the soil surface with a low thermal

conductivity which increases soil temperature consistently (Cooper, 1973). This increase

in soil temperature consistently improves root development in vegetables grown under

mulches. Gupta and Acharya (1993) observed increased root mass under black

polyethylene mulch was attributed to the resultant increase in soil temperature and

nutrient uptake. Niu et al. (2004) concluded that improved productivity was related to

increased root dry weight under mulches and larger rooting systems resulted in greater

ability to take up water and nutrients that led to higher grain yield with mulched wheat.

Plastic mulches improved stand establishment and fruit yields relative to un-mulched

control. Vegetable crops grown under plastic mulches have shown earlier 7 to14 days and

increased yields 2 to 3 times over vegetable crops grown on bare soil (Lamont, 1993).

Mulches ameliorated soil hydrothermal regime, improved vegetative growth, advanced

flowering and fruit yield of tomato plants when compared with bare soil (Agele et al.,

2000). Kirnak and Demirtas (2006) both root and shoot dry weights of cucumber plants

were significantly improved by plastic mulch. Ibarra et al. (2001) concluded that

watermelon plants grown under plastic mulch and row cover showed greater plant

biomass, specific leaf area, relative growth rate and net assimilation rate than bare soil

plants. Similarly time to anthesis (appearance of perfect flowers) was 45 and 55 days

after sowing for black plastic mulch and control plants respectively. The number of

leaves per plant or dry weight per plant better explains the changes in watermelon yield

than net photosynthesis rate (Ibarra-Jimenez et al., 2005). Similarly plant height, number

and length of main roots, fresh and dry weights of roots as well as number of flower were

significantly higher in plants grown on mulch as compared to bare soil (Hasan et al.,

2005). Leaf area ratio and leaf weight ratio were not significantly different in melon

12

grown under plastic mulch but 10-20% higher than those on the bare soil (control) (Lopez

et al., 2000).

2.1.5: Mulch type/color (black and clear plastic mulch)

Mulching with black or clear plastic increased total plant growth and led to an increased

rate of branching and early flowering in tomato (Wein and Minotti, 1988). Hassan et al.

(1995) reported that mulching is practically beneficial in chilli production. They

concluded that increased plant growth for mulched plants may be related to soil moisture

content because plant dry weight was positively correlated with soil temperature and

moisture content. Higher yield of peppers were observed under clear plastic mulch

followed by black and wavelength selective mulch treatments as compare to bare soil;

although weed proliferation was extensive under clear plastic mulch with no added

herbicide (Waterer, 2000). Hallidri (2001) stated that plant height and number of leaves

were higher in black and transparent polythene mulch than control (bare soil) while no

significant difference was observed in case of stem diameter in cucumber. However,

cucumber plants grown on transparent polythene mulch gave the highest number of fruits

per plant and yield. Total plant and leaf fresh weights in plots with black plastic mulch

were higher as compare to bare soil (Palada et al., 1999). Aniekwe et al., (2004)

concluded that leaf area and fresh root tuber yield of cassava varieties was significantly

improved by the application of black plastic mulch with 100% weed control as compared

to bare soil.

Black plastic mulch doubled the yield of tomatoes as well as increasing the amount of

early production for some cultivars when compared with un-mulched control (Abdul-

Baki et al., 1992). Higher tomato yields were reported when black plastic mulch and row

covers were used together is partially due to increase in air and soil temperatures around

the plant growing environment (Znidarcic et al., 2004). Most suitable soil temperature

distribution was observed by the application of clear plastic mulch and it was more

effective on first blossoming and harvesting time, leaf area and total yield in squash,

while lowest plant growth and yield values were observed in bare soil (Tuli and Yesilsoy,

1997). Significant increase was observed in strawberry runners and fruits with the use of

black plastic mulch as compared to clear plastic, white plastic and bare ground treatments

(Himelrick, 1982). A general increase in plant growth and fruit size in hot peppers was

13

observed by the use of plastic mulch while clear plastic mulch increased the early and

total yield by 39% and 19% respectively (Pakyurek et al., 1993). Salau et al. (1991)

reported that mulching significantly enhanced vegetative growth and increased plantain

bunch yield in both first and second year crops. Increase in total yield (first and second

year crops) on an average was about 41% higher with mulched treatments than with the

control. Farias-Larios and Orzoc-Santos (1997) found increased fruit weight (2.94 kg)

and yield 25.5 tonnes ha-1 in watermelon by the application of clear plastic mulch as

compared to un-mulched soil. However no change was observed in total soluble solids of

watermelon fruits by different types of plastic mulches but both clear and white plastic

mulch increased fruit length. Brown et al. (2001) reported that bell peppers grown on

black plastic mulch alone or in combination with drip irrigation increased pepper yields

by 18 and 16 metric tonnes ha-1 respectively when compared with bare soil. Luis et al.

(2002) found that total yield of bell pepper was increased by BPM (black plastic mulch)

alone or combined with row covers by around 10 tones ha-1 compared with control. The

same treatments had a positive effect relative to control in leaf area, specific leaf area and

net assimilation rate. Ibarra-Jimenez et al. (2004) reported that dry weight of cucumber

plants grown under plastic mulch or mulch combined with row covers (at 50 and 110

days after seeding) were significantly different from bare soil plants. Although an early

yield with black plastic mulch was 2.1 times greater when compared with control (10

tonnes ha-1).

2.1.5: Effect of mulch type/color on insects/pests and diseases

Diaz-Perez et al. (2003) studied the relationship between tomato plant growth and fruit

yield with the time of TSWV (tomato spotted wilt virus) symptom appearance on

different colored plastic mulches. They reported that time between transplanting and

appearance of first TSWV symptoms was affected by mulch color and tomato cultivars.

Vegetative top fresh weight, fruit number and total yield increased linearly with the time

plants remained free from symptoms of TSWV in all tomato cultivars. Marketable fruit

yield also increased as the time from transplanting to first appearance of symptoms

increased. Clear plastic mulch has repellent effect on vector; aphids, in Lupinus

angustifolius (Jones, 1991), which has reflective effect and help in reducing the

appearance of viral disease confusing aphids in cantaloupe (Orozco-Santos et al., 1995).

14

Farias-Larios and Orzoc-Santos (1997) concluded that clear plastic mulch could be a

practical management tool for reducing insect populations, virus incidence and increasing

soil temperature, watermelon production and enhancement of fruit quality. Aphids were

less severe on clear plastic mulch than on bare soil and black plastic mulch. Low numbers

of whiteflies on the white and clear plastic mulches during early cycle of culture delayed

virus symptom development. Suwwan et al. (1988) reported that incidence of tomato

yellow leaf curl (TYLC) virus was reduced by the silver plastic mulch. Early and total

fruit counts on both silver and white/black plastic mulches were superior to other

treatments while incidence of sunscald increased significantly but shoot dry weight,

seasonal cracking and incidence of blossom end rot (BER) were not affected by the

mulch treatments. However, Siborlabane (2000) indicated that burnt rice husk and black

plastic mulch were the best among the mulching materials in producing better yield and

quality of fresh market tomato. Black plastic mulch also showed to be highly effective in

controlling weeds and minimizing southern blight and TYLCV incidence.

2.2: Hot peppers as a source of antioxidants

There are growing evidences suggesting that antioxidants may maintain health and

prevent many chronic diseases, such as certain cancers, cardiovascular diseases and other

aging-related diseases (Thompson, 1994). Antioxidants may suppress the formation of

free radicals, quench the existing radicals and reduce the availability of oxygen in

biological system to prevent the oxidative damage of proteins, DNA and lipids in human

body (Jacobs et al., 1995). However, so far direct experimental evidences are lacking

about the beneficial effects of antioxidants which has led to different recommendations

for different populations. For example, the use of supplemental β-carotene has been

identified as a contributing factor to increase risk of lung cancer in smokers (Goodman et

al., 2004) while the risk has not been identified in non-smokers. These studies suggested

that a precaution regarding the use of supplemental β-carotene is not warranted for non-

smokers. If supplementation is desired, the use of a daily multivitamin-mineral

supplement containing antioxidants has been recommended for the general public as the

best advice at this time (Fairfield et al., 2002).

Capsicum cultivars have been identified as potential solanaceous crop with high

antioxidant activity (Ou et al., 2002). Hot pepper is an important vegetable crop both

15

economically and nutritionally because these are excellent sources of natural colors and

antioxidant compounds. Intake of these compounds in food is an important health

protecting factor. They are also helpful in prevention of widespread diseases. A wide

spectrum of antioxidant vitamins, carotenoids, capsaicinoids and phenolic compounds are

present in hot pepper fruits.

2.2.1: Capsaicinoids

Hot peppers are popular food additives valued for their sensory attributes of color,

pungency and aroma in many regions of the world. The fruit is very important

economically due to the vast quantity and the diverse varieties used. The consumption of

hot peppers is mainly due to their pungent flavor which is an important marketing

characteristic and is determined by the production of capsaicinoids within pod.

Capsaicinoids are alkaloid compounds that produce the hot flavor or pungency associated

with eating chillies (Collins et al., 1995). Five analogs make up the collective term

capsaicinoids: capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin

and homocapsaicin. Capsaicin is the predominant capsaicinoid and one of the most

pungent compounds known. Capsaicin and dihydrocapsaicin accounts for more than 90%

of the capsaicinoids in hot peppers and contribute most to pungency (Todd et al., 1977).

Kraikruan et al. (2008) reported that capsaicin concentration was higher than

dihydrocapsaicin and the capsaicin to dihydrocapsaicin ratio was in the range of 1.2:1 to

2.3:1 in nine chilli cultivars studied. Similarly, Zewdie and Bosland (2000) observed a

ratio of about 2:1 in Capsicum frutescens. Quantification of these pungent compounds is

therefore an important index of hot pepper quality (Gibbs and O’Garro, 2004).

Capsaicinoids possess strong physiological and pharmacological activities. In addition to

their widespread nature and use as a neuropharmacological property, the medicinal value

of capsaicin has been evaluated in the treatment of painful conditions such as rheumatic

diseases, cluster headaches, painful diabetic neuropathy and post-herpetic neuralgia. Even

consumed at low amounts in diet, capsaicinoids decrease the myocardial and aortic

cholesterol levels. Today, capsaicinoids are being studied as an effective treatment for a

variety of sensory nerve disorders, including arthritis, cystitis and human

immunodeficiency virus (Perucka and Materska, 2001) and due to antibacterial properties

found to be effective on certain groups of bacteria (Dornates et al., 2000). Capsaicinoids

16

(A)

(B)

Figure 2.1 Chemical structures of Capsaicin (A) and Dihydrocapsaicin (B)

Source: http://en.wikipedia.org/wiki/Capsaicin

17

biosynthesis takes place in the epidermal cells of placenta of Capsicum fruits and

accumulated in blisters along with the epidermis (Suzuki et al., 1980). Number of

enzymes is being involved in this pathway which starts approximately 20 days post-

anthesis (Iwai et al., 1979).

The degree of pungency depends on the Capsicum species, cultivars and their

concentration can be affected by different factors like fruit development stage (Sukrasno

and Yeoman, 1993), genetic character and agro-climatic conditions of each cultivar

(Cisneros-Pineda et al., 2007). Capsaicinoids concentration in hot peppers limits from

0.003 to 0.01% while mild chillies had 0.05 to 0.3% and 0.3-1% was recorded in strong

chillies (Perucka and Oleszek, 2000).

Fixing the stage of harvest for high capsaicin and dihydrocapsaicin concentration would

be helpful in deciding proper harvesting stage for increasing the quality of the produce.

Accurate measurement of pungency has become important because of the increased

demand by consumers for foods; moreover, accurate determination of various

capsaicinoids level is also needed due to their increased use in pharmaceuticals

(Carmichael, 1991). Accumulation of capsaicin starts at an early stage of fruit

development 14 days after flowering (Estrada et al., 1999) and reaching the maximum at

final growth stage of fruit maturity (Contreras-Padilla and Yahia, 1998) and then

decreases by rapid turn over and up to 60% degradation was observed (Iwai et al., 1979)

Gnayfeed et al. (2001) analyzed fruits of four paprika cultivars (K-V2, SZ-178, F-03 and

Cseresznye) harvested at different stages of fruit ripening (green, color break, red, over

ripe and dried) for capsaicinoids determination and found that changes in capsaicinoids

were not similar between different varieties. Total capsaicinoids accumulation in K-V2

and SZ-178 was at color break stage. However, in case of F-03 and Cseresznye the

highest level of capsaicinoids were found at red ripe stage. Loss of capsaicinoids during

maturation and senescence of hot peppers has been related to activity of peroxidase; in

which they differ significantly from each other. Differences in biochemical factors within

varieties can influence the biosynthesis and stability of capsaicinoids during ripening and

drying (Ananthasamy et al., 1960 and Gnayfeed et al., 2001).

Estrada et al. (2000) reported that changes in capsaicinoid concentration increases

steadily with fruit development. Narayanan et al. (1979) observed that capsaicin

18

concentration increased steadily from green fruit to dry pod stage. Robi and

Sreelathakumary (2004) studied the influence of maturity stage at harvest on capsaicin

concentration in different genotypes of Capsicum chinense Jacq. They found significant

differences among genotypes for capsaicin concentration at color changing stage (1.26 to

3.02 %), at red ripe stage (1.32 to 3.18 %) and at withering stage (1.48 to 3.36 %). They

concluded that capsaicin is synthesized and accumulated in capsaicinoid secreting cells in

placenta. The site of capsaicin synthesis and total capsaicin concentration are genetically

controlled. As the fruit matures, placenta also gets matured and capsaicin concentration

increases. At withering stage, moisture contents of the fruits may get reduced when

compared to color changing stage and thus the percentage of capsaicin increases.

Topuz and Ozdemir (2004) reported changes in capsaicinoids of paprika during ten

month of storage period. Under ambient storage, the level of each capsaicinoid in paprika

was significantly decreased with storage. Generally, all capsaicinoid components

decreased almost 30% within ten months of storage and maximum decrease was recorded

in dihydrocapsaicin. Subbulakshmi et al. (1991) reported that the pungency of irradiated

paprika was greater than in that of the control sample. The increase in capsaicinoid

concentration with the effect of irradiation treatments can be explained by changing the

conformation of the molecules and/or accompanying compounds which affects the

extraction yield. Doses up to 5 kGy of gamma radiation led to greater increase in

capsaicin and dihydrocapsaicin levels and an increase of about 10% was found in the

capsaicinoid concentration of paprika (Topuz and Ozdemir, 2004).

2.2.2: Carotenoids

Since ancient times, Capsicum fruits have been used as natural food colorants. An

increasing interest is being paid to the spice red pepper (Capsicum annuum L.) because of

its economical importance and diversified composition. More than 30 different pigments

have been identified in pepper fruits (Matus et al., 1991). These pigments includes green

chlorophylls (a and b); yellow-orange pigments like lutein, zeaxanthin, β-cryptoxanthin,

violaxanthin, antheraxanthin and β-carotene; and the red pigments, capsanthin,

capsorubin and cryptocapsin which are only found in pepper fruit. When carotenoids are

ingested, they show important biological actions such as being antioxidants and free-

radical scavengers as well as reducing the risk of cancer and having a positive effect on

19

the immune response; in addition, some of them (ß- carotene, ß -cryptoxanthin, etc.) have

provitamin A activity (Edge et al., 1997). In addition to this, carotenoids have been

shown to enhance cell-to-cell communication, act as anti-inflammatory, anti-tumor

agents and induce detoxification of enzyme systems (Gonzalez et al., 1998).

A variety of vegetables enriched with carotenoids especially orange, yellow, red and dark

green types (Muller, 1997). Leth et al. (2000) found that 9% of the carotenoid

consumption was obtained from vegetables and 47% and 32% from carrot and tomatoes

respectively. The carotenoid pattern and the pigment concentrations vary within a wide

range depending on cultivars and ripening stage (Minguez-Mosquera et al., 2000). Stage

of maturity at harvest is the one factor that decisively affects carotenoid composition.

Greater variations both qualitative and quantitative were observed in Capsicums (Hart

and Scott, 1995). More than 30 different pigments have been identified in pepper fruits

(Matus et al., 1991). The total carotenoids concentration tend to change during pepper

ripening and the role and stability of carotenoids in spice red pepper depend to a

considerable extent on the genotype (Gnayfeed et al., 2001). The total carotenoid

concentration of immature green sweet pepper decreased from 5.07 to 4.86 mg 100g-1

fresh pepper when reaching maturity (Marin et al., 2004). Kandlakunta et al. (2008)

found that total carotenoids concentration was 2.41 mg 100g-1 in green chillies (85%

moisture content) but the concentration in red chillies is 113 mg 100g-1 (10.1% moisture

content).

Carotenoids are more stable in their natural environment but become sensitive to light as

well as temperature and are readily decomposed after titration and extraction (Minguez-

Mosquera and Hornero-Mendez, 1994). Color loss is a serious problem for the spice

producing industry which is attributed to number of factors i.e. cultivars, moisture

contents and ripeness stage at harvest (Bicas et al., 1992 and Markus et al., 1999).

Change in carotenoids concentration during food storage and processing take place by

peeling, geometric isomerization as well as enzymatic oxidation (Rodriguez-Amaya,

1999; 2002). Lee et al. (1992) found that carotenoid destruction in red peppers is greatly

affected by water activity under prevailing packaging atmosphere and storage

temperature; in addition to this carotenoid stability was improved in red peppers by

lowering the storage temperature. Schweiggert et al. (2007) reported that total carotenoid

20

concentration decreased by 16.7 and 9.6% in chilli and 39.7 and 38.8% in paprika

powders during four months of storage at ambient temperature with and without

illumination respectively. Topuz and Ozdemir (2003) observed that carotenoid reduction

due to irradiation in paprika was possibly caused by an increase in oxidation reaction

under gamma radiation and also secondary oxidative effects of free radical (H2O2, O3 and

OH) formation during radiation. Similarly, they also concluded that carotenoid loss in

paprika was highest in un-irradiated samples than radiated ones.

2.2.3: Ascorbic acid

Ascorbic acid is functional as well as nutritional component of hot pepper fruit and

recognized as an antioxidant and biologically active compound (Simonne et al., 1997;

McCall and Frei, 1999 and Rietjens et al., 2002). Great variations were seen in vitamin

levels due to difference in cultivars, harvest stages, agro-climatic conditions, post harvest

handling and analytical methods (Mozafar, 1994). Hot pepper has high ascorbic acid

concentration as compared to variety of fruits and vegetables which are commonly

recognized as enriched source of this antioxidant (Fawell, 1998). Ascorbic acid

concentration and values was found around 50% higher in red ripe pepper fruits as

compared to green peppers (Sidonia et al., 2005).

Stage of harvest is one of the major factors that determine the compositional quality of

fruits and vegetables. Ascorbic acid concentrations of mature green chilli fruit ranged

from 121.8 to 146.5 mg 100g-1 fresh weight and red succulent fruit had 233.3 mg 100g-1

(Howard et al., 1994 and Lee et al., 1995). Ascorbic acid concentration in chilli peppers

increased after the mature green stage and peaked in red fruit with about 75% moisture

content. However, red fully dry fruit had 15-18% moisture with lowest levels of ascorbic

acid concentration among maturity stages (Osuna-Garcia et al., 1998).

Storage conditions and duration after harvest may also have great impact on the bioactive

compounds. Kalt (2005) reported that ascorbic acid concentration decreased quickly in

many plant products during storage, while carotenoids and flavonoids appear to be more

stable. Ascorbic acid concentration in ground paprika decreased to 10, 20 and 35% of the

original level after 30, 60 and 120 days of storage (Daood et al., 1996). Bibi et al. (2007)

reported that ascorbic acid concentration decreased significantly (7%) at 1 kGy in dried

garlic powder during five months storage. Khattak et al. (2005) observed decrease in

21

ascorbic acid during storage as well as by radiation in bitter gourd. A dose of 1 kGy

caused 12 % loss of ascorbic acid concentration in pre-cut bell pepper (Farkas et al.,

1997). Thayer and Rajswoki (1999) revealed that irradiation oxidized a portion of total

ascorbic acid to dehydro form and both of these forms of vitamins are biologically active

suggesting minimal nutritional impact. Calucci et al. (2008) reported that ascorbic acid

concentration decreased significantly in different aromatic herbs and spices after they

were irradiated at a dose of 10 kGy after three months storage.

2.2.4: Phenolic compounds

Phenolic compounds are naturally synthesized in plants and also considered as secondary

metabolites because of the adaptation to biotic and abiotic stresses (Harborne and

Williams, 2000 and Pitchersky and Gang, 2000.). Plant phenolic compounds emphasis a

great stress and served as powerful antioxidant as well as free radical scavengers, which

protect human body from oxidative damage (Halliwell, 1996). Antioxidant activity of

flavonoids as well as phenolic compounds depends upon the hydroxyl group attached and

their oxidation/reduction properties. (Rice-Evans, 1997). Epidemiological data have

indicated beneficial effects of antioxidant compounds in the prevention of variety of

diseases such as cardiovascular disease, cancer and neurodegenerative disorders

(Hollman and Katan, 1999 and Burda and Oleszek, 2001).

Conforti et al. (2007) concluded that phytochemical changes that occur during maturity

affect antioxidant activity which is an important dietary consideration that may affect the

consumption of peppers. As consumption continues to increase, hot peppers could

provide important amounts of nutritional antioxidants to human diet. Amount of these

antioxidants depends upon genotype, maturity stage and plant part to be consumed as

well as storage and processing conditions. Thus it is necessary to evaluate the appropriate

stage of fruit maturity at harvest as well as its amount to be incorporated in balanced diet.

Maturity is an important factor to determine polyphenols concentration in fruits and

vegetables. Howard et al. (2000) and Materska and Perucka (2005) stated that

concentration of polyphenols increases as the peppers reached maturity while Conforti et

al. (2007) analyzed hot peppers at different stages viz. immature green, green and red and

concluded that total phenolic contents in hot pepper decreases as pepper reaches maturity.

Marin et al. (2004) observed that immature green sweet peppers have high total phenolic

22

contents but green, immature red and red ripe peppers showed 4-5 fold reduction and

concluded that this decrease was not only due to increase in fruit size with advancing

maturity but also to degradation of flavinoids since the size of immature green fruit 135 g

was slightly smaller than mature fruit 188 g.

Being natural bi-product of plant metabolism found in all plant material (Hammer et al.,

1999), polyphenols greatly provide an acceptable antifungal compounds for application

in pre and postharvest conditions, the possible role of natural polyphenols in prevention

of fungal growth as well as toxin production. Recent investigations on polyphenols

reported that their concentration in plants provide systematic resistance to the plant body

as they invaded with fungal attacks (El Modafar et al., 2000 and Siranidou et al., 2002).

In addition, phenolic compounds have also been found to inhibit the production of several

mycotoxins including fumonisins, tricothecenes and aflatoxins (Norton, 1999; Bakan et

al., 2003 and Beekrum et al., 2003). Talcott et al. (2000) revealed that antioxidant

property of polyphenols greatly reduced when they processed at very high temperature or

stored at same conditions as well. Similarly Mustapha and Ghalem (2007) found

significant decrease in total phenolic contents of dates when they were stored at 10°C

after five months storage period. Bircan (2006) reported that olive stored at 30°C and

75% relative humidity for 3 months and tested at one month interval but no aflatoxins

were detected within detection limit. The possible cause of olive fruit not having

aflatoxin production might be the presence of high concentration of polyphenols that

results in complete inhibition of fungal growth. So far no information is available in the

literature on the effect of gamma radiation on total phenolic contents of dried hot peppers

but diverse effects of irradiation on total phenolic contents have been reported on other

spices. Variyar et al. (1998) found increase in phenolic acid concentration after

irradiation in cloves and nutmeg. Similarly, Harrison and Were (2007) also reported

increase in total phenolic contents in almond skin after they were irradiated at 4 kGy.

2.3: Aflatoxin contamination as a concern

Food security and safety is basic human need and it considered as hot issue for national

and international organizations in the recent past. Chemical as well microbial food

hazards are the most important concern now a days and among microbial food and feed

hazards, mycotoxin attracted world’s attention towards fungal invasion to food elements

23

(WHO, 2002). The enigma of food and feed contamination by aflatoxin is the current hot

issue and it has great deal of attention over the last three decades. The frequent incidence

of these toxins in agricultural products have bad impact on the economy of affected

regions, especially in developing countries where proper postharvest techniques are not

sufficient to prevent mold growth. Aflatoxins being highly toxic, mutagenic, teratogenic

as well as carcinogenic compounds that have been considered as causal agent in human

hepatic and extra hepatic carcinogenesis (Massey et al., 1995). Epidemiological studies

revealed that due to hazardous nature of aflatoxin and considered as potential source of

causing liver cancer in human populations mostly exposed to fungal contaminated food

(Groopman et al., 1988). The incidence of human liver cancer increased when they ingest

fungal contaminated food directly as well as indirectly from animals (milk, meat etc)

when fed with contaminated fodder and forage. Moreover, aflatoxin incidence also

associated with kwashiorkor; a disease associated with protein malnutrition in children

(Adhikari et al., 1994).

Aflatoxin is a naturally occurring toxin (Williams et al., 2002) and one of the strongest

carcinogens found in nature (Castegnaro and McGregor, 1998). The filamentous fungi

that are major producers of aflatoxin are Aspergillus flavus (Bankole et al., 2004) and

Aspergillus parasiticus (Begum and Samajpati, 2000 and Erdogen, 2004). Aspergillus

flavus, which is ubiquitous, produces B aflatoxins (Samajphati, 1979) while Aspergillus

parasiticus produces both B and G aflatoxins and has more limited distribution (Garcia-

Villanova et al., 2004). The history and discovery of aflatoxin was the incidence with

deaths of millions of turkey and farm animals by turkey ‘X’ disease in 1960 in United

Kingdom, the most accepted reason for this disaster was Brazilian peanut feed which was

highly infected with Aspergillus flavus.

2.3.1: Chemistry of aflatoxins

Aflatoxins can be classified into two broad groups according to chemical structure which

are difurocoumarocyclopentenone series and difurocoumarolactone (Heathcote, 1984).

They are highly substituted coumarin derivatives that contain a fused dihydrofuran

moiety. There are six major compounds of aflatoxin such as aflatoxin B1 (AFB1),

aflatoxin B2 (AFB2), aflatoxin G1 (AFG2), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1) and

aflatoxin M2 (AFM2). The former four are naturally found aflatoxins and the AFM1 and

24

Figure 2.2 Chemical structures of aflatoxins (Papp et al., 2002)

25

AFM2 are produced by biological metabolism of AFB1 and AFB2 from contaminated feed

used by animals. Aflatoxin B is the aflatoxin which produces a blue color under

ultraviolet while Aflatoxin G produces the green color. AFM1 produces a blue-violet

fluorescence while AFM2 produces a violet fluorescence (Goldblatt, 1969). Aflatoxins

have nearly similar to structures and form of a unique group, which is of highly

oxygenated as well as naturally occurring heterocyclic compounds. The chemical

structures of aflatoxins are shown in Figure 2.2.

The G series of aflatoxin differs chemically from B series by the presence of a β-lactone

ring instead of a cyclopentanone ring. Also a double bond that may undergo reduction

reaction is found in the form of vinyl ether at the terminal furan ring in AFB1 and AFG1

but not in AFB2 and AFG2. However this small difference in structure at the C-2 and C-3

double bond is associated with a very significant change in activity, whereas AFB1 and

AFG1 are carcinogenic and considerably more toxic than AFB2 and AFG2.

2.3.2: Aflatoxins and health

Aflatoxins have been considered as one of the most dangerous contaminant in food and

feed. The contaminated food poses a potential health risk to human such as aflatoxicosis

and cancer (Jeffrey and Williams, 2005). That’s why among mycotoxins, aflatoxin have

received great deal of attention. These are potent toxin and were considered as human

carcinogen by The International Agency for Research on Cancer (IARC) as reported in

World Health Organization monograph (WHO, 1987). These mycotoxins are known to

cause diseases in man and animals called aflatoxicosis (Eaton and Groopman, 1994).

Human exposure to aflatoxins is principally through ingestion of contaminated foods

(Versantroort et al., 2005). In humans, aflatoxins are incriminated source of neonatal

jaundice as well as circumstantial evidence to cause perinatal death and reduction in birth

weight. Aflatoxins have implicated in series of food poisoning that are associated with

serious morbidity with early mortality among young children’s (Hendrickse, 1997).

Inhalation of these toxins may also occur occasionally due to the occupational exposure.

After the intake of contaminated feedstuffs, aflatoxins cause some undesirable effect in

animals, which can range from vomiting, weight loss and acute necrosis to various types

of carcinoma, leading in many cases to death (Bingham et al., 2004). Even at low

concentration, aflatoxins diminish the immune function of animals against infection. Yeh

26

et al. (1989) reported that 91% of the deaths in people were observed due to liver cancer

caused by regular ingestion of fungal contaminated food and their test also found positive

for hepatitis B1 in Southeast China. In views of occurrence and toxicity, AFB1 is

extremely carcinogenic while others are considered as highly carcinogenic (Carlson,

2000). It is immunosuppressive and a potent liver toxin; less than 20 µg of this compound

are lethal to duckling (Hussein and Brasel, 2001).

Besides causing health problems to humans, aflatoxins also cause adverse economic

effect in which it lowers the yield of food production and fiber crops as well as becoming

a major constraint of profitability for food crop producing countries (Rachaputi et al.,

2001). It has been reported that 25% of the world’s food crops are contaminated by

mycotoxins every year resulting in significant economic loss for these countries (Lopez,

2002). Livestock as well as poultry producers are the most victim persons due to

aflatoxin contaminated feeds which ultimately cause significant economic loss by death

of farm animals, destruction of their immune system and reduction in body weight after

intake of contaminated feed.

2.3.3: Occurrence

Series of biotic and abiotic factors are responsible for aflatoxin production but

temperature and relative humidity are the most critical. Studies conducted on hazelnuts

and pistachios manifested that temperature favorable for aflatoxin production is 25–30ºC

and relative humidity 97–99% (Simsek et al., 2002). Water activity, substrate chemical

composition storage duration, insect attack as well as moisture contents also take part in

aflatoxin contamination to some extent (Sakai et al., 1984 and Schatzki and Ong, 2001).

Ross et al. (1979) reported that if both temperature (20–38ºC) and moisture (16–24%) are

favorable for Aspergillus flavus, aflatoxin can be produced within 48 hours but aflatoxin

production can occur at low temperatures i.e. 7–12ºC (Steyn and Stander, 2000). The

fungus responsible for aflatoxin production is cosmopolitan in nature and there are

evidences that aflatoxin may also occur both in pre and postharvest conditions of cereals,

oilseeds, edible nuts and spices (Coker, 1997).

Aspergillus flavus is widely spread in soil. Moldy grains and nuts are commonly

contaminated with the fungus. About half of all known Aspergillus flavus strains produce

mycotoxins. Aflatoxin production stimulated by moisture and high temperature; at least

27

13 types of aflatoxin are produced by different fungal species but the most potent is

aflatoxin B1. The aflatoxin contamination can take place at any stage of food production

from pre-harvest to storage (Wilson and Payne, 1994). Drought stress prior to crop

harvest, improper stage of harvest, insect/pest attack, heavy rains at harvest and further

poor drying of the crop are the principal factors which induce aflatoxin production (Hell,

2000 and Hawkins et al., 2005). However, the humidity, temperature and aeration during

drying and storage remain the most important factors for contamination with aflatoxins.

The development of suitable varieties could result in consistent desirable yields and

reduce weather-related problems concerning quality such as aflatoxin (Keisling et al.,

1999). Crop cultivars are known to have a significant impact on growth of fungi that

produce mycotoxins. In general, varieties that are resistant to fungal attacks during the

growing season are also less likely to become contaminated with mycotoxins, although

mycotoxin problems can still arise during storage (Edwards, 2004).

Fungi are a normal component of food micro flora and may be responsible for spoilage

and production of mycotoxins (Aziz et al., 1998). Fungus might grow on different foods

and feeds under conditions of favorable temperature and relative humidity and produce

aflatoxins during postharvest handling, transportation and storage. Contamination of

spices with aflatoxins can cause potential carcinogenic effects if ingested even in small

amounts (Roy and Chourasia, 1990). Aflatoxin residues in contaminated foods with

Aspergillus flavus remains for a long time even after the death of fungus or disappeared

completely in feed stuff. Aspergillus flavus can thrive best under range of conditions from

hot arid climate to warm humid circumstances. Foods that are particularly susceptible to

being contaminated by aflatoxins include groundnuts, maize, spices, cotton seed and tree

nuts.

Spices are exposed to a wide range of microbial contamination due to poor collection

conditions, unpretentious production process and extended drying times. In addition

spices can be contaminated through dust, waste water and animal/human excreta in

unpackaged spices which are sold in markets and bazaars (Banerjee and Sarker, 2003).

Several studies have reported that spices contaminated with variety of microorganisms

some of which have great potential to produce aflatoxin (Garrido et al., 1992). Toxin

accumulation in spices is an indicative of contamination following harvesting and drying.

28

Now a day’s spices create health problems because they are added to foods without

further processing to sterilize from contamination (Lwellyn et al., 1990).

Red pepper is one of the favorite spice in South Asia (Pakistan) and commonly used for

flavoring, seasoning as well as imparting aroma or color to foods. It is very sensitive to

aflatoxin contamination depends upon atmospheric temperature, relative humidity, drying

and processing conditions (Cokosoyler, 1999). The presence of aflatoxin is major hot

issue for chilli exporters from Pakistan. Pepper powder and paprika are susceptible to

fungal growth. For safety measures, washing of fruits after harvest, contaminated fruits

must be isolated to minimize fungal contamination. Low humidity should be maintained

during fruit drying and moisture contents in paprika must be kept below 11% (water

activity 0.75) to prevent fungal growth (Pitt and Hocking, 1997 and FAO/WHO, 2001).

Red chillies and its powder which is prepared from its dry fruits; both are naturally

susceptible to aflatoxin B1 production (Reddy et al., 2001). During storage, fungus

especially Aspergillus flavus as well as Aspergillus niger found to be present in chillies

(Giridhar and Reddy, 1999). Fresh and sun-dried chillies were infected with species of

Fusarium and A. alternate (Adebanjo and Shopeju, 1994). Harvest should be done at a

proper time (Rahmianna and Ginting, 2003). Paramawati (2003) noted that quick post-

harvest handling could reduce the contamination of aflatoxin in peanut.

Susan and Laurence (1996) concluded that contamination of the spices could have taken

place in the field, during processing, transportation and bad storage conditions. Once

spices and food are contaminated by aflatoxins, it is almost impossible to detoxify them

by normal cooking methods. Cooking food by either conventional gas or microwave oven

does not reduce the level of aflatoxins contamination. In USA, 75% of AFB1

contamination was observed in 12 red pepper samples with a maximum aflatoxin amount

of 30 µg kg-1 (Wood, 1989). Ahmad and Ahmad (1995) collected 176 red hot pepper

samples from different locations in Pakistan and reported that 66% of the samples were

contaminated with AFB1 but aflatoxin contamination was low and only 7 samples

conflicted with 25 µg kg-1 level.

Aydin et al. (2007) tested 50 samples of powdered peppers. They found that AFB1 levels

were below the minimum detection limit in 32 samples while 18 samples had

unacceptable contamination levels higher than the maximum tolerable limit (5 µg kg-1).

29

They concluded that reasons for higher AFB1 levels is result of lying red peppers on soil

surface and asphalt for drying purpose and storage of hot pepper fruits under conditions

of high humidity levels and inadequate control of transportation and shop display

conditions. Colak et al. (2006) reported that 11 out of 30 samples (36.7%) contained total

aflatoxins ranging from 0.8-15.4 µg kg-1 in red pepper. The AFB1 concentrations were in

the range of 2.9-11.2 µg kg-1. The samples obtained from bazaars had the highest

contamination levels (15.4 µg kg-1). Similarly, Yildirim et al. (1997) found total

aflatoxins in 8 out of 34 red pepper samples (23.5%) in the range of 1.6-15.0 µg kg-1.

Zinedine et al. (2006) reported that higher fungal contamination levels were found in red

paprika with 100% positive results in the samples analyzed and average concentration of

AFB1 and total aflatoxins was 2.88-5.23 µg kg-1.

Romagnoli et al. (2007) analyzed 28 samples of spices randomly collected from markets,

shops and bonded ware house in Italy for estimation of aflatoxins and found that higher

level of total aflatoxins was found in whole, dried hot pepper sample; AFB1 26.9 µg kg-1,

AFB2 1.4 µg kg-1, AFG1 1.2 µg/kg and AFG2 1.2 µg kg-1. Only 2% of the samples

contained toxin within non-permissible limits. Russell and Peterson (2007) reported that

chilli production in Pakistan is heavily contaminated with aflatoxin. They collected nine

ground and four pod chilli samples from different cities and found that ground samples

were heavily contaminated with aflatoxins ranges between 6.8-96.2 µg kg-1 as compared

to chilli pods 0.1-6.6 µg kg-1. They concluded that a more comprehensive survey is

required to assist farmers for controlling aflatoxin in Pakistan.

2.3.4: Aflatoxins and storage

Sometimes agricultural commodities contaminated with aflatoxin in field before harvest

where it is usually conflicted with drought stress (Klich, 1987); even more is the fate of

crops stored under circumstances that favor fungal growth. Contamination with

Aspergillus flavus and subsequent production of aflatoxin during storage is considered as

principal hot issue throughout the world (Williams et al., 2004). Development of fungi

during storage in a postharvest commodity is determined also by length of time (Lillehoj

and Zuber, 1988). The longer the storage time, the greater the possibility of

environmental conditions conducive to aflatoxigenisis (Udoh et al., 2000). In storage, the

moisture contents of the substrate, temperature as well as relative humidity are very

30

important factors for fungus proliferation and ultimately presented a major risk for

aflatoxin production (Nakai et al., 2007). The growth of Aspergillus spp. and subsequent

aflatoxin production in storage was favored by high relative humidity (> 85 percent), high

temperature (> 25oC) and insect or rodent activity (CAST, 1989). Ahmad (1993)

observed that aflatoxin contamination in storage was dependent on the storage system

and levels of aflatoxin were lower in closed metal bins which restricted air exchange and

reduced oxygen levels than in gunny bags that allowed air to flow through stored black

gram seeds.

Aflatoxin accumulation was highest in maize stored for 52 weeks during the monsoon, a

season with high relative humidity. A decline of 33% in the level of aflatoxin B1 was

observed during the winter with lower relative humidity (Choudhary and Sinha, 1993).

Chourasia (1995) reported that gunny bags and bare ground had significantly higher

incidences of mycobiota and mycotoxins compared with spices stored in metal

containers, glass containers and wooden boxes. Moreover studies indicated that spice

sample contaminated with A. flavus occur as a result of insect infestation in the field and

during transportation. Insects in such cases operate as biological vectors to transmit and

spread fungal propagates.

Thompson and Henke (2000) studied the effect of climate, storage duration (30, 60 and

90 days) and storage material i.e. metal containers, paper bags, plastic bags and paper

bags inlined with plastic on aflatoxin contamination in corn. Climates included hot 29–

32ºC temperature and both humid 85–88% RH as well as dry 35–40% RH conditions also

with cool climate with 14–18ºC temperature maintained. They concluded that average

aflatoxin levels in corn stored for 30, 60, and 90 days were 41, 11 and 24 mg kg-1

respectively for corn stored in metal containers, 3, 49 and 19 mg kg-1 respectively for

corn stored in plastic bags, and 2, 4 and 20 mg kg-1 respectively for corn stored in paper

bags inside plastic bags. They further reported that levels of aflatoxin in corn stored for

30, 60 and 90 days in hot and humid conditions were 4, 15 and 21 mg kg-1, 40, 5 and 22

mg kg-1, for corn stored in cool and humid conditions, 1, 3 and 39 mg kg-1 respectively

for corn stored in hot and dry conditions and 2, 3 and 19 mg kg-1 respectively for corn

stored in cool and dry conditions.

31

Arrus et al. (2005) studied the effects of relative humidity with 75, 80, 85 and 97% and

temperature at 10, 13, 15, 25 and 30ºC on aflatoxin production in dried Brazilian nuts

with 3.5% moisture contents. Aflatoxin were not detected under detectable limit of 1.75

ng g-1 in nuts stored for 60 days at 10ºC with 97% RH and at 30ºC with 75% RH

conditions. Maximum moisture contents were 4.50 and 9.14 % and water activity 0.78

and 0.92 values (aW) for stored nuts. Results of this study indicated that the limiting

moisture content and aW values required to control aflatoxin production (<4ng g-1) in SW

(Shelled Whole) and WIS (Whole in-Shell) stored at 30ºC for up to 60 d are 4.5, 0.68, 5.0

and 0.75 respectively. Overall studies revealed that increase in relative humidity and

temperature during storage resulted in an increase in aflatoxin and these were the most

significant factors for toxin production in Brazilian nuts. Paramawati et al. (2006) studied

the influence of packaging on the aflatoxin B1 level in peeled peanut during storage. They

observed that compared to polyethylene and vacuum packaging, hermetic (glass, air tight

chamber) packaging provides a good solution to achieve low aflatoxin level after one

month of storage.

2.4: Aflatoxin, microbial load and irradiation

The problem of mycotoxin is most serious issue in developing countries because their

climatic conditions, agricultural practices and storage condition are considered conducive

for fungal proliferation and toxin production (Aziz and Moussa, 2004). Spices such as red

pepper are frequently exposed to insects and microorganisms during cultivation and

storage which might be potential contamination sources in foods even when added in

small amounts. For sanitation, fumigation with ethylene oxide has been widely used.

Despite its strong effect, many countries restrict its use in foods because of possible toxic

residues and health hazards for workers (Fowels et al., 2001). Many studies have shown

that irradiation is a safe process and therefore in 1994 WHO declared that irradiation of

food is safe from nutritional and toxicological point of view (Dwyer et al., 2003). Food

irradiation promises a healthier and safer food to the public by reducing bacterial

spoilage. As many food products are not traded due to insect infestation and microbial

contamination, food irradiation has come up as a solution to all these concerns.

Spices often originate in developing countries where harvest and storage conditions are

inadequately controlled with respect to food hygiene. Thus they may have been exposed

32

to high level of natural contamination by mesophyllic, sporogenic and asporogenic

bacteria, hyphomycetes and faecal coliforms (Bendini et al., 1998).

Food irradiation is a mechanized process of exposing food stuff to carefully controlled

amount of energy in the form of high-speed particles/rays. Interest in irradiation due to

high food infestation losses and spoilage is increasing concern due food borne illness as

well as growing international trade in food commodities that must meet strict quarantine

quality standards (Sadecka, 2007). Irradiation is an emerging new technique in number of

countries where consumption of irradiated food is a major concern in the near future. The

principal concern of food processors is to assure that microbial load in food ingredients

and processing does not allow food spoilage and diminish its microbial safety. Gamma-

radiation is more effective than ethylene oxide fumigation in controlling microbial

contamination without any adverse effect (Byun et al., 1996). However, the process does

have some drawbacks (a number of which are common to alternative processing

technologies). For example, irradiation does not destroy bacterial toxins, viruses or

aflatoxins and can cause significant losses of vitamins, in particular vitamins E and B1,

and of fatty acids in high fat foods (Henson, 1995). Similarly, consumer attitudes to food

irradiation and willingness to purchase irradiated food reflect not only the fundamental

characteristics of the process itself, but also the social, economic and political

environment within which food products in general, and irradiated food products in

particular, are produced, purchased and consumed. There is some evidence that

consumers may not place a high value on the potential benefits of the process which

might otherwise off-set such concerns and support adoption. For example, increased

shelf-life is a benefit of food irradiation which has been emphasized by proponents of the

process. However, there is little evidence to suggest there is an identifiable demand for

further increases in the shelf-life of food products (Foster, 1992).

The process of spice decontamination by gamma-radiation and other dry food

constituents is a viable and effective alternative than other decontamination processes

which have a great deal of application potential in developing as well as developed

countries. In order to strict hygienic conditions in preparation, radiation decontamination

of spices and other dry ingredients with dose of 3–10 kGy to be a reliable technique for

improving it microbial safety (Farkas, 1988). Gamma-radiation is legally permitted in 34

33

countries world wide for microbial decontamination of spices and their powders

(Anonymous, 1996b) while 23 countries commercially using this technology

(Anonymous, 1996a). Dried foods such as red pepper powder are less sensitive to

irradiation than hydrated ones and their irradiation has been authorized at a maximum

dose of 10 kGy and 30 kGy in Korea and in the United States respectively (Olson, 1998).

Dried red pepper and other dry plant materials often contain a variety of microorganisms

and these are affected by different factors including growing and harvesting as well as

postharvest handling (Obuekwe and Ogbimi, 1989). The survival and proliferation of

fungi and bacteria are limited in dry plant materials but Geopfart et al. (1972) reported

that contaminated spices are sources of food spoilage and toxin production. The high load

of aflatoxin producing Aspergillus spp. and saprogenic bacteria isolated from

chilli/pepper is a major concern because this is added for cooking and to raw food (Antai,

1988). A 3-4 kGy dose creates a 2-3 log cycle reduction in viable cell count and sterility

was obtained with 15-20 kGy depending on initial microbial load in ground pepper

(Farkas et al., 1973).

Domsch et al. (1980) studied that contamination of food products with fungal species was

as a result of natural contamination followed by holding dust under humid conditions. An

irradiation dose of 5-10 kGy is sufficient to reduce the population of microbes without

changing essential quality attributes and the spice flavor remained intact up to 7.5 kGy

(Farkas, 1985). Ito et al. (1985) studied the effect of irradiation on 17 different spices and

found that a radiation dose of 5-15 kGy was required to reduce total aerobic bacteria and

4-10 kGy spore forming bacteria to below detectable levels and also eliminated

coliforms. However, untreated spices were stored at 30-35°C and greater than 80%

relative humidity for 1-3 months, fungal count increased up to 108 g-1 in many powdered

spice samples and treated with 4 kGy was sufficient for decontamination. A dose of 10

kGy destroyed all microorganisms in prepackaged samples of red chilli samples with no

effects on spice quality were observed (Munasiri et al., 1987).

Wage et al. (2008) reported that a radiation dose of 10 kGy significantly reduces the

moisture contents from 13.88 to 10.32 in ground black pepper after 6 months storage at

20°C. They further revealed that storage temperature does not change moisture contents

considerably when these were stored at 4 or 20°C. No colony forming units (CFUs) were

34

observed in red chilli after irradiation treatment (Sharma et al., 1989). However, Farag et

al. (1995) revealed that microorganisms in hot peppers were reduced below a detectable

level at a radiation dose of 10-20 kGy. Hilmy et al. (1995) observed no growth of A.

flavus in ground nutmeg stored at relative humidity of 85% or less and peanut under 91-

97%. Mycelium growth and toxin production by moulds were completely inhibited by

irradiation, although the efficacy of irradiation differs with different relative humidity and

media during post-irradiation incubation. During ten months storage, total fungal count in

hot peppers decreased up to the fourth month while afterwards an increase was seen

which could be due to multiplication of irradiation resistant strains. They also reported

that with the increase in storage duration, reduction in fungal population was observed in

samples irradiated at 5 kGy (Onyenekwe and Ogbadu, 1995). Lee et al. (2006) reported

that total fungal count were 4.88 logs colony forming units g-1 in non-radiated red pepper

powder and gamma-radiation decreased TFCs significantly and no growth of fungi,

yeasts and molds was detected in red pepper powder irradiated at 7 kGy.

Onyenekwe and Ogbadu (1995) reported that capsaicin concentration and total available

carbohydrate did not change even after irradiation with 10 kGy in both whole and ground

chilli pepper samples which indicates that capsaicin is radio resistant and storage duration

did not change their concentration. It has been reported that the capsaicinoids are

relatively stable under gamma radiation up to 15 kGy and capsaicin was more stable than

dihydrocapsaicin (Lee et al., 2000). No quality deterioration was observed at a dose of 5-

7.5 kGy in both whole and powdered pepper types after two year of storage in combined

packaging with polyethylene/polycloth sack. However, gamma radiated pepper powder

was more effective for a long-term preservation. At the same time, no growth of yeast

and molds was observed after six months storage in whole red pepper samples as

compared to red pepper powder (1.5 x 101) irradiated at 2.5 kGy (Byun et al., 1996).

Mold growth was completely inhibited in different foods and agricultural products at a

radiation dose of 5 kGy while aflatoxin B1 was detoxified by 82–88% at 10 kGy (Aziz

and Youssef, 2002). There are some reports on aflatoxin decontamination by gamma

radiation in different commodities like Herzallah et al. (2008) reported that a radiation

dose of 5 kGy decrease 10% total aflatoxin contamination in chick feed and it further

decresed to 35% when the samples were irradiated at 25 kGy. Similarly, Aquino et al.

35

(2005) observed that AFB1 and AFB2 were completely reduced at 10 kGy in maize.

Aziz and Moussa (2002) indicated that fungal flora is sensitive to gamma-radiation and

complete inhibition was achieved at a radiation dose of 5 kGy in different fruit samples.

Padro et al. (2003) concluded that percentage infection of peanuts with aflatoxins

decreased significantly by increasing the radiation dose levels from 5-10 kGy and the

molds were completely inhibited at irradiation dose of 10 kGy. Treatment of peanut seeds

with gamma radiation (15, 20, 25 and 30 kGy) destroyed 69-74% of aflatoxin B1 in

sample A and 55-62% in sample B respectively. A reduction in AFB2 (97.6% and 94%)

was more efficient than the reduction of AFB1 (68.95 and 46%) at dose of 2 and 6 kGy in

maize samples. They further observed that a radiation dose of 10 kGy resulted in

complete reduction in AFB1 and AFB2 (Aquino et al., 2005). Aziz et al. (2006) studied

the effect of gamma radiation on mycotoxin contamination in grains during storage. They

concluded that total fungal counts increased 1.4 x 105 – 6.8 x 106 cfu/g grain-1 in non-

radiated grain samples as compared to samples irradiated at 4 kGy (1.1 x 101 – 1.0 x 101

cfu grain-1) after 100 days of storage at room temperature and a radiation dose of 6 kGy

inhibit mold growth completely.

36

CChhaapptteerr 33

MMAATTEERRIIAALLSS AANNDD

MMEETTHHOODDSS The research work was conducted to evaluate the productive and qualitative potential

of three hot pepper hybrids. The study was divided into two parts. In the first part of

study, hot peppers were grown in plastic tunnels to see their productivity under black

and clear plastic mulches (Experiment I) and then antioxidants were quantified in hot

peppers harvested at different stages (Experiment II). The second part of study

involves five month storage studies under different temperatures and packaging

materials to measure the extent of aflatoxin contamination, microbial activity and

quality of these hybrids (Experiment III). After that the effect of gamma radiation on

aflatoxin contaminated samples with respect to microbial load and their quality during

further three month storage was investigated (Experiment IV). The details of the

research plan are described here in after:

3.1 Experiment I: Effect of plastic mulches on hot pepper productivity

Material used in this study was as followed.

3.1.1: Hot pepper hybrids

H1 = Sky Red

H2 = Maha

H3 = Wonder King

3.1.2: Plastic Mulch

M1 = Bare Soil

M2 = Black plastic mulch

M3 = Clear plastic mulch

3.1.3: Layout

The experiment was laid out following Randomized Complete Block Design (RCBD)

with factorial arrangements and replicated three times. There were 9 treatments and 27

37

experimental units. The size of each experimental unit was 9.6 x 0.6m.

3.1.4: Methods

Seeds of commercially cultivated hot pepper hybrids viz. Sky Red, Maha and Wonder

King were obtained from Haji Sons (Pvt.) Seed Distributor, Lahore, Pakistan.

Seedlings were raised in pots and transplanting was done on both sides of raised beds

in simple poly/plastic tunnel on 14 and 18 November during 2005-06 and 2006-07

respectively. Plants were staggered at 0.6 x 0.6m apart. Beds were 0.15m high and

spaced 0.75m apart. Both black and clear plastic mulches used were 30µm thick and

installed on pre-shaped beds manually with drip tape buried 6 cm deep in the center of

beds. According to soil nutrient analysis report, fertilizers were applied @ 180, 200

and 180 kg ha-1 for nitrogen, phosphorus and potash respectively. One third of NPK

was applied during bed preparation prior to applying plastic mulch and drip tape. This

“starter” fertilizer provides some nutrition to the crop during its early growth while

remaining NPK was applied throughout the season as needed by the crop through drip

irrigation. Hoeing and weeding was practiced regularly on plants grown on bare soil.

Plant protection measures followed standard recommendations as and when required.

Fungicides like Trimelotox-forte, Dithane M-45, Mancozeb, Radomil and insecticides

like Imidacloprid, Triguard, Polo and Tracer were applied to control insect and disease

infestation.

The following observations were recorded during the experiment I.

3.1.5: Soil temperature (ºC)

Soil temperature was measured with soil thermometer at depth of 10 cm daily between

12.00 to 1.00 solar hours up to 90 days after transplanting and average was calculated.

3.1.6: Plant height (cm)

Ten plants were selected at random from each experimental unit and their plant height

at first harvest was measured with measuring tape and average height in cm was

computed.

3.1.7: Stem diameter (mm)

Stem diameter of ten randomly selected plants from each experimental unit was

recorded at first harvest with the help of digital vernier caliper at the base of stem and

average stem diameter in mm was computed.

38

3.1.8: Root fresh weight (g)

After final harvest, plants were dug out and roots were cleaned with water and their

fresh weights in g were recorded.

3.1.9: Root dry weight (g)

Roots were placed in an oven for 3 days at 70ºC. Samples were removed and their dry

weights in g were taken with the help of single pan digital balance.

3.1.10: Root length (cm)

After final harvest, length of main root was measured by digging out plants 45 x 50 cm

deep and root length in cm was measured by using measuring tape.

3.1.11: Leaf area per plant (cm2)

Ten gram leaf samples from the top, middle and base of the canopy were collected,

weighed and their leaf area g-1 was calculated with the help of portable leaf area meter

(CI-202 CID Inc.) and converted into leaf area plant-1 by taking leaves fresh weight of

each plant.

3.1.12: Days taken to first flowering (days)

Numbers of days taken to first flowering after transplanting of randomly selected

plants were recorded and the mean values were calculated and analyzed.

3.1.13: Fresh fruit yield per plant (kg)

Fresh fruit weight of randomly selected plants was recorded at each harvest with the

help of single pan digital balance and the data of each harvest was added to get

cumulative yield per plant.

3.1.14: Dry fruit yield per plant (kg)

Fresh fruits were placed in an oven at 65°C for 3-4 days until constant weight was

achieved and their dry weights were recorded.

3.1.15: Harvest index (%)

Harvest index was calculated by the following formula:

Harvest index= fruit yield/fruit yield + vegetative yield x100

3.1.16: Fruit length (cm)

At each harvest, fruit length of 25 selected fruits from each replication was measured

with the help of measuring tape and average was calculated in cm.

39

3.1.17: Fruit diameter (mm)

At each harvest, diameter of 25 selected fruits from each replication was measured

with the help of digital vernier caliper and average was calculated in mm.

3.1.18: Determination of nutrient elements

Leaf analysis was conducted to determine the nutritional status of hot pepper plants at

early fruiting stage and the following procedure was used.

i) Leaf sampling

The newly expanded leaves were collected from each of the ten selected plants in a

replication and combined into one composite sample for nutrient analysis. Leaves were

placed in labeled paper bags and brought to laboratory for analysis without any undue

delay.

ii) Sample preparation

Leaves were lightly scrubbed with hands in tap water to remove surface contaminants,

thoroughly washed with detergent using tap water and then rinsed thoroughly with

distilled water. The leaves were then shade dried to remove the moisture on their

surfaces, placed in labeled perforated paper bags and then shifted to an oven for 48

hours at 65°C. After removal from the oven leaf samples were kept for an hour for

cooling with a drying agent e.g. Silica Gel. Then oven dried leaf samples were taken out

and ground to fine powder in an electric stainless steel grinder. This ground powder

was stored in properly labeled air tight plastic bottles for further analysis.

iii) Estimation of Nitrogen

Nitrogen (N) was determined following digestion method of Chapman and Parker

(1961). It involved the digestion of plant material with concentrated H2SO4 and

digestion mixture comprising K2SO4: Cu2SO4: Fe2SO4 (10: 0.5: 1).

iv) Digestion procedure

One gram of oven dried leaf powder was transferred to Kjeldahl digestion flask along

with 10 g digestion mixture and 30 ml H2SO4 was added into it. The contents were

kept as such for half an hour. The digestion flasks were then placed on heaters. In the

beginning mixture was heated slowly at 100ºC for one hour and then fully heated at

370 + 5ºC till the material was turned into transparent green liquid. On cooling the

contents were transferred to a 250 ml volumetric flask and volume was made up to the

40

mark.

v) Distillation

Aliquot from the digested material was taken for distillation in micro-Kjeldahl

apparatus using 40% NaOH. The nitrogen in the form of ammonia vapors was received

in 10 ml 4% boric acid in 50 ml beaker having some drops of bromocresol green and

methyl red as indicator. The distillate was collected up to about 25 ml was titrated

against 0.1N H2SO4 till the original color of methyl red was restored.

vi) Quantification of nitrogen

From the quantity of acid used in titration, the percentage of the element was

calculated using the following formula:

N (%age) = A – B x C x 0.0014 x 100 D Where

A = Quantity of 0.1N H2SO4 used

B = Blank reading (0.1N H2SO4 used in blank reading)

C = Volume made after digestion (250ml)

D = Volume of digested sample used

100 = for percentage

0.0014 Factor (which is equal to g of N in 1 ml of 0.1N H2SO4).

Blank reading was taken for eliminating the percentage of N present in other chemicals

used to digest the sample.

vii) Estimation phosphorus and potassium

The digestion for estimation of P and K was done according to the method described

by Yoshida et al. (1976).

viii) Digestion procedure

One gram oven dried leaf sample was transferred to 100 ml beaker and 10 ml of tri-

acid mixture comprising HNO3: HClO4: H2SO4 (5:2:1) was added to it. It was covered

with watch glass and left as such for about 2 hours till the initial reactions subsided.

The beakers were heated on hot plate gently until the solid material disappeared, then

heated vigorously till a clear colorless solution resulted. When the volume was reduced

to 1.5 ml it was removed from hot plate and cooled to ambient temperature. Then

distilled water was added to it to completely transfer the contents to 100 ml volumetric

41

flask. The volume was made up to the mark and samples were stored in plastic bottles

for individual analysis of these elements.

ix) Phosphorus estimation

Phosphorus was determined according to the method described by Chapman and

Parker (1961) using spectrophotometer. Color of the sample was developed by adding

5 ml of diluted H2SO4 (1:6), 5 ml of 5% ammonium molybdate and 5 ml of 0.25%

ammonium vanadate. The standard curve was developed by using different

concentration of potassium dihydrogen phosphate. The colored samples were fed in

using IRMECO UV-Vis spectrophotometer Model U2020 and transmittance at a

wavelength of 420 nm was recorded, which was compared with that of standard curve

to find out the quantity of element in ppm which was then converted into percentage

by using the following formula.

P (%age) = ppm on graph x dilution x 100 106 x) Potassium estimation

Potassium was determined by flame photometer according to method described by

Chapman and Parker (1961). Quantity of element was estimated in ppm by comparing

the emission of flame photometer with that of standard curve, which was then

converted into percentage by using the following formula:

K (%age) = ppm on graph x dilution x 100 106

3.2 Experiment II: Quantification of antioxidant constituents in hot peppers

harvested at different stages

Fruits of each hybrid were harvested at different stages from the field, placed in

polyethylene bags and stored immediately in freezer at -20ºC until analyzed. Each

sample had three replications.

3.2.1: Stage designations

Immature green = 15-18 days after fruit development.

Mature green = fully expanded completely green.

Color break stage = fully expanded 50% green, red.

Red ripe = fully expanded completely red.

Dried fruit = fruits were allowed to dry on plant.

42

The following observations were recorded during the experiment II.

3.2.2: Determination of capsaicin and dihydrocapsaicin

Hot pepper pods with pedicels were removed, oven-dried at 60ºC for 2-5 days and then

cooled with Silica Gel. Afterwards ground and kept in plastic bags at 5ºC in cold

storage until further process. All samples were processed within 5 days after grinding.

i) Capsaicinoid standards

These capsaicinoid standards consisted of 50 mg Capsaicin (8-methyl-N-vanillyl-trans-

6-nonenamide) and 50 mg Dihydrocapsaicin (8-methyl-N-vanillylnonanamide) were

purchased from Sigma Chemical Co, St. Louis, MO, USA. All solvents used for

capsaicinoid analysis were of HPLC grade and freshly glass redistilled.

ii) Extraction of capsaicinoids

Samples were extracted by using the following method of Collins et al. (1995) with

little modifications. For capsaicinoid extraction, sample:acetonitrile (1:10; gram:

milliliter) ratio of dried pepper powder was placed in 120 ml glass bottles with Teflon-

lined lids. Bottles were capped and placed in an 80ºC water bath for 4 hours; swirled

manually after every hour. Samples were removed from water bath and cooled at room

temperature. The supernatant content of samples (2-3 ml) was filtered through 0.45 µm

(Millex®-HV filter) using a 5 ml disposable syringe (Millipore, Bedford, MA) into a

HPLC sample vial, capped and stored at 5ºC in refrigerator until analysis. The

capsaicinoid concentrations in samples were expressed as mg 100g-1.

iii) Liquid chromatographic analysis

A Shimadzu (LC-10, Shimadzu, Japan) HPLC system equipped with LC- 10AS multi-

solvent delivery system, a SPD-10A UV-Vis detector at wavelength fixed at 280 nm

and controlled the parameters with system controller unit (SCL-10A). The analysis

was carried out following the conditions: column temperature 30ºC, flow rate of

mobile phase; 1 ml.min-1 and data acquisition was made using Class LC-10 software.

All analyses were performed isocratically using degassed HPLC grade 60%

acetonitrile (Merck, Germany) and 40% water as a mobile phase. The reverse-phase

chromatographic column (Discovery C18 (250 x 4.6 mm, 5 mm), Supelco, Bellefonte,

PA, USA) was used for the detection of capsaicin and dihydrocapsaicin. During HPLC

sample analysis, a standard solution was injected every 10 samples in order to evaluate

43

the retention time verification and instrument calibration.

iv) Quantification of capsaicinoids

A 20 µl aliquot was used for each HPLC injection. Capsaicinoids were identified with

reference to retention time of standards and by spiking the samples with standards.

Standard curves were developed using concentration versus peak area and

capsaicinoids were quantified using linear equation.

3.2.3: Capsaicin to dihydrocapsaicin ratio

The ratio between these capsaicinoids was calculated by dividing capsaicin to

dihydrocapsaicin

3.2.4: Total capsaicinoids

Total capsaicinoids is the sum of capsaicin and dihydrocapsaicin.

3.2.5: Total carotenoids

Two gram hot pepper sample was grounded using mortar and pestle. The grounded

sample was then taken in 100 ml flask. 30 ml hexane-acetone-absolute alcohol-toluene

(10+7+6+7) was added and covered with stopper and swirled for one minute. 2 ml 40%

methanolic KOH into a flask, swirled 1 minute and flask placed in 56°C water bath for

twenty minutes and after that neck of flask cooled to prevent losses of water. Sample was

cooled and let stand in dark for one hour. 30 ml hexane was pipetted into the flask,

swirled one minute and diluted to volume with 10% anhydrous Na2SO4 solution and

shaken vigorously for one minute. Let it to stand in the dark for one hour. The extract

was measured for its absorbance at 436 nm using IRMECO UV-Vis spectrophotometer

Model U2020 and β-carotene was used as standard. Total carotenoids were expressed as

mg 100 g-1 (AOAC, 1990).

3.2.6: Ascorbic acid

Ascorbic acid was quantitatively determined according to 2, 6-dichloroindophenol-dye

method (AOAC, 1970). Hot pepper 10 g fruit sample was ground with 2.5 ml of 20%

metaphosphoric acid and distilled water was then added up to 100 ml mark. 10 ml of

the suspension was titrated with standard 2, 6-dichloroindophenol freshly prepared dye

until light pink color persisted for 15 seconds. Ascorbic acid concentration in each

sample was then calculated as mg 100g-1.

44

3.2.7: Total phenolics

Total phenolic contents of hot peppers were analyzed using the modified Folin-

Ciocalteu reagent method (Pennycooke et al., 2005). About 0.5 g sample was

macerated in 3 ml 80% (v/v) acetone with a mortar and pestle. The samples were

placed into 1.5 ml tightly covered micro-tubes and incubated in darkness at 4ºC

overnight. Samples were centrifuged at 1000 rpm for 2 min. A mixture of 135 µl H2O,

750 µl 1/10 dilution Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA),

and 600 µl 7.5% (w/v) Na2CO3 was added to 50 µl of phenolic extract in 1.5 ml micro-

tubes. After vortexing for 10 s, the mixture was incubated at 45ºC in a water bath for

15 min. Samples were allowed to cool at room temperature before reading the

absorbance at 765 nm using IRMECO UV-Vis spectrophotometer Model U2020. A

blank was prepared from 50 µl 80% (v/v) acetone. Gallic acid standard curve was

prepared from a freshly made 1 mg ml-1 gallic acid (Acros Organics, Belgium) (in

80% (v/v) acetone) stock solution.

3.3 Experiment III: Evaluation of hot peppers for aflatoxin contamination,

microbial load and antioxidant quality under different storage conditions

3.3.1: Packaging and storage of samples

Fully expanded completely red fruits were harvested and dried under sunshine from 9

a.m. to 5 p.m. daily. The dried pods of hot pepper hybrids 250 g were packed in jute

bags and 9 µm thick polyethylene bags (20 x 32 cm). All these samples were stored for

five months under controlled conditions at 20°C, 25°C and 30°C respectively and

analyzed before storage and after every 50 days interval. Each sample had three

replications.

The following observations were recorded during the experiment III.

3.3.2: Moisture contents (%age)

The moisture contents of each sample was determined by using the air forced oven

drying method (indirect distillation at 105°C) according to the method described in

AACC (2000) Method No. 44-15A. The moisture contents of hot peppers was

determined by weighing about 2 g of sample into a weighed moisture dish and drying

at an oven temperature of 105°C till the constant weight of dry material is obtained.

The percentage difference in weight after drying was regarded as the moisture content

45

of the sample, expressed as a percentage of the original sample.

3.3.3: Mycological studies

i) Sterilization of glass ware

After washing with detergent, the sterilization of glass wares such as flasks, Petri

dishes and test tubes were carried out in hot air oven at 171°C for 30 minutes

according to Cappuccino and Sherman (1996).

ii) Media preparation

The media used for microbial growth was prepared according to Aziz et al. (1998) for

total fungal counts and Klich and Pitt (1988) for aspergillus counts.

iii) Sabouraud agar (SA) composition (Aziz et al., 1998)

Dextrose = 40 g

Peptone = 10 g

Agar = 20 g

Distilled water = 1000 ml

After complete mixing of the media it was autoclaved at 121°C, 15 lb/in2 pressure for

15 minute and cooled to 45°C. The pH of media was maintained at 6.4 + 0.2 using

N/10 NaOH and HCl.

iv) Aspergillus flavus parasiticus agar (AFPA) composition (Klich and Pitt, 1988)

Peptone = 10 g

Yeast extract = 20 g

Ferric ammonium citrate = 0.5 g

Dicloran = 1 ml of 0.2% solution in ethanol.

Chloramphenicol = 0.1 g

Agar = 15 g

Distilled water = 1000 ml

All ingredients were sterilized at 121°C for 15 minutes in autoclave. The medium was

cooled to 45°C and pH was maintained at 6.3 + 0.2 using N/10 NaOH and HCl.

v) Preparation of serial dilution

Dilution bottles were used for serial dilution. One gram sample and 9 ml distilled water

was added in dilution bottles and then mixed by inverting three times. It was labeled as

1 in 10 (10-1) dilution. Similarly 1 ml from diluted tube was taken to 2nd test tube

46

which already containing 9 ml of distilled water to obtain 10-2 dilution. By using the

above procedure, other serial dilution i.e. 10-3 and 10-4 were also prepared. For each

dilution, new disposable syringe was used.

vi) Total fungal counts

In each Petri plate 1 ml of the respective dilution and 10 ml of Sabouraud agar medium

were added. Medium and inoculums were immediately mixed by a combination of to

and fro shaking and circular movement lasting 5-7 seconds. Medium was allowed to

solidify. From each 10 petri plates, one control was also used to see the extent of

contamination during inoculation. The petri plates were incubated in inverted position

at 28°C for 24-48 hours.

vii) Aspergillus counts

Ten gram samples were ground and thoroughly mixed with 90 ml of sterile distilled

water. Spore counting was performed by plate count technique on a selective medium

for A. flavus and A. parasiticus (AFPA medium) after incubation for 7 days at 25ºC

using each suspension in a serial dilution from 10-1 up to 10-6 according to method

described by Pitt et al. (1983).

3.3.4: Determination of aflatoxins

i) Aflatoxin standards

These aflatoxin standards Biopure Lot # 06061 Technopark 1A 3430 Tulln, Austria

consisted of total 5 ml (B1 2.02, B2 0.500, G1 2.03 and G2 0.540 µg ml-1) were

purchased from Romer Laboratory Inc., USA. All solvents used for aflatoxin analysis

were of HPLC grade and freshly glass redistilled.

ii) Sample extraction and clean-up

Aflatoxins were analyzed by the procedure developed by Romer Laboratory Inc.,

1301-Stylemaster Drive, Union, MO, USA (Richard, 2000). Hot pepper finely ground

sample 25 g was taken in 250 ml conical flask and 100 ml solvent (acetonitrile: water,

84:16) was added. Flasks were placed in horizontal shaker for 1 hour. The extract was

filtered through Whatman filter paper (No. 3). Of the filtrate, 9 ml was taken in a glass

tube and 70 µl acetic acid was added to acidify the solution. The mixture was then pass

through a Romer MycoSep® column 228 (Romer Laboratory Inc., USA) with a flow

rate of 2 ml per minute. The cleaned-up extract (2 ml) was evaporated to dryness with

47

gentle steam of nitrogen gas in centrifuge glass tube. After drying, samples were

redissolved in 300 µl mobile phase and analyzed by High Performance Liquid

Chromatography (HPLC) according to the method of Alberts et al. (2006). The

concentrations of aflatoxins in samples were expressed as µg kg-1.

iii) Liquid chromatographic analysis

A Shimadzu (LC-10, Shimadzu, Japan) HPLC system equipped with LC- 10AS multi

solvent delivery system, a SPD-10A UV-Vis detector at a wavelength of 365 nm and

controlled the parameters with system controller unit (SCL-10A). The analysis was

carried out following the conditions: column temperature 30°C, flow rate of mobile

phase; 1.5 ml.min-1 and data acquisition was made using Class LC-10 software. All

analyses were performed isocratically using degassed HPLC grade acetonitrile:

methanol: water (22.5:22.5:55 v/v/v) (Merck, Germany) as a mobile phase. The

reverse-phase chromatographic column Discovery C18 (250 x 4.6 mm, 5 mm), Supelco,

Bellefonte, PA, USA was used for the detection of aflatoxins (B1, B2, G1 and G2).

During HPLC sample analysis, a standard solution was injected after every 10 samples

in order to evaluate the retention time verification and instrument calibration.

iv) Quantification of aflatoxins

A 20 µl aliquot was used for each HPLC injection. Aflatoxins were identified with

reference to retention time of standards and by spiking the samples with standards.

Standard curves were developed using concentration versus peak area and aflatoxins

were quantified using linear equation.

3.3.5: Antioxidants determination

Antioxidants were determined as described in experiment II.

3.4 Experiment IV: Effect of gamma-radiation on microbial load and antioxidant

quality of aflatoxin contaminated samples during storage

Sample of dried hot peppers 200 g having maximum microbial and aflatoxin

contamination from experiment III were stored in polyethylene bags and irradiated at

room temperature. Samples were irradiated at 2, 4 and 6 kGy dose levels in a Co60

gamma irradiator (Issle Dovatel. GIK-7-2, Russia), at a dose rate 0.4461 kGy h-1 at

Nuclear Institute for Food and Agriculture (NIFA), Peshawar, Pakistan. These samples

were analyzed immediately and then stored for three months at room temperature. At

48

the end of storage period, all non-radiated and irradiated samples were analyzed for

parameters studied in experiment III. Each treatment had three replications.

3.5: Statistical analysis

Analysis of variance of the data from each attribute was computed using the

STATISTICA Computer Program. The Least Significant Difference test at 5% level of

probability was used to test the differences among mean values (Steel et al., 1997).

49

CChhaapptteerr 44

RREESSUULLTTSS

AANNDD

DDIISSCCUUSSSSIIOONN

4.1 Experiment I: Effect of plastic mulches on growth and yield of hot peppers

4.1.1: Soil temperature (ºC)

Since color of the plastic mulch determined the degree of soil warming and generally

light colored mulches maintained highest values of soil temperature than dark colored

mulches due to light transmission. Data regarding soil temperature under different mulch

treatments revealed that mulched treatments increased soil temperature significantly

during both the years (see figure 4.1.1). Clear plastic mulch maintained highest values of

soil temperature and on an average it was 3.9 and 1.2ºC higher under clear and black

plastic mulch respectively when compared with un-mulched control during the year

2005-06 while it was 3.3 and 1.15ºC higher than un-mulched treatment in 2006-07. These

results are in line with Locher et al. (2005) who found that black plastic mulch caused

1.4ºC increase in soil temperature when compared to the un-mulched control. Similarly

they also reported that soil temperature under light colored plastic mulches (clear, violet,

light green) was 2.5-2.9ºC higher when compared with bare soil.

Each of the different colored mulches used in the production of crops causes different

temperature effects. Different forms of plastic mulches are available varying from woven

plastic to smooth plastic and embossed plastic films. Now-a-days 100% compostable and

biodegradable mulches are also available in advanced countries and are more

environment friendly. In addition to the surface structure, the color and thickness of the

mulch can vary. Each of these variations can have an effect on the plant microclimate and

in particular the soil temperature. Clear plastic mulch is believed to achieve higher soil

temperatures than black plastic. This happens because much of the incident radiation is

absorbed by colored films (Argall and Stewart, 1990) while in case of black plastic mulch

50

Figure 4.1.1: Average soil temperature at a depth of 10cm after transplanting over three months for 15-day periods during the year 2005-06 and 2006-07 at 12.00-1.00 solar hour. (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

0

5

10

15

20

25

30

35

15 30 45 60 75 90

2006-07

Days after transplanting (DAT)

Soil

tem

pera

ture

(ºC

)

0

5

10

15

20

25

30

35

15 30 45 60 75 90

2005-06

Days after transplanting (DAT)

Soil

tem

pera

ture

(ºC

)

BS

BPMCPM

51

it does not pass through the soil. As plastic mulches modify the soil temperature regime

according to their optical properties (Ham et al., 1993). Increase in root zone temperature

by plastic mulch also affect the uptake and translocation of essential nutrients, therefore

influencing root and shoot growth (Tindall et al., 1990). Result from present studies

indicated that plants grown on plastic mulches showed a better vegetative and

reproductive response during both the years when compared with bare soil (control).

These result supports the findings of Tarara (2000) that plastic mulches affect plant

microclimate by modifying the soil energy balance and restricting soil water evaporation,

thereby affecting plant growth and its yield.

4.1.2: Plant height at first harvest (cm)

The year effect on plant height at first harvest was significant with taller plants in 2005-

06 than 2006-07. Significant variations were observed for hybrids and mulch treatments

on this trait (see table 4.1.1). All hybrids differ significantly from each other and on

average in both years, plant height was maximum in Wonder King (97.33 cm) followed

by Maha (85.97 cm) and Sky Red (79.54 cm).

Plastic mulches had significant effect on plant height. When the data was averaged for

two years, plants grown on black plastic mulch produced taller plants (94.32 cm) as

compared with bare soil plants. However, plants from clear plastic mulch were slightly

shorter than black plastic mulch plants but significantly taller than bare soil plants. The

interactive effect of hybrid x mulch was significant in 2005-06 but non-significant in

2006-07 (see figure 4.1.2). Difference in plant height within hybrids was due to their

genetic variability; however effect of plastic mulches on plant height may be attributed to

increase in soil temperature and moisture retention which changes plant microclimate as a

result faster plant growth was observed. Maximum plant height was observed in black

plastic mulch which might be due to absence of weeds when compared with plants grown

on clear plastic mulch. Similar results were reported by Hallidri (2001) who observed that

plant height was maximum in cucumber plants grown on black and transparent polythene

mulch than control (bare soil).

4.1.3: Stem diameter (mm)

Results obtained for stem diameter are presented in table 4.1.2, which clearly showed that

52

Table 4.1.1: Effect of hybrids and mulches on plant height (cm) in hot peppers

Means having different letters differ significantly at 5% probability. NS = Non significant

0

20

40

60

80

100

120

BS BPM CPM

Mulch treatments

Pla

nt h

eigh

t (c

m)

Sky Red

Maha

Wonder King

Figure 4.1.2: Interactive effect of hybrid x mulch on plant height (cm) in hot peppers during the year 2006-07. (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

Plant height (cm)

Treatments

2005-06

2006-07

Two years

mean

Hybrid (H) H1 = Sky Red 82.37 c 76.71 c 79.54 c H2 = Maha 88.27 b 83.66 b 85.97 b H3 = Wonder King 98.81 a 95.86 a 97.33 a Mulch (M) M1 = Bare Soil 81.16 c 76.81 c 78.98 c M2 = Black Plastic 96.63 a 92.01 a 94.32 a M3 = Clear Plastic 91.66 b 87.42 b 89.54 b Year mean 89.82 a 85.41 b Interaction (HxM) * NS

53

year effect was non-significant for stem diameter. However, hot pepper hybrids differ

significantly in both years and when data was averaged for two years; stem diameter was

highest in Sky Red (25.43 mm). Both Maha and Wonder King were statistically alike

with stem diameter 21.97 mm and 22.31 mm respectively.

The effect of plastic mulch on stem diameter was non-significant in the year 2005-06 but

significant in 2006-07. However, when the data was averaged for two years, results were

statistically significant where both plastic mulches behave statistically alike but stem

diameter was slightly higher in plants grown on clear plastic mulch (23.51 mm) than

black plastic mulch (23.41 mm). Stem diameter was lowest in plants grown on bare soil

(22.80 mm). The interaction of hybrid x mulch was non-significant in both years. These

results support the findings of Hallidri (2001) who reported that stem diameter was not

changed by the application of plastic mulch in cucumber plants.

4.1.4: Root fresh weight (g)

Good root growth always plays an important role in above ground plant biomass. Year

effect on root fresh weight was non-significant. However, significant variations were

observed between hybrids and mulch treatments in both years (see table 4.1.3). Among

hybrids, when data was averaged for two years root fresh weight was maximum in hot

pepper hybrid Maha (45.88 g) which was statistically at par with Wonder King (42.40 g).

However, root fresh weight was lowest in Sky Red (36.98 g).

The effect of plastic mulch on root fresh weight was significant and plants grown on both

plastic mulches behaved statistically alike in this regard. When the data was averaged for

two years, plants grown on clear plastic mulch had slightly higher root fresh weight

(45.74 g see table 4.1.3) while root fresh weight was less in case of plants grown on bare

soil (36.96 g). The interactive effect of hybrid x mulch was non-significant in both years.

High soil temperature under plastic mulch improves root growth. These results are being

supported by the findings of Gupta and Acharya (1993) that increased root biomass under

black polyethylene mulch was attributed to the resultant increase in soil temperature and

nutrient uptake.

4.1.5: Root dry weight (g)

Significant effect was observed for this parameter between two years with higher root dry

weight in 2005-06 than 2006-07. All hybrids showed similar response as for root fresh

54

Table 4.1.2: Effect of hybrids and mulches on stem diameter (mm) in hot peppers

Stem diameter (mm)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 25.34 a 25.53 a 25.43 a H2 = Maha 22.24 b 21.70 b 21.97 b H3 = Wonder King 22.51 b 22.12 b 22.31 b Mulch (M) M1 = Bare Soil 23.07 22.52 b 22.80 b M2 = Black Plastic 23.38 23.43 a 23.41 a M3 = Clear Plastic 23.63 NS 23.40 a 23.51 a Year mean 23.36 23.13 NS Interaction (HxM) NS NS

Means having different letters differ significantly at 5% probability. NS = Non significant

Table 4.1.3: Effect of hybrids and mulches on root fresh weight (g) in hot peppers

Root fresh weight (g)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 36.24 b 37.72 c 36.98 b H2 = Maha 48.77 a 43.00 a 45.88 a H3 = Wonder King 44.96 a 39.84 b 42.40 a Mulch (M) M1 = Bare Soil 36.93 c 32.98 c 34.96 b M2 = Black Plastic 44.72 ab 44.44 a 44.58 a M3 = Clear Plastic 48.33 a 43.13 a 45.73 a Year mean 43.33 43.85 NS Interaction (HxM) NS NS

Means having different letters differ significantly at 5% probability. NS = Non significant

55

weight. When the data was averaged for two years, root dry weight was highest in Maha

(20.92 g) followed by Wonder King (19.36 g) and Sky Red (18.38 g), (see table 4.1.4).

However, during the year 2006-07 both Wonder King and Sky Red were statistically at

par for this variable. The effect of plastic mulch on root dry weight was significant and

both plastic mulches were statistically alike in both years. When the data was averaged

for two years, root dry weight was higher in plants grown on black and clear plastic

mulch (22.02 g and 21.27 g) as compared to bare soil plants (15.36 g). The interactive

effect of hybrid x mulch was non-significant in both the years. These results indicate that

it might be due to better soil environment under plastic mulches, where plants had better

root growth which ultimately results in good plant growth above ground. Improved

productivity with mulches was related to the increased root DW (Niu et al., 2004).

Similarly Kirnak and Demirtas (2006) reported that plants grown on plastic mulches had

significantly higher root dry weights as compared to bare soil plants in cucumber.

4.1.6: Root length (cm)

The year effect on root length was significant and was more in 2005-06 than 2006-07.

Significant difference was observed between hybrids in both years where root length was

higher in Sky Red while both Maha and Wonder King behave statistically alike for this

variable see table 4.1.5. When the data was averaged for two years, Sky Red with first

position (29.03 cm) followed by Wonder King (24.28 cm) and Maha (23.69 cm).

As for as effect of plastic mulches is concerned, the difference was non-significant when

both black and clear plastic mulches were compared; however, root length differd

significantly compared with bare soil plants. When the data was averaged for two years,

plants grown on black plastic mulch had more root length 29.43 cm than clear plastic

mulch 28.10 cm while in case of bare soil 18.67 cm root length was recorded. The

interactive effect of hybrid x mulch was non-significant in both the years. The incident

radiation absorbed by mulches can be readily transmitted to the soil surface; as a result

the air near the soil surface is relatively immobile with a low thermal conductivity. Thus,

mulches applied on the soil surface cause a consistent increase in soil temperature which

consistently improves root development in vegetables grown (Cooper, 1973). Hasan et al.

(2005) concluded that length of main roots were significantly higher in tomato plants

grown on plastic mulch than bare soil plants.

56

Table 4.1.4: Effect of hybrids and mulches on root dry weight (g) in hot peppers

Root dry weight (g)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 19.25 c 17.51 b 18.38 c H2 = Maha 22.45 a 19.38 a 20.92 a H3 = Wonder King 20.70 b 18.02 b 19.36 b Mulch (M) M1 = Bare Soil 16.42 b 14.30 b 15.36 b M2 = Black Plastic 23.40 a 20.65 a 22.02 a M3 = Clear Plastic 22.58 a 19.96 a 21.27 a Year mean 20.80 a 18.30 b Interaction (HxM) NS NS

Means having different letters differ significantly at 5% probability.

NS = Non significant

Table 4.1.5: Effect of hybrids and mulches on root length (cm) in hot peppers

Root length (cm)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 30.77 a 27.30 a 29.03 a H2 = Maha 25.12 b 22.26 b 23.69 b H3 = Wonder King 25.96 b 22.61 b 24.28 b Mulch (M) M1 = Bare Soil 19.72 b 17.62 b 18.67 b M2 = Black Plastic 31.42 a 27.45 a 29.43 a M3 = Clear Plastic 30.72 a 27.10 a 28.91 a Year mean 27.28 a 24.05 b Interaction (HxM) NS NS

Means having different letters differ significantly at 5% probability. NS = Non significant

57

4.1.7: Leaf area plant-1 (cm2)

The year effect on leaf area was significant and it was higher during 2005-06 than 2006-

07. The hybrids, plastic mulch and their interactions had significant effect on leaf area

during both years (see table 4.1.6). All hybrids differ significantly for this variable and

when the data was averaged for two years, leaf area plant-1 was significant in Sky Red

(6120.68 cm2) followed by Maha (5913.25 cm2) and Wonder King (5630.62 cm2). This

variation in leaf area between hybrids might be due to the genetic make up of individual

hybrid.

Plastic mulches had significant effect on leaf area and plants grown on black plastic

mulch had significantly higher leaf area than clear plastic mulch and bare soil plants.

When the data was averaged for two years, highest leaf area 6803.98 cm2 was observed in

black plastic mulch plants followed by 6140.87 and 4719.80 cm2 respectively for plants

grown on clear plastic mulch and bare soil. These results are in line with the findings of

Luis et al. (2002) that black plastic mulch alone or combined with row covers had a

positive effect on leaf area in bell peppers relative to the control. Mulching with black or

clear polyethylene increased total plant growth and led to an increased rate of branching

and early flowering in tomato (Wein and Minotti, 1988). Since plant light environment

and soil temperature was affected by mulch surface color. This increase in leaf area might

be attributed to change in plant microclimate by black and clear plastic mulch which

results in better vegetative growth of hot pepper hybrids. However, less leaf area plant-1

under clear plastic mulch might be due to weed competition as compared to black plastic

mulch with no weed growth.

4.1.8: Days taken to first flowering (days)

Non-significant difference was observed between two years for this reproductive trait.

However, significant difference was observed among hybrids for days to first flower after

transplanting and almost similar results were observed for hybrids in both years (see table

4.1.7). When the data was averaged for two years, the hybrid Maha took 76.02 days to

first flower followed by Wonder King 71.67 days while Sky Red start early flowering

than other two with 68.86 days. This difference in days to first flower clearly shows that

this attributes to the genetic ability of individual hybrid in both years.

58

Table 4.1.6: Effect of hybrids and mulches on leaf area plant-1 (cm2) in hot peppers

Leaf area plant-1 (cm2)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 6250.86 a 5990.50 a 6120.68 a H2 = Maha 6039.95 b 5786.56 b 5913.25 b H3 = Wonder King 5770.64 c 5490.60 c 5630.62 c Mulch (M) M1 = Bare Soil 4831.12 c 4608.47 c 4719.80 c M2 = Black Plastic 6983.94 a 6623.83 a 6803.89 a M3 = Clear Plastic 6246.39 b 6035.34 b 6140.87 b Year mean 6020.48 a 5755.88 b Interaction (HxM) * *

Means having different letters differ significantly at 5% probability. NS = Non significant

59

0

1000

2000

3000

4000

5000

6000

7000

8000

BS BPM CPM

Mulch treatments (A)

Lea

f are

a pe

r pl

ant (

cm2)

Sky RedMahaWonder King

0

1000

2000

3000

4000

5000

6000

7000

8000

BS BPM CPM

Much treatments (B)

Lea

f are

a pe

r pl

ant (

cm2)

Figure 4.1.3: Interactive effect of hybrid x mulch on leaf area per plant (cm2) in hot peppers during the year 2005-06 (A) and 2006-07 (B). BS: (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

60

Table 4.1.7: Effect of hybrids and mulches on days taken to first flowering after transplanting (days) in hot pepper

Days taken to first flowering (days)

Treatments

2005-06 2006-07

Two years mean

Hybrid (H) H1 = Sky Red 68.25 c 68.55 c 68.86 c H2 = Maha 75.86 a 76.18 a 76.02 a H3 = Wonder King 72.44 b 70.91 b 71.67 b Mulch (M) M1 = Bare Soil 75.12 a 75.90 a 75.51 a M2 = Black Plastic 72.44 b 71.03 b 71.73 b M3 = Clear Plastic 69.00 c 68.72 c 68.86 c Year mean 72.19 72.22 NS Interaction (HxM) * *

Means having different letters differ significantly at 5% probability. NS = Non significant

61

0

20

40

60

80

100

BS BPM CPM

Mulch treatments (A)

Day

s to

firs

t flo

wer

(day

s)

Sky Red

Maha

Wonder King

0

20

40

60

80

100

BS BPM CPM

Mulch treatments (B)

Day

s to

firs

t flo

wer

(day

s)

Figure 4.1.4: Interactive effect of hybrid x mulch on days taken to first flower (days) during the year 2005-06 (A) and 2006-07 (B). (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

62

Plastic mulches had significant effect on this variable and clear plastic mulch reduced the

number of days to first flower. On an average during both the years, plants grown on

clear plastic mulch took 68.86 days to start flowering followed by black plastic mulch

71.73 days while plants grown without mulch took 75.51 days to start flowering. The

interactive effect of hybrid x mulch was significant in both years (see figure 4.1.4). The

reason for plants starts early flowering under clear plastic mulch might be the higher soil

temperature. Changes in root zone temperature can affect the uptake and translocation of

essential nutrients, therefore influencing root and shoot growth (Tindall et al., 1990).

These results support the findings of Tuli and Yesilsoy (1997) who reported that clear

plastic mulch was found to be more efficient on first blossoming and harvesting time in

squash while lowest plant growth and yield values were observed in bare soil. Similarly

Ibarra-Jimenez et al. (2005) found that time to anthesis in water melon (appearance of

perfect flowers) was 45 and 55 days after sowing for black plastic mulch and control

plants respectively.

4.1.9: Fresh fruit yield plant-1 (kg)

The year effect was significant and fresh fruit yield plant-1 was 10% higher in 2005-06

than 2006-07 (see table 4.1.8). All hybrids differ greatly in terms of fruit yield and when

the data was averaged for two years, highest fruit yield plant-1 was obtained from Wonder

King (1.37 kg) followed by Maha (1.20 kg) and Sky Red (1.09 kg). Plastic mulches had

significant effect on fresh fruit yield plant-1. When the data was averaged for two years,

fruit yield from plants grown on black plastic mulch was 39% greater than un-mulched

treatment. Similarly fresh fruit yield was 35% higher from plants grown on clear plastic

mulch than bare soil. This difference between mulched and un-mulched treatments might

be due to increase in number of fruit set. The interactive effect of hybrid x mulch was

non-significant in 2005-06 but was significant in 2006-07 (see figure 4.1.5). As plastic

mulches improved stand establishment and fruit yield relative to un-mulched (control).

Locher et al. (2005) found that higher yield of sweet peppers were achieved from

mulched treatments due to higher soil temperatures than the un-mulched treatment. The

difference in spectral quality of light reflected from different color of mulches influenced

yield through the regulatory affect of the phytochrome system (Decoteau et al., 1988).

63

Table 4.1.8: Effect of hybrids and mulches on fresh fruit yield plant-1 (kg) in hot pepper

Fresh fruit yield plant-1

(kg)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 1.15 c 1.03 c 1.09 c H2 = Maha 1.26 b 1.13 b 1.20 b H3 = Wonder King 1.42 a 1.31 a 1.37 a Mulch (M) M1 = Bare Soil 0.93 c 0.83 c 0.88 c M2 = Black Plastic 1.51 a 1.35 a 1.43 a M3 = Clear Plastic 1.40 b 1.28 b 1.34 b Year mean 1.28 a 1.16 b Interaction (HxM) NS *

Means having different letters differ significantly at 5% probability. NS = Non significant

0

0.2

0.40.6

0.8

1

1.21.4

1.6

1.8

BS BPM CPM

Mulch treatments

Fre

sh fr

uit y

ield

per

pla

nt (k

g)

Sky RedMahaWonder King

Figure 4.1.5: Interactive effect of hybrid x mulch on fresh fruit yield per plant (kg) in hot peppers during the year 2006-07. (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

64

Mulch ameliorated the hydrothermal regime of the soil, improved vegetative and

flowering performance and significantly increased fruit yield of tomato over bare ground

(Agele et al., 2000). Similarly, vegetable crops grown on plastic mulches have shown

earlier (7 to 14 days) and increased yields (2 to 3 times) over vegetable crops grown on

bare soil (Lamont, 1993). Hassan et al. (1995) reported that mulching is practically

beneficial in chilli production and may be related to soil moisture contents because plant

dry weight was positively correlated with soil temperature and moisture content. Waterer

(2000) observed higher yield of peppers was observed under clear plastic mulch followed

by black and wavelength selective mulch treatments as compared to bare soil (control);

even though weed growth was extensive under the clear mulch with no added herbicide.

4.1.10: Dry fruit yield plant-1 (kg)

Dry fruit yield plant-1 obtained is the final criterion for measuring yield. The year effect

was significant for this trait. The average dry fruit yield plant-1 was 11% greater in 2005-

06 than 2006-07 (see table 4.1.9). Similarly all hybrids differ greatly with respect to dry

fruit yield. When the data was averaged for two years, dry fruit yield plant-1 was highest

in Wonder King (0.23 kg) followed by Maha (0.21 kg) and Sky Red (0.18 kg).

Plastic mulches had significant effect on dry fruit yield plant-1. When the data was

averaged for two years, plants grown on black and clear plastic mulch gave 38% and 30%

more dry fruit yield plant-1 when compared with un-mulched plants. Similarly, the

interactive effect of hybrid x mulch was non-significant in 2005-06 but was significant in

2006-07 (see figure 4.1.6). These results gave clear indication about improved plant

growth with plastic mulches increased hot pepper yields than without mulch.

4.1.11: Harvest index (%)

Year effect was significant on harvest index, a measure of yield efficiency (Harvest index

= fruit yield/fruit yield + vegetative yield) with 66% in 2005-06 and 64% during the year

2006-07. Significant difference was observed among hybrids for harvest index and

almost similar results were observed during both years (see table 4.1.10). When the data

was averaged for two years, both Wonder King and Maha were statistically alike with 67

and 65% harvest index respectively. However, harvest index was lowest in Sky Red

during both years.

65

Table 4.1.9: Effect of hybrids and mulches on dry fruit yield plant-1 (kg) in hot pepper

Dry fruit yield plant-1 (kg)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 0.193 c 0.171 c 0.182 c H2 = Maha 0.232 b 0.201 b 0.216 b H3 = Wonder King 0.245 a 0.225 a 0.235 a Mulch (M) M1 = Bare Soil 0.166 c 0.151 c 0.158 c M2 = Black Plastic 0.272 a 0.232 a 0.252 a M3 = Clear Plastic 0.233 b 0.214 b 0.223 b Year mean 0.223 a 0.199 b Interaction (HxM) NS *

Means having different letters differ significantly at 5% probability. NS = Non significant

0

0.1

0.2

0.3

BS BPM CPM

Mulch treatments

Dry

frui

t yie

ld p

er p

lant

(kg)

Sky RedMahaWonder King

Figure 4.1.6: Interactive effect of hybrid x mulch on dry fruit yield per plant (kg) in hot peppers during the year 2006-07. (BS - Bare soil, BPM - Black plastic mulch and CPM - Clear plastic mulch).

66

Table 4.1.10: Effect of hybrids and mulches on harvest index (%) in hot pepper

Harvest Index (%)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 63 b 62 b 62 b H2 = Maha 66 a 64 ab 65 a H3 = Wonder King 68 a 65 a 67 a Mulch (M) M1 = Bare Soil 63 b 60 b 62 b M2 = Black Plastic 68 a 65 a 66 a M3 = Clear Plastic 66 a 66 a 66 a Year mean 66 a 64 b Interaction (HxM) NS NS

Means having different letters differ significantly at 5% probability. NS = Non significant

67

As for as effect of plastic mulches on harvest index is concerned, both the plastic mulches

were statistically alike and gave greater harvest index than bare soil (control). However,

harvest index was significantly higher during the year 2005-06 than 2006-07. When the

data was averaged for two years, harvest index was 66% on both black and clear plastic

mulch treatments as compared to bare soil 62%. The interactive effect of hybrid x mulch

was non-significant in both the years. The reason for not much difference for harvest

index between hybrids grew under plastic mulches might be due to similar growing

conditions for plants during both years.

4.1.12: Fruit length (cm)

The results pertaining to fruit length are presented in table 4.1.11, which clearly depicted

that year effect was significant. Significant variation between hybrids was seen for this

trait and when the data was averaged for two years, fruit length was highest in Wonder

King (10.60 cm) followed by Maha (8.34 cm) and Sky Red (6.55 cm). The difference in

fruit length between hybrids was due to individuality of each hybrid for their inherited

reproductive characteristics.

The effect of plastic mulches was significant and plants grown on black plastic mulch

produced significantly larger fruits. When the data was averaged for two years, plants

grown on black plastic had fruit length 9.23 cm followed by 8.83 cm and 7.44 cm in

plants grown on clear plastic and bare soil plants respectively. The interactive effect of

hybrid x mulch was non-significant in 2005-06 while significant in 2006-07 (see figure

4.1.7). Since a general increase in plant growth was observed by the use of plastic mulch

which results in better reproductive performance of hot peppers. Pakyurek et al. (1993)

observed increased fruit size in hot peppers grown on plastic mulch along with increased

early and total yield by 39% and 19% respectively with clear plastic mulch. Length of

mature fruit is important as a component of fruit size. The latter is of interest because of

economic consideration in the harvesting and processing of the crop. Mature fruits are

harvested manually and farmer preferred varieties with larger fruits which facilitated the

picking since this operation constitutes about 25% of the costs incurred in hot pepper

production.

68

Table 4.1.11: Effect of hybrids and mulches on fruit length (cm) in hot pepper

Fruit length (cm)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 6.66 c 6.45 c 6.55 c H2 = Maha 8.37 b 8.30 b 8.34 b H3 = Wonder King 10.77 a 10.43 a 10.60 a Mulch (M) M1 = Bare Soil 7.44 c 7.44 c 7.44 c M2 = Black Plastic 9.39 a 9.06 a 9.23 a M3 = Clear Plastic 8.97 b 8.68 b 8.83 b Year mean 8.60 a 8.39 b Interaction (HxM) NS *

Means having different letters differ significantly at 5% probability. NS = Non significant

0

2

4

6

8

10

12

BS BPM CPM

Mulch treatments

Fru

it le

ngth

(cm

)

Sky RedMahaWonder King

Figure 4.1.7: Interactive effect of hybrid x mulch on fruit length (cm) in hot peppers during the year 2006-07. (BS - Bare soil, BPM - Black plastic mulch and CPM - Clear plastic mulch).

69

4.1.13: Fruit diameter (mm)

The year effect on fruit diameter was significant and this was more during the year 2005-

06 than 2006-07 (see table 4.1.12). All hybrids differ significantly for this trait and when

the data was averaged for two years, fruit diameter was greater in Wonder King 11.22

mm followed by Maha and Sky Red which is 10.76 and 9.47 mm respectively. As far as

effect of plastic mulches is concerned, plants grown on black plastic mulch had

maximum fruit diameter 11.27 mm while fruit diameter was lowest in fruits obtained

from bare soil (control) plants (9.31 mm). The interactive effect of hybrid x mulch was

significant in both years (see figure 4.1.8). So far research information regarding this

variable is scarce. From these results, it can be concluded that fruit quality (fruit length

and diameter) in hot peppers can be improved by the application of plastic mulches. The

combined effects of soil temperature, soil moisture and weed suppression not only work

to improve crop growth but they also facilitate hand picking and lead to higher yields and

increased fruit size (Scheerens and Brenneman, 1994).

4.1.14: Leaf nitrogen content at fruit set (%)

The year effect on leaf nitrogen contents at fruit set was significant (see table 4.1.13).

Significant differences were observed among hot pepper hybrids for this nutrient. When

the data was averaged for two years, leaf nitrogen contents were highest in Wonder King

than Maha and Sky Red which were statistically at par. Regarding the effect of plastic

mulch on leaf nitrogen content, plants grown on plastic mulches had significantly higher

nitrogen contents as compared to bare soil plants. Similarly, during the year 2005-06, leaf

nitrogen contents were greater in plants grown on clear plastic mulch while they were

higher in plants from black plastic mulch during the year 2006-07. However, when the

data was averaged for two years, both plastic mulch treatments were statistically alike

with respect to leaf nitrogen. The interactive effect of hybrid x mulch was significant in

2005-06 (see figure 4.1.9) while it was non-significant during the year 2006-07. These

results revealed that plastic mulch improve the nutrient uptake of plants by creating

favorable plant microclimate. These results are in line with the findings of Hassan et al.

(1995) who reported that chilli plants grown on black plastic mulch had significantly

higher leaf nitrogen contents (4.14%) at early fruiting stage as compared to plants grown

on bare soil (control) which is 3.92%. Similarly, Karp et al. (2006) reported that

70

Table 4.1.12: Effect of hybrids and mulches on fruit diameter (mm) in hot peppers

Fruit diameter (mm)

Treatments

2005-06

2006-07

Two years mean

Hybrid (H) H1 = Sky Red 9.47 c 9.17 c 9.45 c H2 = Maha 10.73 b 10.79 b 10.76 b H3 = Wonder King 11.39 a 11.04 a 11.22 a Mulch (M) M1 = Bare Soil 9.18 c 9.43 c 9.31 c M2 = Black Plastic 11.47 a 11.07 a 11.27 a M3 = Clear Plastic 10.94 b 10.49 b 10.72 b Year mean 10.53 a 10.33 b Interaction (HxM) * *

Means having different letters differ significantly at 5% probability. NS = Non significant

71

0

2

4

6

8

10

12

14

BS BPM CPM

Mulch treatments (A)

Fru

it d

iam

eter

(mm

)

Sky Red

MahaWonder King

0

2

4

6

8

10

12

14

BS BPM CPM

Mulch treatments (B)

Fru

it d

iam

eter

(mm

)

Figure 4.1.8: Interactive effect of hybrid x mulch on fruit diameter (mm) in hot peppers during the year 2005-06 (A) and 2006-07 (B). (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

72

Table 4.1.13: Effect of hybrids and mulches on leaf nitrogen (%) at fruit set in hot pepper

Nitrogen (%)

Treatments

2005-06

2006-07

Two years

mean

Hybrid (H) H1 = Sky Red 3.53 b 3.31 c 3.42 b H2 = Maha 3.38 c 3.42 b 3.40 b H3 = Wonder King 3.58 a 3.53 a 3.56 a Mulch (M) M1 = Bare Soil 3.35 c 3.30 c 3.33 b M2 = Black Plastic 3.55 b 3.51 a 3.53 a M3 = Clear Plastic 3.59 a 3.45 b 3.52 a Year mean 3.50 a 3.42 b Interaction (HxM) * NS

Means having different letters differ significantly at 5% probability. NS = Non significant

1

1.5

2

2.5

3

3.5

4

BS BPM CPM

Mulch treatments

Lea

f nit

roge

n (%

)

Sky red

Maha

Wonder King

Figure 4.1.9: Interactive effect of hybrid x mulch on leaf nitrogen content (%) during the year 2005-06. (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

73

mulching treatment significantly influenced nutrient content of leaves in blueberry.

4.1.15: Leaf phosphorus contents at fruit set (%)

The year effect on leaf phosphorus contents at fruit set in hot pepper hybrids was non-

significant (see table 4.1.14). However, hybrids differ significantly in both years but

when the data was averaged for two years, they were non-significant. Leaf phosphorus

contents were significantly higher in both hybrids Wonder King and Maha than Sky Red

and both these were statistically alike during the year 2005-06 where as during the year

2006-07, leaf phosphorus contents were significantly higher in Sky Red than Maha and

Wonder King which were statistically alike. Plastic mulches had significant effect on this

variable and plants grown on clear plastic mulch had significantly higher leaf phosphorus

contents than black plastic mulch and bare soil. When the data was averaged for two

years, leaf phosphorus contents were greater in clear plastic mulch treatment (0.43 %)

followed by black plastic mulch (0.41%) and bare soil (0.37%). The interactive effect of

hybrid x mulch was non-significant in both years. These results support the findings of

Wein et al. (1993) who reported that phosphorus concentration in tomato leaves was

significantly higher in plants grown on clear plastic mulch due to better soil temperature

than plants grown on bare soil (control).

4.1.16: Leaf potash contents at fruit set (%)

The year effect on leaf potash contents at fruit set was significant (see table 4.1.15). All

hybrids differ significantly for leaf potash contents and when the data was averaged for

two years, it was highest in Wonder King followed by Maha and Sky Red. However, leaf

potash contents were 3-4% greater in the year 2005-06 than 2006-07. Significant

differences were observed among plastic mulch treatments with respect to leaf potash

contents as compared to bare soil. When the data was averaged for two years, leaf potash

concentration was significantly greater in plants grown on black plastic mulch. However,

plants grown on clear plastic mulch differ significantly for this variable than bare soil

plants. The interactive effect of hybrid x mulch was significant in both the years (see

figure 4.1.10). These results are in line with the findings of Hassan et al. (1995) who

reported that chilli plants grown on black plastic mulch had significantly higher leaf

potash contents (4.29 %) at early fruiting stage as compared to plants grown on bare soil

(3.85%). Reuter and Robinson (1986) reported that leaf nutrient levels nitrogen (2.9 to

74

Table 4.1.14: Effect of hybrids and mulches on leaf phosphorus (%) at fruit set in hot pepper

Phosphorus (%) Treatments

2005-06

2006-07

Two years

mean

Hybrid (H) H1 = Sky Red 0.38 b 0.42 a 0.40 H2 = Maha 0.42 a 0.39 b 0.41 H3 = Wonder King 0.43 a 0.38 b 0.41 NS Mulch (M) M1 = Bare Soil 0.37 c 0.37 c 0.37 c M2 = Black Plastic 0.42 b 0.39 b 0.41 b M3 = Clear Plastic 0.44 a 0.43 a 0.43 a Year mean 0.41 NS 0.40 NS Interaction (HxM) NS NS

Means having different letters differ significantly at 5% probability. NS = Non significant

Table 4.1.15: Effect of hybrids and mulches on leaf potash (%) at fruit set in hot pepper

Potash (%) Treatments

2005-06

2006-07

Two years

mean

Hybrid (H) H1 = Sky Red 3.60 c 3.47 c 3.53 c H2 = Maha 3.71 b 3.57 b 3.64 b H3 = Wonder King 3.74 a 3.68 a 3.71 a Mulch (M) M1 = Bare Soil 3.56 c 3.45 c 3.50 c M2 = Black Plastic 3.79 a 3.67 a 3.73 a M3 = Clear Plastic 3.71 b 3.59 b 3.65 b Year mean 3.68 a 3.57 b Interaction (HxM) * *

Means having different letters differ significantly at 5% probability. NS = Non significant

75

1

1.5

2

2.5

3

3.5

4

BS BPM CPM

Mulch treatments (A)

Lea

f pot

ash

cont

ents

(%)

Sky RedMahaWonder King

0

0.5

1

1.5

2

2.5

3

3.5

4

BS BPM CPM

Mulch treatments (B)

Lea

f pot

ash

cont

ents

(%)

Figure 4.1.10: Interactive effect of hybrid x mulch on leaf potash content (%) during the year 2005-06 (A) and 2006-07 (B). (BS - Bare soil; BPM - Black plastic mulch and CPM - Clear plastic mulch).

76

4.6%), phosphorus (0.3-0.6%) and potassium (2.6-5.5%) at early fruit stages were

adequate for better vegetative and reproductive response of peppers.

4.1.17. Conclusion

From these results, it can be concluded that vegetative and reproductive traits of hot

pepper hybrid Wonder King significantly performed better than Sky Red and Maha with

few exceptions of root biomass and leaf area. Evaluation of plastic mulches manifested

that quantitative and qualitative attributes of hot pepper hybrids significantly improved

by the application of black plastic mulch. Nutrient uptake pattern in hot pepper hybrids

depicted black > clear > bare soil (control); however, the severity of weed proliferation

and infestation was intensive under clear plastic mulch since complete inhibition of weed

growth was observed under black plastic mulch which could be helpful to boost hot

pepper production in poly/plastic tunnels.

77

4.2 Experiment II: Quantification of antioxidant constituents in hot peppers

harvested at different stages

4.2.1: Capsaicin (mg 100g-1)

Pungency in hot peppers is an important quality attribute due to the presence of

capsaicinoids, including capsaicin and four structurally related compounds namely

nordihydrocapsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin.

Capsaicin is the most abundant capsaicinoid and contributes principally to the

characteristic pungency in hot peppers. Analysis of variance revealed significant (P ≤

0.01) difference for capsaicin concentration in all hot pepper hybrids, harvesting stages

and their interactions (see table 4.2.1). However capsaicin concentration was highest

(average of five harvesting stages) in Sky Red (48.29 mg 100g-1) followed by Wonder

King (33.94 mg 100g-1) and Maha (29.23 mg 100g-1). All hot pepper hybrids studied had

capsaicin concentration in the range of 30 to 50 mg 100g-1 at different stages of harvest

(Meterska and Perucka, 2005).

Fixing the harvesting stage for high capsaicin concentration would be helpful in deciding

the best one for increasing the quality of the produce. The pattern of capsaicin

accumulation in hot pepper hybrids at different harvesting stages is presented in figure

4.2.2. Significant variation in capsaicin concentration was observed in all three hybrids at

all harvesting stages. Result of present studies revealed that capsaicin concentration was

found maximum at mature green stage in Sky Red (61.30 mg 100g-1) followed by

Wonder King (43.93 mg 100g-1), whereas in Maha it was at color break stage (39.13 mg

100g-1). Overall the studies indicated that capsaicin concentration was highest in the

mature green stage and progressively decreased to sun dried stage in all the three hybrids.

Generally capsaicin accumulation was low in the immature green stage in the hybrids

examined in the present study; however once the fruit was fully mature (developed); it

had the highest capsaicin concentration. With the advancement of ripening in hot

peppers, as seen by change in color from green to red, it decreased significantly.

Reduction in capsaicin was highest in Sky Red (45.63%) from mature green stage to

dried fruits. However, decrease in capsaicin from highest concentration was 43.71 and

37.86% in Maha and Wonder King respectively. These changes indicate that differences

in biochemical factors within varieties can influence the biosynthesis and stability of

78

Table 4.2.1: Mean square values from analysis of variance for capsaicin and dihydrocapsaicin concentration of hot pepper hybrids harvested at different stages

S.O.V

D.F

Capsaicin

Dihydrocapsaicin

Hybrids (H)

2

1478.315**

1104.535**

Harvesting Stages

(HS)

4

668.8041**

323.5845**

H x HS

8

35.35294**

40.94897**

Error

30

0.794667

0.549828

**= Significant at 0.01 level

79

(A)

(B)

Figure 4.2.1: Chromatographs of capsaicinoid compounds. A) Standard peaks and B) Peaks of sample

Dihydrocapsaicin

Capsaicin

Dihydrocapsaicin Capsaicin

80

0

10

20

30

40

50

60

70

IG MG CB RR DF

Harvesting Stages

Cap

saic

in m

g/10

0g

SR M WK

Figure 4.2.2: Pattern of capsaicin mg 100g-1 distribution in hot pepper hybrids harvested at different stages. (IG - Immature Green; MG - Mature Green; CB - Color Break; RR - Red Ripe) and DF - Dried fruit). (SR - Sky Red; M - Maha and WK - Wonder King) Vertical bars shows + SD. n= 3 replicates

81

capsaicinoids during ripening and drying (Ananthasamy et al., 1960 and Gnayfeed et al.,

2001). The degree of pungency depends on the Capsicum species, cultivars and its

concentration can be affected by different factors such as the developmental stage of the

fruit (Sukrasno and Yeoman, 1993), intrinsic genetic factors to each cultivar or

alternatively to the environmental conditions where they were cultivated (Cisneros-

Pineda et al., 2007). These results are in line of what described by other authors that

accumulation of capsaicin starts at an early stage of fruit development 14 days after

flowering (Estrada et al., 1999) and reaching the maximum at final growth stage of fruit

maturity (Contreras-Padilla and Yahia, 1998) and then decreases by rapid turn over and

up to 60% degradation was observed (Iwai et al., 1979). It is reported that decrease in

capsaicinoid concentration during maturation and senescence of hot peppers has been

related to activity of peroxidase; in which hot peppers differ significantly from each other

(Gnayfeed et al., 2001).

4.2.2: Dihydrocapsaicin (mg 100g-1)

Dihydrocapsaicin is the second important capsaicinoid. Capsaicin and dihydrocapsaicin

accounts for more than 90% of the capsaicinoids in hot peppers and contribute most to

pungency (Todd et al., 1977). They have been widely studied and are presently used in

the food industry and for medicinal purposes. Analysis of variance depicted significant

differences (P ≤ 0.01) for dihydrocapsaicin concentration in all hot pepper hybrids,

harvesting stages and their interactions (see table 4.2.1). However, dihydrocapsaicin

concentration was highest (average of five harvesting stages) in Sky Red (33.05 mg 100g-

1) followed by Wonder King (19.94 mg 100g-1) and Maha (16.98 mg 100g-1).

Significant variation in dihydrocapsaicin concentration was observed in hot peppers

harvested at different stages. The concentration of dihydrocapsaicin followed the same

trend as did capsaicin concentration in hot pepper hybrids under investigation (see figure

4.2.3). Dihydrocapsaicin reached significantly higher concentration at mature green stage

in Sky Red (43.76 mg 100g-1) and Wonder King (26.16 mg 100g-1) while in Maha it was

maximum (24.20 mg 100g-1) at color break stage. Both Maha and Wonder King were

statistically alike with respect to dihydrocapsaicin concentration at color break stage and

similarly at mature green and red ripe stage. Decrease in dihydrocapsaicin concentration

in Sky Red, Maha and Wonder King was 53.93, 44.68 and 43.09% respectively from

82

0

5

10

15

20

25

30

35

40

45

IG MG CB RR DF

Harvesting Stages

Dih

ydro

caps

aici

n m

g/10

0g

SR M WK

Figure 4.2.3: Pattern of dihydrocapsaicin distribution mg 100g-1 in hot peppers harvested at different stages. (IG - Immature Green; MG - Mature Green; CB - Color Break; RR - Red Ripe and DF - Dried fruit). (SR - Sky Red; M - Maha and WK - Wonder King) Vertical bars shows + SD. n= 3 replicates

83

mature green to dried fruit stages. These results indicated that dihydrocapsaicin

concentration decreases in similar fashion as capsaicin but the concentration of capsaicin

in all the hybrids was higher than dihydrocapsaicin at all harvest stages. These results

supports the findings of Kraikruan et al. (2008) who observed that nine chilli cultivars

studied had higher capsaicin concentration than of dihydrocapsaicin. Normally fruit took

35 to 40 days to reach at fully expanded green stage in all the three hybrids and

dihydrocapsaicin concentration was highest along with capsaicin, afterwards it decreased

significantly. Similar results were observed by Iwai (1979) that capsaicinoids

concentration peaked after 30 to 40 days of flowering and then began to drop.

4.2.3: Capsaicin to dihydrocapsaicin ratio

Analysis of variance revealed significant difference (P ≤ 0.01) for capsaicin to

dihydrocapsaicin ratio in all hot pepper hybrids, harvesting stages and their interactions

(see table 4.2.2). Both the hybrids Maha and Wonder King have ratio of 1.7:1 and were

statistically alike where as capsaicin and dihydrocapsaicin ratio in Sky Red was 1.4:1. As

for as fruit harvest stage is concerned, capsaicin and dihydrocapsaicin ratio was highest at

immature green 1.8:1.

The pattern of capsaicin to dihydrocapsaicin ratio in hot pepper hybrids at different

harvesting stages is presented in figure 4.2.4. It is clear from the figure that capsaicin and

dihydrocapsaicin ratio in Maha and Wonder King was decreased from immature green to

dry fruit stage while in case of Sky Red an increasing trend was observed at same stages.

Capsaicin to dihydrocapsaicin ratio was found significant 2:1 in Wonder King and 1.9:1

was observed in Maha as followed by Sky Red 1.4:1 at immature green stage. All other

stages behaved differently in all hybrids with respect to capsaicin to dihydrocapsaicin

ratio. From these results it can be concluded that variation in capsaicin to

dihydrocapsaicin ratio depends on developmental stage of fruit and genetic make up of

the individual hybrid. These results are in line with Zewdie and Bosland (2000) who

observed a ratio of about 2:1 in Capsicum frutescens and Kraikruan et al. (2008)

observed capsaicin to dihydrocapsaicin ratio in the range of 1.2:1 to 2.3:1 in nine chilli

cultivars.

84

Table 4.2.2: Mean square values from analysis of variance for capsaicin to dihydrocapsaicin ratio and total capsaicinoids in hot pepper hybrids harvested at different stages

S.O.V

D.F

C : DC

Total capsaicinoids

Hybrids (H)

2

0.363602**

5034.317**

Harvesting Stages

(HS)

4

0.079764**

1791.665**

H x HS

8

0.056916**

172.2826**

Error

30

0.006658

3.928169

**= Significant at 0.01 level

85

0

0.5

1

1.5

2

2.5

IG MG CB RR DF

Harvesting Stages

Cap

saic

in to

dih

ydro

caps

aici

n ra

tio

SR M WK

Figure 4.2.4: Pattern of capsaicin to dihydrocapsaicin ratio in hot peppers harvested at different stages. (IG - Immature Green; MG - Mature Green; CB - Color Break; RR - Red Ripe and DF - Dried fruit). (SR - Sky Red; M - Maha and WK - Wonder King) Vertical bars shows + SD. n= 3 replicates.

86

4.2.4: Total Capsaicinoids (mg 100g -1)

In present studies, only capsaicin and dihydrocapsaicin were determined, so the total

capsaicinoids were calculated from the sum of these pungent compounds in each hybrid.

Significant difference (P ≤ 0.01) was observed for total capsaicinoids concentration in all

hot pepper hybrids, harvesting stages and their interactions (see table 4.2.2). Total

capsaicinoids concentration was highest (average of five harvesting stages) in Sky Red

(81.42 mg 100g-1) followed by Maha (54.93 mg 100g-1) and Wonder King (46.26 mg

100g-1). As far as harvest stage is concerned with respect to total capsaicinoid

concentration, mature green (75.71 mg 100g-1) and color break (75.65 mg 100g-1) stages

were on top and statistically alike.

The response of all the three hybrids is highly significant and pattern of total capsaicinoid

accumulation (see figure 4.2.5) revealed that Sky Red showed dominance over Maha and

Wonder King at different harvesting stages. However, highest concentration of capsaicin

and dihydrocapsaicin was observed in Sky Red (105.10 mg 100g-1) at mature green stage

while in Maha and Wonder King it was 70.16 and 63.37 mg 100g-1 at mature green and

color break stage respectively. These results indicated that total capsaicinoids

concentration were higher in early stages (horticultural maturity) and as fruit reaching

maturity (physiological), a progressive decrease was observed in all hybrids examined.

The increase in capsaicinoid concentration during fruit development is not only related to

change in peroxidase activity but is also accompanied by the changes in the different

isoenzymes (Estrada et al., 2000). Sukrasno and Yeoman (1993) reported that capsaicin

and dihydrocapsaicin contents in pepper fruit can be affected by developmental stage and

environmental conditions (Zewdie and Bosland, 2000).

4.2.5: Total Carotenoids (mg 100g-1)

In recent past, great attention is given to the nutritional value of food to know what

contribution of an individual food product is to daily nutritional need and how maturity at

harvest and ripening affect nutritive composition. The intense red color of the ripe hot

peppers and their processed products are due to the presence of carotenoid pigments.

Analysis of variance revealed significant (P ≤ 0.01) differences for total carotenoids

concentration in all hot pepper hybrids, harvesting stages and their interactions (see table

4.2.3). Carotenoids concentration was highest (average of five harvesting stages) in

87

0

20

40

60

80

100

120

IG MG CB RR DF

Harvesting Stages

Tot

al c

apsa

icin

oids

mg/

100g

SR M WK

Figure 4.2.5: Pattern of total capsaicinoids mg 100g-1 distribution in hot peppers harvested at different stages. (IG - Immature Green; MG - Mature Green; CB - Color Break; RR - Red Ripe and DF - Dried fruit). (SR - Sky Red; M - Maha and WK - Wonder King) Vertical bars shows + SD. n= 3 replicates

88

Table 4.2.3: Mean square values from analysis of variance for total carotenoids, ascorbic acid and total phenolic contents of hot pepper hybrids harvested at different stages

S.O.V

D.F

Total carotenoids Ascorbic acid

Total phenolics

Hybrids (H)

2

206.2239**

2514.94**

1910.103**

Harvesting Stages (HS)

4

14641.62**

15848.57**

1670.70**

H x HS

8

119.6284**

47.87594*

900.1382**

Error

30

1.850689

17.57933

8.557778

*, **= Significant at 0.05 and 0.01 levels respectively.

89

0

20

40

60

80

100

120

IG MG CB RR DF

Harvesting Stages

Tot

al c

arot

enoi

ds m

g/10

0g

SR M WK

Figure 4.2.6: Pattern of total carotenoids mg 100g-1 distribution in hot peppers harvested at different stages. (IG - Immature Green; MG - Mature Green; CB - Color Break; RR - Red Ripe and DF - Dried fruit). (SR - Sky Red; M - Maha and WK - Wonder King) Vertical bars shows + SD. n= 3 replicates

90

Wonder King (28 mg 100g-1) followed by Sky Red (22.91 mg 100g-1) and Maha (20.79

mg 100g-1). All hot pepper hybrids had highest carotenoid concentration at dried fruit

stage. The concentration of carotenoids in capsicum fruit depends on various factors like

physiological and morphological characteristic of the cultivar, the level of the genes

governing carotenogenesis, maturity at harvest and growing conditions. Stage of harvest

is the one factor that decisively affects carotenoid composition in hot peppers. Variation

in total carotenoid concentration at different harvesting stages in all three hybrids can be

seen in figure 4.2.6. All hot pepper hybrids were statistically at par for total carotenoid

concentration at immature green, mature green stages and color break stages but they

differ significantly in red ripe and dried fruit stages where Wonder King topped among

the three hybrids. The total carotenoid concentration tended to change during pepper fruit

ripening from immature green to red ripe stage and their concentration increased 4-5 fold

but almost an 8 fold increase was observed from red ripe stage to dried fruit of all the

three hybrids. Significant variation in total carotenoids in fully expanded red fruits might

be due to low flesh content or genetic background of the hybrids. However, in all the

harvest stages, highest carotenoids concentration was observed in Wonder King (112.39

mg 100g-1) as followed by Sky Red (91.08 mg 100g-1) and Maha (83.59 mg 100g-1) in

dried fruits of hot pepper hybrids. Significant increase in total carotenoids from red ripe

to red dried fruit might be due to decrease in moisture content of fruits. Similar results

were observed by Kandlakunta et al. (2008) that total carotenoids concentration was 2.41

mg 100g-1 in green chillies (85% moisture content) but the concentration in red chillies is

113 mg 100g-1 (10.1% moisture content). From these results, it can be concluded that red

chillies contain 4-5 fold more total carotenoids than green chillies and if high level of

total carotenoids are considered, the contribution of red fruit may be substantial for

overall human health. At the same time, consumers should be educated about the benefits

of including fresh and dried hot pepper fruit in the daily diet.

91

4.2.6: Ascorbic Acid (mg 100g-1)

Hot peppers are rich source of ascorbic acid than other vegetables and fruits commonly

recognized as sources of this substance. Significant differences (P ≤ 0.01) were observed

for ascorbic acid concentration in hot pepper hybrids, harvesting stages and by the

interaction (P ≤ 0.05) of these two factors (see table 4.2.3). Ascorbic acid concentration

was highest (average of five ripening stages) in Wonder King (108.89 mg 100g-1)

followed by Sky Red (104.66 mg 100g-1) and Maha (84.64 mg 100g-1). However, all

three hybrids had highest ascorbic acid concentration at red ripe stage.

Harvesting stage is considered as one of the major factor that determines the

compositional quality of fruits and vegetables. Distribution of ascorbic acid concentration

in three hybrids at different harvesting stages is presented in figure 4.2.7. Considerable

variations existed within the hybrids examined and the level of ascorbic acid varied in

immature fruit to fully expanded red fruits from 65.56 to 92.86 mg 100g-1 and 131.63 to

163.46 mg 100g-1 respectively. There is progressive increase in ascorbic acid

concentration with the advancement of fruit maturation and ripening but it decreased

quickly as fruit dry. Maximum ascorbic acid concentration was observed in Wonder King

(163.46 mg 100g-1) followed by Sky Red (155.20 mg 100g-1) and Maha (131.63 mg

100g-1) at red ripe stage. The increase in ascorbic acid concentration from mature green

to red ripe stage in Sky Red is (26.79%), Maha (31.88%) and Wonder King (29.94%)

while decrease in ascorbic acid concentration from red ripe to dried fruits in all three

hybrids was 73.13, 76.11 and 73.03% respectively. Both Sky Red and Wonder King

behaved statistically alike at all harvesting stages except red ripe stage. However, the

hybrid Maha differed significantly from other two hybrids at all maturity stages.

It is reported that red peppers contain highest ascorbic acid concentration among other

important plant food materials including spinach and broccoli (Lee and Kader, 2000).

The results of present studies revealed that as the ripening advanced in hot peppers, the

ascorbic acid concentration reached its maximum towards the red ripe stage and then it

declined. With the advent of red ripe fruit drying, quick decrease in ascorbic acid

concentration might be due to some biochemical changes occur during drying. Ascorbic

acid concentration decreases as moisture decreases from fruits.

92

0

20

40

60

80

100

120

140

160

180

IG MG CB RR DF

Harvesting Stages

Asc

orbi

c A

cid

mg/

100g

SR M WK

Figure 4.2.7: Pattern of ascorbic acid mg 100g-1 distribution in hot peppers harvested at different stages. (IG - Immature Green; MG - Mature Green; CB - Color Break; RR - Red Ripe and DF - Dried fruit). (SR - Sky Red; M - Maha and WK - Wonder King) Vertical bars shows + SD. n= 3 replicates

93

In present studies, red dried fruits had moisture contents between 12 to 13% and contain

the lowest amount of ascorbic acid content among all harvest stages. The above results

are quite interesting in the view of the importance of ascorbic acid content in hot peppers

in determining their dietic value and red ripe fruits are more beneficial to meet the

recommended daily allowance of ascorbic acid than mature green or color break stage.

These results are in line with Howard et al. (1994), Gnayfeed et al. (2001) and Marin et

al. (2004) that as the ripening advanced, ascorbic acid concentration increased in hot

peppers.

4.2.7: Total phenolic contents (mg 100g-1)

In recent years, phenolic compounds have attracted the interest of researchers because

they show promise of being powerful antioxidants that can protect the human body from

free radicals, the formation of which is associated with the normal natural metabolism of

aerobic cells (Halliwell, 1996). Hot peppers were found to be a good source of phenolic

compounds and are ranked fourth after broccoli, spinach and onion with respect to total

phenolic contents (Chu et al., 2002). They also contribute to fruit color, flavor and

pungency in addition to their antioxidant role. Analysis of variance revealed significant

differences (P ≤ 0.01) for total phenolic contents in all hot pepper hybrids, harvesting

stages and their interactions (see table 4.2.3). Total phenolic contents was highest

(average of five harvesting stages) in Sky Red (91.77 mg 100g-1) followed by Maha

(77.71 mg 100g-1) and Wonder King (69.40 mg 100g-1). As for as harvesting stage is

concerned, maximum phenolic contents were observed at color break (90.92 mg 100g-1)

and mature green stage (88.25 mg 100g-1) but both harvesting stages were statistically

alike. Stage of fruit harvest is one of the major factors that determine the total phenolic

contents in fruits and vegetables. The distribution of total phenolic contents in all hot

pepper hybrids at different harvesting stages is presented in figure 4.2.8. All hot pepper

hybrids show greater variation with respect to total phenolic contents accumulation. Total

phenolic contents in Sky Red tend to decrease from immature green stage (114.10 mg

100g-1) to dried fruit stage (59.23 mg 100g-1) where as an increasing trend was observed

in Maha from immature green (58.30 mg 100g-1) to red ripe stage (101 mg 100g-1) and

then it decreases quickly. Similarly in Wonder King maximum concentration of total

phenolics was observed at mature green stage (88.06 mg 100g-1) which was statistically

94

0

20

40

60

80

100

120

IG MG CB RR DF

Harvesting Stages

Tot

al p

heno

lics

mg/

100

g G

AE

SR M WK

Figure 4.2.8: Pattern of total phenolics distribution mg 100g-1 in hot peppers harvested at different stages. (IG - Immature Green; MG - Mature Green; CB - Color Break; RR - Red Ripe and DF - Dried fruit). (SR - Sky Red; M - Maha and WK - Wonder King) Vertical bars shows + SD. n= 3 replicates

95

at par with color break stage. These results give some interesting information about the

pattern of total phenolic contents accumulation in hot pepper hybrids examined at

different harvesting stages. Howard et al. (2000) and Materska and Perucka (2005) stated

that concentration of total phenolic contents increases as the pepper reached maturity

while Conforti et al. (2007) concluded that total phenolic contents in hot pepper

decreases as pepper reaches maturity from immature green to red ripe stage. It can be

concluded from present results that level of total phenolic contents in hot peppers depend

on individual hybrid, stage of harvest and the physiological changes that take place

during maturity process led to change the pigments could have caused the difference in

total phenolic contents in unripe and ripe peppers.

4.2.8. Conclusion

Present investigations revealed that hot pepper hybrids differ significantly with respect to

their antioxidant potential. The concentration of capsaicinoids was higher at mature green

stage in Sky Red followed by Wonder King conversely at color break stage in Maha.

Total carotenoids accumulation was found an 8 fold increase from red ripe to dry fruit

stage, inversely quick decline was observed in ascorbic acid. However, the pattern of

total phenolic contents biosynthesis was found significant at immature green stage in Sky

Red and at color break stage in Maha while in case of Wonder King it was at red ripe

stage.

96

Experiment III. 4.3: Evaluation of hot peppers for aflatoxin contamination,

microbial load and antioxidant quality under different storage conditions

4.3.1: Moisture contents (%age)

Analysis of variance revealed significant (P ≤ 0.01) differences for moisture percentage

in hot pepper hybrids (see table 4.3.1) and it was highest in Wonder King (12.45%)

followed by Maha (12.31%) and Sky Red (11.96%) during five months storage (see

figure 4.3.1). Similarly, there was significant impact (P ≤ 0.05) of packaging on moisture

percentage and samples stored in jute bags had more moisture percentage (12.26%) as

compared to polyethylene bags (12.22%). However, storage temperature and duration

depicted non-significant response in this regard. All interactive effects were also found

non-significant.

4.3.2: Total aflatoxins (µg kg-1)

In today’s changing world, food safety and security have generally remained basic human

needs. Ensuring the safety of food has been a major focus of international and national

action over the last few years. The problem of food and feed contamination with

aflatoxins is of current hot issue and has received a great deal of attention during the last

three decades. The frequent incidence of these toxins in agricultural commodities has a

potential negative impact on the economies of the affected regions, especially in the

developing countries where harvest and postharvest techniques are not sufficient to

prevent mold growth.

Analysis of variance revealed significant (P ≤ 0.01) differences for aflatoxin

contamination in hot peppers during five months storage at different temperatures,

packaging materials and their interactions (see table 4.3.2). All hot peppers differ

significantly with respect to total aflatoxin contamination and it was 96% and 34% higher

in Wonder King than Sky Red and Maha respectively. During the first 100 days of

storage, no contamination was observed in hot peppers stored polyethylene bags at all

temperatures (see figure 4.1.3). However, total aflatoxin detection in polyethylene bags

after 150 days of storage in Sky Red at 25ºC was 0.19 at 30ºC was 0.27 µg kg-1 while in

case of jute bags at 20, 25 and 30ºC temperatures, aflatoxin contamination 0.19, 0.44 and

0.84 µg kg-1 was detected respectively. When Maha was stored in polyethylene bags up

to 150 days of storage at 20, 25 and 30ºC it had total aflatoxin contamination 0.16, 0.46

97

Table 4.3.1: Mean square values from analysis of variance for moisture content (%age) of hot pepper hybrids under different storage conditions

S.O.V D.F Moisture content

(%age)

Hybrid (H) 2 4.698742**

Storage Period (S) 3 0.033882NS

Packaging (P) 1 0.1089*

Temperature (T) 2 0.009187 NS

H x S 6 0.012427 NS

H x P 2 0.013156 NS

H x T 4 0.01529 NS

S x P 3 0.02576 NS

S x T 6 0.12136 NS

P x T 2 0.004448 NS

H x S x P 6 0.005258 NS

H x S x T 12 0.004916 NS

H x P x T 4 0.004559 NS

S x P x T 6 0.00237 NS

H x S x P x T 12 0.001999 NS

Error 144 0.020492

*, ** = Significant 0.05 and 0.01 levels respectively. NS = Non-significant

98

Figure 4.3.1: Moisture content (%age) in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

0

5

10

15

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/Temperature (A)

Moi

stur

e co

nten

ts (%

)

PB JB

0

5

10

15

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/Temperature (B)

Moi

stur

e co

nten

ts (%

)

0

5

10

15

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/temperature (C)

Moi

stur

e co

nten

ts (%

)

99

Table 4.3.2: Mean square values from analysis of variance for total aflatoxins and fungal counts of hot pepper hybrids under different storage conditions

S.O.V D.F Total aflatoxins Total fungal

counts

Hybrid (H) 2 0.905964** 892616.7**

Storage Period (S) 3 4.083723** 781991118**

Packaging (P) 1 2.840523** 86235141**

Temperature (T) 2 0.46547** 4612876**

H x S 6 0.310035** 176123.5**

H x P 2 0.377003** 379850.5**

H x T 4 0.035824** 21820.83**

S x P 3 0.977952** 18692833**

S x T 6 0.210153** 1216451**

P x T 2 0.131367** 2139191**

H x S x P 6 0.16234** 143828.2**

H x S x T 12 0.028924** 9863.04**

H x P x T 4 0.049894** 43789.35**

S x P x T 6 0.046276** 848302.7**

H x S x P x T 12 0.0314** 19297.92**

Error 144 0.001007 1982.176

** = Significant at 0.01 level.

100

G1

(A)

(B)

Figure 4.3.2: Chromatographs of aflatoxins. A) Standard peaks and B) Peaks of sample

G2

B2

B1

G1

101

Figure 4.3.3: Aflatoxin contamination in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

00.20.40.60.8

11.21.41.6

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/Temperature (A)

Tot

al a

flato

xin

µg/k

g

PB JB

00.20.40.60.8

11.21.41.6

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (B)

Tot

al a

flato

xins

µg/

kg

00.20.40.60.8

11.21.41.61.8

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/Temperature (C)

Tot

al a

flat

oxin

s µ

g/kg

102

and 0.40 µg kg-1 while in case of Wonder King under similar conditions it was 0.27, 0.44

and 0.70 µg kg-1 respectively. As for as the response of Maha after 100 days of storage in

jute bags is concerned, the aflatoxin contamination 0.16, 0.43 and 0.60 µg kg-1 was

detected at 20, 25 and 30ºC but when storage duration prolonged up to 150 days then it

was 0.49, 1.12 and 0.97 µg kg-1 at above said conditions. Wonder King was found more

susceptible to aflatoxin contamination when stored for 100 and 150 days in jute bags as

storage medium. Storage of hot peppers in jute bags was found more susceptible for

aflatoxin contamination than polyethylene bags. Higher aflatoxin contamination 1.50 µg

kg-1 was detected in Wonder King at 25ºC while it was 0.19 µg kg-1 in Sky Red at 20ºC

when jute bag is used as storage medium. On an average, aflatoxin contamination was

increased 75% in hot peppers stored in jute bags than polyethylene bags. Temperature

was also found to have significant relation with aflatoxin contamination. Overall results

indicated that contamination was 61% higher in hot peppers stored at 25ºC and 30ºC than

at 20ºC. However, the response of each hybrid was different in all temperature treatments

e.g. aflatoxin contamination in Sky Red was highest at 30ºC while in Wonder King it was

maximum at 25ºC in both packaging materials used in present study.

Hot pepper is one of the favorite spice consumed in Pakistan. Normally they are sun dried

by spreading on floor and turned down several times. In present studies, all hot pepper

hybrids were sun dried from 9 am to 5 pm daily where temperature and humidity levels

were 30-36ºC and 20-52% respectively. Before storage all hot pepper hybrids were tested

to see the levels of total aflatoxins but no contamination was observed which might be

due to low humidity level during drying and careful handling. These results are in line

with Pitt and Hocking (1997) and FAO/WHO (2001) that during hot pepper fruit drying,

low humidity should be maintained to prevent mold growth. The normal planting time of

hot peppers in Punjab starts from mid February and fruit pickings continues up to August.

In the month of July and August, the spell of rains starts which is characteristic feature of

monsoon season. In this season, splash of rains deteriorates the fruit quality because high

humidity accelerates the rottening and dried fruit becomes more susceptible for aflatoxin

contamination. Similarly hot pepper drying during this humid weather might gave

favorable environment for fungal to grow.

In Pakistan, dry hot peppers normally stored in jute bags but in mega stores they are

103

available in polyethylene bags. High levels of aflatoxin contamination observed in jute

bags might be due to jute fabrics which are porous and have tendency to absorb moisture,

have good aeration and mineral oil which is used for the preparation of jute fiber that may

add bad odour in products stored in jute bags. The degree of aeration is also important for

aflatoxin production because mold growth and aflatoxin production are aerobic

processes. Due to this aeration, stored products can easily be contaminated with air borne

spores. Low incidence of aflatoxin contamination in polyethylene bags is due to their

ability of moderate to good resistance to water, no or limited air passage and they also

slow down the microbial growth and imparts no characteristic odor. However, initial

humidity level in polyethylene bags might gave the clue of contamination since no

aeration is there.

So far not much information is published about the incidence of aflatoxin contamination

in hot peppers during storage. Most of the time people just collect the samples from

market and analyzed. Overall results of present studies indicate that aflatoxin

contamination in all three hybrids tested under different storage conditions had mean

levels lower than the existing regulatory limits in the European Commission (EC No.

1881/2006) 10 µg kg-1 for total aflatoxins. It was observed from these results that both

Maha and Wonder King were more susceptible to aflatoxin contamination than Sky Red.

However, their contamination was not so high even temperature conditions were

favorable for fungal growth, but at the same time the moisture contents in the three

hybrids were less than 12.5%. However, it is reported that aflatoxin production can also

take place at temperatures as low as 7–12ºC (Steyn and Stander, 2000). From these

results, it can be concluded that storage of whole dried hot peppers in polyethylene bags

reduced the incidence of aflatoxin contamination even temperature conditions are

favorable for fungal growth during five month storage. Storage at 20ºC was proved to be

the best temperature treatment in present study.

4.3.3: Total fungal counts (TFCs g-1)

Dried red pepper fruits like other dry plant materials, often contain a variety of

microorganisms. The microbial flora is influenced by many factors including growing

and harvesting conditions, postharvest handling and the selective actions of a given

substrate. Analysis of variance revealed significant (P ≤ 0.01) differences for total fungal

104

Figure 4.3.4: Total fungal counts in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

0

1000

2000

3000

4000

5000

6000

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (A)

Tot

al fu

ngal

cou

nts/

g

PB JB

0

1000

2000

3000

4000

5000

6000

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (B)

Tot

al fu

ngal

cou

nts/

g

0

1000

2000

3000

4000

5000

6000

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (C)

Tot

al fu

ngal

cou

nts/

g

105

counts in hot peppers during five months storage at different temperatures, packaging

materials and their interactions (see table 4.3.2). Both packaging materials differ

significantly with respect to total fungal counts (TFCs) but fungal population was greater

in jute bags with a range of 1.8 x 102 to 4.8 x 103 in Sky Red followed by 2.03 x 102 to

5.2 x 103 in Maha while in case of Wonder King it was 2.2 x 102 to 5.3 x 103 recorded in

storage duration upto 150 days. However, TFCs had inverse relation when these hybrids

stored in polyethylene bags (see figure 4.3.4).

TFCs increased as the storage duration prolonged in all hot pepper hybrids at various

temperature regimes. However, TFCs were maximum in hot peppers stored at 25ºC in

jute bags from 4.85 x 103 to 5.39 x 103 after five months of storage which was 65-70%

higher when they were stored in polyethylene bags under similar storage conditions.

Gradual increase in storage temperature as well as duration in both packaging materials

creates most congenial conditions for TFCs proliferation. However, variation in TFCs

may be attributed to relative permeability to atmospheric gases such as oxygen, carbon

dioxide and water activity among both packaging materials. Fungal population increased

in similar fashion in hot pepper hybrids at all temperatures but it was found significant at

25ºC followed by 30ºC. From these results, it can be concluded that TFCs increased as

storage duration extended but their population was significantly less in polyethylene bags

which might be due to restriction of air exchange and reduced oxygen levels. These

results are in line with findings of Ahmad (1993) that low fungal counts in closed metal

bins was due to restriction of air exchange and reduced oxygen levels than gunny bags

that allowed air to flow through stored black gram seeds. Similarly Chourasia (1995)

reported that spices packed in gunny bags and placed on bare ground had significantly

high incidences of mycobiota as compared with spices stored in metal containers, glass

containers and wooden boxes. Overall results indicated that total fungal counts in all hot

pepper hybrids were in the acceptable limits.

4.3.4: Aspergillus flavus/Aspergillus parasiticus count (g-1)

Aspergillus flavus (Bankole et al., 2004) and Aspergillus parasiticus (Begum and

Samajpati, 2000 and Erdogen, 2004); filamentous fungi are major producer of aflatoxins

but the ubiquitous Aspergillus flavus produces B aflatoxins (Samajphati, 1979) while

Aspergillus parasiticus produces both B and G aflatoxins (Garcia-Villanova et al., 2004).

106

Table 4.3.3: Mean square values from analysis of variance for Aspergillus counts of hot pepper hybrids under different storage conditions

S.O.V D.F Aspergillus

counts Capsaicin

Hybrid (H) 2 11596.46** 2537.866**

Storage Period (S) 3 315679.3** 87.17374**

Packaging (P) 1 311676** 4.5443**

Temperature (T) 2 48481.73** 9.435119**

H x S 6 1599.136** 0.460852*

H x P 2 703.1667** 0.007198NS

H x T 4 1296.407** 0.47202*

S x P 3 87249.39** 0.695739**

S x T 6 14761.2** 1.215252**

P x T 2 7473.181** 0.475335NS

H x S x P 6 1256.123** 0.066438NS

H x S x T 12 387.2099** 0.333322*

H x P x T 4 154.7222** 0.086216NS

S x P x T 6 4305.86** 0.073638NS

H x S x P x T 12 348.7346** 0.038871NS

Error 144 72,.9213 0.170733

*, ** = Significant at 0.05 and 0.01 levels respectively. NS = Non-significant

107

Figure 4.3.5: Aspergillus flavus/parasiticus counts in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

0

100

200

300

400

500

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (A)

A.fl

avus

/par

asiti

cus

coun

ts/g

PB JB

0

100

200

300

400

500

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/Temperature (C)

A.fl

avus

/par

asiti

cus

coun

ts/g

0

100

200

300

400

500

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (B)

A.fl

avus

/par

asiti

cus

coun

ts/g

108

Analysis of variance revealed that Aspergillus counts were found significant (P ≤ 0.01) in

hot pepper hybrids during five months storage period at various temperature regimes as

well as packaging materials and their interactions (see table 4.3.3). Both packaging

materials differ significantly with respect to Aspergillus counts but it was greater in jute

bags with a range of 1.8 x 101 to 3.09 x 102 in Sky Red followed by 2.2 x 101 to 4.05 x

102 in Maha while in case of Wonder King 2.5 x 101 to 3.5 x 102 recorded in storage

duration up to 150 days. However, Aspergillus count had inverse relation when these

hybrids stored in low density polyethylene bags (see figure 4.3.5). Aspergillus count

increased in similar fashion like TFCs in hot pepper hybrids as the storage duration

increased at different temperature regimes. However, after five months storage at 25ºC in

jute bags, Aspergillus count was found significant 3.09 x 102 in Sky Red followed by

Maha which had 4.05 x 102 while in case of Wonder King it was observed 3.5 x 102

which was 64-68% higher when polyethylene bags were used. However, significant

increase in Aspergillus count was observed during 50-100 days of storage in both

packaging materials. Gradual increase in temperature under prolonged storage duration

might be the possible reason for conducing the most congenial conditions for Aspergillus

proliferation and aflatoxin production.

Several studies manifested that spices are contaminated with various microorganisms

including toxigenic moulds (especially Aspergillus spp.) which have aflatoxin producing

potential (Garrido et al., 1992). Toxin accumulation in spices is an indicative of a casual

contamination following post harvest handling and drying kinetics. Therefore, spices

pose health problems because they are often added to foods without further processing or

eaten as raw (Lwellyn et al., 1990).

4.3.5: Capsaicin (mg 100g-1)

Pungency is an important factor for determining the commercial quality in hot peppers.

The level of pungency in dried hot peppers depends mainly on the concentration of

capsaicin as well as dihydrocapsaicin. Capsaicin is the most abundant capsaicinoid and

contributes in producing pungency in hot peppers. Significant differences (P ≤ 0.01) were

observed for hot pepper hybrids with respect to their capsaicin concentration during

storage (see table 4.3.3). Results clearly depicted that under prolonged storage duration;

hot pepper hybrids had inverse relation with their capsaicin concentration. However, after

109

Figure 4.3.6: Capsaicin concentration in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

05

10152025303540

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration/Temperature (A)

Cap

saic

in m

g/10

0g

PB JB

05

10152025303540

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration/Temperature (B)

Cap

saic

in m

g/10

0g

05

10152025303540

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration/Temperature (C)

Cap

saic

in m

g/10

0g

110

five months storage period, capsaicin concentration was significantly reduced in Maha

which was 16% followed by 9 and 12% in Sky Red and Wonder King respectively.

Storage duration had significant relation with capsaicin concentration in hot pepper

hybrids but packaging materials shared no relation in this regards.

Storage temperature and duration had inverse relation with capsaicin concentration (see

figure 4.3.5). After five months storage period, capsaicin concentration decreased

significantly 11 and 18% in Sky Red and Maha at 30ºC but in case of Wonder King it

was 14% at 25ºC. However, effect of temperature and packaging material was non-

significant in this regard while it was significant as storage duration prolonged. Capsaicin

concentration decreased significantly in hot peppers hybrids between 50 to100 days at

various temperature regimes. Research information regarding the effect of temperatures

and packaging materials on capsaicin concentration is scarce while Topuz and Ozdemir

(2004) reported that 16% decrease was observed in capsaicin concentration of paprika

after six month storage period at ambient temperature. Present investigations indicated

that with the increase in storage temperature and duration, capsaicin concentration

decreased significantly in hot pepper hybrids.

4.3.6: Dihydrocapsaicin (mg 100g-1)

Dihydrocapsaicin is the second most important capsaicinoid after capsaicin but both

account for more than 90% of the capsaicinoid in hot peppers and contribute most to the

pungency (Todd et al., 1977). Significant differences were observed for hot pepper

hybrids with respect to dihydrocapsaicin concentration during storage (see table 4.3.4).

Results clearly showed that as storage duration increased, dihydrocapsaicin concentration

decreased significantly in hot peppers. However, after five months of storage period,

dihydrocapsaicin concentration decreased significantly 18% in Wonder King followed by

11 and 15% in Sky Red and Maha respectively. Impact of packaging materials as well as

storage duration and temperature was non-significant on dihydrocapsaicin concentration

in hot pepper hybrids while the interactive effect of storage temperature and duration had

significant effect (P ≤ 0.05) in this regard (see figure 4.3.7).

111

Table 4.3.4: Mean square values from analysis of variance for dihydrocapsaicin and total carotenoid concentration of hot pepper hybrids under different storage conditions

S.O.V D.F Dihydrocapsaicin

Total carotenoids

Hybrid (H) 2 937.7108** 16090.31**

Storage Period (S) 3 51.52933** 2306.24**

Packaging (P) 1 1.12338* 78.31298**

Temperature (T) 2 6.982017** 276.108**

H x S 6 0.74873** 21.13008**

H x P 2 0.020226NS 5.869975NS

H x T 4 0.126931NS 10.70013*

S x P 3 0.225742NS 13.88837*

S x T 6 0.874262** 76.09027**

P x T 2 0.257318NS 15.35233*

H x S x P 6 0.030705NS 1.517048NS

H x S x T 12 0.308195* 3.908666NS

H x P x T 4 0.063419NS 2.204348NS

S x P x T 6 0.076293NS 4.375225NS

H x S x P x T 12 0.025883NS 5.062953NS

Error 144 0.165515 3.693399

*, ** = Significant at 0.05 and 0.01 levels respectively. NS = Non-significant

112

Figure 4.3.7: Dihydrocapsaicin concentration in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperature and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

05

10152025

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration/Temperature (A)

Dih

ydro

caps

aici

n m

g/10

0g

PB JB

0

5

10

15

20

25

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration/Temperature (B)

Dih

ydro

caps

aici

n m

g/10

0g

0

5

10

15

20

25

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration/Temperature (C)

Dih

ydro

caps

aici

n m

g/10

0g

113

Dihydrocapsaicin concentration reduced significantly 11% in Sky Red after 100 days of

storage followed by 19 and 21% in Maha and Wonder King after 150 days storage at

30ºC. Topuz and Ozdemir (2004) reported that 15% decrease was observed in

dihydrocapsaicin concentration of paprika after six months storage period at ambient

temperature. In current studies almost similar results were recorded for dihydrocapsaicin

concentration in hot pepper hybrids as well. These results indicated that mechanism of

capsaicin and dihydrocapsaicin reduction varied in all hybrids under different storage

conditions and the hybrid Sky Red proved to be better among these.

4.3.7: Total carotenoids (mg 100g-1)

An increasing interest is being paid to the red hot pepper spice not only because of its

economical importance but also because of its diversified composition. The intense red

color of the ripe peppers and their processed products is due to the presence of carotenoid

pigments. When carotenoids are ingested, they show important biological actions such as

being antioxidants as well as free radical scavengers and reducing the risk of cancer. Hot

pepper hybrids differ significantly with respect to total carotenoids concentration with

highest concentration of carotenoids 111.83 mg 100g-1 was found in Wonder King

followed by Sky Red 91.16 mg 100g-1 while in case of Maha it was 83.53 mg 100g-1.

Analysis of variance depicted that total carotenoid concentration in hot pepper hybrids

had inverse relation with storage duration (see table 4.3.4). Gradual degradation of total

carotenoid concentration in hot pepper hybrids advocated that as storage duration

prolonged up to five months, it was 22% in Maha while in case of Sky Red and Wonder

King, it was 15% and 14% respectively (see figure 4.3.8). As far as the response of hot

pepper hybrids at different temperature regimes is concerned, total carotenoid

concentration reduced 6% in Maha at 30ºC but 4% decrease was observed in case of Sky

Red and Wonder King. Total carotenoid concentration in hot pepper hybrids after five

months storage duration manifested greater stability in Sky Red but in case of Maha and

Wonder King demonstrated 3-4% reduction when jute bag was used as storage medium.

All carotenoids are thermal sensitive in nature and their degradation under present

investigation in hot pepper hybrids after five months storage duration was observed 22%

at 30ºC storage temperature but 16 and 12% reduction was recorded at corresponding

storage temperature of 25ºC and 20ºC.

114

Figure 4.3.8: Total carotenoids concentration in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

0

20

40

60

80

100

120

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (A)

Tot

al c

arot

enoi

ds m

g/10

0g

PB JB

0

20

40

60

80

100

120

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (B)

Tot

al c

arot

enoi

ds m

g/10

0g

020

406080

100120

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (C)

Tot

al c

arot

enoi

ds m

g/10

0g

115

Carotenoids are fairly stable in their natural environment but become sensitive to light

and temperature and are readily decomposed after titration and extraction respectively

(Minguez-Mosquera and Hornero-Mendez, 1994). Present investigations regarding

carotenoids thermal and photo-sensitivity are in line with the findings of Schweiggert et

al. (2007) that total carotenoid concentration during four months storage at ambient

temperature degraded by 9.6 and 16.7 % in chilli and 38.8 and 39.7% in paprika powders

with and without illumination respectively. Similarly variation or loss of carotenoids

during foods processing e.g. peeling as well as storage occurs through geometric

isomerization and enzymatic or non-enzymatic oxidation (Rodriguez-Amaya, 1999;

2002). Lee et al. (1992) reported carotenoid destruction in red peppers was greatly

affected by water activity under prevailing packaging atmosphere and storage

temperature in addition to this carotenoid stability was improved in red peppers by

lowering the storage temperature.

4.3.8: Ascorbic acid (mg 100g-1)

Ascorbic acid is not only well known as being an antioxidant and biologically active

compound but also an important nutritional and functional constituent of hot pepper fruit.

Analysis of variance depicted significant (P ≤ 0.01) differences in hot pepper hybrids

with respect to their ascorbic acid concentration during storage (see table 4.3.5). Changes

in pattern of ascorbic acid concentration during five months storage are presented in

figure 4.3.8 and it clearly showed that as storage duration prolonged, ascorbic acid

concentration in hot pepper hybrids decreased significantly. Bio-degradation of ascorbic

acid concentration in hot pepper hybrids advocated that as storage duration prolonged up

to five months, it was 26% in Wonder King followed by 22% in Sky Red as well as 19%

was recorded in Maha (see figure 4.3.9). Temperature had significant effect on ascorbic

acid concentration in hot pepper hybrids during storage. Ascorbic acid concentration

gradually decreased in hot pepper hybrids with increase in storage temperature and

duration. However, reduction in ascorbic acid concentration at 30ºC was observed 28% in

Sky Red followed by 33% in Maha and it was 35% recorded in Wonder King but the

observed pattern at 20ºC was 15, 12 and 15% in Sky Red, Maha and Wonder King

respectively. Similarly, a steep decrease in ascorbic acid concentration in hot pepper

hybrids was observed between 100-150 days storage period.

116

Table 4.3.5: Mean square values from analysis of variance for ascorbic acid concentration and total phenolic contents of hot pepper hybrids under different storage conditions

S.O.V D.F

Ascorbic acid

Total phenolics

Hybrid (H) 2 1068.297** 13753.68**

Storage Period (S) 3 845.7901** 1064.536**

Packaging (P) 1 39.68082** 22.63336**

Temperature (T) 2 223.1266** 143.317**

H x S 6 15.00209** 62.97654**

H x P 2 0.291718NS 0.176113NS

H x T 4 8.647698** 4.276196**

S x P 3 4.884496** 3.216548**

S x T 6 44.64273** 18.14691**

P x T 2 0.591706NS 1.138589NS

H x S x P 6 0.618977NS 0.214446NS

H x S x T 12 3.195091** 1.988205**

H x P x T 4 1.091039NS 2.319256*

S x P x T 6 0.723533NS 0.403219NS

H x S x P x T 12 0.614792NS 0.474339NS

Error 144 0.622395 0.78118

*, ** = Significant at 0.05 and 0.01 levels respectively. NS = Non-significant

117

Figure 4.3.9: Ascorbic acid concentration in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB - Polyethylene Bag and JB - Jute Bag) during five months storage. Vertical bars show + SD. n= 3 replicates

0

10

20

30

40

50

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/Temperature (A)

Asc

orbi

c ac

id m

g/10

0g

PB JB

0

10

20

30

40

50

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (B)

Asc

orbi

c ac

id m

g/10

0g

0

10

20

30

40

50

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

S torage duration (days)/Temperature (C)

Aso

rbic

aci

d m

g/10

0g

118

The interactive effect of packaging materials and storage duration on ascorbic acid

concentration was found statistically significant. These results revealed that ascorbic acid

is very sensitive to high temperatures during storage and it decreased significantly in all

hot pepper hybrids. So far little information is available regarding the effect of packaging

material, storage temperature and duration on ascorbic acid concentration in dry hot

peppers. These results support the findings of Kalt (2005) that ascorbic acid

concentration decreased quickly in many plant products during storage. Similarly Daood

et al. (1996) reported that ascorbic acid concentration in ground paprika was decreased

10% after 30 days followed by 20 and 35% after 60 and 120 days of storage. Overall

studies indicated that with the increase in storage temperature and duration, gradual

decrease in ascorbic acid concentration was observed in hot peppers. As ascorbic acid

was found to be thermal sensitive, storage at 20ºC proved to be better for storage up to

100 days but after that quick decrease was observed.

4.3.9: Total phenolic contents (mg 100g -1)

Being powerful antioxidants that can protect human body from free radicals, phenolic

compounds have also been found to be inhibitory to the production of several mycotoxins

including fumonisins, tricothecenes and aflatoxins (Norton, 1999; Bakan et al., 2003 and

Beekrum et al., 2003). Hot pepper hybrids differ significantly with respect to their total

phenolic contents with Maha 67.92 mg 100g-1 stand first followed by Sky Red (59.34 mg

100g-1) and Wonder King (41.25 mg 100g-1). Analysis of variance revealed that

significant differences regarding total phenolic contents were observed in hot pepper

hybrids after five months storage at various temperature regimes (see table 4.3.5).

Changes in pattern of total phenolic contents during five months storage are presented in

figure 4.3.10. However, significant reduction in total phenolic contents after five months

storage period was observed in Wonder King 33% at 30ºC but in case of Maha and Sky

Red it was 24 and 12% respectively. Similarly significant decrease of 6-7% in total

phenolic contents was observed between 100-150 days storage period in hot pepper

hybrids. More total phenolic contents stability was observed in polyethylene bags than

jute bags. Overall results revealed that when hot pepper hybrids were stored under

conditions of high storage temperature as well as prolonged storage duration contributed

to substantial decrease in total phenolics contents.

119

Figure 4.3.10: Total phenolic contents in hot peppers viz. Sky Red (A), Maha (B) and Wonder King (C) at different temperatures and packaging materials (PB: Polyethylene Bag and JB: Jute Bag) during five month storage. Vertical bars show + SD. n= 3 replicates

01020304050607080

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration/Temperature (A)

Tot

al p

heno

lics

mg/

100g

PB JB

01020304050607080

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration/Temperature (B)

Tot

al p

heno

lics

mg/

100

g

01020304050607080

0 50 100 150 0 50 100 150 0 50 100 150

20 ºC 25 ºC 30 ºC

Storage duration (days)/Temperature (C)

Tot

al p

heno

lics

mg/

100g

120

Present studies are in line with the findings of Talcott et al. (2000) that the concentration

and oxidation of phenolic acids was accentuated by thermal processing and storage at

higher temperature significantly affect the antioxidant activity. Similarly Mustapha and

Ghalem (2007) found significant decrease in total phenolic contents of dates when they

were stored at 10ºC after five months storage period.

4.3.10. Conclusion

Gradual increase in temperature as well as storage duration had deleterious effect on

antioxidant quality of hot peppers. However, the packaging material and type is very

important for storage pursuits in terms of microbial load due to aeration e.g. jute bag.

Aflatoxin detection in both types of packaging materials was found below levels of

European Union (EU) Legislation but polyethylene packaging approved more hygiene

keeping in mind sanitary and phyto-sanitary measures. Initial moisture contents in hot

peppers before storage play an important role during storage for shelf life extension and

lessen aflatoxin contamination.

121

Experiment IV. 4.4: Effect of gamma-radiation on microbial load and antioxidant

quality of aflatoxin contaminated samples during storage

4.4.1: Moisture contents (%age)

The moisture content plays an important role on the storage stability of produce. Lower

the moisture content, longer the storage stability and shelf life (Amjad and Anjum, 2007).

The moisture is also very important in case of irradiation because water radiolysis takes

place due to irradiation. Lower levels of irradiation dose and moisture content results in

lower water radiolysis. Analysis of variance revealed significant differences (P ≤ 0.01)

with respect to moisture contents in hot peppers and irradiation doses (see table 4.4.1).

Moisture content was higher in Wonder King (12.47%) while it was lower in Sky Red

(11.88%). Significant decrease in moisture contents 12.31 to 12.13% was observed with

the increase in irradiation dose from 2-6 kGy (see figure 4.4.1); however, the interactive

effect of these was non-significant. These results support the findings of Wage et al.

(2008) that radiation at 10 kGy significantly reduces the moisture contents from 13.88 to

10.32 in ground black pepper after 6 months storage at 20°C. They further revealed that

storage temperature does not change moisture contents considerably when these were

stored at 4 or 20°C.

4.4.2: Total aflatoxins (µg kg-1)

With increasing awareness and knowledge about aflatoxins, a lot of efforts have been

made to eliminate aflatoxin completely or to reduce their contents in the foods and

feedstuffs to significantly lower levels because these are potent source of health hazards

to both man and farm animals. Gamma-radiation is a physical means which has been

studied for its efficiency in destroying mycotoxins in agricultural produce and processed

foods. Analysis of variance revealed significant differences (P ≤ 0.01) with respect to

total aflatoxins in hot peppers and radiation doses (see table 4.4.1). However, their

concentration was maximum in Wonder King (1.39 µg kg-1) followed by 1.09 and 0.84

µg kg-1 in Maha and Sky Red respectively. Gamma radiation decresed total aflatoxin

concentration significantly when the samples were irradiated at a dose rate of 6 kGy and

it was 7% as compared with control samples (see figure 4.4.2). However, there were no

significant differences in aflatoxin contamination in hot peppers during three months

storage. So far research information regarding the decontamination of aflatoxins in hot

122

Table 4.4.1: Mean square values from analysis of variance for moisture (%) and total aflatoxins in hot pepper hybrids as affected by gamma radiation and storage

*, ** = Significant at 0.01 levels respectively. NS = Non-significant

S.O.V D.F Moisture contents

Total aflatoxins

Hybrid (H) 2 2.191356** 1.804151**

Storage Period (S) 1 0.006422 NS 0.000556NS

Radiation (R) 3 0.119048** 0.02863**

H x S 2 0.000439 NS 0.000301 NS

H x R 6 0.018993 NS 0.000755 NS

S x R 3 0.012011 NS 0.003237 NS

H x S x R 6 0.001128 NS 0.000294 NS

Error 48 0.012681 0.003247

123

Figure 4.4.1: Effect of gamma radiation on moisture contents in hot peppers during three months storage. n = 3 replicates

0

5

10

15

0 kGy 2 kGy 4 kGy 6 kGy

Radiation dose/Sky Red

Moi

stur

e co

nten

ts (%

age

)

0 days 90 days

0

5

10

15

0 kGy 2 kGy 4 kGy 6 kGy

Radiation dose/Maha

Moi

stur

e co

nten

ts (%

age

)

0

5

10

15

0 kGy 2 kGy 4 kGy 6 kGy

Radiation dose/Wonder King

Moi

stur

e co

nten

ts (%

age

)

124

Figure 4.4.2: Effect of gamma radiation on total aflatoxins in hot peppers during three months storage. n = 3 replicates

0

0.5

1

1.5

0kGy 2kGy 4kGy 6kGy

Radiation dose/S ky Red

Tot

al a

flat

oxin

s µ

g/kg

0 days 90 days

0

0.5

1

1.5

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Tot

al a

flat

oxin

s µ

g/kg

0

0.5

1

1.5

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Tot

al a

flat

oxin

sµg/

kg

125

peppers are scarce. However, there are some reports on aflatoxin decontamination by

gamma radiation in different commodities like Herzallah et al. (2008) reported that a

dose of 5 kGy decrease 10% total aflatoxin contamination in chick feed and it further

decresed to 35% when the samples were irradiated at 25 kGy. Similarly, Aquino et al.

(2005) observed that radiation dose of 10 kGy resulted in complete reduction in AFB1

and AFB2 in maize.

4.4.3: Total fungal counts (TFCs g-1)

Analysis of variance revealed that significant differences were observed for total fungal

counts in hot peppers during three months storage at different doses of irradiation and

their interactions (see table 4.4.2). Among hybrids, total fungal counts were highest in

Wonder King (5.41 x 103) while they were lowest in Sky Red (4.42 x 103). Fungal

population increased significantly in hot peppers from their original level 4.42 x 103 to

5.41 x 103 after three months storage. Radiation treatment significantly reduced the

fungal population with the increase in irradiation doses and these were completely

eliminated at a dose of 6 kGy (see figure 4.4.3). Similarly at 4 kGy, fungal population

reduced significantly from their original level 5.41 x 103 to 2.4 x 101 after three months

storage. A dose rate of 2 kGy reduced total fungal counts from 5.4 x 103 to 5.01x 102

which were 91% less as compared with control samples. However, slight increase in

fungal population was observed in samples irradiated at 2 kGy after three months storage.

This increase could have been due to proliferation of relative irradiation resistant strain or

due to the recovery of injured molds. These results indicated that fungal population is

very sensitive to gamma radiation and it was completely eliminated at 6 kGy in hot

peppers under present investigation. Aziz and Mahrous (2004) reported that a dose rate of

4-6 kGy was required for complete elimination of fungi in different food and feed

products. These results support the findings of Aziz et al. (2006) that fungal counts were

decresed significantly at 2-4 kGy and eliminated completely at 6 kGy. Similarly,

Munasiri et al. (1987) found that a dose of 10 kGy destroyed all microorganisms in

prepackaged red chilli.

4.4.4: Aspergillus flavus/parasiticus counts (g-1)

Analysis of variance revealed that significant differences were observed for Aspergillus

count in hot peppers during three months storage at different doses of irradiation while

126

Table 4.4.2: Mean square values from analysis of variance for total fungal counts and Aspergillus counts in hot pepper hybrids as affected by gamma radiation and storage

S.O.V D.F Total fungal

counts

Aspergillus counts

Hybrid (H) 2 394223.4** 10975.72**

Storage Period (S) 1 109746.1** 6346.889**

Radiation (R) 3 1017390** 1028406**

H x S 2 30431.38* 304.8889NS

H x R 6 346163.7** 7884.722**

S x R 3 58528.72** 3976.185**

H x S x R 6 26478.25** 305.0741NS

Error 48 6719.306 240.5833

*, ** = Significant at 0.05 and 0.01 levels respectively. NS = Non-significant

127

Figure 4.4.3: Effect of gamma radiation on total fungal counts in hot peppers during three months storage. n = 3 replicates

0

1000

2000

3000

4000

5000

6000

0kGy 2kGy 4kGy 6kGy

Radiation dose/Sky Red

Tot

al fu

ngal

cou

nts/

g

0 days 90 days

0

1000

2000

3000

4000

5000

6000

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Tot

al fu

ngal

cou

nts/

g

0

1000

2000

3000

4000

5000

6000

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Tot

al fu

ngal

cou

nts/

g

128

Figure 4.4.4: Effect of gamma radiation on Aspergillus flavus/parasiticus counts in hot peppers during three months storage. n = 3 replicates

0

100

200

300

400

500

600

700

0kGy 2kGy 4kGy 6kGy

Radiation dose/S ky Red

Asp

ergi

llus

flav

us/p

aras

itic

us

coun

ts/g

0 days 90 days

0

100

200

300

400

500

600

700

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Asp

ergi

llus

flav

us/p

aras

itic

us

coun

ts/g

0

100

200

300

400

500

600

700

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Asp

ergi

llus

flav

us/p

aras

itic

us

coun

ts/g

129

the effects of H x S and H x S x R was non-significant (see table 4.4.2). Among hybrids,

Aspergillus counts were higher in Wonder King (5.21 x 102) while these were lower in

Sky Red (3.96 x 102). Fungal population increased significantly in hot peppers from their

original level 3.96 x 102 to 6.14 x 102 after three months storage in control samples where

as radiation treatment significantly reduced Aspergillus counts with the increase in

irradiation doses immediately after radiation and these were completely eliminated at a

dose rate of 6 kGy (see figure 4.4.3). Similarly at 4 kGy, their population reduced

significantly from their original level 6.14 x 103 to 1.13 x 101 after three month storage.

These results follow the same trend as was observed for total fungal counts and

Aspergillus counts were eliminated completely at 6 kGy.

4.4.5: Capsaicin (mg 100g -1)

Significant differences (P ≤ 0.01) were observed for capsaicin concentration in hot

peppers, storage period and irradiation dose. However, the interactive effect among these

was non-significant (see table 4.4.5). Capsaicin concentration was highest in Sky Red

(30.09 mg 100g-1) while it was lowest in Maha (18.36 mg 100g-1). Capsaicin

concentration decreased significantly (4%) after three months storage while significant

increase was observed in capsaicin concentration with the increase in irradiation dose in

hot peppers under investigation (see figure 4.4.5). However, maximum increase was

observed when the samples were irradiated at dose rate of 6 kGy and it was 8% as

compared to non-radiated (control). These results indicated that irradiation up to 6 kGy is

effective for the retention of capsaicin in hot peppers under present investigation and

slight increase in capsaicin concentration was observed at all irradiation doses when

compared with control. These findings are in agreement with Subbulakshmi et al. (1991)

that the pungency of irradiated paprika was greater when compared with non-radiated

control. Similarly, Topuz and Ozdemir (2004) found that increase in capsaicinoid with

the effect of irradiation treatments can be explained by changing the conformation of the

molecules and/or accompanying compounds which affects the extraction yield. Doses up

to 5 kGy of gamma irradiation led to greater increases in capsaicin and dihydrocapsaicin

levels and an increase of about 10% in the capsaicinoid content of paprika.

130

Table 4.4.3: Mean square values from analysis of variance for capsaicin and dihydrocapsaicin concentration in hot pepper hybrids as affected by gamma radiation and storage

S.O.V D.F Capsaicin

Dihydrocapsaicin

Hybrid (H) 2 901.1876** 278.5033**

Storage Period (S) 1 11.44014** 6.23045**

Radiation (R) 3 12.66644** 3.56045**

H x S 2 0.109306NS 0.015879NS

H x R 6 1.251713NS 0.90494NS

S x R 3 2.065694NS 0.61882NS

H x S x R 6 0.674861NS 0.372144NS

Error 48 0.865417 0.331464

** = Significant at 0.01 levels respectively. NS = Non-significant

131

Figure 4.4.5: Effect of gamma radiation on capsaicin concentration in hot peppers during three months storage. n = 3 replicates

5

10

15

20

25

30

35

0kGy 2kGy 4kGy 6kGy

Radiation dose/Sky Red

Cap

saic

in m

g/10

0g

0 days 90 days

0

5

10

15

20

25

30

35

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Cap

saic

in m

g/10

0g

0

5

10

15

20

25

30

35

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Cap

saic

in m

g/10

0g

132

4.4.5: Dihydrocapsaicin (mg 100g-1)

Significant differences (P ≤ 0.01) were observed for dihydrocapsaicin concentration in

hot peppers, storage period and radiation dose. However, the interactive effect of these

was non-significant (see table 4.4.6). Dihydrocapsaicin concentration was highest in Sky

Red (17.63 mg 100g-1) while it was lowest in Maha (11.18 mg 100g-1). Dihydrocapsaicin

concentration decreased significantly (7%) after three months storage while slight

increase was observed in dihydrocapsaicin concentration was observed with increase in

irradiation dose in hot peppers under investigation (see figure 4.4.7). However, maximum

increase was observed when the samples were irradiated at dose rate of 4 and 6 kGy and

it was 7% as compared to non-radiated (control). These results follow the same trend as

was observed for capsaicin concentration. However, stability of capsaicin during storage

was more than dihydrocapsaicin.

4.4.7: Total carotenoids (mg 100g-1)

All hybrids differ significantly (P ≤ 0.01) with respect to total carotenoids concentration

and highest concentration of carotenoids was found in Wonder King (88 mg 100g-1)

followed by Sky Red (66.89 mg 100g-1) and Maha (58.69 mg 100g-1). Carotenoid

concentration decreased significantly with the increase in storage duration and irradiation

dose (see figure 4.4.7). Significant decrease in carotenoid concentration was observed

immediately after irradiation when the samples were irradiated at 6 kGy and it was 7% as

compared to non-radiated control. Similarly carotenoid loss was higher (12 and 14%) in

samples irradiated at 4 and 6 kGy while it was 9% in non-radiated control after three

month storage. However, no significant difference was observed in total carotenoid

concentration when the samples were irradiated at a dose rate of 2 kGy and both were

statistically alike. These results indicated that carotenoids are very sensitive to gamma

radiation and they decreased significantly with increasing radiation dose subjected to

storage. The reduction in carotenoids with increasing irradiation dose may be attributed

to absorbed energy assisted by irradiation doses of 4-6 kGy and/or increase in the rate of

the oxidation reaction. Topuz and Ozdemir (2003) observed that carotenoid reduction due

to irradiation in paprika was possibly caused by an increase in oxidation reaction under

gamma radiation and also secondary oxidative effects of free radical (H2O2, O3 and OH)

formation during radiation. Similarly, they also concluded that carotenoid loss in paprika

133

Figure 4.4.6: Effect of gamma radiation on dihydrocapsaicin concentration in hot peppers during three months storage. n = 3 replicates

0

5

10

15

20

0kGy 2kGy 4kGy 6kGy

Radiation dose/Sky Red

Dih

ycro

caps

aici

n m

g/10

0g

0 days 90 days

0

5

10

15

20

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Dih

ydro

caps

aici

n m

g/10

0g

0

5

10

15

20

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Dih

ydro

caps

aici

n m

g/10

0g

134

Table 4.4.4: Mean square values from analysis of variance for total carotenoids, ascorbic acid and total phenolic contents in hot pepper hybrids as affected by gamma radiation and storage

S.O.V D.F Total carotenoids

Ascorbic acid

Total

phenolics

Hybrid (H) 2 5485.44** 65.03847** 4721.477**

Storage Period (S) 1 426.32** 139.7235** 37.41125**

Radiation (R) 3 77.43648** 12.49681** 8.263472*

H x S 2 2.57625NS 0.221806NS 0.937917NS

H x R 6 10.12495NS 1.349583NS 0.857917NS

S x R 3 17.78037* 0.712731NS 5.409028NS

H x S x R 6 1.677731NS 1.177331NS 0.565694NS

Error 48 6.342083 1.825556 2.6419

*, ** = Significant at 0.05 and 0.01 levels respectively. NS = Non-significant

135

Figure 4.4.7: Effect of gamma radiation on total carotenoids concentration in hot peppers during three months storage. n = 3 replicates

0

20

40

60

80

100

0kGy 2kGy 4kGy 6kGy

Radiation dose/Sky Red

Tot

al c

arot

enoi

ds m

g/10

0g

0 days 90 days

0

20

40

60

80

100

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Tot

al c

arto

enoi

ds m

g/10

0g

0

20

40

60

80

100

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Tot

al c

arot

enoi

ds m

g/10

0g

136

was highest in non-radiated (control) samples than radiated ones.

4.4.8: Ascorbic acid (mg100g-1)

Significant differences (P ≤ 0.01) were observed for ascorbic acid concentration in hot

peppers, storage period and irradiation dose. However, the interactive effect of these was

non-significant (see table 4.4.8). Ascorbic acid concentration was highest in Wonder

King (27.8 mg 100g-1) while it was lowest in Maha (24.6 mg 100g-1). Concentration of

ascorbic acid decreased significantly (10%) after 3 months storage and with the increase

in irradiation dose in hot peppers under investigation (see figure 4.4.8). However,

maximum decrease was observed when the samples were irradiated at dose rate of 4 and

6 kGy and it was 7% as compared to non-radiated (control). So far research information

regarding the effect of irradiation doses on ascorbic acid in dry hot peppers during

storage is scarce. However, Bibi et al. (2007) reported that ascorbic acid concentration

decreased significantly (7%) at 1 kGy in dried garlic powder during five months storage.

From these results, it can be concluded that ascorbic is very sensitive to irradiation and

gradually decrease with the increase in irradiation. Similarly, Calucci et al. (2008) found

that ascorbic acid concentration was decreased significantly in different aromatic herbs

and spices when these were irradiated at a dose of 10 kGy after three months storage.

4.4.9: Total phenolic contents (mg 100g-1)

Hot peppers differ significantly (P ≤ 0.01) with respect to total phenolic contents (see

table 4.4.4) and it was highest in Maha (51.83 mg 100g-1) followed by Sky Red (50.23

mg 100g-1) and Wonder King (26.77 mg 100g-1). Total phenolic contents decreased

significantly (4%) after three months storage while slight increase (P ≤ 0.05) in total

phenolic contents (3%) was observed in samples irradiated at a dose rate of 4 and 6 kGy

in hot peppers under investigation (see figure 4.4.9). However, the interactive effect of

hybrids, storage duration and irradiation was non-significant. These results indicated that

irradiation up to 6 kGy is effective for the retention of total phenolic contents in hot

peppers and slight increase in their concentration was observed at all irradiation doses

when compared with non-radiated control. So far no information is available in the

literature on the effect of gamma radiation on total phenolic contents of dry hot peppers

but diverse effects of radiation on total phenolic contents have been reported on other

spices. Variyar et al. (1998) found increase in phenolic acid concentration after

137

Figure 4.4.8: Effect of gamma radiation on ascorbic acid concentration in hot peppers during three months storage. n = 3 replicates

0

10

20

30

40

0kGy 2kGy 4kGy 6kGy

Radiation dose/Sky Red

Asc

orbi

c ac

id m

g/10

0g

0 days 90 days

0

10

20

30

40

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Asc

orbi

c ac

id m

g/10

0g

0

10

20

30

40

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Asc

orbi

c ac

id m

g/10

0g

138

Figure 4.4.9: Effect of gamma radiation on total phenolic contents in hot peppers during three months storage. n = 3 replicates

0

10

20

30

40

50

60

0kGy 2kGy 4kGy 6kGy

Radiation dose/Sky Red

Tot

al p

heno

lics

mg/

100g

0 days 90 days

0

10

20

30

40

50

60

0kGy 2kGy 4kGy 6kGy

Radiation dose/Maha

Tot

al p

heno

lics

mg/

100g

0

10

20

30

40

50

60

0kGy 2kGy 4kGy 6kGy

Radiation dose/Wonder King

Tot

al p

heno

lics

mg/

100g

139

irradiation in cloves and nutmeg. Similarly, Harrison and Were (2007) also reported

increase in total phenolic contents in almond skin after they were irradiated at 4 kGy.

4.4.10. Conclusion

The greater the irradiation dose used in hot peppers, the lower concentration of

carotenoids and ascorbic acid was found; however, capsaicinoids and total phenolic

contents had positive relation with irradiation dose. Irradiated samples of hot peppers had

7% reduction in aflatoxin contamination as compared to non-radiated (control) but total

fungal population had inverse relation with increasing radiation dose and complete

inhibition was observed when hot peppers were irradiated at 6 kGy and no further fungal

proliferation was seen during three months storage duration.

140

CChhaapptteerr 55

SSUUMMMMAARRYY The objective of the present study was to examine productivity of hot peppers as

influenced by production system as well as to investigate constraints during storage. In

order to pursue the research work, three hot pepper hybrids namely Sky Red, Maha and

Wonder King were forced in poly/plastic tunnels amended with three types of mulches

viz. black, clear plastic and bare soil considered as control during the year 2005-06 and

2006-07. Plastic mulch is an integral component of protected agriculture which has

significant impact on soil warmth as well as stabilizes soil moisture contents. Higher soil

temperature and humidity under plastic mulch act as barrier or insulation to longer

wavelength of light emitted from the soil which creates conducive environment for better

nutrient up take and efficient translocation. Clear plastic mulch increased soil temperature

2.7ºC and 2.15ºC where as with black plastic mulch this increase was 3.9ºC and 3.3ºC

when compared with bare soil during 2005-06 and 2006-07 respectively. Among the

three hot pepper hybrids, Wonder King had better productivity dynamics in terms of

different vegetative and reproductive traits when grown on black plastic mulch. However,

plants grown under clear plastic mulch start flowering 3 and 7 days earlier than black

plastic mulch and bare soil respectively. Fruit yield plant-1 increased 39% by the

application of black plastic mulch followed by 34% under clear plastic mulch than bare

soil; although intensive weed growth was recorded under clear plastic mulch. Efficient

nutrient uptake in terms of nitrogen and potash was observed in plants grown under black

plastic mulch but phosphorus was found under clear plastic mulch.

All hot pepper hybrids as well as their fruit harvesting stages; immature green, mature

green, color break, red ripe and dried fruit had significant effect upon antioxidant

accumulation. Capsaicin to dihydrocapsaicin ratio peaked at 1.7:1 in Wonder King and in

Maha at immature green stage while in Sky Red it was at dried fruit stage. However, the

accumulation of capsaicinoids was higher at mature green stage in Sky Red followed by

Wonder King conversely at color break stage in Maha. Carotenoid accumulation showed

8 fold increase from red ripe to dry fruit stage, inversely quick decline was observed in

141

case of ascorbic acid. However, the pattern of total phenolic contents biosynthesis was

found to peak at the immature green stage in Sky Red but in Maha at color break stage

while in case of Wonder King at red ripe stage.

Dried fruits of these hybrids were stored in two types of packaging materials;

polyethylene and jute bags at different temperature regimes maintained at 20, 25 and

30°C for five months storage periods and analyzed after every 50 days interval for

microbiological and biochemical attributes. Initial moisture content of fruits was 12 ±

0.5%. Aflatoxin contamination was found 96% higher in Wonder King than Sky Red and

34% than Maha.

Storage of hot pepper hybrids at all temperature regimes up to 100 days either in

polyethylene bags or jute bags was found to be safer i.e. having less aflatoxin in case of

Sky Red. Storage of dried fruits in jute bags was found more susceptible for aflatoxin

contamination than polyethylene bags. Likewise temperature was also found to have

significant relation with aflatoxin contamination. Overall results indicated that

contamination was 61% higher in hot peppers stored at 25ºC and 30ºC than at 20ºC.

However, the response of each hybrid was different in all temperature treatments e.g.

aflatoxin contamination in Sky Red was highest at 30ºC while in Wonder King it was

maximum at 25ºC in both types of packaging materials used in present study. On an

average, aflatoxin contamination increased 75% in fruits stored in jute bags than

polyethylene bags. So far not much information is published about the incidence of

aflatoxin contamination in hot peppers during storage. Most of the time people just

collect the samples from market and analyzed but they had no back ground history about

postharvest handling and drying etc. Overall results of present studies argues that

aflatoxin contamination in dried hot pepper fruits tested under various storage regimes

had their lower detection range than the existing regulatory limits in the European

Commission (EC No. 1881/2006) 10 µg kg-1 for total aflatoxins.

Microbiological investigations after five months storage at 25ºC revealed that both total

fungal count (TFC) and Aspergillus count was 65-70% more in jute bags than when

stored in polyethylene bags. Fungal population increased sporadically in dried fruits of

hot pepper hybrids at all temperatures but it was found significant at 25ºC followed by

30ºC in spite of 20ºC. However, significant increase in Aspergillus count was observed

142

during 50-100 days of storage in both types of packaging materials. Hot pepper hybrids

differ significantly with respect to their phytochemical potential e.g. capsaicin,

dihydrocapsaicin, carotenoids, ascorbic acid and total phenolic contents. The fruits of hot

pepper hybrids are thermal-sensitive in nature and their concentration decreased at

various temperature regimes in both packaging materials with extended storage period.

Under prolonged storage duration; hot pepper hybrids had inverse relation with their

capsaicin concentration and it was significantly reduced 16% in Maha followed by 9 and

12% in Sky Red and Wonder King respectively. Storage temperature and duration had

inverse relation with capsaicin concentration but packaging materials shared no relation

in this regards. After five months storage period, capsaicin concentration was

significantly reduced 11 and 18% in Sky Red and Maha at 30ºC but in case of Wonder

King it was reduced 14% at 25ºC. However, effect of temperature and packaging material

was non-significant in this regard while it was significant as storage duration prolonged.

Capsaicin concentration decreased significantly in fruits of hot pepper hybrids between

50-100 days at various temperature regimes. Similarly as storage duration increased,

dihydrocapsaicin concentration significantly decreased in hot peppers. However after five

months of storage period, dihydrocapsaicin concentration significantly reduced 18% in

Wonder King followed by 11 and 15% in Sky Red and Maha, respectively.

Dihydrocapsaicin concentration reduced significantly 11% in Sky Red after 100 days of

storage followed by 19 and 21% in Maha and Wonder King after 150 days storage at

30ºC.

Gradual degradation of total carotenoids concentration in hot pepper hybrids advocated

that as storage duration prolonged up to five months, it was 22% in Maha while in case of

Sky Red and Wonder King, it was 15% and 14% respectively. As far as the response of

hot pepper hybrids at different temperature regimes is concerned, total carotenoids

concentration reduced 6% in Maha at 30ºC but 4% decrease was observed in case of Sky

Red and Wonder King. Total carotenoids concentration in the fruits of hot pepper hybrids

after five months storage duration manifested greater stability in Sky Red but in case of

Maha and Wonder King demonstrated 3-4% reduction when jute bags used as storage

medium. All carotenoids are thermal-sensitive in nature and their degradation under

present investigation in hot pepper hybrids after five months storage duration was

143

observed 22% at 30ºC storage temperature but 16 and 12% reduction was recorded at

corresponding storage temperature of 25ºC and 20ºC storage temperature.

Bio-degradation of ascorbic acid concentration in hot pepper hybrids advocated that as

storage duration prolonged up to five months, it was 26% in Wonder King followed by

22% in Sky Red where as 19% was recorded in Maha. Ascorbic acid concentration

gradually decreased in the fruits with increase in storage temperature and duration.

However, reduction in ascorbic acid concentration at 30ºC was observed 28% in Sky Red

followed by 33% in Maha and it was 35% in Wonder King but the observed pattern at

20ºC was 15, 12 and 15% in Sky Red, Maha and Wonder King, respectively. Similarly a

steep decrease in ascorbic acid concentration in hot pepper hybrids was observed between

100-150 days storage period.

Significant reduction in total phenolic contents after five months storage period at 30ºC

was observed in Wonder King 33% but in case of Maha and Sky Red it was 24 and 12%,

respectively. Similarly significant decrease of 6-7% in total phenolic contents in hot

pepper hybrids was observed between 100-150 days storage period. More total phenolic

content stability was observed in polyethylene bags than jute bags.

Significant decrease 12.31 to 12.13% in moisture contents was observed with the increase

in irradiation dose of zero to 6 kGy. Similarly 7% reduction in total aflatoxins was

achieved at a dose of 6 kGy as compared to control. However, there were no significant

differences in aflatoxin contamination in hot pepper fruits during three month storage.

Radiation treatment significantly reduced the fungal population with the increase in

irradiation doses and these were completely eliminated at a dose of 6 kGy but slight

increase in fungal population was seen in samples irradiated at 2 kGy after three months

storage. Irradiation up to 6 kGy is effective for the retention of capsaicinoids and total

phenolic contents in hot peppers and slight increase in their concentration was observed

at all irradiation doses when compared with control. A 7% increase in capsaicinoids

concentration and 3% for total phenolic contents was observed at 6 kGy in hot pepper

hybrids after three months storage while significant decrease was recorded in control.

However, both carotenoids and ascorbic acid concentration decreased significantly with

the increase in irradiation dose and storage duration as well. Carotenoids loss was higher

(12 and 14%) in samples irradiated at 4 and 6 kGy while it was 9% in non-radiated

144

control after three months storage. However, no significant difference was observed in

total carotenoids concentration when the samples were irradiated at 2 kGy and both were

statistically alike. Concentration of ascorbic acid decreased significantly (10%) after 3

months storage and with the increase in irradiation dose in hot peppers under

investigation. However, maximum decrease was observed when the samples were

irradiated at dose of 4 and 6 kGy and it was 7% as compared to non-radiated (control).

Recommendations:

Ø Poly/plastic tunnels can be used as a tool to boost hot pepper industry in Punjab,

Pakistan as it not only increases yield by 60% opened field conditions but also

reduces the cost/expenditure incurred on the eradication of weeds.

Ø Efficient day light can be used for hot pepper solar drying and then covering properly

at night to avoid the chances of aflatoxin contamination.

Ø Polyethylene in lined with jute bag can be used as a safer packaging medium to

achieve hot pepper commodity with lesser extent of microbial contamination for

mega storage as well as distant transportation.

Future prospects:

Ø Identification and selection of high yielding hot pepper cultivars for poly/plastic

tunnel.

Ø Eco-geographical zoning of Pakistan to boost hot pepper industry for open and

poly/plastic tunnels production.

Ø Standardization of various colored plastic mulches in terms of their spectral qualities

influencing physiological and morphological relationship on hot pepper yield and

yield attributes.

Ø Use of high density plant population in hot pepper production to enhance efficient

land utilization with better quality produce.

Ø Fertigation and standardization of marginal utility of macro and micro nutrients to

achieve better quality yield of hot peppers.

Ø Fixing the maturity stage in hot peppers for maximum accumulation of biochemical

constituents and developing their extraction procedures for efficient utilization in

pharmaceutical industry.

Ø Relationship of phenolic compounds in aflatoxin inhibition.

145

Ø Development of low cost artificial dryers for hot pepper for better quality with greater

color stability.

146

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