FACULTY OF BIOSCIENCE ENGINEERING

INTERUNIVERSITY PROGRAMME (IUPFOOD)

MASTER OF SCIENCE IN FOOD TECHNOLOGY

Major Food Science and Technology

Academic year 2014-2015

CHARACTERIZATION OF SALT-FERMENTED ANCHOVY PASTE FROM THE PHILIPPINES

DULCE FE B. VELASCO

Promoter : Prof. dr. ir. KATLEEN RAES

Tutor : ing. ELLEN NEYRINCK

Master's dissertation submitted in partial fulfilment of the requirements

for the degree of Master of Science in Food Technology

ii

Copyright

The author and promoters give the permission to consult and copy parts of this work for personal

use only. Any other use is under the limitations of copyrights laws, more specifically it is

obligatory to specify the source when using results from this thesis.

Gent, 5 July 2015

The promotor The author

Prof. dr. ir. Katleen Raes Dulce Fe Velasco

iii

Acknowledgement

First and foremost, I wish to express my sincere thanks to my promoter, Professor

Katleen Raes, for providing me with all the necessary facilities for the research. Thank you very

much for the assistance, understanding, constructive suggestions and corrections, and immense

knowledge that greatly improved this paper.

To my tutor, Ellen Neyrinck, my deepest gratitude for valuable guidance and

encouragement throughout my entire laboratory works.

I am also indebted to VLIR-UOS for providing financial assistance for my graduate

studies here in Belgium.

I take this opportunity to express my appreciation to all the IUPFOOD Coordinators,

Prof. Marc Hendrickx, Prof. Koen Dewettinck, Dr. Chantal Smout, ir. Katleen Anthierens,

and Katrien Verbist for their help and support.

Many thanks to my Alma Mater & Employer – Mindanao State University, Main Campus

especially to the College of Fisheries for all the help during the processing of my application

papers and support during the whole duration of my graduate studies.

I am also grateful to a kind-hearted friend, Anna Rose Pilapil, for the encouragement

extended to me. Very big thanks for the guidance which helped me in all the time of research and

writing of this thesis.

To all my friends who, directly or indirectly, have lent their hand in this venture. Thank

you very much for the support and encouragement.

I also thank my parents, spouse and son for the unceasing encouragement, motivation,

support and attention that have greatly uplifted my morale especially at those times when I’m

jaded.

Most importantly, I am very much grateful to my Lord God, Jesus Christ, and Mama

Mary for the good health and wellbeing that were necessary to complete this research paper.

Thank You for the unconditional and amazing love that You’ve given to me.

iv

Abstract

This research comprehensively characterized the salt-fermented anchovy paste or

bagoong from the Philippines which is an important food product in the country. Anchovy and

anchovy paste samples traditionally prepared were obtained in the Philippines and were brought

to Belgium for biochemical analyses. Samples were taken from different batches at different time

points during fermentation of local producers and samples commercially available in markets.

The commercially available anchovy pastes were of 1, 3 and 4-months old. All samples were

analyzed for gross composition, pH, water activity, salt content, non-protein nitrogen (free and

total NPN), fatty acid composition, mineral content and TBARS for lipid oxidation.

The studied bagoong showed a content of DM (38.7±3.52g/100g), protein

(50.03±4.86g/100gDM), fat (5.71±1.54g/100gDM), SAFA (45.73±2.31%), MUFA (16±1.33%),

and PUFA (28.35±2.63%). The samples have an average pH level of 6.56±0.16. The products

were shown to be microbiologically stable with an Aw and NaCl content of 0.82±0.02 and

31.33±6.98g/100gDM respectively. Even if samples were processed in the same country,

different results for other parameters like TBARS (11.76±2.43μgMDA/gDM), total NPN

(117.89±33.55mg/100gDM), free NPN (21.15±3.15mg/100gDM), and mineral content were

observed. The amount and level of proteins, Aw, pH, minerals, PUFA, free and total nitrogen of

the raw anchovy decrease at the start of fermentation period while an opposite is observed for

DM, fats, salt, SFA and TBARS content. Fermentation increases the concentration of DM, salt

content, total and free NPN of the salt-fermented anchovy paste; decreases the content of fats,

proteins and TBARS; and no significant effect in Aw, mineral content and fatty acids was

observed.

Samples taken from different batches at different time points (R, D0, D9, D19 and D28)

of a local producer were microbial characterized. The microbial count increased and is at its

highest on D19 then decreased gradually on D28. Enterobacteriaceae were not detected. Aerobic

bacteria, LAB and halophilic LAB were involved at the start of fermentation whereas

proteolytic, halophilic aerobic bacteria and halophilic yeasts played a role starting at the middle

fermentation period. The fermentation process was predominated with halophilic bacteria.

Halophilic LAB has the highest count. However, no acid fermentation had occurred based on the

values of pH obtained.

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Table of Contents

Copyright............................................................................................................................................... ii

Acknowledgement .............................................................................................................................. iii

Abstract ................................................................................................................................................ iv

List of Abbreviations ..........................................................................................................................vii

List of Figures ................................................................................................................................... viii

List of Tables ....................................................................................................................................... ix

Chapter 1. Introduction ........................................................................................................................ 1

Chapter 2. Review of Related Literature ............................................................................................. 3

A. Fermentation ................................................................................................................................ 3

B. Fish Fermentation ........................................................................................................................ 5

C. Fermented Fish Products in Thailand ......................................................................................... 7

D. Fermented Fish Products in Indonesia ....................................................................................... 8

E. Fermented Fish Products in Myanmar ...................................................................................... 11

F. Fermented Fish Products in Malaysia ....................................................................................... 11

G. Fermented fish products in Vietnam ........................................................................................ 14

H. Fermented Fish Products in Philippines ................................................................................... 15

I. Fermented Fish Paste in the Philippines .................................................................................... 18

Chapter 3. Materials and Methods ..................................................................................................... 20

A. Collected Samples from the Philippines .................................................................................. 20

B. Gross Composition..................................................................................................................... 21

a. Dry Matter Content ................................................................................................................. 21

b. Crude Protein Content ............................................................................................................ 21

c. Crude Fat Content ................................................................................................................... 23

C. pH ................................................................................................................................................ 24

D. Water Activity (Aw) ................................................................................................................... 24

E. Mineral Content .......................................................................................................................... 25

F. Salt Content ................................................................................................................................. 26

G. Fatty Acid Composition ............................................................................................................ 26

a. Anchovy Paste Fatty Acids Extraction .................................................................................. 26

vi

b. Fatty acid Methylation ............................................................................................................ 27

H. Total and Free Non-Protein Nitrogen Content ......................................................................... 29

a. Perchloric Acid Extraction ..................................................................................................... 29

b. Total Non-Protein Nitrogen ................................................................................................... 30

c. Free Non-Protein Nitrogen ..................................................................................................... 30

I. TBARS Analysis ........................................................................................................................ 31

J. Microbiological Analysis............................................................................................................ 33

a. Sampling .................................................................................................................................. 33

b. Microbial Analysis .................................................................................................................. 33

K. Statistical Analysis..................................................................................................................... 34

Chapter 4. Results and Discussion .................................................................................................... 35

A. Raw materials ............................................................................................................................. 35

B. Biochemical and chemical composition of commercially available anchovy pastes ............ 37

a. Chemical Composition ........................................................................................................... 37

b. Mineral Composition .............................................................................................................. 39

c. Fatty Acid Composition .......................................................................................................... 40

d. Non-protein and TBARS composition .................................................................................. 45

C. Characterization of fermented anchovy pastes......................................................................... 47

a. Chemical Characterization ..................................................................................................... 47

i. Chemical Composition ....................................................................................................... 47

ii. Mineral Composition......................................................................................................... 49

iii. Fatty Acid Composition ................................................................................................... 50

iv. Non-protein and TBARS composition……………………………………………….53

b. Microbial Characterization ..................................................................................................... 54

Chapter 5. Conclusions and Recommendation ................................................................................. 58

Works Cited ........................................................................................................................................ 60

vii

List of Abbreviations

ANOVA - analysis of variance

Aw - water activity

BHI - brain heart infusion

Ca - calcium

CFU - colony forming units

Cu - copper

DHA - docosahexaenoic acid

DM - dry matter content

D0 - day 0 of fermentation period

D9 - day 9 of fermentation period

D19 - day 19 of fermentation period

D28 - day 28 of fermentation period

EPA - eicosapentaenoic acid

FAME - fatty acid methyl esters

Fe - iron

FP - anchovy pastes sold in markets/commercially available fish pastes

ICP-AES - Inductively Coupled Plasma-Atomic Emission Spectroscopy

ISO - International Organization for Standardization

K - potassium

LAB - lactic acid bacteria

MDA - malonaldehyde

Mg - magnesium

Mn - manganese

MRS - de Man, Rogosa and Sharpes’ medium

MUFA - monounsaturated fatty acids

Na - sodium

NaCl - sodium chloride salt

NPN - non-protein nitrogen

PCA - plate count agar

PUFA - polyunsaturated fatty acids

R - raw anchovy

SFA - saturated fatty acids

SP - anchovy pastes with different batches produced from one producer

T - timepoints

TBARS - thiobarbituric acid reactive substances

VRBGA - violet red bile glucose agar

Zn - zinc

viii

List of Figures

Figure 1 Raw material for salt-fermented anchovy paste

Figure 2 Fermented anchovy pastes at different time points

Figure 3 Fermented anchovy pastes locally sold in markets

Figure 4 Microbial count of fermented anchovy pastes on different days of fermentation

ix

List of Tables

Table 1 Fish Paste and Fish Sauce Products in Different Countries (Chinte-Sanchez, 2008)

Table 2 Chemical Composition of some Fermented Fish Products in Indonesia (Putro,

1993; Irianto & Irianto, 1998)

Table 3 Quality Standards of Fermented Fish Products in Myanmar (Tyn, 1993)

Table 4 Composition of Fermented Products in Malaysia (Abdul Karim, 1993)

Table 5 Some Physicochemical Parameters of nuoc-mam (Taira, et al., 2007)

Table 6 Chemical Composition of Bagoong and Patis (per 100g edible portion) (Chinte-

Sanchez, 2008)

Table 7 Amino Acid Content of Patis from dilis (mg/100mL) (Chinte-Sanchez, 2008)

Table 8 Chemical Composition of Bagoong dilis (per 100g edible portion) (Chinte-

Sanchez, 2008)

Table 9 Characterization of the samples and manufacturers

Table 10 Chemical composition of anchovy paste traditionally produced in some parts of

the Philippines

Table 11 Mineral composition of anchovy paste traditionally produced in some parts of the

Philippines

Table 12 Fatty acid profile (g/100g FAME) of anchovy paste traditionally produced in

some parts of the Philippines

Table 13 Fatty acid profiles of anchovy pastes obtained from different studies

Table 14 Non-protein nitrogen and TBARS of anchovy paste traditionally produced in

some parts of the Philippines

Table 15 Chemical composition of anchovy pastes in different time points of fermentation

Table 16 Mineral composition of anchovy pastes in different time points of fermentation

Table 17 Fatty acid profiles of anchovy pastes in different stages of fermentation

Table 18 Non-protein nitrogen and TBARS of anchovy paste traditionally produced in

some parts of the Philippines

1

Chapter 1. Introduction

For centuries, food fermentation is a technique used to preserve perishable food products

which includes fruits, cereals, vegetables, milk, meat and fish (Hui et al, 2004). Everywhere in

the world, about 20-40% of the food supply comes from fermented foods and beverages.

Fermentation is a simple and very economical method of food preservation (Chinte-Sanchez,

2008). There are five major fermentation processes: 1) lactic acid fermentation; 2) acetic acid

fermentation; 3) alcoholic fermentation; 4) alkaline fermentation; and 5) adding high amount of

salt. According to Steinkraus (1996), fermentation has five roles. These are:

a) Human diet enrichment through the development of a wide diversity of food flavors,

aromas and textures;

b) Food preservation by lactic acid, acetic acid, alcoholic and alkaline fermentation;

c) Food enrichment with protein, essential amino acids, essential fatty acids, and vitamins;

d) Food detoxification;

e) Energy and cooking time reduction.

Fermented fish products are important traditional foods in many countries of the world,

particularly in the less developed countries. They are nutritious and available for the consumers

at an affordable price. In Asia, fermented small fishes, fish eggs and intestines are widely

consumed (Lee, et al, 1993). In the Philippines, fish fermentation is one of the most common

methods of fish preservation due to its simplicity in technique and low equipment cost. Also due

to its desirable flavor and cheap source of protein, it has become a part of the diet of most

Filipinos. Fermented fish products are fermented through the addition of salt which can be in

high amount (15-20%) or in low amount (less than 10%). The former is steered often by fish

endogenous enzymes while the latter is steered by lactic acid bacteria. The most popular

products of fish fermentation are fish paste and fish sauce. They have salty, slightly cheese-like

flavor and an appetite-stimulating aroma. Fish paste is a naturally fermented whole fish or

shrimp with the addition of 20-25% salt under ambient conditions. On the other hand, fish sauce

is a straw yellow to amber color liquid extracted through the complete hydrolysis of fish/salt

mixture for 9-12 months (Peralta, et al, 2008).

2

Fish paste is an indigenous fermented food in the Philippines and locally known as

bagoong. It is also called kapi in Thailand, mam in Vietnam, ngapi in Burma, padec in Laos,

prahoc in Cambodia, jeotkal in Korea, trassi in Indonesia and shiokara in Japan (Chinte-

Sanchez, 2008). Any species of fish can be used for the fish paste production. In the Philippines,

anchovies, which are small fishes and abundant throughout the country, is typically used as a raw

material for fish paste. Fermented anchovy paste produces a very distinctive salty and fishy

flavor. It is popularly taken not only as side dishes, but also as an ingredient in many types of

salad dressings. It is also used as ingredients in different sauces (Sukuma & Chaiyanan, 2012).

However, even though it is popular throughout Asian countries, particularly in Southeast Asia

including the Philippines, only limited studies have been conducted regarding fish fermentation

unlike other fermentation technologies such as those involving milk and soybeans. Also, as it is

traditionally produced, it is artisanal in nature and developed only by trial and error instead of

scientific methods. Because of these, they are usually characterized by variation in product

quality, efficiency of processes used, and even safety of foods (Lee, et al, 1993). Thus scientific

studies are still required for the understanding and improvement, if necessary, of fermented

anchovy paste.

This study aims to 1) characterize the biochemical and chemical changes that occur in the

traditionally fermented anchovy paste produced in Philippines during the different stages of

fermentation; and 2) get insight in the succession of microbial flora involved in the process to

better understand the fermentation process.

3

Chapter 2. Review of Related Literature

A. Fermentation

Fermentation is derived from the Latin word “fevere” which means “to boil”, as this

phenomenon was associated with the production of carbon dioxide bubbles through the

anaerobic catabolism of sugars by yeasts when producing alcoholic beverages (Chinte-Sanchez,

2008, Chojnacka, 2010). There are various definitions of fermentation depending on the field of

studies. Classical biochemical definition of fermentation is “an anaerobic breakdown of an

organic substrate by an enzyme system in which the final hydrogen acceptor is an organic

compound”. In a broader sense, it is “a metabolic process in which chemicals are brought about

in an organic substrate through the activities of enzymes secreted by microorganisms” (Chinte-

Sanchez, 2008). In its simpler definition, as cited by Chinte-Sanchez (2008), it is the processing

of foods in which a certain typical desirable characteristic of food develops such as flavor,

aroma, and texture as well as to keep its quality by microbial activities. It is also a

biotechnological process whereby foods are produced by controlled biochemical reactions from

agricultural products. From a biochemistry point of view, it is “an energy-generating process in

which organic compounds act as both electron donors and terminal electron acceptors”.

Furthermore, for a microbiologist, it is “a process that involves the application of

microorganisms to carry out enzyme-catalyzed transformation of organic matter”.

Fermentation is one of the oldest techniques in food preservation. It extends the shelf-life

of foods as well as it enhances its flavor and nutritional value (Kilinc et al, 2005). There are a lot

of benefits when foods are fermented. Fermentation adds flavor and aroma to foods; preserves

the raw material; synthesizes desirable constituents such as vitamins, minerals and other

metabolites; increases digestibility, utilization and transportation of essential amino acids thus

improving protein quality; changes the physical state of and impart color to the food; reduces

antinutritional and toxic compounds such as phytates, tannins, cyanogenic glycosides, saponins,

etc.; consumes low energy; uses less capital and operating costs; applies simple technologies;

and improves food safety (Chinte-Sanchez, 2008, Chojnacka, 2010, Sahlin, 1999). However,

fermentation is sensitive and requires careful control to prevent risk of contamination and

4

intoxication that would result in food safety issues (Chojnacka, 2010). Fermented foods can be

eaten cooked or uncooked and even used as condiments (Ishige, 1993).

Most of the ancient fermented foods originated in Central Asia, and then they slowly

spread to China, Europe, and other parts of the world. At that time, preservation of foods by

fermentation was discovered by chance. There was no explanation how it can preserve natural

resources for longer periods. It was during the 19th century that Pasteur discovered fermentation

was associated with microbial activities. Moreover, it were the enzymes, produced by

microorganisms, that were responsible for the (bio)chemical changes that occur during

fermentation. As fermentation changes the physical and chemical characteristics of foods, it does

not reduce the quality of foods but rather improves their nutritional value. Thus, these fermented

foods have become the only source of nutrients for low-income people particularly in less-

developed countries (Chinte-Sanchez, 2008).

Fermentation can be categorized as aerobic or anaerobic based on the oxygen

requirement. In an aerobic fermentation, oxygen is required as hydrogen acceptor while

anaerobic fermentation doesn’t require oxygen but requires other substances to act as its

hydrogen acceptor such as aldehydes or pyruvic acid (Chinte-Sanchez, 2008). In terms of

microorganisms used, fermentation can be performed spontaneously, by back-slopping, or by the

addition of starter cultures. With spontaneous fermentation, the raw material and its initial

treatment encourage the growth of the indigenous flora and a microbial succession takes place.

For back-slopping, a new batch of fermentation is inoculated with part of microorganisms used

from the previous fermentation batch. This type of fermentation produces a higher initial number

of beneficial microorganisms and is faster and more reliable. The addition of a starter culture is

often used to inactivate the indigenous flora present in the raw material allowing only the added

starter microorganism to grow. Starter cultures can be single, multiple or mixed strain. With

single-strain starter, only a single well-defined strain with known technological properties is

added. In the case of multiple-strain starter, 2-6 well-defined strains are added while a mixed-

strain starter consists of a unknown number of undefined strains (Josephsen & Jespersen, 2004).

Microorganisms involved in fermentation are bacteria, yeasts and molds. Bacteria used in

industrial fermentations include strains of the following species: Acetobacter, Streptococcus,

5

Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, Propionibacterium, Brevibacterium,

Bacillus, Microccus, and Staphylococcus. Yeasts include species of Saccharomyces, Candida,

Torulopsis, and Hansenula while molds involved are Aspergillus, Penicillium, Rhizopus, Mucor,

Monascus and Actinomucor (Chojnacka, 2010).

B. Fish Fermentation

Fresh fish is a highly perishable product due to its biological composition. If it is not

immediately utilized or preserved, spoilage will take place. Fish is usually preserved through a

combination of different preservation techniques such as smoking, sun-drying, salting,

fermentation, grilling and frying (Koffi-Nevry & Koussemon, 2012). Among these methods,

fermentation is commonly practiced in Asian countries. Fermented fish products are products of

freshwater and marine finfish, shellfish, and crustaceans which are processed with salt to

undergo fermentation thus, preventing spoilage (Ishige, 1993). Fermented fish differs from salted

fish as there is a change in the original shape of the fish in the partly liquefied product in the

fermentation process (Espejo-Hermes, 1998). Aside from using salt, fish fermentation can be

processed with other ingredients, e.g. in combination with rice, or combined with other methods

such as drying (Beddows, 1997; Ishige, 1993; Chinte-Sanchez, 2008). Fermented fish products

contribute largely to the protein intake of the world’s population (Beddows, 1997). They are of

major importance in Asian countries, like Thailand, Kampuchea, Malaysia, Cambodia,

Philippines and Indonesia, which have a bland rice diet. Through the addition of these products

in the human’s diet, they serve as a major source of protein (Beddows, 1997; Chinte-Sanchez,

2008).

Fish fermentation is the transformation of organic substances into simpler compounds by

the action of either microorganisms or endogenous enzymes (Peralta, et al., 2008; Beddows,

1997), resulting in the production of peptides, amino acids and other nitrogenous compounds.

Peptides and amino acids contribute significantly to the typical flavor and aroma of fermented

products. These peptides and amino acids were found out to be naturally occurring antioxidants

(Peralta et al, 2008).

6

Fermented fish products are categorized as lacto-fermented fish; fish sauces; and fish

pastes, either ground or unground. Lacto-fermented fish is prepared by fermenting fish with rice

or other grains in addition to fish and salt while production of fish paste and fish sauce only

involve mixture of salt and fish and is processed together. Fish paste is the fish which is broken

down chemically through fermentation characterized as a purée or paste, while fish sauce is the

fully hydrolyzed liquid product (Mizutani, et al., 1992). Shrimp, shellfish and crabs can also be

used as raw material. Fish paste and fish sauce are unique in South-east Asia. They have been

developed as substitute for soybeans which are less easily grown in the region. Moreover, highly

salted amino acid or peptide sauces are greatly appreciated in the area due to its umami taste

(Ishige, 1993; Chinte-Sanchez, 2008). Protein hydrolysis in fish sauces and fish pastes is due to

the fish-gut enzymes instead of proteases from bacteria. Generally, bacterial count naturally

present in fish gradually reduces as fermentation goes on in a high salt environment. Halophilic

bacteria or osmotolerant yeasts play a limited role in the development of flavor or aroma (Lee et

al, 1993). Fish pastes and fish sauces vary from different countries in raw materials, flavor, and

physical properties (Table 1) (Chinte-Sanchez, 2008).

Table 1. Fish Paste and Fish Sauce Products in Different Countries

Country Fish/Shrimp Paste Fish/Shrimp Sauce

China

Yu-lu

Cambodia Prahoc Nuoc-mam

Indonesia Trassi; Trassi-ikan; trassi udang Ketjap-kan; Kecap-ikan

Japan Shiokara Shottsuru

Korea Jeotkal Jeot-kuk

Laos Pradec Padec

Malaysia Belachan Budu; Sambal-ikan

Myanmar (Burma) Ngapi Mga ngan-pya-ye; Hymin ngan-pya-ye

Philippines Bagoong/Alamang Patis

Thailand Kapi Nampla

Vietnam Mam-ca; Mam-ton mam Nuoc-mam

Source : (Chinte-Sanchez, 2008)

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C. Fermented Fish Products in Thailand

Thailand is a seasonal tropical climate country which is very dry with only 3-4 months of

rainy season thus, food availability for the whole year is a problem. Because of this, food

storage and preservation become important factors for maintaining food supplies. Fermentation

is one of the techniques practiced by Thai people in preserving their food. These fermented foods

play an important role in Thai diet. Their fermented fish products include nam-pla (fish sauce),

budu (cloudy fish sauce), kapi (shrimp paste) and plaa-raa (salt-fermented fish) which vary

greatly from community to community and between regions handed from generation to

generation. These foods are usually produced for family consumption but nowadays, it has

moved to small factory production. Fish fermented products in Thailand are classified into three

groups: a) fish with a large amount of salt; b) fish with salt and carbohydrate added; and c) fish

with salt and fruit added. Popular products under the first group are nam-pla, kapi and budu.

Plaa-som, plaa-raa, plaa-chao and plaa-paeng-daeng are some products of the second group

while khem-bak-nad and plaa-mum are examples of the third group (Phithakpol, 1993).

For the first group, marine fishes are mostly used. Nam-pla could also be made from

mussels and freshwater fish but the best quality is made from anchovies. The ratio of salt to

fish/shrimp is 1:3 for nam-pla and budu, while 1:3-5 for kapi. The microorganisms responsible

for fermentation of nam-pla were believed to be Staphylococcus, Bacillus and Sarcina sp.; and

Pediococcus halophilus, S. aureus and S. epidermidis for kapi and budu (Phithakpol, 1993). As

cited by Chinte-Sanchez (2008), an initial count of 104 CFU/mL of aerobic bacteria has been

observed in nam-pla. After 3 weeks of fermentation, total bacterial count increases up to 108

CFU/mL then decreases to a non-detectable level after 6 months. She also cited that Bacillus,

Micrococcus, Staphylococcus, and Halobacterium sp. were isolated in nam-pla. The latter is

involved in the maturation of nam-pla. On the other hand, Thienchai & Chaiyanan (2012) have

identified Pediococcus acidilactici, Tetragenococcus halophilus, Lactobacillus plantarum and

Lactobacillus delbrueckii from kapi.

In the second group, freshwater fishes are mainly used but freshwater shrimp could also

be used. Cooked, roasted or fermented rice are added as source of carbohydrate for the

8

microorganisms involved in the fermentation, such as lactic acid bacteria, and for taste

development of the product. Products with cooked rice added include plaa-som, koong-som and

som-fak. The cooked rice can be added whole or minced to fish with salt. Microorganisms

involved are Lactobacillus plantarum, Saccharomyces and Candida sp. Plaa-raa, plaa-chom and

koong-chom uses coarse-ground roasted rice, from glutinous or normal type of rice, added to fish

after few days of salt fermentation. Roasted rice provides browning and specific flavor of the

final product. Pediococcus halophilus, Staphylococcus epidermidis, Micrococcus and Bacillus

sp. were involved in the fermentation process. Plaa-chao and plaa-paeng-daeng are added with

fermented glutinous rice (khaou mak) and ang-kak rice (red rice) to fish respectively. Khaou mak

is prepared by mixing the rice with yeast balls while ang-kak is by rice mixed with mold,

Monascus purpureus, giving the distinct red color and flavor. Microorganisms involved in the

early stage of fermentation of plaa-chao are Bacillus, Staphylococcus, Saccharomyces and

Endomycopsis sp. while Pediococcus cerevisiae is involved in the later stage. For plaa-paeng-

daeng, Lactobacillus, Micrococcus and Saccharomyces sp. were involved. Under the third group,

khem-bak-nad/khem-mak-nad is a salted fish (1:1-5 fish:rock salt ratio) which were cut into long,

thin pieces packed tightly in a container and left overnight. On the next day, chopped pineapple

is added and placed in bottles and then fermented for three months. On the other hand, plaa-

mum is prepared by mixing salt to small-cut pieces of fish (1:3 salt:fish) then mixed with ground

roasted rice, tightly packed in containers for 1.5-2 months after which, fish is repacked in

containers added with chopped papaya and ground galangal (Phithakpol, 1993).

D. Fermented Fish Products in Indonesia

Fermented fish products in Indonesia have gained special popularity in the local markets.

Among the popular fermented fish products in Indonesia are trasi (shrimp or fish paste), pedah

(fatty, partly dried, salty fish), kecap ikan (fish sauce), jambal roti (fermented dry salted marine

catfish) and bekasam (fermented freshwater fish) (Table 2) (Putro, 1993).

Pedah has a reddish brown color, slightly wet and pasty with a flabby texture, and a

cheesy, salty flavor often mixed with a mild rancid flavor. It is made from salting (1:3 salt:fish),

9

drying and fermenting fish, usually from mackerel. It requires salted fish to be wet, stacked in

baskets or other suitable containers for slow dehydration leading to maturation and development

of desirable flavor and texture for about 2-3 months at 29°C. However, the fermentation period

of pedah varies depending on the processor. As cited by Putro (1993), internal organs of fish

stimulate protein and lipid degradation. However, evisceration will facilitate oxidative rancidity

due to a larger surface area. With regards to fatty acids, there were more PUFA losses,

particularly C22:6n-3 and C22:5n-3, during salting while C20:5n-3 was just stable. Most

monounsaturated and saturated fatty acids also decreased except C18:1 and C22:1. Microbial

analysis showed that gram positive cocci predominate in the fermentation process followed by

lactic acid bacteria. He also mentioned that anaerobic condition speeds up the reddish-browning

of the product due to Malliard reaction, hinders oxidative rancidity, facilitate protein breakdown,

and microbial population of pedah.

Trasi is prepared from planktonous shrimp Schizopodes or Mytis sp. and small fishes

such as Stelophorus or Engraulis sp. Trasi udang (shrimp paste) are more popular than trasi ikan

(fish paste). It is used as an appetizer and also eaten with chili, garlic and salt called sambal.

Trasi udang production starts at the time the shrimps are harvested and placed on the fishing

vessels where 10% salt is added, then another 5% salt is added upon arrival on the landing area.

Shrimps are then partially dried under the sun on mats for 1-3 days. When moisture content has

decreased to about 50%, shrimps are kneaded and mixed, then sun-dried and kneaded again. At

the same time, a coloring agent, like carthamine D or rhodamine B, is often added. Using

cylindrical-formed nipa leaves, the paste is pressed and then fermented until the desired trasi

aroma is achieved. However in other places like Java Island, shrimp paste is processed

differently. Raw or pre-cooked shrimps are added with 15% salt and partially sun-dried for only

1 day then minced, mixed and kneaded forming a paste called brabon. This brabon is sun-dried

and kneaded again until it achieves a fine thick homogenous paste which is then pressed in

cylindrical-formed bamboo and fermented until the desired aroma is attained. Trasi ikan is

prepared with the same process as trasi udang but the former is more often added with coloring

agents and has a stronger smell, making it less popular to the locals than the latter. In a study of

trasi powder, the total bacterial count reduces during the 7-day fermentation while lactic acid

bacteria are just constant. In addition, Staphylococcus, Bacillus and Proteus were also present in

10

the product. It was pointed out that changes in total volatile base (TVB) and pH of trasi and trasi

powder was not significant. Yet, trasi powder is less accepted by consumers than the normal

shrimp paste (Putro, 1993).

Kecap ikan is made from oil sardines (Sardinella sp.) where 25-30% salt is added and

then fermented. After fermentation, it is filtered to separate the solid and liquid part. This liquid

part comprises the fish sauce, which is mixed with brown sugar and spices. However, fish sauce

has become less popular in the country due to competition with soy sauce. Bekasam is made

from freshwater fishes. Its production involves evisceration, splitting of fish into butterfly shape,

brining (± 15% w/w) for 3 days, draining, mixing with roasted rice, packing, and then

fermentation. However, the process differs from place to place. Jambal roti is prepared from

marine catfish (Arius sp.) involving salting (pickling), splitting of the pickled fish, drying on

interwoven bamboo mats and fermentation. During the second or third day of drying, fishes are

split again and then turned occasionally. However, the production varies slightly from region to

region. Moisture and salt content of jambal roti varies between 40-50% and 20-25%

respectively. Fermented fish products are household-based processed, thus, the quality of even

same products differs from place to place (Putro, 1993). There has been a need for further studies

for a standardization of the quality and safety fermented fish products.

Table 2. Chemical Composition of some Fermented Fish Products in Indonesia

Components Pedaha

Shrimp

Pasteb

Fish

Saucec

Bekasamd Jambal roti

e

Moisture (g/100g sample) 53.83 3,0-5 66-76 52-66 49.27-49.68

Crude Fat (g/100g sample) 9.63 2-5 0.5-0.7 1-23 0.69-1.19

Crude Protein (g/100g

sample) 52.12 20-40 10-10.5 41-64 54.17-61.86

NaCl (g/100g sample) 19.21 23 25-30 6-17 7.38-8.53

Total Ash (g/100g sample) nd 10-40 21-23 13-28 34.93-38.80

Carbohydrate (g/100g sample) nd 3.5-5 0.3-1.5 nd nd

pH 6.5 nd nd 4.46-4.98 6.57-6.91

Sources : a,b,c As cited by Putro (1993) : aSyachri and Anwar , 1977 ; b Moeljohardjo, 1972; c Poernomo et al., 1984; d Putro, 1993; eIrianto & Irianto, 1998; nd = not determined

11

E. Fermented Fish Products in Myanmar

Fish fermentation is a centuries-old food preservation technique in Myanmar and the

product is commonly known as nga-pi which has become their national food. Nga-pi is prepared

from fish or shrimp, pounded or ground, added with salt and partially sun-dried for 3-4 days. The

mixture is pressed and stored in earthen jars or concrete vats for 3-6 months of maturation. The

liquid formed during fermentation is also utilized and called as ngan-pya-ye (fish sauce). It is

kept in tanks for 3 months to 1 year for aging. After aging, it is boiled for 4-6 hours for partial

sterilization of the sauce and reduction of moisture content up to 55-60%. As cited by Tyn

(1993), nga-pi and ngan-pya-ye are rich in essential amino acids. Due to the fact that fermented

fish has long been existed in Myanmar, and even labeled as their national food, standards were

formulated for the quality of the different products. Table 3 shows the different specifications of

the various fermented fish in Myanmar. Microbiological analysis of hmyin nga-pi and hmyin

ngan-pya-ye reveal that Arachnia propionica, Bacillus sp., Micrococcus sp., and Nocardia

dentocariosa were present in both products. In addition, Staphylococcus epidermidis and

Corynebacterium sp. were also present in hmyin nga-pi (Tyn, 1993).

Table 3. Quality Standards of Fermented Fish Products in Myanmar

Component Fish Paste Fish Sauce Shrimp Paste Shrimp Sauce

Moisture (g/100g sample) 40 55 40 55

Crude Fat (g/100g sample) 1.5 1 1.5 1

Crude Protein (g/100g sample) 18 18 18 18

NaCl (g/100g sample) 25 25 25 25

Source: Tyn, 1993

F. Fermented Fish Products in Malaysia

Post-harvest losses is one of the common and critical problems faced by Malaysian

fishing industry. Through food preservation, such as fermentation, these losses will be

significantly reduced. However, traditional techniques are still being practiced in Malaysian fish

12

fermentation industries which were managed usually by family members of small entrepreneurs

resulting in poor hygiene and quality products. Thus, further studies are required to enhance the

fish fermentation industry of the said country (Abdul Karim, 1993).

Fermented products in Malaysia include budu (fish sauce), kicap ikan, pekasam, belacan

(shrimp paste), and cincaluk. Budu is a dark brown liquid extracted from salt-fermented fish,

particularly from small anchovies (Anchoviella commerson and A. indica), which is rich in salt

and soluble nitrogen compounds with a distinctive odor and flavor. It is mainly used as flavoring

agent and condiment. It is prepared by washing first the anchovies in seawater and then mixed

with salt (1:2-3 salt:fish). The mixture is left to ferment anaerobically in earthenware containers

or concrete vats for 3-12 months at ambient temperature with occasional stirring. During this

period, fish proteolysis is happening along with the development of the typical budu aroma. At

the end of the fermentation period, the formed liquid supernatant is mixed with the fish residues

and boiled in addition of coconut palm sugar, tamarind, and other flavoring ingredients. Palm

sugar eliminates the fishy smell and improves odor and taste of budu, while tamarind reduces the

pH which inhibits putrefactive bacterial growth aside from enhancing the flavor of the product.

After cooling the boiled fish sauce, it is filtered and bottled. Kicap ikan is manufactured in the

same way as budu except that it is made from other types of fish such as goatfish (Upeneus sp.)

and herring (Clupea sp.). Stainless steel containers with tight-fitting lids were used instead of

concrete vats or earthenware containers (Abdul Karim, 1993).

Pekasam is made from fermenting freshwater fish with roasted rice, tamarind and salt

which is usually consumed deep-fat fried or as a side-dish. Marine fish could also be used as raw

material. It is prepared by cleaning the fish, mixing with salt (20-50%) then left overnight,

mixing with roasted rice (50% w/w) and some tamarind after draining, and packed tightly in

earthenware or similar containers for fermentation of 2-4 weeks. Roasted rice aids in masking

the fishy odor, promotes development of the characteristic color of pekasam, and serves as the

source of carbohydrates for the growth of Lactobacilli. During fermentation, there’s an

outgrowth of lactic acid bacteria which lowers the pH, thus, together with the presence of salt,

preserving the product. Organic acids produced, particularly lactic acid, also aid in the flavor

development of pekasam. Fish protein is also broken down into peptides and amines during the

13

fermentation process, where together with acids and other microbial fermentation products, leads

to the development of the typical odor and flavor of pekasam (Abdul Karim, 1993).

Belacan is made from small shrimps (Acetes and Mysid sp.) which is prepared by mixing

shrimps with salt (10-15% w/w), sun-dried (5-8 hours) on mats until 50% moisture content is

reached, minced or pounded into blocks or paste in wooden tub or a similar container, then

fermented for 7 days. Thereafter, the paste is broken down again to small pieces and sun-dried

further for 5-8 hours followed by second mincing, packing tightly into balls or other desired

shape, and then fermented again for a month. These drying, mincing and fermenting processes

can be repeated many times when needed. The finished product is grounded and packed into

desired sizes and shape which usually has a dark color, salty taste and strong shrimp odor. Abdul

Karim (1993) cited that the bacteria involved in belacan fermentation were Bacillus,

Pediococcus, Lactobacillus, Micrococcus, Sarcina, Clostridium, Brevibacterium,

Flavobacterium and Corynebacterium where the predominant bacteria were lactic acid bacteria,

Micrococcus, Bacillus and high salt tolerant species.

Cincaluk is a fermentation product of small shrimps (Acetes sp.) with salt and cooked

rice. It has a pale pinkish color, strong characteristic flavor and salty taste. It is manufactured by

washing the shrimps first in seawater, draining and mixing with salt (20-25%) and cooked rice

(6% w/w weight), packing in covered earthenware or suitable containers, and then a

fermentation of 20-30 days until pink-colored shrimp is achieved. The locals usually use

cincaluk as dips, sauce, and flavoring ingredient, and consumed it with rice (Abdul Karim,

1993).

Table 4. Composition of Fermented Products in Malaysia

Components Budua Pekasam

b Belacan

c

Moisture (g/100g sample) 54.8-76 57.0-73.0 27.0-40.0

Crude Fat (g/100g sample) 0.2-1 3.0-8.0 1.4-2.6

Crude Protein (g/100g sample) 5.8-11.5 15.0-25.0 28.7-40.0

NaCl (g/100g sample) 21.7-28.15 10.0-16.0 13.0-25.3

Ash (g/100g sample) 18.3-20.9 6.0-14.0 20.0-27.6*

pH 5.4-6.2 4.5-6.1 7.2-7.8 Source: aChia Joo Suan (1977) ; bZaiton (1980); cMerican et al. (1980); a,b,c cited by Abdul Karim, 1993; *including salt

14

G. Fermented fish products in Vietnam

The most important fermented fish product in Vietnam is nuoc-mam, a fish sauce It is

consumed largely in the country and mostly added to rice. It has a clear brown color, salty taste

and a distinctive meaty aroma (Beddows, 1998). It is prepared from small fishes, mainly from

clupeids and carangids like Decapterus, Engraulis, Dorosoma, Clupeodes, and Stolephorus sp.,

mixed with high amounts of salt and fermented in earthenware containers for several months

(Prajapati & Nair, 2003, Van Veen, 2012). Amino acids are responsible for the development of

the characteristic flavor of nuoc-mam. The taste-active components of nuoc-mam, as identified

by Park, et al. (2002) were glutamic and aspartic acid, threonine, alanine, valine, histidine,

proline, tyrosine, cystine, methionine, and pyroglumatic acid. Many of these components were

responsible for the umami, sweet, and overall taste of nuoc-mam. Glutamic acid contributes most

of the flavor followed by pyroglutamic acid and alanine. Table 5 shows some physicochemical

characteristics of nuoc-mam. Uchida, et al. (2004) have isolated Bacillus subtilis from

Vietnamese fish sauce while Bacillus vietnamensis sp. nov. were isolated by Noguchi, et al.

(2004).

Another fermented fish product in Vietnam is mam which is a nitrogen-rich fish paste. Its

preparation is comparable with bagoong of the Philippines except that after the removal of the

liquid (nuoc-mam), the fermented fish is coated with rice flour (thinh) and a film of sugar syrup

(chao mam) and again undergo fermentation (Chinte-Sanchez, 2008).

Table 5. Some Physicochemical Parameters of nuoc-mam

NaCl (g/100mL) 26.1

pH 5.3

Total Nitrogen (g/100mL) 2.3

Volatile Basic Nitrogen (mg/100mL) 421 Source : Taira, et al., 2007

15

H. Fermented Fish Products in Philippines

As an archipelago, the Philippines is rich in aquatic resources, particularly fish and

shellfish. Just like any other Asian country, fish fermentation is popular in the Philippines for

preservation of excess of foods brought by seasonality of the fish, and lengthening the shelf-life

of foods. The most common fish fermented products in the Philippines are fish paste (bagoong),

shrimp paste (bagoong alamang), fish sauce (patis), fermented fish-rice mixture (burong isda),

and fermented shrimp-rice mixture (balao-balao). Bagoong, bagoong alamang and patis are

produced at large scale and even exported to other countries while burong isda and balao-balao

are just popular in some parts of the country (Mabesa & Babaan, 1993).

As cited by Chinte-Sanchez (2008), bagoong and patis are manufactured in the same way

although the latter has a longer fermentation period to which fish flesh is allowed to further

disintegrate until it reaches a liquid form. However, in many cases, bagoong and patis are

processed together where the former constitutes the solid part while the latter is the liquid part

(Chinte-Sanchez, 2008). Both products are made from anchovies (Stolephorus commersonni, S.

indicus), sardines (Sardinella fimbriatan, S. longicep), roundscad (Dacapterus macrosoma),

herring (Clupeiodes lila), and mackerel (Rastrilliger neglectus). In the Philippine standard

(1984), bagoong is defined as “a mixture of salt and small fish or small shrimp, with or without

added condiments and flavoring or coloring agents, which have undergone partial or complete

fermentation”. Bagoong na isda is termed when it is prepared from fish, bagoong alamang from

shrimp, and bagoong na sisi from shellfish. Bagoong na isda has a dark grey color, pasty

consistency, and cheesy flavor with traces of fishy odor. Bagoong alamang and bagoong sisi also

have the same characteristics as bagoong na isda. Though the initial color of shrimp is pink, it

turns grayish when fermented. Thus, red food color is added to make bagoong alamang

appealing to consumers. Bagoong, unlike patis, is only hydrolyzed partially making the shape of

the raw material still distinguishable. Bagoong and patis are prepared by mixing salt to fish (1:2-

3 or 2:7 salt:fish) then putting the mixture in vats or concrete tanks, allowing them to ferment

for 30-90 days in the case of bagoong, and 6-12 months for patis at 28-32°C. Extraction of

proteins from the fish is influenced by the pH of the fermentation mixture where a maximum

extraction is obtained at pH 7-9, with a very rapid decrease from pH 6 to pH 5. Protein will also

16

precipitate when salt concentration is 20% or more. Microbial analysis of fermented fish paste

and fish sauce, cited by Chinte-Sanchez (2008), shows that non-salt-tolerant and salt-tolerant

microorganisms are present with halophilic bacteria playing the key role in the fermentation

process. Aerobic gram-positive and gram-negative microorganisms dominate in the initial stage

of fermentation. Bacillus sp. predominates throughout the fermentation period. In patis

fermentation, Bacillus coagulans, B. megaterium and B. subtilis predominate at the early stage

while Bacillus licheniformis, Micrococcus sp., Staphylococcus epidermidis and S. saprophyticus

are found at the later stage of fermentation. The total viable counts decrease rapidly up to the

sixth month of fermentation and slow decline towards the end. The chemical composition of

bagoong and patis is shown in Table 6. Chinte-Sanchez (2008) have cited the changes in pH, salt

concentration, total nitrogen, formaldehyde nitrogen, ammonia nitrogen, amino nitrogen, and

acidity of patis made from Stolephorus sp. mixed with Sardinella and Rastrilliger sp. fermented

for 1 year. The pH ranges from 5.97 to 6.5 and acidity is between 0.67-1.42% which shows no

interdependency between acidity and pH. Salt content is between 26-27% for 3-12 months of

fermentation. Total nitrogen, formol nitrogen, ammonia nitrogen, and amino nitrogen decreases

with fermentation time. There has been an increase in the amino acid content of patis made from

Stolephorus sp. from the first to sixth month of fermentation, with a slight decrease on the ninth

month, and then increases at its peak at the end of fermentation. Table 7 shows the amino acid

content of patis from Stolephorus sp. and mixed fish species at various stages and revealed that

cystine and proline were absent. Cystine is absent possibly because of oxidation or bacterial

action, while proline could have been metabolized during spoilage (Chinte-Sanchez, 2008).

Burong isda is made from mixing rice, boiled dry or cooked to a porridge-like

consistency), fish (freshwater fish, milkfish, Ophicephalus striatus, Tilapia mossambica,

Therapon plumbeus), and salt, with or without angkak (red rice), fermented for days or weeks

depending on the salt concentration. Balao-balao is made in the same way as burong isda except

that fermented shrimp of species Macrobrachium or Peneaus is mixed with rice and salt rather

than fish (Mabesa & Babaan, 1993).

17

Table 6. Chemical Composition of Bagoong and Patis (per 100g edible portion)

Component Bagoong ginamos Bagoong balatohan Bagoong padas patis

Moisture (g) 55.4 30 65.9 66.3

Fat (g) 1.8 3.2 1.7 0.3

Protein (g) 23.4 8.1 9.6 10.6

Ash (g) 19.4 32.8 22.8 21.9

Carbohydrate (g) 0 25.1 0 0.9

Calcium (mg) 821 1770 504 42

Phosphorus (mg) 510 439 435 32

Iron (mg) 8.2 11.9 16.6 9.3

Source : Chinte-Sanchez, 2008

Table 7. Amino Acid Content of Patis from dilis (mg/100mL)

Composition 6 months 12 months

threonine 306 612

isoleucine 404 600

leucine 505 601

lysine 554 1083

methionine 204 339

cystine 0 0

phenylalanine 197 365

tyrosine 31 54

valine 355 770

arginine 102 431

histidine 352 976

alanine 318 625

aspartic 586 960

glutamic 586 960

glycine 165 522

proline 0 0

serine 188 431

Source: Sanchez and Klitsaneephaiboon 1983 cited by Chinte-Sanchez 2008

18

I. Fermented Fish Paste in the Philippines

Fermented fish paste is locally known as bagoong in the country. The final product varies

from region to region. In Tagalog provinces, bagoong is completely fermented, ground, with or

without addition of coloring agent. In Ilocano and Pangasinan provinces, bagoong is partially or

completely fermented with the entire fish still intact. In Visayas or Mindanao, bagoong is just

slightly fermented and without liquid, fishes are still hard and firm and salt is still visible.

Among the types of fishes mentioned above as raw materials for bagoong making, anchovies

(locally called as dilis) are the most preferred one because it results to a product with pleasing

aroma and taste. As discussed earlier, bagoong is manufactured by mixing dry salt with fish (1:2-

3 or 2:7 salt:fish). The fish is cleaned and washed first then dried before adding the salt. The salt-

fish mixture is then placed to vats or tanks for fermentation for 30-90 days at 28-32°C (Chinte-

Sanchez, 2008). However, the process is done traditionally and varies between processors. Some

processors doesn’t wash the fish anymore, especially those freshly harvested fish, and drain the

salt-fish mixture before placing into large plastic drums rather than concrete tanks.

Bagoong is primarily used as a condiment and in some places as a staple food. It contains

8-25% protein making it a good source of protein (Mojica, et al., 2005). Mojica, et al. (2005)

also studied the effect of irradiation in production of fermented fish paste from dilis. Their results

showed that non-irradiated fermented fish paste attains a total plate count of 1.2x102 cfu/g while

3.6x10-1

cfu/g and 9x10-1

cfu/g were obtained from 3kGy and 10kGy irradiated fish paste

respectively. Table 8 shows the different chemical composition of bagoong dilis.

Microbial interaction in bagoong fermentation involves gram-negative rods as initial

flora of fish-salt mixture from fish itself and handlers which were inhibited immediately upon the

addition of salt due to water extraction by osmosis. The halophilic bacteria, in viscera and gills of

fish and those introduced with salt, increase rapidly due to nutrient availability in the brine.

Protein hydrolysis and production of flavors are caused by enzymatic action (endogenous)

produced from Bacillus subtilis and B. coagulans. The following micoorganisms, and the

enzymes they produced, are responsible for fat oxidation leading to the formation of volatile

fatty acids : Bacillus licheniformis, Micrococcus colpogenes, and Staphylococcus epidermidis

19

found at the middle stage of fermentation; Micrococcus roseus, M. varians, and Staphylococcus

saprophyticus at the later stage; and Bacillus pumilus dominating throughout the fermentation

process (Chinte-Sanchez, 2008). It was also cited by Banaay, et al., (2013) that the lactic acid

bacteria, Pediococcus halophilus, was involved in bagoong fermentation.

Table 8. Chemical Composition of Bagoong dilis (per 100g edible portion)

Moisture (g) 67.1

Protein (g) 10.3

Fat (g) 1.9

Ash (g) 20.7

Calcium (mg) 535

Phosphorus (mg) 313

Iron (mg) 10.9

Source: Chinte-Sanchez, 2008

20

Chapter 3. Materials and Methods

A. Collected Samples from the Philippines

Raw anchovies and salt-fermented anchovy pastes weighing 250 grams each, and liquid

extracts at different time points (0, 9, 19 and 28 days) were obtained from a local producer in the

province of Misamis Oriental, Philippines. Three different batches were sampled. Also three

finished products locally sold in the market were purchased from the province of Surigao,

Philippines.

Anchovies that were used for the preparation of the different salt-fermented fish belonged

to the Stolephorus species (figure 1). Traditional way of fermentation is done by placing the

anchovies in a fine mesh strainer to drain the excess of sea water and to remove visible debris.

Still in the strainer, anchovies are mixed with salt in a ratio of 1:3-4 (salt:fish) until the fish

becomes not slimy anymore and left for 30-45 minutes to drain the liquids leached from the fish.

The mixture is then transferred to clean plastic drums covered first with plastic cellophane, then

followed by the cover of the drum. It is then stored at a closed room with a temperature of 28-

31°C for fermentation which takes place for a period of weeks to months. Products can already

be sold to consumers after one week, but mostly, they are sold after at least one month of

fermentation.

Sampling was done from July 16th to August 13th, 2013. All collected samples were

stored under low temperature conditions (-18°C) and preserved at ± 4°C for transportation to

Belgium where they were stored at -20°C until analyses. Raw anchovies and salt-fermented

anchovy pastes were homogenized first before being analyzed.

Figure 1. Raw material for salt-fermented anchovy paste

21

B. Gross Composition

a. Dry Matter Content (ISO 1442-1973)

Material:

Drying oven at 103±2°C

Analytical balance

Desiccator

Preheated sea sand

Aluminum foil cups

Raw anchovies samples or salt-fermented anchovy pastes

Reagents:

99% ethanol

Procedure:

To aluminum foil cups, 15 grams of sea sand was added. The cups were preheated in the

oven at 103°C for one hour, then they were cooled down in a desiccator for at least 45 minutes

and weighed (=M0). To the cups, 5 grams of raw anchovies or salt-fermented anchovy pastes

were added and weighed again (=M1). Samples were then mixed with 5mL of ethanol and placed

in the oven for 3 hours after which they were cooled down in a desiccator for at least 45minutes

and weighed (=M2). Dry matter content was calculated as:

𝐷𝑀 = (𝑀2 − 𝑀0)

(𝑀1 − 𝑀0) 𝑥 100

Where:

DM = dry matter content (g/100 gram sample)

M0 = mass of the preheated sea sand in the aluminum cup (g)

M1 = mass of sand and sample in the aluminum cup before drying (g)

M2 = mass of sand and sample in the aluminum cup after drying (g)

b. Crude Protein Content by Kjeldahl Method (ISO 937-1978)

Material:

Analytical balance

Nitrogen-free paper

Destruction tubes

Digestion system (Büchi)

Distillation unit (Büchi)

Burette

22

Conical flasks (250 mL)

Raw anchovies samples or salt-fermented anchovy paste

Reagents:

Kjeldahl tablets (consisting of 235g Na2SO4, 4g CuSO4.5H2O and 5g selenium powder)

98% Concentrated sulfuric acid (H2SO4)

Tashiro color indicator (1g methyl red and 0.5g methylene blue dissolved in 500mL

ethanol)

Phenolphthalein color indicator

32% Concentrated sodium hydroxide (NaOH)

0.16M Boric acid (10g H3BO3 added with 0.4L distilled water and 0.2L ethanol for 1 liter

solution)

0.1M Hydrochloric acid (HCl)

Procedure:

On a nitrogen-free paper, 1 gram of sample was weighed and placed in a destruction tube,

followed by adding a Kjeldahl tablet and 20mL of H2SO4. The mixture was heated in the

destruction chamber for 1 hour until a clear green solution was obtained. Destruction tubes were

cooled down, after which 50mL distilled water and 4 drops of phenolphthalein indicator were

added. After cleaning the distillation unit with distilled water, the tubes were attached. To each

tube, (32%) NaOH was supplied until the solution turned dark red to brown. A conical flask with

50mL boric acid and 4 drops of tashiro indicator was placed in the outlet of the distillation in

which the distillate was collected. Each sample was distilled for exactly 4 minutes. The purple

boric acid solution turned from purple to gray to green color. The flask with the distillate was

then titrated with 0.1M HCl until the color turned back to purple. The amount of HCl titrated was

recorded. Crude protein content was calculated as:

𝐸 = ((𝑉 − 𝑉𝑏) ∗ 14 ∗ 𝑁 ∗ 6.25

𝑉𝑠 ∗ 𝐷𝑀 ∗ 1000) ∗ 100

Where:

E = crude protein content (g/100gDM)

V = volume of HCl titrated for the sample (mL)

Vb = volume of HCl titrated for the blank (mL)

Vs = weight of the sample (g fresh sample)

N = normality of HCl (mol/L)

14 = molecular weight of nitrogen (g/mol)

6.25 = protein conversion factor

DM = dry matter content of sample (g/100g fresh sample)

23

c. Crude Fat Content by Soxhlet Method (ISO 1444-1973)

Material:

Extraction thimbles

Aluminum foil cup with dried sample (obtained by dry matter determination ISO

1442-1973)

Cotton wool

Soxhlet apparatus

Extraction flasks

Analytical balance

Drying oven 103±2°C

Desiccator

Reagents:

Petroleum ether (bp 40-60°C)

Procedure:

The extraction flasks were first dried at 105°C for 1 hour and were cooled down afterwards

in a desiccator for at least 45 minutes and then weighed (=K1). The aluminum foil cups with

dried samples, obtained by dry matter analysis (ISO 1442-1973), were placed in the extraction

thimbles and covered with cotton wool. In the Soxhlet apparatus, the thimble was placed in the

thimble holder to which petroleum ether was added in such a way that the extraction flask

contained two times the volume of the thimble holder. The apparatus was heated for 6 hours with

petroleum ether. Normally all the fat was extracted after 6 hours as seen at the bottom of the

extraction flask. Petroleum ether was collected and removed until all the extracted fat remained

in a minimum amount of petroleum ether. Extraction flasks were then dried at 103°C for 1 hour

after which they were cooled down in a desiccator and weighed (=K2). Crude fat content was

calculated using the formula:

𝑉 = [(𝐾2 − 𝐾1

𝑀1 − 𝑀0) 𝐷𝑀⁄ ] 𝑥 100

Where:

V = fat content (g/100gDM)

K1 = weight of the empty flask (g)

K2 = weight of the flask with extracted fat (g)

M0 = mass of the preheated sea sand (g) from the dry matter analysis

M1 = mass of sand and sample before drying (g) from the dry matter analysis

DM = dry matter content of sample (g/100g fresh sample)

24

C. pH (Bendall, 1978)

Material:

Ultra Turrax

pH meter (Consort C830)

Plastic sample cups

Raw anchovies samples or salt-fermented anchovy pastes

Reagents:

Bendall solution [9.3mg iodoacetic acid (C2H3IO2), 2mL of 0.1M NaOH and 11.175g

potassium chloride (KCl) were added, diluted to 1L and adjusted to pH 7]

Procedure:

In a plastic sample cup, 5mL of chilled (0°C) Bendall solution was added to 1g of sample

and homogenized for 15 seconds using an Ultra Turrax (13500rpm). The pH of the homogenate

was measured using a pH meter where the temperature of measurement was set to 0°C. The

measurement was done in duplicate.

D. Water Activity (Aw)

Material:

Aqualab, Series 4TE Decagon Devices SN 540001787

Incubator at 22°C

Plastic sample cups

Raw anchovies samples or salt-fermented anchovy pastes

Procedure:

The Aw value was measured using the chilled mirror dewpoint technique at 25°C.

Samples were thawed first and then incubated at 22°C for at least 30 minutes to lower the

temperature difference between the sample and the instrument. Around 80% of the sample cup

was filled with the sample which was then placed in the sealed chamber of the Aqualab

equipment containing a mirror. When the mirror cooled down, a photoelectric cell detected the

change in reflectance from the time when condensation occurred on the mirror. After the sample

and the head-space of the sealed chamber reached the equilibrium, the relative humidity of the

air in the chamber was measured, which also corresponds to the water activity of the sample. At

equilibrium, the temperature was recorded and finally the Aw value was calculated by the

Aqualab equipment using the vapor pressure chart. All samples were measured in duplicate.

25

E. Mineral Content

Material:

ICP-AES (Inductively Coupled Plasma - Atomic Emission Spectroscopy) – VARIAN

VISTA-MPX Muffle oven

Crucibles

Filter paper

Volumetric flasks

Raw anchovies samples or salt-fermented anchovy pastes

Reagents:

70% Concentrated nitric acid (HNO3)

Doubled distilled water (DDW)

Blank (1% Nitric acid solution)

ICP multi-element standard solution (1000mg/l)

Working standards (10, 20, 50, 100, 200 mg/l standards prepared by using the ICP multi-

element standard)

Procedure:

In dried crucibles, 5 grams of samples were weighed. Samples were then burned using a

Bunsen burner to eliminate most of the organic matter. Crucibles were then placed in the muffle

oven at 600°C for at least 17 hours until white ashes were obtained. They were then taken out of

the muffle oven and cooled down at room temperature. After cooling, samples were dissolved in

5mL nitric acid and filtered in a 10mL volumetric flask. Crucibles were washed with DDW and

again poured on the filter. More DDW was added to the flask until the mark and this for the

determination of microelements copper, iron, zinc and manganese. Samples were further diluted

up to 1000 times for the determination of macroelements calcium, magnesium and potassium;

and 2000 times for sodium. The blank, working standards and samples were then analyzed using

ICP-AES containing a plasma and auxiliary flow of 15L/min and 1.5L/min respectively. The

pump rate was 15rpm and the nebulizer pressure was set at 1kPa. Absorbance was measured for

each element at 5 different wavelengths. Mineral content were calculated using the formula:

𝐶𝑜𝑛𝑐𝑚𝑎𝑐𝑟𝑜 =(𝑐 ∗ 𝑑)/1000

𝑉𝑠 ∗ 𝐷𝑀

𝐶𝑜𝑛𝑐𝑚𝑖𝑐𝑟𝑜 =(𝑐 ∗ 𝑑) ∗ 1000/1000

𝑉𝑠 ∗ 𝐷𝑀

Where:

Concmacro = concentration of each macroelement (mg/g DM)

26

Concmicro = concentration of each microelement (µg/g DM)

c = calculated concentration by ICP instrument (mg/L)

d = dilution factor (mL)

Vs = weight of the fresh sample (g)

DM = dry matter content of sample (g/100g fresh sample)

F. Salt Content

Salt content (NaCl) was calculated from the concentration of sodium (Na

+) obtained by

mineral analysis with ICP-AES using the formula:

𝑁𝑎𝐶𝑙 = 𝑎 𝑥 𝑏

𝑐

Where :

NaCl = concentration of NaCl (g/kg dry matter sample)

a = molar mass of NaCl (58.5 g/mol)

b = concentration of Na (mg/g dry matter sample)

c = molar mass of Na (23 g/mol)

G. Fatty Acid Composition

a. Anchovy Paste Fatty Acids Extraction (Folch et al., 1957)

Material:

Glass extraction tubes with screw caps

Analytical balance

100mL Volumetric flasks

Filters

Test tubes with screw caps

Centrifuge

Jet filter pump

Separatory funnels

Rotavapor flasks

Rotating evaporator

Raw anchovies samples or salt-fermented anchovy pastes

Reagents:

Chloroform/Methanol solution (C/M) – 2:1 v/v

0.1% Butylhydroxytoluene (BHT) in chloroform (1g BHT in 1L chloroform)

Chloroform

27

Procedure:

In an extraction tube, 5 grams of frozen sample was weighed to which 25mL C/M

solution and 3mL of 0.1% BHT solution were added. Samples were homogenized for 1 minute

using an Ultra Turrax (13500rpm). The bar of the Ultra Turrax was washed with 10mL C/M

solution which was collected in the extraction tube. The homogenate was left overnight at room

temperature in a dark area. In a 100mL volumetric flask, the homogenate was filtered and

collected. The empty extraction tube, which contained the homogenate, was washed with 10mL

C/M solution twice and poured on the same filter. The filter was rinsed after draining with 5mL

C/M. After 10 minutes, the filtrate was divided into two test tubes containing 15mL of distilled

water each. The volumetric flask was washed with 5mL C/M and the solution was placed in one

of the 2 test tubes. Tubes were then centrifuged at 3000rpm for 10 minutes. Afterwards, the

upper layer of the solution was removed by a water jet filter pump. The remaining content of

both tubes was transferred to one separatory funnel with a glass funnel. The tubes were washed

with 5mL C/M each. Two separated layers were noticeable after 20 minutes and the bottom layer

was collected in a rotavapor flask. The glass funnel and inside of the separatory funnel were

washed with 5mL C/M each. After 20 minutes, to obtain a separation, the bottom layer was again

collected in the rotavapor flask. The extracts were evaporated with the rotating evaporator (water

bath: 40°C, 100rpm). Subsequently, the fat was re-dissolved in 10mL chloroform and put in test

tubes which were stored at -20°C.

b. Fatty acid Methylation (Raes et al., 2001)

Material:

Test tubes

Warm water bath at 50°C

Vortex

Centrifuge

Tip tubes

Pasteur pipette

Evaporator under nitrogen

Reagents:

0.5N NaOH in methanol (20g NaOH dissolved in 1mL MeOH)

HCl in MeOH (0.5L HCl mixed in 0.5L MeOH)

Internal standard (IS) solution (2mg C19:0 per mL hexane)

28

Procedure:

Previously stored extracts were allowed to warm up to room temperature. Then 1mL

extract and 1mL IS were placed in test tubes. The solution was evaporated under nitrogen after

which 3mL of 0.5N NaOH/MeOH was added. After stirring the test tubes with a vortex, the

samples were kept at 50°C for 30 minutes. Afterwards, 2 mL HCl/MeOH was added, vortex and

then kept again at 50°C for 10 minutes. The tubes were shaken and cooled down to room

temperature. Fatty Acid Methyl Esters (FAME) were extracted by the addition of 2mL hexane

and 2mL distilled water to the cooled solution which was then stirred with a vortex and

centrifuged at 2000rpm for 5 minutes. Using a Pasteur pipette, the upper layer was removed and

transferred to a tip tube. Again, 2mL of hexane was added to the remaining solution which was

then stirred with a vortex and centrifuged. The upper layer was removed again and transferred to

the same tip tube by a Pasteur pipette. Hexane was evaporated under nitrogen and finally, FAME

were re-dissolved in 1mL hexane and transferred to vials which were stored at -20°C.

The FAME were analyzed using Gas Chromatography with a column of HP88 Agilent

(60mx0.25mmx0.2µm) and FID detector. The injector and detector temperature, flow rate over

column and injection volume were 250°C, 280°C, 2mL/min and 1µL respectively. The initial

temperature of the column was 120°C which was held for 1 minute and then increased to 175°C

at a rate of 6°C/min. The temperature was again increased to 210°C for 6.5 minutes which was

further increased at a rate of 5°C/min to 230°C. Holding time at 230°C was 5 minutes. Fatty acid

proportion was determined by the formula:

𝑃𝑟𝑜𝑝 = (𝑋

𝑌) ∗ 100

Where:

Prop = proportion of each fatty acid (g/100g FAME)

X = peak area of the specific fatty acid

Y = total amount of area of known and unknown fatty acids (excluding BHT and IS)

29

H. Total and Free Non-Protein Nitrogen Content (Oddy, 1974)

Material:

Ultra Turrax

Filter paper

Boiling water bath

Drying oven at 103±2°C

Spectrophotometer

Sample cups

Raw anchovies samples or salt-fermented anchovy pastes

Reagents:

0.6M Perchloric acid (HClO4) [103mL concentrated HClO4 (70%) diluted to 2L]

8.4M HCl solution

15M NaOH solution

2M NaOH solution

Buffer solution pH 5.8 [mixture of 250mL propionic acid (C3H6O2), 250mL of 2-

methoxyethanol (C3H8O2), 350mL distilled water, and 100mL 15M NaOH,

diluted to 1L]

Ninhydrin solution [5g ninhydrin (C9H6O4) dissolved in 1L buffer solution]

Reductant solution [50mg ascorbic acid (C6H8O6) dissolved in 50ml distilled water]

Leucine stock solution [74.8mg L-leucine (C6H13NO2) dissolved in 100mL distilled

water]

Standard series [0.5, 1.0, 2.0, 3.0, 4.0 and 5.0mL of stock solution diluted to 100mL

distilled water]

60% ethanol solution

a. Perchloric Acid Extraction of Anchovy Paste Samples

Procedure:

In a sample cup, 5g of sample with 40mL of 0.6M perchloric acid (PCA) were

homogenized using an Ultra Turrax at 8000rpm for 1 minute. The bar of the Ultra Turrax was

washed with 10mL of PCA which was collected in the sample cup. The suspension was filtered

with filter paper over a 100mL volumetric flask and the container was rinsed with 30mL PCA

which was again filtered over the flask. PCA was added until the mark. The extracted samples

were stored at -20°C for free and total non-protein nitrogen analysis.

30

b. Total Non-Protein Nitrogen

Procedure:

From each sample, 2 mL of PCA extract was transferred to a test tube to which 5mL of

8.4M HCl was added. Then the solution was mixed, covered and put in the oven (103°C) for 24

hours. After hydrolysis, the samples were neutralized with 2M NaOH to attain a pH between 4

and 10. Samples were then transferred to 50mL volumetric flask and then diluted with distilled

water until the mark. From each sample, 1mL of diluted (100 times with buffer solution)

hydrolyzed PCA extract was transferred in duplicate to test tubes. From each standard solution, 1

ml was transferred to test tubes. Distilled water was used as a blank. To each test tube, 100µL

reductant solution and 1mL of ninhydrin solution were added after which solutions were stirred

using a vortex. Tubes were covered with aluminum foil and placed in a boiling warm water bath

for 20 minutes. After cooling down the tubes with tap water, 5mL of ethanol solution was added,

after which samples were stirred using a vortex. After 15 minutes, the mixture was stirred again

and the absorbance was measured at 570nm. Total NPN was calculated using the following

formula:

𝑇𝑜𝑡𝑎𝑙𝑁𝑃𝑁 =𝑥∗𝑑∗50∗ 100∗100

𝑉𝑠∗𝐷𝑀∗1000

Where:

Total NPN = total α-NH2-N content in dry matter (mg/100gDM)

X = amount of total α-NH2-N obtained from a calibration curve (µg/mL)

d = dilution factor (mL)

Vs = weight of fresh sample used in PCA extract (g)

DM = dry matter (gDM/100g fresh sample)

c. Free Non-Protein Nitrogen

Procedure:

Free NPN was analyzed on the same way as the total NPN except for the hydrolysis part.

From each sample, 1mL of diluted (100 times with buffer solution) PCA extract was transferred

in duplicate to test tubes. From each standard solution, 1 ml was transferred to test tubes.

Distilled water was used as a blank. To each test tube, 100µL reductant solution and 1mL of

ninhydrin solution were added after which samples were stirred using a vortex. Tubes were

covered with aluminum foil placed in a boiling warm water bath for 20 minutes. After cooling

31

down with tap water, 5mL of ethanol solution was added and samples were stirred using a

vortex. After 15 minutes, the mixture was stirred again and the absorbance was measured at

570nm. Free NPN was calculated using the following formula:

𝐹𝑟𝑒𝑒𝑁𝑃𝑁 =𝑥∗𝑑∗100∗100

𝑉𝑠∗𝐷𝑀∗1000

Where:

Free NPN = free α-NH2-N content in dry matter (mg/100gDM)

X = amount of free α-NH2-N obtained from calibration curve (µg/mL)

d = dilution factor (mL)

Vs = weight of fresh sample used in PCA extract (g)

DM = dry matter (gDM/100g fresh sample)

I. TBARS Analysis (Tarladgis, et al., 1964)

Material:

Distillation apparatus (tubes and distillation unit)

Ultra Turrax

Spectrophotometer

Boiling warm water bath

100mL Scott bottle

100mL Volumetric flask

Raw anchovies samples or salt-fermented anchovy pastes

Reagents:

TBA reagent [865 mg 2-thiobarbituric acid placed in a 100mL volumetric flask, covered

with aluminum foil, was dissolved with 75mL of acetic acid in a hot plate

at 70°C. After cooling down at room temperature, 2mL of concentrated

HCl (37%) was added and filled up to the mark with acetic acid.]

BHT solution [1.5g of 2,6-di-tertiary-butyl-4-methylphenol dissolved with ethanol in a

100mL volumetric flask and stored in a dark bottle at room temperature]

4M HCl solution

Standard stock solution [14.4µL of 1,1,3,3-tetramethoxypropane dissolved in 100mL

distilled water]

Standard working solution [20 times dilution of the stock solution (1mL standard stock

solution dissolved in 20mL distilled water)]

32

Standard series :

concentration (nmol/5mL) µL working solution mL water

(blank) 0.0 0.0 5.00

2.19 50 4.95

4.37 100 4.90

8.74 200 4.80

13.12 300 4.70

17.49 400 4.60

21.86 500 4.50

Procedure:

In a 100mL scott bottle, 10g of sample was weighed and then 40mL distilled water and

1mL BHT solution was added. The sample was homogenized using an Ultra Turrax (13000rpm)

for 30 seconds and then transferred to the distillation tube. The bottle was again added with

30mL of distilled water and homogenized for a while with Ultra Turrax to rinse its dispersing

unit, and then transferred again to the distillation tube. To the distillation tube containing the

homogenate, 3mL of 4M HCl was added. After cleaning the distillation unit with distilled water,

the tube was attached. Distillate was collected in a 100mL volumetric flask until the mark. From

the collected distillate, 5mL was transferred to test tubes in duplicate. Also, 5ml standard

solutions were transferred in tubes. To the tubes with sample distillates, or with standard

solutions, 1mL TBA reagent was added, then stirred in a vortex and put in a boiling warm water

bath for 35 minutes. After cooling down the tubes at room temperature with tap water, the

absorbance was measured at 532nm. Samples were diluted 3 times before the absorbance was

read. The concentration of TBARS was calculated with the formula:

𝑇𝐵𝐴𝑅𝑆 = 𝑐 𝑥 20 𝑥 𝑑 𝑥 72

𝑚 𝑥 𝐷𝑀∗1000

Where:

TBARS = TBARS content (µg malonaldehyde/gDM)

c = calculated concentration of TBARS obtained from the calibration

curve (nmol malonaldehyde/5mL distillate of sample)

d = dilution factor (mL)

72 = molar mass of malonaldehyde (µg/µmol)

m = weight of fresh sample (g)

DM = dry matter (gDM/100g fresh sample)

33

J. Microbiological Analysis

Materials:

Crushed ice

8mL sterile Glycerol

10mL sterile Brain Heart Infusion (BHI) medium

9mL physiological water

Plate Count Agar (PCA)

de Man, Rogosa Sharpe (MRS) Agar (MRS Broth added with Bacteriological Agar)

Nutrient Agar (Nutrient Broth added with Bacteriological Agar)

Malt Extract Agar (Malt Extract Broth added with Bacteriological Agar)

Violet Red Bile Glucose Agar (VRBGA)

Sodium Chloride (NaCl)

Potassium Sorbate

Casein

Incubators

Test tubes

Plates

Drigalski spatula

Ethanol

Procedure:

a. Sampling:

From the liquid part of the fermented fish products collected, 2mL was taken and placed

into falcon tubes containing 8mL glycerol. These glycerol stocks were transported from the

Philippines to Belgium with the same procedure done with the collected fermented anchovy

pastes and stored at -20°C upon arrival at UGent Kortrijk Campus laboratory until analyzed.

b. Microbial Analysis

Glycerol stock was thawed under chilled condition with crushed ice until liquid becomes

less viscous. The glycerol stock was vortex and 250µL of the glycerol stock was transferred to

BHI medium, and then incubated for 4 hours at 30°C. Dilution series until 10-5

of the BHI-

cultured broth was made. From the cultured broth (100) and dilutions 10

-1, 10

-3 and 10

-5, 0.1mL

was taken and plated (spread plate) on the different agar plates. Total aerobic plate count was

analyzed using PCA and incubated for 48 hours at 37°C, while for the total halophilic plate count

PCA with 10% NaCl was used and incubated for 14 days at 37°C. For the total lactic acid

34

bacteria, MRS agar with 1.4g/L potassium sorbate was used and incubated for 72 hours at 30°C.

Total halophilic lactic acid bacteria was analyzed using the former medium added with 10%

NaCl and also incubated for 72 hours at 30°C. For the total proteolytic bacteria, nutrient agar

supplemented with 10% NaCl and 1% casein was used and incubated at 37°C for 48 hours.

Halophilic yeast counts were analyzed on Malt extract agar added with 10% NaCl, and at pH of

4.8 and incubated at 25°C for 72 hours. Total enterobacteriaceae were analyzed with VRBA and

incubated for 24 hours at 37°C. Media used were sterilized first before putting into plates except

for the VRBA. All the visible colonies were counted and represented as colony forming units

(CFU). The number of microorganisms were calculated using the formula:

𝑋 = 𝐴 𝑥 𝑉

𝑙

Where :

X = number of microorganisms (CFU/mL)

A = number of colonies on the plate (CFU)

V = reciprocal of the dilution factor

l = volume of inoculum (mL)

K. Statistical Analysis

Data were analyzed statistically using SPSS Statistics 21 software (IBM Corporation,

2012). Normality of data distribution and equality of variance were considered using

Kolmogorov-Smirnov and Modified Levene tests. One-way ANOVA (parametric) and Kruskal

Wallis (non-parametric) tests were used for hypothesis testing. Significant differences between

the results from the different time points and producers of anchovy paste were determined. In

conjunction with ANOVA, Tukey test was used to find which means were significantly different

from each other. All the used statistical tools were set at 5% level of significance.

35

Chapter 4. Results and Discussion

A. Raw materials

Salt-fermented anchovy paste samples were traditionally produced. Three different

batches from one local producer (SP) were sampled for different time points (T) of fermentation

(raw fish =R; day0=D0; day9=D9; day19=D19; day28=D28) (figure 2). Three locally salt-

fermented anchovy pastes, sold in markets (FP) were also purchased (figure 3). Descriptions of

the samples are presented in table 9. Names of the producers and their years of experience in

making bagoong are also included.

Table 9. Characterization of the samples and manufacturers

Name of Manufacturer Date/Time

Manufactured Region of Origin

Other

Characteristics

SPA. Gimaylan Salted Fish

Processors Organization (6 years experience)

July 2013

8AM

Misamis Oriental

1:3.6 salt:fish

ratio

fish used was

caught on the previous day and

stored at -10°C

before processing

unwashed fish

liquid drained

SPB. Gimaylan Salted Fish Processors Organization

(6 years experience)

July 2013

10AM

1:3.6 salt:fish

ratio

used freshly

caught fish from local fishermen

unwashed fish

liquid drained

SPC. Gimaylan Salted Fish Processors Organization

(6 years experience)

July 2013

1PM

1:3.6 salt:fish

ratio

fish used from earlier catch of

local fishermen

unwashed fish

liquid drained

FPA. Escalona (20 years experience)

May 2013 Surigao del Norte

salt and fish ratio

not specified

FPB. Millan

(5 years experience) July 2013 Surigao del Sur

FPC. Durando (17 years experience)

April 2013 Surigao del Sur

36

Figure 2. Fermented anchovy pastes at different time points

Figure 3. Fermented anchovy pastes locally sold in markets

37

B. Biochemical and chemical composition of commercially available anchovy pastes

a. Chemical Composition

Gross composition of the different purchased anchovy pastes are given in table 10. Dry

matter content of FP varied between 36-40g/100g and no significant differences between

samples were observed (p˃0.05). The DM content of the samples is slightly higher than the

typical anchovy paste (bagoong dilis) mentioned by Chinte-Sanchez (2008) which has a value of

32.9g/100g. It is also within the range of fish pastes reported by Montaño, et al. (2001) from dilis

available in a main public market in the northern part of the Philippines, which has a mean value

of 39.2g/100g, and within the Philippine Standards for bagoong which was 40g/100g (Chinte-

Sanchez, 2008). However, it has lower values compared to nga-pi of Myanmar which is

60g/100g (Tyn, 1993), terasi and pedah of Indonesia which is 50-65 and 53-56g/100g

respectively (Putro, 1993; Aryanta, 2000), and belacan of Malaysia which has 60-73g/100g of

DM content.

Crude fat content of FP varied from 4-6g/100gDM which is similar to what Chinte-

Sanchez (2008) has stated with the value of 5.78g/100gDM. Also, it has higher levels than the

one obtained by Montaño, et al. (2001) which has 1.53g/100gDM, fish pastes from Myanmar

containing 2.5g/100gDM (Tyn, 1993), and fish paste from Malaysia with 1.9-4.3g/100gDM of

fats (Abdul Karim, 1993). However, it has lower values compared to pedah which is

20.86g/100gDM (Putro, 1993) but just of the same range with terasi from Indonesia which has a

value of 2.5-6.4g/100gDM (Aryanta, 2000). The analyzed fat content of FP showed no

significant differences (p˃0.05) between the different producers.

There were also no significant differences (p˃0.05) observed between crude protein of

FP, varying between 34-44g/100gDM. These levels are a bit higher to the Philippine Standards

for Bagoong which is 31.25g/100gDM, and as mentioned by Chinte-Sanchez (2008) which is

31.31g/100gDM. It is also higher than nga-pi (30g/100gDM) of Myanmar (Tyn, 1993). On the

other hand, these levels are comparable with terasi and belacan of Indonesia and Malaysia (31-

69 and 39.3-66.7g/100gDM respectively) (Aryanta, 2000; Abdul Karim, 1993).

38

Table 10. Chemical composition of anchovy paste traditionally produced in some parts of the

Philippines

Sample

FPA FPB FPC p-value

DM (g/100g) 35.9±0.54 40.4±1.33 40.2±1.57 0.060

Crude Fat (g/100gDM) 6.47±1.92 4.55±0.38 4.45±0.05 0.240

Crude Protein (g/100gDM) 39.4±1.45 43.8±4.3 34.4±4.58 0.180

Aw 0.80±0.001 0.75±0.002 0.74±0.002 0.102

pH* 5.33±0.11a 6.39±0.02

b 6.55±0.14

c 0.007

NaCl (g/100gDM) 37.0±10.8 46.3±3.28 34.6±4.29 0.341

Results expressed as mean ± standard deviation; n=2;*-n=4

Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

Other parameters analyzed were water activity (Aw), NaCl content and pH (table 10). The

Aw of FP ranged between 0.74-0.80. It coincides to the studies of Sumino, et al., (1999,2003) on

bagoong which has an average Aw of 0.77 and belacan of Malaysia with an average Aw of 0.74.

On the other hand, terasi of Indonesia and kapi of Thailand got an average Aw of 0.718 and 0.71

respectively(Sumino, et al., 1999). There were no significant differences (p˃0.05) in Aw for the

different producers of FP. NaCl content of FP varied between 34-46g/100gDM and no

significant differences (p˃0.05) were observed between different producers. These values were

lower than the one obtained by Montaño, et al., (2001) which is 59.69g/100gDM, and the

Philippine Standards for Bagoong which is 50-62.5g/100gDM (Chinte-Sanchez, 2008).

However, the analyzed salt content is in agreement with terasi of Indonesia as reported by

Aryanta (2000) which contains 23-40g/100gDM, belacan of Malaysia (Abdul Karim, 1993)

containing 18-42g/100gDM, and nga-pi of Myanmar with 42g/100gDM of salt (Tyn, 1993). The

pH of FP varied from 5.3-6.6 and was significantly different (p<0.05) from each other. It is in

agreement with budu (fish sauce from anchovies) of Malaysia which has a pH of 5.4-6.2 (Abdul

Karim, 1993). In contrast, the belacan (from shrimp) of Malaysia and terasi (from fish and/or

shrimp) of Indonesia has a rather neutral pH of 7.2-7.6 and 7.5 respectively (Abdul Karim, 1993;

Surono & Hosono, 1994). The pH of fermented fish varies depending on the biological

39

properties of fish. Furthermore, different species of fish affects its biological properties resulting

to production of different kinds of peptides and amino acids (Ng, et al., 2011). This could be the

reason why the reported pH values of belacan and terasi differs with the sampled anchovy

pastes. On the other hand, budu, which is a fish sauce, acquired same pH level due to the fact

that it was also produced from anchovies. The pH of FP, though from the same raw materials,

also varies from each other because of their difference in the ripening period. FPA, which is 3-

month old, has lower pH than FPB which is fermented only for 1 month. The decrease in pH is

due to the production of acids (Kilinc, et al., 2005; Ng, et al., 2011). Moreover, as fermentation

continues and protein hydrolysis starts, proteins are degraded resulting to increase in pH due to

the production of N-compounds (Kilinc, et al., 2005; Ng, et al., 2011). Such N-compounds

produced are ammonia and trimethylamine (TMA) which have basic properties that will react

with acidic compounds forming a more stable compound (Ng, et al., 2011).

b. Mineral Composition

Mineral content of the product is shown in table 11. Minerals are divided into micro- and

macroelements. In this study, microelements analyzed include copper (Cu), iron (Fe), manganese

(Mn) and zinc (Zn). On the other hand, macroelements analyzed were magnesium (Mg),

potassium (K), calcium (Ca) and sodium (Na). Microelements analyzed resulted to non-

detectable values for Cu and Mn while Fe and Zn varied between 15.8-18.9 and 5-28µg/gDM

respectively. No significant difference (p˃0.05) was observed in Fe while Zn content of FPA was

significantly lower compared to FPB and FPC (p-value<0.05). For the macroelements, values

obtained were between 1.25-11, 12-16, 36-68 and 136-182mg/gDM for Mg, K, Ca and Na

respectively. There was no significant difference (p˃0.05) between producers for K and Na

analyzed. Whereas, Mg of the different FP varies significantly from each other (p-value<0.05)

and Ca of FPA differs significantly (p-value<0.05) from FPC. The amount of Ca in this study is

higher than the one reported by Chinte-Sanchez (2008) whose value was 16.26g/kgDM for

bagoong dilis. The value of K for FP (500-600mg/100g) is higher than those studied by Sumino,

et al. (1999, 2003) which had a mean K content of 157±30mg/100g in bagoong,

466±144mg/100g in terasi, 116mg/100g in kapi and 207mg/100g in belacan. According to

Chinte-Sanchez (2008), bagoong dilis have an Fe level of 331.31µg/gDM which is much higher

40

than the ones obtained in this study. Also the Zn in the analyzed FP (1.52-11.7mg/kg) is higher

than the shrimp pastes obtained by (Pilapil, et al., 2015) whose values were 5.94-6.92mg/kg. The

difference in the mineral content of the studied anchovy pastes from other studies is due to the

fact that composition of fish varies with regards to their diet, feed rate, genetic strain, age, size,

sex, sexual maturity, and seasonal differences (Sankar, et al., 2013; Bakhiet, et al., 2013). Also,

FP attained a high Na content due to the added salt for the fermentation to take place.

Table 11. Mineral composition of anchovy paste traditionally produced in some parts of the

Philippines

Mineral Sample

FPA FPB FPC p-value

Microelement

(μg/g DM)

Cu ND ND ND

Fe 18.9±6.25 ND 15.8±5.14 0.643

Mn ND ND ND

Zn 5.39±1.71a 28.1±0.39

b 20.9±6.01

b 0.018

Macroelement

(mg/g DM)

Mg 1.25±0.003a 1.95±0.18

b 11±1.38

c <0.001

K 15.9±10.03 14.9±2.0 12.5±1.56 0.215

Ca 36.4±1.69a 49.5±6.91

ab 67.7±8.58

b 0.037

Na 146±42.35 182±12.88 136±16.86 0.341

Results expressed as mean ± standard deviation; n=2. ND=below the detection limit. Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

c. Fatty Acid Composition

Fatty acids can be saturated and unsaturated. Saturated fatty acids (SFA) contain no

double bond in their carbon chain, while unsaturated fatty acids have double bonds in their

carbon chain. Unsaturated fatty acids are classified as monounsaturated (MUFA) and

polyunsaturated (PUFA) containing only one double bond and two or more double bonds

respectively. In tables 12 and 13, SFA present in the analyzed anchovy pastes are lauric acid

(C12:0), tridecanoic acid (C13:0), myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic

41

acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), heneicosanoic

acid (C21:0), and behenic acid (C22:0). MUFA consists of myristoleic acid (C14:1), 10-

pentadecenoic acid (C15:1), palmitoleic acid (C16:1), 10-heptadecenoic acid (C17:1), oleic acid

(C18:1), and gadoleic acid (C20:1). Linoleic acid (C18:2c), γ-linolenic acid (C18:3n-6), α-

linolenic acid (C18:3ω-3), 11,14-eicosadienoic acid (C20:2), arachidonic acid (C20:4),

eicosapentaenoic acid (C20:5ω-3; EPA), docosapentaenoic acid (C22:5ω-3), and

docosahexaenoic acid (C22:6ω-3; DHA) are among the PUFA.

Total SFA, MUFA and PUFA obtained from FP ranges from 44.5-53.4, 14-18, and 18.9-

31.3g/100g FAME respectively. These fatty acids differ significantly (p-value<0.05) where FPB

differs from FPA and FPC. According to Montaño, et al. (2001) and Peralta, et al. (2008), long

periods of fermentation doesn’t affect the highly unsaturated fatty acids such as EPA and DHA.

But, it can be observed from this study that with longer fermentation periods, SFA and MUFA

decreases while PUFA increases. However, the differences in FP could not be clearly identified

due to lack of information towards how these products are processed. Table 13 shows the fatty

acid profile of raw and different anchovy pastes obtained from different studies. The SFA

content of FP obtained is concurrent with the results of Sumino, et al. (1999, 2003) for

bagoong,belacan and kapi, however, the one reported by Montaño, et al. (2001) has a higher

value. It can also be observed that the raw anchovy obtained by Sankar, et al. (2013) has a lower

SFA value than the fermented anchovies. For MUFA, FP is in agreement with bagoong reported

by Sumino, et al. (1999) but a little bit lower than the studies of Montaño, et al. (2001), belacan

and kapi of Sumino, et al.(1999,2003) and raw anchovy from Sankar, et al. (2013). The PUFA

content of FP is higher compared to the one reported by Montaño, et al. (2001) but lower than

the bagoong and belacan studied by Sumino, et al. (1999,2003) and raw anchovy by Sankar, et

al. (2013). However, it is just in agreement with the PUFA of kapi reported by Sumino, et al.

(1999).

PUFA can be divided into omega3 (ω3) and omega6 (ω6) fatty acids. Total ω3 obtained

in this study range a value between 14.6-26.9g/100g FAME which is higher than the one

obtained from the study of Montaño, et al. (2001) which has 4.7g/100gFAME. It is in agreement

with kapi and belacan of Thailand and Malaysia (Sumino, et al., 1999; Sumino, et al., 2003) but

42

lower than the bagoong from the Philippines obtained by Sumino, et al. (1999) (Table 13). Total

ω6 of FP varies from 4.31-4.49g/100gFAME and these results coincide with kapi studied by

Sumino, et al (1999). They are also higher than the ones obtained by Montaño (2001) and

belacan and bagoong of Sumino, et al (1999,2003) (Table 13). Total ω6 shows no significant

difference (p-value˃0.05) from the different producers while total ω3 differs significantly (p-

value<0.05) where FPB has lower value than FPA and FPC.

43

Table 12. Fatty acid profile (g/100g FAME) of anchovy paste traditionally produced in some

parts of the Philippines

FAME (g/100g) Sample

FPA FPB FPC p-value

C 12:0 0.17±0.003 0.23±0.02 1.17±0.28 0.102

C 13:0 0.08±0.00 0.17±0.01 0.17±0.04 0.063

C 14:0 5.42±0.07a 6.75±0.13

b 5.38±0.34

ac 0.012

C 15:0 1.36±0.03 2.08±0.04 1.35±0.06 0.180

C 16:0 26.4±0.73a 27.9±0.26

ab 23.5±1.04

ac 0.022

C 17:0 2.42±0.08a 2.63±0.009

ab 2.26±0.12

ac 0.046

C 18:0 8.35±0.07a 12.94±0.3

b 10.1±0.26

c 0.001

C 20:0 0.31±0.004a 0.23±0.002

b 0.25±0.02

b 0.007

C21:0 0.1±0.004 0.22±0.19 0.09±0.006 0.368

C22:0 0.15±0.008 0.24±0.01 0.22±0.04 0.076

C 14:1 0.21±0.002 0.32±0.2 0.31±0.02 0.643

C 15:1 0.27±0.03a 0.34±0.005

ab 0.54±0.04

c 0.005

C 16:1 4.48±0.14a 7.13±0.45

b 3.82±0.22

ac 0.003

C 17:1 0.85±0.09a 1.28±0.02

b 0.88±0.02

ac 0.013

C 18:1 8.1±0.18a 8.8±0.05

ab 7.68±0.31

ac 0.028

C20:1 1.1±0.02a 0.49±0.02

b 0.72±0.07

c 0.002

C 18:2ω-6 1.73±0.05 1.51±0.1 1.54±0.15 0.245

C 18:3ω-6 0.41±0.06 0.45±0.009 0.51±0.2 0.798

C20:2 ω-6 0.15±0.03 0.4±0.06 0.32±0.13 0.111

C20:4 ω-6 2.07±0.02 1.95±0.1 2.12±0.09 0.230

C18:3ω-3 0.9±0.02a 0.68±0.07

b 0.85±0.04

ab 0.045

C20:5ω-3 6.09±0.06a 2.77±0.16

b 4.38±0.35

c 0.002

C22:5ω-3 1.35±0.2 0.98±0.33 1.67±0.38 0.233

C22:6ω-3 18.6±0.04a 10.2±0.89

b 16.7±0.61

ac 0.002

Total SFA 44.8±1.00a 53.4±0.98

b 44.5±2.2

ac 0.012

Total MUFA 15±0.46a 18.4±0.74

b 14±0.69

ac 0.009

Total PUFA 31.3±0.47a 18.9±1.73

b 28.1±1.92

ac 0.003

Total ω3 26.9±0.28a 14.6±1.46

b 23.6±0.63

ac 0.002

Total ω6 4.36±0.16 4.31±0.269 4.49±0.57 0.669

Results expressed as mean ± standard deviation; n=2.

Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

44

Table 13. Fatty acid profile of anchovy pastes obtained from different studies

FAME (g/100g) Montaño

(2001)

Sankar, et al.,

(2013)*

Belacan (Sumino,

et al., 2003)

Kapi (Sumino,

et al., 1999)

Bagoong

(Sumino, et al.,

1999)

C 12:0 1.3±1.1 0.03±0 nd nd nd

C 13:0 nd 0.08±0 nd nd nd

C 14:0 11.3±0.2 1.8±0.11 4.2±1.1 6.0±3.7 5.7±1.7

C 15:0 nd 1.29±0.12 nd nd nd

C 16:0 38.4±1.5 8.79±0.34 30.3±2.0 33.4±6.0 26.2±5.3

C 17:0 nd 1.06±0.3 nd nd nd

C 18:0 16.6±0.2 8.09±0.45 10.3±2.2 13.0±2.1 10.2±1.2

C 20:0 1.08±0.07 nd nd nd nd

C 21:0 nd nd nd nd nd

C 22:0 0.9±0.1 nd nd nd nd

C 14:1 nd nd nd nd nd

C 15:1 nd nd nd nd nd

C 16:1 8.3±0.2 3.96±0.04 8.6±2.0 7.7±1.7 4.4±1.6

C 17:1 nd 0.91±0.07 nd nd nd

C 18:1 12.3±0.4 8.94±0.56 13.0±6.3 13.3±4.0 8.3±2.1

C 20:1 1.0±0.2 6.08±0.45 nd nd 3.4±0.9

C 20:4 0.50±0.17 2.12±0.33 nd nd nd

C 18:3ω-3 0.14±0.12 2.32±0.15 nd nd nd

C 20:5ω-3 0.5±0.4 4.97±0.39 14.3±3.4 7.9±3.0 9.3±2.7

C 22:5ω-3 0.05±0.08 nd nd nd nd

C 22:6ω-3 1.7±0.2 22.5±1.15 10.7±2.9 8.9±4.3 30.3±6.8

C 18:2ω-6 0.63±0.03 0.45±0.04 2.9±3.9 4.4±1.7 1.5±0.4

C 18:3ω-6 nd nd nd nd nd

C 20:2ω-6 0.12±0.11 12±0.56 nd nd nd

Total SFA 71.5±2.0 24±1.2 44.8±5.3 52.4±11.8 42.1±8.2

Total MUFA 21.3±0.5 23.9±1.09 21.6±8.3 23.9±6.6 16.1±4.6

Total PUFA 7.3±1.8 51.9±1.99 33.5±11.8 23.7±10.3 41.8±10.6

Total ω3 4.7±0.9 29.8 25±6.3 16.8±7.3 39.6±9.5

Total ω6 2.6±0.9 15.9 2.9±3.9 4.4±1.7 1.5±0.4

Results expressed as mean ± standard deviation. nd=not determined. * = unfermented

45

d. Non-protein and TBARS composition

Non-protein nitrogen (NPN) content of the anchovy pastes was also analyzed (table 14).

NPN are products of protein hydrolysis which includes peptides, free amino acids, amines and

ammonia, which are responsible for the flavor and aroma development of fermented products

(Jiang, et al., 2007; Majumdar & Basu, 2010; Kim, et al., 2003; Peralta, et al., 2008). Total NPN

includes all the substances produced during the 24-hour acid hydrolysis while free NPN concerns

the α-amino nitrogen readily present (not peptide-bound) in the product. Total NPN of FP varied

between 76-107mg/100gDM). This is lower compared to the fermented fish reported by

Majumdar & Basu (2010) which has a total NPN content of 540mg/100gDM. Even though that

there was no significant differences (p˃0.05) between producers, it can be observed that FPB has

higher total NPN than FPA and FPC which is of the same pattern acquired with their crude

protein content from table 10. This high value for NPN may have contributed to the high pH

value of FPB (6.39). High pH of FPC (6.55) may be attributed to other factors than NPN. Free

NPN content of FP varied between 12-20mg/100gDM where FPA is significantly higher

(p<0.05) than FPB and FPC. The difference between producers might not be clear as the process

of production of these fermented products is also unclear, but possible reasons could be due to

their salt:fish ratio and to whether the raw fish have been washed or unwashed prior to

processing. Salt affects protein hydrolysis where the higher salt concentration, the faster is the

proteolytic activity. This is in contrast to the study of Kim, et al. (2003) where lower salt

concentration results to a higher hydrolyzing activities of proteases and peptidases in shrimp

byproducts. According to the study of Giri, et al. (2009), washing step removed the extractive

nitrogen resulting to a lower free NPN content. Aside from that, levels of extractable nitrogen

increases rapidly during fermentation period for unwashed fish due to oxidation and higher

decomposition rate of protein. The free NPN content of the fermented fish in this study is still

lower than the ones obtained by Majumdar & Basu (2010) which has 163mg/100gDM free NPN

content. The difference in NPN content of FP to the other study is due to variability in raw

materials used, production process and maturation period of the fermentation process (Faithong,

et al., 2010). The NPN content increases with fermentation period (Majumdar, et al., 2006;

Kilinc, et al., 2005). In the study of Majumdar & Basu (2010), Indian shad was used and

fermentation period is 4-6 months thus, acquiring a higher level of NPN.

46

Another chemical parameter analyzed in this study was thiobarbituric acid reactive

substances (TBARS). TBARS is used as an indicator of lipid oxidation in meat and fish products

(Irwin & Hedges, 2004). Values of TBARS analyzed for FP (table 14) ranged from 10.33-

21.73µgMDA/gDM which showed significant difference (p<0.05) between producers. FPC,

which has a maturation period of 4 months, has a lower value than FPA and FPB with ripening

periods of 3 and 1 month respectively. It can be observed that there is a decreasing trend of TBA

with fermentation period, however, FPA and FPB doesn’t differ significantly from each other.

According to Peralta, et al. (2008), prolonging the fermentation period results in the production

of substances that contribute to increase in antioxidant activity and the ability to suppress lipid

oxidation, thus, lowering the TBA value. This would explain why FPB have higher TBARS

values compared to FPA and FPC since FPB was just fermented for 1 month. Comparing FPA

and FPC which has longer ripening period, FPA attain a higher TBARS content. This is

attributed to its higher content of PUFA and Fe. PUFAs are highly susceptible to lipid oxidation

(Rashid, et al., 1992; Binsas, et al., 2008) while copper acts as prooxidant (Ladikos &

Lougovois, 1990).

Table 14. Non-protein nitrogen and TBARS of anchovy paste traditionally produced in some

parts of the Philippines

Composition Sample

A B C p-value

Free NPN (mg/100gDM) 20.2±0.25a 12.4±4.04

b 16.9±0.74

b 0.018

Total NPN (mg/100gDM) 79.3±4.06 107±42 76.9±10.92 0.397

TBARS (µgMDA/gDM) 18.8±0.42a 20±1.95

ab 13.2±3.26

c 0.025

Results expressed as mean ± standard deviation; n=4.

Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

47

C. Characterization of fermented anchovy pastes

Salt-fermented anchovy pastes from different batches produced by a local producer (SP)

were analyzed as a function of time (T). The sampled SPA, SPB and SPC showed no significant

difference (p-value˃0.05) with each other in the different time points for all measured

parameters. Therefore, the following analysis is done where the three sampled anchovy pastes

are used as replicates.

a. Chemical Characterization

i. Chemical composition

The chemical composition of SP as a function of T is shown in table 15. Dry matter (DM)

content of SP varied between 22-48g/100g. There was significant difference (p<0.05) between

the raw fish to the different days of fermentation. It can be observed that DM increased from R

to D19. A raw fish contains high amount of moisture as moisture is one of its major components

thus, having a low DM (Giri, et al., 2009). The moment when the salt was added and then

drained (D0), the free water in fish is removed through the draining. As fermentation goes on,

more water is removed through osmosis by the action of the salt (Majumdar, et al., 2006; Petrus,

et al., 2013; Chinte-Sanchez, 2008).

Crude fat content varied from 4.1-8.3g/100gDM for SP. The analyzed pastes showed no

significant differences (p˃0.05) between T. This shows that the fat content is hardly affected by

the fermentation process. But comparing the start and end of fermentation (D0 to D28), a

decrease in fat content is attained which is consistent with the study of Kilinc, et al., (2005) on

fish sauce processing. Petrus, et al., (2013) also cited a decrease in fat content during

fermentation due to the leaching process of fish muscle in correlation with salt penetration.

Analyzed values of crude protein in SP ranged between 42.5-93.1g/100gDM, in which R

significantly differed from the other T (p < 0.05). Fish is rich and a good source of protein, thus,

having a high value of protein in R of SP (Kristinsson & Rasco, 2000). At D0, protein decreases

48

drastically due to the draining where soluble proteins are also removed together with the water.

As fermentation goes on, protein also decreases due to protein hydrolysis and leaching out in the

brine (El-Sebaiy & Metwalli, 1989).

Other parameters analyzed were water activity (Aw), pH and NaCl content (table 15).

The Aw of SP ranged between 0.82-0.99. There was no significant difference (p˃0.05) in T.

There may not differ significantly with the statistical analysis, but it can be observed that there

was a decrease in Aw from R to D0 and then a constant Aw to the proceeding T. The decrease in

Aw is attributed to the removal of moisture and the addition of salt which has an osmotic effect

on the product. A decrease in moisture is accompanied with the increase in salt and ash content

(El-Sebaiy & Metwalli, 1989; Majumdar, et al., 2006; Petrus, et al., 2013). NaCl content of SP

ranged between 5.8-37.1g/gDM which has significant difference (p<0.05) between R and D0.

Salt content increases due to the addition of NaCl to start the fermentation. Salt content began to

increase up to D9, a decrease on D19 and then an increase afterwards. But comparing the

beginning and end of fermentation in this study (D0 and D28), the result showed an increase in

the salt content. This is because of osmosis where moisture is removed in replace of salt that has

been absorbed into the fish flesh (Majumdar, et al., 2006; Petrus, et al., 2013). High salt content

enhances the shelf-life of the fermented fish due to lower Aw, inhibiting the growth of spoilage

microorganisms (Chinte-Sanchez, 2008; Majumdar & Basu, 2010; Montaño, et al., 2001). For

pH of SP, analyzed values varied from 6.7-7.0 where there was no significant differences

(p˃0.05) between T. This is in contrast to the study of Surono & Hosono (1994) on fermented

fish product terasi where the pH level rises from 6.0 to 6.5 and later reduces to 4.5. Upon further

fermentation, the pH increased to 7.8. It has been said that organic acids, such as lactic acid, are

produced during fermentation resulting to lowering the pH. Moreover, protein degradation

occurs during fermentation which releases nitrogenous compounds, such as amines and ammonia

resulting to an increase in pH (Ng, et al., 2011; Kilinc, et al., 2005; Surono & Hosono, 1994). In

the present study, there’s only a minimal change in pH and it is of a rather neutral pH. This result

is similar to the one reported by Sarojnalini & Suchitra (2009) on starter fermented ‘Ngari’ with

a pH level of 6.74 after 40 days of fermentation. The fermented anchovy paste attain a rather

neutral pH possibly because most of the organic acids produced during fermentation is in its salt

form which is comparable to the shrimp paste studied by Mizutani, et al. (1992). Comparing the

49

start and end of fermentation (D0 and D28), a slight increase in pH from 6.73 to 7.01 has been

observed. This suggests that protein degradation occurs during the fermentation period of SP.

However, the pH of the fermented fish at the end of fermentation is not higher than 7.0 due to its

strong buffering capacity (Visessanguan & Chaikaew, 2014).

Table 15. Chemical composition of anchovy pastes in different time points of fermentation

T DM

(g/100g)

Crude Fat

(g/100gDM)

Crude Protein

(g/100gDM) Aw pH

*

NaCl

(g/100gDM)

R 22±0.69a 6.44±1.47 93.1±2.04

a 0.99±0.004 6.82±0.18 5.80±2.33

a

D0 38±1.97b 8.29±2.76 49±2.75

b 0.82±0.02 6.73±0.14 30.5±11.50

b

D9 39±3.19b 6.11±1.06 52.8±2.54

b 0.82±0.03 6.72±0.18 37.1±8.14

b

D19 48±9.68b 4.12±0.95 42.5±9.58

b 0.82±0.03 6.94±0.19 22.2±4.48

b

D28 45.9±9.18b 5.24±3.71 45.2±11.65

b 0.82±0.03 7.01±0.31 37.1±11.04

b

p-value 0.004 0.153 0.045 0.112 0.091 0.005

Results expressed as mean ± standard deviation; n=3;*-n=6

Values within the column with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

ii. Mineral Composition

Mineral content of SP is shown in table 16. Microelements analyzed resulted in values of

9-15, 8-26, 2-5, and 26-75 µg/g DM for Cu, Fe, Mn and Zn respectively. There were no

significant differences (p˃0.05) observed between T for Cu and Mn contents. However, Fe and

Zn contents varied significantly (p<0.05) between R and D0. For the macroelements, values

obtained were between 1.7-3.8, 11.8-18.6, 39-48 and 23-146 mg/gDM for Mg, K, Ca and Na

respectively. There were no significant differences (p˃0.05) between T for all macroelements

analysed except Na where it differs significantly (p<0.05) with R and D0. In the study of Sankar,

et al. (2013) on fresh anchovy, the macroelements Na and Ca are lower than SP whose values

obtained were 16.57, 18.7m/gDM respectively. However, Sankar, et al. (2013) reported a higher

K content in the fresh anchovy which is 54mg/gDM. Also in their study, the microelements Cu

and Mn are lower than the one obtained in SP with varying values of 0.85 and 0.42mg/gDM

correspondingly. It can be observed that all minerals, except Na, decreased from R to D0. The

decrease is attributed to the draining process while the increase in Na is due to the addition of

50

salt (NaCl). Comparing the start and the end of fermentation, there is no significant differences

in the content of the microelements. This suggests that microelements were not affected by

fermentation. However, this was in contrast with the study of Bakhiet, et al., (2013) on salted

Hydrocynus spp. who reported a decrease in Cu and Fe and an increase in Zn due to salting and

fermentation. On the other hand, there was an increase for the macroelements from the start to

the end of fermentation. The analysis was in agreement with the study of Kim, et al., (2003)

where an increase of Ca, Fe and Mg was also observed from the salt-fermented shrimp sauce.

Whereas in the study of Bakhiet, et al., (2013), Ca and Mg were not affected by salting and

fermentation while a decrease in K is observed due to washing.

Table 16. Mineral composition of anchovy pastes in different time points of fermentation

T Microelement (µg/gDM) Macroelement (mg/gDM)

Cu Fe Mn Zn Mg K Ca Na

R 14.6±7.62 26.4±7.30a 5.31 75.2±20.83

a 3.81±1.31 18.6±7.48 44.1±4.09 22.8±9.16

a

D0 10 8.11±5.12ab

2.36 34.3±5.91b 1.71±0.74 11.8±7.75 39±18.24 119±45.21

b

D9 10.9±1.10 11.8±4.05ab

2.51±0.29 35.3±9.54b 2.72±0.87 14.8±5.01 51.2±17.27 145±32.01

b

D19 9.47±0.50 9.93±4.65b 2.14±0.08 26.5±1.63

b 1.75±0.64 12.4±6.49 41.6±11.42 87.3±17.62

ab

D28 10.6 8.38±5.98b 2.52 31.8±5.18

b 2.38±1.12 13.6±6.62 48.7±25.12 145±43.39

b

p-value 0.543 0.014 0.295 0.001 0.122 0.752 0.892 0.005

Results expressed as mean ± standard deviation; n=3. Values within the column with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

iii. Fatty Acid Composition

Total SFA, MUFA and PUFA obtained from SP are shown in table 17. Their values

ranged from 38.9-49.5, 14.6-17.4, and 24-35.8g/100g FAME respectively. SFA was the

predominant group of fatty acids in the sampled anchovy paste where the major SFA is C16:0. In

the study of Sankar, et al. (2013) on medium-sized anchovy, SFA was also the predominating

fatty acid group. Another study of fermented anchovy paste also showed SFA as the major fatty

acid group (Montaño, et al., 2001). Sankar, et al., (2013) and Montaño, et al. (2001) reported that

C16:0 is the major SFA which coincides with the study of El-Sebaiy & Metwalli (1989) on

51

fermented Bouri fish. The major PUFA was C22:6ω-3 which is also in agreement with the

studies of Sankar, et al. (2013), Montaño, et al. (2001) and El-Sebaiy & Metwalli, (1989). There

was a significant increase (p-value<0.05) in the total SFA from 39.7g/100gFAME at R to

46.5g/100gFAME at D0, while a significant decrease (p-value<0.05) was observed in the total

PUFA from 34.7g/100gFAME at R to 26.3g/100gFAME at D0. On the other hand, there was no

significant difference (p-value˃0.05) in total MUFA between R (14.8g/100gFAME ) and D0

(16.6g/100gFAME). The increase in total SFA from R to D0 suggests that the addition of salt

increased the SFA content. This could be attributed to the halophilic bacteria inherent in the salt.

Oren (2003) has stated that halophilic bacteria mainly contain common straight-chain saturated

and monounsaturated fatty acids in their membrane lipids such as C16:0, C16:1 and especially

C18:1. The increase in SFA is also supported by the different studies shown in table 13 where

total SFA of the unfermented anchovy has a lower value than the fermented ones. On the other

hand, the decrease in total PUFA from R to D0 is attributed to the decrease in C22:6ω-3,

C20:5ω-3 and C 18:3ω-6. Table 13 also showed that unfermented anchovy has more PUFA than

the fermented ones. The decrease is an effect of lipid oxidation, wherein PUFAs are degraded

into free fatty acids, during draining as the products were exposed to air. It has been known that

PUFA are highly prone to oxidation (Rashid, et al., 1992; Binsas, et al., 2008). Comparing the

start and end of fermentation, there was no significant difference in the total SFA, MUFA and

PUFA. This suggests that fatty acids are not affected by fermentation which is also in consistent

to the one reported by Montaño, et al. (2001) on fermented shrimp paste. Looking at the

individual PUFA, C22:6ω-3 (DHA) has the highest value. This indicate that the fermented

anchovy paste is rich in DHA which is essential for the normal functioning and development of

retina and brain of humans, particularly in infants (Sankar, et al., 2013).

Total ω3 of SP obtained in this study varied between 21.4-29.4g/100g FAME in which R

(29.4g/100gFAME) significantly decrease (p-value <0.05) to D0 (21.4g/100gFAME). This

decrease is attributed to the decrease in its individual ω3, C20:5ω-3 and C22:6ω-3. This decrease

is also supported by the studies of Sankar, et al. (2013), Montaño, et al. (2001) and belacan and

kapi from Sumino, et al. (1999,2003) in table 13. Their studies showed a lower total ω3 value in

the fermented anchovy pastes than the unfermented anchovy. Comparing D0 to D28, no

significant differences (p-value˃0.05) were observed. Total ω6 of SP varies from 4.8-

52

5.36g/100gFAME and the results showed no significant differences (p-value˃0.05) from the

different T.

Table 17. Fatty acid profile of anchovy pastes in different stages of fermentation

FAME

(g/100g)

Time point (T) p-value

R D0 D9 D19 D28

C 12:0 0.14±0.03 0.17±0.01 0.18±0.007 0.17±0.03 0.17±0.04 0.536

C 13:0 0.12±0.02 0.12±0.04 0.11±0.03 0.09±0.03 0.15±0.02 0.210

C 14:0 3.83±0.17 4.14±0.51 4.46±0.22 4.28±0.17 4.67±0.59 0.149

C 15:0 1.62±0.10 1.82±0.20 1.73±0.23 1.62±0.13 1.89±0.23 0.349

C 16:0 22.2±1.47a 26.2±1.06

b 24.9±0.44

ab 25.9±1.22

b 25.1±1.6

ab 0.018

C 17:0 2.22±0.17 2.50±0.13 2.41±0.24 2.34±0.07 2.55±0.14 0.167

C 18:0 9.16±0.89 11±1.09 10.5±0.89 10.3±0.94 11.4±0.60 0.089

C 20:0 0.23±0.02 0.29±0.005 0.28±0.05 0.35±0.06 0.30±0.05 0.067

C21:0 0.06±0.03 0.09±0.01 0.10±0.03 0.12±0.04 0.10±0.06 0.472

C22:0 0.17±0.05 0.23±0.02 0.25±0.03 0.23±0.06 0.23±0.05 0.353

C 14:1 0.25±0.11 0.29±0.10 0.37±0.19 0.33±0.06 0.33±0.16 0.845

C 15:1 0.30±0.17 0.34±0.17 0.47±0.07 0.46±0.15 0.39±0.05 0.489

C 16:1 4.35±0.48 4.70±0.53 4.57±0.66 4.51±0.16 5.02±0.31 0.509

C 17:1 1.26±0.16 1.33±0.09 1.24±0.08 1.26±0.20 1.37±0.06 0.697

C 18:1 7.98±0.78 9.48±1.15 8.72±1.06 9.45±0.73 8.61±0.51 0.259

C20:1 0.73±0.24 0.49±0.11 0.52±0.06 0.58±0.08 0.69±0.10 0.179

C 18:2ω-6 1.59±0.20 1.67±0.11 1.62±0.04 1.60±0.02 1.72±0.20 0.735

C 18:3ω-6 0.53±0.16a 0.45±0.12

b 0.45±0.07

ab 0.72±0.24

ab 0.45±0.11

ab 0.039

C20:2ω-6 0.19±0.03 0.29±0.10 0.24±0.02 0.21±0.02 0.21±0.02 0.277

C20:4ω-6 3.05±0.33 2.45±0.11 2.49±0.14 2.82±0.25 2.51±0.34 0.062

C18:3ω-3 0.86±0.23 0.74±0.17 0.76±0.13 0.75±0.14 0.94±0.08 0.507

C20:5ω-3 4.55±0.19a 3.33±0.22

b 3.69±0.13

ab 4.0±0.30

ab 3.65±0.72

ab 0.025

C22:5ω-3 1.81±0.44 1.80±0.72 1.63±0.70 1.84±0.29 2.13±0.70 0.885

C22:6ω-3 22.1±1.29a 15.6±1.77

b 17.9±0.87

ab 18.4±1.07

ab 16.8±4.16

ab 0.039

Total SFA 39.7±2.97a 46.5±3.07

b 44.9±2.16

b 45.4±2.76

b 46.6±3.36

b 0.003

Total MUFA 14.9±1.93 16.6±2.14 15.9±2.13 16.6±1.37 16.4±1.18 0.068

Total PUFA 34.7±2.87a 26.3±3.31

b 28.8±2.08

ab 30.3±2.33

ab 28.4±6.34

ab 0.018

Total ω3 29.4±2.15a 21.4±2.88

b 24±1.82

ab 24.9±1.80

ab 23.5±5.66

ab 0.024

Total ω6 5.36±0.72 4.86±0.44 4.8±0.27 5.35±0.53 4.89±0.67 0.644

Results expressed as mean ± standard deviation; n=3. Values within the row with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

53

iv. Non-protein and TBARS composition

Total and free NPN content of SP are shown in table 18. Total NPN varied between 54-

386 mg/100gDM where there was a significant decrease (p<0.05) from R to D0. Total NPN

increases from D0 to D9and then rest stable. Free NPN content varied between 9-

38mg/100gDM. The analysis showed significant differences (p<0.05) between T. Free NPN

content decreased from R to D0 and then began to increase until the end of fermentation. The

decrease in NPN (total and free) can be due to the removal of some NPN as a result of the

draining at D0. On the other hand, the increase in total and free NPN from the start to the end of

fermentation is the result of protein hydrolysis (Ng, et al., 2011; Kilinc, et al., 2005).

Values of TBARS analyzed in SP ranged between 3-18µgMDA/gDM. There was an

increase in TBARS content when salt was added at D0 then follows a decreasing trend from D9

to D28. The increase is due to the exposure of lipids to air when they were drained. Then as

fermentation goes on, TBARS decreases due to anaerobic condition restricting the reaction of

oxygen and lipids. In the study of Montaño, et al. (2001) on fermented shrimp paste, it was said

that oxidation was significantly suppressed during fermentation. Peralta, et al. (2008) also

observed an increase in antioxidant activity in the fermented shrimp paste causing PUFAs to

remain intact even at longer fermentation period thus not much of it is involved in lipid

oxidation.

Table 18. Non-protein nitrogen and TBARS of anchovy paste traditionally produced in some

parts of the Philippines

Time point Free NPN

(mg/100gDM) Total NPN

(mg/100gDM)

TBARS

(µgMDA/gDM)

R 31.9±4.05a 234±94.37

a 9.90±2.96

a

D0 10.1±0.90b 88.9±35.18

b 14.7±3.70

ab

D9 22±5.05c 125±50.64

b 8.65±2.99

ac

D19 25.4±5.09ac

116±17.25b 4.70±2.09

c

D28 30.3±5.04ad

116±13.95b 4.12±2.03

c

p-value < 0.001 0.006 < 0.001

Results expressed as mean ± standard deviation; n=6. Values within the column with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

54

b. Microbial Characterization

The liquid part of the fermented anchovy pastes with different T were also collected and

analysed for microbial characterization. The total Enterobacteriaceae, aerobic bacteria,

halophilic aerobic bateria, lactic acid bacteria (LAB), halophilic LAB, proteolytic bacteria and

halophilic yeasts were analysed.

The analysis showed no counts of Enterobacteriaceae. Enterobacteriaceae are gram-

negative, non-spore-forming bacteria of which some of them are important human and animal

pathogens such as Escherichia, Shigella, Salmonella and Yersinia. They cause foodborne

diseases as well as food spoilage. They are used as an indicator organism for poor hygiene,

inadequate processing or post-process contamination of foods (Baylis et al., 2011). With this

result, it suggests that fermentation process was done hygienically.

*points on the detection limit line are below the detection limit*

Figure 4. Microbial Count of fermented anchovy pastes on different days of fermentation

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

D0 D9 D19 D28

Mic

rob

ial C

ou

nt

(CFU

/mL)

Fermentation Day

LAB halophilic LAB halophilic yeast

total proteolytic total aerobic total halophilic aerobic

detection limit

55

Figure 4 showed the growth of the different microorganisms involved during

fermentation of SP. The total aerobic count increased up to 106CFU/mL from D0 to D19 and

then slightly decreased to 105CFU/mL with D28. In the case of total halophilic bacteria, they

were below the detection limit at the start of fermentation. But they gradually increased to

106CFU/mL at D19 and then decreased slightly to 10

5CFU/mL with D28. Chinte-Sanchez (2008)

has cited similar results obtained from nampla where the total bacterial count increased up to 108

CFU/mL on the third week of fermentation, and then a slow decrease after six months was

observed. Also during the first 10 days of fermentation in the study on Indonesian fermented fish

sauce by Ijong & Ohta (1996), total bacterial count increased significantly then gradually

decreased thereafter. According to Jiang et al. (2007), there is a high bacterial count in the early

stage of fermentation because salt has not yet completely dissolved and penetrated into the fish

flesh. After several months, non-halophilic bacteria disappeared while halotolerant and

halophilic bacteria remain. These halotolerant and halophiles played a main role in fermentation.

Lactic acid bacteria (LAB) and halophilic LAB were already present even from the start

of the fermentation. Their count both increase from 103 to 10

6CFU/mL and 10

4 to 10

7CFU/mL

respectively until D19 but then both decreased to 105CFU/mL at D28. It was the halophilic LAB

which has a higher count than LAB. This shows that halophilic LAB are the dominant

microorganisms involved during the fermentation process. In the study of Ijong & Ohta (1996)

on Indonesian fermented fish sauce, LAB increased during the first 10 days of fermentation then

gradually decreased thereafter. A similar result was also obtained in the study of Kilinc, et al.

(2005) on fish sauce where LAB increased until day 8 of fermentation corresponding to a

decrease in pH. Afterwards, LAB count decreased. LAB are mesophilic but some species are

able to grow at low temperature (as low as 5°C) and high temperature (as high as 45°C)

(Sivasankar, 2005). LAB produces lactic acid from carbohydrates lowering the pH of food. They

also produce bacteriocins inhibiting growth of pathogens and spoilage microorganisms.

Moreover, they modify the raw material to obtain new sensory properties as well as improving

the shelf-life of fish products (Thienchai & Chaiyanan, 2012). LAB also contributes to the flavor

development of fish sauces (Kilinc, et al., 2005). Leuconostoc, Lactobacillus, Streptococcus and

Pediococcus are among the species which belong to the group of LAB. The increase in the

56

number of LAB should correspond to the decrease in pH due to the production of lactic acid.

However, in this study, there is a minimal change in the pH. Though there is an increase of LAB

in the sample, the lactic acid produced by these microorganisms may not be enough to decrease

the pH compared to the alkalinity caused by proteolysis of the fish samples. This is due to the

buffering capacity of the fermented fish and organic acids produced are in salts form

(Visessanguan & Chaikaew, 2014; Mizutani, et al., 1992).

For proteolytic bacteria and halophilic yeasts, they were not yet detected at the start of

fermentation but both started to increase up to 106CFU/mL on D19, and then a gradual decrease

was observed to 104CFU/mL on D28. Proteolytic bacteria are bacteria which produces protease

that split proteins into peptides and amino acids. By the breakdown of proteins, the structures of

food products are changed (Sivasankar, 2005; Brown, 2011;). Species of these bacteria include

aerobic, facultative and spore-forming organisms. Clostridium, Bacillus, Pseudomonas and

Proteus are the dominant species belonging to this group (Sivasankar, 2005). It was observed in

this study that at D19, where the count of proteolytic bacteria was at its highest, the pH started to

increase slightly. Higher pH allows the bacteria to become dominant and also favors the

breakdown of proteins that releases amine compounds (Visessanguan & Chaikaew, 2014).

Yeasts help in the synthesis of essential amino acids such as glutamic acid and lysine

which enhances the flavor of fish paste (Chinte-Sanchez, 2008). Yeasts were also identified by

Thapa,et al. (2004) in their study on ngari, a fermented fish product. The yeasts involved were

Candida and Saccharomycopsis. In the study of Sanni, et al. (2002) on fermented fish momoni,

they have identified the yeasts Debaryomyces hansenii and Hansenula anomala. Likewise,

Crisan and Sands (1975) reported Candida clausenii to be present in patis.

It can be observed that numerous microorganisms identified in the study are halophilic

bacteria. Halophilic bacteria require a certain minimal salt (NaCl) concentration for their growth.

They can survive even at high salt concentration depending on the type of the species which

could be slightly, moderately or extremely halophilic or halotolerant. Species belonging to this

type of bacteria include Bacillus, Micrococcus, Vibrio, Moraxella, Halobacterium,

Corynebacterium, Streptococcus and Clostridium (Sivasankar, 2005). According to Chinte-

57

Sanchez (2008), the microorganisms which have a main role in fermentation of fish paste were

the halophilic bacteria. These bacteria increased rapidly because of the favorable growth

condition from the nutrients in brine. She also described that Bacillus, and not halophilic

LAB,where the predominant bacteria throughout the fermentation suggesting that Bacillus have

an active role in the process. This is also supported by Sanni (2002) stating that Bacillus were the

predominant bacterial flora in the fermented fish. In the study of Sarojnalini & Suchitra (2009,

2012), Bacillus and Micrococcus were the predominant microorganisms involved in the

fermentation of ngari. These bacteria have proteolytic and lipolytic activity which contribute to

the development of the typical odour and flavour of the final product (Sarojnalini & Suchitra,

2009,2012). Enzymes from Bacillus subtilis and B. coagulans have minimal role in proteolysis

during the early stage but are mainly responsible for the production of flavors (Chinte-Sanchez,

2008). Different authors enumerated halophilic bacteria like Bacillus, Micrococcus,

Pediococcus, Moraxella, LAB and some mould and yeasts dominate in the ripening of fermented

fish products. They are important for fish tissue breakdown and generation of aroma and flavour

(Majumdar & Basu, 2010; Aryanta, 2000).

58

Chapter 5. Conclusions and Recommendation

The anchovy paste products in the Philippines are microbiologically stable. Due to its low

Aw and high salt content, spoilage microorganisms are inhibited. The product is predominant in

SFA and C16:0 is the major SFA. It is also rich in PUFA, particularly in DHA which is essential

for infant’s brain development. Moreover, it is also rich in proteins and minerals (Na, Ca, K, Zn,

Fe) which are essential to human diet.

Gross composition of the anchovy pastes from the different producers didn’t vary

significantly. Though they were processed in different techniques, they still conform to the

standards for bagoong set by the Philippine Standard Association. However, there is significant

difference in the pH, fatty acid, NPN and TBARS to which the cause might be attributed to the

ripening period.

The amount and level of proteins, Aw, pH, minerals, PUFA, ω3, free and total nitrogen of

the raw anchovy decrease at the start of fermentation period. On the other hand, an opposite is

observed for DM, fats, salt, SFA and TBARS content. The difference is caused by the draining,

addition of salt and exposure of the product to air during draining.

Fermentation increases the concentration of DM, salt content, total and free NPN of the

salt-fermented anchovy paste. This is due to the effect of osmosis and proteolysis. In contrast,

fermentation decreases the content of fats, proteins and TBARS which is caused by fat

degradation, protein hydrolysis and restriction of aerobic condition. Fermentation does not affect

significantly the Aw, mineral content and fatty acids of the salt-fermented anchovy paste.

The microbial count of the fermented anchovy paste increases and is at its highest on D19

then decrease gradually on D28. Enterobacteriaceae are not detected in the studied fermented

anchovy pastes which show that the product is processed hygienically. Aerobic bacteria, LAB

and halophilic LAB are involved at the start of fermentation. Proteolytic, halophilic aerobic

bacteria and halophilic yeasts play a role starting at the middle period of fermentation. The

fermentation of the anchovy pastes is predominated by halophilic bacteria. Halophilic LAB

59

attain the highest count however, the pH of the fermented product doesn’t decrease that much

because most of the acids produced where in its salt form. The fermented fish also has a neutral

pH rather than higher pH because of the buffering capacity of the fermented fish. Based on the

values of pH obtained and literatures gathered, it can be concluded that it is Bacillus species that

play a vital role in the fermentation process.

Further studies regarding the salt-fermented anchovy pastes particularly on the

identification of the species of halophilic bacteria involved in the fermentation process are

deemed necessary. Also, a study should be taken on the characterization of the amino acids and

biogenic amines of the salt-fermented anchovy pastes during the different stages of fermentation

as these were not analyzed by the present study. Since the samples were stored for a year already

before analyzed, it would be interesting to conduct a similar study with samples gathered earlier

to compare and verify the results obtained in this study. It would also be interesting to conduct a

similar research on salt-fermented anchovy pastes from the other areas of the Philippines.

60

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