ANTIOXIDATION ACTIVITY A - Mahidolmulinet11.li.mahidol.ac.th/e-thesis/scan/4136715.pdf · Prof Molsiri Veerothai who was the external ... Some dietary sources of plant phenolic compounds

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ANTIOXIDATION ACTIVITY OF OKARA TEMPE' A

FERMENTED PRODUCT WIT}di RHIZOPUS OLIGOSPORUS

KANITTAWANTHAWIN2

A THESIS SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQIIIREMENTS FOR

THE DEGREE OF MASTER OF SCIENCE

(FOOD AND NUTRITION FOR DEVELOPMENT)

FACULTY OF GRADUATE STI]DIES

MAHIDOL I.]NIVERSITY

2002

rsBN 974-04-2668-9COPYRIGHT OF MAHIDOL I.NIYERSITY

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.......1.1.11.,r.,.. l:,:::I.\i.ij..lilil.ii1i

Copyright by Mahidol University

ThesisEntitled

ANTIOXIDATION ACTIVITY OF OKARA TEMPE, AFERMENTf,D PRODUCT WITH RHIZOPUS OLIGOSPORUS

KqOt.y-.. -Wa*LwinMiss.Kanitta WanthawinCandidate

Ph.D.(Food Science)Maior-Advisor

[email protected]. Prapasri Puwastein,Ph.D.(Food Technology)Co-Advisor

Assist.Prof. Sittiwat tertsiri,

Assoc.Prof.Rassmidara Hoonsawat,

Ph.D.(Agricultural Science)Co-Advisor

AylLfio'fr*$D ,

n.'"1. p'"i:. s""g; gilrj;,;,Ph.D.(Pharmacy Chemistry)ChairMaster of Science Programme inFood and Nutrition for DevelopmentInstitute of Nutrition

Ph.D.DeanFaculty of Graduate Studies


ThesisEntitled

ANTIOXIDATION ACTIVITY OF OKARA TEMPE, AFER]VIENTED PRODUCT WITH RH IZOPAS OLI G OSPORUS

was submitted to the Faculty of Graduate Studies, Mahidol UniversityFor the degree of Master of Science (Food and Nutrition for Development)

on29 October,2002

.Konilq-' U".+hor"ryr-Miss.Kanitta WanthawinCandidate

fr,,-L A/h

Ph.D.(Food Science)Chair

4y"'/A"tu

Ph.D.DeanFaculty of Graduate StudiesMahidol University

Assoc.Prof. Prapasri Puwastein,Ph.D.(Food Technology)Member

Ph.D.(Agricultural Science)Member

$rrLd"{G"tr--.;;'6. P; i;il',k $'i;;j;,,Ph.D.@harmacy Chemistry)DirecterInstitute of NutritionMahidol University

vrffi^)Molsiri Veerothai, Ph.D.

lJ--/


ACKNOWLEDGEMENT

I would like to express my deep appreciation and sincere thanks to my advisor,

Dr. Anadi Nititdthamyong, for her supervision, helpfi.rl guidance and encouragement

which has enabling me to the successful completion of this thesis.

I would like to express sincere appreciation to my co-advisor, Assoc. Prof.

Prapasri Puwastien and Assist. Prof. Sittiwat Lertsiri for their helpfrrl guidance,

cornments, invaluable suggestion, discussion and throughout encouragement the

course of the study.

I would also like to state my sincere appreciation to Associate Prof. Visith

Chavasit, Associate Prof. Pongtom Sungpuag, Asst. Prof. Ratchanee Kongkachuichai,

Miss. Renu Tavichat'oritayakul and Mrs. Yupapom Nakngamanog for their help and

suggestion.

I do greatly appreciate to Assist. Prof Molsiri Veerothai who was the external

examiner ofthe thesis defense for her kindness in valuable advice and guidance.

My special thanks go to all staff members of Food Science and Technology

Laboratory, Food Microbiology Laboratory and Food Chemistry Laboratory and all

the staff at the lnstitute of Nutriton, Mahidol University for co-operation and

assistance.

I would like to thank the Greenspot (Thailand) Co., Ltd., and the Thai Vegetable

Oil Company for the contribution of the okara and soybean oil used in this study.

I would like to thank all my friends for their help and encouragement.

Finally, I am grateful to my family for their financial support, entirely care, and

love. The usefulness of this thesis, I dedicate to my father, my mother and all the

teachers who have taught me since my childhood.

Kanitta Wanthawin


Fac. ofGrad. Studies, Mahidol Univ. Thesis / iv

4136715 NUFN/I\,f: MAJOR : FOOD AND NUTRITION FOR DEVELOPMENT ;

M.Sc.(FOOD AND NUTRITION FOR DEVELOPMENT)

KEY WORDS : TEMPE / OKARA / ANTIOXIDANT ACTTVITY / PHENOLICCOMPOUND

KANITTA WANTIIAWIN : ANTIOXIDATION ACTIVITY OF OKARATEMPE, A FERMENTED PRODUCT WITH RHIZOPUS OLIGOSPORUS. THESISADVISORS: ANADI NITITHAMYONG, Ph.D., PRAPASRI PUWASTEIN, Ph.D.,SITTIWAT LERTSIRI, Ph.D. 109 p. ISBN 974-04-2668-9

Antioxidants in food have attracted special interest because they can protectthe human body from fiee radicals and retard oxidative rancidity in food. Regarding

tempe, it has already been reported that this fermented soybean product is very stable

to rancidity development, and possesses antioxidative activity. Okar4 a by-productfrom the soybean milk industry could be considered as a raw material to preparc

okara tempe and its antioxidant should be investigated. Hence, the study aimed toprepare okara tempe and test for antoxidant activity by the liposome model and an oilstorage test.

Fried okara tempe from okara tempe stored in a refrigerator for differentperiods of time still exhibited antioxidant activity. However, fiying and storage couldreduce their activity compared to fresh tempe. The contents of vitamin E decreased

whereas tannin content increased during storage. Okara tempe extmct from a 48 hourfermentation period showed the highest antioxidant activity of four fermentationperiods (0, 24, 48 afi 72 hours). Vitamin E content was constant during thefermentation periods. The content of tanrfn in 100 g sample was22.37,14.90, 16.04

mg and not detected respectively throughout the fermentation time. Moreover, the

total phenolic compounds contents correlated well with their antioxidation activityand ranged from 16.38 to 83.38 (gallic acid equivalences).

In the oil storage test, antioxidant activity of okara tempe extract from a 48hour fermentation periods provided the highest antioxidant activity similar to that inthe liposome model.


Fac. ofGrad. Studies, Mahidol Univ.

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Thesis / v


CONTENTS

ACKNOWLEDGEMENTS

ABSTRACT

LIST OF TABLES

LIST OF FIGURES

LIST OF APPENDIXS

LIST OF ABBREVIATION

CHAPTER

I

II

Page

iii

iv

viii

x

xii

xiii

ilI

INTRODUCTION

LITERATURE REVIEW

2.1 Free radical and lipid oxidation

2.2 Antioxidant

2.3 Tempe

MATERIALS AND METHODS

3.1 Chemicals and materials

3.2 Preparation of soybean and okara tempe

3.3 Characteristic of soybean and okara tempe

Effects ofheat and shelflife on the antioxidant propertyof okara tempe

Effects of fermentation period on antioxidant activity of

okara tempe

Antioxidant activity of okara tempe extmct on soybean oil

I

3

3

18

32

42

42

44

45

47

49

3.4

3.5

3.6 Copyright by Mahidol University

vll

CONTENTS

(coNrrNUED)

RESULTS

4.1 Characteristic of soybean tempe and okara tempe

4.2 Influence ofheat and shelf life on antioxidant property

of okara tempe

4.3 Influence of fermentation period of okara tempe on it

antioxidant activity 55

4.4 Antioxidant activity of okara tempe extract on soybean oil 64

DISCUSSION 71

5.1 Production of okara tempe 72

5.2 Antioxidant activity of fried okara tempe 72

5.2 Antioxidant activity of okara tempe extract and its

potential application

CONCLUSION

50

50

51

76

81

REFERENCES

APPENDIX

BIOGRAPTTY

83

94

r09


LIST OF TABLES

Table

l. Cellular free radical targets

2. Example of free radicals

3. Lipid peroxidation-induced diseases and effects

4. Analytical method to determine the degree of oxi&tion of fats and oils

5. Some biologically important antioxidants

6. Advantages and disadvantages of natural antioxidants compared to

synthetic antioxidants

7. Some dietary sources of plant phenolic compounds

8. Nutrient composition oftempe and soybean

9. Active substances identified from tempe

10. Sensory acceptability scores of soybean tempe and okara tempe

I 1. TBAR formation of fried okara tempe exhat

12. Vitamin E contents of fried okara tempe

I 3. The total tannin content in fried okara tempe

14. TBAR formation of the reaction mixtures containing lot 1, 2 and 3

okara tempe extract

I 5 . Effect of fermentation period on Vitamin E, tannin and total

phenolic content

Page

4

5

14

t9

2l

24

31

34

35

50

52

54

55

56

16. Average peroxide values from soybean oil heatrnents at 60oC in the dark 65


LIST OF TABLES

(coNrrNUED)

Table

17. Weight of fresh okara tempe (before freeze drying), weight of powdered

okara tempe (after freeze drying) and evaporated dry weight of okara tempe

(after extraction) 94


LIST OF FIGURES

Figure

1 . Diagram representation of the initiation and propagation

reaction of lipid peroxidation .

Malondaldehyde, its tautomeric forms (enol, enolate) and

the proposed molecular formation, as a result ofperoxidation

ofpolyunsaturated lipids containing more than two double bonds

Classical free radical mediated autoxidation

Overall mechanism of lipid oxidation

Some synthetic antioxidants

Formulas of eight members oftocopherol and tocotrienol series

Structure of ascorbic aid and its oxidation and degradation products

Four important aglycone isoflavones produced during tempe fermentation

and possible hansformation to Factor-Il.

Formation of egocalaiferal (vitamin D2) from ergosteral

Antioxidant activity of methanolic exuacts from fried okara tempe

as measure by TBAR method

Antioxidant of okara tempe extract in liposome oxidation

system as measured by TBAR method.

Antioxidant activity of okara tempe exhact and BHT at same

concentration (500 pglml)in liposome oxidation system

Page

10

3.

4.

5.

6.

7.

8.

ll.

t2

t6

t7

23

25

28

36

389.

10.

60

53

12.

6l


xl

LIST OF FIGI]RES

(coNrrNUED)

Figure

13. Peroxide value of soybean oil treatments stored at 60oC

in the dark with okara tempe extmcts at 0.010/0

14. Peroxide value of soybean oil treatments stored at 60oC

in the dark with okara tempe extracts at 0.02%o

15. Peroxide value of soybean oil treatments stored at 60"C

in the dark with okara tempe extracts at 0.030lo

Page

67

68

70


LIST OF APPENDXES

APPENDIX

A. Dry weight of okara tempe

B. Phosphate buffer preparation

C. Determination of vitamin E by high performance liquid chromatography

D. Determination of iron-binding phenolic group [Tannin and catechin]

E. Peroxide value

F. Cleaning of labware for TBARS analysis

G. The picture shows the appearance of soybean tempe and okara tempe

H. The picture show the appearance of freeze-dried okara tempe at different

fermentation periods

Page

94

96

97

100

105

106

t07

108


AOAC

OC

et al.

h

min

M

MDA

mg

lrg

ml

rnM

pM

MLV

OD

rpm

SW

TBAR

TBARS

LIST OF'ABBREVIATIONS

The Association of Official Analltical Chemists

degree Celsius

et.Alii (Latin), and other

gram

hour

min

molar

malondialdehyde

milligram

microgram

milliliter

millimolar

micromolar

multi lamellar vesicles

optical density

revolutions per minute

small unilarmellar vesicles

thiobarbituric acid reactivity

thiobarbituric acid reactive substances


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / I

CHAPTERI

INTRODUCTION

Lipid oxidation is of great concem to the food industry because it leads to the

development of 'ndesirable

off-flavors, the loss of nutritional values such as vitamin

A, D and E, and essential fatty acids. It may arso form some toxic compounds and

colored products. During lipid oxidation, antioxidants act in various ways, i.e. chain

breakers (free-radical inhibitors), peroxide decomposers, metal inactivators, or oxygen

scavengers. These properties of antioxidants have important roles in preventing lipid

oxidation in food products and living systems. Incorporation of antioxidants in food

not only provide the wholesomeness of food but also reduce the risk of chronic and

degenerative diseases. These include ischaemia-reperfusion i"juv, chronic

inflammation, arteriosclerosis, aging, rheumatoid arthritis and cancer (l).

synthetic antioxidants such as butylated hydroxyanisole @HA), butyrated

hydroxytoluene @HT), tertiary-butylhydro-quinone (TBHe) and propyl gallate @G)

may be added to food products to retard the lipid oxidation (2). However, the use of

synthetic antioxidants is under a strict regulation due to the potential health hazards

(3). Therefore, a search for natual antioxidants as an altemative to synthetic ones is

of great interest of many among researchers.

The role of natural antioxidants in several soybean and Asian fermented soybean

products have been studied. Among these, tempe showed remarkably skongCopyright by Mahidol University

Kanitta Wanthawin Intsoduction / 2

antioxidant activity (4). It has already been reported that this fermented soybean

product is very stable to rancidity and has a good effect on preventing health problems

related to lipid oxidation.

This study was designed to assess the effectiveness of a natural antioxidant

produced from okara tempe in liposome model and oil storage model. At the same

time the vitamin E, tannin and phenolic content of okara temp€ were also determined.

okara, a high volume by-product of the soybean milk industry, has a low market

value. It may be used as an animal feed, burnt as waste or dumpt as a landfill material.

Therefore, studying the antioxidant activity from tre okara tempe could add some

economic value to the okara. It may also introduce the okara tempe as a functional

food and a natural antioxidant.

General obiective

To prepare okara tempe extract and test for antoxidant activities in some model

systems.

Specific obiective

1. To produce tempe from okara by fermentationwith Rhizopus oligosporus.

2. To estimate the ef[ect of processing (ch ling and firying) on antioxidant

activities of okara tempe.

3. To detrmine the optimum fermentation period for the okara tempe production.

4. To estimate the antioxidant action of okara tempe extract in liposome system

and soybean oil storage test.

5. To determine total phenolic compounds and vitamin E content in okara tempe.


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nurition for Development) / 3

CHAPTER II

LITERATT]RE REYIEW

2.1 FREE RADICAL AND LIPID OXIDATION

FREE RADICAL

Free radical may be defined as any chemical species that has an odd number of

electrons. It contains one or more unpaired electron (s), which is an electron that

occupies an atomic or molecular orbital by itself(1, 5-6). The presence ofone or more

unpaired electron (s) usually causes free radicals to be athacted slightly to magnetic

field (i.e. to be paramagretic) and sometimes makes them highly reactive (1, 5).

Due to high reactivity, free radicals and reactive oxygen species are capable of

causing reversible or irreversible damage to biochemical compounds (Table l), i.e.

nucleic acids, proteins and free amino acids, lipids and lipoproteins, carbohydrates and

connective tissue macromolecules (1,5, 1 4- I 6).

Radical can easily be formed when covalent bond is broken. If one electron from

each of the pair shared remains with each atom, in a process known as hemolytic

fission (l) follows.

A: B > A'+B'

Type of free radicals

There are numerous types of free radicals tlat can be formed in the biological

systems. The major free radical species of interest are those of oxygen centered free


Kanitta Wanthawin

Table 1. Cellular free radical targets (15).

Literatue Review / 4

Targets Consequences

Small molecules

Unsaturated and

containing

Nucleic acid bases

Carbohydrates

Unsaturated lipids

Cofactors

Neurotransmitters

Antioxidants

Macromolecules

Protein

DNA

Hyaluronic acid

Protein denaturation and cross-linking enzyme inhibition

Cell cycle changes, mutations

Cel[ surface reporter changes

Cholesterol and fatty acid oxidation

Lipid crossJinking

Organelle and cell permeabilility changes

Decreased nicotinamide and flavin-containing cofactor

availability and activity, ascorbate oxidation, porphyrin

oxidation

Decreased neurotransmitter availability and activity, including

serotonin, epinephrine

Decreased availability, including cr-tocopherol and p- carotene

Peptide chain scission, denaturation

Strand scission, base modification

Change in synovial fluid viscosity

radicals or ROS. The most common ROS include: superoxide anion (O2'), hydroxyl

radicals (OH'), hydrogenperoxide (HzOz) and peroxyl radicals @OO). Superoxide

anions are formed when oxygen acquires the electron, leaving the molecule with only

one unpaired electron. Hydroxyl radicals are short-lived, but the most damaging

radicals with in the body. There are wide ranges of free radical that can be generated

in living systems. Table 2 illustrates examples of these compounds. Thiyl radical

@S'), a group of radicals with an unpaired electron residing on sulphur, can be formed

from endogenous thiol compo,nd (e.g. glutathione) and subsequent hemolyticCopyright by Mahidol University

Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / 5

cleavage of disulfide bonds in proteins. A carbon-centered radical or carbon-free

radical involve in oxidation of lipid and usually react fast with 02 to make peroxyl

radicals (1, 5, 7).

Table 2. Example of free radicals (1).

Name Formula Comments/examples

Hydrogen atom

Trichloromethyl

Superoxide

Hydroxyl

ThiyUperthiyl

Peroxyl, alkoyl

Oxides of nitrogen

Nitrogen-centeredradicals

Transition-metalions

H.

cc13'

RS. /RSS.

RO2" RO'

NO" NO2'

c6HN=N'

Fe, Cu, etc.

Oz''

orf

The simplest free radical

A carbon-centered radical (the unpairedelectron resides on carbon).CCl3' is formed during metabolism ofCCla in the liver and contributes to thetoxic effects ofthis solvent. Carbonradicals usually react rapidly with 02 tomake peroxyl radicals, e.g.

CCl3'+ 02 ---'tCl3O2'An oxygen-centered radical

A highly reactive oxygen-centeredradical; attacks all biomolecules

A group of radicals that have unpairedelectrons residing on sulphur

Orygen-centered radicals formed (amongotler routes) during the breakdownof organic peroxides and reaction of carbonradicals with 02 (RO2')

Nitric oides is formed in vivo from theamino acid L-arginine; nitrogen dioxide ismade when N0' reacts with O2'-, both arefound in polluted air and smoke frombuming organic materials, e.g. cigarettesmoke

Formed during oxidation ofphenylhydrazine by erythrocytese.g. phenyldiazine radical

Ability to change oxidation numbers byone allows them to accept/donate singleelectrons; hence they are often powerfulcatalysts in free-radicals reactions


e

Oz ---------)

Kanitta Wanthawin Literah[e Review / 6

Biological sources of free radical

l. Reduction of 02

o,2- , H2O2 and OH- are considered to be generated by reduction of molecular

ox]gen (O2) in living organisms, usually univalent pathway of reduction and

sequentially production of some toxic intermediates. Thus molecular O2 can be

reduced by one electron giving a superoxide radical (o2) which can be firrther reduced

to hydrogen peroxide QI2O2) and hydroxyl radical (OH) and finally to water (8)

according to the scheme below.

e' + 2,r{ e-+tf e-+ Ifoz-

----+Hzoz----------+olf

-----------rHzo

2. Transition metal

The hydroxyl radical is the reactive oxygen species, which is found iz vivo. lt

can be formed from Oz- and HzOz via the ion-catalyzed Harber-Weiss reaction (a) or

the interaction of copper or iron in the Fenton reaction (b). These reactions are

significant as the substrates are found within the body and could easily interact (1, 5,

e-11).

(a) Haber-Weiss

H2o2 + o2'- Fe/cu catalvst >o2 + oH'+ otf

(b) Fenton

Fe2* + H2o2 Fe3*+oH-+oH'Copyright by Mahidol University

Fac. of Grad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 7

3. Radiation and photolysis

Radical chain reaction can be initiated by ionizing radiation and light. Radiolysis

is the breaking of one or more interatomic bond(s) due to exposure to high energy

radiation (e.g. x-ray, y-ray), by homolytic clevage of water (c). Radiolysis of water or

aqueous solution produce the cation radical (HzO)*', hydrate electrons (e- aq), (tt). and

(HO)', all very active to yield a host of charge and neutral secondary radicals (1, 5).

G)Radiolysis

HzO

---------| OH', H', e- aq,H2O2,H2

W light can photolyse chemical bonds as a result of energy absorption by a

molecule. It can cause bond homolysis in H2O2 and so generate hydroxyl radical

oH'(d).

(d)

UV lisht

oH + (oH)'

4. Microbial killing by phagocytic cell

Some of the production of free radicals in vivo may be accidental, but much is

functional. Phagoclic cells, neutrophils or macrophages, defend against foreign

organism by generating O2- and nitric oxide as part ofkilling mechanism (12, l3).

5. A group of cellular enzfmes metabolism

The superoxide anion appears to play a central role some, because other reactive

intermediates are formed from it. Superoxide is formed upon one-electron reduction

of oxygen mediated by enzyme such as NADPH oxidase or xanthine oxidase. The

HzOz


Kanitta Wanthawin Literature Review / 8

half life of Oz'- in tissues is dependent on the presence of enzyme superoxide

dismutase (SOD) in different cellular compartrnents. SOD catalyzes the dismutation

of superoxide anion (O2) to hydrogen peroxide (HzOz) and molecular oxygen (1, 5,

9,11,14).

Oz''

2o2- + 2r{ soD > H2o2+ e2

H2O2 is a secondary product of one-electron autoxidaion, via spontaneous or

enzymatically catalyzed dismutation of Oz'-. H2O2 is also a natural primary product of

miscellaneous oxidases, mainly the peroxisomal oxidase and some mitochondrial

enzymes. The decomposition of hydrogen peroxide to water and oxygen can be

catalyzed by catalase and glutathione peroxidase (1, 5, I l, l4).

e--------->Oxidase

Oz

2H2O2

H2O2 + 2GSHGlutathione peroxidase

2H2O + 02

2 H2O + GSSG

LIPID OXIDATION

Lipid oxidation can be divided into biological lipid oxidation and dietary lipid

oxidation (2).

Biological lipid oxidation

In cellular systems, lipid peroxidation can occur mainly in biomembranes, where

the contents of unsaturated fatty acids are relatively high. Polyunsaturated fatty acids

are essential biomolecules. They play an important role in cellular metabolism and


Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / 9

cell structure. The natural unsaturated fatty acids have only cls carbon-carbon double

bonds starting from the ninth C upward, each double bond being separated from the

other by an allylic methylene (CH2) group. Because of their peculiar chemical

structure, unsaturated fatty acids can readily react with free radicals and undergo

peroxidation.

Lipid peroxidation

Lipid peroxidation is a complex process which occurs in the presence of oxygen

and transition metal ions or enzymes. Lipid peroxidation is defined as the oxidative

deterioration of pollunsaturated lipid. There are usually three stages in the oxidation

process: initiation, propagation and termination. These processes can become

autocatalyic after initiation and yield lipid peroxides, lipid alcohols, and aldehydes

(Figure 1) (1, 5, 10, 20-21).

Initiation

The initiation of chain reaction occurs through the abstraction of a hydrogen

atom from an allylic group (CH2) of polyusaturated fatty acid side chain GfD by a

reactive free radical @') such as hydroxyl radical. Abstraction of hydrogen atom

leaves behind an unpaired electron on carbon (carbon-centered radical or lipid radical,

L)

LH+R. --}

L.+RH

Propagation

The second step is a series of propagation reaction. Carbon-centered radical (L)

or lipid radical usually reacts with molecular oxygen which binds to the radical to

form peroxyl radical (LOO').


Kanitta Wanthawin Literature Review / l0

lnitiation ofperoxidation

Removal ofH (can occur atseveral positions in the chain)

Major reactionIfabstractionAdjacent membrane

l. Oxidation of cholesterol2. Atta.k on membrane proteins3-Reaction oftwo peroxyl ndicals tocause singlet oxygen formation

A-A--A-/ ---1

| ,o,".,,., I a*oon-""n,"r"0

I rearranqement

I radicats

\,n-AJ---ll,,2

\n-\:Aotrryryilipid

Lipid hydroperoxide plusa new carbon-centeredradical that continues thechain reaction

Figure 1. Diagram representation of the initiation and propagation reaction of lipid

peroxidation (1).

Oz'Lipid peroxylradical


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / I I

The peroxyl radicals can attack membrane protein and damage receptor and

enzymes. ln addition, they can abstract another hydrogen atom from adjacent

pol,,unsaturated fatty acid to give the corresponding lipid hydroperoxide (LOO[I) as

well another lipid radical (L). The remained carbon-centered radical can react with a

second molecule of 02 to generate new peroxyl radical.

l'+Oz LOO'

LoO'+LH -----------> LooH+L'

The lipid hydroperoxide (LOOID can be broken down to a variety of radical

species in the presence oftransition metal ions, such as copper and iron (22).

LOOH + Fe3+ ------} LOO'+ Fe2* + Ff

LooH + Fe2+

-_| Lo'+ Fe3t + oH-

Termination

Propagation of the radical chain reaction takes place continuously until the

substrate is depleted. The process can be intemrpted by an antioxidant (AtI) or free

radicals produced combinding with each other. The terminations are shown below.

LOO' (or L) + AII---------il,OOH (or LH) + A'

LOO'(orL)+L' LOOLoTLz

LOO'+LOO' -----------) LOOL+Oz

Peroxidation of fatty acids containing three or more double bonds produce

malondialdehyde (MDA) (Figure 2). The production of malondialdehyde involves the

formation ofhydroperoxides, p cleavage to yield hydroperoxylaldehydes and finally


Kanitta Wanthawin

A

CH<H

Enolate

o-cH : .r-- (

tHvHtl

o -.4-=..'^\ *

l))-.-r'."tcoH

Figure 2. Malondialdehyde, its tautomeric forms (enol, enolate) and the proposed

molecular formation, as a result of peroxidation of polyunsaturated lipids containing

more than two double bonds (5).

B

ra ,/,1- ront.'

H ,,H{--} scr- cu : cu- c(o \o

o- c c- o\.r,/

Malondialdehyde

t

J u *o,.,

H

I o,.i,,iont

--<:

Literature Review / 12


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 13

a second p scission. Both malondialdehyde and acrolein radicals can combine with

hydroxyl radical (OH) to form the enol (5).

Measuring lipid peroxidation

The lipid peroxidation is a complex process and occws in multiple stages. Hence

many techniques are available for measuring the rate of peroxidation of membrane

lipids, lipoproteins or fatty acids. It can be evaluated using diflerent tests and different

mechanisms, which measuring primary and secondary breakdown products. The most

frequently measured products are volatile compound, thiobarbituric acid reactive

substances (TBARS) as secondary products. The TBARS assay measures the amount

of malondialdehyde (MDA) which is the end product of peroxidative decomposition

of pollunsaturated fatty acids (1, 5,23-25).

Health implications of biological lipid oxidation

Oxidative stress is a general term used to describe a state of darnage caused by

reactive oxygen species (17-18). This damage can affect specific molecules or the

entire organism. Although the cells are protected from these reactive oxygen species

by a number of cellular defense mechanisms, some lipid peroxidation does occur in

biomembranes under certain conditions that overcome the cell defense system. Lipid

peroxidation in membranes destroys the membrane structure and causes loss in

function of cell organelles. Receptors present in the membrane are also released or

inactivated. The secondary effects of lipid peroxidation are the initiation of new free-

radical reactions, thereby inducing changes in DNA and inflammatory reactions.

Furthermore, it activates a process of cell death by degradation of cellular components

and inactivates the cellular defense systems (1, 2,5).



Induction of lipid peroxidation has been linked to the number of diseases (l).

list of diseases related to the process of lipid peroxidation is given in Table

Although these diseases are linked to some kinds of oxidative stress, reactive oxygen

species may be responsible for biological toxicity, and lipid peroxidation can occur as

a consequence of these changes.

Table 3. Lipid peroxidation-induced diseases and effects (2).

A

J.

Diseases Remarks

LHemochromatosis Organ damage due to Fe overload leading to increaied lipid

peroxidation.

2.Keshan diseases Selenium deficiency causes a decrease in glutathione peroxidase

activity leading to increased lipid peroxidation.

3.Rheumatoid arthritis Due to Fe-induced lipid peroxidation.

4.Artherosclerosis Lipid peroxides and the reaction products oflipid peroxidation such

as hydroxyalkenals alter low-density lipoproteins (LDLs), which

are important in the development of the artherosclerotic lesion.

S.Ischaemia Occurs during reperfusion injury ofheart and brain; also results in

lipid peroxidation, probably by transformation ofxanthine oxidase

and by production of reactive orygen species.

6.Aging May be due to lipid peroxidation, but has been confirmed in

erythrocytes.

T.Carcinogenesis Wide speculation about t}te involvement of lipid peroxidation in

carcinogenesis; this is due to genotoxic effects of lipid peroxides.



Dietary lipid oxidation

Lipids in almost all foodstuffs are in the form of triglycerides, which are esters of

fatty acids and glycerides. These natural fatty acids contain straight-chain even-

number aliphatic carboxylic acids, which may be saturated or unsaturated with up to

six double bonds. The latter are normally arranged along the chain, separated from

each other by methylene groups and with cis conformation. It has been well

established that the carbon chain length and the degree of unsaturation of the fatty

acids are most critical in the determining the oxidative stability of the lipids.

Unsaturation of fatty acids makes lipid susceptible to oxygen attack leading to

complex chemical changes that eventually manifest themselves in the development of

off-flavors in food. This process known as autoxidation is a free radical mediated

process (2, 26).

Autoxidation

This is the process in food and bulk lipids which leads to rancidity. Rancidity is

the spoiled off-flavor obtained by subjective organoleptic appraisal of food.

In autoxidation, the lipid is converted to an intermediate which subsequently will

be converted to the derived lipid. In rancidity it is the derived lipid that give the off-

flavor whilst, in many of the analytical techniques used to follow oxidation, it is the

intermediate which is monitored.

Lipid + Intermediate + Derived lipid

The first step in autoxidation is called initiation step (Figure 3). The mechanisms

for this step have not been fully elucidated but they produce free radicals, e.g. both

oxygen and carbon free radicals, e.g. peroxj RO2', alkoxy RO' and alkyl R'.


Initiation

RH+O2 ------f R'+ RO2'+ OH' + H2O'

Propagation

R'+ 02

RO2'+RHBranching

ROOH

2 ROOH

Termination

__________|Roz.

ROOH +R'

--->

RO'+OH'

-----------> Roo'+ Ro'+ H2o

(l)

Q)

(3)

(4)

2R' --------->

R'+ RO2' --------->

RO2'+ RO2' ------+

RoR

ROOR

ROOR+ 02

(s)

(6)

o)

Where R = Fatty Acid Radical

ROOH Fatty Acid Hydro peroxide

Peoxyl raical

Alkoxyl radical

Roz'

RO'

Figure 3. Classical free radical mediated autoxidation (7).

Kanitta Wanthawin Literaturc Review / 16

Having produced a free radical, it can react in equation I with oxygen in a very

fast reaction with 1( = 10e lmol-rs-r. If the peroxy radical is formed it can attack

another lipid molecule or the starting molecule to remove a hydrogen. It is this

hydroperoxide which is the intermediate mentioned earlier.


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / l7

The reason v/hy these changes are so destructive is that the hydroperoxide can

break down to give two free radicals (either alkoxide or hydroxyl) or it can yield

peroxy free radical, hydroxyl free radical and water. These branching steps lead to

proliferation of free radicals which may go back to aid the propagation steps and the

reaction becomes autocatalytic. The reaction can be terminated in a low oxygen

environment by equation 5 and in a high oxygen environment by equations 6 afi7 e,

7,26-27 ).

Lipid oxidation reactions cause sensory quality changes in food, including

rancidity or off- flavor. It is generally treated as the most frequently occurring form of

lipid deterioration, which leads to polymerization, reversion, and a number of other

reactions causing reduction in the shelf life and nutritive values of the food products.

The overall mechanisms of lipid oxidation is presented in Figure 4.



Measuring lipid oxidation

The extent of lipid oxidation can be measued by chemical, sensory and

instrumental methods. The principles of some analytical methods are presented in

Table 4. Fat and oil can be evaluated in terms of peroxide value. Peroxides are the

main initial products of autoxidation. They can be measured by techniques based on

their ability to liberate iodine from potassium iodide, or by oxidizing ferrous to ferric

ions. The peroxide value is applicable for following the peroxide formation at the

early stage of oxidation. It should be performed at stable temperature because this

method is extremely sensitive to temperature changes (2, 29).

2.2 ANTIOXIDAI\IT

Unfortunately, the word antioxidant means different thing to different people.

Food technologists use antioxidant to inhibit lipid peroxidation and consequent

rancidity in food material. Food scientists, definition is implicitly restricted to chain-

breaking inhibitors of lipid oxidation such as cr-tocopherol. However, free radicals

generated in vivo damage many other targets (Table l), including protein, DNA and

small molecules. Hence a broader definition ofan antioxidant is 'any substance that,

when present at low concentrations compared to those of an oxidizible substrate,

significantly delays or prevents oxidation of that substrate (l ,l 8, 30).

Antioxidant can act at many different stages in the oxidative sequences such as

o removing oxygen or decreasing local 02 concentmtion

o removing catalytic metal ions


M.Sc. @ood and Nutrition for Development) / 19

o0)

o0)0o

oo)

z

0)

d()

IJ.ia\z

, -\J L)I

FO

-tqr C,

d =X.z2\.?-!c lVcpal2̂tillîq IqJ^rUl:E4 -L),/a -i) 'tJ-lc)t\lo

+r!r

Orzl4:E++r \J+\_r'\ EF '\2-"i"/lac, .i

z

0z)JO

I

1oz

0z)J(J

I

4

o

otl

+o

fr'-ozIztOI

&

tszI

Eo

Udz+

(J

I

g+€+I

oI

.5(JI'n

+ -* -VYvk

oool!xUIt_d,Yg^..,clli^l

'r. q/XOv)vI 2: ? uti!l-\lP iE-O IY'i

""lû).-:l + Glr r"i zU v c..lll+dc(,5

,^ lo0)

.:.,I E L: hoo cr EHqE () d >'tc L ti 6 i+.

>ro + 6 bo- trXE e= :6I9:i -ox xboE!E 9E E}Ef' - >oa

q)

GI

.: a)

o3o

-oOtribDoxEO

Or 'E>aH6N

o

o.o(nU

(l)

F

o

9>=o)(,tpoo:l<d


c.l

o

cd

,d

o

RI

XooorL)

bI)(l)

o€(D

0)oo

oo

o

(B

€)

6tF



removing key ROS/RON such as O2'',H2O2, HOCI, singlet 02 or ONOO-

scavenging initiating radicals such as OH', RO', RO2'

breaking the chain ofan initiated sequence

Many antioxidants have more than one mechanism of actions. cells have formidable

defenses against oxidative damage, many of which may at first sight not seem to be

antioxidant. Antioxidant protection can operate at different levels within the cell such

as (31)

preventing radical formation

intercepting formed radicals

repairing oxidative damage

increasing elimination of damaged molecules

o promoting the death of cells with excessively damaged DNA so prevent

transformed cells

Antioxidative defenses

The deleterious effects of ROS, RNS, RIS, and RNC are controlled by

antioxidative defenses, which are usually divided into two groups: enzymatic and

nonerzymatic antioxidant (Table 5) (1, 5, 30, 32).

The human body has an elaborate antioxidant defense system. Antioxidants are

manufactured within the body and can also be extracted from the food humans eat

such as fruits, vegetables, seeds, nuts, meats, and oil. There are two lines of

antioxidant defense within the cell. The first line, found in the fat-soluble cellular

membrane, consisting of vitamin E, beta carotene, and coenzyme e.


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) I 2l

Table 5. Some biologically important antioxidants (30,32).

Mode of action

Enzymatic antioxidant

Superoxide dismutase Cata$ic removal from cell of 02

Catalase Catalytic removal from cell of H2O2 at high concentrations

(catalatic activity). Has a peroxidatic activity when methanol,

ethanol, formate, and nitric are electron donors.

Glutathione peroxidase Catalytic removal of FI2o2 and lipid hydroperoxides.

Can effectively remove low steady-state levels of H2O2

Nonenzymatic antioxidants

Vitamin E Lipid soluble, chain breaking antioxidant. May also protect

lipoprotein lipids in the plasma.

Beta carotene Singlet orygen and Olf radical scavenge; inhibitor of lipid

peroxidations under certain condition.

Vitamin C Free radical scavenger, singlet oxygen quencher, regeneration

of vitamin E.

Glutathione Catalytic removal of hydrogen peroxide, hydroxyl radicals

quencher, singlet oxygen quencher, regeneration of vitamin E

and vitamin C.

Transferrin Binds ferric ions.

Lactoferrin Secreted by phagocytic cells, binds fenic ions and retains

them at low pH.


Kanitta Wanthawin Literatue Review / 22

of these, vitamin E is considered the most potent chain breaking antioxidant within

membrane of the cell.

The second line, inside the cell wall, oxygen scavenger are present. These include

vitamin C, glutathione peroxidase, superoxide dismutase, and catalase.

Use of antioxidant in food products

The incorporation of antioxidants in fat and oils or in foods that contain fat and

oils is effectively helpful in inhibiting the oxidation of lipid. The use of antioxidants

in food products significantly retards deterioration and extends the shelf life of many

products.

Kinds of food antioxidants

o Synthetic antioxidants. Synthetic antioxidants are the phenolic type. The

differences in their antioxidant activities are related to their chemical structures which

also influence their physical properties such as volatility, solubility and thermal

stability. The commercially available and currently used synthetic antioxidants are

butylated hydroxyanisole @HA), butylated hydroxytoluene @HT), tert-bttyl

hydroquinone (TBHQ), and propyl gallate @igure 5) (2).

o Natural antioxidants. Wide ranges of natural antioxidants have been shown to

occur in many vegetables, fruits, tea, and herbs (7). In recent years, consumers and

food manufacturers have been opting for products with'all natural' labels. The area

of natural antioxidants developed enormously in the past decade mainly because of the

increasing limitations on the use of synthetic antioxidants and the enhanced public

awareness on health issues. consumers prefer natural antioxidants because they are

considered safe (33, 34). Table 6 presents some of advantages and disadvantages of

natural antioxidants compared to synthetic antioxidants.Copyright by Mahidol University

Fac. of Grad. Studies, Mahidol Univ.

(CHr)r

M.Sc. (Food and Nutrition for Development) / 23

(CH:):

Hydrorytoluene (BHT)

CH',

/erl-butyl hydroquinone (TBHQ)

(cH3)3c

Butylated

Hydroryanisole @HA)

Propyl Gallate

Figure 5. Some synthetic antioxidants.

OH

r\,YOH



Table 6. Advantages and disadvantages of natural antioxidants compared to synthetic

antioxidants(2).

Some of antioxidant agents

Vitamin E

Vitamin E is a lipid-soluble antioxidant, which is the major natural antioxidant in

food and is important for the stability of vegetable oils. It occurs in eight different

forms: cr-, F-, y-, ^d 6-tocopherols and cr-, p1 y-, and Stocotrienols @igure 6). Their

antioxidant effrcacy decreases in order 5 > y , F > "c, whereas c-tocopherol is most

effective as vitamin E (35-38).

Vitamin E is the principal component of the secondary defense mechanisms

against free-radicals. In fact, it is the only natural physiological lipid-soluble

antioxidant that can inhibit lipid peroxidation in cell membrane (1, 39).

Advantages Disadva"tages

Readily accepted by the consumer, as Usually more-xpensive ifuurifred and

considered to be safe and not a less effrcient if not purified.

'chemical' Properties of different preparations vary ifNo safety tests required by legislation ifa not purified.

component ofa food, that is 'generally Safety often not known

recognized as safe' (GRAS) May impart color, aftertaste, or flavor

to product.


Fac. ofcrad. Studies, Mahidol Univ.

Tocopherols

CH:

R1

H

Tocotrienols

M.Sc. @ood and Nutrition for Development) / 25

CH3 CH:

CHdCH,CH,CL CH,3H

R,

Tocopherols Tocotrienols Rr R2

(t

p

v

6

(x

B

v

5

CH:

H

CH:

H

CH3

CH:

H

Figure 6. Formulas of eight members oftocopherol and tocotrienol series (l).


Kanitta Wanthawin

o-Tocopherol action

Literature Review / 26

Poll,nsaturated lipids (L$ can form alkyl radicals (L) when they become oxidized

in the presence of an initiator (X), generally an alkoxy radical (Lo') produced by

decomposition of hydroperoxides in the presence of trace metal (eq l). These alkyl

radicals react very rapidly with oxygen to form peroxyl radicals (Loo') (eq 2), which

react with more lipids to produce hydroperoxides (LoorD (eq 3). o-Tocopherol

inhibits this free radical oxidation by reacting with peroxyl radicals to stop chain

propagation (eq 4), and with the alkoxyl radicals to inhibit the decomposition of the

hydroperoxides and decrease the formation of aldehydes (eq 5). Thus, cr,-tocopherol

behaves as a chain-breaking antioxidant by competing with substrate (LFD for the

chain-carrying peroxyl radicals, normally present in highest concentration in the

system (eq 3). The tocopherol radical can form non-radical products, including

dimers, stable peroxides, alkyl or unsaturated derivatives, whereby the antioxidant is

regenerated (35-38, 40).

LH+X' -----|

L'+XH

t'+Oz ------+ LOO'

LOO' +LH------+ LOOH+L'

LOO'+AH----> LOOH+A'

LO'+411 ------f LOH+A'

vitamin E is not volatile as butylated hydroxloluene @HT) and butylated

hydroxyanisole @HA). It does not cause off-flavor as tertiary butylhydroquinone

(TBHQ). Therefore, tocopherols are now widely used as safe antioxidants.

(l)

Q)

(3)

(4)

(s)



Vitamin C (Ascorbic acid)

Vitamin C, a water-soluble antioxidant, effectively scavenges a vaxiety of free

radicals for example oz'-, Hzoz, oH', Hocl, aqueous peroxyl radicals, and singlet

oxygen (41). It has,nique 2,3-enediol moiety in the five-member ring and possess a

strong eletron-donating ability. Donation of one electron by ascorbate gives the

semidehydroascorbate radical, which can be further oxidized to dehydroascorbate

(36). The semidehydroascorbate radical is not particularly reactive and mainly

undergoes a disproportionation reaction. The reaction is that two molecules of

semidehydroascorbate yield ascorbate and dehydroascorbate. Dehydroascorbate is

unstable and broken down rapidly in a very complex way, eventually oxalic acid and

L-t}reonic acid are produced @igure 7) (36).

Ascorbic acid (AscH) react rapidly with both superoxide radical (O2) and

peroxyl radical (LOO') and even more rapidly with hydroxyl radicals (OH') and

hydrogenperoxides (H2O2) to give the semidehydroascorbate radical (Aac.) and

dehydroascorbate (DIIA) (35, 3 8)

AscH- + OH' ---------+ H2O + Asc'-

AscH- * 02' HzOz * Asc''

AscH-+ LOO._|' LH + Asc.-

AscH- + H2O2 + H* ------| 2H2O + DHA

However, it may also act as prooxidant by reacting with trace metal ions to give

hydrogenperoxide (H2O2) and hydroxyl radical (OH').


Kanitta WanthawinLiterature Review / 28

-e_* *- -rro

DHA

HJ,-{}F#HrlA

ooAsc'-

o_-c- oHIo-cIo-cI

H-C-oHI

I

CH2OH

Diketo-L-gulonic acid

OCOHI

IIo- c_ oH

oxalic acid

o < ---oHI

H -C

--__oHI

HO ---c -l{I

CH2OH

L-threonic acid

Figure 7. Structure of ascorbic aid and its oxidation and degradation products (36).



Because of autoxidation of ascorbic acid, many attempts have been made to develop

ascorbic acid derivatives to increase resistance to autoxidation. A ripophilic group

was introduced to the hydroxyl at position 2 or 3 of ascorbic acid giving 2-0-

alkylascorbic acid and 3-0-alkylascorbic acid, respectivery (4s). The ripophilic group

in ascorbic acid might exert a site-specific to active oxygen species. The reaction is

not only by maintaining an interaction with membrane phospholipids but also by

suppressing superoxide production of membrane-associated superoxide generating

system.

Ascorbic acid is noted for its complex multi-functional effects. Depending on

conditions ascorbic acid can act as an antioxidant, a metal chelator, a pro-oxidant, a

reducing agent or an oxygen scavenger (40).

Carotenoids

The carotenoids including p-carotene, y-carotene and lycopene are lipid soluble

antioxidants (35, 38). p-carotene is the most prominent representative of this

lipophilic class of compounds. It is referred to as pro-vitamin A because of its ability

to be metabolized in animals to vitamin A. p-carotene is effective as an antioxidant by

quenching singlet oxygen or free radicals that are formed during lipid oxidation, such

as the lipid radicals formed by hydrogen abstraction from an allylic cHz group, the

peroxyl and the hydroxyl radicals (5, 48-49) and scavenging of reactive oxygen

species (e.g. oxyhalides, sulfite and fenton reaction-generated radicals) (35, 3s-39).

In addition, B-carotene is efficient in a chain termination at low partial oxygen

pressures. In the presence of peroxyl radicals, p-carotene produces a carbon --centered


Kanitta Wanthawin

carotenyl radical (p-car)', which in tle absence of oxygen,

terminator (1, 5).

Literahre Review / 30

is an elficient chain

p-carotene + ROO'_____; $_car).

(p-car)' + ROO' inactive products

In the presence of oxygen the carotenyl radical reacts reversibly with oxygen to

yield a chain-propagation species, the p-carotene peroxyl radical (p-car-oo)', which

triggers f,rther oxidation.

G-car)'+ OZ ------f

(P-car-OO)'

Phenolic compounds

Phenolic compo,nds are widely distributed in plants (rabre 7), which are

important in contributing to flavor and color of many fruits and vegetable products.

The term phenolic compound embraces a wide range of substances, which possess an

aromatic ring bearing one or more hydroxyl substituents. They frequently occur

attached to sugar (glycosides) and methoxyl groups (42, 43).

Many polyphenols other than vitamin E exert powerful antioxidant effect in vitro,

inhibiting lipid peroxidation by acting as chain-breaking peroxyl-radical scavenger.

Phenols with two adjacent -oH groups or other chelating structures can also bind

transition metal ions (especially iron and copper) to form less active free-radical

promoters. This chelating ability can interfere with metal absorption in the diet.

Phenols can also directly scavenge ROS, such as OH., ONOOH and HOCI.


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / 3l

Table 7. Some dietary sources ofplant phenolic compounds.

Flavanols

epicatechin

catechin

epigallocatechin

epicatechin gallate

epigallocatechin gallate

Flavanones

naringin

taxifolin

Flavonols

kaempferol

quercetin

mlricetin

Flavones

chrysin

apigenin

Anthocyanidins

malvidin

cyanidin

apigenidin

Phenylpropanoids

caffeic acid

p-coumaric

chlorogenic acid

green and black teas

red wine

peel of citrus fruits

citrus fruits

broccoli, radish, grapefruit, black tea

onion, lettuce, broccoli, cranberry, apple skin, berries, olive,

te4 grapes, red wine

cranberry, grapes, red wine

fruit skin

celery, parsley

red grapes, red wine

cherry, raspberry, strawberry,grapes

colored fruits aad peels

white grapes, white wine, olives, olive oil, spinach, cabbage,

asparagus, coffee

white grapes, white wine, tomatoes, spinach, cabbage,

asparagus

apples, pears, cherries, plums, peaches, apricots, blueberries,

tomatoes, anise


Kanitta Wanthawin Literatue Review / 32

Thus, like p-carotene, many plant phenolics are good inhibitors of ripid peroxidation.

sometimes, however, like vitamin c, phenols can reduce transition metal ions and

exert pro-oxidant effect in vitro (l).

several plant phenols (flavonoids) can inhibit LDL oxidation such as isoflavones

glycosides (e.g' genistein and daidzein) (44). They have antioxidant activity in a

variety of in vitro assay systems (45) and a recent study showed that genistein and

dai&ein oppose estrogen action and inhibit protein kinase (46). Like other phenors,

flavonoids are often powerful inhibitors of lipid peroxidation, RoS/RNS scavengers,

metal ion binding agents and inhibitors of lipoxygenase and cyclooxygenase enzlrnes.

In isolated cells, some flavonoids have been reported to exert anti-cancer effects,

prevent expression of adhesion molecules and inhibit replication of Hrv. In whole

animals, administration of flavonoids has been reported to exert various anti-

inflammatory and anti-cancer effects (1).

The high antioxidant activity of prant phenolic compounds is considered

attractive to the food industry prompting their use as replacements for synthetic

antioxidants (47).

2.3 TEMPE

Tempe is a traditional food that is produced through fermentation process based

on soybean as substrate. During fermentation process, prominent fu..gi Rhizopus

oligosporus grows throughout dehulled and cooked soybean and formed compact

cake.

Tempe is considered as highry nutritious food, primarily as a source of protein.

It is also rich in other nutrients such as carbohydrates, fats, vitamins and minerals,


Fac. ofGrad. (Food and Nutrition for Development) / 33

with relatively high content of fiber (50).

active substances and it is easy to produce.

and easy to cook.

Tempe potency

ln addition, tempe contains a number of

It is also inexpensive food, with good taste

The benefits of tempe for human can be distinguished into two categories, firstly

as sources of nutrients, and secondly ,rs sources of active substances which are

potentially useful for health (50). Due to these advantages, tempe is prospectively

used as a firnctional food.

Nutritive value of tempe

During tempe fermentation, microorganisms enrtic./,arly Rhizopr,rs sp.) act in the

transformation for constituents of soybean. During the process> various components

are hydrolyzed into simple compounds. Hence, as regard to nutritive values, tempe

possesses several advantages such as being easily digested, rich in unsaturated fatty

acid and vitamins, and containing less anti-nutritive substances.

The constituents of tempe compared to the raw material (soybean) are presented

in Table 8.

Active substances

Besides nutrient constituents, tempe is also rich in active substances that are

produced during tempe fermentation by micoorganisms. First of all, it may be

important to define the term'active substances'. In general, they constitute secondary

metabolites, which can potentially influence metabolism and benefit for health.

cunently, several active substances in tempe and their potency for pharmaceutical and

medical uses are identified as shown in Table 9-

I !35f '

I t5Copyright by Mahidol University

Table 8. Nutrient composition of soybean and tempe (51).


48.2

23.6

28.5

3.7

6.1

14.0

6.5

50.2

19.3

30.2

7.2

3.6

34.0

39.0

19.5

7.5

9.9

3.2

1.6

28.0

(Bl) (me)

iacin @3) (mg)

0.5

0.15

0.67

0.46

0.08

34

2s-30

0.15

0.15

0.85

4.35

1.0

0.47

7r.0

140-170

5.0

0.28

0.65

2.52

0.52

0.83

53.0

0.1

3.9

42

254

781

11

347

724

9

142

240

5


No. Active substances Potency / function References

Isofl avones: daidzein, glycitine

genistein and Factor-Il

Antioxidant, antihemolysis

antifu ngi and anticancer

Gyorry, et al. (1964),

Murat4 et al (1964), Jha

i198s)

2 Unsaturated fatty acids :

oleic acid, linoleic acid

md linolenic acid

Antioxidant,

hypocholesteremic

Winamo and Reddy

(1986), Herring, et al.

(leeo)

3 Fat soluble - Vitamin :

vitamin E (mixed by cr-tocopheral and B-carotene(provitamin A))

Antioxidant, antihemolysis,

cells propagation & cells

protection, metabolisms

Bisping, et al. (1993)

4 Antibacterial compound Inhibition the growth of

several bacteria

Wang, et al. (1969)

5 Ergosterol Hlpocholesteremic,

orovitamin D

Bisping, et al. (1993)

6 Vitamin B complex :

Ihiamine, Riboflavin,

Niacin, panthothenic acid,

Cyanocobalamine, Folacin

Metabolisms (co-enzyme),

antianemia pemicious

Muratq et al (1967), Liem,

et al (1979), Bisping, et al.

(1ee3)

7 Enzlnnes : Protease, lipase,

rmylase, glycosidase,

iuperoxide dismutase

Metabolism / Hydrolysis Steinkraus (1983)

A.suti (1995)


Table 9. Active substances identified from tempe.

Source: Sudarmaji S et al, 1997 (50)

Isoflavones

Isoflavones are secondary metabolic compounds fo,nd in soybean in conjugated

form through o-glycosidic bond to sugar. In this case isoflavone conjugate is inactive.

During tempe fermentation the isoflavone conjugates are transformed by micro-

organism, so that aglycone isoflavones are released and be active substances. TheCopyright by Mahidol University


responsible microorganisms for transformation of isoflavone to aglycone form, consist

of not only the prominent ft,,gi Rhizopus oligosporus, but also other microorganisms

such as yeast or bacteria which considered as contaminant microorganisms (52).

There are 4 important aglycones (Figure 8) produced during tempe fermentation, i.e.

dai&ine, genestein, glycetein and Factor-Il (6, 7, 4, trihydroxy isoflavone).

Genistein Factor II'Qls*", -D7--,oHo

Daizein Glycitein

H

H CH:

Figure 8. Four important aglycone isoflavones produced during tempe fermentation,

and possible transformation to Factor-Il.

Unsaturated fatty acid

Lipid is metabolized during tempe fermentation, due to the activity of

microorganisms, finally fatty acids are liberated (around 40-50%), while the lipid in

soybean as well as in tempe are relatively constant, (arcwd, 20-23%). Fatty acids in

soybean are charapterized as rich in unsaturated fatty acids (around g0%). However, y-Copyright by Mahidol University


linolenic is not identified. The main unsaturated fatty acids consist of oleic

linoleic acid and crJinolenic acid. The concentration of oleic acid and linoleic

increases proportionarly with duration of fermentation time, but o-rinolenic

decreases, and optimal concentration is achieved in 24 hours of fermentation (53).

acid,

acid

acid

Unsaturated fatty acids are well known rerated to the health, particularly to the

health of heart and circulatory system. The mode of action of unsaturated fattv acids

can be described through the following mechanisms:

- Hypocholesteremic effect in blood serum

- Effect on permeability and fluidity of membrane cells, particularry brood vessel

- Inhibition of constriction processes ofblood vessel

- Decrease in platelet aggregation

- Inhibition of thromboxan formation from arachidonic acid

some unsaturated fatty acids are considered as essential nutrient, since there are

not synthesized in the body, so that they should be supplied from food.

Ergosterol

Ergosterol is a steroid compound produced by yeast and fungi, including

Rhizopus oligosporus (52). However, the property of ergosterol is opposite to

cholesterol, so that the presence of ergosterol can arso minimize the negative effect ofcholesterol' Ergosteror is also potent on the fluidity of cen membrane since ergosterol

can be integrated into cell membrane component along with fatty acids.

In addition, ergosterol acts as a precursor of vitamin D-2 (ergocalciferor). This

vitamin is important for the bone growth and the formation of paratlormon.

Chemical conversion of ergosterol (vitamin D2) to ergostrol is described in

Figure 9. Copyright by Mahidol University


cHl cH3

I I ,ca, r CHT

IH{{H=CH{H{H \ cH

H{-CH{H.CH{H3

W------------)

Ergosterol Ergocalciferol (vitamin D2)

Figure 9. Formation of egocalciferol (vitamin D2) from ergosterol.

Antibacterial compound

Antibacterial compounds in tempe, derived from water extraction of tempe, are

particularly active to Gram-positive bacteria (50). This potency has been applied in

Indonesia for curing children suffering from dianhea, and it even can be noticed tiat

tempe formula improve weight gain.

Vitamins

Vitamins are compo,nds, which are required in small quantity, however they are

essential for the maintenance of physiological and metabolic activities. vitamins

should be supplied from food, due to incapability to synthesize in the body.

Tempe is relatively rich in vitamins. The origin of vitamins in tempe comes from

the raw material (soybean) and from the synthesis by microorganism activities. The

/:H!\"

3



complicity of microorganisms during tempe fermentation produced different vitamins

and lead to enrichment of its nutritive value.

During tempe fermentation the content of vitamin B which include vitamin B 2

(riboflavin), pantothenic acid, niacin, vitamin B6 (pyridoxine), and vitamin B 12

(cyanocobalamine), increases, with an exception for thiamine (vitamin B1). Tempe as

a source of vitamin B12 constitutes a particular added value, since vitamin Bl2 is not

commonly found in vegetarian food. It is produced by bacteria which are considered

as contaminants bacteria such x Kebsiella pneumoniae, citrobacter .freundii, etc..

while the other vitamin B are produced by Rhizopus sp.

Besides vitamin B which are categorized into water-soluble vitamins, tempe also

contains a group of fat soluble vitamins, particularly vitamin A and D. vitamin A is

produced by R. oligosporus in the form of B-carotene as a precursor, while vitamin D

is produced in the form of ergosterol as a precursor. vitamin E was also recently

identified in tempe in the form of tocopherol. This vitamin is also acting as an

antioxidant.

Tempe as a source of superoxide dismutase

Superoxide dismutases are metalloenzymes. The enzyme superoxide dismutase

removes superoxide radicals and plays a sigrrificant role in inhibiting biological lipid

oxidation. It appears that SoD is essential to normal aerobic life (54). soD may also

contribute to the stability of food products. The combination of superoxide dismutase

and catalase reduces the development of oxidized flavor in heat-heated high linoleic

acid milk. There are three types of SoD, Fe-SoD, that is found in prokaryotes, Mn-

soD found in both prokaryotes and eukaryotes, wh e cuZn-soD is found only in

eukaryotes. Superoxide dismutases are caled primary scavenger because theyCopyright by Mahidol University


calalyze the dismutation of the toxic superoxide radicals to hydrogen peroxide (10, 55-

s6).

Superoxide dismutases have been detected in some foods particularry of plant

origins, they are found in fresh food so,rces e.g. spinach leaves, tomato fruit, mung

bean, com, cabbage (l). The presence of SoD in the foodstuff may be conelated with

their quality and freshness.

superoxide dismutases have been found in soybean inoculated with Rhizopus

oligosporus, Rhizopus oryze and commercial culture from the Indonesian Science

Institute (50). An increase of soD during tempe fermentation is possible due to the

protective action against superoxide radicals which is produced during mold gror+th.

The presence of SoD in soybean tempe indicated that fermentation by mold has a

good effect to the development of bioactive substances which involve in the defense

mechanism system against oxidation (50).

Antioxidative co[stituents of tempe

Tempe is well known for its antioxidative constituents. The research in this field

was initiated by isolation of a new isoflavone (6, 7, 4-trihydroxyisoflavone) from

tempe (57).

Isoflavone have been found to have antioxidant activity both in vitro (5g-59), and

iz vivo studies. It was found that tempe is able to inhibit lipid peroxidation. This was

expected both as the direct antioxidation effect of isoflavanoids in tempe and through

the iron binding capability of isoflavanoids into chelated comprexes, which then

inhibits iron, catalyst of lipid peroxidation (60).


Fac. ofGrad. Studies, Mahidol Univ. M. Sc. @ood and Nutrition for Development) / 4 I

It can be definitivery said that the antioxidative activity of tempe is the result of

the synergistic effects of the substances. The known antioxidants of tempe taking part

in the synergistic reaction are vitamin E, vitamin E-dimer (57), mixture of amino

acids, isoflavanoids and 3-hydroxyanthranilic acid (4).

Antioxidant property of tempe

The role of tempe as a health food has been extensively researched. A diet of

tempe given to rats for four months showed to successively prevent arteriosclerosis.

Moreover, consuming 120 g tempe a day for two weeks has been found to be able to

reduce blood cholesterol levels (61).

During the fermentation process an antioxidant is synthesized within tempe,

known as factor II (6, 7, 4-trihydroxyisoflavone) (62). This antioxidant was shown to

be a potent antioxidant in lipid./aqueous systems and can bind iron and prevent it fiom

catalynng oxidative reaction. The crude tempe oil has also been reported to be more

stable to oxidation compared with the oil from unfermented soybeans. This tempe oil

showed its antioxidative effect when added to soybean, cotton seed, and safflower oils

and lards (64). Tempe also contains alpha and gamma tocopherols (vitamin E), as

antioxidants which protect soybean oil against oxidative damage (61).

Tempe is not only a source of protein, but it is also a good source of minerals.

During fermentation, available calcium increases; iron availability increases and also

zinc (62). The increase in bioavailability which occurs during tempe fermentation can

be partially explained by the action of phltase which reducing phyic acid (63). The

minerals in tempe involve in redox reaction and protect against cell oxidation.


Kanitta WanthawinMaterials and methods / 42

CIIAPTER III

MATERIALS AI\D METHODS

3.1 Chemicals and materials

AII chemicals used in this study were analltical grade and food grade or the best

grade available.

3.1.1 Soybean tempe preparation

Soybean whole seed was purchased from a locar market in Bangkok, Thailand.

The strain of Rhizopus oligosporus was obtained from Thailand lnstitute of scientific

and rechnological Research (TISTR), and potato dextrose agar was purchased from

Merck Company @armstadt, German).

3.1.2 Okara tempe preparation

Soybean residues (Okara) was provided by Green Spot Company (Thailand) Co.,

Ltd. The shain of Rhizopus origosporus was obtained from Tha and rnstitute of

scientific and Technological Research (TIsrR), and potato dextrose agz, was

purchased from Merck Company @armstadt, German).


Fac. ofGrad. Studies, Mahidol. Univ. M.Sc. @ood and Nutrition for Development / 43

3.1.3 Liposome model preparation

Egg yolk lecithin egg was obtained from Fluka company (Buchs, switzerland).

Iron (t)sulfateheptahydrate (FeSoa.7H2o) and L(+)-ascorbic acid were purchased

from Merck Company @armstadt, Germany).

3.1.4 Thiobarbituric reactive substance (TBARS) measurement

Trichloroacetic acid (TCA) was purchased fiom Merck Company @armstadt,

Germany). Malonaldehyde bis-dimethyl acetates (MDA) was obtained from Aldrich

chemical company. Butylated hydroxytoluene @HT) and thiobarbituric acid (TBA)

were purchased from Fluka Company (Buchs, Switzerland).

3.1.5 Vitamin E analysis

Ethanol, potassium hydroxide (KO[I), sodium chloride (NaCl), L(+) ascorbic

acid, diisopropyl ether were obtained from Merck company @armstadt, Germany)

standard alpha tocopherol, Disodium sulphide (Na2S), Glycerar were p,rchased from

Fluka Company @uchs, Switzerland).

3.1.6 Tannin measurement

Tannic acid-molecular weight 170r, catechin-morecular weight l77g and gum

arabic were purchased from Signr.a Chemical Company (St. Louis, Mo, USA). Urea

was obtained from Univar company (sydney, Australia). DMF (dimethylformamide)

was supplied by BDH Company (poole, England). Hydrochrolic acid (HCL), Ferric

ammonium sulfate (NlIaFe(SOa)2) by Merck Company @armstadt, Germany).


Kanitta Wanthawin

3.1.7 Total phenolic content (TpC) measurement

Materials and methods / 44

Gallic acid was obtained from Sigma Chemical Company (St. Louis, MO, USA).

Folin ciocalteu reagent and sodium carbonate (Na2co3) were purchased from Merck

Company @armstadt, Germany).

3.1.8 Peroxide value determination

Acetic acid (CH3COOH), chloroform (CHCI3), potassium iodide (KI), and

sodium thiosulphate (Na2s2o3) were purchased from Merck company @armstadt,

Germany).

3.2 Preparation of soybean and okara tempe

soybean and okara tempe was prepared by a modification method of Matsuo (65)

and Keeratisuthisathron (66).

3.2.1 Soybean tempe

soybeans were used in the fermentation procedure. whole soybeans were cleaned

to remove dirt, stone, weed seeds, damaged and possibly decomposed beans as well as

any other foreign matters. After creaning they were soaked ovemight in tap water (1

kg of soybean per 3 I of water) to loosen the hulls. The soaked soybean were drained

and manually dehulled and the hull were separated by floatation, accompanied by

gentle stirring the beans. The soybeans were then autoclaved at l2l"c for 15 min,

drained and cooled to 37oc and inoculated with Rhizopus origosporus spores at

approximately 1 x107 spores per 100 g of soybean (dry weight) and mixed thoroughly.

The spore suspension ofa 3-day-ord culfxe of Rhizopus origosporus grown on potato


Fac. of crad. Studies, Mahidol. Univ. M.Sc. @ood and Nutrition for Development / 45

dextrose agar slant was used in this procedure. The inoculated soybeans were packed

in plastic bag with small holes and fermented at room temp€rature (30oc) for 24 h.

3.2.2 Okara tempe

okara was pressed to expel water to decrease moisture to about 75 %. It was then

autoclaved at l2l"c for 15 min, drained and cooled to 37.c and inoculated with

Rhizopus oligosporus spores at approximately 1 xr07 spores per 100 g of soy okara

(dry weight) and mixed thoroughly. The spore suspension of a 3-day-ord curture of

Rhizopus oligosporus grown on potato dextrose agar slant was used in this procedure.

The inoculated okara was packed in plastic bag with small holes and fermented at

room temperature (30"c) for four different fermentation periods, 0 h, 24h, 4g h nd,72

h.

3.3 Comparison of characteristic of soybean tempe aud okara tempe

3.3.1 Sensory evaluation

Soybean tempe and okara tempe that has been fermented at room temperature

(30oc) for 24 h were sliced and deep fried in soybean oir at r62c on a laboratory

scale. The fried salted tempes were given to 20 panelists for sensory evaluation. Taste,

colour, flavour, texture and overall acceptability were rated on a 9-point hedonic scale,

with 9 for like extremely and I for dislike extremely.

3.3.2 StatisticalAnalysis

Data were analyzed with SpSS progam version 10. significance within sets of

data was determined by one-way analysis of variance (p<0.05).


Kanitta Wanthawin Materials and methods / 46

3.4 Effects of heat and shelf life on the antioxidant property of okara tempe

3.4.1 Sample preparation

Five grams of fried okara tempe (FOT), ttrat had been fermented at room

temperature (30oc) for 24 h, kept for l, 4 days and 7 days in a refrigerator were

ground and extracted with 50 ml of methanol in a shaking incubator at 25.c for g hr.

The extracts were filtered through whatrnan filter paper No.l. The remaining residue

was re-extracted under the same condition, and the combined filtrates were evaporate

to dryness at 40oC by using rotary vacuum evaporator @yela Tokyo fukakiki Co.,

LTd) under reduced pressure and then weighed (67).

3.4.2 Preparation of lecithin-liposome model

Twenty grams of egg yolk lecithin were dissolved in 100 ml of chloroform.

Stock solution of lecithin (6 mt) was evaporated in 250 ml round-bottom flask with a

rotary evaporator under vacuum to form a dried thin film on the inner surface of the

flask and purged with nitrogen gas. The dried lecithin film was resuspended in 40 ml,

10 mM phosphate buffer pH 7.4 (see Appendix B) to obtain the final concentration of

lecithin in buffer at 30 mg/ml. Then lecithin solution was mixed well by using a vortex

mixer (Scientific lndustry, USA) and sonicated in ultrasonic cleaner (Farmingdale,

NY, USA) for 30 min (67) to produce sonicate vesicles (SUV) (6g).

3.4.3 Liposomeoxidation

Liposome suspension 30 mglml (l ml) was incubated with 600 pM FeSOa (0.5

ml)/ 600 pM ascorbic acid (0.5 ml) at 37'c in the presence or absence of 1 ml For

extracts and fresh okara tempe extract at various concentrations (250, 500 and 1000

pglml)for2h(69). Copyright by Mahidol University

Fac. ofGrad. Studies, Mahidol. Univ. M.Sc. @ood and Nurition for Development / 47

3.4.4 Measurement of liposome oxidation (TBAR method)

Liposome oxidation was terminated by adding 0.r ffil of 2%ilv BHT methanolic

solution. The reaction mixtures were estimated as thiobarbituric acid reactive

substances (TBARS) by adding 2 rnl rBA solution (15% wlv trichloroacetic acid,

0.37%o w/v thiobarbituric acid in 0.25 N HCI) and heating for 15 min in boiling water.

The reactions were measured the absorbance at 535 nm. Malonaldehyde bis (dimethyl

acetal; MDA) was used as a standard reference (70).

3.4.5 Determination of vitamin E

vitamin E was analyzed using high performance riquid chromatography method

according to AOAC (71) as shown in Appendix C.

3.4.6 Determination of iron-binding phenolic groups

Tannin was determined by using a modified method of Brune e/ al e2). As

shown in Appendix D.

3'5 Effects of fermentation period on antioxidant activity of okara tempe extract

3.5.1 Sample preparation

Okara tempe with different fermentation peri od, (0, 24, 4g and 72 h)was prepared

in the laboratory. Each sample was lyoph ized and vacuum packed in heat-sealed

plastic bag and stored at 5oC until used.


Kanitta Wanthawin Materials and methods / 48

3.5.2 Sample extraction

okara tempe (5 g) was extracted with 50 mr of methanol in a shaking incubator at

25"c for 8 h. The extract was filtered through whatrnan firter paper No.l . The

remaining residue was re-extracted under the same condition, and the combined

filtrates were evaporated to dryness at 40oc by using a rotary vacuum evaporator

@yela Tokyo Rikakiki co., LTd, japan) under reduced pressure and then weighed to

determine the yield (67).

3.5.3 Determination of antioxidant effect on liposome

Liposome suspension 30 mg/ml (1 ml) was incubated with 600 pM FeSOa (0.5

ml/ 600 pM ascorbic acid (0.5 ml) in the presence of extracts okara tempe extracts

from different fermentation peri od (0,24, 48,72 h) and various concentrations (0, 250,

500, 750 and 1000 prg/ml) the mixture was incubated at 37"C for 2 h (69). TBAR

formation was measured after terminating the reaction.

3.5.4 Determination of vitamin E and iron-binding phenolic groups

vitamin E and tannin were determined using the same methods as described in

Section 3.4.5 and 3.4.6.

3.5.5 Determination of total phenolic compounds

The method used for the determination of total phenols using Folin ciocalteu

reagent was adapted from McDonald et al (73). A diluted of extract or phenolic

standard was mixed with Forin ciocalteu reagent (5 ml, r:10 diluted with water) and

aqueous Na2co3 (4 nrl, 1 M). The solution was heated in a 45oc water bath for 15

min and the total phenols were determined colorimetrically at 765 '-^.

The standard


Fac. ofGrad. Studies, Mahidol. Univ. M.Sc. (Food and Nurition for Development / 49

cr'[ve was prepared using solutions of gallic acid in methanol. Total phenol values are

expressed as gallic acid equivalents.

3.6 Antioxidant activity of okara tempe extracted in soybean oil.

3.6.1 Oil storage test

Oil storage test was performed according to Tian et al, (74). Soybean oil was

provided by Thai vegetable oil company. The oil contained no additives

(antioxidants). All tests were carried out on duplicate oil samples. okara tempe

extract (0.01, 0.02 and 0.0370) was added to soybean oil and the oil was stored at 60oc

in the dark for l0 days. Additional treatments for the test included tertiary butyl

hydroquinone (TBHQ), Q.02yo) as a positive control, and a negative control

containing no additive. The samples were stored in open beakers (50 rnl). The oils

were sampled every two days for determination peroxide value by modification of

titration method according to AOAC (71) as shown in Appendix E.


Kanittta WanthawinResults / 50

CHAPTERIV

REST]LTS

4.I Characteristics of soybean tempe and okara tempe

Tempe was prepared from soaked soybean seeds as okara with an initiatal

moisture content approximately 75 %o.

Table I 0 presents the results of sensory acceptability of okara tempe compared to

soybean tempe. No significant differences (p>0.05) were found in acceptability mean

scores for color, odor, taste, texture and overall acceptance between the two kinds of

tempe.

Table 10. sensory acceptability scores from in-house panel consumer test of soybean

tempe and okara tempe.l

Mean(SD) from CRB design, n:15

\ine-point hedonic scale (9 : like extremely, 5 = neither like nor dislike, l : dislike

extremely)

3In the same column without superscripts indicates no significant difference @>0.05)

Sample Colol'3 odols I aste-" Texture2'3 Overall acceptance2'3

Soybean Tempe 6.27

(1.34\

6.00

(1.46)

5.53

0.19)

5.67

(1.40)

s.20

(r.47')

Okara Tempe 6.53

(1.4r)

5.67

(1.63)

5.20

(1.01)

5.33

(1.s4)

5.60

(1.60)


Fac. ofGrad. Studies, Mahidol Univ. M.SC. (Food and Nutrition for Development) / 5l

4.2 Influence of heat and storage time on the antioxidant property of okrra

tempe

4.2.1 Changes of antioxidative activity during storage and frying

The antioxidative activity of methanolic extracts offresh (24 h) okara tempe and

fried okara tempe with different storage time in a refrigerator ( I day, 4 days and,l

days), was investigated in metal-induced lecithin-liposome oxidation (FeSoy'ascorbic

acid)' Liposomes are simply vesicles in which buffer solution is enclosed by a cell

membrane composed of lipid molecules (usually phospholipid).

The TBAR formation of the liposome oxidation in metal induced system

(Fe2*/ascorbic acid) with the presence of three lots of fried okara tempe extract from

okara tempe with different storage time in a refrigerator and at levels of concentration

(250, 500 and 1000) pglml compared with fresh okara tempe and BHT was measured.

The results are shown in Table 11. There were no significant differences (p>0.05) in

the TBAR formation of the three lots of fried okara tempe extmct.

The average values for antioxidant activity of fried okara tempe extmcts are

shown in Figure 10. There were significant differences G<0.05) between the three

storage time. The MDA equivalence increased with the length of storage at all

concentrations. All treatrnents of the same concentration (500pg/ml) had significantly

(p<0.05) higher TBAR values when compared with fiesh okara tempe and BHT.

There were significant differences (p<0.05) between three levels of concentration of

extract. Treatments with crude extracts at concentration 250, 500 and 1,000 pglml

were had significanfly (p<0.05) lower TBAR values when compared with the


Kanitna Wanthawin Results / 52

Table 11. TBAR formation of the reaction mixture containing lot 1,2 and 3 fried

okara tempe extract. t

MEAN1SD of the analysis in triplicates

values in the same line without superscripts indicate no significant diflerence(p>0.05)

2Frot:fresh okara tempe extract

'FoT=fried okara tempe extract

BHT=butylated hydroxltoluene

Sample MDA equivalence (mM)Lot I Lot 2 Lot 3 Average

Frol 500 pg

BHT 500 pg

0.85+0.01

0.83+0.01

0.86+0.017

0.81+0.002

0.86+0.028

0.80+0.026

0.86

0.81

FOTjld0pg

FoT3 1d250pg

FoT3 ld50opg

FoT3 ldlooopg

1.38+0.02

0.9910.03

1.01+0.02

1.05+0.01

1.38+0.005

1.00+0.01

1.02+0.01

1.05+0.02

1.35+0.018

0.99+0.01

1.01+0.01

1.06+0.01

1.37

0.99

l.0l

1.05

Fof4d0pgFoT34d250pg

FoT34d500ps

FOT34d1000pg

1.38+0.01

1.16+0.01

1.08+0.01

1.07+0.02

1.38+0.01

1.15+0.01

1.08+0.01

1.08+0.01

1.35+0.02

1 l7+0.01

1.09+0.01

1.09+0.01

1.37

1.16

1.08

1.08

FOTjTd0pg

FoT37d25opg

For3Td5oopg

FoT37d10oopg

1.38+0.01

1.48+0.01

1.2510.01

1.16+0.01

1.38+0.01

1.37L0.02

1.25+0.02

1.14+0.01

1.36+0.02

1.37+0.01

1.25+0.02

l.t4+0.02

1.37

1.40

t.2s

1.15


1.6

F 1.42E r.z€)9r

.E

.E 0'8

3 o.oq)

3o*a o.2

0

Fac. ofGrad. Studies, Mahidol Univ. M.SC. @ood and Nutrition for Development) / 53

Figure 10. Antioxidant activity of methanolic extracts from fried okara tempe as

measure by TBAR method.I

1000 l?oa-+1dConcentration [microg/ml]

-+-4d+7d-r{* Frot--x-BHT

lAverage values from 3 lots

corresponding treatments with no extracts except for extracts from fried okara tempe

after 7 days of refrigerated storage at 250 lrghn concentration. The results showed

that fried okara tempe, fresh okara tempe and BHT inhibited the autoxidation of

liposome, which could act as anti-oxidant. The antioxidant activity of the fried okara

tempe extracts was less than fresh okara tempe extract and BHT. The antioxidants

showed much higher activity at r day than 4 days and 7 days of storage time in a

refrigerator, respectively.

-


Kanittta Wanthawin Results / 54

4.2.2 The vitamin e content of fried okara tempe

The antioxidant property of tempe may be the synergistic effect of some of the

tempe constituents such as the amount tocopherol present. The vitamin E content in

fried okara tempe was measured using HPLC analysis method of AOAC. Table 12

shows the vitamin E contents of three lots of fried okara tempe. From the results there

were no significant differences (p>0.05) in the vitamin E contents among three lots of

fried okara tempe. Results also revealed that storage times of fried okara tempe had no

significant (P0.05) efect on vitamin E content of fried okara tempe. When the

samples were defatted, vitamin E content markedly diminished indicating that the

vitamin E measured may come mainly from soybean oil and was also lost during

defatting.

Table 12. Vitamin e analysis of fried okara tempe.t.2.3

are means of analysis in duplicates. ND : Not detected, FOT= fried okaratempe

from tempe at different storage time in a refrigerator

2Means in the same column with different superscripts indicate significant difference (p<0.05)

3Means in the same line with the same superscripts indicate no significant difference (p>0.05)

Sample Vitamin E pgll 0Ogsample

Lot 1 Lot2 Lot 3 Average

Include

oil

FOT 1d32.64u 33.65u 33.49n 33.26

FOT4d32.05u 33.02' 32.66u 32.58

FOTTd29.82u 33.00 u

31 .89 n31.57

Not include

oil

FOTId0.54 b 0.57b 0.540 b

0.55FOT4d

ND" ND" ND" NDFOTTd

ND" ND" ND" ND


Fac. ofGrad. Studies, Mahidol Univ. M.SC. @ood and Nutrition for Development) / 55

4.2.3 Iron-binding phenolic groups in fried okara tempe

Table 13 shows the level of total tannin in fried okara tempe. From the results,

the total tannin content was not significantly different (p0.05) among three lots of

fried okara tempe. on the other hand, tannin content seem to increase with storage

time tom I to 7 days.

Table 13. The total tannin content in fried okara tempe.r,2,3

Results are means ofduplicate analysis

2Means in the same line with different superscripts indicate significant difference (p<0.05)

3Mean in the same column witlr different superscripts indicate significant difference (p<0.05)

FOT = fried okara tempe from tempe at different storage time in a refrigerator

4.3 Determination of influence of fermentation period of okara tempe on

antioxidant activity

4.3.1 Determination of antioxidant effect on liposome

Methanolic extraction of liophilised okara tempe was used in this part of the

study. The TBAR formation of the liposome oxidation in metal induced system with

the presence of three lots of okara tempe extracts at different fermentation time and

with four dosage 250, 500, 750 and 1000 pglml was investigated (as shown in Table

t4).

Sample Tannin content (mg/100 g sample)

Lot 1 I LotZ Lot3 I AVf,RAGE

FOTIdg.37"b g.0gub 7 31ub 7.92

FOT4d10.38* 10.99"" 10.79* 10.68

FOTTd12.55^d 12.ggud D.45"d 12.65


Results / 56

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Kanitta WanthawinResults / 60

BHT was used :rs a positive control. From the results, the TBAR formation of three

lots of okara tempe extracts shown no significance differences (p>0.05).

The data of rBAR forrnation of okara tempe extracts from fresh okara (not

fermented) okara tempe in metal induced liposome oxidation systems was shown in

Figure 1 L The extract of fresh okara induced liposome oxi&tion and significantly

0<0.05) enhanced rBAR formation when compared with okara tempe extracts and

control (without any extract). These results suggested that extract of fresh okara could

possible enhance autoxidation of liposome, possible by acting as a pro-oxidant.

Figure 11 Antioxidant of okara tempe extract in liposome oxidation system as

measured by TBAR method.1

0 200 400 600 8oo 10oo 1200

Concentration of OTE [miroy'm[

L_=

---.- OTE 0 h

--+- oTE 24 h

--.r- OTE 48 h

-

---r+- OTE 72 h

lAverage from tlree lotsl


Fac. ofGrad. Studies, Mahidol Univ. M.SC. @ood and Nutrition for Development) / 6l

On the other hand, the systems containing okara tempe extract from okara tempe

fermented for 24, 48 and 72 hours significantly decreased (p<0.05) the TBAR

formation when compared with control. These extracts exhibited possible inhibition

of autoxidation of liposome, by acting as an antioxidant.

As shown in Figure 12, there were no significant differences (p0.05) in

antioxidative activity of the okara tempe extract tom okara tempe fermented for 24

and 72 hours. while extracts of okara tempe fermented for 4g hours exhibited the

highest activity. The activities of all samples were also found to be comparable to that

of BHT. Moreover, there was a slight increase in the inhibition of rBAR formation

with increasing level of concentration of extract (250, 500, 750 and 1000 pglml).

x'igure 12. Antioxidant activity of okara tempe extract and BHT at sarne concentration

(500 pglml) in liposome oxidation system. I

OTEo hr OTE24 hr OTE48 hr OTE72 btS.mple conceDtrrtion .tsoonicrog/ml

'Average from three lots


Kanitta WanthawinResults / 62

4.3.2 Determination of vitamin e content

Table 15 shows the vitamin E content in okara tempe (not fried), the vitamin E

content of three lots of okara tempe showed no significant differences (p>0.05) for all

fermentation times.

The total content of vitamin E remained constant during 72 hours of fermentation

with Rhizopus oligosporus with an average value of 3.56, 3.26, 3.37 and 3.37 mg/r00

g of freeze-dried okara tempe sample from 0,24, 4g and 72 ho,rs of fermentation.

respectively.

4.3.3 Iron-binding phenolic groups in okara tempe

Table 15 shows the total tannin content in okara tempe (not-fried). From the

results, the total tannin content of three lots of okara tempe showed no significance

differences (p>0.05). However, there were significant differences (p<0.05) among 4

fermentation periods of okara tempe. At the beginning of fermentation (0 hour) the

tannin was the highest content e2.37 mgl100g) among all okara tempe samples. Then

total tarurin content of the okara tempe decreased significantly (p<0.05) with

fermentation time until tannin was not detected in okara tempe fermented for 72 hours.

4.3.4 Total phenolic groups in okara tempe

The total phenolic compounds in methanolic okara tempe extracts were measured

using Folin-ciocauteu Method. It is well known that plant polyphenolic extracts act as

free radical scavengers and as antioxidants (106).


. @ood and Nutition for Development) / 63

Table 15. Effect of fermentation period on Vitarnin E, tannin and total phenolic

content.3'4

duplicate analysis, triplicate analysis

Means in the same line without superscripts indicate no significant difference (p>0.05),

OT 0 hr = okara tempe fermented for 0 hour

OT 24 hr : okara tempe fermented for 24 hours



nd= not detect

Sample

VitaminEr

us,/100e

Tanninl

mg/l00e

Total Phenoliccompound2

[gallic acid eq uivalencesl

oT0h

lot 1 3.35

3.58

3.74

23.72

21.48

21.90

16.23

16.50

16.42

lot2

lot 3

average 3.56 22.37' 16.38"

oT 24h

lot I 3.29

3.40

3.09

15.19

14.18

15.34

68.76

69.60

68.04

lot 2

lot 3

average 3.26 14.90b 68.801b

oT48h

lot I 3.54

3.32

3.24

15.92

t5.64

16.57

83.13

84.67

83.84

lot2

lot 3

average 3.37 16.04b 83.88"

oT 72h

lot I 3.26

3.47

3.37

nd

nd

nd

71.96

71.73

72.44

lot2

lot 3

average 3.37 nd" 72.O4d

Results are means of are means of


Kanitta Wanthawin Results / 64

Table 15 shows the total phenolic compound in okara tempe. The content ofthese

compounds in tlree lots of okara tempe extracts showed no significant differences

0>0.05). The extract of okara tempe fermented for 48 hours contained the highest

polyphenol content, followed by that of okara tempe fermente d 72,24 and 0 hour.

Total phenolic compound of the okara tempe fermente d for 0,24,48 and 72 hours

were 16.38, 68.80, 83.88 and 72.04, respectively. There seem to be a good correlation

between the level of total phenol compounds in extracts of okara tempe and the

antioxidant activity as shown in Table 14.

Determination the antioxidant activity of okara tempe extract in soybean

The ability of okara tempe extract in the inhibition of soybean oil oxidation was

determined by measuring the peroxide value @V) in the oil. The exhacts were

obtained from okara tempe of four different fermentation time 0,24,48 and 72 hours

add to the oil at different dosage, 0.01%, 0.02o/o and 0.03%. TBHQ was used as a

positive control whereas a negative control contained no additive. From the Table 16

peroxide values of soybean oil with three lots okara tempe extract were not

signifrcantly different (p>0.05). The okara tempe extracts at 0.01%o was less effective

as an artioxidant in soybean oil than at 0.02 and 0.03%. No significant differences

tp>0.05) were found in the last two levels of extracts.

4.4

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Fac. ofGrad. Studies, Mahidol Univ. M.SC. @ood and Nurition for Development) / 65

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In Figure 13 shows the results of soybean oil treated with okara tempe extracts at

0.01%, stored at 60oC in the dark for 10 days. The PV of the control (no additive) and

the sample with okara tempe extract from fresh tempe or 0 hour fermentation were

higher than PV of all treatments after 2 days storage. Until day 4, no significant

differences were found among the treatments containing okara tempe extracts from

okara tempe fermented for 24, 48 and 72 hours. The treatments containing okara

tempe extracts from okara tempe fermented for 24 and 72 hours had a sigtificantly

higher PV than treatnents containing the okara tempe extracts from okara tempe

fermented for 48 hours and TBHQ after 4 days of storage. On day 6, the treatrnents

containing the okara tempe extacts from okara tempe fermented for 48 hours were

significantly lower in PV than treafinents containing the other okara tempe extracts.

The treatment containing 0.02% of TBHQ was significantly lower in PV than all other

treatrnents throughout storage.

Figure 14 shows the results of soybean oil treated with okara tempe extracts at

0.02%, stored at 60oC in the dark for 10 days. The PV of the control was higher than

PV of all treatments after 2 days storage. Until day 4, no sigrificant differences were

found among the treatments containing okara tempe extracts from okara tempe

fermented for 0,24,48 and 72 hours and TBHQ. After day 2 the treatrnent containing

okara tempe extracts from fresh tempe or 0 hour fermentation had a significantly

higher PV than the other treatments containing okara tempe extracts. The treatments

containing okara tempe extracts from okara tempe fermented for 24 and 72 hours had

a sigpifrcantly higher PV than treaunents containing the okara tempe extracts from

okara tempe fermented for 48 hours and TBHQ after 4 days of storage. The heatrnent


Fac. ofGrad. Studies, Mahidol Univ. M.Sc.(Food and Nutrition for Development) / 67

containing 0.02% of TBHQ was signiflcantly lower in PV than did all other heatnents

after day 4.

Figure 13. Peroxide value of soybean oil treatrnents stored at 60oC in the dark with

okara tempe extracts at 0.01 o/o.

Peoxide values of soybean oil 0.01% OTE

Days stored at60 C in the dark

'I

:'6_g 40qoE

930toE'X

20EID(L

12104

---a- control (no addlUve)

--a-- 0 h

---C- 24 |

--X- aa n

---*.- 72 h

---l}- TBHO 0.020,6

rAverage from three lots

TBHQdertiary butyl hydroquinoner


Kanitta wandlawin Results / 68

Figure 14. Peroxide value of soybean oil heatments stored at 60.C in the dark with

okara tempe extracts at 0.02%.

Peroxide values of soybean oil0.02%OTE

Days stored at 60 C in the dark

50

=o.ô)-ooE

top920o(L

rAverage from three lots

TBHQ:tertiary butyl hydroquinonel

--a- control (no additive)

-{-oh

-'*-24h

-X- ce h

--*-72h

--a- TBHe o.ozlo


Fac. ofGrad. Studies, Mahidol Univ. M.Sc.(Food and Nutrition for Development) / 69

During storage of soybean oil treated with okma tempe extracts at 0.03% stored

at 60'C in the dark for 10 days (Figure 15), the control treaunent was sigrrificantly

higher in PV than were all of treafinents. The treatment containing 0.02% of TBHQ

and okara tempe extracts from okara tempe fermented for 48 hours were significantly

lower in PV than all other treatrnents throughout storage. After day 4, the treatrnents

containing okara tempe extracts from fresh tempe for 0 hour and okara tempe extracts

from okara tempe fermente d for 24 hours had a significantly higher PV than

treatments containing the okara tempe extracts from okara tempe fermented for 48 and

72 hours and TBHQ. The treatrnents containing okara tempe extracts from okara

tempe fermented for 72 hours had a significantly higher PV than treatments containing

the okara tempe extracts from okara tempe fermented for 48 hours and TBHQ after 6 d

of storage.



Figure 15. Peroxide value of soybean oil treatrnents stored at 60"c in the dark with

okara tempe extracts at 0.0370.

;oo)

ot!)Eo5(!

o!'=oo)(L

60

50

40

30

20

10

0

Days stored at60 C in the dark

Peroxide values of soybean oil 0.03% OTE

_--<)- control (no additive)

--!- o h

--*-24h

--X- a8 h

--*- 72h

--{-T8HO 0.02%

lAverage from three lots

TBHQ=tertiary butyl hydroquinoner


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and nutrition for Development) / 7l

CIIAPTER V

DISCUSSION

Reactive oxygen substances (Ros) play an important rore in vital processes ofan

organism' They attack polyunsaturated fatty acids of cell membranes, causing their

oxidation and finally cell damage. Lipid peroxides formed in these reactions may

accelerate aging and also are considered to be responsibre in many diseases, including

artherosclerosis and cancer (1, 5, 14-15). Since antioxidants of dietary origin may

play an important role in preventing tissue damage stimulated by free radical

reactions, a growing interest conceming these natural sources of antioxidants is

observed.

It has long been known that soybeans and their products contain natural

antioxidants' It is interesting to note that fermented foods from soybeans ( i.e. tempe,

miso, natto, shoyu) do not lose their antioxidative properties, but in fact show

increased antioxidative activity (4 ,7g,79).

Tempe is a natural product used widely for centuries in the Far East, especialry

Indonesia and is received with great interest in the United State as a cheap basic

foodstuff for nutrition. Moreover, tempe was reported to possess many active

substances including antioxidant. Jha et al reported antioxidative effect of tempe in

rats (61)' Gyorgy described a procedure to prorong shelfJife of meat product by

adding tempe as an antioxidant (gl).


Kadtta Wanthawin Discussion / 72

5.1 Production of okara tempe

The traditional tempe is produced from soybean. However, tempe has been made

with a wide variety of legumes and grains such as faba bean, wheat, barley, rice, oat,

maize, wild rice, cowpea, mungbean, okara, and Mucuna (50, 51, 61, 78, 80-82)

instead of soybean.

In the first stage of this study okara tempe was produced and compared with

soybean tempe for the acceptance by sensory evaluation. Previous findings stated that

those of the panelists reported texture, taste and odor of soybean tempe to be better

than okara tempe as a result poor quality of okara product owing to difficulty in

controlling its high moisture content (83). Nevertheless, this difference did not

influence their preference because the result showed that the organoleptic quality and

overall acceptance of okara tempe and soybean tempe that had been deep-fried and

salted were not significantly different. Hence, it may be assumed that okara is suitable

to use as a soybean substitute in making of tempe. In this study, prior to tempe

production water was pressed out from okara to obtain a moisture content around 75

oZ. The value was approximately equal to that found in soaked soybean. The resulting

okara tempe was of acceptable quality and rated 5.60 (meaning like slightly) for

overall acceptance by the panelists.

5.2 Antioddant activity of fried okara tempe

Fried okara tempe was used to estimate the antioxidation activity by using

liposome model to demonstrate that okara tempe can be used as a functional food.

The basis for this experiment was because deep frying is among the most cornmon

preparation methods for tempe before consumption.


Fac. ofCrad. Studies, Mahidol Univ. M.Sc. (Food and nutrition for Development) / 73

Phospholipids, named as derivatives of phosphatidic acid such as

phosphatidylcholine (ecithin), are believed to be present in high amounts in cell

membranes. In order to study the antioxidant activity of fried okara tempe extracts in

biological systems, the phospholipids, prepared as liposome, was used as the model

system to evaluate the inhibitory activity against lipid oxidation in cell membranes

(67). In this experiment the method of liposome preparation was carried out according

to the hand-shaken metrod to prepare of multilamellar vesicles (MLV) incorporated

with sonication process to produce small unilamellar vesicles (SUV) (g4, g5). MLV

have a wide range in sizes, and low entrapment e{ficiency. In order to reduce their

size, sonication process was used to prepare SUV, which are more suitable for large

volume of liposome production.

oxidative damage to biological membrane is modulated by many factors such as

xanthine oxidase and organic hydroperoxide. Ros have been used to induce

membrane lipid peroxidation. In this study, metal-induced system @eSoy'ascorbate)

was used to induced lipid peroxidation of liposome (8g).

Metal-induced system leads to the formation of hydroxyl radical (OH.), which is

an extremely potent oxidizing agent and frequently proposed as the initiating RoS of

lipid peroxidation (86, 87). The mechanisms of system were shown as follow.

FeSOy'ascorbate

oz + e- ------->

2Oz' + 2f -=)Fe2* + I{2O2 ---->Fe3* + ass6r'6gte ------->

oz'

H2O2 + 02

Fe3* + oH' + oH-

Fe2* + 4sly6.oascorbateCopyright by Mahidol University

Kanitta Wanthawin Discussion / 74

Malondialdehyde (MDA), an end product of the lipid peroxidation process, was

used as an index of peroxidation in general experiments of oxidative measurement.

The TBAR assay, a colorimetric method to detect MDA, was used in this study. one

molecule of MDA reacts with two molecules of rBA, and a pink pigrnent with an

absorption maximum at 535 nm was produce d (1, 11,23-24).

As shown in Figure 10, the sample of fiesh okara tempe and BHT exhibited

better antioxidative activity. In this study, the order of decreasing antioxidation

activity was fresh okara =BHT(p>0.05)>FOTE I day storage>FOTE 4 day

storage>ForE 7 day storage (ForE=fried okara tempe extracts) at the same

concentration (500 pglml). This means that the antioxidant activity of okara tempe

was reduced by frying and longer storage time. Although the antioxidant activity of

deep fat fried okara tempe that was stored for l, 4, and 7 day in a refrigerator was

weaker than that of fresh okara, it could still prevent lipid oxidation compared to the

sample in which their methanolic extract was absent. The result implied that fried

okara might help to protect against damage to cell membrane. only the fried okara

tempe after storage for 7 days in a refrigerator at 250 pg/ml had a significantly higher

MDA level. ForE from okara tempe stored for 7 days acted as pro-oxidant due to it

ability to enhance the hydroxyl radical formatio n. yen et al rcported similar evidence

in the tea extract (89). The extract showed the both of anti and pro-oxidative activities

especially presented the pro-oxidative activity at lower dose.

vitamin E and phenolic compounds, such as isoflavone and 3-hydroxyanthanilic

acid, were known as the potent antioxidant substances in tempe (4, 50, 94). From

Table 12, fried okara tempe also contains in vitamin E. The amounts of vitamin E in

fried okara tempe was significantly higher (p<0.05) than those in defatted samples.Copyright by Mahidol University

Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food and nutrition for Development) / 75

vegetable firying oils are an excellent source of vitamin E. Therefore, a significant

increase of vitamin E content of fried okara tempe compared with defatted sample

might partially be from an increase in fat uptake during frying (93). For the storage

time, the result suggested that storage time affected vitamin E contents in fried okara

tempe. During storage a gradual decrease in the vitamin E was observed. The

decrease could be attributed to the opening of heterocyclic ring of certain tocopherols

by atrnospheric oxidation to give compound such as cr-tocoquinone which possesses

no antioxidant activity (95).

It is well known that pollphenolic groups contributed to antioxidant activity in

tempe. Tannin is high molecular weight pollphenolic compound, which presented an

inhibitory eflect on nonheme iron absorption. Gilloely and colleague (19g3)

suggested that tannin could bind nonheme iron to form insoluble iron-tannate

complexes that are poorly absorbed (96). From these adverse effects of tarmin if okara

tempe is to be promoted as healthy food with regard to its antioxidant activity, the

tannin content in fried okara tempe should be determined.

Results in Table 13 presented that not all phenolic compounds are tannins but

tannins was one of the phenolic compounds found in fried okara tempe. The tannin

contents were found to significantly increase (p<0.05) ftom I day (1.923

mg/l00gsample), 4 days (10.68 mg/t00gsample) to 7 days (12.67 mg/tO0gsample)

storage in a refrigerator. The increase was probably due to the hydrolysis of the

tannin-protein and tannin-enzyme complexes to release assayable tann in (g2,97).

From the results in this experiment, it was noted that fried okara tempe processed

antioxidant activity particularly when it was stored for a short period time. Long

storage caused a decrease in the activity. Hence, consumption of this food productCopyright by Mahidol University

Kanitta Wanthawin Discussion / ?5

could provide some health benefits to the consumer, similar to regular soybean tempe

(46,62,63). Moreover, it appeared that the antioxidant activity did not conelate well

with the vitamin E or tarurin content. In order to flfther investigate this point, the total

phenolic compounds content was also determined in the following step ofthe study.

5.3 Antioxidant activity of okara tempe extract ard its potential application

The next series of the experiment, lyophilized okara tempe was used due to its

higher stability and longer shelf life than fresh okara tempe.

In general, study on antioxidant activity of tempe depended on two conditions,

they are solvents extraction and fermentation periods. Esaki et al (1996) reported that

the potent antioxidants, which were produced during the incubation of fermented

soybeans with R. oligosporus, tended to be dissolved in methanol (4). Fermentation of

soybean was reported to generate phenolic components, which were high polarity in

similar to methanol. Therefore, methanol was selected as a solvent in the preparation

of okara extract.

The effect of fermentation periods of okara tempe on liposome oxidation was

studied. The results of the experiment (Figure I l) confirmed that the increase in the

antioxidant property was from fermentation process. Extract of fresh okara or 0 h

fermentation enhanced lipid oxidation and could significantly increase the TBAR

formation (p<0.05) when compared with okara tempe fermente d for 24, 4g and 72

hours in every concentration used (250, 500, 750 1000 pglml). Nevertheless, in the

absence of okara tempe, TBAR or MDA levels was significantly higher (p<0.05) than

when okara tempe extract was present in every concentration. These results revealed

that okara tempe extracts might be protective against damage to cell membrane since

they reduced the level of lipid oxidation. The antioxidative activity had progressivelyCopyright by Mahidol University

Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and nutition for Development) / 77

increased during fermentation of okara tempe, and the 2 days (4g h) fermented sample

had the strongest activity (Figure 12). The antioxidative activity in the 4 days (72 h)

fermentation extract dropped slightly to the level similar to I day e4 h) sample. It

was, therefore presumed that the antioxidative components in okara tempe

accumulated during the fermentation process then began to decompose after 2 days.

Halliwell and Gutteridge (1) indicated that free radical scavengers do not inhibit

the peroxidation of some membrane lipids (iposome or microsome) induced by

Fenton reaction. Damage to the membrane might result from the site-specific effect

caused by direct binding of ionic iron with tle membrane (1, 90-91). yenet al e000\

presented that EDTA chelated the iron ion on the membrane and showed a weak

scavenging effect in liposome model. On the other hand, o-tocopherol scavenged the

peroxyl radicals formed from the lipid peroxidation in the inner membrane and yield

better scavenging effect (90, 92). Therefore, the antioxidant activity of antioxidants in

a membrane system depends on their ability not only to donate a hydrogen atom but

also to incorporate into membrane. It may be possible that some components in okara

tempe could be incorporated into the membrane to provide antioxidant activity.

During okara tempe fermentation the content of vitamin E of okara tempe

remained constant. According to Denter I et al (1998'1, the total amount of vitamin E

remained constant but the content of free tocopherals decreased (98). These findings

and the fact that unfermented soybeans contain vitamin E only in free, not esterified

form suggested that vitamin E was bound by the activities of the mould during

fermentation, possibly for stabilization of membranes and protection against oxidation

(ee).



Furthermore, Total tannin levels of okara tempe was investigated. Tannins are

potential biological antioxidant because of their potential to affect protein digestibility,

metal ion availability and radical scavenging (97, 100). The fermentation periods

influenced the tannin content of okara tempe, as shown in Table 14. The content of

tarurin was reduce fiom 22.37, 14.90 and 16.04 mg/100 g of freeze dried okara tempe

to not detected in sample of fresh, 24 h, 48h, and 72 h fermented okara respectively.

The result suggested that tannin content could be reduced by fermentation (97, 101).

Similar finding were reported in the fermentation cowpeas (vigna unguiculata\ on the

nutritional quality of the cowpea meal which showed that 72 hours fermentation

increased the content of protein, ash and lipid levels while decreased the levels of

tannin and phytate (102).

Moreover, the correlation between total phenols and antioxidant activity of okara

tempe was investigated. ln general, the higher polyphenolic extraction yield

corresponded witl higher antioxidant activity, due to the combined action of the

present substances. The additive or synergistic effects of polyphenols made the

antioxidant activity of the crude extracts higher than that of isolated compounds or

simulated extract (103). Among 4 fermentation periods, 2 days fermentation okara

tempe had the highest antioxidant activity. According to a previous study 3-

hydroxyanthranilic acids (HAA), which is a co-antioxidant for n-tocopheral and able

to inhibit lipoprotein and plasma lipid oxidation in human, increased during the

fermentation of tempe and reached a maximum content at the stage of2 days (4, 104).

This may be a reason why a correlation was observed between the total between the

total polyphenolic compounds and antioxidative activity of okara tempe that had been

fermented for different periods.Copyright by Mahidol University

Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and nutrition for Development) / 79

For the potential application of antioxidant activity of okara tempe in food

matrix, use of okara tempe extract was compared with rBHQ in an oil storage test.

Methanol extracts from the okara tempe fermented for 4 different periods was added at

0.01, 0.02 and 0.03%o to soybean oil and tle result was followed by monitoring the

oxidative stability of the oil. The systems were oxidized in an accelerated condition,

60oc at atrnospheric pressure and the formation of the hydroperoxide or peroxide was

measured as peroxide value.

Significant differences (p<0.05) were observed among the 3 levels (0.01, 0.02

and 0.03o/o ) of extract used in this investigation. Extracts of okara tempe at 0.01%

was less effective than those of the other levels. Nevertheless 0.01% of extracts still

protective to oil compared to the no additive control. All of four okara tempe extracts

(0, 24, 48, 72h fermentation) showed a similar pattem of increase in pV with the

progression of storage time. Because of no differences in antioxidant pattem were

found within all of extracts, it was confirmed that 0.01% of okara tempe extracts was

less effective than 0.02 and 0.03 o% and thus the antioxidant activity exhibited a dose-

response relationship.

Extracts from fresh okara (fermented 0 h) appeared to possess the lowest

antioxidant activity (p<0.05) compared to those of other fermented periods. The

results indicated that okara tempe fermente d for 24 and 72 hours possessed greater

antioxidant activity than unfermented okara tempe. However, it was less effective in

inhibiting oxidative rancidity development in soybean oils than TBHe and okara

tempe extract from 48 hours fermentation. These results confirmed tlle results of the

earlier trial that the active components may start to decreas e after z days of

fermentation. Copyright by Mahidol University


It was also observed that fresh okara tempe extract acted as a pro-oxidant in the

liposome model whereas it showed some antioxidant effect when used in soybean oil.

Earlier research reported that some components in soybean such as genistein, the

major isoflavone showed varying degree of activity depending on concentration.

Genistein lost the control on TBAR formation at higher (>100 pM) concentration in

FeSoy'Ascorbateal2o2 (105). It can predict that there are some components in fresh

okara such as genistein in soybean can be show pro-oxidant activity in liposome,

where as possessed little antioxidative activity in soybean oil system (4).

It was of additional interest to note tlat apart from its strong antioxidant

property okara tempe extract from 48 hours fermentation did not impart any visible

color perceivable odor to the oils at the levels used. It was lightly colored, dissolved

instantly in oil upon vortex to form an emulsion which remained completely dispersed

in oils. These qualities of okara tempe extract fiom 48 hours fermentation indicated

that it could be a potential natural antioxidant for use in vegetable oil or products

containing fat and oil.

Tempe is considered as a potential food to develop as a functional food due to its

antioxidation activity (51). According to this study the highest antioxidant activity

was found in okara tempe fermented for 48 hours. However, it was not suitable for

direct consumption because when the fermentation time increased (>24 hours), the

appearance of okara tempe tumed undesirable. Nevertheless okara tempe that was

fermented for 24 hours still possessed antioxidant activity. Therefore, the production

of tempe from okara for promoting consumption presented good potential in terms of

health benefit. Its application to food system is also worth considering for use as a

natural food additive. Copyright by Mahidol University

2.

3.

Fac. ofGrad. Studies, Mahidol. Univ. M.Sc. @ood and nukitionfor development) / 8l

CHAPTERYI

CONCLUSIONS

1. Utilization of okara as a raw material to produce okara tempe by Rhizopus

oligosporus was possible. Fried okara tempe was acceptable to the panelist as like

slightly.

Fried okara tempe could be beneficial to consumer because its methanolic extmcts

inhibit the TBAR formation in metal-induced lecithin liposome oxidation model

which indicated that it possessed antioxidation activity.

Fried and stored in a refrigerator reduced antioxidation activity of okara tempe. In

this study fried okara tempe storage also caused a reduction of vitamin E but

increased the tannin and catechin contents.

4. optimum fermentation periods of okara tempe to give the highest antioxidant

activity was 48 hours.

5. During fermentation vitamin E content of okara tempe did not change, while the

tarurin decreased. Furthermore this study showed that the total phenolic

compounds content of okara tempe extracts were conelated well with their

antioxidative activity.

6. In oil storage test, extracts from fermented okara exerted an effective antioxidant

effect upon soybean oil. The order of increasing antioxidant activity was okara

extract (OE) 0 h fermented > okara tempe extracts (OTE) 24 h = OTE 72h> OTE

48 h = TBHQ. There was also a dose-response relationship between theCopyright by Mahidol University

Kanitta Wanthawin Conclusions / 82

concentmtion of extracts used and their activity within the range applied in the trial

(0.01-0.03olo).



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Kanitta Wanthawin Appendix / 94

APPENDIX A

Dry weight of okara tempe

Tablel7. Weight of fresh okara tempe (before freeze drying), weight of powdered

okara tempe (after freeze drying) and evaporated dry weight of okara tempe (after

extraction).

Okara tempe

at different

fermentation

period

Fresh okara tempe(g)

(before freeze drying)

Powdered okara

tempe(g)

(after freeze drying)

Evaporated dry weight

of okara tempe(g)

(afier extraction)

Lot I

-0h

-24h

-48h

- 72h

250.90

?50.35

250.98

250.11

44.49

46.74

43.72

41.73

4.00

10.33

7 .28

8.56

l-ot2

-0h

-24h

-48h

- 72h

250.37

25t.64

251.00

2s't.04

47;14

43.6s

41.94

4.Zt

10.80

7.75

8.82

Irt 3

-0h

-24h

-48h

- 72h

2s1.24

250.42

250.24

250.60

44.56

46.23

44.28

41.83

4.22

10.25

7.54

8.76


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development ) / 95

Tablel7. Weight of fresh okara tempe (before freeze drying), weight of powdered

okara tempe (after fteeze drying) and evaporated dry weight of okara tempe (after

extraction).

Okara

tempe at

different

fermentation

period

Fresh okara tempe(g)

(before freeze drying)

Powdered okara

tempe(g)

(afier freeze drying )

Evaporated dry weight

of okara tempe(g)

(after extraction )

Mean of 3 lots

-0h 2so.82 44.13 4.14

-24h 250.80 46.90 10.46

-48h 250.74 43.88 '1 .52

-72h 250.58 4 t.83 8.71



APPEI\IDIXB

Phosphate buffer preparation

Potassium phosphate buffer pH 7.4 preparation

The l0 mM phosphate buffer pH 7.4 was prepared in deionized water. 0.5 M of

potassium dihydrogen orthophosphate (KH2PO4) 3.36 ml and 0.5 M of dipotassium

hydrogen phosphate 16 ml were mix together to I liter. The 10 mM

phosphate buffer checked the pH value with a pH meter.


Fac. of Grad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development ) / 97

APPENDIX C

Determination of vitamin E by high performance liquid

chromatography

Principle

After homogenisation and saponification of the material under investigation in a

solution of ethanolic potassium hydroxide, the tocopherol (vitamin E) released is

totally extracted with organic solvents. Separation and determination of the

tocopherol content and done with part of extuact by reversed-phase HPLC.

Measurement is carried out against an extemal vitamin E standard that has undergone

the same procedure as the sample.

Reagent:

1. Ethanol,95%(vlv)

2. Alcoholic potassium hydroxide (KO[D 2 N: dissolve 5.6 g KOH pellets in 10 ml

deionized water and dilute to 50 ml with 95 Yo ethanol, freshly prepared.

3. Ascorbic acid, l|Yo (Vv): dissolve 10 g ascorbic acid in 100 ml deionized water,

freshly prepared.

4. NazS -glycerol solution: dissolve 120 g sodium sulfide hydrate (Na2S) in 200 ml

deionized water and mix this solution with700 ml glycerol.

5. Diisopropyl ether.

6. KOH 5%o (w/v): dissolve 5 g KOH in 100 ml deionized water.

7. Standard vitamin E: alp ha-tocopherclCopyright by Mahidol University

Kanitta Wanthawin

8. n-heptane.

9. 2%lPA in n-heptane.

70. l0o/o sodium chloride.

Procedure:

Saponifi cation and extraction

Column:

UV/VIS detector:

Mobile phase:

Flow rate:

phenomenex, 250 x 4.60 mm 5 pm

UV at 294 nm

2% IPA in n-heptane

1.5 mUmin

Appendix / 98

Weight tin duplicate) 2 g samples in a brown 250 ml saponification flask. Add l0

ml l0 %o w/v ascorbic acid solution, 5 ml Na2S-glycemlsolution, 50 ml 2N KOH

solution. Mix rurtil there is no dry sample left in the flask and reflux on a boiling

water for 30 minutes. Cool in ice- bath and add 70 ml diisopropyl ether and mix.

After separation of two layers, transfer the upper layer into a brown 250 ml separating

funnel that already contain 50 ml 5Yo KOH solution. Re-extract the sample in

saponification flask 2 times with 35 ml diisopropyl ether and combine the upper layer

in separating funnel. Shake the separating firnnel and let the layers separate and

discard the lower layer. Wash the ether exhact with 80-100 ml of l0% NaCl and wash

further with 80-100 ml water until the discard water is alkaline free (about 3 times).

Dry the ether with the strips of filter paper and transfer all ether into a 250 ml brown

round bottom flask. Dry ether by mean of rotary vacuum evaporator and blow with N2

gas and dissolved the residue in a known volume of n-heptane.

HPLC condition for vitamin E


of crad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development ) / 99

Injection volume:


Kanitta Wanthawin Appendix / I 00

APPENDIXD

Determination of iron-binding phenolic group

[Tannin and catechin]

Principle

Phenolic compounds are extracted from food sample by dimethylformamide

(DMF) in an acetate buffer. A ferric ammonium sulfatereagent is add and the resulting

color is red spectrophotometricaly at

absorbance maxima of Fe-catechol and

reagent blanks are substracted.

Reagent:

1. Acetate buffer (0.1 M, pH 7.4)

two wavelengths corresponding to

Fe-galloyl complexes. Food blanks

the

and

Solution A: add ll.5 ml acetate acid (CH3COOH) to 1000 ml by deionized

water-

Solution B: 16.4 gram of sodium acetate (CH3COONa) is dissolved and

diluted to 1000 ml by deionized water.

Mixture solution A and B (Acetate buffer): Mix 305 ml of solution A and 195 ml of

solution B, adjust the pH to 4.4 with

sodium hydroxide and diluted to 1000

ml with deionized water


Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development ) / l0l

2. 50o/o Dimethylformamide acetate buffer (50 % DMF-acetate solution): carefully

mix 500 ml dimethylformamide with 500 ml 0.1 M, pH 4.4 acetate buffer.

3. 50% Urea in acetate buffer: Dissolve 250 garnurea (H2NCONH2) in 500 ml 0.1

M, pH 4.4, acetate buffer.

4. 1%;o Arabic gum: Dislove 1 gram Arabic gum in 100 ml deionized water.

5. 5%o feric Ammonium Sulfide (FAS): Dissolve 5 gram(NlIaFe(SO4)2.12 HzO) in

100 ml 1 M hydrochloric acid (HCl).

6. Food blank reagent: prepare by mixing, just before us;

89 parts of 50% urea in 0.1 M. acetate buffer solution

l0 parts of 1% Arabic gum

l partof 5%of I MHCl

7. FAS reagent (Iron reagent): Prepare by mixing, just before use

89 parts of 50% urea in 0.1 M. acetate buffer solution

10 parts ofl% Arabic gum

I part of 5%o ferric-ammonium sulphate

Apparatus:

l Spectrophotimeter

2. Vortex mixer

3. Automatic pipette

Procedure:

1. Weight 2-5 gram food sample into a 125 ml Erlenmeyer flask

2. Add 50 ml 50% DMF-acetate solution.

3. Sample flask was covered with parafilm and shaken in a shaking machine for 16

hours at room temperatureCopyright by Mahidol University

5.


4. Remove sample food shaker and flter through a paper filter lWhatman No.541)

2 ml of the filtrate is vigorously shaken with 8 ml FAS-reagent in a 15 ml test tube.

After 15 min the sample is read at 578 and 680 nm against a reagent blank

consisting of 2 ml DMF-acetate and 8 ml FAS-reagent.

The food blank is prepared by mixing 2 ml of filtrate with 8 ml of food blank

reagent in 15 ml test tube. Read against a blank consisting of 2 ml DMF-acetate

and 8 ml food blank reagent at both wavelengths.

7. Values for food blank are substracted from

wavelength. The result net extinction values

calculation.

polyphenol extinction at each

at 578 and 680 nm are use in

8. Standard solutions containing tannic acid (TA) and catechin (C) are read with

unknown samples, reblank and food blanks in each series.

Standard preparation

l. Standard tannin: Dissolve 0.5 gram of tannic (SiGma Cat. No. T-0125) in

50% DMF acetate and make up to 50 ml volume.

2. Standara catechin: Dissolve 0.5 gram of tannic acid (SiGma CaL No. C-1788)

in 50% DMF acetate and make up to 50 ml volume.



Working standard (conc. 25-400 pg/ml):

Concentration (pglml)

25

M.Sc. (Food and Nutrition for Development ) / 103

50

100

200

400

Vol. Pipette (ml)

1.25

0.25

0.5

1.0

2.0

The food blank absorbance is subtracted from the food sample absorbance at 578

nm and 680 nm. The absorbance spectra of two kinds of Fe-phenolic complexes

overlapped. The content of galloyl and catechol groups in the sample is, therefore,

calculated using linear regression equations for the four standard curves, tannic acid

(galloyl groups) and catechin (catecholl groups) at two wavelengths. Thresulting

equation set is readily solved with a programmable calculator.

l. abbreviations

A. Unknown samples;

N578 : Net sample extinction at 578 nm

N680 = Net sample extinction at 680 nm

SW = Sample weight in gram

Standard solutions

St. ext. TA 578 = Standard extinction tannic accid at 578 nm

St. ext. TA 680 = Standard extinction tannic accid at 680 nm

St. ext. C 578 = Standard extinction catechin at 578 nm

St. ext. C 680 = Standard extinction catechin at 680 nmCopyright by Mahidol University

Kanitta Wanthawin

St. conc. TA

St. conc. C

T ext. 578

T ext. 680

2. Calculations

Appendix / 104

: Standard concentration tannic acid pglml

= Standard concentration catechin pglml

= True extinction at 578 nm (tannin extinction)

= True extinction at 680 nm (tannin extinction)

A. Extinction rations of standard;

Kl : St. ext. C 578St. extTTEO-

K2 = St. ext. TA 578St. exr TA-680

B. Calculation of content of tannin and catechin in unknown samples

Step I. Calculation oftrue extinctions;

T ext. 578 = N578 -K, Ns78

l-Kr Kz

T ext. 680 = N68o-K, N68o

1-Kr Kz

Step II. Calculation of amounts of catechin equations (mg/100g) and

tannin equivalents (mg/l 00g)

Tannin equivalents: St. conc. TA x 50 x T ext. 578 x 100(mg/100g) SW x St. ext. TA 578

Catechin equivalents = St. conc. Cx 50x Cext x 100(mg/100g) SW x St. ext. 680


Fac. of Grad. Studies, Univ. M.Sc. (Food and Nutrition for Development) /105

APPENDIXE

Peroxide value

Peroxide value was determined by modification of tritration method 1711. Three

gram of oil was dissolved in 30 ml of glacial acetic acid and chloroform mixture (3:2,

v/v). Add 0.5 ml saturated potassium iodide solution, let stand with occasional

shaking I min, and add 30 ml of co2 free distilled water. Slowly tritrate with 0.01 N

sodium thiosulfate solution (NazSzo:.5Hzo) with vigorous shaking until yellow color

is almost gone. Add 0.5 rnl l% starch solution as an indicator, and continue titration,

shaking vigorously to release alr 12 from chroroform layer, until blue color just

disappears. Peroxide value was expressed as milliequivalent peroxide per kilogram

fat.



APPENDIXF

Cleaning of labware for TBARS analysis

Labware is first washed with detergent in the normal manner. Soak the

detergent-washed labware in 2.5Yo v/v HCI for at least 2 hours, or ovemight. Then

thoroughly rinse labware tlree times with deionised water. Drain labware well

between washing.


Fac. of Grad- Studies, Mahidol Univ. M.Sc. lTood and Nutrition for Development) / 102

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NAME

DATE OF'BIRTH

PLACE OF BIRTH

INSTITUTIONS ATTENDED

HOMEADDRESS

M.Sc. (Food & Nutrition for Development) / 109

BIOGRAPHY

Miss Kanitta Wanthawin

30 September 1974

Bangkok, Thailand

Srinakharinwirot University, l9g4_1g97

Bachelor of Science (Food Science and

Nutrition)

Mahidol University, 1998_2002:

Master of Science (Food and Nutrition

for Development)

35/24 Soi. Mettanue prachasongkroh Rd.,

Dindaeng Bangkok 10320

Tel.0-2245-4624

-


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