lttK toqnLO OA
9\d
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
\.iiiil ct:':r1;liri,oi
.......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
[emailprotected]. 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
Copyright by Mahidol University
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--/
Copyright by Mahidol University
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ.
4136715 NUFN/M : dlt liyt : o't1.11:[[aylflyutnt:
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Thesis / v
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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 hom*olytic 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 hom*olysis 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
Copyright by Mahidol University
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
Copyright by Mahidol University
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').
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
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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).
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Kanitta Wanthawin Literature Review / l4
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.
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / 15
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'.
Copyright by Mahidol University
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.
Copyright by Mahidol University
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.
Copyright by Mahidol University
Kanitta Wanthawin Literature Review / l8
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
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M.Sc. @ood and Nutrition for Development) / 19
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Fac. ofGrad. Studies, Mahidol Univ.
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Copyright by Mahidol University
Kanitta Wanthawin Literature Review / 20
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.
Copyright by Mahidol University
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.
Copyright by Mahidol University
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
Copyright by Mahidol University
Kanitta Wanthawin Literature Review / 24
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.
Copyright by Mahidol University
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).
Copyright by Mahidol University
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)
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 27
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').
Copyright by Mahidol University
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).
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 29
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
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
Copyright by Mahidol University
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,
Copyright by Mahidol University
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).
Kanitta Wanthawin Literature Review / 34
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
Copyright by Mahidol University
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)
Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 35
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
Kanitta Wanthawin Literature Review / 36
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
Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / 37
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
Kanitta Wanthawin Literature Review / 38
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
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / 39
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
Kanitta Wanthawin Literature Review / 40
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).
Copyright by Mahidol University
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.
Copyright by Mahidol University
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).
Copyright by Mahidol University
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).
Copyright by Mahidol University
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
Copyright by Mahidol University
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).
Copyright by Mahidol University
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 f*ckakiki 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.
Copyright by Mahidol University
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
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)
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
1.6
F 1.42E r.z€)9r
.E
.E 0'8
3 o.oq)
3o*a o.2
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.
-
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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).
Copyright by Mahidol University
. @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
OT 48 hr : okara tempe fermented for 48 hours
OT 72 hr : okara tempe fermented for 72 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
Copyright by Mahidol University
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
oil
Copyright by Mahidol University
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aF Copyright by Mahidol University
Kanitta Wanthawin Results / 66
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
Copyright by Mahidol University
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
Copyright by Mahidol University
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.^o)-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
Copyright by Mahidol University
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.
Copyright by Mahidol University
Kanitta Wanthawin Results / 70
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
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
Copyright by Mahidol University
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).
Copyright by Mahidol University
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.
Copyright by Mahidol University
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).
Copyright by Mahidol University
Kanitta Wanthawin Discussion / 78
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
Kanitta Wanthawin Discussion / 80
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).
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 83
REFERENCES
1. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 3rd ed.
Oxford Clarendon: Oxford University; 1999.
2. Madhavi DL, Salunkhe DK, Deshpande SS. Food Antioxidants. New york:
Marcel Dekker; 1996.
3. Hettiarachchy NS, Glenn KC, Gnanasanbandam R, Johnson MG. Natural
antioxidant extract from fenugreck. JFood Sci 1996;61:516-19.
4. Esaki H, Onozaki H, Kawakishi S, Osawa T. New antioxidant isolated from
tempeh. J Agric Food Chem 1996;44: 696-700.
5. Roberfroid M, Calderon PB. Free radicals and oxidation phenomena in biological
systems. New York: University of Catholique de Louvain Brussels; 1995
6. Mason RP, Chignell CF. Free radicals in pharmacology and toxicology selected
topics. Pharmacol Rev 1982;33: 189-2ll
7. Hamilton RJ, Kalu C, Prisk E, Padly FB, Pierce H. Chemistry of free radicals in
lipids. Food Chem 1997;60(PA): g3-99.
8. Boonyarat C. Synthetic of nicotinyl amide and chroman amide derivatives
Bangkok:inhibits of lipid peroxidation II [M.S Thesis in pharmacology].
Faculty of Graduate Studies, Mahidol Univercity; 1996.
9. Stahl W, Sies H. Biochemistry of oxidative stress. Inst physiol Chem 2001; 50:
10' chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs.
Physiol Rev 1979; 59: 527 -605.Copyright by Mahidol University
Kanitta Wanthawin
1 l. Halliwell B, Gutteridge JMC, editors. Method in
References / 84
enzymology. Oxford:
Acadamic Press; 1990.
12. Weiss SJ, Lobuglio AF. Phagoclte-generated oxygen metabolism and cellular
injury. Lab krvest 1982;47:5-18.
1 3.ai{'uvrf u:6:rfiarl. ierrfiudruoonfirnfu lu :u.rn'nuai qrr:.ltu'yfin{1, u::0r1;nr:.
n?r!fr1?ildrill.trndryimurto.ruruavol?n5rd; ii1l.riu{qlorq. n!uylt{{ : nruy
rnfrrqran{ umriflu1fruufl'aa; zs3gi r4i1 so-64.
14. Wem SW, Lucchesii BR. Free radicals and ischemic tissue injury. Tips 1990;
ll:161-6.
15. Freeman BA, Crapo JD. Biology of disease free radicals and tissue injury. Lab
Invest 1982; 47 : 412-26.
16. Borek C. Antioxidant health effects of aged garlic extract. J Nutr 2001; 131:
1010s-5s.
17. Temple NJ. Antioxidants and diseases: more question than answers. Nutr Res
2000: 20(Pt 3): 449 .
18. Aruoma OI, Spencer JPE, Wanen D, Jenner P, Butter J, Halliwell B.
Characterization of food antioxidants, illustrated using commercial garlic
and ginger preparations. Food Chem 1997;60@2):149-56.
19. Bray TM. Dietary antioxidants and assessment of oxidative stress. Nutrition
2000, 16: 578-81.
20. Porter NA. Chemistry of lipid peroxidation. Methods Enzymol l9B4; 105:273-
305.
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 85
21. Diplock AT. Antioxidant nutrient and disease prevention: an overview. Am J cli
Nutr 1991; 53: 189-93.
22. Rosen GM, Britigan BE, Halpem HJ. Free radical biology and detection by spin
trapping. Oxford: Oxford Univercity Press; 1999.
23. Punchard NA, Kelly Fj, editor s. Free radical a practical approach. Oxford:
Oxford University Press; 1996.
24. Draper HM, Itadly M. Malondialdehyde determination as index of lipid
peroxidation. Method Enzl,rnol 1990; 186: 421-31.
25. Moure A, Cruz JM, Franco D, Domingurz JM, Sineiro J, Pominguez H, etal.
Natural antioxidants from residual sources. Food Chem. 2001 ; 72: 145-7l.
26. Char S HW. Autoxidation of unsaturated lipids. London: Acadamic Press;
1987.
27. Allen J, Anjelo ST. Lipid oxidation. Washington, D.C.: American Chemical
Society;1992.
28. Lee SK, Mei L, Decker EA. Lipid oxidation in cooked turkey as affected by
added antioxidant enzyme. J Food Sci 1996 6l(Pt 4\:726-28.
29. Allen JC, Hamilton Rj. Rancidity in foods. 3rd ed. London: Chapman & Hall;
1994.
30. Aruoma OI. Free radicals and antioxidant strategies in sports. J Nutr Biochem
1994;,5:370-81.
31. Gilbert DL, Carol CA. Reactive oxygen species in biological systems. New
York: Kluwer Acadamic; 1999.
32. Maxwell SRJ. Prospects for the use of antioxidant therapies. Drug 1995;49 345-
61. Copyright by Mahidol University
Kanitta Wanthawin References / 86
33. Asamarai AM, Addis PB, Epley RI, Krick TP. Wild rice hull antioxidant. J
Agric Food Chem 1996; 44:126-30.
34. Aluz M, Zanora R, Hidalgo Fj. Natural antioxidants produces in oxidized
lipid/amino acid browning reaction. JAOCS 1995;72 (ptlZ): l17l-75.
35. Basu Tk, Temple NJ, Gars ML, editors. Antioxidants in human health and
disease. New York: CABI; 1999.
36. Machlin Lj, editor. Handbook of vitamins. 2nd ed. New York: Marcel Dekker;
1991.
37. Packer L. Protective role of vitamin E in biological systems. Am J Clin Nutr
1991;53: 1050s-5s.
38. Roberts DCK. Antioxidant vitamin. Curtin (ACT): Pharmaceutical Society of
Australia; 1998.
39. Mascio PD, Murphy ME, Sies H. Antioxidant defense systems: the role of
' carotenoid, tocopherols, and thiols. Am J ClinNutr 1991:53: 194s-200s
40. Frankel EN. Antioxidants in lipid foods and their impact on food quality. Food
Chem 1996; 57@ 1): 51-57 .
41. Helmul S. Oxidative stress: oxidants and antioxidants. London: Acadamic Press:
1991.
42. Cardenas E, Packer L. Handbook of antioxidants. New York: Marcel Dekker:
1996.
43. Sies H. Antioxidants in disease mechanisms and therapy. London: Acdamic
Press; 1997.
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ. M.Sc. @ood and Nutrition for Development) / 87
44. Hangson JM, Croft KD, Puddey IB, Mori TA, Beilin LJ. Soybean isoflavonoids
and their metabolic products inhibit in vitro lipoprotein oxidation in serum.
J Nuh Biochem 1996:7:664-69.
45. Cook NC, Samman S. Flavonoids chemistry, metabolism cardio protection
effects and dietary sources. J Nutr Biochem 1996; 7:66-76.
46. Cassidy A, Bingham S, Setchell KDR. Biological effects of a diet soy protein rich
in isoflavones on the menstrural cycle of premenopausal women. Am J
Clin Nutr 1994; 60: 333-340.
47. Balasinska B, Troszy A. Total antioxidative activity of evening primrose
(Oenothera paradoxa) cake extract measured in vivo by liposome model
and murine Ll 2 1 0 cell. J Agric Food Chem 1998; 43 : 3558-63.
48. Kennedy TA, Liebler DC. Peroxyl radical scavenging by p-carotene in lipid
bilayers. J Biol Chem 1992;267:4658-63.
49. Motencen A, Skibsted LH. Importance of carotenoid structure in radical-
scavenging reaction. J Agric Food Chem 1997; 45:2970-77.
50. Sudarmaji S, Supramo, Raharjo S. Reinventing the hidden miracle of tempe.
Indonesia: Indonisian Tempe Foundation; 1997.
51. Steinhaus KH. Handbook of indigenous fermented food. New York: Marcel
Dekker; 1996.
52. Bisping B, Baumann U, Keuth S, Denter J, Wiesel L, Rehm HJ. Tempe
fermentation formation of protease and vitamin and some ecological aspect.
Tempe workshop. Febuary 15-16; BPP Teknologi. Jakarta;1993.
Copyright by Mahidol University
55.
56.
Kanitta Wanthawin References / 88
53. Wagenknecht AC, Mattrick LR, Lewin LM, Hand DB, Steinkraus KH. Change
in soybean lipide during tempeh fermentation. J Food Sci 1960; 26:373-
76.
54. Asuti M. Superoxide dismutase in tempe an antioxidant enzyme and its
implication on health and disease. J Nutr 1997; 122:. 625-26.
Fridovichl. Superoxide dismutase. Annu Rev Biochem 1975;44:147-59.
Benov L, Fridovich E. Functional significance of the CuZnSOD in E. Coli. Arch
Biochem Biophys 1996; 327 : 249-53.
57. Hoppe MB, Chandra J, Egge H. Structure of an antioxidant from fermented
soybeans tempe. JAOCS 1997; 74:477-99.
58. Pratt DE, Birac PM. Source of antioxidant activity of soybean and soy products.
J Food Sci 1979;44: 1720-22.
59. Record IR, Dreosti IE, Maclnemey JK. The antioxidant of genistein in vitro. J
Nutr Biochem 1995; 60@ 6): 481-85.
60. Asuti M. The role of tempe on lipid profile and lipid peroxidation. AM J Clin
Nutr 1998; 1522s.
61. Aganoff J. The complete handbook of tempe. New York: The American Soybean
Association; 1999.
62. Myliopawho SML, Gorden F,Gorden D. Bioavailability of zinc in fermented
soybean. J Food Sci 1989; 53: 460-63.
63. Kasaoka S, Asuti M, Uehara M, Suzuki K, Goto S. Effect of Indonesian
lipid peroxidation infermented soybean tempe on iron availability and
animic rat. J Agric Food Chem 1997;45: 195-98.
Copyright by Mahidol University
64.
Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development) / 89
Gyorgy P, Murata K, Suginmoto Y. Studied on antioxidant activity of tempe oil.
JAOCS 1974; 5l:377-79.
Matsuo, M., Hitomi, E. Suppression of plasma cholesteral elevation by okara
tempe in Ral Biosci. Biotech. Biochem 19931'57 (Pt 7): 1188-90.
Keeratisuthisathron, A. Nutritive value of protein in soybean residue fermented
wtth Rhizopus oligosporus arid Bacollus subtilis. [M.S thesis in
Phamacology]. Bangkok: Faculty of Graduate studies, Chulalongkom
University; 1996.
Duh PD, Du PC, Yen GC. Action of methanolic extract of mung bean hulls as
inhibitors of lipid peroxidation and nonJipid oxidative damage. Food and
chemical toxicology 1999; 37: 1055-61.
Prasad R, Manual on Membrane lipids. lth ed. New York: Springer-Verlag
Berlin; 1990.
Yen, GC, Hsieh CL. Antioxidant activity of extracts from du-zhong (Eucommia
ulmoides) toward various lipid peroxidation models in vitro. J Agric Food
Chem. 1998; 46: 3952-57.
Burge JA, Aust SD. Microsomal lipid peroxidation. In: Moldave k, Grossman L,
editor. Method in enzymology vol. 30 (Nucleic acids and protein synthesis:
part F). Califomia: Academic Press; 1978; p.302-10).
AOAC Intemational. Official Method of Analysis of AOAC intemational. l6th
ed.; 1995.
Brune M, Hallberg L, Skanberg AB. Determination of iron-binding phenolic
$oups in food. J Food Sci 1991; 56(Pt 1): 128-31.
65.
66.
67.
68.
69.
70.
71.
Copyright by Mahidol University
Kanitta Wanthawin References / 90
73' MacDonald S, Prenzler PD, Antolovich M, Robards K. phenolic content and
antioxidant activity ofolive extracts. Food Chem 2001:73 73_g4.
74. Tian LL, white PJ. Antioxidant activity of oat extract in soybean and cottonseed
oil. JAOCS 1994; 7t(Pt l0): 1079-86.
75. Miller DM, Buetbrer GR, Aust SD. Transition metals as catalysts of autoxidation
reactions. Free Rad Bio & Med 1990; 8: 95-108.
76. Belitz FID, Grosch W. Food Chemistry. New york: Springer-Verlag Berlin
Heidenberg; 1987.
77. Wettasinghe M, Shahidi F. Antioxidant activity of performed cooked cured-meat
pigment in B-carotene/linoleate model system. Food Chem 1992; 58(pt 3):
203-07.
78. Berghofer E, Grzeskowiak B, Mundigler N, Sentall WB, Walcak J. Antioxidant
of faba bean, soybean and oat tempeh. Intemational J of Food and Nutr
1998;49:45-54.
79. Esaki H, Onozaki H, Kawakichi S, Osawa T. Antioxidant activity and isolation
from soybeans fermented w:tth Aspergillus spp. J Agic Food Chem 1997;
45(6):2020-24.
80. Egounlety M, Aworh OC, Akingbala JO, Houben JH, Nago MC. Nutritional and
sensory evaluation of tempe-maize based weaning foods. Intemational J of
Food and Nutr 2002: 53: 15-27.
81. Gyorgy p. Food product containing tempeh. US Patent 3.681 .095. 1972.
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ.
82. Mugula JK, Lyimo M. Evaluation
fingermillef based tempe as
Intemational J of Food and Nutr
M.Sc. (Food and Nutrition for Development) / 9l
of nutritional quality and acceptability of
potent weaning foods in Tanzania.
1999;50:275-82.
83.
84.
Iftaidej L. Studied on tempe and tempe like products. Food 1976; g (3): 2l_2g.
New RRC. Liposome a practical approach. New york: Oxford University press;
1990.
85. Prasad R. Manual on Membrane lipids. New york: Springer-Verlag Berlin
Heidenberg; 1995.
86. Ohyashiki T, Nunomura M. A marked stimulation of Fe3*-dependent lipid
peroxidation in phospholipid liposomes under acidic condition. Biochem
Biophys Acta 2000; 1484 241-50.
87. Ohyashiki T, Karino T, Matsui K. Stimulation of Fe2* induced lipid peroxidation
in phosphatidylcholine liposome by aluminium ions at physiological pH.
Biochem Biophys Acta 1993; I 170: 182-88.
88. Donovan JL, Meyer AS, Waterhouse AL. Phenolic composition and antioxidant
activity of prunes and prune juice @runus domestica). J Agric Food Chem
1998: 46: 1247-52.
89. Yen GC, Chen HY, Peng HH. Antioxidant and pro-oxidant ef[ect of various teas
extmcts. J Agric Food Chem 19971' 45: 30-4.
90. Yen GC, Chuang DY. Antioxidant properties of water extracts from Cassra toa L.
in the relation to the degree of roasting. J Agric Food Chem 2000; 4g:
2760-65.
91. Benzie FF. Lipid peroxidation a review of causes, consequences, measurement
and dietary influences. Intemational J of Food and Nutr 1996; 47 : 233-61 .Copyright by Mahidol University
Kanitta Wanthawin References / 92
92. Ratty Ak, Sunamoto J, Das Np. Interaction of flavonoids with 1,1- diphenyl-2-
picrydrazyl free radical, liposomal membranes and soybean lipoxygenase-l.
Biochem Phamacol 1 988; 37 : 9t9-95.
93. Fillion L, Henry CJK. Nutrient losses and gain during firying: a review.
Intemational J of Food and Nutr 1998; 49: 157-16g.
94. Hoppe MB, Jha HC, Egge H. Structure of an antioxidant from fermented sovbean
(tempe). JAOCS 1997; 7 4(Pt 4): 477 -79.
95. Patterson HBW. Handling and storage of oilseeds, oils, fats and meal. New
York: Elsevier Science; 1989.
96. Gillooly M, Bothwell TH, Torrance DJ, MacPhali Ap, Derman Dp, Bezwoda
WR" Mills W, Carlton RW. The effects of organic acids, phytates and
polyphenols on absorption of iton from vegetables. Br J Nutr 1983; 49:
331-42.
97. Hagerman AE. Tannin chemistry [online]; 2002. Available from:
htto ://www.users.muohio.edu/hagermae/tannin.pdf.
[Accessed 2002 Sep 29]
98. Denter J, Rehm HJ, Bisping B. Change in the content of fat-soluble vitamins and
provitamins during tempe fermentation. Intemational J of Food Micro
1998;'45:129-34.
99. Stillwell W, Ehringer W, Wassall SR. Interaction of cr-tocopheral with fatty
Biochem Biophys Acta 1992; 1105: 237-acids in membranes and ethanol.
44.
Copyright by Mahidol University
Fac. ofGrad.
100. cheynier v, Larbarbe B, Moutounet M. Estimation of procyanin chain length.
In: Packer L. Methods in Enzynology vol. 335@lavonoids and other poly
phenols). Califomia: Acadamic press;2001. p. g2-96.
101' obizoba IC, Egbuna HI. Effect of germination and fermentation on nutritional
quality of bambara n:ut (Voandzeia subterranean L. Thouars) and its
product (milk). Plant Food for Human Nutrition 1992 42: 13-23.
lO2.Nnam NM. Evaluation of nutritional quality of fermented cowpea (Vigna
unguiculata) flours. Ecology of Food and Nutrition 1995;71:514-lg.
103. Moure A, Franco D, Sineiro J, Dominguez H, Nunez MJ, Lema JM. Evaluation
of extracts fuom Gevuina avellana hulls as antioxidants. J Agric Food
Chem 2000; 48: 3890-97.
104. Thomus S\ Witting PK, Stocker R. 3-Hydroanthranillicacid is an efficient, cell-
derived co-antioxidant for cr-tocopheral, inhibiting human low density
lipoprotein and plasma lipid peroxidation. J Bio Chem 1996; 27l!,t 5l)l
32714-21.
105. Record IR, Dreosti IE, Mclnerney JK. The antioxidation activity of genistein in
vitro. Nutr Biochem 1995; 6: 481-85.
l06.Sato M, Ramarathnam N, Suzuki Y, Ohkubo T, Takeuchi M, Ochi H. Variental
differences in the phenolic content and superoxide radical scavenging
potential of wines from different sourses. J. Agric Food Chem 1996, 44,37-
41.
;r;r, ;;;-i-" Sc (Food and Nutrition for Deveropment) / e3
Copyright by Mahidol University
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
Copyright by Mahidol University
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 ftee*ze 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
Copyright by Mahidol University
Kanitta Wanthawin Appendix / 96
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.
Copyright by Mahidol University
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 hom*ogenisation 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
Copyright by Mahidol University
of crad. Studies, Mahidol Univ. M.Sc. (Food and Nutrition for Development ) / 99
Injection volume:
Copyright by Mahidol University
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
Copyright by Mahidol University
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.
Kanitta Wanthawin Appendix / 102
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.
Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ.
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
Copyright by Mahidol University
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.
Copyright by Mahidol University
Kanitta Wanthawin Appendix / 106
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.
Copyright by Mahidol University
Fac. of Grad- Studies, Mahidol Univ. M.Sc. lTood and Nutrition for Development) / 102
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Copyright by Mahidol University
Fac. ofGrad. Studies, Mahidol Univ.
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
-
Copyright by Mahidol University