1. Effect of genistein on the reduction of Maillard reaction in heated mixed protein-lactose suspension
Presented by
Md. Anisur Rahman Mazumder and Parichat Hongsprabhas
Good morning every body, I am Md. Anisur Rahman Mazumder, PhD student. Department of Food Science and Technology, Kasetsart University, Bangkok Thailand. Today I like to present some parts research titled as “Effect of genistein on reduction of Maillard reaction in heated mixed protein-lactose suspension “
Department of Food Science and Technology
Faculty of Agro-Industry, Kasetsart University

2. Development of functional food Soy product???
I am in Danger of
bone loss
Suffering malnutrition
hormone-dependent breast & prostate cancers
symptoms of menopause
Any solution?
Development of functional food Soy product???
yes
Both male and female populations are increasing throughout the world including Bangladesh and Thailand, but the problem is women are suffering much more malnutrition than men and also in threaten of breast and cervix cancer It is also known that women have symptoms of menopause when they reach a certain age.  Women lose bone mass more rapidly than men.
Researches revealed that soy isoflavones and their glycosides are associated with a lower incidence of cardiovascular disease, hormone-dependent breast and prostate cancers, colon cancer, menopausal symptoms and osteoporosis
osteoporosis
soy isoflavones & their
glycosides reduce
cancers
menopausal symptoms
cardiovascular disease

3. The demand soy milk are increased
High amounts of protein, iron , unsaturated fatty acids
and niacin
Low amounts of fat, Carbohydrates & calcium as compared
with cow’s and human milk
Genistein, the major isoflavone in soy products
inhibitor of tyrosine kinase activity
The demand and intake of soy milk and soy products are increased in Bangladesh for several reasons : High amounts of protein, iron, unsaturated fatty acids and niacin, Low amounts of fat, carbohydrates, and calcium as compared with cow’s milk and human milk. Genistein, the major isoflavone in soybeans, is a powerful inhibitor of tyrosine kinase activity in vitro and, thus, inhibits cell growth proliferation. It has also been shown to inhibit cell cycle progression and normalizes normalizes transformed cells, both of which are consistent witha protective effect against cancer.
inhibits cell growth proliferation
inhibit cell cycle progression and normalizes

4. Why worry about Maillard reaction?
Aesthetics
Overly browned foods are not asthetically attractive
Nutritional effects
Amino acids that participants in the Maillard reactions are lost
Mutagenicity
There is still the possibility that highly browned food may contain potential mutagens
Oh, yes
Oh, No
Possible toxicants
Acrylamide
Overly browned foods are not asthetically attractive and may not be accepted
Some researchers find mutagenic products in browned model systems. But not yet settled in the literature. But there is still the possibility that highly browned food may contain potential mutagens
Amino acids that participants in The Maillard reactions are lost from a nutritional piont of view
Furan
Heterocyclic amines
3-Methyl Imidazolone
3-Mono Chloro Propan Diol

5. Maillard reaction (MR)
Non-enzymatic browning
Complex set of reactions between amines usually from proteins and carbonyl group from
Reducing sugar
Aldehyde/keto group of oxidized lipids
Aldehyde/keto group of oxidized polyphenols
Maillard reaction: it’s a non-enzymatic browning reaction and complex set of reaction between amines, usually from proteins, and carbonyl group, from reducing sugars, aldehyde or ketone group of oxidized lipids and polyphenols and dehydroascorbic acid of oxidized ascorbic acids. The consequences are the formation of many desirable and undesirable products most of them associated with flavor and color compounds.
Bittner, 2006
Dehydroascorbic acid of oxidized ascorbic acid
Gregory, 1996; Handwerk and Coleman, 1988;
Namiki, 1988; Pischetsrieder et al., 1998; Yin, 1996

6. Advanced stages Final stages Initial stages
In initial stage of Maillard reaction, condensation of carbonyl group of reducing sugar with a free amino group to give a condensation product N-substituted glycosylamine, which rearranges to form the Amadori rearrangement product (ARP). The subsequent degradation of the Amadori product is dependent on the pH of the system. At pH 7 or below, it undergoes mainly 1,2-enolisation with the formation of furfural (when pentoses are involved) or hydroxymethylfurfural (HMF) (when hexoses are involved). At pH >7 the degradation of the Amadori compound is thought to involve mainly 2,3 enolisation, where reductones, and a variety of fission products, including acetol, pyruvaldehyde and diacetyl are formed. All these compounds are highly reactive and take part in further reactions. Dicarbonyl compounds will react with amino acids with the formation of aldehydes and a-aminoketones. This reaction is known as the Strecker degradation. Subsequently, in an advanced stage, a range of reactions takes place, cyclizations, dehydrations, retroaldolisations, rearrangements, isomerisations and further condensations, which ultimately, in a final stage, lead to the formation of brown nitrogenous polymers and co-polymers, known as melanoidins.
Final stages
Fig. 1: Maillard reaction scheme adopted from Hodge

7. Reactive carbonyl species (RCSs)
MRPs and MR intermediates in food
Associated with increased biological exposure of
carbonyl stress and AGEs formation
3 Deoxyglucosone
Methylglyoxal
Glyoxal
Glycolaldehyde
Maillard reaction products (MRPs) have been broadly studied in foods for decades because of their importance on flavor/acceptability, the nutritional value and toxicity. MRPs and MR intermediates in food, such as RCSs, have also been associated with increased biological exposure of carbonyl stress and AGEs formation. Examples of RCSs generated by the MR (in food) or by glycation (in vivo) include compounds such as 3-deoxyglucosone, methylglyoxal, glyoxal and glycolaldehyde, among others. Reactive carbonyl species glyoxal, methylglyoxal, and 3-deoxyglucosone, generated by the Maillard reaction, are important intermediates for flavor and color formation.
Figure 2: RCS in Maillard reaction

8. Dietary phenolic compounds reported to reduce RCSs
Certain polyphenols trapped RCS, indicating a new
chemical characteristic of polyphenols
Jiang and Peterson, 2010
The phenolic compounds can control MR pathways
By scavenging RCSs via adduct formation
Genistein
Epicatechin
Catechin A C B
Certain polyphenols were recently shown to have trapping activity for reactive carbonyl species, indicating a new chemical characteristic of polyphenols . Trapping reactive carbonyl species by polyphenols can alter Maillard reaction pathways. For example, some flavor compounds normally generated in the Maillard reaction, such as pyrazine, methylpyrazine, 2,5-dimethylpyrazine, and trimethylpyrazine, were inhibited when epicatechin was added to a glucose-glycine model system.
Figure 3: Dietary phenolic compounds

9. Hypothesis
Genistein could act as an anti-browning agent
in the heated protein-lactose suspensions
Objectives
To investigate the effect of genistein and genistein concentration as anti-browning agent in the heated soy protein isolate (SPI) and whey protein concentrate (WPC) suspensions in the presence of lactose

10. Experiment
1.1 Effect of protein and lactose concentration on advanced glycation end product (AGE) formation
Fluorescence intensity* was measured before and after heating of protein (WPC) suspension at 90oC for 30 min
1.2 Effect of genistein on AGE formation of heated protein suspensions in the presence of lactose
For MR both amino group and carbonyl group is important, but we try to make confirm which one has more pronounced effect on AGE formation. So the 1st experiment was Effect of protein and lactose concentration on advanced glycation end product (AGE) formation of heated protein suspensions. WPC was used as protein source for this experiment. AGE formation measured as fluorescence intensity was measured before and after heating of WPC suspension at 90o C for 30 min.
Therefore, the influence of protein concentration and the presence of genistein on the formation of AGEs were further investigated. Effect of genistein on AGE formation of heated protein suspensions in the presence of lactose. Again Fluorescence intensity* was measured before and after addition of genistein in the heated protein suspension at 90oC for 30 min
Fluorescence intensity* was measured with or without genistein in the heated protein suspension at 90oC for 30 min
* Pan and Melton, 2007

11. 1.3 Determination of brown pigment (BR) formation
WPC, SPI ± Genistein
Heat at 90oC for 30 min
Cooled (25oC)
Diluted to 0.08% protein w/v
BR= absorbance at 420 nm- absorbance at 620 nm
Modified from Hongsprabhas et al., 2011
1.4 Effect of genistein concentration on AGE formation of
heated protein suspensions in the presence of lactose
Determination of brown pigment formation. Protein suspensions were prepared in distilled water, heated at 90 °C for 30 min, cooled to room temperature (25 °C) and evaluated for brown pigment using a diluted 0.08% protein (w/v) suspension by measuring the absorbance at 420 nm with a UV–visible spectrophotometer and corrected for turbidity by subtracting the absorbance at 620 nm.
The roles of genistein concentration on the formation of AGEs were further investigated in heated WPC-lactose suspension. Effect of genistein concentration on AGE formation of heated protein suspensions in the presence of lactose. Fluorescence intensity* was measured after addition of linear genistein concentration in the heated protein suspension at 90oC for 30 min.
Fluorescence intensity* was measured after addition of different concentration of genistein in the heated protein suspension at 90oC for 30 min
* Pan and Melton, 2007

12. Before heating
After heating
1.1 Effect of protein and lactose concentration on advanced glycation end product (AGE) formation
Figure 4 shows the effect of lactose concentration on AGE formation. In x axis is total lactose concentration (w/v) and in Y axis is advanced glycation end products (AGE) measured as fluorescence intensity, a) before heat and b) after heat. Heating mixed WPC-lactose at 90 C for 30 min increased the advanced glycation end product (AGE) formation, measured as fluorescence intensity, as shown in Figure 1. The 2-way ANOVA also showed that protein concentration had more pronounced effects than did lactose concentration in AGE formation. Therefore, the influence of protein concentration and the presence of genistein on the formation of AGEs were further investigated.
Total lactose (% w/v)
Total lactose (% w/v)
Protein concentration had shown more pronounced effect than lactose during AGE formation
Fig. 4 Effect of protein and lactose concentration on AGE formation
(measured as fluorescence intensity)

13. 1.2 Effect of genistein on AGE formation of heated protein suspensions in the presence of 5% lactose
Protein concentration showed more pronounced effect than lactose contestation. So, the lactose concentration is fixed and focused on protein concentration and added genistein. Figure 5 shows the effect of genistein on AGE formationPaired compared t-test showed that The presence of added genistein at the genistein to protein ratio of 1:1000 did not have significant effect on AGE formation in heated WPC-lactose suspensions. However, added genistein could reduce AGE formation in heated SPI-lactose suspension. It should be noted that the magnitude of AGE formation in heated SPI-lactose was much lower than that in heated WPC-lactose suspension.
Paired compare test between no genistein an added genistein
Fig. 5 Effect of genistein addition on AGE formation in heated
protein-lactose mixtures containing 5 % lactose: (a)
WPC and (b) SPI

14. Genistein was more effective in SPI than WPC; may be due to:
Amino acids profile
Denaturation temperature of protein
Indigenous genistein
The efficiency of added genistein on SPI over WPC was likely due to amino acid composition, denaturation temperature of both protein source and/or the presence of indigenous genistein in SPI and needs further clarification.

15. Amino acids with ԑ- and end terminal amino groups are the
most reactive in the Maillard reaction
lysyl residue in protein molecules have ԑ- amino group
The most reactive functional group in milk proteins
the ԑ-amino group of lysyl
During Maillard reaction lysyl residure are the most reactive amino acids due to its ԑ- amino groups in protein molecules. Usually the ԑ-amino group of lysyl residue is the preferential site for initiation of the Maillard reaction during processing as well as storage of foods which contains protein and carbohydrates. The ԑ-amino group of lysyl residue was most reactive functional group in milk proteins along with indolyl group of tryptophan, the imidazole group of histidine, the guanidino group of arginine and the α-amino group of proteins and free amino acids. So its very much necessary to analyze the amino acid profile of both protein.
the indolyl group of tryptophan
the imidazole group of histidine
the guanidino group of arginine

16. Indigenous genistein Heat denauration involves
Unfolding of the protein
Which exposed reactive groups for Maillard reaction
Indigenous genistein
the magnitude of AGE formation in heated SPI-
lactose was much lower than that in heated WPC-
lactose suspension
Protein denaturation is the unfolding of the tertiary and secondary structure with out unfolding of peptide bond. Generally, the globular structure of native protein maintains by some force like hydrogen boding, van der wals force, hydrophobic and electrostatic interactions. Heating is the physical means by which bonding can be ruptured and protein unfold into random configuration which exposed reactive side for Maillard reaction.
The magnitude of AGE formation in heated SPI- lactose and WPC-lactose suspension indicates that there is a great effect of indigenous genistein on AGE and brown pigment formation. For clear understanding, the analysis of denaturation temperature and indigenous genistein contents is necessary.

17. 1.3 Effect of genistein on brown pigment formation
The presence of genistein prior to heat treatment reduce brown pigment formation (p<0.05)
Figure 6 shows the effect of genistein on brown pigment formation. Although the added genistein did not have significant effect on AGE formation in heated WPC-lactose suspension, it could significantly reduce brown pigment formation in heated WPC-lactose suspension. The role of added genistein on the reduction of brown pigment formation in heated SPI-lactose, however, was not pronounced as it did on AGE formation. This was probably due to the low AGE formation in heated SPI-lactose during intermediate stage to proceed to full Maillard brown pigment formation.
Fig. 6 Effect of protein source and genistein addition on the reduction
of brown pigment in heated protein-lactose mixtures containing
5.5% final lactose

18. 1.4.1 Effect of genistein concentration on AGE formation of
heated protein (4% w/v) suspensions in the presence
of lactose (5% w/v)
Increasing the genistein concentration significantly reduce the AGE formation (p<0.05)
Figure 6 a) shows the Effect of genistein concentration on the reduction of AE formation in heated protein-lactose mixtures containing 5 % total lactose. Y axis- AGE formation measured as fluorescence intensity and X-axis genistein concentration mg% w/v. The roles of genistein concentration on the formation of AGEs were further investigated in heated WPC-lactose suspension. It was found that increasing the genistein concentration significantly reduced the AGE formation during heating of WPC-lactose suspensions in non-linear fashion.
Fig. 6 a) Effect of genistein concentration on the reduction of AGE
formation in heated protein-lactose mixtures containing 5 %
total lactose

19. 1.4.2 Dose-response effect of genistein on AGE formation
Increasing dose-response slopes and decreasing genistein concentration reflect the reduction of AGE formation
.This equation Y=Bottom + (Top-Bottom)/(1+10^((X-LogIC50))) represents the response, measured as AGE formation, when genistein bind to reactive group involved Maillard reaction. The bottom response was the lowest AGE formation at high genistein concentration while the top response was the highest AGE formation at zero genistein concentration.
Increasing dose-response slopes and decreasing genistein concentration reflect that AGE formation decreased significantly with increasing genistein concentration. From best-fit values (R2 = ), it was calculated that the IC50 of genistein in inhibiting the AGE formation in heated WPC-lactose (4% protein and 5% lactose) was mg %. Genistein likely acted as an agonist that binds to a reactive group and causes an inhibitory response. The curve will go downhill with increasing agonist concentration.
Fig. 6 b) Dose response curve fitting for effect of genistein
concentration on the reduction of AGE formation

20. The presence of genistein could reduce the Maillard
reaction by two possible ways:
trapping of dicarbonyl compounds A C B A C A C B B
The presence of genistein could reduce the Maillard reaction by two ways: (i) trapping of dicarbonyl compounds and/or (ii) attraction for amino residue of both carbonyl group of genistein and lactose. Genistein might trap α-dicarbonyl compound by electrophillic aromatic substitution reaction; where the major mono- and di- α dicabonyl conjugated adducts of genistein could be formed. The carbon (C) positions 6 and 8 of the A ring of genistein is major active sites for trapping α-dicabonyls. On the other hand, carbonyl group of both genistein and lactose has attraction for amino group of proteins, which helps to reduce MRPs formation during heating of WPC-lactose and SPI-lactose.
Methylglyoxal
Genistein
Mono-MGO adduct
Di-MGO adduct
Lv et al., 2011
attraction for amino residue of both carbonyl group of
genistein and lactose

21. Conclusions Genistein reduce the AGEs and browning pigment formation
Genistein can act as an anti-browning agent in heated
protein suspension

22. Acknowledgements Kasetsart University for PhD scholarship
Siew Sngim Karnchanchari Research Leadership Award for research grants

23. for your kind attention
Thank you very much
for your kind attention