Otieno, Daniel Obed (2007) Stability of bioactive isoflavones and glycolytic enzymes produced by probiotic bacteria in soy based food during processing and storage. PhD thesis, Victoria University.

Abstract

Micro-organisms possess endogenous enzymes, however, the stability of these enzymes during storage in soymilk has not been studied. β-glucosidase is an important enzyme that could be used in the bioconversion of the predominant soy isoflavone glycosides to their bioactive aglycone forms. Fifteen probiotic micro-organisms that included Bifidobacterium sp., Lactobacillus acidophilus and Lactobacillus casei were screened for β-glucosidase activity using ρ- nitrophenyl-β-D-glucopyranoside as a substrate. Six strains were selected on the basis of β- glucosidase activity produced during fermentation of soymilk. The stability of the enzyme activity was assessed during incubation for up to 48 h and during storage for 8 weeks at frozen(-80°C), refrigerated (4°C), room (25°C) and incubation (37°C) temperatures. L. casei strains showed the highest β-glucosidase activity after 24 h of incubation followed by L. acidophilus strains, while Bifidobacterium strains showed least activity. However, β-glucosidase from Bifidobacterium animalis BB12 showed the best stability during the 48 h fermentation. Lower storage temperatures (-80°C and 4°C) showed significantly higher (P<0.05) β-glucosidase activity and better stability than that at higher temperatures (25°C and 37°C). The stability of β-glucosidase from these microorganisms should be considered for enzymic biotransformation during storage, of isoflavone β-glycosides to bioactive isoflavone aglycone forms with potential health benefits. Three strains of L. acidophilus, two strains of L. casei and one strain of Bifidobacterium were screened for β-glucosidase activity using ρ-nitrophenyl-β-D-glucopyranoside as a substrate and their potential for the breakdown of isoflavone glycosides to the biologically active aglycones in soymilk. Isoflavones quantification with HPLC and β-glucosidase activity was performed after 0, 12, 24, 36, and 48 h of incubation in soymilk at 37°C. All 6 microorganisms produced β-glucosidase, which hydrolysed the predominant isoflavone β-glycosides. There was a significant increase in the concentration of isoflavone aglycones and a subsequent decrease (P < 0.05) in the concentration of isoflavone glycosides in fermented soymilk. Based on the concentration of isoflavones during peak β-glucosidase activity, the hydrolytic potential was evaluated. L. acidophilus 4461 had the highest aglycone concentration of 76.9% after 24 h of incubation, up from 8% in unfermented soymilk (at 0 h). It also had the best isoflavone hydrolytic index of 2.01, signifying its relative importance in altering the biological activity of soymilk. Soymilk fermented containing soy isoflavones with B. animalis Bb12 was stored at various temperatures (-80°C, 4°C, 25°C and 37°C) for 8 weeks and the concentration of isoflavones determined weekly using RP-HPLC. The first order kinetic model was used to assess the degradation of each isoflavone isomer at each storage temperature. During storage at various temperatures, concentrations of individual isoflavone isomers appeared to be significantly stable (P<0.01). Interestingly, the aglycones showed much smaller degradation constants as compared to the glycosides at all the storage temperatures. Genistein and daidzein were much more stable than glycitein and had almost similar degradation pattern, despite differences in their concentrations in the fermented soymilk. It was, however, observed that 4°C was the most suitable storage temperature for the product as there was a minimal degradation of bioactive isoflavone aglycones. Three selected L. acidophilus strains were used in the fermentation of soymilk and then stored separately at various temperatures (-80°C, 4°C, 25°C and 37°C) for 8 weeks and the concentration of isoflavones determined weekly using RP-HPLC with diode array uv visible detector. The decreasing concentration of isoflavones in soymilk during storage due to degradation was found to fit first order kinetic model. Isoflavone aglycones as well as isoflavone glycosides largely appeared to be stable during storage (P<0.01). Interestingly, the aglycone forms showed much smaller degradation as compared to glycoside forms at all the storage temperatures. Of the isoflavone aglycones, daidzein was most stable followed by genistein, while glycitein was least stable. Isoflavone aglycones such as glycitein, daidzein and genistein showed smaller degradation constants in fermented soymilk at lower storage temperatures (-80°C and 4°C) and higher degradation constants at higher storage temperatures (25°C and 37°C) with each strain. In contrast, glycosides glycitin and daidzin showed higher degradation at lower storage temperatures (-80°C and 4°C) and lower degradation at higher storage temperatures (25°C and 37°C). Storage temperature was therefore found to be very important in regulating the rate of degradation of soy isoflavones in fermented soymilk. The degradation of each isoflavone compound in soymilk fermented with 2 Lactobacillus casei strains and stored at various storage temperatures (-80°C, 4°C, 25°C and 37°C) was evaluated and again found to fit the first order kinetic model. All isoflavone compounds in the soymilk appeared to be generally stable during storage (P< 0.01) at all storage temperatures. Aglycone forms however, had smaller degradation constants compared to glycosides at all storage temperature in the presence of each of the micro-organisms. Specifically, aglycones showed a unique trend of smaller degradation at lower storage temperatures (-80ºC and 4ºC) than at higher temperatures (25ºC and 37ºC). Glycoside genistin was least stable at all storage temperatures compared to other isoflavones, while aglycone daidzein was the most stable. L. casei 2607 in fermented soymilk stored at 4ºC after 8 weeks gave the least degradation for daidzein of a mere 3.78% loss from 9.53 to 9.17 ng/µL. L. casei 2607 showed greater hydrolytic potential than L. casei ASCC 290 as denoted by higher degradation of isoflavone glycosides in fermented soymilk at lower storage temperatures. The optimum storage temperature offering least degradation of bioactive isoflavone aglycones in fermented soymilk was found to be 4ºC. Liquid chromatography coupled with positive electro spray ionisation tandem mass spectrometry (MS/MS) and diode array detection was used for the quantitation and characterisation of isoflavones in fermented and unfermented soymilk made from soy protein isolate SUPRO 590. Bifidobacterium animalis ssp. lactis Bb12 was used for the fermentation of soymilk. The isoflavones were found to produce characteristic radical ions as well as molecules of H20, CO2, a sugar unit, and an alcohol through collision-induced fragmentation. Product ion fragments revealed unique fragmentation pathways for each isoflavone compound. Characteristic fragmentation of different isoflavones were unequivocally identified and differentiated. The occurrence of aldehydes such as pentanal, ethanal and methanal was shown to be specifically linked with isoflavone aglycones, daidzein, genistein and glycitein, respectively. Main glycosides such as genistin, daidzin and glycitin as well as the acetyl-, and malonyl forms also showed respective aglycone ions in their spectra fragmentation. Thus positive ion fragmentation was important in the unequivocal confirmation of isoflavones and for revealing the occurrence of other related compounds such as aldehydes in the soymilk. Comparison of endogenous β-glucosidases and β-galactosidases in selected probiotic bacteria as hydrolysing enzymes in the breakdown of the predominant isoflavone glycosides in soymilk into bioactive isoflavone aglycones is critical for an optimised processing of a probiotic functional food. β-glucosidase activity and β-galactosidase activity of probiotic organisms including L. acidophilus ATCC 4461, L. casei 2607 and B. animalis ssp. lactis Bb12 in soymilk was evaluated and correlated with the increase in concentration of isoflavone aglycones during fermentation. The concentrations of isoflavone compounds in soymilk were monitored using a Varian model HPLC with an amperometric electrochemical detector. In all microorganisms, β-glucosidase activity was found to be greater than that of β-galactosidase. The aglycone concentration in the soymilk with L. acidophilus 4461, L. casei 2607 and B. animalis ssp. lactis Bb12, increased by 5.37, 5.52 and 6.10 fold, respectively after 15 h of fermentation at 37ºC. The maximum hydrolytic potential was also observed at 15 h of fermentation for the three micro-organisms coinciding with peak activities of the two enzymes. β-glucosidase activity was found to be more than 15 times higher than that of β-galactosidase activity in the soymilk for each microorganism during fermentation. It appears β-glucosidase played a greater role in isoflavone hydrolysis. It is important to determine critical parameters such as which hydrolysing enzyme have a greater impact in the development of a probiotic functional food beverage. In this case it is essential to enhance β-glucosidase activity for its greater role in improving the biological activity of soymilk during processing. Having established that endogenous β-glucosidase plays a greater role in isoflavone biotransformation, it was essential to compare endogenous and exogenous β-glucosidases for their role in isoflavone biotransformation. β-glucosidase activity of probiotic organisms including Bifidobacterium animalis ssp. lactis Bb12, Lactobacillus acidophilus ATCC 4461 and Lactobacillus casei 2607 in soymilk was evaluated and found to relate to the increase in concentration of isoflavone aglycones during fermentation. The concentrations of isoflavone compounds in soymilk were monitored using a Varian model HPLC with an Amperometric electrochemical detector. The aglycone composition, also known as aglycone equivalent ratio, has been considered to be important for delivery of health benefits of isoflavones, was also monitored during fermentation of soymilk. Comparison of the hydrolytic effectiveness of both exogenous and endogenous enzyme during 4 h incubation in soymilk was conducted using the Otieno-Shah (O-S) index. Results showed that exogenous enzyme exhibited faster rate of isoflavone glycoside hydrolysis than that by endogenous enzyme. Highest O-S indices were obtained after 4, 3 and 2 h of incubation with enzyme solution having β-glucosidase activity of 0.288 UmL-1, 0.359 UmL-1, and 0.575 UmL-1 resulting into aglycone concentration increments of 5.87, 6.07 and 5.94 fold, respectively. Conversely, aglycone concentration in the soymilk with B. animalis ssp. lactis Bb12, L. casei 2607 and L. acidophilus 4461 increased by 3.43, 2.72 and 3.03 fold, respectively after 4 h of fermentation at 37ºC. Also, the O-S index of endogenous enzyme was much lower than that of the exogenous enzyme over the same 4 h incubation period. Optimum aglycone equivalent ratios coincided with highest O-S indices and highest aglycone concentrations in soymilk hydrolysed with exogenous enzyme. The same correlation of O-S indices and highest aglycone concentrations occurred for endogenous enzyme during the 24 h of fermentation. Therefore, obtaining highest aglycone concentration as well as optimum aglycone equivalent ratio could provide a critical beginning point in clinical trials for maximum realisation of the unique health benefits of soy isoflavones. Screening for β-glucosidase activities of probiotic bacteria in soymilk as well as comparing their hydrolytic potentials with that exogenous β-glucosidase could find wide applications in the development of different aglycone rich functional soy beverages.

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