M: Food Microbiology & Safety JFS M: Food Microbiology and Safety Effect of Lactulose on Biotransformation of Isoflavone Glycosides to Aglycones in Soymilk by Lactobacilli T.T. PHAM AND N.P. SHAH ABSTRACT: Lactobacillus acidophilus 4461, L. acidophilus 4962, L. casei 290, and L. casei 2607 were used to hy- drolyze isoflavone glycosides (IG) to biologically active forms—isoflavone aglycones (IA)—in soymilk (SM) prepared from soy protein isolate (SPI) and soymilk supplemented with 0.5% (w/v) of lactulose (SML). L. acidophilus 4461 utilized the highest level of lactulose (3.01 mg/mL) and L. acidophilus 4962 utilized the least (0.86 mg/mL) at 24 h of incubation. The pH values decreased to 4.00 to 5.00 in SML, while they remained relatively high (6.15 to 6.36) in SM. Supplementation with lactulose significantly (P < 0.05) enhanced the viable counts of all the 4 Lactobacillus strains. At the end of incubation, the viable counts of Lactobacillus ranged from 8.08 to 8.25 log CFU/mL in SML compared to 6.99 to 7.11 log CFU/mL in SM. Supplementation with lactulose increased the biotransformation of IG to IA after 6 h of incubation. The presence of lactulose in the medium enhanced the biotransformation level of IG to IA by Lactobacillus up to 21.9%. The hydrolysis level of malonyl genistin and acetyl genistin in SML was much higher than in SM by all the 4 probiotic organisms. The biotransformation of IG to IA occurred rapidly during the 1st 12 h of incubation in both SML and SM. Among the 4 Lactobacillus strains, L. acidophilus 4461 biotransformed the highest level (88.8%) of IG to IA in SML compared to 68.2% in SM after 24 h of incubation. Keywords: aglycones, biotransformation, isoflavones, Lactobacillus, lactulose Introduction L actulose (β -D galactose 1 → 4 α-D fructose) is produced dur- ing the heat treatment of lactose as a result of an isomerisation reaction (Lobry de Bruyn–Alberda van Ekenstein rearrangement) (Chavez-Servin and others 2006). Lactulose has been considered as a bifidogenic factor, which is able to proliferate healthy intesti- nal microflora (Salminen and Salminen 1997; Gonzales and others 2003). Lactulose was also reported to enhance the β -glucosidase and β -galactosidase activities of intestinal microflora, including lactobacilli and bifidobacteria (Juskiewicz and Zdunczyk 2002). Recently, Pham and Shah (2008) reported that both of these en- zymes were able to hydrolyze inactive isoflavone glycosides (IG) to isoflavone aglycones (IA), which are biologically active forms (Pham and Shah 2008). The IA group includes daidzein, glycitein, genistein, biochanin A, and formononetin (Hughes and others 2003). As the chemical structure of IA is similar to that of estro- gen, they are also classified as phytoestrogens as they are able to bind to estrogen receptor sites and therefore mimic the function of estradiol and relieve menopausal symptoms (Setchell and Cassidy 1999). However, in nature as well as in nonfermented soy products, IA comprise a minor fraction (1.6% to 16.1%) of total isoflavone compounds ranging from 0.5 to 1.7 mg/g (King and Bignell 2000). To achieve health benefits, the amount of IA required is 30 to 40 mg/d (Malnig and Brown 2007). Although IG are hydrolyzed to IA in the gastrointestinal tract by gut microflora, the rate of hydrolysis varies with an individual and remained unclear (Hughes and others MS 20070779 Submitted 10/15/2007, Accepted 1/8/2008. Authors are with School of Molecular Sciences, Faculty of Health, Engineering and Sci- ence, Victoria Univ., Werribee Campus, P.O. Box 14428, Melbourne, Vic- toria 8001, Australia. Direct inquiries to author Shah (E-mail: Nagen- dra.Shah@vu.edu.au). 2003; Sugano 2005). The natural sources of isoflavones are soybean, lentils, chickpeas, and red clover. Therefore, it is important to pro- vide food with a considerable amount of IA. To transform IG to IA, the β -glucosidic linkage between a β - glycoside and an isoflavone aglycone in an isoflavone glycoside molecule must be cleaved. Several groups of probiotic organisms have been used to convert IG to IA in soymilk (Tsangalis and oth- ers 2002; Chien and others 2006; Otieno and others 2006; Wei and others 2007). However, the biotransformation rate of IG to IA by probiotic bacteria in general was considerably low in fermented soymilk and β -glucosidase was claimed to be the only enzyme re- sponsible for the biotransformation (Tsangalis and others 2002; Chien and others 2006). Only 6.4% of the total IG in soymilk was fermented by B. longum after 32 h of fermentation at 37 ◦ C (Chien and others 2006). However, it is now realized that β -galactosidase is also responsible for the biotransformation of IG to IA (Pham and Shah 2008). Thus, in order to enhance the biotransformation level, soymilk (SM) could be supplemented with lactulose, which is reported to enhance β -glucosidase and β -galactosidase activities (Juskiewicz and Zdunczyk 2002). In addition, SM is normally pre- pared from soy protein isolate (SPI), which is made from defatted soy flour with most of the nonprotein components, including fats and carbohydrates, removed. Furthermore, SM prepared from SPI did not support the growth of probiotic organisms (Kamaly 1997; Pham and Shah 2007). As a bifidogenic factor, lactulose is expected to enhance the growth of probiotic organisms in SM supplemented with lactulose (Gonzales and others 2003). However, there is no re- port about the fermentation of IG in soymilk supplemented with lactulose. Therefore, the objective of this study was to investigate the effect of lactulose on the growth of Lactobacillus, the predom- inant probiotic group, and their biotransformation ability of IG to IA in fermented soymilk. M158 JOURNAL OF FOOD SCIENCE—Vol. 73, Nr. 3, 2008 C  2008 Institute of Food Technologists doi: 10.1111/j.1750-3841.2008.00687.x Further reproduction without permission is prohibited