Enrichment of Two Isoflavone Aglycones in Black Soymilk by Immobilized β‑Glucosidase on Solid Carriers Kuan-I Chen, † Yi-Chen Lo, † Nan-Wei Su, ‡ Cheng-Chun Chou, † and Kuan-Chen Cheng* ,†,§ † Graduate Institute of Food Science & Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, Taiwan ‡ Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan § Institute of Biotechnology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, Taiwan ABSTRACT: A catalytic system for deglycosylation of isoflavone in black soybean milk was established. β-Glucosidase which was covalently immobilized onto the glass microspheres exhibited a significant efficiency for the conversion of pNPG to p- nitrophenol over other carriers. The optimum temperature for pNPG hydrolysis was 40 °C, and complete reaction can be reached in 30 min. Operational reusability was confirmed for more than 40 batch reactions. Moreover, the storage stability verification demonstrated that the glass microsphere catalytic system was capable of sustaining its highest catalytic activity for 40 days. The kinetic parameters, including rate constant (K) at which isoflavone glycosides deglycosylation were determined, the time (τ 50 ) in which 50% of isoflavone glycosides deglycosylation was reached, and the time (τ complete ) required to achieve complete isoflavone glycosides deglycosylation, were 0.35 ± 0.04 min -1 , 2.04 ± 0.25 min, and 30 min (for daidzin) and 0.65 ± 0.03 min -1 , 1.19 ± 0.08 min, and 20 min (for genistin), respectively. HPLC results revealed that this enzyme system took only 30 min to reach complete isoflavone deglycosylation and the aglycone content in the total isoflavones in black soymilk was enriched by 51.42 ± 0.17% under a 30 min treatment by the glass microsphere enzymatic system. KEYWORDS: isoflavone deglycosylation, β-glucosidase, black soymilk, glass microspheres, immobilized enzyme system ■ INTRODUCTION Isoflavone, a well-known phytoestrogen, is a unique subgroup of flavonoids found most abundantly in cotyledon and hypocotyls of soybeans and soy derived foods. 1 The studies of isoflavone have recently drawn tremendous attention due to its potential health-enhancing benefits, 2 including reduction in cardiovascular disease, 3 cancer prevention, 4 osteoporosis prevention, 5 and high in antioxidant activities. 6 The amount of isoflavone has been reported to be ∼1-5 mg/g in dry-soybean and ∼10 mg/100 g in soy milk. 7 Studies have also revealed that isoflavones in their aglycone forms exhibit higher biological activity 8,9 and are more metabolically active that can be absorbed faster in greater amounts than their glycosides. 10,11 These results suggested that the intake of isoflavone aglycone-rich soy foods might be more effective for the purpose of healthy enhancement. For this reason, the enrichment of isoflavone aglycones in soy foods before consumption attracts growing attention. 12 Soymilk is one of the traditional food beverages consumed popularly in Asian countries over the past decades. In recent years, soymilk has been consumed as a milk substitute by premenopausal women, milk allergy and lactose intolerance patients, and vegans. 13,14 Many studies have been carried out to enhance the total isoflavone aglycone content in soymilk fermented with probiotic microorganisms by means of endogenous β-glucosidase activity that can hydrolyze glucoside moieties. 12,15 The function of β-glucosidase is to remove the glucoside conjugates resulting in the accumulation of isoflavone aglycones. Because of the advantage of stabilizing micro- organisms firmly on solid surfaces, immobilization of micro- organisms becomes a common approach during soybean fermentation. 16-18 Despite of the fact that fermentation is beneficial for isoflavone bioconversion, some limitations still exist, such as the fact that the process is time-consuming and the difficulty in scaling up in food industry. Recently, immobilized enzyme systems have been established in food industry to obtain specific products by catalyzing their corresponding substrates. Immobilized biocatalyst has several advantages over free enzyme system, including easy separation from reaction solution, reusability for reducing costs, continuous processing, and long-term stability. 19 The most famous example in food industry is the production of fructose syrup from glucose converted by immobilized glucose isomerase. 20,21 Diano et al. also reported a bed reactor packed with polyacrylonitrile (PAN) beads coated with pectin enzymes for the clarification of apple juice. 22 Moreover, some microscale 23 or nanoscale 24 materials have also been applied in the research of enzyme immobilization. In the present study, we evaluated the feasibility of soybean milk isoflavone conversion using an immobilized β-glucosidase enzyme system. In this respect, β-glucosidase was immobilized on four different carriers (glass microspheres, nylon pellets, cellulose beads, and PAN beads) for deglycosylation of isoflavone in a more direct and faster manner, and 4-nitrophenyl β-D-glucuronide (pNPG) was used as an indicator. The carrier which possessed the most efficient catalytic behavior among these four solid carriers was chosen and applied to deglycosylation of isoflavone in black soymilk. The kinetic parameters of the best catalytic system for isoflavone deglycosylation were further determined as a function of different operative conditions. The efficiency of this immobilized Received: August 22, 2012 Revised: November 27, 2012 Accepted: November 28, 2012 Published: November 28, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 12540 dx.doi.org/10.1021/jf304405t | J. Agric. Food Chem. 2012, 60, 12540-12546