Production of Α- and Β-Galactosidases from Bifidobacterium Longum Subsp

J. Microbiol. Biotechnol. (2014), 24(5), 675–682 http://dx.doi.org/10.4014/jmb.1402.02037 Research Article jmb Production of α- and β-Galactosidases from Bifidobacterium longum subsp. longum RD47 Yoo Ri Han1, So Youn Youn1, Geun Eog Ji1,2*, and Myeong Soo Park3* 1Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University, Seoul 151-742, Republic of Korea 2Research Center, BIFIDO Co., Ltd., Hongcheon 205-804, Republic of Korea 3Department of Hotel Culinary Arts, Yeonsung University, Anyang 430-749, Republic of Korea Received: February 19, 2014 Revised: March 7, 2014 Approximately 50% of people in the world experience abdominal flatulence after the intake of Accepted: March 8, 2014 foods containing galactosides such as lactose or soybean oligosaccharides. The galactoside hydrolyzing enzymes of α- and β-galactosidases have been shown to reduce the levels of galactosides in both the food matrix and the human gastrointestinal tract. This study aimed to First published online optimize the production of α- and β-galactosidases of Bifidobacterium longum subsp. longum March 10, 2014 RD47 with a basal medium containing whey and corn steep liquor. The activities of both o *Corresponding authors enzymes were determined after culturing at 37 C at pH 6.0 for 30 h. The optimal production of G.E.J. α- and β-galactosidases was obtained with soybean oligosaccharides as a carbon source and Phone: +82-2-880-8749; proteose peptone no. 3 as a nitrogen source. The optimum pH for both α- and β-galactosidases Fax: +82-2-884-0305; E-mail: [email protected] was 6.0. The optimum temperatures were 35oC for α-galactosidase and 37oC for β- M.S.P. galactosidase. They showed temperature stability up to 37oC. At a 1 mM concentration of Phone: +82-31-441-1347; Fax: +82-31-441-1347; metal ions, CuSO4 inhibited the activities of α- and β-galactosidases by 35% and 50%, E-mail: [email protected] respectively. On the basis of the results obtained in this study, B. longum RD47 may be used for the production of α- and β-galactosidases, which may reduce the levels of flatulence factors. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2014 by Keywords: Bifidobacteria, α- and β-galactosidases, soybean oligosaccharides, proteose peptone The Korean Society for Microbiology no. 3, whey and corn steep liquor and Biotechnology Introduction catalyze the hydrolysis of terminally joined galactosidic residues in simple galactose-including oligosaccharides as There has been a growing interest in probiotics, prebiotics, well as in complex polysaccharides [20]. Because α- or their combined use as synbiotics to enhance human galactosidase is not synthesized by humans, the presence health [25]. Probiotics is defined as “living microbial diet of oligosaccharides can impede the digestion of nutrients supplements which beneficially affect the host by improving and lead to flatulence [6]. Therefore, α-galactosidase can be its intestinal balance” [7]. Among them, bifidobacteria have useful for eliminating the α-galactosyl residue in the some potential health-promoting properties in that they soybean oligosaccharides and thus promote the nutrition of maintain the intestinal microbial balance by regulating legume and bean foods. antimicrobial activity [3], preventing diarrheal diseases [26] β-Galactosidase, known as a lactase that hydrolyzes and upper gastrointestinal tract diseases [17], alleviating lactose into glucose and galactose, is a commercially lactose-intolerance symptoms, and stimulating immune important enzyme in the food industry for alleviating the responses [21]. problems associated with lactose crystallization in frozen Bifidobacteria possess glycohydrolases, including α- and concentrated desserts [16]. A half of the world’s population β-galactosidases, which are capable of metabolizing lacks this enzyme, leading to the development of lactose various carbohydrates [4]. These galactosidase enzymes intolerance or maldigestion [28]. The principle symptoms May 2014 ⎪ Vol. 24⎪ No. 5 676 Han et al. of lactose intolerance are flatulence, bloating, diarrhea, and Enzyme Preparation and Assay abdominal pain. The incubated bacteria were collected by centrifugation (10,000 ×g Several studies have shown an effect of α- and β- for 3 min at 4oC) and the harvested pellet was washed twice with galactosidase administration on intestinal gas production 50 mM sodium phosphate buffer (pH 6.0). The pellet was and the occurrence of gas-related symptoms [5, 19]. Di resuspended in the phosphate buffer (pH 6.0) and disrupted with a cell sonicator (VCX 400; Sonics & Material Inc., Newtown, CT, Stefano et al. [5] reported that the oral administration of α- USA) for 10 min to extract intracellular enzymes. The disrupted galactosidase was proved to be effective for controlling bacterial solution was centrifuged at 10,000 ×g for 10 min at 4oC excessive gas production and reducing gas-related symptoms and the supernatant was used as crude enzyme extract for assay after a meal rich in fermentable carbohydrates. Lin et al. of α- or β-galactosidase. Eighty microliters of the crude enzyme [19] also showed that the intake of β-galactosidase solution was added to 20 µl of 5 mM p-nitrophenyl- α- or β- improves the in vivo digestion of lactose through the galactopyranoside substrate and the mixture was incubated at o enhanced gastrointestinal digestion of lactose and the 37 C. The reaction was stopped by adding 100 µl of 1 M Na2CO3. reduced production of gas. Therefore, the efficient production Enzyme activities were determined by monitoring the amount of of α- and β-galactosidases from microorganisms would be the released p-nitrophenol (pNP) from p-nitrophenyl- α- or β- valuable for industrial, biotechnological, and further galactopyranoside at 405 nm in a spectrophotometer at 37oC. One medicinal applications. unit (U) of enzyme activity was defined as the amount of enzyme o Previous studies have demonstrated that bifidobacteria that liberated 1 µmol of pNP per minute at 37 C and pH 6.0. can produce α- or β-galactosidases [10, 12, 13, 18, 22]. Van Effects of Carbon and Nitrogen Sources Laere et al. [27] reported that β-galactosidase from B. To investigate the effects of various carbon sources on the adolescentis preferentially hydrolyzes galactooligosaccharides. production of α- and β-galactosidases, glucose, galactose, fructose, However, the production of both α- and β-galactosidases maltose, arabinose, sucrose (all from Sigma), lactose (Trade TCI by the same strain has not yet been reported. Considering Mark, Japan), or soybean oligosaccharides (Xian Rongsheng the economic aspects as well as the efficiency of the Biotechnology Co., Ltd, China) at 2% concentration was added production of the two enzymes, the utilization of a medium into the basal medium. For the assessment of nitrogen source, the with a renewable source would reduce the cost of industrial basal medium containing 2% soybean oligosaccharides (SBO applications. medium) was supplemented with various nitrogen sources (at 2%). The aim of this paper was to assess the optimal culture Yeast extract, malt extract, proteose peptone no. 3, beef extract (all conditions and obtain high levels of α- and β-galactosidase Difco products), and gelatin (Sigma) were used as nitrogen sources. activities from B. longum RD47 in a low-cost medium and then to characterize these enzymes. Effects of pH and Temperature on the Activities and Stability of α- and β-Galactosidases For determination of the effect of pH on the crude enzyme Materials and Methods activities, assay was done at a pH range of 5.0-7.5 with 50 mM sodium phosphate buffer (pH 5.0-7.5) at 37oC. The effect of Microorganisms and Culture Conditions temperature was evaluated at 30oC to 55oC in 50 mM sodium B. longum RD47, which was shown to produce the greatest level phosphate buffer. To determine the thermostability at various of α- and β-galactosidases in a preliminary study, was used in the temperatures, the enzyme solution in 50 mM sodium phosphate present study. B. longum RD47 was activated by two successive buffer (pH 6.0) was incubated at different temperatures (37oC, precultures in MRS medium (Difco, USA) with 0.05% (w/v) L- 45oC, and 50oC) for 1, 1.5, and 2 h and then subjected to α- and cysteine-HCl (Sigma, USA) and was grown under anaerobic β-galactosidase activity assay at 37oC. conditions at 37oC for 18 h. Then, the activated bacteria were again cultured at 37oC and pH 6.5 for 30 h in the basal medium containing Effects of Metal Ions on the Activities of α- and β-Galactosidases 10% whey, 10% corn steep liquor (CSL), and 0.05% cysteine-HCl. Enzyme assays were performed in the presence of various Preparation of Media metal ions (1 mM), including KCl, NaCl, Na2SO4, MgSO4, MnCl2, Ten percent (w/v) whey solution was prepared and the pH was ZnSO4, CuSO4, FeSO4, CaCl2, MnSO4, and MgCl2. The relative activity of the enzyme was compared with the activity obtained in adjusted to 5.4 with 95% H2SO4 (Samchun, Seoul, Korea). In order o to precipitate proteins, it was heated at 121oC for 15 min and 50 mM sodium phosphate buffer (pH 6.0) at 37 C for 30 min. filtered through a Whatman No. 1 filter paper. The deproteinized whey solution was then supplemented with 10% CSL (Sigma) and Hydrolysis of Substrates 0.05% L-cysteine-HCl and the pH was adjusted to 6.5 with 5 M The reaction mixture containing α- or β-galactosidase from NaOH. Then, it was sterilized in an autoclave at 121oC for 15 min. B. longum RD47 and 10 mM raffinose or lactose in 50 mM sodium J. Microbiol. Biotechnol. Production of α- and β-Galactosidases from Bifidobacterium longum 677 phosphate buffer (pH 6.0) was incubated at 37oC for 0.5-24 h. Thin layer chromatography (TLC) was performed on a precoated silica gel plate (Silica gel 60F; Merck, Darmstadt, Germany).

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