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

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

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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). The mobile phase was composed of n-propanol, ethyl acetate, and water at a volume ratio of 7:1:2. Detection of components was achieved by spraying with 10% H2SO4 and subsequent heating at 95oC for 10 min.

Statistical Analysis of Data The data generated from this study were subjected to one-way analysis of variance (ANOVA) at 5% level of significance using SPSS 18.0. Means were separated by Duncan’s multiple range tests.

Results and Discussion

Screening of Microorganisms and Selection of a Basal Medium In previous studies, 43 lactic acid bacteria were assessed with respect to the production of α- and β-galactosidases. Among them, B. longum RD47 showed the highest production Fig. 1. Effects of the carbon sources on the production of α- of both α- and β-galactosidases, and it was selected for (A) and β-galactosidases (B) from B. longum RD47. further investigation (data not shown). The present study The basal medium contained 10% deproteinized whey, 10% CSL, and was carried out in a medium containing 10% CSL with 0.05% L-cysteine-HCl. Two percent of various carbon sources were various concentrations of deproteinized whey. The production supplemented in the basal medium. Determination of enzyme activity o of α- and β-galactosidases was maximal when 10% was made at 30 h at 37 C. Bars indicate the standard error of the mean. The bars bearing different lowercase letters are significantly deproteinized whey was added. different according to Duncan’s multiple range test (p < 0.05).

Effects of Carbon Sources on α- and β-Galactosidase Production The effects of various carbon sources, including glucose, In the case of β-galactosidase, B. longum RD47 grown on galactose, fructose, lactose, maltose, arabinose, sucrose, the basal medium showed the highest activity among and soybean oligosaccharides, on the activities of α- and β- various carbon sources at 30 h of cultivation at 37oC galactosidases from B. longum RD47 are shown in Fig. 1. It (Fig. 1B). Although the presence of galactose showed 9.6% was found that α-galactosidase in B. longum RD47 grown lower activity than the basal medium, it was not significant on soybean oligosaccharides showed the highest activity of (p > 0.05). Hsu et al. [11] reported that lactose is the best 0.3 U/ml at 30 h of cultivation at 37oC (Fig. 1A). Soybean carbon source for inducing the maximum production of β- oligosaccharides are composed of raffinose-family carbohydrates galactosidase by bifidobacteria. A similar result showed such as raffinose, stachyose, and verbascose. Previous that 4% lactose is the best condition for the synthesis of β- studies reported high levels of α-galactosidase activities galactosidase [12, 15]. The concentration of lactose in from bifidobacteria on raffinose [29]. Raffinose including deproteinized whey may have been enough for the optimal the α-galactosyl group linkage may have induced the production of β-galactosidase from B. longum RD47, as gal operon genes encoding α-galactosidase gene [1]. The whey contains lactose. The reduction of β-galactosidase results of this study were compatible with reports by Xiao production in a medium supplemented with 5% or more et al. [29] and Amaretti et al. [2], which indicated that the lactose can be attributed to the increased concentration of intracellular activity of α-galactosidase from bifidobacteria internally released glucose, which represses the biosynthesis in a medium containing raffinose was higher than that of β-galactosidase [14]. Our study also indicated that an of glucose. It is expected that soybean oligosaccharides addition of different carbon sources into a basal medium having raffinose, stachyose, and verbascose are efficient inhibited the activity of β-galactosidase from B. longum inducers for the synthesis of α-galactosidase. RD47.

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Effects of Nitrogen Sources on α- and β-Galactosidase RD47 produced the highest activity of β-galactosidase Production (0.5 U/ml) with proteose peptone no. 3, followed by The SBO medium was added with various nitrogen gelatin. Malt extract showed the lowest activity for both α- sources, such as gelatin, yeast extract, malt extract, proteose and β-galactosidases, which might be due to the high level peptone no. 3, and beef extract, to assess the effects on the of maltose existing in the malt extract, which repressed production of α- and β-galactosidases from B. longum both the enzymes as a carbon source (Fig. 1). RD47. In the case of α-galactosidase activity, the addition The results of the nitrogen sources showed that B. longum of extra nitrogen sources showed no additive effects. The RD47 grown on proteose peptone no. 3 had a significantly SBO medium containing CSL as a nitrogen source showed higher (p < 0.05) β-galactosidase activity than other nitrogen the best result, followed by proteose peptone no. 3, which sources. B. longum RD47 showed high levels of activities was not significant (p > 0.05) (Fig. 2A). for both enzymes in both the carbohydrate (soybean On the other hand, the α-galactosidase activity from oligosaccharides) and the nitrogen source (proteose peptone B. longum RD47 grown in the presence of yeast extract was no. 3) experiments. repressed. These findings are not in agreement with Alazzeh According to the results of this study, soybean et al. [1] or Gote et al. [8], who found that yeast extract was oligosaccharides did not have a significant effect on the the best nitrogen source for α-galactosidase. CSL, which is activity of β-galactosidase from B. longum RD47. However, in the SBO medium, is an important ingredient of various the effects of soybean oligosaccharides and proteose growth media and can replace yeast extract as a rich source peptone no. 3 were noted on the activity of β-galactosidase of nutrients such as organic nitrogen or vitamins. from B. longum RD47 compared with a SBO medium. According to Shaikh et al. [23], nitrogen sources can affect the microbial biosynthesis of β-galactosidase. B. longum

Fig. 2. Effects of nitrogen sources on the production of α- (A) and β-galactosidases (B) from B. longum RD47. The SBO medium contained 10% whey, 10% CSL, 2% soybean oligosaccharides, and 0.05% L-cysteine-HCl. Two percent of various nitrogen sources were supplemented. Determination of enzyme activity was made at 30 h at 37oC. Bars indicate the standard error of the Fig. 3. Effect of pH on the enzyme activities of α- (A) and β- mean. The bars bearing different lowercase letters are significantly galactosidases (B) from B. longum RD47. different according to Duncan’s multiple range test (p < 0.05). Fifty mM of sodium phosphate buffer (pH 5-7.5) was used.

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Effects of pH and Temperature on α- and β-Galactosidase than 80% activity after 1 h of incubation (Fig. 5). On the Activities other hand, α- and β-galactosidase activities decreased The activities of α- and β-galactosidases at different pH quickly at 45oC and 50oC after holding for 0.5 h. levels are shown in Figs. 3A and 3B. The optimum pH of α- and β-galactosidase activity in crude extract from B. longum Effects of Various Metal Ions on α- and β-Galactosidase RD47 was found at pH 6.0 in 50 mM sodium phosphate Activities buffer. On the other hand, it was expected that α- and β- The effects of various metal ions on α- and β-galactosidase galactosidase activities would decrease, except in pH 6.0. activities in crude extract from B. longum RD47 are shown Usually, bifidobacterial activities of α- and β-galactosidases in Figs. 6A and 6B. α-Galactosidase activity was inhibited show an optimum pH in a weak acidic range (6.0-7). In by all metal ions at a concentration of 1 mM (Fig. 6A). β- contrast, the activities of α- and β-galactosidases from Galactosidase activity was slightly enhanced by ZnSO4,

Aspergillus spp. are in a more acidic range (3.5-5) [9, 24]. KCl, Na2SO4, MgSO4, MnCl2, and NaCl metal ions but not The highest activities of α- and β-galactosidase were significantly (p > 0.05) (Fig. 6B). Commonly, the activities o o shown at 35 C and 37 C, respectively, and decreased over of the two enzymes were strongly inhibited by CuSO4. The the optimal temperature (Fig. 4). Specifically, the activities enzyme activities of α- and β-galactosidases remained at of the two enzymes were markedly decreased at 55oC. The about 65% and 50% of the original activity level, respectively. two enzymes were fairly stable at 37oC, retaining more Hydrolysis of Substrates The time course of the hydrolysis of raffinose is shown in Fig. 7. Complete hydrolysis of raffinose was observed within 3 h of incubation at 37oC (lane 8). The synthetic substrates

Fig. 4. Effect of temperature on the enzyme activities of α- (A) Fig. 5. Thermal stability of α- (A) and β-galactosidases (B) and β-galactosidases (B) from B. longum RD47. from B. longum RD47.

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of p-nitrophenyl-α-galactopyranoside were hydrolyzed by α-galactosidase. Over time, the reaction products found in an analysis of the TLC showed that sucrose and galactose were the main products. Similarly, the time course of the lactose hydrolysis is shown in Fig. 7. Most of the lactose was hydrolyzed within 3 h of incubation at 37oC (lane 21). The synthetic substrate p-nitrophenyl-β-galactopyranoside was hydrolyzed by β-galactosidase. Over time, the reaction products of β-galactosidase found in the TLC analysis showed that galactose and glucose were the main products. The results of the hydrolysis of the substrates indicated the feasibility of utilizing α- and β-galactosidases from bifidobacteria. In conclusion, this work indicated that the media were desirable with a mixture of a carbon source (soybean oligosaccharides) and a nitrogen source (proteose peptone no. 3) to obtain high α and β-galactosidase activities. In particular, soybean oligosaccharides were essential for the effective production of α-galactosidase. This apparent effect of both soybean oligosaccharides and proteose peptone no. 3 was also noted in the production of β-galactosidase from B. longum RD47. Bifidobacteria having stable α- and β-galactosidase activities at the optimum temperature and pH levels similar to those of the human intestine would be well harmonized with the intestinal environment. Raffinose and lactose are indigestible for mammals and can cause flatulence or gas production in Fig. 6. Effects of various metal ions on α- (A) and β- the intestinal tract. Therefore, foods containing raffinose galactosidase (B) activities from B. longum RD47. and lactose at low levels are preferred for the galactoside- The bars indicate the standard error of the mean. The bars bearing intolerant consumers. The results of this study show different lowercase letters are significantly different according to several types of benefits. The findings could be helpful for Duncan’s multiple range test (p < 0.05). the development of soymilk beverages and milk beverages

Fig. 7. Thin-layer chromatography of hydrolysis products from raffinose with α-galactosidase (1-11) and from lactose with β-galactosidase (12-22) from B. longum RD47. Lanes: standard materials for raffinose (1), glucose (2, 13), galactose (3, 14), sucrose (4), lactose (12), and hydrolysis product of raffinose after 0 h (5), (6) 0.5 h, (7) 1 h, (8) 3 h, (9) 7 h, (10) 9 h, and (11) 24 h, respectively, and hydrolysis product of lactose after 0 h (15), 0.5 h (16), 1 h (17), 1.5 h (18), 2 h (19), 2.5 h (20), 3 h (21), and 12 h (22) of incubation with the enzyme, respectively.

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for those who are lactose-intolerant, as the possibility of derived oligosaccharides and other carbohydrates on α- applying the findings to the practical production of foods galactosidase and α-glucosidase activity in Bifidobacterium was demonstrated. This is supported by previous studies, adolescentis. Lett. Appl. Microbiol. 46: 73-79. which reported that controlling excessive gas production 11. Hsu CA, Yu RC, Chou CC. 2005. Production of β- and reducing gas-related symptoms can be achieved with galactosidase by bifidobacteria as influenced by various culture conditions. Int. J. Food Microbiol. 104: 197-206. α-galactosidase [5] and β-galactosidase [19]. 12.Hsu CA, Yu RC, Chou CC. 2006. Purification and With the increasing attention on the role of bifidobacteria characterization of a sodium-stimulated β-galactosidase from owing to its health benefits, the use of B. longum RD47, Bifidobacterium longum CCRC 15708. World J. 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