Production of ${\alpha}$- and ${\beta}$-Galactosidases from Bifidobacterium longum subsp. longum RD47

Abstract

Approximately 50% of people in the world experience abdominal flatulence after the intake of foods containing galactosides such as lactose or soybean oligosaccharides. The galactoside hydrolyzing enzymes of ${\alpha}$- and ${\beta}$-galactosidases have been shown to reduce the levels of galactosides in both the food matrix and the human gastrointestinal tract. This study aimed to optimize the production of ${\alpha}$- and ${\beta}$-galactosidases of Bifidobacterium longum subsp. longum RD47 with a basal medium containing whey and corn steep liquor. The activities of both enzymes were determined after culturing at $37^{\circ}C$ at pH 6.0 for 30 h. The optimal production of ${\alpha}$- and ${\beta}$-galactosidases was obtained with soybean oligosaccharides as a carbon source and proteose peptone no. 3 as a nitrogen source. The optimum pH for both ${\alpha}$- and ${\beta}$-galactosidases was 6.0. The optimum temperatures were $35^{\circ}C$ for ${\alpha}$-galactosidase and $37^{\circ}C$ for ${\beta}$-galactosidase. They showed temperature stability up to $37^{\circ}C$. At a 1 mM concentration of metal ions, $CuSO_4$ inhibited the activities of ${\alpha}$- and ${\beta}$-galactosidases by 35% and 50%, respectively. On the basis of the results obtained in this study, B. longum RD47 may be used for the production of ${\alpha}$- and ${\beta}$-galactosidases, which may reduce the levels of flatulence factors.

Introduction

There has been a growing interest in probiotics, prebiotics, or their combined use as synbiotics to enhance human health [25]. Probiotics is defined as “living microbial diet supplements which beneficially affect the host by improving its intestinal balance” [7]. Among them, bifidobacteria have some potential health-promoting properties in that they maintain the intestinal microbial balance by regulating antimicrobial activity [3], preventing diarrheal diseases [26] and upper gastrointestinal tract diseases [17], alleviating lactose-intolerance symptoms, and stimulating immune responses [21].

Bifidobacteria possess glycohydrolases, including α- and β-galactosidases, which are capable of metabolizing various carbohydrates [4]. These galactosidase enzymes catalyze the hydrolysis of terminally joined galactosidic residues in simple galactose-including oligosaccharides as well as in complex polysaccharides [20]. Because α- galactosidase is not synthesized by humans, the presence of oligosaccharides can impede the digestion of nutrients and lead to flatulence [6]. Therefore, α-galactosidase can be useful for eliminating the α-galactosyl residue in the soybean oligosaccharides and thus promote the nutrition of legume and bean foods.

β-Galactosidase, known as a lactase that hydrolyzes lactose into glucose and galactose, is a commercially important enzyme in the food industry for alleviating the problems associated with lactose crystallization in frozen concentrated desserts [16]. A half of the world’s population lacks this enzyme, leading to the development of lactose intolerance or maldigestion [28]. The principle symptoms of lactose intolerance are flatulence, bloating, diarrhea, and abdominal pain.

Several studies have shown an effect of α- and β- galactosidase administration on intestinal gas production and the occurrence of gas-related symptoms [5, 19]. Di Stefano et al. [5] reported that the oral administration of α- galactosidase was proved to be effective for controlling excessive gas production and reducing gas-related symptoms after a meal rich in fermentable carbohydrates. Lin et al. [19] also showed that the intake of β-galactosidase improves the in vivo digestion of lactose through the enhanced gastrointestinal digestion of lactose and the reduced production of gas. Therefore, the efficient production of α- and β-galactosidases from microorganisms would be valuable for industrial, biotechnological, and further medicinal applications.

Previous studies have demonstrated that bifidobacteria can produce α- or β-galactosidases [10, 12, 13, 18, 22]. Van Laere et al. [27] reported that β-galactosidase from B. adolescentis preferentially hydrolyzes galactooligosaccharides. However, the production of both α- and β-galactosidases by the same strain has not yet been reported. Considering the economic aspects as well as the efficiency of the production of the two enzymes, the utilization of a medium with a renewable source would reduce the cost of industrial applications.

The aim of this paper was to assess the optimal culture conditions and obtain high levels of α- and β-galactosidase activities from B. longum RD47 in a low-cost medium and then to characterize these enzymes.

 

Materials and Methods

Microorganisms and Culture Conditions

B. longum RD47, which was shown to produce the greatest level of α- and β-galactosidases in a preliminary study, was used in the present study. B. longum RD47 was activated by two successive precultures in MRS medium (Difco, USA) with 0.05% (w/v) Lcysteine-HCl (Sigma, USA) and was grown under anaerobic conditions at 37℃ for 18 h. Then, the activated bacteria were again cultured at 37℃ and pH 6.5 for 30 h in the basal medium containing 10% whey, 10% corn steep liquor (CSL), and 0.05% cysteine-HCl.

Preparation of Media

Ten percent (w/v) whey solution was prepared and the pH was adjusted to 5.4 with 95% H2SO4 (Samchun, Seoul, Korea). In order to precipitate proteins, it was heated at 121℃ for 15 min and filtered through a Whatman No. 1 filter paper. The deproteinized whey solution was then supplemented with 10% CSL (Sigma) and 0.05% L-cysteine-HCl and the pH was adjusted to 6.5 with 5 M NaOH. Then, it was sterilized in an autoclave at 121℃ for 15 min.

Enzyme Preparation and Assay

The incubated bacteria were collected by centrifugation (10,000 ×g for 3 min at 4℃) and the harvested pellet was washed twice with 50 mM sodium phosphate buffer (pH 6.0). The pellet was resuspended in the phosphate buffer (pH 6.0) and disrupted with a cell sonicator (VCX 400; Sonics & Material Inc., Newtown, CT, USA) for 10 min to extract intracellular enzymes. The disrupted bacterial solution was centrifuged at 10,000 ×g for 10 min at 4℃ and the supernatant was used as crude enzyme extract for assay of α- or β-galactosidase. Eighty microliters of the crude enzyme solution was added to 20 μl of 5 mM p-nitrophenyl- α- or β- galactopyranoside substrate and the mixture was incubated at 37℃. The reaction was stopped by adding 100 μl of 1 M Na2CO3. Enzyme activities were determined by monitoring the amount of the released p-nitrophenol (pNP) from p-nitrophenyl- α- or β- galactopyranoside at 405 nm in a spectrophotometer at 37℃. One unit (U) of enzyme activity was defined as the amount of enzyme that liberated 1 μmol of pNP per minute at 37℃ and pH 6.0.

Effects of Carbon and Nitrogen Sources

To investigate the effects of various carbon sources on the production of α- and β-galactosidases, glucose, galactose, fructose, maltose, arabinose, sucrose (all from Sigma), lactose (Trade TCI Mark, Japan), or soybean oligosaccharides (Xian Rongsheng Biotechnology Co., Ltd, China) at 2% concentration was added into the basal medium. For the assessment of nitrogen source, the basal medium containing 2% soybean oligosaccharides (SBO medium) was supplemented with various nitrogen sources (at 2%). Yeast extract, malt extract, proteose peptone no. 3, beef extract (all Difco products), and gelatin (Sigma) were used as nitrogen sources.

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 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 37℃. The effect of temperature was evaluated at 30℃ to 55℃ in 50 mM sodium phosphate buffer. To determine the thermostability at various temperatures, the enzyme solution in 50 mM sodium phosphate buffer (pH 6.0) was incubated at different temperatures (37℃, 45℃, and 50℃) for 1, 1.5, and 2 h and then subjected to α- and β-galactosidase activity assay at 37℃.

Effects of Metal Ions on the Activities of α- and β-Galactosidases

Enzyme assays were performed in the presence of various metal ions (1 mM), including KCl, NaCl, Na2SO4, MgSO4, MnCl2, ZnSO4, CuSO4, FeSO4, CaCl2, MnSO4, and MgCl2. The relative activity of the enzyme was compared with the activity obtained in 50 mM sodium phosphate buffer (pH 6.0) at 37℃ for 30 min.

Hydrolysis of Substrates

The reaction mixture containing α- or β-galactosidase from B. longum RD47 and 10 mM raffinose or lactose in 50 mM sodium phosphate buffer (pH 6.0) was incubated at 37℃ for 0.5-24 h. Thin layer chromatography (TLC) was performed on a precoated ilica 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 95℃ 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 of both α- and β-galactosidases, and it was selected for further investigation (data not shown). The present study was carried out in a medium containing 10% CSL with various concentrations of deproteinized whey. The production of α- and β-galactosidases was maximal when 10% deproteinized whey was added.

Effects of Carbon Sources on α- and β-Galactosidase Production

The effects of various carbon sources, including glucose, galactose, fructose, lactose, maltose, arabinose, sucrose, and soybean oligosaccharides, on the activities of α- and β- galactosidases from B. longum RD47 are shown in Fig. 1. It was found that α-galactosidase in B. longum RD47 grown on soybean oligosaccharides showed the highest activity of 0.3 U/ml at 30 h of cultivation at 37℃ (Fig. 1A). Soybean oligosaccharides are composed of raffinose-family carbohydrates such as raffinose, stachyose, and verbascose. Previous studies reported high levels of α-galactosidase activities from bifidobacteria on raffinose [29]. Raffinose including the α-galactosyl group linkage may have induced the gal operon genes encoding α-galactosidase gene [1]. The results of this study were compatible with reports by Xiao et al. [29] and Amaretti et al. [2], which indicated that the intracellular activity of α-galactosidase from bifidobacteria in a medium containing raffinose was higher than that of glucose. It is expected that soybean oligosaccharides having raffinose, stachyose, and verbascose are efficient inducers for the synthesis of α-galactosidase.

Fig. 1.Effects of the carbon sources on the production of α- (A) and β-galactosidases (B) from B. longum RD47. The basal medium contained 10% deproteinized whey, 10% CSL, and 0.05% L-cysteine-HCl. Two percent of various carbon sources were supplemented in the basal medium. Determination of enzyme activity was made at 30 h at 37℃. Bars indicate the standard error of the mean. The bars bearing different lowercase letters are significantly different according to Duncan’s multiple range test (p < 0.05).

In the case of β-galactosidase, B. longum RD47 grown on the basal medium showed the highest activity among various carbon sources at 30 h of cultivation at 37℃ (Fig. 1B). Although the presence of galactose showed 9.6% lower activity than the basal medium, it was not significant (p > 0.05). Hsu et al. [11] reported that lactose is the best carbon source for inducing the maximum production of β- galactosidase by bifidobacteria. A similar result showed that 4% lactose is the best condition for the synthesis of β- galactosidase [12, 15]. The concentration of lactose in deproteinized whey may have been enough for the optimal production of β-galactosidase from B. longum RD47, as whey contains lactose. The reduction of β-galactosidase production in a medium supplemented with 5% or more lactose can be attributed to the increased concentration of internally released glucose, which represses the biosynthesis of β-galactosidase [14]. Our study also indicated that an addition of different carbon sources into a basal medium inhibited the activity of β-galactosidase from B. longum RD47.

Effects of Nitrogen Sources on α- and β-Galactosidase Production

The SBO medium was added with various nitrogen sources, such as gelatin, yeast extract, malt extract, proteose peptone no. 3, and beef extract, to assess the effects on the production of α- and β-galactosidases from B. longum RD47. In the case of α-galactosidase activity, the addition of extra nitrogen sources showed no additive effects. The SBO medium containing CSL as a nitrogen source showed the best result, followed by proteose peptone no. 3, which was not significant (p > 0.05) (Fig. 2A).

On the other hand, the α-galactosidase activity from B. longum RD47 grown in the presence of yeast extract was repressed. These findings are not in agreement with Alazzeh et al. [1] or Gote et al. [8], who found that yeast extract was the best nitrogen source for α-galactosidase. CSL, which is in the SBO medium, is an important ingredient of various growth media and can replace yeast extract as a rich source of nutrients such as organic nitrogen or vitamins.

According to Shaikh et al. [23], nitrogen sources can affect the microbial biosynthesis of β-galactosidase. B. longum RD47 produced the highest activity of β-galactosidase (0.5 U/ml) with proteose peptone no. 3, followed by gelatin. Malt extract showed the lowest activity for both α- and β-galactosidases, which might be due to the high level of maltose existing in the malt extract, which repressed both the enzymes as a carbon source (Fig. 1).

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 37℃. Bars indicate the standard error of the mean. The bars bearing different lowercase letters are significantly different according to Duncan’s multiple range test (p < 0.05).

The results of the nitrogen sources showed that B. longum RD47 grown on proteose peptone no. 3 had a significantly higher (p < 0.05) β-galactosidase activity than other nitrogen sources. B. longum RD47 showed high levels of activities for both enzymes in both the carbohydrate (soybean oligosaccharides) and the nitrogen source (proteose peptone no. 3) experiments.

According to the results of this study, soybean oligosaccharides did not have a significant effect on the activity of β-galactosidase from B. longum RD47. However, the effects of soybean oligosaccharides and proteose peptone no. 3 were noted on the activity of β-galactosidase from B. longum RD47 compared with a SBO medium.

Effects of pH and Temperature on α- and β-Galactosidase Activities

The activities of α- and β-galactosidases at different pH levels are shown in Figs. 3A and 3B. The optimum pH of α- and β-galactosidase activity in crude extract from B. longum RD47 was found at pH 6.0 in 50 mM sodium phosphate buffer. On the other hand, it was expected that α- and β- galactosidase activities would decrease, except in pH 6.0. Usually, bifidobacterial activities of α- and β-galactosidases show an optimum pH in a weak acidic range (6.0-7). In contrast, the activities of α- and β-galactosidases from Aspergillus spp. are in a more acidic range (3.5-5) [9, 24].

Fig. 3.Effect of pH on the enzyme activities of α- (A) and β-galactosidases (B) from B. longum RD47. Fifty mM of sodium phosphate buffer (pH 5-7.5) was used.

The highest activities of α- and β-galactosidase were shown at 35℃ and 37℃, respectively, and decreased over the optimal temperature (Fig. 4). Specifically, the activities of the two enzymes were markedly decreased at 55℃. The two enzymes were fairly stable at 37℃, retaining more than 80% activity after 1 h of incubation (Fig. 5). On the other hand, α- and β-galactosidase activities decreased quickly at 45℃ and 50℃ after holding for 0.5 h.

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

Fig. 5.Thermal stability of α- (A) and β-galactosidases (B) from B. longum RD47

Effects of Various Metal Ions on α- and β-Galactosidase Activities

The effects of various metal ions on α- and β-galactosidase activities in crude extract from B. longum RD47 are shown in Figs. 6A and 6B. α-Galactosidase activity was inhibited by all metal ions at a concentration of 1 mM (Fig. 6A). β- Galactosidase activity was slightly enhanced by ZnSO4, KCl, Na2SO4, MgSO4, MnCl2, and NaCl metal ions but not significantly (p > 0.05) (Fig. 6B). Commonly, the activities of the two enzymes were strongly inhibited by CuSO4. The enzyme activities of α- and β-galactosidases remained at about 65% and 50% of the original activity level, respectively.

Fig. 6.Effects of various metal ions on α- (A) and β- galactosidase (B) activities from B. longum RD47. The bars indicate the standard error of the mean. The bars bearing different lowercase letters are significantly different according to Duncan’s multiple range test (p < 0.05).

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 37℃ (lane 8). The synthetic substrates 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 37℃ (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.

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.

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 the intestinal tract. Therefore, foods containing raffinose and lactose at low levels are preferred for the galactosideintolerant consumers. The results of this study show several types of benefits. The findings could be helpful for the development of soymilk beverages and milk beverages for those who are lactose-intolerant, as the possibility of applying the findings to the practical production of foods was demonstrated. This is supported by previous studies, which reported that controlling excessive gas production and reducing gas-related symptoms can be achieved with α-galactosidase [5] and β-galactosidase [19].

With the increasing attention on the role of bifidobacteria owing to its health benefits, the use of B. longum RD47, having effective α- and β-galactosidase activities, may lead to feasible industrial, biotechnological, and medicinal applications.