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J. Microbiol. Biotechnol. (2017), 27(8), 1392–1400 https://doi.org/10.4014/jmb.1702.02058 Research Article Review jmb

Synthesis of β-Galactooligosaccharide Using Bifidobacterial β-Galactosidase Purified from Recombinant Escherichia coli So Young Oh1, So Youn Youn1, Myung Soo Park2,3, Hyoung-Geun Kim4, Nam-In Baek4, Zhipeng Li1, and Geun Eog Ji1,2*

1Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University, Seoul 08826, Republic of Korea 2Research Center, BIFIDO Co. Ltd., Hongcheon 25117, Republic of Korea 3Department of Hotel Culinary Art, Yeonsung University, Anyang 14011, Republic of Korea 4Graduate School of Biotechnology and Oriental Medicine Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea

Received: February 23, 2017 Revised: April 28, 2017 Galactooligosaccharides (GOSs) are known to be selectively utilized by , which Accepted: May 21, 2017 can bring about healthy changes of the composition of intestinal microflora. In this study, β-GOS were synthesized using bifidobacterial β-galactosidase (G1) purified from recombinant E. coli with a high GOS yield and with high productivity and enhanced bifidogenic activity. o First published online The purified recombinant G1 showed maximum production of β-GOSs at pH 8.5 and 45 C. A May 24, 2017 matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the

*Corresponding author major peaks of the produced β-GOSs showed MW of 527 and 689, indicating the synthesis of Phone: +82-2-880-8749; β-GOSs at degrees of polymerization (DP) of 3 and DP4, respectively. The were Fax: +82-2-884-0305; identified as β-D-galactopyranosyl-(1 → 4)-O-β-D-galactopyranosyl-(1 → 4)-O-β-D-glucopyranose, E-mail: [email protected] and the were identified as β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl- (1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranose. The maximal production yield of GOSs was as high as 25.3% (w/v) using purified recombinant β-galactosidase and 36% (w/v) of as a substrate at pH 8.5 and 45oC. After 140 min of the reaction under this condition, 268.3 g/l of GOSs was obtained. With regard to the effect, all of the tested Bifidobacterium except for B. breve grew well in BHI medium containing β-GOS as a sole carbon source, whereas lactobacilli and Streptococcus thermophilus scarcely grew in the same medium. Only Bacteroides fragilis, Clostridium ramosum, and Enterobacter cloacae among the 17 pathogens tested grew in BHI medium containing β-GOS as a sole carbon source; the remaining pathogens did not grow in the same medium. Consequently, the β-GOS are expected to contribute to the beneficial change of intestinal microbial flora. pISSN 1017-7825, eISSN 1738-8872

Copyright© 2017 by Keywords: β-Galactooligosaccharides, Bifidobacterium longum subsp. longum RD47, prebiotics, The Korean Society for Microbiology and Biotechnology β-galactosidase, recombinant Escherichia coli

Introduction beneficial bacteria, especially the Bifidobacterium and Lactobacillus species [4, 5], and inhibit the adhesion of A prebiotic is defined as “a selectively fermented ingredient pathogens to intestinal epithelial cells [6-9]. Therefore, that allows specific changes, both in the composition and/ GOSs bring about a healthy change of the composition of or activity in the gastrointestinal microflora and that the intestinal microbial flora [10]. This change of the gut confers benefits upon a host’s well- being and health” [1]. microbiota has positive effects on the immune response , (FOSs), , and and on metabolic syndrome markers [11-14]. Furthermore, galactooligosaccharides (GOSs) are included as prebiotics the fermentation products of GOSs in the colon by the gut [2, 3]. GOSs selectively promote the growth of intestinal microbiota are short-chain fatty acids such as acetate,

J. Microbiol. Biotechnol. β-Galactooligosaccharide Synthesis via Bifidobacterial β-Galactosidase 1393

propionate, butyrate, and lactate, which supply energy to Table 1. Bacterial strains and plasmids. the colonic epithelium, lower the colonic pH, stimulate calcium Strain or plasmid Relevant characteristics and magnesium absorption, and decrease proinflammatory B. longum RD47 Source of a response markers [5, 15-18]. β-galactosidase gene (g1) Over the years, galactosidases have been mainly used E. coli BL21 Expression host for the hydrolysis of lactose in milk [19-21]. However, pColdI Ampr, cloning galactosidases also have transgalactosylation activities that vector (containing can yield GOSs. To date, GOSs have mainly been synthesized polyhistidine-tagged with galactosidases gained from Aspergillus spp. and sequence) Kluyveromyces spp. [22-24]. Recently, there has been growing Prevotella intermedia KCTC 5694 Intestinal pathogens interest in recombinant DNA technology to improve the Listeria monocytogenes KCTC 3569 production efficiency of GOSs [25]. Staphylococcus aureus KCTC 1916 Bifidobacterium is dominant in breast-fed infants and has Salmonella typhimurium ATCC 14028 been used as a probiotic. it has also been approved as a Bacteroides fragilis KCTC 5013 “generally recognized as safe” substance [4, 26]. Enterococcus faecalis KCTC 3511 Various strains of Bifidobacterium contain β-galactosidase, Gardnerella vaginalis KCTC 5096 and some of them efficiently produce GOS [18]. Moreover, Clostridium perfringens KCTC 3269 GOSs synthesized by β-galactosidase from Bifidobacterium C. ramosum KCTC 3323 have unique product profiles and are more selectively utilized Eubacterium rectale KCTC 5835 by Bifidobacterium [2, 27, 28]. The successful application of C. leptum KCTC 5155 recombinant E. coli enhances the synthesized amount of Ruminococcus gnavus KCTC 5920 GOSs because recombinant DNA technology improves the Bacteroides coprocola KCTC 5443 activity, stability, and expression yield of β-galactosidase E. coli KCTC 1039 [29]. In our previous study, Bifidobacterium longum subsp. Enterobacter cloacae KCTC 2361 longum RD47 with relatively high transgalactosylation C. butyricum KCTC 1871 activity was isolated and the maximal production condition B. cellulosilyticus KCTC 5800 of crude β-galactosidase was preliminarily studied [30]. Lactobacillus rhamnosus KCTC 3237 Intestinal beneficial In this paper, we report the synthesis, purification, and bacterial strains characterization of β-GOSs using bifidobacterial β-galactosidase L. delbrueckii subsp. bulgaricus KCTC 3635 purified from recombinant E. coli. In addition, we investigate L. casei KFRI 699 the optimal synthesis conditions of β-GOSs and the prebiotic L. plantarum KFRI 708 effects of the purified β-GOSs on the major types of intestinal Lactococcus lactis KCTC 2013 bacteria. Bifidobacterium breve ATCC 15700 B. adolescentis ATCC 15705 Materials and Methods B. thermophilum KCCM 12097

Microorganisms and Culture Conditions B. bifidum ATCC 29521 The strains of bacteria and plasmids used in this study are listed B. longum RD47 in Table 1. The bacteria were cultured anaerobically in MRS broth B. bifidum BGN4 (Difco, USA) supplemented with 0.05% L-cysteine·HCl at 37oC for B. catenulatum KCTC 3221 18 h. B. breve KCTC 3419 Escherichia coli was cultured aerobically on Luria-Bertani (LB) B. animalis KCTC 3219 broth (BD Difco LB broth, Becton-Dickinson Company, USA) at B. infantis KCTC 3249 37oC for 15 h, with 100 μg/ml ampicillin (Bio Basic Inc., Canada) B. longum BORI supplemented when needed. S. salivarius subsp. thermophilus KCTC 3779 Expression of Cloned Bifidobacterial β-Galactosidase in E. coli For the cloning of the β-galactosidase gene, the genomic DNA of B. longum RD47 was isolated with a DNeasy Blood & Tissue Kit was used as the polymerase chain reaction (PCR) template. (Qiagen, USA) according to the manufacturer’s instructions and The PCR products of the open reading frame of the β-galactosidase

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gene were ligated into the pColdI Vector system (Takara, Japan) sulfuric acid and ethanol (1:9 (v/v)), and dried in an oven (120°C) with His-tag and transformed into E. coli BL21. The recovered for 5 min. plasmids were confirmed to have the bifidobacterial β-galactosidase gene by DNA sequencing. When the optical density of the Purification of β-GOS recombinant E. coli at 600 nm reached 0.4-0.6, the expression of β-GOS was purified using activated charcoal (Samchun Pure the cloned gene was induced by adding 1 mM isopropyl 1-thio-β- Chemical Co., Ltd, Korea). The activated charcoal was loaded D-galactopyranoside (Tokyo Chemical Industry Co., Ltd., Japan). into a column (50 × 5 cm Glass Econo-Column; Bio-Rad). After The recombinant E. coli was then incubated aerobically at 37°C for equilibrating the column, the reaction product was loaded into the 21 h. column. After washing the column with DW and 10% (v/v) ethanol, the β-GOSs adsorbed onto the activated charcoal were Preparation of Crude and Purification extracted with 50% (v/v) ethanol. The eluted fraction was After cultivation, 50 ml of the culture was centrifuged (8,000 ×g, concentrated by a speed vacuum concentrator (ScanSpeed 40; 10 min, 4°C) and washed in 50 mM sodium phosphate buffer (PB, LaboGene, Denmark). pH 6.5), after which the supernatant was discarded. The washed cells were resuspended in 1 ml of a binding buffer (PRO-Hunt Estimation of Saccharides His-Bind Buffer Kit 100; Elpis Biotech, Korea) and were disrupted The were quantified by an YL9100 HPLC system with a sonicator (Q500 sonicator; Q-Sonica, USA) for 1.0 sec with equipped with an YL9170 RI detector and with Autochro-3000 1.0 sec off intervals for 10 min, after which they were centrifuged. data system software (Younglin, Korea). The supernatant was obtained as the cell-free extract and was used to purify β-galactosidase using His-Bind Agarose resin and a Structural Characterization of β-GOS PRO-Hunt His-Bind Buffer Kit (Elpis Biotech) according to the The mass of the purified β-GOS was characterized by matrix- manufacturer’s instructions. The eluted fractions were concentrated assisted laser desorption ionization time-of-flight mass spectrometry using a centrifugal filter (30K Amicon Ultra centrifugal filter; (MALDI-TOF MS, AB Sciex TOF/TOF 5800; AB Sciex, USA) using Millipore, USA) and were resuspended in 1 ml PB. The purity was 2,5-dihydroxybenzoic acid as the matrix at the NCIRF of Seoul confirmed by SDS-PAGE with a 10% (w/w) acrylamide gel. The National University. Mass spectra were obtained over the m/z purified enzyme was mixed with a protein sample buffer (protein range of 300-1,500. The structure of the purified β-GOS was 2× sample buffer; Elpis Biotech), boiled for 5 min, and electrophoresed analyzed with a high resolution 600 MHz NMR spectrometer at 100 V for 30 min and then at 200V for 40 min with a power (AVANCE 600; Bruker, Germany) using 1D (¹H, ¹³C) and 2D supply (PowerPac Basic Power Supply; Bio-Rad, USA). The gel (HSQC, HMBC) with D2O as a solvent. was stained with a Stain Solution (Brilliant Blue R250 Stain Solution; Elpis Biotech) and destained with distilled water (DW). Prebiotic Effect of β-GOS The bacterial cells cultured in BHI medium were centrifuged Determination of the Hydrolytic Activity of the Purified (15,000 ×g, 10 min, 4°C) and washed in 50 mM of a sodium β-Galactosidase phosphate buffer (pH 6.5) to remove residual and The relative enzyme activity was measured by the release of and were then resuspended in PB. Cell suspensions (1% (v/v)) para-nitrophenol from para-nitrophenyl (pNP) β-D-galactopyranoside were inoculated in BHI broth without (BHI broth without (Sigma Aldrich, USA). An enzyme solution (80 μl) was added to dextrose; MB Cell, Korea), and 200 μl of this suspension was 20 μl of 5 mM pNP β-D-galactopyranoside suspended in 50 mM of immediately added to a 96-well microplate (96-well cell culture a sodium phosphate buffer (pH 6.5). The mixture was subsequently plate 3595; Corning, USA) containing 22 μl of 9% (w/v) β-GOS. incubated at 37°C for 1 h. The reaction was stopped by adding The cultures were anaerobically incubated at 37°C and the optical

100 μl of 1 M Na2CO3. The activity was measured in a 96-well density (OD) was measured at 600 nm [31]. microplate at 405 nm using a spectrophotometer (Model 680 Microplate reader; Bio-Rad). Stability Measurement of β-GOS The purified β-GOSs and FOSs were dissolved in buffers with Determination of the Transgalactosylation Activity pH values of 3.0, 4.1, 7.1, and 10.2 until the final concentration of The activity of the synthesized β-GOS was determined with solutions becomes 10% (w/v). Each of the GOS and FOS solution lactose (40% (w/v)) as the substrate. The mixture of purified was maintained at 70°C and 100°C for 120 min. enzyme and substrate was incubated at 37°C, and the reaction was terminated by boiling for 10 min. The product was determined by Results thin-layer chromatography (TLC) and then spotted onto a silica gel plate (20 × 20 cm TLC silica gel 60 F254; EMD Millipore Co., Gene Cloning of β-Galactosidases from B. longum RD47 Germany). The plate was developed with a 1-propanol:DW:ethyl- In our previous study, the full genome sequence of acetate (7:2:1) solvent solution, visualized using a solution of

J. Microbiol. Biotechnol. β-Galactooligosaccharide Synthesis via Bifidobacterial β-Galactosidase 1395

B. longum RD47 revealed three β-galactosidase genes (G1, G3, and G4) which were cloned and characterized for hydrolysis and GOS production, respectively. Among them, G1 showed the best productivity for GOS. The sizes of these genes were 3,072 bp (g1). G1 contained 1,024 amino acids and had a calculated molecular mass of 112.64 kDa. In this study, the ORF of G1 was subcloned into pCold1 for high expression and efficient purification using His-tag as described in the Materials and Methods section.

Purification of The purified enzyme sample was loaded onto SDS-PAGE gel, and the purified β-galactosidase showed a single band of approximately 100 kDa, which corresponds with the molecular mass of 112.64 kDa deduced from the gene sequence (Fig. 1).

SDS-PAGE of β-galactosidase from recombinant E. coli Fig. 1. Effects of pH and Temperature on the Hydrolytic Activity BL21(G1) purified by means of the His-tag purification The optimal pH for the hydrolytic activity of purified β- method. galactosidase was found to be pH 8.5 when investigated in M, protein marker; lane 1, crude enzyme; lanes 2-5, fractions of the elution step of purification. the pH range of 7.4-8.9 (Fig. 2A). At the optimal pH, the

Fig. 2. Effects of pH and temperature on the hydrolytic activity (A), (B) and transgalactosylation (C), (D) of β-galactosidase from recombinant E. coli BL21(G1) purified by means of the His-tag purification method.

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Table 2. Analysis of the yield and productivity of β-galactooligosaccharide (β-GOS). Initial lactose (g/l) Residual lactose (g/l) GOS yield (%) Productivity (g/l h) Bifidobacterial β-galactosidase from recombinant E. coli 360 83.9 25.3 115.2 maximal hydrolytic activity of β-galactosidase was found and Enterobacter cloacae among the tested pathogens grew to be 40°C (Fig. 2B). well (Fig. 4B).

Effects of pH and Temperature on Transgalactosylation Stability Measurement of β-GOS Activity Stability measurement of β-gos was determined by TLC. The investigated range of the optimal pH for the The glycosidic linkages of FOS were totally hydrolyzed transgalactosylation activity of β-galactosidase was identical after 120 min at pH 3.0 at 100°C and were partly hydrolyzed to that used to assess the hydrolytic activity, and the maximum after 120 min at pH 3.0 at 70°C and at pH 4.1 at 100°C production of β-GOS was observed at pH 8.5 (Fig. 2C). The (Fig. 5B). On the other hand, the glycosidic linkages of GOS effect of the temperature for the transgalactosylation were much more stable than those of the FOS. The production of the purified β-GOS was studied in the range glycosidic linkages of GOS were maintained stably after of 31.5-50°C and at pH 8.5. The optimal production of 120 min at pH 3.0, 4.1, 7.0, and 10.2 at 70°C and 100°C β-GOS was found to occur at 45°C (Fig. 2D). (Fig. 5A).

Analysis of the Yield and Productivity of β-GOS Discussion The maximal production yield of GOS was found to be as high as 25.3% (w/v) using purified recombinant There is growing interest in industrially useful micro- β-galactosidase and 36% (w/v) of lactose as a substrate at pH 8.5 and 45°C (Table 2). After 140 min of the reaction under this condition, 268.3 g/l of GOS was obtained. The yield and productivity were calculated as follows.

Yield (%) = [β-GOS (g/l)/initial lactose concentration (g/l)]*100 Productivity (g/l h) = β-GOS (g/l)/reaction time (h) [32]

Structural Characterization of β-GOS The peaks of the β-GOS according to the MALDI-TOF- MS analysis were at m/z 527 and 689, which indicated that the synthesized β-GOS consisted of DP3 and DP4, respectively (Fig. 3A). The trisaccharides according to the NMR analysis were identified as β-D-galactopyranosyl- (1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranose, and the tetrasaccharides were identified as β-D-galactopyranosyl- (1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl- (1→4)-O-β-D-glucopyranose (Fig. 3B).

Prebiotic Effect of β-GOS All of the tested Bifidobacterium species except for B. breve grew sufficiently in BHI medium containing β-GOS as a sole carbon source (Fig. 4A). On the other hand, Listeria monocytogenes, Salmonella typhimurium, Enterococcus faecalis, Gardnerella vaginalis, Ruminococcus gnavus, and Bacteroides coprocola scarcely grew in BHI medium under identical Fig. 3. MALDI-TOF-MS result of the purified β- conditions. Only Bacteroides fragilis, Clostridium ramosum, galactooligosaccharide (A) and its deduced structure (B).

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Fig. 4. Growth of intestinal pathogens (A) and beneficial bacterial strains (B) in BHI medium without carbon source, with glucose, and with β-galactooligosaccharide (β-GOS) as the sole carbon source. (A): 1. Prevotella intermedia; 2. Listeria monocytogenes; 3. S. aureus; 4. Salmonella typhimurium; 5. B. fragilis; 6. E. faecalis; 7. G. vaginalis; 8. C. perfringens; 9. Clostridium ramosum; 10. Eubacterium rectale; 11. Clostridium leptum; 12. Ruminococcus gnavus; 13. B. coprocola; 14. E. coli; 15. Enterobacter cloacae; 16. C. butyricum; 17. B. cellulosilyticus. (B): 1. Lactobacillus rhamnosus; 2. L. delbrueckii subsp. bulgaricus; 3. L. casei; 4. L. plantarum; 5. Lactococcus lactis; 6. Bifidobacterium breve; 7. B. adolescentis; 8. B. thermophilum; 9. B. bifidum; 10. B. longum RD47; 11. B. bifidum BGN4; 12. B. catenulatum; 13. B. breve; 14. B. animalis; 15. B. infantis; 16. B. longum BORI; 17. Streptococcus salivarius subsp. thermophilus.

Stabilities of purified galactooligosaccharide (GOS) (A) organisms to improve the production efficiency and prebiotic Fig. 5. and control (FOS) (B) after 120 min effect of GOS. For these purposes, we attempted to find the incubation at 70°C and 100°C at pH 3.0, 4.1, 7.1, and 10.2. optimal condition for GOS production with a bifidobacterial β-galactosidase purified from recombinant E. coli and to characterize the production efficiency and prebiotic effects. strain. In an earlier work, the optimum pH and temperature β-Galactosidases from various sources of microorganisms of β-galactosidase activity from a cell-free extract of B. longum have different features [33]. Even β-galactosidases from the RD47 were pH 6.0 and 37°C [30], whereas the corresponding same species (B. longum) have different optimum pH levels optimum pH and temperature of purified β-galactosidase and temperatures with regard to β-galactosidase activity. from E. coli were pH 8.5 and 40°C. As described before, The optimum pH and temperature for β-galactosidase there are three β-galactosidase genes (G1, G3, and G4) in activity from B. longum BCRC 15708 were pH 6.8 and 45°C, B. longum RD47, with two (G3 and G4) showing relatively whereas the corresponding optimum pH and temperature high hydrolytic activity levels as compared with the levels for β-galactosidase activity from B. longum RD47 were of transgalactosylation activity. Thus, the hydrolytic activity pH 6.0 and 37°C [4, 30]. This indicates that probiotic of a cell-free extract of B. longum RD47 reflects those of G3 activity such as β-galactosidase activity depends on the and G4. It is interesting that G1 showed optimal hydrolytic

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Table 3. NMR results of purified β-galactooligosaccharide (β-GOS).

Carbon No. β-GOS (D2O) Lactose (D2O) D1 Proton No. β-GOS (D2O) α-glc-1 94.7 92.0 2.7 α-glc-1 5.23, d, J=3.7 Hz α-glc-2 71.2 70.3 0.9 α-glc-2 3.93, overlapped α-glc-3 72.9 71.4 1.5 α-glc-3 3.71, overlapped α-glc-4 81.1 78.6 2.5 α-glc-4 3.71, overlapped α-glc-5 72.9 71.6 1.3 α-glc-5 3.71, overlapped -glc-662.8 α-glc-662.8 60.1 2.7 α-glc-63.97, overlapped, 3.89, overlapped β-glc-1 98.6 96.0 2.6 β-glc-1 4.68, d, J=8.0 Hz β-glc-2 74.0 74.0 0.0 β-glc-2 3.31, d, J=8.0, 8.0 Hz β-glc-3 74.2 74.6 -0.4 β-glc-3 3.71, overlapped β-glc-4 80.9 78.4 2.5 β-glc-4 3.71, overlapped β-glc-5 77.2 75.0 2.2 β-glc-5 3.76, overlapped β-glc-6 62.9 60.3 2.6 β-glc-63.97, overlapped, 3.89, overlapped gal-1’ 105.3 103.1 2.2 gal-1’ 4.53, d, J=7.9 Hz gal-2’ 71.4 71.2 0.2 gal-2’ 3.93, overlapped gal-3’ 73.8 72.7 1.1 gal-3’ 3.71, overlapped gal-4’ 84.7 68.8 15.9 gal-4’ 3.84, overlapped gal-5’ 77.8 75.6 2.2 gal-5’ 3.76, overlapped gal-6’ 62.9 61.3 1.6 gal-6’ 3.77, overlapped gal-1’’ 107.3 gal-1’’ 4.62, d, J=7.7 Hz gal-2’’ 71.4 gal-2’’ 3.93, overlapped gal-3’’ 75.3 gal-3’’ 3.65, overlapped gal-4’’ 84.7 gal-4’’ 3.84, overlapped gal-5’’ 77.9 gal-5’’ 3.71, overlapped gal-6’’ 62.9 gal-6’’ 3.77, overlapped gal-1’’’ 107.3 gal-1’’’ 4.62, d, J=7.7 Hz gal-2’’’ 71.2 gal-2’’’ 3.93, overlapped gal-3’’’ 75.3 gal-3’’’ 3.65, overlapped gal-4’’’ 73.0 gal-4’’’ 3.72, overlapped gal-5’’’ 77.9 gal-5’’’ 3.71, overlapped gal-6’’’ 62.8 gal-6’’’ 3.77, overlapped activity at a relatively alkali condition when compared compared with the transgalactosylation activity of other with other β-galactosidases from B. adolescentis DSM20083, β-galactosidases from B. infantis HL96, which showed the which showed optimal hydrolytic activity at pH 6.0 and optimal level of activity at 60°C [35]. 50°C [34]. There are various reports on how β-galactosidases When the production yield and productivity of GOS produced from multiple probiotic bacteria express their using β-galactosidase from B. longum RD47 were assessed, maximum enzyme activity under somewhat acidic conditions it showed a higher production yield and greater productivity [35, 36]. In this study, G1 with high transgalactosylation of GOS than β-galactosidases from yeast and fungi [32, 37, activity was chosen to produce GOS. GOSs are produced 38]. Furthermore, it showed higher productivity when by transgalactosylation during the hydrolysis of lactose compared with recombinant β-galactosidase from B. infantis [33]. The optimal pH and temperature for hydrolytic activity HL96 [35], for which the productivity was measured at are nearly identical to the optimum pH and temperature 12.7 g/l h. for transgalactosylation activity. The optimal production of The synthesized β-GOS was used with all bifidobacteria β-GOS was achieved at 45°C. This is relatively low when tested, excluding B. breve KCTC 3419, but it was not used

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with most of the tested pathogens. Apparently, the synthesized galactooligosaccharide mixture increases the bifidobacterial GOS possessed a structure that can be preferentially used population numbers in a continuous in vitro fermentation with the β-galactosidase of Bifidobacterium. An additional system and in the proximal colonic contents of pigs in vivo. reason for this prebiotic effect may be the possession of a J. Nutr. 135: 1726-1731. higher level of β-galactosidase by Bifidobacterium as compared 6. Quintero M, Maldonado M, Perez-Munoz M, Jimenez R, Fangman T, Rupnow J, et al. 2011. Adherence inhibition of with those of other intestinal bacterial species [39-41]. Cronobacter sakazakii to intestinal epithelial cells by prebiotic The glycosidic linkages of GOS and FOS were stable in . Curr. Microbiol. 62: 1448-1454. alkaline and neutral conditions and at 100°C condition. 7. Sinclair HR, de Slegte J, Gibson GR, Rastall RA. 2009. However, the glycosidic linkages of GOS were much more Galactooligosaccharides (GOS) inhibit Vibrio cholerae toxin stable than those of the FOS in acidic condition such as pH binding to its GM1 receptor. J. Agric. Food Chem. 57: 3113-3119. 3.0 and 4.1. The weakness of glycosidic linkages of FOS in 8. Shoaf K, Mulvey GL, Armstrong GD, Hutkins RW. 2006. acidic condition was previously reported [42]. Prebiotic galactooligosaccharides reduce adherence of Aside from this prebiotic effect of selectively growing enteropathogenic Escherichia coli to tissue culture cells. intestinal beneficial bacteria, GOSs have another prebiotic Infect. Immun. 74: 6920-6928. effect mechanism. GOSs have been shown to inhibit the 9. Searle LE, Cooley WA, Jones G, Nunez A, Crudgington B, adherence of Cronobacter sakazakii, enteropathogenic E. coli, Weyer U, et al. 2010. Purified galactooligosaccharide, derived and Vibrio cholerae toxins [6-8]. Moreover, GOSs have been from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar shown to affect intestinal immunity [11, 13, 43]. Accordingly, Typhimurium adhesion and invasion in vitro and in vivo. J. the β-GOS produced in this study warrants further study of Med. Microbiol. 59: 1428-1439. other possible prebiotic effects. 10. Cardelle-Cobas A, Corzo N, Olano A, Peláez C, Requena T, In summary, the newly produced β-GOS can cause Ávila M. 2011. Galactooligosaccharides derived from lactose beneficial changes of intestinal microbial flora and may and lactulose: influence of structure on Lactobacillus, have other prebiotic effects as well. Streptococcus and Bifidobacterium growth. Int. J. Food Microbiol. 149: 81-87. Acknowledgments 11. Vulevic J, Juric A, Tzortzis G, Gibson GR. 2013. A mixture of trans-galactooligosaccharides reduces markers of metabolic This work was carried out with support from the “National syndrome and modulates the fecal microbiota and immune Research Foundation of Korea(NRF) grant funded by the function of overweight adults. J. Nutr. 143: 324-331. Korea government(MSIP) (Project No.2017R1A2B2012390),” 12. Vulevic J, Juric A, Walton GE, Claus SP, Tzortzis G, Toward RE, et al. 2015. Influence of galacto- mixture (B-GOS) from the Rural Development Administration, and from the on , immune parameters and metabonomics “Promoting Regional specialized Industry (Project No. in elderly persons. Br. J. Nutr. 114: 586-595. R0004140)” project of the Ministry of Trade, Industry 13. Vulevic J, Drakoularakou A, Yaqoob P, Tzortzis G, Gibson GR. and Energy (MOTIE), and from the Korea Institute for 2008. 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