Synthesis of Stachyobifiose Using Bifidobacterial α-Galactosidase

Jan 24, 2018 - Research Center, BIFIDO Co. Ltd., Kangwon 250-804, Republic of Korea. ∥ Department of Hotel Culinary Arts, Yeonsung University, Gyeon...
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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Synthesis of Stachyobifiose Using Bifidobacterial α‑Galactosidase Purified from Recombinant Escherichia coli So Young Oh,† So Youn Youn,‡ Myeong Soo Park,§,∥ Nam In Baek,⊥ and Geun Eog Ji*,†,§ †

Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University, Seoul 151-742, Republic of Korea ‡ Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs, Gimcheon, Gyeongsangbuk-do 39660, Republic of Korea § Research Center, BIFIDO Co. Ltd., Kangwon 250-804, Republic of Korea ∥ Department of Hotel Culinary Arts, Yeonsung University, Gyeonggi 14011, Republic of Korea ⊥ Graduate School of Biotechnology and Oriental Medicine Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea ABSTRACT: The prebiotic effects of GOS (galactooligosaccharides) are known to depend on the glycosidic linkages, degree of polymerization (DP), and the monosaccharide composition. In this study, a novel form of α-GOS with a potentially improved prebiotic effect was synthesized using bifidobacterial α-galactosidase (α-Gal) purified from recombinant Escherichia coli. The carbohydrate produced was identified as α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→2)-[α-D-galactopyranosyl-(1→6)-O-β-Dfructofuranoside] and was termed stachyobifiose. Among 17 nonprobiotics, 16 nonprobiotics showed lower growth on stachyobifiose than β-GOS. In contrast, among the 16 probiotics, 6 probiotics showed higher growth on stachyobifiose than β-GOS. When compared with raffinose, stachyobifiose was used less by nonprobiotics than raffinose. Moreover, compared with stachyose, stachyobifiose was used less by Escherichia coli, Enterobacter cloacae, and Clostridium butyricum. The average amounts of total short-chain fatty acids (SCFA) produced were in the order of stachyobifiose > stachyose > raffinose > β-GOS. Taken together, stachyobifiose is expected to contribute to beneficial changes of gut microbiota. KEYWORDS: stachyobifiose, Bif idobacterium longum subsp. longum RD 47, prebiotics, α-galactosidase, recombinant Escherichia coli

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INTRODUCTION

magnesium absorption, decrease proinflammatory response markers, and decrease adiposity and body weight.1,8,11−14 GOS with various glycosidic linkages and degrees of polymerization (DP) have been synthesized using various galactosidases.10 Commercial GOS are usually synthesized with galactosidases from fungi such as Aspergillus spp. and Kluyveromyces spp.10,15−17 GOS synthesized by β-galactosidase (β-Gal) from Bif idobacterium have unique product profiles and are utilized more selectively by Bif idobacterium18−20 than GOS synthesized with galactosidases from fungi. Similar to β-Gal, α-galactosidase (α-Gal) can also be used to synthesize GOS. However, the synthesis of GOS in an α-anomeric configuration by α-Gal has rarely been discussed.21 In our previous study, Bifidobacterium longum subsp. longum RD 47 with relatively high transgalactosylation activity was isolated, and the maximal production condition of crude α-Gal was studied.22 In this paper, we report the synthesis of novel α-GOS using α-Gal of B. longum subsp. longum RD 47 purified from recombinant E. coli. The structure of the novel α-GOS has not yet been reported in other articles, and the term stachyobifiose was assigned to it. In addition, the ability to ferment stachyobifiose by major types of intestinal bacteria was compared to raffinose, stachyose, and β-GOS.

The gut microbiota is involved in promoting the health of the host, and an optimal gut microbiota composition contributes to this. On the other hand, dysbiosis of the gut microbiota composition causes not only intestinal diseases but also systemic diseases such as obesity and certain disorders of the central nervous system (CNS).1,2 The gut microbiota composition is specific to the host but can be changed by alterations in the diet.3 The gut of breast-fed infants consists of abundant Bif idobacterium. A Bif idobacterium-rich gut has protective effects with regard to immune-related diseases.4 Moreover, an acidic environment is created by the metabolite such as short-chain fatty acids (SCFA) produced by Bif idobacterium, which inhibits the growth of nonprobiotics.5 Prebiotics such as GOS (galactooligosaccharides), FOS (fructooligosaccharides), XOS (xylooligosaccharides), and inulin are defined as selectively fermented ingredients that confer benefits upon the host’s well-being and health by selectively stimulating the growth and/or activity of specific species of gut microbiota.6 In particular, GOS are well-known to increase the populations of beneficial intestinal bacteria selectively, especially the Bif idobacterium and Lactobacillus species,7,8 whereas it is not commonly used by harmful intestinal bacteria.9 Therefore, GOS can bring about a healthy change of the composition of the gut microbiota.10 Furthermore, GOS are fermented by the gut microbiota into SCFA such as acetate, propionate, butyrate, and lactate, which lower the colonic pH, stimulate calcium and © XXXX American Chemical Society

Received: October 11, 2017 Revised: December 26, 2017 Accepted: January 9, 2018

A

DOI: 10.1021/acs.jafc.7b04703 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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was added to induce the expression of the cloned gene and incubated aerobically at 37 °C for 21 h. Preparation of Crude Enzyme and Purification. After cultivation, 50 mL of the culture was centrifuged (8,000 × g, 10 min, 4 °C) and washed in 50 mL of sodium phosphate buffer (50 mM, pH 6.5), after which the supernatant was discarded. The washed cells were resuspended in 1 mL of a binding buffer (PRO-Hunt His-Bind Buffer Kit 100, Elpis Biotech, Korea) and disrupted with a sonicator (Q500 sonicator, Q-Sonica, USA) for 1.0 s with 1.0 s off intervals for 10 min, on ice. The supernatant was obtained as the cell-free extract by centrifugation (16,000 × g, 5 min, 4 °C) and used to purify α-Gal using His-Bind Agarose resin and a PRO-Hunt His-Bind Buffer Kit (Elpis Biotech, Korea) according to the manufacturer’s instructions. The eluted fractions were concentrated using a centrifugal filter (30K Amicon Ultra centrifugal filter, Millipore, USA) and were resuspended in 1 mL of PB. The purity was confirmed by SDS-PAGE with a 10% (w/w) acrylamide gel. The purified enzyme was mixed with a protein sample buffer (protein 2× sample buffer, Elpis Biotech, Korea), boiled for 5 min, and electrophoresed at 100 V for 30 min and then at 200 V for 40 min with a power supply (PowerPac Basic Power Supply, Bio-Rad, USA). The gel was stained with a Stain Solution (Brilliant Blue R250 Stain Solution, Elpis Biotech, Korea) and destained with distilled water (DW). Determination of the Hydrolytic Activity of the Purified αGalactosidase. The relative enzyme activity was measured by the release of para-nitrophenol from para-nitrophenyl (pNP) α-D-galactopyranoside (Sigma-Aldrich, USA). An enzyme solution (80 μL) was added to 20 μL of 5 mM pNP α-D-galactopyranoside in 50 mM of a sodium phosphate buffer (PB, pH 6.5). The mixture was subsequently incubated at 37 °C for 1 h. The reaction was stopped by adding 100 μL of 1 M Na2CO3. The activity was measured in a 96-well microplate at 405 nm using a spectrophotometer (Model 680 Microplate reader, Bio-Rad, USA). Determination of the Transgalactosylation Activity. The stachyobifiose was synthesized using raffinose (40%, w/v) as a substrate and the purified α-Gal. Raffinose and purified enzyme were mixed at a ratio of 5:1, which was incubated at 37 °C, and the reaction was terminated by boiling for 10 min. The product was analyzed by

MATERIALS AND METHODS

Microorganisms and Culture Conditions. The strains of bacteria and plasmids used in this study are described below (Table 1).

Table 1. Bacterial Strains and Plasmids strain or plasmid B. longum RD47 E. coli BL21 pColdI

relevant characteristics source of a α-Gal gene (g7) expression host ampR, cloning vector (containing polyhistidine-tagged sequence) nonprobiotics

Prevotella intermedia KCTC 5694 Listeria monocytogenes KCTC 3569 Staphylococcus aureus KCTC 1916 Salmonella typhimurium ATCC 14028 Bacteroides f ragilis KCTC 5013 Enterococcus faecalis KCTC 3511 Gardnerella vaginalis KCTC 5096 Clostridium perf ringens KCTC 3269 Clostridium ramosum KCTC 3323 Eubacterium rectale KCTC 5835 Clostridium leptum KCTC 5155 Ruminococcus gnavus KCTC 5920 Bacteroides coprocola KCTC 5443 Escherichia coli KCTC 1039 Enterobacter cloacae KCTC 2361 Clostridium butyricum KCTC 1871 Bacteroides cellulosilyticus KCTC 5800 Lactobacillus rhamnosus KCTC 3237 probiotics L. delbrueckii subsp. bulgaricus KCTC 3635 L. casei KFRI 699 L. plantarum KFRI 708 Lactococcus lactis KCTC 2013 Bifidobacterium breve ATCC 15700 B. adolescentis ATCC 15705 B. thermophilum KCCM 12097 B. bif idum ATCC 29521 B. longum RD 47 B. bif idum BGN4 B. catenulatum KCTC 3221 B. breve KCTC 3419 B. animalis KCTC 3219 B. infantis KCTC 3249 B. longum BORI

Table 2. Summary of Purification of α-Galactosidase from Recombinant E. coli

crude αgalactosidase purified αgalactosidase

The probiotic bacteria were cultured anaerobically in MRS broth (H2 5%, CO2 5%, N2 90%) (Difco, USA) supplemented with 0.05% L-cysteine·HCl at 37 °C for 18 h. The nonprobiotics were cultured anaerobically in BHI broth (Difco, USA) at 37 °C for 18 h. Recombinant Escherichia coli were cultured aerobically in Luria− Bertani (LB) broth (BD Difco LB broth, Becton-Dickinson company, USA) at 37 °C for 16 h, and 100 μg/mL ampicillin (Bio Basic Inc., Canada) was supplemented when needed. Expression of Cloned Bifidobacterial α-Galactosidase in E. coli. For the cloning of the α-Gal gene, the genomic DNA of B. longum RD47 was isolated with a DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions and was used as the polymerase chain reaction (PCR) template. The α-Gal gene was amplified with the forward primer (5′-CGG ATC CAT GGC CGA GAA CAC CTC) and the reverse primer (5′-CCA ATT GTC AGC TTA ACG CCA CAG CCT TG). The PCR products of the open reading frame of the α-Gal gene were ligated into the pColdI Vector system (Takara) with His-tag and transformed into E. coli BL21. The recovered plasmids were confirmed to have the bifidobacterial α-Gal gene by DNA sequencing. When the OD600 of the recombinant E. coli reached 0.4−0.6, 1 mM isopropyl 1-thio-β-D-galactopyranoside (Tokyo Chemical Industry Co., Ltd., Japan)

total activity (U)

total protein (mg)

specific activity (U/mg)

purification fold

overall (yield %)

1526.36

66.48

22.96

1

100

882.04

2.25

391.67

17.06

57.79

Figure 1. SDS-PAGE of α-galactosidase from recombinant E. coli BL21 purified by means of the His-tag purification method. M, protein marker; lane 1, crude enzyme; lane 2, fraction of purification residue; lanes 3−5, fraction of washing step of purification; lanes 6−9, fraction of elution step of purification. B

DOI: 10.1021/acs.jafc.7b04703 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry thin-layer chromatography (TLC, 20 × 20 cm TLC silica gel 60 F254, EMD Millipore Co., Germany). 1 μL of the product (8%, w/v) was loaded on TLC. The plate was run with a 1-propanol:DW (Distilled Water):ethyl acetate (7:2:1) solvent solution, visualized using a solution of sulfuric acid and ethanol (1:9, v/v), and dried in an oven (120 °C) for 5 min. Purification of Stachyobifiose. The stachyobifiose was purified using activated charcoal (Samchun Pure Chemical Co., Ltd., Korea). The activated charcoal was loaded into a column (50 × 5 cm Glass Econo-Column, Bio-Rad, USA). After DW flowed to equilibrate the column, the reaction product was loaded onto the column. After washing the column with DW and 10% (v/v) ethanol, the stachyobifiose adsorbed onto the activated charcoal was extracted using 50% (v/v) ethanol. The stachyobifiose was further purified by preparative HPLC (JAI, Japan) using a column (2.0 × 25 cm YMC-Pack polyamine II column, YMC, Japan) and RI detector (JAI, Japan). The eluted fractions were concentrated by a speed vacuum concentrator (ScanSpeed 40, LaboGene, Denmark) and were resuspended in 1 mL of PB. Structural Characterization of Stachyobifiose. The mass of the purified stachyobifiose was characterized by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-

TOF MS, AB Sciex TOF/TOF 5800, AB Sciex, USA) using 2,5dihidroxybenzoic acid as the matrix at the NCIRF of Seoul National University. Mass spectra were obtained over the m/z range of 300−1500. The structure of the purified stachyobifiose was analyzed with a high resolution 600 MHz NMR spectrometer (AVANCE 600, Bruker, Germany) using 1D (1H, 13C) and 2D (HSQC, HMBC) with D2O as a solvent. Effect of Prebiotics on Growth of Intestinal Bacteria. In order to analyze the growth characteristics of representative intestinal bacterial cells on stachyobifiose, the bacterial cells listed in Table 1 cultured in BHI medium were centrifuged (15,000 × g, 10 min, 4 °C) and washed in 50 mM of a sodium phosphate buffer (PB, pH 6.5) to remove residual sugar and nutrients and then resuspended in the same volume of PB as the original culture medium. Cell suspensions (1%, v/v) were inoculated in BHI broth without glucose (BHI Broth without Dextrose, MB cell, Seoul, Korea), and 200 μL of this suspension was immediately added to a 96-well microplate (96-well cell culture plate 3595, Corning, USA) containing 22 μL of 9% (w/v) stachyobifiose. The cultures were anaerobically incubated at 37 °C, and the optical density (OD) was measured at 600 nm.23

Figure 2. Effects of the pH and temperature on the hydrolytic activity (A, B) and transgalactosylation (C, D) of α-galactosidase from recombinant E. coli BL21 (G7) purified by means of the His-tag purification method. C

DOI: 10.1021/acs.jafc.7b04703 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry SCFA Analysis. Samples from the culture broth above were centrifuged at 10,000 × g for 5 min, and supernatant was injected onto an YL9100 HPLC system equipped with an YL9170 RI detector and with Younglin Autochro-3000 data system software (Younglin, Korea). SCFAs were separated on an Aminex HPX-87H Ion Exclusion column (300 × 7.8 mm, 9 μm, Bio-Rad, USA) and run isocratic with 5 mM sulfuric acid at a flow rate of 0.6 mL per min and a temperature of 35 °C.

Structural Identification of Stachyobifiose. The peak of stachyobifiose according to a MALDI-TOF-MS analysis was m/z 689, which indicated that the synthesized stachyobifiose consisted of DP4 (Figure 3a). The tetrasaccharide was identified as α-D-galactopyranosyl(1→6)-O-α-D-glucopyranosyl-(1→2)-[α-D-galactopyranosyl(1→6)-O-β-D-fructofuranoside] by NMR (Figure 3b). Ability To Ferment Stachyobifiose. Among the tested probiotic strains, none of the Lactobacillus strains showed growth on stachyobifiose (Figure 4b). All of the tested Bifidobacterium strains, except for B. bifidum ATCC 29521, B. bifidum BGN4, and B. breve KCTC 3419, grew well in a BHI medium containing stachyobifiose as the sole carbon source (Figure 4b). On the other hand, only Bacteroides f ragilis KCTC 5013, Clostridium butyricum KCTC 1871, and Bacteroides cellulosilyticus KCTC 5800 among the tested nonprobiotics showed competitive growth on stachyobifiose compared to their growth with glucose (Figure 4a). SCFA Production during Fermentation. The profiles of SCFA in cultures of B. breve ATCC 15700, B. adolescentis ATCC 15705, B. longum subsp. longum RD 47, B. catenulatum KCTC 3221, B. animalis KCTC 3219, and B. infantis KCTC 3249 are shown in Table 3. The total SCFA in all of the sugar-added cultures was significantly higher than the total SCFA in control cultures. Compared with β-GOS, the production of total SCFA in α-GOS was higher in most samples, except for B. animalis KCTC 3219. Among the α-GOS samples, the total amount of SCFA was found to differ depending on the strain. However, the average amount of total SCFA for the six strains was in the order of stachyobifiose > stachyose > raffinose.

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RESULTS Cloning of the α-Galactosidase Gene from B. longum RD47. In our previous study, the full genome sequence of B. longum RD47 revealed one α-Gal gene (G7), which was cloned and characterized with respect to substrate hydrolysis and GOS production. The size of this gene was 2,307 bp nucleotides (GenBank accession number MF782363). G7 was predicted to contain 769 amino acids and to have a calculated molecular weight of 84.59 kDa. In this study, the ORF of G7 was subcloned into pCold1 for high expression and efficient purification using His-tag. Purification of Enzymes. After his-tag purification of recombinant α-Gal, the specific activity levels of 391.7 U/mg and purification fold of 17.1 were obtained (Table 2), respectively. When the purified enzyme sample was loaded onto SDS-PAGE gel, it showed a single band between 70 and 100 kDa, which corresponds to the predicted molecular mass of 84.59 kDa induced from the gene sequence (Figure 1). Effects of the pH and Temperature on the Hydrolytic Activity. The optimal pH for the hydrolytic activity of purified α-Gal was found to be pH 6.5 when investigated in the pH range of 5.7−7.6 (Figure 2a). At the optimal pH, the maximal hydrolytic activity of α-Gal was found at 31.5 °C (Figure 2b). Effects of the pH and Temperature on the Transgalactosylation Activity. The investigated range of the optimal pH for the transgalactosylation activity of α-Gal was identical to that used to assess the hydrolytic activity, and the maximum production of stachyobifiose was not significantly affected by pH (Figure 2c). The effect of temperature for the transgalactosylation production of purified stachyobifiose was studied in the range of 22−42 °C and at a pH of 6.5. The optimal production of stachyobifiose was found to occur in the temperature range of 22−32 °C (Figure 2d).

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DISCUSSION There is a growing interest in industrially useful enzymes to improve the prebiotic effect of GOS. We synthesized novel α-GOS using α-Gal of B. longum subsp. longum RD 47 purified from recombinant E. coli. The structure of the novel α-GOS has not yet been reported in other articles and assigned the term stachyobifiose.

Figure 3. MALDI-TOF-MS result of purified stachyobifiose and its deduced structure. D

DOI: 10.1021/acs.jafc.7b04703 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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cell-free extract of B. longum RD 47 (84.59 kDa) nearly coincided with the molecular mass of α-Gal from bifidobacteria. The optimum pH values of α-Gal from Lactobacillus reuteri, B. adolescentis, B. breve, and B. longum RD 47 were 5.0, 5.5, 5.5−6.5, and 6.5, respectively.28−30 In our previous work, the optimum pH and temperature of α-Gal activity from a cell-free extract of B. longum RD 47 were pH 6.0 and 35 °C,22 which are similar to the optimum pH (6.5) and temperature (31.5 °C) of bifidobacterial α-Gal purified from recombinant E. coli, as the genome of B. longum RD 47 has only one identifiable α-Gal gene.9 The prebiotic effect of GOS depends on the degree of polymerization (DP), the type of linkage, and the monosaccharide composition.10 In our previous study, the structure of β-GOS synthesized using β-Gal from B. longum RD 47 was identical to that of commercial GOS(β-D-galactopyranosyl-(1→4)-O-β-Dgalactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-Dglucopyranose).9 This β-GOS was used by most of the intestinal beneficial bacteria as well as various harmful bacteria, indicating that the selective utilization of this β-GOS among them is not very high. However, β-GOS is considered a prebiotic despite it not having a perfect selective utilization profile. Therefore, to determine whether a substrate is prebiotic, it should be determined not only by in vitro but also by more experiments. However, stachyobifiose was selectively used by most of the Bifidobacterium strains tested. On the other hand, among 17 nonprobiotic bacteria, only three (Bacteroides f ragilis KCTC 5013, Clostridium butyricum KCTC 1871, and Bacteroides cellulosilyticus KCTC 5800) showed noticeable growth on α-GOS. Compared to the stachyobifiose of DP4, raffinose of DP3 was used better by Prevotella intermedia KCTC 5694, Escherichia coli KCTC 1039, Enterobacter cloacae KCTC 2361, and Clostridium butyricum KCTC 1871 among the tested nonprobiotics. Therefore, stachyobifiose showed a stronger selective fermentation ability than DP3 α-GOS. On the other hand, in β-GOS, Bif idobacteria grew better in DP3 GOS than in DP4 GOS.31 Both stachyobifiose and stachyose are DP4 and have identical monosaccharide compositions, but stachyobifiose is more steric. Among nonprobiotics, most of the strains showed poor growth on stachyobifiose except for Bacteroides f ragilis KCTC 5013 (Figure 4a). On the other hand, probiotics strains showed nearly different amounts of growth on both stachyobifiose and stachyose, except for B. bif idum BGN4 (Figure 4b). It was interesting to note that nearly identical B. breve strains (Figure 4b; #6 and 13) showed different growth characteristics on different sugars. This indicates that the probiotic characteristics depend not only on the genus-species level but also on the strain level.32 The different ability to ferment between stachyose and stachyobifiose may have arisen due to the different steric structures, with stachyobifiose not easily used by nonprobiotic bacteria and stachyose used by Escherichia coli KCTC 1039, Enterobacter cloacae KCTC 2361, and Clostridium butyricum KCTC 1871. Similarly, stronger prebiotic effects were obtained from steric β-GOS with β-1→6 linkages compared to those by linear GOS with β-1→4 linkages.10,31 SCFA is a metabolite produced by gut microbiota, representing therefore an important factor that affects the gut environment.33 SCFA can lower the colonic pH, stimulate calcium and magnesium absorption, decrease proinflammatory response markers, and decrease adiposity and body weight.1,8,11−14 The total amount of SCFA was affected by the monosaccharide composition, the type of linkage, and the DP of GOS. The production of total short-chain fatty acids (SCFA) from α-GOS by six probiotic strains was higher than that from β-GOS in most samples. These findings are based on the monosaccharide

Figure 4. Growth of intestinal nonprobiotics (A) and probiotics (B) in BHI medium without a carbon source, with glucose, and with β-GOS, stachyobifiose, raffinose, and stachyose as a sole carbon source. (A): 1. Prevotella intermedia KCTC 5694; 2. Listeria monocytogenes KCTC 3569; 3. Staphylococcus aureus KCTC 1916; 4. Salmonella typhimurium ATCC 14028; 5. Bacteroides fragilis KCTC 5013; 6. Enterococcus faecalis KCTC 3511; 7. Gardnerella vaginalis KCTC 5096; 8. Clostridium perf ringens KCTC 3269; 9. Clostridium ramosum KCTC 3323; 10. Eubacterium rectale KCTC 5835; 11. Clostridium leptum KCTC 5155; 12. Ruminococcus gnavus KCTC 5920; 13. Bacteroides coprocola KCTC 5443; 14. Escherichia coli KCTC 1039; 15. Enterobacter cloacae KCTC 2361; 16. Clostridium butyricum KCTC 1871; 17. Bacteroides cellulosilyticus KCTC 5800. (B): 1. Lactobacillus rhamnosus KCTC 3237; 2. L. delbrueckii subsp. bulgaricus KCTC 3635; 3. L. casei KFRI 699; 4. L. plantarum KFRI 708; 5. Lactococcus lactis KCTC 2013; 6. Bif idobacterium breve ATCC 15700; 7. B. adolescentis ATCC 15705; 8. B. thermophilum KCCM 12097; 9. B. bif idum ATCC 29521; 10. B. longum RD 47; 11. B. bifidum BGN4; 12. B. catenulatum KCTC 3221; 13. B. breve KCTC 3419; 14. B. animalis KCTC 3219; 15. B. infantis KCTC 3249; 16. B. longum BORI.

The chemical structure of GOS, determined by the specific enzymes employed, affects the prebiotic properties.10 Like β-GOS, α-GOS are reported to be selectively consumed by intestinal beneficial bacteria and can inhibit the adherence of nonprobiotics.24,25 α-GOSs have been produced by enzymes from Pycnoporus cinnabarinus, Lactobacillus reuteri, and Candida guilliermondii.21,26,27 However, research on the production and effects of α-GOS by α-Gal from Bif idobacterium is scant. Therefore, we attempted to find the optimal condition for the production of stachyobifiose with bifidobacterial α-Gal purified from recombinant E. coli and to characterize the ability to ferment stachyobifiose by major types of intestinal bacteria. Compared with the molecular masses of α-Gal (79, 80, and 64 kDa, respectively) reported from B. adolescentis,28 B. breve,29 and Lactobacillus reuteri,30 the molecular mass of α-Gal from a E

DOI: 10.1021/acs.jafc.7b04703 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 3. Summary of SCFA Production during Fermentation B. breve ATCC 15700 lactate acetate propionate isobutyrate total SCFA B. adolescentis ATCC 15705 lactate acetate propionate isobutyrate total SCFA B. longum subsp. longum RD 47 lactate acetate propionate isobutyrate total SCFA B. catenulatum KCTC 3221 lactate acetate propionate isobutyrate total SCFA B. animalis KCTC 3219 lactate acetate propionate isobutyrate total SCFA B. infantis KCTC 3249 lactate acetate propionate isobutyrate total SCFA

DW

Glu

β-GOS

stachyobifiose

raffinose

stachyose

1.7 7.2 2.8 9.4 21.1

36.8 71.5 11.7 6.0 126.0

43.7 8.4 22.7 6.3 81.1

51.8 36.8 0.8 11.5 100.9

80.3 31.5 22.8 6.1 140.7

46.5 25.6 12.3 5.2 89.6

2.6 6.4 19.8 7.5 36.2

12.8 33.2 40.9 6.2 93.2

19.0 6.0 3.1 8.5 36.6

54.1 56.6 3.8 14.3 128.9

74.3 54.1 16.2 11.8 156.5

41.4 43.6 2.8 9.1 96.8

2.8 22.7 22.9 8.2 56.5

74.9 19.7 0.7 7.1 102.4

24.7 31.0 3.0 8.2 66.8

74.9 32.7 4.1 15.1 126.8

50.5 20.6 9.4 5.3 85.8

81.6 35.7 10.0 9.9 137.1

1.5 6.8 18.0 4.8 31.1

46.2 24.9 3.7 5.9 80.7

37.0 25.2 16.7 6.0 85.0

56.4 27.2 3.9 9.8 97.3

44.7 23.2 19.7 3.2 90.7

63.6 31.2 25.1 5.7 125.6

6.8 21.0 23.8 7.7 59.3

132.1 12.3 0.7 8.5 153.5

26.6 22.7 124.6 6.5 180.4

54.6 34.0 20.2 8.2 117.0

36.6 26.7 19.4 5.1 87.8

49.9 39.5 9.5 10.7 109.6

1.8 32.0 15.7 9.4 59.0

23.4 37.1 23.3 8.6 92.4

30.9 38.6 16.0 9.2 94.7

70.5 50.4 10.5 15.4 146.8

51.2 35.3 19.0 6.2 111.7

84.5 43.3 12.8 12.6 153.2

Human Ecology, Seoul National University, Seoul 151-742, Republic of Korea.

composition and the type of linkage of GOS. Among the α-GOS samples, the total amount of SCFA varies depending on the strain, as noted above. However, the average amounts of the total SCFA of the six strains were in the order of stachyobifiose > stachyose > raffinose. The average amounts of the total SCFA of the six strains from the stachyose and stachyobifiose of DP4 was higher than that of raffinose of DP3. The results are consistent with the results for β-GOS.31 Moreover, a greater average amount of the total SCFA of the six strains was obtained from stachyobifiose than from stachyose. Lactic acid and acetic acid mainly contributed to total concentration of SCFA in β-GOS, whereas only lactic acid mainly contributed to total concentration of SCFA in α-GOS. The structure of GOS is reportedly associated with the inhibition of the intestinal adherence of nonprobiotics.34,35 Moreover, resistance to ileal digestion depends on the monomer and on the linkage type of GOS.36 Accordingly, stachyobifiose may be further studied as a potential prebiotic.

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ORCID

So Young Oh: 0000-0002-7966-439X Funding

This work was carried out with support from the National Research Foundation of Korea (NRF) (grant no. 2017R1A2B2012390) funded by the Korea government (MSIP), the High Value-added Food Technology Development Program (No. 317043-3), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET), Ministry of Agriculture, Food and Rural Affairs (MAFRA), and the Technological Innovation R&D Program (No. S2463318) funded by the Small and Medium Business Administration, Republic of Korea. Notes

The authors declare no competing financial interest.

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AUTHOR INFORMATION

REFERENCES

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Corresponding Author

*Phone: +82-2-880-8749. Fax: +82-2-884-0305. E-mail: geji@ snu.ac.kr. Corresponding author address: Research Institute of F

DOI: 10.1021/acs.jafc.7b04703 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.7b04703 J. Agric. Food Chem. XXXX, XXX, XXX−XXX