Article pubs.acs.org/JAFC
In Vitro Fermentation of Lactulose by Human Gut Bacteria Bingyong Mao,†,§ Dongyao Li,†,§ Jianxin Zhao,† Xiaoming Liu,† Zhennan Gu,†,‡ Yong Q. Chen,†,‡ Hao Zhang,*,†,‡ and Wei Chen*,†,‡ †
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China ‡ Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, People’s Republic of China S Supporting Information *
ABSTRACT: Lactulose has been known as a prebiotic that can selectively stimulate the growth of beneficial bifidobacteria and lactobacilli. Recent studies have indicated that Streptococcus mutans, Clostridium perfringens, and Faecalibacterium prausnitzii are also able to utilize lactulose. However, the previous studies mainly focused on the utilization of lactulose by individual strains, and few studies were designed to identify the species that could utilize lactulose among gut microbiota. This study aimed to identify lactulose-metabolizing bacteria in the human gut, using in silico and traditional culture methods. The prediction results suggested that genes for the transporters and glycosidases of lactulose are well distributed in the genomes of 222 of 453 strains of gastrointestinal-tract bacteria. The screening assays identified 35 species with the ability to utilize lactulose, of which Cronobacter sakazakii, Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas putida were reported for the first time to be capable of utilizing lactulose. In addition, significant correlations between lactulose and galactooligosaccharide metabolism were found. Thus, more attention should be paid to bacteria besides bifidobacteria and lactobacilli to further investigate the relationship between functional oligosaccharides and gut bacteria. KEYWORDS: lactulose, intestine, microbiota, oligosaccharides, utilization
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INTRODUCTION Nondigestible carbohydrates in the human diet are capable of reaching the large intestine without digestion or absorption, where they supply energy for gut bacteria and modify the composition of gut microbiota. These carbohydrates include nonstarch polysaccharides, termed “dietary fiber”, and some oligosaccharides, among which lactulose, fructooligosaccharides, and galactooligosaccharides (GOS) are regarded as prebiotics.1 According to Gibson et al.,1 prebiotics are functional oligosaccharides that can selectively stimulate the growth of beneficial bacteria, chiefly Bifidobacterium and Lactobacillus, in the host.2−7 Dietary lactulose and fructooligosaccharides have been reported to protect the host from enteric pathogens and reduce the risk of colon cancer.8,9 However, Clostridium perfringens, Clostridium difficile, and Streptococcus faecalis have also been found to ferment lactulose,10 and pathogenic Escherichia coli BEN2908 has been shown to be capable of metabolizing fructooligosaccharides,11 which could cause human disease at high quantities. These results suggest that oligosaccharides may be utilized by nonprobiotic bacteria in the gut. Fermentation properties of oligosaccharides derived from lactulose or raffinose and human milk oligosaccharides by fecal microbiota have been assessed in vitro by determination of short-chain fatty acids and gas production.6,7,12,13 However, no detailed study has been conducted of the intestinal bacteria that are capable of utilizing oligosaccharides, and it remains unknown which species in the gut are able to use oligosaccharides. Bacteria can grow on oligosaccharides only when the latter are degraded into monosaccharides and subsequently metabolized for energy production. Therefore, transporters and © 2014 American Chemical Society
glycoside hydrolases are key factors in the utilization of oligosaccharides.14,15 Gänzle reviewed the metabolism of oligosaccharides in lactobacilli16 and found metabolism to be limited by transport, because glycoside hydrolases are intracellular. This may differentiate lactobacilli from Bifidobacterium spp. Lactulose is a simple disaccharide composed of galactose and fructose by a β-1,4-glycosidic bond and has been known as “bifidus factor” since the 1950s.17 Lactulose does not occur naturally, and it may be produced by chemical isomerization of lactose or enzymatic synthesis.18 Commercial lactulose can reach a purity of 99%, which is suitable for bacterial isolation as a single carbon source. Therefore, in the current study the utilization of lactulose by gut microbiota was investigated. A traditional culture method was used to identify lactulosemetabolizing bacteria, and the search for key GOS enzymes, cross-checked against data on body site specialized reference genomes gathered for the Human Microbiome Project (HMP), was carried out in silico.
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MATERIALS AND METHODS
Chemicals and Reagents. Lactulose (purity by HPLC 99.3%, water by Karl Fischer titration 0.7%) was purchased from EMD Chemicals, Inc. (San Diego, CA), and GOS (≥98%) was purchased from Shanghai Dingjie Technology, Ltd. (Shanghai, China). Received: Revised: Accepted: Published: 10970
July 24, 2014 October 13, 2014 October 23, 2014 October 23, 2014 dx.doi.org/10.1021/jf503484d | J. Agric. Food Chem. 2014, 62, 10970−10977
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Bacterial Strains and Growth Media. Bifidobacterium dentium KCTC 3361, Clostridium leptum KCTC 5155, Enterococcus saccharolyticus KCTC 3643, Mitsuokella multacida KCTC 5453, Pediococcus acidilactici KCTC 15064, Clostridium nexile KCTC 5578, Clostridium scindens KCTC 5591, Ruminococcus gnavus KCTC 5920, and Weissella paramesenteroides KCTC 3531 were purchased from the Korean Collection for Type Cultures. Megamonas funiformis JCM 14723 was purchased from the Japan Collection of Microorganisms. Marvinbryantia formatexigens DSM 14469 and Ruminococcus obeum DSM 25238 were purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures). Lactobacillus acidophilus ATCC 4356, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus fermentum ATCC 14931, and Lactobacillus plantarum subsp. plantarum ATCC 14917 were kindly provided by Professor Heping Zhang (Inner Mongolia Agricultural University, China). Bifidobacterium adolescentis CICC 6070 was purchased from the China Center of Industrial Culture Collection. Clostridium perfringens ATCC 13124 was purchased from the American Type Culture Collection. Lactobacillus reuteri CCFM 8217 and Leuconostoc mesenteroides CCFM 8220 were obtained from the Culture Collection of Food Microorganisms, Jiangnan University. The medium used to isolate the bacteria was modified from gut microbiota medium (GMM),19 which was modified from the supplemented tryptone−yeast−glucose medium by Gordon and coworkers19 to culture fecal microbiota. The modified GMM contained 4 g of lactulose, 2 g of peptone, 1 g of yeast extract, 2 g of KH2PO4, 0.002 g of MgSO4·7H2O, 0.08 g of NaCl, 8 mg of CaCl2, 0.73 mg of FeSO4·7H2O, 1.2 mg of hematin, 10 mL of ATCC vitamin mix, 10 mL of ATCC trace mineral mix, 0.5 mL of Tween 80, and 0.5 g of Lcysteine per liter. The pH was adjusted to 7.0 ± 0.1 by use of NaOH, and the medium was sterilized at 115 °C for 20 min. Lactulose was filter-sterilized through a 0.22-μm filter and added to the autoclaved medium as the sole carbon source. Solid medium was obtained by adding 18 g of agar/L of liquid medium. A total of 15 mL of 0.5% bromocresol purple was added to the GMM agar medium as the indicator of pH change. Database Preparation. Site-specialized protein multifasta (PEP) files for reference genomes can be downloaded from the HMP Data Analysis and Coordination Center. These files record all HMP isolates that have completed sequencing and annotation. The gastrointestinaltract PEP file (382 rows) was downloaded from http://hmpdacc.org/ HMRGD/. A customized script was used to eliminate redundance. The output of HMP’s mothur community profiling of metagenomic 16S rRNA sequence data generated by a low-stringency approach (http://hmpdacc.org/HMMCP/) was also used for reference. One hundred seven of the genera depicted in the mothur community profiling output were not found in the list of reference genomes. Therefore, the 98 available genome coding sequences for these 107 genera were downloaded from the National Center for Biotechnology Information and merged into the gastrointestinal-tract PEP file, followed by BLAST database formatting. Genes Responsible for Uptake and Hydrolysis of Galactooligosaccharides. By use of literature mining, 41 genes were identified as responsible for the transport or catabolism of GOS (Table 1). However, no genes have been reported to be responsible for the metabolism of lactulose. Like lactulose, GOS is formed by a β-1,4glycosidic bond. Therefore, the genes related to GOS metabolism were used in this study to predict potential lactulose-metabolizing bacteria. Alignment of Transporters and Glycosidases against the Local Protein Database. BLASTP searches were run against the local protein database with transporters and glycoside hydrolases used as queries. When the alignment length was found to exceed 70% of the transporter/glycoside hydrolase sequence and the identity was greater than 40%, the strain was inferred to have homologous proteins for corresponding functions. Isolation and Identification of Lactulose-Metabolizing Bacteria. Fresh fecal samples were collected from five healthy donors aged 25−29 years. The fecal samples were placed in prereduced 0.1 M phosphate-buffered saline (PBS; 80 g of NaCl, 2 g of KCl, 14.4 g of Na2PO4, 2.4 g of KH2PO4, and 0.5 g of L-cysteine per liter; pH 7.0),
Table 1. Genes Responsible for Transport and Catabolism of Galactooligosaccharides gene
annotation
Bifidobacterium lactis Bl-0420 lacS-balac_0475 MFS permease balac_0485 permease balac_0486 permease lacZ-balac_0476 β-galactosidase balac_0484 β-galactosidase Bifidobacterium longum LMG1319721 bl1523 permease bl0260 permease bl0261 permease bl0259 β-galactosidase bl0976 galactoside transporter bl0978 β-galactosidase Bifidobacterium longum subsp. infantis ATCC1569722 blon_0267 permease blon_0268 β-galactosidase blon_2331 permease blon_2332 permease blon_2334 β-galactosidase blon_2016 β-galactosidase blon_2123 β-galactosidase blon_2416 β-galactosidase blon_2414a solute binding protein blon_2453a β-galactosidase Bifidobacterium breve UCC200324 bbr_0417 galC solute binding protein bbr_0418 galD permease bbr_0419 galE permease bbr_0420 galG β-galactosidase bbr_1551 lacS galactoside symporter bbr_1552 lacZ β-galactosidase bbr_0530 gosC solute binding protein bbr_0527 gosD permease bbr_0528 gosE permease bbr_0529 gosG β-galactosidase Geobacillus stearothermophilus T-625 ganA extracellular endo-β-1,4-galactanase precursor ganE solute binding protein ganF permease ganG permease ganB β-galactosidase Lactobacillus acidophilus26 lacS-lba1463 permease Lactobacillus acidophilus NCFM27 lacZ-lba1462 β-galactosidases lacL-lba1467 β-galactosidases lacM-lba1468 β-galactosidases Bifidobacterium bifidum NCIMB4117128 bbgII β-galactosidase a
This entry from ref 23.
with 15 mL/g of feces per sample.19 The samples were dispersed and suspended by vortexing with sterilized glass beads for 5 min. Serial dilution was performed with 0.1 M PBS, and the diluted (10−3) samples (0.1 mL) were plated. Six plates were used for each fecal sample, as recommended by Gordon and co-workers,19 and the medium without lactulose was used for negative controls. The plates were incubated in an anaerobic workstation (Whitley DG250, Don Whitley Scientific Limited, West Yorkshire, U.K.) at 37 °C for 24 h under an atmosphere of 80% N2, 10% CO2, and 10% H2. Colonies 10971
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Figure 1. Distribution of homologue proteins of transporters and glycosidases for lactulose (vertical axis) among GIT bacteria (horizontal axis). Positive results for transporters and glycosidases are indicated in red and green, relatively. with yellow surroundings on the GMM plates were picked up, reinoculated into liquid medium, and cultured anaerobically. The bacteria were isolated and purified by the standard streak-plate technique. The isolated bacteria were Gram-stained and identified by amplification and sequencing of 16S rRNA. The polymerase chain reaction (PCR) procedure was as follows: 95 °C for 5 min; 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min 30 s, 30 cycles; 72 °C for 10 min; 12 °C for 10 min. The resulting PCR product was sequenced by Sangon Biotech (Shanghai) Co., Ltd. Characterization of Bacterial Growth on Lactulose. Bacteria were cultured anaerobically in GMM at 37 °C for 24 h, with lactulose as the sole carbon source. Absorbance at 600 nm (OD600) and pH were recorded every 2 h to characterize the growth. The amount of lactulose in the culture supernatant was determined by the method developed by Zhang et al.29 Fermentation of Galactooligosaccharides by LactuloseMetabolizing Bacteria. Bacteria capable of metabolizing lactulose were inoculated on GMM plates with GOS as the sole carbon source and bromocresol purple as the indicator. GMM plates without GOS were set as negative controls. The bacteria on the GMM−GOS plates that were capable of growing and coloring their surroundings yellow were considered to have the ability to metabolize GOS.
Table 2. Identified Lactulose-Metabolizing Bacteria in Healthy Adult Fecesa genus and species Cronobacter sakazakii Enterobacter aerogenes Enterobacteriaceae bacterium Enterococcus casseliflavus Enterococcus faecalis Enterococcus faecium Enterococcus gallinarum Escherichia coli Escherichia fergusonii Klebsiella pneumoniae
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Klebsiella sp.
RESULTS Homologous Proteins for Lactulose Transporters and Glycosidases in 453 Gastrointestinal-Tract Bacteria Strains. The results of aligning the transporters and glycosidases with the local protein database are shown in Figure 1. Surprisingly, homologue proteins for the glycosidases appeared in 222 of the 453 strains, which accounted for 56 of the 114 genera. In contrast, homologue proteins for the transporters were found in only 90 strains, accounting for 29 of the 114 genera. These 90 strains also possessed homologue proteins for the glycosidases. In general, lactulose metabolism requires transporters and galactosidases. In addition to Bifidobacterium and Lactobacillus, 27 genera with homologue proteins for both transporters and glycosidases were found (Figure 1), indicating that lactulose may not be exclusively a stimulant of probiotics.30 However, it remains unclear whether the predicted bacteria are able to utilize lactulose; further investigation is required. Utilization of Lactulose by Cronobacter sakazakii, Enterococcus casseliflavus, Enterococcus faecalis, and Klebsiella pneumoniae. Compared with GOS, lactulose has an accurate chemical structure and metabolic process, and its purity can reach 99.3%. In the current study, fecal samples collected from five healthy adults were plated on GMM with lactulose as the sole carbon source. Ninety-six strains belonging to 18 species of nine genera were identified (Table 2). To our knowledge, Cronobacter, Enterococcus, Klebsiella, and Pseudomonas are first reported in our study to be capable of utilizing lactulose. One strain from each species was selected and its growth on lactulose was characterized (Figure 2). OD600
Klebsiella variicola Pseudomonas putida Streptococcus anginosus Streptococcus equinus Streptococcus sanguinis Bifidobacterium pseudocatenulatum Bifidobacterium sp.
strains CCFM 8305, 8306, 8307, 8308, 8309, 8310, 8311, 8312 CCFM 8313 CCFM 8314, 8315 CCFM 8316, 8317, 8318, 8319 CCFM 8320 CCFM 8321, 8322, 8323, 8324 CCFM 8325 CCFM 8326, 8327, 8328, 8329, 8330, 8331, 8332, 8333, 8334, 8335, 8336, 8337, 8338, 8339, 8340, 8341, 8342 CCFM 8343, 8344, 8345, 8346 CCFM 8347, 8348, 8349, 8350, 8351, 8352, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361, 8362, 8363, 8364, 8365, 8366, 8367, 8368, 8369, 8370 CCFM 8371, 8372, 8373, 8374, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382, 8383, 8384, 8385, 8386 CCFM 8387 CCFM 8388 CCFM 8389 CCFM 8390, 8391 CCFM 8392, 8393, 8394 CCFM 8395, 8396, 8397, 8398 CCFM 8399, 8400
a
16S rRNA nucleotide acid sequences of the identified strains are shown in Table S1 in Supporting Information.
increased during the 24-h culture period, along with a decrease in pH and a reduction in the lactulose amount, which provided direct proof of lactulose utilization. In addition, the ability of 21 strains to metabolize lactulose was assessed, and 17 strains were proven to be able to grow on lactulose (Table 3 and Figure 3). Thirteen of the 15 strains with homologue proteins for both transporters and glycosidases were capable of utilizing lactulose, indicating that searching for transporter and glycosidase homology may be an effective method of predicting lactulose-metabolizing bacteria. Galactooligosaccharide Utilization by Lactulose-Metabolizing Bacteria. Due to the structural similarity of lactulose and GOS, the 18 strains isolated (Figure 2) and the 17 strains found to be capable of metabolizing lactulose (Table 3) were tested for their ability to ferment GOS. The results showed that 32 strains were capable of GOS utilization (Table 10972
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Figure 2. Growth of isolated strains on GMM medium with lactulose as the sole carbon source. OD600 (○), pH (●), and the amount of lactulose (×) were recorded every 2 h, and the strains are presented as follows: (A) C. sakazakii CCFM 8306, (B) E. aerogenes CCFM 8313, (C) Enterobacteriaceae bacterium CCFM 8315, (D) E. casseliflavus CCFM 8316, (E) E. faecalis CCFM 8320, (F) E. faecium CCFM 8321, (G) E. gallinarum CCFM 8325, (H) E. coli CCFM 8331, (I) E. fergusonii CCFM 8343, (J) K. pneumoniae CCFM 8358, (K) Klebsiella sp. CCFM 8371, (L) K. variicola CCFM 8387, (M) P. putida CCFM 8388, (N) S. anginosus CCFM 8389, (O) S. equinus CCFM 8390, (P) S. sanguinis CCFM 8392, (Q) B. pseudocatenulatum CCFM 8395, and (R) Bifidobacterium sp. CCFM 8399. 10973
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lactulose in the gut. The gut is a complex ecosystem, with about 1014 bacteria categorized into more than 1000 species,32 and the aim of the current study was to identify lactulosemetabolizing bacteria within this ecosystem. Transporters and glycosidases are of great importance to the utilization of oligosaccharides. Thus far, however, no encoding genes for lactulose metabolism have been identified. Therefore, the genes for GOS metabolism acquired through literature mining were used instead. Moreover, the unculturability of most gut microbes has limited our understanding of the metabolic characteristics of individual strains. To date, HMP has failed to reach its goal of isolating 3000 reference genomes from human body sites. In the current study, the site-specialized reference genome data provided by HMP were combined with the output of mothur community profiling to build a local protein database. As shown in Figure 1, the genes for lactulose metabolism appeared in 222 GIT bacteria strains, of which 90 strains possessed homologue proteins for both transporters and glycosidases. It is reasonable to infer from the existence of transporters in a given strain that the strain contains corresponding glycosidases. If no corresponding glycosidases were available, the strain would be unlikely to contain transporters. To identify lactulose-metabolizing bacteria in the gut, 18 species were isolated from fecal samples (Table 2), and 17 strains were proven to have the ability to utilize lactulose (Table 3). This indicates that bioinformatic analysis is an effective tool for predicting lactulose-metabolizing bacteria, thereby narrowing the scope of experimental research. Unlike the results of previous studies,10,31,33 the findings of the current study indicated for the first time that the genera Cronobacter, Enterococcus, Klebsiella, and Pseudomonas are able to utilize lactulose. To the best of our knowledge, C. sakazakii, E. aerogenes, Enterobacteriaceae bacterium, E. casseliflavus, E. faecalis, E. faecium, E. gallinarum, E. fergusonii, K. pneumoniae, Klebsiella sp., K. variicola, P. putida, S. anginosus, S. equinus, and B. pseudocatenulatum have never been reported to be capable of utilizing lactulose. Therefore, lactulose was shown to have the potential to stimulate the growth of many commensal bacteria in addition to bifidobacteria and lactobacilli. More gut bacteria capable of utilizing lactulose may exist than previously supposed. Additionally, lactulose exhibited similar metabolic characteristics to GOS (Table 4), which may be derived from lactulose.34 Therefore, lactulose may be considered a type of GOS with a lower degree of polymerization, and lactulose and GOS may possess similar genes for transporters or galactosidases. In the human gut, competition for limited food takes place among the bacteria, and those capable of utilizing prebiotic substrates might have advantages during the competition with other bacteria.35 In conclusion, the combination of a homology search and the traditional isolation method assisted in the identification of lactulose-metabolizing bacteria. Thirty-five species were found, most of which were reported for the first time to be capable of utilizing lactulose. The commensal gut bacteria Cronobacter, Enterococcus, Escherichia, Klebsiella, Pseudomonas, and Streptoccoccus were found to be able to ferment lactulose. In addition, significant correlations were observed between lactulose and GOS metabolism. This work offers greater insight into the utilization of oligosaccharides by gut bacteria. To further explore the relationship between functional oligosaccharides
Table 3. Bacteria Obtained on the Basis of Alignment Analysisa strain
source
transporters
glycosidases
lactulose
Bifidobacterium adolescentis CICC 6070 Bifidobacterium dentium KCTC 3361 Clostridium leptum KCTC 5155 Clostridium perfringens ATCC 13124 Enterococcus saccharolyticus KCTC 3643 Eubacterium rectale ATCC 33656 Lactobacillus acidophilus ATCC 4356 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 Lactobacillus fermentum ATCC 14931 Lactobacillus plantarum subsp. plantarum ATCC 14917 Lactobacillus reuteri CCFM8217 Leuconostoc mesenteroides CCFM8220 Marvinbryantia formatexigens DSM 14469 Megamonas funiformis JCM 14723 Mitsuokella multacida KCTC 5453 Pediococcus acidilactici KCTC 15064 Weissella paramesenteroides KCTC 3531 Clostridium nexile KCTC 5578 Clostridium scindens KCTC 5591 Ruminococcus gnavus KCTC 5920 Ruminococcus obeum DSM 25238
CICC
Y
Y
+
KCTC
Y
Y
+
KCTC
N
Y
+
ATCC
N
Y
+
KCTC
N
Y
+
KCTC
Y
Y
+
Zhang
Y
Y
+
Zhang
N
Y
+
Zhang
Y
Y
+
Zhang
Y
Y
+
CCFM
Y
Y
+
CCFM
Y
Y
+
DSM
Y
Y
+
JCM
Y
Y
+
KCTC
Y
Y
+
KCTC
Y
Y
+
KCTC
Y
Y
+
KCTC
N
Y
−
KCTC
N
Y
−
KCTC
Y
Y
−
DSM
Y
Y
−
a
Symbols: Y, positive for alignment search; N, negative for alignment search; +, positive growth on lactulose; −, negative growth on lactulose.
4). Although lactulose is a disaccharide composed of fructose and galactose, while GOS is a complex with different degrees of galactose polymerization, they may share the same transporters or glycosidases. Moreover, the 17 lactulose-metabolizing strains obtained from the alignment search contained homologue proteins for transporters and glycosidases that have been reported to be responsible for metabolizing GOS. Thus, there seems to be a significant correlation between lactulose and GOS.
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DISCUSSION Lactulose has generally been regarded as a special substrate used by probiotic bifidobacteria and lactobacilli. However, Faecalibacterium prausnitzii, Bacteroides oralis, E. coli, and C. perfringens have also been reported to utilize lactulose in vitro.10,31 It remains unclear which species are able to utilize 10974
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Figure 3. Growth of obtained strains on GMM medium with lactulose as the sole carbon source. OD600 (○), pH (●), and the amount of lactulose (×) were recorded every 2 h, and the strains are presented as follows: (A) B. adolescentis CICC 6070, (B) B. dentium KCTC 3361, (C) C. leptum KCTC 5155, (D) C. perfringens ATCC 13124, (E) E. saccharolyticus KCTC 3643, (F) E. rectale ATCC 33656, (G) L. acidophilus ATCC 4356, (H) L. delbrueckii subsp. bulgaricus ATCC 11842, (I) L. fermentum ATCC 14931, (J) L. plantarum subsp. plantarum ATCC 14917, (K) L. reuteri CCFM 8217, (L) L. mesenteroides CCFM 8220, (M) M. formatexigens DSM 14469, (N) M. funiformis JCM 14723, (O) M. multacida KCTC 5453, (P) P. acidilactici KCTC 15064, and (Q) W. paramesenteroides KCTC 3531. 10975
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Journal of Agricultural and Food Chemistry Table 4. Growth of Lactulose-Metabolizing Strains on Galactooligosaccharidesa strain
GOS
strain
+ + +
B. adolescentis CICC 6070 B. dentium KCTC 3361 C. leptum KCTC 5155
+ + −
+ + − + +
+ + + + +
E. fergusonii CCFM8343 K. pneumoniae CCFM8358
+ +
Klebsiella sp. CCFM8371 K. variicola CCFM8387 P. putida CCFM8388 S. anginosus CCFM8389 S. equinus CCFM8390 S. sanguinis CCFM8392 B. pseudocatenulatum CCFM8395 Bifidobacterium sp. CCFM8399
+ + + + + − +
C. perfringens ATCC 13124 E. saccharolyticus KCTC 3643 E. rectale ATCC 33656 L. acidophilus ATCC 4356 L. delbrueckii subsp. bulgaricus ATCC 11842 L. fermentum ATCC 14931 L. plantarum subsp. plantarum ATCC 14917 L. reuteri CCFM8217 L. mesenteroides CCFM8220 M. formatexigens DSM 14469 M. funiformis JCM 14723 M. multacida KCTC 5453 P. acidilactici KCTC 15064 W. paramesenteroides KCTC 3531
a
+ + + + + + + + +
+
and gut bacteria, more attention should be paid to bacteria other than bifidobacteria and lactobacilli.
ASSOCIATED CONTENT
S Supporting Information *
One table listing 16S rRNA nucleotide acid sequences of the identified strains. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
(1) Gibson, G. R.; Probert, H. M.; Loo, J. V.; Rastall, R. A.; Roberfroid, M. B. Dietary modulation of the human colonic microbiota: Updating the concept of prebiotics. Nutr. Res. Rev. 2004, 17, 259−275. (2) Roberfroid, M. Prebiotics: The concept revisited. J. Nutr. 2007, 130, 830S−837S. (3) Panesar, P. S.; Kumari, S. Lactulose: Production, purification and potential applications. Biotechnol. Adv. 2011, 29, 940−948. (4) Buddington, R. K.; Williams, C. H.; Chen, S. C.; Witherly, S. A. Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. Am. J. Clin. Nutr. 1996, 63, 709−716. (5) Rycroft, C. E.; Jones, M. R.; Gibson, G. R.; Rastall, R. A. A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. J. Appl. Microbiol. 2001, 91, 878−887. (6) Cardelle-Cobas, A.; Olano, A.; Corzo, N.; Villamiel, M.; Collins, M.; Kolida, S.; Rastall, R. A. In vitro fermentation of lactulose-derived oligosaccharides by mixed fecal microbiota. J. Agric. Food Chem. 2012, 60, 2024−2032. (7) Boler, B. M. V.; Serao, M. C. R.; Faber, T. A.; Bauer, L. L.; Chow, J.; Murphy, M. R.; Fahey, G. C., Jr. In vitro fermentation characteristics of select nondigestible oligosaccharides by infant fecal inocula. J. Agric. Food Chem. 2013, 61, 2109−2119. (8) Sahota, S. S.; Bramley, P. M.; Menzies, I. S. The fermentation of lactulose by colonic bacteria. J. Gen. Microbiol. 1982, 128, 319−325. (9) Porcheron, G.; Kut, E.; Canepa, S.; Maurel, M. C.; Schouler, C. Regulation of fructo-oligosaccharide metabolism in an extra-intestinal pathogenic Escherichia coli strain. Mol. Microbiol. 2011, 81, 717−733. (10) Buddington, K. K.; Donahoo, J. B.; Buddington, R. K. Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumor inducers. J. Nutr. 2002, 132, 472−477. (11) Wollowski, I.; Rechkemmer, G.; Pool-Zobel, B. L. Protective role of probiotics and prebiotics in colon cancer. Am. J. Clin. Nutr. 2001, 73, 451S−455S. (12) Marcobal, A.; Barboza, M.; Froehlich, J. W.; Block, D. E.; German, J. B.; Lebrilla, C. B.; Mills, D. A. Consumption of human milk oligosaccharides by gut-related microbes. J. Agric. Food Chem. 2010, 58, 5334−5340. (13) Hernandez-Hernandez, O.; Côté, G. L.; Kolida, S.; Rastall, R. A.; Sanz, M. L. In vitro fermentation of alternansucrase raffinose-derived oligosaccharides by human gut bacteria. J. Agric. Food Chem. 2011, 59, 10901−10906. (14) Schluter, J.; Foster, K. R. The evolution of mutualism in gut microbiota via host epithelial selection. PLoS Biol. 2012, 10, 1−9. (15) Diard, M.; Garry, L.; Selva, M.; Mosser, T.; Denamur, E.; Matic, I. Pathogenicity-associated islands in extraintestinal pathogenic Escherichia coli are fitness elements involved in intestinal colonization. J. Bacteriol. 2010, 192, 4885−4893. (16) Ganzle, M. G.; Follador, R. Metabolism of oligosaccharides and starch in lactobacilli: A review. Front. Microbiol. 2012, 3, 340. (17) Petuely, F. Lactobacillus bif idus flora produced in artificially-fed infants by bifidogenic substances (bifidus factor). Z. Kinderheilkd. 1957, 79, 174−179. (18) Panesar, P. S.; Kumari, S. Lactulose: Production, purification and potential applications. Biotechnol. Adv. 2011, 29, 940−948. (19) Goodman, A. L.; Kallstrom, G.; Faith, J. J.; Reyes, A.; Moore, A.; Dantas, C.; Gordon, J. I. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6252−6257. (20) Andersen, J. M.; Barrangou, R.; Hachem, M. A.; Lahtinen, S. J.; Goh, Y. J.; Svensson, B.; Klaenhammeret, T. R. Transcriptional
+, positive growth on GOS; −, negative growth on GOS.
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ABBREVIATIONS
GOS, galactooligosaccharides; HMP, Human Microbiome Project; GMM, gut microbiota medium; GIT, gastrointestinal tract
GOS
C. sakazakii CCFM8306 E. aerogenes CCFM8313 Enterobacteriaceae bacterium CCFM8315 E. casseliflavus CCFM8316 E. faecalis CCFM8320 E. faecium CCFM8321 E. gallinarum CCFM8325 E. coli CCFM8331
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AUTHOR INFORMATION
Corresponding Authors
*Telephone/fax 86-510-85912155; e-mail zhanghao@jiangnan. edu.cn. *Telephone/fax 86-510-85912155; e-mail chenwei66@ jiangnan.edu.cn. Author Contributions §
B.M. and D.L. contributed equally to this work.
Funding
This work was supported by the National Science Fund for Distinguished Young Scholars (31125021), Key Projects in the National Science and Technology Pillar Program during the 12th five-year plan period (2013BAD18B01, 2013BAD18B02, 2012BAD28B07, 2012BAD28B08), the 111 Project B07019, and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank all adults who provided fecal samples. We also thank Professor H. Zhang for providing Lactobacillus acidophilus ATCC 4356, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus fermentum ATCC 14931, and Lactobacillus plantarum subsp. plantarum ATCC 14917. 10976
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analysis of oligosaccharide utilization by Bif idobacterium lactis Bl-04. BMC Genomics 2013, 14, 312. (21) González, R.; Klaassens, E. S.; Malinen, E.; de Vos, W. M.; Vaughan, E. E. Differential transcriptional response of Bif idobacterium longum to human milk, formula milk, and galactooligosaccharide. Appl. Environ. Microb. 2008, 74, 4686−4694. (22) Garrido, D.; Ruiz-Moyano, S.; Jimenez-Espinoza, R.; Eom, H. J.; Block, D. E.; Mills, D. A. Utilization of galactooligosaccharides by Bif idobacterium longum subsp. infantis isolates. Food Microbiol. 2013, 33, 262−270. (23) Kim, J. H.; An, H. J.; Garrido, D.; German, J. B.; Lebrilla, C. B.; Millset, D. A. Proteomic analysis of Bif idobacterium longum subsp. infantis reveals the metabolic insight on consumption of prebiotics and host glycans. PLoS One 2013, 8, 1−13. (24) O’Connell, M. M.; Kinsella, M.; Fitzgerald, G. F.; van Sinderen, D. Transcriptional and functional characterization of genetic elements involved in galacto-oligosaccharide utilization by Bif idobacterium breve UCC2003. Microb. Biotechnol. 2013, 6, 67−79. (25) Tabachnikov, O.; Shoham, Y. Functional characterization of the galactan utilization system of Geobacillus stearothermophilus. FEBS J. 2013, 28, 950−964. (26) Andersen, J. M.; Barrangou, R.; Hachem, M. A.; Lahtinen, S.; Goh, Y. J.; Svensson, B.; Klaenhammer, T. R. Transcriptional and functional analysis of galactooligosaccharide uptake by lacS in Lactobacillus acidophilus. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 17785−17790. (27) Andersen, J. M.; Barrangou, R.; Hachem, M. A.; Lahtinen, S. J.; Goh, Y. J.; Svensson, B.; Klaenhammer, T. R. Transcriptional analysis of prebiotic uptake and catabolism by Lactobacillus acidophilus NCFM. PLoS One 2012, 7, 1−12. (28) Goulas, T.; Goulas, A.; Tzortzis, G.; Gibson, G. R. Expression of four beta-galactosidases from Bif idobacterium bif idum NCIMB41171 and their contribution on the hydrolysis and synthesis of galactooligosaccharides. Appl. Microbiol. Biotechnol. 2009, 84, 899− 907. (29) Zhang, Z.; Wang, H.; Yang, R. J.; Jiang, X. A novel spectrophotometric method for quantitative determination of lactulose in food industries. Int. J. Food Sci. Technol. 2010, 45, 258−264. (30) Roberfroid, M. B. Prebiotics: Preferential substrates for specific germs? Am. J. Clin. Nutr. 2001, 73, 406S−409S. (31) Cecchini, D. A.; Laville, E.; Laguerre, S.; Robe, P.; Leclerc, M.; Doré, J.; Henrissat, B.; Remaud-Siméon, M.; Monsan, P.; PotockiVéronèse, G. Functional metagenomics reveals novel pathways of prebiotic breakdown by human gut bacteria. PLoS One 2013, 8, No. e72766. (32) Whitman, W. B.; Coleman, D. C.; Wiebe, W. J. Prokaryotes: The unseen majority. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6578− 6583. (33) Moynihan, P. J.; Ferrier, S.; Blomley, S.; Wright, W. G.; Russell, R. R. Acid production from lactulose by dental plaque bacteria. Lett. Appl. Microbiol. 1998, 27, 173−177. (34) Marín-Manzano, M. C.; Abecia, L.; Hernandez-Hernandez, O.; Sanz, M. L.; Montilla, A.; Olano, A.; Rubio, L. A.; Moreno, F. J.; Clemente, A. Galacto-oligosaccharides derived from lactulose exert a selective stimulation on the growth of Bif idobacterium animalis in the large intestine of growing rats. J. Agric. Food Chem. 2013, 61, 7560− 7567. (35) Ignatova, T.; Iliev, I.; Kirilov, N.; Vassileva, T.; Dalgalarrondo, M.; Haertlé, T.; Chobert, J. M.; Ivanova, I. Effect of oligosaccharides on the growth of Lactobacillus delbrueckii subsp. bulgaricus strains isolated from dairy products. J. Agric. Food Chem. 2009, 57, 9496− 9502.
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In Vitro Fermentation of Lactulose by Human Gut Bacteria Bingyong Mao,†,§ Dongyao Li,†,§ Jianxin Zhao,† Xiaoming Liu,† Zhennan Gu,†,‡ Yong Q. Chen,†,‡ Hao Zhang,*,†,‡ and Wei Chen*,†,‡ †
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China ‡ Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, People’s Republic of China S Supporting Information *
ABSTRACT: Lactulose has been known as a prebiotic that can selectively stimulate the growth of beneficial bifidobacteria and lactobacilli. Recent studies have indicated that Streptococcus mutans, Clostridium perfringens, and Faecalibacterium prausnitzii are also able to utilize lactulose. However, the previous studies mainly focused on the utilization of lactulose by individual strains, and few studies were designed to identify the species that could utilize lactulose among gut microbiota. This study aimed to identify lactulose-metabolizing bacteria in the human gut, using in silico and traditional culture methods. The prediction results suggested that genes for the transporters and glycosidases of lactulose are well distributed in the genomes of 222 of 453 strains of gastrointestinal-tract bacteria. The screening assays identified 35 species with the ability to utilize lactulose, of which Cronobacter sakazakii, Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas putida were reported for the first time to be capable of utilizing lactulose. In addition, significant correlations between lactulose and galactooligosaccharide metabolism were found. Thus, more attention should be paid to bacteria besides bifidobacteria and lactobacilli to further investigate the relationship between functional oligosaccharides and gut bacteria. KEYWORDS: lactulose, intestine, microbiota, oligosaccharides, utilization
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INTRODUCTION Nondigestible carbohydrates in the human diet are capable of reaching the large intestine without digestion or absorption, where they supply energy for gut bacteria and modify the composition of gut microbiota. These carbohydrates include nonstarch polysaccharides, termed “dietary fiber”, and some oligosaccharides, among which lactulose, fructooligosaccharides, and galactooligosaccharides (GOS) are regarded as prebiotics.1 According to Gibson et al.,1 prebiotics are functional oligosaccharides that can selectively stimulate the growth of beneficial bacteria, chiefly Bifidobacterium and Lactobacillus, in the host.2−7 Dietary lactulose and fructooligosaccharides have been reported to protect the host from enteric pathogens and reduce the risk of colon cancer.8,9 However, Clostridium perfringens, Clostridium difficile, and Streptococcus faecalis have also been found to ferment lactulose,10 and pathogenic Escherichia coli BEN2908 has been shown to be capable of metabolizing fructooligosaccharides,11 which could cause human disease at high quantities. These results suggest that oligosaccharides may be utilized by nonprobiotic bacteria in the gut. Fermentation properties of oligosaccharides derived from lactulose or raffinose and human milk oligosaccharides by fecal microbiota have been assessed in vitro by determination of short-chain fatty acids and gas production.6,7,12,13 However, no detailed study has been conducted of the intestinal bacteria that are capable of utilizing oligosaccharides, and it remains unknown which species in the gut are able to use oligosaccharides. Bacteria can grow on oligosaccharides only when the latter are degraded into monosaccharides and subsequently metabolized for energy production. Therefore, transporters and © 2014 American Chemical Society
glycoside hydrolases are key factors in the utilization of oligosaccharides.14,15 Gänzle reviewed the metabolism of oligosaccharides in lactobacilli16 and found metabolism to be limited by transport, because glycoside hydrolases are intracellular. This may differentiate lactobacilli from Bifidobacterium spp. Lactulose is a simple disaccharide composed of galactose and fructose by a β-1,4-glycosidic bond and has been known as “bifidus factor” since the 1950s.17 Lactulose does not occur naturally, and it may be produced by chemical isomerization of lactose or enzymatic synthesis.18 Commercial lactulose can reach a purity of 99%, which is suitable for bacterial isolation as a single carbon source. Therefore, in the current study the utilization of lactulose by gut microbiota was investigated. A traditional culture method was used to identify lactulosemetabolizing bacteria, and the search for key GOS enzymes, cross-checked against data on body site specialized reference genomes gathered for the Human Microbiome Project (HMP), was carried out in silico.
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MATERIALS AND METHODS
Chemicals and Reagents. Lactulose (purity by HPLC 99.3%, water by Karl Fischer titration 0.7%) was purchased from EMD Chemicals, Inc. (San Diego, CA), and GOS (≥98%) was purchased from Shanghai Dingjie Technology, Ltd. (Shanghai, China). Received: Revised: Accepted: Published: 10970
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Bacterial Strains and Growth Media. Bifidobacterium dentium KCTC 3361, Clostridium leptum KCTC 5155, Enterococcus saccharolyticus KCTC 3643, Mitsuokella multacida KCTC 5453, Pediococcus acidilactici KCTC 15064, Clostridium nexile KCTC 5578, Clostridium scindens KCTC 5591, Ruminococcus gnavus KCTC 5920, and Weissella paramesenteroides KCTC 3531 were purchased from the Korean Collection for Type Cultures. Megamonas funiformis JCM 14723 was purchased from the Japan Collection of Microorganisms. Marvinbryantia formatexigens DSM 14469 and Ruminococcus obeum DSM 25238 were purchased from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures). Lactobacillus acidophilus ATCC 4356, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus fermentum ATCC 14931, and Lactobacillus plantarum subsp. plantarum ATCC 14917 were kindly provided by Professor Heping Zhang (Inner Mongolia Agricultural University, China). Bifidobacterium adolescentis CICC 6070 was purchased from the China Center of Industrial Culture Collection. Clostridium perfringens ATCC 13124 was purchased from the American Type Culture Collection. Lactobacillus reuteri CCFM 8217 and Leuconostoc mesenteroides CCFM 8220 were obtained from the Culture Collection of Food Microorganisms, Jiangnan University. The medium used to isolate the bacteria was modified from gut microbiota medium (GMM),19 which was modified from the supplemented tryptone−yeast−glucose medium by Gordon and coworkers19 to culture fecal microbiota. The modified GMM contained 4 g of lactulose, 2 g of peptone, 1 g of yeast extract, 2 g of KH2PO4, 0.002 g of MgSO4·7H2O, 0.08 g of NaCl, 8 mg of CaCl2, 0.73 mg of FeSO4·7H2O, 1.2 mg of hematin, 10 mL of ATCC vitamin mix, 10 mL of ATCC trace mineral mix, 0.5 mL of Tween 80, and 0.5 g of Lcysteine per liter. The pH was adjusted to 7.0 ± 0.1 by use of NaOH, and the medium was sterilized at 115 °C for 20 min. Lactulose was filter-sterilized through a 0.22-μm filter and added to the autoclaved medium as the sole carbon source. Solid medium was obtained by adding 18 g of agar/L of liquid medium. A total of 15 mL of 0.5% bromocresol purple was added to the GMM agar medium as the indicator of pH change. Database Preparation. Site-specialized protein multifasta (PEP) files for reference genomes can be downloaded from the HMP Data Analysis and Coordination Center. These files record all HMP isolates that have completed sequencing and annotation. The gastrointestinaltract PEP file (382 rows) was downloaded from http://hmpdacc.org/ HMRGD/. A customized script was used to eliminate redundance. The output of HMP’s mothur community profiling of metagenomic 16S rRNA sequence data generated by a low-stringency approach (http://hmpdacc.org/HMMCP/) was also used for reference. One hundred seven of the genera depicted in the mothur community profiling output were not found in the list of reference genomes. Therefore, the 98 available genome coding sequences for these 107 genera were downloaded from the National Center for Biotechnology Information and merged into the gastrointestinal-tract PEP file, followed by BLAST database formatting. Genes Responsible for Uptake and Hydrolysis of Galactooligosaccharides. By use of literature mining, 41 genes were identified as responsible for the transport or catabolism of GOS (Table 1). However, no genes have been reported to be responsible for the metabolism of lactulose. Like lactulose, GOS is formed by a β-1,4glycosidic bond. Therefore, the genes related to GOS metabolism were used in this study to predict potential lactulose-metabolizing bacteria. Alignment of Transporters and Glycosidases against the Local Protein Database. BLASTP searches were run against the local protein database with transporters and glycoside hydrolases used as queries. When the alignment length was found to exceed 70% of the transporter/glycoside hydrolase sequence and the identity was greater than 40%, the strain was inferred to have homologous proteins for corresponding functions. Isolation and Identification of Lactulose-Metabolizing Bacteria. Fresh fecal samples were collected from five healthy donors aged 25−29 years. The fecal samples were placed in prereduced 0.1 M phosphate-buffered saline (PBS; 80 g of NaCl, 2 g of KCl, 14.4 g of Na2PO4, 2.4 g of KH2PO4, and 0.5 g of L-cysteine per liter; pH 7.0),
Table 1. Genes Responsible for Transport and Catabolism of Galactooligosaccharides gene
annotation
Bifidobacterium lactis Bl-0420 lacS-balac_0475 MFS permease balac_0485 permease balac_0486 permease lacZ-balac_0476 β-galactosidase balac_0484 β-galactosidase Bifidobacterium longum LMG1319721 bl1523 permease bl0260 permease bl0261 permease bl0259 β-galactosidase bl0976 galactoside transporter bl0978 β-galactosidase Bifidobacterium longum subsp. infantis ATCC1569722 blon_0267 permease blon_0268 β-galactosidase blon_2331 permease blon_2332 permease blon_2334 β-galactosidase blon_2016 β-galactosidase blon_2123 β-galactosidase blon_2416 β-galactosidase blon_2414a solute binding protein blon_2453a β-galactosidase Bifidobacterium breve UCC200324 bbr_0417 galC solute binding protein bbr_0418 galD permease bbr_0419 galE permease bbr_0420 galG β-galactosidase bbr_1551 lacS galactoside symporter bbr_1552 lacZ β-galactosidase bbr_0530 gosC solute binding protein bbr_0527 gosD permease bbr_0528 gosE permease bbr_0529 gosG β-galactosidase Geobacillus stearothermophilus T-625 ganA extracellular endo-β-1,4-galactanase precursor ganE solute binding protein ganF permease ganG permease ganB β-galactosidase Lactobacillus acidophilus26 lacS-lba1463 permease Lactobacillus acidophilus NCFM27 lacZ-lba1462 β-galactosidases lacL-lba1467 β-galactosidases lacM-lba1468 β-galactosidases Bifidobacterium bifidum NCIMB4117128 bbgII β-galactosidase a
This entry from ref 23.
with 15 mL/g of feces per sample.19 The samples were dispersed and suspended by vortexing with sterilized glass beads for 5 min. Serial dilution was performed with 0.1 M PBS, and the diluted (10−3) samples (0.1 mL) were plated. Six plates were used for each fecal sample, as recommended by Gordon and co-workers,19 and the medium without lactulose was used for negative controls. The plates were incubated in an anaerobic workstation (Whitley DG250, Don Whitley Scientific Limited, West Yorkshire, U.K.) at 37 °C for 24 h under an atmosphere of 80% N2, 10% CO2, and 10% H2. Colonies 10971
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Figure 1. Distribution of homologue proteins of transporters and glycosidases for lactulose (vertical axis) among GIT bacteria (horizontal axis). Positive results for transporters and glycosidases are indicated in red and green, relatively. with yellow surroundings on the GMM plates were picked up, reinoculated into liquid medium, and cultured anaerobically. The bacteria were isolated and purified by the standard streak-plate technique. The isolated bacteria were Gram-stained and identified by amplification and sequencing of 16S rRNA. The polymerase chain reaction (PCR) procedure was as follows: 95 °C for 5 min; 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min 30 s, 30 cycles; 72 °C for 10 min; 12 °C for 10 min. The resulting PCR product was sequenced by Sangon Biotech (Shanghai) Co., Ltd. Characterization of Bacterial Growth on Lactulose. Bacteria were cultured anaerobically in GMM at 37 °C for 24 h, with lactulose as the sole carbon source. Absorbance at 600 nm (OD600) and pH were recorded every 2 h to characterize the growth. The amount of lactulose in the culture supernatant was determined by the method developed by Zhang et al.29 Fermentation of Galactooligosaccharides by LactuloseMetabolizing Bacteria. Bacteria capable of metabolizing lactulose were inoculated on GMM plates with GOS as the sole carbon source and bromocresol purple as the indicator. GMM plates without GOS were set as negative controls. The bacteria on the GMM−GOS plates that were capable of growing and coloring their surroundings yellow were considered to have the ability to metabolize GOS.
Table 2. Identified Lactulose-Metabolizing Bacteria in Healthy Adult Fecesa genus and species Cronobacter sakazakii Enterobacter aerogenes Enterobacteriaceae bacterium Enterococcus casseliflavus Enterococcus faecalis Enterococcus faecium Enterococcus gallinarum Escherichia coli Escherichia fergusonii Klebsiella pneumoniae
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Klebsiella sp.
RESULTS Homologous Proteins for Lactulose Transporters and Glycosidases in 453 Gastrointestinal-Tract Bacteria Strains. The results of aligning the transporters and glycosidases with the local protein database are shown in Figure 1. Surprisingly, homologue proteins for the glycosidases appeared in 222 of the 453 strains, which accounted for 56 of the 114 genera. In contrast, homologue proteins for the transporters were found in only 90 strains, accounting for 29 of the 114 genera. These 90 strains also possessed homologue proteins for the glycosidases. In general, lactulose metabolism requires transporters and galactosidases. In addition to Bifidobacterium and Lactobacillus, 27 genera with homologue proteins for both transporters and glycosidases were found (Figure 1), indicating that lactulose may not be exclusively a stimulant of probiotics.30 However, it remains unclear whether the predicted bacteria are able to utilize lactulose; further investigation is required. Utilization of Lactulose by Cronobacter sakazakii, Enterococcus casseliflavus, Enterococcus faecalis, and Klebsiella pneumoniae. Compared with GOS, lactulose has an accurate chemical structure and metabolic process, and its purity can reach 99.3%. In the current study, fecal samples collected from five healthy adults were plated on GMM with lactulose as the sole carbon source. Ninety-six strains belonging to 18 species of nine genera were identified (Table 2). To our knowledge, Cronobacter, Enterococcus, Klebsiella, and Pseudomonas are first reported in our study to be capable of utilizing lactulose. One strain from each species was selected and its growth on lactulose was characterized (Figure 2). OD600
Klebsiella variicola Pseudomonas putida Streptococcus anginosus Streptococcus equinus Streptococcus sanguinis Bifidobacterium pseudocatenulatum Bifidobacterium sp.
strains CCFM 8305, 8306, 8307, 8308, 8309, 8310, 8311, 8312 CCFM 8313 CCFM 8314, 8315 CCFM 8316, 8317, 8318, 8319 CCFM 8320 CCFM 8321, 8322, 8323, 8324 CCFM 8325 CCFM 8326, 8327, 8328, 8329, 8330, 8331, 8332, 8333, 8334, 8335, 8336, 8337, 8338, 8339, 8340, 8341, 8342 CCFM 8343, 8344, 8345, 8346 CCFM 8347, 8348, 8349, 8350, 8351, 8352, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361, 8362, 8363, 8364, 8365, 8366, 8367, 8368, 8369, 8370 CCFM 8371, 8372, 8373, 8374, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382, 8383, 8384, 8385, 8386 CCFM 8387 CCFM 8388 CCFM 8389 CCFM 8390, 8391 CCFM 8392, 8393, 8394 CCFM 8395, 8396, 8397, 8398 CCFM 8399, 8400
a
16S rRNA nucleotide acid sequences of the identified strains are shown in Table S1 in Supporting Information.
increased during the 24-h culture period, along with a decrease in pH and a reduction in the lactulose amount, which provided direct proof of lactulose utilization. In addition, the ability of 21 strains to metabolize lactulose was assessed, and 17 strains were proven to be able to grow on lactulose (Table 3 and Figure 3). Thirteen of the 15 strains with homologue proteins for both transporters and glycosidases were capable of utilizing lactulose, indicating that searching for transporter and glycosidase homology may be an effective method of predicting lactulose-metabolizing bacteria. Galactooligosaccharide Utilization by Lactulose-Metabolizing Bacteria. Due to the structural similarity of lactulose and GOS, the 18 strains isolated (Figure 2) and the 17 strains found to be capable of metabolizing lactulose (Table 3) were tested for their ability to ferment GOS. The results showed that 32 strains were capable of GOS utilization (Table 10972
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Figure 2. Growth of isolated strains on GMM medium with lactulose as the sole carbon source. OD600 (○), pH (●), and the amount of lactulose (×) were recorded every 2 h, and the strains are presented as follows: (A) C. sakazakii CCFM 8306, (B) E. aerogenes CCFM 8313, (C) Enterobacteriaceae bacterium CCFM 8315, (D) E. casseliflavus CCFM 8316, (E) E. faecalis CCFM 8320, (F) E. faecium CCFM 8321, (G) E. gallinarum CCFM 8325, (H) E. coli CCFM 8331, (I) E. fergusonii CCFM 8343, (J) K. pneumoniae CCFM 8358, (K) Klebsiella sp. CCFM 8371, (L) K. variicola CCFM 8387, (M) P. putida CCFM 8388, (N) S. anginosus CCFM 8389, (O) S. equinus CCFM 8390, (P) S. sanguinis CCFM 8392, (Q) B. pseudocatenulatum CCFM 8395, and (R) Bifidobacterium sp. CCFM 8399. 10973
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lactulose in the gut. The gut is a complex ecosystem, with about 1014 bacteria categorized into more than 1000 species,32 and the aim of the current study was to identify lactulosemetabolizing bacteria within this ecosystem. Transporters and glycosidases are of great importance to the utilization of oligosaccharides. Thus far, however, no encoding genes for lactulose metabolism have been identified. Therefore, the genes for GOS metabolism acquired through literature mining were used instead. Moreover, the unculturability of most gut microbes has limited our understanding of the metabolic characteristics of individual strains. To date, HMP has failed to reach its goal of isolating 3000 reference genomes from human body sites. In the current study, the site-specialized reference genome data provided by HMP were combined with the output of mothur community profiling to build a local protein database. As shown in Figure 1, the genes for lactulose metabolism appeared in 222 GIT bacteria strains, of which 90 strains possessed homologue proteins for both transporters and glycosidases. It is reasonable to infer from the existence of transporters in a given strain that the strain contains corresponding glycosidases. If no corresponding glycosidases were available, the strain would be unlikely to contain transporters. To identify lactulose-metabolizing bacteria in the gut, 18 species were isolated from fecal samples (Table 2), and 17 strains were proven to have the ability to utilize lactulose (Table 3). This indicates that bioinformatic analysis is an effective tool for predicting lactulose-metabolizing bacteria, thereby narrowing the scope of experimental research. Unlike the results of previous studies,10,31,33 the findings of the current study indicated for the first time that the genera Cronobacter, Enterococcus, Klebsiella, and Pseudomonas are able to utilize lactulose. To the best of our knowledge, C. sakazakii, E. aerogenes, Enterobacteriaceae bacterium, E. casseliflavus, E. faecalis, E. faecium, E. gallinarum, E. fergusonii, K. pneumoniae, Klebsiella sp., K. variicola, P. putida, S. anginosus, S. equinus, and B. pseudocatenulatum have never been reported to be capable of utilizing lactulose. Therefore, lactulose was shown to have the potential to stimulate the growth of many commensal bacteria in addition to bifidobacteria and lactobacilli. More gut bacteria capable of utilizing lactulose may exist than previously supposed. Additionally, lactulose exhibited similar metabolic characteristics to GOS (Table 4), which may be derived from lactulose.34 Therefore, lactulose may be considered a type of GOS with a lower degree of polymerization, and lactulose and GOS may possess similar genes for transporters or galactosidases. In the human gut, competition for limited food takes place among the bacteria, and those capable of utilizing prebiotic substrates might have advantages during the competition with other bacteria.35 In conclusion, the combination of a homology search and the traditional isolation method assisted in the identification of lactulose-metabolizing bacteria. Thirty-five species were found, most of which were reported for the first time to be capable of utilizing lactulose. The commensal gut bacteria Cronobacter, Enterococcus, Escherichia, Klebsiella, Pseudomonas, and Streptoccoccus were found to be able to ferment lactulose. In addition, significant correlations were observed between lactulose and GOS metabolism. This work offers greater insight into the utilization of oligosaccharides by gut bacteria. To further explore the relationship between functional oligosaccharides
Table 3. Bacteria Obtained on the Basis of Alignment Analysisa strain
source
transporters
glycosidases
lactulose
Bifidobacterium adolescentis CICC 6070 Bifidobacterium dentium KCTC 3361 Clostridium leptum KCTC 5155 Clostridium perfringens ATCC 13124 Enterococcus saccharolyticus KCTC 3643 Eubacterium rectale ATCC 33656 Lactobacillus acidophilus ATCC 4356 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 Lactobacillus fermentum ATCC 14931 Lactobacillus plantarum subsp. plantarum ATCC 14917 Lactobacillus reuteri CCFM8217 Leuconostoc mesenteroides CCFM8220 Marvinbryantia formatexigens DSM 14469 Megamonas funiformis JCM 14723 Mitsuokella multacida KCTC 5453 Pediococcus acidilactici KCTC 15064 Weissella paramesenteroides KCTC 3531 Clostridium nexile KCTC 5578 Clostridium scindens KCTC 5591 Ruminococcus gnavus KCTC 5920 Ruminococcus obeum DSM 25238
CICC
Y
Y
+
KCTC
Y
Y
+
KCTC
N
Y
+
ATCC
N
Y
+
KCTC
N
Y
+
KCTC
Y
Y
+
Zhang
Y
Y
+
Zhang
N
Y
+
Zhang
Y
Y
+
Zhang
Y
Y
+
CCFM
Y
Y
+
CCFM
Y
Y
+
DSM
Y
Y
+
JCM
Y
Y
+
KCTC
Y
Y
+
KCTC
Y
Y
+
KCTC
Y
Y
+
KCTC
N
Y
−
KCTC
N
Y
−
KCTC
Y
Y
−
DSM
Y
Y
−
a
Symbols: Y, positive for alignment search; N, negative for alignment search; +, positive growth on lactulose; −, negative growth on lactulose.
4). Although lactulose is a disaccharide composed of fructose and galactose, while GOS is a complex with different degrees of galactose polymerization, they may share the same transporters or glycosidases. Moreover, the 17 lactulose-metabolizing strains obtained from the alignment search contained homologue proteins for transporters and glycosidases that have been reported to be responsible for metabolizing GOS. Thus, there seems to be a significant correlation between lactulose and GOS.
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DISCUSSION Lactulose has generally been regarded as a special substrate used by probiotic bifidobacteria and lactobacilli. However, Faecalibacterium prausnitzii, Bacteroides oralis, E. coli, and C. perfringens have also been reported to utilize lactulose in vitro.10,31 It remains unclear which species are able to utilize 10974
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Figure 3. Growth of obtained strains on GMM medium with lactulose as the sole carbon source. OD600 (○), pH (●), and the amount of lactulose (×) were recorded every 2 h, and the strains are presented as follows: (A) B. adolescentis CICC 6070, (B) B. dentium KCTC 3361, (C) C. leptum KCTC 5155, (D) C. perfringens ATCC 13124, (E) E. saccharolyticus KCTC 3643, (F) E. rectale ATCC 33656, (G) L. acidophilus ATCC 4356, (H) L. delbrueckii subsp. bulgaricus ATCC 11842, (I) L. fermentum ATCC 14931, (J) L. plantarum subsp. plantarum ATCC 14917, (K) L. reuteri CCFM 8217, (L) L. mesenteroides CCFM 8220, (M) M. formatexigens DSM 14469, (N) M. funiformis JCM 14723, (O) M. multacida KCTC 5453, (P) P. acidilactici KCTC 15064, and (Q) W. paramesenteroides KCTC 3531. 10975
dx.doi.org/10.1021/jf503484d | J. Agric. Food Chem. 2014, 62, 10970−10977
Journal of Agricultural and Food Chemistry Table 4. Growth of Lactulose-Metabolizing Strains on Galactooligosaccharidesa strain
GOS
strain
+ + +
B. adolescentis CICC 6070 B. dentium KCTC 3361 C. leptum KCTC 5155
+ + −
+ + − + +
+ + + + +
E. fergusonii CCFM8343 K. pneumoniae CCFM8358
+ +
Klebsiella sp. CCFM8371 K. variicola CCFM8387 P. putida CCFM8388 S. anginosus CCFM8389 S. equinus CCFM8390 S. sanguinis CCFM8392 B. pseudocatenulatum CCFM8395 Bifidobacterium sp. CCFM8399
+ + + + + − +
C. perfringens ATCC 13124 E. saccharolyticus KCTC 3643 E. rectale ATCC 33656 L. acidophilus ATCC 4356 L. delbrueckii subsp. bulgaricus ATCC 11842 L. fermentum ATCC 14931 L. plantarum subsp. plantarum ATCC 14917 L. reuteri CCFM8217 L. mesenteroides CCFM8220 M. formatexigens DSM 14469 M. funiformis JCM 14723 M. multacida KCTC 5453 P. acidilactici KCTC 15064 W. paramesenteroides KCTC 3531
a
+ + + + + + + + +
+
and gut bacteria, more attention should be paid to bacteria other than bifidobacteria and lactobacilli.
ASSOCIATED CONTENT
S Supporting Information *
One table listing 16S rRNA nucleotide acid sequences of the identified strains. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
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+, positive growth on GOS; −, negative growth on GOS.
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ABBREVIATIONS
GOS, galactooligosaccharides; HMP, Human Microbiome Project; GMM, gut microbiota medium; GIT, gastrointestinal tract
GOS
C. sakazakii CCFM8306 E. aerogenes CCFM8313 Enterobacteriaceae bacterium CCFM8315 E. casseliflavus CCFM8316 E. faecalis CCFM8320 E. faecium CCFM8321 E. gallinarum CCFM8325 E. coli CCFM8331
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Article
AUTHOR INFORMATION
Corresponding Authors
*Telephone/fax 86-510-85912155; e-mail zhanghao@jiangnan. edu.cn. *Telephone/fax 86-510-85912155; e-mail chenwei66@ jiangnan.edu.cn. Author Contributions §
B.M. and D.L. contributed equally to this work.
Funding
This work was supported by the National Science Fund for Distinguished Young Scholars (31125021), Key Projects in the National Science and Technology Pillar Program during the 12th five-year plan period (2013BAD18B01, 2013BAD18B02, 2012BAD28B07, 2012BAD28B08), the 111 Project B07019, and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank all adults who provided fecal samples. We also thank Professor H. Zhang for providing Lactobacillus acidophilus ATCC 4356, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus fermentum ATCC 14931, and Lactobacillus plantarum subsp. plantarum ATCC 14917. 10976
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