Article pubs.acs.org/JAFC
Lactulose Differently Modulates the Composition of Luminal and Mucosal Microbiota in C57BL/6J Mice Bingyong Mao,†,‡,⊥ Dongyao Li,†,⊥ Chunqing Ai,§ Jianxin Zhao,†,‡ Hao Zhang,†,‡ and Wei Chen*,†,‡,∥ †
State Key Laboratory of Food Science and Technology, School of Food Science and Technology and ‡International Joint Research Laboratory for Probiotics, Jiangnan University, 214122 Wuxi, People’s Republic of China § National Engineering Research Center of Seafood, School of Food Science and Technology, Dalian Polytechnic University, 116034 Dalian, People’s Republic of China ∥ Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University, 102488 Beijing, People’s Republic of China S Supporting Information *
ABSTRACT: In this study, C57BL/6J mice were fed diets supplemented with different proportions of lactulose (0%, 5%, and 15%) for 2 weeks to study its effects on the luminal and mucosal microbiota. The luminal and mucosal samples of cecum and colon were investigated. After high-lactulose treatment (15%), pH of the luminal contents decreased from 6.90−7.72 to 5.95− 6.21 from the cecum to distal colon, and the amount of total short-chain fatty acids in the cecum was significantly increased. The luminal content was mostly dominated by Firmicutes, Actinobacteria, and Bacteroidetes, while the mucus was dominated by Firmicutes, Proteobacteria, and Bacteroidetes. The abundance of Actinobacteria was significantly increased in the content, and Proteobacteria was the most abundant phylum (∼50%) in the mucus after high-lactulose treatment. At the genus level, Bifidobacterium and Akkermansia were both significantly increased in the content, and Helicobacter was the most abundant in the mucus. KEYWORDS: lactulose, mucosal, luminal, Helicobacter, Bif idobacterium
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oides,16−18 and few studies were conducted on the global and detailed effects of lactulose on luminal and mucosal microbiota. As an alternative to the traditional culture methods, denaturing gradient gel electrophoresis, and fluorescent in situ hybridization, the 16S rDNA amplicon sequencing technique is an effective tool with which to investigate global changes in microbial communities and develop new target genera for further studies.19 C57BL/6 mice are typically and commonly used for gut microbiota research, and the microbiota of the luminal contents were different and not uniformly distributed along the stomach, small intestine, and large intestine.20 Information on the mucosal microbiota was not given in that study. In addition, the physiological conditions of the cecum and colon as a bioreactor and fermenter were not available (e.g., pH, short-chain fatty acids (SCFAs), and ammonia). In this study, C57BL/6J mice were fed low and high doses of lactulose. The contents and mucus of the cecum and colon (equally divided into the proximal, middle, and distal parts) were collected for analysis. The physiological conditions were characterized, and the luminal and mucosal microbiota were assessed by 16S rDNA amplicon analysis to evaluate the effects of lactulose. A detailed map of the spatial ecology of the cecum and colon was made to elucidate the host-microbe interactions and
INTRODUCTION The human gut is colonized by a large mixture of microbial species,1 and the relationship between gut microbiota and a range of diseases has been investigated extensively.2,3 The composition of the microbiota is dependent on both individual genetic and environmental factors (e.g., diet),4 and diet dominates host genotype in shaping the microbial ecology.5 Previous studies have focused on fecal microbiota due to its accessibility for sampling,6−9 and fewer investigations have been carried out on luminal or mucosal samples. However, fecal microbiota differ phylogenetically from the luminal and mucosal communities.10,11 In the colon, the epithelium is covered by two layers of mucus; the tight inner mucus layer is nearly sterile, and the loose outer mucus is densely colonized by commensal bacteria.12,13 The outer mucus is an initial native defense between colon epithelium and commensal bacteria and retains many bacterial species, of which only a few species have sufficient catabolic enzymes to use mucus glycans as an alternative carbon source. Thus, mucus bacteria is a valuable target to study the host−microbe interactions. Lactulose has been regarded as a “bifidus factor” since the 1950s,14 selectively increasing the bifidobacterial counts, and it has exerted beneficial health properties to humans, such as inhibiting the growth of pathogenic Salmonella and treating constipation, hepatic encephalopathy, and tumor prevention.15 Lactulose is a disaccharide that is indigestible and used by the gut bacteria. Owing to the limitation of experimental techniques, previous studies were mainly focused on given groups of bacteria, including bifidobacteria, lactobacilli, clostridia, and bacter© 2016 American Chemical Society
Received: Revised: Accepted: Published: 6240
May 22, 2016 July 13, 2016 July 20, 2016 July 20, 2016 DOI: 10.1021/acs.jafc.6b02305 J. Agric. Food Chem. 2016, 64, 6240−6247
Article
Journal of Agricultural and Food Chemistry Table 1. Concentration of SCFAs in Cecal, Colonic, and Fecal Contents of Mice in Different Groupsa SCFAs, μmol/gb cecal content acetic acid propanoic acid isobutyric acid butanoic acid isopentanoic acid totald colonic content proximal acetic acid propanoic acid isobutyric acid butanoic acid isopentanoic acid total middle acetic acid propanoic acid isobutyric acid butanoic acid isopentanoic acid total distal acetic acid propanoic acid isobutyric acid butanoic acid isopentanoic acid total
Lactulose-High
Lactulose-Low
Pc
50.72 ± 6.69 12.27 ± 2.58 1.66 ± 0.32a 12.77 ± 5.27 1.58 ± 0.78 78.69 ± 5.22
48.61 ± 12.40 13.55 ± 4.76 1.44 ± 0.18a 12.61 ± 6.93 1.16 ± 0.44 77.20 ± 18.94
70.34 ± 14.91 6.87 ± 5.39 0.29 ± 0.33b 12.85 ± 1.27 0.42 ± 0.51 90.92 ± 21.53
NS NS 0.05). dTotal = acetic acid + propanoic acid + isobutyric acid + butanoic acid + isopentanoic acid.
a c
sterile EP tubes on ice. The mice were returned to their former cages after feces collection, and the feces were stored at −80 °C until analysis. The pH was measured using a Mettler Toledo pH meter with an InLab Solids pH electrode (Mettler Toledo International Inc., Columbus, OH). At the end of the experiment, the animals were sacrificed, and blood was collected through the retro-orbital vein prior to sacrifice. Serum was obtained by centrifugation at 3000 rpm for 15 min (Eppendorf 5424R) and stored at −80 °C until analysis. The cecum and colon were excised and immediately weighed as a whole to give the total weight. The colon was divided equally into three parts as the proximal, middle, and distal colon. The contents of the cecum and the proximal, middle, and distal colon were gently extruded and collected on ice, and the pH was measured. Part of the contents was immediately processed to analyze the SCFAs, and the remainder was stored at −80 °C for later analysis. The mucus of the cecum and colon were obtained as described by Gustafsson22 and stored at −80 °C for further microbiota analysis. Determination of Lactulose and Glucose in Cecal and Colonic Contents. Lactulose in the cecal and colonic contents was extracted by the method developed by Dotz23 with some modifications. The samples were thawed, dispersed in water (0.02 g/mL), and gently stirred for 1.5 h at 20 °C before centrifugation at 18000g for 10 min. The amount of lactulose in the supernatant was determined according to the method developed by Zhang et al.,24 and the amount of glucose was determined using a glucose oxidase assay kit (Rongsheng Biotech Co., Ltd., Shanghai, China). Glucose was measured to exclude its interference on the amount of lactulose. Determination of SCFAs in Cecal and Colonic Contents. The cecal and colonic samples (20 mg) were resuspended in 500 μL of saturated NaCl solution and acidified with 20 μL of sulfuric acid (10%); 800 μL of diethyl ether was added to extract the fatty acids, and then the
could be helpful for further studies on gut microbiota and the health of the host.
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MATERIALS AND METHODS
Chemicals and Reagents. Lactulose (purity of 99.3% by HPLC) was purchased from EMD Chemicals, Inc. (San Diego, CA). AIN-93-VX Vitamin Mix and AIN-93G Mineral Mix were purchased from MP Biomedicals, LLC (Santa Ana, CA). Animal Experiment Design. Male C57BL/6J mice (6 weeks old) (Shanghai Laboratory Animal Center, Shanghai, China) were randomly assigned to the three dietary treatments (n = 8 for each treatment). The mice were co-housed and maintained in an IVC rodent caging system under a strict 12 h light−dark cycle in a temperature- and humiditycontrolled room and fed the diets and water ad libitum (Table S1). All of the protocols were reviewed and approved by the Ethics Committee of Jiangnan University (JN no. 20140926-1226-35), and the procedures were in accordance with the European Community guidelines (Directive 2010/63/EU) for the care and use of experimental animals. The experiments were carried out as shown in Figure S1. The mice were allowed to adapt to the new environment for 1 week and then fed the various diets for 2 weeks. Lactulose was added to the control diet, partially replacing the dextrose component. The treatments were as follows: (1) control diet; (2) low-lactulose diet (5%); and (3) highlactulose diet (15%). Details on the diet composition are given in Table 1, and the composition was based on the diet developed by Campbell et al.21 All of the mice were weighed daily. Besides the three treatments, three mice were co-housed and fed the control diet, and they were sacrificed after 1 week on the control diet to isolate the lactulosemetabolizing bacteria from the cecal and colonic samples. Sample Collection. At the end of the adaption period, the mice were placed into individual clean cages, and their feces were collected in 6241
DOI: 10.1021/acs.jafc.6b02305 J. Agric. Food Chem. 2016, 64, 6240−6247
Article
Journal of Agricultural and Food Chemistry mixture was centrifuged at 18000g for 15 min. A total of 0.25 g of Na2SO4 was then added to the supernatant to remove the inside water, and the concentration of SCFAs (acetic, propionic, isobutyric, butyric, and isovaleric acids) was analyzed by gas chromatography−mass spectrometry (GCMS-QP2010 Ulra system, Shimadzu Corporation, Japan), which was equipped with a Rtx-Wax column (length of 30 m and i.d, of 0.25 μm). The carrier gas was helium (2 mL/min), and the injection volume was 1 μL with a split ratio of 10:1. The injection temperature was set at 240 °C, and the GC temperature program was as follows: begin at 100 °C, increase to 140 °C at the rate of 7.5 °C/min, followed by 60 °C/min to 200 °C, and hold at 200 °C for 3 min. The ion source temperature was set at 220 °C. The analytes were detected using the full scan mode. The concentration of SCFAs was calculated using the external standard method and expressed as μmol/g sample. Determination of Ammonia in Cecal and Colonic Contents. The samples were resuspended in water (20 mg/mL) and mixed, and the supernatant was obtained by centrifugation at 18000g for 15 min. The ammonia concentration was measured according to the colorimetric method developed by Chaney and Marbach.25 Serum Metabolic Parameters. Biochemical parameters in the serum were determined using a hematological autoanalyzer (BS-480, Mindray Medical International Limited, Shenzhen, China), including glucose, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (C-LDL), high-density lipoprotein cholesterol (C-HDL), IgA, IgM, and IgG. 16S rDNA Sequencing and Bioinformatic Analysis. Bacterial genomic DNA was extracted from the cecal and colonic samples using a FastDNA Spin Kit for soil (MP Biomedicals, Santa Ana, CA) according to the manuals. The V4 region of the 16S rDNA gene was PCRamplified from the genomic DNA as described previously.19,26 PCR products were excised from a 1.5% agarose gel, recovered using GeneClean Turbo (MP Biomedicals), and quantitied by Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Grand Island, NY). Libraries were prepared using TruSeq DNA LT Sample Preparation Kit (Illumina, catalog no. FC-121-2001) and sequenced on Illumina MiSeq with the MiSeq Reagent Kit (500 cycles PE, catalog no. MS-102-2003). Processing of sequence reads were performed with the QIIME package. The raw sequences with a lower quality score (50%) in the mucus samples after treatment with lactulose (the -High group). Among the Actinobacteria, Bifidobacterium was the most abundant genus, and its abundance notably increased from 50% in the luminal samples in the Lactulose-High group (Student’s t test, P < 0.05). Bifidobacterium was found at a lower abundance (
