Study design
To better understand the interactions of different molecular weight kappa carrageenan polysaccharides (KCPs) with human gut microbiota and host health, the degradation of KCP, SKCO and KCO by human gut microbiota were evaluated in vitro first, KCO-degrading bacterial was then isolated from human fecal samples, and their effects of KCO and KCO plus KCO-degrading bacterial on germ free mouse were investigated.
Preparation Of Carrageenan Polysaccharides And Oligosaccharides
KCP were obtained from Yantai Runlong Marine Biological Products Co., Ltd., China. Two types of carrageenan oligosaccharides were obtained after hydrolysis with dilute HCl (SKCO or 732 resin plus HCl (KCO). For SKCO preparation, 10 mg/ml carrageenan was adjusted to pH 1.26 with 0.1 M HCl and then heated to 37 °C for 3 hours. For KCO preparation, 30 mg/ml carrageenan was dissolved at 70 °C, and treated with 732 resin for 4 hours. The hydrolytic product was neutralized with 1 M NaOH and put into a dialysis bag (MW cutoff was 200–500 Da) to remove the salt. The final product was obtained by rotary evaporation and lyophilization. The MW of KCP, SKCO, and KCO was measured using high-performance liquid chromatography (HPLC) (Agilent 1260, U.S.) with a Shodex OH pak SB-804 HQ column, detected using a refractive index detector and multiangle laser light scattering. The average MWs of KCP, SKCO, and KCO were 450 kDa, 100 kDa, and 4.5 kDa, respectively.
Electrospray ionization mass spectrometry (ESI-MS) for sugar structure analysis
The derivatives generated from digestion of CGN oligosaccharides were determined by gel filtration chromatography and analyzed using negative-ion electrospray ionization mass spectrometry (ESI-MS). In brief, after removing the bacteria by centrifugation, the supernatants were separated on a Superdex Peptide 10/300 column. The sequence of each fraction was determined on a Thermo LTQ Orbitrap XL instrument (Thermo Finnigan Corp, U.S.). Samples were then dissolved in CH3CN/H2O (1:1, v/v) at a concentration of 10 pmol/µl and 5 µl was injected. Solvent volatilization temperature and capillary temperatures were 275 °C. The sheath flow gas flow rate was 8 arb. The flow rate was 8 µl/min during ESI-MS analysis. Helium was used as collision gas, with a collision energy of 20–25 eV.
Origin Of Human Fecal Samples
Eight healthy human volunteers (from Hangzhou, China), between 24 and 27 years of age, were recruited for the current study. The donors had not received antibiotics or pro- or prebiotic treatment for at least three months prior to sample collection. All of the volunteers provided informed, written consent, and the study was approved by the Ethics Committee of the Zhejiang Academy of Agricultural Sciences.
Batch culture fermentation of KCP, SKCO, and KCO with human fecal slurries
Batch culture fermentation was conducted using the procedure described by Lei et al. [56]. Briefly, basic growth medium VI contained the following components (g/l): yeast extract, 4.5; tryptone, 3.0; peptone, 3.0; bile salts No. 3, 0.4; L-cysteine hydrochloride, 0.8; hemin, 0.05; NaCl, 4.5; KCl, 2.5; MgCl2.6H2O, 0.45; CaCl2.6H2O, 0.2; KH2PO4, 0.4; Tween 80, 1 ml; and 2 ml of a solution of trace elements. To assess the degradation and utilization by human fecal microbiota, either 1.0 g of KCP, 5.0 g of SKCO, and 8.0 g of KCO were added as the sole carbon source. Due to the viscosity of KCP and SKCO, the amount of KCP and SKCO in the medium was reduced compared to KCO. The media were adjusted to pH 6.5 before autoclaving. Fresh fecal samples were homogenized in stomacher bags with 0.1 M anaerobic phosphate-buffered saline (PBS) (pH 7.0) to make 10% (wt/vol) slurry. Large food residues were removed by passing the mixture through a 0.4 mm sieve. The human fecal slurry (7 ml) was inoculated into a bottle containing 63 ml of growth medium, and the bottle was incubated at 37 °C for 72 h in an anaerobic chamber (anaerobic workstation AW 500, Electrotek Ltd., U.K.). Fermentation products were collected at different time points for further analysis. The pH value after 48 h fermentation was measured by a pH probe (Eutech, Singapore).
Thin-layer Chromatography (tlc) And Total Carbohydrate Analysis
The degradation of KCP, SKCO, and KCO was detected by TLC analysis. Briefly, 0.2 µL of sample was loaded on pre-coated silica gel-60 TLC aluminum plates (Merck, Germany). After development with a solvent system consisting of formic acid/n-butanol/water (6:4:1, v:v:v), the plate was soaked in orcinol reagent and carbohydrates were visualized by heating at 120 °C for 3 min.
The total carbohydrate concentration in the fermentation samples was determined using the phenol–sulfuric acid method, as described previously, using D-galactose as standard. Results are expressed as the mean amount of remaining carbohydrate relative to the total amount detected at 0 h.
Quantitation Of Bacterial Groups By Real-time Pcr
Quantification of bacterial DNA was performed using an ABI PRISM 7500 Real-Time PCR Detection System (Applied Biosystems) according to the manufacturer’s instructions. A 20-µl amplification reaction was performed with 10 µl Thunderbird SYBR qPCR Mix (Toyobo Co., Ltd, Osaka, Japan), 0.04 µl 50 × ROX reference dye, 0.5 mM of each primer, 1 µl DNA template (20 ng/µl), and distilled water. Amplifications were performed with the following PCR program: one cycle at 95 °C for 1 min, 40 cycles at 95 °C for 15 s, an appropriate annealing temperature for 35 s, and 72 °C for 35 s. Fluorescence was measured after the extension phase of each cycle. Melt curve analyses were performed by slowly heating the PCR mixtures from 55 °C to 95 °C. These served as end point assays and were used to confirm PCR specificity. Six pairs of primers were selected for quantification of the total number of the following bacteria: Bacteroides–Prevotella group, Bifidobacterium, Clostridium cluster XI, Enterobacteriaceae, Lactobacillus, and Desulfovibrio. The primer sequences and annealing temperature for each primer pair are shown in Supplementary Table S3. The quantitative measurement of unknown samples was achieved using standard curves made with known concentrations of plasmid DNA containing the respective amplicons for each set of primers.
Short-chain Fatty Acid (scfa) Analysis
Production of short-chain fatty acids (SCFAs) was determined by HPLC with an Aminex HPX-87H Exclusion Column. In brief, fermentation products were centrifuged at 14, 000 rpm for 15 min, and the supernatant was used for measurement. The detection condition included 5 mM H2SO4 used as mobile phase at a flow rate of 0.6 ml/min. The column temperature was 50 °C and a refractive index detector was used at a wavelength of 215 nm.
Animal Experiments
Twenty-four three-week-old germ-free Kunming mice were randomly divided into four groups. KCO degrading bacteria (5 × 108 in 0.5 ml) were inoculated intragastrically to the GN and GNK groups on day 0. Bacterial colonization was allowed for 56 days. Fecal pellets were collected after 4 weeks from each group to check the bacterial composition and population by 16 s rRNA gene clone library sequencing. After 56 days, 5% KCO was given in drinking water in the GNK and GK groups for an additional 8 weeks. The group without any treatment (GF) was used as control. Mice were sacrificed by cervical dislocation, and serum, liver, cecal content, and gut tissue samples were collected. All the animals were handled in strict compliance with current regulations and guidelines concerning the use of laboratory animals in China. The procedures were approved by the Zhejiang Academy of Agricultural Science and Third Military Medical University.
Rna-seq Analysis
To identify genes that were differentially expressed (DEGs) in response to KCOs and KCO-degrading bacteria in germ-free mice at the transcription level, total RNA was isolated from rectum samples for RNA-Seq analysis. RNA quality was assessed by Nanodrop and electrophoresis. Samples with RNA integrity numbers ≥ 8 were prepared using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA). Multiplex amplification was used to prepare cDNA with a paired-end read length of 100 bases using an Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA, USA). RNA-Seq was performed by Shanghai Personal Biotechnology Co., Ltd. (http://www.personalbio.cn/en/).
Quality control assessment of RNA-Seq data was completed using the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for high-throughput sequencing before and after RNA-Seq alignment. RNA-Seq data were analyzed using the DNABOX suite (http://www.dnabox.cn/) of sequence analysis programs, including Bowtie, TopHat, and Cufflinks. Using TopHat, the resulting FASTQ files were aligned to the NCBI reference mouse genome (Mus_musculus.GRCm38.75) to identify known transcripts. Mapped reads were processed using the Cufflinks algorithm to calculate fragments per kilobase of exon per million mapped reads (FPKM), which accurately reflects the RNA transcript number normalized for RNA length and total number of mapped reads. DEGs with a P-value < 0.05 were identified and subjected to GO and KEGG analysis using the online tool DAVID (https://david.ncifcrf.gov/).
He Staining
The duodenum, jejunum, ileum, colon, and rectum samples were immediately removed, immersed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 24 hours, dehydrated with dH2O for 30 minutes, rinsed in 75% ethanol for 1 hour, 95% ethanol for 1 hour twice, 100% ethanol for 1 hour twice, and 100% xylene for 20 minutes twice, and then immersed in paraffin at 58–60 °C for 3 hours. After the tissue was paraffin-embedded, 4-µm coronal serial sections were cut using a Microm HM-340E microtome (Microm, Walldorf, Germany). The sections were subjected to HE staining and mounted with neutral balsam. Subsequently, they were examined by microscopy to observe the changes in ulcer size and infiltration of inflammatory cells. Injuries to colon tissue were scored as previously described.
Immunohistochemical (ihc) Analysis
The specimens were stained with the EnVision™ two-step strategy and high-temperature antigen retrieval (pressure cooker, Supor Co, China). In brief, 4-µm-thick paraffin-embedded sections were deparaffinized twice (10 minutes each in 100% xylene) and then hydrated with 100% ethanol for 5 minutes twice, with 95% ethanol for 3 minutes, and with 80% ethanol for 5 minutes. After two 5-minute soakings in distilled water, the slides were put into the pressure cooker filled with 1000 ml of boiling sodium citrate buffer (pH 9.0) and heated under pressure. After steaming, the pressure cooker was removed from the heat source and cooled down to room temperature with tap water. The slides were then rinsed twice for 3 minutes with PBS. The slides were incubated with 3% hydrogen peroxide for 10 minutes and rinsed twice in PBS for 3 minutes. Primary antibody (anti-CD3, 1:100, clone SP7, ab16669, Abcam; anti-VCAM1, 1:200, clone EPR5047, ab134047, Abcam; anti-PECAM1, 1:1000, clone EPR17259, ab182981, Abcam; anti-phospho-p38 MAPK (Thr180 and Tyr182), 1:400, clones D3F9 and 4511, Cell Signaling Technology) were applied for 60 minutes in a moist chamber at 37 °C. After rinsing twice for 3 minutes with PBS, the slides were incubated with HRP polymer for 30 minutes at 37 °C. After adding diaminobenzidine (DAB) chromagen, the slides were observed and examined for color change under a light microscope. This was followed by counterstaining with hematoxylin for 1 minute and rinsing with tap water for 1 minute. Two slides were treated with PBS instead of primary antibody and served as the negative control. Sections were observed using the double-blind method by a pathological physician. In each section, cells were selected in five randomly selected fields to calculate the percentage of positive cells.
Statistical analysis
All values are presented as mean ± SEM. Statistical analyses were performed in SPSS v18.0 (SPSS Inc., Chicago, IL, United States). Data on SCFA and total carbohydrate concentrations were analyzed using two-way analysis of variance (ANOVA) and the Tukey multiple comparison tests with the Data Processing System (DPS) v16.5 [59]. Metabolites with variable importance in the projection (VIP) values > 1.0 were considered changed. Metabolomics were analyzed with a Student’s t-test.