Chitooligosaccharides (COS) have garnered great attention in the field of human healthcare. The prebiotic activities and antiglycation of COS were investigated using a combination of in vitro and in vivo studies. COS supplementation dramatically increased the levels of acetic acid, while reducing the concentrations of propionic and butyric acids.
Carbohydrate Polymers 242 (2020) 116413 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Effect of chitooligosaccharides on human gut microbiota and antiglycation a b, c a c a T b Wei Liu , Xiaoqiong Li *, Zhonglin Zhao , Xionge Pi , Yanyu Meng , Dibo Fei , Daqun Liu , Xin Wanga a State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR China b Institute of Food Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR China c College of Sciences, Henan Agricultural University, Zhengzhou 450002, PR China A R T I C LE I N FO A B S T R A C T Keywords: AGEs Antiglycation Chitooligosaccharides Gut microbiota Prebiotics SCFA Chitooligosaccharides (COS) have garnered great attention in the field of human healthcare The prebiotic activities and antiglycation of COS were investigated using a combination of in vitro and in vivo studies COS supplementation dramatically increased the levels of acetic acid, while reducing the concentrations of propionic and butyric acids It also decreased the total bacterial population; however, it did not affect diversity and richness of the gut microbiota In addition, COS modulated the gut microbiota composition by increasing Bacteroidetes, decreasing Proteobacteria and Actinobacteria, and lowering the Firmicutes/Bacteroidetes ratio COS promoted the generation of beneficial Bacteroides and Faecalibacterium genera, while suppressing the pathogenic Klebsiella genus The antiglycation activity of COS and acetic acid was dose-dependent Furthermore, COS prevented the decrease of serum Nε-(carboxymethyl) lysine (CML) level caused by CML ingestion in a mouse model of diet-induced obesity To improve host health, COS could be potential prebiotics in food products Introduction Prebiotics are substrates that nourish beneficial bacteria in host microorganisms (Gibson et al., 2017) Chitooligosaccharides (COS), the oligomers of β-(1-4)-linked D-glucosamine, are compounds prepared from chitosan, a N-deacetylated derivative of chitin (Thadathil & Velappan, 2014) COS are water-soluble compounds characterized by polymerization degrees (DP) less than 20 and an average molecular weight (Mw) below 3.9 kDa (Liaqat & Eltem, 2018) Their biological activity, including the antimicrobial, anti-tumor, antioxidant, anti-inflammatory, immunoregulatory, anti-obesity, anti-diabetics, anti-Alzheimer's disease, and anti-hypertension functions (Liaqat & Eltem, 2018; Muanprasat & Chatsudthipong, 2017; Vinsova & Vavrikova, 2011) Due to the properties of COS, they have been regarded as new potential prebiotics, and applied in various industries such as food, agriculture and medicine (Lee, Park, Jung, & Shin, 2002; Liaqat & Eltem, 2018) The biological functions of COS may be affected by interactions with gut microbiota that are considered as an extra organ influencing host health However, the results from the studies available in the literature regarding the relationship between COS and gut microbiota are not consistent In mice, one in vivo study demonstrates that COS (DP = 2-6, Mw < kDa, 200 mg kg-1 d-1) treatment promotes the population of Bacteroidetes, but inhibits the Proteobacteria phylum At the genus level, COS treatment reduces the population of probiotic Lactobacillus, Bifidobacterium, and harmful Desulfovibrio bacteria, while increasing the abundance of Akkermansia (Zhang, Jiao, Wang, & Du, 2018) Meanwhile, in vitro fermentation assessments conducted in the same study show that COS decreases the number of Escherichia/Shigella pathogens (Zhang et al., 2018) In the diabetic db/db mice model, COS of the same Mw relieve the gut dysbiosis by promoting Akkermansia and suppressing Helicobacter (Zheng et al., 2018) However, in pigs, dietary supplementation of COS (Mw =1.5 kDa) increases the number of bifidobacteria and lactobacilli, without affecting the Escherichia coli counts (Yang et al., 2012) In yet another study, COS (Mw =1 kDa) supplementation is shown to decrease the proportion of Escherichia coli in pig colonic content, while increasing short chain fatty acids (SCFA) concentrations and the number of beneficial bacterial species such as Bifidobacterium spp., Faecalibacterium prausnitzii, Lactobacillus spp., Prevotella, Fusobacterium prausnitzii, and Roseburia, and SCFA Abbreviations: AGEs, advanced glycation end products; CML, Nε-(carboxymethyl) lysine; COS, Chitooligosaccharides; DIO, diet-induced obesity; GC, gas chromatography; HF, high fat; LDA, Linear discriminant analysis; OTU, operational taxonomic unit; PCoA, principal-coordinate analysis; QIIME, quantitative insights into microbial ecology; q-PCR, quantitative real-time PCR; RDA, Redundancy analysis; SCFAs, short chain fatty acids ⁎ Corresponding author at: No 198 Shiqiao Road, Hangzhou 310021, PR China E-mail address: 0707lianlan@gmail.com (X Li) https://doi.org/10.1016/j.carbpol.2020.116413 Received November 2019; Received in revised form 30 April 2020; Accepted 30 April 2020 Available online 11 May 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 242 (2020) 116413 W Liu, et al had not received any medications, including antibiotics, for at least three months prior to sample collection The written informed consent obtained from each volunteer was approved by the Ethics Committee of the Zhejiang Academy of Agricultural Sciences The collected fresh fecal samples were kept in an anaerobic jar and processed within h concentrations (Kong, Zhou, Lian, Liu, & Tan, 2014) To the best of our knowledge, only limited reports have investigated the role of COS on human gut microbiota The results of these studies are also inconsistent, due to the diversity of COS sources, Mw, and enterotype backgrounds, as well as to the dissimilar experimental settings Vernazza, Gibson, and Rastall (2005) show that COS (Mw < 5000 Da) fermentation stimulates the growth of bacteroides, without affecting bifidobacterial It also increases the concentration of butyric acid More recently, Mateos-Aparicio, Mengíbar, and Heras (2016) reported that the biofunctionality of COS compounds is closely related to their main physico-chemical characteristics (Mw and acetylation degree) COS compounds (Mw = kDa, 1% w/v) with many acetylated residues increase Lactobacillus/Enterococcus population and the production of SCFAs (mainly acetic acid), while significantly deacetylated COS compounds (Mw = kDa) might decrease some human microbiota populations This suggests that the COS compounds corresponding to acetylated chitosans are not potential prebiotics, while those produced from deacetylated chitosans could induce a colonic microbiota imbalance Despite the importance of COS, the molecular mechanism of their biological activity remains unclear Pathogenic pathways involved in the development of obesity, diabetes, chronic inflammation, Alzheimer's disease, and cancer are promoted by the advanced glycation end products (AGEs) generated via non-enzymatic reactions of reducing sugars and amino groups (Xue et al., 2014) Therefore, AGEs inhibition constitutes a potential therapeutic approach for the prevention of obesity and other chronic diseases Although the antiglycation activity of certain polysaccharides and their degradation products has been reported (Zhu et al., 2019), the efficiency of COS compounds in preventing obesity and age-related dysfunctions via the antiglycation mechanism has not yet been investigated Such investigation is essential, particularly considering that different COS compounds are expected to show varying antiglycation activity, due to differences in structural characteristics, such as Mw and degree of esterification To confirm the prebiotic potency of COS in humans and to clarify the mechanism of the compounds’ beneficial activity, we investigated the effect of COS on human gut microbiota by conducting16S rRNA gene high-throughput sequencing we also used a BSA/glucose system and a mouse model of high-fat (HF) diet-induced obesity (DIO) to evaluate the antiglycation effect of COS on AGEs formation and accumulation 2.3 In vitro batch culture fermentation The batch cultures were fermented in 10 mL vials containing mL of basal medium VI, as per the method described previously (Wu et al., 2017) To evaluate the effect of COS compounds on human feces, a filter-sterilized (0.2-μm PTFE membrane) stock COS solution (100 mg mL-1) was added to the fecal samples at a final concentration of 30 mg mL-1 prior to fermentation A control medium containing no COS was also prepared The medium was autoclaved at 115 °C for 15 min, and the initial pH was adjusted to 6.5 To prepare the inoculum, fresh fecal samples (0.8 g) were suspended in 8.0 mL of 0.1 M anaerobic phosphate-buffered saline (pH = 7.0) using an automatic fecal homogenizer (Halo Biotechnology Co LTD., Jiangsu, China) to make 10% (w/v) slurries Batch fermentation was performed by inoculating 1% of the fecal slurry into each vial at 37 °C for 24 h Aliquots (1 mL) of the culture broth were taken from the vials at 4, 8, 12, and 24 h for further analysis The cultures were centrifuged, and the precipitates were collected and stored at -20 °C before use 2.4 Fecal SCFAs quantification The concentrations of SCFAs, such as acetic, propionic and butyric acids in the culture filtrates were measured on a gas chromatograph (GC, Shi-madzu, GC-2010 Plus, Japan) equipped with a DB-FFAP column (Agilent Technologies, USA) and an H2 flame ionization detector Trans-2-butenoic acid was used as an internal standard (Bai et al., 2017) 2.5 Fecal DNA extraction and quantitative real-time PCR Microbial genomic DNA was extracted from the culture broth at 24 h, using a QIAamp DNA Stool Mini Kit, according to the manufacturer's instructions (Qiagen, Germany) The concentration of extracted DNA, stored at −80 °C, was determined using a NanoDrop 2000 UV spectrophotometer (Thermo Scientific, Wilmington, USA), and the integrity and size of this DNA were confirmed by agar gel electrophoresis (1.0%) Quantitative real-time PCR (qPCR) assessments were used to quantify the copy numbers of the bacterial 16 s rRNA gene, using an ABI PRISM 7500 Real-Time PCR Detection System (Applied Biosystems) with the 341 F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’) primer pair DNA standards of bacteria were prepared by serial dilutions of the pGEM-T Easy Vector (Promega) containing the 16S rRNA gene of Escherichia coli The PCR reaction and amplification were performed according to the method described in a previous study (Yin et al., 2013) Materials and methods 2.1 Chitooligosaccharides COS with deacetylation degrees greater than 95% and average Mw below kDa were kindly provided by Dalian Glycobio Co., Ltd (Dailian, China) The monosacchaaride composition in COS was confirmed by pre-column (1-phenyl-3-methyl-5-pyrazolone) PMP derivatization high-performance liquid chromatograph (HPLC) method, using an Agilent 1260 HPLC system (Waldbronn, Germany) with a Thermo-C18 column (4.6 mm × 250 mm, 5μm) The DP of COS was determined by Acchrom S6000 HPLC system (Acchrom, China) equipment with an Acchrom XAmide column (4.6 mm × 250 mm × μm) The relative abundance of COS oligomers was calculated from their peak area of each oligosaccharide component using COS oligomer standards as external standard (Qingdao Marine Biomedical Research Institute Inc., Testing Center) 2.6 16S rRNA gene pyrosequencing and bioinformatic analysis The V3–V4 region of the bacterial 16S rRNA gene was amplified using the primers 338 F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGG TWTCTAAT-3’) The amplicons were extracted and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified with a QuantiFluor™ST (Promega, USA) Next generation sequencing (2 × 300 paired-end) was performed on an Illumina MiSeq platform, according to the standard protocols of Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China) The raw sequence data were deposited in the NCBI Sequence Read Archive (SRA) database under accession number SRR8361789 After sequencing, raw fastq files were demultiplexed and quality-filtered by QIIME software package (version 1.17) Operational 2.2 Collection of fecal samples from human volunteers A total of eleven healthy human volunteers living in Hangzhou, China and aged between 34 and 60 years participated in the study All donors of fecal samples were in good health and physical condition, followed a normal Chinese diet, presented no digestive diseases, and Carbohydrate Polymers 242 (2020) 116413 W Liu, et al taxonomic units (OTUs) were clustered at 97% similarity using UPARSE (version 7.1 http://drive5.com/uparse/), and chimeric sequences were identified and removed by UCHIME Taxonomic annotation of OTUs was performed with RDP Classifier against the SILVA database v 128, with a confidence threshold of 0.7 Rare OTUs (< 0.001%) were removed in order to reduce sampling heterogeneity for further alpha and beta diversity calculations Alpha (observed OTUs (sobs), Chao, Shannon and Simpson) and beta diversity (weighted and unweighted UniFrac-based principal coordinate (PCoA) analyses were performed using the R software and vegan package (version 3.3.1) The linear discriminant analysis (LDA) effect size (LEfSe) method was used to identify the effect of each differentially abundant taxon and distinguish the one with the greatest biological activity among the two groups (Segata et al., 2011) Additionally, redundancy analyses (RDA) and Spearman correlations were used to associate abundant differential taxa with SCFAs A Wilcoxon rank-sum test and Welch’s t-test were used to compare the data, and the significance value was set to 0.05 according to the Km factor ratio of and 37 for mice (20 g) and humans (60 kg), respectively (Reagan‐Shaw, Nihal, & Ahmad, 2008) Body weight and food intake were monitored every week After weeks treatment, blood was collected from the tail vein and centrifuged at 2000 g for 15 Serum was then separated and stored at −20 °C All animal study protocols were approved by Zhejiang Academy of Agricultural Sciences (approval number: ZAAS2020041) 2.7 Antiglycation activity assay Except for the bioinformatic information, all data recorded in this study were analyzed using the SPSS 12.0 software (IBM Corp., Armonk, N.Y., USA) The data obtained for CTR and COS were compared using the Student’s t-test (qualitative data, equal variance), or Welch’s t-test (qualitative data, unknown variance) The differences between fluorescence intensity, AGEs inhibition and CML concentration were assessed by using an analysis of variance (ANOVA) with a (post hoc) Turkey test Differences with p values less than 0.05 were considered statistically significant 2.8.1 CML quantification CML is of used as a marker of AGEs formulation Serum CML content was measured with mouse CML ELISA kit (Andy gene Co., Ltd, Shanghai, China) following manufacturer’s instructions The serum was diluted with kit-provided diluent to fall within the measurable concentration range of the kit, and measured in duplicate The CML ELISA kit is a colorimetric immunoassay comparing samples to a standard curve 2.9 Statistical analysis The effect of COS and acetic acid in inhibiting the formation of AGEs was evaluated using the BSA/glucose system, based on a previously reported method (Meng, Xiao, & Zhang, 2019), with some modifications Briefly, 10 mL of 20 mg mL-1 BSA, mL of 0.5 M glucose, 0.02% sodium azide, and 0.2 M phosphate buffer (pH = 7.4) were mixed and reacted with mL samples of COS compounds (concentrations of 2, 4, and mg mL-1) or acetic acid (8.3, 25, and 75 mM) dissolved in phosphate buffer (0.2 M, pH = 7.4) A Mixture containing aminoguanidine (AG, mg mL-1) instead of COS was used as the positive control, whereas the negative control contained neither AG nor COS Samples containing only COS (concentrations of 2, 4, and mg mL-1) were also run, so as to measure any fluorescence emissions caused by endogenous substances in these samples All mixtures were incubated in the dark at 37 °C for weeks Subsequently, 0.5 mL of the glycated solution were diluted with PBS (0.2 M, pH = 7.4) to a final volume of 10 mL The AGEs content in the diluted solutions was determined using fluorospectrophotometry, at excitation and emission wavelengths of 370 and 440 nm, respectively (SpectraMax® M5, Molecular Devices, Sunnyvale, United States) The percentage of antiglycation activity was calculated according to the following equation: % antiglycation = [(ANC−Asample)/ANC] × 100, where ANC and Asample represent the absorbance values of the negative control and COS, acetic acid or AG groups, respectively Results 3.1 Structure characterization of COS The analysis of monosaccharide composition showed that the content of glucosamine in COS was 100%, and glucosamine was the only monosaccharide presented in COS (Fig S1) The COS were found to be composed of 2–8 DP oligomers (Mw ≈ 856 Da), with 33.6% disaccharide, 16.9% trisaccharide, 15.8% tetrasaccharide, 12.4% pentasaccharide, 8.3% hexasaccharide, 7.1% heptasaccharide, and 5.9% octasaccharide, respectively (Fig 1) 3.2 Effect of COS on SCFAs production SCFAs were produced during the fermentation of COS in human fecal samples (Fig 2) Noticeably, the concentration of acetic acid was significantly higher in the COS group than that in the CTR group during the fermentation However, the amounts of propionic and butyric acid produced in the CTR group were much greater than those observed for the COS group 2.8 Animal experiments DIO and control C57BL/6 J mice (male, 18-week-old, DIO: 38.4 ± 4.1 g, control: 27.0 ± 1.8 g) were purchased from GemPharmatech Co., Ltd (Jiangsu, China) To generate DIO models, HF diet (60% kcal/fat, D12492, Research Diets) were introduced at 6week-old and fed for 12 consecutive weeks before purchase All mice were housed in an air-conditioned room at 20 − 22 °C with alternating 12 h cycles of light and dark, and with free access to pellet food and water After 10 days of acclimatization, mice in NC group (n = 6) were continued to feed a normal diet (10% kcal/fat, MD12031), and orally gavaged with 300 μL sterile physiological saline (SPS) The rest of DIO mice were continued to feed a HF diet, and randomly assigned to groups (n = 7): 1) HC, mice orally gavaged with equivalent volume of SPS; 2) COS, mice orally gavaged with 500 mg kg-1 body weight COS dissolved in SPS; 3) CML, mice orally gavaged with 10 mg kg-1 body weight Nε-carboxymethyllysine (CML) (≥97%, Perfemiker Co., Ltd Shanghai, China) dissolved in SPS; and 4) C + M, mice orally gavaged with equivalent concentration of COS and CML dissolved in SPS The dose of 500 mg/kg/day COS and 10 mg/kg/day CML was equivalent to consumption of 2.4 g COS and 48.8 mg CML/day by a 60 kg human 3.3 Effect of COS on the microbial abundances The total copy numbers of the bacterial 16S rRNA gene in fecal samples were estimated by quantitative real-time PCR (qPCR) As shown in Fig 3, after 24 h of incubation, the copy numbers of the gene significantly decreased in the COS group compared to the CTR group (8.01 vs 6.92 Log10 copies mL-1, p < 0.001) Such an inhibitory effect of COS on fecal bacteria was expected 3.4 Effect of COS on the composition of the bacterial community A total of 1,124,106 high-quality sequences with a minimum of 30,602 sequences per sample (mean = 52,096; read length = 265 to 509) were obtained Based on the 97% sequence similarity criterion, the sequences were assigned to 339 OTUs, representing 11 phyla and 185 genera After removing the rare OTUs (< 0.001% of total sequences), Carbohydrate Polymers 242 (2020) 116413 W Liu, et al Fig Relative contents of COS mixture by peak area ratio method using HPLC analysis COS with the polymerization degree 2-8, their weight percentages were 3.12%, 11.30%, 19.82%, 17.90%, 34.35%, 3.06%, and 10.45%, respectively 269 and 266 OTUs were retained in the CTR and COS groups, respectively Veen analysis shows that 220 out of 315 OUTs (∼70%) are shared by both groups, compared to 49 and 46 unique OTUs in the CTR and COS groups, respectively (Fig S3) Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria were the prominent phyla identified in the tested samples (Fig 4A) After 24 h of fermentation in the presence of COS, phylum Bacteroidetes remarkably increased in abundance from 15.5 to 53.0% (p < 0.001), while the abundances of phyla Proteobacteria and Actinobacteria significantly decreased from 41.2% to 16.8% and from 2.0% to 0.7%, respectively (p < 0.05) (Fig 4B) Furthermore, COS treatment reduced the ratio of Firmicutes to Bacteroidetes (F/B) from 2.27 in the CTR group to 0.56 in the COS group (Fig S4) The bacterial genera detected at ≥ 1% average relative abundance were showed in Fig 4C Among these genera, Bacteroides, Faecalibacterium, and Alistipes were found to be more abundant in the COS group (41.4%, 2.8%, and 1.1%, respectively) than in the CTR group (12.8%, 0.2%, and 0.1%, respectively, p < 0.05) The abundance of Klebsiella, on the other hand, was reduced from 5.6% to 0.7% upon COS treatment (p < 0.05) Interestingly, COS increased the abundance of Prevotella from 0.1% to 7.1%, while decreasing the amount of Escherichia-Shigella from 28.7% to 13.1% after COS treatment; however, the variations showed no significant effect (Fig 4D) Fig Effect of COS on the population of the bacterial community The abundance of bacteria was estimated by quantitative PCR, based on the copy numbers of the bacterial 16S rRNA gene in the fermentation culture at 24 h Data are presented as mean ± SD (n = 11, ***p < 0.001) Fig Acetic (A), propionic (B), and butyric acids (C) concentration produced during in vitro fecal fermentation Data are represented as the means ± SEM (n = at 4, and 12 h, n = 11 at 24 h) Values with significant correlations at the same fermentation time are marked by *p < 0.05; **p < 0.01; ***p < 0.001 Carbohydrate Polymers 242 (2020) 116413 W Liu, et al Fig Effect of COS on microbial composition, alpha, and beta-diversity Relative abundance of bacterial phyla (A) and genera (C) Lanes CTR1–CTR11 and COS1–COS11 correspond to the samples in the CTR and COS groups, respectively Heat maps of the mean relative abundances of the prominent phyla (B) and genera (D) Wilcoxon rank-sum test was used to compare bacterial abundances at phylum and genus levels between the CTR and COS groups Significant differences are marked by *p < 0.05; **p < 0.01; ***p < 0.001 The cladogram of Linear discriminant analysis (LDA) effect size (LEfSe) analysis of microbial abundance from phylum to genus level (E) LDA score assessments of the size of differentiation between the CTR and COS groups, with a score threshold of 4.0 (F) Bacterial richness (observed OTUs (Sobs) and Chao index) and diversity comparison (Shannon and Simpson index) between the two groups (G) Principal-coordinate analysis (PCoA) based on weighted UniFrac distances (H) and unweighted UniFrac distances (I) of samples from CTR and COS Carbohydrate Polymers 242 (2020) 116413 W Liu, et al 3.7 The antiglycation activity of COS and acetic acid in vitro The linear discriminant analysis (LDA) effect size (LEfSe) method was used to identify the classified bacterial taxa with significant abundance differences between the CTR and COS groups As shown in Fig 4E, 23 bacterial clades present statistically significant differences with an LDA score of 4.0 (Fig 4F) In accordance with Wilcoxon ranksum test, COS stimulated the growth of Bacteroides and Faecalibacterium, while suppressing Klebsiella The antiglycation activity was conducted in vitro for COS and acetic acid As shown in Fig 6A, COS exhibited strong autofluorescence, with intensities increasing as a function of COS concentration When added to the incubation system, COS inhibited the formation of AGEs, with greater inhibition observed at higher concentrations (Fig 6B) The solution with the highest COS concentration (8 mg mL-1) showed the strongest suppression of AGEs formation (85.57% ± 15.19%) during the 14 days of incubation, followed by the solutions with intermediate (4 mg mL-1, 51.22% ± 14.87%) and low (2 mg mL-1, 20.10% ± 8.02%) COS concentrations The AG solution (4 mg mL-1) yielded the least AGEs suppression (13.48% ± 9.52%, p < 0.05), and the inhibition percentages observed for this solution are similar to those recorded for mg mL-1 COS For acetic acid, the highest dose (75 mM) had the lowest fluorescence intensity and greatest AGEs inhibition rate of 79.06% at day (Fig 6C&D) However, lower doses (25 and 8.3 mM) of acetic acid did not show antiglycation activity 3.5 Effect of COS on alpha and beta bacterial communities Before calculating the alpha diversity indices, the samples were rarefied to 28,204 sequences in order to account for the unequal numbers of sequences between the groups The results presented in Fig 4G showed that the CTR and COS groups exhibited relatively similar community diversity (Shannon and Simpson) and richness indices (sobs and chao), however the richness values of the COS group were constantly higher than those of the CTR group Collectively, the data indicate that COS did not affect bacterial alpha diversity As for beta diversity, weighted UniFrac-based principal-coordinate analysis (PCoA) demonstrated a distinct clustering of bacterial composition in the two groups (with the expectation of COS1) (Fig 4H) Contrarily, no clear visual separation between the COS and CTR groups could be observed in the plot of unweighted UniFrac distances (Fig 4I) However, the ANOSIM of both, weighted and unweighted UniFrac, showed that the treatments are significantly different (p = 0.001) These results indicate that COS treatment markedly affected the beta diversity of the bacterial community 3.8 Effect of COS on serum CML concentration Comparison of serum CML levels in different groups was conducted to investigate the effects of COS on AGEs accumulation (Fig 7) After administration of free CML for consecutive weeks, serum CML level in CML mice was lower than that in NC group (27.5 vs 20.5 μg/L, p = 0.0312) Two mice in CML group died during the experiment, and the rest of CML mice showed reduced weight gain (Fig S6) However, due to the short-term intake of COS and CML, no differences were observed in the comparison between other groups 3.6 The relationship between bacterial community and SCFAs profiles Discussion The correlation between the prominent gut microbes and SCFAs was detected using redundancy (RDA) and Spearman’s correlation analyses The results presented in Fig indicate that, as expected, enriched Bacteroidetes in the COS group are positively correlated with the concentration of acetic acid (Fig 5A) This relationship was confirmed by the statistically significant positive correlation between the Bacteroides and acetic acid (rs = 0.525, p < 0.05) (Fig 5B) Faecalibacterium (rs = 0.601, p < 0.01) and Blautia (rs = 0.442, p < 0.05) of the Firmicutes also showed positive correlations with acetic acid Moreover, the Fusobacterium and Klebsiella genera enriched in the CTR group are positively related to propionic and butyrate acids, respectively COS have garnered a lot of attention in the field of human healthcare, particularly for the treatment and prevention of obesity and diabetes However, the detailed mechanisms of the anti-obesity and antidiabetics activities of COS remain unclear (Karadeniz & Kim, 2014) In vitro gut fermentation remains an irreplaceable tool for screening as well as studying the mechanisms of action of prebiotics Being host-free, it makes an ideal system in which to study microbial perturbations resulting from prebiotic interventions, as microbial changes can be measured without host interference (Elzinga, van der Oost, de Vos, & Smidt, 2019; Payne, Zihler, Chassard, & Lacroix, 2012) In this study, Fig The correlation between microbial structure and SCFA indices Redundancy analysis (RDA) of the prominent phyla responding to SCFA (A); A heatmap of Spearman’s correlation between the prominent genera and SCFA (B) The intensity of the colors represents the degree of association (red, positive correlation; blue, negative correlation) Significant correlations are marked by *p < 0.05; **p < 0.01; ***p < 0.001 Carbohydrate Polymers 242 (2020) 116413 W Liu, et al Fig Inhibition of advanced glycation end products (AGEs) formation by chitooligosaccharides (COS) and acetic acid Fluorescence intensity of total fluorescent AGEs (A) and (C); The antiglycation activities of Aminoguanidine (AG) and COS (B) and acetic acid (D) Fluorescence intensity was determined at 370 (excitation) and 440 nm (emission) The lowercase letters over each bar for comparison between treatment and fraction for a given incubation time Bars with different letters differ significantly (p < 0.05) (Koliada et al., 2017; Ley, Turnbaugh, Klein, & Gordon, 2006), it is expected that COS supplementation may prevent obesity by reducing the F/B ratio and reshaping the structure of gut microbiota Coincidentally, COS treatment relieves gut dysbiosis in diabetic mice by suppressing Firmicutes and Helicobacter, while promoting Bacteroidetes and Akkermansia (Zheng et al., 2018) At the genus level, COS supplementation increased Bacteroides, Faecalibacterium, Alistipes, and Prevotella The phylum Bacteroidetes possess a broad saccharolytic potential (Martens, Koropatkin, Smith, & Gordon, 2009), and are represented mainly by the genera Bacteroides and Prevotella genera in the human gut These genera are normally symbiotic and saccharolytic, and they produce acetate, propionate, and succinate as the major metabolic end products (Downes, Sutcliffe, Booth, & Wade, 2007; Robert, Chassard, Lawson, & Bernalier-Donadille, 2007) The increase in the abundance of Bacteroides and Prevotella is probably due to the effect of COS in facilitating their proliferation Faecalibacterium prausnitzii, a major representative of the Faecalibacterium genus in the Firmicutes phylum, represents more than 5% of the total bacterial population in healthy human gut microbiota (Miquel et al., 2013) It produces butyrate and other SCFAs in the colonic epithelium, possesses anti-inflammatory properties, and protects against colorectal cancer and inflammatory bowel diseases (Lopez-Siles, Duncan, Garcia-Gil, & Martinez-Medina, 2017) Chinese subjects diagnosed with type diabetes (Zhang et al., 2013) or other gut diseases, exhibit depleted levels of F prausnitzii Therefore, this bacterium is considered as a new generation of probiotic (Martín et al., 2017) Moreover, the interesting phenomenon observed from our study that an increase in the abundance of Faecalibacterium was accompanied with Bacteroides enrichment, probably due to the cross-feeding interactions between the two members Bacteroides produces acetate, whereas Faecalibacterium consumes acetate to produce butyrate; thus, the two genera are metabolically complementary (Wrzosek et al., 2013) The effect of COS consumption in increasing the population of F prausnitzii has also been observed in a pig model (Kong et al., 2014) Based on these results, it could be postulated that COS are potential prebiotics SCFAs, the most abundant products of the bacterial fermentation of undigested dietary fibers, mainly consist of acetate, propionate, and butyrate (typically occurring in the ratio of 3:1:1) These compounds drive the crosstalk between the host and gut microbiota, and the Fig CML concentration in serum (μg/L) in mice in NC, HC, COS, CML and C + M groups Data are presented as mean ± SD (n = 5-7, *p < 0.05) the impact of COS on human gut microbial ecology and metabolic endproducts was investigated using the in vitro batch fermentation model The antimicrobial activities of COS against various microorganisms are well known (Kittur, Vishu Kumar, Varadaraj, & Tharanathan, 2005; Liaqat & Eltem, 2018) In our study, COS treatment not only reduced the whole bacterial flora population, but also reduced abundances of Proteobacteria and Klebsiella, as well as to a relatively decreased Escherichia-Shigella proportion Similar results are reported by Zhang et al (2018), from which the inhibitory effect of bacterial populations could be enhanced through increasing concentrations of COS (0.1-3 g L-1), but declined with extended treatment time Klebsiella (Paczosa & Mecsas, 2016) and Escherichia-Shigella (Li et al., 2006), key pathogens of the Proteobacteria phylum, are major contributors to infections worldwide The antibacterial activity of COS against E coli and K pneumoniae is confirmed in yet another study (Fernandes et al., 2010) Although COS significantly inhibited the growth of the bacterial community as a whole, it did not affect the alpha diversity Beta diversity analysis, on the other hand, showed a relatively strong influence of COS treatment on gut microbiota composition The concurrent increase in Bacteroidetes and decrease in Proteobacteria were in line with previous findings concerning the administration of g L-1 COS in mice fecal fermentation (Zhang et al., 2018) Knowing that obese individuals and mice exhibit increased Firmicutes/Bacteroidetes (F/B) ratios Carbohydrate Polymers 242 (2020) 116413 W Liu, et al a mouse model of DIO CML, commonly known as a biomarker of oxidative stress, is a major antigenic AGE structure Accumulation of CML in adipose tissue of obese subjects is able to activate inflammatory signaling pathways contributing to obesity-related insulin resistance, and meanwhile lends to a decreased circulating CML blood levels (Gaens et al., 2015) In line with this finding, we observed a lower circulating CML serum levels in DIO mice after a short-term consumption of free CML, which indicate that CML ingestion promotes AGEs accumulation in adipose tissue Nevertheless, no significant change in serum CML level was found when ingestion of COS and CML simultaneously Therefore, COS may prevent the accumulation of AGEs in adipose tissue to a certain extent However, no statistical decrease of CML in blood is found in rat fed with long-term CML-rich diets (Li et al., 2015) Whether blood CML may serve as an inversely correlated marker of CML accumulation in adipose tissue need to be further verified mechanism by which gut microbiota affects the host’s physiological and pathological processes is partly mediated by SCFAs (Koh, De Vadder, Kovatcheva-Datchary, & Bäckhed, 2016; Rooks & Garrett, 2016) According to Mateos-Aparicio et al (2016), low Mw COS compounds promote the production of total SCFAs and increase the concentration of acetate In vitro fermentation assessments show that the continuous accumulation of acetate and butyrate in samples of mice feces with COS is significantly greater than that in the control sample (-Zhang et al., 2018) Consistently, our results indicate that acetate was the main organic acid product of fermentation, and that its concentration significantly increased when the fecal samples were supplemented with COS Contrarily, COS appreciably inhibited the production of propionate and butyrate Acetate is a co-substrate used by cross-feeding species to produce butyrate It exhibits anti-inflammatory effects, and contributes to the biosynthesis of cholesterol and fatty acids (Rivière, Selak, Lantin, Leroy, & De Vuyst, 2016) Acetate is mainly consumed by two butyrate-producing bacteria, namely, Faecalibacterium prausnitzii and Roseburia intestinalis/Eubacterium rectale (Duncan et al., 2004) Knowing that Faecalibacterium was enriched upon COS treatment, we assume that the effect of high COS concentration in reducing the amounts of propionate and butyrate is transient, and probably due to an initial decline in the absolute number of butyrate-producing bacteria However, the gut microbiota is resilient and will likely recover with extended incubation time (Zhang et al., 2018) The onset of obesity and age-related disorders, such as cancer and Alzheimer’s disease, is associated with the accumulation of AGEs (Yamagishi, Nakamura, Suematsu, Kaseda, & Matsui, 2015) It remains unclear whether COS protect against these diseases through antiglycation mechanism The analyses performed herein confirmed that highly deacetylated COS compounds exhibited a strong inhibitory effect on the formation of AGEs in the BSA/glucose system, which was consistent with previously reported results (Zhang, Yu, Zhang, Zhao, & Dong, 2014) Nevertheless, Wang et al (2018) show that, in a complex real food system, COS and lysine undergo non-enzymatic glycation reactions, resulting in formation of CML Whereas in the presence of transglutaminase, the formation of AGEs is inhibited by transglutaminase and COS-induced glycosylation Such inhibition may be attributed to a competing reaction wherein the carbonyl groups in the reducing sugar bind to the amino groups of COS, thereby limiting the combination of glucose and BSA Furthermore, our results indicate that the antiglycation activity of COS depends on the concentrations of these compounds This is consistent with the observations of Zhang et al (2014), who show that high antiglycation activity is correlated with high glucosamine acid content Therefore, the wide bioactivity range of COS can be partly explained by the strong antiglycation effect of COS So far, inconsistent findings have been reported regarding the impact of AGEs on the gut microbiota composition, which may be due to the different glycated substrates used (Snelson & Coughlan, 2019) Nevertheless, intestinal microflora and its metabolites (e.g SCFAs) may in turn have an impact on the formation of AGEs Acetic acid, which was dramatically boosted (75.71 mM at 24 h) by COS fermentation was evaluated for its impact on the AGEs formation Interestingly, we found that high-dose (75 mM), but not low-dose acetic acid significantly prevented AGEs formation Besides, the inhibitory effect of acetic acid on AGEs formation tended to wane with time It has been reported that new AGEs formation during cooking can be inhibited following exposure to acidic solutions of lemon juice or vinegar (acetic acid), due to a low or acidic pH arrests AGEs development (Uribarri et al., 2010) Similarly, an acidified environment in the gut may also limit new AGEs formation Qu et al (2017) show that dietary AGEs exhibit an adverse effect on gut microbiota by reducing their diversity and richness Both high-dose COS and acetate acid showed strong antiglycation activities Therefore, synergistic effect of them on antiglycation may benefit the gut microbial ecology as a whole and contribute to the prevention of obesity and age-related dysfunctions We further extended these in vitro observations to in vivo ones using Conclusions COS supplementation in a batch fermentation model did not significantly influence the richness and diversity of the bacterial community; however, it dramatically altered the structure and functions of microbiota Furthermore, COS treatment reduced the population of the bacterial community as a whole and increased the production of acetic acid It also enhanced the abundance of phylum Bacteroidetes and genus Bacteroides which were positively correlated with acetic acid Finally, COS compounds modulated the bacterial composition by increasing the abundance of beneficial genus Faecalibacterium and decreasing the levels of harmful genus Klebsiella COS and acetic acid inhibited AGEs formation in BSA/glucose systems via the antiglycation effect Furthermore, COS may prevent AGEs accumulation in adipose tissue caused by CML ingestion in a mouse model of DIO Based on these results, we supposed that the prebiotic and antiglycation properties of COS might have a synergistic effect on benefiting the gut microbial ecology and contribute to the prevention of obesity and agerelated dysfunctions Author contributions XQL, WL, and ZLZ conceived the study and designed the experiments WL, XEP, DQL, and WCW conducted the experiments XQL, WL, and XW analyzed the data and wrote the paper with input from all the other authors All authors read and approved the final version of this manuscript CRediT authorship contribution statement Wei Liu: Conceptualization, Methodology, Data curation, Writing original draft, Resources Xiaoqiong Li: Conceptualization, Writing review & editing, Visualization Zhonglin Zhao: Resources, Methodology Xionge Pi: Methodology, Resources Yanyu Meng: Methodology, Resources Dibo Fei: Methodology, Resources Daqun Liu: Methodology, Resources Xin Wang: Supervision, Funding acquisition Declaration of Competing Interest The authors declare that they have no conflicts of interest Acknowledgements This research was financially 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formation, and H2O2 -induced oxidative damage International Journal of Biological ... synergistic effect on benefiting the gut microbial ecology and contribute to the prevention of obesity and agerelated dysfunctions Author contributions XQL, WL, and ZLZ conceived the study and designed... of them on antiglycation may benefit the gut microbial ecology as a whole and contribute to the prevention of obesity and age-related dysfunctions We further extended these in vitro observations... the production of total SCFAs and increase the concentration of acetate In vitro fermentation assessments show that the continuous accumulation of acetate and butyrate in samples of mice feces