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Axe on Youtube 2. During in vitro fermentation of COS by mice gut microbiota, total bacterial population significantly decreased after 8-h COS treatment but was returned to the normal level after extended incubation. Consumption of COS and production of SCFAs suggested that COS were utilized by the microbe, although the consumption of chitosan pentasaccharides was obviously slower than others.

In vivo animal study indicated that COS reduced population of probiotic genera Lactobacillus , Bifidobacterium and harmful genus Desulfovibrio , and increased abundance of genus Akkermansia. Phylum Proteobacteria was significantly inhibited by COS both in the animal model and in vitro fermentation.

Our findings suggested that COS could reform the community structure of gut microbiota. The relationship among COS, gut microbiota and host health deserve further study. Chitin the second most abundant biopolymer in the world. Chitosan oligosaccharides COS , a mixture of oligomers of beta-1, 4-linked D-glucosamine, are prepared from chitosan, a N-deacetylated derivative is of chitin Thadathil and Velappan, The water-soluble and low toxicity of COS assists their versatile biological activities Muanprasat and Chatsudthipong, COS have been reported to possess various properties like anti-microbial Choi et al.

Therefore, COS have good applicable values in food Du et al. Gut microbiota can be considered as an extra organ with remarkable dynamics that influence the host gut health. Thousands of genes that encode carbohydrate-active enzymes CAZymes have been identified in the human gut microbiome El Kaoutari et al.

However, the human genome encodes, at most, only 17 enzymes for the digestion of food glycans, specifically starch, sucrose, and lactose and no polysaccharide lyases Cantarel et al. COS as potential non-digestible oligosaccharides for the host, could be metabolized by gut microbiota.

Dietary supplementation of COS has been found to improve gut barrier function, increase the population of Bifidobacterium spp.

and Lactobacillus spp. On the other hand, various studies reported that supplementation of COS significantly increased the abundance of Escherichia spp.

in rats Shang et al. Moreover, supplementation of COS and resistant starch mixtures alleviated metabolic disorders through synergistic actions, including positive manipulations on gut microbiota, lipid metabolism, and thickness of colonic mucosa layer in the rat Shang et al.

Potential functions of oligosaccharides on gut microbiota could be tested using in vitro batch fermentation models that could exclude interferences from the host Rastall, ; Payne et al. Limited number of studies had investigated the effect of COS on the gut microbiota in vitro and provided controversial conclusions.

Lee et al. in pure cultures. On the contrary, Vernazza et al. This inconsistency through different researches might be due to different sources of COS and experimental settings Lee et al.

Based on these studies, the relationship among COS, gut microbiota and host gut health had been preliminarily established. However, the mechanism and causality of this relationship was still poorly explored. As an important part of the mechanism, the direct impact of COS on the gut microbiota needed to be further studied.

To investigate the effect of COS on the gut microbiota, we have determined the structure of microbiota in mice feces incubated with COS in in vitro batch cultures and colon contents in mice feeding with COS.

To achieve this goal, COS with determined degree of polymerization DP and deacetylation DD was prepared. Batch cultures of mice feces were carried out with different concentrations of COS during in vitro anaerobic incubation.

Consumption of COS as well as production of short chain fatty acids SCFAs and biogas were determined. Furthermore, the influence of COS upon both fecal microbiota in vitro and gut microbiota in animal was assessed.

These studies are an important step toward a better understanding of the COS—microbe—host relationship. The content of COS was determined by UPLC-ESI-Q-TOF-MS Waters Corp.

COS with different DP from 2 to 6 were shown as COS-2, COS-3, COS-4, COS-5, and COS-6, respectively. Aqueous solutions 5 μL of COS 10 mg mL -1 were injected into HPLC for analysis. Mass spectrometry was applied for the qualitative analysis under negative ion modes.

The MS parameters were set up as follows: capillary voltage, 3 kV; cone voltage, 30 V; source temperature, °C; desolvation temperature, °C; gas flows of cone and desolvation, 50 and L h Mass spectrometry data were processed using Masslynx 4.

COS samples were also analyzed using Acchrom S HPLC system Acchrom, China equipment with an Acchrom XAmide column 4. The flow rate was 0. The relative abundance of COS oligomers was evaluated by determining the peak area of each oligosaccharide component using COS oligomer standards as external standard Dalian Glycobio Co.

Each sample was analyzed for three times. Fresh feces 0. Anaerobic incubations were prepared by adding 3 mL of seed liquid into 27 mL of fresh GMM in mL glass bottles. The stock solution of COS g L -1 was sterilized by filtration, then added into samples to final concentrations of 0.

Same volume of GMM was added to the control treatment. Bottles were sealed with butyl stoppers, then flushed with N 2 for 3 min and incubated at 37°C. After 72 h incubation, 3 mL cultured samples of the primary culture G1 were transferred into the 27 mL GMM to get the second subculture G2.

The 3rd subculture G3 was conducted after h incubation of the G2. All tests were carried out in triplicate for each treatment. Gaseous samples μL were collected from the headspace of the bottle and injected into GC with a pressure-lock precision analytical syringe Baton Rouge, LA, United States and concentrations of H 2 , CH 4 , and CO 2 were analyzed using a gas chromatograph GCA Agilent Technologies, United States , equipped with thermal conductivity detector TCD , flame ionization detector FID and Electrical Conductivity Detector ECD Zhang et al.

Fermentation samples were centrifuged at 14, × g for 10 min and the supernatants were filtered through 0. The sediments were stored at °C for DNA extraction.

SCFAs including formate, acetate, butyrate and L-lactate were analyzed using an Acchrom S high performance liquid chromatography HPLC system Acchrom, China equipment with a C18 column 4. Five microliter sample was injected into the HPLC system for each run.

The pH of mobile phase was adjusted to 2. Cultured samples were taken from incubations at indicated time points.

Microbial DNA was extracted following the protocol of FastDNA SPIN Kit with bead-beating using FastPrep MP Biomedicals, CA, United States. Standards with a fold dilution series from 8. The standard CD diet Aoke Xieli Co. After feeding for 5 months at 22°C with a 12—12 h dark—light cycle, all mice were euthanized and colon contents were sampled.

Samples were immediately frozen in liquid nitrogen and stored at °C until DNA extraction. The animal experiments were approved by the Animal Ethical Experimentation Committee of Chinese Academy of Sciences permission number: SYXK The V3—V4 region of bacterial 16S rRNA genes was subjected to PCR amplification.

Purified amplicons were sequenced on the Illumina MiSeq platform by Allwegene Technology Inc. Beijing, China. Raw sequences were analyzed by QIIME software package as described previously Caporaso et al.

Alpha and beta diversity were examined as well as redundancy analysis RDA and principal coordinate analysis PCoA.

Linear discriminant analysis effect size LEfSe was used to estimate taxonomic abundance and characterize differences between groups Segata et al. All 16S rRNA pyrosequencing datasets had been deposited in GenBank Sequence Read Archive SRA database Accession number: SRP and SRP All statistical analyses were carried out using GraphPad Prism version 6, GraphPad Software Inc.

Data were presented as means ± SD. Data of the H 2 and SCFAs concentrations and bacterial population abundance were subjected to the parametric ANOVA analysis, along with the Tukey—Kramer test, to determine the significant differences between the treatments.

LEfSe analysis of the treatment groups was performed on the basis of the results of the Kruskal—Wallis and Wilcoxon tests and the threshold on the logarithmic linear discriminant analysis LDA score was 2. In the first 33 h, 0. Then, the degradation was slow down with only 0. The change of COS was not recorded in samples treated with 0.

To determine the degradation of different COS components by mice fecal microbiota, changes in the relative abundance of each COS oligomer were characterized through fermentation. Contents of major components of the original COS sample were COS-2 DP2 3.

Interestingly, degradation of COS-5 was obviously slower than other components Figure 1E. FIGURE 1. Dynamics of COS polymers during COS treatment on mice feces. The concentration of COS-total A , and the level of COS-2 B , COS-3 C , COS-4 D , COS-5 E , and COS-6 F in mice feces treated with 0.

COS with different degree of polymerization DP from 2 to 6 were shown as COS-2, COS-3, COS-4, COS-5, and COS-6, respectively. Data were collected at 0, 8, 24, 32, 48, and 72 h of the treatment. H 2 , lactate and SCFAs were produced during the fermentation of COS in mice fecal samples Figure 2.

H 2 , acetate and butyrate showed a continuous accumulation in the samples with COS treatment Figures 2A,C,E. The concentration of H 2 , acetate and butyrate was significantly higher in treatments with 1 and 3 g L -1 COS than that in 0 and 0.

At the end of incubation, the partial pressure of H 2 was higher in COS treated samples 3. Acetate was the most abundant SCFAs and reached 36, In the control, acetate increased to Formate accumulated during the first 24 h, then decreased to about 6.

The highest concentrations of formate were However, the accumulation of formate only reached a relative low level in the control and 0. In addition, the production of propionate could not be detected during the incubation.

FIGURE 2. Production of H 2 and SCFAs by the mice fecal microbe during COS treatment. The concentration of H 2 A , formate B , acetate C , lactate D , and butyrate E was analyzed in mice feces treated with 0, 0.

In all tested groups, lactate accumulated during the first 24 h, then dropped till the end of the incubation Figure 2D. The highest concentration of lactate was above 32 mM in the control and samples treated with 0. Thus, the lactate production was significantly inhibited by COS in relative high concentrations Supplementary Figure S3D.

It suggested that lactate-producing microbe in the fecal sample was inhibited by COS. The total bacterial 16S rRNA gene copy numbers were estimated by quantitative PCR Table 1.

After 8 h incubation, the copy numbers of bacterial 16S rRNA gene significantly decreased from 3. However, copy numbers of bacterial 16S rRNA gene under these treatments returned to the same level as the control group after 72 h incubation.

Besides, the bacterial copy numbers were barely affected by the treatment with 0. Thus, the inhibitory effect of COS on the fecal bacteria was dependent on the concentration of COS as expected. However, the population of the bacterial community quickly recovered with the extended incubation time likely due to the degradation of COS as shown above.

To determine the composition of the bacterial community, bacterial 16S rRNA gene pyrosequencing was performed. Alfa-diversity of bacterial community of samples showed that, as the sequencing depth increased, the number of observed species also increased Supplementary Figure S4A.

There was no significant difference in richness as indicated by rarefaction of observed species and diversity between the control and samples treated with 0. RDA was conducted to examine the relationships between environmental properties and bacterial population distribution of the samples Figure 3A.

Content change of COS, COS-5, H 2 , acetate and butyrate explained The concentration of COS was positively associated with the H 2 partial pressure and the concentration of acetate and butyrate.

However, there was a negative correlation between COS and lactate concentration. UniFrac-based principal coordinates analysis PCoA revealed a distinct clustering of bacteria composition for each experimental group Figure 3B.

Those results indicated that the addition of more than 1 g L -1 COS markedly affected bacterial population distribution.

FIGURE 3. Redundancy analysis RDA and UniFrac-based PCoA analysis on bacterial diversity influenced by COS treatment. RDA for samples from primary culture G1 with COS total , COS-5, H 2 , SCFA and acetate as environmental variables A. The length of each arrow indicated the contribution of the corresponding parameter to the structural variation.

UniFrac-based PCoA using the weighted version for samples from the primary culture and subculture B. Mice stool incubated with 0, 0. Phylogenetic analysis indicated that all tested samples comprised mainly phylum Proteobacteria , Firmicutes , Bacteroidetes , and Actinobacteria Supplementary Figure S5.

The influence of COS on the relative abundance of bacterial community at the genus level was analyzed Figure 4. The bacterial community structure in the 0.

On the other hand, various studies reported that supplementation of COS significantly increased the abundance of Escherichia spp. in rats Shang et al. Moreover, supplementation of COS and resistant starch mixtures alleviated metabolic disorders through synergistic actions, including positive manipulations on gut microbiota, lipid metabolism, and thickness of colonic mucosa layer in the rat Shang et al.

Potential functions of oligosaccharides on gut microbiota could be tested using in vitro batch fermentation models that could exclude interferences from the host Rastall, ; Payne et al. Limited number of studies had investigated the effect of COS on the gut microbiota in vitro and provided controversial conclusions.

Lee et al. in pure cultures. On the contrary, Vernazza et al. This inconsistency through different researches might be due to different sources of COS and experimental settings Lee et al. Based on these studies, the relationship among COS, gut microbiota and host gut health had been preliminarily established.

However, the mechanism and causality of this relationship was still poorly explored. As an important part of the mechanism, the direct impact of COS on the gut microbiota needed to be further studied.

To investigate the effect of COS on the gut microbiota, we have determined the structure of microbiota in mice feces incubated with COS in in vitro batch cultures and colon contents in mice feeding with COS.

To achieve this goal, COS with determined degree of polymerization DP and deacetylation DD was prepared. Batch cultures of mice feces were carried out with different concentrations of COS during in vitro anaerobic incubation. Consumption of COS as well as production of short chain fatty acids SCFAs and biogas were determined.

Furthermore, the influence of COS upon both fecal microbiota in vitro and gut microbiota in animal was assessed.

These studies are an important step toward a better understanding of the COS—microbe—host relationship. The content of COS was determined by UPLC-ESI-Q-TOF-MS Waters Corp. COS with different DP from 2 to 6 were shown as COS-2, COS-3, COS-4, COS-5, and COS-6, respectively.

Aqueous solutions 5 μL of COS 10 mg mL -1 were injected into HPLC for analysis. Mass spectrometry was applied for the qualitative analysis under negative ion modes.

The MS parameters were set up as follows: capillary voltage, 3 kV; cone voltage, 30 V; source temperature, °C; desolvation temperature, °C; gas flows of cone and desolvation, 50 and L h Mass spectrometry data were processed using Masslynx 4. COS samples were also analyzed using Acchrom S HPLC system Acchrom, China equipment with an Acchrom XAmide column 4.

The flow rate was 0. The relative abundance of COS oligomers was evaluated by determining the peak area of each oligosaccharide component using COS oligomer standards as external standard Dalian Glycobio Co. Each sample was analyzed for three times.

Fresh feces 0. Anaerobic incubations were prepared by adding 3 mL of seed liquid into 27 mL of fresh GMM in mL glass bottles. The stock solution of COS g L -1 was sterilized by filtration, then added into samples to final concentrations of 0. Same volume of GMM was added to the control treatment.

Bottles were sealed with butyl stoppers, then flushed with N 2 for 3 min and incubated at 37°C. After 72 h incubation, 3 mL cultured samples of the primary culture G1 were transferred into the 27 mL GMM to get the second subculture G2.

The 3rd subculture G3 was conducted after h incubation of the G2. All tests were carried out in triplicate for each treatment.

Gaseous samples μL were collected from the headspace of the bottle and injected into GC with a pressure-lock precision analytical syringe Baton Rouge, LA, United States and concentrations of H 2 , CH 4 , and CO 2 were analyzed using a gas chromatograph GCA Agilent Technologies, United States , equipped with thermal conductivity detector TCD , flame ionization detector FID and Electrical Conductivity Detector ECD Zhang et al.

Fermentation samples were centrifuged at 14, × g for 10 min and the supernatants were filtered through 0. The sediments were stored at °C for DNA extraction.

SCFAs including formate, acetate, butyrate and L-lactate were analyzed using an Acchrom S high performance liquid chromatography HPLC system Acchrom, China equipment with a C18 column 4. Five microliter sample was injected into the HPLC system for each run.

The pH of mobile phase was adjusted to 2. Cultured samples were taken from incubations at indicated time points. Microbial DNA was extracted following the protocol of FastDNA SPIN Kit with bead-beating using FastPrep MP Biomedicals, CA, United States.

Standards with a fold dilution series from 8. The standard CD diet Aoke Xieli Co. After feeding for 5 months at 22°C with a 12—12 h dark—light cycle, all mice were euthanized and colon contents were sampled. Samples were immediately frozen in liquid nitrogen and stored at °C until DNA extraction.

The animal experiments were approved by the Animal Ethical Experimentation Committee of Chinese Academy of Sciences permission number: SYXK The V3—V4 region of bacterial 16S rRNA genes was subjected to PCR amplification.

Purified amplicons were sequenced on the Illumina MiSeq platform by Allwegene Technology Inc. Beijing, China. Raw sequences were analyzed by QIIME software package as described previously Caporaso et al.

Alpha and beta diversity were examined as well as redundancy analysis RDA and principal coordinate analysis PCoA. Linear discriminant analysis effect size LEfSe was used to estimate taxonomic abundance and characterize differences between groups Segata et al.

All 16S rRNA pyrosequencing datasets had been deposited in GenBank Sequence Read Archive SRA database Accession number: SRP and SRP All statistical analyses were carried out using GraphPad Prism version 6, GraphPad Software Inc.

Data were presented as means ± SD. Data of the H 2 and SCFAs concentrations and bacterial population abundance were subjected to the parametric ANOVA analysis, along with the Tukey—Kramer test, to determine the significant differences between the treatments.

LEfSe analysis of the treatment groups was performed on the basis of the results of the Kruskal—Wallis and Wilcoxon tests and the threshold on the logarithmic linear discriminant analysis LDA score was 2.

In the first 33 h, 0. Then, the degradation was slow down with only 0. The change of COS was not recorded in samples treated with 0. To determine the degradation of different COS components by mice fecal microbiota, changes in the relative abundance of each COS oligomer were characterized through fermentation.

Contents of major components of the original COS sample were COS-2 DP2 3. Interestingly, degradation of COS-5 was obviously slower than other components Figure 1E. FIGURE 1. Dynamics of COS polymers during COS treatment on mice feces. The concentration of COS-total A , and the level of COS-2 B , COS-3 C , COS-4 D , COS-5 E , and COS-6 F in mice feces treated with 0.

COS with different degree of polymerization DP from 2 to 6 were shown as COS-2, COS-3, COS-4, COS-5, and COS-6, respectively. Data were collected at 0, 8, 24, 32, 48, and 72 h of the treatment. H 2 , lactate and SCFAs were produced during the fermentation of COS in mice fecal samples Figure 2.

H 2 , acetate and butyrate showed a continuous accumulation in the samples with COS treatment Figures 2A,C,E. The concentration of H 2 , acetate and butyrate was significantly higher in treatments with 1 and 3 g L -1 COS than that in 0 and 0. At the end of incubation, the partial pressure of H 2 was higher in COS treated samples 3.

Acetate was the most abundant SCFAs and reached 36, In the control, acetate increased to Formate accumulated during the first 24 h, then decreased to about 6. The highest concentrations of formate were However, the accumulation of formate only reached a relative low level in the control and 0.

In addition, the production of propionate could not be detected during the incubation. FIGURE 2. Production of H 2 and SCFAs by the mice fecal microbe during COS treatment. The concentration of H 2 A , formate B , acetate C , lactate D , and butyrate E was analyzed in mice feces treated with 0, 0.

In all tested groups, lactate accumulated during the first 24 h, then dropped till the end of the incubation Figure 2D. The highest concentration of lactate was above 32 mM in the control and samples treated with 0.

Thus, the lactate production was significantly inhibited by COS in relative high concentrations Supplementary Figure S3D. It suggested that lactate-producing microbe in the fecal sample was inhibited by COS. The total bacterial 16S rRNA gene copy numbers were estimated by quantitative PCR Table 1.

After 8 h incubation, the copy numbers of bacterial 16S rRNA gene significantly decreased from 3. However, copy numbers of bacterial 16S rRNA gene under these treatments returned to the same level as the control group after 72 h incubation.

Besides, the bacterial copy numbers were barely affected by the treatment with 0. Thus, the inhibitory effect of COS on the fecal bacteria was dependent on the concentration of COS as expected.

However, the population of the bacterial community quickly recovered with the extended incubation time likely due to the degradation of COS as shown above.

To determine the composition of the bacterial community, bacterial 16S rRNA gene pyrosequencing was performed. Alfa-diversity of bacterial community of samples showed that, as the sequencing depth increased, the number of observed species also increased Supplementary Figure S4A.

There was no significant difference in richness as indicated by rarefaction of observed species and diversity between the control and samples treated with 0. RDA was conducted to examine the relationships between environmental properties and bacterial population distribution of the samples Figure 3A.

Content change of COS, COS-5, H 2 , acetate and butyrate explained The concentration of COS was positively associated with the H 2 partial pressure and the concentration of acetate and butyrate.

However, there was a negative correlation between COS and lactate concentration. UniFrac-based principal coordinates analysis PCoA revealed a distinct clustering of bacteria composition for each experimental group Figure 3B.

Those results indicated that the addition of more than 1 g L -1 COS markedly affected bacterial population distribution. FIGURE 3.

Redundancy analysis RDA and UniFrac-based PCoA analysis on bacterial diversity influenced by COS treatment. RDA for samples from primary culture G1 with COS total , COS-5, H 2 , SCFA and acetate as environmental variables A. The length of each arrow indicated the contribution of the corresponding parameter to the structural variation.

UniFrac-based PCoA using the weighted version for samples from the primary culture and subculture B. Mice stool incubated with 0, 0.

Phylogenetic analysis indicated that all tested samples comprised mainly phylum Proteobacteria , Firmicutes , Bacteroidetes , and Actinobacteria Supplementary Figure S5. The influence of COS on the relative abundance of bacterial community at the genus level was analyzed Figure 4.

The bacterial community structure in the 0. FIGURE 4. Relative abundance of bacterial community in mice fecal samples at genus level. Mice fecal samples treated with 0, 0. The trend of changed proportions of certain bacterial groups in the mice fecal sample under COS treatment was further intensified by subcultures Figures 4B,C.

Furthermore, COS significantly stimulated the growth of Parabacteroides through transfer of subcultures. The relative abundance of Parabacteroides was 10 times higher in 3 g L -1 COS treated group than that in the control.

Linear discriminant analysis LDA effect size LEfSe was performed to identified bacteria strains which exhibited obvious changes in relative abundance under COS treatment Supplementary Figures S6 , S7. Six different taxa were identified in samples treated with 1 g L -1 COS and 10 taxa in samples with 3 g L -1 COS Supplementary Figures S6A,B.

COS treatments significantly increased proportions of Enterococcus and Lactobacillales Supplementary Figures S6E,F. It is important to note that the relative abundance of Lactobacillus decreased significantly from 2 to 0.

The gut microbiota was dominated by four major phyla, Firmicutes , Actinobacteria , Bacteroidetes , and Proteobacteria in the control mice Figure 5A. Moreover, the abundance of the phylum Bacteroidetes in the COS group showed a trend of increase, however, it was not significantly different from that in the control.

In addition, the relative abundance of Mucispirillum , vadinBB60 and Acetatifactor were below 0. FIGURE 5. Effect of COS treatment on the structure of gut bacterial community in mice.

Relative abundance of bacterial community at phylum level A genus level B. Significant differences of the bacteria taxa in mice with or without COS treatments based on Linear discriminant analysis effect size LEfSe C. The threshold logarithmic LDA score was 3. Gut microbiota is associated with not only food digestion and metabolism, but also gut heath and several diseases Clemente et al.

COS have shown versatile health-related biological functions such as immune-stimulation, anti-microbial activity Liaqat and Eltem, Those biological functions might be related to interaction with gut microbiota. An important result of our present studies is a comprehensive and comparative analysis of the microbial community from mice fecal samples treated with different concentrations of COS both in vitro and in animal model.

Addition of 1 g L -1 or higher concentration of COS significantly inhibited the growth of fecal bacteria in vitro. However, following the elongation of the incubation time, the total number of bacteria was quickly returned to the same level of bacteria in the untreated control Table 1 , likely due to the consumption of COS by the microbe Figures 1 , 2.

Interestingly, the utilization rate was different among COS oligomers, in which COS-5 was obviously not favored by the fecal microbe. At the phylum level, the abundance of Bacteroidetes was the significantly increased, while Proteobacteria was remarkably decreased under 3 g L -1 COS treatment at the end of the 3rd subculture.

was increased under COS treatments in vitro. The trend was reinforced by increased concentration of COS and times of subculture. Changes on the production of several kinds of SCFA from in vitro fermentation were also identified.

The relationship among COS, microbe and metabolites was also preliminarily established. In the mouse model, the abundance of phylum Firmicutes and Proteobacteria was remarkably decreased in the colon contents of mice fed with COS.

At the genus level, supplementation of COS obviously increased the abundance of beneficial bacteria Akkermansia , and greatly decreased the relative abundance of harmful bacteria Desulfovibrio phylum Proteobacteria. Chitosan oligosaccharides have been shown to exhibit inhibitory effects against both Gram-positive and Gram-negative bacteria with the minimum inhibitory concentration MIC of 0.

Our studies also found that copy numbers of bacterial 16S rRNA genes in samples treated with 0. However, there was no significant difference in 16S rRNA genes copy numbers between the COS treated groups and the control after incubation for 72 h.

Same trend was observed when Lactobacillus spp. strains were treated with COS in pure cultures Supplementary Figure S8. Thus, the inhibitory effects of COS on bacteria declined in accordance with the extension of the treatment time. Consistently, the concentration of COS in the sample dropped down quickly Figure 1 , indicating the consumption of COS by the microbe.

In addition, cellulose as a limited fermentability control is helpful for confirming COS as microbiota-accessible carbohydrates.

Degree of polymerization of COS is tightly associated with its bio-activities Kittur et al. In the present study, COS oligomers showed different degrading dynamics by the fecal microbe: slower degradation of COS with higher DP Figure 1.

The transient accumulation of COS with lower DP might be due to the degradation of larger oligomers. Interestingly, degradation of COS-5 was obviously slower than others, suggesting the utilization of COS-5 was relatively difficult or unfavorable for the microbe Figure 1E.

In fact both direct cellular uptake and extracellular enzymatic degradation might contribute to COS consumption by gut microbe. Studies showed that gut bacteria was able to directly uptake a variety of oligosaccharides through specific transporters Abou Hachem et al.

A recent study showed that cell uptake of COS dimer COS-2 was faster than trimer COS-3 by S. coelicolor Viens et al. Furthermore, chitosanase have been found in a variety of microorganisms, and was usually expressed extracellularly Jo et al.

Interestingly, studies on GH8 and GH46 chitosanase families indicated that the substrates associated with them were both composed of six subsites Liu et al. Moreover, the rate of COS degradation by some chitosanase obviously decreased with a decrease in the substrate size, with a fold difference on the degradation rate between COS-6 and COS-5 Jo et al.

Thus, COS-5 might be unfavorable for both extracellular chitosanase degradation and cellular uptake of gut microbe.

This observation brought an important proof for the structural and functional specificity of each COS oligomer. Thus, to better understand the structural-functional relationship of COS, purified COS oligomers should be used in the future studies.

The inhibitory effect was enhanced with increased concentration of COS. Metagenomic studies had noted associations between certain species of Bacteroides and diabetes Johnson et al. Qin et al.

The Proteus spp. bacteria were mostly known as opportunistic human pathogens associated with complicated urinary tract and wound infections as well as nosocomial infections Armbruster and Mobley, On the other hand, the relative abundance of Parabacteroides spp. was significantly increased under 3 g L -1 COS treatments Supplementary Figure S6.

Members of the phylum Bacteroidetes , including genus Parabacteroides , participated in provisioning the host with energy harvested through the fermentation of indigestible polysaccharides to produce SCFAs Johnson et al. Moreover, colonic hydrogen concentration in rats was positively correlated with the abundance of genus Parabacteroides Nishimura et al.

Those results indicated that Parabacteroides spp. might contribute to the increased concentration of hydrogen and SCFAs through COS treatments Figures 1 — 3.

Butyrate was an important substrates in the degradation of non-digestible carbohydrates, rather than by protein fermentation under anaerobic conditions Louis and Flint, During the anaerobic incubation, we did not detect the production of methane by GC analysis. In addition, there was no electron acceptors, such as nitrate or sulfate in the culture medium.

Therefore, butyrate significantly accumulated in the in vitro incubation system. In the host in vivo environment, SCFAs reaching total concentrations of 50— mM in the human large intestine could be taken up efficiently by the gut mucosa and have important positive impacts upon host physiology Koh et al.

Especially, butyrate could be used preferentially as an energy source by the gut mucosa and most often considered to benefit health, including protection against colorectal cancer Koh et al.

Thus, these results suggested that COS could positively manipulate the composition of microbial community and stimulate the production of SCFAs, which might bring benefits to gut health. In animal study, COS as a potential non-digestable oligosaccharide for the host, could be metabolized by gut microbiota.

Supplementation of COS greatly decreased the abundance of phylum Proteobacteria Supplementary Figure S5 , consistent with the result of in vitro study. The relative abundance of genus Desulfovibrio phylum Proteobacteria , which may contribute to colorectal cancer development Carbonero et al.

Supplementation of COS also significantly inhibited probiotic genus Lactobacillus Figure 5 , consistent with the result of in vitro study. The probiotic genus Bifidobacterium was also significant inhibited in mice fed with COS.

These results were consistent with previous observations Mateos-Aparicio et al. However, COS greatly elevated the abundance of beneficial genus Akkermansia in mice gut Figure 5. Genus Akkermansia was involved in the enterocytes proliferation of the colonic wounds and reversing high-fat diet induced metabolic disorders, including fat-mass gain, adipose tissue inflammation and insulin resistance Everard et al.

It has to be noted that those beneficial effects of bacteria Akkermansia were in accordance with the biological activities of COS Liaqat and Eltem, There is currently no recommended maximum amount established in the United States 2. While studies have shown chitosan supplementation to be generally safe in adults, the doses studied range widely, from 0.

But staying below that 3-gram maximum set by European safety authorities may be a good reference 2. Check the supplement label to see how much chitosan is in one serving remember that one serving may include multiple capsules and how many servings are recommended per day. Add everything up to see the total daily dose.

When looking for a supplement, always verify that it has been third-party tested. Third-party testing ensures that the supplement meets certain purity and potency standards.

Look for a seal on the packaging from an organization such as NSF International , USP , or ConsumerLab. These seals are typically good indicators of supplement quality.

Talk with a healthcare professional before taking a chitosan supplement. If weight loss is your goal, they may be able to provide more personalized recommendations that are better suited for that purpose. Chitosan is a widely available supplement promoted for weight loss. While some research indicates that it may be somewhat effective in conjunction with a calorie-restricted diet and exercise, more research is needed 2 , 8.

Always proceed with caution when starting a new supplement regimen, and ensure that the benefits outweigh the potential risks. Where chitosan is concerned, its benefits for weight loss are inconclusive. Try this today: Sustainable weight loss is best achieved through a whole-food diet , physical activity, and — importantly — social support.

Our experts continually monitor the health and wellness space, and we update our articles when new information becomes available. Shellfish, such as shrimp, clams, scallops, and lobster, are highly nutritious powerhouses.

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How do chitosan supplements work? Benefits of chitosan supplements. Risks of chitosan supplements. Dosage and safety. The bottom line. Just one thing Try this today: Sustainable weight loss is best achieved through a whole-food diet , physical activity, and — importantly — social support.

Was this helpful? How we reviewed this article: History. Sep 7, Written By Molly Knudsen, MS, RDN. Medically Reviewed By Adrienne Seitz, MS, RD, LDN. Share this article. Read this next. What Is Shellfish? Everything You Need to Know.

Research shows that chitosan-based compounds may be beneficial for wound healing. They have exhibited antimicrobial activity, fighting gram-negative and gram-positive bacteria.

For this reason, chitosan topicals, such as gels and sprays, are used in wound dressing to reduce swelling and fight infections. Recent investigations suggest that chitosan and its derivatives exhibit anti-tumor activity.

It has been shown, in both in vitro and in vivo models, to elevate the production of T-lymphocytes, or white blood cells. Researchers also found that chitooligosaccharides, which are degraded products of chitosan, have proved to repress tumor growth in mice with lung cancer.

It may cause adverse side effects in some people, possibly leading to digestive complaints like upset stomach, gas and constipation. Chitosan may also reduce the absorption of essential nutrients , including vitamins A, D, E and K and calcium, which may contribute to nutrient deficiencies.

People who are allergic to shellfish may not tolerate chitosan supplements. If you experience allergy symptoms, like rash, throat irritation and stomach pains, discontinue use immediately.

Chitosan should not be taken by people who are on blood thinners, such as warfarin, as it may increase the blood thinning effects.

When buying a supplement or topical, choose from a reputable, trustworthy brand that has clear directions and indicates the dosage. Do not exceed the recommended dose. Chitosan supplements are available online and in vitamin stores.

Read the directions carefully for usage and dosage. There is no standard recommended dose for chitosan, and more evidence is needed to make a scientifically proven recommendation.

Trials on weight loss have involved three- to four-gram doses, with insignificant results. For blood pressure, taking up to three grams of a table salt product containing chitosan may be effective. DigestShield® makes that dream easier!

We include a healthy dose of vegetarian-derived, ultra-low molecular weight chitosan in every capsule of DigestShield®. The molecular weight is important because in clinical trials, the lower the molecular weight of the chitosan used, the greater its binding capability.

The chitosan found in typical shrimp or crab shell-derived chitosan, used in glucosamine chondroitin formulas is approximately , Da. Studies have shown that chitosan binds to fat in the gut and prevents it from being absorbed as well. The study concluded that cricket powder supported growth of the probiotic bacterium, Bifidobacterium animalis, which increased 5.

Yet, the results suggest that the chitin in cricket powder may contribute to a healthier digestive system.

In a study conducted in , on animal models, the authors concluded:. In Yoshimi Shibata. told Sciencedaily how crab and other crustacean shells, may hold key to preventing, treating inflammatory bowel disease. In , his team discovered that Chitin protects the layer of our guts called the epithelial barrier, which protects the guts from physical and chemical damage, infection, dehydration, and heat loss.

A study conducted on animal models indicates that dietary chitin is an effective preparation for treating colitis. In the cricket study from the previous chapter, the researchers suggest that chitin can lower tumor necrosis factor alpha TNFα.

Excess production of TNFα can cause many bad diseases such as cancer, AD, major depression, IBD and psoriasis. Like any other insoluble fiber, chitin sits in the gastrointestinal tract, absorbing fluid and sticking to other byproducts of digestion that are ready to be formed into the stool.

Insoluble fiber adds bulk to the stool and appears to help food pass more quickly through the stomach and intestines. Helping prevent intestinal blockage and constipation or reduced bowel movements.

Insoluble fiber physically fills up space in the stomach and intestines, increasing the sensation of being full. And of course, it may help you live longer. If you want to increase your fiber intake and possibly increase the number of healthy bacteria in your gut, you may want to give chitin a try.

Many people who have an allergic reaction towards shellfish or edible insects, are in fact allergic to chitin.