Effects of Variation in the Community Structures of Human Gut Microbiota on the Utilization of Marine Oligosaccharides in Food

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1 Effects of Variation in the Community Structures of Human Gut Microbiota on the Utilization of Marine Oligosaccharides in Food Dr. Xin Wang Zhejiang Academy of Agricultural Science (ZAAS), Hangzhou, China

2 Significant variation in the community compositions presented in the individual human gut Up to microbes composing of 1000 to 1150 phylotypes are inhabited in human gut. At least 160 bacterial phylotypes are existed in each gut. A significant variation does exist in terms of the composition of colonic microbiota. Nature, Qin. et al. 2010, 464,59

3 Can we manipulate the gut microbiota? Genetic background Food/feed ingredients Composition of gut microbiota Exposure at early life Reducing the colonization of pathogens Pre- and probiotics Safety and functions Factors affecting the community structures of gut microbiota:

4 Marine polysaccharides have been widely used as food additives 20,000 tons of agarose are produced annually worldwide and mainly used to the food industry. Annual requirement for k-carrageenan is estimated 80,000 tones.

5 Chemical structures of marine and other polysaccharides Traditional Chinese Foods, eating in the form of raw or making jelly Agarose : linear polysaccharide composed of 3,6-anhydro-L-galactose and D-galactose Alginate: linear polymer composed of mannuronic acid unit (M) and guluronic acid unit (G) k-carrageenan: repeating dissacharide sequence of α-d-galactopyranose and β-d-galactopyranose, containing various degree of sulphate hemi ester groups Safety debate

6 Evaluation of special functions of oligo- and poly-saccharides Oligo-, polysaccharides Healthy volunteers Fecal Microbiota Whether the sugar can be degraded by the individuals Yes/No Which bacterium is the primary degrader Good/Bad Chemical structures of derivatives Whether desulfurization, deamination, decarboxylation occur during the degradation Effects on the host health

7 Significant variation is existed in the degradation of agaro-oligosaccharide (AO) among the individual fecal samples 1# 2# 3# 4# 5# 6# 7# 8# 9# 10# 11# There are three heterotypes of fermentation in the degradation of agarooligosaccharide 24h 轻微轻微降解降解 轻微降解 降解 降解 轻微 降解 轻微降解 Type 1:Completely degraded after 24h fermentation Type 48h 2:Partially 降解降解 degraded 降解降解 after 降解 48h fermentation 降解轻微降解 Type 3:Undegraded after 48h fermentation 轻微降解 轻微降解 降解降解降解 降解降解降解

8 Identification and isolation of AO degrading bacteria Degraded feces undegraded Cut off band DNA extraction PCR with Eub338 Sequencing B. uniformis B. uniformis L8 (small colonies) E. coli (large colonies)

9 NMR analysis of the residual fragments of AO after 48h fermentation by B. uniformis L8 Fraction Found ions Theoretical Assignment (Charge) H form DP Sequences A (-1) G 323.1(-1) A-G A (-1) G-A-G A (-1) G-A-G- A-G A (-1) G-A-G-A-G- A-G A: 3,6-anhydro-L-galactose; G: D-galactose

10 Synergetic effects of B. uniformis L8, Bifidobacterium and E. coli B2 on the degradation of AO Bifidobacteria only degrade AO with DP<3 % of total carbohydrates in the medium D-galactose 1 Glucose 2 PYG+AO 3 B. adolescentis B. infantis B. longum B. bifidum Galactose 8 AO3 9 AO5 10 AO Medium 5g/L E. Coli B2 B.uniformis L8+B2 Medium B. uniformis L8 generates galactose as the end product which further utilized by E. coli

11 EFFECT OF CARRAGEENAN-OLIGOSACCHARIDES (KCO) ON THE GUT MICROBIOTA -Carrageenan 卡拉胶

12 Carrageenan with large molecular weights can not be degraded by human fecal slurries -carrageenan 450 kda -carrageenan 100 kda

13 Degradation of KCO by human fecal slurries 4.5 kda Degradation of KCO were observed in 6 of 8 human fecal slurries with varied degradation rates

14 KCO E. coli Bact. 38F6 Bact.+E. coli Isolation and identification of KCO degradation bacteria Spread Fecal no.8 on selective agar plates Pick up single colonies TLC analysis Isolate degradation strains 38F6,38F10 Bacteroides sp. 38F6 16S rrna sequencing

15 Structural analysis of end products Sequence analysis using spray ionization mass spectrometry Fractions 38F6 S7D No. K6 No. K8 Found ions (charge) Assignment DP Sequences B 1 C 1 D 1 E (-1) 1 G4S,salt B 2 C2 D 2 E (-1) 2 A-G4S B 3 C 3 D 4 E (-2) 3 G4S-A-G4S B 4 C (-2) 4 A-G4S-A-G4S B 5 C 5 D 6 E (-3) 5 G4S-A-G4S-A-G4S B 6 C (-4) 7 G4S-A-G4S-A-G4S-A-G4S Main end products: -KCO (DP=7, 5 and 3). κ-neocarrabiose and -tetraose (DP=4 and 2)

16 Identification of carrageenanase produced by Bacteroides sp. 38F6 We identified carrageenanase gene in the genome of Bacteroides sp. 38F6 dp2 dp2 dp4 4 : κ- -neocarratetraose 4J:derivatives from κ- neocarratetraose 6 : κ-neocarrahexaose 6J: derivatives from κ-neocarrahexaose - 1,4 glycosidase

17 Bacterial isolates that can degrade marine and other oligosaccharides Marine and oligosaccharides Alginate chondroitin sulfate Heparin Agarose K-carrageenan oligosaccharides Degraded by human fecal slurries Degraded by 6 fecal samples Degraded by 6 fecal samples Degraded by 6 fecal samples Degraded by 2 in 6 fecal samples Degraded by 4 in 6 fecal samples Identified bacterial strains B. ovatus B. thetaiotaomicrom Clostridium hathewayi B. thetaiotaomicrom B. ovatus B. uniformis Bacteroides sp. Dialister sp. Li. et al. Plos One, 2014 Shang et al. J. Biological Macromolecule. 2015, Li. et al. Anaerobes, 2015

18 Fecal isolates that can degrade chondroitin sulfate A Sample No. Number of colonies during the isolation process colonies CSAdegrading % of positive colonies Number of CSA-degrading strains B. thetai. B. thetai8 C. hathe. B. ovatus Total

19 Impacts of oligo-, polysaccharides on the host health Can be degraded by feces Oligo-,polysaccharides Can t be degraded by feces Effects on human gut microbiota Effects on mucin secretion Identification of metabolites Immune response or inflammatory res. Effects on the fat metabolism Effects on the host health

20 Conclusions: Marine oligosaccharides such as AO, KCO could be degraded at various rates by fecal microbiota collected from different persons; Bacteroides spp. are the primary degrader for marine oligosaccharides. However, different Bacteroides species presented in the human gut are responsible for the degradation of different marine oligosaccharides Synergistic effects between two bacterial species occurred in the degradation of marine oligosaccharides

21 Those work are conducted by: Zhejiang Academy of Agricultural Science (ZAAS) and Ocean University of China (OUC) Contributors: Dr. Guangli yu Dr. Yeshi Yin Dr. Liying Zhu Ms. Miaomiao Li Mr. Guangsheng Li 谢谢!