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1 48 1 Vol.48, No OCEANOLOGIA ET LIMNOLOGIA SINICA Jan., 2017 * 1 翀 ( ; ),, 48h, TLC PAGE, PMP-HPLC, GC :, (10 20kDa),, ; (>20kDa),, (),,,, ; ; ; ; Q539 doi: /hyhz (fucoidan), α-l-, (Pomin et al, 2008;, 2012; Wijesinghe et al, 2012) (, 2012),,,,,,, (Barthe et al, 2004), (Buddington et al, 2002), 1000, Gill (2006), Czjzek (Hehemann et al, 2010), Z. galactanovorans *, 2015GSF ;, ; -, U ,, xjlxiejieling@163.com :,,, zhaoxue@ouc.edu.cn : , :

2 1 : 51 (Thomas et al, 2012) Li (, 2014; Li et al, 2014),, (SCFAs),,, (, 2006) (BCFAs),,,,, (, 2014),,,,,, 1 1.1,, 10kDa (HMWF) 1.2 Sigma ; 6 ( ) 1.3 MLS-3750, SANYO ; Agilent 1100, ; DYY-6C, ; Agilent 1260, ; LTQ-Orbitrap XL, thermo ; Agilent 6890, g 0.08g 15mL, 2mol/L NaOH ph 7.5, 3mL/h 4.5%H 2 O 2, 60 C 4h(pH ), Chelex 100 Cu Da,, (LMWF)(, 2011) ( 3 # L- ) 5g/L HMWF LMWF, ph mL 25mL,, 115 C 40min ( Nos.1 5), 2, 0.1mol/L (ph 7.0) 20%(w/v), 0.5mL, HMWF LMWF,, 37 C 48h, 0h 48h 1mL mol/L NaCl 10mg/mL, : Agilent 1100 ; : TSK-gel G2500 PWxl (7.8cm 30cm); : (G1362A RID); : 0.2mol/L NaCl; : 0.5mL/min; : 40 C; : 1kDa (MW164.16) 5000Da Da, 180Da 2500Da 4600Da 7100aD 10000Da Agilent GPC (TLC) 12000r/min 10min,, 1.2µL, 0h, : : =6:4:1(v:v:v),, - (0.2mL 0.1mL HCl 0.2g

3 mL 85% 10mL ), (, 2007) (PAGE) 12000r/min 10min, 10µL, 50% % 15%, 200V, 30min 5%(w/v) (2% ) 1h, 2% (, 2014) (HPLC) 12000r/min 10min, 0.4mL 5mL, 2mL 2mol/L N 2, 110 C 8h,,, 3, 100µL, 0.3mol/L NaOH, 200µL, 6000r/min 10min 90µL 10µL 20mmol/L,, 10 20mmol/L 10µL, PMP : EC-C18 (4.6mm 150mm, 5μm); : (245nm); : 0.3mL/min; : 30 C; : A: (ph mol/L KH 2 PO 4 ): =82:18, B: (ph mol/L KH 2 PO 4 ): =65:35; : 0 40min; : 5% 50%; 10µL(, 2009) (GC) 2mL 12000r/min 10min,, 1/10 50% H 2 SO 4, 3, 35min, 12000r/min 10min,,, (Zhang et al, 2012): : N 2 ; : ; : 3mL/min; : HP-INNOWAS; : 80 C 0.5min, 8 C / min 150 C, 1min, 20 C/min 200 C, 5min; : 250 C; : 1µL; : FID; : 250 C Excel 2007, ( X S) 2 2.1, 500kDa,, 1, 7.74kDa, 1.02,, 500Da,,, 1 Fig.1 The HPLC chromatogram showing the molecular weight of LMWF : 5000Da: 5000Da ; Da: Da 2.2, TLC PAGE 2 TLC 2a TLC, 48h, No.1 No.2 No.5, No.3 No.4 2b TLC,, 48h,,,,, 3 PAGE, 10kDa 40kDa 3a PAGE, 48h,

3 PAGE analysis on the LMWF and HMWF after being degraded by Nos.

4 1 : 53 2 TLC Nos.1 5 Fig.2 TLC analysis on the LMWF and HMWF after being degraded by Nos.1 5 human gut microbiota 注 : 1: 岩藻糖单糖 ; 2: 硫酸软骨素酶解产物 ; Nos.1 5: 为 Nos PAGE Nos.1 5 Fig.3 PAGE analysis on the LMWF and HMWF after being degraded by Nos.1 5 human gut microbiota : 1: 40kDa ; 2: 10kDa ; 3: ; a-4: ; b-4: ; 5 9: Nos kDa, 10 20kDa 3b PAGE, 20kDa, 48h,, TLC PAGE,, 10 20kDa, 2.3 1, 48h,,,, 48h, TLC PAGE,,, 1,,, h,,,

5 54 48 Tab.2 Tab.1 表 1 人肠道菌群酵解对岩藻聚糖硫酸酯的单糖组成的影响 Changes in monosaccharide composition of fucoidan of different molecular weights after human gut microbial degradation : (%) 表 2 人肠道微生物厌氧酵解岩藻聚糖硫酸酯后短链脂肪酸的变化 The contents of short-chain fatty acids in fucoidan medium after human gut microbiota fermentation in anaerobic culture system in vitro (µg/ml) (µg/ml) 0h 48h 0h 48h 50.91± ± ± ± ± ± ± ± ± ± ± h,,, (, 2014),, 3, 10 20kDa,,,,,, 20kDa Li(, 2014; Li et al, 2014),,, - -,

6 1 : 55, ( ),,,,, (, 1999),, ( ),, (, 2014) 4,,,,,,, , 39(S1): 63 68,, , 6(4): ,,, , 33(5): , κ-. :, 104,,, , 24(1): 38 40, 69,,, , 47(6): ,,, , 35(1): ,,, , 30(3): 25 30,,, , 35(18): 93 97,,, , 28(6): Buddington K K, Donahoo J B, Buddington R K, Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumor inducers. The Journal of Nutrition, 132(3): Barthe L, Woodley J, Lavit M et al, In vitro intestinal degradation and absorption of chondroitin sulfate, a glycosaminoglycan drug. Arzneimittelforschung, 54(5): Gill S R, Pop M, DeBoy R T et al, Metagenomic analysis of the human distal gut microbiome. Science, 312(5778): Hehemann J H, Correc G, Barbeyron T et al, Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature, 464(7290): Li M M, Li G S, Zhu L Y et al, Isolation and characterization of an agaro-oligosaccharide (AO)- hydrolyzing bacterium from the gut microflora of Chinese individuals. PLoS One, 9(3): e91106 Pomin V H, Mourão P A S, Structure, biology, evolution, and medical importance of sulfated fucans and galactans. Glycobiology, 18(12): Thomas F, Barbeyron T, Tonon T et al, Characterization of the first alginolytic operons in a marine bacterium: from their emergence in marine Flavobacteriia to their independent transfers to marine Proteobacteria, and human gut Bacteroides. Environmental Microbiology, 14(9): Wijesinghe W A J P, Jeon Y J, Biological activities and potential industrial applications of fucose rich sulfated polysaccharides and fucoidans isolated from brown seaweeds: a review. Carbohydrate Polymers, 88(1): Zhang X, Zhao Y F, Zhang M H et al, Structural changes of gut microbiota during berberine-mediated prevention of obesity and insulin resistance in high-fat diet-fed rats. PLoS One, 7(8): e42529

7 56 48 HUMAN GUT MICROBES DEGRADE AND UTILIZE FUCOIDAN AND ITS OLIGOSACCHARIDES FROM LAMINARIA JAPONICA IN VITRO XIE Jie-Ling 1, SHI Xiao-Chong 2, SHI Jiao-Xia 1, Yu Tong 1, LI Ba-Fang 1, ZHAO Xue 1 (1. College of Food Science and Engineering, Ocean University of China, Qingdao , China; 2. College of Marine Life Sciences, Ocean University of China, Qingdao , China) Abstract To investigate the degradation and utilization of fucoidan from Laminaria japonica of different molecular weights with human gut microbiota, we sampled human gut microbiota from five volunteers in vitro. High- and low-molecular-weight fucoidan (coded HMWF and LMWF, respectively) were fermented with the microbiota in anaerobic culture system in vitro for 48h. TLC and PAGE were used to analyze the degradation of fucoidan. PMP-HPLC and GC were used to analyze the monosaccharide composition and short chain fatty acids in fermentation product, respectively. Results show that the content of oligosaccharides and low-molecular-weight fucoidan (10 20kDa) decreased markedly, while the monosaccharide composition of degradation products remained unchanged, indicating that the LMWF could be degraded entirely and utilized by human gut microbiota in anaerobic culture system in vitro. However, only small amounts of HMWF (>20kDa) could be degraded to oligosaccharides, and the proportions of galactose and mannose in the fermentation product decreased significantly. GC analysis showed that both LMWF and HMWF could be degraded to acetic acid, propionic acid and butyric acid (short-chain fatty acids). Branched-chain fatty acids (isobutyric acid and isovaleric acid) and valeric acid could be detected in HMWF medium only. Therefore, human gut microbiota can degrade and utilize complex fucoidan oligosaccharides and low-molecular-weight fractions from L. japonica in anaerobic culture system in vitro; however, it could be difficult to degrade a large-molecular-weight fucoidan with human gut microbiota. All sugars in the fucoidan oligosaccharides and large-molecular-weight fucoidan could be utilized by the microbiota to produce short-chain fatty acids, which is good for intestinal tract health. However, only LWMF could inhibit the produce of branched-chain fatty acids with human gut microbiota. Key words fucoidan; human gut microbiota; fucoidan oligosaccharides; degradation; short chain fatty acids

s 15 contact s W Si 3 N μm N / m 4 AFM 95% AFM WPOJ UPOJ WPOJ UPOJ 260 ~ 280 nm WPOJ-DS UPOJ-DS 1 cm 1 cm DEAE-ce

s 15 contact s W Si 3 N μm N / m 4 AFM 95% AFM WPOJ UPOJ WPOJ UPOJ 260 ~ 280 nm WPOJ-DS UPOJ-DS 1 cm 1 cm DEAE-ce 32 1 2013 2 Journal of Chinese Electron Microscopy Society Vol. 32 No. 1 2013-02 1000-6281 2013 01-0054-08 1 1* 2 1 1 1. 2. 710062 AFM SEM WPOJ-DS UPOJ-DS WPOJ-DS β- UPOJ-DS AFM WPOJ-DS UPOJ-DS SEM UPOJ-DS