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In vitro fermentation of sulfated polysaccharides from E. prolifera and L. japonica by human fecal microbiota
Accepted Manuscript Title: In vitro fermentation of sulfated polysaccharides from E. prolifera and L. japonica by human fecal microbiota Author: Qing Kong Shiyuan Dong Jian Gao Chaoyu Jiang PII: DOI: Reference:
S0141-8130(16)30574-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.036 BIOMAC 6209
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-3-2016 1-6-2016 12-6-2016
Please cite this article as: Qing Kong, Shiyuan Dong, Jian Gao, Chaoyu Jiang, In vitro fermentation of sulfated polysaccharides from E.prolifera and L.japonica by human fecal microbiota, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.06.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro fermentation of sulfated polysaccharides from E. prolifera and L. japonica by human fecal microbiota
Qing Kong*, Shiyuan Dong, Jian Gao, Chaoyu Jiang
School of Food Science and Engineering, Ocean University of China, Shandong 266003, China
Running title: in vitro fermentation of sulfated polysaccharides
To whom all correspondence should be addressed: Dr. Qing Kong School of Food Science and Engineering Ocean University of China Qingdao, Shandong 266003, China Tel: +86-532-8203-1851 Fax: +86-532-8203-2272 Email: [email protected]
1
ABSTRACT In
vitro
fermentation of the
sulfated polysaccharides
from seaweeds
Enteromorpha prolifera and Laminaria japonica and their prebiotic effects on human fecal microbiota were investigated in this study. The sulfated polysaccharides were fermented in vitro for 48 h by human fecal cultures. When 0.8 g MWCOL (polysaccharides MWCO<30 kD) from L. japonica was fermented, the pH in fecal cultures decreased from 6.5 to 5.1 and the levels of short chain fatty acids, such as acetic, butyric and lactic acids all significantly increased. After 48 h fermentation, 0.8 g MWCOL showed good effect on modulating the gut microflora balance, because the beneficial strains (Lactobacillus and Bifidobacterium) were both significantly higher than those in control group (p<0.05). As far as we know, this is the first report that consumption of sulfated polysaccharides from E. prolifera and L. japonica is beneficial to the ecosystem of the intestinal tract by increasing the populations of probiotics and short chain fatty acids. Furthermore, our reports indicated that molecular weight of sulfated polysaccharide from marine algae is related to its prebiotic effects.
Key words: Enteromorpha prolifera; Laminaria japonica; in vitro fermentation
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1. Introduction Recent years green algae belonging to Enteromorpha prolifera had been frequently involved in algal proliferation in China’s Qingdao coastal areas [1], so it’s urgent to find better resource use to protect environment and reduce processing cost. Many researches proved that sulfate polysaccharides from Enteromorpha species which are a group of sulfated heteropolysaccharides, possessed potential antioxidant activities and some of them had been shown as potent antioxidants [2,3]. The polysaccharides from E. prolifera had a high hypolipidaemic activity and could be a suitable alternative hypolipidaemic source for humans [4,5]. Furthermore, the polysaccharides from E. prolifera also exhibited potent immunomodulatory properties and degraded polysaccharide selenide showed stronger inhibitory effect on Escherichia coli and plant pathogenic fungi [6,7]. The brown seaweed, Laminaria japonica, is common seafood in China and many other countries, and documented as a drug in traditional Chinese medicine for over a thousand years. The sulfated polysaccharide (fucoidan) extracted from L. japonica is also a heteropolysaccharide, mainly composed of fucose, galatose, and sulfate, with smaller amounts of mannoses, glucuronic acid, glucose, rhamnose, arabinose and xylose. Fucoidan was active as an antioxidant, being strong in scavenging superoxide radical and also hydroxyl radical [8,9]. Some researches reported that sulfated polysaccharides from brown seaweeds had a potential protective effect against psychological stress-induced vascular endothelial cells damage in rat, and possessed high antitumor activity and inhibited proliferation and colony formation of breast cancer and melanoma cell lines [10,11]. To sum up, sulfated polysaccharide in seaweed contains diverse biological activity in potential medicinal value, such as anticoagulant, antithrombotic, anti-inflammatory, antitumour, contraceptive, antiviral, and antioxidant [12]. However, the effects of sulfated polysaccharide on gut health remained unclear. It is reported that agaro-oligosaccharide and agarose from marine red algae can be degraded and utilized at various rates by fecal microbiota [13]. The human gut is inhabited by 1014 microbes,
3
including of 1000 to 1150 phylotypes [14], so we hypothesized that sulfated polysaccharides also could be utilized in human gut and show prebiotic effect. In this study, the effects of the sulfated polysaccharides from E. prolifera and L. japonica on human fecal microbiota and production of short chain fatty acids (SCFA) were investigated.
2. Materials and methods 2.1 Materials E. prolifera was collected and L. japonica was cultured in the beach of Qingdao in August, 2015. The fresh seaweed were both washed with tap water, air dried, ground into powder and kept in plastic bags at room temperature before being used.
2.2 Preparation of polysaccharide from E. prolifera Scheme for experimental procedure is shown in Fig. 1. Preparation of polysaccharide from E. prolifera was according to Li et al. [2]. Briefly, dry algae powder (100 g) was extracted with distilled water (7500 mL) at 90℃ for 4 h under continuous stirring. After filtered through gauze, the hot liquid supernatant was centrifuged and filtered by siliceous earth. The residue was washed with additional water and the liquid was collected. The residue was extracted once more under the same procedure. The combined solution was concentrated to about 1000 mL under reduced pressure. The polysaccharides were precipitated by the addition of 4000 mL ethanol then lyophilized to yield white powdered polysaccharides.
2.3 Preparation of fucoidans from L. japonica Preparation of polysaccharides from L. japonica was according to Wang et al. [8]. Briefly, dry algae powder (100 g) was autoclaved in 3500 mL water at 120℃ for 2 h. The hot solution was separated from algae residues by successive filtration through gauze and siliceous earth. The solution was concentrated to about 800 mL under reduced pressure, and then added 4 g MgCl2 and 246 mL of 85% (v/v) ethanol to
4
eliminate algin. The precipitate was filtrated through siliceous earth. Finally, the fucoidans were precipitated by addition of 4000 mL of 95% (v/v) ethanol, and then dried at 80℃. The crude fucoidans were filtrated using an ultrafiltration YM-3 membrane (MWCO: 30 kD, Bio-Rad, Sweden), and two fractions of fucoidans were collected and lyophilized (MWCOL, MWCO<30 KD and MWCOH, MWCO>30 KD).
2.4 In vitro digestion In vitro digestion refers to simulated upper gastrointestinal digestion, i.e., mouth, stomach, and small intestine, which was carried out according to Lebet et al. [15] with a slight modification. All steps were carried out at 37℃ under continuous agitation on a magnetic stirrer. Firstly, 5 g of sulfated polysaccharides were suspended in phosphate buffer (20 mM, pH 6.9). Then 0.25 ml of human salivary α-amylase suspension (A-1031, Sigma Chemicals, St Louis, USA) was added and the mixture was incubated for 15 min. Secondly, the pH of mixture was adjusted to 2.0 with HCl, and 1.25 mL of porcine pepsin suspension (P-7012, Sigma Chemicals, St Louis, USA) was added and the mixture was incubated for 30 min to simulate the gastric digestion. Thirdly, the pH of mixture was adjusted to 6.9 with NaOH, and then a final incubation of 90 min was conducted after the addition of 5 mL of porcine pancreatin suspension (P-7345, Sigma Chemicals, St Louis, USA). The digested mixture was immediately freeze-dried.
2.5 In vitro fermentation In vitro fermentation refers to simulated lower gastrointestinal fermentation, i.e., large intestine, which was carried out according to Rose et al. [16] with some modifications. After in vitro digestion, 0.2 g and 0.8 g of polysaccharide from E. prolifera and L. japonica were weighed into each of 3 serum tubes for each replicate (i.e., one tube per replicate for each sampling period; 12, 24, and 48 h), respectively. Fermentations were carried out in duplicate, and duplicate blank tubes contained no polysaccharide. Anaerobic carbonate-phosphate buffer was prepared and sterilized by 5
autoclaving for 20 min at 121℃. Immediatedly following autoclaving, 0.25 mg/L of cysteine hydrochloride was added as a reducing agent, and carbon dioxide was bubbled through the buffer. During utilisation, a constant stream of carbon dioxide was bubbled through the buffer to maintain anaerobiosis. 8 mL of this buffer was added, along with 100 μL of Oxyrase for Broth (Oxyrase, Inc., Mansfield, OH, USA) to each tube. The Oxyrase was added to scavenge any residual oxygen, and the tubes were sealed anaerobically (by flushing headspace with carbon dioxide) with a rubber stopper and metal crimp cap and placed at 4℃ overnight to hydrate. The next morning, feces were collected from three healthy volunteers who had not taken antibiotics in the last 3 months. The feces were pooled and homogenized with 3 parts sterile anaerobic carbonate-phosphate buffer and then filtered through four layers of cheesecloth. Tubes were opened and 1 mL of filtrate was used to inoculate each tube under constant carbon dioxide flushing. The tubes were then resealed and incubated at 37℃ with gentle shaking. At predetermined time intervals (0, 12, 24, or 48 h), the tubes were opened and microbial activity was halted by the addition of 0.4 mL of 2.75 mg/mL copper sulfate. The pH was recorded, and aliquots of slurry were removed for SCFA quantification or frozen (-20℃) until analysis.
2.6 Fermentation analyses For SCFA quantification, 0.4 mL of fermentation slurry was combined with 0.1 mL of 5% phosphoric acid. They were mixed with a vortex mixer, and allowed to rest for 30 min. Samples were then centrifuged at 12,000 rpm for 10 min, and 1 μl aliquot was injected onto a GC (6890, Agilent, Santa Clara, CA, USA) under the following conditions: glass column packed with Porapak Q (1.5 m×1.5 mm), N2 as the carrier gas, temperatures of the flame ionization detector (FID) and injection port set at 260℃, and column temperature at 215℃. The concentration of SCFA was determined according to a standard calibration curve. For determination of bacterial concentrations, fermentation slurries were removed aseptically, immediately placed into an anaerobic chamber, and dissolved in sterile
6
pre-reduced PBS. Starting from the lowest concentration, 50μL of serial 10-fold dilutions were plated and cultured on different media in triplicate using the spread plate method. Agar media used for enumeration of each group were: Enterococcus spp., Azide agar containing polypeptone (10 g l-1), yeast extract (5 g l-1), sodium chloride (5 g l-1), K2HPO4 (4 g l-1), KH2PO4 (1.5 g l-1), sodium azide (0.5 g l-1), esculin (1 g l-1), 0.05% crystal violet (0.4 ml l-1), ammonium ferric citrate (0.5 g l-1), agar (20 g l-1), pH at 8.0; Enterobacter spp., eosin methylene blue agar (EMB) (Beijing Land Bridge Technology Co., Ltd., Beijing, China); Lactobacillus spp., LBs agar containing peptone (10 g l-1), beef extract (10 g l-1), yeast extract (2 g l-1), glucose (20 g l-1), tween-80 (1 ml l-1), K2HPO4 (2 g l-1), sodium acetate (5 g l-1), ammonium citrate tribasic (2 g l-1), MgSO4 (0.2 g l-1), MnSO4 (0.05 g l-1), agar (25 g l-1), pH at 6.5; Bifidobacterium spp., BBL agar containing peptone (15 g l-1), yeast extract (2 g l-1), glucose (20 g l-1), soluble starch (0.5 g l-1), sodium chloride (5 g l-1), 5% cysteine HCl (10 ml l-1), tomato extract (400 ml l-1), tween-80 (1 ml l-1), liver extract (80 ml l-1), agar (20 g l-1), pH at 7.0 [17]. Azide agar and EMB agar were plated out aerobically and incubated at 37℃ for 48 h, and all other agars were incubated in the anaerobic cabinet at 37℃ for 48 h. Colonies were counted on the dilution with discernible numbers (30-300). The cultures were checked for purity and given preliminary identifications on the basis of their Gram reaction, cell and colony morphologies and limited biochemical profiles [18].
2.7 Statistical analysis All data were presented as the mean±SD. Unpaired and paired t-tests were performed using SAS version 8.1. P<0.05 was the level of significance.
3. Results and discussion 3.1 pH change of the fermentation The change of pH was a reflection for fermentation. As shown in Fig. 2, the initial pH (6.4) of the fermentation cultures was slightly acidic, and this was
7
correlated to the initial amount of SCFA in the human fecal cultures as shown in Fig. 3-5. A decreased pH (6.5-5.1) was observed for the fermentation of the polysaccharides from 0 h to 48 h compared with the control (pH 6.5-5.8). It is interesting to note that the pH in fermented culture added with polysaccharides was lower than that in the control at all time points within 48 h. It could be related to higher levels of SCFA produced in polysaccharides fermentation compared to the control. The pH of fermented culture added with 0.8 g MWCOL and 0.8 g MWCOH was significantly lower than that in the control (p<0.01), which demonstrated that the sulfated polysaccharides from L. japonica were more easily fermented by fecal bacteria.
3.2 SCFA production during in vitro fermentation Concentrations of individual short chain fatty acids produced by the sulfated polysaccharides were very different (Fig. 3-5). Acetate was the primary fermentation product, with butyrate being of secondary abundance, although only slightly more than lactic acid. 0.8 g MWCOL (MWCOL<30 kD) from L. japonica produced significantly more acetate and lactic acid than any of other tested samples (p<0.01), whereas 0.8 g MWCOH (MWCOL>30 kD) from L. japonica produced significantly more butyrate than other samples (p<0.01). More sulfated polysaccharides from L. japonica added, more short chain fatty acids produced. This indicates that the sulfated polysaccharides from L. japonica were fermentable, with efficient conversion of carbohydrate to SCFAs by the fecal bacteria. This may be beneficial due to the numerous trophic effects SCFAs have on the colonic environment [19]. 0.8 g polysaccharides from E. prolifera didn’t produce significantly more SCFAs than the control (p>0.05), suggesting that polysaccharides from E. prolifera were difficult to be fermented by fecal bacteria.
3.3 Microbial Counts during in vitro fermetnation The populations of Enterobacter species were all significantly higher than that in control group after sulfated polysaccharides fermentation (p<0.01) (Table 1). The 8
population of Enterococcus in 0.8 g MWCOL from L. japonica group was significantly higher than that in control (p<0.05) after 48 h fermentation. 0.8 g MWCOL from L. japonica group showed good effect on modulating the gut microflora balance, because the beneficial strains (Lactobacillus and Bifidobacterium) were both significantly higher than those in control group (p<0.05) after 48 h fermentation. Besides, the population of Enterococcus in 0.8 g MWCOH from L. japonica group was also significantly higher than that in control (p<0.01) after 48 h fermentation. Sulfated polysaccharides from L. japonica showed better prebiotic effect than the sulfated polysaccharides from E. prolifera. The intestinal microbiotas of humans and animals comprise hundreds of different types of microorganisms which play an important role in host nutrition and health. Among them, bifidobacteria and lactobacilli are believed to be of health-promoting bacteria to suppress potentially pathogenic bacteria in the gut [20]. Furthermore, as probiotics, bifidobacteria and lactobacilli have demonstrated an ability to influence the immune system, reduce inflammation and/or eliminate or reduce unwanted pro-inflammatory molecules from foods [21]. In this study, the sulfated polysaccharides from L. japonica were found to have prebiotic effect. After 48 h fermentation of 0.8 g sulfated polysaccharides (MWCOH and MWCOL) from L. japonica by fecal bacteria, the fermented cultures contained more SCFAs (including acetate, butyrate, and lactic acid, etc.) to lower the pH values below 5.4, and the populations of Lactobacillus and Bifidobacterium in 0.8 g MWCOL from L. japonica group were both significantly higher than those in control group (p<0.05). In this environment of lower pH and more probiotics, harmful bacteria, such as Salmonella sp., Escherichia coli, Clostridium perfringens, etc., are difficult to survive [22]. Our results were consistent with Marzorati et al. [23]. After addition of a dietary supplement containing a mixture of plant polysaccharides including Undaria pinnatifida fucoidans in a three-vessel colon model, the increase in Bifidobacterium was seen, both via plate counts and quantitative PCR. But it was a mixture of plant-derived polysaccharides, no conclusions can be made regarding to prebiotic activity of the seaweed fucoidan component. Our results also showed low molecular 9
weight (MWCO<30 kD) sulfated polysaccharides from L. japonica had better prebiotic activity than high molecular weight sulfated polysaccharides. In conclusion, though some seaweed polysaccharides, such as alginate oligosaccharides [24], laminarin [25,26], fucoidan [27,28], Undaria pinnatifida and Porphyra ternera extracts [29], etc. had been examined their prebiotic potential, as far as we know, this is the first report that consumption of sulfated polysaccharides from E. prolifera and L. japonica is beneficial to the ecosystem of the intestinal tract by increasing the populations of probiotics and short-chain fatty acids. Furthermore, our reports indicated that molecular weights of sulfated polysaccharides from marine algae had influence on their prebiotic effects. Further animal studies are required to evaluate the beneficial effects of different molecular weights of sulfated polysaccharides from E. prolifera and L. japonica on gut health.
Acknowledgements The study was supported by the research grant from Shinan District Municipal Science and Technology Commision (2013-12-003-YY) of Qingdao, Shandong Province, People's Republic of China and National Natural Science Foundation of China (31471657).
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References [1] J. Zhao, P. Jiang, Z.Y. Liu, W. Wei, H.Z. Lin, F.C. Li, J.F. Wang, S. Qin, Chinese Sci. Bull. 58(2013) 2298-2302. [2] B. Li, S. Liu, R.E. Xing, K.C. Li, R.F. Li, Y.K. Qin, X.Q. Wang, Z.H. Wei, P.C. Li, Carbohydr. Polym. 92(2013) 1991-1996. [3] Z.S. Zhang, X.M. Wang, M.X. Zhao, H.M. Qi, Int. J. Biol. Macromol. 69(2014) 39-45. [4] Z.H. Tang, H.W. Gao, S. Wang, S.H. Wen, S. Qin, Int. J. Biol. Macromol. 58(2013) 186-189. [5] Z.L. Teng, L. Qian, Y. Zhou, Int. J. Biol. Macromol. 62(2013) 254-256. [6] H.T. Lü, Y.J. Gao, H. Shan, Y.T. Lin, Carbohydr. Polym. 107(2014) 98-102. [7] J.T. Wei, S.X. Wang, G. Liu, D. Pei, Y.F. Liu, Y. Liu, D.L. Di, Int. J. Biol. Macromol. 64(2014) 1-5. [8] J. Wang, Q.B. Zhang, Z.S. Zhang, Z.E. Li, Int. J. Biol. Macromol. 42(2008) 127-132. [9] X. Zhao, C.H. Xue, Z.J. Li, Y.P. Cai, H.Y. Liu, H.T. Qi, J. Appl. Phycol. 16(2004) 111-115. [10] O.S. Vishchuk, S.P. Ermakova, T.N. Zvyagintseva, Carbohydr Res 346(2011) 2769-2776. [11] J. Li, S.Y. Wang, X.M. Yang, G.B. Pang, H. Zheng, B. Shen, G.H. Li, D.C. Shi, J.N. Wang, L.Y. Feng, M.L. Li, W.Y. Wei, W. Qin, L. Xie, J. Ethnopharmacol. 151(2014) 601-608. [12] I. Wijesekara, R. Pangestuti, S.K. Kim, Carbohydr. Polym. 84(2011) 14-21. [13] M.M. Li, G.S. Li, L.Y. Zhu, Y.S. Yin, X.L. Zhao, C. Xiang, G.L. Yu, X. Wang, PLOS ONE 9(2014) e91106. [14] L.V. Hooper, M.H. Wong, A. Thelin, L. Hansson, P.G. Falk, J.I. Gordon, Science 291(2001) 881-884. [15] V. Lebet, E. Arrigoni, R. Amado, LWT-Food Sci. Technol. 31(1998) 509-515. [16] D.J. Rose, J.A. Patterson, B.R. Hamaker, J. Agr. Food Chem. 58(2010) 493-499.
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[17] Q. Kong, G.Q. He, J.L. Jia, Q.L. Zhu, H. Ruan, Curr. Microbiol. 62(2011) 512-517. [18] G.R. Gibson, E.R. Beatty, X. Wang, J.H. Cummings, Gastroenterology, 108(1995) 975-982. [19] J.M.W. Wong, R. de Souza, C.W.C. Kendall, A. Emam, D.J.A. Jenkins, J. Clin. Gastroenterol. 40(2006) 235-243. [20] E.E. Vaughan, B. Mollet, W.M. deVos, Curr. Opin. Biotechnol. 10(1999) 505-510. [21] S. Bengmark, Pharmacol. Res. 69(2013) 87-113. [22] R.G. Crittenden, Prebiotics, in: G.W. Tannock (Ed.), Horizon Scientific Press, UK, 1999, pp. 141-156. [23] M. Marzorati, A. Verhelst, G. Luta, R. Sinnott, W. Verstraete, T. Van de Wiele, S. Possemiers, Int. J. Food Microbiol. 139(2010) 168-176. [24] Y. Wang, F. Han, B. Hu, J.B. Li, W.G. Yu, Nutr. Res. 26(2006) 597-603. [25] T. Kuda, T. Yano, N. Matsuda, M. Nishizawa, Food Chem. 91(2005) 745-749. [26] A.M. Neyrinck, A. Mouson, N.M. Delzenne, Int. Immunopharmacol. 7(2007) 1497-1506. [27] P. Reilly, J.V. O’Doherty, K.M. Pierce, J.J. Callan, J.T. O’Sullivan, T. Sweeney, Animal 2(2008) 1465-1473. [28] D.A. Gahan, M.B. Lynch, J.J. Callan, J.T. O’Sullivan, J.V. O’Doherty, Animal 3(2009) 24-31. [29] M. Gudiel-Urbano, I. Goñi, Nutr. Res. 22(2002) 323-331.
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Fucoidans from L. japonica
Polysaccharide from E. prolifera
In vitro digestion (simulated upper gastrointestinal digestion) In vitro fermentation (simulated lower gastrointestinal fermentation) Fermentation analysis
Determination of bacterial concentration: Enterococcus (Azide agar) Enterobacter (EMB agar) Lactobacllus (LBs agar) Bifidobacterium (BBL agar)
Short chain fatty acids quantification by GC: Acetate acid Butyrate acid Lactic acid
pH
Fig. 1. Schematic diagram for experimental procedure.
14
time (h)
48
0.8g MWCOL 0.2g MWCOL 0.8g MWCOH 0.2g MWCOH 0.8g E. prolifera 0.2g E. prolifera Control
24
12
0
5
5.4
5.8 pH
6.2
6.6
Fig. 2. Changes of the pH in fermented cultures added with sulfated polysaccharides at different time during fermentation. Data are mean±standard deviations of three independent experiments.
15
acetate concentration (mg/mL)
0.7 Control 0.2g E. prolifera 0.8g E. prolifera 0.2g MWCOH 0.8g MWCOH 0.2g MWCOL 0.8g MWCOL
0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24
48
fermentation time (h)
Fig. 3. Acetate concentrations from in vitro fermentation of sulfated polysaccharides. Error range of all values were between ± 5 - 20%.
16
butyrate concentration (mg/mL)
0.5 Control 0.2g E. prolifera 0.8g E. prolifera 0.2g MWCOH 0.8g MWCOH 0.2g MWCOL 0.8g MWCOL
0.4
0.3
0.2 0
12 24 fermentation time (h)
48
Fig. 4. Butyrate concentrations from in vitro fermentation of sulfated polysaccharides. Error range of all values were between ± 5 - 20%.
17
lactic acid concentration (mg/mL)
0.35 Control 0.2g E. prolifera 0.8g E. prolifera 0.2g MWCOH 0.8g MWCOH 0.2g MWCOL 0.8g MWCOL
0.3 0.25 0.2 0.15 0.1 0.05 0 0
12 24 48 fermentation time (h)
Fig. 5. Lactic acid concentrations from in vitro fermentation of sulfated polysaccharides. Error range of all values were between ± 5 - 20%.
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Table 1 Microbial flora population of the fermentation slurries Organisms (log CFU/mL) Group
Enterococcus
Enterobacter
Lactobacillus
Bifidobacterium
12h
24h
48h
12h
24h
48h
12h
24h
48h
12h
24h
48h
Control
6.45
6.41
6.35
5.95
5.87
5.71
7.86
7.85
7.70
7.01
6.86
6.52
0.2 g E. prolifera
7.84
8.01
7.86
6.59
7.88
7.71
7.91
8.12
7.99
7.22
7.41
7.40
**
**
8.02
7.91
8.14
8.65
8.64
7.51
7.82
7.62
**
**
7.79
7.73
7.98
8.16
8.14
7.23
7.91
7.76
**
**
7.95
7.65
8.03
8.42
8.21
7.33
8.24
8.21
**
**
**
**
7.66
7.51
8.52
8.46
**
**
**
**
8.14
8.03
8.85
8.61
**
**
**
**
0.8 g E. prolifera
0.2
g
MWCOH
8.04
6.56
8.52
6.77
8.32
6.71
7.12
6.61
(MWCO>30 kD) 0.8
g
MWCOH
7.75
7.92
7.72
7.03
(MWCO>30 kD) 0.2
g
MWCOL
7.03
7.11
6.92
6.62
(MWCO<30 kD) 0.8
g
MWCOL
(MWCO<30 kD)
7.86
8.05
7.96
7.21*
8.02
8.27
8.32
8.11
9.01
8.98
*
*
7.48
7.99
Data (mean±SD) are expressed as the logarithm of the bacterial count in 1 mL of fermentation slurries (log CFU/mL). *p<0.05 vs. control group; **p<0.01 vs. control group. Error range of all values are between ± 4 - 10%. The actual error values were omitted in the table.
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S0141-8130(16)30574-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.036 BIOMAC 6209
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-3-2016 1-6-2016 12-6-2016
Please cite this article as: Qing Kong, Shiyuan Dong, Jian Gao, Chaoyu Jiang, In vitro fermentation of sulfated polysaccharides from E.prolifera and L.japonica by human fecal microbiota, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.06.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro fermentation of sulfated polysaccharides from E. prolifera and L. japonica by human fecal microbiota
Qing Kong*, Shiyuan Dong, Jian Gao, Chaoyu Jiang
School of Food Science and Engineering, Ocean University of China, Shandong 266003, China
Running title: in vitro fermentation of sulfated polysaccharides
To whom all correspondence should be addressed: Dr. Qing Kong School of Food Science and Engineering Ocean University of China Qingdao, Shandong 266003, China Tel: +86-532-8203-1851 Fax: +86-532-8203-2272 Email: [email protected]
1
ABSTRACT In
vitro
fermentation of the
sulfated polysaccharides
from seaweeds
Enteromorpha prolifera and Laminaria japonica and their prebiotic effects on human fecal microbiota were investigated in this study. The sulfated polysaccharides were fermented in vitro for 48 h by human fecal cultures. When 0.8 g MWCOL (polysaccharides MWCO<30 kD) from L. japonica was fermented, the pH in fecal cultures decreased from 6.5 to 5.1 and the levels of short chain fatty acids, such as acetic, butyric and lactic acids all significantly increased. After 48 h fermentation, 0.8 g MWCOL showed good effect on modulating the gut microflora balance, because the beneficial strains (Lactobacillus and Bifidobacterium) were both significantly higher than those in control group (p<0.05). As far as we know, this is the first report that consumption of sulfated polysaccharides from E. prolifera and L. japonica is beneficial to the ecosystem of the intestinal tract by increasing the populations of probiotics and short chain fatty acids. Furthermore, our reports indicated that molecular weight of sulfated polysaccharide from marine algae is related to its prebiotic effects.
Key words: Enteromorpha prolifera; Laminaria japonica; in vitro fermentation
2
1. Introduction Recent years green algae belonging to Enteromorpha prolifera had been frequently involved in algal proliferation in China’s Qingdao coastal areas [1], so it’s urgent to find better resource use to protect environment and reduce processing cost. Many researches proved that sulfate polysaccharides from Enteromorpha species which are a group of sulfated heteropolysaccharides, possessed potential antioxidant activities and some of them had been shown as potent antioxidants [2,3]. The polysaccharides from E. prolifera had a high hypolipidaemic activity and could be a suitable alternative hypolipidaemic source for humans [4,5]. Furthermore, the polysaccharides from E. prolifera also exhibited potent immunomodulatory properties and degraded polysaccharide selenide showed stronger inhibitory effect on Escherichia coli and plant pathogenic fungi [6,7]. The brown seaweed, Laminaria japonica, is common seafood in China and many other countries, and documented as a drug in traditional Chinese medicine for over a thousand years. The sulfated polysaccharide (fucoidan) extracted from L. japonica is also a heteropolysaccharide, mainly composed of fucose, galatose, and sulfate, with smaller amounts of mannoses, glucuronic acid, glucose, rhamnose, arabinose and xylose. Fucoidan was active as an antioxidant, being strong in scavenging superoxide radical and also hydroxyl radical [8,9]. Some researches reported that sulfated polysaccharides from brown seaweeds had a potential protective effect against psychological stress-induced vascular endothelial cells damage in rat, and possessed high antitumor activity and inhibited proliferation and colony formation of breast cancer and melanoma cell lines [10,11]. To sum up, sulfated polysaccharide in seaweed contains diverse biological activity in potential medicinal value, such as anticoagulant, antithrombotic, anti-inflammatory, antitumour, contraceptive, antiviral, and antioxidant [12]. However, the effects of sulfated polysaccharide on gut health remained unclear. It is reported that agaro-oligosaccharide and agarose from marine red algae can be degraded and utilized at various rates by fecal microbiota [13]. The human gut is inhabited by 1014 microbes,
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including of 1000 to 1150 phylotypes [14], so we hypothesized that sulfated polysaccharides also could be utilized in human gut and show prebiotic effect. In this study, the effects of the sulfated polysaccharides from E. prolifera and L. japonica on human fecal microbiota and production of short chain fatty acids (SCFA) were investigated.
2. Materials and methods 2.1 Materials E. prolifera was collected and L. japonica was cultured in the beach of Qingdao in August, 2015. The fresh seaweed were both washed with tap water, air dried, ground into powder and kept in plastic bags at room temperature before being used.
2.2 Preparation of polysaccharide from E. prolifera Scheme for experimental procedure is shown in Fig. 1. Preparation of polysaccharide from E. prolifera was according to Li et al. [2]. Briefly, dry algae powder (100 g) was extracted with distilled water (7500 mL) at 90℃ for 4 h under continuous stirring. After filtered through gauze, the hot liquid supernatant was centrifuged and filtered by siliceous earth. The residue was washed with additional water and the liquid was collected. The residue was extracted once more under the same procedure. The combined solution was concentrated to about 1000 mL under reduced pressure. The polysaccharides were precipitated by the addition of 4000 mL ethanol then lyophilized to yield white powdered polysaccharides.
2.3 Preparation of fucoidans from L. japonica Preparation of polysaccharides from L. japonica was according to Wang et al. [8]. Briefly, dry algae powder (100 g) was autoclaved in 3500 mL water at 120℃ for 2 h. The hot solution was separated from algae residues by successive filtration through gauze and siliceous earth. The solution was concentrated to about 800 mL under reduced pressure, and then added 4 g MgCl2 and 246 mL of 85% (v/v) ethanol to
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eliminate algin. The precipitate was filtrated through siliceous earth. Finally, the fucoidans were precipitated by addition of 4000 mL of 95% (v/v) ethanol, and then dried at 80℃. The crude fucoidans were filtrated using an ultrafiltration YM-3 membrane (MWCO: 30 kD, Bio-Rad, Sweden), and two fractions of fucoidans were collected and lyophilized (MWCOL, MWCO<30 KD and MWCOH, MWCO>30 KD).
2.4 In vitro digestion In vitro digestion refers to simulated upper gastrointestinal digestion, i.e., mouth, stomach, and small intestine, which was carried out according to Lebet et al. [15] with a slight modification. All steps were carried out at 37℃ under continuous agitation on a magnetic stirrer. Firstly, 5 g of sulfated polysaccharides were suspended in phosphate buffer (20 mM, pH 6.9). Then 0.25 ml of human salivary α-amylase suspension (A-1031, Sigma Chemicals, St Louis, USA) was added and the mixture was incubated for 15 min. Secondly, the pH of mixture was adjusted to 2.0 with HCl, and 1.25 mL of porcine pepsin suspension (P-7012, Sigma Chemicals, St Louis, USA) was added and the mixture was incubated for 30 min to simulate the gastric digestion. Thirdly, the pH of mixture was adjusted to 6.9 with NaOH, and then a final incubation of 90 min was conducted after the addition of 5 mL of porcine pancreatin suspension (P-7345, Sigma Chemicals, St Louis, USA). The digested mixture was immediately freeze-dried.
2.5 In vitro fermentation In vitro fermentation refers to simulated lower gastrointestinal fermentation, i.e., large intestine, which was carried out according to Rose et al. [16] with some modifications. After in vitro digestion, 0.2 g and 0.8 g of polysaccharide from E. prolifera and L. japonica were weighed into each of 3 serum tubes for each replicate (i.e., one tube per replicate for each sampling period; 12, 24, and 48 h), respectively. Fermentations were carried out in duplicate, and duplicate blank tubes contained no polysaccharide. Anaerobic carbonate-phosphate buffer was prepared and sterilized by 5
autoclaving for 20 min at 121℃. Immediatedly following autoclaving, 0.25 mg/L of cysteine hydrochloride was added as a reducing agent, and carbon dioxide was bubbled through the buffer. During utilisation, a constant stream of carbon dioxide was bubbled through the buffer to maintain anaerobiosis. 8 mL of this buffer was added, along with 100 μL of Oxyrase for Broth (Oxyrase, Inc., Mansfield, OH, USA) to each tube. The Oxyrase was added to scavenge any residual oxygen, and the tubes were sealed anaerobically (by flushing headspace with carbon dioxide) with a rubber stopper and metal crimp cap and placed at 4℃ overnight to hydrate. The next morning, feces were collected from three healthy volunteers who had not taken antibiotics in the last 3 months. The feces were pooled and homogenized with 3 parts sterile anaerobic carbonate-phosphate buffer and then filtered through four layers of cheesecloth. Tubes were opened and 1 mL of filtrate was used to inoculate each tube under constant carbon dioxide flushing. The tubes were then resealed and incubated at 37℃ with gentle shaking. At predetermined time intervals (0, 12, 24, or 48 h), the tubes were opened and microbial activity was halted by the addition of 0.4 mL of 2.75 mg/mL copper sulfate. The pH was recorded, and aliquots of slurry were removed for SCFA quantification or frozen (-20℃) until analysis.
2.6 Fermentation analyses For SCFA quantification, 0.4 mL of fermentation slurry was combined with 0.1 mL of 5% phosphoric acid. They were mixed with a vortex mixer, and allowed to rest for 30 min. Samples were then centrifuged at 12,000 rpm for 10 min, and 1 μl aliquot was injected onto a GC (6890, Agilent, Santa Clara, CA, USA) under the following conditions: glass column packed with Porapak Q (1.5 m×1.5 mm), N2 as the carrier gas, temperatures of the flame ionization detector (FID) and injection port set at 260℃, and column temperature at 215℃. The concentration of SCFA was determined according to a standard calibration curve. For determination of bacterial concentrations, fermentation slurries were removed aseptically, immediately placed into an anaerobic chamber, and dissolved in sterile
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pre-reduced PBS. Starting from the lowest concentration, 50μL of serial 10-fold dilutions were plated and cultured on different media in triplicate using the spread plate method. Agar media used for enumeration of each group were: Enterococcus spp., Azide agar containing polypeptone (10 g l-1), yeast extract (5 g l-1), sodium chloride (5 g l-1), K2HPO4 (4 g l-1), KH2PO4 (1.5 g l-1), sodium azide (0.5 g l-1), esculin (1 g l-1), 0.05% crystal violet (0.4 ml l-1), ammonium ferric citrate (0.5 g l-1), agar (20 g l-1), pH at 8.0; Enterobacter spp., eosin methylene blue agar (EMB) (Beijing Land Bridge Technology Co., Ltd., Beijing, China); Lactobacillus spp., LBs agar containing peptone (10 g l-1), beef extract (10 g l-1), yeast extract (2 g l-1), glucose (20 g l-1), tween-80 (1 ml l-1), K2HPO4 (2 g l-1), sodium acetate (5 g l-1), ammonium citrate tribasic (2 g l-1), MgSO4 (0.2 g l-1), MnSO4 (0.05 g l-1), agar (25 g l-1), pH at 6.5; Bifidobacterium spp., BBL agar containing peptone (15 g l-1), yeast extract (2 g l-1), glucose (20 g l-1), soluble starch (0.5 g l-1), sodium chloride (5 g l-1), 5% cysteine HCl (10 ml l-1), tomato extract (400 ml l-1), tween-80 (1 ml l-1), liver extract (80 ml l-1), agar (20 g l-1), pH at 7.0 [17]. Azide agar and EMB agar were plated out aerobically and incubated at 37℃ for 48 h, and all other agars were incubated in the anaerobic cabinet at 37℃ for 48 h. Colonies were counted on the dilution with discernible numbers (30-300). The cultures were checked for purity and given preliminary identifications on the basis of their Gram reaction, cell and colony morphologies and limited biochemical profiles [18].
2.7 Statistical analysis All data were presented as the mean±SD. Unpaired and paired t-tests were performed using SAS version 8.1. P<0.05 was the level of significance.
3. Results and discussion 3.1 pH change of the fermentation The change of pH was a reflection for fermentation. As shown in Fig. 2, the initial pH (6.4) of the fermentation cultures was slightly acidic, and this was
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correlated to the initial amount of SCFA in the human fecal cultures as shown in Fig. 3-5. A decreased pH (6.5-5.1) was observed for the fermentation of the polysaccharides from 0 h to 48 h compared with the control (pH 6.5-5.8). It is interesting to note that the pH in fermented culture added with polysaccharides was lower than that in the control at all time points within 48 h. It could be related to higher levels of SCFA produced in polysaccharides fermentation compared to the control. The pH of fermented culture added with 0.8 g MWCOL and 0.8 g MWCOH was significantly lower than that in the control (p<0.01), which demonstrated that the sulfated polysaccharides from L. japonica were more easily fermented by fecal bacteria.
3.2 SCFA production during in vitro fermentation Concentrations of individual short chain fatty acids produced by the sulfated polysaccharides were very different (Fig. 3-5). Acetate was the primary fermentation product, with butyrate being of secondary abundance, although only slightly more than lactic acid. 0.8 g MWCOL (MWCOL<30 kD) from L. japonica produced significantly more acetate and lactic acid than any of other tested samples (p<0.01), whereas 0.8 g MWCOH (MWCOL>30 kD) from L. japonica produced significantly more butyrate than other samples (p<0.01). More sulfated polysaccharides from L. japonica added, more short chain fatty acids produced. This indicates that the sulfated polysaccharides from L. japonica were fermentable, with efficient conversion of carbohydrate to SCFAs by the fecal bacteria. This may be beneficial due to the numerous trophic effects SCFAs have on the colonic environment [19]. 0.8 g polysaccharides from E. prolifera didn’t produce significantly more SCFAs than the control (p>0.05), suggesting that polysaccharides from E. prolifera were difficult to be fermented by fecal bacteria.
3.3 Microbial Counts during in vitro fermetnation The populations of Enterobacter species were all significantly higher than that in control group after sulfated polysaccharides fermentation (p<0.01) (Table 1). The 8
population of Enterococcus in 0.8 g MWCOL from L. japonica group was significantly higher than that in control (p<0.05) after 48 h fermentation. 0.8 g MWCOL from L. japonica group showed good effect on modulating the gut microflora balance, because the beneficial strains (Lactobacillus and Bifidobacterium) were both significantly higher than those in control group (p<0.05) after 48 h fermentation. Besides, the population of Enterococcus in 0.8 g MWCOH from L. japonica group was also significantly higher than that in control (p<0.01) after 48 h fermentation. Sulfated polysaccharides from L. japonica showed better prebiotic effect than the sulfated polysaccharides from E. prolifera. The intestinal microbiotas of humans and animals comprise hundreds of different types of microorganisms which play an important role in host nutrition and health. Among them, bifidobacteria and lactobacilli are believed to be of health-promoting bacteria to suppress potentially pathogenic bacteria in the gut [20]. Furthermore, as probiotics, bifidobacteria and lactobacilli have demonstrated an ability to influence the immune system, reduce inflammation and/or eliminate or reduce unwanted pro-inflammatory molecules from foods [21]. In this study, the sulfated polysaccharides from L. japonica were found to have prebiotic effect. After 48 h fermentation of 0.8 g sulfated polysaccharides (MWCOH and MWCOL) from L. japonica by fecal bacteria, the fermented cultures contained more SCFAs (including acetate, butyrate, and lactic acid, etc.) to lower the pH values below 5.4, and the populations of Lactobacillus and Bifidobacterium in 0.8 g MWCOL from L. japonica group were both significantly higher than those in control group (p<0.05). In this environment of lower pH and more probiotics, harmful bacteria, such as Salmonella sp., Escherichia coli, Clostridium perfringens, etc., are difficult to survive [22]. Our results were consistent with Marzorati et al. [23]. After addition of a dietary supplement containing a mixture of plant polysaccharides including Undaria pinnatifida fucoidans in a three-vessel colon model, the increase in Bifidobacterium was seen, both via plate counts and quantitative PCR. But it was a mixture of plant-derived polysaccharides, no conclusions can be made regarding to prebiotic activity of the seaweed fucoidan component. Our results also showed low molecular 9
weight (MWCO<30 kD) sulfated polysaccharides from L. japonica had better prebiotic activity than high molecular weight sulfated polysaccharides. In conclusion, though some seaweed polysaccharides, such as alginate oligosaccharides [24], laminarin [25,26], fucoidan [27,28], Undaria pinnatifida and Porphyra ternera extracts [29], etc. had been examined their prebiotic potential, as far as we know, this is the first report that consumption of sulfated polysaccharides from E. prolifera and L. japonica is beneficial to the ecosystem of the intestinal tract by increasing the populations of probiotics and short-chain fatty acids. Furthermore, our reports indicated that molecular weights of sulfated polysaccharides from marine algae had influence on their prebiotic effects. Further animal studies are required to evaluate the beneficial effects of different molecular weights of sulfated polysaccharides from E. prolifera and L. japonica on gut health.
Acknowledgements The study was supported by the research grant from Shinan District Municipal Science and Technology Commision (2013-12-003-YY) of Qingdao, Shandong Province, People's Republic of China and National Natural Science Foundation of China (31471657).
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References [1] J. Zhao, P. Jiang, Z.Y. Liu, W. Wei, H.Z. Lin, F.C. Li, J.F. Wang, S. Qin, Chinese Sci. Bull. 58(2013) 2298-2302. [2] B. Li, S. Liu, R.E. Xing, K.C. Li, R.F. Li, Y.K. Qin, X.Q. Wang, Z.H. Wei, P.C. Li, Carbohydr. Polym. 92(2013) 1991-1996. [3] Z.S. Zhang, X.M. Wang, M.X. Zhao, H.M. Qi, Int. J. Biol. Macromol. 69(2014) 39-45. [4] Z.H. Tang, H.W. Gao, S. Wang, S.H. Wen, S. Qin, Int. J. Biol. Macromol. 58(2013) 186-189. [5] Z.L. Teng, L. Qian, Y. Zhou, Int. J. Biol. Macromol. 62(2013) 254-256. [6] H.T. Lü, Y.J. Gao, H. Shan, Y.T. Lin, Carbohydr. Polym. 107(2014) 98-102. [7] J.T. Wei, S.X. Wang, G. Liu, D. Pei, Y.F. Liu, Y. Liu, D.L. Di, Int. J. Biol. Macromol. 64(2014) 1-5. [8] J. Wang, Q.B. Zhang, Z.S. Zhang, Z.E. Li, Int. J. Biol. Macromol. 42(2008) 127-132. [9] X. Zhao, C.H. Xue, Z.J. Li, Y.P. Cai, H.Y. Liu, H.T. Qi, J. Appl. Phycol. 16(2004) 111-115. [10] O.S. Vishchuk, S.P. Ermakova, T.N. Zvyagintseva, Carbohydr Res 346(2011) 2769-2776. [11] J. Li, S.Y. Wang, X.M. Yang, G.B. Pang, H. Zheng, B. Shen, G.H. Li, D.C. Shi, J.N. Wang, L.Y. Feng, M.L. Li, W.Y. Wei, W. Qin, L. Xie, J. Ethnopharmacol. 151(2014) 601-608. [12] I. Wijesekara, R. Pangestuti, S.K. Kim, Carbohydr. Polym. 84(2011) 14-21. [13] M.M. Li, G.S. Li, L.Y. Zhu, Y.S. Yin, X.L. Zhao, C. Xiang, G.L. Yu, X. Wang, PLOS ONE 9(2014) e91106. [14] L.V. Hooper, M.H. Wong, A. Thelin, L. Hansson, P.G. Falk, J.I. Gordon, Science 291(2001) 881-884. [15] V. Lebet, E. Arrigoni, R. Amado, LWT-Food Sci. Technol. 31(1998) 509-515. [16] D.J. Rose, J.A. Patterson, B.R. Hamaker, J. Agr. Food Chem. 58(2010) 493-499.
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[17] Q. Kong, G.Q. He, J.L. Jia, Q.L. Zhu, H. Ruan, Curr. Microbiol. 62(2011) 512-517. [18] G.R. Gibson, E.R. Beatty, X. Wang, J.H. Cummings, Gastroenterology, 108(1995) 975-982. [19] J.M.W. Wong, R. de Souza, C.W.C. Kendall, A. Emam, D.J.A. Jenkins, J. Clin. Gastroenterol. 40(2006) 235-243. [20] E.E. Vaughan, B. Mollet, W.M. deVos, Curr. Opin. Biotechnol. 10(1999) 505-510. [21] S. Bengmark, Pharmacol. Res. 69(2013) 87-113. [22] R.G. Crittenden, Prebiotics, in: G.W. Tannock (Ed.), Horizon Scientific Press, UK, 1999, pp. 141-156. [23] M. Marzorati, A. Verhelst, G. Luta, R. Sinnott, W. Verstraete, T. Van de Wiele, S. Possemiers, Int. J. Food Microbiol. 139(2010) 168-176. [24] Y. Wang, F. Han, B. Hu, J.B. Li, W.G. Yu, Nutr. Res. 26(2006) 597-603. [25] T. Kuda, T. Yano, N. Matsuda, M. Nishizawa, Food Chem. 91(2005) 745-749. [26] A.M. Neyrinck, A. Mouson, N.M. Delzenne, Int. Immunopharmacol. 7(2007) 1497-1506. [27] P. Reilly, J.V. O’Doherty, K.M. Pierce, J.J. Callan, J.T. O’Sullivan, T. Sweeney, Animal 2(2008) 1465-1473. [28] D.A. Gahan, M.B. Lynch, J.J. Callan, J.T. O’Sullivan, J.V. O’Doherty, Animal 3(2009) 24-31. [29] M. Gudiel-Urbano, I. Goñi, Nutr. Res. 22(2002) 323-331.
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Fucoidans from L. japonica
Polysaccharide from E. prolifera
In vitro digestion (simulated upper gastrointestinal digestion) In vitro fermentation (simulated lower gastrointestinal fermentation) Fermentation analysis
Determination of bacterial concentration: Enterococcus (Azide agar) Enterobacter (EMB agar) Lactobacllus (LBs agar) Bifidobacterium (BBL agar)
Short chain fatty acids quantification by GC: Acetate acid Butyrate acid Lactic acid
pH
Fig. 1. Schematic diagram for experimental procedure.
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time (h)
48
0.8g MWCOL 0.2g MWCOL 0.8g MWCOH 0.2g MWCOH 0.8g E. prolifera 0.2g E. prolifera Control
24
12
0
5
5.4
5.8 pH
6.2
6.6
Fig. 2. Changes of the pH in fermented cultures added with sulfated polysaccharides at different time during fermentation. Data are mean±standard deviations of three independent experiments.
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acetate concentration (mg/mL)
0.7 Control 0.2g E. prolifera 0.8g E. prolifera 0.2g MWCOH 0.8g MWCOH 0.2g MWCOL 0.8g MWCOL
0.6 0.5 0.4 0.3 0.2 0.1 0 0
12
24
48
fermentation time (h)
Fig. 3. Acetate concentrations from in vitro fermentation of sulfated polysaccharides. Error range of all values were between ± 5 - 20%.
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butyrate concentration (mg/mL)
0.5 Control 0.2g E. prolifera 0.8g E. prolifera 0.2g MWCOH 0.8g MWCOH 0.2g MWCOL 0.8g MWCOL
0.4
0.3
0.2 0
12 24 fermentation time (h)
48
Fig. 4. Butyrate concentrations from in vitro fermentation of sulfated polysaccharides. Error range of all values were between ± 5 - 20%.
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lactic acid concentration (mg/mL)
0.35 Control 0.2g E. prolifera 0.8g E. prolifera 0.2g MWCOH 0.8g MWCOH 0.2g MWCOL 0.8g MWCOL
0.3 0.25 0.2 0.15 0.1 0.05 0 0
12 24 48 fermentation time (h)
Fig. 5. Lactic acid concentrations from in vitro fermentation of sulfated polysaccharides. Error range of all values were between ± 5 - 20%.
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Table 1 Microbial flora population of the fermentation slurries Organisms (log CFU/mL) Group
Enterococcus
Enterobacter
Lactobacillus
Bifidobacterium
12h
24h
48h
12h
24h
48h
12h
24h
48h
12h
24h
48h
Control
6.45
6.41
6.35
5.95
5.87
5.71
7.86
7.85
7.70
7.01
6.86
6.52
0.2 g E. prolifera
7.84
8.01
7.86
6.59
7.88
7.71
7.91
8.12
7.99
7.22
7.41
7.40
**
**
8.02
7.91
8.14
8.65
8.64
7.51
7.82
7.62
**
**
7.79
7.73
7.98
8.16
8.14
7.23
7.91
7.76
**
**
7.95
7.65
8.03
8.42
8.21
7.33
8.24
8.21
**
**
**
**
7.66
7.51
8.52
8.46
**
**
**
**
8.14
8.03
8.85
8.61
**
**
**
**
0.8 g E. prolifera
0.2
g
MWCOH
8.04
6.56
8.52
6.77
8.32
6.71
7.12
6.61
(MWCO>30 kD) 0.8
g
MWCOH
7.75
7.92
7.72
7.03
(MWCO>30 kD) 0.2
g
MWCOL
7.03
7.11
6.92
6.62
(MWCO<30 kD) 0.8
g
MWCOL
(MWCO<30 kD)
7.86
8.05
7.96
7.21*
8.02
8.27
8.32
8.11
9.01
8.98
*
*
7.48
7.99
Data (mean±SD) are expressed as the logarithm of the bacterial count in 1 mL of fermentation slurries (log CFU/mL). *p<0.05 vs. control group; **p<0.01 vs. control group. Error range of all values are between ± 4 - 10%. The actual error values were omitted in the table.
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