从单菌株组学到宏基因组学:Lactobacillus casei Zhang益生功能
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摘要
干酪乳杆菌Zhang(Lactobacillus casei Zhang, LCZ)是一株研究充分的益生菌,受传统蒙医学的启发,分离筛选自内蒙古牧民的自然发酵酸马奶.其研究经历了3个主要阶段,单菌株组学体外评价阶段,肠道优势菌结合益生功能体内评价阶段,益生功能评价与肠道菌群组学结合的阶段.LCZ经过耐酸耐胆盐筛选,具有较好的体外黏附特性和体外调节巨噬细胞活性的作用.接着进行了菌株全基因组的测定和解析,抗生素抗性实验显示其具有较强的基因组稳定性.生长过程、酸应激和胆盐胁迫的蛋白组学鉴定出一系列与益生菌生长和抗性相关的功能蛋白.进一步体内试验表明,LCZ已经被证明具有调节肠道菌群的丰度和比例、抗脂质过氧化和增加抗氧化酶活性、调节细胞和体液免疫以及肿瘤免疫功能、改善血脂和肝脏脂质代谢、缓解口服糖耐量受损和预防二型糖尿病、保护肝脏和预防肝损伤、抑制肝移植瘤生长和诱变结肠癌发生发展等多项功能.小规模人群试验表明, LCZ可以维持肠道菌群的丰富度,增加有益菌的丰度和减少潜在有害菌的丰度.更重要的是,通过调节肠道菌群预防上呼吸道感染和改善部分衰老指标的功能在中等规模人体随机临床试验中也得到了验证.益生菌LCZ未来将在改善肠道菌群方面发挥更大的益处,具有开发为预防和辅助改善疾病的保健食品/特医食品的潜能.
Lactobacillus casei Zhang(LCZ) is a well-studied probiotic bacterium that was inspired by traditional Mongolian medicine and isolated from naturally fermented koumiss in Inner Mongolia. There are three major stages of LCZ research and development. It contains single strain omics and in vitro evaluation stage(Stage 1), gut dominated microbes and in vivo evaluation stage(Stage 2) as well as metagenomics and clinical evaluation stage(Stage 3). LCZ was screened out from acid resistance, bile salt tolerance and processed favorable effects of in vitro cell adhesion characteristics and regulation of macrophage activity. And then, the whole genome of LCZ was determined and analyzed, which is the first identified Lactobacillus genome in China at 2008. Antibiotics resistance experiments showed a strong stability of LCZ genome under amoxicillin or gentamycin exposure through 2000 generations of laboratory evolution. A set of functional proteins such as NagA and NagB were identified from LCZ proteomics of growth process, acid stress or bile salt tolerance. Furthermore in vivo tests, LCZ has been demonstrated to process several beneficial effects including regulating microbiota abundance and ratios, lipid peroxidation inhibition and increasing antioxidant enzymes activities, regulating cellular and humoral immunity and tumor associated immune function, hepatoprotective effect and inhibition of liver transplanted tumor growth. Researchers have long appreciated the benefits of the gut microbiota to health. Moreover, LCZ was confirmed to promote the intestinal Bacteroides abundance and thereby alleviating impaired glucose tolerance and preventing type 2 diabetes mellitus by Q-PCR methods. Using the third generation PacBio sequencing technology, LCZ could delay the colon cancer progression in mice via altered gut microbiota composition and microbial acetic acid production. Detailedly, Alloprevotella rava and Parabacteroides merdae are the precise potential health-promoting species increased by LCZ intake. In addition to modulate gut microbiota composition, probiotic LCZ also manipulate bile acids, affects its conversation and induced tissue influx of chloride ion, which can impact the expression of chloride ion related proteins(eg.CLCN3 and CFTR). Small-scale human trial indicated that LCZ could maintain the abundance of gut microbiota, increase the number of beneficial bacteria and reduce potential harmful bacteria via 454 pyrosequencing. In 2018, a large-scale, randomized, double-blind and placebo-controlled study was conducted by LCZ administration in Malaysian population. In adult subjects, it confirmed that LCZ could prevent upper respiratory tract infection including reducing the number of days for pharyngeal and general flu symptoms as well as nasal symptoms. In elderly populations, LCZ not only reduced nasal symptoms but also alleviated aged index including improving the mean corpuscular hemoglobin concentration and erythrocyte sedimentation rate. These are attributing to microbiota modulation and its mediated immunity such as increased anti-inflammatory IL-4. It is suggested that LCZ will exert more benefits in microbiota regulation and became a promising health food/special medical food for preventive or therapeutic effects.
Lactobacillus casei Zhang(LCZ) is a well-studied probiotic bacterium that was inspired by traditional Mongolian medicine and isolated from naturally fermented koumiss in Inner Mongolia. There are three major stages of LCZ research and development. It contains single strain omics and in vitro evaluation stage(Stage 1), gut dominated microbes and in vivo evaluation stage(Stage 2) as well as metagenomics and clinical evaluation stage(Stage 3). LCZ was screened out from acid resistance, bile salt tolerance and processed favorable effects of in vitro cell adhesion characteristics and regulation of macrophage activity. And then, the whole genome of LCZ was determined and analyzed, which is the first identified Lactobacillus genome in China at 2008. Antibiotics resistance experiments showed a strong stability of LCZ genome under amoxicillin or gentamycin exposure through 2000 generations of laboratory evolution. A set of functional proteins such as NagA and NagB were identified from LCZ proteomics of growth process, acid stress or bile salt tolerance. Furthermore in vivo tests, LCZ has been demonstrated to process several beneficial effects including regulating microbiota abundance and ratios, lipid peroxidation inhibition and increasing antioxidant enzymes activities, regulating cellular and humoral immunity and tumor associated immune function, hepatoprotective effect and inhibition of liver transplanted tumor growth. Researchers have long appreciated the benefits of the gut microbiota to health. Moreover, LCZ was confirmed to promote the intestinal Bacteroides abundance and thereby alleviating impaired glucose tolerance and preventing type 2 diabetes mellitus by Q-PCR methods. Using the third generation PacBio sequencing technology, LCZ could delay the colon cancer progression in mice via altered gut microbiota composition and microbial acetic acid production. Detailedly, Alloprevotella rava and Parabacteroides merdae are the precise potential health-promoting species increased by LCZ intake. In addition to modulate gut microbiota composition, probiotic LCZ also manipulate bile acids, affects its conversation and induced tissue influx of chloride ion, which can impact the expression of chloride ion related proteins(eg.CLCN3 and CFTR). Small-scale human trial indicated that LCZ could maintain the abundance of gut microbiota, increase the number of beneficial bacteria and reduce potential harmful bacteria via 454 pyrosequencing. In 2018, a large-scale, randomized, double-blind and placebo-controlled study was conducted by LCZ administration in Malaysian population. In adult subjects, it confirmed that LCZ could prevent upper respiratory tract infection including reducing the number of days for pharyngeal and general flu symptoms as well as nasal symptoms. In elderly populations, LCZ not only reduced nasal symptoms but also alleviated aged index including improving the mean corpuscular hemoglobin concentration and erythrocyte sedimentation rate. These are attributing to microbiota modulation and its mediated immunity such as increased anti-inflammatory IL-4. It is suggested that LCZ will exert more benefits in microbiota regulation and became a promising health food/special medical food for preventive or therapeutic effects.
引文
1 Cani P D,Delzenne N M.The gut microbiome as therapeutic target.Pharmacol Ther,2011,130:202-212
2 Panwar H,Calderwood D,Grant I R,et al.Lactobacillus strains isolated from infant faeces possess potent inhibitory activity against intestinal alpha-and beta-glucosidases suggesting anti-diabetic potential.Eur J Nutr,2014,53:1465-1474
3 Larsen B,Monif G R.Understanding the bacterial flora of the female genital tract.Clin Infect Dis,2001,32:e69-e77
4 Karimi G,Sabran M R,Jamaluddin R,et al.The anti-obesity effects of Lactobacillus casei strain Shirota versus Orlistat on high fat dietinduced obese rats.Food Nutr Res,2015,59:29273
5 Ashraf R,Shah N P.Immune system stimulation by probiotic microorganisms.Crit Rev Food Sci Nutr,2014,54:938-956
6 Dos Reis S A,da Concei??o L L,Siqueira N P,et al.Review of the mechanisms of probiotic actions in the prevention of colorectal cancer.Nutr Res,2017,37:1-19
7 Dong J,Zhang Y,Zhang H.Chapter 2:Health properties of traditional fermented Mongolian milk foods.In:Liong M T,ed.Beneficial Microorganisms in Food&Nutraceutical.Netherlands:Springer,2015
8 Kozhakhmetov S,Tynybayeva I,Baikhanova D,et al.Metagenomic analysis of koumiss in Kazakhstan.Cent Asian J Glob Health,2014,3(Suppl):163
9 Yao G,Yu J,Hou Q,et al.A perspective study of koumiss microbiome by metagenomics analysis based on single-cell amplification technique.Front Microbiol,2017,8:165
10 Chen Y,Wang Z,Chen X,et al.Identification of angiotensin I-converting enzyme inhibitory peptides from koumiss,a traditional fermented mare’s milk.J Dairy Sci,2010,93:884-892
11 Wu R,Wang L,Wang J,et al.Isolation and preliminary probiotic selection of lactobacilli from koumiss in Inner Mongolia.J Basic Microbiol,2009,49:318-326
12 Piano M D,Morellic L,Strozzib G P,et al.Probiotics:From research to consumer.Dig Liver Dis,2006,38(Suppl 2):S248-S255
13 Guo Z,Wang J,Yan L,et al.In vitro comparison of probiotic properties of Lactobacillus casei Zhang,a potential new probiotic,with selected probiotic strains.LWT-Food Sci Technol,2009,42:1640-1646
14 Hamon E,Horvatovich P,Bisch M,et al.Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics.J Proteome Res,2012,11:109-118
15 Zhang W,Yu D,Sun Z,et al.Complete genome sequence of Lactobacillus casei Zhang,a new probiotic strain isolated from traditional homemade koumiss in Inner Mongolia,China.J Bacteriol,2010,192:5268-5269
16 Wu R,Wang W,Yu D,et al.Proteomics analysis of Lactobacillus casei Zhang,a new probiotic bacterium isolated from traditional home-made koumiss in Inner Mongolia of China.Mol Cell Proteomics,2009,8:2321-2338
17 Wu R,Zhang W,Sun T,et al.Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress.Int J Food Microbiol,2011,147:181-187
18 Wu R,Sun Z,Wu J,et al.Effect of bile salts stress on protein synthesis of Lactobacillus casei Zhang revealed by 2-dimensional gel electrophoresis.J Dairy Sci,2010,93:3858-3868
19 Koskenniemi K,Laakso K,Koponen J,et al.Proteomics and transcriptomics characterization of bile stress response in probiotic Lactobacillus rhamnosus GG.Mol Cell Proteomics,2011,10:M110.002741
20 Alcántara C,Zú?iga M.Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23.Microbiology,2012,158:1206-1218
21 Guo H,Pan L,Li L,et al.Characterization of antibiotic resistance genes from Lactobacillus isolated from traditional dairy products.JFood Sci,2017,82:724-730
22 Zhang W,Guo H,Cao C,et al.Adaptation of Lactobacillus casei Zhang to gentamycin involves an alkaline shock protein.Front Microbiol,2017,8:2316
23 Ya T,Zhang Q,Chu F,et al.Immunological evaluation of Lactobacillus casei Zhang:A newly isolated strain from koumiss in Inner Mongolia,China.BMC Immunol,2008,9:68
24 Wang Y,Xie J,Wang N,et al.Lactobacillus casei Zhang modulate cytokine and toll-like receptor expression and beneficially regulate poly I:C-induced immune responses in RAW264.7 macrophages.Microbiol Immunol,2013,57:54-62
25 Tuo Y,Du R T,Zhang H P.Antitumor effect and mechanism of probiotic Lb.casei Zhang on H22 tumor cell bearing mice(in Chinese).Cancer Res Prev Treatment,2010,37:463-465[托娅,杜瑞亭,张和平.益生菌Lb.casei Zhang对H22荷瘤小鼠的抗肿瘤作用及机制.肿瘤防治研究,2010,37:463-465]
26 Zhang Y,Du R,Wang L,et al.The antioxidative effects of probiotic Lactobacillus casei Zhang on the hyperlipidemic rats.Eur Food Res Technol,2010,231:151-158
27 Zhang Y,Du R T,He Q W,et al.Effect of Lactobacillus casei Zhang administration on liver lipids metabolism of high-fat diet induced hypercholesterolemia rats(in Chinese).Sci Agric Sin,2012,45:943-950[张勇,杜瑞亭,何秋雯,等.饲喂益生菌Lactobacillus casei Zhang对高脂日量大鼠肝脏脂质代谢的作用.中国农业科学,2012,45:943-950]
28 Zhong Z,Zhang W,Du R,et al.Effect of Lactobacillus casei Zhang on global gene expression in the liver of hypercholesterolemic rats.Eur J Lipid Sci Technol,2012,114:244-252
29 Wang Y,Li Y,Xie J,et al.Protective effects of probiotic Lactobacillus casei Zhang against endotoxin-and d-galactosamine-induced liver injury in rats via anti-oxidative and anti-inflammatory capacities.Int Immunopharmacol,2013,15:30-37
30 Zhang Y,Wang L,Zhang J,et al.Probiotic Lactobacillus casei Zhang ameliorates high-fructose-induced impaired glucose tolerance in hyperinsulinemia rats.Eur J Nutr,2014,53:221-232
31 Zhang Y,Guo X,Guo J,et al.Lactobacillus casei reduces susceptibility to type 2 diabetes via microbiota-mediated body chloride ion influx.Sci Rep,2014,4:5654
32 Kwok L Y,Wang L,Zhang J,et al.A pilot study on the effect of Lactobacillus casei Zhang on intestinal microbiota parameters in Chinese subjects of different age.Benef Microbes,2014,5:295-304
33 Zhang J,Wang L,Guo Z,et al.454 pyrosequencing reveals changes in the faecal microbiota of adults consuming Lactobacillus casei Zhang.FEMS Microbiol Ecol,2014,88:612-622
34 Zhang Y,Ma C,Zhao J,et al.Lactobacillus casei Zhang and vitamin K2 prevent intestinal tumorigenesis in mice via adiponectin-elevated different signaling pathways.Oncotarget,2017,8:24719-24727
35 Hor Y Y,Lew L C,Lau A S Y,et al.Probiotic Lactobacillus casei Zhang(LCZ)alleviates respiratory,gastrointestinal&RBC abnormality via immuno-modulatory,anti-inflammatory&anti-oxidative actions.J Funct Foods,2018,44:235-245
36 Zhou Q,Wang J,Guo Z,et al.Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in mare milk during storage.Int J Dairy Technol,2009,62:249-254
37 Wang J,Guo Z,Zhang Q,et al.Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and bovine milk during storage.J Dairy Sci,2009,92:2468-2476
38 Wang H K,Dong C,Chen Y F,et al.A new probiotic cheddar cheese with high ACE-inhibitory activity andγ-aminobutyric acid content produced with koumiss-derived Lactobacillus casei Zhang.Food Tech Biotechnol,2010,48:1-3
39 Zhang H P,Zhang Q J,Menghe B,et al.Effect of oral administration of L.casei Zhang on T-lymphocyte subclass,serum IgG and intestinal mucous SIgA of mouse(in Chinese).Chin Dairy Ind,2006,34:4-8[张和平,张七斤,孟和毕力格,等.L.casei Zhang对小鼠T淋巴细胞亚群及血清IgG和肠黏膜SIgA的影响.中国乳品工业,2006,34:4-8]
40 Sivananthan K,Petersen A M.Review of Saccharomyces boulardii as a treatment option in IBD.Immunopharmacol Immunotoxicol,2018,17:1-11
2 Panwar H,Calderwood D,Grant I R,et al.Lactobacillus strains isolated from infant faeces possess potent inhibitory activity against intestinal alpha-and beta-glucosidases suggesting anti-diabetic potential.Eur J Nutr,2014,53:1465-1474
3 Larsen B,Monif G R.Understanding the bacterial flora of the female genital tract.Clin Infect Dis,2001,32:e69-e77
4 Karimi G,Sabran M R,Jamaluddin R,et al.The anti-obesity effects of Lactobacillus casei strain Shirota versus Orlistat on high fat dietinduced obese rats.Food Nutr Res,2015,59:29273
5 Ashraf R,Shah N P.Immune system stimulation by probiotic microorganisms.Crit Rev Food Sci Nutr,2014,54:938-956
6 Dos Reis S A,da Concei??o L L,Siqueira N P,et al.Review of the mechanisms of probiotic actions in the prevention of colorectal cancer.Nutr Res,2017,37:1-19
7 Dong J,Zhang Y,Zhang H.Chapter 2:Health properties of traditional fermented Mongolian milk foods.In:Liong M T,ed.Beneficial Microorganisms in Food&Nutraceutical.Netherlands:Springer,2015
8 Kozhakhmetov S,Tynybayeva I,Baikhanova D,et al.Metagenomic analysis of koumiss in Kazakhstan.Cent Asian J Glob Health,2014,3(Suppl):163
9 Yao G,Yu J,Hou Q,et al.A perspective study of koumiss microbiome by metagenomics analysis based on single-cell amplification technique.Front Microbiol,2017,8:165
10 Chen Y,Wang Z,Chen X,et al.Identification of angiotensin I-converting enzyme inhibitory peptides from koumiss,a traditional fermented mare’s milk.J Dairy Sci,2010,93:884-892
11 Wu R,Wang L,Wang J,et al.Isolation and preliminary probiotic selection of lactobacilli from koumiss in Inner Mongolia.J Basic Microbiol,2009,49:318-326
12 Piano M D,Morellic L,Strozzib G P,et al.Probiotics:From research to consumer.Dig Liver Dis,2006,38(Suppl 2):S248-S255
13 Guo Z,Wang J,Yan L,et al.In vitro comparison of probiotic properties of Lactobacillus casei Zhang,a potential new probiotic,with selected probiotic strains.LWT-Food Sci Technol,2009,42:1640-1646
14 Hamon E,Horvatovich P,Bisch M,et al.Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics.J Proteome Res,2012,11:109-118
15 Zhang W,Yu D,Sun Z,et al.Complete genome sequence of Lactobacillus casei Zhang,a new probiotic strain isolated from traditional homemade koumiss in Inner Mongolia,China.J Bacteriol,2010,192:5268-5269
16 Wu R,Wang W,Yu D,et al.Proteomics analysis of Lactobacillus casei Zhang,a new probiotic bacterium isolated from traditional home-made koumiss in Inner Mongolia of China.Mol Cell Proteomics,2009,8:2321-2338
17 Wu R,Zhang W,Sun T,et al.Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress.Int J Food Microbiol,2011,147:181-187
18 Wu R,Sun Z,Wu J,et al.Effect of bile salts stress on protein synthesis of Lactobacillus casei Zhang revealed by 2-dimensional gel electrophoresis.J Dairy Sci,2010,93:3858-3868
19 Koskenniemi K,Laakso K,Koponen J,et al.Proteomics and transcriptomics characterization of bile stress response in probiotic Lactobacillus rhamnosus GG.Mol Cell Proteomics,2011,10:M110.002741
20 Alcántara C,Zú?iga M.Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23.Microbiology,2012,158:1206-1218
21 Guo H,Pan L,Li L,et al.Characterization of antibiotic resistance genes from Lactobacillus isolated from traditional dairy products.JFood Sci,2017,82:724-730
22 Zhang W,Guo H,Cao C,et al.Adaptation of Lactobacillus casei Zhang to gentamycin involves an alkaline shock protein.Front Microbiol,2017,8:2316
23 Ya T,Zhang Q,Chu F,et al.Immunological evaluation of Lactobacillus casei Zhang:A newly isolated strain from koumiss in Inner Mongolia,China.BMC Immunol,2008,9:68
24 Wang Y,Xie J,Wang N,et al.Lactobacillus casei Zhang modulate cytokine and toll-like receptor expression and beneficially regulate poly I:C-induced immune responses in RAW264.7 macrophages.Microbiol Immunol,2013,57:54-62
25 Tuo Y,Du R T,Zhang H P.Antitumor effect and mechanism of probiotic Lb.casei Zhang on H22 tumor cell bearing mice(in Chinese).Cancer Res Prev Treatment,2010,37:463-465[托娅,杜瑞亭,张和平.益生菌Lb.casei Zhang对H22荷瘤小鼠的抗肿瘤作用及机制.肿瘤防治研究,2010,37:463-465]
26 Zhang Y,Du R,Wang L,et al.The antioxidative effects of probiotic Lactobacillus casei Zhang on the hyperlipidemic rats.Eur Food Res Technol,2010,231:151-158
27 Zhang Y,Du R T,He Q W,et al.Effect of Lactobacillus casei Zhang administration on liver lipids metabolism of high-fat diet induced hypercholesterolemia rats(in Chinese).Sci Agric Sin,2012,45:943-950[张勇,杜瑞亭,何秋雯,等.饲喂益生菌Lactobacillus casei Zhang对高脂日量大鼠肝脏脂质代谢的作用.中国农业科学,2012,45:943-950]
28 Zhong Z,Zhang W,Du R,et al.Effect of Lactobacillus casei Zhang on global gene expression in the liver of hypercholesterolemic rats.Eur J Lipid Sci Technol,2012,114:244-252
29 Wang Y,Li Y,Xie J,et al.Protective effects of probiotic Lactobacillus casei Zhang against endotoxin-and d-galactosamine-induced liver injury in rats via anti-oxidative and anti-inflammatory capacities.Int Immunopharmacol,2013,15:30-37
30 Zhang Y,Wang L,Zhang J,et al.Probiotic Lactobacillus casei Zhang ameliorates high-fructose-induced impaired glucose tolerance in hyperinsulinemia rats.Eur J Nutr,2014,53:221-232
31 Zhang Y,Guo X,Guo J,et al.Lactobacillus casei reduces susceptibility to type 2 diabetes via microbiota-mediated body chloride ion influx.Sci Rep,2014,4:5654
32 Kwok L Y,Wang L,Zhang J,et al.A pilot study on the effect of Lactobacillus casei Zhang on intestinal microbiota parameters in Chinese subjects of different age.Benef Microbes,2014,5:295-304
33 Zhang J,Wang L,Guo Z,et al.454 pyrosequencing reveals changes in the faecal microbiota of adults consuming Lactobacillus casei Zhang.FEMS Microbiol Ecol,2014,88:612-622
34 Zhang Y,Ma C,Zhao J,et al.Lactobacillus casei Zhang and vitamin K2 prevent intestinal tumorigenesis in mice via adiponectin-elevated different signaling pathways.Oncotarget,2017,8:24719-24727
35 Hor Y Y,Lew L C,Lau A S Y,et al.Probiotic Lactobacillus casei Zhang(LCZ)alleviates respiratory,gastrointestinal&RBC abnormality via immuno-modulatory,anti-inflammatory&anti-oxidative actions.J Funct Foods,2018,44:235-245
36 Zhou Q,Wang J,Guo Z,et al.Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in mare milk during storage.Int J Dairy Technol,2009,62:249-254
37 Wang J,Guo Z,Zhang Q,et al.Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and bovine milk during storage.J Dairy Sci,2009,92:2468-2476
38 Wang H K,Dong C,Chen Y F,et al.A new probiotic cheddar cheese with high ACE-inhibitory activity andγ-aminobutyric acid content produced with koumiss-derived Lactobacillus casei Zhang.Food Tech Biotechnol,2010,48:1-3
39 Zhang H P,Zhang Q J,Menghe B,et al.Effect of oral administration of L.casei Zhang on T-lymphocyte subclass,serum IgG and intestinal mucous SIgA of mouse(in Chinese).Chin Dairy Ind,2006,34:4-8[张和平,张七斤,孟和毕力格,等.L.casei Zhang对小鼠T淋巴细胞亚群及血清IgG和肠黏膜SIgA的影响.中国乳品工业,2006,34:4-8]
40 Sivananthan K,Petersen A M.Review of Saccharomyces boulardii as a treatment option in IBD.Immunopharmacol Immunotoxicol,2018,17:1-11