筛选具有抑制引起腹泻致病菌黏附功能的益生菌
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摘要
筛选能够抑制引起腹泻致病菌黏附的益生菌,并且对其适应肠道环境:耐酸、耐胆盐和黏附性能进行评价。从中国西部地区传统发酵食品以及人类粪便样品中分离的14株益生菌作为筛选菌株。对这14株益生菌进行抑制致病菌黏附的筛选,最终筛选出7株使致病菌黏附数量降低到1.5×104CFU/m L的益生菌进行耐受肠道环境能力评价。实验结果显示这7株益生菌在p H2的条件下处理90min后存活的益生菌依然能够达到107CFU/m L左右,在0.3%的胆盐环境中处理2h后菌数降低到原来的百分之一,单层细胞上黏附的益生菌数J5、G15和F0533能够达到106CFU/m L,显示出了较强的黏附能力。经过综合分析筛选出F0533、IN4125、G15、J5和M7益生菌,经16S r DNA基因技术鉴定分别为Lactobacillus rhamnosus、Lactobacillus rhamnosus、Lactobacillus paracasei、Lactobacillus casei和Lactobacillus paracasei。
In this study,fourteen selected Lactobacillus isolated from human intestinal and ferment milk were assessed the ability to inhibit the infection of enteropathogens. The Lactobacillus strains were screened on the basis of probiotic characteristics(i.e.,resistance to low p H and bile salts,adhesion to the human gastrointestinal tract). By an in vitro system simulating gastric transit,it indicated that the three probiotic strains had the ability to tolerate gastroenteric environment and the adhesive capacity to HT-29 cells. It was demonstrated that the number of 7 probiotics strains could reach about 107CFU/m L after treatment of 90 min in p H2 and the number of bacteria strains decreased to one percent of the original after treatment of 0.3% of the bile salt environment 2h.Adhesion of probiotics on number of J5,G15 and F0533 could reach 106CFU/m L,which showed strong adhesion ability. Among the selected strains,five selected Lactobacillus showed a high probiotic potential and could be used in health-promoting food products. After analyzing the sequence of the 16 S r DNA regions of these strains,five potential probiotic F0533,IN4125,G15,J5 and M7 were Lactobacillus rhamnosus,Lactobacillus rhamnosus,Lactobacillus paracasei,Lactobacillus casei and Lactobacillus paracasei.
In this study,fourteen selected Lactobacillus isolated from human intestinal and ferment milk were assessed the ability to inhibit the infection of enteropathogens. The Lactobacillus strains were screened on the basis of probiotic characteristics(i.e.,resistance to low p H and bile salts,adhesion to the human gastrointestinal tract). By an in vitro system simulating gastric transit,it indicated that the three probiotic strains had the ability to tolerate gastroenteric environment and the adhesive capacity to HT-29 cells. It was demonstrated that the number of 7 probiotics strains could reach about 107CFU/m L after treatment of 90 min in p H2 and the number of bacteria strains decreased to one percent of the original after treatment of 0.3% of the bile salt environment 2h.Adhesion of probiotics on number of J5,G15 and F0533 could reach 106CFU/m L,which showed strong adhesion ability. Among the selected strains,five selected Lactobacillus showed a high probiotic potential and could be used in health-promoting food products. After analyzing the sequence of the 16 S r DNA regions of these strains,five potential probiotic F0533,IN4125,G15,J5 and M7 were Lactobacillus rhamnosus,Lactobacillus rhamnosus,Lactobacillus paracasei,Lactobacillus casei and Lactobacillus paracasei.
引文
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[21]Richardson A J,Mc Kain N,Wallace R J.Ammonia Production by Human Faecal Bacteria,and the Enumeration,Isolation and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids[J].BMC Microbiology,2013,13(1):6-13.
[2]O’Reilly C E,Jaron P,Ochieng B,et al.Risk Factors for Death among Children Less Than 5 Years Old Hospitalized with Diarrhea in Rural Western Kenya,2005-2007:A Cohort Study[J].PLo S Medicine,2012,9(7):e1001256.
[3]Liu L,Li Q,Lee R A,et al.Trends in Causes of Death among Children under 5 in Bangladesh,1993-2004:An Exercise Applying a Standardized Computer Algorithm to Assign Causes of Death Using Verbal Autopsy Data[J].Popul Health Metr,2011,9(1):43-55.
[4]Scallan E,Mahon B E,Hoekstra R M,et al.Estimates of Illnesses,Hospitalizations and Deaths Caused by Major Bacterial Enteric Pathogens in Young Children in the United States[J].The Pediatric Infectious Disease Journal,2013,32(3):217-221.
[5]Walker C L F,Rudan I,Liu L,et al.Global Burden of Childhood Pneumonia and Diarrhoea[J].The Lancet,2013,381(9875):1405-1416.
[6]BARR W,SMITH A.Acute Diarrhea[J].American Family Physician,2014,89(3):538-552.
[7]Trejo F M,Minnaard J,Perez P F,et al.Inhibition of Clostridium difficile Growth and Adhesion to Enterocytes by Bifidobacterium Supernatants[J].Anaerobe,2006,12(4):186-193.
[8]Ramiah K,van Reenen C A,Dicks L M.Surface-Bound Proteins of Lactobacillus plantarum 423 That Contribute to Adhesion of Caco-2 Cells and Their Role in Competitive Exclusion and Displacement of Clostridium sporogenes and Enterococcus faecalis[J].Research in Microbiology,2008,159(6):470-475.
[9]Mishra V,Prasad D.Application of in Vitro Methods for Selection of Lactobacillus Casei Strains as Potential Probiotics[J].International Journal of Food Microbiology,2005,103(1):109-115.
[10]Bao Y,Zhang Y,Zhang Y,et al.Screening of Potential Probiotic Properties of Lactobacillus fermentum Isolated from Traditional Dairy Products[J].Food Control,2010,21(5):695-701.
[11]Liu Z,Jiang Z,Zhou K,et al.Screening of Bifidobacteria with Acquired Tolerance to Human Gastrointestinal Tract[J].Anaerobe,2007,13(5):215-219.
[12]Ruiz-Moyano S,Martín A,Benito M J,et al.Screening of Lactic Acid Bacteria and Bifidobacteria for Potential Probiotic Use in Iberian Dry Fermented Sausages[J].Meat Science,2008,80(3):715-721.
[13]Kim P I,Jung M Y,Chang Y-H,et al.Probiotic Properties of Lactobacillus and Bifidobacterium Strains Isolated from Porcine Gastrointestinal Tract[J].Applied Microbiology and Biotechnology,2007,74(5):1103-1111.
[14]Maragkoudakis P A,Zoumpopoulou G,Miaris C,et al.Probiotic Potential of Lactobacillus Strains Isolated from Dairy Products[J].International Dairy Journal,2006,16(3):189-199.
[15]Sonnenburg J L,Chen C T,Gordon J I.Genomic and Metabolic Studies of the Impact of Probiotics on a Model Gut Symbiont and Host[J].PLo S Biology,2006,4(12):e413.
[16]Parkes G C,Brostoff J,Whelan K,et al.Gastrointestinal Microbiota in Irritable Bowel Syndrome:Their Role in Its Pathogenesis and Treatment[J].The American Journal of Gastroenterology,2008,103(6):1557-1567.
[17]Remus D M,Kleerebezem M,Bron P A.An Intimate Tête--Tête—How Probiotic Lactobacilli Communicate with the Host[J].European Journal of Pharmacology,2011,668:S33-S42.
[18]Goldenberg J Z,Ma S S,Saxton J D,et al.Probiotics for the prevention of Clostridium difficile-associated diarrhea in adults and children[J].Status and Date:New,published in,2013,5.
[19]Ou J,De Lany J P,Zhang M,et al.Association between Low Colonic Short-Chain Fatty Acids and High Bile Acids in High Colon Cancer Risk Populations[J].Nutrition and Cancer,2012,64(1):34-40.
[20]Zhou Z,Topping D L,Morell M K,et al.Changes in Starch Physical Characteristics Following Digestion of Foods in the Human Small Intestine[J].British Journal of Nutrition,2010,104(4):573-580.
[21]Richardson A J,Mc Kain N,Wallace R J.Ammonia Production by Human Faecal Bacteria,and the Enumeration,Isolation and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids[J].BMC Microbiology,2013,13(1):6-13.