具有肠道动力及肠道菌群调节功能乳杆菌的筛选及功能评价
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摘要
便秘作为常见的胃肠道疾病,常伴有肠道动力及肠道菌群的异常。本研究通过测定45株乳杆菌的体外生理特性,并筛选出3株进行体内功能性评价,探讨乳杆菌对肠道动力以及肠道菌群的调节能力。结果表明:干酪乳杆菌LC9、发酵乳杆菌LF37和鼠李糖乳杆菌LR45的耐酸耐胆盐存活率高于10%,均能利用低聚果糖、低聚木糖、低聚半乳糖,且短链脂肪酸产量高于1.2 mmol/L。此外,3株乳杆菌均能促进小鼠的小肠推进率,缩短首粒排黑便时间,增加粪便水分质量分数,加快肠道运动,同时能提高小鼠粪便菌群中有益菌的相对丰度,改善菌群结构。综上,这3株菌对小鼠肠道功能有一定的调节作用,具有潜在的应用价值。
Constipation is a common gastrointestinal disease frequently accompanied by abnormal intestinal motility andflora composition. The in vitro physiological characteristics of 45 strains of Lactobacillus were tested in the current study.Out of these, three strains were screened for in vivo functionality evaluation. Meanwhile, we evaluated the regulatory effect of Lactobacillus on intestinal motility and flora. The results indicated that survival rates of the selected strains,Lactobacillus casei LC9, Lactobacillus fermentum LF37 and Lactobacillus rhamnosus LR45, were all higher than10% when exposed to acid and bile salt. All three strains were able to utilize oligofructose, xylo-oligosaccharide and galacto-oligosaccharide and the production of short chain fatty acids by each strain exceeded 1.2 mmol/L. Furthermore, a series of improved bowel functions were observed in the probiotic group, as manifested by the increased intestinal propulsion rate, the reduced first black stool defecation time, the augmented water content in feces, and the enhanced intestinal motility.The selected Lactobacillus could increase the relative abundance of beneficial bacteria in the mouse fecal flora and improve the structure of the fecal flora. The above results indicate that the three screened strains can regulate intestinal functions in mice, indicating their application potential.
Constipation is a common gastrointestinal disease frequently accompanied by abnormal intestinal motility andflora composition. The in vitro physiological characteristics of 45 strains of Lactobacillus were tested in the current study.Out of these, three strains were screened for in vivo functionality evaluation. Meanwhile, we evaluated the regulatory effect of Lactobacillus on intestinal motility and flora. The results indicated that survival rates of the selected strains,Lactobacillus casei LC9, Lactobacillus fermentum LF37 and Lactobacillus rhamnosus LR45, were all higher than10% when exposed to acid and bile salt. All three strains were able to utilize oligofructose, xylo-oligosaccharide and galacto-oligosaccharide and the production of short chain fatty acids by each strain exceeded 1.2 mmol/L. Furthermore, a series of improved bowel functions were observed in the probiotic group, as manifested by the increased intestinal propulsion rate, the reduced first black stool defecation time, the augmented water content in feces, and the enhanced intestinal motility.The selected Lactobacillus could increase the relative abundance of beneficial bacteria in the mouse fecal flora and improve the structure of the fecal flora. The above results indicate that the three screened strains can regulate intestinal functions in mice, indicating their application potential.
引文
[1]SHIN J E,JUNG H K,LEE T H,et al.Guidelines for the diagnosis and treatment of chronic functional constipation in Korea[J].Journal of Neurogastroenterology and Motility,2016,22(3):383.DOI:10.5056/jnm15185.
[2]MUGIE S M,BENNINGA M A,LORENZO C D.Epidemiology of constipation in children and adults:a systematic review[J].Best Practice&Research in Clinical Gastroenterology,2011,25(1):3-18.DOI:10.1016/j.bpg.2010.12.010.
[3]刘桂章.功能性便秘体质的辨识及中医治疗[J].临床医学,2012,32(1):126-126.DOI:10.3969/j.issn.1003-3548.2012.01.078.
[4]ALAME A M,BAHNA H.Evaluation of constipation[J].Clinics in Colon&Rectal Surgery,2012,25(1):5-11.DOI:10.1055/s-0032-1301753.
[5]中华医学会消化病学分会胃肠动力学组,中华医学会外科学分会结直肠肛门外科学组.中国慢性便秘诊治指南(2013,武汉)[J].中华消化杂志,2013,33(5):605-612.DOI:10.3760/cma.j.issn.0254-1432.2013.05.002.
[6]刘海宁,陈玉琢,吴昊,等.肠道菌群与功能性便秘的研究进展[J].复旦学报(医学版),2015,42(4):564-568.DOI:10.3969/j.issn.1672-8467.2015.04.024.
[7]GE Xiaolong,ZHAO Wei,DING Chao,et al.Potential role of fecal microbiota from patients with slow transit constipation in the regulation of gastrointestinal motility[J].Scientific Reports,2017,7(1):1-11.DOI:10.1038/s41598-017-00612-y.
[8]ZHAO Yanfang,MA Xiuqiang,WANG Rui,et al.Epidemiology of functional constipation and comparison with constipation‐predominant irritable bowel syndrome:the systematic investigation of gastrointestinal diseases in China(SILC)[J].Alimentary Pharmacology&Therapeutics,2011,34(8):1020-1029.DOI:10.1111/j.1365-2036.2011.04809.x.
[9]刘广进.浅谈肛肠冲洗器治疗便秘、肛裂和痔疮[J].当代医学(学术版),2007(4):41-42.DOI:10.3969/j.issn.1009-4393.2007.07.020.
[10]罗文舟.功能性便秘患者中医体质类型分布的初步研究[D].南京:南京中医药大学,2017:1-37.
[11]韩亚超,何永高,张新红,等.益生菌及其益生作用机理[J].阜阳职业技术学院学报,2014,25(2):57-59.
[12]KIM G,DEEPINDER F,MORALES W,et al.Methanobrevibacter smithii is the predominant methanogen in patients with constipationpredominant IBS and methane on breath[J].Digestive Diseases&Sciences,2012,57(12):3213-3218.DOI:10.1007/s10620-012-2197-1.
[13]PICARD C,FIORAMONTI J,FRANCOIS A,et al.Review article:bifidobacteria as probiotic agents-physiological effects and clinical benefits[J].Alimentary Pharmacology&Therapeutics,2005,22(6):495-512.DOI:10.1111/j.1365-2036.2005.02615.x.
[14]HIGASHIKAWA F,NODA M,AWAYA T,et al.Improvement of constipation and liver function by plant-derived lactic acid bacteria:a double-blind,randomized trial[J].Nutrition,2010,26(4):367-374.DOI:10.1016/j.nut.2009.05.008.
[15]曹永强.干酪乳杆菌N1115产酸特性及其在发酵乳中的应用[D].北京:北京工商大学,2016:1-46.
[16]翟齐啸.乳酸菌减除镉危害的作用及机制研究[D].无锡:江南大学,2015:1-110.
[17]毛丙永.功能性低聚糖对肠道细菌的影响及机制[D].无锡:江南大学,2015:1-88.
[18]刘思思.乳酸菌利用低聚糖的特性及在肠道中的作用[D].无锡:江南大学,2015:1-47.
[19]International Organization For Standardization,International Dairy Federation.Milk and milk products-determination of the minimal inhibitory concentration(MIC)of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria(LAB)[EB/OL].[2018-08-16].http://agris.fao.org/openagris/search.do?recordID=XF2015015812.
[20]卫生部卫生法制与监督司.保健食品检验与评价技术规范[M].北京:卫生部卫生法制与监督司,2003:1-311.
[21]王琳琳.双歧杆菌对便秘的影响及其作用机理研究[D].无锡:江南大学,2017:1-115.
[22]LI Xiangfei,XU Qi,JIANG Tian,et al.A comparative study of the antidiabetic effects exerted by live and dead multi-strain probiotics in the type 2 diabetes model of mice[J].Food&Function,2016,7(12):4851-4860.DOI:10.1039/c6fo01147k.
[23]MANDAL H,JARIWALA R,BAGCHI T.Isolation and characterization of Lactobacilli from human faeces and indigenous fermented foods for their potential application as probiotics[J].Canadian Journal of Microbiology,2015,62(4):349-359.DOI:10.1139/cjm-2015-0576.
[24]LI Chun,WANG Chunlin,SUN Yu,et al.Microencapsulation of Lactobacillus rhamnosus GG by transglutaminase cross‐linked soy protein isolate to improve survival in simulated gastrointestinal conditions and yoghurt[J].Journal of Food Science,2016,81(7):M1726-M1734.DOI:10.1111/1750-3841.13337.
[25]DYEZ-MUNICIO M,KOLIDA S,HERRERO M,et al.In vitro faecal fermentation of novel oligosaccharides enzymatically synthesized using microbial transglycosidases acting on sucrose[J].Journal of Functional Foods,2016,20:532-544.DOI:10.1016/j.jff.2015.11.032.
[26]MACFARLANE G T,STEED H,MACFARLANE S.Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics[J].Journal of Applied Microbiology,2008,104(2):305-344.DOI:10.1111/j.1365-2672.2007.03520.x.
[27]GOH Y J,KLAENHAMMER T R.Genetic mechanisms of prebiotic oligosaccharide metabolism in probiotic microbes[J].Annual Review of Food Science and Technology,2015,6(6):137-156.DOI:10.1146/annurev-food-022814-015706.
[28]G?NZLE M G,FOLLADOR R.Metabolism of oligosaccharides and starch in Lactobacilli:a review[J].Frontiers in Microbiology,2012,3:340.DOI:10.3389/fmicb.2012.00340.
[29]MORRISON D J,TOM P.Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism[J].Gut Microbes,2016,7(3):189-200.DOI:10.1080/19490976.2015.11340822.
[30]FELD L,BIELAK E,HAMMER K,et al.Characterization of a small erythromycin resistance plasmid pLFE1 from the food-isolate Lactobacillus plantarum M345[J].Plasmid,2009,61(3):159-170.DOI:10.1016/j.plasmid.2009.01.002.
[31]?T?EPETOVA J,TAELMA H,SMIDT I,et al.Assessment of phenotypic and genotypic antibiotic susceptibility of vaginal Lactobacillus sp[J].Journal of Applied Microbiology,2017,123(2):524-534.DOI:10.1111/jam.13497.
[32]李少英.奶牛源性双歧杆菌和乳杆菌的分离鉴定及耐药性研究[D].呼和浩特:内蒙古农业大学,2008:1-111.
[33]邵玉宇.干酪乳杆菌和植物乳杆菌耐药分子机制的研究[D].呼和浩特:内蒙古农业大学,2015:1-115.
[34]HERRMANN J,HERMES R,BREVES G.Transepithelial transport and intraepithelial metabolism of short-chain fatty acids(SCFA)in the porcine proximal colon are influenced by SCFA concentration and luminal pH[J].Comparative Biochemistry&Physiology Part A Molecular&Integrative Physiology,2011,158(1):169-176.DOI:10.1016/j.cbpa.2010.10.018.
[35]GEERLINGS S,KOSTOPOULOS I,BELZER C,et al.Akkermansia muciniphila in the human gastrointestinal tract:when,where,and how?[J].Microorganisms,2018,6(3):1-26.DOI:10.3390/microorganisms6030075.
[36]EVERARD A,LAZAREVIC V,GAIA N,et al.Microbiome of prebiotic-treated mice reveals novel targets involved in host response during obesity[J].The ISME Journal,2014,8(10):2116-2130.DOI:10.1038/ismej.2014.45.
[37]KANG D W,DIBAISE J K,ILHAN Z E,et al.Gut microbial and short-chain fatty acid profiles in adults with chronic constipation before and after treatment with lubiprostone[J].Anaerobe,2015,33:33-41.DOI:10.1016/j.anaerobe.2015.01.005.
[38]李川.植物乳杆菌NCU116的益生功能及其作用机制[D].南昌:南昌大学,2015:1-147.
[2]MUGIE S M,BENNINGA M A,LORENZO C D.Epidemiology of constipation in children and adults:a systematic review[J].Best Practice&Research in Clinical Gastroenterology,2011,25(1):3-18.DOI:10.1016/j.bpg.2010.12.010.
[3]刘桂章.功能性便秘体质的辨识及中医治疗[J].临床医学,2012,32(1):126-126.DOI:10.3969/j.issn.1003-3548.2012.01.078.
[4]ALAME A M,BAHNA H.Evaluation of constipation[J].Clinics in Colon&Rectal Surgery,2012,25(1):5-11.DOI:10.1055/s-0032-1301753.
[5]中华医学会消化病学分会胃肠动力学组,中华医学会外科学分会结直肠肛门外科学组.中国慢性便秘诊治指南(2013,武汉)[J].中华消化杂志,2013,33(5):605-612.DOI:10.3760/cma.j.issn.0254-1432.2013.05.002.
[6]刘海宁,陈玉琢,吴昊,等.肠道菌群与功能性便秘的研究进展[J].复旦学报(医学版),2015,42(4):564-568.DOI:10.3969/j.issn.1672-8467.2015.04.024.
[7]GE Xiaolong,ZHAO Wei,DING Chao,et al.Potential role of fecal microbiota from patients with slow transit constipation in the regulation of gastrointestinal motility[J].Scientific Reports,2017,7(1):1-11.DOI:10.1038/s41598-017-00612-y.
[8]ZHAO Yanfang,MA Xiuqiang,WANG Rui,et al.Epidemiology of functional constipation and comparison with constipation‐predominant irritable bowel syndrome:the systematic investigation of gastrointestinal diseases in China(SILC)[J].Alimentary Pharmacology&Therapeutics,2011,34(8):1020-1029.DOI:10.1111/j.1365-2036.2011.04809.x.
[9]刘广进.浅谈肛肠冲洗器治疗便秘、肛裂和痔疮[J].当代医学(学术版),2007(4):41-42.DOI:10.3969/j.issn.1009-4393.2007.07.020.
[10]罗文舟.功能性便秘患者中医体质类型分布的初步研究[D].南京:南京中医药大学,2017:1-37.
[11]韩亚超,何永高,张新红,等.益生菌及其益生作用机理[J].阜阳职业技术学院学报,2014,25(2):57-59.
[12]KIM G,DEEPINDER F,MORALES W,et al.Methanobrevibacter smithii is the predominant methanogen in patients with constipationpredominant IBS and methane on breath[J].Digestive Diseases&Sciences,2012,57(12):3213-3218.DOI:10.1007/s10620-012-2197-1.
[13]PICARD C,FIORAMONTI J,FRANCOIS A,et al.Review article:bifidobacteria as probiotic agents-physiological effects and clinical benefits[J].Alimentary Pharmacology&Therapeutics,2005,22(6):495-512.DOI:10.1111/j.1365-2036.2005.02615.x.
[14]HIGASHIKAWA F,NODA M,AWAYA T,et al.Improvement of constipation and liver function by plant-derived lactic acid bacteria:a double-blind,randomized trial[J].Nutrition,2010,26(4):367-374.DOI:10.1016/j.nut.2009.05.008.
[15]曹永强.干酪乳杆菌N1115产酸特性及其在发酵乳中的应用[D].北京:北京工商大学,2016:1-46.
[16]翟齐啸.乳酸菌减除镉危害的作用及机制研究[D].无锡:江南大学,2015:1-110.
[17]毛丙永.功能性低聚糖对肠道细菌的影响及机制[D].无锡:江南大学,2015:1-88.
[18]刘思思.乳酸菌利用低聚糖的特性及在肠道中的作用[D].无锡:江南大学,2015:1-47.
[19]International Organization For Standardization,International Dairy Federation.Milk and milk products-determination of the minimal inhibitory concentration(MIC)of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria(LAB)[EB/OL].[2018-08-16].http://agris.fao.org/openagris/search.do?recordID=XF2015015812.
[20]卫生部卫生法制与监督司.保健食品检验与评价技术规范[M].北京:卫生部卫生法制与监督司,2003:1-311.
[21]王琳琳.双歧杆菌对便秘的影响及其作用机理研究[D].无锡:江南大学,2017:1-115.
[22]LI Xiangfei,XU Qi,JIANG Tian,et al.A comparative study of the antidiabetic effects exerted by live and dead multi-strain probiotics in the type 2 diabetes model of mice[J].Food&Function,2016,7(12):4851-4860.DOI:10.1039/c6fo01147k.
[23]MANDAL H,JARIWALA R,BAGCHI T.Isolation and characterization of Lactobacilli from human faeces and indigenous fermented foods for their potential application as probiotics[J].Canadian Journal of Microbiology,2015,62(4):349-359.DOI:10.1139/cjm-2015-0576.
[24]LI Chun,WANG Chunlin,SUN Yu,et al.Microencapsulation of Lactobacillus rhamnosus GG by transglutaminase cross‐linked soy protein isolate to improve survival in simulated gastrointestinal conditions and yoghurt[J].Journal of Food Science,2016,81(7):M1726-M1734.DOI:10.1111/1750-3841.13337.
[25]DYEZ-MUNICIO M,KOLIDA S,HERRERO M,et al.In vitro faecal fermentation of novel oligosaccharides enzymatically synthesized using microbial transglycosidases acting on sucrose[J].Journal of Functional Foods,2016,20:532-544.DOI:10.1016/j.jff.2015.11.032.
[26]MACFARLANE G T,STEED H,MACFARLANE S.Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics[J].Journal of Applied Microbiology,2008,104(2):305-344.DOI:10.1111/j.1365-2672.2007.03520.x.
[27]GOH Y J,KLAENHAMMER T R.Genetic mechanisms of prebiotic oligosaccharide metabolism in probiotic microbes[J].Annual Review of Food Science and Technology,2015,6(6):137-156.DOI:10.1146/annurev-food-022814-015706.
[28]G?NZLE M G,FOLLADOR R.Metabolism of oligosaccharides and starch in Lactobacilli:a review[J].Frontiers in Microbiology,2012,3:340.DOI:10.3389/fmicb.2012.00340.
[29]MORRISON D J,TOM P.Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism[J].Gut Microbes,2016,7(3):189-200.DOI:10.1080/19490976.2015.11340822.
[30]FELD L,BIELAK E,HAMMER K,et al.Characterization of a small erythromycin resistance plasmid pLFE1 from the food-isolate Lactobacillus plantarum M345[J].Plasmid,2009,61(3):159-170.DOI:10.1016/j.plasmid.2009.01.002.
[31]?T?EPETOVA J,TAELMA H,SMIDT I,et al.Assessment of phenotypic and genotypic antibiotic susceptibility of vaginal Lactobacillus sp[J].Journal of Applied Microbiology,2017,123(2):524-534.DOI:10.1111/jam.13497.
[32]李少英.奶牛源性双歧杆菌和乳杆菌的分离鉴定及耐药性研究[D].呼和浩特:内蒙古农业大学,2008:1-111.
[33]邵玉宇.干酪乳杆菌和植物乳杆菌耐药分子机制的研究[D].呼和浩特:内蒙古农业大学,2015:1-115.
[34]HERRMANN J,HERMES R,BREVES G.Transepithelial transport and intraepithelial metabolism of short-chain fatty acids(SCFA)in the porcine proximal colon are influenced by SCFA concentration and luminal pH[J].Comparative Biochemistry&Physiology Part A Molecular&Integrative Physiology,2011,158(1):169-176.DOI:10.1016/j.cbpa.2010.10.018.
[35]GEERLINGS S,KOSTOPOULOS I,BELZER C,et al.Akkermansia muciniphila in the human gastrointestinal tract:when,where,and how?[J].Microorganisms,2018,6(3):1-26.DOI:10.3390/microorganisms6030075.
[36]EVERARD A,LAZAREVIC V,GAIA N,et al.Microbiome of prebiotic-treated mice reveals novel targets involved in host response during obesity[J].The ISME Journal,2014,8(10):2116-2130.DOI:10.1038/ismej.2014.45.
[37]KANG D W,DIBAISE J K,ILHAN Z E,et al.Gut microbial and short-chain fatty acid profiles in adults with chronic constipation before and after treatment with lubiprostone[J].Anaerobe,2015,33:33-41.DOI:10.1016/j.anaerobe.2015.01.005.
[38]李川.植物乳杆菌NCU116的益生功能及其作用机制[D].南昌:南昌大学,2015:1-147.