甘露二糖、甘露三糖体外抑制大肠杆菌对IPEC-1细胞的黏附作用研究
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摘要
试验以猪小肠上皮细胞(IPEC-1)为模型,探讨甘露二糖和甘露三糖抑制大肠杆菌对IPEC-1细胞黏附、侵袭和损伤的影响。在IPEC-1细胞培养基中分别加入甘露二糖和甘露三糖,使培养基中甘露二糖和甘露三糖终浓度分别为0(对照组)、5、10、20μg/mL和40μg/mL。采用平板计数法检测甘露二糖和甘露三糖对IPEC-1黏附与侵袭的影响;建立大肠杆菌诱导细胞损伤模型,采用乳酸脱氢酶(LDH)法检测甘露寡糖对细胞损伤的修复作用。试验结果表明:与对照组相比,甘露二糖和甘露三糖能显著抑制大肠杆菌对IPEC-1细胞的黏附与侵袭,且随着甘露二糖和甘露三糖浓度的增加,其抑制效果增强,具有剂量效应,甘露二糖、甘露三糖浓度在40μg/mL时,其对大肠杆菌的黏附抑制达到最强;甘露二糖、甘露三糖能降低大肠杆菌对IPEC-1细胞的损伤。甘露二糖和甘露三糖能抑制致病性大肠杆菌对IPEC-1细胞的黏附、侵袭和损伤,保证细胞的完整性。
The effect of mannobiose and mannotriose on adhesion, invasion and injury of E.coli to IPEC-1 cells in vitro were investigated in this experiment. The mannobiose and mannotriose were supplied into the cell culture medium of IPEC-1 separately, and keep the final concentrations of mannobiose and mannotriose in the medium were 0(control), 5, 10, 20 μg/mLand 40 μg/mL, respectively. The effects of mannobiose and mannotriose on the adhesion and invasion of IPEC-1 were detected by plate counting. The injury cell model of E. coli was established and the repair effect of manno-oligosaccharide on cell damage was detected by lactate dehydrogenase(LDH). The results showed that: The adhesion and invasion of E. coli to IPEC-1 cells were inhibited significantly in mannobiose and mannotriose treatments compared with the control group, and increased with the dose of mannobiose and mannotriose. The best inhibition ability of adhesion to E. coli was attained at 40 μg/mL of mannobiose and mannotriose. Manno-oligosaccharide could reduce the damage rate of IPEC-1 cells. The experiment revealed that mannobiose and mannotriose could inhibit the adhesion, invasion and damage of E.coli to IPEC-1 cells in vitro.
The effect of mannobiose and mannotriose on adhesion, invasion and injury of E.coli to IPEC-1 cells in vitro were investigated in this experiment. The mannobiose and mannotriose were supplied into the cell culture medium of IPEC-1 separately, and keep the final concentrations of mannobiose and mannotriose in the medium were 0(control), 5, 10, 20 μg/mLand 40 μg/mL, respectively. The effects of mannobiose and mannotriose on the adhesion and invasion of IPEC-1 were detected by plate counting. The injury cell model of E. coli was established and the repair effect of manno-oligosaccharide on cell damage was detected by lactate dehydrogenase(LDH). The results showed that: The adhesion and invasion of E. coli to IPEC-1 cells were inhibited significantly in mannobiose and mannotriose treatments compared with the control group, and increased with the dose of mannobiose and mannotriose. The best inhibition ability of adhesion to E. coli was attained at 40 μg/mL of mannobiose and mannotriose. Manno-oligosaccharide could reduce the damage rate of IPEC-1 cells. The experiment revealed that mannobiose and mannotriose could inhibit the adhesion, invasion and damage of E.coli to IPEC-1 cells in vitro.
引文
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[21]赵玉蓉,王耀东.牛膝多糖抑制沙门氏菌侵染猪小肠上皮细胞的研究[J].动物营养学报,2018(12):5083-5088.
[22]Ebersbach T,Andersen J B,Anders Bergstrom,et al.Xylooligosaccharides inhibit pathogen adhesion to enterocytes in vitro[J].Research in Microbiology,2012,163(1):22-27.
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[28]孙盛明,谢骏,朱健,等.饲料中添加甘露寡糖对团头鲂幼鱼生长性能、抗氧化能力和肠道菌群的影响[J].动物营养学报,2014(11):2273-3379.
[29]Jahanian E,Mahdavi A H,Asgary S,et al.Effect of dietary supplementation of mannanoligosaccharides on growth performance,ileal microbial counts,and jejunal morphology in broiler chicks exposed to aflatoxins[J].Livestock Science,2016,157(2):123-130.
[30]Dimitroglou A,Merrifield D L,spring P,et al.Effects of mannanoligosaccharide(MOS)supplementation on growth performance,feed utilisation,intestinal histology and gut microbiota of gilthead sea bream(Sparusaurata)[J].Aquaculture,2010,300(1/4):182-188.
[31]Spitzer F,Speiser S,Vahjen W,et al.Effect of different feed ingredients and additives on IPEC-J2 cells challenged with an enterotoxigenic Escherichia coli strain[J].Cytotechnology,2016,68(4):1463-1471.
[32]马相杰,汪立平,赵勇,等.甘露寡糖对罗非鱼幼鱼肠道微生物的影响[J].微生物学通报,2010,37(5):708-713.
[33]杜英男,鞠贵春,薛军,等.果聚糖和甘露寡糖对水貂肠道菌群影响的研究[J].经济动物学报,2007,11(2):83-86.
[34]Castill S L,Heredia N,Contreras J F,et al.Extracts of edible and medicinal plants in inhibition of growth,adherence,and cytotoxin production of Campylobacter jejuni and Campylobacter coli[J].Journal of Food Science,2011,76(6):6.
[35]吴士.魔芋甘露低聚糖与枯草芽孢杆菌对肠上皮细胞损伤的协同修复作用[D].武汉:华中农业大学,2017.
[36]施小凤,傅晋翔,朱彦,等.乳酸脱氢酶、羟丁酸脱氢酶检测在巨幼细胞性贫血诊治中的意义[J].苏州大学学报(医学版),2009(6):1179-1181.
[37]冯莉,安雪娇,齐妍,等.魔芋低聚糖对三硝基苯磺酸致小鼠溃疡性结肠炎的保护作用[J].食品工业科技,2015,36(1):349-352.
[38]张帅.魔芋甘露低聚糖与枯草芽孢杆菌对LPS诱导肠道氧化损伤的保护作用及机制研究[D].武汉:华中农业大学,2018.
[2]Halas V,Nochta I.Mannan oligosaccharides in nursery pig nutrition and their potential mode of action[J].Animals An Open Access Journal from Mdpi,2012,2(2):261-274.
[3]Ganan M,Carrascosa A V,Pascualteresa S D,et al.Inhibition by yeastderived mannoproteins of adherence to and invasion of caco-2 cells by campylobacter jejuni[J].J.Food Prot.,2009,72(1):55-59.
[4]Santin E,Maiorka A,MacarI M,et al.Performance and intestinal mucosa development of broiler chickens fed diets containing Saccharomyces cerevisiae cell wall[J].Journal of Applied Poultry Research,2001,10(3):236-244.
[5]Mcsweegan E,Walaker R I.Identification and characterization of two campylobacter jejuniadhesins for cellular and mucous substrates[J].Infection&Immunity,1986,53(1):141.
[6]Shoaf K,Mulvey G L,Armstrong G D,et al.Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells[J].Infection and Immunity,2007,74(12):6920-6928.
[7]Russell R G,JR B D C.Cell association and invasion of Caco-2 cells by campylobacter jejuni[J].Infection&Immunity,1994,62(9):3773-3779.
[8]Baurhoob,Letellier A,Zhao X,et al.Cecal populations of lactobacilli and bifidobacteria and Escherichia coli populations after in vivo Escherichia coli challenge in birds fed diets with purified lignin or mannanoligosaccharides[J].Poult.Sci.,2007,86(12):2509-2516.
[9]李溪,王金荣,赵银丽,等.甘露寡糖对乳酸粪球菌和沙门氏菌细胞凝集的影响研究[J].饲料研究,2016(3):1-7.
[10]王金荣,谢金鹏,苏兰利,等.甘露寡糖对大肠杆菌体外凝集作用的研究[C]//第七届中国猪营养学术研讨会论文集,2015:270.
[11]姚晓红,孙宏,吴逸飞,等.甘露寡糖对枯草芽孢杆菌在肉鸡肠道内的排出及肠道菌群的影响[J].中国畜牧杂志,2011(17):56-59.
[12]秦清娟,张媛,刘倍毓,等.半干法酶解制备魔芋葡甘低聚糖工艺及其抗氧化性能研究[J].食品工业科技,2013,34(23):186-191.
[13]孙芝兰.卷曲乳杆菌益生特性及黏附机理研究[D].济南:山东大学,2012.
[14]王定发,黄代勇,晏邦富.甘露寡糖对犊牛血液免疫球蛋白的研究[J].饲料研究,2004(3):41-42.
[15]Ganan M,Carrascosa A V,S.De Pascual-teresa,et al.Effect of mannoproteins on the growth,gastrointestinal viability,and adherence to caco-2 cells of lactic acid bacteria[J].Journal of Food Science,2012,77(3):5.
[16]Hori K,Matsumoto S.Bacterial adhesion:From mechanism to control[J].Biochemical Engineering Journal,2010,48(3):424-434.
[17]Ramirez-Hemandeza,Rupnow J,Hutkins R W.Adherence reduction of Campylobacter jejuni and Campylobacter coliStrains to HEp-2 cells by mannan oligosaccharides and a high-molecular-weight component of cranberry extract[J].Journal of Food Protection,2015,78(8):1496-1505.
[18]潘晓东.若干寡糖的功能特性及对肠道生理生态调控机制的研究[D].杭州:浙江大学,2009.
[19]Halsa V,Nochta I.Mannan oligosaccharides in nursery pig nutrition and their potential mode of action[J].Animals An Open Access Journal from Mdpi,2012,2(2):261-274.
[20]Lane J A,Marino KK,Rudd P M,et al.Methodologies for screening of bacteria-carbohydrate interactions:anti-adhesive milk oligosaccharides as a case study[J].J.Microbiol.Methods,2012,90(1):53-59.
[21]赵玉蓉,王耀东.牛膝多糖抑制沙门氏菌侵染猪小肠上皮细胞的研究[J].动物营养学报,2018(12):5083-5088.
[22]Ebersbach T,Andersen J B,Anders Bergstrom,et al.Xylooligosaccharides inhibit pathogen adhesion to enterocytes in vitro[J].Research in Microbiology,2012,163(1):22-27.
[23]Lane J A,Mario K,Naughton J,et al.Anti-infective bovine colostrum oligosaccharides:Campylobacter jejuni as a case study[J].International Journal of Food Microbiology,2012,157(2):182-188.
[24]Ganan M,Collins M,Rastall R,et al.Inhibition by pectic oligosaccharides of the invasion of undifferentiated and differentiated Caco-2 cells by Campylobacter jejuni[J].International Journal of Food Microbiology,2010,137(2/3):181-185.
[25]Park B K,Kim M M.Applications of chitin and its derivatives in biological medicine[J].International Journal of Molecular Sciences,2010,11(12):5152-5164.
[26]Lodhi G,Kim Y S,Hwang J W,et al.Chitooligosaccharide and its derivatives:preparation and biological applications[J].Bio.Med.Research International,2014,2014(1):654913.
[27]计成,王晓霞,易中华,等.果寡糖和枯草芽孢杆菌对肉鸡肠道菌群数量、发酵粪中氨气和硫化氢散发量及营养素利用率的影响[C]//全国家禽营养与饲料科技研讨会,2007.
[28]孙盛明,谢骏,朱健,等.饲料中添加甘露寡糖对团头鲂幼鱼生长性能、抗氧化能力和肠道菌群的影响[J].动物营养学报,2014(11):2273-3379.
[29]Jahanian E,Mahdavi A H,Asgary S,et al.Effect of dietary supplementation of mannanoligosaccharides on growth performance,ileal microbial counts,and jejunal morphology in broiler chicks exposed to aflatoxins[J].Livestock Science,2016,157(2):123-130.
[30]Dimitroglou A,Merrifield D L,spring P,et al.Effects of mannanoligosaccharide(MOS)supplementation on growth performance,feed utilisation,intestinal histology and gut microbiota of gilthead sea bream(Sparusaurata)[J].Aquaculture,2010,300(1/4):182-188.
[31]Spitzer F,Speiser S,Vahjen W,et al.Effect of different feed ingredients and additives on IPEC-J2 cells challenged with an enterotoxigenic Escherichia coli strain[J].Cytotechnology,2016,68(4):1463-1471.
[32]马相杰,汪立平,赵勇,等.甘露寡糖对罗非鱼幼鱼肠道微生物的影响[J].微生物学通报,2010,37(5):708-713.
[33]杜英男,鞠贵春,薛军,等.果聚糖和甘露寡糖对水貂肠道菌群影响的研究[J].经济动物学报,2007,11(2):83-86.
[34]Castill S L,Heredia N,Contreras J F,et al.Extracts of edible and medicinal plants in inhibition of growth,adherence,and cytotoxin production of Campylobacter jejuni and Campylobacter coli[J].Journal of Food Science,2011,76(6):6.
[35]吴士.魔芋甘露低聚糖与枯草芽孢杆菌对肠上皮细胞损伤的协同修复作用[D].武汉:华中农业大学,2017.
[36]施小凤,傅晋翔,朱彦,等.乳酸脱氢酶、羟丁酸脱氢酶检测在巨幼细胞性贫血诊治中的意义[J].苏州大学学报(医学版),2009(6):1179-1181.
[37]冯莉,安雪娇,齐妍,等.魔芋低聚糖对三硝基苯磺酸致小鼠溃疡性结肠炎的保护作用[J].食品工业科技,2015,36(1):349-352.
[38]张帅.魔芋甘露低聚糖与枯草芽孢杆菌对LPS诱导肠道氧化损伤的保护作用及机制研究[D].武汉:华中农业大学,2018.