自然发酵豆酱中降胆固醇乳酸菌的筛选鉴定及对大鼠血清胆固醇的影响
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摘要
采用不同培养基从分离自自然发酵豆酱样品中的285株乳酸菌种中分离筛选出3株能够耐受人工胃肠环境,具有降胆固醇作用的乳酸菌,编号分别为DQ17-2、DD1-1、KY1-1,对胆固醇的去除率分别为34. 56%、27. 12%和29. 20%,最后通过形态学观察、生理生化试验、16S r DNA序列鉴定法确定3株乳酸菌均为植物乳杆菌。将菌株DQ17-2重新命名为植物乳杆菌WW(L. plantarum WW),为了进一步验证其在体内的降胆固醇作用,将32只5周龄雄性SD大鼠随机分为4组:普通对照组(A)、高脂模型组(B)、脱脂乳对照组(C)、菌液干预组(D)。饲喂12周后,采血,测定血清总胆固醇(totalcholesterol,TC)、高密度脂蛋白胆固醇(highdensity lipoprotein-cholesterol,HDL-C)、低密度脂蛋白胆固醇(lowdensity lipoprotein-cholesterol,LDL-C)、甘油三酯(triglyceride,TG)含量。结果表明,饲喂12周后成功构建高脂模型,与B组和C组相比,D组血清TC、TG、LDL-C含量明显降低(P <0. 05),HDL-C含量明显升高(P <0. 05)。该实验筛选出一株在体内外均具有降胆固醇作用的L. plantarum WW,为今后降胆固醇产品的研发提供原料与依据。
Three lactic acid bacteria strains,DQ17-2,DD1-1 and KY1-1,being resistant to the artificial gastrointestinal environment and cholesterol-lowering effect( the cholesterol removal rates were 34. 56%,27. 12% and29. 20%,respectively),were selected from 285 isolates of lactic acid bacteria,which were isolated from the naturally fermented soybean sauces using three different media. They were identified to be Lactobacillus plantarum by morphology,physiological and biochemical tests and 16 S r DNA sequencing. The strain DQ17-2 was renamed as In order to further verify its in vivo cholesterol-lowering effect,L. plantarum WW cell suspends were fed 32 5-week-old male SD rats within randomly 4 groups [Control group( A),High fat group( B),Skim milk control group( C) and L. plantarum WW fed group( D) ]. After feeding for 12 weeks,the levels of total cholesterol( TC),high density lipoprotein-cholesterol( HDL-C),low density lipoprotein-cholesterol( LDL-C) and triglyceride( TG) in serum were examined. The results showed that the model of hyperlipidemia was successfully established and the levels of TC,TG,and LDL-C in group D were significantly lower than those of group B and C( P < 0. 05) and the level of HDL-C was significantly increased( P < 0. 05). In conclusion,L. plantarum WW had both in vivo and in vitro cholesterol-lowering effect,which provides a new strategy for development of cholesterol-lowering products.
Three lactic acid bacteria strains,DQ17-2,DD1-1 and KY1-1,being resistant to the artificial gastrointestinal environment and cholesterol-lowering effect( the cholesterol removal rates were 34. 56%,27. 12% and29. 20%,respectively),were selected from 285 isolates of lactic acid bacteria,which were isolated from the naturally fermented soybean sauces using three different media. They were identified to be Lactobacillus plantarum by morphology,physiological and biochemical tests and 16 S r DNA sequencing. The strain DQ17-2 was renamed as In order to further verify its in vivo cholesterol-lowering effect,L. plantarum WW cell suspends were fed 32 5-week-old male SD rats within randomly 4 groups [Control group( A),High fat group( B),Skim milk control group( C) and L. plantarum WW fed group( D) ]. After feeding for 12 weeks,the levels of total cholesterol( TC),high density lipoprotein-cholesterol( HDL-C),low density lipoprotein-cholesterol( LDL-C) and triglyceride( TG) in serum were examined. The results showed that the model of hyperlipidemia was successfully established and the levels of TC,TG,and LDL-C in group D were significantly lower than those of group B and C( P < 0. 05) and the level of HDL-C was significantly increased( P < 0. 05). In conclusion,L. plantarum WW had both in vivo and in vitro cholesterol-lowering effect,which provides a new strategy for development of cholesterol-lowering products.
引文
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[24]武俊瑞.东北传统发酵特色食品中主要微生物多样性研究[D].沈阳:沈阳农业大学,2013.
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[29] HU Xu,WANG Tao,LI Wei,et al. Effects of NS Lactobacillus strains on lipid metabolism of rats fed a high-cholesterol diet[J]. Lipids in Health and Disease,2013,12(1):67.
[30] RAO K P,CHENNAPPA G,SURAJ U,et al. Probiotic potential of Lactobacillus strains isolated from sorghumbased traditional fermented food[J]. Probiotics&Antimicrobiology Protection,2015,7(2):146-156.
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[32]于平,汪晓辉.植物乳杆菌Lp T1和Lp T2大鼠体内降胆固醇特性[J].微生物学报,2012,52(1):124-129.
[33]张晓磊.益生菌发酵豆乳对高脂饮食小鼠降血脂及缓解肝损伤作用[D].长春:吉林农业大学,2016.
[34] LI C,NIE S P,DING Q,et al. 2014. Cholesterol-lowering effect of Lactobacillus plantarum NCU116 in a hyperlipidaemic rat model[J]. Journal of Functional Foods,8(8):340-347.
[2] KIM Y,RYU B H,KIM J,et al. Characterization of a novel SGNH-type esterase from Lactobacillus plantarum[J].International Journal of Biological Macromolecules,2017,96:560-568.
[3]肖仔君,钟瑞敏,陈惠音,等.植物乳杆菌的生理功能与应用[J].中国食品添加剂,2005(2):87-89.
[4] PEREIRA,D I A,GIBSON G R. Effects of consumption of probiotics and prebiotics on serum lipid levels in humans[J]. Critical Reviews in Biochemistry and Molecular Biology,2002,37(4):259-281.
[5]王俊国,孟和毕力格,包秋华,等.植物乳杆菌LIP-1对高脂血症大鼠血脂的调节作用[J].中国食品学报,2013,13(2):6-12.
[6]齐炳理,张兴昌,孙婷,等.植物乳杆菌发酵乳对高脂血症大鼠血脂的影响[J].食品研究与开发,2015,36(20):17-19.
[7] AHMED R M,ELSANHOTY R M,AL-SAMAN A A,et al. Hypocholesterolaemic effect of probiotic yogurt enriched with barleyβ-glucan in rats fed on a high-cholesterol diet[J]. Mediterranean Journal of Nutrition&Metabolism,2017,10(1):1-12. DOI:10. 3233/MNM-16114.
[8] LE BLANC J G,GARRO M S,SILVESTRONI A,et al.Reduction of alpha-galactooligosaccharides in soymilk by Lactobacillus fermentum CRL 722:In vitro and in vivo evaluation of fermented soymilk[J]. Appl Microbiol,2004,97(4):876-881.
[9] CHIEN Y L,WU L Y,LEE T C,et al. Cholesterol-lowering effect of phytosterol-containing lactic-fermented milk powder in hamsters[J]. Food Chemistry,2010,119(3):1 121-1 126.
[10] KIM S J,PARK S H,SIN H S,et al. Hypocholesterolemic Effects of Probiotic Mixture on Diet-Induced Hypercholesterolemic Rats[J]. Nutrients,2017,9(3):293.
[11] LYE H S,KATO T,LOW W Y,et al. Lactobacillus fermentum FTDC 8312 combats hypercholesterolemia via alteration of gut microbiota[J]. Journal of Biotechnology,2017,262:75-83.
[12] OLIVEIRA P M,BROSNAN B,FUREY A,et al. Lactic acid bacteria bioprotection applied to the malting process.Part I:Strain characterization and identification of antifungal compounds[J]. Food Control,2015,51:433-443.
[13]胡梦坤,岳喜庆.植物乳杆菌LP1103的筛选及其降胆固醇作用机理的研究[J].食品科学,2008,29(6):226-229.
[14] DING Wu-rong,SHI Chao,CHEN Ming,et al. Screening for lactic acid bacteria in traditional fermented Tibetan yak milk and evaluating their probiotic and cholesterollowering potentials in rats fed a high-cholesterol diet[J].Journal of Functional Foods,2017,32:324-332.
[15]王友湘,陈庆森,阎亚丽.用于乳酸菌分离鉴定的几种培养基的筛选及应用[J].食品科学,2007,28(9):374-378.
[16]凌代文,东秀珠.乳酸细菌分类鉴定及试验方法[M].北京:中国轻工业出版社,1999,(1):1-25.
[17]郭东起,侯红漫,刘阳,等.降胆固醇益生乳酸菌的体外筛选[J].中国乳品工业,2007,8(35):10-14.
[18]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[19]冯广达,陈美标,羊宋贞,等.用于PCR扩增的细菌DNA提取方法比较[J].华南农业大学学报,2013,34(3):440-442.
[20]李铁. Gen Bank数据库检索及其应用[J].中华医学图书情报杂志,2008,17(5):49-51.
[21] LIONG M T,SHAH N P. Bile salt deconjugation and BSH activity of five bifidobacterial strains and their cholesterol co-precipitating properties[J]. Food Research International,2005,38(2):135-142.
[22] KIMOTO H,OHMOMO S,OKAMOTO T. Cholesterol removal from media by lactococci[J]. Journal of Dairy Science,2002,85(12):3 182-3 188.
[23] ZHANG X L,WU Y F,WANG Y S,et al. The protective effects of probiotic-fermented soymilk on high-fat dietinduced hyperlipidemia and liver injury[J]. Journal of Functional Foods,2017,30:220-227.
[24]武俊瑞.东北传统发酵特色食品中主要微生物多样性研究[D].沈阳:沈阳农业大学,2013.
[25]武俊瑞,岳喜庆,石璞,等. PCR-DGGE分析东北自然发酵酸菜中乳酸菌多样性[J].食品与生物技术学报,2014,33(2):127-130.
[26] WASSENAAR T M,KLEIN G. Safety aspects and implications of regulation of probiotic bacteria in food and food supplements[J]. Journal of Food Protection,2008,71(8):1 734-1 741.
[27] FAO/WHO. Guidelines for the evaluation of probiotics in food. Report of a joint FAO/WHO working group on drafting guidelines for evaluation of probiotics in food,London,Ontario. Canada. 2002.
[28] MANSON J E,TOSTESON H,RIDKER P M,et al. The primary prevention of myocardial infaction[J]. New England Journal of Medicine,1992,326(21):1 406-1 416.
[29] HU Xu,WANG Tao,LI Wei,et al. Effects of NS Lactobacillus strains on lipid metabolism of rats fed a high-cholesterol diet[J]. Lipids in Health and Disease,2013,12(1):67.
[30] RAO K P,CHENNAPPA G,SURAJ U,et al. Probiotic potential of Lactobacillus strains isolated from sorghumbased traditional fermented food[J]. Probiotics&Antimicrobiology Protection,2015,7(2):146-156.
[31] HUANG Ying,WANG Xiao-jun,WANG Jin-feng,et al.Lactobacillus plantarum strains as potential probiotic cultures with cholesterol-lowering activity[J]. Journal of Dairy Science,2013,96(5):2 746-2 753.
[32]于平,汪晓辉.植物乳杆菌Lp T1和Lp T2大鼠体内降胆固醇特性[J].微生物学报,2012,52(1):124-129.
[33]张晓磊.益生菌发酵豆乳对高脂饮食小鼠降血脂及缓解肝损伤作用[D].长春:吉林农业大学,2016.
[34] LI C,NIE S P,DING Q,et al. 2014. Cholesterol-lowering effect of Lactobacillus plantarum NCU116 in a hyperlipidaemic rat model[J]. Journal of Functional Foods,8(8):340-347.