粪肠球菌R-WL发酵培养基的优化
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摘要
本试验旨在优化培养基提高粪肠球菌(Enterococcus faecalis)R-WL发酵液的菌体密度,为高效、优质的微生态制剂产品研制奠定基础。首先通过生长曲线的测定、培养条件和培养基成分的单因素试验,确定菌种RWL的最适培养条件为:时间14 h、pH 8.5、温度37℃、接种量3%,最适碳源为乳糖,浓度为10 g/L,最适氮源为蛋白胨+酵母膏,浓度为30 g/L。然后通过Plackett-Burman试验分析培养基中对粪肠球菌发酵影响最重要的主要因素为蛋白胨、乙酸钠和柠檬酸二铵,再通过最陡爬坡试验获得主要因素的最适范围,最后通过Box-Behnken试验和响应面分析得到主要因素的最适添加量。结果表明:最优发酵培养基配方为:乳糖10 g/L、酵母膏15 g/L、蛋白胨12.18 g/L、乙酸钠4.5 g/L、柠檬酸二铵4.5 g/L、硫酸镁0.58 g/L、硫酸锰0.25 g/L、磷酸氢二钾2 g/L、轻质碳酸钙3 g/L,初始pH 8.5。通过试验验证,优化后发酵培养基的发酵菌液活菌数可达(6.44±0.10)×109cfu/mL,比原MRS培养基活菌数提高了71.28%。
This experiment aimed to improve the concentration of Enterococcus faecalis R-WL by optimizing fermentation medium,and to lay the foundation for efficient quality probiotics.First,measured the growth curve assay and single factor test for culture condition and medium composition.The optimum incubation time,pH,temperature,inoculation amount of R-WL were 14 h,8.5,37 ℃,3%,the optimum carbon and nitrogen sources were lactose and peptone + yeast extract,the concentration were 10 g/L and 30 g/L,respectively.Plackett-Burman test was applied to analyze the main factors of fermentation medium were peptone,sodium acetate and diammonium citrate,and then the main factors were tested through the steepest ascent method to get the optimal range.Finally,the optimum addition of main factors was obtained by Box-Behnken test and the response surface methodology. The optimal formulation of fermentation medium were:lactose 10 g/L,yeast extract15 g/L,peptone 12.18 g/L,sodium acetate 4.5 g/L,diammonium citrate 4.5 g/L,magnesium sulphate 0.58 g/L,manganese sulfate 0.25 g/L,potassium hydrogen phosphate 2 g/L,light calcium carbonate 3 g/L,pH 8.5.Enterococcus faecalis were cultured by the optimized fermentation medium,and the viable count reached(6.44±0.10)×109 cfu/mL,which increased by 71.28% compared with the number of viable count in MRS culture.
This experiment aimed to improve the concentration of Enterococcus faecalis R-WL by optimizing fermentation medium,and to lay the foundation for efficient quality probiotics.First,measured the growth curve assay and single factor test for culture condition and medium composition.The optimum incubation time,pH,temperature,inoculation amount of R-WL were 14 h,8.5,37 ℃,3%,the optimum carbon and nitrogen sources were lactose and peptone + yeast extract,the concentration were 10 g/L and 30 g/L,respectively.Plackett-Burman test was applied to analyze the main factors of fermentation medium were peptone,sodium acetate and diammonium citrate,and then the main factors were tested through the steepest ascent method to get the optimal range.Finally,the optimum addition of main factors was obtained by Box-Behnken test and the response surface methodology. The optimal formulation of fermentation medium were:lactose 10 g/L,yeast extract15 g/L,peptone 12.18 g/L,sodium acetate 4.5 g/L,diammonium citrate 4.5 g/L,magnesium sulphate 0.58 g/L,manganese sulfate 0.25 g/L,potassium hydrogen phosphate 2 g/L,light calcium carbonate 3 g/L,pH 8.5.Enterococcus faecalis were cultured by the optimized fermentation medium,and the viable count reached(6.44±0.10)×109 cfu/mL,which increased by 71.28% compared with the number of viable count in MRS culture.
引文
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[11] Djellouli M,Martinez-Alvarez O,Arancibia MY,et al.Effect of seafood peptones on biomass and metabolic activity by Enterococcus faecalis DM19[J].Lwt-Food Science and Technology,2017,81:94~100.
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[15] Li P H,Niu Q,Wei Q T,et al.Microbial shifts in the porcine distal gut in response to diets supplemented with Enterococcus Faecalis as alternatives to antibiotics[J].Scientific Reports,2017,7:1~10.
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[18] Lopes R P,Mota M J, Gomes A M,et al.Application of High Pressure with Homogenization,Temperature,Carbon Dioxide,and Cold Plasma for the Inactivation of Bacterial Spores:A Review[J].Comprehensive Reviews in Food Science and Food Safety,2018,17(3),532~535.
[19] Montgomery.实验设计与分析[M].傅钰生,张健,王振羽,等,译.6版.北京:人民邮电出版社,2009:366~379.
[20] Perumal V,Repally A,Dasari A,et al.Partial purification and characterization of bacteriocin produced by Enterococcus faecalis DU10 and its probiotic attributes[J].Preparative Biochemistry&Biotechnology,2016,46(7):686~694.
[21] Silva B C,Jung LRC,Sandes SHC,et al.In vitro assessment of functional properties of Lactic acid bacteria isolated from faecal microbiota of healthy dogs for potential use as probiotics[J]. Beneficial Microbes,2013,4(3):267~275.
[22] Song D,Wang Y W,Hou Y J,et al.The effects of dietary supplementation of microencapsulated Enterococcus faecalis and the extract of Camellia oleifera seed on growth performance,immune functions,and serum biochemical parameters in broiler chickens[J].Journal of Animal Science,2016,94(8):3271~3277.
[23] Yoo H,Rheem I,Rheem S,et al.Optimizing medium components for the maximum growth of Lactobacillus plantarum JNU2116 Using response surface methodology[J].Korean Journal for Food Science of Animal Resources,2008,38(2):240~250.
[24] Zeng J,Wang CT,Zhang FS,et al.Effect of probiotics on the incidence of ventilator-associated pneumonia in critically ill patients:a randomized controlled multicenter trial[J].Intensive Care Medicine,2016,42(6):1018~1028.
[25] Zhang G Y,Mills D A,Block D E.Development of chemically defined media supporting high-cell-density growth of Lactococci,Enterococci,and Streptococci[J].Applied and Environmental Microbiology,2009,75(4):1080~1087.
[2]刘香英,孙洪蕊,范海茹,等.益生性植物乳杆菌K25高密度培养工艺优化[J].Food Science and technology,2018,43(3):20~24.
[3]闵伟红,崔学祥,方丽,等.碱性中和与补料分批高密度培养保加利亚乳杆菌的研究[J].食品科学,2009,30(1):217~221.
[4]张广敏,王炜,包慧芳,等.植物乳杆菌Lp-2的高密度发酵[J].中国生物工程杂志,2009,29(6):68~73.
[5] Allameh S K,Ringo E,Yusoff F M,et al.Dietary supplement of Enterococcus faecalis on digestive enzyme activities,short-chain fatty acid production,immune system response and disease resistance of Javanese carp(Puntius gonion otus,Bleeker 1850)[J].Aquaculture Nutrition,2017,23(2):331~338.
[6] Amachawadi R G,Giok F,Shi X R,et al.Antimicrobial resistance of Enterococcus faecium strains isolated from commercial probiotic products used in cattle and swine[J].Journal of Animal Science,2018,96(3):912~920.
[7] Ba H V,Seo H W,Cho S H,et al.Utilisation possibility of Enterococcus faecalis isolates from neonate's faeces for production of fermented sausages as starter cultures[J].International Journal of Food Science and Technology,2017,52(7):1660~1669.
[8] Ben B O,Morandi S,Cremonesi P,et al.Biotechnological potential,probiotic and safety properties of newly isolated enterocin-producing Enterococcus lactic strains[J].Lwt-Food Science and Technology,2018,92:361~370.
[9] Box G E P.Statistics for experiments:an Introduction to Design,Data Analysis and Model Building[M].New York:Wiley,1990.
[10] Chen M F,Weng K F,Huang S Y,et al.Pretreatment with a heat-killed probiotic modulates monocyte chemoattractant protein-1and reduces the pathogenicity of influenza and enterovirus 71 infections[J].Mucosal Immunology,2017,10(1):215~227.
[11] Djellouli M,Martinez-Alvarez O,Arancibia MY,et al.Effect of seafood peptones on biomass and metabolic activity by Enterococcus faecalis DM19[J].Lwt-Food Science and Technology,2017,81:94~100.
[12] Hassani A,Zarnkow M, Becker T.Optimisation of fermentation conditions for probiotication of sorghum wort by Lactobacillus acidophilus LA5[J].International Journal of Food Science and Technology,2015,50(10):2271~2279.
[13] Hwanhlem N,Ivanova T,Biscola V,et al.Bacteriocin producing Enterococcus faecalis isolated from chicken gastrointestinal tract originating from Phitsanulok,Thailand:Isolation,screening,safety evaluation and probiotic properties[J].Food Control,2017,78:187~195.
[14] Kwon S,Lee P C,Lee E G,et al.Production of lactic acid by Lactobacillus rhamnosus with vitamin supplemented soybean hydrolysate[J].Enzyme MicrobialTechnology,2000,26(2~4):209~215.
[15] Li P H,Niu Q,Wei Q T,et al.Microbial shifts in the porcine distal gut in response to diets supplemented with Enterococcus Faecalis as alternatives to antibiotics[J].Scientific Reports,2017,7:1~10.
[16] Liu C Q,Zhu Q,Chang J,et al.Effects of Lactobacillus casei and Enterococcus faecalis on growth performance,immune function and gut microbiota of suckling piglets[J].Archives Of Animal Nutrition,2017,71(2):120~133.
[17] Liu X T,Wang Y,Wang H L,et al.Effect of a liquid culture of Enterococcus faecalis CGMCC 1.101 cultivated by a high density process on the performance of weaned piglets[J].Livestock Science,2014,170:100~107.
[18] Lopes R P,Mota M J, Gomes A M,et al.Application of High Pressure with Homogenization,Temperature,Carbon Dioxide,and Cold Plasma for the Inactivation of Bacterial Spores:A Review[J].Comprehensive Reviews in Food Science and Food Safety,2018,17(3),532~535.
[19] Montgomery.实验设计与分析[M].傅钰生,张健,王振羽,等,译.6版.北京:人民邮电出版社,2009:366~379.
[20] Perumal V,Repally A,Dasari A,et al.Partial purification and characterization of bacteriocin produced by Enterococcus faecalis DU10 and its probiotic attributes[J].Preparative Biochemistry&Biotechnology,2016,46(7):686~694.
[21] Silva B C,Jung LRC,Sandes SHC,et al.In vitro assessment of functional properties of Lactic acid bacteria isolated from faecal microbiota of healthy dogs for potential use as probiotics[J]. Beneficial Microbes,2013,4(3):267~275.
[22] Song D,Wang Y W,Hou Y J,et al.The effects of dietary supplementation of microencapsulated Enterococcus faecalis and the extract of Camellia oleifera seed on growth performance,immune functions,and serum biochemical parameters in broiler chickens[J].Journal of Animal Science,2016,94(8):3271~3277.
[23] Yoo H,Rheem I,Rheem S,et al.Optimizing medium components for the maximum growth of Lactobacillus plantarum JNU2116 Using response surface methodology[J].Korean Journal for Food Science of Animal Resources,2008,38(2):240~250.
[24] Zeng J,Wang CT,Zhang FS,et al.Effect of probiotics on the incidence of ventilator-associated pneumonia in critically ill patients:a randomized controlled multicenter trial[J].Intensive Care Medicine,2016,42(6):1018~1028.
[25] Zhang G Y,Mills D A,Block D E.Development of chemically defined media supporting high-cell-density growth of Lactococci,Enterococci,and Streptococci[J].Applied and Environmental Microbiology,2009,75(4):1080~1087.