研究报告

真空冷冻干燥保护剂对植物乳杆菌67黏附能力的影响

  • 陈大卫 ,
  • 郭聪聪 ,
  • 任晨瑜 ,
  • 程月 ,
  • 陈春萌 ,
  • 瞿恒贤 ,
  • 瓦云超 ,
  • 燕宪涛 ,
  • 关成冉 ,
  • 张臣臣 ,
  • 郑英明 ,
  • 钱建亚 ,
  • 顾瑞霞
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  • 1(扬州大学 食品科学与工程学院,江苏省乳品生物技术与安全控制重点实验室,江苏 扬州,225127)
    2(江苏宇航食品科技有限公司,江苏 东台,224000)
博士,副教授(顾瑞霞教授为通信作者,E-mail:guruixia1963@163.com)

收稿日期: 2022-10-24

  修回日期: 2022-12-02

  网络出版日期: 2023-06-05

基金资助

国家自然科学基金面上项目(32272362,31972094);江苏省自然科学基金面上项目(BK20211325);江苏省高等学校自然科学研究重大项目(19KJA140004);市校合作共建科技创新平台项目(YZ2020265);江苏省乳业生物工程技术中心开放课题项目(ZK2018002)

Effect of vacuum freeze-drying protective agent on adhesion of Lactobacillus plantarum 67

  • CHEN Dawei ,
  • GUO Congcong ,
  • REN Chenyu ,
  • CHENG Yue ,
  • CHEN Chunmeng ,
  • QU Hengxian ,
  • WA Yunchao ,
  • YAN Xiantao ,
  • GUAN Chengran ,
  • ZHANG Chenchen ,
  • ZHENG Yingming ,
  • QIAN Jianya ,
  • GU Ruixia
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  • 1(Yangzhou University, Jiangsu Province Key Lab of Dairy Biotechnology and Safety Control, College of Food Science and Engineering, Yangzhou 225127, China)
    2(Jiangsu Yuhang Food Technology Co., Ltd., Dongtai 224000, China)

Received date: 2022-10-24

  Revised date: 2022-12-02

  Online published: 2023-06-05

摘要

黏附是乳酸菌在机体内发挥益生作用的关键,研究冻干保护剂对乳酸菌黏附能力的影响有助于益生菌产品的开发及其功能的进一步挖掘。研究单一保护剂对真空冷冻干燥后植物乳杆菌67的黏附率和存活率等的影响,筛选出保护效果较优的单一保护剂;并以黏附率和存活率等为指标优化保护剂配方,探究优化的复合保护剂对冻干后菌株67表面微观形态、黏附Caco-2细胞能力及其抑制肠道致病菌黏附Caco-2细胞能力的改善作用。结果表明,菌株67在分别去除了脱脂乳(skim milk,S)、菊粉(inulin,L)、蔗糖(sucrose,C)、谷氨酸钠(sodium glutamate,G)及海藻糖(trehalose,T)的5种复合保护剂中冻干后,对Caco-2细胞的黏附率均显著低于其他复合保护剂(P<0.05);在分别去除L、G和山梨醇后,存活率均显著低于其他复合保护剂(P<0.05)。菌株67分别在140 g/L S、80 g/L L、60 g/L C、90 g/L G及100 g/L T中冻干后对Caco-2细胞的黏附率均大于5.85%,显著高于各自保护剂的其他浓度(P<0.05);分别在120 g/L S、100 g/L L、100 g/L C、70 g/L G及80 g/L T中冻干后的存活率均大于18.34%,显著高于各自保护剂的其他浓度(P<0.05)。菌株67在含有160 g/L S、100 g/L L、40 g/L C、70 g/L G及120 g/L T的优化复合保护剂中冻干后,其黏附率和存活率分别为28.73%和93.52%,均显著高于未优化的20.47%和76.31%(P<0.05);较未优化的复合保护剂而言,菌体形态变化较小、未破裂、未塌缩,能更紧密地黏附于Caco-2细胞上,且冻干前后的黏附素均为表面蛋白;同时,菌株抑制金黄色葡萄球菌和大肠杆菌黏附Caco-2细胞的能力也得到了显著提高(P<0.05)。S、L、C、G及T对冻干后植物乳杆菌67的黏附率和存活率产生较大的影响,优化后的复合保护剂对冻干后植物乳杆菌67的黏附能力、存活能力及其抑制肠道致病菌黏附肠上皮细胞的能力有良好的保护作用。

本文引用格式

陈大卫 , 郭聪聪 , 任晨瑜 , 程月 , 陈春萌 , 瞿恒贤 , 瓦云超 , 燕宪涛 , 关成冉 , 张臣臣 , 郑英明 , 钱建亚 , 顾瑞霞 . 真空冷冻干燥保护剂对植物乳杆菌67黏附能力的影响[J]. 食品与发酵工业, 2023 , 49(9) : 16 -26 . DOI: 10.13995/j.cnki.11-1802/ts.034090

Abstract

Adhesion is the key for lactic acid bacteria (LAB) to play a probiotic role in the body, the research of the effect of freeze-dried protective agents on the adhesion of LAB will help to develop probiotic products and further explore their functions. The effects of the single protectant on the adhesion rate and survival rate of Lactobacillus plantarum 67 after vacuum freeze-drying were studied, and a single protective agent with better protection effect were selected, and the composite protective agent was optimized according to the adhesion rate and survival rate. The improvement effect of the optimized composite protective agent on the surface microscopic morphology, the ability to adhere to Caco-2 cells, and the ability to inhibit intestinal pathogens from adhering to Caco-2 cells of strain 67 after freeze-drying were explored. The results showed that the adhesion rate to Caco-2 cells of strain 67 was significantly decreased after freeze-drying in 5 kinds of composite protectors that removed skim milk (S), inulin (L), sucrose (C), sodium glutamate (G) and trehalose (T), respectively (P<0.05), and the survival rate was significantly decreased after freeze-drying in 3 kinds of composite protectors that removed L, G and sorbitol respectively (P<0.05). The adhesion rate of strain 67 to Caco-2 cells were greater than 5.85% after freeze-drying in 140 g/L S, 80 g/L L, 60 g/L C, 90 g/L G and 100 g/L T respectively, which significantly higher than other concentrations of their respective protective agents (P<0.05), and the survival rate were greater than 18.34% after freeze-drying in 120 g/L S, 100 g/L L, 100 g/L C, 70 g/L G and 80 g/L T respectively (P<0.05), which significantly higher than other concentrations of their respective protective agents. The adhesion rate and the survival rate of strain 67 were 28.73% and 93.52% respectively when the optimized composite protectant that contain 160 g/L S, 100 g/L L, 40 g/L C, 70 g/L G and 120 g/L T, and which significantly higher than not optimization that were 20.47% and 76.31% (P<0.05). Compared with the not optimization composite protectant, the change of cell morphology was smaller, and the strain 67 adhere to Caco-2 cells was more closely and without breaking, without collapsing, and the adhesin was surface proteins before and after freeze-drying. At the same time, the ability of strain 67 to inhibit the adhesion of Staphylococcus aureus and Escherichia coli to Caco-2 cells was significantly improved (P<0.05). S, L, C, G and T have a great impact on the adhesion rate and the survival rate of L. plantarum 67, and the optimized composite protective agent has a good protective effect on the adhesion ability, the survival ability and the ability to inhibit intestinal pathogenic bacteria from adhering to intestinal epithelial cells of L. plantarum 67 after freeze-drying.

参考文献

[1] TUO Y F, YU H L, AI L Z, et al.Aggregation and adhesion properties of 22 Lactobacillus strains[J].Journal of Dairy Science, 2013, 96(7):4 252-4 257.
[2] ALP D, KULEAŞAN H.Adhesion mechanisms of lactic acid bacteria:Conventional and novel approaches for testing[J].World Journal of Microbiology & Biotechnology, 2019, 35(10):156.
[3] GAO X, WANG Z X, LI X, et al.A new Lactobacillus gasseri strain HMV18 inhibits the growth of pathogenic bacteria[J].Food Science and Human Wellness, 2022, 11(2):247-254.
[4] AYYASH M M, ABDALLA A K, ALKALBANI N S, et al.Invited review: Characterization of new probiotics from dairy and nondairy products—Insights into acid tolerance, bile metabolism and tolerance, and adhesion capability[J].Journal of Dairy Science, 2021, 104(8):8 363-8 379.
[5] WU H Q, XIE S, MIAO J F, et al.Lactobacillus reuteri maintains intestinal epithelial regeneration and repairs damaged intestinal mucosa[J].Gut Microbes, 2020, 11(4):997-1 014.
[6] RAO V B, FEISS M.The bacteriophage DNA packaging motor[J].Annual Review of Genetics, 2008, 42:647-681.
[7] VAN DE GUCHTE M, SERROR P, CHERVAUX C, et al.Stress responses in lactic acid bacteria[J].Antonie Van Leeuwenhoek, 2002, 82(1-4):187-216.
[8] KANDIL S, EL SODA M.Influence of freezing and freeze drying on intracellular enzymatic activity and autolytic properties of some lactic acid bacterial strains[J].Advances in Microbiology, 2015, 5(6):371-382.
[9] ZÁRATE G, NADER-MACIAS M E.Viability and biological properties of probiotic vaginal lactobacilli after lyophilization and refrigerated storage into gelatin capsules[J].Process Biochemistry, 2006, 41(8):1 779-1 785.
[10] CHENG Z Y, YAN X, WU J Y, et al.Effects of freeze drying in complex lyoprotectants on the survival, and membrane fatty acid composition of Lactobacillus plantarum L1 and Lactobacillus fermentum L2[J].Cryobiology, 2022, 105:1-9.
[11] OLUWATOSIN S O, TAI S L, FAGAN-ENDRES M A.Sucrose, maltodextrin and inulin efficacy as cryoprotectant, preservative and prebiotic-towards a freeze dried Lactobacillus plantarum topical probiotic[J].Biotechnology Reports, 2022, 33:e00696.
[12] 黄玉军, 姚瑶, 周帆, 等.益生菌干预频次及周期对高脂血症大鼠血清抗氧化能力的影响[J].现代食品科技, 2020, 36(1):1-7.
HUANG Y J, YAO Y, ZHOU F, et al.Effect of probiotic intervention frequency and cycle on serum antioxidant capacity in rats with hyperlipidemi[J].Modern Food Science and Technology, 2020, 36(1):1-7.
[13] 陈春萌. 消化应激对乳酸菌黏附能力的影响[D].扬州:扬州大学, 2021.
CHEN C M.Effect of oro-gastrointestinal stress on adhesion of lactic acid bacteria[D].Yangzhou:Yangzhou University, 2021.
[14] 陈大卫, 程月, 任晨瑜, 等.乳杆菌耐消化应激能力及消化应激对其肠道黏附能力的影响[J].食品科学, 2022, 43(14):143-150.
CHEN D W, CHENG Y, REN C Y, et al.Capability of Lactobacillus to tolerate digestive stress and effect of digestive stress on its intestinal adhesion capacity[J].Food Science, 2022, 43(14):143-150.
[15] 于平, 胡淳玉, 黄星星, 等.产肌醇的植物乳杆菌ZJ2868菌粉制备工艺[J].中国食品学报, 2021, 21(9):142-149.
YU P, HU C Y, HUANG X X, et al.Preparation process of inositol-producing Lactobacillus plantarum ZJ2868 powder[J].Journal of Chinese Institute of Food Science and Technology, 2021, 21(9):142-149.
[16] DIANAWATI D, MISHRA V, SHAH N P.Survival of microencapsulated probiotic bacteria after processing and during storage:A review[J].Critical Reviews in Food Science and Nutrition, 2016, 56(10):1 685-1 716.
[17] 李大鹏, 高玉荣.益生菌植物乳杆菌G1-28复合冻干发酵剂制备及保藏条件研究[J].食品工业科技, 2021, 42(10):100-104.
LI D P, GAO Y R.Preparation and preservation technology of probiotic Lactobacillus plantarum G1-28 composite lyophilized starter[J].Science and Technology of Food Industry, 2021, 42(10):100-104.
[18] 赵延胜, 肖香, 周兴华, 等.冻干保护剂影响植物乳杆菌代谢途径的研究[J].现代食品科技, 2016, 32(8):103-108;147.
ZHAO Y S, XIAO X, ZHOU X H, et al.Influence of cryoprotectants on the metabolic pathways of Lactobacillus plantarum[J].Modern Food Science and Technology, 2016, 32(8):103-108;147.
[19] BROECKX G, VANDENHEUVEL D, CLAES I J J, et al.Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics[J].International Journal of Pharmaceutics, 2016, 505(1-2):303-318.
[20] CAPELA P, HAY T K C, SHAH N P.Effect of cryoprotectants, prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt[J].Food Research International, 2006, 39(2):203-211.
[21] LU W J, FU N, WOO M W, et al.Exploring the interactions between Lactobacillus rhamnosus GG and whey protein isolate for preservation of the viability of bacteria through spray drying[J].Food & Function, 2021, 12(7):2 995-3 008.
[22] JEFFERY C J.Intracellular/surface moonlighting proteins that aid in the attachment of gut microbiota to the host[J].AIMS Microbiology, 2019, 5(1):77-86.
[23] FU N, CHEN X D.Towards a maximal cell survival in convective thermal drying processes[J].Food Research International, 2011, 44(5):1 127-1 149.
[24] CASTRO H P, TEIXEIRA P M, KIRBY R.Evidence of membrane damage in Lactobacillus bulgaricus following freeze drying[J].Journal of Applied Microbiology, 1997, 82(1):87-94.
[25] DIANAWATI D, SHAH N P.Survival, acid and bile tolerance, and surface hydrophobicity of microencapsulated B.animalis ssp.lactis Bb12 during storage at room temperature[J].Journal of Food Science, 2011, 76(9):M592-M599.
[26] 袁峥, 赵瑞香, 牛生洋, 等.酸胁迫下嗜酸乳杆菌菌体形态的扫描电镜观察[J].食品工业科技, 2012, 33(24):199-201.
YUAN Z, ZHAO R X, NIU S Y, et al.Observation of the mycelia morphology of Lactobacillus acidophilus with scanning electron microscope under acid stress[J].Science and Technology of Food Industry, 2012, 33(24):199-201.
[27] PEIGHAMBARDOUST S H, GOLSHAN TAFTI A, HESARI J.Application of spray drying for preservation of lactic acid starter cultures:A review[J].Trends in Food Science & Technology, 2011, 22(5):215-224.
[28] CARVALHO A S, SILVA J, HO P, et al.Relevant factors for the preparation of freeze-dried lactic acid bacteria[J].International Dairy Journal, 2004, 14(10):835-847.
[29] ADACHI T, KAKUTA S, AIHARA Y, et al.Visualization of probiotic-mediated Ca2+ signaling in intestinal epithelial cells in vivo[J].Frontiers in Immunology, 2016, 7:601.
[30] SAVEDBOWORN W, TEAWSOMBOONKIT K, SURICHAY S, et al.Impact of protectants on the storage stability of freeze-dried probiotic Lactobacillus plantarum[J].Food Science and Biotechnology, 2019, 28(3):795-805.
[31] YUSTE A, AROSEMENA E L, CALVO M À.Study of the probiotic potential and evaluation of the survival rate of Lactiplantibacillus plantarum lyophilized as a function of cryoprotectant[J].Scientific Reports, 2021, 11(1):19078.
[32] ROMANO N, MARRO M, MARSAL M, et al.Fructose derived oligosaccharides prevent lipid membrane destabilization and DNA conformational alterations during vacuum-drying of Lactobacillus delbrueckii subsp.bulgaricus[J].Food Research International, 2021, 143:110235.
[33] POLOMSKA X, WOJTATOWICZ M, ZAROWSKA B, et al.Freeze-drying preservation of yeast adjunct cultures for cheese production[J].Polish Journal of Food and Nutrition Sciences, 2012, 62(3):143-150.
[34] AMINE K M, CHAMPAGNE C P, SALMIERI S, et al.Effect of palmitoylated alginate microencapsulation on viability of Bifidobacterium longum during freeze-drying[J].LWT - Food Science and Technology, 2014, 56(1):111-117.
[35] CARVALHO A S, SILVA J, HO P, et al.Protective effect of sorbitol and monosodium glutamate during storage of freeze-dried lactic acid bacteria[J].Le Lait, 2003, 83(3):203-210.
[36] LIÉVIN V, PEIFFER I, HUDAULT S, et al.Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity[J].Gut, 2000, 47(5):646-652.
[37] IRAPORDA C, RUBEL I A, MANRIQUE G D, et al.Influence of inulin rich carbohydrates from Jerusalem artichoke (Helianthus tuberosus L.) tubers on probiotic properties of Lactobacillus strains[J].LWT, 2019, 101:738-746.
[38] PATIL A, MUNOT N, PATWEKAR M, et al.Encapsulation of lactic acid bacteria by lyophilisation with its effects on viability and adhesion properties[J].Evidence-Based Complementary and Alternative Medicine:ECAM, 2022, 2022:4651194.
[39] TYMCZYSZYN E E, DEL ROSARIO DÍAZ M, GÓMEZ-ZAVAGLIA A, et al.Volume recovery, surface properties and membrane integrity of Lactobacillus delbrueckii subsp.bulgaricus dehydrated in the presence of trehalose or sucrose[J].Journal of Applied Microbiology, 2007, 103(6):2 410-2 419.
[40] JAWAN R, ABBASILIASI S, TAN J S, et al.Influence of type and concentration of lyoprotectants, storage temperature and storage duration on cell viability and antibacterial activity of freeze-dried lactic acid bacterium, Lactococcus lactis Gh1[J].Drying Technology, 2022, 40(9):1 774-1 790.
[41] VELLY H, BOUIX M, PASSOT S, et al.Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp.lactis TOMSC161[J].Applied Microbiology and Biotechnology, 2015, 99(2):907-918.
[42] WANG J F, JIANG S M, HUANG J Q, et al.Optimization of initial cation concentrations for L-lactic acid production from fructose by Lactobacillus pentosus cells[J].Applied Biochemistry and Biotechnology, 2021, 193(5):1 496-1 512.
[43] PAPADIMITRIOU K, ALEGRÍA Á, BRON P A, et al.Stress physiology of lactic acid bacteria[J].Microbiology and Molecular Biology Reviews:MMBR, 2016, 80(3):837-890.
[44] YADAV A K, TYAGI A, KAUSHIK J K, et al.Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen[J].Microbiological Research, 2013, 168(10):639-645.
[45] JEFFERY C.Intracellular proteins moonlighting as bacterial adhesion factors[J].AIMS Microbiology, 2018, 4(2):362-376.
[46] CASTALDO C, VASTANO V, SICILIANO R A, et al.Surface displaced Alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein[J].Microbial Cell Factories, 2009, 8:14.
[47] CANDELA M, BERGMANN S, VICI M, et al.Binding of human plasminogen to Bifidobacterium[J].Journal of Bacteriology, 2007, 189(16):5 929-5 936.
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