WO2022236527A1 - 一种防止不锈钢制品表面有害细菌生物膜形成的方法 - Google Patents

一种防止不锈钢制品表面有害细菌生物膜形成的方法 Download PDF

Info

Publication number
WO2022236527A1
WO2022236527A1 PCT/CN2021/092585 CN2021092585W WO2022236527A1 WO 2022236527 A1 WO2022236527 A1 WO 2022236527A1 CN 2021092585 W CN2021092585 W CN 2021092585W WO 2022236527 A1 WO2022236527 A1 WO 2022236527A1
Authority
WO
WIPO (PCT)
Prior art keywords
stainless steel
lactobacillus plantarum
biofilm
steel product
lactobacillus
Prior art date
Application number
PCT/CN2021/092585
Other languages
English (en)
French (fr)
Inventor
李沛军
张子夜
徐宝才
罗慧婷
朱苗苗
肖晴
陈从贵
Original Assignee
合肥工业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 合肥工业大学 filed Critical 合肥工业大学
Priority to PCT/CN2021/092585 priority Critical patent/WO2022236527A1/zh
Publication of WO2022236527A1 publication Critical patent/WO2022236527A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
    • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
    • C23C26/00—Coating not provided for in groups C23C2/00 - C23C24/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the application specifically relates to a method for preventing the formation of harmful bacterial biofilms on the surface of stainless steel products, which belongs to the field of food science and technology.
  • Food is rich in nutrients, which provide excellent growth conditions for the growth of bacteria. Bacteria often adhere to the surface of processing and conveying equipment, and then form biofilm. Bacterial biofilms are highly resistant to the environment, and are dozens or even thousands of times more resistant to chemical fungicides than planktonic bacteria, so it is difficult to completely remove them. In the process of food processing and transmission, harmful bacterial biofilm will not only cause damage to the surface of food processing, transmission equipment/device, material conveying pipeline, etc., but also a potential source of pollution. By migrating to the food system, it becomes the source of food corruption . According to statistics, 27% of food contamination is caused by processing equipment. At present, the presence of harmful bacterial biofilms has been detected from the surface of various food processing and conveying stainless steel instruments.
  • the purpose of this application is to provide a method for preventing the formation of harmful bacterial biofilm on the surface of stainless steel products, so as to overcome the deficiencies in the prior art.
  • the embodiment of the present application provides a method for preventing the formation of harmful bacterial biofilm on the surface of stainless steel products, which includes:
  • a Lactobacillus plantarum liquid is provided, the bacterial liquid contains Lactobacillus plantarum with a viable cell count of 10 6 to 10 9 CFU/mL, and the Lactobacillus plantarum is preserved in the China General Microorganism Culture Collection Center (CGMCC), The deposit number is CGMCC No.16939, and the deposit date is December 14, 2018;
  • CGMCC China General Microorganism Culture Collection Center
  • the surface of the stainless steel product is fully contacted with the bacterial liquid, and placed at 4 to 37° C. for more than 12 hours, so that the lactobacillus plantarum forms a biofilm of the plantarum lactobacillus on the surface of the stainless steel product.
  • the method specifically includes: immersing stainless steel products in the bacteria liquid, and placing them at 4-37°C for 12-48 hours, so that the Lactobacillus plantarum can form Lactobacillus plantarum organisms on the surface of the stainless steel products. membrane.
  • the method specifically includes: evenly spraying the bacterial solution on the surface of the stainless steel product at an inoculation amount of 10-50 mL/m 2 , and placing it at 4-37°C for 12-48 hours, so that the plant Lactobacillus plantarum biofilm formed on the surface of stainless steel products.
  • the method further includes: after the Lactobacillus plantarum biofilm is formed, cleaning and removing the planktonic bacteria on the surface of the stainless steel product, or removing the planktonic bacteria on the surface of the stainless steel product without washing.
  • the removal of planktonic bacteria without cleaning can bring convenience to industrial use and reduce links; at the same time, because planktonic Lactobacillus plantarum is a beneficial bacterium, it also has a beneficial effect on food semi-finished or finished products such as freshness preservation.
  • the method specifically includes:
  • the inoculum amount is 3-4V/V%, culture at 37°C for 12-18h, and continuously transfer culture for 3 times to obtain the third-generation fermentation broth;
  • the concentration of the bacterial liquid is adjusted so that the number of live cells of Lactobacillus plantarum in it is 10 6 -10 9 CFU/mL, and the bacterial liquid of Lactobacillus plantarum is obtained .
  • the MRS medium comprises the following components calculated in parts by weight: 10 parts of caseinase digest, 10 parts of beef extract powder, 4 parts of yeast extract, 20 parts of glucose, 5 parts of sodium acetate, citric acid 2 parts of triammonium, Tween-801.08 parts, 2 parts of dipotassium hydrogen phosphate, 0.2 part of magnesium sulfate heptahydrate, 0.05 part of manganese sulfate tetrahydrate, the rest contains distilled water, and the mass volume ratio of caseinase digest and distilled water is 1g : 100 mL; the pH value of the MRS medium is 5.7-6.2.
  • the stainless steel article is a stainless steel appliance for food processing and/or conveyance.
  • the material of the stainless steel product includes austenitic stainless steel, austenitic-ferritic stainless steel, ferritic stainless steel or martensitic stainless steel, etc., but is not limited thereto.
  • the embodiment of the present application also provides the application of Lactobacillus plantarum with preservation number CGMCC No.16939 in the preparation of biological antibacterial film on the surface of stainless steel products. ), the date of deposit is December 14, 2018.
  • the embodiment of the present application also provides the use of Lactobacillus plantarum with the preservation number CGMCC No.16939 in preventing the formation of harmful bacteria biofilms on the surface of stainless steel products.
  • CGMCC the preservation number
  • Fig. 1a-Fig. 1b is the influence figure that adopts different lactic acid bacteria to form biofilm in advance to the formation of Clostridium perfringens biofilm in the embodiment of the present application, among the figure a represents the number of cells of Clostridium perfringens in the biofilm, and b represents preventing Clostridium perfringens biofilm formation efficiency;
  • Fig. 2 is the influence figure of adopting plantarum biofilm and sake Lactobacillus biofilm to the cell activity in the process of Clostridium perfringens biofilm formation process in the embodiment of the application;
  • Fig. 3 is the impact diagram of intracellular active oxygen level (ROS) in the formation process of Clostridium perfringens biofilm adopting Lactobacillus plantarum biofilm and Lactobacillus sake biofilm in the embodiment of the present application;
  • ROS active oxygen level
  • Fig. 4 adopts plantarum biofilm and sake Lactobacillus biofilm in the embodiment of the application to the impact figure of cell superoxide dismutase (SOD) activity in the process of Clostridium perfringens biofilm formation;
  • SOD cell superoxide dismutase
  • Fig. 5 is the influence diagram of the cell quorum sensing signal during the formation of Clostridium perfringens biofilm using Lactobacillus plantarum biofilm and Lactobacillus sake biofilm in the embodiment of the present application;
  • Fig. 6a-Fig. 6c are the cells AhpC (a), LuxS (b) and Spo0A (c) in the process of biofilm formation of Clostridium perfringens using Lactobacillus plantarum biofilm and Lactobacillus sake biofilm in the embodiment of the present application Influence plot of gene expression.
  • Embodiment 1 A method for preventing the formation of harmful bacterial biofilms on the surface of stainless steel products, comprising the steps of:
  • Lactobacillus plantarum Preserved in the China General Microorganism Culture Collection Center (CGMCC), the preservation number is CGMCC No.16939, and the preservation date is December 14, 2018, hereinafter referred to as "Lactobacillus plantarum"
  • CGMCC China General Microorganism Culture Collection Center
  • Lactobacillus plantarum In the MRS (Man Rogosa and Sharp) medium, the inoculation amount was 4% (V/V), cultured at 37°C for 12 hours, and continuously transferred for 3 times to obtain the third-generation fermentation broth.
  • the aforementioned MRS medium consists of 10.0g of caseinase digest, 10.0g of beef extract powder, 4.0g of yeast extract, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of triammonium citrate, Tween-801.08g, dipotassium hydrogen phosphate 2.0g, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate tetrahydrate and 1000mL of distilled water are mixed evenly, and the pH value is adjusted to 5.7-6.2.
  • Embodiment 2 The obtained Lactobacillus plantarum biofilm in Example 1 is used to prevent the stainless steel surface from forming a Clostridium perfringens biofilm, specifically comprising the following steps:
  • the activated Clostridium perfringens was inoculated into the brain heart infusion broth (BHI) at an inoculum size of 4% (V/V), cultured at 37°C for 12 hours, and continuously transferred for 3 times to obtain the third generation Fermentation broth.
  • the stainless steel sheet carrying the Lactobacillus plantarum biofilm was placed in a petri dish containing chicken broth thawed loss juice (MTLB) medium, and Clostridium perfringens was inoculated therein to make the final concentration 10 2 CFU/mL, Aerobic culture at 25°C for 72h.
  • MTLB chicken broth thawed loss juice
  • Embodiment 3 index determination
  • biofilm cell suspension Lactobacillus plantarum-product Clostridium perfringens biofilm cell suspension
  • the cultured bacterial solution was diluted with AB medium at a ratio of 1:5000.
  • the culture solution was mixed and used as the test sample, negative control and medium control respectively.
  • Amplification was performed with a Bio-Rad fluorescent quantitative PCR instrument, and the PCR amplification program was as follows: 95°C for 3min; 95°C for 2s, 55°C for 20s, 40 cycles; 65°C for 5s. Three replicate wells were made for each gene, and the relative gene expression was calculated using the 2 - ⁇ CT method.
  • the inventors of this case also prepared Pediococcus pentosaceus, Lactobacillus pentosus, Lactobacillus fermentum and Lactobacillus sake respectively with reference to the method of Example 1.
  • biofilms of 4 kinds of lactic acid bacteria and further referring to Example 2, the aforementioned Pediococcus pentosaceae biofilms, Lactobacillus pentosaceus biofilms, Lactobacillus fermentum biofilms and Lactobacillus sake biofilms that were previously formed were used to prevent gas production Clostridium perfringens biofilm formation.
  • Fig. 1a and Fig. 1b it can be seen that the pre-formed biofilms of different lactic acid bacteria have different preventive effects on the formation of Clostridium perfringens biofilms. Both 48h and 72h showed a good effect of preventing the formation (P ⁇ 0.05), and the efficiency of preventing the formation of Clostridium perfringens biofilm after 72 hours of cultivation was as high as 91.4%.
  • C.perfringens corresponds to Clostridium perfringens biofilm
  • P.pentosaceus-C.perfringens corresponds to Pediococcus pentosaceus pre-formed biofilm after inoculation of Clostridium perfringens Bacteria form biofilm
  • L.plantarum-C.perfringens corresponds to Lactobacillus plantarum pre-formed biofilm and then inoculated Clostridium perfringens to form biofilm
  • L.pentosus-C.perfringens corresponds to Lactobacillus pentosus pre-formed biofilm and then inoculated Clostridium perfringens forms biofilm
  • L.fermentum-C.perfringens corresponds to preformed biofilm of Lactobacillus fermentum and then inoculates Clostridium perfringens to form biofilm
  • L.sakei-C.perfringens corresponds
  • the preformed biofilm of Lactobacillus plantarum can significantly reduce the aggregation of bacteria.
  • most of the bacteria adhering to the stainless steel sheet were dead after 72 hours, indicating that the pre-formed biofilm of Lactobacillus plantarum may lead to the death of Clostridium perfringens, thereby reducing the formation of its biofilm.
  • the AI-2 signal intensity of Lactobacillus plantarum group was significantly higher than that of Lactobacillus sake group and Clostridium perfringens group, and within 72 hours, the number of biofilm cells of Lactobacillus plantarum was significantly lower than that of Lactobacillus sake and Clostridium perfringens group.
  • Clostridium perfringens P ⁇ 0.05. This shows that, for the tested strains, the strength of the quorum sensing signal was inversely correlated with the number of biofilm cells.
  • the AI-2 signal in the Lactobacillus plantarum-Clostridium perfringens group was significantly higher than that in the Clostridium perfringens group, indicating that the quorum sensing between biofilm cells was enhanced to prevent the formation of C. perfringens biofilm .
  • Cp corresponds to the biofilm of Clostridium perfringens
  • Lp-Cp corresponds to the pre-formed biofilm of Lactobacillus plantarum and then inoculates the biofilm of Clostridium perfringens
  • Ls-Cp corresponds to the pre-formed biofilm of Lactobacillus sake Post-inoculation with Clostridium perfringens to form biofilms.
  • the Lactobacillus plantarum of the present application can form a biofilm on the surface of the stainless steel device in advance, thereby effectively preventing other harmful bacteria from forming a biofilm on the surface of the stainless steel device. It is a green and safe method for preventing the formation of a harmful bacterial biofilm. method.
  • the Lactobacillus plantarum biofilm formed on its surface may migrate into food or its semi-finished products, and its bioprotective properties also have a positive effect on product quality and storage.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Environmental Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Pest Control & Pesticides (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Dentistry (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Agronomy & Crop Science (AREA)
  • Materials Engineering (AREA)
  • Mechanical Engineering (AREA)
  • Metallurgy (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本申请公开了一种防止不锈钢制品表面有害细菌生物膜形成的方法，包括：提供植物乳杆菌菌液，所述菌液包含一定量的植物乳杆菌Lactobacillus plantarum，所述植物乳杆菌保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏编号为CGMCC No.16939，保藏日期为2018年12月14日；使不锈钢制品表面与所述菌液充分接触，并在一定温度下放置，从而使所述植物乳杆菌在不锈钢制品表面形成植物乳杆菌生物膜。本申请利用植物乳杆菌预先在不锈钢制品表面形成生物膜，进而有效防止其他有害细菌生物膜的形成，是一种绿色安全的防止有害细菌生物膜形成的方法。

Description

一种防止不锈钢制品表面有害细菌生物膜形成的方法 技术领域
本申请具体涉及一种防止不锈钢制品表面有害细菌生物膜形成的方法，属于食品科学技术领域。
背景技术
食品中富含丰富的营养物质，为细菌生长提供了极佳的生长条件，其加工、传送器械表面常有细菌粘附，进而形成生物膜。细菌生物膜对环境有很强的抗性，对化学杀菌剂的抵抗性是浮游细菌的几十倍乃至几千倍，因此难以彻底清除。在食品加工、传送过程中，有害细菌生物膜不仅会对食品加工、传送设备/装置表面、物料输送管道等造成损伤，更是一种潜在的污染源，通过迁移到食品体系，成为食品腐败的源头。据统计，27％的食品污染是由于加工设备造成的。目前，已经从多种食品加工、传送的不锈钢器械表面检测到了有害细菌生物膜的存在。
有害细菌生物膜的防治主要有物理方法和化学方法。物理方法主要是使用超声波、电击等方法，不适用于大型的食品生产设备。化学方法主要使用化学杀菌剂，这种方法很容易造成杀菌剂残留，成为威胁食品安全的隐患。因此，寻找新的途径，尤其是绿色安全的生物途径来消除生物膜危害，成为行业的研究热点和难点。
发明内容
本申请的目的在于提供一种防止不锈钢制品表面有害细菌生物膜形成的方法，以克服现有技术中的不足。
为实现上述发明目的，本申请采用如下技术方案：
本申请实施例提供了一种防止不锈钢制品表面有害细菌生物膜形成的方法，其包括：
提供植物乳杆菌菌液，所述菌液包含活细胞数为10 6～10 9CFU/mL的植物乳杆菌Lactobacillus plantarum，所述植物乳杆菌保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏编号为CGMCC No.16939，保藏日期为2018年12月14日；
使不锈钢制品表面与所述菌液充分接触，并在4～37℃放置12h以上，从而使所述植物 乳杆菌在不锈钢制品表面形成植物乳杆菌生物膜。
在一些实施方式，所述的方法具体包括：将不锈钢制品浸泡于所述菌液内，并在4～37℃放置12～48h，从而使所述植物乳杆菌在不锈钢制品表面形成植物乳杆菌生物膜。
在一些实施方式，所述的方法具体包括：将所述菌液按10～50mL/m 2的接种量均匀喷洒在不锈钢制品表面，并在4～37℃放置12～48h，从而使所述植物乳杆菌在不锈钢制品表面形成植物乳杆菌生物膜。
在一些实施方式，所述的方法还包括：在形成所述植物乳杆菌生物膜后，清洗去除所述不锈钢制品表面的浮游菌体，或不清洗去除所述浮游菌体。其中，不清洗去除浮游菌体可以给工业使用带来方便，减少环节；同时因为浮游的植物乳杆菌是有益菌，对食品半成品或成品还有保鲜等有益作用。
在一些实施方式，所述的方法具体包括：
将活化好的所述植物乳杆菌接种于MRS培养基中，接种量为3～4V/V％，37℃培养12～18h，连续转接培养3次，得到第三代发酵菌液；
从所述第三代发酵菌液离心分离出菌体并洗涤后，再调整菌液浓度至其中的植物乳杆菌活细胞数为10 6～10 9CFU/mL，获得所述植物乳杆菌菌液。
在一些实施方式，所述MRS培养基包含按照重量份计算的如下组分：酪蛋白酶消化物10份、牛肉膏粉10份、酵母提取物4份、葡萄糖20份、乙酸钠5份、柠檬酸三铵2份、吐温-801.08份、磷酸氢二钾2份、七水合硫酸镁0.2份、四水合硫酸锰0.05份，其余部分包含蒸馏水，且酪蛋白酶消化物与蒸馏水的质量体积比为1g：100mL；所述MRS培养基的pH值为5.7～6.2。
在一些实施方式，所述不锈钢制品为食品加工和/或传送用的不锈钢器械。
在一些实施方式，所述不锈钢制品的材质包括奥氏体型不锈钢、奥氏体-铁素体型不锈钢、铁素体型不锈钢或马氏体型不锈钢等，但不限于此。
本申请实施例还提供了保藏编号为CGMCC No.16939的植物乳杆菌Lactobacillus plantarum于制备不锈钢制品表面生物抗菌膜剂中的用途，所述植物乳杆菌保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏日期为2018年12月14日。
本申请实施例还提供了保藏编号为CGMCC No.16939的植物乳杆菌Lactobacillus plantarum于防止不锈钢制品表面形成有害菌生物膜中的用途，所述植物乳杆菌保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏日期为2018年12月14日。
与现有技术相比，本申请至少具有如下优点：
(1)利用前述植物乳杆菌在不锈钢制品表面预先形成的生物膜能够有效阻止有害细菌在不锈钢制品表面形成生物膜，效率达到90％以上；
(2)利用前述植物乳杆菌在不锈钢制品表面形成的生物膜存在迁移进入到食品或其半成品中的可能，其生物保护特性对食品品质及贮藏性有积极作用；
(3)利用前述植物乳杆菌在不锈钢制品表面形成生物膜的工艺简便，无须改变食品生产工艺，且不会对后续的食品生产造成安全隐患，是一种绿色安全的防止有害细菌生物膜形成的方法。
附图说明
图1a-图1b为本申请实施例中采用不同乳酸菌预先形成生物膜对产气荚膜梭菌生物膜形成的影响图，图中a表示生物膜中产气荚膜梭菌细胞数量，b表示阻止产气荚膜梭菌生物膜形成效率；
图2为本申请实施例中采用植物乳杆菌生物膜和清酒乳杆菌生物膜对产气荚膜梭菌生物膜形成过程中细胞活性的影响图；
图3为本申请实施例中采用植物乳杆菌生物膜和清酒乳杆菌生物膜对产气荚膜梭菌生物膜形成过程中细胞内活性氧水平(ROS)的影响图；
图4为本申请实施例中采用植物乳杆菌生物膜和清酒乳杆菌生物膜对产气荚膜梭菌生物膜形成过程中细胞超氧化物歧化酶(SOD)活性的影响图；
图5为本申请实施例中采用植物乳杆菌生物膜和清酒乳杆菌生物膜对产气荚膜梭菌生物膜形成过程中细胞群体感应信号的影响图；
图6a-图6c为本申请实施例中采用植物乳杆菌生物膜和清酒乳杆菌生物膜对产气荚膜梭菌生物膜形成过程中细胞AhpC(a)、LuxS(b)和Spo0A(c)基因表达的影响图。
具体实施方式
下面结合附图对本申请的较佳实施例进行详细阐述，以使本申请的优点和特征能更易于被本领域技术人员理解，从而对本申请的保护范围做出更为清楚明确的界定。
下述实施例中的具体实施方法，如无特殊说明，均为常规方法；所用的原料、试剂，如无特殊说明，均来自商店或试剂销售公司。
实施例1一种防止不锈钢制品表面有害细菌生物膜形成的方法，包括如下步骤：
(1)植物乳杆菌的培养
将活化的植物乳杆菌Lactobacillus plantarum(保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏编号为CGMCC No.16939，保藏日期为2018年12月14日，如下简称“植物乳杆菌”)接种于MRS(Man Rogosa and Sharp)培养基中，接种量为4％(V/V)，37℃培养12h，连续转接培养3次，得到第三代发酵菌液。
(2)植物乳杆菌生物膜的制备
将第三代发酵菌液离心分离，用磷酸盐缓冲液(PBS，0.1M，pH值＝5.7)洗涤沉淀，重悬菌体并用MRS培养基调整菌液浓度至植物乳杆菌活细胞数为10 6CFU/mL。将食品级304不锈钢片浸泡其中，25℃放置48h，用无菌生理盐水洗去不锈钢片表面的浮游菌体，制得植物乳杆菌生物膜。
前述MRS培养基由酪蛋白酶消化物10.0g、牛肉膏粉10.0g、酵母提取物4.0g、葡萄糖20.0g、乙酸钠5.0g、柠檬酸三铵2.0g、吐温-801.08g、磷酸氢二钾2.0g、七水合硫酸镁0.2g、四水合硫酸锰0.05g和蒸馏水1000mL混合均匀，调节pH值至5.7～6.2制成。
实施例2实施例1所获植物乳杆菌生物膜用于防止不锈钢表面形成产气荚膜梭菌生物膜，具体包括如下步骤：
(1)产气荚膜梭菌的培养
将活化后的产气荚膜梭菌接种于脑心浸液肉汤(BHI)中，接种量为4％(V/V)，37℃培养12h，连续转接培养3次，得到第三代发酵菌液。将第三代发酵菌液离心分离，取沉淀，用PBS(0.1M，pH＝7.4)洗涤沉淀中的杂质，并调整菌液浓度至产气荚膜梭菌数为10 3CFU/mL。
(2)植物乳杆菌预先形成生物膜对产气荚膜梭菌生物膜形成的防止作用
将载有植物乳杆菌生物膜的不锈钢片置于含有鸡肉汁解冻损失汁液(MTLB)培养基的培养皿中，向其中接种产气荚膜梭菌，使其最终浓度为10 2CFU/mL，25℃需氧培养72h。
MTLB培养基的制备方法：将鸡胸肉置于干净的容器中，用5kg的重物平压于其表面，在-20℃下冷冻24h，随后将其置于4℃下解冻16h，收集鸡肉渗出液，离心后(8000g，20min)收集上清，测定其蛋白浓度后分装，置于-80℃冰箱中保存。使用前分别用0.45μm和0.22μm的无菌微孔滤膜过滤除菌，并用PBS(0.1M，pH值＝7.4)将蛋白浓度调整至5mg/mL，制得。
实施例3指标测定
(1)产气荚膜梭菌生物膜细胞数量的测定
用无菌生理盐水清洗不锈钢片表面以去除未粘附的浮游菌体，将其放置在装有10mL无菌生理盐水的均质袋中，拍打120s，所得上清液即为植物乳杆菌-产气荚膜梭菌生物膜细胞悬液(以下简称生物膜细胞悬液)。吸取1mL悬液进行10倍梯度稀释，选取合适的梯度涂布 至胰月示-亚硫酸盐-环丝氨酸琼脂基础(TSC)平板中，在37℃下厌氧培养48h后计数。植物乳杆菌防止产气荚膜梭菌形成生物膜的效率计算公式如下：
Figure PCTCN2021092585-appb-000001
(2)生物膜细胞活性的测定
用无菌生理盐水清洗不锈钢片表面以去除浮游菌体，室温条件(25℃)下，使用LIVE/DEAD荧光染液将不锈钢表面完全覆盖，使其与粘附的生物膜细胞在避光条件下作用15min。随后，使用无菌生理盐水洗涤不锈钢片以去除多余的荧光染料，室温避光放置，自然干燥后，使用激光共聚焦显微镜观察粘附菌体的活性。绿色荧光为生物膜内具有完整细胞膜的菌体(即活菌)；红色荧光为生物膜中细胞膜受损伤的菌体(即死菌)。
(3)ROS含量的测定
取1mL前述生物膜细胞悬液，加入1μL 10mM的2，7-二氯荧光黄双乙酸盐(DCFH-DA)溶液，混合均匀后，37℃避光孵育20min，每隔5min振摇一次，使探针和细胞充分接触。孵育完成后用PBS(0.1M，pH＝7.4)洗涤细胞三次，以充分去除未进入细胞内的DCFH-DA，取100μL混合液于黑色酶标板中，在488nm激发波长、525nm发射波长下进行荧光强度测定。以Rosup刺激产气荚膜梭菌生物膜细胞作为阳性对照组。相对ROS含量计算公式如下：
Figure PCTCN2021092585-appb-000002
(4)SOD活性的测定
取1mL前述生物膜细胞悬液，12000g离心5min，取菌体沉淀，利用《总SOD检测试剂盒》(NBT法)进行细胞内总SOD活性的测定。
(5)AI-2活力的测定
将活化的哈维氏弧菌BB170接种于自诱导培养基(AB)中，于25℃培养至OD600＝1。将培养后的菌液用AB培养基以1∶5000比例稀释。取1mL前述生物膜细胞悬液、AB培养基和MTLB培养基，使用0.22μm的无菌滤膜过滤，将过滤后的上清液以1∶9的比例与稀释后的哈维氏弧菌BB170培养液混合，分别作为待测样品、阴性对照和介质对照。取200μL混合液于黑色透明底酶标板中，在0～6h内，每隔半小时测一次荧光强度，以阴性对照荧光强度值达到最小的时间点为基准，计算相对AI-2活性，公式如下：
Figure PCTCN2021092585-appb-000003
(6)AhpC、LuxS与Spo0A表达量的测定
取1mL前述生物膜细胞悬液，用细菌总RNA提取试剂盒提取其总RNA。选择16S rRNA基因为内参基因，涉及引物序列参见表1。按照FastKing RT Kit(gDNase)说明书将RNA反转录为cDNA。配置20μL反应体系用于荧光定量PCR扩增，包括：10μL 2X SG Fast qPCR Master Mix，0.4μL的上下游引物，1μL的cDNA和8.2μL的无菌ddH 2O。用Bio-Rad荧光定量PCR仪进行扩增，PCR扩增程序如下：95℃3min；95℃2s，55℃20s，40个循环；65℃5s。每个基因做3个复孔，应用2 -ΔΔCT法计算基因相对表达量。
表1引物序列
Figure PCTCN2021092585-appb-000004
(7)数据统计分析
所有的实验重复三次，结果表示为平均值±标准差。实验数据统计采用Statistix8.1软件包中的Linear Models程序，差异显著性(P＜0.05)分析使用Tukey HSD程序进行。
同时，作为对照，本案发明人还参考实施例1的方式，分别制备了戊糖片球菌(Pediococcus pentosaceus)、戊糖乳杆菌(Lactobacillus pentosus)、发酵乳杆菌(Lactobacillus fermentum)和清酒乳杆菌(Lactobacillus sakei)4种乳酸菌的生物膜，进而还参考实施例2将预先形成的前述戊糖片球菌生物膜、戊糖乳杆菌生物膜、发酵乳杆菌生物膜和清酒乳杆菌生物膜用于防止产气荚膜梭菌生物膜形成。以及，还参考实施例3测定了预先形成的前述戊糖片球菌生物膜、戊糖乳杆菌生物膜、发酵乳杆菌生物膜和清酒乳杆菌生物膜对产气荚膜梭菌生物膜数量的影响。同时，还参考实施例3测定了预先形成的前述清酒乳杆菌生物膜对生物膜细胞活性、ROS含量、SOD活性、AI-2活力和基因(AhpC、LuxS与Spo0A)表达量的影响。
以下将结合附图具体说明本申请所述植物乳杆菌及前述4种乳酸菌防止不锈钢制品表面有害细菌生物膜形成的效果。
参阅图1a和图1b，可以看到，不同乳酸菌预先形成的生物膜对产气荚膜梭菌生物膜形成的防止效果不同，植物乳杆菌生物膜对产气荚膜梭菌生物膜在24h、48h、72h时均表现出很好的阻止形成效果(P＜0.05)，经72h培养后阻止产气荚膜梭菌生物膜形成的效率高达91.4％。 该图1a、图1b及图2-图5中，C.perfringens对应产气荚膜梭菌生物膜；P.pentosaceus-C.perfringens对应戊糖片球菌预先形成生物膜后接种产气荚膜梭菌形成生物膜；L.plantarum-C.perfringens对应植物乳杆菌预先形成生物膜后接种产气荚膜梭菌形成生物膜；L.pentosus-C.perfringens对应戊糖乳杆菌预先形成生物膜后接种产气荚膜梭菌形成生物膜；L.fermentum-C.perfringens对应发酵乳杆菌预先形成生物膜后接种产气荚膜梭菌形成生物膜；L.sakei-C.perfringens对应清酒乳杆菌预先形成生物膜后接种产气荚膜梭菌形成生物膜。
参阅图2，根据荧光染色结果，植物乳杆菌预先形成生物膜可显著减少细菌的聚集。在该处理组中，72h后不锈钢片上粘附的细菌大多呈死亡状态，说明植物乳杆菌预先形成的生物膜可能会导致产气荚膜梭菌死亡，进而减少其生物膜的形成。
再请参阅图3和图4，可以看到，清酒乳杆菌-产气荚膜梭菌处理组的ROS和SOD水平与产气荚膜梭菌组无显著差异(P＞0.05)，表明经清酒乳杆菌预先形成生物膜后，细菌对氧化应激的压力更加耐受。相比之下，植物乳杆菌在不锈钢表面预先形成生物膜会导致生物膜细胞内的ROS和SOD水平在48h内显著提升(P＜0.05)，表明在此情况下生物膜细胞内氧化应激明显增强。
参阅图5，植物乳杆菌组的AI-2信号强度显著高于清酒乳杆菌组与产气荚膜梭菌组，且在72h内，植物乳杆菌的生物膜细胞数量显著低于清酒乳杆菌和产气荚膜梭菌(P＜0.05)。这表明，对于受试菌株，群体感应信号强度与生物膜细胞数量呈负相关。植物乳杆菌-产气荚膜梭菌组中AI-2信号显著高于产气荚膜梭菌组，说明生物膜细胞之间的群体感应增强，阻止了产气荚膜梭菌生物膜的形成。
参阅图6a-图6c，荧光定量PCR结果发现，植物乳杆菌预先形成的生物膜可使产气荚膜梭菌生物膜细胞氧化应激相关基因AhpC与群体感应相关基因LuxS表达量上调，产孢与生物膜形成相关基因Spo0A表达量下调。这与上述研究结果(图3～图5)相符，说明植物乳杆菌生物膜能够通过引起产气荚膜梭菌氧化应激反应，增强群体感应，从而阻止产气荚膜梭菌生物膜的形成。该图6中，Cp对应产气荚膜梭菌生物膜；Lp-Cp对应植物乳杆菌预先形成生物膜后接种产气荚膜梭菌形成生物膜；Ls-Cp对应清酒乳杆菌预先形成生物膜后接种产气荚膜梭菌形成生物膜。
通过类似实验可以看到，相较于由戊糖片球菌、戊糖乳杆菌、发酵乳杆菌等预先形成的生物膜，本申请所述植物乳杆菌预先形成的生物膜阻止产气荚膜梭菌生物膜形成的作用展现出明显优势。
以上图1a-图6c中不同大写字母表示相同处理时间不同处理组的差异显著(P＜0.05)； 不同小写字母表示相同处理组不同处理时间的差异显著(P＜0.05)。
由以上实施例可知，本申请的植物乳杆菌能够预先在不锈钢器械表面形成生物膜，进而有效防止其他有害细菌在该不锈钢器械表面形成生物膜，是一种绿色安全的防止有害细菌生物膜形成的方法。另外，对于应用于食品行业的不锈钢器械来说，在其表面形成的植物乳杆菌生物膜存在迁移进入到食品或其半成品中的可能，其生物保护特性对产品品质及贮藏性也有积极作用。
尽管已参考说明性实施例描述了本申请，但所属领域的技术人员将理解，在不背离本申请的精神及范围的情况下可做出各种其它改变、省略及/或添加且可用实质等效物替代所述实施例的元件。另外，可在不背离本申请的范围的情况下做出许多修改以使特定情形或材料适应本申请的教示。因此，本文并不打算将本申请限制于用于执行本申请的所揭示特定实施例，而是打算使本申请将包含归属于所附权利要求书的范围内的所有实施例。
Figure PCTCN2021092585-appb-000005
Figure PCTCN2021092585-appb-000006

Claims (10)

  1. 一种防止不锈钢制品表面有害细菌生物膜形成的方法，其特征在于包括：
    提供植物乳杆菌菌液，所述菌液包含活细胞数为10 6～10 9CFU/mL的植物乳杆菌Lactobacillus plantarum，所述植物乳杆菌保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏编号为CGMCC No.16939，保藏日期为2018年12月14日；
    使不锈钢制品表面与所述菌液充分接触，并在4～37℃放置12h以上，从而使所述植物乳杆菌在不锈钢制品表面形成植物乳杆菌生物膜。
  2. 根据权利要求1所述的方法，其特征在于具体包括：将不锈钢制品浸泡于所述菌液内，并在4～37℃放置12～48h，从而使所述植物乳杆菌在不锈钢制品表面形成植物乳杆菌生物膜。
  3. 根据权利要求1所述的方法，其特征在于具体包括：将所述菌液按10～50mL/m 2的接种量均匀喷洒在不锈钢制品表面，并在4～37℃放置12～48h，从而使所述植物乳杆菌在不锈钢制品表面形成植物乳杆菌生物膜。
  4. 根据权利要求1-3中任一项所述的方法，其特征在于还包括：在形成所述植物乳杆菌生物膜后，清洗去除所述不锈钢制品表面的浮游菌体，或不清洗去除所述浮游菌体。
  5. 根据权利要求1所述的方法，其特征在于具体包括：
    将活化好的所述植物乳杆菌接种于MRS培养基中，接种量为3～4V/V％，37℃培养12～18h，连续转接培养3次，得到第三代发酵菌液；
    从所述第三代发酵菌液离心分离出菌体并洗涤后，再调整菌液浓度至其中的植物乳杆菌活细胞数为10 6～10 9CFU/mL，获得所述植物乳杆菌菌液。
  6. 根据权利要求5所述的方法，其特征在于：所述MRS培养基包含按照重量份计算的如下组分：酪蛋白酶消化物10份、牛肉膏粉10份、酵母提取物4份、葡萄糖20份、乙酸钠5份、柠檬酸三铵2份、吐温-801.08份、磷酸氢二钾2份、七水合硫酸镁0.2份、四水合硫酸锰0.05份，其余部分包含蒸馏水，且酪蛋白酶消化物与蒸馏水的质量体积比为1g∶100mL；所述MRS培养基的pH值为5.7～6.2。
  7. 根据权利要求1所述的方法，其特征在于：所述不锈钢制品为食品加工和/或传送用的不锈钢器械。
  8. 根据权利要求1所述的方法，其特征在于：所述不锈钢制品的材质包括奥氏体型不锈钢、奥氏体-铁素体型不锈钢、铁素体型不锈钢或马氏体型不锈钢。
  9. 保藏编号为CGMCC No.16939的植物乳杆菌Lactobacillus plantarum于制备不锈钢制品表面生物抗菌膜剂中的用途，所述植物乳杆菌保藏于中国普通微生物菌种保藏管理中心 (CGMCC)，保藏日期为2018年12月14日。
  10. 保藏编号为CGMCC No.16939的植物乳杆菌Lactobacillus plantarum于防止不锈钢制品表面形成有害菌生物膜中的用途，所述植物乳杆菌保藏于中国普通微生物菌种保藏管理中心(CGMCC)，保藏日期为2018年12月14日。
PCT/CN2021/092585 2021-05-10 2021-05-10 一种防止不锈钢制品表面有害细菌生物膜形成的方法 WO2022236527A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/092585 WO2022236527A1 (zh) 2021-05-10 2021-05-10 一种防止不锈钢制品表面有害细菌生物膜形成的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/092585 WO2022236527A1 (zh) 2021-05-10 2021-05-10 一种防止不锈钢制品表面有害细菌生物膜形成的方法

Publications (1)

Publication Number Publication Date
WO2022236527A1 true WO2022236527A1 (zh) 2022-11-17

Family

ID=84028635

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/092585 WO2022236527A1 (zh) 2021-05-10 2021-05-10 一种防止不锈钢制品表面有害细菌生物膜形成的方法

Country Status (1)

Country Link
WO (1) WO2022236527A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109679880A (zh) * 2019-01-28 2019-04-26 合肥工业大学 一种用于低盐肉制品的植物乳杆菌保鲜剂
CN110191945A (zh) * 2016-08-03 2019-08-30 波比奥泰克股份公司 乳酸菌及其在细菌生物膜形成的预防性、抑制性和/或减少性处理中的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110191945A (zh) * 2016-08-03 2019-08-30 波比奥泰克股份公司 乳酸菌及其在细菌生物膜形成的预防性、抑制性和/或减少性处理中的应用
CN109679880A (zh) * 2019-01-28 2019-04-26 合肥工业大学 一种用于低盐肉制品的植物乳杆菌保鲜剂

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALBERTO AGUAYO-ACOSTA, EDUARDO FRANCO, ANGEL MERINO, JORGE DÁVILA-AVIÑA, JORGE VIDAL, NORMA HEREDIA, SANTOS GARCIA: "P1-05: Culture Supernatants of Lactobacillus Plantarum Reduces Sporulation, and Biofilm Formation, of Clostridium Perfringens by Downregulating Transcription of Agr-like Quorum Sensing Genes", JOURNAL OF FOOD PROTECTION, INTERNATIONAL ASSOCIATION FOR FOOD PROTECTION, US, vol. 82, no. Suppl. A, 1 July 2019 (2019-07-01) - 24 July 2019 (2019-07-24), US , pages 78, XP009541034, ISSN: 0362-028X, DOI: 10.4315/0362-028X-82.sp1.1 *
SPERANZA, B. ; SINIGAGLIA, M. ; CORBO, M.R.: "Non starter lactic acid bacteria biofilms: A means to control the growth of Listeria monocytogenes in soft cheese", FOOD CONTROL, BUTTERWORTH, LONDON, GB, vol. 20, no. 11, 1 November 2009 (2009-11-01), GB , pages 1063 - 1067, XP026099738, ISSN: 0956-7135, DOI: 10.1016/j.foodcont.2009.01.006 *
WANG NI; YUAN LEI; SADIQ FAIZAN AHMED; HE GUOQING: "Inhibitory effect of Lactobacillus plantarum metabolites against biofilm formation by Bacillus licheniformis isolated from milk powder products", FOOD CONTROL, BUTTERWORTH, LONDON, GB, vol. 106, 17 June 2019 (2019-06-17), GB , XP085797621, ISSN: 0956-7135, DOI: 10.1016/j.foodcont.2019.106721 *
ZHANG HUAN, LI PEIJUN, TIAN XINGLEI, CHEN QIAN, KONG BAOHUA: "Antimicrobial Activities of Lactic Acid Bacteria and Staphylococcus xylosus against Clostridium perfringens", FOOD SCIENCE, BEIJING FOOD SCIENCE RESEARCH INSTITUTE, CN, vol. 41, no. 6, 1 January 2020 (2020-01-01), CN , pages 86 - 92, XP093004307, ISSN: 1002-6630, DOI: 10.7506/spkx1002-6630-20190123-302 *

Similar Documents

Publication Publication Date Title
Liu et al. Induction and recovery of the viable but nonculturable state of hop-resistance Lactobacillus brevis
Li et al. Identification of rhizospheric actinomycete Streptomyces lavendulae SPS-33 and the inhibitory effect of its volatile organic compounds against Ceratocystis Fimbriata in postharvest sweet potato (Ipomoea Batatas (L.) Lam.)
Sukmawati et al. Biocontrol activity of Aureubasidium pullulans and Candida orthopsilosis isolated from Tectona grandis L. phylloplane against Aspergillus sp. in post-harvested citrus fruit
CN104611251B (zh) 一株具有广谱抑菌活性的乳酸菌及其应用
CN115927066B (zh) 一种对尖孢镰刀菌具有拮抗作用的贝莱斯芽胞杆菌及其应用
CN101857843A (zh) 诱导提高生防酵母对果实病害防治效力方法及所用培养基
Maggio et al. Interactions between L. monocytogenes and P. fluorescens in dual-species biofilms under simulated dairy processing conditions
CN107354102B (zh) 一种耐高糖季也蒙毕赤酵母菌株及其应用
CN109234204A (zh) 一种泡菜发酵剂及其制备和应用方法
CN110004096A (zh) 一株植物乳杆菌及其应用
Lu et al. Characterization, high-density fermentation, and the production of a directed vat set starter of lactobacilli used in the food industry: A Review
Pan et al. Biocontrol ability and action mechanism of Meyerozyma guilliermondii 37 on soft rot control of postharvest kiwifruit
CN113201462B (zh) 一株埃切假丝酵母zb432及其应用
WO2022236527A1 (zh) 一种防止不锈钢制品表面有害细菌生物膜形成的方法
CN111264817B (zh) 直投式乳酸菌发酵剂在榨菜腌制中的应用
Zhang et al. Exploring the efficacy of biocontrol microbes against the fungal pathogen Botryosphaeria dothidea JNHT01 isolated from fresh walnut fruit
Wang et al. Microbiological and Physico-Chemical Characteristics of Black Tea Kombucha Fermented with a New Zealand Starter Culture
CN113249252B (zh) 一种防止不锈钢制品表面有害细菌生物膜形成的方法
Rezende et al. Occurrence of Salmonella spp. in persimmon fruit (Diospyrus kaki) and growth of Salmonella enteritidis on the peel and in the pulp of this fruit
CN101824462B (zh) 一种食品中弯曲菌的定量检测方法
CN102604851B (zh) 一株具有降胆固醇及产胞外多糖能力的乳酸乳球菌
CN110643543B (zh) 一种富含高活性乳酸菌的黄酒制备工艺
CN103820544B (zh) 一种水产品中腐败希瓦氏菌的分离和检测方法
Radouane et al. Exploring the Bioprotective Potential of Halophilic Bacteria against Major Postharvest Fungal Pathogens of Citrus Fruit Penicillium digitatum and Penicillium italicum
CN102154462A (zh) 香蕉细菌性软腐病害病原菌分子快速检测的方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21941106

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21941106

Country of ref document: EP

Kind code of ref document: A1