CN113249252A - Method for preventing harmful bacteria and biofilm formation on surface of stainless steel product - Google Patents
Method for preventing harmful bacteria and biofilm formation on surface of stainless steel product Download PDFInfo
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Abstract
The invention discloses a method for preventing harmful bacteria and biofilm from forming on the surface of a stainless steel product, which comprises the following steps: providing a Lactobacillus plantarum bacterial liquid, wherein the bacterial liquid contains a certain amount of Lactobacillus plantarum, the Lactobacillus plantarum is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC No.16939, and the preservation date is 2018, 12 months and 14 days; and fully contacting the surface of the stainless steel product with the bacterial liquid, and placing at a certain temperature, so that the lactobacillus plantarum forms a lactobacillus plantarum biofilm on the surface of the stainless steel product. The method utilizes the lactobacillus plantarum to form the biofilm on the surface of the stainless steel product in advance, thereby effectively preventing the formation of other harmful bacteria biofilms, and being a green and safe method for preventing the formation of the harmful bacteria biofilms.
Description
Technical Field
The invention particularly relates to a method for preventing a harmful bacteria biofilm from forming on the surface of a stainless steel product, belonging to the technical field of food science.
Background
The food is rich in nutrient substances, provides excellent growth conditions for the growth of bacteria, and the surfaces of processing and conveying devices of the food are always adhered with bacteria to form a biological film. Bacterial biofilms are very resistant to the environment, tens to thousands of times as resistant to chemical disinfectants as planktonic bacteria, and are therefore difficult to remove completely. In the food processing and conveying processes, the harmful bacterial biofilm can not only cause damage to the surfaces of food processing and conveying equipment/devices, material conveying pipelines and the like, but also is a potential pollution source and becomes a source of food spoilage by migrating to a food system. Statistically, 27% of the food contamination is due to processing equipment. The presence of harmful bacterial biofilms has been detected on the surface of stainless steel instruments used in a variety of food processing and delivery applications.
The control of harmful bacterial biofilms is mainly carried out by physical methods and chemical methods. The physical method mainly uses ultrasonic waves, electric shocks and the like, and is not suitable for large-scale food production equipment. The chemical method mainly uses chemical bactericides, and the method easily causes bactericide residues and becomes hidden danger threatening the food safety. Therefore, finding new approaches, especially green and safe biological approaches to eliminate the biofilm hazard, becomes an industry research hotspot and difficulty.
Disclosure of Invention
The invention aims to provide a method for preventing harmful bacteria and biofilm from forming on the surface of a stainless steel product so as to overcome the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the embodiment of the invention provides a method for preventing harmful bacteria and biofilm from forming on the surface of a stainless steel product, which comprises the following steps:
providing a lactobacillus plantarum bacterial liquid, wherein the bacterial liquid contains 10 viable cell numbers6～109The Lactobacillus plantarum is characterized in that the Lactobacillus plantarum is CFU/mL, is preserved in China general microbiological culture collection center (CGMCC), has a preservation number of CGMCC No.16939, and has a preservation date of 2018, 12 months and 14 days;
and fully contacting the surface of the stainless steel product with the bacterial liquid, and standing at the temperature of 4-37 ℃ for more than 12 hours, so that the lactobacillus plantarum forms a lactobacillus plantarum biofilm on the surface of the stainless steel product.
In some embodiments, the method specifically comprises: and soaking the stainless steel product in the bacterial liquid, and standing at the temperature of 4-37 ℃ for 12-48 h, so that the lactobacillus plantarum forms a lactobacillus plantarum biofilm on the surface of the stainless steel product.
In some embodiments, the method specifically comprises: the bacterial liquid is mixed according to the ratio of 10-50 mL/m2The inoculation amount of the lactobacillus plantarum is uniformly sprayed on the surface of a stainless steel product, and the stainless steel product is placed at the temperature of 4-37 ℃ for 12-48 hours, so that the lactobacillus plantarum forms a lactobacillus plantarum biofilm on the surface of the stainless steel product.
In some embodiments, the method further comprises: and after the lactobacillus plantarum biofilm is formed, cleaning and removing the planktonic bacteria on the surface of the stainless steel product, or not cleaning and removing the planktonic bacteria. Wherein, the floating thalli can be cleaned without washing, thereby bringing convenience to industrial use and reducing links; meanwhile, the floating lactobacillus plantarum is beneficial bacteria, and has beneficial effects of fresh keeping and the like on semi-finished products or finished products of food.
In some embodiments, the method specifically comprises:
inoculating the activated lactobacillus plantarum in an MRS culture medium, wherein the inoculation amount is 3-4V/V%, culturing for 12-18 h at 37 ℃, and continuously transferring and culturing for 3 times to obtain a third-generation zymocyte liquid;
centrifugally separating the thallus from the third-generation zymophyte liquid, washing, and adjusting the concentration of the bacteria liquid until the number of viable cells of the lactobacillus plantarum in the bacteria liquid is 106～109And CFU/mL to obtain the lactobacillus plantarum bacterial liquid.
In some embodiments, the MRS medium comprises the following components in parts by weight: 10 parts of casein digest, 10 parts of beef extract powder, 4 parts of yeast extract, 20 parts of glucose, 5 parts of sodium acetate, 2 parts of triammonium citrate, 801.08 parts of tween-8, 2 parts of dipotassium phosphate, 0.2 part of magnesium sulfate heptahydrate and 0.05 part of manganese sulfate tetrahydrate, and the balance of distilled water, wherein the mass-volume ratio of the casein digest to the distilled water is 1 g: 100 mL; the pH value of the MRS culture medium is 5.7-6.2.
In some embodiments, the stainless steel article is a stainless steel food processing and/or conveying appliance.
In some embodiments, the material of the stainless steel product includes, but is not limited to, austenitic stainless steel, austenitic-ferritic stainless steel, or martensitic stainless steel.
The embodiment of the invention also provides application of the Lactobacillus plantarum with the preservation number of CGMCC No.16939 in preparing the biological antibacterial film agent on the surface of the stainless steel product, wherein the Lactobacillus plantarum is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation date of 2018, 12 months and 14 days.
The embodiment of the invention also provides application of the Lactobacillus plantarum with the preservation number of CGMCC No.16939 in preventing harmful bacteria biofilm from forming on the surface of a stainless steel product, wherein the Lactobacillus plantarum is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation date of 2018, 12 and 14 days.
Compared with the prior art, the invention has at least the following advantages:
(1) the biological film formed on the surface of the stainless steel product in advance by using the lactobacillus plantarum can effectively prevent harmful bacteria from forming the biological film on the surface of the stainless steel product, and the efficiency reaches more than 90%;
(2) the biological membrane formed on the surface of the stainless steel product by the lactobacillus plantarum has the possibility of transferring into food or semi-finished products thereof, and the biological protection property of the biological membrane has positive effects on the quality and the storability of the food;
(3) the process for forming the biofilm on the surface of the stainless steel product by using the lactobacillus plantarum is simple and convenient, does not need to change the food production process, does not cause potential safety hazard to subsequent food production, and is a green and safe method for preventing the harmful bacteria from forming the biofilm.
Drawings
FIGS. 1 a-1 b are graphs showing the effect of pre-biofilm formation by different lactic acid bacteria on Clostridium perfringens biofilm formation in examples of the invention, wherein a represents the number of Clostridium perfringens cells in the biofilm and b represents the efficiency of preventing Clostridium perfringens biofilm formation;
FIG. 2 is a graph showing the effect of using Lactobacillus plantarum and Lactobacillus sake biofilms on cell activity during Clostridium perfringens biofilm formation in an example of the invention;
FIG. 3 is a graph of the effect of using Lactobacillus plantarum and Lactobacillus sake biofilms on intracellular Reactive Oxygen Species (ROS) during Clostridium perfringens biofilm formation in an example of the invention;
FIG. 4 is a graph showing the effect of using Lactobacillus plantarum and Lactobacillus sake biofilms on the activity of cellular superoxide dismutase (SOD) during the formation of Clostridium perfringens biofilm in accordance with an embodiment of the present invention;
FIG. 5 is a graph showing the effect of using Lactobacillus plantarum and Lactobacillus sake biofilms on cell population induction signals during Clostridium perfringens biofilm formation in an example of the invention;
FIGS. 6 a-6 c are graphs showing the effect of using Lactobacillus plantarum and Lactobacillus sake biofilms on the expression of AhpC (a), LuxS (b), and SpoOA (c) genes in the formation of Clostridium perfringens biofilm in accordance with examples of the present invention.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
The specific implementation methods in the following examples are conventional methods unless otherwise specified; the raw materials and reagents used are from stores or reagent selling companies unless otherwise specified.
Example 1a method for preventing the formation of harmful bacterial biofilms on the surface of stainless steel articles, comprising the steps of:
(1) culture of Lactobacillus plantarum
Inoculating activated Lactobacillus plantarum (preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.16939 and the preservation date of 2018, 12 months and 14 days, hereinafter referred to as Lactobacillus plantarum) into an MRS (Man Rogosa and Sharp) culture medium, wherein the inoculation amount is 4% (V/V), culturing at 37 ℃ for 12 hours, and continuously transferring and culturing for 3 times to obtain a third-generation zymocyte liquid.
(2) Preparation of lactobacillus plantarum biofilm
Centrifuging the third generation fermented bacterial liquid, washing the precipitate with phosphate buffer solution (PBS, 0.1M, pH 5.7), resuspending the thallus and adjusting the bacterial liquid concentration with MRS culture medium until the viable cell count of Lactobacillus plantarum is 106CFU/mL. Soaking a food-grade 304 stainless steel sheet in the solution, standing at 25 ℃ for 48h, and washing off planktonic bacteria on the surface of the stainless steel sheet by using sterile physiological saline to obtain the lactobacillus plantarum biomembrane.
The MRS culture medium is prepared by uniformly mixing 10.0g of casein 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, 801.08g of tween-801.08 g, 2.0g of dipotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate tetrahydrate and 1000mL of distilled water, and adjusting the pH value to 5.7-6.2.
Example 2 the lactobacillus plantarum biofilm obtained in example 1 was used to prevent clostridium perfringens biofilm formation on stainless steel surfaces, and specifically included the following steps:
(1) culture of clostridium perfringens
Inoculating activated Clostridium perfringens into brain Heart infusion Broth (BHI)The inoculation amount is 4% (V/V), the culture is carried out for 12h at 37 ℃, and the transfer culture is continuously carried out for 3 times, so as to obtain the third generation zymocyte liquid. Centrifuging the third generation zymocyte liquid, collecting precipitate, washing impurities in the precipitate with PBS (0.1M, pH 7.4), and adjusting the concentration of the liquid until the Clostridium perfringens number is 103CFU/mL。
(2) Prevention effect of pre-formed biofilm of lactobacillus plantarum on clostridium perfringens biofilm formation
Placing the stainless steel sheet loaded with Lactobacillus plantarum biofilm in a culture dish containing chicken broth thawing loss juice (MTLB) culture medium, inoculating Clostridium perfringens to the culture dish to obtain final concentration of 102CFU/mL, aerobic culture at 25 ℃ for 72 h.
The preparation method of the MTLB culture medium comprises the following steps: placing chicken breast in a clean container, pressing with 5kg weight on its surface, freezing at-20 deg.C for 24 hr, thawing at 4 deg.C for 16 hr, collecting chicken exudate, centrifuging (8000g, 20min), collecting supernatant, determining protein concentration, packaging, and storing at-80 deg.C in refrigerator. Was sterilized by filtration through sterile microporous membranes of 0.45 μ M and 0.22 μ M, respectively, before use, and the protein concentration was adjusted to 5mg/mL with PBS (0.1M, pH 7.4).
Example 3 index measurement
(1) Determination of the number of biofilm cells of clostridium perfringens
And (3) cleaning the surface of the stainless steel sheet by using sterile normal saline to remove non-adhered floating thalli, placing the stainless steel sheet in a homogenizing bag filled with 10mL of sterile normal saline, and beating for 120s to obtain supernatant, namely the lactobacillus plantarum-clostridium perfringens biomembrane cell suspension (hereinafter referred to as biomembrane cell suspension). 1mL of suspension is sucked for 10-fold gradient dilution, a proper gradient is selected and coated on a Marseilles-sulfite-cycloserine agar base (TSC) plate, and the obtained product is subjected to anaerobic culture at 37 ℃ for 48 hours and then counted. The calculation formula of the efficiency of lactobacillus plantarum in preventing clostridium perfringens from forming a biofilm is as follows:
(2) determination of biofilm cell Activity
Washing the surface of the stainless steel sheet with sterile normal saline to remove floating thalli, completely covering the surface of the stainless steel with LIVE/DEAD fluorescent dye solution at room temperature (25 ℃), and allowing the stainless steel sheet to act with the adhered biomembrane cells for 15min under the condition of keeping out of the sun. Subsequently, the stainless steel sheet was washed with sterile physiological saline to remove excess fluorescent dye, left at room temperature in the dark, and after natural drying, the activity of the adhered cells was observed using a confocal laser microscope. Green fluorescence is a thallus (i.e. viable bacteria) with an intact cell membrane in a biological membrane; the red fluorescence is a thallus with damaged cell membrane in the biological membrane (i.e. dead bacteria).
(3) Determination of ROS content
1mL of the biofilm cell suspension is taken, 1 mu L of 10mM 2, 7-dichloro-fluorescent yellow diacetate (DCFH-DA) solution is added, after uniform mixing, the mixture is incubated for 20min in a dark place at 37 ℃, and the mixture is shaken every 5min to ensure that the probe is fully contacted with the cells. After completion of incubation, the cells were washed three times with PBS (0.1M, pH 7.4) to sufficiently remove DCFH-DA that had not entered the cells, and 100 μ L of the mixed solution was placed in a black microplate and fluorescence intensity measurement was performed at 488nm excitation wavelength and 525nm emission wavelength. And using Rosu stimulated clostridium perfringens biomembrane cells as a positive control group. The relative ROS content calculation is as follows:
(4) determination of SOD Activity
Collecting 1mL of the above biomembrane cell suspension, centrifuging at 12000g for 5min, collecting thallus precipitate, and measuring total SOD activity in cells by using total SOD detection kit (NBT method).
(5) Determination of AI-2 Activity
Activated Vibrio harveyi BB170 was inoculated into an autoinduction medium (AB) and cultured at 25 ℃ until OD600 ═ 1. Diluting the cultured bacterial liquid with AB culture medium at a ratio of 1: 5000. 1mL of the biomembrane cell suspension, the AB culture medium and the MTLB culture medium are taken, a sterile filter membrane with the diameter of 0.22 mu m is used for filtration, and the filtered supernatant is mixed with the diluted Vibrio harveyi BB170 culture solution according to the proportion of 1: 9 and is respectively used as a sample to be detected, a negative control and a medium control. And (3) putting 200 mu L of mixed solution into a black transparent-bottom enzyme label plate, measuring the fluorescence intensity once every half hour within 0-6 h, and calculating the relative AI-2 activity by taking the time point when the negative control fluorescence intensity value reaches the minimum as a reference, wherein the formula is as follows:
(6) measurement of expression levels of AhpC, LuxS and Spo0A
1mL of the biofilm cell suspension is taken, and the total RNA of the biofilm cell suspension is extracted by using a bacterial total RNA extraction kit. The 16S rRNA gene was selected as the reference gene and the primer sequences involved are shown in Table 1. RNA was reverse transcribed into cDNA according to the FastKing RT Kit (gDNase) instructions. Configuring a 20 mu L reaction system for fluorescent quantitative PCR amplification, comprising: 10 μ L of 2 XSG Fast qPCR Master Mix, 0.4 μ L of upstream and downstream primers, 1 μ L of cDNA and 8.2 μ L of sterile ddH2And O. The amplification was performed using a Bio-Rad fluorescent quantitative PCR instrument, and the PCR amplification procedure was as follows: 3min at 95 ℃; 2s at 95 ℃, 20s at 55 ℃ and 40 cycles; 5s at 65 ℃. Making 3 multiple wells for each gene, and application 2-ΔΔCTThe relative expression level of the gene is calculated by the method.
TABLE 1 primer sequences
(7) Statistical analysis of data
All experiments were repeated three times and the results are expressed as mean ± standard deviation. Experimental data statistics were performed using the Linear Models program in the Statistix8.1 software package, and the differential significance (P < 0.05) analysis was performed using the Tukey HSD program.
Meanwhile, as a control, the present inventors also prepared biofilms of 4 kinds of lactic acid bacteria, Pediococcus pentosaceus (Pediococcus pentosaceus), Lactobacillus pentosus (Lactobacillus pentosus), Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus sake (Lactobacillus sakei), respectively, referring to the manner of example 1, and further used the previously formed biofilms of Pediococcus pentosaceus, Lactobacillus fermentum and Lactobacillus sake to prevent clostridium perfringens biofilm formation, referring to example 2. And, the effect of the previously formed foregoing pediococcus pentosaceus biofilm, lactobacillus pentosus biofilm, lactobacillus fermentum biofilm, and lactobacillus sakei biofilm on the number of clostridium perfringens biofilms was also determined with reference to example 3. Meanwhile, the effects of the previously formed biofilm of Lactobacillus sake on biofilm cell activity, ROS content, SOD activity, AI-2 activity and gene (AhpC, LuxS and Spo0A) expression were also determined with reference to example 3.
The effect of the lactobacillus plantarum of the present invention and the aforementioned 4 kinds of lactic acid bacteria on preventing the formation of harmful bacteria biofilm on the surface of stainless steel products will be described in detail below with reference to the accompanying drawings.
Referring to fig. 1a and fig. 1b, it can be seen that the biofilm formed by different lactic acid bacteria in advance has different effects on the formation of clostridium perfringens biofilm, the lactobacillus plantarum biofilm has good effect (P is less than 0.05) on the formation of clostridium perfringens biofilm at 24h, 48h and 72h, and the efficiency of inhibiting the formation of clostridium perfringens biofilm after 72h culture is as high as 91.4%. Perfringens corresponds to clostridium perfringens biofilm in fig. 1a, 1b and 2-5; P.pentosaceus-C.perfringens is inoculated with clostridium perfringens to form a biofilm after the biofilm is formed in advance by the pediococcus pentosaceus; L.plantarum-C.perfrankens forms a biofilm in advance corresponding to lactobacillus plantarum, and then clostridium perfringens is inoculated to form the biofilm; L.pentosus-C.perfringens inoculating clostridium perfringens to form a biofilm after the biofilm is formed in advance by lactobacillus pentosus; L.fermentum-C.pcrfringens is inoculated with clostridium perfringens to form a biofilm after the lactobacillus fermentum forms a biofilm in advance; l. sakei-C. perfringens is inoculated with clostridium perfringens to form a biofilm after the biofilm is formed in advance by lactobacillus sakei.
Referring to fig. 2, according to the fluorescent staining results, the lactobacillus plantarum pre-biofilm formation significantly reduced bacterial aggregation. In the treatment group, most of the bacteria adhered to the stainless steel sheet are dead after 72 hours, which indicates that the biofilm formed by lactobacillus plantarum may cause the death of clostridium perfringens, and further reduces the formation of the biofilm.
Referring again to fig. 3 and 4, it can be seen that there was no significant difference in ROS and SOD levels in the lactobacillus sake-clostridium perfringens treated group from the clostridium perfringens group (P > 0.05), indicating that the bacteria were more resistant to oxidative stress after biofilm formation by lactobacillus sake. In contrast, the pre-biofilm formation by lactobacillus plantarum on the stainless steel surface resulted in a significant increase in intracellular ROS and SOD levels in the biofilm (P < 0.05) within 48h, indicating a significant increase in intracellular oxidative stress in the biofilm in this case.
Referring to FIG. 5, the AI-2 signal intensity of the Lactobacillus plantarum group was significantly higher than that of the Lactobacillus sake group and Clostridium perfringens group, and the biofilm cell number of Lactobacillus plantarum was significantly lower than that of the Lactobacillus sake and Clostridium perfringens within 72h (P < 0.05). This indicates that, for the test strains, the quorum-sensing signal intensity is inversely related to the number of biofilm cells. The AI-2 signal was significantly higher in the lactobacillus plantarum-clostridium perfringens group than in the clostridium perfringens group, indicating increased quorum sensing between biofilm cells, preventing the formation of clostridium perfringens biofilms.
Referring to fig. 6 a-6 c, the results of the fluorescence quantitative PCR show that the biofilm formed by lactobacillus plantarum can enable the expression level of the clostridium perfringens biofilm cell oxidative stress related gene AhpC and the quorum sensing related gene LuxS to be up-regulated, and the expression level of the sporulation and biofilm formation related gene SpoOA to be down-regulated. This is consistent with the above findings (fig. 3-5), indicating that lactobacillus plantarum biofilms are capable of preventing the formation of clostridium perfringens biofilms by causing clostridium perfringens oxidative stress, enhancing quorum sensing. In this fig. 6, Cp corresponds to clostridium perfringens biofilm; the lactobacillus plantarum corresponding to the Lp-Cp is inoculated with clostridium perfringens to form a biofilm after the biofilm is formed in advance; and (3) inoculating clostridium perfringens to form the biofilm after the biofilm is formed in advance by the lactobacillus sake corresponding to the Ls-Cp.
It can be seen by similar experiments that the pre-formed biofilm of lactobacillus plantarum of the invention exhibits a significant advantage in preventing clostridium perfringens biofilm formation compared to pre-formed biofilms of pediococcus pentosaceus, lactobacillus pentosus, lactobacillus fermentum, etc.
The different capital letters in FIGS. 1 a-6 c above indicate significant differences (P < 0.05) between the different treatment groups at the same treatment time; different lower case letters indicate significant differences in treatment times (P < 0.05) for the same treatment groups.
From the above examples, it can be seen that the lactobacillus plantarum of the present invention can form a biofilm on the surface of a stainless steel appliance in advance, and further effectively prevent other harmful bacteria from forming a biofilm on the surface of the stainless steel appliance, so that the lactobacillus plantarum is a green and safe method for preventing the harmful bacteria from forming a biofilm. In addition, for stainless steel instruments applied to the food industry, the lactobacillus plantarum biofilm formed on the surface of the stainless steel instruments has the possibility of migrating into food or semi-finished products thereof, and the biological protection property of the lactobacillus plantarum biofilm also has a positive effect on the quality and the storability of the products.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. A method for preventing the formation of harmful bacteria and biofilms on the surface of a stainless steel product is characterized by comprising the following steps:
providing a lactobacillus plantarum bacterial liquid, wherein the bacterial liquid contains 10 viable cell numbers6～109The Lactobacillus plantarum is characterized in that the Lactobacillus plantarum is CFU/mL and is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC No.16939, and the preservation date is CGMCC No.1693912 months and 14 days 2018;
and fully contacting the surface of the stainless steel product with the bacterial liquid, and standing at the temperature of 4-37 ℃ for more than 12 hours, so that the lactobacillus plantarum forms a lactobacillus plantarum biofilm on the surface of the stainless steel product.
2. The method according to claim 1, characterized in that it comprises in particular: and soaking the stainless steel product in the bacterial liquid, and standing at the temperature of 4-37 ℃ for 12-48 h, so that the lactobacillus plantarum forms a lactobacillus plantarum biofilm on the surface of the stainless steel product.
3. The method according to claim 1, characterized in that it comprises in particular: the bacterial liquid is mixed according to the ratio of 10-50 mL/m2The inoculation amount of the lactobacillus plantarum is uniformly sprayed on the surface of a stainless steel product, and the stainless steel product is placed at the temperature of 4-37 ℃ for 12-48 hours, so that the lactobacillus plantarum forms a lactobacillus plantarum biofilm on the surface of the stainless steel product.
4. The method according to any one of claims 1-3, further comprising: and after the lactobacillus plantarum biofilm is formed, cleaning and removing the planktonic bacteria on the surface of the stainless steel product, or not cleaning and removing the planktonic bacteria.
5. The method according to claim 1, characterized in that it comprises in particular:
inoculating the activated lactobacillus plantarum in an MRS culture medium, wherein the inoculation amount is 3-4V/V%, culturing for 12-18 h at 37 ℃, and continuously transferring and culturing for 3 times to obtain a third-generation zymocyte liquid;
centrifugally separating the thallus from the third-generation zymophyte liquid, washing, and adjusting the concentration of the bacteria liquid until the number of viable cells of the lactobacillus plantarum in the bacteria liquid is 106～109And CFU/mL to obtain the lactobacillus plantarum bacterial liquid.
6. The method of claim 5, wherein: the MRS culture medium comprises the following components in parts by weight: 10 parts of casein digest, 10 parts of beef extract powder, 4 parts of yeast extract, 20 parts of glucose, 5 parts of sodium acetate, 2 parts of triammonium citrate, 801.08 parts of tween-8, 2 parts of dipotassium phosphate, 0.2 part of magnesium sulfate heptahydrate and 0.05 part of manganese sulfate tetrahydrate, and the balance of distilled water, wherein the mass-volume ratio of the casein digest to the distilled water is 1 g: 100 mL; the pH value of the MRS culture medium is 5.7-6.2.
7. The method of claim 1, wherein: the stainless steel product is a stainless steel apparatus for food processing and/or conveying.
8. The method of claim 1, wherein: the stainless steel product is made of austenitic stainless steel, austenitic-ferritic stainless steel, ferritic stainless steel or martensitic stainless steel.
9. The Lactobacillus plantarum with the preservation number of CGMCC No.16939 is used for preparing a biological antibacterial film agent on the surface of a stainless steel product, the Lactobacillus plantarum is preserved in China general microbiological culture collection center (CGMCC) with the preservation date of 2018, 12 months and 14 days.
10. The Lactobacillus plantarum with the preservation number of CGMCC No.16939 is used for preventing harmful bacteria biofilms from forming on the surfaces of stainless steel products, and is preserved in China general microbiological culture collection center (CGMCC) with the preservation date of 2018, 12 months and 14 days.
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