Lactobacillus rhamnosus FJS004 for inhibiting food-borne pathogenic bacteria as well as lactein and application of lactobacillus rhamnosus FJS004
By providing a Lactobacillus rhamnosus FJS004 and its lactic acid bacterial lactic acid bacterial metabolites that can destroy cell membranes and inhibit various biochemical processes, the problem of insufficient activity of lactic acid bacteria metabolites in the prior art in inhibiting various food-borne pathogenic bacteria is solved, and effective inhibition and bactericidal of various pathogenic bacteria is achieved, with broad-spectrum antibacteriality and stability.
Patent Information
- Application Number
- CN202510106418.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-05-13
AI Technical Summary
The existing lactic acid bacteria and their metabolites have insufficient broad-spectrum activity in inhibiting a variety of food-borne pathogenic bacteria, and it is difficult to effectively inhibit a variety of pathogenic bacteria at the same time.
A strain of Lactobacillus rhamnosus FJS004 and its prepared lactic acid bacteria can effectively inhibit or kill a variety of foodborne pathogenic bacteria by destroying cell membrane integrity, promoting nucleic acid and protein leakage, inhibiting RNA synthesis and cell wall synthesis.
Lactobacillus rhamnosus FJS004 and its lactic acid bacterial cellulose have good antibacterial or bactericidal effects on E. coli, Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Enterobacteria sakazaki, showing broad-spectrum antibacterial properties and good temperature and pH stability.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of probiotics, and in particular to Lactobacillus rhamnosus FJS004 for inhibiting foodborne pathogens, and lactobacillus and applications thereof. Background Art
[0002] Foodborne diseases seriously threaten human health. According to the World Health Organization (WHO), billions of people are affected by foodborne diseases every year, a considerable portion of which are caused by foodborne pathogens. Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Enterobacter sakazakii are common foodborne pathogens.
[0003] At present, common methods of inhibiting these foodborne pathogens include physical sterilization (such as heating and irradiation), chemical preservation (such as adding preservatives), etc. However, physical sterilization may affect the quality and nutritional content of food, the safety of chemical preservatives has also attracted much attention, and long-term use may cause pathogens to develop drug resistance.
[0004] Lactic acid bacteria and their metabolites have potential application value as a natural biological inhibition method. Generally speaking, lactobacilli produced by lactic acid bacteria have better antibacterial activity, better stability, and are more convenient to use than lactic acid bacteria. However, the sources of lactobacilli are very wide and varied, and they are usually composed of different amino acids and groups, which leads to great differences in the structure of lactobacilli and obvious differences in their antibacterial properties. Even for the same strain of bacteria, the activity of lactobacilli prepared under different extraction conditions will be different. At present, lactic acid bacteria and their metabolites that can have good inhibitory effects on multiple foodborne pathogens at the same time, and even can cause the death of foodborne pathogens, are rare, and the ability to broadly inhibit the activity of foodborne pathogens is insufficient. Summary of the invention
[0005] In view of the technical problem that lactic acid bacteria and their metabolites in the prior art are insufficient in their ability to broadly inhibit the activity of pathogenic bacteria, the present invention provides a strain of Lactobacillus rhamnosus FJS004 for inhibiting foodborne pathogens. Lactobacillus rhamnosus FJS004 and its lactobacillus have good antibacterial or bactericidal effects on Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Enterobacter sakazakii.
[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a Lactobacillus rhamnosus ( Lactobacillus rhamnosus) FJS004 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 26, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.26253.
[0007] In a second aspect, the present invention provides a Lactobacillus rhamnosus FJS004 lactic acid bacteriocin prepared from the above-mentioned Lactobacillus rhamnosus FJS004; the preparation method is as follows: inoculate Lactobacillus rhamnosus FJS004 in an MRS liquid culture medium at an inoculation rate of 1%, culture and activate at 37°C for 24 hours to obtain an activated liquid, inoculate the activated liquid in an MRS liquid culture medium at an inoculation rate of 1%, culture at 37°C for 12-48 hours, and collect the supernatant by centrifugation to obtain Lactobacillus rhamnosus FJS004 lactic acid bacteriocin.
[0008] In a third aspect, the present invention provides the use of the above-mentioned Lactobacillus rhamnosus FJS004 or / and Lactobacillus rhamnosus FJS004 lactic acid bacteria in the preparation of a preparation for inhibiting foodborne pathogens, wherein the foodborne pathogens include one or more of Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Enterobacter sakazakii.
[0009] Furthermore, Lactobacillus rhamnosus FJS004 lactic acid bacteria inhibit or kill foodborne pathogens by destroying the integrity of cell membranes.
[0010] Furthermore, damaging the integrity of the cell membrane includes damaging the cell membrane integrity of foodborne pathogens by increasing the activity of intracellular superoxide dismutase.
[0011] Furthermore, Lactobacillus rhamnosus FJS004 lactic acid bacteria inhibit or kill foodborne pathogens by promoting the leakage of intracellular nucleic acids.
[0012] Furthermore, Lactobacillus rhamnosus FJS004 lactic acid bacteria inhibit or kill foodborne pathogens by promoting the leakage of intracellular proteins.
[0013] Furthermore, Lactobacillus rhamnosus FJS004 lactic acid bacteria inhibit or kill foodborne pathogens by inhibiting RNA synthesis.
[0014] Furthermore, Lactobacillus rhamnosus FJS004 lactobacillus inhibits or kills foodborne pathogens by inhibiting cell wall synthesis.
[0015] Furthermore, the preparation refers to a food, health food or medicine containing Lactobacillus rhamnosus FJS004 or its metabolite components as ingredients for inhibiting foodborne pathogens; the metabolite components include Lactobacillus rhamnosus FJS004 lactic acid bacteria and Lactobacillus rhamnosus FJS004 postbiotics; Lactobacillus rhamnosus FJS004 or its metabolite components exist in the form of powder, granules or liquid in the ingredients for inhibiting foodborne pathogens.
[0016] The beneficial effects of the present invention are: The Lactobacillus rhamnosus FJS004 and its lactobacillus provided by the present invention can simultaneously play a good antibacterial or bactericidal effect on eight food-borne pathogens, including Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Enterobacter sakazakii, and have a broad-spectrum antibacterial property. Experimental studies have shown that the Lactobacillus rhamnosus FJS004 lactobacillus can inhibit bacteria by destroying the cell membrane integrity of food-borne pathogens, promoting the leakage of intracellular nucleic acid substances, promoting the leakage of intracellular proteins, inhibiting RNA synthesis and / or inhibiting cell wall synthesis. Moreover, the Lactobacillus rhamnosus FJS004 lactobacillus has good temperature stability and pH stability, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0018] Figure 1 It is a bar graph of the antibacterial effect of the strain to be identified on eight indicator bacteria.
[0019] Figure 2 This is a photo of the inhibition zone of the strain to be identified against Escherichia coli.
[0020] Figure 3 This is a photograph of the inhibition zone of the strain to be identified against Staphylococcus aureus.
[0021] Figure 4 This is a photograph of the inhibition zone of the strain to be identified against Enterobacter sakazakii.
[0022] Figure 5 is the phylogenetic tree of Lactobacillus rhamnosus FJS004.
[0023] Figure 6 This is the result of the bile salt resistance of Lactobacillus rhamnosus FJS004.
[0024] Figure 7 It is the antibacterial effect of Lactobacillus rhamnosus FJS004 lactobacillus on indicator bacteria.
[0025] Figure 8 It is the pH stability result of lactobacillus rhamnosus FJS004 lactobacillus.
[0026] Fig. 9 This is the temperature stability result of lactobacillus rhamnosus FJS004 lactobacillus.
[0027] Fig.10 Figure 2 is the effect of Lactobacillus rhamnosus FJS004 lactic acid bacteria on the leakage of indicator bacteria nucleic acid substances, wherein (a) is the effect of Lactobacillus rhamnosus FJS004 lactic acid bacteria on the leakage of Escherichia coli nucleic acid substances, E.CG is the control group of Escherichia coli not treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria, and E.EG is the experimental group of Escherichia coli treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria; (b) is the effect of Lactobacillus rhamnosus FJS004 lactic acid bacteria on the leakage of Staphylococcus aureus nucleic acid substances, S.CG is the control group of Staphylococcus aureus not treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria, and S.EG is the experimental group of Staphylococcus aureus treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria.
[0028] Fig.11 Figure 2 is the effect of Lactobacillus rhamnosus FJS004 lactic acid bacteria on the protein leakage of indicator bacteria, wherein (a) is the effect of Lactobacillus rhamnosus FJS004 lactic acid bacteria on the protein leakage of Escherichia coli, E.CG is the control group of Escherichia coli not treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria, and E.EG is the experimental group of Escherichia coli treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria; (b) is the effect of Lactobacillus rhamnosus FJS004 lactic acid bacteria on the protein leakage of Staphylococcus aureus, S.CG is the control group of Staphylococcus aureus not treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria, and S.EG is the experimental group of Staphylococcus aureus treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria.
[0029] Fig.12 Figure 5 is the result of PI staining flow cytometry analysis, where (a), (b), (c), and (d) are the scatter plots of Escherichia coli cell signals treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria for 0, 3, 6, and 9 h, respectively; (e), (f), (g), and (h) are the scatter plots of Staphylococcus aureus cell signals treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria for 0, 3, 6, and 9 h, respectively.
[0030] Fig.13These are the results of fluorescence spectrum scanning, where (a) is the Escherichia coli control group, (b) is the experimental group of Escherichia coli treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria; (c) is the Staphylococcus aureus control group, and (d) is the experimental group of Staphylococcus aureus treated with Lactobacillus rhamnosus FJS004 lactic acid bacteria.
[0031] Fig.14 This is the effect of lactobacillus rhamnosus FJS004 on the intracellular SOD activity of indicator bacteria. In the figure, CG is the indicator bacteria control group, and EG is the experimental group of indicator bacteria treated with lactobacillus rhamnosus FJS004. DETAILED DESCRIPTION
[0032] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
[0033] Example 1 Isolation, screening and identification of bacterial strains 1. Strain isolation and purification (1) Sampling: yogurt, sauerkraut juice and sausage, January 2018, Qingdao, Shandong Province; (2) Strain isolation: Take 25g of sausage sample under sterile environment, cut into pieces, add to 225mL of sterile saline, and homogenize with a slapping homogenizer. Take another 25mL of yogurt sample and 25mL of sauerkraut juice sample, add to 225mL of sterile saline and homogenize. Then, dilute the three samples 10 times, select the appropriate dilution, and evenly spread them on MRS solid medium containing calcium carbonate. Isolate the acid-producing strains with a calcium-soluble ring around them. The acid produced by the acid-producing strains can react with calcium carbonate, and a calcium-soluble ring is produced around the colonies. Repeated purification was performed to obtain purified primary screening strains, and a total of 83 Gram-positive bacteria that can produce acid were isolated.
[0034] The formula of MRS solid medium containing calcium carbonate is as follows: peptone 10 g, beef extract 10 g, yeast powder 5 g, dipotassium hydrogen phosphate 2 g, diammonium citrate 2 g, sodium acetate 2 g, glucose 20 g, Tween-80 1 mL, calcium carbonate 20 g, agar 15 g, and distilled water 1000 mL.
[0035] 2. Screening of strains with inhibitory effects on foodborne pathogens (1) Activation of primary screening strains The 83 initially screened strains were inoculated into MRS liquid culture medium (calcium carbonate and agar were not added to the MRS culture medium formula containing calcium carbonate) at an inoculation rate of 1%, and cultured and activated at 37°C for 24 hours to obtain an activated solution.
[0036] (2) Expansion of screening strains Take 1 mL of the activated liquid of the primary screened strain, add it into 100 mL of MRS liquid culture medium (calcium carbonate and agar are not added to the MRS culture medium formula containing calcium carbonate) according to a 1% inoculation amount, and culture it at 37°C for 12 hours to obtain a fermentation liquid.
[0037] (3) Screening of primary screening strains with inhibitory effects on foodborne pathogens Using Staphylococcus aureus (CGMCC 1.8721) and Escherichia coli (CGMCC 1.8723) as indicator bacteria, the 83 initial screening strains were initially screened for antibacterial effects, specifically: LB solid medium (agar content of 1.5%) was used as the bottom medium, and poured onto the plate after melting. Then LB semi-solid medium (agar content of 0.7%) was melted, and 5% indicator bacteria was added after cooling. The medium was fully shaken and 10 mL was poured onto the bottom medium. After complete solidification, the Oxford cup was set aside, and 0.1 mL of fermentation liquid was injected into the Oxford cup. After culturing at 37°C for 24 hours, the size of the inhibition zone was measured, and the ratio of the diameter of the inhibition zone (D) to the diameter of the Oxford cup (d) was used to represent the antibacterial effect. The results showed that the ratio of the diameter of the inhibition zone of 5 strains to the indicator bacteria exceeded 2.00, which was significantly different from other strains, showing a better antibacterial effect. These 5 strains were preserved for further study.
[0038] Eight foodborne pathogenic bacteria, including Escherichia coli (CGMCC 1.8723), Salmonella (CICC 10982), Shigella (CICC 21534), Staphylococcus aureus (CGMCC 1.8721), Listeria monocytogenes (CICC 21633), Pseudomonas aeruginosa (CICC 21636), Bacillus cereus (ATCC 14579) and Enterobacter sakazakii (ATCC 29544), were used as indicator bacteria. The fermentation broths of the above five strains were screened for antibacterial effects according to the initial screening method. The results are as follows: Figure 1 As shown in the figure, a strain isolated from sauerkraut juice (hereinafter referred to as the strain to be identified) showed a strong inhibitory effect on eight foodborne pathogens. Figure 1-Figure 4 shown.
[0039] 3. Identification and preservation (1) Microscopic examination The strain to be identified was subjected to Gram staining and the colony morphology was observed under a microscope, and it was found that the strain was a Gram-positive bacillus.
[0040] (2) Physiological and biochemical identification The strain to be identified was subjected to physiological and biochemical identification using a microbiochemical kit, and was inoculated into biochemical identification tubes of esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, and raffinose, respectively, and cultured at 37°C for 24 hours for identification. As shown in Table 1, the identification results were consistent with the biochemical reaction characteristics of Lactobacillus rhamnosus.
[0041] Table 1 Physiological and biochemical reaction results of the strains to be identified
[0042] Note: + represents a positive reaction, - represents a negative reaction.
[0043] (3) Molecular biological identification The template DNA of the strain was extracted using a genome extraction kit (EasyPure® Genomic DNA Kit, Beijing Quanshijin Biotechnology Co., Ltd.), and 1 μL of the template solution was added to 50 μL of the PCR mixture to amplify the 16S rDNA of the strain to be identified. 6 μL of the PCR product was subjected to agarose gel electrophoresis at 120 V / min for 30 min, and the electrophoresis bands were observed using a gel imaging analysis system. The PCR product was sequenced and analyzed by Shanghai Shenggong Biotechnology Co., Ltd. After sequencing, the 16S rDNA sequence of the strain to be identified was obtained, with a length of 1463 kb. The sequencing results were searched using BLAST on NCBI, and the gene sequences were aligned and compared with the sequences in the GenBank database for homology to construct a phylogenetic tree. Figure 5 As shown in Figure 2, the bacteria were classified as Lactobacillus rhamnosus Lactobacillus rhamnosus, Named as Lactobacillus rhamnosus FJS004.
[0044] The Lactobacillus rhamnosus ( Lactobacillus rhamnosus )The 16SrDNA gene sequence of FJS004 is:
[0045] Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) FJS004 was sent to the General Microbiology Center of China Microorganism Culture Collection Administration for preservation. The preservation date is December 26, 2022, and the preservation number is CGMCC No.26253.
[0046] Example 2 Determination of bile salt resistance of Lactobacillus rhamnosus FJS004 The activated solution of Lactobacillus rhamnosus FJS004 was added to the MRS liquid culture medium containing 1%, 2%, and 3% bile salts at a 1% inoculation rate, and the OD600 absorbance was measured at regular intervals. Figure 6 As shown, the growth of Lactobacillus rhamnosus FJS004 in bile salts showed an overall upward trend over time. The higher the bile salt concentration, the lower the absorbance value, which indicates that Lactobacillus rhamnosus FJS004 has a strong tolerance to bile salts.
[0047] Example 3 Preparation of crudely extracted lactobacillus rhamnosus FJS004 lactobacillus The activated liquid of Lactobacillus rhamnosus FJS004 was inoculated at 1% into MRS liquid culture medium (calcium carbonate and agar were not added to the MRS culture medium formula containing calcium carbonate) and fermented at 37°C for 48 hours, then centrifuged at 10000 r / min for 10 minutes at 4°C, and the supernatant was collected as the crude extract of Lactobacillus rhamnosus FJS004 lactic acid bacteria for the following experiments (including Example 4, Example 5 and Example 6).
[0048] Example 4 Lactobacillus rhamnosus FJS004 lactobacillus has an inhibitory effect on foodborne pathogens Eight foodborne pathogenic bacteria, including Escherichia coli (CGMCC 1.8723), Salmonella (CICC 10982), Shigella (CICC 21534), Staphylococcus aureus (CGMCC 1.8721), Listeria monocytogenes (CICC 21633), Pseudomonas aeruginosa (CICC 21636), Bacillus cereus (ATCC 14579) and Enterobacter sakazakii (ATCC 29544), were used as indicator bacteria. The antibacterial effect of the lactobacillus rhamnosus FJS004 lactic acid bacteria was tested according to the method for screening the primary screening strains with inhibitory effect on foodborne pathogens in (3) of Example 1.
[0049] Results Figure 7 As shown in the figure, the lactobacillus rhamnosus FJS004 lactic acid bacteria have good antibacterial effects on eight foodborne pathogens including Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Enterobacter sakazakii.
[0050] Example 5 Test on the stability of antibacterial ability of lactobacillus rhamnosus FJS004 (1) pH stability Use 1 mol / L HCl or NaOH to adjust the lactobacillus rhamnosus FJS004 lactobacillus to 2.5, 3.5, 4.5, 5.5, 6.5, and 7.5, respectively. Use Escherichia coli as the indicator bacteria to conduct antibacterial tests. After culturing at 37°C for 24 hours, observe and record the results. Figure 8 As shown, the activity of Lactobacillus rhamnosus FJS004 lactic acid bacteria is higher under low pH conditions. When the pH in the stomach and intestine is acidic, Lactobacillus rhamnosus FJS004 lactic acid bacteria is more likely to play a role in the stomach and intestine.
[0051] (2) Temperature stability According to the method for screening the primary screening strains with inhibitory effect on foodborne pathogens in Example 1 (3), an antibacterial test was performed on the high-temperature treated Lactobacillus rhamnosus FJS004 lactic acid bacteria, that is, during the operation, 0.1 mL of fermentation liquid injected into the Oxford cup was replaced with 0.1 mL of high-temperature treated Lactobacillus rhamnosus FJS004 lactic acid bacteria.
[0052] The high temperature treatment process is as follows: take Lactobacillus rhamnosus FJS004 lactic acid bacteria and pack them into several sterile test tubes, and place them in constant temperature water baths at 60℃, 70℃, 80℃, 90℃, 100℃ and 121℃ (boiling water bath) respectively. After placing them for 30 minutes, cool them with running water immediately, and then conduct antibacterial tests after cooling to room temperature. Escherichia coli is used as the indicator bacteria in the antibacterial test. The results are as follows: Fig. 9 As shown in the figure, the lactobacillus rhamnosus FJS004 lactobacillus treated with high temperature has different degrees of antibacterial activity against Escherichia coli, which is different from the antibacterial effect of lactobacillus rhamnosus FJS004 lactobacillus treated with high temperature on Escherichia coli ( Figure 7 ) Compared with the control group, after high temperature treatment at 60-80℃, the antibacterial activity of Lactobacillus rhamnosus FJS004 lactic acid bacteria remained above 90%, after high temperature treatment at 90-100℃, the antibacterial activity remained between 80%-90%, and after high temperature treatment at 121℃, the antibacterial activity still remained above 66%. It can be seen that Lactobacillus rhamnosus FJS004 lactic acid bacteria have high thermal stability.
[0053] Example 6 Study on the antibacterial mechanism of lactobacillus rhamnosus FJS004 Among foodborne pathogens, Escherichia coli, Salmonella, Shigella, Pseudomonas aeruginosa and Enterobacter sakazakii are Gram-positive bacteria, while Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus are Gram-negative bacteria. Through experimental research on the antibacterial mechanism of Lactobacillus rhamnosus FJS004 lactic acid bacteria on these eight foodborne pathogens, similar conclusions were obtained. Now, Escherichia coli and Staphylococcus aureus are used as representative indicator bacteria of Gram-negative and Gram-positive foodborne pathogens, respectively, and the experimental process and results are made public.
[0054] (1) Promotes the leakage of nucleic acid substances in indicator bacteria cells Take Lactobacillus rhamnosus FJS004 lactic acid bacteria, mix it with the indicator bacteria suspension in equal proportion, and culture it at 37℃ for a certain period of time as the experimental group. Take out equal volumes of supernatant after 0h, 2h, 4h, 6h, and 8h, measure the absorbance at 260nm, and draw a curve to study the effect of Lactobacillus rhamnosus FJS004 lactic acid bacteria on the leakage of indicator bacteria nucleic acid substances. Use the indicator bacteria suspension without mixing Lactobacillus rhamnosus FJS004 lactic acid bacteria as the control group.
[0055] like Fig.10 As shown in the figure, after being treated with lactobacillus rhamnosus FJS004 lactobacillus, the absorbance values of the experimental groups (E.EG and S.EG) at 260nm continued to increase, indicating that the content of nucleic acid substances in the test samples continued to increase. It may be that the integrity of the cell membrane of the indicator bacteria was destroyed, and holes were dissolved in a certain way, resulting in the continuous leakage of nucleic acid substances.
[0056] (2) Promoted the leakage of intracellular proteins of indicator bacteria Take Lactobacillus rhamnosus FJS004 lactic acid bacteria, mix it with the indicator bacteria suspension in equal proportion, and culture it at 37℃ for a certain period of time as the experimental group. Take equal volumes of supernatant after 0h, 2h, 4h, 6h, and 8h, and use the Coomassie Brilliant Blue method to determine the leakage of the indicator bacteria intracellular protein. Use the indicator bacteria suspension without mixing Lactobacillus rhamnosus FJS004 lactic acid bacteria as the control group.
[0057] like Fig.11 As shown, with the extension of time, the protein content in the experimental groups (E.EG and S.EG) continued to increase, indicating that the cell membrane of the indicator bacteria was damaged, the leakage of intracellular proteins of the indicator bacteria gradually increased, and the life activities of the indicator bacteria were inhibited.
[0058] (3) Destruction of the integrity of the indicator bacteria cell membrane Flow cytometric analysis: Take the lactobacillus rhamnosus FJS004 lactic acid bacteria, mix it with the bacterial suspension of the indicator bacteria in equal proportions, and culture it at 37°C. Take the bacterial suspension of 0h, 3h, 6h and 9h, centrifuge it at 8000r / min for 10min at 4°C, and harvest the indicator bacterial cells, and wash them three times with physiological saline. Subsequently, the indicator bacterial cells were stained by adding propidium iodide (PI) solution, and the mortality rate of the indicator bacterial cells was evaluated by flow cytometry.
[0059] PI is often used to cross damaged cell membranes and embed into DNA to distinguish between live cells and necrotic cells. Fig.12 It can be seen that after being treated with rhamnosus lactobacillus FJS004 lactobacillus for a certain period of time (3h, 6h and 9h), the PI fluorescence intensity (Q1) of the indicator bacteria cells was high, which indicated that rhamnosus lactobacillus FJS004 lactobacillus destroyed the cell membrane of the indicator bacteria and induced an increase in the PI fluorescence intensity, and the fluorescence intensity continued to increase with the increase in treatment time. After being treated with rhamnosus lactobacillus FJS004 lactobacillus for 3h, 6h and 9h, the mortality rates of Escherichia coli were 12.9%, 44.3% and 75.3%, respectively, and the mortality rates of Staphylococcus aureus were 16.5%, 55.8% and 78.8%, respectively. However, almost no dead cells were observed in the control group (cultured for 0h). It can be seen from the mortality rate that rhamnosus lactobacillus FJS004 lactobacillus not only inhibited the growth of indicator bacteria, but also caused the death of most indicator bacteria. This may be because it destroyed the integrity of the indicator bacteria cell membrane, increased the permeability, and the leakage of key internal components reached a certain extent, which eventually led to the death of the indicator bacteria.
[0060] Fluorescence spectroscopy analysis: The lactobacillus rhamnosus FJS004 lactobacillus was mixed evenly with the indicator bacterial suspension and cultured at 37°C. The bacterial solution cultured for 12 hours was centrifuged at 8000r / min for 10 minutes at 4°C to harvest the indicator bacterial cells. Subsequently, fluorescein diacetate (FDA) and propidium iodide (PI) were added to stain the indicator bacterial cells. The fluorescence intensity of the stained indicator bacteria at an excitation wavelength of 450nm was detected using a fluorescence spectrum scanner. The indicator bacterial suspension that was not treated with lactobacillus rhamnosus FJS004 lactobacillus was used as a control.
[0061] Fig.13 This is a fluorescence spectrum diagram after staining two indicator bacteria with FDA and PI. It can be seen from the figure that the indicator bacteria that have not been treated with rhamnosus lactobacillus FJS004 lactobacillus have a single peak near 520nm, which is the absorption peak of FDA. However, after the indicator bacteria were treated with rhamnosus lactobacillus FJS004 lactobacillus, new absorption peaks appeared at 604nm for both indicator bacteria. The new absorption peak is a typical absorption peak of PI, which further shows that rhamnosus lactobacillus FJS004 lactobacillus has destroyed the integrity of the indicator bacteria cell membrane.
[0062] (4) Destruction of superoxide dismutase (SOD) in indicator bacteria cells The lactobacillus rhamnosus FJS004 lactobacillus and the indicator bacterial suspension were mixed evenly and cultured at 37°C. The bacterial suspension cultured for 12 hours was taken, ultrasonically disrupted at 4°C, and centrifuged at 10000r / min at 4°C for 10 minutes. The supernatant was taken and the intracellular superoxide dismutase (SOD) activity was determined according to the instruction manual of the kit (purchased from Nanjing Jiancheng Bioengineering Institute). The indicator bacterial suspension without lactobacillus rhamnosus FJS004 lactobacillus was used as the control group.
[0063] SOD activity can reflect the body's ability to remove oxygen free radicals. When cells are damaged, in order to ensure cell activity, the activity of SOD with detoxification ability will increase. Fig.14 It can be seen that under the action of Lactobacillus rhamnosus FJS004 lactic acid bacteria, the SOD activity of the experimental groups of the two indicator bacteria increased significantly, indicating that Lactobacillus rhamnosus FJS004 lactic acid bacteria are toxic to both indicator bacteria, causing the cell membrane to undergo lipid peroxidation and be destroyed, thereby damaging the bacteria.
[0064] (5) Minimum inhibitory concentration (MIC) of four antibiotics against Escherichia coli and Staphylococcus aureus The minimum inhibitory concentration of indicator bacteria was determined to evaluate the antibacterial effects of four antibiotics: kanamycin, nalidixic acid, penicillin, and rifamycin.
[0065] The activated indicator bacteria were centrifuged at 4°C and 8000 r / min for 10 min, washed three times with PBS buffer, and the bacterial concentration was adjusted to 1×10 4 CFU / mL. Then prepare an antibiotic solution with a concentration of 2048μg / mL, and dilute it with LB medium in multiples to 1024μg / mL, 512μg / mL, 256μg / mL, 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, and 1μg / mL, a total of 12 antibiotic solutions.
[0066] Use a pipette to successively pipette 100 μL of indicator bacteria solution and 100 μL of antibiotic solution into the same well of a 96-well plate as the experimental group. The growth control group consists of 100 μL of indicator bacteria solution and 100 μL of LB medium, and the blank control group consists of 200 μL of LB medium. Set up three parallel wells for the experiment, culture at 37°C for 24 h, and use an enzyme reader to measure the absorbance at 600 nm.
[0067] Table 2 Minimum inhibitory concentration of indicator bacteria to four antibiotics (μg / mL)
[0068] (6) Sublethal bacterial damage repair experiments and target research Take the lactobacillus rhamnosus FJS004 lactobacillus, mix it with the indicator bacteria suspension at a ratio of 1:3, and culture it at 37°C for 12 hours to obtain the experimental mixture. Dilute the experimental mixture 10 times with physiological saline, then take 100 μL of the dilution from 2-3 appropriate gradients and apply it to LB solid culture medium, culture it at 37°C for 24 hours for sublethal damage repair, as the antibiotic-free group; at the same time, apply the experimental mixture to LB solid culture medium containing antibiotics (the antibiotics and LB solid culture medium are mixed and poured on the plate, the concentration of antibiotics used is shown in Table 3), and culture it at 37°C for 24 hours, as the antibiotic-containing group. Calculate the inhibition recovery level of the E.EG group (the indicator bacteria added are Escherichia coli) and the S.EG group (the indicator bacteria added are Staphylococcus aureus), and the calculation formula is as follows:
[0069] Similarly, the indicator bacterial suspension was cultured at 37°C for 12 hours as the control solution. The control solution was diluted with physiological saline in a 10-fold gradient, and then 100 μL of the dilution was taken from 2-3 appropriate gradients and spread on LB solid culture medium, and cultured at 37°C for 24 hours for sublethal damage repair, as the antibiotic-free group; at the same time, the control solution was spread on LB solid culture medium containing antibiotics (the antibiotics were mixed with LB solid culture medium and poured on the plate, and the concentrations are shown in Table 3), and cultured at 37°C for 24 hours, as the antibiotic-containing group. The above formula was used to calculate the inhibition recovery level of the E.CG group (the indicator bacteria added were Escherichia coli) and the S.CG group (the indicator bacteria added were Staphylococcus aureus).
[0070] The results of the inhibition recovery level of indicator bacteria are summarized in Table 4.
[0071] Table 3 Concentrations (μg / mL) of four antibiotics added to LB medium and their targets
[0072] Table 4 Inhibition recovery level of indicator bacteria (%)
[0073] Sublethally damaged bacteria have the ability to repair themselves and return to normal cell levels, and the repaired cells can self-divide and grow. The inhibitory targets of the four antibiotics, kanamycin, nalidixic acid, penicillin and rifamycin, on indicator bacteria are known (see Table 3). Based on the above MIC experimental results, we screened the content of antibiotics to be added in this experiment to ensure that its content would not inhibit the growth and reproduction of indicator bacteria that have not been treated with rhamnosus lactobacillus FJS004 lactobacillus, but would inhibit the sublethal damage repair of indicator bacteria treated with rhamnosus lactobacillus FJS004 lactobacillus, that is, the rhamnosus lactobacillus FJS004 lactobacillus in the experimental group put the indicator bacteria in a sublethal damage repair state. After contacting a certain amount of antibiotics, if the target of the antibiotic on the indicator bacteria is the same as the target of the rhamnosus lactobacillus FJS004 lactobacillus on the indicator bacteria, the sublethal damage repair of the indicator bacteria will be inhibited, and normal growth and reproduction will no longer be possible.
[0074] As shown in Table 4, the inhibition recovery levels of E.CG and S.CG are close to 0 (lower than 0.12). The reason is that the indicator bacteria in the antibiotic-containing groups of E.CG and S.CG are in a normal growth and reproduction state due to the low antibiotic concentration, and the colony count of the indicator bacteria is equivalent to that of the corresponding antibiotic-free groups.
[0075] When penicillin and rifamycin were added, the inhibition recovery levels of E.EG and S.EG were significantly greater than those of the corresponding E.CG and S.CG. The reason was that the indicator bacteria in their antibiotic-containing groups were in a state of sublethal damage repair after being treated with Lactobacillus rhamnosus FJS004 lactobacillus. The targets of penicillin and rifamycin on the indicator bacteria were the same as those of Lactobacillus rhamnosus FJS004 on the indicator bacteria, which could inhibit the sublethal damage repair of the indicator bacteria. The colony count of the indicator bacteria was lower than that of the corresponding antibiotic-free group, and the inhibition recovery level was improved. It can be seen that the targets of Lactobacillus rhamnosus FJS004 lactobacillus are cell wall and RNA, and it has an antibacterial effect by inhibiting the synthesis of cell wall and RNA. On the contrary, there was no significant difference in the inhibition recovery levels between E.CG and E.EG, and between S.CG and S.EG of nalidixic acid and kanamycin, indicating that Lactobacillus rhamnosus FJS004 lactobacillus does not have an antibacterial effect by inhibiting protein synthesis and DNA synthesis.
[0076] Example 7 Preparation of Lactobacillus rhamnosus FJS004 bacterial powder Lactobacillus rhamnosus FJS004 was inoculated into MRS liquid culture medium, cultured and activated at 37°C for 24 hours, then inoculated into MRS liquid culture medium at a 1% inoculum amount, and expanded cultured at 37°C for 12 hours to obtain Lactobacillus rhamnosus FJS004 expanded culture fluid, which was spray-dried and compounded with inulin to obtain Lactobacillus rhamnosus FJS004 powder, the number of viable bacteria in the powder was 10 billion / g.
[0077] Example 8 Preparation of Lactobacillus rhamnosus FJS004 bacterial powder Lactobacillus rhamnosus FJS004 was inoculated into MRS liquid culture medium, cultured and activated at 37°C for 24 hours, then inoculated into MRS liquid culture medium at a 1% inoculum amount, and expanded cultured at 37°C for 24 hours to obtain Lactobacillus rhamnosus FJS004 expanded culture fluid, which was freeze-dried and compounded with xylooligosaccharides to obtain Lactobacillus rhamnosus FJS004 powder, the number of viable bacteria in the powder being 100 billion / g.
[0078] Example 9 Preparation of yogurt containing Lactobacillus rhamnosus FJS004 The above experiment shows that Lactobacillus rhamnosus FJS004 can inhibit the growth of eight food-borne pathogens. Adding Lactobacillus rhamnosus FJS004 to yogurt can improve the health and safety of yogurt. Streptococcus thermophilus and Lactobacillus bulgaricus in traditional yogurt are not easy to colonize in the large intestine. Lactobacillus rhamnosus FJS004 has a better antibacterial effect and is easier to colonize in the intestine, which can effectively improve the probiotic effect of yogurt.
[0079] The preparation method of yogurt containing Lactobacillus rhamnosus FJS004 is as follows: Heat the raw milk to about 50℃, add 8% white sugar, continue to heat up to 65℃, filter, enter the homogenizer, homogenize under 8.0-10.0MPa pressure, sterilize at 95℃ for 5-10min, immediately cool to 42℃, add bacteria (Lactobacillus rhamnosus FJS004: Streptococcus thermophilus: Lactobacillus bulgaricus = 1:1:1, inoculation amount is 8%), ferment at 42℃ for 6h, after complete solidification, refrigerate at 4℃ for 12h, and you can get yogurt containing Lactobacillus rhamnosus FJS004.
[0080] Example 10 Preparation of a probiotic beverage containing Lactobacillus rhamnosus FJS004 The reconstituted milk is made from skim milk powder as the main raw material, 6% white sugar is added, the temperature is raised to 85°C, stirred and dissolved for 30 minutes, filtered, and put into a homogenizer, homogenized at a pressure of 15-20.0MPa, sterilized at 65°C for 30 minutes, immediately cooled to 42°C, and bacteria (Lactobacillus rhamnosus FJS004: Streptococcus thermophilus: Lactobacillus bulgaricus = 1:1:1, inoculation amount is 8%) are added, fermented for 3-4 hours, stirred for 10 minutes, demulsified, and made into a probiotic fermentation liquid. The probiotic fermentation liquid is mixed with water at a ratio of 1:1 to obtain a probiotic fermentation dilution, 0.05% citric acid, 2% white sugar and 0.15% stabilizer CMC-Na are added to the probiotic fermentation dilution according to the mass ratio, and after sufficient stirring, it is homogenized with a homogenizer at a homogenization pressure of 10MPa, and then filled and sterilized to obtain a probiotic beverage containing Lactobacillus rhamnosus FJS004.
[0081] Example 11 Preparation of postbiotics from Lactobacillus rhamnosus FJS004 Postbiotics are a general term for probiotics and metabolites after probiotics are processed, including bacteria and metabolites. Recent studies have found that postbiotics have similar effects to probiotics, but compared with probiotics, they have higher tolerance to gastric acid and bile salts and are easy to preserve. Lactobacillus rhamnosus FJS004 was inoculated into MRS liquid culture medium at 1% inoculation and cultured at 37°C for 24-48h. The obtained Lactobacillus rhamnosus FJS004 culture solution was sterilized at 63°C for 30min, and sprayed or freeze-dried to obtain Lactobacillus rhamnosus FJS004 postbiotics that can be used as natural preservatives.
[0082] Example 12 Preparation of yogurt containing Lactobacillus rhamnosus FJS004 postbiotics Heat the raw milk to 50°C, add 8% white sugar, continue to heat to 65°C, filter, homogenize, sterilize at 95°C for 5-10 minutes, immediately cool to 42°C, add bacteria and postbiotics (bacteria thermophilic Streptococcus: Lactobacillus bulgaricus = 1:1, inoculation amount is 8%; Lactobacillus rhamnosus FJS004 postbiotic addition amount is 8%), ferment at 42°C for 6h, after complete solidification, refrigerate at 4°C for ripening, and you can get yogurt containing Lactobacillus rhamnosus FJS004 postbiotics.
[0083] Example 13 Preparation of a probiotic beverage containing Lactobacillus rhamnosus FJS004 postbiotics Reconstituted milk is made from skim milk powder as the main raw material. After adding 6% white sugar, the temperature is raised to 85℃ and stirred for 30 minutes to dissolve. Then the mixture is filtered and homogenized in a homogenizer at a pressure of 15-20.0MPa. The mixture is sterilized at 65℃ for 30 minutes and immediately cooled to 42℃. Bacteria (Thermophilic Streptococcus: Bulgarian Lactobacillus = 1:1, inoculation amount is 8%) are added. After fermentation for 3-4 hours, the mixture is stirred for 10 minutes, demulsified, and the probiotic fermentation liquid is made. The probiotic fermentation liquid is mixed with water at a ratio of 1:1 to obtain a probiotic fermentation dilution. According to the mass ratio, 0.05% citric acid, 2% white sugar, 0.15% stabilizer CMC-Na and 12% postbiotics of Lactobacillus rhamnosus FJS004 are added to the probiotic fermentation dilution. After being fully stirred, the mixture is homogenized in a homogenizer at a homogenization pressure of 10MPa. Then the mixture is filled and sterilized.
[0084] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.
Claims
1. A strain of Lactobacillus rhamnosus FJS004 for inhibiting foodborne pathogens, characterized in that: Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) FJS004 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 26, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26253.
2. A lactobacillus rhamnosus FJS004 lactobacillus, characterized in that The lactobacillus rhamnosus FJS004 is prepared according to claim 1; the preparation method is as follows: inoculating Lactobacillus rhamnosus FJS004 in an MRS liquid culture medium at an inoculation rate of 1%, culturing and activating at 37°C for 24 hours to obtain an activated liquid, inoculating the activated liquid in an MRS liquid culture medium at an inoculation rate of 1%, culturing at 37°C for 12-48 hours, and collecting the supernatant by centrifugation to obtain Lactobacillus rhamnosus FJS004 lactobacillus.
3. Use of the Lactobacillus rhamnosus FJS004 according to claim 1 or / and the Lactobacillus rhamnosus FJS004 lactobacillus according to claim 2 in the preparation of a preparation for inhibiting foodborne pathogens, characterized in that: Foodborne pathogens include one or more of Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus and Enterobacter sakazakii.
4. The use according to claim 3, characterized in that Lactobacillus rhamnosus FJS004 lactobacillus inhibits or kills foodborne pathogens by destroying the integrity of cell membranes.
5. The use according to claim 4, characterized in that Disruption of cell membrane integrity includes disruption of cell membrane integrity of foodborne pathogens by increasing intracellular superoxide dismutase activity.
6. The use according to claim 3, characterized in that Lactobacillus rhamnosus FJS004 lactic acid bacteria inhibit or kill foodborne pathogens by promoting the leakage of intracellular nucleic acids.
7. The use according to claim 3, characterized in that Lactobacillus rhamnosus FJS004 lactobacillus inhibits or kills foodborne pathogens by promoting intracellular protein leakage.
8. The use according to claim 3, characterized in that Lactobacillus rhamnosus FJS004 lactobacillus inhibits or kills foodborne pathogens by inhibiting RNA synthesis.
9. The use according to claim 3, characterized in that Lactobacillus rhamnosus FJS004 lactobacillus inhibits or kills foodborne pathogens by inhibiting cell wall synthesis.
10. The use according to claim 3, characterized in that Preparations refer to foods, health foods or medicines that contain Lactobacillus rhamnosus FJS004 or its metabolites as ingredients for inhibiting foodborne pathogens; metabolite ingredients include Lactobacillus rhamnosus FJS004 lactic acid bacteria and Lactobacillus rhamnosus FJS004 postbiotics; Lactobacillus rhamnosus FJS004 or its metabolites exist in the form of powder, granules or liquid in the ingredients for inhibiting foodborne pathogens.