Application of lactobacillus paracasei Jlus 660321 in bacteriostasis

Abstract

The invention belongs to the field of microorganisms, and particularly relates to application of lactobacillus paracasei Jlus 660321 in the aspect of bacteriostasis, the lactobacillus paracasei Jlus 660321 is classified and named as lactobacillus paracasei, is preserved in the China General Microbiological Culture Collection Center (CGMCC), has the preservation number of CGMCC No.33962, is separated and screened from naturally fermented dairy products, and has the advantages that the lactobacillus paracasei Jlus 660321 can be used for preparing the lactobacillus paracasei; then, after ultraviolet radiation mutagenesis, a series of screening experiments are adopted to obtain the strain. The strains inhibited by the strain comprise bacteria and fungi, and the bacteria comprise one or more of escherichia coli, staphylococcus aureus and salmonella. The fungi comprise one or more of botrytis cinerea, trichothecium roseum, aspergillus niger, fusarium verticillium, Penicillium Polish, Penicillium adalidaea and Penicillium oxalicum; the method can be used for preparing products such as antibacterial drugs, food and health-care products, and has wide development prospects and application values.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a Lactobacillus paracasei Jlus660321 with broad-spectrum antibacterial function and application thereof in inhibiting bacteria. BACKGROUND

[0002] Foodborne pathogenic bacteria are pathogenic bacteria that can cause food poisoning or food as a transmission medium, and are an important source of food safety problems. Globally, a large number of people suffer from diseases every year due to the intake of food contaminated by foodborne pathogenic bacteria (such as Salmonella, Escherichia coli, and Staphylococcus aureus). At the same time, food spoilage problems caused by bacterial or mold (including Penicillium, Aspergillus, etc.) contamination cannot be ignored. About 1 / 3 of the world's food is wasted every year due to microbial contamination, and some microorganisms also produce toxins, which directly threaten human health. These factors seriously affect the healthy development of mankind.

[0003] At present, antibiotics and chemical preservatives are widely used to inhibit the growth of these microorganisms. However, chemical preservatives have safety risks and may adversely affect the flavor and quality of food. Overuse of antibiotics may accelerate the formation of drug-resistant bacteria. At the same time, the use of these chemicals may also affect the activity of some beneficial bacteria in food, and even affect the fermentation process of fermented food. Lactobacillus paracasei is a kind of lactic acid bacteria that widely exists in the intestinal tract of healthy animals, but the research on Lactobacillus paracasei in inhibiting fungi is less, and not every Lactobacillus paracasei has the effect of inhibiting fungi. SUMMARY

[0004] In order to solve the above technical problems, the application of Lactobacillus paracasei in inhibiting bacteria is developed, and the present application provides a Lactobacillus paracasei Jlus660321 and application thereof in inhibiting bacteria.

[0005] The Lactobacillus paracasei Jlus660321 provided in the present application is classified and named as Lactobacillus paracasei, which is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, and has a preservation date of March 24, 2025 and a preservation number of CGMCC No. 33962. The 16S rDNA sequence thereof is shown as SEQ ID No. 1.

[0006] The Lactobacillus paracasei Jlus660321 provided in the present application is isolated and screened from naturally fermented dairy products, and then obtained through a series of screening experiments after ultraviolet radiation mutagenesis.

[0007] The application provides application of Lactococcus lactis Jlus 660321 in inhibition of bacteria according to the bacteriostatic function of the Lactococcus lactis Jlus 660321, and the inhibited bacteria include bacteria and fungi, and the bacteria include gram-positive bacteria and gram-negative bacteria.

[0008] Further, the bacteria include one or more of Escherichia coli, Staphylococcus aureus and Salmonella, and the fungi include one or more of Botrytis cinerea, Trichothecium roseum, Aspergillus niger, Fusarium verticilliodes, Penicillium polonicum, Penicillium chrysogenum and Penicillium oxalicum.

[0009] Further, based on the bacteriostatic function of the Lactococcus lactis Jlus 880314, the application also provides application of the Lactococcus lactis Jlus 660321 in preparation of an antibacterial drug, and the Lactococcus lactis Jlus 660321 has a significant inhibitory effect on growth of gram-negative bacteria and / or fungi.

[0010] In another aspect, the application also provides application of the Lactococcus lactis Jlus 660321 in preparation of a product for relieving enteritis, and the enteritis is caused by gram-negative bacteria and / or fungi.

[0011] In another aspect, the application also provides application of the Lactococcus lactis Jlus 660321 in preparation of a product for treating gastrointestinal microecological imbalance of human or animals, and the gastrointestinal microecological imbalance is caused by gram-negative bacteria and / or fungi.

[0012] The product is one or more of a drug, a nutrient or a health product.

[0013] The application has the following beneficial effects:

[0014] The Lactococcus lactis Jlus 660321 provided by the application has excellent and extensive bacteriostatic function in a bacteriostatic experiment, and can effectively inhibit growth of bacteria and fungi such as Escherichia coli, Staphylococcus aureus, Salmonella, Botrytis cinerea, Trichothecium roseum, Aspergillus niger, Fusarium verticilliodes, Penicillium polonicum, Penicillium chrysogenum and Penicillium oxalicum. Therefore, the Lactococcus lactis Jlus 660321 provided by the application can be used for preparing bacteriostatic drugs, foods, health products and other products, and has wide development prospect and application value. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is a strain microscopic examination photo of the Lactococcus lactis Jlus 660321;

[0016] Figure 2 The figure is a growth curve change result of Staphylococcus aureus in Example 2.

[0017] Figure 3 Staphylococcus aureus colony count change results of Example 2 of the present application;

[0018] Figure 4 Staphylococcus aureus cell morphology change results of Example 2 of the present application;

[0019] Figure 5 Escherichia coli growth curve change results of Example 2 of the present application;

[0020] Figure 6 Escherichia coli colony count change results of Example 2 of the present application;

[0021] Figure 7 Escherichia coli cell morphology change results of Example 2 of the present application;

[0022] Figure 8 Salmonella growth curve change results of Example 2 of the present application;

[0023] Figure 9 Salmonella colony count change results of Example 2 of the present application;

[0024] Figure 10 Salmonella cell morphology change results of Example 2 of the present application;

[0025] Figure 11 Botrytis cinerea growth rate change results of Example 3 of the present application;

[0026] Figure 12 Trichothecium sanguinolentum growth rate change results of Example 3 of the present application;

[0027] Figure 13 Aspergillus niger growth rate change results of Example 3 of the present application;

[0028] Figure 14 Fusarium verticillioides growth rate change results of Example 3 of the present application;

[0029] Figure 15 Penicillium polonicum growth rate change results of Example 3 of the present application;

[0030] Figure 16 Penicillium chrysogenum growth rate change results of Example 3 of the present application;

[0031] Figure 17 Penicillium oxalicum growth rate change results of Example 3 of the present application. DETAILED DESCRIPTION

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0033] The materials, reagents, etc. used in the following examples, unless otherwise specified, were obtained from commercial sources.

[0034] Example 1,

[0035] The isolation screening, identification and safety evaluation of Lactobacillus paracasei Jlus 660321 are as follows:

[0036] I. Isolation screening:

[0037] After diluting the traditional fermented milk sample with physiological saline, it was spread on MRS plates containing calcium carbonate and cultured at 37°C for 48 h. Then single colonies with obvious calcium dissolution rings and different morphologies were picked from the plates and cultured in MRS liquid medium at 37°C for 24 h. Then the process of solid medium streak culture-single colony picking in liquid medium culture was repeated three times. Then the bacterial liquid after isolation and purification was subjected to gram staining microscopy and hydrogen peroxide enzyme experiment. Gram-positive bacilli and hydrogen peroxide enzyme-negative bacteria were selected for the next experiment. After irradiating each bacterial liquid with 254 nm ultraviolet radiation for 30 s, it was spread on MRS plates containing bile salts and calcium carbonate, and colonies with obvious calcium dissolution rings and larger morphology were picked and cultured in MRS liquid medium at 37°C for 24 h. Then the process of solid medium streak culture-single colony picking in liquid medium culture was repeated three times. Finally, a Lactobacillus paracasei Jlus 660321 was obtained.

[0038] Preservation instructions of the obtained Lactobacillus paracasei Jlus 660321:

[0039] Strain name: Lactobacillus paracasei;

[0040] Latin name: Lactobacillus paracasei;

[0041] Reference biological material (strain): Jlus 660321;

[0042] Preservation agency: China General Microbiological Culture Collection Center;

[0043] Abbreviation of the preservation agency: CGMCC;

[0044] Address: No. 3, Beichen West Road, Chaoyang District, Beijing;

[0045] Preservation date: March 24, 2025;

[0046] Preservation center registration number: CGMCC No. 33962;

[0047] II. Strain identification:

[0048] The frozen glycerol-preserved bacteria were activated for three generations using MRS medium, and then isolated on MRS solid plates using a four-zone division method. The plates were incubated at 37°C for 24 h, and the colony morphology was observed and the colonies were picked for Gram staining and microscopic observation.

[0049] The results showed that the strain presented white round colonies with smooth edges and surfaces. The Gram staining result was positive, and the strain was rod-shaped, single, paired or in chains, and did not produce spores. Figure 1 The microscopic examination results of the strain are shown in the following figure.

[0050] The strain DNA was extracted using a DNA kit and subjected to PCR amplification and 16S rDNA sequencing. According to the sequencing results, the strain was Lactococcus lactis, and its 16S rDNA sequence is shown as SEQ ID No. 1.

[0051] The culture temperature of Lactococcus lactis Jlus 660321 was 37°C; the natural pH; the medium composition: 10 g of proteose peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 ml of Tween 80, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 15 g of agar, and 1 L of distilled water.

[0052] III. Safety evaluation:

[0053] The activated Lactococcus lactis Jlus 660321 bacterial solution was inoculated onto the surface of MRS solid medium containing 5% defibrillated sheep blood, and the plate was incubated at 37°C for 24 h. The hemolysis of the plate was observed.

[0054] The results showed that the Lactococcus lactis Jlus 660321 was γ-hemolytic, i.e., not hemolytic, in the blood plate culture medium, which proved its biological safety.

[0055] Example 2,

[0056] I. Source of experimental bacteria:

[0057] The Staphylococcus aureus used in the experiment was from the China Industrial Microbial Culture Collection Center, with the number CICC 10001; the Escherichia coli was from Jilin University, with the number ATCC O157: H7; and the Salmonella enterica was from the American Type Culture Collection, with the number ATCC 13076.

[0058] II. Effect of Lactococcus lactis Jlus 660321 on bacterial growth:

[0059] After the frozen bacteria were thawed, they were streaked on solid medium and incubated at 37°C for 24 h. Then, single colonies were picked and cultured in liquid medium to the logarithmic phase, and each harmful bacteria was diluted to 10 6 CFU / mL using TSB medium. Lactobacillus paracasei Jlus 660321 was inoculated in MRS medium and incubated at 37°C for 48 h, and the supernatant was filtered through a 0.22 μm filter to obtain a sterile fermentation broth. The harmful bacteria dilution and the Lactobacillus paracasei Jlus 660321 fermentation broth were mixed in equal amounts and incubated at 37°C for 24 h, and the absorbance value was measured every 0.5 h to draw the growth curve of the harmful bacteria. Then, the bacteria after incubation were diluted and spread on the surface of TSA plates, and incubated at 37°C for 24 h to observe the change in bacterial count. In addition, the bacteria after incubation were collected, fixed with glutaraldehyde, dehydrated and dried, and observed for changes in morphology using a scanning electron microscope after gold spraying. The control group was MRS medium without inoculation of Lactobacillus paracasei Jlus 660321 and was subjected to the same operation to compare the inhibitory effect of Lactobacillus paracasei Jlus 660321 on harmful bacteria.

[0060] Figures 2 to 4 Lactobacillus paracasei Jlus 660321 against Staphylococcus aureus; Figures 5 to 7 Lactobacillus paracasei Jlus 660321 against Escherichia coli; Figures 8 to 10 Lactobacillus paracasei Jlus 660321 against Salmonella. After treatment with Lactobacillus paracasei Jlus 660321 fermentation broth, the growth of the three experimental bacteria was completely inhibited. The results of the plate dilution and spread method showed that the inhibition rate of Lactobacillus paracasei Jlus 660321 fermentation broth on the three bacteria was more than 99.9%. The results of scanning electron microscopy showed that the Staphylococcus aureus in the control group was full and round, and Escherichia coli and Salmonella, as gram-negative bacteria, slightly shrunk during dehydration, but the bacterial bodies were still intact and the contents were sufficient. After treatment with Lactobacillus paracasei Jlus 660321 fermentation broth, the Staphylococcus aureus cells ruptured, the Escherichia coli cells showed obvious damage on the surface, and the Salmonella became shriveled. This indicates that the metabolites of Lactobacillus paracasei Jlus 660321 can not only inhibit the growth of harmful bacteria, but also damage the integrity of their cell membranes, causing cell death, and exhibit excellent bactericidal activity.

[0061] Example 3,

[0062] I. Source of experimental fungi:

[0063] Botrytis cinerea used in the experiment was from Shanghai Center for Collection and Cultivation of Microorganisms, with the number AS3.3789; Trichothecium roseum, Aspergillus niger, Penicillium polonicum and Penicillium adametzioides were isolated from cherry tomatoes; Fusarium verticillioides was from rotten kiwi fruit; and Penicillium oxalicum was from deteriorated citrus.

[0064] II. Effect of Lactobacillus paracasei Jlus 660321 on fungal growth

[0065] After the frozen fungal spore suspension was thawed, it was coated on solid culture medium and cultured at 25°C to activate, and B. cinerea was cultured for 5 days, T. roseum and F. verticillioides were cultured for 3 days, and A. niger, P. polonicum, P. adametzioides and P. oxalicum were all cultured for 24 h. Lactobacillus paracasei Jlus 660321 was inoculated in MRS medium and cultured at 37°C for 48 h, and the supernatant was obtained by passing the 0.22 μm filter membrane to obtain the sterile fermentation broth. After the PDA medium (additional 1.5% agar) was sterilized and not solidified, it was mixed with the fermentation broth of Lactobacillus paracasei Jlus 660321 in equal volume, and then the medium was poured. After activation, each fungal plate was punched (9 mm), and then the small agar blocks of each fungus were transferred to the surface of the prepared PDA plate containing the fermentation broth for culture. The control group selected MRS medium without inoculation of Lactobacillus paracasei Jlus 660321 and was subjected to the same operation. The mycelial expansion of each group of fungi was observed, and the inhibitory effect of Lactobacillus paracasei Jlus 660321 metabolites on spoilage fungi was compared.

[0066] Figures 11 to 17The fermentation broth of Lactobacillus paracasei Jlus 660321 was used to inhibit the growth of each group of fungi. Because the growth rates of each fungus were different, the growth results of Aspergillus niger were measured on the 3rd day, the growth results of Trichothecium sanguinaris were measured on the 4th day, the growth results of Fusarium verticillioides were measured on the 5th day, the growth results of Botrytis cinerea and Penicillium oxalicum were measured on the 6th day, and the growth results of Penicillium polonicum and Penicillium adametzi were measured on the 7th day. Lactobacillus paracasei Jlus 660321 had obvious inhibitory effect on the seven strains of mold in the experiment, and could completely inhibit the growth of Botrytis cinerea, Trichothecium sanguinaris, Penicillium adametzi and Penicillium oxalicum (the inhibition rate was about 100%). It could directly reduce the growth area of Aspergillus niger from 42.0 mm to 36.5 mm (the inhibition rate was about 16.7%), reduce the diffusion diameter of Penicillium polonicum from 28.6 mm to 21.3 mm (the inhibition rate was about 37.4%), and reduce the growth area of Fusarium verticillioides from 41.3 mm to 29.0 mm (the inhibition rate was about 38.0%). In general, Lactobacillus paracasei Jlus 660321 also had obvious inhibitory effect on the growth of various fungi that could cause food spoilage. The above results show that Lactobacillus paracasei Jlus 660321 can exhibit excellent antibacterial activity against various pathogenic bacteria and spoilage fungi, and can be used to prepare antibacterial drugs and food additives for extending the shelf life of food.

[0067] Although the present application has been described above with the aid of the general description and the specific embodiments, the above description and the specific embodiments are only part of the embodiments of the present application; on the basis of the present application, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, some modifications or improvements made on the basis of the present application all fall within the scope of the present application.

Claims

1. The application of Lactobacillus paracasei Jlus 660321, characterized by: The Lactobacillus paracasei Jlus 660321 is classified and named Lactobacillus paracasei. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 24, 2025, and the accession number is CGMCC No. 33962. The Lactobacillus paracasei Jlus 660321 is used to prepare antimicrobial drugs, which are used to inhibit bacteria and / or fungi.

2. The application according to claim 1, characterized in that: The bacteria are one or more of Escherichia coli, Staphylococcus aureus, and Salmonella; the fungi are one or more of Botrytis cinerea, Trichoderma pinkis, Aspergillus niger, Fusarium rotundifolium, Penicillium polonum, Penicillium adapalene, and Penicillium oxalate.

3. The application according to claim 1, characterized in that: Application in the preparation of products for relieving enteritis caused by Gram-negative bacteria and / or fungi.

4. The application according to claim 1, characterized in that: Application in the preparation of products for treating gastrointestinal microecological imbalances in humans or animals caused by Gram-negative bacteria and / or fungi.

5. The application according to claim 3 or 4, characterized in that: The product is one or more of the following: pharmaceuticals, nutritional products, and health products.

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