Lactobacillus paracasei GY1 as well as screening method and application thereof

Abstract

The invention belongs to the technical field of biological application, and particularly relates to lactobacillus paracasei GY1 as well as a screening method and application thereof. The lactobacillus paracasei is lactobacillus paracasei GY1 separated and screened from mulberry soil samples, the preservation number is CGMCC No.30931, and the lactobacillus paracasei is classified and named as lactobacillus paracasei in China General Microbiological Culture Collection Center on June 11, 2024, and the preservation number is CGMCC No.30931; the strain is a safe strain with good probiotic potential, and the nucleotide sequence of 16S rDNA sequencing of the strain is as shown in SEQ ID NO. 1. By optimizing the culture conditions of the screened strains, the bacteriostasis rate of the obtained supernate on staphylococcus aureus is higher than 95%, a new thought is provided for the field of food preservation of dairy products and the like, and good market prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of food preservative preparation, and particularly relates to a Lactobacillus paracasei GY1 and a screening method and application thereof. Background Art

[0002] Dairy products can provide abundant nutrients such as protein, carbohydrates and vitamins for human body and are highly favored. However, because dairy products are nutritious, they become potential hosts of a large number of bacteria. Wherein, Staphylococcus aureus (Staphylococcusaureus) is a typical bacterium common in a kind of dairy products, is a kind of Gram-positive bacteria, facultative anaerobic bacteria, belongs to Staphylococcus, and is round or oval in shape, and it has strong tolerance to drying, heat, high salt and antibiotics, and can produce multiple toxins, such as enterotoxins, hemolytic toxins and exfoliative toxins, etc., wherein enterotoxins are the main causes of food poisoning, because enterotoxins still remain in dairy products after high temperature treatment, and multiple diseases can be caused in human body after mistakenly eating. In addition, Staphylococcus aureus can also cause multiple skin infections, such as furuncle, carbuncle and impetigo, etc., which seriously endanger human health. After mistakenly eating the food infected by Staphylococcus aureus, it is easy to cause the symptoms such as nausea, vomiting, abdominal pain and diarrhea in human body (Molecular Medical Microbiology, 2, 2002, 839-888).

[0003] In recent years, due to the abuse of antibiotics and other products, the drug resistance problem of Staphylococcus aureus has become increasingly prominent. Therefore, the emergence of an antibacterial product with excellent inhibitory properties against Staphylococcus aureus will help alleviate the current phenomenon and has important research significance in the fields of food, medical care, etc.

[0004] Traditional food preservation relies heavily on chemical preservatives to extend the shelf life (Trends in Food Science & Technology, 129, 2022, 421-439). Microbial growth is the main cause of food spoilage, and chemical preservatives can extend the shelf life to a certain extent by blocking microbial growth. However, chemical preservatives not only harm health, but also cause the iterative increase of the resistance of spoilage microorganisms. Long-term use may cause similar "antibiotic abuse" and lead to "no medicine available", so it is necessary to find new food preservatives. At present, antimicrobial peptides used for food preservation have received great attention due to their good biosafety (Biomaterials Advances, 155, 2023, 213684). Antimicrobial peptides have the advantages of broad spectrum, high safety and wide sources, which make them have unique application value in the antibacterial field and are increasingly becoming new alternatives to antibiotics and other antibacterial drugs. Studies have shown that Lactobacillus paracasei can produce antimicrobial peptides that have broad-spectrum antibacterial properties against bacteria, fungi and viruses (LWT-Food Science and Technology, 143, 2021, 111125). The fermentation products of Lactobacillus paracasei contain a large amount of antimicrobial peptides. Therefore, the discovery of new Lactobacillus paracasei is of great value to the field of food preservation. Summary of the invention

[0005] In view of the deficiencies in the prior art, the invention aims to provide a lactobacillus paracasei GY1 and a screening method and application thereof. The invention obtains the lactobacillus paracasei GY1 by screening mulberry soil and taking the inhibitory performance on Staphylococcus aureus as an index. After optimizing the carbon source, nitrogen source, pH, temperature and carbon source concentration in the fermentation process, the inhibition rate of the lactobacillus paracasei GY1 on Staphylococcus aureus can be as high as 95%. The invention is a strain with probiotic potential and good application prospect.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0007] A Lactobacillus paracasei GY1, characterized in that the Lactobacillus paracasei GY1 is isolated from mulberry soil, is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on June 11, 2024, with a deposit number of CGMCC No.30931, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Lactobacillus paracasei Lactobacillus paracasei The nucleic acid sequence of 16S rDNA sequencing of the Lactobacillus paracasei GY1 is shown in SEQ ID NO.1.

[0008] As an improvement, the colony of the Lactobacillus paracasei GY1 is white, moist, and round, with a diameter of 0.03 to 0.1 mm, a short rod-shaped cell morphology, a length of 3 to 4.5 μm, a width of 0.3 to 0.5 μm, and a smooth end. It is a Gram-positive bacterium, does not produce bioamines, does not produce DNAse, has strong self-aggregation, has tolerance in a bile salt environment of less than 0.4% and an osmotic pressure of less than 4%, grows in simulated gastric juice or simulated intestinal juice, and is resistant to gastric acid.

[0009] The Lactobacillus paracasei GY1 is isolated from mulberry soil using a gradient dilution separation method and an Oxford cup method. The screening method comprises the following steps:

[0010] Step 1, medium preparation

[0011] MRS liquid medium, LB liquid medium;

[0012] Step 2, taking appropriate amounts of mulberry soil and inoculating them in MRS liquid medium and LB liquid medium, respectively, culturing at 37°C, 220rpm, culturing for 72h, centrifuging, taking the supernatant, using Staphylococcus aureus as an indicator bacteria, using the Oxford cup method for preliminary screening, and then storing the strains with inhibition zones in a -80°C refrigerator at a volume ratio of 1:1 with 40% glycerol;

[0013] Step 3, take out the strain from the -80°C refrigerator, and spread and activate it on MRS and LB solid plates after gradient dilution, pick a single colony with an inoculation loop and inoculate it into MRS liquid culture medium and LB liquid culture medium respectively, culture at 37°C, 220rpm, culture for 3 days, centrifuge, take the supernatant, and use Staphylococcus aureus as an indicator bacteria to perform rescreening using the Oxford cup method;

[0014] Step 4, same as step 3, the -80°C strain is gradiently diluted and spread on MRS and LB solid plates for further separation and purification. After confirming that it is pure bacteria, it is identified.

[0015] As an improvement, the centrifugal speed in step 2 is 5000-12000 rpm, and the centrifugation is 2-10 min; the specific operation of the Oxford cup method is: take 10-100 μL of the supernatant, dilute the Staphylococcus aureus gradient, and then take 50-100 μL to apply on the MRS solid culture medium.

[0016] The invention discloses a Lactobacillus paracasei GY1 fermentation liquid, which is prepared by fermenting and culturing the above Lactobacillus paracasei GY1 to separate a supernatant.

[0017] As an improvement, the fermentation culture conditions are as follows: the carbon source is glucose, fructose, maltose, sucrose, β-cyclodextrin, starch, xylan, xylose, lactose, chitosan, trehalose and arabinose, the nitrogen source is yeast powder, soy protein, wheat protein, peptone, silkworm pupa powder and whey protein, the pH is 3-11, the temperature is 25-40°C, and the carbon source concentration is 20-120 g / L.

[0018] A further improvement is that when the fermentation culture conditions are: sucrose as the carbon source, yeast powder as the nitrogen source, pH 7, temperature 37°C, and carbon source concentration 40g / L, the resulting supernatant has an inhibitory activity of 95% or more against GY1 Staphylococcus aureus.

[0019] Application of the above Lactobacillus paracasei GY1 and / or the above Lactobacillus paracasei GY1 fermentation liquid in inhibiting the growth of Staphylococcus aureus.

[0020] A food preservative comprises the above-mentioned Lactobacillus paracasei GY1 and / or the above-mentioned Lactobacillus paracasei GY1 fermentation liquid.

[0021] Application of the above food preservatives in dairy products or meat products.

[0022] Beneficial Effects

[0023] Compared with the prior art, the invention discloses a lactobacillus paracasei GY1 and a screening method and application thereof. The invention screens mulberry soil and screens the strains by taking the inhibitory performance on Staphylococcus aureus as an index to obtain the lactobacillus paracasei GY1, which has a preservation number of CGMCC No.30931. After optimizing the carbon source, nitrogen source, pH, temperature and carbon source concentration in the fermentation process, the antibacterial rate of the lactobacillus paracasei GY1 on Staphylococcus aureus can be as high as 95%. Furthermore, through a probiotic experiment, it is determined that the lactobacillus paracasei GY1 is a strain with probiotic potential and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the colony characteristic diagram of Lactobacillus paracasei GY1;

[0025] Figure 2 This is a cell morphology characteristic diagram of Lactobacillus paracasei GY1;

[0026] Figure 3 To investigate the effects of different carbon sources on the production of antibacterial substances by Lactobacillus paracasei GY1;

[0027] Figure 4 To study the effect of different nitrogen sources on the production of antibacterial substances by Lactobacillus paracasei GY1;

[0028] Figure 5To study the effect of different fermentation pH on the production of antibacterial substances by Lactobacillus paracasei GY1;

[0029] Figure 6 The effect of different fermentation temperatures on the production of antibacterial substances by Lactobacillus paracasei GY1;

[0030] Figure 7 The effect of different carbon source concentrations on the production of antibacterial substances by Lactobacillus paracasei GY1. DETAILED DESCRIPTION

[0031] MRS liquid culture medium: glucose 2%, yeast extract powder 0.5%, peptone 1%, beef extract 1%, dipotassium hydrogen phosphate 0.2%, triammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.058%, manganese sulfate 0.025%, Tween 80 1mL, pH 6.2, all are mass-volume fractions.

[0032] MRS solid medium, i.e., MRS liquid medium, was added with 1.5% (W / V) agar.

[0033] LB liquid medium: sodium chloride 1%, peptone 1%, yeast extract 0.5%, all in mass-volume fractions.

[0034] LB solid medium is LB liquid medium with 1.5% (W / V) agar added.

[0035] Embodiment 1 Screening of Lactobacillus paracasei

[0036] Mulberry soil (the selected mulberry soil comes from the sericulture base of the Sericulture Research Institute of Jiangsu University of Science and Technology) was inoculated in appropriate amounts into MRS liquid culture medium and LB liquid culture medium, respectively, and cultured at 37°C, 220rpm for 72h to obtain a fermentation broth enriched with lactic acid bacteria, centrifuged, and the supernatant was taken. Staphylococcus aureus was used as an indicator bacterium and the Oxford cup method was used for primary screening. The strains with inhibition zones were stored in a -80°C refrigerator with 40% glycerol at a volume ratio of 1:1; the preserved frozen tubes were taken out and gradiently diluted on MRS and LB solid plates and then coated for activation. Single colonies were picked up with an inoculation loop and inoculated into MRS and LB liquid culture media, respectively, and cultured at 37°C, 220rpm for 3 days, centrifuged, and the supernatant was taken for rescreening using the Oxford cup method; the strains that were screened and could effectively inhibit Staphylococcus aureus were further gradiently diluted and coated for purification. After obtaining pure bacteria, the genome was extracted, amplified, and 16S rDNA was identified.

[0037] The colony of the Lactobacillus paracasei GY1 is white, moist and round, with a diameter of 0.03-0.1 mm, a short rod-shaped cell morphology, a length of 3-4.5 μm, a width of 0.3-0.5 μm, and a smooth end. The bacteria belong to Gram-positive bacteria, do not produce bioamines, do not produce DNAse, have strong self-aggregation, have tolerance in a bile salt environment lower than 0.4% and an osmotic pressure lower than 4%, grow in simulated gastric juice or simulated intestinal juice, and are resistant to gastric acid.

[0038] The selected colonies were amplified by PCR, and further analyzed by 16S rDNA sequencing and strain identification:

[0039] Use an inoculation loop to dip a small amount of pure bacteria in the frozen tube and streak it on an LB solid medium plate and culture it in a 37℃ incubator for 12h. Then use an inoculation loop to pick a single colony and inoculate it in 5mL LB. Culture it at 37℃, 220rpm for 24h to obtain the fermentation broth. Take 200μL of fermentation broth and centrifuge it at 12000rpm for 2 minutes, remove the supernatant, add 50μL of sterile deionized water to the bacterial mud at 70Hz, break it twice for 120s, and centrifuge it again at 12000rpm for 1min. Take 2μL of supernatant and add it to the PCR reaction system (25μL) for amplification. The reaction system includes: 2μL template, 0.5μL of universal primers F27 and R1492, 12.5μL of sterile deionized water, and 9.5μL Prime STAR Max. PCR amplification conditions: 98℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 90s, 30 cycles; 72℃ extension for 10min. The strains with obvious bands in 1% agarose gel electrophoresis of PCR products were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S sequence determination. Finally, it was confirmed that the GY1 strain with the largest inhibition zone had a homology of 99% with Lactobacillus paracasei, so the strain was Lactobacillus casei.

[0040] The nucleic acid sequence of 16S rDNA sequencing of the selected colony is shown in SEQ ID NO.1.

[0041] SEQ ID NO.1

[0042]

[0043] The selected strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 11, 2024, with a deposit number of CGMCC No.30931 and a taxonomic name of Lactobacillus paracasei. Lactobacillus paracasei , named Lactobacillus paracasei GY1.

[0044] Example 2 Antibacterial rate experiment

[0045] 1) Use an inoculation loop to pick up a small amount of Staphylococcus aureus from the frozen tube and streak it on an LB solid medium plate and incubate it at 37°C in an incubator for 12 hours. Then use an inoculation loop to pick up a single colony and inoculate it into 5 ml LB. Incubate it at 37°C, 220 rpm, and incubate it for 24 hours to obtain the primary seed. Then inoculate 10% of the colony into fresh LB medium and incubate it at 37°C, 220 rpm, and incubate it for 12 hours. Detect the bacterial concentration OD 600 The value is about 0.5;

[0046] 2) In order to prevent Staphylococcus aureus from dying naturally due to lack of nutrients during the culture process, the fermentation liquid was taken out after culture, centrifuged at 5000 rpm for 5 min at room temperature, the supernatant was immediately removed, and an appropriate amount of fresh LB was added to resuspend the bacteria. After resuspension, the OD 600 The value is about 0.45;

[0047] 3) Antibacterial experiment: This experiment uses 96-well cell culture plates to culture the test strains and detect the antibacterial rate. 185 μL of the resuspended bacterial suspension and 15 μL of fermentation supernatant were added to each well. In addition, the control group was set to 185 μL of bacterial suspension and 15 μL of MRS.

[0048] All 96-well plates need to be shaken for 5 minutes to mix the bacterial solution thoroughly, and the absorbance value at a wavelength of 600nm is measured using an ELISA reader, that is, the bacterial growth at T0. Subsequently, the 96-well cell culture plate is placed at 37°C for 12 hours. After that, the 96-well cell culture plate is shaken for 5 minutes to mix evenly, and the absorbance value at 600nm is measured and recorded as TF.

[0049] The antibacterial activity is expressed as inhibition percentage (%), and the calculation formula (1) is as follows:

[0050]

[0051] Where TF sample and TF blank The OD values ​​of the experimental group and the control group after 12 days of culture were 600 Value; T0 sample and T0 blank The OD of the experimental group and the control group at the beginning 600 value.

[0052] Example 3 Probiotic Function of Lactobacillus paracasei GY1

[0053] In order to evaluate the safety performance and probiotic potential of Lactobacillus paracasei GY1, the self-aggregation ability, salt concentration tolerance, gastric acid tolerance and survival ability of GY1 in simulated gastrointestinal fluid were analyzed. Lactobacillus paracasei GY1 almost completely settled within 12 hours, indicating that it has a strong self-aggregation ability, which is conducive to colonization in the intestine, so as to better play its role in regulating the balance of intestinal flora and promoting intestinal health; Lactobacillus paracasei GY1 can survive under high osmotic pressure, so it can tolerate the concentration of physiological saline in the intestinal environment, so as to better maintain the balance of intestinal microecology and play its role in regulating intestinal function; GY1 can survive normally in both pH 2 and 3 environments, that is, it can smoothly reach the intestine in the gastric acid environment, play its role in regulating intestinal flora and promoting intestinal health; GY1 can also survive in complex gastrointestinal fluid environments.

[0054] According to the test method described in the article Enhanced DPPH Radical Scavenging Activity and Enriched γ-Aminobutyric Acid in Mulberry Juice Fermented by the Probiotic Lactobacillusbrevis S3, Lactobacillus paracasei GY1 has the ability to tolerate bile salt environments below 0.4% and osmotic pressures below 4%, grows in simulated gastric fluid or simulated intestinal fluid, and is resistant to gastric acid. Therefore, Lactobacillus paracasei GY1 is a strain with good probiotic potential.

[0055] Example 4 Effect of different culture conditions on the production of antimicrobial substances by Lactobacillus paracasei GY1

[0056] The initial fermentation conditions were MRS medium, the carbon source was 20 g / L glucose, the nitrogen source was 10 g / L peptone, 5 g / L yeast powder, 10 g / L beef extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L triammonium citrate, 5 g / L sodium acetate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 1 mL / L Tween 80, and acetic acid was used to adjust pH to 6.2.

[0057] Based on the above initial fermentation conditions, the effects of different carbon sources on the production of antimicrobial peptides by Lactobacillus paracasei GY1 were investigated.

[0058] Use an inoculation loop to dip a small amount of Lactobacillus paracasei GY1 from a -80℃ glycerol tube and streak it on an MRS plate, then invert it and culture it in a 37℃ incubator for 48h. Pick an appropriate amount of colonies from the activated solid culture medium and inoculate it into 5mL MRS liquid culture medium, and culture it at 37℃, 220rpm for 24h to obtain seed culture solution; take the seed culture solution and inoculate it into MRS liquid culture medium at a 10% inoculation amount, and culture it at 37℃, 220rpm for 15h as the secondary seed solution; the secondary seed solution is still inoculated at a 10% inoculation amount in fermentation medium with glucose, fructose, maltose, sucrose, β-cyclodextrin, starch, xylan, xylose, lactose, chitosan, trehalose and arabinose as the sole carbon source, and culture it at 37℃, 220rpm for 48h. The optimal carbon source is determined by detecting the growth of the bacteria and the antibacterial activity against Staphylococcus aureus.

[0059] On the basis of the optimal carbon source, the effects of 7 different organic nitrogen sources, including yeast powder, soy protein, wheat protein, beef extract, peptone, silkworm pupa powder and whey protein, on the growth and bacteriocin production of Lactobacillus paracasei GY1 were further studied. The bacterial activation method was similar to the optimization of the carbon source. The final fermentation was still inoculated with 10% of the inoculum into the fermentation broth containing 7 different nitrogen sources for fermentation for 48 hours, and the fermentation broth was still measured by the method of Example 2. In addition to exploring the effect of the culture medium composition on the antibacterial ability of the target strain, the effects of pH (3, 5, 7, 9, 11), temperature (25°C, 30°C, 37°C, 40°C) and carbon source concentration (20g / L, 40g / L, 60g / L, 80g / L, 100g / L, 120g / L) on its production of antimicrobial peptides were also studied in turn.

[0060] Through experiments, we know that the effects of different carbon sources on the production of antimicrobial peptides by Lactobacillus paracasei GY1 are as follows: Figure 3 As shown in the figure, when sucrose is used as the only carbon source, its inhibitory ability against Staphylococcus aureus is significantly strong and the price is reasonable, so sucrose is selected as the optimal carbon source.

[0061] Effects of different nitrogen sources on the production of antimicrobial peptides by Lactobacillus paracasei GY1 Figure 4 As shown, when yeast powder is the only organic nitrogen source, the antibacterial rate can reach 90%, which is highly significant, so yeast powder is selected as the optimal nitrogen source.

[0062] Effects of different fermentation pH on the production of antimicrobial peptides by Lactobacillus paracasei GY1 Figure 5 As shown, the antibacterial ability of the cell-free supernatant after fermentation in fermentation broths with different pH values ​​against Staphylococcus aureus is slightly different. When the pH value is 7, the antibacterial ability is significantly high. Therefore, pH 7 is selected as the optimal pH fermentation condition.

[0063] Effects of different fermentation temperatures on the production of antimicrobial peptides by Lactobacillus paracasei GY1 Figure 6 As shown, at room temperature, the antibacterial ability is significantly low, and the inhibition rate is less than 30%. When the temperature is 37°C and 40°C, the inhibition rate is significantly improved. Combined with the concept of energy saving and environmental protection, 37°C is selected as the most suitable fermentation temperature.

[0064] Effects of different carbon source concentrations on the production of antimicrobial peptides by Lactobacillus paracasei GY1 Figure 7 As shown in the figure, with the increase of carbon source concentration, the antibacterial ability of Lactobacillus paracasei GY1 first increased significantly and then tended to be flat, so 40g / L was selected as the optimal fermentation carbon source concentration.

[0065] After single-factor condition optimization, the lactobacillus paracasei GY1 of the invention has a carbon source of 40 g / L sucrose, a nitrogen source of yeast powder, a pH of 7, a temperature of 37 DEG C, and an antibacterial rate of a fermentation supernatant of up to 95%, which lays a foundation for the application of the lactobacillus paracasei GY1 and its fermentation products in the inhibition of Staphylococcus aureus and provides a new idea for the food preservation industry such as dairy products.

[0066] The present invention and its embodiments are described schematically above, and the description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by it and designs a structural method and an embodiment similar to the technical solution without creativity without departing from the purpose of the invention, they shall all fall within the protection scope of the present invention.

Claims

1. A Lactobacillus paracasei GY1, characterized in that The Lactobacillus paracasei GY1 was isolated from mulberry soil and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 11, 2024. The deposit number is CGMCC No. 30931. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Lactobacillus paracasei Lactobacillus paracasei The nucleic acid sequence of 16S rDNA sequencing of the Lactobacillus paracasei GY1 is shown in SEQ ID NO.

1.

2. Lactobacillus paracasei GY1 according to claim 1, characterized in that, The colony of the Lactobacillus paracasei GY1 is white, moist, and round, with a diameter of 0.03-0.1 mm. The cell morphology is short rod-shaped, 3-4.5 μm long, 0.3-0.5 μm wide, and has a smooth end. It is a Gram-positive bacterium, does not produce bioamines, does not produce DNAse, has strong self-aggregation, and has the ability to tolerate a bile salt environment below 0.4% and an osmotic pressure below 4%. It grows in simulated gastric fluid or simulated intestinal fluid and is resistant to gastric acid.

3. Lactobacillus paracasei GY1 according to claim 1, characterized in that, The Lactobacillus paracasei GY1 is isolated from mulberry soil using a gradient dilution separation method and an Oxford cup method. The screening method comprises the following steps: Step 1, medium preparation MRS liquid medium, LB liquid medium; Step 2, take appropriate amount of mulberry soil and inoculate it into MRS liquid medium and LB liquid medium respectively, culture at 37°C, 220 rpm, culture for 72 h, centrifuge, take the supernatant, use Staphylococcus aureus as indicator bacteria, use Oxford cup method for primary screening, and then use 40% glycerol to store the strains with inhibition zone in a -80°C refrigerator at a volume ratio of 1:1 for standby use; Step 3, take out the strain from the -80°C refrigerator, and spread it on MRS and LB solid plates for activation after gradient dilution. Use an inoculation loop to pick a single colony and inoculate it into MRS liquid culture medium and LB liquid culture medium respectively, culture at 37°C, 220 rpm, and incubate for 3 days. Centrifuge, take the supernatant, and use Staphylococcus aureus as an indicator bacteria to perform rescreening using the Oxford cup method; Step 4, same as step 3, the -80°C strain is gradiently diluted and spread on MRS and LB solid plates for further separation and purification. After confirming that it is pure bacteria, it is identified.

4. Lactobacillus paracasei GY1 according to claim 3, characterized in that, The centrifugation speed in step 2 is 5000~12000 rpm, and the centrifugation is 2~10 min; the specific operation of the Oxford cup method is: take 10~100 μL of the supernatant, dilute the Staphylococcus aureus gradient, and then take 50~100 μL to apply on MRS solid culture medium.

5. A Lactobacillus paracasei GY1 fermentation broth, characterized in that The Lactobacillus paracasei GY1 according to claim 1 is used for fermentation culture to separate the supernatant.

6. A Lactobacillus paracasei GY1 fermentation liquid according to claim 5, characterized in that, The fermentation culture conditions are as follows: the carbon source is glucose, fructose, maltose, sucrose, β-cyclodextrin, starch, xylan, xylose, lactose, chitosan, trehalose and arabinose, the nitrogen source is yeast powder, soy protein, wheat protein, peptone, silkworm pupa powder and whey protein, the pH is 3-11, the temperature is 25-40°C, and the carbon source concentration is 20-120 g / L.

7. A Lactobacillus paracasei GY1 fermentation liquid according to claim 6, characterized in that, When the fermentation conditions were: sucrose as carbon source, yeast powder as nitrogen source, pH 7, temperature 37°C, and carbon source concentration 40 g / L, the inhibitory activity of the obtained supernatant against GY1 Staphylococcus aureus reached 95% or more.

8. Use of the Lactobacillus paracasei GY1 according to claims 1-4 and / or the Lactobacillus paracasei GY1 fermentation liquid according to claims 5-7 in inhibiting the growth of Staphylococcus aureus.

9. A food preservative, characterized in that: The invention comprises the Lactobacillus paracasei GY1 according to claims 1-4 and / or the Lactobacillus paracasei GY1 fermentation liquid according to claims 5-7.

10. Use of the food preservative according to claim 9 in dairy products or meat products.

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