Lactococcus lactis ZB11 with bacteriostatic activity, cell-free fermentation supernatant and preparation method of cell-free fermentation supernatant

Abstract

The invention relates to the technical field of fermentation, in particular to lactococcus lactis ZB11 with bacteriostatic activity, cell-free fermentation supernate and a preparation method of the cell-free fermentation supernate. The lactococcus lactis ZB11 is preserved in Guangdong Province Microbial Culture Collection Center, the address is Guangdong Province Institute of Microbiology, Academy of Sciences, Guangdong Province, the address is No.59 building, No.100 Courtyard, Xianlie Middle Road, Guangzhou City, the preservation date is July 26, 2024, and the preservation number is GDMCC No: 64913. When the lactococcus lactis disclosed by the invention is prepared into cell-free fermentation supernate, the use of chemical preservatives in fermented meat products can be reduced, the shelf life of the meat products can be prolonged, and the cell-free fermentation supernate disclosed by the invention is a natural green antibacterial material and provides guarantee for food safety.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, and in particular to a strain of Lactococcus lactis ZB11 with antibacterial activity and a cell-free fermentation supernatant and a preparation method thereof. Background Art

[0002] Traditional fermented meat products (beef jerky, bacon, sausage, etc.) have unique flavor, taste, color and texture, and are deeply loved by people. Studies have found that the microorganisms produced during the pickling process of fermented meat products will ferment them, thus forming fermented meat products with distinct regional characteristics and long-term preservation. The microbial flora in fermented meat products is mainly composed of bacteria such as Staphylococcus and lactic acid bacteria, as well as yeast and mold, and the formation of its flavor substances also depends on the endogenous microbial flora in the meat and the competition between fermentation microorganisms and miscellaneous bacteria in the natural environment to achieve fermentation. After a long period of maturation, the surface of bacon products is covered with a large number of microorganisms, including some filamentous fungi that produce mycotoxins. This makes it possible for traditional fermented meat products to be contaminated with a certain degree of mycotoxins, increasing the risk of people being exposed to mycotoxins.

[0003] With the continuous improvement of consumption concepts, food safety issues have become a hot topic of concern. In order to prevent deterioration and extend the shelf life, fermented meat products commonly found in the market usually add antibacterial agents to inhibit the proliferation of microorganisms on the basis of high-temperature sterilization. Commonly used preservatives include potassium sorbate, sodium D-isoascorbate, nisin, sodium benzoate, etc. However, excessive intake of these food additives can also cause adverse effects on the human body. Therefore, the development of a natural, pollution-free food antibacterial agent with good antibacterial effect is of great significance to further ensure food safety and extend the shelf life of food.

[0004] Lactic acid bacteria are a type of Gram-positive, non-spore-forming, catalase-negative, anaerobic or facultative anaerobic bacteria that produce a large amount of lactic acid during carbohydrate metabolism. They include 4 classes, 7 orders, 18 families, 39 genera, and at least 653 species. According to their morphology, they can be divided into bacilli and cocci. In terms of fermentation type, they can be divided into homotypic fermentation and heterotypic fermentation. As a recognized safe strain, lactic acid bacteria not only improve the flavor of food and increase the nutritional value, but also produce a series of antibacterial compounds such as organic acids, hydrogen peroxide, fatty acids, antimicrobial peptides, cyclic dipeptides, exopolysaccharides, volatile compounds, etc. during the fermentation process. The above substances can improve the sensory properties of food, increase the richness of volatile flavor substances in food, and extend the shelf life of food. Studies have shown that the antimicrobial compounds produced by lactic acid bacteria can effectively act as a natural barrier to prevent food spoilage caused by bacteria and fungi. Lactic acid bacteria that produce bacteriocins are widely used in fermented meat products, dairy products, and food preservation.

[0005] Isolating and screening food-borne lactic acid bacteria with antibacterial ability from traditional fermented meat products has become a research hotspot in this field. Domestic studies have shown that the fermentation supernatant of Lactobacillus plantarum LP-1, Bacillus rhamnosus LR-1 and Lactobacillus paracasei LC-1 can effectively inhibit Penicillium patulum and Aspergillus flavus isolated from moldy steamed bread. The steamed bread made by adding 40% Lactobacillus plantarum LP-1 fermentation supernatant to the dough only showed mold spots on the 4th day, which was 2 days longer than the blank control. Ding Ning et al. screened 139 lactic acid bacteria from cereals, among which 3 strains of Pediococcus acidilactici and 5 strains of Pediococcus pentosaceus had significant inhibitory effects on Aspergillus flavus and Fusarium graminearum. Du Hechao et al. found that Lactobacillus plantarum PC4-5 had a good inhibitory effect on Escherichia coli, Staphylococcus aureus, Salmonella enteritidis and Bacillus subtilis, and was suitable for the preservation of acidic foods. Ferrara et al. showed that inoculating P. nalgiovense on the surface of sausages can prevent the colonization of toxin-producing fungi. Meftah et al. found that C. zeylanoides strains can inhibit the growth of P. nordicum.

[0006] Existing studies on the antibacterial effect of lactic acid bacteria have mostly focused on pathogenic bacteria such as Bacillus, Salmonella, Staphylococcus aureus, and Escherichia coli, while studies on the inhibition of common fungi in meat products (Aspergillus ochraceus, Penicillium, etc.) by lactic acid bacteria are relatively rare. Therefore, exploring lactic acid bacteria with high antifungal ability is of great significance to the safety of fermented foods such as fermented meat products. Summary of the invention

[0007] The object of the present invention is to provide a strain of Lactococcus lactis ZB11 with antibacterial activity and a cell-free fermentation supernatant and a preparation method thereof.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a strain of Lactococcus lactis ZB11 with antibacterial activity. The Lactococcus lactis ZB11 has a Latin name of Lactococcus lactis ZB11, is preserved in Guangdong Microbiological Culture Collection Center, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a preservation date of July 26, 2024 and a preservation number of GDMCC No: 64913.

[0010] The present invention also provides the use of the Lactococcus lactis ZB11 in preparing a product with fungus inhibition activity.

[0011] Preferably, the fungus is one or more of Penicillium grisofulvumDierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides.

[0012] The present invention also provides a cell-free fermentation supernatant with antibacterial activity, wherein the cell-free fermentation supernatant is obtained by fermenting the Lactococcus lactis ZB11.

[0013] The present invention also provides the use of the cell-free fermentation supernatant in preparing a product for inhibiting fungal activity in food.

[0014] Preferably, the fungus is one or more of Penicillium grisofulvumDierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides.

[0015] The present invention also provides a method for preparing a cell-free fermentation supernatant, comprising the following steps:

[0016] (1) inoculating the Lactococcus lactis ZB11 into MRS solid culture medium, culturing at 36-38° C. for 20-28 hours, and obtaining a single colony;

[0017] (2) inoculating the single bacterial colony described in step (1) into MRS liquid culture medium, and culturing at 36-38° C. for 20-28 h to obtain a seed solution; inoculating the seed solution into MRS liquid culture medium, and culturing at 36-38° C. for 20-28 h to obtain a working bacterial solution;

[0018] (3) inoculating the working bacterial liquid into MRS broth medium, culturing at 36-38° C. for 20-28 hours to obtain a fermentation liquid; centrifuging the fermentation liquid, collecting the supernatant, filtering, and taking the filtrate to obtain a cell-free fermentation supernatant.

[0019] Preferably, in step (3), the inoculation amount of the working bacterial solution inoculated into the MRS broth medium is 1-2% of the volume of the MRS broth medium.

[0020] Preferably, the centrifugal speed in step (3) is 4500-5500 rpm, and the centrifugal time is 8-12 min.

[0021] Preferably, the filtration in step (3) is membrane filtration; the pore size of the membrane is ≤0.22 μm. The present invention provides a strain of Lactococcus lactis ZB11 with antibacterial activity and a cell-free fermentation supernatant and a preparation method thereof. The present invention isolates Lactococcus lactis ZB11 from the traditional fermented meat product Zhenba bacon. The Latin name is: Lactococcus lactisZB11, which is preserved in the Guangdong Provincial Microbiological Culture Collection Center, located at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a preservation date of July 26, 2024 and a preservation number of GDMCC No: 64913. Lactococcus lactis ZB11 is prepared into a cell-free fermentation supernatant. The cell-free fermentation supernatant is used as an antibacterial agent for fermented meat products, and can inhibit the growth of Penicillium grisofulvum Dierckx, Talaromycesverruculosus, Aspergillus ochraceus, and Fusarium verticillioides. The cell-free fermentation supernatant of the present invention does not belong to a chemical preservative, but is a natural green antibacterial material. By adding the cell-free fermentation supernatant of Lactococcus lactis, the use of chemical preservatives in fermented meat products can be reduced, and the shelf life of meat products can be extended. The raw materials of the present invention are easy to obtain, and the harm of preservatives to human health is reduced while ensuring the microbial safety of the product, thereby providing a guarantee for food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The inhibition results of 4 fungi after adding different amounts of CFS for 72 hours (the upper left shows the inhibition result against Penicillium griseofulvum, the upper right shows the inhibition result against Penicillium verrucosus, the lower left shows the inhibition result against Aspergillus ochraceus, and the lower right shows the inhibition result against Fusarium moniliforme).

[0023] Collection Instructions

[0024] Lactococcus lactis ZB11, Latin name: Lactococcus lactis ZB11, is deposited in Guangdong Microbiological Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a deposit date of July 26, 2024 and a deposit number of GDMCC No: 64913. DETAILED DESCRIPTION

[0025] The present invention provides a strain of Lactococcus lactis ZB11 with antibacterial activity. The Lactococcus lactis ZB11 has a Latin name of Lactococcus lactis ZB11, is preserved in Guangdong Microbiological Culture Collection Center, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a preservation date of July 26, 2024 and a preservation number of GDMCC No: 64913.

[0026] The present invention also provides the use of the Lactococcus lactis ZB11 in preparing a product with fungus inhibition activity.

[0027] In the present invention, the fungus is one or more of Penicillium grisofulvum Dierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides.

[0028] The present invention also provides a cell-free fermentation supernatant with antibacterial activity, wherein the cell-free fermentation supernatant is obtained by fermenting the Lactococcus lactis ZB11.

[0029] The present invention also provides the use of the cell-free fermentation supernatant in preparing a product for inhibiting fungal activity in food.

[0030] In the present invention, the fungus is one or more of Penicillium grisofulvum Dierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides.

[0031] The present invention also provides a method for preparing a cell-free fermentation supernatant, comprising the following steps:

[0032] (1) inoculating the Lactococcus lactis ZB11 into MRS solid culture medium, culturing at 36-38° C. for 20-28 hours, and obtaining a single colony;

[0033] (2) inoculating the single bacterial colony described in step (1) into MRS liquid culture medium, and culturing at 36-38° C. for 20-28 h to obtain a seed solution; inoculating the seed solution into MRS liquid culture medium, and culturing at 36-38° C. for 20-28 h to obtain a working bacterial solution;

[0034] (3) inoculating the working bacterial liquid into MRS broth medium, culturing at 36-38° C. for 20-28 hours to obtain a fermentation liquid; centrifuging the fermentation liquid, collecting the supernatant, filtering, and taking the filtrate to obtain a cell-free fermentation supernatant.

[0035] In the present invention, the MRS solid culture medium uses water as a solvent and includes 66.2 g / L of MRS agar culture medium. In the present invention, the MRS agar culture medium is purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd. In the present invention, the MRS liquid culture medium uses water as a solvent and also includes 52.44 g / L of MRS broth culture medium and 10 g / L of glucose. In the present invention, the broth culture medium is purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0036] In the present invention, the culture temperature in steps (1) to (3) is preferably 37° C., and the culture time is preferably 24 h. In the present invention, the inoculation amount of the working bacterial liquid inoculated into the MRS broth culture medium in step (3) is 1 to 2% of the volume of the MRS broth culture medium, preferably 1%.

[0037] In the present invention, the centrifugal speed in step (3) is 4500-5500 rpm, preferably 5000 rpm, and the centrifugal time is 8-12 min, preferably 10 min.

[0038] In the present invention, the filtration in step (3) is membrane filtration; the pore size of the membrane is ≤0.22 μm, preferably 0.22 μm.

[0039] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The MRS solid medium in the embodiment of the present invention uses water as a solvent and includes 66.2 g / L of MRS agar medium. The MRS agar medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd. The MRS liquid medium uses water as a solvent and also includes 52.44 g / L of MRS broth medium and 10 g / L of glucose. The broth medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0041] Penicillium grisofulvum Dierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides described in the examples of the present invention were all purchased from Beina Biotech - Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC).

[0042] Example 1

[0043] Lactic acid bacteria with antibacterial activity were isolated and screened from Zhenba bacon, and a lactic acid bacteria strain with strong inhibitory effect on Penicillium grisofulvum Dierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides was obtained. After whole genome sequencing and 16SRNA identification, it was named Lactococcus lactis ZB11. Experiments have shown that the strain is a homotypic fermentation type, no virulence gene was detected, and it is a safe food-grade lactic acid bacteria.

[0044] The Lactococcus lactis ZB11, Latin name: Lactococcus lactis ZB11, is deposited in the Guangdong Microbial Culture Collection Center, located at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a deposit date of July 26, 2024 and a deposit number of GDMCC No: 64913.

[0045] Example 2

[0046] Take the glycerol bacteria preserved at -80°C in Example 1, use an inoculating loop to pick up the bacterial solution, streak in a zigzag shape on an MRS solid culture medium, and culture at 37°C for 24 hours to obtain a single colony. After the streak culture is completed, add 1.2mL of MRS liquid culture medium to a 1.5mL centrifuge tube, use an inoculating loop to pick up a single colony in a 1.5mL centrifuge tube, and culture at 37°C for 24 hours to obtain a seed solution. Inoculate the seed solution into an MRS liquid culture medium (the inoculation amount of the seed solution is 1% of the volume of the MRS liquid culture medium), and culture at 37°C for 24 hours to obtain a working bacterial solution. Inoculate the working bacterial solution into an MRS broth culture medium at an inoculation amount of 1vt%, and culture at 37°C for 24 hours to obtain a fermentation solution, and measure the OD value of the fermentation solution. 600 The value was 2.0. The supernatant was collected by centrifugation at 5000 pm at 4 °C for 10 min in a high-speed low-temperature refrigerated centrifuge. Then, impurities were filtered out using a 0.22 μm water filter membrane to obtain the Lactococcus lactis cell-free fermentation supernatant (CFS). The 50 mL centrifuge tube was sealed with a sealing film and stored at -20 °C.

[0047] PDA solid medium: 80 g potatoes, 200 mL water, boil the potatoes in water, filter, and take the filtrate to obtain potato boiled liquid. Add 2.0 g glucose, 0.3 g potassium hydrogen phosphate, 0.15 g magnesium sulfate heptahydrate, and 2.0 g agar powder to every 100 mL potato boiled liquid, sterilize, and obtain PDA solid medium.

[0048] PDA liquid culture medium: 80 g potatoes, 200 mL water, boil the potatoes in water, filter, and take the filtrate to obtain potato boiled liquid. Add 2.0 g glucose, 0.3 g dipotassium hydrogen phosphate, and 0.15 g magnesium sulfate heptahydrate to every 100 mL potato boiled liquid, sterilize, and obtain PDA liquid culture medium.

[0049] Fungal activation and spore suspension preparation: Referring to the instructions for use of four fungi (Penicillium grisofulvumDierckx, Talaromyces verruculosus, Aspergillus ochraceus, Fusarium verticillioides), freeze-dried fungal powder was added with sterile water to prepare a fungal suspension, which was then spread on PDA solid medium. After culturing at 28°C for 6 days, 8 mL of sterile water was added to the fungal plate, and the spores were slowly scraped off with a sterile coating stick. The spore suspension was then filtered with two layers of sterile gauze and collected in a 10 mL sterile centrifuge tube to obtain a fungal spore suspension (1.0 × 10 7 cfu / mL). Store at 4℃ for future use.

[0050] Five groups were set up, including 0% CFS, 10% CFS, 20% CFS, 40% CFS and 80% CFS. The experiment was carried out in a 96-well plate, and the culture system in each well of the 96-well plate was 200 μL. 0% CFS group: 190 μL PDA liquid medium and 10 μL fungal spore suspension were added; 10% CFS group: 20 μL CFS, 170 μL PDA liquid medium, and 10 μL fungal spore suspension were added; 20% CFS group: 40 μL CFS, 150 μL PDA liquid medium, and 10 μL fungal spore suspension were added; 40% CFS group: 80 μL CFS, 110 μL PDA liquid medium, and 10 μL fungal spore suspension were added; 80% CFS group: 160 μL CFS, 30 μL PDA liquid medium, and 10 μL fungal spore suspension were added. CFS, PDA liquid culture medium, and fungal spore suspension were inoculated into 96-well plates according to the above proportions and cultured in a 28°C incubator. The absorbance (OD) at 600 nm was measured at 24 h, 48 h, and 72 h. 600 ), the 0% CFS group was used as the control group and recorded as OD control, 4 groups with different concentrations of CFS were added as experimental groups, recorded as OD LAG The antibacterial rate was calculated using the following formula:

[0051]

[0052] The results of the inhibition rate are shown in Table 1, and the results of the inhibition rate for 72 hours are shown in Figure 1. Figure 1 shown.

[0053] Table 1 Inhibition rate of 4 fungi with different addition amounts of CFS

[0054]

[0055]

[0056] Table 1 and Figure 1 It showed that the fungi did not reach the stable growth period after 24 hours of cultivation. After 72 hours, the antibacterial rate of 80% CFS added to the four fungi could reach more than 80%.

[0057] It can be seen from the above embodiments that the present invention provides a strain of Lactococcus lactis ZB11 with antibacterial activity and a cell-free fermentation supernatant and a preparation method thereof. The present invention isolates Lactococcus lactis ZB11 from the traditional fermented meat product Zhenba bacon, the Latin name is: The Latin name is: Lactococcus lactis ZB11, which is preserved in the Guangdong Microbiological Culture Collection Center, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, the preservation date is July 26, 2024, and the preservation number is GDMCC No: 64913. Lactococcus lactis ZB11 is prepared into a cell-free fermentation supernatant. The cell-free fermentation supernatant is used as an antibacterial agent for fermented meat products, and can inhibit the growth of Penicillium grisofulvum Dierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides. The cell-free fermentation supernatant of the present invention does not belong to a chemical preservative, but is a natural green antibacterial material. By adding the cell-free fermentation supernatant of Lactococcus lactis, the use of chemical preservatives in fermented meat products can be reduced, and the shelf life of meat products can be extended. The raw materials of the present invention are easy to obtain, and the harm of preservatives to human health is reduced while ensuring the microbial safety of the product, thereby providing a guarantee for food safety.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactococcus lactis ZB11 having antibacterial activity, characterized in that: The Lactococcus lactis ZB11, Latin name: Lactococcus lactis ZB11, is preserved in the Guangdong Microbial Culture Collection Center, located at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a preservation date of July 26, 2024 and a preservation number of GDMCC No: 64913.

2. Use of the Lactococcus lactis ZB11 according to claim 1 in the preparation of a product having antifungal activity.

3. The use according to claim 2, characterized in that: The fungus is one or more of Penicillium grisofulvum Dierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides.

4. A cell-free fermentation supernatant having antibacterial activity, characterized in that: The cell-free fermentation supernatant is obtained by fermenting the Lactococcus lactis ZB11 according to claim 1.

5. Use of the cell-free fermentation supernatant according to claim 4 in preparing a product for inhibiting fungal activity in food.

6. The use according to claim 5, characterized in that: The fungus is one or more of Penicillium grisofulvum Dierckx, Talaromyces verruculosus, Aspergillus ochraceus, and Fusarium verticillioides.

7. A method for preparing a cell-free fermentation supernatant, characterized in that: The steps include: (1) inoculating the Lactococcus lactis ZB11 described in claim 1 into MRS solid culture medium and culturing at 36-38° C. for 20-28 h to obtain a single colony; (2) inoculating the single bacterial colony described in step (1) into MRS liquid culture medium, and culturing at 36-38° C. for 20-28 h to obtain a seed solution; inoculating the seed solution into MRS liquid culture medium, and culturing at 36-38° C. for 20-28 h to obtain a working bacterial solution; (3) inoculating the working bacterial liquid into MRS broth medium, culturing at 36-38° C. for 20-28 hours to obtain a fermentation liquid; centrifuging the fermentation liquid, collecting the supernatant, filtering, and taking the filtrate to obtain a cell-free fermentation supernatant.

8. The preparation method according to claim 7, characterized in that: In step (3), the inoculation amount of the working bacterial solution when inoculated into the MRS broth medium is 1-2% of the volume of the MRS broth medium.

9. The preparation method according to claim 8, characterized in that: The centrifugal speed in step (3) is 4500-5500 rpm, and the centrifugal time is 8-12 min.

10. The preparation method according to any one of claims 7 to 9, characterized in that: The filtration in step (3) is membrane filtration; the pore size of the membrane is ≤0.22 μm.

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