Complex probiotics with broad-spectrum antibacterial activity, metabolite preparation of complex probiotics and application of complex probiotics in fresh keeping of meat products

Abstract

The invention discloses a compound probiotic with broad-spectrum bacteriostatic activity, a metabolite preparation of the compound probiotic and application of the metabolite preparation in meat product preservation, the compound probiotic is lactobacillus plantarum and lactobacillus fermentum which are preserved in the China Center for Type Culture Collection, and the preservation numbers are respectively CCTCC NO: M 20241253 and CCTCC NO: M20241254. The safe, efficient and cheap compound probiotic preparation is obtained, is simple to prepare, has a remarkable synergistic antibacterial effect, can be applied to food processing and preservation of cold storage, and particularly can remarkably inhibit quality deterioration and food safety of chilled fresh meat caused by proliferation and metabolism of putrefying bacteria and pathogenic bacteria in cold chain circulation; and the shelf life of fresh meat products can be prolonged. Meanwhile, the preparation can be independently prepared or compounded to prepare a preservative or a biological control agent, and the formation of putrefying bacteria and pathogenic bacterium biofilms on the surfaces of equipment and raw material solids can be effectively inhibited and reduced.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a composite probiotic with broad-spectrum antibacterial activity and a metabolite preparation thereof and an application of the composite probiotic in preserving meat products. Background Art

[0002] As a low-temperature fresh meat product, chilled meat undergoes the process of stiffening, thawing and maturation in a low-temperature environment from slaughter to market. It is tender, juicy, fresh in soup, easy to chew and easy to digest. Chilled meat has a high moisture content and is rich in nutrients. Pseudomonas and other putrefactive microorganisms carried by animals, as well as a series of quality fission problems such as protein degradation, fat oxidation and quality deterioration caused by excessive numbers of putrefactive microorganisms in the slaughtering, processing, storage and transportation environments are very likely to occur. At the same time, foodborne pathogens such as Salmonella and Coccus aureus in fresh meat can cause harm to human health. At present, the more common preservation methods for chilled meat are low-temperature preservation and the addition of chemical synthetic preservatives, but the preservation effect and safety are relatively low.

[0003] At present, the antibacterial method used to control bacterial growth in the production environment during the production and processing of meat products is mainly chemical disinfectants. However, the chemical reagent residues are harmful to the health of humans and animals and will pollute the environment. In recent years, due to the urgent need for green antibacterial food, natural antibacterial substances such as allicin and tea polyphenols have been extracted from animals and plants as biological antibacterial agents. Although the above antibacterial agents can meet the needs of green environmental protection, they have the disadvantages of high manufacturing costs, easy changes in food flavor and quality, and poor bactericidal effects. Therefore, it is urgent to develop new biological antibacterial agents.

[0004] At present, probiotics and their preparations have been widely used to inhibit harmful bacteria and regulate the microecological balance of the host and the environment. Lactic acid bacteria (LAB) is a new type of biological preservative. Its main body of action, lactic acid bacteria and its metabolites have good antibacterial activity. As a biological antibacterial agent, it has the advantages of high efficiency, broad spectrum, stability, safety, and no residue. By inhibiting the physiological activity of microorganisms and reducing the number of microorganisms, it can achieve the purpose of stabilizing food quality, delaying food corruption, extending the shelf life of food, and improving food safety. Among them, Lactobacillus plantarum, as an important lactic acid bacteria, has a high number of live bacteria, can produce a large amount of acid, and the pH value in the stable solution does not increase. At the same time, it can produce a unique biological preservative "lactobacillus" during the breeding process. At the same time, Lactobacillus fermentum, as an important probiotic, is widely present in fermented seeds and vegetables, whiskey, milk, cheese, handmade fermentation agents, human saliva, human and animal intestines, etc. Although lactic acid bacteria antimicrobial agents have achieved good application results in the fields of dairy products, meat products, fruits and vegetables, aquatic products, etc., single lactic acid bacteria have a limited shelf life and a single antibacterial spectrum, and there are few studies on synergistic composite lactic acid bacteria as preservatives. Therefore, it is of great economic significance and social value to study composite lactic acid bacteria preparations to improve their antibacterial activity and extend the shelf life and safety of foods such as fresh meat. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a broad-spectrum antibacterial synergistic composite probiotic and its metabolite preparation, which can be used in food production and refrigeration preservation, especially can inhibit the spoilage of fresh meat caused by pathogens during transportation and storage, thereby extending the shelf life of the product.

[0006] The present invention also provides a preparation method and application of the composite probiotic metabolite preparation, which can effectively inhibit the corruption and quality deterioration caused by the reproduction and metabolism of pathogenic bacteria in meat products under cold conditions, and provide a safe, efficient and inexpensive biological preparation for preserving cold fresh meat and extending its shelf life.

[0007] Technical solution: To achieve the above purpose, the composite probiotics with broad-spectrum antibacterial activity described in the present invention, the two lactic acid bacteria contained in the composite probiotics in the present invention are isolated from kimchi samples in Yangzhou City, Jiangsu Province, and are identified as Lactobacillus plantarum and Limosilactobacillus fermentum, respectively, and have a broad-spectrum antibacterial effect. These two probiotics Lactobacillus plantarum YZU-LP52 and Limosilactobacillus fermentum YZU-LF56 are preserved in the China Center for Type Culture Collection (abbreviated as CCTCC), with a preservation address: Wuhan University, Wuhan; preservation number: CCTCC NO: M 20241253, CCTCC NO: M20241254; preservation date: June 24, 2024.

[0008] The present invention also provides a composite probiotic metabolite preparation with broad-spectrum antibacterial activity, wherein the composite probiotic metabolite preparation is formed by metabolites produced by the interaction of two probiotic strains of Lactobacillus plantarum (Lactiplantibacillus plantarum) YZU-LP52 and Lactobacillus fermentum (Limosilactobacillusfermentum) YZU-LF56 after mixed culture.

[0009] The composite probiotics and metabolite preparations thereof in the present invention have the following characteristics:

[0010] (1) The two strains of lactic acid bacteria in the composite probiotic preparation have good bile salt and acid resistance.

[0011] (2) The two strains of lactic acid bacteria in the composite probiotic preparation are non-hemolytic and have good safety.

[0012] (3) The two strains of lactic acid bacteria in the composite probiotic preparation have a synergistic and broad-spectrum antibacterial effect against Listeria monocytogenes, Salmonella, Pseudomonas fluorescens, Staphylococcus aureus, and Bacillus.

[0013] (4) Composite probiotics and their metabolite preparations can delay the impact of spoilage bacteria on the quality of fresh meat.

[0014] Among them, two strains of lactic acid bacteria were inoculated in the culture medium at a concentration of 10 7 -10 9 CFU / mL, the two cultured lactic acid bacteria were mixed and inoculated into the culture medium for cultivation, and then the bacterial supernatant was obtained by centrifugation. The culture temperature of the two lactic acid bacteria was 30-37°C, and the culture medium was MRS culture medium.

[0015] The composite probiotics with broad-spectrum antibacterial activity and metabolite preparations thereof described in the present invention are used in inhibiting or reducing spoilage bacteria and pathogenic bacteria.

[0016] Among them, the composite probiotics with broad-spectrum antibacterial activity and their metabolite preparations are used to inhibit or reduce spoilage bacteria and pathogenic bacteria in the production environment or storage and transportation equipment of fresh meat or meat products.

[0017] Among them, the composite probiotics with broad-spectrum antibacterial activity and their metabolite preparations are used to inhibit the reproduction and metabolism of spoilage bacteria and pathogenic bacteria in the processing of fresh meat and low-temperature cold chain processes.

[0018] Among them, the composite probiotics with broad-spectrum antibacterial activity and their metabolite preparations are used alone or mixed with existing antibacterial agents to prepare spray liquids, rinse liquids, preservatives or biological microbial control agents for inhibiting spoilage bacteria and pathogenic bacteria.

[0019] Among them, the composite probiotics with broad-spectrum antibacterial activity and their metabolite preparations are made into spray liquids or rinse liquids, which are used for spraying or washing the production environment and production equipment to reduce the contamination of spoilage bacteria and pathogenic bacteria during the cold chain processing of meat; or added during the preparation and storage to inhibit the metabolism and reproduction of spoilage bacteria and pathogenic bacteria during the processing and low-temperature storage of fresh meat.

[0020] Among them, the spoilage bacteria and pathogenic bacteria include Pseudomonas fluorescens, Staphylococcus aureus, Salmonella monocytogenes, Pseudomonas fluorescens, Salmonella typhimurium, Bacillus anthracis, anaerobic Bacillus, Bacillus nanhaiensis, and Staphylococcus aureus.

[0021] The present invention prepares the above-mentioned composite probiotics into a microecological preparation, which is used as a preservative in the refrigeration and preservation of meat products. The preparation method is as follows: the above-mentioned two activated lactic acid bacteria are inoculated into 100mL MRS liquid culture medium according to a volume ratio of 1:1, and the supernatant obtained after culturing in a 37°C constant temperature incubator for 24 hours and low-temperature centrifugation is the composite probiotic metabolite preparation. At the same time, the prepared composite probiotic metabolite preparation is used for antibacterial test, stored at 4°C, and reserved.

[0022] Application of the composite probiotics or metabolite preparation thereof in the preservation of chilled fresh meat.

[0023] Among them, the composite probiotics or their metabolite preparations are used to inhibit meat-derived spoilage and pathogenic bacteria such as Pseudomonas fluorescens, Staphylococcus aureus, and Salmonella in fresh cold meat.

[0024] Among them, the composite probiotics or their metabolite preparations are used to inhibit typical meat-derived spoilage and pathogenic bacteria such as Listeria monocytogenes, Pseudomonas fluorescens, Staphylococcus aureus, Salmonella, Bacillus, etc. in the preservation of meat products and their production environment or storage and transportation equipment.

[0025] Among them, the composite probiotics or their metabolite preparations are used to inhibit the growth and reproduction of meat-derived spoilage bacteria and pathogenic bacteria such as Listeria monocytogenes, Pseudomonas fluorescens, Staphylococcus aureus, Salmonella, and Bacillus during the processing and low-temperature storage of fresh meat.

[0026] The composite probiotics and metabolite preparations thereof described in the present invention are used in the preparation of spraying liquids, leaching liquids, preservatives or biological bacteria control agents for inhibiting spoilage and pathogenic bacteria such as Listeria monocytogenes, Pseudomonas fluorescens, Staphylococcus aureus, Salmonella, Bacillus, etc.

[0027] Among them, the composite probiotics or their metabolite preparation isolates or cultures are used alone or mixed with existing antibacterial agents to prepare spray solutions, rinse solutions, preservatives or biological microbial control agents for inhibiting spoilage and pathogenic bacteria such as Listeria monocytogenes, Pseudomonas fluorescens, Staphylococcus aureus, Salmonella, Bacillus, etc.

[0028] Among them, the composite probiotics or their metabolite preparations are made into spray liquids or rinse liquids, which are used for spraying or washing the production environment and production equipment, so as to reduce the load of spoilage and pathogenic bacteria such as Pseudomonas fluorescens, Staphylococcus aureus, and Salmonella in the meat processing environment, as well as the formation of biofilms; or the composite probiotic metabolite preparations are used as preservatives and added during the preparation and storage to inhibit the metabolism and reproduction of spoilage and pathogenic bacteria such as Listeria monocytogenes, Pseudomonas fluorescens, Staphylococcus aureus, Salmonella, and Bacillus during the processing and low-temperature storage of fresh meat.

[0029] The single lactic acid bacteria strain involved in the present invention can produce a certain inhibitory effect on spoilage bacteria and pathogenic bacteria, while the composite probiotics and their metabolite preparations have a better antibacterial effect. This significant antibacterial advantage shows obvious synergistic effect and broad-spectrum antibacterial effect.

[0030] Beneficial effect: Compared with the prior art, the composite probiotics and metabolite preparations proposed by the present invention have good stability, high safety, and very significant synergistic enhancement and broad-spectrum antibacterial characteristics, which can inhibit the growth and reproduction of important spoilage and pathogenic bacteria such as Listeria monocytogenes, Pseudomonas fluorescens, Staphylococcus aureus, Salmonella, Bacillus, etc. in cold fresh meat, and are simple to prepare, cheap and safe. The composite probiotic metabolite preparation of the present invention is easily formulated into a spray or rinse solution by traditional methods, and the environment or utensils are sterilized, which can effectively control the growth and biofilm formation of various spoilage bacteria and pathogens in various matrices, thereby reducing the risk of contamination of cold fresh products by spoilage bacteria and pathogens in the production environment. The safety of the strain is proved by experiments such as hemolysis and drug resistance, and it can be directly applied to meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the evolutionary tree analysis diagram of Lactiplantibacillusplantarum YZU-LP52.

[0032] Figure 2 This is the evolutionary tree analysis diagram of Limosilactobacillusfermentum YZU-LF56.

[0033] Figure 3 To evaluate the hemolytic safety of two strains of lactic acid bacteria. Figure 3 A is the hemolytic zone of YZU-LP52 and Bacillus cereus 12-1; Figure 3 B is the hemolytic zone of YZU-LF56 and Bacillus cereus 12-1.

[0034] Figure 4 The pH changes of fresh meat at different storage days. The yellow columns in the figure are the pH changes of untreated fresh meat during storage at 4°C; the light green columns are the pH changes of fresh meat after being treated with YZU-LF56 probiotic metabolites during storage at 4°C; the dark green columns are the pH changes of fresh meat after being treated with YZU-LP52 probiotic metabolites during storage at 4°C; the blue columns are the pH changes of fresh meat after being treated with composite probiotic metabolites during storage at 4°C.

[0035] Figure 5 The figure shows the changes in the total number of colonies of fresh meat under different storage days. The green column in the figure shows the changes in the total number of colonies of fresh meat during storage at 4°C without treatment; the khaki column shows the changes in the total number of colonies of fresh meat after being treated with the probiotic metabolites of the YZU-LF56 group during storage at 4°C; the bright yellow column shows the changes in the total number of colonies of fresh meat after being treated with the probiotic metabolites of the YZU-LP52 group during storage at 4°C; the orange column shows the changes in the total number of colonies of fresh meat after being treated with the metabolites of the composite probiotics during storage at 4°C.

[0036] Figure 6 The dark blue columns in the figure show the changes in TVB-N of fresh meat during storage at 4°C without treatment; the dark green columns show the changes in TVB-N of fresh meat after treatment with probiotic metabolites from the YZU-LF56 group during storage at 4°C; the light green columns show the changes in TVB-N of fresh meat after treatment with probiotic metabolites from the YZU-LP52 group during storage at 4°C; and the yellow columns show the changes in TVB-N of fresh meat after treatment with compound probiotic metabolites during storage at 4°C.

[0037] Figure 7 The changes in the degree of protein hydrolysis of fresh meat after different storage days.

[0038] Figure 8 The yellow curve in the figure shows the change of L* color value of fresh meat during storage at 4℃ without treatment; the green curve shows the change of L* color value of fresh meat after being treated with probiotic metabolites of YZU-LP52 group during storage at 4℃; the dark green curve shows the change of L* color value of fresh meat after being treated with probiotic metabolites of YZU-LF56 group during storage at 4℃; the purple curve shows the change of L* color value of fresh meat after being treated with probiotic metabolites of composite group during storage at 4℃.

[0039] Fig. 9 The a* chromaticity value of fresh meat at different storage days. The black curve in the figure shows the change of a* chromaticity value of fresh meat during storage at 4℃ without treatment; the red curve shows the change of a* chromaticity value of fresh meat after being treated with probiotic metabolites of YZU-LP52 group during storage at 4℃; the blue curve shows the change of a* chromaticity value of fresh meat after being treated with probiotic metabolites of YZU-LF56 group during storage at 4℃; the green curve shows the change of a* chromaticity value of fresh meat after being treated with probiotic metabolites of composite group during storage at 4℃.

[0040] Fig.10 The yellow curve in the figure shows the change of b* chromaticity value of fresh meat during storage at 4℃ without treatment; the green curve shows the change of b* chromaticity value of fresh meat after being treated with probiotic metabolites of YZU-LP52 group during storage at 4℃; the dark green curve shows the change of b* chromaticity value of fresh meat after being treated with probiotic metabolites of YZU-LF56 group during storage at 4℃; the purple curve shows the change of b* chromaticity value of fresh meat after being treated with probiotic metabolites of YZU-LF56 group during storage at 4℃. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0042] Unless otherwise specified, the raw materials and reagents in the present invention are commercially available.

[0043] The indicator bacteria used in the test of the present invention, such as Listeria monocytogenes Z21, Pseudomonas fluorescens J124, Salmonella typhimurium N25, Salmonella typhimurium P22, Bacillus anthracis C423, anaerobic Bacillus N15, Bacillus nanhaiensis M6, Staphylococcus aureus Q21, Thermomyces thermophilus L35, Escherichia coli P34, etc., are separated from meat products by conventional methods, and are all wild strains provided by Yangzhou University.

[0044] Example 1

[0045] Isolation, purification and identification of lactic acid bacteria

[0046] Kimchi was purchased randomly from the Yangzhou vegetable market, and the samples were placed in a 37°C constant temperature incubator for 12 h. Then 100 μL of the enriched liquid was diluted in sterile saline in a 10-fold gradient to 10 -1 ~10 -6 . Select the diluted samples in turn and spread them on MRS-CaCO3 solid culture medium. After culturing in a constant temperature incubator under anaerobic conditions at 37°C for 48h to 72h, pick out single colonies with obvious calcium dissolution rings from the MRS-CaCO3 solid culture medium, and then separate and culture them through three streaking. Further, use the Gram staining method to spread the strains without foreign bacteria after separation on MRS solid culture medium, culture them in a constant temperature incubator under anaerobic conditions at 37°C for 24h to 36h, observe the morphology of the grown single colonies, pick out single colonies for Gram staining, and observe the morphological characteristics of Gram-positive bacteria under a microscope and record them. The obtained strains were separated by streaking three times until there were no foreign bacteria under the colony microscopy. It was preliminarily determined that two strains were obtained for strain identification.

[0047] The preliminary identification results are as follows: Gram staining microscopy results of the two isolated strains were positive, rod-shaped, without flagella and spores. The colony morphology of the two lactic acid bacteria on MRS solid medium after culturing at 37℃ for 48h was characterized by milky white protrusions, neat edges, and smooth and moist surfaces. The main physiological and biochemical characteristics are as follows: catalase negative, nitrate reduction negative, gelatinase negative, HS negative, acid production using glucose positive, and gas production using glucose negative; they were named YZU-LP52 and YZU-LF56 respectively.

[0048] A single colony was inoculated into 10 mL of MRS liquid medium and cultured at 37°C and 150 r / min for 12 h, then centrifuged and the supernatant was discarded. Genes were extracted using a bacterial genome kit, 16S rDNA fragments were amplified, and then sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing analysis, and the obtained sequences were compared in the NCBI database. Among them, the PCR amplification primers are:

[0049] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3';

[0050] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0051] The PCR reaction conditions were: 94°C, pre-denaturation for 5 min; 95°C, denaturation for 30 s; 59°C, annealing for 50 s; 72°C, extension for 90 s; 30 cycles; 72°C, extension for 10 min. The 16S rDNA sequences (5'-3') of the two lactic acid bacteria strains are shown in SEQ ID NO.1-2 below:

[0052] (1) 16S rDNA sequence of YZU-LP52:

[0053]

[0054]

[0055] (2) 16S rDNA sequence of YZU-LF56:

[0056]

[0057]

[0058] The 16S rDNA gene sequencing results were compared with the GenBank database by NCBI BLAST and identified as Lactobacillus plantarum and Lactobacillus fermentum ( Figure 1 and Figure 2 ), and combined with their physiological and biochemical characteristics, they were named Lactiplantibacillus plantarum YZU-LP52 and LimosilactobacillusfermentumYZU-LF56, and were deposited in China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China on June 24, 2014, with the deposit numbers CCTCC NO:M 20241253 and CCTCC NO:M 20241254, respectively.

[0059] Example 2

[0060] Test of synergistic and broad-spectrum antibacterial ability of composite probiotic metabolite preparations

[0061] (1) Preparation of indicator bacteria

[0062] Ten single colonies of indicator bacteria were inoculated into LB liquid medium, cultured overnight at 37°C, and the concentration was adjusted to 10 with sterile saline.8 CFU / mL, stored at 4℃ for future use.

[0063] (2) Preparation of composite probiotic metabolite preparations

[0064] The two lactic acid bacteria cultured in Example 1 (with an initial bacterial solution concentration of 10 9 CFU / mL) was inoculated into MRS liquid at a ratio of 1:1 (v / v) and a volume ratio of 5%, and cultured in a constant temperature incubator at 37°C for 24 h. After centrifugation at 10000g and 4°C for 10 min, the supernatant of the bacterial liquid was taken for antibacterial test and stored at 4°C for later use.

[0065] At the same time, the supernatant of two lactic acid bacteria cultured separately was designed, and the bacterial concentration was 10 9 CFU / mL, the volume of bacterial solution was the same as the volume after mixing the above two bacteria, and the culture conditions were the same.

[0066] (3) Determination of antibacterial ability

[0067] Place a sterile Oxford cup on a culture dish, then take 100 μL of indicator bacteria and LB semi-solid medium, mix them evenly and pour them into the culture dish. After the culture medium solidifies, take out the Oxford cup, add 100 μL of the supernatant of two strains of lactic acid bacteria and the metabolite preparation of the composite probiotics in the well, and observe the diameter of the inhibition zone for each indicator bacteria after culturing at 37 ° C for 24 hours. The results of the inhibition zone and its synergistic index show that the composite probiotic metabolite preparation exhibits an antibacterial effect on all indicator bacteria, and the antibacterial effect on most indicator bacteria shows a broad-spectrum antibacterial and synergistic effect (Table 1). Among them, the synergistic index (Fractional Inhibitory Concentration Index, FICI) is an index used to evaluate the synergistic effect of two or more antibacterial substances when used in combination, and its calculation formula is FICI = MIC (mixed bacteria A and B) / MIC (A single bacteria) + MIC (mixed bacteria A and B) / MIC (B single bacteria). FICI≤0.50 indicates synergistic effect; 0.50<FICI≤1.00 indicates additive effect; 1.00<FICI<2.00 indicates irrelevant effect; FICI≥2.00 indicates antagonistic effect.

[0068] Table 1 Synergistic and broad-spectrum antibacterial effects of composite probiotic metabolite preparations

[0069]

[0070]

[0071] Example 3

[0072] Safety and tolerability evaluation of strains in composite probiotic metabolite preparations

[0073] (1) Hemolysis analysis

[0074] Take 5 μL of seed solution (10 8 CFU / mL) were spotted on a blood plate (Guangdong Huankai Microbiology Co., Ltd., catalog number: 024072), and after culturing in a 37°C incubator for 24 hours, the surrounding area was observed for hemolysis. A wild-type Bacillus cereus 12-1 from Yangzhou University was selected as a positive control. The results showed that strain YZU-LP52 ( Figure 3 A) and YZU-LF56( Figure 3 B) There was no hemolytic zone on the blood plate, while the positive control Bacillus cereus 12-1 produced an obvious hemolytic zone, indicating that the two strains of the composite probiotics had no hemolytic ability.

[0075] (2) Drug sensitivity test

[0076] The two lactic acid bacteria identified in the example were tested for drug resistance using the disc diffusion method. The concentration of the activated two bacterial solutions was adjusted to 10 8 CFU / mL, dip a sterile cotton swab in an appropriate amount of bacterial solution, evenly spread it on the MRS agar plate, use sterile tweezers to evenly place the drug-sensitive paper on it, and culture it in a constant temperature incubator at 37°C for 24 hours, and record the diameter of the inhibition zone of each drug-sensitive sheet. The drug resistance results are shown in Table 2: Lactobacillus plantarum YZU-LP52 is intermediate to three antibiotics: minocycline, penicillin, and cephalothin, and is sensitive to 12 antibiotics: cefotaxime, clindamycin, oxacillin, ceftazidime, nitrofurantoin, co-trimoxazole, erythromycin, clarithromycin, rifampicin, cefoxitin, chloramphenicol, and tetracycline. The fermented lactobacillus YZU-LF56 was intermediate to two antibiotics, erythromycin and ceftazidime, and was sensitive to 13 antibiotics, including minocycline, cefotaxime, penicillin, cephalothin, clindamycin, oxacillin, nitrofurantoin, cotrimoxazole, clarithromycin, rifampicin, cefoxitin, chloramphenicol, and tetracycline. The above results showed that the two lactic acid bacteria were sensitive to antibiotics.

[0077] Table 2 Evaluation of the tolerance of two lactic acid bacteria to different antibiotics

[0078]

[0079] (3) Acid resistance test

[0080] The two lactobacilli identified in the example (10 9CFU / mL) were inoculated in 5mL MRS medium with pH values ​​of 2, 2.5, 3, and 5.6 (control) at an inoculum size of 5% by volume, and mixed by pipetting and then cultured in a 37°C constant temperature incubator for 4h. After mixing (0h) and culturing for 2h and 4h, 100 μL of bacterial solution were taken respectively and gradiently diluted in a finger-shaped tube containing 900 μL PBS (pH=6.78) solution. After dilution to a suitable gradient, 100 μL of dilution was spread on an MRS agar plate, and counted after culturing for 24h in a 37°C constant temperature incubator. As a result, compared with the control group (pH=5.6), the viable count of plant lactobacillus YZU-LP52 in the pH 2 and pH 2.5 groups showed a trend of first decreasing and then increasing, indicating that plant lactobacillus YZU-LP52 has acid resistance at pH 2 (Table 3). As shown in Table 4, compared with the control group (pH=5.6), when the pH was 2, the number of live bacteria of fermented Lactobacillus YZU-LP56 remained stable, while when the pH was 2.5, the number of live bacteria of fermented Lactobacillus increased, indicating that the fermented Lactobacillus YZU-LF56 has acid resistance at pH 2.

[0081] Table 3 Stability of Lactobacillus plantarum YZU-LP52 at different pH

[0082]

[0083] Table 4 Stability of fermented Lactobacillus YZU-LF56 at different pH

[0084]

[0085] (4) Bile salt resistance test

[0086] The two lactic acid bacteria identified in the example (10 9 CFU / mL) were inoculated in MRS medium containing 0% (control), 0.1%, 0.3%, and 0.5% bile salts at a volume ratio of 5%, and then mixed with a pipette and cultured in a 37°C constant temperature incubator for 4 hours. After mixing (0h) and culturing for 2h and 4h, 100μL of bacterial solution was taken and diluted in a finger-shaped tube containing 900μL PBS (pH=6.78) solution in a gradient manner. After diluting to an appropriate gradient, 100μL of the dilution was spread on an MRS agar plate, and cultured in a 37°C constant temperature incubator for 24h before counting. The results showed that the growth of the two lactic acid bacteria in 0.1% bile salts showed a trend of first decreasing and then increasing, while they did not grow in 0.3% and 0.5% bile salts. Therefore, Lactobacillus plantarum YZU-LP52 and Lactobacillus fermentum YZU-LF56 can tolerate 0.1% bile salt (Table 5, Table 6), proving that the strains of the present invention help them survive and reproduce better in the intestine and have probiotic properties.

[0087] Table 5 Stability test of Lactobacillus plantarum YZU-LP52 at different bile salt concentrations

[0088]

[0089]

[0090] Table 6 Stability test of Lactobacillus fermentum YZU-LP56 at different bile salt concentrations

[0091]

[0092] Example 4

[0093] Application of compound probiotic metabolite preparation in the preservation of fresh meat

[0094] Take two lactic acid bacteria (both bacterial solution concentrations are 10 9 CFU / mL) was inoculated into MRS liquid at a ratio of 1:1 (v / v) and a 5% inoculum size, and cultured in a 37°C constant temperature incubator for 24 h. After centrifugation at 10000g and 4°C for 10 min, the supernatant of the bacterial liquid was collected and stored at 4°C for later use.

[0095] At the same time, the supernatant of two lactic acid bacteria cultured separately was designed, and the bacterial concentration was 10 9 CFU / mL, the volume of bacterial solution was the same as the volume after mixing the above two bacteria, and the culture conditions were the same.

[0096] Under sterile conditions, fresh pork hind leg meat was peeled, fascia and excess fat were removed, and the meat was cut into two portions, each 100 g. The prepared supernatant of the composite probiotics and the supernatant of the two single bacteria were evenly spread on the pork hind leg meat as the experimental composite group, YZU-LP52 group, and YZU-LF56 group. The pork hind leg meat samples without any treatment were used as the control group. The samples were refrigerated at 4°C for 8 days, and samples were taken and analyzed every 2 days.

[0097] (1) Effect of the prepared composite probiotic supernatant on pH changes in pork

[0098] pH is one of the important indicators for judging the microbial spoilage of meat and meat products. The effect of the prepared compound probiotic supernatant on the pH change of pork was tested with reference to GB 5009.237-2016 "National Food Safety Standard Determination of Food pH". The reference standards are: first-level freshness (pH 5.7-6.2), second-level freshness (pH 6.2-6.7), and spoiled meat (pH>6.7). Figure 4The results showed that with the increase of storage time, the pH of the cold fresh meat in the control group and the three treatment groups increased slowly, but the pH increase rate of the three treatment groups was significantly lower than that of the control group. The pH values ​​of the samples of the YZU-LP52 and YZU-LF56 groups reached the second-level freshness on the sixth day, while the pH of the compound group samples was 5.71, which was still within the range of the first-level freshness of the national standard. On the eighth day, the pH of the control group samples was 6.88, while the pH of the compound group samples was 6.03, which was much lower than 6.7, indicating that the preservation effect of the composite probiotic metabolite preparation was better than that of the single strain metabolite preparation, and it could slow down the spoilage of cold fresh meat by inhibiting the increase of pH.

[0099] (2) Effect of the prepared composite probiotic supernatant on the total colony count in pork

[0100] The total colony count is an indicator used to determine the degree of bacterial contamination and sanitary quality of food. The detection method refers to GB4789.2-2016 "National Food Safety Standard for Food Microbiological Inspection Colony Count Determination". According to the determination of my country's chilled pork industry standard, the total colony count of chilled fresh meat cannot be greater than 1.0×10 6 CFU / g. Reference evaluation standard: First-class freshness <1.0×10 4 CFU / g, secondary freshness <1.0×10 6 CFU / g, spoiled meat>1.0×10 6 CFU / g. Figure 5 The results show that with the increase of storage time, the total number of colonies in the control group and the three treatment groups of fresh meat increased, while the rate of increase of the total number of colonies in the three treatment groups was significantly lower than that of the control group. The total number of colonies in the control group samples reached 5.9Log CFU / g on the second day, close to deterioration; while the colony counts of the YZU-LP52 and YZU-LF56 samples on the sixth day were 6.31Log CFU / g and 6.28Log CFU / g, basically deteriorating. The composite group reached 5.71Log CFU / g on the sixth day, which was significantly lower than the control group. This shows that the composite probiotic metabolite preparation of the present invention can effectively inhibit the number of colonies in fresh pork and ensure that the shelf life of pork reaches 6 days at 4°C.

[0101] (3) Effect of the prepared composite probiotic supernatant on the changes of pork TVB-N

[0102] TVB-N is one of the commonly used indicators for evaluating the freshness of meat. The national standard stipulates that TVB-N of fresh meat should be ≤15mg / 100g. The detection method refers to GB5009.228-2016 "National Food Safety Standard Determination of Volatile Basic Nitrogen in Food". Figure 6The changes of TVB-N of chilled pork in each treatment group during storage. It can be seen that the TVB-N values ​​of chilled fresh meat in the control group and the three treatment groups gradually increased with the extension of storage time. The TVB-N growth rate of pork in the three experimental groups was significantly lower than that in the control group, and the TVB-N values ​​of the control group samples were always higher than those of the other three experimental groups. The TVB-N of chilled pork in the control group reached 15.1 mg / 100 g on the 4th day, which was the first to exceed the standard. The samples of YZU-LP52 and YZU-LF56 exceeded the national standard on the 6th day, while the composite group exceeded the national standard for chilled fresh meat on the 8th day, indicating that the composite probiotic metabolite preparation significantly increased the shelf life of chilled fresh meat samples by 5 days compared with the control group, and increased the shelf life by two days compared with the single strain metabolite preparation.

[0103] (4) Effect of the prepared composite probiotic supernatant on the degree of pork protein hydrolysis

[0104] The degree of protein hydrolysis in cold fresh meat was analyzed by SDS-PAGE, and the sample preparation method was the same as above. Then the myofibrillar protein in the sample was extracted, the protein precipitate was mixed with the buffer in a 1:1 ratio, and then 80μL of the mixed sample solution and 20μL of loading buffer were placed in a 100℃ metal bath and boiled for 10 minutes. After natural cooling, SDS-PAGE analysis was performed. Electrophoresis conditions: The initial voltage during electrophoresis is 80V. When the sample front just enters the separation gel, the voltage is increased to 120V. The electrophoresis time is about 2h. After staining with Coomassie Brilliant Blue R250 mixed dye solution for 1h, wash the gel with clean water, add decolorizing solution and decolorize repeatedly on a decolorizing shaker. Figure 7 The results showed that the 45kDa (actin) bands in the control group and the experimental group became lighter over time, but the protein bands in the experimental group did not change significantly. It was concluded that the composite probiotic metabolite preparation could delay the hydrolysis of protein in fresh meat.

[0105] (5) Effect of the prepared composite probiotic supernatant on the chromaticity change of pork

[0106] The control group and the three experimental groups of cold fresh meat samples were cut into small pieces of about 1cm×1cm×1cm, and then the color index (L* value, a* value, b* value) of the fresh meat during the cold storage process was measured immediately with a CR-400 automatic colorimeter. The L* value represents the brightness value, and the larger the L* value, the better the glossiness of the meat. Figure 8The changes in the glossiness of fresh pork in each treatment group during storage. It can be seen that the glossiness of the four groups of fresh meat gradually decreased with the extension of storage time. Among them, the glossiness decline rate of pork in the three experimental groups was significantly lower than that of the control group, and the glossiness of the control group samples was always lower than that of other experimental groups. The glossiness of the composite group was higher than that of the YZU-LP52 and YZU-LF56 groups. This shows that the composite probiotic metabolite preparation acts as a protective barrier for fresh meat, reduces the contact between the surface of fresh meat and the air, inhibits the growth and reproduction of a large number of corrupt bacteria, and thus reduces the oxidative discoloration of fresh meat. The a* value represents the redness value, which mainly depends on the color and oxidation state of myoglobin in the meat. From Fig. 9 It can be seen that the redness value of the 0-8d control group varies greatly throughout the refrigeration period, while the YZU-LP52, YZU-LF56 and composite group samples have a smaller change range than the control group samples, and the composite group has the smallest change range, indicating that the application of this composite probiotic metabolite preparation in the preservation of fresh meat can form a barrier on the surface of fresh meat, resulting in prolonged color stability and slightly slowing down the oxidation of meat. The b* value indicates yellowness, and a high b* value indicates that the oxidation of meat is aggravated and a large number of bacteria are multiplying. Fig.10 The yellowness value of fresh pork in each treatment group during storage is shown in Figure 2. The yellowness value of fresh pork in the control group and the three treatment groups gradually increased with the extension of storage time. The growth rate of the yellowness value of pork in the YZU-LP52, YZU-LF56 and composite groups was significantly lower than that in the control group, and the yellowness value of the composite sample was always lower than that in the YZU-LP52 and YZU-LF56 groups. This shows that the application of the composite probiotic metabolite preparation has a good effect in delaying the oxidation of fresh meat.

Claims

1. A composite probiotic with broad-spectrum antibacterial activity, characterized in that: The two lactic acid bacteria in the composite probiotics are Lactobacillus plantarum YZU-LP52 and Lactobacillus fermentum YZU-LF56, which were deposited in the China Center for Type Culture Collection on June 24, 2024, with the deposit numbers CCTCC NO:M 20241253 and CCTCC NO:M 20241254, respectively.

2. A composite probiotic metabolite preparation with broad-spectrum antibacterial activity, characterized in that: The composite probiotic metabolite preparation is formed by metabolites produced by the interaction of two strains of lactic acid bacteria after mixed culture. The two strains of lactic acid bacteria are Lactobacillus plantarum YZU-LP52 and Lactobacillus fermentum YZU-LF56, which were deposited in the China Center for Type Culture Collection on June 24, 2024, and the deposit numbers are CCTCC NO:M 20241253 and CCTCC NO:M 20241254, respectively.

3. A method for preparing the composite probiotic metabolite preparation with broad-spectrum antibacterial activity according to claim 2, characterized in that: The two strains of lactic acid bacteria were mixed and inoculated into liquid culture medium at a concentration of 10 8 CFU / mL or above, take the supernatant of the composite probiotic solution after centrifugation, mix, and save for later use.

4. The preparation method according to claim 3, characterized in that: Preferably, the two cultured lactic acid bacteria are mixed and inoculated into a culture medium for culture, and then the culture supernatant is obtained by centrifugation. The culture temperature of the two lactic acid bacteria is 30-37° C., and the culture medium is MRS culture medium.

5. Use of the composite probiotic with broad-spectrum antibacterial activity according to claim 1 or the metabolite preparation according to claim 2 in inhibiting or reducing spoilage bacteria and pathogenic bacteria.

6. The use according to claim 5, characterized in that: The composite probiotics or metabolite preparations with broad-spectrum antibacterial activity are used to inhibit or reduce spoilage bacteria and pathogenic bacteria in the production environment or storage and transportation equipment of fresh meat or meat products.

7. The use according to claim 5, characterized in that: The composite probiotics or metabolite preparations with broad-spectrum antibacterial activity are used in inhibiting the reproduction and metabolism of spoilage bacteria and pathogenic bacteria in the processing of fresh meat and the low-temperature cold chain process.

8. The use according to claim 5, characterized in that: The composite probiotics or metabolite preparation with broad-spectrum antibacterial activity is used alone or in combination with existing antibacterial agents to prepare spray liquids, rinse liquids, preservatives or biological microbial control agents for inhibiting spoilage bacteria and pathogenic bacteria.

9. The use according to claim 5, characterized in that: The composite probiotic or metabolite preparation with broad-spectrum antibacterial activity is made into a spray liquid or a rinse liquid, which is used for spraying or washing the production environment and production equipment to reduce the contamination of spoilage bacteria and pathogenic bacteria during the cold chain processing of meat; or it is added during the preparation and storage to inhibit the metabolism and reproduction of spoilage bacteria and pathogenic bacteria during the processing and low-temperature storage of fresh meat.

10. The use according to claim 5, characterized in that: The spoilage bacteria and pathogenic bacteria include Listeria monocytogenes, Pseudomonas fluorescens, Salmonella typhimurium, Bacillus anthracis, anaerobic Bacillus, Bacillus nanhaiensis, and Staphylococcus aureus.

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