Stable natural polypeptide broad-spectrum bacteriostatic agent as well as preparation method and application thereof

Abstract

The invention discloses a stable natural polypeptide broad-spectrum bacteriostatic agent as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) preparing a polypeptide bacteriostatic agent culture medium 1; (2) preparing a polypeptide bacteriostatic agent culture medium 2; (3) inoculating streptococcus thermophilus in the PPBM1, and culturing to obtain a PPBM1 culture; inoculating bifidobacterium animalis subsp. Lactis to the PPBM2, and culturing to obtain a PPBM2 culture; and (4) mixing the PPBM1 culture and the PPBM2 culture under a sterile condition, continuing to culture, centrifuging and filtering to obtain a filtrate, namely the stable natural polypeptide broad-spectrum bacteriostatic agent. The bacteriostatic agent disclosed by the invention has safety and is not easy to generate drug resistance; the antibacterial agent has the advantages that the antibacterial agent is a natural antibacterial agent, has the safety performance of no hemolysis and no allergy, has relatively strong stability, has good stability under strong ultraviolet rays, has heat resistance, acid resistance, cholate resistance and broad-spectrum bacteriostasis, and is a safe and harmless natural antibacterial agent with wide application.

Description

Technical Field

[0001] The invention discloses a preparation and application method of a natural polypeptide antibacterial agent with broad-spectrum antibacterial activity and high stability, and belongs to the field of applied biotechnology. Technical Background

[0002] From the perspective of safety and health, an important approach to finding new natural antibacterial agents with broad-spectrum antibacterial activity and high stability, while avoiding antibiotic antibacterial agents to avoid drug resistance, is to select polypeptide antibacterial agents.

[0003] As consumers pay more attention to the safety of disinfectants and antibacterial agents and have a growing negative perception of antibiotic resistance, people are increasingly interested in developing safe and non-toxic biological antibacterial agents. Therefore, it is crucial to find new natural antibacterial agents with broad-spectrum antibacterial activity and high stability and apply them to food preservation and other fields.

[0004] Previous research results show that antibacterial substances such as bacteriocins, organic acids (such as lactic acid, acetic acid and butyric acid) and hydrogen peroxide produced by probiotics during the fermentation process can be used to inhibit common spoilage bacteria and pathogens in fruits, vegetables and fresh meat. [1-2] The physical and chemical methods can be used to select and breed Lactobacillus acidophilus NX2-6 to produce antibacterial agents. [3] Lactic acid bacteria form a competitive relationship with harmful microorganisms. In the process of colonizing mucosal epithelial cells, the number of harmful microorganisms is reduced through competition, reducing the occurrence of diseases. [4-5] Lactobacillus casei can produce extracellular polysaccharides to inhibit the adhesion of Staphylococcus aureus and thus achieve antibacterial effects [6] .

[0005] Probiotics and antibacterial agents can be applied to foods such as fruits and vegetables, fresh meat, aquatic products, dairy products, etc. In addition, probiotics and antibacterial agents are also used in medical and feed applications. Lactic acid bacteria can inhibit pathogenic bacteria in the intestines and reduce the risk of related diseases. At the same time, they can enhance gastrointestinal motility, promote food digestion, and enhance the body's immunity. [7-9] .

[0006] However, at present, probiotic antibacterial agents are not very stable and their antibacterial spectrum is not broad.

[0007] References:

[0008] [1]WHO,FAO.Report of a joint FAO / WHO expert consultation onevaluation of health and nutritional properties of probiotics in foodincluding powder milk with live lactic acid bacteria[R].Amerian córdoba parkhotel córdoba argentina:WHO&FAO,2001.

[0009] [2] Gao Yuqi. Research on the breeding and application of high-yield bacteriocin strains of Lactobacillus acidophilus NX2-6[D]. Nanjing: Nanjing Agricultural University, 2015.

[0010] [3] Shan Chengjun, Xia Xiudong, Liu Xiaoli, et al. Isolation, purification and characterization of Lactobacillus panis C-M2 bacteriocin[J]. Food Science, 2017, 38(20): 20-26.

[0011] [4] Qu Yanfeng, Yue Yingxue, Wang Bingran, et al. Research progress on adhesion characteristics of lactic acid bacteria and their immunomodulatory effects[J]. Food Science and Technology, 2019, 44(12): 12-16.

[0012] [5]Bhat MI,Kapila S,Kapila R.Lactobacillus fermentum(MTCC-5898)supplementation rendersprophylactic action against Escherichia coliimpairedintestinal barrier function through tightjunction modulation[J].LWT,2020,123(C):10911.

[0013] [6]Merghni A, Dallel I, Noumi E, et al. Antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus aureusstrains[J]. MicrobialPathogenesis, 2017, 104:84-89.

[0014] [7]Xu Bingzheng, Wang Ying, Liang Xiaoyue, et al. Research progress on application of lactic acid bacteria bacteriocins[J]. Journal of Heilongjiang Bayi Agricultural University, 2015(1):60-63.

[0015] [8] Thirumurugan A, Ramachandran S, Gobikrishnan S. Optimization of medium components for maximizing bacteriocin production by Lactobacillusplantarum ATM11 using statistical design [J]. International food researchjournal, 2015, 22(3): 1272-1279.

[0016] [9] Zhu Shuang, Zhang Aizhong, Jiang Ning, et al. Bacteriocins and their application in animal production [J]. Journal of Animal Nutrition, 2014, 26(2): 327-333. Summary of the invention

[0017] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a stable natural polypeptide broad-spectrum antibacterial agent.

[0018] The second object of the present invention is to provide a method for preparing a stable natural polypeptide broad-spectrum antibacterial agent.

[0019] The third object of the present invention is to provide the use of the above-mentioned stable natural polypeptide broad-spectrum antibacterial agent in the preparation of health products, food, preservatives, feed or skin anti-inflammatory agents.

[0020] The fourth object of the present invention is to provide a composition containing the above-mentioned stable natural polypeptide broad-spectrum antibacterial agent.

[0021] The fifth object of the present invention is to provide the use of the above composition in the preparation of environmental disinfectants.

[0022] The technical solution of the present invention is summarized as follows:

[0023] A method for preparing a stable natural polypeptide broad-spectrum antibacterial agent comprises the following steps:

[0024] (1) Preparation of polypeptide antibacterial agent medium 1: weigh 4-30 g of rice bran powder, 3-20 g of whole milk powder, 2-20 g of yeast extract, 2-10 g of NaCl, and 5-30 g of glucose, add water to 1 L, and sterilize. Polypeptide antibacterial agent medium 1 is referred to as PPBM1;

[0025] (2) Preparation of polypeptide antibacterial agent medium 2: weigh 4-30 g rice bran powder, 4-20 g whole milk powder, 2-20 g beef extract, 5-30 g glucose, 1-2 g triammonium citrate, 1-5 g sodium acetate, 0.1-1 g MgSO4, 0.05-2 g MnSO4, 1-2 g K2HPO4, add water to 1 L, sterilize, polypeptide antibacterial agent medium 2 is referred to as PPBM2;

[0026] (3) Inoculating Streptococcus thermophilus CICC 20369 in PPBM1 and culturing to obtain PPBM1 culture; inoculating Bifidobacterium animalis subsp. lactis CICC 24210 in PPBM2 and culturing to obtain PPBM2 culture;

[0027] (4) Under sterile conditions, the PPBM1 culture and the PPBM2 culture are mixed in a volume ratio of 1:0.1-10, and the culture is continued for 24-240 hours. The mixture is centrifuged at 5000-10000 r / min for 10-30 minutes, and filtered. The filtrate is a stable natural polypeptide broad-spectrum antibacterial agent.

[0028] Preferably, the step (3) comprises: inoculating thermophilic Streptococcus CICC 20369 in PPBM1, culturing for 24-48 hours, heating to 42° C. and culturing for 1-3 hours to obtain PPBM1 culture; inoculating animal Bifidobacterium lactis subsp. CICC 24210 in PPBM2 and culturing for 24-48 hours to obtain PPBM2 culture.

[0029] Preferably, the step (4) is: under sterile conditions, PPBM1 culture and PPBM2 culture are mixed in a volume ratio of 1:0.33, and cultured for 48 hours, centrifuged at 8000 r / min for 20 minutes, and filtered. The filtrate is a stable natural polypeptide broad-spectrum antibacterial agent.

[0030] The preparation method is a stable natural polypeptide broad-spectrum antibacterial agent.

[0031] The above-mentioned stable natural polypeptide broad-spectrum antibacterial agent is used in the preparation of health products, food, preservatives, feed or skin anti-inflammatory agents.

[0032] The invention discloses a composition containing a stable natural polypeptide broad-spectrum antibacterial agent.

[0033] The application of the above-mentioned stable natural polypeptide broad-spectrum antibacterial agent composition in the preparation of environmental disinfectants.

[0034] Advantages of the present invention

[0035] The invention discloses a stable natural polypeptide broad-spectrum antibacterial agent, which is safe and avoids negative effects such as the development of drug resistance of the inhibited microorganisms to the medical antibiotics during the use of the (medical antibiotic) antibacterial agent; and has the safety performance of being non-hemolytic and non-allergic, and relatively strong stability, including good stability under strong ultraviolet rays, heat resistance (still stable after boiling at 100°), acid resistance (strong acidity of pH 2), and resistance to 3% bile salts. The broad-spectrum antibacterial agent includes inhibiting Escherichia coli, Staphylococcus aureus, Staphylococcus aureus, Candida albicans, etc., and is a safe and harmless natural antibacterial agent with a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effect of different pH on the activity of antibacterial agents. DETAILED DESCRIPTION

[0037] The present invention will be further described below by means of specific examples.

[0038] In the following examples, polypeptide antibacterial agent culture medium 1 is referred to as PPBM1, and polypeptide antibacterial agent culture medium 2 is referred to as PPBM2.

[0039] Streptococcus thermophilus CICC 20367, purchased from China Industrial Microbiological Culture Collection Center in June 2020. Provider contact information: www.chian-cicc.org

[0040] Bifidobacterium animalis subsp. lactis CICC 24210 was purchased from China Industrial Microbiological Culture Collection Center in June 2020. Provider contact information: www.chian-cicc.org

[0041] The Escherichia coli (E. coli) (ATCC 25922) disclosed in the present invention (commercially available);

[0042] Staphylococcus albus (S. albus) (ATCC 8032) (commercially available);

[0043] Staphylococcus aureus (S. aureus) (ATCC 12600) (commercially available);

[0044] Candida albicans (C.albicans) (ATCC 76615 (commercially available)). The present invention discloses the above bacteria and fungi for a better understanding of the present invention, but does not limit the present invention.

[0045] Example 1

[0046] A method for preparing a stable natural polypeptide broad-spectrum antibacterial agent comprises the following steps:

[0047] (1) Preparation of polypeptide antibacterial agent medium 1: weigh 10 g rice bran powder, 10 g whole milk powder, 10 g yeast extract, 5 g NaCl, 10 g glucose, add water to 1 L, and sterilize;

[0048] (2) Preparation of polypeptide antibacterial agent medium 2: weigh 15 g rice bran powder, 10 g whole milk powder, 10 g beef extract, 10 g glucose, 1.5 g triammonium citrate, 2 g sodium acetate, 0.5 g MgSO4, 0.5 g MnSO4, 1.2 g K2HPO4, add water to 1 L, and sterilize;

[0049] (3) Inoculate PPBM1 with Streptococcus thermophilus CICC 20369, culture for 36 h, heat to 42°C and culture for 2 h to obtain PPBM1 culture; inoculate PPBM2 with Bifidobacterium animalis subsp. lactis CICC 24210 and culture for 36 h to obtain PPBM2 culture;

[0050] (4) Under sterile conditions, the PPBM1 culture and the PPBM2 culture were mixed at a volume ratio of 1:0.33, and the culture was continued for 48 hours. The mixture was centrifuged at 8000 r / min for 20 minutes and filtered. The filtrate was a stable natural polypeptide broad-spectrum antibacterial agent.

[0051] Example 2

[0052] A method for preparing a stable natural polypeptide broad-spectrum antibacterial agent comprises the following steps:

[0053] (1) Preparation of polypeptide antibacterial agent medium 1: weigh 4 g rice bran powder, 20 g whole milk powder, 2 g yeast extract, 10 g NaCl, 5 g glucose, add water to 1 L, and sterilize;

[0054] (2) Preparation of polypeptide antibacterial agent medium 2: weigh 4 g rice bran powder, 20 g whole milk powder, 2 g beef extract, 30 g glucose, 1 g triammonium citrate, 5 g sodium acetate, 0.1 g MgSO4, 2 g MnSO4, 1 g K2HPO4, add water to 1 L, and sterilize;

[0055] (3) Inoculate PPBM1 with Streptococcus thermophilus CICC 20369, culture for 24 h, heat to 42°C and culture for 1 h to obtain PPBM1 culture; inoculate PPBM2 with Bifidobacterium animalis subsp. lactis CICC 24210 and culture for 24 h to obtain PPBM2 culture;

[0056] (4) Under sterile conditions, the PPBM1 culture and the PPBM2 culture were mixed at a volume ratio of 1:0.1, and the culture was continued for 24 hours. The mixture was centrifuged at 5000 r / min for 30 minutes and filtered. The filtrate was a stable natural polypeptide broad-spectrum antibacterial agent.

[0057] Example 3

[0058] A method for preparing a stable natural polypeptide broad-spectrum antibacterial agent comprises the following steps:

[0059] (1) Preparation of polypeptide antibacterial agent medium 1: weigh 30 g rice bran powder, 3 g whole milk powder, 20 g yeast extract, 2 g NaCl, 30 g glucose, add water to 1 L, and sterilize;

[0060] (2) Preparation of polypeptide antibacterial agent medium 2: weigh 30 g rice bran powder, 4 g whole milk powder, 20 g beef extract, 5 g glucose, 2 g triammonium citrate, 1 g sodium acetate, 1 g MgSO4, 0.05 g MnSO4, 2 g K2HPO4, add water to 1 L, and sterilize;

[0061] (3) Inoculate PPBM1 with Streptococcus thermophilus CICC 20369, culture for 48 hours, heat to 42°C and culture for 3 hours to obtain PPBM1 culture; inoculate PPBM2 with Bifidobacterium animalis subsp. lactis CICC 24210 and culture for 48 hours to obtain PPBM2 culture;

[0062] (4) Under sterile conditions, the PPBM1 culture and the PPBM2 culture were mixed at a volume ratio of 1:10, and the culture was continued for 240 hours. The mixture was centrifuged at 10,000 r / min for 10 minutes and filtered. The filtrate was a stable natural polypeptide broad-spectrum antibacterial agent.

[0063] Example 4

[0064] A method for verifying the thermal stability of a stable natural polypeptide broad-spectrum antibacterial agent (hereinafter referred to as antibacterial agent):

[0065] The antibacterial agent prepared in Example 1 was heated at 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 30 min, and then naturally cooled to room temperature;

[0066] Escherichia coli, Staphylococcus aureus and Staphylococcus aureus were used as indicator bacteria and evenly spread on a universal MRS solid culture medium. The culture was carried out for 4 hours, and holes with a diameter of 0.5 cm were made. 200 ul of the antibacterial agent to be tested, which had been heated and then cooled to room temperature, were added to the holes. The culture was carried out at a constant temperature of 37°C for 48 hours, and the growth of bacteria under the action of the antibacterial agent was observed to evaluate the antibacterial effect of the antibacterial agent after heating.

[0067] After being treated at different temperatures, the antibacterial properties of the antibacterial agent (see Table 1) indicate that the antibacterial agent prepared in Example 1 has good thermal stability. Generally speaking, the thermal stability of a protein is inversely proportional to its molecular weight, so it is proved that the substance produced by the antibacterial agent of the present invention is a small molecule protein or amino acid polypeptide substance with strong thermal stability.

[0068] Table 1 Antibacterial activity of antibacterial agents at different temperatures

[0069]

[0070] Note: The diameter of the inhibition zone is the mean ± square error (n = 3)

[0071] The antibacterial agent prepared in Example 1 in this example was replaced by the antibacterial agents prepared in Example 2 and Example 3, respectively, and their thermal stability was similar to that of the antibacterial agent prepared in Example 1.

[0072] Example 5

[0073] UV stability test of antibacterial agent

[0074] The antibacterial agent prepared in Example 1 was irradiated under 200-253.5 nm UVC disinfection ultraviolet light, and the irradiation time was 2 h, 4 h, 6 h, 8 h, and 10 h respectively;

[0075] Escherichia coli, Staphylococcus aureus, and Staphylococcus aureus were used as indicator bacteria and evenly coated on a universal MRS solid culture medium. After incubation for 4 hours, holes with a diameter of 0.5 cm were made, and 200ul of the antibacterial agent irradiated with ultraviolet light for different times was added to the holes. The antibacterial agent was cultured at a constant temperature of 37°C for 48 hours, and the growth of bacteria under the action of the antibacterial agent was observed to evaluate the antibacterial effect of the antibacterial agent after ultraviolet irradiation. (See Table 2).

[0076] Table 2 Effect of ultraviolet irradiation at different times on the antibacterial activity of antibacterial agents

[0077]

[0078] Note: The diameter of the inhibition zone is the mean ± square error (n = 3)

[0079] The antibacterial agent prepared in Example 1 in this example was replaced by the antibacterial agents prepared in Example 2 and Example 3, respectively, and their ultraviolet stability was similar to that of the antibacterial agent prepared in Example 1.

[0080] Example 6

[0081] Bile salt tolerance characteristics and verification methods:

[0082] Adding ox bile salt to the antibacterial agent prepared in Example 1, the mass content of ox bile salt was prepared to be 0%, 1.0%, 2.0% and 3.0% respectively;

[0083] Escherichia coli, Staphylococcus aureus and Staphylococcus albus were used as indicator bacteria and evenly spread on a universal MRS solid culture medium. The culture was carried out for 4 hours. A hole with a diameter of 0.5 cm was made. 200 ul of an antibacterial agent containing ox bile salts with different mass concentrations was added to the hole. The cells were cultured at 37°C for 48 hours. The growth of bacteria under the action of the antibacterial agent was observed to evaluate the antibacterial effect of the antibacterial agent containing ox bile salts.

[0084] The results showed that the antibacterial effect of the antibacterial agent did not change under the condition of 0%-2.0% ox bile salt, and the antibacterial effect was still greater than 70% under the condition of 3% ox bile salt, indicating that the antibacterial effect of the antibacterial agent remained stable in an environment with high bile salt content.

[0085] The antibacterial agents prepared in Example 1 in this example were replaced by the antibacterial agents prepared in Example 2 and Example 3, respectively, and their bile salt tolerance was similar to that of the antibacterial agent prepared in Example 1.

[0086] Example 7

[0087] The antibacterial agent has acid tolerance characteristics, and its verification method is:

[0088] The pH of the antibacterial agent prepared in Example 1 was adjusted to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 with hydrochloric acid and sodium hydroxide, respectively. The antibacterial agent without pH adjustment was the blank control (CK).

[0089] Escherichia coli, Staphylococcus aureus, and Staphylococcus aureus were used as indicator bacteria and evenly spread on a universal MRS solid culture medium. The culture was carried out for 4 hours, and holes with a diameter of 0.5 cm were made. 200ul of the antibacterial agent for adjusting pH were added to the holes. The culture was carried out at a constant temperature of 37℃ for 48 hours. The growth of bacteria under the action of the antibacterial agent was observed to evaluate the antibacterial effect of the antibacterial agent with different pH values. The untreated strain was used as a control. Figure 1 .

[0090] The antibacterial agent prepared in Example 1 in this example was replaced by the antibacterial agents prepared in Example 2 and Example 3, respectively, and their acid tolerance was similar to that of the antibacterial agent prepared in Example 1.

[0091] Since the antibacterial agent of the present invention is acid-resistant, it can be applied to acidic lactic acid bacteria, acidic feed particles, acidic foods, and acidic health products.

[0092] Example 8

[0093] Antibacterial agent hemolysis safety experiment:

[0094] The antibacterial agent prepared in Example 1 was inoculated on a Columbia blood agar plate (purchased from the market) by the conventional plate streaking method. After culturing at 37°C for 24 hours, no transparent circle or grass green ring appeared around the colony by naked eye observation, indicating no hemolysis. The positive control selected Escherichia coli with strong hemolytic properties, and the experiment was repeated 3 times independently. Escherichia coli had transparent circles and grass green rings; while the antibacterial agent prepared in Example 1 had no transparent circles or grass green rings. The safety assessment of the antibacterial agent was safe.

[0095] Table 3 Antibacterial agent hemolysis safety experiment

[0096] Transparent circle Grass green ring Escherichia coli culture have have Antibacterial Agents none none

[0097] Note: The experiment was repeated 3 times independently.

[0098] The safety evaluation of the antibacterial agents prepared in Examples 2 and 3 was that they were safe.

[0099] Example 9

[0100] Verification method for the safety of antibacterial agents without skin irritation.

[0101] The skin patch test was used to verify that 51 people were randomly divided into three groups, with 17 people in each group. 0.5 ml of the antibacterial agent prepared in Example 1, Example 2 and Example 3 was placed on a 1 cm 2 Each person applies four layers of gauze of different sizes to the skin behind the ear, back, and inner forearm, covering with 2cm 2 The airtight cellophane was fixed with adhesive tape, and the skin at the test site of the patch test was observed to see if any allergic reaction occurred. The patch test results showed that all three groups had no allergy or irritation and could be used as skin anti-inflammatory agents.

[0102] Example 10

[0103] The broad-spectrum resistance of pathogenic bacteria to antibacterial agents is tested in accordance with national disinfection technical specifications.

[0104] The antibacterial agent of Example 1 is tested on Escherichia coli, Staphylococcus aureus, Candida albicans, etc. according to the 2002 edition of "Technical Specifications for Disinfection" promulgated by the Ministry of Health of the People's Republic of China. The bactericidal rates of the antibacterial agent on Escherichia coli, Staphylococcus aureus, and Candida albicans after 10 minutes of action are: Escherichia coli>90.01%, Staphylococcus aureus>90.05%, and Candida albicans>98.9%. It can be added to health products, food, preservatives, feed, and skin anti-inflammatory agents for use, and the final concentration of addition is 3%-25% and has an effect.

[0105] The antibacterial agents prepared in Example 2 and Example 3 were used to replace the antibacterial agent prepared in Example 1 in this example for testing, and the antibacterial agents had similar effects.

[0106] Embodiment 11

[0107] A composition containing a stable natural polypeptide broad-spectrum antibacterial agent, the antibacterial agent of Example 1, benzalkonium bromide (purchased from the market, English name: BenzyldodecyldimethylammoniumBromide, alias: dodecyldimethylbenzyl ammonium bromide) and water.

[0108] The composition contains 2% benzalkonium bromide by mass concentration (can also be any value between 1% and 3%), 10% by mass concentration of a stable natural polypeptide broad-spectrum antibacterial agent (can be in the range of 3% to 25%), and the remainder is water (the composition is called an environmental disinfectant). According to the 2002 edition of the Technical Specification for Disinfection, Escherichia coli, Staphylococcus aureus, Candida albicans, etc. are tested, and the sterilization rates of the composition for Escherichia coli, Staphylococcus aureus, and Candida albicans after 2 minutes of action are: Escherichia coli>99.98, Staphylococcus aureus>99.98, and Candida albicans>99.98.

[0109] Therefore, the composition can be safely sprayed in cars, airplanes, and indoors as an environmental disinfectant, and has a high killing rate for pathogenic bacteria on surfaces and in the air. The environmental disinfectant has a disinfecting effect on pathogenic bacteria in the air of public places such as hospitals, waiting rooms, and airport lounges.

[0110] The composition consisting of the antibacterial agent prepared in Example 1 of this embodiment, benzalkonium bromide and water can be replaced by the antibacterial agents prepared in Example 2 and Example 3 respectively, and can also be used as an environmental disinfectant, and the application scope is the same as that of this embodiment.

Claims

1. A method for preparing a stable natural polypeptide broad-spectrum antibacterial agent, characterized in that The steps include: (1) Preparation of polypeptide antibacterial agent medium 1: weigh 4-30 g of rice bran powder, 3-20 g of whole milk powder, 2-20 g of yeast extract, 2-10 g of NaCl, and 5-30 g of glucose, add water to 1 L, and sterilize. Polypeptide antibacterial agent medium 1 is referred to as PPBM1; (2) Preparation of polypeptide antibacterial agent medium 2: weigh 4-30 g rice bran powder, 4-20 g whole milk powder, 2-20 g beef extract, 5-30 g glucose, 1-2 g triammonium citrate, 1-5 g sodium acetate, 0.1-1 g MgSO4, 0.05-2 g MnSO4, 1-2 g K2HPO4, add water to 1 L, sterilize, polypeptide antibacterial agent medium 2 is referred to as PPBM2; (3) Inoculating Streptococcus thermophilus CICC 20369 in PPBM1 and culturing to obtain PPBM1 culture; inoculating Bifidobacterium animalis subsp. lactis CICC 24210 in PPBM2 and culturing to obtain PPBM2 culture; (4) Under sterile conditions, the PPBM1 culture and the PPBM2 culture are mixed in a volume ratio of 1:0.1-10, and the culture is continued for 24-240 hours. The mixture is centrifuged at 5000-10000 r / min for 10-30 minutes, and filtered. The filtrate is a stable natural polypeptide broad-spectrum antibacterial agent.

2. The preparation method according to claim 1, characterized in that The step (3) is as follows: inoculating thermophilic Streptococcus CICC 20369 in PPBM1, culturing for 24-48 hours, heating to 42° C. and culturing for 1-3 hours to obtain PPBM1 culture; inoculating animal Bifidobacterium lactis subspecies CICC 24210 in PPBM2 and culturing for 24-48 hours to obtain PPBM2 culture.

3. The preparation method according to claim 1, characterized in that The step (4) is: under sterile conditions, PPBM1 culture and PPBM2 culture are mixed in a volume ratio of 1:0.33, and cultured for 48 hours, centrifuged at 8000 r / min for 20 minutes, filtered, and the filtrate is a stable natural polypeptide broad-spectrum antibacterial agent.

4. A stable natural polypeptide broad-spectrum antibacterial agent prepared by the preparation method of any one of claims 1 to 3.

5. Use of a stable natural polypeptide broad-spectrum antibacterial agent according to claim 4 in the preparation of health products, food, preservatives, feed or skin anti-inflammatory agents.

6. A composition comprising a stable natural polypeptide broad-spectrum antibacterial agent according to claim 4.

7. Use of the stable natural polypeptide broad-spectrum antibacterial composition according to claim 4 in the preparation of environmental disinfectants.

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