Bacteriocin Thurisin A2 and its application

Abstract

The present invention belongs to the field of microbial applications, and specifically relates to a bacteriocin Thurisin A2 and its application. The bacteriocin Thurisin A2 has an amino acid sequence as shown in SEQ ID NO.1, and can be synthesized by a polypeptide solid-phase synthesis method or a liquid-phase polypeptide synthesis method, or the coding gene of the bacteriocin Thurisin A2 can be expressed in an engineered bacterium. It has a simple structure, is safe and low-toxic, is easy to synthesize, can effectively kill Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and Listeria monocytogenes, and has no hemolytic activity against eukaryotic animal red blood cells. It has biosafety and can be used to prepare food preservatives, feed additives, antibacterial drugs, etc., and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of microbial applications, and particularly relates to bacteriocin Thurisin A2 and applications thereof. Background Art

[0002] Foodborne diseases caused by food spoilage pose a serious threat to human health and hinder sustainable economic and social development. The 2015 World Health Organization (WHO) report, "Estimates of the Global Burden of Foodborne Disease," indicates that approximately 600 million people worldwide become ill each year from consuming contaminated food, and 420,000 die (including 125,000 children under the age of five). The 2018 World Bank report, "The Economic Burden of Foodborne Disease," indicates that foodborne diseases contribute to $110 billion in lost productivity and healthcare costs in low- and middle-income countries.

[0003] Among the many factors that affect food safety, microbial contamination ranks first. The food processing industry often uses food preservatives to inhibit or kill microorganisms in food, prevent food spoilage, and extend the shelf life of food. Food preservatives are divided into chemical food preservatives and natural food preservatives based on their source. Although traditional chemical preservatives have a good preservative effect, improper use can have certain side effects, and long-term excessive intake can cause certain damage to human health. Natural food preservatives are different from chemical food preservatives. They are a type of food preservative made from substances secreted or present in organisms with antibacterial effects that are artificially extracted or processed. Natural food preservatives are natural substances, some of which are components of food themselves. They are non-toxic, residue-free, highly effective, and do not affect the taste of food. They are a type of food preservative with important development prospects. As consumers increasingly pursue natural, green and healthy food, finding safe, non-toxic and highly effective natural preservatives from animals, plants and microorganisms is a major research direction in the development of food science. It is also an important measure to improve the efficiency and safety of the food industry and has important research and application value.

[0004] Bacteriocins are a class of peptides or proteins with antimicrobial activity produced by bacteria during their growth process through ribosomal biosynthesis. Bacteriocins possess numerous excellent properties: highly potent antimicrobial activity both in vitro and in vivo; non-toxic to humans and animals, and easily degraded by proteases in the animal digestive tract without residue; a unique antimicrobial spectrum with highly selective bactericidal effects; ease of biomodification to meet diverse human needs; thermal stability and acid and alkali resistance for convenient storage; inherently odorless, without affecting the texture or flavor of food or feed; and the ability to lower sterilization temperatures and shorten heat treatment times, thereby improving the nutritional value, flavor, and color of food. Therefore, bacteriocins hold broad development and application prospects in food additives, feed additives, and pharmaceuticals.

[0005] The technical problem to be solved by the present invention is to discover new bacteriocins with excellent properties. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a bacteriocin Thurisin A2 having an amino acid sequence as shown in SEQ ID NO. 1. The bacteriocin Thurisin A2 of the present invention can be synthesized by solid-phase polypeptide synthesis or liquid-phase polypeptide synthesis, or the encoding gene of the bacteriocin Thurisin A2 can be expressed in an engineered bacterium.

[0007] The second object of the present invention is to provide the use of the bacteriocin Thurisin A2 in the preparation of antibacterial and / or antiseptic products.

[0008] Furthermore, the antimicrobial and / or antiseptic product inhibits any one or more of Staphylococcus aureus, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes.

[0009] The third object of the present invention is to provide an antibacterial and / or antiseptic product comprising the bacteriocin ThurisinA2.

[0010] A fourth object of the present invention is to provide the use of the bacteriocin Thurisin A2 in the preparation of drugs for treating and / or preventing bacterial infectious diseases.

[0011] Furthermore, the bacteria are any one or more of Staphylococcus aureus, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes.

[0012] A fifth object of the present invention is to provide a drug for treating and / or preventing bacterial infectious diseases, comprising the bacteriocin Thurisin A2.

[0013] The present invention has the following beneficial effects:

[0014] The bacteriocin Thurisin A2 provided by the present invention has a simple structure, is safe, low-toxic, and easy to synthesize. It is highly effective in killing Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Clostridium perfringens, and has no hemolytic activity against eukaryotic animal red blood cells, demonstrating biosafety. Thurisin A2 can be used to prepare food preservatives, feed additives, antibacterial drugs, and other products, demonstrating broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the hemolytic activity and cytotoxicity test results of bacteriocin Thurisin A2 in Example 3. DETAILED DESCRIPTION

[0016] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0017] Example 1: Acquisition of bacteriocin Thurisin A2

[0018] The bacteriocin Thurisin A2 proposed in the present invention is derived from the Bacillus thuringensis XIN-LX43 (Bacillus thuringensis XIN-LX43) polypeptide sequence database we constructed. The bacterium was deposited in the China Center for Type Culture Collection (CCTCC) on June 15, 2021, with the deposit number CCTCC NO: M 2021719; the deposit address is Wuhan University, Wuhan, China. The amino acid sequence of the bacteriocin Thurisin A2 (shown in SEQ ID NO.1) is as follows: MVAFLRIVGQLGAKAASWAWANKGRVLGWIRDGMAIEWIINKINDMVS. Through online BlastP alignment analysis (Protein BLAST: search protein databases using a protein query (nih.gov)), the bacteriocin Thurisin A2 has no homology with the amino acid sequences of identified and reported bacteriocins, indicating that it is a novel bacteriocin, which is the first time studied, identified and reported by the inventor. According to the amino acid sequence shown, the corresponding antibacterial peptide was synthesized by Qiangyao Biotechnology Co., Ltd. (Shanghai) with a purity of >95%.

[0019] Example 2: Determination of the antibacterial activity of bacteriocin Thurisin A2 against various foodborne pathogens

[0020] Using physiological saline as the diluent, a series of bacteriocin solutions (0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μM) were prepared by the two-fold dilution method. The minimum inhibitory concentration (MIC) of Thurisin A2 at different concentrations against various foodborne pathogens was determined using the agar diffusion method. An appropriate amount of indicator bacteria (approximately 5 × 10 5 cfu / mL), mix thoroughly, and pour onto plates. Select Bacillus cereus CMCC 63301, Bacillus cereus CMCC 63303, Clostridium perfringens ATCC 13124, Listeria monocytogenes ATCC 19111, Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 6538, and Staphylococcus aureus ATCC 43300 as indicator bacteria. After solidification, punch wells using a 6mm borer. Add approximately 50 μL of sample to each well, incubate the plate at 4°C for approximately 2 hours to allow the sample to fully diffuse. Then, incubate at 30°C for 12 hours and observe the antibacterial effect (observe for the appearance of a transparent zone of inhibition). The minimum inhibitory concentrations of Thurisin A2 against Bacillus cereus CMCC 63301, Bacillus cereus CMCC 63303, Clostridium perfringens ATCC 13124, Listeria monocytogenes ATCC 19111, Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 6538, and Staphylococcus aureus ATCC 43300 were determined to be 0.5 μM, 0.5 μM, 2 μM, 1 μM, 0.5 μM, 0.5 μM, and 2 μM, respectively (Table 1).

[0021] Table 1 Determination of the minimum inhibitory concentration of Thurisin A2 against four types of foodborne pathogens

[0022]

[0023] Example 3: Determination of hemolytic activity of bacteriocin Thurisin A2

[0024] Collect 3 mL of fresh blood from a healthy individual (with anticoagulant added), centrifuge at 3000 g for 5 minutes, discard the plasma and collect the red blood cells. Wash the collected red blood cells three times with physiological saline and resuspend the blood cells at a ratio of 2% (V / V). Add 100 μL of red blood cell resuspension to a sterile 96-well cell culture plate, and then add different concentrations of bacteriocin solution, with 3 parallels for each bacteriocin concentration. Use 0.1% Tritonx-100 as a positive control for complete hemolysis, and a physiological salt group as a negative control for no hemolysis. Incubate at 37°C for 2 hours and centrifuge at 3000 g for 10 minutes. Transfer the supernatant after centrifugation to a new sterile 96-well cell culture plate, and use a microplate reader to detect the light absorbance value at 405 nm to calculate the hemolytic activity of the bacteriocin.

[0025] The hemolytic activity of bacteriocin Thurisin A2 is as follows Figure 1 As shown, the bacteriocin exhibited only 1.1% hemolytic activity at a high concentration of 64 μM, indicating that its hemolytic activity was very low and biosafe.

[0026] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0027] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications. Sequence Listing <110> Huaibei Normal University <120> Bacteriocin Thurisin A2 and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 48 <212> PRT <213> Artificial sequence <400> 1 Met Val Ala Phe Leu Arg Ile Val Gly Gln Leu Gly Ala Lys Ala Ala 1 5 10 15 Ser Trp Ala Trp Ala Asn Lys Gly Arg Val Leu Gly Trp Ile Arg Asp 20 25 30 Gly Met Ala Ile Glu Trp Ile Ile Asn Lys Ile Asn Asp Met Val Ser 35 40 45

Claims

1. A use of bacteriocin Thurisin A2 in the preparation of antibacterial and / or antiseptic products, characterized in that: The amino acid sequence of the bacteriocin Thurisin A2 is shown in SEQ ID NO.

1. The antibacterial and / or antiseptic product inhibits any one or more of Staphylococcus aureus, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes.

2. Use of the bacteriocin Thurisin A2 according to claim 1 in the preparation of a medicament for treating and / or preventing bacterial infectious diseases, characterized in that: The bacteria are any one or more of Staphylococcus aureus, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes.

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