A strain of Bacillus Velez G14 and its application

Abstract

The present invention provides a strain of Bacillus Velez G14 and its application. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on July 16, 2024, with a deposit number of GDMCC NO: 64871, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The Bacillus Velez G14 strain and its fermentation broth of the present invention can not only effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, but also have better genetic stability, acid-base stability, temperature stability, enzyme stability, ultraviolet stability and storage stability.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a Bacillus Velezii G14 strain and its application. Background Art

[0002] Pasteurella multocida can cause serious diseases in many animals, such as pleuropneumonia, fowl cholera, hemorrhagic disease, sepsis, etc. These diseases are characterized by explosive epidemics and often cause huge losses to the breeding industry. In addition, Pasteurella multocida is also a pathogen of zoonotic diseases. Humans can be infected by contacting the secretions, excretions or corpses of infected animals, which may cause lung diseases (such as pneumonia, bronchiectasis, etc.), urinary system diseases (such as urethritis, cystitis, etc.), nervous system diseases (such as meningitis, brain abscess, etc.), kidney diseases (such as nephritis, pyelonephritis, etc.), liver diseases (such as liver abscess, liver failure, etc.) and many other diseases. Therefore, it is very important to find a means to effectively inhibit Pasteurella multocida.

[0003] Microbial control, as a green control technology, effectively inhibits Pasteurella multocida through organisms or their metabolites, etc. It has the advantages of being non-toxic, non-residue, non-killing natural enemies, non-drug resistance, safe and environmentally friendly, highly economical, and having good control effects. It stands out among the existing methods for inhibiting Pasteurella multocida. However, there are still few known microorganisms that can be used to inhibit Pasteurella multocida. Summary of the invention

[0004] The present invention aims to provide a Bacillus velezensis G14 strain for inhibiting a variety of harmful bacteria including Pasteurella multocida, thereby providing a new microbial selection for inhibiting Pasteurella multocida.

[0005] The first object of the present invention is to provide a Bacillus velezensis G14 strain.

[0006] The second object of the present invention is to provide the use of the above-mentioned Bacillus Velez G14 strain or its fermentation liquid in inhibiting Pasteurella multocida.

[0007] The third object of the present invention is to provide the use of the above-mentioned Bacillus Velez G14 strain or its fermentation liquid in inhibiting Enterobacter aerogenes.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned Bacillus Velez G14 strain or its fermentation liquid in inhibiting Enterococcus faecalis.

[0009] The fifth object of the present invention is to provide the use of the above-mentioned Bacillus Velez G14 strain or its fermentation liquid in inhibiting Pseudomonas aeruginosa.

[0010] The sixth object of the present invention is to provide the use of the above-mentioned Bacillus Velez G14 strain or its fermentation broth in the preparation of Pasteurella multocida inhibitors, Enterobacter aerogenes inhibitors, Enterococcus faecalis inhibitors and / or Pseudomonas aeruginosa inhibitors.

[0011] The seventh object of the present invention is to provide bacteriocin obtained by fermentation of the above-mentioned Bacillus Velez G14 strain.

[0012] The eighth object of the present invention is to provide the use of the above bacteriocin in inhibiting Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and / or Pseudomonas aeruginosa.

[0013] The ninth objective of the present invention is to provide the use of the above bacteriocin in the preparation of Pasteurella multocida inhibitors, Enterobacter aerogenes inhibitors, Enterococcus faecalis inhibitors and / or Pseudomonas aeruginosa inhibitors.

[0014] The tenth object of the present invention is to provide a Pasteurella multocida inhibitor, an Enterobacter aerogenes inhibitor, an Enterococcus faecalis inhibitor and / or a Pseudomonas aeruginosa inhibitor.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] The present invention provides a Bacillus velezensis G14 strain, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 16, 2024, with a deposit number of GDMCC NO: 64871, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0017] The above-mentioned Bacillus Velez G14 strain and its fermentation liquid can not only effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, but also have better genetic stability, acid-base stability, temperature stability, enzyme stability, ultraviolet stability and storage stability in its antibacterial performance. Therefore, the use of the above-mentioned Bacillus Velez G14 strain or its fermentation liquid in inhibiting Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis, Pseudomonas aeruginosa, and in preparing Pasteurella multocida inhibitors, Enterobacter aerogenes inhibitors, Enterococcus faecalis inhibitors and / or Pseudomonas aeruginosa inhibitors should all be within the protection scope of the present invention.

[0018] The bacteriocin extracted and purified from the fermentation broth of the above-mentioned Bacillus Velez G14 strain is a Class III bacteriocin, which has a structure close to lysozyme C, and the bacteriocin can effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa. Therefore, the bacteriocin obtained by fermentation of the above-mentioned Bacillus Velez G14 strain, the use of the above-mentioned bacteriocin in inhibiting Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and / or Pseudomonas aeruginosa, the use of the above-mentioned bacteriocin in the preparation of Pasteurella multocida inhibitors, Enterobacter aerogenes inhibitors, Enterococcus faecalis inhibitors and / or Pseudomonas aeruginosa inhibitors, and a Pasteurella multocida inhibitor, Enterobacter aerogenes inhibitor, Enterococcus faecalis inhibitor and / or Pseudomonas aeruginosa inhibitor using the above-mentioned Bacillus Velez G14 strain or its fermentation broth, or the above-mentioned bacteriocin as an active ingredient should also be within the protection scope of the present invention.

[0019] The present invention has the following beneficial effects:

[0020] The Bacillus Velezii G14 strain and fermentation liquid thereof can not only effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, but also have better antibacterial performance in genetic stability, acid-base stability, temperature stability, enzyme stability, ultraviolet stability and storage stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the phylogenetic tree of strain G14.

[0022] Figure 2 Gel chromatography column chromatogram.

[0023] Figure 3 This is an ion exchange column chromatogram.

[0024] Figure 4 a is the inhibition zone, Figure 4 b is a gel electrophoresis diagram, where "L" represents antibacterial protein and "M" represents marker. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0026] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0027] 1. Preparation of culture medium

[0028] Composite salt mother solution: 1g KNO 3 , 0.5g K 2 HPO4 , 0.1 g NH 4 NO 3 、0.5g MgSO 4 7H 2 O, 0.5 g NaCl, 0.01 g FeSO 4 , 0.001 g MnCl 2 ·H 2 O, 0.001g ZnSO 4 7H 2 O was added into deionized water and the volume was made up to 10 mL with deionized water.

[0029] M10 culture medium: dissolve 10 g soluble starch and 0.5 g hydrolyzed casein in 10 mL of complex salt mother solution, add 14 g agar, and dilute to 1000 mL with deionized water.

[0030] 2. Determination of antibacterial ability of samples to be tested

[0031] Pasteurella multocida (batch number: CVCC393), Enterobacter aerogenes (batch number: ATCC13048), Enterococcus faecalis (batch number: ATCC29212), and Pseudomonas aeruginosa (batch number: ATCC9027) were used as indicator bacteria. The indicator bacteria were inoculated into LB solid culture medium, which was then perforated with a sterile perforator, and the samples to be tested were inoculated into the wells. After culturing at 37°C for 24 hours, the diameter of the inhibition zone around the wells was measured.

[0032] Example 1 Isolation, screening, identification and preservation of strain G14

[0033] 1. Isolation of strain G14

[0034] The roots, stems and leaves of mangrove plants were collected from the Guangdong Zhanjiang Mangrove Nature Reserve, packed in sealed bags and stored at 25°C. Washed with 5% (v / v) sodium hypochlorite solution, soaked in 75% (v / v) ethanol solution for 3 minutes, and then rinsed repeatedly with sterile water. After drying, fully ground with an electric homogenizer to make a tissue stock solution. Then take 0.1mL and evenly spread it on M10 culture medium, cultured at 28°C for 7 days, selected single colonies, and stored in an environment of -80°C.

[0035] 2. Screening and identification of strain G14

[0036] The selected single colony was inoculated into LB liquid culture medium and cultured at 28°C and 180 rpm for 24 h. Sterile LB broth was then added to the culture medium to dilute to 10 5 CFU / mL, and obtain the bacterial solution.

[0037] The inhibitory ability of the bacterial solution against the four indicator bacteria was determined, and the strain G14 with antibacterial activity was screened out. After extracting its DNA, the whole genome was sequenced. The sequencing results were compared and analyzed in the NCBI (https: / / blast.ncbi.nlm.nih.gov / ) database, and the phylogenetic tree obtained was as follows: Figure 1 As shown, the results showed that strain G14 was Bacillus velezensis.

[0038] 3. Deposit of strain G14

[0039] The Bacillus velezensis G14 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 16, 2024, with the deposit number GDMCC NO: 64871, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0040] Example 2 Antibacterial performance of strain G14 and test of its antibacterial performance stability

[0041] 1. Antibacterial properties of strain G14

[0042] The strain G14 was inoculated into 13 culture media (with water as solvent) as shown in Table 1, cultured at 37°C and 180 rpm for 24 h, and then diluted to 10% by adding sterile LB broth into the culture medium. 5 The inhibitory ability of the bacterial solution to the four indicator bacteria was determined, and the results are shown in Table 1.

[0043] Table 1

[0044]

[0045]

[0046] The results showed that strain G14 grown in these 13 culture media could effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, indicating that strain G14 of the present invention not only has excellent antibacterial performance, but also has strong environmental adaptability.

[0047] 2. Stability of antibacterial properties of strain G14

[0048] Strain G14 was inoculated into LB liquid medium and cultured at 30°C and 180rpm for 24h as the first generation; then streaked on LB solid medium and cultured at 30°C for 24h as the second generation; single colonies were picked and inoculated into LB liquid medium and cultured at 30°C and 180rpm for 24h as the third generation; then streaked on LB solid medium and cultured at 30°C for 24h as the fourth generation. And so on, until the 11th generation was cultured. At the 1st, 3rd, 5th, 7th, 9th and 11th generations, 0.6mL of culture solution was spread on 30mL LB solid medium, and after cultured at 37°C for 24h, the inhibitory ability of strain G14 against four indicator bacteria was determined. The results are shown in Table 2.

[0049] Table 2

[0050]

[0051] The results showed that after multiple subcultures, strain G14 was still able to effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, indicating that the antibacterial performance of strain G14 of the present invention has better genetic stability.

[0052] Example 3 Stability test of antibacterial performance of fermentation broth of strain G14

[0053] 1. Preparation of fermentation broth of strain G14

[0054] Strain G14 was inoculated into the fermentation medium (water as solvent, mannitol 20 g / L, fish peptone 5 g / L, KH 2 PO 4 0.5g / L, K 2 HPO 4 0.5g / L, MnSO 4 ·H 2 O 0.25g / L), after shaking culture in a shaker at 30°C and 180rpm for 24h, the culture solution was placed in a centrifuge at 4°C and 8000×g for 30min, the bacterial precipitate was filtered to remove the bacterial precipitate, and the supernatant obtained by centrifugation was filtered and sterilized with a filter membrane with a pore size of 0.22μm, and then the volume of the system was concentrated by 10 times with a dialysis bag with a molecular weight cutoff of 1kDa to obtain the fermentation broth of the strain G14.

[0055] 2. Stability of antibacterial properties of strain G14 fermentation broth after treatment at different pH values

[0056] The pH of the fermentation broth of strain G14 was adjusted to 5, 5.5, 6, 7, 8, and 9 with 1 mol / L HCl solution and NaOH solution, respectively. After being placed at 4°C for 12 h, the pH was adjusted to 7. The inhibitory ability of the fermentation broth of strain G14 on the four indicator bacteria was measured. The results are shown in Table 3.

[0057] Table 3

[0058]

[0059] The results showed that after being treated with acid and alkali solutions of different pH values, the fermentation broth of strain G14 could still effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, indicating that the antibacterial performance of the fermentation broth of strain G14 of the present invention has better acid-base stability.

[0060] 3. Stability of antibacterial properties of strain G14 fermentation broth after treatment at different temperatures

[0061] The fermentation broth of strain G14 was placed in a water bath at 30, 40, and 60°C for 30 min, and the inhibitory ability of the fermentation broth of strain G14 on the four indicator bacteria was measured. The results are shown in Table 4.

[0062] Table 4

[0063]

[0064] The results showed that after being treated at different temperatures, the fermentation broth of strain G14 could still effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, indicating that the antibacterial performance of the fermentation broth of strain G14 of the present invention has better temperature stability.

[0065] IV. Stability of antibacterial properties of strain G14 fermentation broth after different enzyme treatments

[0066] Trypsin, pepsin and proteinase K were added to the fermentation broth of strain G14 at a concentration of 1 mg / mL, respectively. After being water-bathed at 37°C for 1 hour, the inhibitory ability of the fermentation broth of strain G14 against the four indicator bacteria was measured. The results are shown in Table 5.

[0067] Table 5

[0068]

[0069] The results showed that after being treated with different enzymes, the fermentation broth of strain G14 could still effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa, indicating that the antibacterial performance of the fermentation broth of strain G14 of the present invention has better enzyme stability.

[0070] 5. Stability of antibacterial properties of strain G14 fermentation broth after UV irradiation for different time periods

[0071] The fermentation liquid of strain G14 was placed 15 cm away from the UV lamp and irradiated with a 15W UV lamp for 1, 2, 3, and 4 hours, respectively. The inhibitory ability of the fermentation liquid of strain G14 against the four indicator bacteria was measured. The results are shown in Table 6.

[0072] Table 6

[0073]

[0074] The results showed that the fermentation broth of strain G14 could still effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa after being exposed to ultraviolet light for different lengths of time, indicating that the antibacterial performance of the fermentation broth of strain G14 of the present invention has better ultraviolet stability.

[0075] VI. Stability of antibacterial properties of strain G14 fermentation broth after storage for different time periods at different temperatures

[0076] The fermentation broth of strain G14 was placed in an environment of 4°C and -20°C, and stored for 0, 3, 6, 9, and 12 days, respectively. The inhibitory ability of the fermentation broth of strain G14 against the four indicator bacteria was measured. The results are shown in Table 7.

[0077] Table 7

[0078]

[0079]

[0080] The results showed that the fermentation broth of strain G14 could still effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa after being stored for different periods of time in environments with different temperatures, indicating that the antibacterial performance of the fermentation broth of strain G14 of the present invention has better storage stability.

[0081] Example 4 Bacteriocin obtained by fermentation of strain G14 and its antibacterial properties

[0082] 1. Preparation of fermentation broth of strain G14

[0083] Strain G14 was inoculated into the fermentation medium (water as solvent, mannitol 20 g / L, fish peptone 5 g / L, KH 2 PO 4 0.5g / L, K 2 HPO 4 0.5g / L, MnSO 4 ·H 2 O 0.25g / L), after shaking culture in a shaker at 30°C and 180rpm for 24h, the culture solution was placed in a centrifuge at 4°C and 8000×g for 30min, the bacterial precipitate was filtered to remove the bacterial precipitate, and the supernatant obtained by centrifugation was filtered and sterilized with a filter membrane with a pore size of 0.22μm, and then the volume of the system was concentrated by 10 times with a dialysis bag with a molecular weight cutoff of 1kDa to obtain the fermentation broth of the strain G14, which was stored in a refrigerator at -20°C for future use.

[0084] The inhibitory ability of the fermentation broth of strain G14 on four indicator bacteria was determined, and the results showed that it had inhibitory effects on all four indicator bacteria.

[0085] 2. Crude Extraction of Antimicrobial Proteins

[0086] At 4°C, the fermentation broth of strain G14 was salted out with a 60% saturated ammonium sulfate solution, and then centrifuged at 8000×g for 30 minutes to collect the precipitate obtained by centrifugation. The precipitate was dissolved with 20 mL of sterile PBS buffer (pH=5.5), and then dialyzed with a 1 kDa molecular weight cutoff dialysis bag (to remove ammonium sulfate), and then filtered and sterilized with a 0.22 μm pore size filter membrane to obtain a crude antimicrobial protein extract.

[0087] The inhibitory ability of the crude extract of antimicrobial protein on four indicator bacteria was determined, and the results showed that it had inhibitory effects on all four indicator bacteria.

[0088] 3. Preliminary separation and purification of antimicrobial proteins

[0089] The crude extract of antibacterial protein was separated and purified using a protein purifier ACHROM Firin plus (the chromatography column was a Tideroe-GF75 gel chromatography column, the mobile phase was deionized water, the flow rate was 0.55 mL / min, and the detection wavelength was 280 nm). The obtained gel chromatography column chromatogram was as shown in Figure 2 As shown, three purification peaks can be seen.

[0090] The eluates from all separation and purification processes were collected and dispensed into tubes of 1 mL each. The inhibitory abilities of all the eluates against the four indicator bacteria were determined. The results showed that the protein that had inhibitory effects on the four indicator bacteria was in the eluate corresponding to the third purification peak, i.e., this part of the eluate was the preliminary purification product of the antibacterial protein and was stored in a -20°C refrigerator for future use.

[0091] 4. Purification of Antimicrobial Proteins

[0092] The preliminary purified product of the antimicrobial protein was further purified using a protein purifier ACHROM Firin plus (the exchange column was a Titrap Q HP ion exchange column equilibrated with a 0.05 mol / L sterile NaCl solution, the mobile phase was deionized water, the flow rate was 3 mL / min, and the detection wavelength was 280 nm). The ion exchange column chromatogram obtained was as shown below: Figure 3 As shown, three purification peaks can be seen.

[0093] Collect all the eluates from the purification process and dispense them into 1 tube per 1 mL. Determine the inhibitory ability of all the eluates against the four indicator bacteria. The results show that the protein that has inhibitory effect on the four indicator bacteria is in the eluate corresponding to the third purification peak, that is, this part of the eluate is the antibacterial protein (the inhibition zone is shown in Figure 2). Figure 4 a), and stored in a -20°C refrigerator for later use.

[0094] 5. SDS-PAGE analysis of antimicrobial proteins

[0095] The antimicrobial protein was detected by SDS-PAGE gel electrophoresis. Figure 4 As shown in b, it can be seen that the molecular weight of the purified antibacterial protein band is 10-17 kDa, that is, the antibacterial protein is a class III bacteriocin.

[0096] VI. Mass spectrometry analysis of antimicrobial proteins

[0097] Cut the antimicrobial protein into 1 mm pieces 3 The rectangular colloid particles were placed in a test tube containing 1.5 mL of ultrapure water and sent to a biological company for protein decolorization, dehydration, reductive alkylation, enzyme cleavage, peptide extraction, LC-MS / MS analysis, and analyzed and processed by Byonic software. The results are shown in Table 8.

[0098] Table 8

[0099] Protein name score Sequence coverage % Molecular weight / Da Bifunctional polymyxin resistance protein 266.9 25 74186 Lysozyme C 95.46 26 16537 Immunoglobulins 77.03 11 13143 Immunoglobulin mass constant α2 62.73 5 42334 Immunoglobulin mass constant α1 62.73 5 42849 Immunoglobulin-2 heavy chain 62.73 2 48934 Lysosome-associated membrane glycoprotein 60.67 3 43865

[0100] The results showed that the structure of the antimicrobial protein was similar to that of lysozyme C, and it could effectively inhibit Pasteurella multocida, Enterobacter aerogenes, Enterococcus faecalis and Pseudomonas aeruginosa.

[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Bacillus velez Bacillus velezensis ) G14 strain, characterized in that The Bacillus Velez G14 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 16, 2024, with the collection number GDMCCNO: 64871, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The fermentation liquid obtained by fermenting the Bacillus Velez G14 strain according to claim 1, characterized in that: The preparation method of the fermentation broth is as follows: strain G14 is inoculated into a fermentation medium, and after shaking and culturing in a shaker at 30°C and 180 rpm for 24 hours, the culture solution is placed in a centrifuge at 4°C and 8000×g for centrifugation for 30 minutes, bacterial precipitation is filtered out, and the supernatant obtained by centrifugation is filtered and sterilized with a filter membrane with a pore size of 0.22 μm, and then the volume of the system is concentrated by 10 times with a dialysis bag with a molecular weight cutoff of 1 kDa to obtain the fermentation broth; wherein the fermentation medium uses water as a solvent and contains 20 g / L mannitol, 5 g / L fish peptone, 0.5 g / L KH2PO4, 0.5 g / L K2HPO4, and 0.25 g / L MnSO4·H2O.

3. Use of the Bacillus Velez G14 strain according to claim 1 or the fermentation broth according to claim 2 in the preparation of a Pasteurella multocida inhibitor, an Enterobacter aerogenes inhibitor, an Enterococcus faecalis inhibitor and / or a Pseudomonas aeruginosa inhibitor.

4. The crude antimicrobial protein extract obtained by fermentation of the Bacillus Velez G14 strain according to claim 1, characterized in that: The preparation method comprises: at 4°C, salting out the fermentation broth according to claim 2 with a 60% saturation ammonium sulfate solution, centrifuging at a speed of 8000×g for 30 min, and collecting the precipitate obtained by centrifugation; The precipitate was dissolved in 20 mL of sterile PBS buffer at pH 5.5, dialyzed with a dialysis bag with a molecular weight cutoff of 1 kDa, and then sterilized by filtration with a filter membrane with a pore size of 0.22 μm to obtain a crude antibacterial protein extract.

5. Use of the crude antimicrobial protein extract according to claim 4 in the preparation of a Pasteurella multocida inhibitor, an Enterobacter aerogenes inhibitor, an Enterococcus faecalis inhibitor and / or a Pseudomonas aeruginosa inhibitor.

6. A Pasteurella multocida inhibitor, an Enterobacter aerogenes inhibitor, an Enterococcus faecalis inhibitor and / or a Pseudomonas aeruginosa inhibitor, characterized in that: The active ingredient is the Bacillus Velez G14 strain described in claim 1, the fermentation liquid described in claim 2 or the crude antibacterial protein extract described in claim 4.

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