阅读说明：本技术 一种新型复合制剂及其在细菌性病害中的应用 (Novel composite preparation and application thereof in bacterial diseases ) 是由 何四龙 谢晓莉 丛郁 徐旭凌 肖逍 丁良 于浩 樊小九 许文建 徐天舜 许小康 于 2020-07-04 设计创作，主要内容包括：本发明公开了一种新型复合制剂及其在细菌性病害中的应用,属于细菌性病害防治技术领域,所述新型复合制剂为噬菌体和益生菌配伍制剂,该复合制剂在弧菌病害、大肠杆菌病、沙门氏菌病、爱德华氏病菌、假单细胞菌病、屈挠杆菌病、链球菌病、巴氏杆菌病、布氏杆菌病、克雷伯氏菌病、嗜水气单胞菌病、金黄色葡萄球菌病、绿脓杆菌病、由茄科雷尔氏菌侵染引起的青枯病、由假单孢杆菌侵染引起的斑点病、由黄单孢杆菌侵染引起的叶枯病和溃疡病、由假单孢杆菌侵染引起的青枯病、由欧文氏杆菌侵染引起的腐烂病、由癌肿野杆菌侵染引起的畸型病等细菌性病害中有良好的应用效果。(The invention discloses a novel composite preparation and application thereof in bacterial diseases, belonging to the technical field of bacterial disease control, wherein the novel composite preparation is a compatible preparation of bacteriophage and probiotics, the compound preparation has good application effect in vibrio diseases, colibacillosis, salmonellosis, Edwardsiella, pseudomonad, flexobacillosis, streptococcosis, pasteurellosis, brucellosis, Klebsiella, Aeromonas hydrophila, Staphylococcus aureus, Pseudomonas aeruginosa, bacterial wilt caused by infection of Ralstonia solanacearum, spot disease caused by infection of pseudomonad, leaf blight and ulcer disease caused by infection of Xanthomonas, bacterial wilt caused by infection of pseudomonad, rot disease caused by infection of Erwinia europaea, teratocarcinosis caused by infection of wild bacillus oncoma and other bacterial diseases.)

1. A novel composite preparation and application thereof in bacterial diseases are characterized in that: the novel composite preparation is a phage and probiotic compatible preparation, and is applied to bacterial diseases such as vibriosis, colibacillosis, salmonellosis, edwardsiella, pseudomonobasic mycosis, flexobacillosis, streptococcosis, pasteurellosis, brucellosis, klebsiella, aeromonas hydrophila, staphylococcus aureus, pseudomonas aeruginosa, bacterial wilt caused by infection of ralstonia solanacearum, spot disease caused by infection of pseudomonas, leaf blight and ulcer disease caused by infection of xanthomonas, bacterial wilt caused by infection of pseudomonas, rot disease caused by infection of erwinia europaea, and teratocarcinosis caused by infection of phyma.

2. The novel complex formulation and its use in bacterial diseases according to claim 1, wherein the novel complex formulation is a probiotic and phage compatible formulation.

3. The novel compound preparation and the application thereof in bacterial diseases according to claim 2, wherein the component ratio of the phage to the probiotics is 1-5: 5-1.

4. A novel complex formulation and its use in bacterial diseases according to claim 3, characterized in that the bacteriophage is a single bacteriophage or a mixture of multiple bacteriophages.

5. A novel combination according to claim 3 and its use in bacterial diseases wherein the probiotic is a single probiotic or a mixture of probiotics.

6. The novel composite preparation according to claim 4, wherein the bacteriophage is one or more of Vibrio parahaemolyticus phage, Vibrio alginolyticus phage, Vibrio harveyi phage, Vibrio cholerae phage, Escherichia coli phage, Salmonella phage, Xanthomonas campestris phage, Ralstonia solanacearum phage, Staphylococcus aureus phage, Aeromonas hydrophila phage, Streptococcus agalactiae phage, Streptococcus iniae phage, Pseudomonas aeruginosa phage, Klebsiella pneumoniae phage, Acinetobacter baumannii phage, Propionibacterium acnes phage, Agrobacterium tumefaciens phage, Corynebacterium michigani phage, Listeria phage, Campylobacter jejuni phage, or Clostridium perfringens phage.

7. The novel complex formulation as claimed in claim 5, wherein said probiotic bacteria is selected from the group consisting of Streptococcus thermophilus, Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, Bacillus lentus, Bacillus thuringiensis, Bacillus natto, Bacillus laterosporus, Bacillus mucilaginosus, Bacillus cereus, Bacillus firmus, Bacillus belgii, Bacillus coagulans, Bacillus polymyxa, Bacillus mycoides, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus buchneri, Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus brevis, Bacillus licheniformis, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus delbrussensis, Lactobacillus delbrusselenii, Lactobacillus delbrusse, One or more of lactobacillus cellobiosus, lactobacillus sake and lactobacillus salivarius, lactococcus lactis subspecies lactis, lactococcus lactis subspecies cremoris, lactococcus lactis diacetyl subspecies lactis, enterococcus faecalis, enterococcus faecium, enterococcus lactis, pediococcus acidilactici, pediococcus pentosaceus, leuconostoc mesenteroides, leuconostoc citreum, propionibacterium propionicum, propionibacterium freudenreichii subspecies schanicolae, staphylococcus parvum, staphylococcus carnosus, saccharomyces cerevisiae, pichia, candida utilis, kluyveromyces marxianus, rhodotorula cerealis, candida guilliermondii or saccharomyces boulardii.

8. The novel complex formulation and its use in bacterial diseases according to claim 6, characterized in that the bacteriophages are prepared by the following steps in sequence: seed culture, enrichment culture, sterilization purification and/or drying.

9. The novel complex formulation and its use in bacterial diseases according to claim 7, characterized in that the probiotic bacteria are prepared by the following steps in sequence: seed culture, fermentation culture, thallus collection and/or drying.

Technical Field

The invention relates to the field of bacterial disease control, in particular to a novel composite preparation and application thereof in bacterial diseases.

Background

In recent years, bacterial diseases have caused a great economic loss in the fields of aquaculture, fur-bearing animal breeding, poultry and livestock breeding, crop cultivation, and the like, and researchers have been searching for new solutions to the problem of bacterial diseases, and have been gaining favor as bacteriophages specific to bacteria and green non-stressful probiotics, for example:

patent CN201710953780.5 discloses a cocktail scheme for preventing and treating vibrio in aquaculture by using 2 strains of vibrio parahaemolyticus phage and 3 strains of vibrio alginolyticus phage; patent CN200510009788.3 discloses that natural metabolite peptides of Lactobacillus paracasei HD1-7 are used as preservatives to be applied to prevention and treatment of pathogenic bacteria such as Escherichia coli; patent CN201910354887.7 discloses a fresh-cut fruit and vegetable fresh-keeping agent compounded by bacteriostatic substances produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens; patent CN202010093203.5 discloses a Bacillus amyloliquefaciens TBA03 and application thereof, which can effectively prevent and control the diseases of Ralstonia solanacearum and relieve the diseases of Ralstonia solanacearum.

The technical schemes do not carry out deep research aiming at the use of the compatibility of the bacteriophage and the probiotics, the scheme of single compatibility of the bacteriophage or the probiotics is difficult to achieve the expectation of comprehensive prevention and control of bacterial diseases, and the technical blank still exists on the aspect of preparing the composite preparation by the compatibility of the bacteriophage and the probiotics.

Disclosure of Invention

Aiming at the vacancy in the prior art, the invention aims to provide a novel composite preparation and application thereof in bacterial diseases by taking compatibility of bacteriophage and probiotics as a technical breakthrough, and has strong process operability, universality and popularization.

In order to achieve the purpose, the invention provides the following technical scheme:

a novel composite preparation and an application thereof in bacterial diseases, wherein the novel composite preparation is a phage and probiotic compatible preparation, and the novel composite preparation is applied to bacterial diseases such as vibrio diseases, colibacillosis, salmonellosis, Edwardsiella, pseudomonas, flexobacillosis, streptococcosis, pasteurellosis, brucellosis, Klebsiella, Aeromonas hydrophila, staphylococcus aureus, Pseudomonas aeruginosa, bacterial wilt caused by infection of Ralstonia solanacearum, spot disease caused by infection of Pseudomonas, leaf blight and ulcer disease caused by infection of Xanthomonas, bacterial wilt caused by infection of Pseudomonas, Venturi rot caused by infection of Europe bacillus, and teratocarcinosis caused by infection of Staphyloma.

Furthermore, the novel compound preparation is a compatible preparation of probiotics and bacteriophage.

Further, the ratio of the components of the phage to the probiotics is 1-5: 5-1.

Further, the bacteriophage is a single bacteriophage or a mixture of multiple bacteriophages.

Further, the probiotics are single probiotics or a mixture of multiple probiotics.

Further, the bacteriophage is one or more of vibrio parahemolyticus bacteriophage, vibrio alginolyticus bacteriophage, vibrio harveyi bacteriophage, vibrio cholerae bacteriophage, escherichia coli bacteriophage, salmonella bacteriophage, xanthomonas sobria bacteriophage, ralstonia bacteriophage of solanaceae, staphylococcus aureus bacteriophage, aeromonas hydrophila bacteriophage, streptococcus agalactiae bacteriophage, streptococcus iniae bacteriophage, pseudomonas aeruginosa bacteriophage, klebsiella pneumoniae bacteriophage, acinetobacter baumannii bacteriophage, propionibacterium acnes bacteriophage, agrobacterium tumefaciens bacteriophage, corynebacterium michiganensis, listeria bacteriophage, campylobacter jejuni bacteriophage or clostridium perfringens bacteriophage.

Further, the probiotic bacteria are Streptococcus thermophilus, Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, Bacillus lentus, Bacillus thuringiensis, Bacillus natto, Bacillus laterosporus, Bacillus mucilaginosus, Bacillus cereus, Bacillus firmus, Bacillus belgii, Bacillus coagulans, Bacillus polymyxa, Bacillus mycoides, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus buchneri, Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus sake, and Lactobacillus salivarius, Lactobacillus lactis subsp Lactococcus lactis diacetyl subspecies, enterococcus faecalis, enterococcus faecium, enterococcus lactis, pediococcus acidilactici, pediococcus pentosaceus, leuconostoc mesenteroides, leuconostoc citreum, propionibacterium propionicum, propionibacterium freudenreichii subspecies schleri, staphylococcus parvum, staphylococcus carnosus, saccharomyces cerevisiae, pichia pastoris, candida utilis, kluyveromyces marxianus, rhodotorula marinus, saccharomyces cerevisiae, candida guilliermondii or saccharomyces boulardii.

Further, the phage is prepared by the following steps in sequence: seed culture, enrichment culture, sterilization purification and/or drying.

Further, the probiotics are prepared by the following steps in sequence: seed culture, fermentation culture, thallus collection and/or drying.

In conclusion, the invention has the following beneficial effects: the novel composite preparation is obtained by the compatibility of the bacteriophage and the probiotics, so that the advantages between the bacteriophage and the probiotics can be enhanced, the overall bacteriostatic or bactericidal effect is improved, the growth environment is improved, and the composite preparation has a good application prospect when being applied to bacterial disease prevention and control.

Detailed Description

The following examples serve to further illustrate the invention, but do not limit the effective scope of the invention in any way.

In the following examples, the reference numbers of the strains are the same as the name numbers of the company, and some strains are obtained from the market.

The preservation unit of Vibrio parahaemolyticus phage VP46(Vibrio parahaemolyticus phase VP46) is China center for type culture Collection, and the address is Wuhan university, Wuhan Lophania mountain Lound, Wuhan City, Hubei province, zip code 430072; the preservation date is 2016, 5 and 26 months; the preservation number is CCTCC NO: m2016290.

The preservation unit of Vibrio parahaemolyticus phage VP48(Vibrio parahaemolyticus phaseVP 48) is China center for type culture Collection, and the address is Wuhan university, Wuhan Lophania mountain Lound, Wuhan City, Hubei province, postfix 430072; the preservation date is 2016, 5 and 26 months; the preservation number is CCTCC NO: m2016291.

The preservation unit of Vibrio parahaemolyticus phage VP7(Vibrio parahaemolyticus phaseVP 7) is China center for type culture Collection, and the address is Wuhan university, Wuhan Lophania mountain Lound, Wuhan City, Hubei province, postfix 430072; the preservation date is 2016, 5 and 26 months; the preservation number is CCTCC NO: m2016289.

The preservation unit of Staphylococcus aureus bacteriophage BP-13A (Staphylococcus aureus phaseBP-13A) is China center for type culture Collection, and the address is Wuhan university, postfix 430072, at Wuchan Lophania mountain Wuhan university, Wuhan city, Hubei province; the preservation date is 2016 year, 9 month and 28 days, and the preservation number is CCTCC NO: m2016535.

The preservation unit of the Ralstonia solanacearum phage GP3(Ralstonia solanacearum phase GP3) is the China center for type culture Collection, and the address is Wuhan university, Logania Hayama mountain, Wuhan, Hubei province, postcode 430072; the preservation date is 2016, 11 and 10 months; the preservation number is CCTCC NO: m2016635.

In the following examples, test strains are commercially available, the test strains are in an open state, and scientists can request from relevant entities.

The preservation number of the lactobacillus plantarum strain is CCTCC AB 2013128; the preservation number of the bacillus subtilis strain is CCTCC AB 13003; the preservation number of the enterococcus faecium is CCTCC AB 2010212; the number of the candida utilis is CCTCC AY 91011.

In the following examples, the following examples are given,

the formula of the TSB liquid culture medium is as follows: tryptone 15g, soybean peptone 5g, sodium chloride 5g, and distilled water 1000 mL.

The MRS liquid culture medium comprises the following components in percentage by weight: 20g of glucose, 10g of peptone, 5g of beef extract powder, 4g of yeast extract powder, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1g of tween and 1000mL of distilled water.

The LB liquid culture medium has the formula: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride and 1000mL of distilled water.

The formula of YEPD liquid culture medium is as follows: 10g of yeast extract, 20g of peptone, 20g of glucose and 1000mL of distilled water.