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CN111789132A - Novel composite preparation and application thereof in bacterial diseases - Google Patents

Novel composite preparation and application thereof in bacterial diseases Download PDF

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Publication number
CN111789132A
CN111789132A CN202010633920.2A CN202010633920A CN111789132A CN 111789132 A CN111789132 A CN 111789132A CN 202010633920 A CN202010633920 A CN 202010633920A CN 111789132 A CN111789132 A CN 111789132A
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China
Prior art keywords
phage
lactobacillus
bacillus
infection
bacterial diseases
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Pending
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CN202010633920.2A
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Chinese (zh)
Inventor
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
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Phagelux Nanjing Biotechnology Co ltd
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Phagelux Nanjing Biotechnology Co ltd
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Priority to CN202010633920.2A priority Critical patent/CN111789132A/en
Publication of CN111789132A publication Critical patent/CN111789132A/en
Priority to CN202110753905.6A priority patent/CN113383798A/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
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    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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    • A—HUMAN NECESSITIES
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    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
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Abstract

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.

Description

Novel composite preparation and application thereof in bacterial diseases
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.
Example 1:
preparation of Vibrio parahaemolyticus phage
Seed culture: inoculating the activated vibrio parahaemolyticus host bacteria into a TSB culture medium, inoculating the vibrio parahaemolyticus host bacteria at the inoculation ratio of 2%, culturing at 220rpm and 37 ℃ for 4h, and obtaining vibrio parahaemolyticus host bacteria seed liquid.
Enrichment culture: inoculating the vibrio parahaemolyticus phage and host bacteria into TSB culture medium, and allowing infection complex MOI value to be 5 × 10-6The inoculation ratio of the host bacteria is 5 percent, and the host bacteria are cultured at 190rpm and 37 ℃ for 5 hours.
And (3) degerming and purifying: after fermentation, centrifuging the fermentation liquid at 7000rpm for 10min, collecting supernatant, sterilizing and purifying the supernatant with 0.22 μm filter membrane to obtain vibrio parahaemolyticus bacteriophage aqueous solution with titer of 5.7 × 10 for VP46, VP48 and VP710PFU/mL、4.8×1010PFU/mL and 4.5X 1010PFU/mL。
And (3) drying: adding a protective agent according to the component ratio of 3:1, wherein the protective agent comprises the following components in percentage by weight: 15 parts of skimmed milk powder, 3 parts of glycerol, 4 parts of sodium glutamate and 5 parts of sucrose, mixing uniformly, drying, crushing and sieving to obtain phage powder, wherein the titer of each of VP46, VP48 and VP7 is 1.1 × 1012PFU/g、9.5×1011PFU/g and 7.7X 1011PFU/g。
Example 2:
preparation of Staphylococcus aureus phage
Seed culture: inoculating the activated staphylococcus aureus host bacteria into a TSB culture medium, and culturing for 6h at the temperature of 36 ℃ at the speed of 190rpm according to the inoculation proportion of 1% to obtain staphylococcus aureus host bacteria seed liquid.
Enrichment culture: inoculating staphylococcus aureus bacteriophage and host bacteria into TSB cultureIn the nutrient medium, the MOI value of the multiplicity of infection is 3X 10-5The inoculation ratio of the host bacteria is 10 percent, and the host bacteria are cultured for 6 hours at 190rpm and 36 ℃.
And (3) degerming and purifying: after fermentation, centrifuging the fermentation liquid at 8000rpm for 8min, collecting supernatant, sterilizing and purifying the supernatant with 0.22 μm filter membrane to obtain Staphylococcus aureus bacteriophage aqueous solution with BP-13A titer of 5.1 × 1010PFU/mL。
And (3) drying: adding a protective agent according to the component ratio of 3:1, wherein the protective agent comprises the following components in percentage by weight: 15 parts of skimmed milk powder, 3 parts of glycerol, 4 parts of sodium glutamate and 5 parts of sucrose, mixing uniformly, drying, crushing and sieving to obtain phage powder with BP-13A titer of 9.3 multiplied by 1011PFU/g。
Example 3:
preparation of phage against Ralstonia solanacearum
Seed culture: inoculating the activated ralstonia solanacearum host bacteria into a TSB culture medium, and culturing at 30 ℃ for 16h at the inoculation ratio of 5% and 150rpm to obtain the ralstonia solanacearum host bacteria seed liquid.
Enrichment culture: inoculating Ralstonia solanacearum bacteriophage and host bacteria into TSB culture medium, wherein MOI value of infection complex number is 1 × 10-2The inoculation ratio of the host bacteria is 8 percent, the host bacteria are cultured at 160rpm and 30 ℃ for 12 hours.
And (3) degerming and purifying: after fermentation, centrifuging the fermentation liquid at 7600rpm for 13min, collecting supernatant, sterilizing and purifying the supernatant with 0.22 μm filter membrane to obtain Ralstonia solanacearum bacteriophage aqua with GP3 titer of 2.6 × 1010PFU/mL。
And (3) drying: adding a protective agent according to the component ratio of 3:1, wherein the protective agent comprises the following components in percentage by weight: 15 parts of skimmed milk powder, 3 parts of glycerol, 4 parts of sodium glutamate and 5 parts of cane sugar, mixing uniformly, drying, crushing and sieving to obtain phage powder, wherein the titer of GP3 is 1.9 multiplied by 1011PFU/g。
Example 4:
preparation of Lactobacillus plantarum
Seed culture: inoculating the activated lactobacillus plantarum into an MRS culture medium, inoculating the lactobacillus plantarum into the MRS culture medium according to the inoculation proportion of 10%, and performing static culture at 35 ℃ for 12 hours to obtain lactobacillus plantarum seed liquid.
Fermentation culture: inoculating the lactobacillus plantarum seed solution into an MRS culture medium, and culturing at 50rpm and 35 ℃ for 10h at the inoculation ratio of 5%.
And (3) collecting thalli: after fermentation is finished, the fermentation liquor is centrifuged for 10min at 10000rpm, and thalli and fermentation metabolite clear liquid are collected.
And (3) drying: the thallus is added with a protective agent according to the component ratio of 3:1, and the protective agent comprises the following components in percentage by weight: 10 parts of skimmed milk powder, 5 parts of glycerol, 2 parts of sodium glutamate and 3 parts of sucrose, mixing uniformly, drying, crushing and sieving to obtain the lactobacillus plantarum powder with viable count of 1.8 multiplied by 1011CFU/g。
Example 5:
preparation of Bacillus subtilis
Seed culture: inoculating the activated bacillus subtilis into an LB culture medium, and culturing at the temperature of 37 ℃ for 5h at 200rpm according to the inoculation proportion of 10% to obtain a bacillus subtilis seed solution.
Fermentation culture: inoculating the bacillus subtilis seed solution into an LB culture medium, wherein the inoculation proportion is 8%, and the bacillus subtilis seed solution is cultured at 230rpm and 37 ℃ for 24 hours.
And (3) collecting thalli: after fermentation is finished, the fermentation liquor is centrifuged for 10min at 10000rpm, and thalli and fermentation metabolite clear liquid are collected.
And (3) drying: the thallus is added with a protective agent according to the component ratio of 3:1, and the protective agent comprises the following components in percentage by weight: 10 parts of skimmed milk powder, 5 parts of glycerol, 2 parts of sodium glutamate and 3 parts of sucrose, mixing uniformly, drying, crushing and sieving to obtain bacillus subtilis powder with viable count of 5.6 multiplied by 1010CFU/g。
Example 6:
preparation of enterococcus faecium
Seed culture: inoculating the activated enterococcus faecium into a TSB culture medium, and culturing at 37 ℃ for 6h at 200rpm according to the inoculation proportion of 2% to obtain enterococcus faecium seed liquid.
Fermentation culture: inoculating enterococcus faecium seed liquid into TSB culture medium, inoculating at a ratio of 6%, culturing at 200rpm and 37 deg.C for 9 hr.
And (3) collecting thalli: after fermentation is finished, the fermentation liquor is centrifuged for 10min at 10000rpm, and thalli and fermentation metabolite clear liquid are collected.
And (3) drying: the protective agent is added into the thallus according to the component ratio of 3:1The proportion of the protective agent is as follows: 10 parts of skimmed milk powder, 5 parts of glycerol, 2 parts of sodium glutamate and 3 parts of sucrose, mixing uniformly, drying, crushing and sieving to obtain enterococcus faecium powder with viable count of 8.4 multiplied by 1010CFU/g。
Example 7:
preparation of candida utilis
Seed culture: inoculating the activated candida utilis into a YEPD culture medium, and culturing at 31 ℃ for 16h at 180rpm according to the inoculation proportion of 15% to obtain candida utilis seed liquid.
Fermentation culture: inoculating Candida utilis seed liquid into YEPD culture medium, inoculating at 10%, 200rpm, and culturing at 31 deg.C for 24 hr.
And (3) collecting thalli: after fermentation is finished, the fermentation liquor is centrifuged for 15min at 8500rpm, and thalli and fermentation metabolite clear liquid are collected.
And (3) drying: the thallus is added with a protective agent according to the component ratio of 3:1, and the protective agent comprises the following components in percentage by weight: 10 parts of skimmed milk powder, 5 parts of glycerol, 2 parts of sodium glutamate and 3 parts of sucrose, mixing uniformly, drying, crushing and sieving to obtain candida utilis powder with viable count of 6.8 multiplied by 1010CFU/g。
Example 8:
the vibrio parahaemolyticus phage for the toxicity attack experiment of the vibrio parahaemolyticus phage and lactobacillus plantarum compound preparation on the growth influence of the penaeus vannamei boone is prepared and obtained in the embodiment 1; the Vibrio parahaemolyticus phage was prepared in example 4.
The test process comprises the following steps: adding 1x10 final concentration into culture pond of Penaeus vannamei Boone5The challenge test of the CFU/mL vibrio parahaemolyticus liquid on the penaeus vannamei boone is carried out, the test is divided into a compound preparation group, a phage group, a probiotic group, a common group and a negative control group, the observation is continuously carried out for 2 weeks, the death and culling rate of the penaeus vannamei boone is observed and counted every day, and the results are detailed in table 1. The compound preparation is prepared by counteracting toxic substance and adding 1x10 final concentration6The compound preparation is sprinkled after being uniformly mixed with water, the component ratio of phage and probiotic in the compound preparation is 4:1, the vibrio parahaemolyticus phage is powder, the lactobacillus plantarum is powder, and the component ratio of the vibrio parahaemolyticus phage VP46, VP48 and VP7 is 1:1: 1; the phage group is used for counteracting and addingAdding to final concentration of 1x106PFU/mL vibrio parahaemolyticus phage is added with water, mixed uniformly and sprinkled, the vibrio parahaemolyticus phage is powder, and the component ratio of VP46, VP48 and VP7 is 1:1: 1; the probiotic group is used for counteracting toxic substance and adding final concentration of 1x106Adding water into the CFU/mL probiotics, uniformly mixing, and splashing, wherein the lactobacillus plantarum is powder; the common group is counteracting toxic pathogen without adding preparation; the negative control group was not treated for challenge. The mortality and culling rate (the number of dead culling of penaeus vannamei/total number of penaeus vannamei) × 100%.
TABLE 1 Effect of Complex preparations on the mortality and culling Rate of Penaeus vannamei Boone
Figure BDA0002569866330000061
Figure BDA0002569866330000071
As shown in table 1, the ratio of the death and culling rate of the composite preparation group to that of the normal group of penaeus vannamei boone is lower, which indicates that the composite preparation has an obvious bacteriostatic effect on vibrio, and the composite preparation group has a better result in terms of controlling the death and culling rate than the phage group and the probiotic group, and further indicates that the novel composite preparation obtained by matching the phage and the probiotic can enhance the synergistic effect of the phage and the probiotic in sterilization or bacteriostasis, is beneficial to improving the water quality environment of the penaeus vannamei boone, has no other toxic or side effect on the penaeus vannamei boone, and has higher safety.
Example 9:
application of a composite preparation of a ralstonia solanacearum phage, bacillus subtilis and candida utilis to ginger blast, the ralstonia solanacearum phage is prepared in example 3, and the bacillus subtilis thallus is prepared in example 5; candida utilis was prepared in example 7.
The test process comprises the following steps: the final concentration of the infusion solution in the root of ginger is 1x105The CFU/mL of the ralstonia solanacearum bacterial liquid is used for carrying out an offensive toxicity test on the ginger, the test is divided into a negative control group, a compound preparation group and a common group, the negative control group, the compound preparation group and the common group are continuously observed for 2 weeks, the necrosis rate of the ginger is observed and counted every day, and the results are detailed in a table 2. The compound preparation is composed ofToxin and add final concentration of 1 × 106The compound preparation is added with water and is uniformly mixed, and then root irrigation is carried out, wherein the component ratio of phage to probiotics in the compound preparation is 1:1, the phage of Ralstonia solanacearum is an aqueous solution, bacillus subtilis is powder, Candida utilis is a metabolite aqueous solution, and the component ratio of the bacillus subtilis to Candida utilis is 3: 1; the phage group was challenged with the addition of 1 × 10 final concentration6PFU/mL solanaceae ralstonia phage, watering and mixing uniformly, and irrigating root, wherein the solanaceae ralstonia phage is an aqueous solution; the probiotic group is used for counteracting toxic substance and adding final concentration of 1x106Adding water into CFU/mL probiotics, uniformly mixing, and then irrigating roots, wherein the bacillus subtilis is powder, and the candida utilis is metabolite water; the general group was challenged without complex formulation; the negative control group was not treated for challenge. The ratio of the death rate is (number of dead ginger/total number of ginger) × 100%.
TABLE 2 Effect of the Complex formulation on ginger necrosis Rate
Figure BDA0002569866330000072
Figure BDA0002569866330000081
As shown in table 2, the compound preparation group has a lower ginger necrosis rate than the common group, which indicates that the compound preparation has significant bacteriostatic action on ralstonia solanacearum, and the compound preparation group has a better result in controlling the necrosis rate than the phage group and the probiotic group, further indicates that the novel compound preparation obtained by matching the phage and the probiotic can enhance the synergistic action of the phage and the probiotic in sterilization or bacteriostasis, is beneficial to improving the soil environment at the root of ginger, has no other toxic action on ginger, and has higher safety.
Example 10:
application of staphylococcus aureus bacteriophage, enterococcus faecium, bacillus subtilis and lactobacillus plantarum composite preparation to piglet oily skin disease
Staphylococcus aureus phage was prepared from example 2; enterococcus faecium was prepared from example 6; bacillus subtilis was prepared from example 5; lactobacillus plantarum was prepared as in example 4.
The test process comprises the following steps: grinding the skin of piglets with sand paper to slight wound, and applying 1 × 10 to the wound5The challenge test of the CFU/mL staphylococcus aureus on piglets is carried out, the test is divided into a negative control group, a compound preparation group and a common group, the negative control group, the compound preparation group and the common group are continuously observed for 2 weeks, the incidence rate of the piglet epidermis is observed and counted every day, and the result is detailed in a table 3. The compound preparation is prepared by counteracting toxic substance and adding 1x10 final concentration6The composite preparation is applied after being uniformly mixed with water, the component ratio of phage to probiotics in the composite preparation is 1:3, staphylococcus aureus phage is powder, enterococcus faecium is a fermentation metabolism water aqua, bacillus subtilis is powder, lactobacillus plantarum is a fermentation metabolism water aqua, and the component ratio of enterococcus faecium, bacillus subtilis and lactobacillus plantarum is 1:5: 3; the phage group was challenged with the addition of 1 × 10 final concentration6Adding water into PFU/mL staphylococcus aureus bacteriophage, uniformly mixing, and coating, wherein the staphylococcus aureus bacteriophage is powder; the probiotic group is used for counteracting toxic substance and adding final concentration of 1x106CFU/mL probiotics are added with water, evenly mixed and smeared, enterococcus faecium is a fermentation metabolism water aqua, bacillus subtilis is powder, and lactobacillus plantarum is a fermentation metabolism water aqua; the general group was challenged without complex formulation; the negative control group was not treated for challenge. The incidence rate is (number of piglets suffering from oil skin disease/total number of piglets) × 100%.
TABLE 3 Effect of the Complex formulation on the incidence of oil skin disease in piglets
Time of day Compound preparation set Phage group Probiotic group General group Negative control group
1DAY 0％ 0％ 0％ 0％ 0％
2DAY 0％ 0％ 1％ 1％ 0％
3DAY 1％ 1％ 1％ 2％ 0％
4DAY 1％ 2％ 2％ 3％ 0％
5DAY 2％ 2％ 4％ 5％ 0％
6DAY 3％ 3％ 5％ 6％ 0％
7DAY 5％ 5％ 6％ 7％ 1％
8DAY 6％ 7％ 8％ 8％ 1％
9DAY 7％ 8％ 9％ 10％ 2％
10DAY 8％ 9％ 11％ 12％ 2％
11DAY 8％ 9％ 13％ 15％ 3％
12DAY 9％ 10％ 15％ 17％ 4％
13DAY 10％ 10％ 18％ 20％ 5％
14DAY 10％ 11％ 22％ 25％ 5％
As shown in table 3, the compound preparation group has a lower piglet morbidity compared with the ordinary group, which indicates that the compound preparation has an obvious bacteriostatic action on staphylococcus aureus, and the compound preparation group has a better result in controlling the piglet morbidity compared with the phage group and the probiotic group, and further indicates that the novel compound preparation obtained by matching the phage and the probiotic can enhance the synergistic action of the phage and the probiotic in sterilization or bacteriostasis, is beneficial to improving the skin repair of piglet wounds, has no other toxic action on piglets, and has higher safety.
By integrating the embodiments 1-10, the prepared novel composite preparation can fully embody the respective advantages of the bacteriophage and the probiotics, can play a remarkable control effect on bacterial diseases, is mild in process, free of stress reaction, green, environment-friendly and high in safety, and has a wide prospect in the application field of bacterial diseases.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims (9)

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.
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Application publication date: 20201020