CN116463258B - Bacillus coagulans King27 for preventing diarrhea of pets, microbial inoculum, preparation method and application - Google Patents
Bacillus coagulans King27 for preventing diarrhea of pets, microbial inoculum, preparation method and application Download PDFInfo
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- CN116463258B CN116463258B CN202310387894.3A CN202310387894A CN116463258B CN 116463258 B CN116463258 B CN 116463258B CN 202310387894 A CN202310387894 A CN 202310387894A CN 116463258 B CN116463258 B CN 116463258B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/02—Local antiseptics
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- C12N1/20—Bacteria; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of microbial application, in particular to bacillus coagulans King27 for preventing diarrhea of pets, a microbial inoculum, a preparation method and application. Bacillus coagulans (Bacillus coagulans) King27 is preserved in China general microbiological culture Collection center (CGMCC No. 25556) at 8 months and 19 days of 2022, and has a preservation address of Beicheng Kogyan North Xielu No. 1 and 3. The strain can effectively inhibit common pathogenic bacteria causing diarrhea of pets. Meanwhile, experiments prove that the microbial inoculum prepared by bacillus coagulans King27 is added into the feeding feed for pets, so that seasonal diarrhea caused by cooling of the pets and diarrhea caused by lactose intolerance can be effectively prevented, the diarrhea rate of the pets is greatly reduced, and the microbial inoculum has important significance for daily maintenance of health of the pets and prevention of diarrhea of the pets.
Description
Technical Field
The invention relates to the technical field of microbial application, in particular to bacillus coagulans King27 for preventing diarrhea of pets, a microbial inoculum, a preparation method and application.
Background
Dogs and cats are the most common household pets at present, but the digestive system of the dogs and cats is very fragile and sensitive, and many tiny factors can cause disturbance of the environment in the intestinal tract, so that the pets can have symptoms of diarrhea, vomiting and the like. Common causes of diarrhea in pets in daily life include the following: 1. changing the milk powder of the pet after weaning, and causing diarrhea due to lactose intolerance; 2. the intestines and stomach are cooled caused by the change of the ambient temperature, and seasonal diarrhea appears; 3. the diet or environment causes bacteria to invade the gastrointestinal system and bacterial infection causes diarrhea.
At present, the diarrhea of the pets is mainly treated by oral antibiotic medicines, and the diarrhea caused by bacterial infection is mainly treated, but the antibiotic medicines are easy to cause dysbacteriosis of the intestinal tracts of the pets, and the antibiotic medicines can cause dysbacteriosis of the intestinal tracts of the pets in the intestinal tracts of the pets, so that the sick pets are difficult to completely recover. The use of antibacterial drugs has the same drawbacks of ecological safety, drug residue, drug resistance, antibiotic-associated diarrhea, etc., and in recent years, the world is in a feed "forbidden" mode, so it is urgent to seek a safe and effective antibiotic substitute.
Chinese patent CN114437993A discloses Lactobacillus reuteri and application thereof in preparation of preparations for preventing cat diarrhea, and has antibacterial effect on pathogenic bacteria causing cat diarrhea commonly found in salmonella, campylobacter jejuni, shigella and the like. Chinese patent CN113057253B relates to a composite probiotic pet food for regulating intestines and stomach, which is prepared by self-making probiotic additives and is matched with specific traditional Chinese medicine extracts and catechins to cooperatively act, so that beneficial microorganisms in the intestines of the pet can be proliferated, and harmful bacteria in the intestines can be inhibited, the microbial population structure and quantity in the intestines of the pet are improved, and the intestines and stomach of the pet are regulated effectively. The probiotic preparation or the compound probiotic preparation which is used for preventing and treating the intestinal diseases of the pets and is available or reported on the market mainly aims at the diarrhea of the pets caused by harmful bacteria infection, has single function, and has no report on the probiotic products with the prevention effect on cold diarrhea and lactose intolerance diarrhea in daily life of the pets.
Disclosure of Invention
Aiming at the problems that the existing probiotics preparation for preventing or treating pet diarrhea is single in function and cannot achieve the expected prevention and treatment effects, the invention provides bacillus coagulans King27 for preventing pet diarrhea, a metabolic preparation and application thereof, and the strain can not only strongly inhibit intestinal pathogens, but also prevent seasonal diarrhea of pets and lactose intolerant diarrhea caused by changing milk powder of pets after weaning.
In a first aspect, the present invention provides a bacillus coagulans King27 for preventing diarrhea in pets, wherein bacillus coagulans (Bacillus coagulans) King27 is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 25556 and a preservation address of North-West-Lu No. 1 and No. 3 in the Korean region of Beijing city at 8-19 days of 2022.
Bacillus coagulans is facultative anaerobe, can grow in both aerobic and anaerobic environments, can adapt to low-oxygen intestinal environments, has higher tolerance to acid and bile, can perform lactic acid fermentation, can reduce the pH value of intestinal tracts, inhibit harmful bacteria, and can promote the growth and reproduction of beneficial bacteria such as bifidobacteria. Bacillus coagulans can form spores, and is favorable for restoring microecological balance of gastrointestinal tracts compared with other bacillus which does not produce lactic acid. After bacillus coagulans spores are fermented, a large amount of antibacterial coagulants are produced, and the growth of aerobic pathogenic bacteria can be effectively inhibited.
In a second aspect, the invention provides a bacillus coagulans King27 microbial inoculum for preventing diarrhea in pets, comprising a bacterial concentrate of the bacillus coagulans King 27.
In a third aspect, the invention provides a preparation method of the bacillus coagulans King27 microbial inoculum, which specifically comprises the following steps:
(1) Activating the frozen and preserved bacillus coagulans King27 strain, inoculating the single activated colony into an MRS liquid culture medium, and culturing for 18-24 hours at 40-45 ℃ and 200r/min to obtain seed liquid;
(2) Inoculating the seed liquid into a primary fermentation culture medium, aerating, and performing shake culture at the temperature of 40-45 ℃ and the speed of 200r/min for 22-26 hours to obtain a primary fermentation liquid; transferring the first-stage fermentation liquid into a second-stage fermentation medium, aerating, and performing shake cultivation for 45-50 h at the temperature of 40-45 ℃ and the speed of 200r/min to obtain a second-stage fermentation liquid;
(3) Centrifuging the secondary fermentation liquor, collecting thalli, washing the thalli by using sterile physiological saline, and re-suspending the thalli in 15% (w/w) reconstituted skim milk to obtain suspension; freeze-drying the bacterial suspension to obtain bacterial powder;
(4) Mixing the fungus powder with glucose to obtain the fungus agent.
Further, the specific operation of the activation in step (1) is: the frozen and preserved Bacillus coagulans King27 strain was streaked on MRS plate medium, and cultured at 42℃for 48 hours under aerobic conditions.
Further, in the step (2), the seed liquid is inoculated into the primary fermentation culture medium with an inoculation amount of 4-6%.
Further, in the step (2), the preparation methods of the primary fermentation medium and the secondary fermentation medium are as follows:
40g/L of glucose, 10g/L of yeast extract, 15g/L of peptone, 3g/L of dipotassium hydrogen phosphate, 0.05g/L of manganese sulfate, 4g/L of ammonium chloride and 18g/L of calcium carbonate, and sterilizing the culture medium for 20min at 121 ℃, and cooling the culture medium to 40-45 ℃ for standby.
In a fourth aspect, the invention provides an application of the bacillus coagulans King27 in preparing a product for preventing diarrhea caused by intestinal pathogenic bacteria of pets.
In a fifth aspect, the invention provides the use of bacillus coagulans King27 as described above for the preparation of a product for preventing seasonal diarrhea caused by cooling of a pet.
In a sixth aspect, the invention provides the use of bacillus coagulans King27 as described above for the preparation of a product for preventing diarrhea caused by lactose intolerance in pets.
The invention has the beneficial effects that:
the bacillus coagulans King27 provided by the invention can effectively inhibit common pathogenic bacteria causing diarrhea of pets. Meanwhile, experiments prove that the microbial inoculum prepared by bacillus coagulans King27 is added into the feeding feed for pets, so that seasonal diarrhea caused by cooling of the pets and diarrhea caused by lactose intolerance can be effectively prevented, the diarrhea rate of the pets is greatly reduced, and the microbial inoculum has important significance for daily maintenance of health of the pets and prevention of diarrhea of the pets.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The various media components used in the following examples were composed and prepared as follows:
MRS plate medium: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 801mL of tween-801 mL, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water; mixing the above materials, sterilizing at 121deg.C for 20min at natural pH, pouring sterilized culture medium into a plate, and cooling.
MRS liquid medium: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water, and after the raw materials are mixed, sterilizing for 20min at 121 ℃, and cooling for standby.
LB liquid medium: weighing 25.0g of LB culture medium dry powder, dissolving in 1000mL of distilled water, sterilizing at 121 ℃ for 15min for later use; LB media dry powder was purchased from Qingdao sea Bo biotechnology Co.
Nutrient agar plate medium: 10g of peptone, 3g of beef powder, 5g of sodium chloride, 15g of agar and 1000mL of distilled water, mixing the above raw materials, sterilizing for 15min at 121 ℃, pouring the sterilized culture medium into a plate, and cooling for later use.
Nutrient broth medium: 5g of peptone, 3g of beef extract and 1000mL of distilled water, and after the raw materials are mixed, sterilizing for 15min at 121 ℃, and cooling for standby.
Primary fermentation medium and secondary fermentation medium: 40g/L of glucose, 10g/L of yeast extract, 15g/L of peptone, 3g/L of dipotassium hydrogen phosphate, 0.05g/L of manganese sulfate, 4g/L of ammonium chloride and 18g/L of calcium carbonate, sterilizing the culture medium for 20min at 121 ℃, and cooling the culture medium to 40-45 ℃ for standby.
The campylobacter jejuni ATCC 33291, escherichia coli cic 10899, clostridium difficile ATCC 43593 used in the following examples were purchased from the chinese industrial microorganism collection.
Example 1 isolation and identification of species
1. Strain sources: pickled Chinese cabbage, 2022, is collected from Chang-tui county, feilong, ling City, liaoning, and 2 months.
2. Strain screening and purification
(1) Sampling: cutting pickled Chinese cabbage into small pieces for standby;
(2) Preparing a sample:
(1) placing sterilized normal saline (0.85%) into a sterile triangular flask, adding pickled Chinese cabbage therein, and oscillating for use;
(2) diluting the solution obtained in the step (1) to obtain samples with different concentration gradients, respectively 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 The reference numerals are 1#, 2#, 3#, 4#, 5#, 6#, 7#, respectively, for standby;
(3) Culturing: coating the solutions No. 1, no.2, no. 3, no. 4, no. 5, no. 6 and No. 7 in the step (2) in an MRS flat plate culture medium by using a coater, and culturing for 48 hours at 42 ℃ under the aerobic condition;
(4) Colonies were selected according to the following colony characteristics:
the colony diameter is 1-2 mm, and the colony is opaque, complete in edge, milky white, glossy and soft in texture on the culture medium.
(5) Separation and purification
Selecting 5 single colonies according to the colony characteristics of the step (4), inoculating to the culture medium of the step (3) by a streaking method, culturing for 48 hours at 42 ℃ under aerobic conditions, selecting single colonies, and placing in a glycerol tube for preservation at-70 ℃.
2. Authentication
And (3) sending the single colony separated and purified in the step (5) to an identification unit: the China center for industrial microorganism strain preservation management, the primer sequence used in the identification process is as follows:
27F：AGAGTTTGATCMTGGCTCAG
1492R：GGTTACCTTGTTACGACTT
the obtained gene sequence is:
GTGCGCACCTTTTAAAAGCTTGCTTTTAAAAGGTTAGCGGCGCACGCGTGAGTAACACGTGGGCAACCTGCCTGTAAGATCGGGATAACGCCGGGAAACCGGGGCTAATACCGGATAGTTTTTTCCTCCGCATGGAGGAAAAAGGAAAGACGGCTTTTGCTGTCACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGCCTTCGGGTCGTAAAACTCTGTTGCCGGGGAAGAACAAGTGCCGTTCGAACAGGGCGGCGCCTTGACGGTACCCGGCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTCTTAAGTCTGATGTGAAATCTTGCGGCTCAACCGCAAGCGGTCATTGGAAACTGGGAGGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCTCCCTGGAGACAGGGCCTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCCAATCCCAGAAAACCATTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCT
identification result: the strain was identified as Bacillus and presumably as Bacillus coagulans (Bacillus coagulans).
The identified strain is named as bacillus coagulans King27, and is sent to China general microbiological culture collection center (CGMCC) for preservation, and the preservation information is as follows;
strain name: bacillus coagulans King27; classification naming: bacillus coagulans Bacillus coagulans; preservation date: 2022, 8, 19; address: beijing, chaoyang area, north Chenxi way No. 1, no. 3, post code: 100101; preservation number: CGMCC No.25556.
Example 2 bacteriostasis Capacity experiment of Bacillus coagulans King27 fermentation broth
1. The bacillus coagulans King27 broth was tested for its bacteriostatic ability against 3 common pathogenic bacteria causing diarrhea (campylobacter jejuni ATCC 33291, escherichia coli CICC 10899, clostridium difficile ATCC 43593).
The experimental steps are as follows:
(1) Taking out the bacillus coagulans King27 strain from the ultralow temperature refrigerator at the temperature of minus 80 ℃, and carrying out streak culture on an MRS flat plate culture medium, wherein the culture is carried out at the temperature of 42 ℃ for 48 hours in an aerobic way; single colonies were picked from the dishes from which the colonies were grown and inoculated in MRS liquid medium by aseptic manipulation, and cultured at 42℃for 18 hours at 200 r/min.
(2) Inoculating the activated bacillus coagulans King27 into MRS liquid culture medium, and performing shake culture at 42 ℃ and 200r/min for 24 hours to obtain bacillus coagulans King27 fermentation broth.
(3) The campylobacter jejuni ATCC 33291, the escherichia coli CICC 10899 and the clostridium difficile ATCC 4359 are respectively inoculated into a nutrient broth culture medium according to the inoculation amount of 2 percent, the culture medium is subjected to stationary culture at the constant temperature of 37 ℃ for 24 hours, and then the campylobacter jejuni ATCC 33291, the escherichia coli CICC 10899 and the clostridium difficile ATCC 43593 are inoculated into an LB liquid culture medium and subjected to shake culture at the temperature of 42 ℃ for 8 hours to obtain an indicator bacterium liquid.
(4) The antibacterial effect was evaluated by the oxford cup method, and the solutions of the indicator bacteria of Campylobacter jejuni ATCC 33291, the indicator bacteria of Escherichia coli CICC 10899 and the indicator bacteria of Clostridium difficile ATCC 43593 were respectively diluted to about 1X 10 with sterile physiological saline 8 For CFU/mL use, 0.1mL of the bacterial suspension solution is sucked on a nutrient agar plate culture medium, and the culture medium is uniformly coated by a sterile coating rod. After the indicator bacteria liquid on the surface of the flat plate is uniformly coated and absorbed, placing a sterile oxford cup on the surface of a flat plate culture medium, adding 0.2mL of bacillus coagulans King27 fermentation liquor into the cup, performing diffusion culture for 4 hours in a refrigerator at the temperature of 4 ℃, performing aerobic culture at the constant temperature of 37 ℃ for 24 hours, and measuring the diameter of a bacteriostasis ring. The average diameter of the inhibition zone was used to evaluate the size of the inhibition activity, wherein the oxford cup had an inner diameter of about 6mm, and the results are shown in table 1, with 3 replicates per group.
TABLE 1 diameter of inhibition zone (mm) of Bacillus coagulans King27 fermentation broth against 3 indicator bacteria
Conclusion: the bacillus coagulans King27 fermentation liquid has strong inhibition capability on campylobacter jejuni ATCC 33291, escherichia coli CICC 10899 and clostridium difficile ATCC 43593, wherein the inhibition effect on campylobacter jejuni ATCC 33291 and escherichia coli CICC 10899 is the strongest.
2. The antibacterial performance of the bacillus coagulans King27 fermentation broth is further studied, influences of some acidic metabolites (lactic acid, acetic acid and the like), hydrogen peroxide and the like on the antibacterial effect are eliminated, and the primary judgment of the metabolites of the bacteria is carried out.
(1) Influence of different pretreatment pH values on bacteriostasis capacity of bacillus coagulans fermentation liquor
The pH values of the bacillus coagulans King27 fermentation broth are respectively 2, 6 and 10 by using 0.5mol/L hydrochloric acid and 0.5mol/L sodium hydroxide, the pH value is adjusted back to 6 after the treatment for 1h at 37 ℃ to avoid the influence of different pH values on indicator bacteria, oxford cup bacteriostasis tests are carried out, the indicator bacteria are adopted for tests, the diameter of a bacteriostasis zone is observed, the influence on the bacteriostasis capacity of the bacillus coagulans King27 fermentation broth after the pretreatment of different pH values is examined, and the results are shown in Table 2.
TABLE 2 diameter of zone of inhibition (mm) for King27 fermentation broths at different pretreatment pH values
Conclusion: under the pretreatment of different pH values, fermentation liquor of bacillus coagulans King27 has different effects on the size of inhibition zones of 3 different indicator bacteria. When the pH value of the pretreatment is 6, the diameter of the bacteriostasis circle of the bacillus coagulans King27 fermentation liquor on 3 indicator bacteria is maximum, and the too high or too low pH value can destroy the structure of bacteriostasis substances in the fermentation liquor and influence the bacteriostasis effect of the bacteria.
(2) Influence of different temperatures on bacteriostasis ability of Bacillus coagulans King27 fermentation liquor
After the bacillus coagulans King27 fermentation broth is treated for 30min under the conditions of different temperature gradients (-20 ℃,4 ℃,37 ℃, 80 ℃ and 121 ℃) respectively, an oxford cup bacteriostasis test is carried out to determine the bacteriostasis capability, the influence of different temperature conditions on the bacteriostasis capability of the bacillus coagulans King27 fermentation broth is examined by observing the diameter of a bacteriostasis zone, and the results are shown in Table 3.
TABLE 3 diameter of zone of inhibition (mm) for King27 fermentation broths at different temperatures
Conclusion: the Bacillus coagulans King27 fermentation broth treated at 37℃had the highest bacteriostatic activity, indicating that this temperature is the optimal temperature for the Bacillus coagulans King27 to antagonize other strains.
(3) Influence of different enzymes on bacteriostasis ability of Bacillus coagulans King27 fermentation liquor
Adjusting the pH value to the optimal pH value of pepsin, trypsin, proteinase K and catalase by using 0.5mol/L hydrochloric acid and 0.5mol/L sodium hydroxide, respectively reacting the bacillus coagulans King27 fermentation liquor with various enzymes for 30min under the water bath condition of 37 ℃, adjusting the pH value to 6, taking the bacillus coagulans King27 fermentation liquor which is not subjected to enzyme treatment as a control group, carrying out oxford cup bacteriostasis test, and evaluating whether bacteriostasis substances in the bacillus coagulans King27 fermentation liquor are protein substances or not by observing the diameters of bacteriostasis circle of the control group and the test group, wherein the results are shown in Table 4.
TABLE 4 diameter of inhibition zone (mm) of King27 fermentation broths after treatment with different enzymes
| Campylobacter jejuni (Fr.) karst | Escherichia coli | Clostridium difficile | |
| Control group | 20 | 18 | 16 |
| Pepsin | 6 | 7 | 6 |
| Trypsin, trypsin and its preparation method | 5 | 6 | 8 |
| Proteinase K | 7 | 3 | 5 |
| Catalase enzyme | 14 | 17 | 15 |
After various proteases (pepsin, trypsin and proteinase K) are adopted to treat the bacillus coagulans King27 fermentation broth, the diameter of a bacteriostasis ring of a treatment group is obviously lower than that of a control group (P < 0.05); and after the catalase treatment is adopted, the antibacterial activity of the treated group is not significantly different from that of the control group (P is more than 0.05). The bacillus coagulans King27 fermentation liquor does not contain hydrogen peroxide or has extremely low content, and can play a role in bacteriostasis, and the bacillus coagulans King27 fermentation liquor is possibly a bacteriocin substance of extracellular proteins.
EXAMPLE 3 preparation of Bacillus coagulans King27 inoculant
(1) Taking out the bacillus coagulans King27 strain from the ultralow temperature refrigerator at the temperature of minus 80 ℃, and carrying out streak culture on an MRS flat plate culture medium, wherein the culture is carried out at the temperature of 42 ℃ for 48 hours in an aerobic way; single colonies were picked from the dishes from which the colonies were grown and inoculated in MRS liquid medium by aseptic manipulation, and cultured at 42℃for 18 hours at 200r/min to obtain seed solutions.
(2) Inoculating the seed liquid into a primary fermentation culture medium according to the inoculation amount of 5%, and performing shaking culture at 42 ℃ for 24 hours at 200r/min to obtain primary fermentation liquid; and (3) transferring the primary fermentation broth into a secondary fermentation medium, and performing shaking culture at 42 ℃ and 200r/min for 48 hours to obtain a secondary fermentation broth.
(3) Centrifuging the secondary fermentation liquor, collecting thalli, washing the thalli by using sterile physiological saline, and re-suspending the thalli in 15% (w/w) reconstituted skim milk to obtain suspension; freeze-drying the bacterial suspension to obtain bacterial powder;
(4) Mixing the fungus powder with glucose to prepare a fungus agent; glucose was purchased from the precious organism stock company, inc; in this example, the number of cells in the microbial inoculum prepared was 2X 10 7 CFU/g。
EXAMPLE 4 preparation of Bacillus coagulans King27 inoculant
(1) Taking out the bacillus coagulans King27 strain from the ultralow temperature refrigerator at the temperature of minus 80 ℃, and carrying out streak culture on an MRS flat plate culture medium, wherein the temperature is 40 ℃ and the aerobic culture is carried out for 48 hours; single colonies were picked from the dishes from which the colonies were grown and inoculated in MRS liquid medium by aseptic manipulation, and cultured at 40℃for 24 hours at 200r/min to obtain seed solutions.
(2) Inoculating the seed liquid into a primary fermentation culture medium according to the inoculation amount of 6%, and performing shaking culture at 40 ℃ for 26 hours at 200r/min to obtain primary fermentation liquid; transferring the first-stage fermentation liquid into a second-stage fermentation medium, aerating, and performing shake culture at 40 ℃ and 200r/min for 45h to obtain a second-stage fermentation liquid.
(3) Centrifuging the secondary fermentation liquor, collecting thalli, washing the thalli by using sterile physiological saline, and re-suspending the thalli in 15% (w/w) reconstituted skim milk to obtain suspension; freeze-drying the bacterial suspension to obtain bacterial powder;
(4) Mixing the fungus powder with glucose to prepare a fungus agent; glucose was purchased from the precious organism stock company, inc; in this example, the number of cells in the microbial inoculum prepared was 1X 10 7 CFU/g。
EXAMPLE 5 preparation of Bacillus coagulans King27 inoculant
(1) Taking out the bacillus coagulans King27 strain from the ultralow temperature refrigerator at the temperature of minus 80 ℃, and carrying out streak culture on an MRS flat plate culture medium, wherein the temperature is 45 ℃ and the aerobic culture is carried out for 48 hours; single colonies were picked from the dishes from which the colonies were grown and inoculated in MRS liquid medium by aseptic manipulation, and cultured at 45℃for 20 hours at 200r/min to obtain seed solutions.
(2) Inoculating the seed liquid into a primary fermentation culture medium according to the inoculation amount of 4%, and performing shaking culture at 45 ℃ for 22 hours at 200r/min to obtain primary fermentation liquid; and (3) transferring the primary fermentation broth into a secondary fermentation medium, and performing shaking culture at 45 ℃ for 50 hours by using a shaking table at 200r/min to obtain a secondary fermentation broth.
(3) Centrifuging the secondary fermentation liquor, collecting thalli, washing the thalli by using sterile physiological saline, and re-suspending the thalli in 15% (w/w) reconstituted skim milk to obtain suspension; freeze-drying the bacterial suspension to obtain bacterial powder;
(4) Mixing the fungus powder with glucose to prepare a fungus agent; glucose was purchased from the precious organism stock company, inc; in this example, the number of cells in the microbial inoculum prepared was 1.5X10 7 CFU/g。
Example 6 clinical trials of Bacillus coagulans King27 preparation for preventing diarrhea in summer
Summer is the high incidence of pet diarrhea, and seasonal variation, the air conditioning room and outdoor difference in temperature are big, easily cause the pet to catch a cold, cause the seasonal diarrhea of pet.
The method comprises the steps of selecting 40 healthy small dogs and 40 healthy small cats of different varieties, randomly dividing the small dogs and the small cats into 4 groups, respectively, namely a control group, a probiotic 1 group, a probiotic 2 group and a probiotic 3 group, wherein the control group is used for feeding feeds normally, the probiotic 1 group, the probiotic 2 group and the probiotic 3 group are respectively added with bacillus coagulans King27 bacterial agents prepared in the embodiment 3, the embodiment 4 and the embodiment 5, the addition amount is 10g/kg, the small dogs and the cats are continuously fed for 30 days at 20-35 ℃ in 6-7 months in northern areas, the test period is 60 days, and the diarrhea condition of each group of dogs and cats is recorded every day.
The diarrhea cases were represented clinically as follows: poor spirit, inappetence or abolishment of pets, pale mucous membrane, tension and pain of abdomen, increased stool frequency, rarefaction, water sample or rice soup sample, and sometimes mixed with egg flower, yellow green color; there is also less stool, but mixed oil mucus, purulent blood, stinking stool, obvious abdominal pain, tenesmus, and diarrhea accompanied with symptoms of fever, elevated body temperature, dehydration, etc.
The incidence of diarrhea in pets is calculated according to the records, and the calculation formula is as follows:
wherein, the number of diarrhea dogs and cats in the test period is the sum of the number of diarrhea dogs and cats in each day.
The results were as follows:
table 5 6-7 month incidence of diarrhea in pets
| For 0-30 days | 31-60 days | |
| Control group | 8.0％ | 9.7％ |
| Probiotic group 1 | 3.5％ | 4.2％ |
| Probiotic group 2 | 3.7％ | 3.8％ |
| Probiotic group 3 | 4.0％ | 3.7％ |
Conclusion: during the period of feeding the probiotics, the diarrhea rate of the probiotics group is obviously lower than that of a control group, the diarrhea incidence rate of pets is effectively reduced, and after the probiotics are stopped being added, the diarrhea rate of the probiotics group is increased, but is obviously lower than that of the control group, so that the diarrhea incidence rate of the pets can be effectively reduced and seasonal diarrhea of the pets can be prevented by adding the bacillus coagulans King27 microbial inoculum.
EXAMPLE 7 clinical trials of Bacillus coagulans King27 preparation for the prevention of diarrhea due to lactose intolerance
Lactose intolerance, also known as lactose dyspepsia or lactose malabsorption, refers to the absence of lactose degrading lactase in cats. Milk powder can be drunk by a cat after weaning, and the milk powder contains lactose, so that the lactose can be decomposed into lactic acid in the intestinal tract of the cat by bacteria to destroy the alkaline environment of the intestinal tract, so that a large amount of alkaline digestive juice is secreted by the intestinal tract to neutralize the lactic acid, and diarrhea finally occurs.
Generally, kittens can wean about a month, but even if breast milk is broken at this time, their intestines and stomach are very fragile and sensitive, and diarrhea caused by lactose intolerance easily occurs when milk powder is added vigorously. Lactose intolerance can cause abdominal distention, diarrhea and serious dehydration, and is generally manifested by soft stool or diarrhea after drinking milk powder.
The method comprises the steps of selecting 40 young cats which are just weaned, randomly dividing the young cats into 4 groups, namely a control group, a test 1 group, a test 2 group and a test 3 group, feeding milk powder normally to the control group, feeding milk powder of the test 1 group, the test 2 group and the test 3 group, adding bacillus coagulans King27 microbial inoculum prepared in the examples 3, 4 and 5 to the feeding milk powder of the test 1 group, the test 2 group and the test 3 group, continuously feeding the young cats for 28 days, wherein the adding amount is 5g/kg, the test period is 28 days, and recording diarrhea condition of each group of young cats every day.
The diarrhea incidence rate of kittens was calculated from the records, and the calculation formula was as follows:
wherein, the number of the diarrhea young cats in the test period is the sum of the number of the diarrhea young cats per day.
The results were as follows:
TABLE 6 incidence of diarrhea in milk powder replacement after weaning young cats
| Incidence of diarrhea (%) | |
| Control group | 6.25 |
| Test 1 group | 2.86 |
| Test 2 groups | 2.64 |
| Test 3 groups | 2.72 |
Conclusion: the obvious diarrhea rate of the test group is lower than that of the control group, and the addition of the bacillus coagulans King27 microbial inoculum can effectively prevent diarrhea caused by milk powder replacement after weaning of young cats.
The application method of the bacillus coagulans King27 microbial inoculum provided by the invention can be added into the feeding feed or the feeding milk powder in the embodiment, or can be placed in a bowl for free licking, or can be mixed with a small amount of dry food or can, or can be taken by using cool white boiled water below 40 ℃.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (9)
1. Bacillus coagulans for preventing diarrhea of petsBacillus coagulans) King27, wherein Bacillus coagulans King27 is preserved in China general microbiological culture Collection center (CGMCC) No.25556 at 2022, 8 and 19, and has a preservation address of North Chen Xiyu No. 1 and 3 in the Chaoyang area of Beijing city.
2. A bacillus coagulans King27 microbial agent for preventing diarrhea in pets, comprising the bacillus coagulans King27 microbial powder of claim 1.
3. The method for preparing the bacillus coagulans King27 microbial inoculum according to claim 2, which is characterized by comprising the following steps:
(1) Activating a frozen and preserved bacillus coagulans King27 strain, inoculating the single activated colony into an MRS liquid culture medium, and culturing at 40-45 ℃ for 18-24 hours at 200r/min to obtain seed liquid;
(2) Inoculating the seed liquid into a primary fermentation culture medium, and performing shaking culture at 40-45 ℃ and 200r/min for 22-26 hours to obtain a primary fermentation liquid; transferring the first-stage fermentation liquid into a second-stage fermentation medium, and performing shaking culture at 40-45 ℃ for 45-50 hours at 200r/min to obtain a second-stage fermentation liquid;
(3) Centrifuging the secondary fermentation liquor, collecting thalli, washing the thalli by using sterile physiological saline, and re-suspending the thalli in 15% (w/w) reconstituted skim milk to obtain suspension; freeze-drying the bacterial suspension to obtain bacterial powder;
(4) Mixing the fungus powder with glucose to obtain the fungus agent.
4. A method according to claim 3, wherein the specific operation of activation in step (1) is: the frozen and preserved Bacillus coagulans King27 strain was streaked on MRS plate medium, and cultured at 42℃for 48 hours under aerobic conditions.
5. The method according to claim 3, wherein in the step (2), the seed liquid is inoculated into the primary fermentation medium in an amount of 4% -6%.
6. The process according to claim 3, wherein in the step (2), the primary fermentation medium and the secondary fermentation medium are prepared as follows:
40g/L of glucose, 10g/L of yeast extract, 15g/L of peptone, 3g/L of dipotassium hydrogen phosphate, 0.05g/L of manganese sulfate, 4g/L of ammonium chloride and 18g/L of calcium carbonate, sterilizing the culture medium for 20min at 121 ℃, and cooling the culture medium to 40-45 ℃ for standby.
7. Use of bacillus coagulans King27 according to claim 1 for the preparation of a product for the prevention of diarrhea caused by intestinal pathogens in pet dogs and cats.
8. Use of bacillus coagulans King27 according to claim 1 for the preparation of a product for preventing seasonal diarrhea caused by cooling of pet dogs and cats.
9. Use of bacillus coagulans King27 according to claim 1 for the preparation of a product for preventing diarrhea caused by lactose intolerance in pet dogs and cats.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001034168A1 (en) * | 1999-11-08 | 2001-05-17 | Ganeden Biotech, Inc. | Inhibition of pathogens by bacillus coagulans strains |
| CN115094012A (en) * | 2022-08-18 | 2022-09-23 | 北京农学院 | Preparation method and application of bacillus coagulans BC-HYC strain microbial inoculum |
| CN115354002A (en) * | 2022-09-20 | 2022-11-18 | 微康益生菌(苏州)股份有限公司 | Bacillus coagulans BC07, application thereof, bacteriostatic agent, medicine, food and bacteriostatic method |
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| FR2999191B1 (en) * | 2012-12-12 | 2016-02-05 | Lesaffre & Cie | PROBIOTIC STRAINS FOR THE TREATMENT AND / OR PREVENTION OF DIARRHEA |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001034168A1 (en) * | 1999-11-08 | 2001-05-17 | Ganeden Biotech, Inc. | Inhibition of pathogens by bacillus coagulans strains |
| CN115094012A (en) * | 2022-08-18 | 2022-09-23 | 北京农学院 | Preparation method and application of bacillus coagulans BC-HYC strain microbial inoculum |
| CN115354002A (en) * | 2022-09-20 | 2022-11-18 | 微康益生菌(苏州)股份有限公司 | Bacillus coagulans BC07, application thereof, bacteriostatic agent, medicine, food and bacteriostatic method |
Non-Patent Citations (2)
| Title |
|---|
| Bacillus coagulans GBI-30, 6086 limits the recurrence of Clostridium difficile-Induced colitis following vancomycin withdrawal in mice;Leo R Fitzpatrick 等;《Gut pathogens》;第4卷(第1期);全文 * |
| 凝结芽孢杆菌分离筛选及复合益生菌剂的应用研究;戴青;《中国优秀硕士学位论文全文数据库》;全文 * |
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