CN110628683A - Probiotic compound live bacterium preparation for preventing and treating chicken diarrhea and preparation method thereof - Google Patents
Probiotic compound live bacterium preparation for preventing and treating chicken diarrhea and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a probiotic composite live bacterial preparation for preventing and treating chicken diarrhea and a preparation method thereof, wherein the preparation of the probiotic composite live bacterial preparation for preventing and treating chicken diarrhea is completed through the processes of preparation of lactobacillus plantarum freeze-dried powder, preparation of bifidobacterium pseudolongum freeze-dried powder, preparation of saccharomyces cerevisiae freeze-dried powder and compounding; the invention can effectively reduce the antibiotic-associated diarrhea rate of chickens, reduce the death rate of chickens cultured in a chicken farm, improve the daily gain of the chickens, improve the intestinal flora of the chickens, promote the proliferation of probiotics in the intestinal tract, maintain the balance of the intestinal flora, improve the immunity of organisms, is suitable for popularization and application in a large-scale chicken farm and realizes the aim of healthy culture.
Description
Technical Field
The invention relates to the technical field of production of microecological preparations, in particular to a probiotic compound live bacterium preparation for preventing and treating chicken diarrhea and a preparation method thereof.
Background
The application of the microecological preparation has the advantages that: increasing the probiotic flora in the organism of the patient and laying a solid foundation for the survival of probiotics such as bifidobacterium, lactobacillus and the like in the intestinal tract; the growth speed of pathogenic bacteria is hindered, and the reduction of toxin in the blood of a patient is facilitated; improving the natural barrier function of intestinal mucosa and improving the function of non-immune defense in the intestinal tract; increasing the permeability of the intestinal tract of the patient and improving the state of the micro-ecology in the intestinal tract; increase the function of endogenous bacteria in the metabolic process, and the like. When the microecological preparation is applied to treatment of antibiotic-associated diarrhea, the microecological preparation can play a role in maintaining the ecological balance of the intestinal tract of a patient by increasing the immune defense effect in the intestinal tract of the patient, exerting the level of a medicament and reducing the concentration of harmful bacteria in the intestinal tract.
Diarrhea is a common symptom of various intestinal diseases caused by various causes, and is generally divided into 2 types of infectious diarrhea and non-infectious diarrhea in clinic. Infectious diarrhea accounts for about 80%, and people are used to treatment of infectious diarrhea by using antibiotics, but because of massive blind use of the antibiotics, beneficial bacteria in intestinal tracts are inhibited, the microecological balance of the intestinal tracts is broken, flora imbalance is caused, the metabolic regulation level and physiological function of intestinal epithelial cells are disordered, and the diarrhea is further aggravated, namely, the diarrhea is caused (AAD).
The normal flora in the intestinal tract of the chicken comprises streptococcus, bifidobacterium, lactobacillus, corynebacterium and the like, and the normal flora has important significance for the development of the immune system of the organism and the maintenance of the nitrogen balance of the organism, the synthesis of vitamins, the absorption of fat and inorganic salt, the utilization of carbohydrate and the like, so the existence of the normal flora is an indispensable condition for maintaining the life activity of the organism and ensuring the health of the organism. At present, antibiotics commonly used in the poultry breeding process are as follows: penicillin, chloramphenicol, oxytetracycline, cephalosporins, quinolones and the like, and if antibiotics are used for a long time, the antibiotics are likely to cause drug resistance of some conditional pathogenic bacteria to abnormally proliferate, so that intestinal flora imbalance is caused to cause antibiotic-related diarrhea. The chicken has weak self-healing capacity and long disease course for antibiotic-associated chronic diarrhea, is difficult to recover obviously in a short period, and further directly influences the health of the chicken, and if a microecological preparation is supplemented properly, the recovery of chicken intestinal flora can be promoted, so that the diarrhea symptom is relieved.
In the process of poultry cultivation and treatment of intestinal diseases, intestinal symbiotic bacteria play an important role. Although effective in treating intestinal diseases of poultry, the application of antibacterial drugs has brought about a series of effects with the large and unreasonable use of the antibacterial drugs, and the effects comprise: antibacterial residue, reduced food quality safety, overgrowth of fungi and few bacteria in normal flora, antibacterial drug-related diarrhea, promotion of drug-resistant strain generation, enhancement of pathogenic sensitivity of pathogenic bacteria, interference of nutrition metabolism and direct drug poisoning effect, etc. Of these, the effects of antibiotic-associated diarrhea-induced dysbacteriosis are most direct and significant. The application of a large amount of antibacterial agents in poultry industry reduces or even eliminates the anaerobic strains sensitive to the antibacterial agents, causes the mass propagation and diffusion of the aerobic bacteria and the pathogenic bacteria, thereby causing the imbalance of the intestinal flora of poultry and further causing related symptoms such as diarrhea, anorexia and the like. The mechanism of the probiotics is that the ecological restoration of the flora in the digestive tract of the animals can be promoted through exogenous supplementation, biological oxygen deprivation, nutrition aiding digestion, immunity enhancement and the like, so the indigenous probiotics are also given during or after the treatment of the antibacterial drugs to help the intestinal flora to restore the balance, thereby achieving the effect of treating the flora imbalance caused by antibiotic-related diarrhea.
Disclosure of Invention
The invention aims to provide a probiotic compound live bacterium preparation for preventing and treating chicken diarrhea and a preparation method thereof, aiming at the conditions that in the intensive breeding of poultry, due to the fact that a large amount of antibacterial drugs or long-term use of the antibacterial drugs causes the disturbance of intestinal flora of chicken flocks, the reduction of diversity of flora and the increase of drug-resistant bacteria, and further causes antibiotic-related diarrhea.
In order to achieve the purpose, the invention provides the following technical scheme: a probiotic compound live bacteria preparation for preventing and treating chicken diarrhea is prepared by respectively fermenting and freeze-drying lactobacillus plantarum strain, bifidobacterium pseudolongum strain and saccharomyces cerevisiae strain and compounding, wherein the compounding ratio of the lactobacillus plantarum strain, the bifidobacterium pseudolongum strain and the saccharomyces cerevisiae strain is 10:10:1, and the number of live bacteria of the lactobacillus plantarum and the bifidobacterium pseudolongum is not less than 1.0 multiplied by 109CFU/g, viable count of yeast is not less than 1 × 108CFU/g, and the balance being glucose.
The invention also aims to disclose a preparation method of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea, which is characterized in that the preparation of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea is completed through the preparation of lactobacillus plantarum freeze-dried powder, the preparation of bifidobacterium pseudolongum freeze-dried powder, the preparation of saccharomyces cerevisiae freeze-dried powder and the compounding process; the method comprises the following specific steps:
(1) preparation of lactobacillus plantarum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.0 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.0 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6-8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14-16 hours, the tank pressure is 0.05MPa, feeding is carried out in a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained through centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and placed at the temperature of 2-8 ℃, the preparation of the lactobacillus plantarum freeze-dried powder is finished for later use, and the viable count of the obtained lactobacillus plantarum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(2) Preparation of bifidobacterium pseudolongum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.4 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.4 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6-8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14-16 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained by centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and the mixture is placed at the temperature of 2-8 ℃, so that the preparation of the bifidobacterium pseudolongum freeze-dried powder is finished for standby application, the viable count of the obtained bifidobacterium pseudolongum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(3) Preparing saccharomyces cerevisiae freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 6.5 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone and 4 percent of trace salt, and the pH value is adjusted to 6.5 for standby;
(III) seeding tank culture Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6-8 hours for later use;
d. fermentation in a fermentation tank:inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14-16 hours, the tank pressure is 0.05MPa, feeding is carried out in a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained through centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and placed at the temperature of 2-8 ℃, the preparation of the saccharomyces cerevisiae freeze-dried powder is finished for standby use, and the viable count of the obtained saccharomyces cerevisiae freeze-dried powder is not less than 1.0 multiplied by 108CFU/g；
(4) Compounding: and mixing the prepared lactobacillus plantarum freeze-dried powder, the prepared bifidobacterium pseudolongum freeze-dried powder and the prepared saccharomyces cerevisiae freeze-dried powder according to the ratio of 10:10:1, thus completing the preparation of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea.
Further, the trace salt formula is as follows: 0.2g/L CaCl2, 0.48g/L MgSO4, 10g/L NaHCO3, 1.0g/L KH2PO4, 1.0g/L K2HPO4, 2.0g/L NaCl.
Further, the formula of the feed in the fermentation step of the fermentation tank is 3 times of the carbon-nitrogen source of the fermentation medium.
Further, the protective agents in the fermentation step of the fermentation tank are as follows: 5% of cane sugar, 3% of skim milk, 1% of gelatin and the balance of water.
Compared with the prior art, the invention has the beneficial effects that: the lactobacillus plantarum and the bifidobacterium pseudolongum in the invention are screened from intestinal tracts of healthy chickens, are common strains in intestinal tracts of healthy chickens, and have the functions of improving intestinal flora, increasing the number of probiotics beneficial to organisms, reducing the number of pathogenic bacteria or conditional pathogenic bacteria, regulating the dynamic balance of the intestinal flora and enhancing the immunity of the organisms; after the saccharomyces cerevisiae from the fruit peel and the two bacteria enter the intestinal tract together, an anaerobic environment required for growth can be provided for the other side to promote the growth of the saccharomyces cerevisiae; the invention can effectively reduce the antibiotic-associated diarrhea rate of chickens, reduce the death rate of chickens cultured in a chicken farm, improve the daily gain of the chickens, improve the intestinal flora of the chickens, promote the proliferation of probiotics in the intestinal tract, maintain the balance of the intestinal flora, improve the immunity of organisms, is suitable for popularization and application in a large-scale chicken farm and realizes the aim of healthy culture.
Drawings
FIG. 1 is a flow chart of the preparation process of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
FIG. 1 shows a flow chart of the preparation process of the present invention.
Example 1
A probiotic compound viable bacteria preparation for preventing and treating chicken diarrhea is prepared by fermenting and freeze-drying Lactobacillus plantarum strain, Bifidobacterium pseudolongum strain and Saccharomyces cerevisiae strain respectively, and compounding, wherein the compounding ratio of the Lactobacillus plantarum strain, the Bifidobacterium pseudolongum strain and the Saccharomyces cerevisiae strain is 10:10:1, and the viable bacteria number of the Lactobacillus plantarum and the Bifidobacterium pseudolongum is not less than 1.0 x 109CFU/g, viable count of yeast is not less than 1 × 108CFU/g, and the balance being glucose.
A preparation method of a probiotic composite live bacterial preparation for preventing and treating chicken diarrhea comprises the steps of preparing lactobacillus plantarum freeze-dried powder, preparing pseudo bifidobacterium longum freeze-dried powder, preparing saccharomyces cerevisiae freeze-dried powder and compounding to complete the preparation of the probiotic composite live bacterial preparation for preventing and treating chicken diarrhea; the method comprises the following specific steps:
(1) preparation of lactobacillus plantarum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.0 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.0 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing the slant with sterilized normal saline, inoculating into a shake flask culture medium according to the inoculation amount of 1%, and standing and culturing for 16 hours at 37 ℃ for later use;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, bacterial sludge is obtained by centrifugation after the fermentation is finished, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and the mixture is placed at the temperature of 2 ℃, so that the preparation of the lactobacillus plantarum freeze-dried powder is finished for later use, the viable count of the obtained lactobacillus plantarum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(2) Preparation of bifidobacterium pseudolongum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.4 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.4 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing the slant with sterilized normal saline, inoculating into a shake flask culture medium according to the inoculation amount of 1%, and standing and culturing for 16 hours at 37 ℃ for later use;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, bacterial sludge is obtained by centrifugation after the fermentation is finished, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and the mixture is placed at the temperature of 2 ℃, so that the preparation of the bifidobacterium pseudolongum freeze-dried powder is finished for standby use, the viable count of the obtained bifidobacterium pseudolongum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(3) Preparing saccharomyces cerevisiae freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 6.5 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone and 4 percent of trace salt, and the pH value is adjusted to 6.5 for standby;
(III) seeding tank culture Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained by centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and placed under the condition of 2 ℃, the preparation of the saccharomyces cerevisiae freeze-dried powder is finished for later use, the viable count of the obtained saccharomyces cerevisiae freeze-dried powder is not less than 1.0 multiplied by 108CFU/g；
(4) Compounding: and mixing the prepared lactobacillus plantarum freeze-dried powder, the prepared bifidobacterium pseudolongum freeze-dried powder and the prepared saccharomyces cerevisiae freeze-dried powder according to the ratio of 10:10:1, thus completing the preparation of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea.
The formula of the trace salt is as follows: 0.2g/L CaCl2, 0.48g/L MgSO4, 10g/L NaHCO3, 1.0g/L KH2PO4, 1.0g/L K2HPO4, 2.0g/L NaCl.
The formula of the feed supplement in the fermentation step of the fermentation tank is 3 times of the carbon-nitrogen source of the fermentation medium.
The protective agent in the fermentation step of the fermentation tank is as follows: 5% of cane sugar, 3% of skim milk, 1% of gelatin and the balance of water.
Example 2
A probiotic compound viable bacteria preparation for preventing and treating chicken diarrhea is prepared by fermenting and freeze-drying Lactobacillus plantarum strain, Bifidobacterium pseudolongum strain and Saccharomyces cerevisiae strain respectively, and compounding, wherein the compounding ratio of the Lactobacillus plantarum strain, the Bifidobacterium pseudolongum strain and the Saccharomyces cerevisiae strain is 10:10:1, and the viable bacteria number of the Lactobacillus plantarum and the Bifidobacterium pseudolongum is not less than 1.0 x 109CFU/g, viable count of yeast is not less than 1 × 108CFU/g, and the balance being glucose.
A preparation method of a probiotic composite live bacterial preparation for preventing and treating chicken diarrhea comprises the steps of preparing lactobacillus plantarum freeze-dried powder, preparing pseudo bifidobacterium longum freeze-dried powder, preparing saccharomyces cerevisiae freeze-dried powder and compounding to complete the preparation of the probiotic composite live bacterial preparation for preventing and treating chicken diarrhea; the method comprises the following specific steps:
(1) preparation of lactobacillus plantarum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.0 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.0 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing the slant with sterilized normal saline, inoculating into a shake flask culture medium according to the inoculation amount of 1%, and standing and culturing at 37 ℃ for 18 hours for later use;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 16 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained by centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and placed under the condition of 8 ℃, and the lactobacillus plantarum is finishedPreparing lyophilized powder, wherein the viable count of the obtained Lactobacillus plantarum lyophilized powder is not less than 1.0 × 109CFU/g；
(2) Preparation of bifidobacterium pseudolongum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.4 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.4 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing the slant with sterilized normal saline, inoculating into a shake flask culture medium according to the inoculation amount of 1%, and standing and culturing at 37 ℃ for 18 hours for later use;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is performed for 16 hours, the tank pressure is 0.05MPa, feeding is performed in a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained through centrifugation, a protective agent with 2 times of mass is added under an aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and the mixture is placed at 8 ℃, so that the preparation of the bifidobacterium pseudolongum freeze-dried powder is completed for standby application, the viable count of the obtained bifidobacterium pseudolongum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(3) Preparing saccharomyces cerevisiae freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 6.5 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone and 4 percent of trace salt, and the pH value is adjusted to 6.5 for standby;
(III) seeding tank culture Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, anaerobic culture is carried out for 16 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, bacterial sludge is obtained by centrifugation after the fermentation is finished, a protective agent with 2 times of mass is added under an aseptic condition, uniformly mixing, freeze-drying, crushing and bagging are carried out, the preparation of the saccharomyces cerevisiae freeze-dried powder is finished under the condition of 8 ℃, the preparation is reserved, and the viable count of the obtained saccharomyces cerevisiae freeze-dried powder is not less than 1.0 multiplied by 108CFU/g；
(4) Compounding: and mixing the prepared lactobacillus plantarum freeze-dried powder, the prepared bifidobacterium pseudolongum freeze-dried powder and the prepared saccharomyces cerevisiae freeze-dried powder according to the ratio of 10:10:1, thus completing the preparation of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea.
The formula of the trace salt is as follows: 0.2g/L CaCl2, 0.48g/L MgSO4, 10g/L NaHCO3, 1.0g/L KH2PO4, 1.0g/L K2HPO4, 2.0g/L NaCl.
The formula of the feed supplement in the fermentation step of the fermentation tank is 3 times of the carbon-nitrogen source of the fermentation medium.
The protective agent in the fermentation step of the fermentation tank is as follows: 5% of cane sugar, 3% of skim milk, 1% of gelatin and the balance of water.
Example 3
A probiotic compound viable bacteria preparation for preventing and treating chicken diarrhea is prepared by fermenting and freeze-drying Lactobacillus plantarum strain, Bifidobacterium pseudolongum strain and Saccharomyces cerevisiae strain respectively, and compounding, wherein the compounding ratio of the Lactobacillus plantarum strain, the Bifidobacterium pseudolongum strain and the Saccharomyces cerevisiae strain is 10:10:1, and the viable bacteria number of the Lactobacillus plantarum and the Bifidobacterium pseudolongum is not less than 1.0 x 109CFU/g, viable count of yeast is not less than 1 × 108CFU/g, and the balance being glucose.
A preparation method of a probiotic composite live bacterial preparation for preventing and treating chicken diarrhea comprises the steps of preparing lactobacillus plantarum freeze-dried powder, preparing pseudo bifidobacterium longum freeze-dried powder, preparing saccharomyces cerevisiae freeze-dried powder and compounding to complete the preparation of the probiotic composite live bacterial preparation for preventing and treating chicken diarrhea; the method comprises the following specific steps:
(1) preparation of lactobacillus plantarum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.0 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.0 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing the slant by using sterilized normal saline, inoculating the slant into a shake flask culture medium according to the inoculation amount of 1%, and standing and culturing for 17 hours at 37 ℃ for later use;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 7 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 15 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, bacterial sludge is obtained by centrifugation after the fermentation is finished, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and the mixture is placed at the temperature of 5 ℃, so that the preparation of the lactobacillus plantarum freeze-dried powder is finished for later use, the viable count of the obtained lactobacillus plantarum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(2) Preparation of bifidobacterium pseudolongum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.4 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.4 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing the slant by using sterilized normal saline, inoculating the slant into a shake flask culture medium according to the inoculation amount of 1%, and standing and culturing for 17 hours at 37 ℃ for later use;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 7 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, anaerobic culture is carried out for 15 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, bacterial sludge is obtained by centrifugation after the fermentation is finished, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and the mixture is placed at 5 ℃, so that the preparation of the bifidobacterium pseudolongum freeze-dried powder is finished for standby application, the viable count of the obtained bifidobacterium pseudolongum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(3) Preparing saccharomyces cerevisiae freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 6.5 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone and 4 percent of trace salt, and the pH value is adjusted to 6.5 for standby;
(III) seeding tank culture Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
b. shake flask strain amplification: after the slant is opened, reducing the slant by using sterilized normal saline, inoculating the slant into a shake flask culture medium according to the inoculation amount of 1%, and standing and culturing for 17 hours at 37 ℃ for later use;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 7 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into fermentation tank culture medium at an inoculation amount of 8%, wherein the fermentation temperature is 37 deg.CControlling the pH value to be 6.5, stirring at a rotating speed of 50r/min, carrying out anaerobic culture for 15 hours, feeding materials in a fed-batch mode, centrifuging to obtain bacterial sludge after fermentation is finished, adding a protective agent with the mass of 2 times of that of the bacterial sludge under an aseptic condition, uniformly mixing, freeze-drying, crushing and bagging, and placing at 5 ℃ to finish the preparation of the saccharomyces cerevisiae freeze-dried powder for later use, wherein the viable count of the obtained saccharomyces cerevisiae freeze-dried powder is not less than 1.0 multiplied by 108CFU/g；
(4) Compounding: and mixing the prepared lactobacillus plantarum freeze-dried powder, the prepared bifidobacterium pseudolongum freeze-dried powder and the prepared saccharomyces cerevisiae freeze-dried powder according to the ratio of 10:10:1, thus completing the preparation of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea.
The formula of the trace salt is as follows: 0.2g/L CaCl2, 0.48g/L MgSO4, 10g/L NaHCO3, 1.0g/L KH2PO4, 1.0g/L K2HPO4, 2.0g/L NaCl.
The formula of the feed supplement in the fermentation step of the fermentation tank is 3 times of the carbon-nitrogen source of the fermentation medium.
The protective agent in the fermentation step of the fermentation tank is as follows: 5% of cane sugar, 3% of skim milk, 1% of gelatin and the balance of water.
Example 4
The lactobacillus plantarum strain culture solution has the effects on escherichia coli and salmonella pullorum;
preparing MH culture medium and solid culture medium thereof, and PTYG liquid culture medium and solid culture medium thereof for later use, wherein MH is a commercial culture medium, and the PTYG culture medium comprises the following components: tryptone 5.0g/L, yeast extract powder 10.0g/L, glucose 10.0g/L, L-cysteine hydrochloride 0.5g/L, pH 7.0, 115 ℃ sterilization for 20 minutes, its solid medium added with 2.0% agar powder. The Lactobacillus plantarum strain was activated by using a PTYG solid medium, then inoculated into 200mL/250mL of a PTYG liquid medium, and cultured by standing at 37 ℃ for 24 hours in a constant temperature incubator for use. Respectively diluting activated escherichia coli and salmonella pullorum by 1000 times with sterile normal saline, coating the diluted escherichia coli and salmonella pullorum on an MH solid flat plate, respectively and uniformly placing 2 sterile oxford cups into a culture dish by using forceps, respectively sucking 200 mu l of PYTG culture medium and lactobacillus plantarum culture solution, placing the obtained product in the oxford cups, carrying out overnight culture at 37 ℃, and observing an inhibition zone, wherein the PTYG culture medium has no inhibition effect on the escherichia coli and the salmonella pullorum, the diameter of the inhibition zone of the lactobacillus plantarum culture solution on the escherichia coli is 16-17 mm, and the diameter of the inhibition zone on the salmonella pullorum is 21-22 mm. The results show that the lactobacillus plantarum strain has a good antibacterial effect on escherichia coli and salmonella pullorum.
Example 5
The effect of Bifidobacterium pseudolongum strain culture solution on Escherichia coli and Salmonella pullorum;
preparing MH culture medium and solid culture medium thereof, and PTYG liquid culture medium and solid culture medium thereof for later use, wherein MH is a commercial culture medium, and the PTYG culture medium comprises the following components: tryptone 5.0g/L, yeast extract powder 10.0g/L, glucose 10.0g/L, L-cysteine hydrochloride 0.5g/L, pH 7.4, 115 ℃ sterilization for 20 minutes, its solid medium added with 2.0% agar powder. The Bifidobacterium pseudolongum B456 strain was activated with a PTYG solid medium, then inoculated into 200mL/250mL of a liquid medium, and subjected to static culture at 37 ℃ for 48 hours in a constant temperature incubator for use. Respectively diluting activated escherichia coli and salmonella pullorum by 1000 times with sterile normal saline, coating the diluted solutions on an MH solid plate, respectively and uniformly placing 2 sterile oxford cups into a culture dish by using forceps, respectively sucking 200 mu l of PYTG culture medium and bifidobacterium pseudodurans culture solution, placing the bottles in the oxford cups, performing overnight culture at 37 ℃, and observing an inhibition zone, wherein the results show that the PTYG culture medium has no inhibition effect on the escherichia coli and the salmonella pullorum, the diameter of the inhibition zone of the bifidobacterium pseudodurans culture solution on the escherichia coli is between 16mm and 17mm, and the diameter of the inhibition zone of the salmonella pullorum is between 23mm and 24 mm. The result shows that the bifidobacterium pseudolongum strain has better bacteriostatic effect on escherichia coli and salmonella pullorum.
Example 6
In order to test the effect of the probiotic compound live bacteria preparation on preventing and treating chicken antibiotic-associated diarrhea, animal experiments are carried out. The composite live bacteria preparation is freeze-dried strain prepared in example 1, example 2 and example 3Mixing the powders, wherein the content of Bacillus bifidus and lactobacillus should not be less than 1.0 × 109CFU/g, yeast should not be less than 1.0 × 108CFU/g, the remainder being made up by glucose. 150 SPF chicks of 5-7 days old were randomly divided into 5 treatment groups of 30 animals each, with 3 replicates per treatment group. During molding, the normal control group is only filled with sterile normal saline, the natural recovery group and 3 experimental groups are filled with cefoperazone and ampicillin sodium antibiotic mixed solution, the method of combined medication and gradual increase of dosage is adopted, the stomach is filled for 1 time every day, 500 mu l of the medicine is taken every time, and the first day: cefoperazone 0.0048g + ampicillin sodium 0.2g, the next day: cefoperazone 0.0048g + ampicillin sodium 0.2g, day three: cefoperazone 0.0096g + ampicillin sodium 0.4g, day four: cefoperazone 0.0144g + ampicillin sodium 0.6g, day five: 0.0192g of cefoperazone and 0.8g of ampicillin sodium, and the chicken antibiotic-induced intestinal flora imbalance diarrhea model can be successfully constructed by continuously giving antibiotics for induction for 5-6 days.
After the model construction is successful, the experimental groups are respectively given to the bifidobacterium, lactobacillus and yeast composite live bacteria preparations of example 1, example 2 and example 3, the administration period is 6-8 days, the clinical symptoms of the chicken are observed during the administration period, the data of the weight, the state of the excrement and the like are recorded, and the change condition of the excrement flora is known in different modes.
Table 1 effect of probiotic complex live bacterial formulation on test chicken body weight (g,n＝10)
note: ". mark" in table 1 indicates P <0.05 compared to normal control group; the "#" in table 1 indicates P <0.05, which indicates the significance of the difference, compared to the natural recovery group.
TABLE 2 Effect of probiotic Complex Living bacteria preparation on the stool status of test chickens
Note: stool status: "-" indicates as formed; "+" indicates formation but slightly more moisture; "+ +" indicates a viscous loose stool; "+ + + +" indicates water-like stool.
TABLE 3 Effect of probiotic Complex Living bacteria preparation on microbial Numbers of SPF Chicken feces
| Group of | Enterobacter | Enterococcus | Lactobacillus strain | Bifidobacterium |
| Normal group | 7.58±0.23 | 6.4±0.34 | 8.09±0.3 | 8.09±0.15 |
| Natural recovery group | 8.61±0.32* | 6.44±0.22 | 6.58±0.39* | 6.31±0.36* |
| Example 1 | 7.65±0.08# | 6.53±0.4 | 8.15±0.24# | 7.77±0.27# |
| Example 2 | 7.42±0.37# | 6.62±0.37 | 7.93±0.25# | 7.49±0.39# |
| Example 3 | 7.47±0.37# | 6.46±0.28 | 7.82±0.49# | 7.78±0.28# |
Note: in table 3 "+" indicates P <0.05 compared to control; in table 3 "#" indicates P <0.05 compared to the natural recovery group, P indicating the significance of the difference.
As can be seen from the tables 1, 2 and 3, no significant difference exists among 3 experimental groups, the probiotic composite viable bacteria preparation has stable treatment effect, can effectively treat antibiotic-associated diarrhea of chickens, reduces the defecation rate, promotes the feed intake, is beneficial to healthy growth of the chickens, helps the chickens to recover the quantity of intestinal flora, increases the diversity of flora, promotes the dynamic balance of the intestinal flora, improves the quantity of beneficial bacteria such as lactobacillus, bifidobacterium and the like, reduces the quantity of pathogenic bacteria such as escherichia coli, klebsiella, salmonella and the like, and improves the immunity of the chickens only resisting the invasion of pathogenic bacteria or opportunistic pathogenic bacteria.
The lactobacillus plantarum and the bifidobacterium pseudolongum in the invention are screened from intestinal tracts of healthy chickens, are common strains in intestinal tracts of healthy chickens, and have the functions of improving intestinal flora, increasing the number of probiotics beneficial to organisms, reducing the number of pathogenic bacteria or conditional pathogenic bacteria, regulating the dynamic balance of the intestinal flora and enhancing the immunity of the organisms; after the saccharomyces cerevisiae from the fruit peel and the two bacteria enter the intestinal tract together, an anaerobic environment required for growth can be provided for the other side to promote the growth of the saccharomyces cerevisiae; the invention can effectively reduce the antibiotic-associated diarrhea rate of chickens, reduce the death rate of chickens cultured in a chicken farm, improve the daily gain of the chickens, improve the intestinal flora of the chickens, promote the proliferation of probiotics in the intestinal tract, maintain the balance of the intestinal flora, improve the immunity of organisms, is suitable for popularization and application in a large-scale chicken farm and realizes the aim of healthy culture.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (5)
1. A probiotic compound live bacteria preparation for preventing and treating chicken diarrhea is characterized in that: the lactobacillus plantarum strain, the bifidobacterium pseudolongum strain and the saccharomyces cerevisiae strain are respectively fermented, freeze-dried and compounded to form the lactobacillus plantarum strain, the bifidobacterium pseudolongum strain and the saccharomyces cerevisiae strain, the compounding ratio of the lactobacillus plantarum strain, the bifidobacterium pseudolongum strain and the saccharomyces cerevisiae strain is 10:10:1, and the viable count of the lactobacillus plantarum and the bifidobacterium pseudolongum is not less than 1.0 multiplied by 109CFU/g, viable count of yeast is not less than 1 × 108CFU/g, and the balance being glucose.
2. A method for preparing a probiotic compound live bacterial preparation for preventing and treating chicken diarrhea according to claim 1, which is characterized in that: the preparation of the probiotic composite live bacteria preparation for preventing and treating the chicken diarrhea is completed through the preparation of lactobacillus plantarum freeze-dried powder, the preparation of bifidobacterium pseudolongum freeze-dried powder, the preparation of saccharomyces cerevisiae freeze-dried powder and the compounding process; the method comprises the following specific steps:
(1) preparation of lactobacillus plantarum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.0 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.0 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6-8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14-16 hours, the tank pressure is 0.05MPa, feeding is carried out in a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained through centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and placed at the temperature of 2-8 ℃, the preparation of the lactobacillus plantarum freeze-dried powder is finished for later use, and the viable count of the obtained lactobacillus plantarum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(2) Preparation of bifidobacterium pseudolongum freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 7.4 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is adjusted to 7.4 for standby;
(III) seeding tank culture Medium: 1.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride and 4 percent of trace salt, and the pH value is controlled to be 6.5 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 2.0 percent of yeast extract powder, 2.0 percent of peptone, 0.05 percent of L-cysteine hydrochloride, 4 percent of trace salt and 6.5 percent of pH value for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6-8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14-16 hours, the tank pressure is 0.05MPa, feeding is carried out by adopting a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained by centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and the mixture is placed at the temperature of 2-8 ℃, so that the preparation of the bifidobacterium pseudolongum freeze-dried powder is finished for standby application, the viable count of the obtained bifidobacterium pseudolongum freeze-dried powder is not less than 1.0 multiplied by 109CFU/g；
(3) Preparing saccharomyces cerevisiae freeze-dried powder:
a. preparation of culture media of all levels:
(I) slant culture medium: fixing the culture medium for PTYG, and adjusting the pH value to 6.5 for later use;
(II) Shake flask culture Medium: 1.0 percent of glucose, 1.0 percent of yeast extract powder, 1.0 percent of peptone and 4 percent of trace salt, and the pH value is adjusted to 6.5 for standby;
(III) seeding tank culture Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
(IV) fermenter Medium: 2.0 percent of glucose, 1.5 percent of yeast extract powder, 1.5 percent of peptone and 4 percent of trace salt, and the pH value is controlled to be 6.0 for standby;
b. shake flask strain amplification: after the slant is opened, reducing with sterilized normal saline, inoculating into shake flask culture medium according to 1% of inoculum size, and standing and culturing at 37 deg.C for 16-18 hr;
c. fermenting in a seeding tank: inoculating the shake flask bacterial liquid into a seed tank culture solution according to the inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the tank pressure is 0.05MPa in the fermentation process, and the anaerobic culture is carried out for 6-8 hours for later use;
d. fermentation in a fermentation tank: inoculating the fermentation broth into a fermentation tank culture medium according to an inoculation amount of 8%, wherein the fermentation temperature is 37 ℃, the pH value is controlled to be 6.5, the stirring speed is 50r/min, the anaerobic culture is carried out for 14-16 hours, the tank pressure is 0.05MPa, feeding is carried out in a fed-batch mode, after the fermentation is finished, bacterial sludge is obtained through centrifugation, a protective agent with 2 times of mass is added under the aseptic condition, the mixture is uniformly mixed, freeze-dried, crushed and bagged, and placed at the temperature of 2-8 ℃, the preparation of the saccharomyces cerevisiae freeze-dried powder is finished for standby use, and the viable count of the obtained saccharomyces cerevisiae freeze-dried powder is not less than 1.0 multiplied by 108CFU/g；
(4) Compounding: and mixing the prepared lactobacillus plantarum freeze-dried powder, the prepared bifidobacterium pseudolongum freeze-dried powder and the prepared saccharomyces cerevisiae freeze-dried powder according to the ratio of 10:10:1, thus completing the preparation of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea.
3. The preparation method of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea according to claim 2 is characterized by comprising the following steps of: the formula of the trace salt is as follows: 0.2g/L CaCl2, 0.48g/L MgSO4, 10g/L NaHCO3, 1.0g/LKH2PO4, 1.0g/L K2HPO4, 2.0g/L NaCl.
4. The preparation method of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea according to claim 2 is characterized by comprising the following steps of: the formula of the feed supplement in the fermentation step of the fermentation tank is 3 times of carbon and nitrogen sources of the fermentation medium.
5. The preparation method of the probiotic compound live bacteria preparation for preventing and treating the chicken diarrhea according to claim 2 is characterized by comprising the following steps of: the protective agent in the fermentation step of the fermentation tank is as follows: 5% of cane sugar, 3% of skim milk, 1% of gelatin and the balance of water.
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