CN112741210A - Biological preparation for improving animal organism immunity function and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of feed additives, in particular to a preparation method of a biological agent for improving the immune function of an animal body; comprises the following steps: activating clostridium butyricum strains; transferring the activated clostridium butyricum strain into a fermentation tank filled with a seed culture medium, culturing for 18-20 h at 32-34 ℃ to obtain a primary seed solution, transferring the primary seed solution into the seed tank filled with the fermentation culture medium, and culturing for 18-20 h at 32-34 ℃ to obtain a secondary seed solution; transferring the secondary seed solution into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 8% to obtain a culture solution, and adding a pH regulator in the culture process to obtain a fermentation liquid when the microscopic spore formation rate is more than 10%; concentrating the fermentation liquor; and (5) freeze drying. The clostridium butyricum biological preparation can regulate the intestinal microecological balance, inhibit the growth of intestinal pathogenic bacteria escherichia coli, salmonella and shigella, enhance the immunity of organisms and promote the weight gain of animals.

Description

Biological preparation for improving animal organism immunity function and preparation method thereof

Technical Field

The invention relates to the technical field of feed additives, in particular to a biological agent for improving the immune function of an animal body and a preparation method thereof.

Background

Since the 20 th century, various antibiotics and hormones have been widely used as feed additives, and although they play an indispensable role in the production of livestock and poultry, the prevention and treatment of epidemic diseases, the promotion of animal growth, and the like, the use of antibiotics has more and more problems due to long-term use and abuse, which mainly appear in that: drug residues and toxicity; the use of a large number of broad-spectrum antibiotics in fed livestock results in rapid bacterial resistance; the long-term use of antibiotics disturbs the mutual restriction pattern of microbial communities in microbial systems in livestock and poultry bodies, and then destroys the microecological balance in the livestock and poultry bodies, so that the digestion function is disordered, and various digestive tract diseases are caused; reducing the immune function of the animal. In order to overcome the disadvantages, researchers in all countries in the world continuously strive to find and develop a novel additive which is safe, has no toxic or side effect and no residue, can promote the growth of animals, can prevent and treat livestock and poultry diseases and can replace antibiotics. The animal microecological preparation (microbial feed additive) is produced based on the safety problem of feed antibiotics, can inhibit pathogenic bacteria, is nontoxic to livestock and poultry products, promotes growth, improves the quality and flavor of the livestock and poultry products, is favorable for ecological environment and export trade, and is an optimal solution for future green feed.

Regarding the kind of strains used for probiotics, 43 safe strains that can be directly fed to animals were issued by the U.S. Food and Drug Administration (FDA) and the feed consortium (AAFCO) in 1989. In the catalog of feed additive varieties allowed to be used by the No. 105 publication published by the Ministry of agriculture in China in 6 months 1999, only 12 feed-grade microbial additive strains are available. The 2005 edition (revised 318 bulletin catalog) feed additive variety catalog allowed the use of microbial species to increase to 18. The 2013 version of feed additive variety catalog allows the use of microbial strains added to 35, including Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp Lactobacillus paracasei, Bacillus coagulans, Bacillus brevis (original name: Bacillus laterosporus), Phaffia rhodozyma, etc. Clostridium butyricum is listed in catalog of new feed and new feed additive varieties in monitoring period 7 months in 2009.

Clostridium butyricum cell rod-shaped, 0.6-1.2X 3.0-7.0 μm, single paired, short-chain, occasional filamentous thallus. With the circum-flagellum movement, the spore is oval, eccentric to the secondary, and has no spore outer wall and accessory silk. Is an anaerobic gram-positive bacillus which becomes negative in old cultures. Poor or no growth on nutritious watercress; good growth on glucono-delta-agar. The surface bacterial colony is irregular, has a diameter of 1-3 μm, is slightly convex, is white to cream color, and has a glossy to matte surface. Poor or no growth in broth; grow well and produce gas in broth media containing fermentable carbohydrates. Clostridium butyricum is a normal flora of the human and animal intestinal tract and is also widely present in soil, cheese and natural yoghurt.

Clostridium butyricum has been successfully used in livestock breeding of piglets, chicks and the like as a beneficial microorganism and has received good effects. The research shows that: the clostridium butyricum can regulate the microecological balance of intestinal tracts, inhibit the growth of intestinal pathogenic bacteria escherichia coli, salmonella and shigella, and simultaneously remarkably promote the growth of intestinal beneficial bacteria lactobacillus acidophilus, bifidobacterium, enterococcus faecalis and the like. Enhancing the immunity of organisms, promoting the growth of animals, reducing the dosage of antibiotics and improving the economic benefit. The produced probiotic substance butyric acid is a main nutrient substance for energy metabolism and normal growth of colonic epithelial cells, and meanwhile, clostridium butyricum can produce short-chain fatty acids such as acetic acid and propionic acid, decompose harmful substances such as amines and indole hydrogen sulfide, improve the health quality and improve the environment. Clostridium butyricum is receiving more and more attention as a safe, reliable, green and environment-friendly probiotic, and is widely applied to the fields of water purification, aquaculture, feed additives and the like.

Therefore, the biological preparation for improving the immune function of the animal body and the preparation method are provided.

Disclosure of Invention

The invention aims to provide a biological agent for improving the immune function of an animal body and a preparation method thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the preparation method of the biological agent for improving the immune function of the animal body comprises the following steps:

(1) activating clostridium butyricum strains;

(2) transferring activated clostridium butyricum strains into a fermentation tank filled with a seed culture medium, and culturing for 18-20 h at 32-34 ℃ to obtain a primary seed solution, wherein the clostridium butyricum strains in the primary seed solution account for 8 vt%, then transferring the primary seed solution into a seed tank filled with a fermentation culture medium, and culturing for 18-20 h at 32-34 ℃ to obtain a secondary seed solution, wherein the primary seed solution in the secondary seed solution accounts for 8 vt%;

(3) transferring the secondary seed solution into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 8% to obtain a culture solution, culturing the culture solution at 32-34 ℃ and 0.05-0.06 MPa for 18-20 h, controlling the pH value of the culture solution at 6.0-6.5 by adding a pH regulator in the culture process, intermittently stirring, and obtaining a fermentation liquid when the formation rate of spores is higher than 10% through microscopic examination;

(4) concentrating the fermentation liquor, namely concentrating the fermentation liquor by 5 times by using a disc centrifuge to obtain a fermentation concentrated solution;

(5) freeze-drying, namely adding a carrier protective agent into the fermentation concentrated solution and freeze-drying, wherein the addition amount of the carrier protective agent is 20g/L, and freeze-drying treatment is carried out in five stages, wherein the first stage is frozen for 4h at the temperature of minus 40 ℃, the second stage is frozen for 10h at the temperature of minus 10-40 ℃, the third stage is frozen for 8h at the temperature of minus 10-minus, the fourth stage is dried for 16h at the temperature of minus 30-plus, and the fifth stage is dried for 3h at the temperature of minus 30-plus 32 ℃ to obtain the clostridium butyricum biological preparation;

wherein the seed culture medium comprises 10 g/L-15 g/L glucose, 8 g/L-10 g/L peptone, 3 g/L-5 g/L beef extract, 12 g/L-15 g/L agar, 1 g/L-3 g/L yeast powder, 0.4 g/L-0.6 g/L monopotassium phosphate and 0.2 g/L-0.3 g/L calcium carbonate, and the pH value of the seed culture medium is 7.1-7.2;

the fermentation medium comprises 20-25 g/L of corn starch, 3-6 g/L of yeast extract, 1-3 g/L of peptone, 1-2 g/L of beef extract, 0.2-0.5 g/L of monopotassium phosphate, 0.5-1 g/L of sodium bicarbonate, 0.1-0.2 g/L of manganese sulfate and 0.05-0.1 g/L of calcium chloride, and the pH value of the fermentation medium is 7.1-7.2.

Specifically, the carrier protective agent in the step (5) comprises 3-5 g/L of trehalose, 6-8 g/L of soybean protein powder and 4-8 g/L of skimmed milk powder.

Specifically, the pH regulator in the step (3) is 10 wt% potassium hydroxide solution.

Specifically, the fermentation medium is prepared in proportion and then sterilized for 30min at 121 ℃, after sterilization is finished, the temperature is reduced, nitrogen is introduced for pressure maintaining, the tank pressure in a fermentation tank is controlled to be 0.05-0.06 MPa, and the heated and activated secondary seed liquid is inoculated when the temperature is reduced to the culture temperature.

Specifically, the water content of the clostridium butyricum biological preparation in the step (5) is less than or equal to 5 percent.

Specifically, the step (3) of intermittent stirring includes the following steps: stirring for 1-2 min every 2h, and controlling the stirring speed to be 75-100 r/min.

The biological preparation for improving the immune function of the animal body is prepared by the preparation method.

The invention has the beneficial effects that:

(1) the carrier protective agent is added in the freeze-drying stage of the clostridium butyricum biological preparation, so that the consumption of live oxygen bacteria in the clostridium butyricum biological preparation in the freeze-drying process can be effectively reduced, the storage period and the survival rate of the clostridium butyricum biological preparation can be effectively improved, and the raw material cost of the carrier protective agent is low;

(2) the seed culture medium and the fermentation culture medium used in the invention have simple components and low cost, and are suitable for large-scale production of factories;

(3) the clostridium butyricum biological preparation can regulate intestinal microecological balance, inhibit the growth of intestinal pathogenic bacteria escherichia coli, salmonella and shigella, prevent diarrhea of pigs caused by the escherichia coli, the salmonella, clostridium perfringens and the like, enhance the immunity of organisms, promote the weight gain of animals and improve the economic benefit.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.

Example 1

The preparation method of the biological agent for improving the immune function of the animal body comprises the following steps:

(1) activating clostridium butyricum strains: inoculating clostridium butyricum strains into a seed culture medium, and culturing and growing for 18-20 h at the temperature of 32-34 ℃ in a constant-temperature incubator to realize activation treatment of the clostridium butyricum strains;

(2) transferring the activated clostridium butyricum strain into a fermentation tank filled with a seed culture medium according to the inoculation amount of 8%, and culturing at 32-34 ℃ for 18-20 h to reach the logarithmic phase, thereby obtaining a first-stage seed solution; then transferring the primary seed solution to a fermentation tank filled with a seed culture medium according to the inoculation amount of 8%, and culturing for 18-20 h at 32-34 ℃ to reach a logarithmic phase, so as to obtain a secondary seed solution, wherein the culture end points of the primary seed solution and the secondary seed solution are determined by microscopic examination;

(3) transferring the secondary seed liquid into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 8% to obtain a culture liquid, wherein the fermentation culture medium is sterilized at 121 ℃ for 30min after being prepared in proportion, cooling and introducing nitrogen for pressure maintaining after the sterilization is finished, controlling the tank pressure in the fermentation tank to be 0.05-0.06 MPa, and transferring the heated and activated secondary seed liquid when the temperature is reduced to the culture temperature; culturing the culture solution at 32-34 ℃ and 0.05-0.06 MPa for 18-20 h, controlling the pH value of the culture solution at 6.0-6.5 in a mode of online monitoring and automatic addition of 10 wt% potassium hydroxide solution as a pH regulator in the culture process, stirring for 1-2 min every 2h, controlling the stirring speed at 75-100 r/min, and obtaining fermentation liquor when the microscopic spore formation rate is more than 10%;

(4) concentrating the fermentation liquor, namely concentrating the fermentation liquor by 5 times by using a disc centrifuge to obtain a fermentation concentrated solution;

(5) freeze drying, namely adding a carrier protective agent into the fermentation concentrated solution and freeze drying, wherein the addition amount of the carrier protective agent is 20g/L, and the carrier protective agent comprises 3 g/L-5 g/L of trehalose, 6 g/L-8 g/L of soybean protein powder and 4 g/L-8 g/L of skimmed milk powder; freezing and drying treatment is carried out in five stages, wherein the first stage is frozen for 4 hours at minus 40 ℃, the second stage is frozen for 10 hours at minus 10-40 ℃, the third stage is frozen for 8 hours at minus 10-minus, the fourth stage is dried for 16 hours at minus 30-plus, and the fifth stage is dried for 3 hours at minus 30-plus 32 ℃ to obtain the clostridium butyricum biological preparation, wherein the water content of the clostridium butyricum biological preparation is less than or equal to 5%;

wherein the seed culture medium comprises 10g/L glucose, 8g/L peptone, 3g/L beef extract, 12g/L agar, 1g/L yeast powder, 0.4g/L monopotassium phosphate and 0.2g/L calcium carbonate, and the pH value of the seed culture medium is 7.1-7.2;

the fermentation medium comprises 20g/L of corn starch, 3g/L of yeast extract, 1g/L of peptone, 1g/L of beef extract, 0.2g/L of monopotassium phosphate, 0.5g/L of sodium bicarbonate, 0.1g/L of manganese sulfate and 0.05g/L of calcium chloride, and the pH value of the fermentation medium is 7.1-7.2.

Example 2

The preparation method of the biological preparation for improving the immune function of the animal body in the embodiment is the same as that of the embodiment 1, except that the seed culture medium comprises 15g/L glucose, 10g/L peptone, 5g/L beef extract, 15g/L agar, 3g/L yeast powder, 0.6g/L potassium dihydrogen phosphate and 0.3g/L calcium carbonate, and the pH value of the seed culture medium is 7.1 to 7.2; the fermentation medium comprises 25g/L of corn starch, 6g/L of yeast extract, 3g/L of peptone, 2g/L of beef extract, 0.5g/L of monopotassium phosphate, 1g/L of sodium bicarbonate, 0.2g/L of manganese sulfate and 0.1g/L of calcium chloride, and the pH value of the fermentation medium is 7.1-7.2.

Example 3

The preparation method of the biological preparation for improving the immune function of the animal body in the embodiment is the same as that of the embodiment 1, except that the seed culture medium comprises 13g/L glucose, 9g/L peptone, 4g/L beef extract, 14g/L agar, 2g/L yeast powder, 0.5g/L potassium dihydrogen phosphate and 0.25g/L calcium carbonate, and the pH value of the seed culture medium is 7.1 to 7.2; the fermentation medium comprises 23g/L of corn starch, 5g/L of yeast extract, 2g/L of peptone, 1.8g/L of beef extract, 0.4g/L of monopotassium phosphate, 0.7g/L of sodium bicarbonate, 0.15g/L of manganese sulfate and 0.06g/L of calcium chloride, and the pH value of the fermentation medium is 7.1-7.2.

Comparative example 1

The biological preparation for improving the immune function of the animal body in the comparative example is prepared by the same method as that of example 1, except that the seed culture medium comprises 9g/L glucose, 7g/L peptone, 2g/L beef extract, 10g/L agar, 0.5g/L yeast powder, 0.3g/L potassium dihydrogen phosphate and 0.1g/L calcium carbonate, and the pH value of the seed culture medium is 7.1 to 7.2; the fermentation medium comprises 18g/L of corn starch, 2g/L of yeast extract, 0.5g/L of peptone, 0.5g/L of beef extract, 0.1g/L of monopotassium phosphate, 0.4g/L of sodium bicarbonate, 0.05g/L of manganese sulfate and 0.03g/L of calcium chloride, and the pH value of the fermentation medium is 7.1-7.2.

Comparative example 2

The biological preparation for improving the immune function of the animal body in the comparative example is prepared by the same method as that of example 1, except that the seed culture medium comprises 16g/L glucose, 11g/L peptone, 6g/L beef extract, 17g/L agar, 5g/L yeast powder, 0.8g/L potassium dihydrogen phosphate and 0.4g/L calcium carbonate, and the pH value of the seed culture medium is 7.1 to 7.2; the fermentation medium comprises 30g/L of corn starch, 8g/L of yeast extract, 5g/L of peptone, 3g/L of beef extract, 0.6g/L of monopotassium phosphate, 2g/L of sodium bicarbonate, 0.3g/L of manganese sulfate and 0.2g/L of calcium chloride, and the pH value of the fermentation medium is 7.1-7.2.

Comparative example 3

In the comparative example, a clostridium butyricum strain standard (clostridium butyricum strain) is adopted for carrying out a control experiment, the clostridium butyricum strain standard is 9014-01-1, and the content of active substances is as follows: 100 percent, procurement to Weifang Rui Biotechnology Co., Ltd.

Clostridium butyricum in vitro bacteriostatic test

Performing plate culture on the clostridium butyricum biological preparation, inoculating the clostridium butyricum biological preparation into a liquid culture medium, culturing for 18-20 h under the anaerobic condition at the temperature of 32-34 ℃, taking escherichia coli, shigella, salmonella and clostridium perfringens as target bacteria, and determining the antibacterial activity of the clostridium butyricum biological preparation by using a tube-disc method, wherein the result is shown in table 1.

TABLE 1 Clostridium butyricum in vitro bacteriostatic test Effect

As can be seen from Table 1, the results show that the Clostridium butyricum biological agent has good bacteriostatic activity on Escherichia coli, Shigella, Salmonella and Clostridium perfringens.

Influence of clostridium butyricum biological preparation on production performance and diarrhea rate of weaned piglets

Test animals: 30 +/-1 day old Du X big X long ternary piglet

And (3) experimental design: the test adopts single factor design, 200 healthy piglets with basically consistent weight at 30 +/-1 day of age are selected and randomly divided into 5 groups. The control group is fed with basic ration, the example 2 and the comparative example 3 are selected for testing, the example 2 and the comparative example 3 are added on the basis of the basic ration, the adding amount is 500g/t, and the testing period is 30 d.

Feeding management: during the test period, the feed is fed at 08:00, 12:00 and 20:00 every day, the feeding amount is preferably small amount of residual feed in a trough, the feed and drinking water are freely taken and drunk in the whole period, and the immunization prevention and feeding management are carried out according to the conventional method. Diarrhea statistics is carried out at 15:00 every day, health conditions of piglets are observed, and the number of sick, dead and washed piglets and reasons of the sick and dead piglets are recorded.

Index measurement: recording the number of the piglets, diarrhea and death and elimination conditions of each repeated piglet every day, counting the feed intake and diarrhea rate once a week, and calculating the average daily feed intake; weighing the piglets on the day after the test is finished, counting the feed intake in the whole period, and calculating the feed-meat ratio in the whole period, the diarrhea rate and the death and culling rate of the piglets.

And (3) determining the cecal microorganisms: changes in the number of escherichia coli, salmonella, clostridium perfringens.

The test results are shown in tables 2 and 3.

TABLE 2 influence of Clostridium butyricum biologics on the productivity and diarrhea rate of weaned piglets

Numbering	Daily average weight gain (g/d)	Daily average feed intake (g/d)	Material to weight ratio	Diarrhea Rate (%)
					Control group	218.72	456.54	1.85	9.26
Example 2	252.33	427.12	1.43	4.59
					Comparative example 3	231.25	450.09	1.66	6.82

TABLE 3 influence of Clostridium butyricum biologics on the cecal flora of weaned piglets

Numbering	Escherichia coli (logcfu/g)	Salmonella (logcfu/g)	Clostridium perfringens (logcfu/g)
				Control group	6.73	6.65	8.41
Example 2	5.19	5.46	7.69
				Comparative example 3	5.91	6.24	8.15

As shown in tables 1 and 2, the addition of 0.1% of example 2 to the daily ration significantly reduced the diarrhea rate of weaned piglets, improved the productivity, and significantly reduced the number of Escherichia coli, Salmonella, and Clostridium perfringens in the cecum.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims (7)

1. The preparation method of the biological agent for improving the immune function of the animal body is characterized by comprising the following steps:

(1) activating clostridium butyricum strains;

(2) transferring activated clostridium butyricum strains into a fermentation tank filled with a seed culture medium, and culturing for 18-20 h at 32-34 ℃ to obtain a primary seed solution, wherein the clostridium butyricum strains in the primary seed solution account for 8 vt%, then transferring the primary seed solution into a seed tank filled with a fermentation culture medium, and culturing for 18-20 h at 32-34 ℃ to obtain a secondary seed solution, wherein the primary seed solution in the secondary seed solution accounts for 8 vt%;

(3) transferring the secondary seed solution into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 8% to obtain a culture solution, culturing the culture solution at 32-34 ℃ and 0.05-0.06 MPa for 18-20 h, controlling the pH value of the culture solution at 6.0-6.5 by adding a pH regulator in the culture process, intermittently stirring, and obtaining a fermentation liquid when the formation rate of spores is higher than 10% through microscopic examination;

(4) concentrating the fermentation liquor, namely concentrating the fermentation liquor by 5 times by using a disc centrifuge to obtain a fermentation concentrated solution;

(5) freeze-drying, namely adding a carrier protective agent into the fermentation concentrated solution and freeze-drying, wherein the addition amount of the carrier protective agent is 20g/L, and freeze-drying treatment is carried out in five stages, wherein the first stage is frozen for 4h at the temperature of minus 40 ℃, the second stage is frozen for 10h at the temperature of minus 10-40 ℃, the third stage is frozen for 8h at the temperature of minus 10-minus, the fourth stage is dried for 16h at the temperature of minus 30-plus, and the fifth stage is dried for 3h at the temperature of minus 30-plus 32 ℃ to obtain the clostridium butyricum biological preparation;

wherein the seed culture medium comprises 10 g/L-15 g/L glucose, 8 g/L-10 g/L peptone, 3 g/L-5 g/L beef extract, 12 g/L-15 g/L agar, 1 g/L-3 g/L yeast powder, 0.4 g/L-0.6 g/L monopotassium phosphate and 0.2 g/L-0.3 g/L calcium carbonate, and the pH value of the seed culture medium is 7.1-7.2;

the fermentation medium comprises 20-25 g/L of corn starch, 3-6 g/L of yeast extract, 1-3 g/L of peptone, 1-2 g/L of beef extract, 0.2-0.5 g/L of monopotassium phosphate, 0.5-1 g/L of sodium bicarbonate, 0.1-0.2 g/L of manganese sulfate and 0.05-0.1 g/L of calcium chloride, and the pH value of the fermentation medium is 7.1-7.2.

2. The method for preparing a biological agent for improving the immune function of an animal body according to claim 1, wherein the carrier protective agent in the step (5) comprises 3-5 g/L of trehalose, 6-8 g/L of soybean protein powder and 4-8 g/L of skimmed milk powder.

3. The method for preparing a biological agent for enhancing immune function of an animal according to claim 1, wherein the pH regulator in step (3) is 10 wt% potassium hydroxide solution.

4. The method for preparing the biological agent for improving the immune function of the animal body according to claim 1, wherein the fermentation medium is prepared in proportion and then sterilized at 121 ℃ for 30min, after the sterilization is finished, the temperature is reduced, nitrogen is introduced for pressure maintaining, the tank pressure in the fermentation tank is controlled to be 0.05-0.06 MPa, and the heated and activated secondary seed solution is inoculated when the temperature is reduced to the culture temperature.

5. The method for preparing the biological agent for improving the immune function of the animal body according to claim 1, wherein the water content of the clostridium butyricum biological agent in the step (5) is less than or equal to 5 percent.

6. The method for preparing a biological agent for improving immune function of an animal body according to claim 1, wherein the step (3) comprises the following steps: stirring for 1-2 min every 2h, and controlling the stirring speed to be 75-100 r/min.

7. Biological agent for improving the immune function of an animal body, characterized in that the biological agent of clostridium butyricum is prepared by the preparation method of any one of claims 1 to 6.