Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems of antibiotic residue, easy failure of vaccine immunity, poor prevention and treatment effect of the existing non-antibiotic additive and the like in the prior art, the invention provides the feed additive for treating piglet diarrhea.
The invention also provides a preparation method and application of the feed additive for preventing piglet diarrhea.
The technical scheme is as follows: in order to achieve the purpose, the invention provides a feed additive for preventing piglet diarrhea, which comprises the following raw materials in parts by weight: 8-12 parts of bacillus subtilis microcapsule, 8-12 parts of bacillus amyloliquefaciens microcapsule, 10-15 parts of astragalus polysaccharide, 5-8 parts of antimicrobial peptide No. 1, 5-8 parts of antimicrobial peptide No. 2 and 45-64 parts of corn flour.
Wherein the bacillus subtilis is bacillus subtilis JCL16 with a preservation number of CCTCC NO: and M2018336.
Wherein the bacillus amyloliquefaciens is the bacillus amyloliquefaciens CPLK1314 with the preservation number of CCTCC NO: M2017658.
Wherein the preparation of the bacillus subtilis microcapsule comprises the following steps: activating the bacillus subtilis preserved on the inclined plane, culturing the activated bacillus subtilis to obtain seed fermentation liquor, inoculating the seed fermentation liquor for fermentation, and collecting the fermentation liquor; centrifuging the fermentation liquor, removing supernatant, collecting and washing thallus precipitates, uniformly mixing the thallus precipitates with a sodium alginate solution, injecting the mixture into a calcium carbonate solution to obtain a bacterium-containing mixed solution, slowly adding the bacterium-containing mixed solution into soybean oil containing tween, stirring, slowly stirring, adding glacial acetic acid, standing, centrifugally deoiling, collecting microcapsules, washing to obtain wet capsules, and freeze-drying to obtain the freeze-dried bacillus subtilis microcapsules.
Wherein the preparation of the bevel bacillus amyloliquefaciens microcapsule comprises the following steps: activating strains of the bacillus amyloliquefaciens preserved on the inclined plane, culturing the activated bacillus amyloliquefaciens to obtain seed fermentation liquor, inoculating the seed fermentation liquor, fermenting and collecting the fermentation liquor; centrifuging the fermentation liquor, removing supernatant, collecting and washing thallus precipitates, uniformly mixing the thallus precipitates with a sodium alginate solution, injecting the mixture into a calcium carbonate solution to obtain a bacterium-containing mixed solution, slowly adding the bacterium-containing mixed solution into soybean oil containing tween, stirring, slowly stirring, adding glacial acetic acid, standing, centrifugally deoiling, collecting microcapsules, washing to obtain wet capsules, and freeze-drying to obtain the freeze-dried bacillus amyloliquefaciens microcapsule.
Preferably, the antimicrobial peptide No. 1 is obtained by separating fermentation liquor after fermentation culture of bacillus subtilis CCTCC NO: M2018336.
Preferably, 5-8 parts of the antimicrobial peptide 2 is obtained by separating fermentation liquor after fermentation culture of bacillus amyloliquefaciens CCTCC NO: M20117658.
The preparation method of the feed additive for preventing the piglet diarrhea comprises the following steps:
uniformly mixing the bacillus subtilis microcapsule, the bacillus amyloliquefaciens microcapsule, the astragalus polysaccharide, the antimicrobial peptide No. 1, the antimicrobial peptide No. 2 and the corn flour to obtain the feed additive for preventing piglet diarrhea.
The feed additive for preventing piglet diarrhea is applied to preparation of a feed or a medicament for preventing and treating piglet diarrhea.
Wherein 10g of additive is added into 500g of feed.
The invention provides a feed additive formula for preventing and treating piglet diarrhea, wherein bacillus subtilis and bacillus amyloliquefaciens are added into an organism to regulate a microecological flora structure in an intestinal tract of the organism and can activate the immune function of the organism, and antibacterial peptide has activities such as antibacterial activity, antiviral activity and the like, polysaccharide has an enhancement effect on the specific and nonspecific immune functions of the organism, and the three are singly used for treating piglet diarrhea, so that the three are mixed in proportion through experiments to obtain a better effect of preventing and treating piglet diarrhea.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the feed additive for preventing and treating piglet diarrhea has the advantages of high viable count, greenness, safety, no toxic or side effect and the like, can effectively prevent and treat piglet diarrhea, and reduces the use amount of antibiotics.
2. The bacillus subtilis and the bacillus amyloliquefaciens have the effects of improving the intestinal microbial environment, improving the immunity of organisms and the like, can be used as a vaccine adjuvant and a vaccine for combined use, and can promote the growth and development of piglets, enhance the immunity of the piglets, reduce the diarrhea rate and the like.
3. The astragalus polysaccharide, the bacillus subtilis and the antibacterial peptide have synergistic effect, jointly enhance the immune response of organisms, and improve the activity of immune cells to promote the increase of immune globulin of piglets, thereby improving the immunity of the piglets.
4. The bacillus subtilis and the bacillus amyloliquefaciens are wrapped in the microcapsule, so that the bacterium inactivation caused by gastric acid can be avoided, the bacterium can be released at a fixed point, and the bacterium survival rate is improved.
5. The preparation method is simple and efficient, has high bioavailability, and can realize better effect of preventing and treating piglet diarrhea
Detailed description of the invention
The present invention is further illustrated by the following examples.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.
The Bacillus subtilis strain JCL16 of the invention is preserved in China center for type microorganism preservation, the preservation time is 06 months 01 years in 2018, the preservation number is M2018336, and the strain is publicly preserved in the applicant's prior patent CN 111411053A.
The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens subsp. plantarum) strain CPLK131 is preserved in a China center for type microorganism preservation with the preservation time of 2017, 11 and 06 days, the preservation number is CCTCC NO: M2017658, and the strain is already publicly preserved in the applicant's prior patent CN 109182197A.
Slant culture medium: 10g of beef extract peptone, 5g of yeast extract, 2g of sodium chloride, 0.1g of glucose, 5g of sodium lactate and 20g of agar, and dissolving in 1000mL of distilled water.
Seed culture medium and fermentation medium: 10g of beef extract peptone, 5g of yeast extract, 2g of sodium chloride, 0.1g of glucose and 5g of sodium lactate, and the beef extract peptone, the yeast extract and the sodium chloride are dissolved in 1000mL of distilled water.
Example 1
And (3) preparing the bacillus subtilis microcapsule.
The well preserved bevel bacillus subtilis strain CCTCC NO: m2018336 is inoculated in LB slant culture medium, cultured for 24h at 37 ℃ and activated. Inoculating the activated bacillus subtilis strain into a sterilized LB seed culture medium, and culturing for 24h at 37 ℃ at 200r/min in a constant-temperature shaking incubator to obtain seed fermentation liquor; 50L of fermentation tank is filled with 35L of LB culture medium, the seed fermentation liquor is inoculated according to the inoculation amount with the volume fraction of 5 percent, and fermentation is carried outThe control conditions are as follows: the temperature is 37 ℃, the number of the living bacteria to be detected reaches 1.0 multiplied by 109At cfu/mL, the fermentation broth is collected.
Centrifuging the fermentation liquor for 15min at 5000r/min, removing the supernatant, collecting the thallus precipitate, washing the thallus precipitate for a plurality of times by using 0.9% sterile normal saline, uniformly mixing the thallus precipitate with 2.0% (w/v) of sodium alginate solution in a ratio of 1:4(w/v), and mixing the mixed solution of the bacillus subtilis and the sodium alginate by using a sterile syringe according to a ratio of 1: 3(v: v) is injected into 2.0% (w/v) calcium carbonate solution to obtain bacteria-containing mixed solution, then the bacteria-containing mixed solution is slowly added into soybean oil containing tween 1.0% (v/v) according to the volume ratio of 1:2, the soybean oil is stirred for 15min at the temperature of 4 ℃ at the stirring speed of 400r/min, then glacial acetic acid is added under slow stirring to adjust the pH value to 5.5, the mixture is kept stand for 2h, then centrifugal deoiling is carried out at 8000r/min, microcapsules are collected, wet capsules are obtained after 3 times of washing by physiological saline, the wet capsules are pre-frozen for 12h at the temperature of minus 20 ℃, then the wet capsules are put into a vacuum freeze dryer at the temperature of minus 60 ℃ for freeze drying for 48h, and the freeze-dried bacillus subtilis microcapsules are obtained.
Example 2
And (3) preparing the bacillus amyloliquefaciens microcapsule.
The well preserved strain of the slant bacillus amyloliquefaciens with CCTCC NO of M20117658 is inoculated in a slant LB culture medium and cultured for 24 hours at 37 ℃ to activate the strain. Inoculating the activated bacillus amyloliquefaciens strain into a sterilized seed LB culture medium, and culturing for 24 hours at 37 ℃ at 200r/min in a constant-temperature shaking incubator to obtain seed fermentation liquor; 50L of fermentation tank LB culture medium 35L, inoculating the seed fermentation liquor according to the inoculation amount of 5% of volume fraction, the fermentation control conditions are as follows: the temperature is 37 ℃, the number of the living bacteria to be detected reaches 1.0 multiplied by 109At cfu/mL, the fermentation broth is collected.
Centrifuging the fermentation liquor for 15min at 5000r/min, removing supernatant, collecting thallus precipitate, washing the thallus precipitate for several times by using 0.9% sterile normal saline, uniformly mixing the thallus precipitate with 2.0% (w/v) of sodium alginate solution in a ratio of 1:4(w/v), and mixing the mixed solution of the bacillus amyloliquefaciens and the sodium alginate by using a sterile injector according to a ratio of 1: 3(v: v) is injected into 2.0% (w/v) calcium carbonate solution to obtain bacteria-containing mixed solution, then the bacteria-containing mixed solution is slowly added into soybean oil containing tween 1.0% (v/v) according to the volume ratio of 1:2, the soybean oil is stirred for 15min at the temperature of 4 ℃ at the stirring speed of 400r/min, then glacial acetic acid is added under slow stirring to adjust the pH value to 5.5, the mixture is kept stand for 2h, then centrifugal deoiling is carried out at 8000r/min, microcapsules are collected, wet capsules are obtained after 3 times of washing by physiological saline, the wet capsules are pre-frozen for 12h at the temperature of minus 20 ℃, then the wet capsules are put into a vacuum freeze dryer at the temperature of minus 60 ℃ for freeze drying for 48h, and the freeze-dried bacillus amyloliquefaciens microcapsules are obtained.
Example 3
Preparation method of antibacterial peptide No. 1
The well preserved bevel bacillus subtilis strain CCTCC NO: m2018336 is inoculated in a slant culture medium, cultured for 24h at 37 ℃ and activated. Inoculating the activated Bacillus subtilis strain into sterilized seed culture medium, and culturing at 37 deg.C for 24 hr at 200r/min in a constant temperature vibration incubator to obtain seed fermentation liquid with bacteria content of 107-108cfu/mL; fermenting and culturing in a 50L fermentation tank, fermenting and canning 35L of culture medium in a 50L fermentation tank, inoculating the seed fermentation liquid according to the inoculation amount of 5 percent, wherein the fermentation control conditions are as follows: the temperature is 37 ℃, and the time is 120h, thus obtaining the fermentation liquor.
The fermentation broth was centrifuged at 5000r/min for 15min and the supernatant was adjusted to pH2.0 with concentrated hydrochloric acid and precipitated overnight. Centrifuging at 5000r/min for 25min to obtain precipitate, extracting the precipitate with 10% methanol for 2 times (2 hr each time), and mixing the methanol extracts. Concentrating the methanol extract under reduced pressure, and freeze drying to obtain antibacterial peptide No. 1.
Example 4
Preparation method of antimicrobial peptide No. 2
The well preserved strain of the slant bacillus amyloliquefaciens with CCTCC NO of M20117658 is inoculated in a slant LB culture medium and cultured for 24 hours at 37 ℃ to activate the strain. Inoculating the activated Bacillus amyloliquefaciens strain into sterilized seed LB culture medium, and culturing at 37 deg.C for 24 hr at 200r/min in a constant temperature shaking incubator to obtain seed fermentation liquid with bacteria content of 107-108cfu/mL; fermenting and culturing in a 50L fermentation tank, filling 35L LB culture medium in a 50L fermentation tank, inoculating the seed fermentation liquid according to the inoculation amount of 5 percent, wherein the fermentation control conditions are as follows: the temperature is 37 ℃, and the time is 120h, thus obtaining the fermentation liquor.
The fermentation broth was centrifuged at 5000r/min for 15min and the supernatant was adjusted to pH2.0 with concentrated hydrochloric acid and precipitated overnight. Centrifuging at 5000r/min for 15min to obtain precipitate, extracting the precipitate with 1/10 volume of methanol for 2 times, each for 2 hr, and mixing the methanol extracts. Concentrating the methanol extract under reduced pressure, and freeze drying to obtain antibacterial peptide No. 2.
Example 5
8 parts of a bacillus subtilis microcapsule; 12 parts of bacillus amyloliquefaciens microcapsules; 13 parts of astragalus polysaccharide; 5 parts of antibacterial peptide No. 1; 8 parts of antibacterial peptide No. 2; 54 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 6
12 parts of bacillus subtilis microcapsules; 8 parts of bacillus amyloliquefaciens microcapsules; 10 parts of astragalus polysaccharide; 8 parts of antibacterial peptide No. 1; 6 parts of antibacterial peptide No. 2; 56 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 7
10 parts of a bacillus subtilis microcapsule; 10 parts of bacillus amyloliquefaciens microcapsules; 15 parts of astragalus polysaccharide; 6 parts of antibacterial peptide No. 1; 5 parts of antibacterial peptide No. 2; 54 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 8
8 parts of a bacillus subtilis microcapsule; 8 parts of bacillus amyloliquefaciens microcapsules; 10 parts of astragalus polysaccharide; 5 parts of antibacterial peptide No. 1; 5 parts of antibacterial peptide No. 2; 45 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 9
12 parts of bacillus subtilis microcapsules; 12 parts of bacillus amyloliquefaciens microcapsules; 15 parts of astragalus polysaccharide; 8 parts of antibacterial peptide No. 1; 8 parts of antibacterial peptide No. 2; 64 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Test example 1
1. Test animals and test design
Selecting 140 healthy weaned piglets (28 +/-2) with day age, randomly dividing the selected piglets into 6 groups according to the principles of same breed and birth times, similar weight and half of the male parent and the female parent, and repeating for 2 times and 10 times for each group. The basal diets of each group were identical, and the trial started with 28 days old weaned piglets, for a period of 28 days.
The feed is fed by mixing the powder with water, and the feed is prepared by adding water and mixing with water. The piglets are fed regularly, and the feeding time is respectively 8, 12, 16 and 20 points per day. Piglets had free access to water during the trial period and piggery management was performed as per the trial piggery routine.
Continuously observing the defecation condition of piglets from 7:00 in the morning to 21:00 in the evening every day, carrying out sensory evaluation on the feces discharged by each piglet every time, counting the feces condition of the piglets recorded every day (according to 0-3 grade records, 0 grade-dry, 1 grade-soft, 2 grade-dilute and 3 grade-water samples, wherein 0 grade and 1 grade are normal, and 2 grade and 3 grade are diarrhea), calculating the diarrhea rate, and recording and evaluating the growth performance index ratios of feed intake, daily gain, feed meat and the like. The diarrhea rate (diarrhea rate ═ number of diarrhea heads/(number of piglets × number of test days) ] × 100%) and the final statistics were calculated as shown in table 1:
2. test feed
Group A: basal ration, commercially available, for routine feed (containing only conventional ingredients) for piglets.
Group B: conventional feed the additive of example 5 was added at 10g per 500g feed.
Group C: conventional feed the additive of example 6 was added at 10g per 500g feed.
Group D: conventional feed the additive of example 7 was added at 10g per 500g feed.
Group E: conventional feed additive (except for astragalus polysaccharide, the other components are the same as in example 5), and 10g of the additive is added into every 500g of feed.
And F group: conventional feed additives (except for Bacillus subtilis and Bacillus amyloliquefaciens, and the other components are the same as in example 5) are added into every 500g of feed by 10 g.
Group G: the conventional feed additive (except for antimicrobial peptide No. 1, the other components are the same as in example 5), and 10g of the additive is added to 500g of feed.
Group H: the conventional feed additive (except for antimicrobial peptide No. 2, the other components were the same as in example 5), and 10g of the additive was added per 500g of the feed.
Group I: the conventional feed additive (except for antimicrobial peptide No. 1 and antimicrobial peptide No. 2, the rest components are the same as in example 5), and 10g of the additive is added to 500g of feed.
Group J: conventional feed additive (Bacillus subtilis, Bacillus amyloliquefaciens culture fluid is centrifuged to obtain bacterial mud, the mud is not made into microcapsules, the mud is freeze-dried and added into the additive, and the rest components are the same as the components in the example 5), and 10g of the additive is added into 100g of feed.
TABLE 1 Effect of feed additives on piglet diarrhea
As can be seen from Table 1, the feed additive for preventing diarrhea of piglets has the obvious effect of reducing the diarrhea rate of the piglets. The diarrhea rate of the group E, the group F, the group G, the group H and the group I is far higher than that of the group B, C, D, which indicates that certain synergistic effect exists between astragalus polysaccharide and bacillus subtilis and bacillus amyloliquefaciens. The experimental results of group G show that the diarrhea rate of the two bacteria which are not prepared into the microcapsule is higher than that of the two bacteria which are prepared into the capsule, and the two bacteria are wrapped in the microcapsule, so that the inactivation of the viable bacteria can be reduced.