CN113801825A - Bacillus subtilis for producing active peptide and application thereof - Google Patents

Bacillus subtilis for producing active peptide and application thereof Download PDF

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Publication number
CN113801825A
CN113801825A CN202111225475.7A CN202111225475A CN113801825A CN 113801825 A CN113801825 A CN 113801825A CN 202111225475 A CN202111225475 A CN 202111225475A CN 113801825 A CN113801825 A CN 113801825A
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bacillus subtilis
active peptide
fermentation
producing
feed additive
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黄贞胜
庄淑芳
谢晓露
林斌
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Xiamen Haijiacheng Biotechnology Co ltd
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Xiamen Haijiacheng Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus subtilis for producing active peptide and application thereof, wherein the bacillus subtilis is preserved in the Guangdong province microbial strain preservation center, the preservation date is 2021 year, 9 months and 9 days, and the preservation number is GDMCC No. 61925.

Description

Bacillus subtilis for producing active peptide and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus subtilis for producing active peptide and application thereof.
Background
Growth promoting health agents such as antibiotics, chemical synthetic drugs, hormones and the like are widely used in animal feed and breeding for many years, so that the serious drug residue in livestock and aquatic products, the quality reduction of the livestock and poultry products, the enhancement of the drug resistance of bacteria and the increase of drug-resistant strains are caused, and great threat is brought to the safety and the ecological environment of human beings. Due to the side effects of antibiotics, especially the concern of consumers on food safety in recent years, all countries in the world forbid or limit the use of antibiotics in many times, and accelerate the development and popularization of novel safe and efficient green feed additives so as to reduce or replace pharmaceutical additives such as antibiotics.
Due to the unique physical and chemical properties and biological functions, the microecologics become a research hotspot in the field of novel green feed additives. At present, soybean peptide is seen in domestic markets, is mostly imported products, is high in price and is mainly produced by an enzyme method. The enzyme method is to degrade soybean protein into small peptide by using protease such as plant protease like papain, bromelain and the like or animal protease like trypsin, pepsin and the like, but the soybean peptide produced by the method is easy to generate unacceptable fishy smell or bitter taste, has high production cost and is not suitable for being applied in animal husbandry; compared with the enzyme method, the microbial fermentation method can combine the enzyme production and the enzymolysis of the microorganisms into a whole, shortens the working procedures, reduces the cost, and the soybean peptide product has natural fragrance and good taste.
The bacillus subtilis can generate a large amount of protease, amylase, lipase and other beneficial factors in the metabolic process, can be quickly colonized after entering an organism, forms a good micro-ecological environment in intestinal tracts, and has the effects of enhancing the digestion capacity of animals, degrading anti-nutritional factors, increasing the utilization rate of feed, enhancing the immunity of the animals and the like. The feed-grade microbial additive strains which are published by the ministry of agriculture in China and can be directly fed to animals comprise bacillus subtilis, which is taken as safe probiotics and is widely applied to the field of feeds.
Disclosure of Invention
Therefore, the bacillus subtilis for producing the active peptide and the application thereof are needed, and the active peptide produced by fermenting the bacillus subtilis can improve the absorption and utilization rate of animals on nutrient elements in feed and improve the yield and production capacity of milk, meat and eggs.
In order to achieve the purpose, the invention provides the active peptide producing bacillus subtilis which is preserved in Guangdong province microorganism culture collection center, wherein the preservation date is 2021, 9 months and 9 days, and the preservation number is GDMCC No. 61925.
The application of the bacillus subtilis for producing the active peptide is applied to the fermentation preparation of the active peptide bacillus subtilis feed additive.
Further, the preparation of the active peptide bacillus subtilis feed additive comprises the following steps:
(1) selecting an activated Bacillus subtilis from nutrient agar slant culture medium, inoculating to seed culture medium at 25-35 deg.C and 100--1Obtaining seed liquid after 12-36 h;
(2) taking vegetable protein as a fermentation raw material, adding water to prepare the fermentation raw material with the weight percentage concentration of 5-20%, adjusting the pH value to 6.0-7.0, then performing sterilization treatment at the high temperature of 121 ℃, and cooling to 30-35 ℃ to obtain a liquid fermentation culture medium;
(3) inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 2-4%, and fermenting in a fermentation tank at 35 + -2 deg.C for 24-48 hr;
(4) and (4) uniformly mixing the fermentation liquor obtained in the step (3) with a carrier, and performing spray drying to obtain the active peptide bacillus subtilis feed additive. And carrying out spray drying on hypha and the active peptide in the fermentation liquor together, so that the prepared feed additive comprises the active peptide and bacillus subtilis.
Further, the formula of the seed culture medium is as follows: yeast extract 4-10g/L, sodium chloride 3-10g/L, peptone 5-10g/L, and distilled water in balance, and pH is 7.0.
Further, the vegetable protein is soy protein and/or gluten.
Further, the soybean protein comprises soybean protein isolate, soybean meal, soybean cake and soybean, and the gluten comprises corn protein, corn protein powder and wheat protein.
Further, the carrier is dextrin and/or starch.
Furthermore, the dextrin is at least one of maltodextrin and cyclodextrin, and the starch is at least one of corn starch, wheat flour and modified starch.
Furthermore, the addition amount of the active peptide feed additive in the feed is 100-500 g/t.
The technical scheme has the following beneficial effects:
in the invention, the screened bacillus subtilis strain can produce active peptide with high yield by fermentation, and the fermentation liquor and the hypha are sprayed and dried together to be used as a feed additive, the active peptide feed additive can effectively improve the absorption and utilization rate of the animal to nutrients (such as protein, mineral substances, vitamins and the like), improve the yield and the production capacity of milk, meat and eggs, improve the quality and the flavor of the milk, the meat and the eggs, stimulate the development of immune organs of the animal, improve the immunity and reduce the morbidity, can be widely used for economic animals such as poultry, cattle, sheep, pigs, aquatic products and the like, and can be used as a high-quality active peptide bacillus subtilis feed additive such as a substitute of antibiotics and the like.
Drawings
FIG. 1 is a growth curve of a strain selected from Vibrio parahaemolyticus antagonistic strains.
FIG. 2 is a diagram of the inhibition zone of screened active peptide of the strain to antagonize Vibrio parahaemolyticus.
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
In the case of the example 1, the following examples are given,
the mutagenesis and screening method of bacillus subtilis for producing active peptide comprises the following steps:
1. the strain is screened by using collected soil to prepare bacterial suspension, 200ul of the bacterial suspension is evenly coated on an LB solid culture medium flat plate in a sterile environment, and the strain is cultured for 24 hours in a constant temperature incubator at 37 ℃.
LB solid medium: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, 15g of agar powder and 1L of distilled water, adjusting the pH to 7.3 +/-0.2, and carrying out autoclaving at 121 ℃ for 20 min.
2. After the surface of the culture medium is full of single colonies through strain purification and identification, suspected single colonies of the bacillus subtilis are picked according to the appearance characteristics of the strains and are respectively inoculated into an LB liquid culture medium, and the single colonies are cultured for 18 hours at the temperature of 37 ℃ and 200r/min through a shaking table.
Diluting the cultured bacterial liquid to 10-2，10-3And (3) gradient concentration, respectively sucking 100ul of the bacterial suspension, coating the bacterial suspension on an LB solid culture medium plate, and culturing in a constant temperature incubator at 37 ℃ for 24 h. Obtaining a purified single colony, carrying out PCR amplification on the single colony, then carrying out sequencing identification, and selecting colony bacteria liquid determined to be bacillus subtilis to store in a refrigerator at the temperature of-20 ℃ for later use.
LB liquid medium: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride and 1L of distilled water, adjusting the pH to 7.3 +/-0.2, and carrying out autoclaving at 121 ℃ for 20 min.
3. Screening and selecting single bacillus subtilis colonies of the vibrio parahaemolyticus antagonistic strain, respectively inoculating the single bacillus subtilis colonies into an LB liquid culture medium, and culturing for 18h at the temperature of 37 ℃ in a shaking table and at the speed of 200 r/min. Inoculating the bacillus subtilis and the fermentation supernatant to an LB solid culture medium coated with vibrio parahaemolyticus, and culturing in a constant-temperature incubator at 37 ℃ for 24 hours. Selecting the strain having antagonism with the vibrio parahaemolyticus according to the growth condition of the strain and the formation condition of the inhibition zone.
4. Measuring the strain growth curve, respectively taking strains having antagonistic action with Vibrio parahaemolyticus, inoculating in 100ml LB liquid culture medium, and shake culturing at 37 deg.C and 230 r/min. Drawing the growth curve of each bacillus subtilis strain by an absorptiometry method, sampling by a liquid-moving machine every 2h, measuring the OD value with the wavelength of 600nm, recording, continuously measuring for 24h, and drawing the growth curve of the strain (shown in figure 1).
5. Preparation of mutant strain suspension according to growth curve of strain, taking 2ml of bacterial liquid at 4000r/min in logarithmic phase of strain, centrifuging for 5min, washing with sterile water for 2 times, discarding supernatant, adding sterile water again to obtain bacterial suspension, measuring bacterial number with blood counting plate, diluting with sterile water in gradient manner to obtain strain with bacterial number of 108The left and right bacterial suspensions were used for subsequent use.
EMS mutagenesis and ethionine-resistant plate selection in step 5 108Adding 0.5mol/L EMS into the bacterial suspension, shaking at 25 ℃ on a shaker and 200r/min for 50min, and adding 6 percent sodium thiosulfate solution of the final volume to terminate the reaction. 4000r/min, centrifuging for 5min, discarding the supernatant, washing with sterile water for 2 times, diluting, spreading on 3.0g/L ethionine resistant plate, and culturing in a constant temperature incubator at 37 deg.C for 24 h.
Ethionine-resistant plates: adding the filter-sterilized ethionine solution into the sterilized LB solid culture medium.
7. The vibrio parahaemolyticus antagonism screening selects a bacterial colony growing on an ethionine plate, after liquid culture, the thallus and a fermentation supernatant are inoculated on a solid culture medium coated with the vibrio parahaemolyticus, and the solid culture medium is cultured for 24 hours in a constant temperature incubator at 37 ℃.
UV mutagenesis and ethionine resistant plate screening the strain plate antagonistic to Vibrio parahaemolyticus in step (7) was subjected to mutagenesis for 90s at a light source of 8wUV cm. Continuously culturing the mutagenized plate, selecting the strain, and inoculating the strain in an LB liquid culture medium for shake culture.
9. And (3) carrying out antagonistic screening on vibrio parahaemolyticus, namely centrifuging the bacterial liquid cultured in the step (8), inoculating the thallus and the fermentation supernatant on a solid culture medium coated with the vibrio parahaemolyticus, and culturing for 24h in a constant-temperature incubator at 37 ℃. Selecting strains with large antagonism, performing amplification culture with LB liquid culture medium, and preserving at-20 deg.C.
10. The filtration of the passage stability of the bacillus subtilis is to cultivate the selected bacillus subtilis by a shaking table, inoculate the thallus and the fermentation supernatant on a solid culture medium coated with the vibrio parahaemolyticus, continuously carry out generations, detect the stability of the antagonistic vibrio parahaemolyticus, and select a strain with a bacteriostatic zone, as shown in figure 2.
The selected Bacillus subtilis strain is preserved in Guangdong province microorganism culture collection center with the preservation date of 2021, 9 months and 9 days, and the preservation number is GDMCC No. 61925.
Example 2
The method for producing the bacillus subtilis feed rich in active peptide by fermenting the bacillus subtilis with the preservation number of GDMCC No.61925 comprises the following steps:
the preparation of the active peptide bacillus subtilis feed additive comprises the following steps:
(1) selecting an activated Bacillus subtilis from nutrient agar slant culture medium, inoculating to seed culture medium at 25-35 deg.C and 100--1And obtaining a seed solution within 12-36h, wherein the formula of a seed culture medium is as follows: yeast extract 4-10g/L, sodium chloride 3-10g/L, peptone 5-10g/L, and distilled water in balance, wherein the pH is 7.0;
(2) taking vegetable protein as a fermentation raw material, adding water to prepare the fermentation raw material with the weight percentage concentration of 5-20%, adjusting the pH value to 6.0-7.0, then performing sterilization treatment at the high temperature of 121 ℃, and cooling to 30-35 ℃ to obtain a liquid fermentation culture medium;
(3) inoculating the planting solution into a liquid fermentation culture medium according to the inoculation amount of 2-4%, stirring and fermenting, wherein the temperature is kept at 35 +/-2 ℃ during fermentation, and the fermentation period is 24-48 hours;
(4) and (4) uniformly mixing the fermentation liquor obtained in the step (3) with a carrier, and performing spray drying to obtain a finished product of the active peptide bacillus subtilis feed additive.
The carrier of this example is a mixture of maltodextrin and corn starch.
In this example, soy protein isolate and zein were selected as fermentation raw materials.
Example 3
The difference from the example 2 is that the soybean meal, the soybean cake and the corn protein powder are selected as the fermentation raw materials.
Example 4
The difference from example 2 is that soybean and wheat protein were used as fermentation raw materials.
Example 5
The difference from the example 2 is that cyclodextrin wheat flour and modified starch are used as carriers.
Example 6
160 21-day-old weaned piglets were randomly divided into 4 groups of 4 replicates each, 10 replicates each, with a trial period of 4 weeks. The groups of tests were treated as follows:
test group 1: commercial feed +100g/t of the active peptide Bacillus subtilis feed additive of example 2;
test group 2: commercial feed +300g/t of the active peptide Bacillus subtilis feed additive of example 2;
test group 3: commercial feed +500g/t of the active peptide Bacillus subtilis feed additive of example 2;
control group: commercial feed;
weighing the weight of each group of piglets with an empty stomach in the morning at the beginning and the end of the test, counting the feed intake of each repeated group of pigs in the test process, and strictly recording the number of diarrhea pigs in each treatment group, wherein the influence on the growth performance of the piglets is shown in table 1.
The diarrhea rate (%). sigma (number of diarrhea piglets × number of diarrhea days)/(total number of piglets × total number of test days) × 100%.
TABLE 1 Effect of active peptide Bacillus subtilis feed additive on growth Performance of weaned piglets
Figure BDA0003313753190000071
As can be seen from Table 1, when the active peptide Bacillus subtilis is added into the feed, the average daily gain is obviously increased compared with the control group, wherein the average daily gain effect of the test group 2, namely the test group added with 300g/t of the active peptide Bacillus subtilis to the basic feed, is most obvious, and the feed conversion ratio is smaller than that of the other three test groups. The diarrhea rate of the test group added with the active peptide bacillus subtilis is obviously lower than that of the control group, and the diarrhea rate of the test group 2 is the lowest. Therefore, the active peptide bacillus subtilis feed additive disclosed by the invention can effectively improve the metabolic absorption of animals on nutrient components in the feed, and can also improve the immunity of the animals and reduce the occurrence of diarrhea.
Example 7
600 laying hens with similar laying rate at 37 weeks are randomly divided into 3 groups of 200 laying hens, each group is tested for 2 months, and the treatment of each group is as follows:
test group 1: commercial chicken feed +100g/t of the active peptide bacillus subtilis feed additive of example 2;
test group 2: commercial chicken feed +300g/t of the active peptide bacillus subtilis feed additive of example 2;
test group 3: commercial chicken feed +500g/t of the active peptide Bacillus subtilis feed additive of example 2;
control group: commercial chicken feed;
in the test process, the chickens are fed for 3 times a day, the health of the chickens is observed, the daily egg laying amount, the egg weight and the feed intake in the test period are recorded, and the egg laying rate, the feed-egg ratio and the average egg weight are calculated. The effect of use is shown in table 2.
TABLE 2 Effect of active peptide Bacillus subtilis feed additive in layer chicken feeding
Test group 1 Test group 2 Test group 3 Blank control group
Laying rate (%) 80.27±2.16 89.03±2.53 82.36±2.13 78.08±3.8
Feed egg ratio (%) 2.62±0.08 2.24±0.06 2.52±0.07 2.91±0.2
Average egg weight (g) 63.62±0.94 64.58±0.83 63.91±0.53 63.46±0.95
As can be seen from Table 2, the active peptide Bacillus subtilis feed additive prepared by the invention can improve the conversion utilization rate of the laying hens on the feed and also obviously improve the laying rate of the laying hens. In the test group 2, the effect of adding 300g/t of active peptide bacillus subtilis is most obvious.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.
Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.

Claims (9)

1. The bacillus subtilis for producing the active peptide is characterized by being preserved in Guangdong province microbial strain collection center with the preservation date of 2021, 9 months and 9 days and the preservation number of GDMCC No. 61925.
2. The use of the active peptide-producing bacillus subtilis strain of claim 1 in the preparation of a feed additive comprising the active peptide-producing bacillus subtilis strain by fermentation.
3. The use of bacillus subtilis for producing an active peptide as claimed in claim 2, wherein the preparation of the active peptide bacillus subtilis feed additive comprises the steps of:
(1) pick a ringThe activated Bacillus subtilis of claim 1, inoculated into a seed medium at a temperature of 25-35 ℃ and a pressure of 100--1Obtaining seed liquid after 12-36 h;
(2) taking vegetable protein as a fermentation raw material, adding water to prepare the fermentation raw material with the weight percentage concentration of 5-20%, adjusting the pH value to 6.0-7.0, then performing sterilization treatment at the high temperature of 121 ℃, and cooling to 30-35 ℃ to obtain a liquid fermentation culture medium;
(3) inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 2-4%, and fermenting, wherein the temperature is kept at 35 +/-2 ℃ during fermentation, and the fermentation period is 24-48 hours;
(4) and (4) uniformly mixing the fermentation liquor obtained in the step (3) with a carrier, and performing spray drying to obtain the active peptide bacillus subtilis feed additive.
4. The use of bacillus subtilis for producing active peptide according to claim 3 wherein the seed medium is formulated as: yeast extract 4-10g/L, sodium chloride 3-10g/L, tryptone 5-10g/L, and distilled water in balance, and the pH is 7.0.
5. Use of bacillus subtilis for the production of active peptides according to claim 3 wherein said vegetable protein is soy protein and/or gluten.
6. The use of claim 3 comprising Bacillus subtilis for the production of active peptides, wherein the soy protein comprises soy protein isolate, soybean meal, soybean cake, soy, and the gluten comprises zein, zein flour, and wheat protein.
7. The use of bacillus subtilis for the production of an active peptide according to claim 3 wherein said carrier is dextrin and/or starch.
8. The use of bacillus subtilis for producing an active peptide according to claim 7 wherein said dextrin is at least one of maltodextrin and cyclodextrin, and said starch is at least one of corn starch, wheat flour and modified starch.
9. The use of claim 2 comprising active peptide-producing Bacillus subtilis as a feed additive in an amount of 100-500 g/t.
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Cited By (1)

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CN115553402A (en) * 2022-10-25 2023-01-03 山东深海生物科技股份有限公司 Biological polypeptide preparation capable of improving nonspecific immunity function of haliotis discus hannai and application thereof

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Application publication date: 20211217