CN115094012B - Preparation method and application of bacillus coagulans BC-HYC strain microbial inoculum - Google Patents
Preparation method and application of bacillus coagulans BC-HYC strain microbial inoculum Download PDFInfo
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Abstract
The new separated bacillus coagulans BC-HYC strain has the stress resistance of heat resistance, artificial stomach resistance, intestinal juice resistance, bile salt resistance and the like, and has better stress resistance compared with the bacillus coagulans BC-CUI strain separated by the inventor in the early stage. Further, the bacillus coagulans BC-HYC strain is prepared into a microbial inoculum which can be used for producing fermented feed, the prepared fermented feed is acidic, the content of small peptides is relatively higher, the average daily gain value and the average feed intake of animals can be improved by adding the biological fermented feed on the basis of basic daily ration, the feed conversion ratio is reduced, and the economic benefit of cultivation is improved; in addition, the microbial inoculum can be used for preventing diarrhea of piglets, producing feeds and feeding weaned piglets, and can also obviously improve daily average weight gain and feed intake of the weaned piglets, reduce feed-meat ratio and improve the economic benefit of cultivation.
Description
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a bacillus coagulans BC-HYC strain microbial inoculum.
Background art:
from No. 1 of 7 months in 2020, feed production enterprises suspend production of commercial feeds containing growth-promoting drug feed additives (except traditional Chinese medicines), commonly called resistance banning orders, and mark that China comprehensively enters a new age of feed resistance-free. The issue and implementation of resistance banning causes a great number of phenomena in the feed market, and how to smoothly transition to the nonreactive era under the background of complicated animal epidemic diseases, especially the normalization of African swine fever, has become the focus and challenge of the attention of the whole animal husbandry industry, various replacement products are emerging, the animal husbandry biological industry taking biotechnology as the leading factor is accelerating to form, and the high-efficiency and rapid development of the animal husbandry is promoted. The microbial feed additive is considered as one of potential substitutes of antibiotics, and has the effects of improving growth performance, regulating immune response, balancing intestinal microbial flora and the like. Lactic acid bacteria are used as probiotic strains in livestock and poultry breeding for a long time, wherein lactobacillus acidophilus, lactobacillus plantarum, enterococcus faecalis, bifidobacterium and the like are more used. The yeast can produce fragrance, so that the palatability is improved, but the preparation has poor stress resistance and low survival rate of live bacteria, and the feeding effect of the preparation is influenced. The bacillus probiotics can be directly added into the compound feed due to strong spore stress resistance.
Bacillus coagulans is a facultative anaerobic gram-positive bacterium, belonging to the genus Bacillus. The surface of the thallus has no flagellum, is in hair shape, has the length of about 2.5-5.0 μm and the width of about 0.6-1.0 μm; spore formation, such as hammer; prefers neutral pH bias acid, and the optimum temperature is 37-45 deg.C. During the growth process, various saccharides can be utilized to generate L-lactic acid through homolactic fermentation, the L-lactic acid has the same health care effect as lactic acid bacteria, and has the characteristics of acid resistance (pH is more than 2.42), heat resistance (80 ℃), salt resistance (1-8% NaCl), easy culture and storage resistance of bacillus. Bacillus coagulans bacterium (A), (B) and (C)Bacillus coagulans) Is bacillus probiotics secreting lactic acid, has the characteristics of bacillus and the advantages of lactic acid bacteria due to the dual property, has been approved by countries including America, european Union and the like as a novel probiotic,the use of the bacillus coagulans has been approved in 2004 in China, and the bacillus coagulans is widely applied to food, animal husbandry, medicine industry and aquaculture industry. In recent years, bacillus coagulans is a research hotspot of feed microbial additives as probiotic lactic acid bacteria capable of forming spores due to good stress resistance and probiotic characteristics, and is formally listed in feed additive variety catalog in agricultural rural departments in 2013. Many research results show that the bacillus coagulans has various biological characteristics, such as antibiosis, intestinal flora balance regulation, anti-inflammation, organism immunity improvement, digestion and absorption promotion, piglet diarrhea improvement and the like, and the single bacillus coagulans added into the feed can obviously improve the growth performance of livestock and poultry by inhibiting main intestinal harmful bacteria such as escherichia coli, salmonella and the like; research also shows that the bacillus coagulans can improve the breeding benefit of the laying fowl by adjusting the balance of intestinal flora, promoting the intestinal health, absorbing nutrient substances and the like. The bacillus coagulans has the functions of regulating the flora balance in intestinal tracts, promoting nutrient metabolism and improving immunity of common lactic acid bacteria, and also has the biological characteristics of bile salt resistance, gastrointestinal digestive juice resistance, heat stability and the like. The bacillus coagulans is added into animal feed, the bacillus coagulans is in a sporulated thallus form, can smoothly pass through granulation conditions and an animal gastric juice environment and enter duodenum, then spores germinate to form a nutrient body, and then the nutrient body enters small intestine to start to rapidly grow and reproduce, and exert biological functions. Therefore, it is a great topic of great national attention to develop strains with strong stress resistance and beneficial animal health, enrich the resource library of bacillus coagulans, and develop effective probiotic preparations.
The invention content is as follows:
in order to solve the technical problems, the invention aims to provide a bacillus coagulans microbial inoculum and a preparation method and application thereof.
The invention adopts the following technical scheme:
the invention provides a bacillus coagulans microbial inoculum, wherein the bacillus coagulans is a bacillus coagulans BC-HYC strain, is preserved in China general microbiological culture Collection center at 23.02/23.2022, and has the following names in terms of the names: bacillus coagulansBacteriaBacillus coagulansThe preservation number is: CGMCC No.24423.
The invention also claims a preparation method of the bacillus coagulans microbial inoculum, which comprises the steps of strain activation, preparation of primary seed liquid, preparation of secondary seed liquid, preparation of fermentation liquid and spray drying, and is characterized in that the bacillus coagulans is a bacillus coagulans BC-HYC strain, is preserved in the China general microbiological culture Collection center at 23.02/23.2022, and has the following names in terms of the classification names: bacillus coagulansBacillus coagulansThe preservation number is: CGMCC No.24423.
Preferably, the preparation method of the bacillus coagulans microbial inoculum is characterized by comprising the following steps of:
(1) Activating strains: streaking a strain plate of the preserved bacillus coagulans BC-HYC strain to an MRS plate culture medium, and culturing for 24h in an incubator at 42 ℃ to activate the preserved strain;
(2) Preparing a first-level seed solution: picking single colony from plate, inoculating in triangular flask containing MRS liquid culture medium, shake culturing at 42 deg.C, culturing at 120rpm for 12 hr to obtain OD 600 The value is more than or equal to 0.8, and first-stage seed liquid is obtained;
(3) Preparing a secondary seed solution: inoculating the prepared first-stage seed liquid into MRS liquid culture medium according to the volume ratio of 1%, performing shake culture at 42 deg.C, and culturing at 120rpm for 8 hr to make OD 600 The value is more than or equal to 0.7, and secondary seed liquid is obtained;
(4) Preparing fermentation liquor: inoculating the secondary seed liquid into a fermentation culture medium according to the volume ratio of 2%, then placing the secondary seed liquid into a production tank for fermentation, filtering after 12h of fermentation, and performing gradient dilution on the filtrate to obtain the secondary seed liquid with the concentration of more than or equal to 10 8 cfu/g strain liquid;
(5) Adding 25% zeolite powder into the prepared fermentation liquid, mixing, and spray drying to obtain product with water content of less than or equal to 5% and viable count of more than or equal to 1 × 10 10 CFU/g of bacterial powder.
Wherein the fermentation medium in the step (4) comprises 1% of peanut cake powder, 2% of soybean meal, 2% of corn flour, 0.1% of dipotassium phosphate, 0.02% of magnesium sulfate heptahydrate, 2% of molasses and the balance of water, the percentages are mass volume percentages, the pH value is 6.5-7.0, and the fermentation medium is sterilized by high-pressure steam at 121 ℃ for 20 minutes.
The invention also claims a method for preparing fermented feed by using the microbial inoculum, which is characterized by comprising the following steps: the preparation method of the fermented feed comprises the following steps:
firstly, crushing corn flour, bran, bean pulp, rice bran and zeolite powder in advance, screening the crushed corn flour, bran, bean pulp, rice bran and zeolite powder by a 1.5mm sieve, and then uniformly mixing the corn flour, the bran, the bean pulp, the rice bran and the zeolite powder according to the weight ratio of 10;
step two, weighing 50 parts by weight of water, and dissolving the bacillus coagulans microbial inoculum described in the embodiment 4 in the water to obtain a fermentation agent, wherein the microbial inoculum is weighed according to 1g/kg of fermentation base material;
and step three, uniformly mixing the leavening agent and the fermentation base material, carrying out aerobic fermentation for 72 hours at the temperature of 40 ℃ and the air humidity of 60-65%, and then drying until the water content is less than or equal to 8% to obtain the fermented feed.
The invention also provides a fermented feed, which is characterized by being prepared by the method.
The invention also provides application of the microbial inoculum in preparation of feed, which is characterized in that the microbial inoculum is added into the feed, so that the content of bacillus coagulans BC-HYC strain powder in the feed is more than or equal to 10 11 CFU/kg。
Based on the technical scheme, the invention has the following advantages and beneficial effects:
the bacillus coagulans BC-HYC strain newly separated by the invention has the stress resistance of heat resistance, artificial stomach resistance, intestinal juice resistance, bile salt resistance and the like, and has better stress resistance compared with the bacillus coagulans BC-CUI strain separated by the inventor in the early stage. Further, the bacillus coagulans BC-HYC strain is prepared into a microbial inoculum which can be used for producing fermented feed, the prepared fermented feed is acidic, the content of small peptides is relatively higher, the average daily gain value and the average feed intake of animals can be improved by adding the biological fermented feed on the basis of basic daily ration, the feed conversion ratio is reduced, and the economic benefit of cultivation is improved; in addition, the microbial inoculum can be used for preventing piglet diarrhea, producing feeds and feeding weaned pigs, can also obviously improve daily weight gain and feed intake of the weaned pigs, reduce feed-meat ratio and improve the economic benefit of cultivation.
The attached drawings of the specification:
FIG. 1: separation of bacillus coagulans BC-HYC strain: a, a gram staining result graph; and B, streaking the plate for purification.
The specific implementation mode is as follows:
example 1.
And (3) separating and identifying the BC-HYC strain of the bacillus coagulans.
Isolation of B.coagulans BC-HYC Strain:
5g of intestinal contents of healthy chicks bred in a laying hen farm of Beijing Hongyao scientific and technological development Limited company are taken, placed in 100mL of sterilized enrichment medium (1 g/L potassium sorbate is added in MRS liquid medium and 10g/L alpha-methyl glucoside is added), placed in a constant temperature incubator at 42 ℃ for culture for 72h, and shaken once every 24h to obtain enrichment liquid. Performing water bath on the enriched solution at 90 ℃ for 15min, performing 10-time gradient dilution, selecting 10-time, 100-time and 1000-time dilutions, respectively sucking 100 mu L of the dilutions, coating in an MRS plate, repeating 3 dilutions, and performing inverted culture at 42 ℃ for 24h. The suspected colony is picked for gram staining, microscopic examination is carried out, a gram-positive rod-shaped spore-forming bacterium is obtained by separation, the bacterium is subjected to plate streaking for 3 times to purify the strain, and a single colony is taken for preservation (shown in figure 1).
Identification of Bacillus coagulans BC-HYC strain:
1.2.1 Biochemical identification:
according to Bergey's Manual of bacteria identification and ' Manual of common bacteria identification ', bacillus coagulans BC-HYC strain is subjected to biochemical tests such as indole test, growth at 55 ℃, growth of 7% sodium chloride, citrate utilization, V-P test, gelatin liquefaction, nitrate reduction, tyrosine hydrolysis, glucose, xylose, arabinose, mannitol fermentation and lysozyme growth. The specific results are shown in Table 1 below.
TABLE 1 physiological and biochemical characteristics of the B.coagulans strain BC-HYC
Note that: "+" indicates positive; "-" indicates negative.
Sequence determination
Amplifying a 16s rDNA fragment of the bacterium by using a bacterial 16s rDNA universal primer (an upstream primer is 5 '-AGAGTTTGATCCTGGCTCAG-3' and a downstream primer is 5 '-GGTTACCTTGACTT-3'), sequencing to obtain a sequence fragment, analyzing and comparing the obtained sequence with a 16s rDNA sequence in Genbank, and combining a physiological and biochemical test result to further identify the isolate as the bacillus coagulans, wherein the homology of the bacterium and bacillus coagulans DSM2314 (CP 033687) published by NCBI is as high as 98.8%.
Preservation of strains
The separated strain is preserved in the general microbiological center of China Committee for culture Collection of microorganisms at 23.02/2022, and is named as BC-HYC strain with classification name: bacillus coagulansBacillus coagulansThe preservation number is: CGMCC No.24423 with the preservation address as follows: the institute of microbiology, national academy of sciences No. 3, xilu No. 1, beijing, chaoyang, beijing.
Example 2.
And (3) carrying out stress resistance test on the BC-HYC strain of the bacillus coagulans.
Preparation of fermentation broth
2.1.1 Activating strains:
the preserved Bacillus coagulans BC-HYC strain (CGMCC NO. 24423) and BC-CUI strain (CGMCC NO.6151, described in the inventor's earlier granted patent ZL 201310008503.9) are streaked and inoculated into an MRS culture medium respectively for culture for 24h at 37 ℃ in an anaerobic manner.
Seed liquid preparation
And (3) picking the activated single colony 2 ring, inoculating the single colony to an MRS culture medium, and performing shake culture at 37 ℃ and 150rpm for 24 hours.
Preparation of fermentation broth
Inoculating the seed solution into an MRS culture medium according to the inoculation amount of 5%, and performing shake culture at 37 ℃ and 150rpm for 24h to respectively obtain a bacillus coagulans BC-HYC strain fermentation liquor and a bacillus coagulans BC-CUI strain fermentation liquor.
Temperature sensitivity test:
taking fermentation liquor, respectively carrying out heat preservation treatment for 10min at 25 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃ and 100 ℃, taking a 25 ℃ treatment group as a control group, carrying out gradient dilution on each group of bacterial liquid, uniformly coating the bacterial liquid on an MRS culture medium, carrying out anaerobic fermentation at 37 ℃ for 48h, then carrying out colony counting, determining the viable count and the survival rate of each treatment group, and repeating the test for 3 times for each group. The specific results are shown in Table 2 below.
TABLE 2 comparison of the Heat resistance of the B.coagulans strain BC-HYC with the strain BC-CUI
Based on the results in the table 2, the bacillus coagulans BC-HYC strain obtained by separation has more excellent heat resistance, the survival rate of the bacillus coagulans BC-HYC strain is still up to 91.0% after the bacillus coagulans BC-HYC strain is treated at the temperature of 95 ℃, the bacillus coagulans BC-HYC strain is higher than a BC-CUI strain separated earlier by the inventor, the survival rate of the bacillus coagulans BC-CUI strain is increased by 11.4% compared with the BC-CUI strain, the bacillus coagulans BC-CUI strain has more excellent heat resistance, the application range of the bacillus coagulans BC-HYC strain is wider, the bacillus coagulans BC-CUI strain is more suitable for feed additives and feed processing processes, and the influence of high-temperature processing on the activity of the strain is avoided.
Artificial stomach, intestinal juice and bile salt resistance test:
taking 1mL of fermentation liquor, adding 9mL of artificial gastric juice with pH =2 as a test group, simultaneously taking 1mL of fermentation liquor, adding 9mL of physiological saline as a control group, standing and culturing at 37 ℃ for 4h, performing gradient dilution on the distribution of the two groups of fermentation liquor, uniformly coating the two groups of fermentation liquor on an MRS culture medium, and counting after anaerobic culture at 37 ℃ for 48 h.
Taking 1mL of fermentation liquid, adding 9mL of artificial intestinal fluid with the pH value of =7.6 as a test group, adding 9mL of physiological saline as a control group into 1mL of fermentation liquid, performing static culture at 37 ℃ for 12h, performing gradient dilution on the distribution of the two groups of fermentation liquids, uniformly coating the two groups of fermentation liquids on an MRS culture medium, performing anaerobic culture at 37 ℃ for 48h, and counting.
Survival (%) = viable cell number of artificial gastric fluid (or artificial intestinal juice) treated group/viable cell number of control group × 100%.
Adding 1mL of fermentation liquor into 9mL of 0.3%, 0.5%, 0.7% and 1% bile salt solution, adding 9mL of physiological saline as a control group into 1mL of fermentation liquor, standing and culturing at 37 ℃ for 4h, performing gradient dilution on the distribution of the two groups of fermentation liquor, uniformly coating the two groups of fermentation liquor on an MRS culture medium, performing anaerobic culture at 37 ℃ for 48h, and counting.
Survival (%) = viable cell number in cholate-treated group/viable cell number in control group × 100%.
TABLE 3 test results of resistance to artificial stomach, intestinal juice and bile salts
Based on the tests, the newly separated bacillus coagulans BC-HYC strain has better tolerance to artificial gastric acid and artificial intestinal juice, the survival rate of the treated artificial gastrointestinal juice is up to more than 90%, and the newly separated bacillus coagulans BC-HYC strain also has better tolerance to bile salt, the activity of the bacillus coagulans BC-HYC strain is basically not influenced by 0.3% treatment of the bile salt, and the survival rate of the bacillus coagulans BC-HYC strain is still up to 92.02% after 1% treatment of the bile salt. In contrast, compared with the Bacillus coagulans BC-CUI strain, the Bacillus coagulans BC-HYC strain obtained by the separation method has relatively better tolerance to gastric acid, intestinal juice and bile salt.
Example 3:
probiotic activity test of the strain Bacillus coagulans BC-HYC.
Safety test of strains
3.1.1 Hemolysis test:
culturing Bacillus coagulans BC-HYC strain in liquid culture medium for 24h, and diluting with sterile physiological saline to 10% 8 And (5) absorbing 0.1mL of diluted bacteria liquid to an MRS agar plate containing 5% rabbit blood by CFU/mL, and uniformly coating. Culturing at 37 deg.C for 48h, and observing existence of hemolytic loop.
Mouse toxicity test:
culturing with liquid culture medium of Bacillus coagulans BC-HYC strain for 24h, and storing in refrigerator at 4 deg.C. Selecting20 healthy mice, 10 of which were control groups, were drunk with clean purified water; in addition, 10 test groups were provided, and a culture solution of Bacillus coagulans BC-HYC strain was added to each drinking water to give a concentration of about 10 8 CFU/mL, for 10 consecutive days, the growth of the mice during the trial and whether lesions were present in the internal organs of the mice after 10 days.
In the hemolysis test, the Bacillus coagulans BC-HYC strain was spread on MRS agar plates containing 5% rabbit blood and no hemolysis ring formation was observed. In the mouse toxicity test, the mice in the control group and the test group are not dead and have no abnormal expression after being continuously fed for 10 days, and no abnormality and lesion of each organ and digestive tract are found after the mice are dissected. The results show that the bacillus coagulans BC-HYC strain is safe to mice and has no toxic or side effect.
And (3) bacteriostatic activity test:
bacterial strains for bacteriostatic activity test: staphylococcus aureus (ATCC 25923), escherichia coli K88 (CGMCC 1.2385), salmonella pullorum (CVCC 533), shigella dysenteriae (CGMCC 1.1869) were purchased, stored and provided by the Beijing college of agriculture laboratory.
The invention adopts an Oxford cup method to carry out bacteriostasis determination on common pathogenic bacteria, and the specific method comprises the following steps:
(1) Common pathogenic bacteria were activated in test tubes containing 10mL of nutrient broth: staphylococcus aureus (ATCC 25923), escherichia coli K88 (CGMCC 1.2385), salmonella pullorum (CVCC 533), shigella dysenteriae (CGMCC 1.1869), and culturing at 37 deg.C under shaking for 24 hr;
(2) Preparing a double-layer flat plate: taking a flat plate with the diameter of 90mm, injecting 15mL of sterilized nutrient agar, horizontally placing the flat plate to solidify the nutrient agar to be used as a bottom layer, uniformly mixing the nutrient agar (cooled to about 50 ℃) with a proper amount of indicator bacterium liquid (50 mL of culture medium plus 5 mL) cultured at 37 ℃ and 24h, sucking 15mL, pouring the mixture on the culture medium of the bottom layer, horizontally placing the culture medium to solidify the culture medium to be used as a bacterium layer;
(3) Adding a sample: and clamping the sterilized Oxford cups by using sterile forceps, opening a dish cover, and placing 3 Oxford cups in each culture dish for adding 100 mu L of aureomycin, oxytetracycline and Bacillus coagulans bacteria liquid respectively. Then, 24h was incubated at 37 ℃ and then the Diameter of the Zone of Inhibition (DIZ) was measured. And determining the diameter of the inhibition zone according to the average value of the maximum value and the minimum value of the diameter of the inhibition zone, and determining the inhibition effect and comparing and analyzing the inhibition effect. The specific results are shown in table 4 below:
table 4: determination result of bacteriostatic activity test
According to the diameter value of the used oxford cup (7.8 mm), 16.0 mm, which is about 2 times the outer diameter of the oxford cup, is divided into "sensitive" and "highly sensitive", so the set standard is determined: "insensitive", DIZ =7.8 mm; sensitive, namely, the 7.8 mm woven fabric DIZ is less than or equal to 16.0 mm; "highly sensitive", DIZ >16.0 mm.
Based on the tests, the bacillus coagulans BC-HYC strain disclosed by the invention has good bacteriostatic activity on staphylococcus aureus, escherichia coli K88, salmonella pullorum and shigella dysenteriae, and has better bacteriostatic activity compared with the bacillus coagulans BC-CUI strain obtained by early separation by the inventor. Therefore, the bacillus coagulans BC-HYC strain has relatively high bacteriostatic activity, and can be used for preparing medicines for inhibiting pathogenic bacteria such as staphylococcus aureus, escherichia coli K88, salmonella pullorum, shigella dysenteriae and the like.
Enzyme-producing ability measurement:
3.4.1 preparation of crude enzyme solution: respectively taking a single colony of bacillus coagulans BC-HYC strain, a bacillus coagulans BC-CUI strain and a bacillus coagulans CGMCC 1.10823 (purchased from CGMCC and stored and provided by Beijing academy of agriculture) to inoculate in an MRS seed culture medium, carrying out shake culture at 180rpm at 37 ℃ for 24h, transferring to a fermentation culture medium according to 1 percent of inoculum concentration, and carrying out shake culture at 180rpm at 37 ℃ for 48h to obtain fermentation liquor. The spore formation rate in the fermentation liquor reaches more than 90 percent, the fermentation supernatant is taken and centrifuged for 5min at 10000rpm, and the supernatant is reserved; adding 60% ammonium sulfate powder into the crude enzyme solution under ice bath condition, and precipitating at 4 deg.C to obtain protein in the crude enzyme solution to obtain precipitate; and re-dissolving the obtained precipitate with Tris-HCl buffer solution, and dialyzing at 4 ℃ to obtain concentrated enzyme solution.
And (3) enzyme activity determination: respectively adopting amylase kit (MAK 0009) and protease kit (PF 0100) purchased from sigma; the activities of amylase, protease and lactase in the concentrated enzyme solution are measured by a diglycosidase measuring kit (lactase) (colorimetric method) (A082-1-1) purchased from Nanjing institute of bioengineering. The results are as follows:
TABLE 5 results of enzyme activity measurement
Based on the test results, the strain BC-HYC strain separated by the method can secrete amylase, protease and lactase, and the activity of the amylase is the highest, and compared with the BC-CUI strain separated earlier, the BC-HYC strain separated by the method has similar lactase activity, but has higher amylase activity and protease activity compared with BC-CUI strain and CGMCC 1.10823 strain.
Acid productivity test
Selecting a single bacillus coagulans colony to 500mL of sterilized MRS culture solution under an aseptic condition, carrying out static culture at a constant temperature of 40 ℃, fermenting for 24 hours, and detecting the content of organic acids such as lactic acid, acetic acid, propionic acid and butyric acid in fermentation liquor by using a liquid chromatograph, wherein the specific results are shown in Table 6.
TABLE 6 measurement results of organic acid content (mg/L) in fermentation broth
Based on the results, the BC-HYC strain obtained by separation has better acid production capacity, the lactic acid content in the fermentation liquor reaches 98500mg/L and is much higher than that of BC-CUI strain and CGMCC 1.10823 strain, and the butyric acid content is only 9.2mg/L although lower, but is more than 1 time higher than that of other strains compared with other two strains. Therefore, the bacillus coagulans separated by the method has better acid production capacity, and particularly, the yield of lactic acid and butyric acid is obviously higher than that of the other two bacillus coagulans. Therefore, the bacillus coagulans BC-HYC strain has good acid production capacity and can be used for producing lactic acid.
Example 4.
A Bacillus coagulans BC-HYC strain microbial inoculum and a preparation method thereof.
The preparation method of the microbial inoculum comprises the following steps:
(1) Activating strains: streaking a strain plate of the preserved bacillus coagulans BC-HYC strain to an MRS plate culture medium, and culturing for 24h in an incubator at 42 ℃ to activate the preserved strain;
(2) Preparing a first-level seed solution: picking single colony from plate, inoculating in triangular flask containing MRS liquid culture medium, shake culturing at 42 deg.C, culturing at 120rpm for 12 hr to obtain OD 600 The value is more than or equal to 0.8, and first-class seed liquid is obtained;
(3) Preparing a secondary seed solution: inoculating the prepared first-stage seed liquid into MRS liquid culture medium according to the volume ratio of 1%, performing shake culture at 42 deg.C, and culturing at 120rpm for 8 hr to make OD 600 The value is more than or equal to 0.7, and secondary seed liquid is obtained;
(4) Preparing fermentation liquor: inoculating the secondary seed liquid into a fermentation culture medium according to the volume ratio of 2%, then placing the secondary seed liquid into a production tank for fermentation, filtering after 12h of fermentation, and performing gradient dilution on the filtrate to obtain the secondary seed liquid with the concentration of more than or equal to 10 8 cfu/g strain liquid;
(5) Adding 25% zeolite powder into the prepared fermentation liquid, mixing, and spray drying to obtain product with water content of less than or equal to 5% and viable count of more than or equal to 1 × 10 10 CFU/g of bacterial powder.
Wherein the fermentation medium in the step (4) comprises 1% of peanut cake powder, 2% of soybean meal, 2% of corn flour, 0.1% of dipotassium phosphate, 0.02% of magnesium sulfate heptahydrate, 2% of molasses and the balance of water (the percentages are mass volume percentages), the pH value is 6.5-7.0, and the fermentation medium is sterilized by high-pressure steam at 121 ℃ for 20 minutes.
Example 5.
Application of Bacillus coagulans microbial inoculum in preparing fermented feed.
Firstly, crushing corn flour, bran, bean pulp, rice bran and zeolite powder in advance, screening the crushed corn flour, bran, bean pulp, rice bran and zeolite powder by a 1.5mm sieve, and then uniformly mixing the corn flour, the bran, the bean pulp, the rice bran and the zeolite powder according to the weight ratio of 10;
step two, weighing 50 parts by weight of water, and dissolving the bacillus coagulans microbial inoculum described in embodiment 4 in the water to obtain a fermentation agent, wherein the microbial inoculum is weighed according to 1g/kg of fermentation base material;
and step three, uniformly mixing the leavening agent and the fermentation base material, carrying out aerobic fermentation for 72 hours at the temperature of 40 ℃ and the air humidity of 60-65%, and then drying until the water content is less than or equal to 8% to obtain the fermented feed.
Through determination, the content of small peptides in the fermented feed prepared by the bacillus coagulans microbial inoculum is up to 22.14 percent, and the pH value is 4.0; when the bacillus coagulans BC-CUI strain is adopted to replace the bacillus coagulans BC-HYC strain, the content of small peptides in the prepared fermented feed is 17.65%, and the pH value is 4.6. Therefore, the microbial inoculum disclosed by the invention has better biological activity, can better promote the degradation of macromolecular components in feed raw materials, has better acidification, reduces the pH value of feed, and inhibits the growth of mixed bacteria.
Example 6.
Selecting 150 broilers, randomly dividing into three groups, wherein the group 1 is a blank control group, and feeding the broilers by adopting a conventional daily ration; group 2 was a test group to which 10% of the biofermented feed prepared based on the bacillus coagulans BC-HYC strain of example 5 was added on a regular daily basis; group 3 was a control group to which 10% of the biofermented feed prepared based on Bacillus coagulans BC-CUI strain was added on a regular daily ration basis (in the same manner as in example 5), and during the feeding period, the diet was sufficiently supplied and the chicks were fed freely and had water. The test lasts for 30 days, the average daily feed intake, the average daily weight gain and the feed-meat ratio of each group are calculated, and the specific results are shown in the following table 7:
TABLE 7 influence of fermented feed on broiler Productivity
Based on the results, the biological fermentation feed is added on the basis of basic daily ration, so that the average daily gain value and the average feed intake of animals can be improved, the feed conversion ratio is reduced, the economic benefit of cultivation is improved, and particularly, compared with a blank control group, the biological fermentation feed prepared by adding the BC-HYC strain can improve the average daily gain by 13%, and the weight gain effect is obvious.
Example 7.
9 healthy 28 +/-2 days-old weaned healthy Du multiplied by long multiplied by big commercial hybrid piglets are randomly divided into 3 groups according to the principle of half each male and half each female, wherein the group 1 is a blank control group and fed with basic ration; groups 2 and 3 were fed separately with additive 10 9 CFU/kg、10 11 CFU/kg basic daily ration of Bacillus coagulans BC-HYC strain powder. Feeding is stopped at 8 pm on 14 th day of the experiment, water is supplied as usual, pigs are weighed on empty stomach at 8 pm on the next morning, and the average daily gain (total gain/(number of heads x 14)), average daily feed intake (total feed intake/(number of heads x 14)) and feed conversion efficiency of the experiment are calculated.
TABLE 8 influence of Bacillus coagulans BC-HYC strain powder on the growth performance of weaned piglets
| Average daily gain (g) | Average daily food intake (g) | Meat ratio of materials | |
| Group 1 (blank) | 185.34 | 315.67 | 1.70 |
| Group 2 (10) 9 CFU/kg） | 205.50 | 335.40 | 1.63 |
| Group 3 (10) 11 CFU/kg） | 223.34 | 341.50 | 1.53 |
The results show that: 10 11 The growth performance of the weaned piglets can be obviously improved by the bacillus coagulans BC-HYC strain powder added in the amount of CFU/kg. In the whole test period, the daily gain and the feed intake of the group added with the bacillus coagulans BC-HYC are higher than those of a blank control group, and the material-weight ratio is obvious. Experiments prove that the bacillus coagulans BC-HYC strain added into the daily ration of piglets can obviously improve the feed intake of the piglets, improve the growth speed and daily gain of the piglets, promote the utilization rate of feed, greatly improve the production performance of the pigs and improve the economic benefit of breeding.
Claims (5)
1. A preparation method of a bacillus coagulans microbial inoculum comprises strain activation, preparation of primary seed liquid, preparation of secondary seed liquid, preparation of fermentation liquid and spray drying, and is characterized in that the bacillus coagulans is a bacillus coagulans BC-HYC strain, and is preserved in China general microbiological culture Collection center at 23.02.23.2022 years, with classification names: bacillus coagulansBacillus coagulansThe preservation number is: CGMCC No.24423;
the BC-HYC strain can secrete amylase, protease and lactase;
the preparation method of the bacillus coagulans microbial inoculum comprises the following steps:
(1) Activating strains: streaking a strain plate of the preserved bacillus coagulans BC-HYC strain to an MRS plate culture medium, and culturing for 24h in an incubator at 42 ℃ to activate the preserved strain;
(2) Preparing a first-level seed solution: picking single colony from plate, inoculating in triangular flask containing MRS liquid culture medium, shake culturing at 42 deg.C, culturing at 120rpm for 12 hr to obtain OD 600 The value is more than or equal to 0.8, and first-stage seed liquid is obtained;
(3) Preparing a secondary seed solution: inoculating the prepared first-stage seed liquid into MRS liquid culture medium at volume ratio of 1%, shake culturing at 42 deg.C, and culturing at 120rpm for 8 hr to make OD 600 The value is more than or equal to 0.7, and secondary seed liquid is obtained;
(4) Preparing fermentation liquor: inoculating the secondary seed liquid into a fermentation culture medium according to the volume ratio of 2%, then placing the secondary seed liquid into a production tank for fermentation, filtering after 12h of fermentation, and performing gradient dilution on the filtrate to obtain the secondary seed liquid with the concentration of more than or equal to 10 8 cfu/g strain liquid;
(5) Adding 25% zeolite powder into the prepared fermentation liquid, mixing, and spray drying to obtain product with water content of less than or equal to 5% and viable count of more than or equal to 1 × 10 10 CFU/g of bacterial powder.
2. The process according to claim 1, wherein the fermentation medium in the step (4) comprises 1% of peanut cake flour, 2% of soybean meal, 2% of corn flour, 0.1% of dipotassium phosphate, 0.02% of magnesium sulfate heptahydrate, 2% of molasses and the balance of water, wherein the above percentages are mass volume percentages, the pH is 6.5-7.0, and the fermentation medium is autoclaved at 121 ℃ for 20 minutes.
3. A method for preparing a fermented feed by using the microbial agent prepared by the method of any one of claims 1 to 2, characterized in that: the preparation method of the fermented feed comprises the following steps:
firstly, crushing corn flour, bran, bean pulp, rice bran and zeolite powder in advance, screening the crushed corn flour, bran, bean pulp, rice bran and zeolite powder by a 1.5mm sieve, and then uniformly mixing the corn flour, the bran, the bean pulp, the rice bran and the zeolite powder according to the weight ratio of 10;
step two, weighing 50 parts by weight of water, and dissolving the bacillus coagulans microbial inoculum in the water to obtain a fermentation agent, wherein the microbial inoculum is weighed according to 1g/kg of fermentation base material;
and step three, uniformly mixing the leavening agent and the fermentation base material, carrying out aerobic fermentation for 72 hours at the temperature of 40 ℃ and the air humidity of 60-65%, and then drying until the water content is less than or equal to 8% to obtain the fermented feed.
4. A fermented feed, characterized in that it is produced by the method of claim 3.
5. The application of the microbial inoculum prepared by the method according to any one of claims 1-2 in preparing feed, characterized in that the microbial inoculum is added into the feed, so that the content of bacillus coagulans BC-HYC strain powder in the feed is more than or equal to 10 11 CFU/kg。
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