CN112725222A

CN112725222A - Bacillus subtilis Q3 for producing complex enzyme, culture method and application

Info

Publication number: CN112725222A
Application number: CN202011520077.3A
Authority: CN
Inventors: 朱丽萍; 颜世敢
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-04-30
Anticipated expiration: 2040-12-21
Also published as: ZA202102965B; CN112725222B

Abstract

The invention discloses a bacillus subtilis for producing complex enzyme, a culture method and application. A Bacillus subtilis Q3 producing complex enzyme is preserved in China center for type culture Collection in 2017 at 17.11.13.address eight-way Wuhan university No. 299 in Wuchang district, Wuhan city, Hubei province, with the strain preservation number of CCTCC NO: and M2017701. The bacillus subtilis disclosed by the invention is separated from leaf mold, can produce cellulase and pectinase, and has an inhibiting effect on common pathogenic bacteria. The Bacillus subtilis can be used as animal feed additive. The application of the Q3 bacteria for fermenting the echinacea purpurea can improve the release of effective components in the echinacea purpurea, improve the growth promotion, the immunity enhancement and the disease prevention and treatment effects of the echinacea purpurea. The bacterium and the echinacea fermentation preparation have good market application prospect.

Description

Bacillus subtilis Q3 for producing complex enzyme, culture method and application

Technical Field

The invention belongs to the technical field of microorganisms and the field of traditional Chinese medicine fermentation, and particularly relates to a bacillus subtilis Q3 capable of producing complex enzyme, a culture method and application.

Background

The Chinese herbal medicine is a treasure from China, has been used for thousands of years in China, and plays an important role in preventing and treating human diseases. The Chinese herbal medicine contains effective components with the functions of enhancing immunity, resisting bacteria and viruses, promoting growth and the like, and can be used for conditioning, health care and disease prevention and treatment of human and animals. With the increase of work and life pressure of human beings and the acceleration of rhythm, many people are in states of tension, anxiety, dysphoria and irregular life, which causes sub-health, immune metabolism disorder and even illness, and at the moment, the Chinese herbal medicine has the advantages of conditioning, health care and disease prevention. With the limited use of antibiotics in the domestic and foreign breeding industry, the Chinese herbal medicine is used as an antibiotic substitute for preventing and treating livestock and poultry diseases, and has wide application prospect.

The cell walls of the Chinese herbal medicines contain a large amount of cellulose, pectin and other components, so that the release of effective components in cells is hindered, and the using effect of the Chinese herbal medicines is influenced. The technology for promoting the wall breaking of the plant cells can improve the separation of the effective components in the Chinese herbal medicine. At present, the technology for breaking the wall of the Chinese herbal medicine comprises superfine grinding, stewing, organic solvent extraction, ultrasonic crushing, enzymolysis, microbial fermentation and the like, wherein the microbial fermentation method is favored because of the advantages of high efficiency, environmental protection and the like. There are a considerable number of bacteria and fungi in nature that secrete enzymes to degrade plant cell walls. Most of microorganisms commonly used for degrading plant cell walls at present are fungi capable of producing cellulase, such as trichoderma, penicillium, and sporotrichum. The plant cell wall contains components such as pectin and the like besides cellulose, and the cell wall breaking effect of the Chinese herbal medicine is not ideal when the microorganism which can only produce cellulase is used. Therefore, the method screens the microorganisms with high-yield complex enzyme, improves the efficiency of degrading the cell walls of the Chinese herbal medicines by the microorganisms, promotes the separation of effective components in the Chinese herbal medicines, can improve the using effect of the Chinese herbal medicines, and is favorable for promoting the modernization of the Chinese herbal medicines and the application of the Chinese herbal medicines in the medical and breeding industries.

Based on the defects of the prior art, microorganisms which produce complex enzyme and have good effect of breaking cell walls of plants need to be separated. The Bacillus subtilis has wide enzyme production spectrum and wide application, can form spores, has strong stress resistance, and is an important microorganism source for screening the wall breaking of Chinese herbal medicines.

Echinacea purpurea is perennial herb of Echinacea of Compositae, contains phenolic acids, polysaccharides, alkylamides, unsaturated ketones, volatile oil, alkaloids, polyacetylenes and other active ingredients, has effects of enhancing immunity, resisting virus, resisting inflammation, resisting oxidation and the like, and can be used for enhancing immunity of human and livestock and preventing and treating diseases. The phenolic acid chicoric acid is the most important effective component in echinacea purpurea and is commonly used for evaluating the quality of echinacea purpurea preparations.

The invention has the following beneficial effects: a bacillus subtilis Q3 strain with high yield of cellulase and pectinase is separated and identified, and a microbial inoculum is prepared by adopting liquid fermentation culture and solid fermentation culture and is used as an animal feed additive and can promote the growth of animals; the fermented Echinacea preparation prepared by fermenting Echinacea with Q3 strain can promote the release of effective components in Echinacea, thereby enhancing the effects of resisting diseases, promoting growth and enhancing immunity of Echinacea.

Disclosure of Invention

Problems to be solved

Aiming at the problem of poor treatment effect of microorganisms for breaking the cell wall of Chinese herbal medicines in the prior art, the invention provides a bacillus subtilis Q3 for producing cellulase and pectinase, and further provides a bacillus subtilis Q3 culture method and a fermented echinacea Chinese herbal medicine preparation prepared by fermenting echinacea by using bacillus subtilis Q3.

Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a bacillus subtilis Q3 for producing complex enzyme, a culture method and application.

The technical scheme of the invention is as follows:

a Bacillus subtilis Q3 producing complex enzyme is preserved in China center for type culture Collection in 2017 at 17.11.13.address eight-way Wuhan university No. 299 in Wuchang district, Wuhan city, Hubei province, with the strain preservation number of CCTCC NO: and M2017701.

The Bacillus subtilis Q3 capable of producing the complex enzyme is characterized by being separated from leaf mold, producing cellulase and pectinase at high yield, and having an inhibiting effect on common pathogenic bacteria such as escherichia coli, salmonella, staphylococcus aureus and the like.

The invention relates to application of Bacillus subtilis Q3 in promoting animal growth, improving immunity and preventing and treating diseases.

The growth-promoting animal feed additive is characterized by comprising an effective dose of Bacillus subtilis Q3 as an active ingredient.

The growth-promoting animal feed additive is characterized in that a Q3 microbial inoculum and feed are uniformly mixed according to a certain proportion, so that the number of Q3 bacteria in the feed reaches 1 hundred million CFU/kg.

The growth-promoting animal feed additive is characterized in that the culture method of the bacillus subtilis Q3 is as follows:

(1) activating strains: inoculating bacillus subtilis Q3 in a nutrient broth slant culture medium by streak, and placing the culture medium in a constant-temperature incubator at the temperature of 32-35 ℃ for activated culture for 20-24 hours;

(2) preparing a seed solution: selecting activated Q3 bacteria by using an inoculating loop, inoculating the bacteria into a nutrient broth liquid culture medium, and carrying out aerobic shaking culture at the temperature of 32-35 ℃ for 20-24 h, wherein the rotating speed of a shaking table is 150-200 rpm;

(3) preparing a Q3 liquid microbial inoculum: the spore-forming liquid culture medium is prepared by adding glucose into distilled water per 1000mL²5g, 0.5g of yeast extract, 17g of corn steep liquor, 3g of sodium chloride, 0.2g of magnesium sulfate, 5.0g of calcium phosphate, 0.0001g of manganese sulfate, 0.0001g of ferrous sulfate, 7.2 of pH value adjustment, 30min of high-pressure steam sterilization at 121 ℃, inoculating Q3 seed liquid when the temperature of a culture medium is reduced to room temperature, carrying out aerobic oscillation culture at 32-35 ℃ for 48-72 h, rotating the shaking table at 150-210 rpm, stopping fermentation when the spore content of Q3 in a fermentation liquid is more than or equal to 500 ten thousand CFU/mL, and preparing Q3 bacterial liquid which is a Q3 liquid microbial inoculum;

(4) preparing a Q3 solid microbial inoculum: the formula of the solid fermentation culture medium comprises 10% of corn flour, 85% of wheat bran, 5% of soybean meal, 0.8g of calcium chloride and 0.02g of monopotassium phosphate, the water content is adjusted to be 40% -60%, the wheat bran, the soybean meal, the calcium chloride and the monopotassium phosphate are mixed, the mixture is subpackaged in a high-temperature resistant container, high-pressure steam sterilization is carried out for 30min at the temperature of 121 ℃, after the temperature of the culture medium is reduced to room temperature, Q3 seed solution is inoculated, the bacterial inoculation amount is 10% -20% (v/w), the mixture is sprayed and inoculated while being mixed, the culture is placed in a constant-temperature incubator at the temperature of 32-35 ℃ for 3-4 days, the material is turned over once every 12h, the fermented Q3 solid culture is placed in a 55 ℃ oven to be dried until the water content is lower than 15%, the dried culture is crushed by.

The fermented Chinese herbal medicine for promoting animal growth, improving immunity and preventing diseases is characterized by comprising effective dose of Bacillus subtilis Q3 serving as an active ingredient.

The fermented Chinese herbal medicine for promoting animal growth, improving immunity and preventing diseases is characterized in that the echinacea is fermented by the strain Q3.

The fermented Chinese herbal medicine for promoting animal growth, improving immunity and preventing diseases is characterized in that the purple coneflower fermented by the strain Q3 and feed are mixed uniformly according to the proportion of 0.1-0.3% (w/w) and fed.

The fermented Chinese herbal medicine for promoting animal growth, improving immunity and preventing diseases is characterized in that the preparation method of the fermented echinacea preparation comprises the following steps: taking echinacea purpurea powder, adjusting the water content to be 40-60%, subpackaging into a high-temperature resistant container, sterilizing for 30min by high-pressure steam at the temperature of 121 ℃, spraying and inoculating Q3 seed liquid into the sterilized echinacea purpurea powder when the materials are cooled to room temperature, spraying and inoculating the seed liquid with the bacterial inoculation amount of 10-20% (v/w), stirring and uniformly mixing, carrying out solid state fermentation culture at the temperature of 32-35 ℃ for 3-5 days, turning over the materials once every 12h, after the fermentation is finished, drying the fermented echinacea purpurea in a drying oven at the temperature of 55 ℃ until the water content is lower than 15%, preparing fermented echinacea purpurea powder, subpackaging and storing at room temperature.

Advantageous effects

The invention discloses a bacillus subtilis Q3 for highly producing cellulase and pectinase, which can degrade cellulose and pectin in plant cell walls and improve the release of effective components in plant cells, thereby improving the bioavailability of plant resources and being used as an animal feed additive. The invention also discloses a liquid fermentation culture and solid fermentation culture method of the bacillus subtilis Q3, and the prepared liquid microbial inoculum and solid microbial inoculum with high bacterial content can be used as animal feed additives, can promote animal growth and improve feed conversion rate. The invention also discloses echinacea purpurea fermented by the bacillus subtilis Q3, wherein the echinacea purpurea powder can improve the separation of active ingredients such as chicoric acid and the like in the echinacea purpurea after being fermented by the Q3 bacteria, and obviously improve the effects of promoting the growth of animals, improving the immunity, resisting diseases and the like of the echinacea purpurea.

Drawings

FIG. 1A transparent circle of Bacillus subtilis Q3 on a CMC-Congo red agar plate.

FIG. 2 colony morphology of Bacillus subtilis Q3 on nutrient broth agar plates.

Detailed Description

The technical solution of the present invention is further described below with reference to specific examples, but the scope of the present invention includes but is not limited thereto.

The Bacillus subtilis Q3 described in the examples is stored in the China center for type culture Collection in 2017 at 11 months and 17 days, and the strain preservation number is CCTCC NO: and M2017701.

Example 1: screening and identification of cellulase-producing bacillus subtilis

Weighing 2.0g of leaf mold, placing in a sterilized conical flask, adding appropriate amount of sterilized normal saline, soaking for 3h, centrifuging at low speed, and collecting supernatant. Diluting the supernatant with sterilized normal saline at a ratio of 10 times, and selecting sample solution (such as 10 times of the diluted sample solution)^-4、10^-5、10^-6) Each of the plates was plated on Congo red agar plates containing 1% sodium carboxymethylcellulose, and 100. mu.l of each sample solution was inoculated. The culture was incubated at 35 ℃ for 72 hours under aerobic conditions, and it was observed whether a transparent circle was formed around the colony. Picking single colony capable of generating transparent ring on cellulose-congo red agar plate, inoculating nutrient broth, and culturing at 35 deg.C under aerobic condition for 24 hr to obtain pure culture of isolate. The bacterial separation test result shows that the strain with the number Q3 produces the largest transparent circle, which indicates that the strain produces cellulase and has high enzyme activity. Smear of bacterial liquid of Q3 bacterium, gram stain, and observation under optical microscopeThe bacterium is a purple bacterium, which indicates that the bacterium is a gram-positive bacterium. The strain Q3 is dipped by an inoculating loop, streaked on a nutrient broth solid plate for culture, and aerobically and isothermally cultured at 35 ℃ for 24h, wherein the colony is large, round, rough in surface and white.

Genomic DNA of the strain Q3 was extracted using a bacterial genomic DNA extraction kit according to the instructions. PCR amplification was performed with 16S rDNA universal PCR amplification primers 27F and 1492R. The PCR amplification system was 25. mu.L, containing 2. mu.L of bacterial DNA, 2.5. mu.L of 10 XPCR buffer (containing Mg)²⁺) dNTPs of 2 mu L, upstream and downstream primers of 0.5 mu L and Taq DNA polymerase of 0.5 mu L respectively, and distilled water is added to 25 mu L. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, extension at 72 ℃ for 1min for 30 cycles, and finally extension at 72 ℃ for 5 min. The PCR amplification product was sent to a sequencing company for gene sequencing. The gene sequence is uploaded to a Genbank database, and the accession number of the obtained gene sequence is MW 334967. Logging in NCBI website, inputting gene sequence into BLAST online analysis platform, and performing sequence comparison analysis. The 16S rDNA sequence alignment analysis result shows that the Q3 bacterium is bacillus subtilis.

Example 2: enzymatic characteristic analysis of cellulase and pectinase produced by bacillus subtilis Q3

Preparation of a crude enzyme solution of bacillus subtilis Q3: preparing nutrient broth culture medium, sterilizing with high pressure steam at 121 deg.C for 30min, cooling to room temperature, adding Q3 seed solution at an inoculum size of 6% (v/v), placing in a shaking table, culturing at 35 deg.C under aerobic shaking for 72h, and rotating at 180 rpm. Centrifuging the bacterial liquid at 10000rpm for 10min, and obtaining the supernatant as the crude enzyme liquid. And respectively measuring the enzyme activities of the cellulase and the pectinase in the crude enzyme solution.

(1) Analysis of enzyme activity of cellulase in crude enzyme solution of Bacillus subtilis Q3

The filter paper enzyme activity represents the total enzyme activity after the synergistic action of three enzyme components of the cellulase. The enzyme activity of the cellulase is determined by a filter paper method. According to the specification of the filter paper method for measuring the activity of the feeding cellulase of GB/T23881-2009.

1g of glucose was weighed and dissolved in 100mL of 0.05mol/L citrate buffer solution at pH 5.7 to prepare a 10mg/mL glucose standard solution. Get 25mL with plug scaleAnd 7 test tubes, accurately sucking 0mL, 0.04 mL, 0.06 mL, 0.08 mL, 0.12 mL, 0.16 mL and 0.2mL of glucose solution into each test tube respectively, adding distilled water to 2.0mL respectively, adding 2mL of DNS reagent into each test tube respectively, carrying out boiling water bath for 5 minutes, taking out, immediately cooling to room temperature by using cold water, adding distilled water to a constant volume of 25mL, shaking uniformly, and measuring the OD value at a wavelength of 540 nm. As absorbance OD₅₄₀Values are plotted on the abscissa and glucose standard curves are plotted on the ordinate versus the number of milligrams of sugar in the glucose standard solution.

50mg of a shredded filter paper strip was added to a 25mL graduated tube, 0.05mol/L citrate buffer lmL pH 5.7 was added, and the mixture was equilibrated in a 37 ℃ water bath for 10 min. Then 0.5mL of properly diluted enzyme solution and 5mL of water are sequentially added, the temperature is kept in a water bath at 37 ℃ for 1 hour, 2mL of DNS reagent is added, the mixture is boiled in the water bath for 5 minutes, the mixture is taken out and immediately cooled to the room temperature by cold water, distilled water is added to the mixture to be constant volume to 25mL, the mixture is shaken up, and the absorbance is measured at 540 nm. Meanwhile, the inactivated enzyme solution after boiling at 100 ℃ for l0min is used as a contrast, and the background is subtracted. Finding out the corresponding glucose content from the glucose standard curve according to the absorbance, and calculating the enzyme activity value according to the gram number of the generated glucose. Filter paper enzyme activity calculation formula: x is (M × n × 1000)/(t × M). X is the enzyme activity of the filter paper enzyme, and the unit is U/g. And m is the content of glucose found from a glucose standard curve. And n is the total dilution multiple of the enzyme solution. t is the reaction time in min. M is the molar mass of glucose, 180.2 g/moL.

The determination result shows that the cellulase activity in the crude enzyme solution of the bacillus subtilis Q3 is 662U/g.

(2) Enzyme activity analysis of crude enzyme liquid pectinase of bacillus subtilis Q3

And (4) carrying out enzyme activity determination on the pectinase according to a method of an industrial standard QB/T4482-2013. Adding 2mL of 0.2% polygalacturonic acid substrate into a 25mL test tube with a plug scale, preheating for 5min at 45 ℃, adding 20uL of enzyme solution, uniformly mixing, carrying out water bath at 45 ℃ for 15min, sequentially adding 3mL of 0.03moL of phosphoric acid reagent, and measuring the OD value at 235 nm. Meanwhile, the inactivated enzyme solution after boiling at 100 ℃ for l0min is used as a contrast, and the background is subtracted. The enzyme activity calculation formula of the pectinase is as follows: x ═ X (a × N × V1 × 1000000)/(t × b × V2 × 1000 × 4600). X is the enzyme activity of pectinase, and the unit is U/g. A is the absorbance of the diluted enzyme solution. N is the dilution factor of the enzyme solution. t is the reaction time in min. b is the thickness of the cuvette in cm. V1 is the total volume of the reaction system in mL. V2 is the volume of the diluted enzyme solution in mL.

The enzyme activity of the pectinase in the crude enzyme solution of the bacillus subtilis Q3 is calculated to be 279U/g.

Example 3: research on bacteriostatic effect of bacillus subtilis Q3

The inhibition effect of the bacillus subtilis on common pathogenic bacteria is measured by an Oxford cup method. Selecting a single bacterial colony of the bacillus subtilis Q3, inoculating the single bacterial colony to a nutrient broth culture medium, carrying out shaking culture at 35 ℃ for 24h, centrifuging the bacterial liquid at 10000rpm for 10min, and taking the supernatant. Picking out single colonies of Escherichia coli ATCC25922, salmonella CMCC50956 and staphylococcus aureus ATCC25923 respectively, inoculating the single colonies into a nutrient broth culture medium, and carrying out shake culture at 37 ℃ for 24 h. Respectively sucking 0.1mL of bacterial liquid of various pathogenic bacteria, uniformly coating on a nutrient broth solid plate, placing a sterilized Oxford cup (with the outer diameter of 8mm) on the solid plate by using an aseptic forceps, and lightly pressing to make the bottom of the cup tightly attached to the solid plate. 0.15mL of Bacillus subtilis Q3 supernatant was added to each Oxford cup. Then placing the culture dish at the constant temperature of 37 ℃ for culturing for 24h, and measuring the diameter of the inhibition zone.

The results of bacteriostatic tests show that the diameters of bacteriostatic rings of the liquid supernatant of the bacillus subtilis Q3 on escherichia coli, salmonella and staphylococcus aureus are 12 mm, 13 mm and 11.6mm respectively, and that Q3 has an inhibitory effect on common pathogenic bacteria.

Example 4: culture of bacillus subtilis Q3 and preparation of microbial inoculum thereof

Activating strains: the single bacterial colony of the bacillus subtilis Q3 is picked by an inoculating loop, streaked and inoculated in a nutrient broth slant culture medium, and placed in a constant temperature incubator of 35 ℃ for activation culture for 20 h.

Preparing a seed solution: a loop of activated Q3 strain is picked up by using an inoculating loop, inoculated into 100mL of nutrient broth liquid culture medium, and subjected to shaking culture at 35 ℃ for 24h, and the rotating speed of a shaking table is 180 rpm.

(1) Liquid state fermentation culture and liquid microbial inoculum preparation

The formula of the spore-forming liquid culture medium is that 25g of glucose, 0.5g of yeast extract, 17g of corn steep liquor, 3g of sodium chloride, 0.2g of magnesium sulfate, 5.0g of calcium phosphate, 0.0001g of manganese sulfate and 0.0001g of ferrous sulfate are added into per 1000mL of distilled water, and the pH value is adjusted to 7.2. Sterilizing with high pressure steam at 121 deg.C for 30 min.

After the temperature of the liquid culture medium is reduced to room temperature, inoculating Q3 seed liquid according to the inoculation amount of 10% (v/v), carrying out aerobic shaking culture at 35 ℃ for 60h, and rotating the shaking table at 180 rpm.

The Q3 bacterial liquid prepared by the method is the spore bacterial agent. The number of spores was checked by plate dilution coating. And (3) carrying out water bath at 80 ℃ for 15min, and detecting the spore content by using a plate dilution method, wherein the number of spores reaches 800 ten thousand CFU/mL.

And (3) storage: and (3) refrigerating the Q3 bacterial liquid in a refrigerator at 4 ℃ or storing the bacterial liquid in a cool place at room temperature.

(2) Solid fermentation culture and preparation of powdered microbial inoculum

Preparing a solid fermentation culture medium: the method comprises the steps of adjusting the water content to 50% according to 10% of corn flour, 85% of wheat bran, 5% of soybean meal, 0.8g of calcium chloride and 0.02g of monopotassium phosphate, naturally adjusting the pH value, uniformly mixing, preparing a solid fermentation culture medium, subpackaging the solid fermentation culture medium in a high-temperature resistant plastic box, sterilizing for 30min by high-pressure steam at 121 ℃, and cooling to room temperature.

Solid fermentation culture: uniformly spraying the Q3 seed solution into a solid fermentation culture medium according to the inoculation amount of 20%, uniformly mixing, and culturing in a constant-temperature incubator at 35 ℃ for 3 days, wherein the stirring is carried out once every 12 h.

Drying and crushing: drying the fermented Q3 solid culture in a 55 ℃ oven until the water content is lower than 15%, pulverizing the dried culture with a pulverizer, sieving with a 60-mesh sieve to obtain powdery microbial inoculum, subpackaging in sealed plastic bags, and storing in dry and cool places.

The viable count of Q3 bacteria in the powder was determined by a plate dilution coating method. The bacteria counting result shows that the viable count of Q3 in the powder reaches 620 ten thousand CFU/g.

Example 5: application effect observation of broiler fed with bacillus subtilis Q3 microbial inoculum

100 broiler chicks of 1 day old are selected and averagely divided into 2 groups, and the average body weight of the two groups is close to that of the two groups. In the treatment group, 20mL of bacillus subtilis Q3 bacterial liquid is added into each kilogram of complete feed, so that the number of spores in the feed is 1.6 hundred million CFU/kg. The control group was fed with commercial complete feed. The two groups of test chickens were fed under the same conditions except for the difference in the feed. The breeding period is 42 days, the ground is adopted for flat breeding, water and food are freely drunk, and the illumination is 24 hours. The temperature in the chicken house was 33 ℃ initially tested and subsequently decreased by 1 ℃ every three days until it reached 26 ℃. The vaccine immunization is carried out according to the conventional immunization program. The number of the test chickens, the total weight and the average weight of the test chickens in each group are recorded at the beginning of the test, the number of dead chickens and the feed intake are recorded every day, the test is performed for 12 hours without water prohibition after the test is finished, the weight of each group of chickens is weighed, and the death rate, the average weight gain and the feed-meat ratio of the chickens in each group during the test are calculated. Slaughtering test chickens, collecting cecal contents, extracting genome DNA of cecal microorganisms by using a fecal genome extraction kit, performing metagenome sequencing, and analyzing the diversity of intestinal flora of the test chickens. The results are shown in Table 1.

TABLE 1 influence of Bacillus subtilis Q3 microbial inoculum on growth performance of broiler chicken fed with the strain

The test result of the influence of the bacillus subtilis Q3 microbial inoculum on the growth performance of the broiler chicken shows that the death and culling rates of the Q3 microbial inoculum feeding group and the control group are respectively 4% and 6%, the average daily gain is respectively 55.5 g/chicken and 49.1 g/chicken, and the feed-meat ratio is respectively 1.86 and 2.06. The result of a contrast feeding test shows that the Q3 microbial inoculum is safe to chickens and has the effects of promoting growth and resisting diseases.

The sequencing result of the caecum microorganism metagenome shows that the number of lactobacillus, corynebacterium and enterococcus in the caecum of the chicken can be increased to different degrees by feeding the Q3 microbial inoculum, wherein the number of lactobacillus and enterococcus is obviously increased (P is less than 0.05); and the number of bacteroides, streptococcus and escherichia coli is reduced, wherein the number of bacteroides and escherichia coli is obviously reduced (P < 0.05).

Example 6: bacillus subtilis Q3 fermented echinacea and application effect observation thereof

Removing impurities from Echinacea purpurea flower floc and rhizome, drying, pulverizing, and sieving with 60 mesh sieve to obtain Echinacea purpurea powder. Uniformly spraying tap water into echinacea purpurea powder, adjusting the water content to be 50%, subpackaging into high-temperature-resistant plastic baskets, sterilizing for 30min by high-pressure steam at 121 ℃, cooling the materials to room temperature, weighing bacillus subtilis Q3 seed solution according to the proportion of 15% (v/w), spraying and inoculating into the sterilized echinacea purpurea powder, uniformly stirring while spraying, and performing solid state fermentation culture at 35 ℃ for 4 days, wherein the materials are turned over once every 12 h. After fermentation, placing the fermented Echinacea purpurea in a 55 ℃ oven to dry until the water content is lower than 15%, preparing into fermented Echinacea purpurea powder, subpackaging, and storing at room temperature.

Taking fermented echinacea powder and unfermented echinacea powder with the same mass, and mixing the fermented echinacea powder and the unfermented echinacea powder according to a material-water ratio of 1: adding distilled water into 10(w/v), soaking overnight in a refrigerator at 4 ℃, centrifuging for 10min at 10000rpm, and filtering the supernatant through a 0.45 mu m filter membrane to obtain a sample to be detected. RP-HPLC method is used for analyzing the content of chicoric acid in echinacea purpurea, and standard curve method is used for calculating the content of chicoric acid in fermentation liquor. The high pressure liquid chromatograph is Shimadzu LC-20A type, the ultraviolet detector is SPD-20A type, the detection wavelength is 330nm, and the reversed phase chromatographic column is Venusil^TMXBP C18 column (250mm × 4.6mm, 5 μm), column temperature 35 ℃, sample size 10 μ L, flow rate 1mL/min, mobile phase acetonitrile: 0.2% phosphoric acid in water (28:72), isocratic elution. The analysis result shows that the yield of the chicoric acid in the fermented echinacea is 76.2 percent, the yield of the chicoric acid in the unfermented echinacea is 43.8 percent, and the difference of the two is obvious (P is less than 0.05) through the biological statistical analysis. Experimental results prove that the purple coneflower fermented by the Q3 strain can promote the separation of the effective component chicoric acid.

300 chicks of 1 day old are averagely divided into 3 groups, each group comprises 100 chickens, the weight of each group of chickens is equivalent, one group of chickens are fed with the feed added with the fermented echinacea, the other group of chickens are fed with the feed added with the unfermented echinacea, and the blank control group of chickens are fed with the common commercial complete feed. The fermented Echinacea feed and the unfermented Echinacea feed are added into the complete feed according to the proportion of 0.3% (w/w) respectively. The three groups of test chickens were fed under the same conditions except for the different feeds. The breeding period is 42 days, the ground is used for flat breeding, water and food are freely drunk, and the illumination is 24 hours. The temperature in the chicken house was 33 ℃ initially tested and subsequently decreased by 1 ℃ every three days until it reached 26 ℃. The vaccine immunization is carried out according to the conventional immunization program. The number of the test chickens, the total weight and the average weight of the test chickens in each group are recorded at the beginning of the test, the number of dead chickens and the feed intake are recorded every day, the test is performed for 12 hours without water prohibition after the test is finished, the weight of each group of chickens is weighed, and the death and culling rate, the average weight gain and the feed-meat ratio of the chickens in each group during the test are calculated. At the end of the test, slaughtering the test chickens, picking spleens, weighing and counting the spleen body weight index of each group of chickens.

The results of the feeding test of fermented Echinacea are shown in Table 2. The mortality and washout rates of the fermented echinacea feeding group, the unfermented echinacea feeding group and the blank control group are respectively 2%, 4% and 6%, the average daily gain is respectively 63.1, 56.4 and 49.2 g/mouse, the feed-meat ratio is respectively 1.72, 1.83 and 2.06, and the spleen body weight index is respectively 196.1, 178.8 and 169.3 mg/kg. The results of contrast feeding tests show that the echinacea purpurea has the effects of promoting the growth of animals, enhancing the immunity and resisting diseases, and the growth promotion, the immunity enhancement and the disease resistance of the fermented echinacea purpurea are superior to those of the unfermented echinacea purpurea.

TABLE 2 Bacillus subtilis Q3 fermentation echinacea purpurea feeding broiler chicken test results

Claims

1. A Bacillus subtilis Q3 producing complex enzyme is preserved in China center for type culture Collection in 2017 at 17.11.13.address eight-way Wuhan university No. 299 in Wuchang district, Wuhan city, Hubei province, with the strain preservation number of CCTCC NO: and M2017701.

2. The Bacillus subtilis Q3 for producing complex enzyme according to claim 1, which is characterized by being separated from leaf mold, producing cellulase and pectinase and having an inhibitory effect on common pathogenic bacteria.

3. The application of the Bacillus subtilis Q3 capable of producing the complex enzyme in the aspects of promoting animal growth, improving immunity and preventing diseases according to the claim 1.

4. A growth-promoting animal feed additive characterized by comprising as an active ingredient an effective amount of Bacillus subtilis Q3 according to claim 1.

5. The growth-promoting animal feed additive according to claim 4, wherein the bacterial agent Q3 is mixed with feed in a proportion such that the number of bacteria Q3 in the feed is 1 hundred million CFU/kg.

6. The compound enzyme-producing bacillus subtilis and the application of the compound enzyme-producing bacillus subtilis as claimed in claims 3, 4 and 5, wherein the culture method of the bacillus subtilis Q3 is as follows:

(1) activating strains: the bacillus subtilis Q3 of claim 1 is streaked and inoculated in a nutrient broth slant culture medium, and is placed in a constant temperature incubator at 32-35 ℃ for activation culture for 20-24 h;

(2) preparing a seed solution: selecting activated Q3 bacteria by using an inoculating loop, inoculating the bacteria into a nutrient broth liquid culture medium, carrying out shake culture at the temperature of 32-35 ℃ for 20-24 h, and rotating the rotating speed of a shaking table at 150-200 rpm;

(3) preparing a Q3 liquid microbial inoculum: the formula of the spore production liquid culture medium is that 25g of glucose, 0.5g of yeast extract, 17g of corn steep liquor, 3g of sodium chloride, 0.2g of magnesium sulfate, 5.0g of calcium phosphate, 0.0001g of manganese sulfate, 0.0001g of ferrous sulfate, 7.2 of pH value adjustment, 30min of high-pressure steam sterilization at 121 ℃, Q3 seed liquid is inoculated after the temperature of the culture medium is reduced to room temperature, the bacterial inoculation amount is 5-10% (v/v), aerobic oscillation culture is carried out for 48-72 h at 32-35 ℃, the rotation speed of a shaking table is 150-200 rpm, fermentation is stopped when the spore content of Q3 in the fermentation liquid is more than or equal to 500 ten thousand CFU/mL, and the prepared Q3 bacterial liquid is Q3 liquid microbial inoculum;

(4) preparing a Q3 solid microbial inoculum: the solid fermentation culture medium comprises 10% of corn flour, 85% of wheat bran, 5% of soybean meal, 0.8g of calcium chloride and 0.02g of monopotassium phosphate, the water content is adjusted to be 40% -60%, the wheat bran, the soybean meal, the calcium chloride and the monopotassium phosphate are mixed uniformly, the mixture is subpackaged in a high-temperature resistant container, high-pressure steam sterilization is carried out for 30min at the temperature of 121 ℃, after the temperature of the culture medium is reduced to room temperature, Q3 seed solution is inoculated, the bacterial inoculation amount is 10% -20% (v/w), the mixture is sprayed and inoculated while being mixed, the culture is placed in a constant-temperature incubator at the temperature of 32-35 ℃ for 3-4 days, the material is turned over once every 12h, the Q3 solid culture after fermentation is placed in an oven at the temperature of 55 ℃ and dried until the water content is lower than 15%, the dried culture is.

7. A fermented Chinese herbal medicine for promoting animal growth, enhancing immunity and preventing diseases, which is characterized by comprising an effective amount of Bacillus subtilis Q3 as claimed in claim 1 as an active ingredient.

8. The fermented Chinese herbal medicine for promoting animal growth, improving immunity and preventing diseases according to claims 3 and 7, which is characterized in that the echinacea is fermented by the strain Q3.

9. The fermented Chinese herbal medicine for promoting animal growth, improving immunity and preventing diseases according to claims 3, 7 and 8, which is characterized in that the fermented echinacea and the feed are mixed uniformly according to the proportion of 0.1-0.3% (w/w) and fed.

10. The fermented Chinese herbal medicine for promoting animal growth, improving immunity and preventing diseases according to claims 3, 7, 8 and 9, which is characterized in that the preparation method of the fermented echinacea comprises the following steps: taking echinacea purpurea powder, adjusting the water content to be 40-60%, subpackaging the echinacea purpurea powder in a high-temperature resistant container, sterilizing the echinacea purpurea powder for 30min by high-pressure steam at the temperature of 121 ℃, inoculating Q3 seed liquid when the temperature of the material is reduced to room temperature, inoculating 10-20% (v/w) of the bacterial inoculum size, uniformly mixing while spraying, carrying out solid state fermentation culture at the temperature of 32-35 ℃ for 3-5 days, turning over the material once every 12h, and after the fermentation is finished, drying the fermented echinacea purpurea in a 55 ℃ oven until the water content is lower than 15% to prepare the fermented echinacea purpurea powder.