CN110438028B - Min pig source Bacillus beleisi GX-1 for degrading cellulose - Google Patents
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Abstract
The invention discloses a Min pig source Bacillus belgii GX-1 for degrading cellulose. Is preserved in China center for type culture Collection (CCTCC for short), the preservation date is 11 months and 29 days in 2018, the preservation number is M2018841, and the culture is classified and named as Bacillus velezensis GX-1. The strain can produce cellulase, can efficiently degrade cellulose, and is used for biodegradation of cellulose. The cellulase produced by the GX-1 strain has better thermal stability, and can keep better enzyme activity within the range of 40-70 ℃; the strain GX-1 has the advantages of rapid growth, high enzyme yield, low requirements on culture conditions and enzyme production conditions, and easiness in colonization in pig intestinal tracts; it has important significance for the development of livestock breeding industry and feed fermentation industry.
Description
Technical Field
The invention belongs to the field of agricultural and livestock application, and particularly relates to a Min pig source Bacillus velezensis GX-1 for degrading cellulose.
Background
The cellulose is the most abundant and cheap renewable resource on the earth, although China is a big agricultural country with abundant cellulose resources, the cellulose resources are not reasonably utilized, and great waste is caused, the reasonable utilization of the cellulose in the breeding industry can greatly reduce the use of conventional energy feed (such as corn) in daily ration, most fungi with higher temperature requirements are mainly microorganisms including fungi, bacteria, actinomycetes and the like in the nature, the fungi are easy to produce extracellular cellulase, easy to extract and separate, high in enzyme activity and good in degradation effect, for example, Saroj separates 15 strains of thermophilic fungi capable of growing at 50 ℃, aspergillus fumigatus JCM 10253 has higher screened activity of streptomyces fumarose and β -glucosidase and activity of xylanase, the extremely high bacteria have lower propagation speed and no strong enzyme production capacity, the fungi have high pH value, are selected from streptomyces fumaroeus JCM 10253 in soil, the streptomyces typus and the actinomycetes JCM strain with high pH value, and have high resistance to the pH value of the bacteria such as bacillus acidogen, the bacillus typhus strain has high resistance to the pH value of bacillus acidogen, the bacteria has the resistance to the pH value of the bacteria which is high temperature of the bacillus typhus strain of the bacillus typhus JCM and the bacillus typhus strain is high temperature, the strain of the strain which is selected from the strain of.
Disclosure of Invention
Based on the practical problems and requirements in the livestock breeding production process, the invention aims to provide the Min pig source Bacillus velezensis GX-1 for degrading cellulose, which can generate high-content cellulase, can efficiently degrade cellulose, sodium carboxymethylcellulose and the like, and is beneficial to the digestibility of the cellulose by animals, thereby improving the utilization of cellulose resources by the animals.
The technical characteristics of the invention are as follows: a civilian pig source Bacillus belief (Bacillus velezensis) GX-1 for degrading cellulose is preserved in China center for type culture Collection at the address: in the Wuhan university school of eight-channel 299 # in Wuhan district, Wuhan City, Hubei province, the postal code is as follows: 430072, with a preservation date of 2018, 11 months and 29 days, and a preservation number of: CCTCC NO is M2018841, and is named as Bacillus velezensis GX-1 in classification.
The invention also has the following technical characteristics:
1. the above described cellulose degrading Bacillus belgii GX-1 of Min swine origin has the biological characteristics of: white wax, white colony, irregular edge, dryness, and wrinkles.
2. The fermentation culture method of the Min pig source Bacillus belgii GX-1 for degrading cellulose comprises the following steps: taking OD6001-5% of the seed solution 1.0 into the fermentation medium, and shake culturing at 31-41 deg.C, 180 rpm and pH 4.0-7.0. Wherein the formula of the fermentation medium is as follows: 5g/L of yeast powder, 10g/L of trypsin vein, 10g/L of sodium chloride, 10g/L of CMC-Na and 6 +/-0.5 of pH.
3. The fermentation culture method of the Min pig source Bacillus belgii GX-1 for degrading cellulose is preferably carried out at 35 ℃, at pH 5.0 and with the inoculation amount of 2%.
4. The Bacillus belgii GX-1 of Min swine origin for degrading cellulose is characterized in that the cellulose degradation condition of the produced cellulase is 40-70 ℃, the pH value is 4.0-7.0, and the reaction time is 5-40 min.
5. The Min pig source Bacillus belgii GX-1 for degrading cellulose is characterized in that the temperature of cellulase generated by the Min pig source Bacillus belgii GX-1 for degrading cellulose is preferably 55 ℃, the pH value is 4.5, the reaction time is 25min, and the enzyme activity is 41.18U/mL.
6. The application of the Min pig source Bacillus belgii GX-1 for degrading cellulose in the preparation of fibrous feed.
The invention has the advantages and beneficial effects that: the strain GX-1 provided by the invention has strong capability of producing cellulase, the produced cellulase can keep high activity, the cellulose can be efficiently degraded, the requirement on enzyme production conditions is not high, and the strain is easy to fix in the intestinal tract of a pig; the strain has important significance for the utilization of fibrous feed resources and the development of the feed fermentation industry and the livestock breeding industry. The cellulase produced by the GX-1 strain has better thermal stability, and can keep better enzyme activity within the range of 40-70 ℃; the strain GX-1 has the advantages of rapid growth, high enzyme yield, low requirements on culture conditions and enzyme production conditions, and easiness in colonization in pig intestinal tracts; it has important significance for the development of livestock breeding industry and feed fermentation industry.
Drawings
FIG. 1 shows the form of GX-1 cells;
FIG. 2 phylogenetic tree of strain GX-1;
FIG. 3 glucose standard curve;
FIG. 4 influence of pH on enzyme production by the bacterium GX-1;
FIG. 5 effect of temperature on enzyme production by GX-1;
FIG. 6 influence of inoculum size on enzyme production by strain GX-1;
FIG. 7 effect of temperature on cellulase enzyme activity;
FIG. 8 influence of pH on cellulase enzyme activity;
FIG. 9 effect of reaction time on cellulase activity;
Detailed Description
Example 1: strain screening and identification
The Min pig is one of eight excellent pig breeds in China, has the advantages of good meat quality, coarse feeding resistance, more farrowing, strong cold resistance, strong disease resistance and the like, is suitable for grazing feeding due to the coarse feeding resistance, and can eat a large amount of green coarse feed and wild herbs and wild vegetables. There are studies showing that: under the condition of feeding three daily rations with 9%, 12% and 15% of crude fiber, the contents of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, lactate dehydrogenase and creatine kinase of Min pigs and big white pigs are increased, but the increase degree of Min pigs is smaller; in addition, the influence on the tissues and organs such as livers, hearts, muscles and the like of the Min pigs is far less than that of the Min pigs fed with the daily ration with high crude fiber content. Therefore, we speculate that the intestinal tract of the Min pig is populated with a large number of microorganisms capable of utilizing plant cellulose. The strain of the embodiment is obtained by screening and separating the feces of adult Min pigs, and the specific process is as follows:
weighing 1.0g of the feces of the Min pig, transferring the feces of the Min pig into a triangular flask containing 100mL of sterile water, and oscillating the feces of the Min pig in an electric heating constant-temperature oscillating water bath kettle at the temperature of 80 ℃ for 30 min. The mixture was spread on an LB + 1% CMC plate using sodium carboxymethylcellulose as the sole carbon source by plate coating and cultured at 37 ℃ for 24 hours. Adding a proper amount of 0.1% Congo red solution into a culture dish, dyeing for 1h, then discarding the dye solution, selecting a bacterial colony with a transparent ring from a flat plate, carrying out primary screening, continuously selecting a bacterial colony with a transparent ring, and carrying out streaking separation and purification on an LB (Langmuir-Blodgett) flat plate to obtain a single bacterial colony. And respectively measuring the cellulase activity of the obtained single colonies, and re-screening, wherein the strain with the best cellulose degradation effect and the highest cellulase activity is selected and named as GX-1.
The biological characteristics of strain GX-1 found in this example include: white wax, white colony, irregular edge, dry, wrinkled (see fig. 1). The cellulase has strong capability, and the produced cellulase can keep high activity and can realize efficient degradation on cellulose.
The physiological and biochemical characteristics of the strains of the invention are as follows (see table 1 below):
TABLE 1 physiological and biochemical characteristics of strain GX-1
Note: "+" represents positive, and "-" represents negative
The molecular identification process and results are shown below:
the total DNA of the strain is extracted by adopting a Shanghai genome DNA extraction reagent SK8255, and 16S rDNA PCR amplification is carried out by an SK8131 kit to obtain a 1492bp16S rDNA fragment.
Entrusting Shanghai biological engineering technology service Limited company to complete 16S rDNA sequencing, wherein sequencing primers are as follows: an upstream primer: 5'-CAGAGTTTGATCCTGGCTAGGAGGTGATCCAGCCGCA-3', respectively; a downstream primer: 5 '-AGTTTGATCMTGGCTCAGGGTTACCTTGTTACGACTT-3'. Obtaining a DNA sequencing result, comparing the DNA sequencing result on a ribosome database http:// rdp.cme.msu.edu/index.jsp, comparing and analyzing the DNA sequencing result with a strain 16S rDNA sequence with the closest homology, drawing a phylogenetic tree of the strain, and comparing the phylogenetic tree with the strain similarity to reach 99 percent (shown in figure 2).
Through morphological observation, physiological and biochemical reaction and 16S rDNA molecular identification, the strain is determined to be a new species of Bacillus subtilis (Bacillus velezensis), and the Bacillus subtilis in the digestive tract of the min pig is found to have cellulose degradation capability for the first time and is named as Bacillus subtilis (Bacillus velezensis) GX-1.
Example 2: cellulase Activity assay
The method comprises the following steps: preparing a crude enzyme solution: OD of activated culture broth600Adjusting to 1.0 to obtain the seed liquid for test; then inoculating the seed solution OD (equal to 1.0) into a 50mL shake flask fermentation medium in an inoculation amount of 1.0%, fermenting and culturing for 24h at 37 ℃ and 220rpm, centrifuging the fermentation broth for 15min at 5000rpm and 4 ℃, and obtaining the supernatant as a crude enzyme solution; drawing a glucose standard curve: preparing 1mg/mL glucose mother liquor, respectively taking 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4mL of the glucose mother liquor in a clean colorimetric tube, supplementing 2mL of ultrapure water when the volume is less than 2mL of the glucose mother liquor, then adding 2.5mL of DNS solution, shaking up, carrying out boiling water bath for 5min, rapidly cooling, then using purified water to fix the volume to 10mL, and mixing uniformly. 100 μ L of the sample was measured at a wavelength of 540nm in a microplate reader to determine OD value, and the absorbance OD value measured at 540nm was plotted as ordinate and glucose content (mg) as abscissa to draw a standard curve (see FIG. 3). Determination of cellulase (CMCA) enzyme activity: adding 1mL of crude enzyme solution and 1.5mL of 1% CMC-Na phosphate buffer solution into a clean colorimetric tube, and preserving the temperature for 30min in a water bath kettle at 50 ℃; then, 2.5mL of DNS solution is quickly added and shaken up to terminate the reaction, and the mixture is subjected to boiling water bath for 5 min; after rapid cooling, the volume is determined to 10mL by purified water, and the OD value is measured at the wavelength of 540 nm. And finding out the corresponding glucose content on a standard glucose curve according to the value of the absorbance. The amount of enzyme required for the substrate to produce 1. mu.g of glucose per hour is one enzyme activity unit (U). The invention tests the activity of the cellulase of the strain GX-1, and finds that the CMC enzyme activity of a 24-hour culture solution of the strain GX-1 is 37.24U/mL.
Example 3: influence of different culture conditions on growth and reproduction of strain GX-1
The invention provides a fermentation culture method of the strain GX-1, which specifically comprises the following steps: the shaking culture method in the laboratory: taking OD 6001% of the seed solution 1.0 was inoculated into the fermentation medium and cultured with shaking at 31-41 ℃ and 180 rpm at pH 4.0-7.0. Wherein the formula of the fermentation medium is as follows: 5g/L of yeast powder, 10g/L of trypsin vein, 10g/L of sodium chloride, 10g/L of CMC-Na,pH＝6±0.5。
(1) effect of different initial pH on Strain GX-1: FIG. 4 shows the enzyme production profile of Bacillus belgii GX-1 according to the present embodiment with respect to the initial pH. The enzyme activity of the strain is relatively stable when the initial pH value is between 4 and 7, and the enzyme activity reaches the highest value when the pH value is 5.0, so that the initial pH value of 5.0 is the optimal initial pH value for producing the enzyme by the Bacillus belief GX-1.
(2) Effect of different temperatures on the strain GX-1: FIG. 5 shows the enzyme production profile of B.belgii GX-1 according to this embodiment with temperature. The enzyme activity reaches the maximum value at the temperature of 35 ℃, and is 22.03U, so the temperature is the optimum temperature for producing the enzyme by the Bacillus beiLeisi GX-1 at the temperature of 35 ℃.
(3) Effect of different inoculum sizes on the strain GX-1: FIG. 6 shows the enzyme production profile of Bacillus belgii GX-1 according to the present embodiment with the amount of inoculation. When the inoculation amount is 2%, the enzyme activity reaches the maximum value, and the enzyme activity is 26.39U, so that the inoculation amount of 2% is the optimal inoculation amount of the Bacillus beiLeisi GX-1 for producing enzyme.
(4) Temperature tolerance of strain GX-1: inoculating the strain frozen at-80 ℃ to an LB liquid culture medium for activation for 8h, transferring 1 percent of the strain to the LB liquid culture medium, respectively placing the strain at 70 ℃ and 80 ℃ overnight, and then culturing for 24h, wherein the result shows that the strain GX-1 can survive in a dormant mode at high temperature.
Example 4: research on enzymatic characteristics of cellulase produced by strain GX-1
(1) Detecting the thermal stability of the cellulase produced by the Bacillus belgii GX-1: the reaction temperature profile is shown in FIG. 7. The optimal temperature for the CMCase reaction is 55 ℃. When the temperature is raised to 70 ℃, the enzyme activity of the cellulase is still more than 80% of the optimum temperature enzyme activity, which indicates that the cellulase produced by GX-1 has good thermal stability.
(2) And (3) detecting the pH tolerance of the cellulase produced by the Bacillus belgii GX-1: FIG. 8 shows the pH change curve of the Bacillus belgii GX-1 enzyme reaction according to this embodiment. The optimal pH for the CMCase reaction is 4.5.
(3) Detecting the effective time of the cellulase produced by the Bacillus belgii GX-1 on the cellulose reaction: FIG. 9 shows the reaction time of Bacillus belgii GX-1. The CMCase reaction is carried out for 25min, and the enzyme activity is highest, and then the enzyme activity and the substrate activity are gradually reduced along with the prolonging of the reaction time.
Example 5: preparation of Bacillus beleisi GX-1 microbial inoculum
In the embodiment, the preparation of the microbial inoculum is carried out by using the frozen bacillus belgii GX-1 strain, and the specific operation method comprises the following steps: inoculating the strain GX-1 into a reaction system (a formula: 5g/L of yeast powder, 10g/L of trypsin vein, 10g/L of sodium chloride, 10g/L of CMC-Na, pH 6 +/-0.5) for activation culture for 8h, then transferring the activated culture into the reaction system (a suggested system: 50ml) according to the inoculation ratio of 2%, wherein the culture conditions are as follows: the temperature is 35 ℃, the pH value is 5, after shaking culture is carried out for 24h, a GX-1 microbial inoculum can be obtained, the cellulase activity of the GX-1 microbial inoculum is measured, the enzyme activity is 41.18U/mL (the short-term storage condition of the microbial inoculum is 4 ℃, the suggested storage time is 48h, and the long-term storage condition is-40 ℃).
Example 6: influence of short-term drenching of strain GX-1 fermentation seed liquid on diversity of intestinal flora of SD rat
The invention utilizes the strain GX-1 and adopts a single-factor random block experimental design, 24 healthy SD female rats with 3 weeks of age, similar weight and SPF level are selected and randomly divided into 2 groups, each group has 6 repetitions, and each repetition has 2 rats. Pre-feeding for 3 days, wherein the control group (A group) is infused with 0.2ml/d of normal saline, and the test group (B group) is infused with 0.2ml/d of bacterial liquid; the gavage is carried out for 1 time every day, and the gavage is continuously carried out for 7 days. Free feeding, on the 21 st day of the experiment, killing mice in the A group and the B group, taking caecum chyme, and freezing and storing the caecum chyme for DNA extraction, library construction and sequencing, data processing, OTU division and diversity analysis;
the effect of short-term drenching of Bacillus belgii GX-1 on levels of cecal microbiota in SD rats is shown in Table 2. The results show that: the ratio of drenching Bacillus belgii GX-1 to SD rat cecal microorganisms actinomycetomycota and proteobacteria is obviously reduced (P < 0.05).
TABLE 2 Effect of short term drenching of Bacillus belgii GX-1 on levels of Microdochium cecum in SD rats
Note: the same row of data is marked with different letters to show significant difference (P <0.05), and no letter to show insignificant difference (P > 0.05).
Example 7: influence of long-term drenching of strain GX-1 fermentation seed liquid on intestinal morphological development of SD rat
In this example, 24 healthy SD rats of 3 weeks of age and similar body weight were selected and randomly divided into 2 groups of 6 replicates each, each of which was 2 replicates, using strain GX-1 and a single-factor randomized block experimental design. The normal saline solution is 0.2mL/d for the control group, and the bacterial liquid is 0.2mL/d for the test group (the concentration of the bacterial liquid is 1 multiplied by 10)8CFU/mL); the administration is performed 1 time per day for 20 days. Free feeding, all rats were sacrificed on test day 21 for sampling. The abdominal cavity was opened quickly and the jejunum, ileum were collected. 2cm sections of the intestine were cut and fixed in 10% formalin for intestinal HE staining for intestinal morphology. Taking out the intestinal section fixed in 10% formalin, washing, clearing, waxing, paraffin embedding, blocking, slicing, dewaxing and other treatment, hematoxylin and eosin staining, dewatering and sealing. Under an optical microscope, a typical visual field, i.e., 5 with intact villus morphology, was selected from each jejunum tissue section and photographed. Jejunal villus height and crypt depth were measured using Image-Pro Plus 6.0 Image analysis software, and the ratio was calculated from the measured villus height and crypt depth. The height of the villus is the distance between the topmost end of the villus and the midpoint of the connecting line of the villus joints of the recesses at the two ends, and the depth of the recess is the distance between the midpoint of the connecting line of the villus joints of the recesses at the two ends and the mucosa substrate.
The effect of drenching Bacillus beleisi GX-1 on morphological development of jejunum and ileum of SD rats is shown in Table 3. The burysibacillus beilesensis GX-1 is infused in the whole period, so that the villus height of the jejunum of a rat is obviously improved (P <0.05), and the crypt depth of the ileum is obviously reduced (P < 0.05). There is a tendency for the depth of the jejunal crypt to decrease (P >0.05) and for the height of the ileal villus to increase (P > 0.05).
TABLE 3 influence of Bacillus beleisi GX-1 on the morphological development of the intestinal tract of SD rats
Note: the same row of data is marked with different letters to show significant difference (P <0.05), and no letter to show insignificant difference (P > 0.05).
Example 8: effect of short-term drenching of Bacillus beleisis M2 on blood biochemical and blood immune factors of 35-day-old SD rats
In the embodiment, 24 healthy SD female rats (Beijing Wittison Hua) with 3 weeks of age, similar body weight and SPF level are selected by using strain GX-1 through a single-factor random grouping test design and are randomly divided into 2 groups, each group is 6 in repetition, 2 rats are repeated, a control group (group A) is infused with physiological saline 0.2mL/d, a test group (group B) is infused with bacterial liquid 0.2mL/d, the stomach is infused for 1 time every day, the stomach is infused for 7d continuously, the food is collected freely, a pre-feeding period is carried out for 3 days, one rat is selected randomly in each cage on the 11 th day of a formal period test and is killed, a rat blood sample is collected by a heparin sodium anticoagulation tube (an eyeball blood sampling method is adopted), the rat is centrifuged for 10min at 3500r/min, the plasma is separated, total cholesterol, triglyceride, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol are collected by a GPO-enzyme method, glucose determination is carried out by a glucose oxidase method, blood immune factors are determined by an enzyme-linked immunosorbent assay (IgA), IgG, IL-3583, IL-84 and IL concentration research kits are constructed according to the operation instructions of southern PAP and southern PAP research.
The effect of short-term drenching of bacillus belgii M2 on blood biochemical and blood immune factors in 35-day-old SD rats is shown in tables 4 and 5: short term infusion of Bacillus belgii reduced the concentration of total cholesterol, blood glucose, low density lipoprotein cholesterol in plasma compared to the control group, but the difference was not significant (P > 0.05); the concentration of high density lipoprotein cholesterol in plasma is improved, but the difference is not significant (P > 0.05); the triglyceride concentration was significantly reduced (P < 0.01).
TABLE 4 Effect of short-term drenching of Bacillus belgii M2 on the biochemistry of blood in 35 day-old SD rats
Note: the same row of data is marked with different letters to show significant difference (P <0.05), and no letter to show insignificant difference (P > 0.05).
TABLE 5 Effect of short term drenching of Bacillus belgii M2 on blood immune factors of 35 day old SD rats
Note: the same row of data is marked with different letters to show significant difference (P <0.05), and no letter to show insignificant difference (P > 0.05).
Example 9: effect of full-term drenching of Bacillus beleisis M2 on apparent digestibility of nutrients in SD rat diets
The test uses a total manure collection method, and manure collection is started at the 18 th day of the test. The food intake of each group of test SD rats was accurately recorded every day and feces excreted every day by each group of test SD rats was accurately collected for 3 days continuously. Weighing feces collected every day, adding hydrochloric acid solution with the mass being 10% of the fresh weight of the feces, fully and uniformly mixing, storing at-20 ℃, after the test is finished, fully and uniformly mixing the feces collected from the same group, selecting about 100g of fresh feces samples in each group, firstly drying in a 120 ℃ drying oven for 15min to inactivate the enzyme, cooling to 65 ℃ to dry, fully dampening for 24h, weighing until the weight is constant, and crushing to prepare a sample to be tested. Measuring the content of the crude protein by adopting a Kjeldahl method; measuring the content of crude fat by using an ether extraction method; energy is measured by a Parr 6300 type oxygen bomb calorimeter; ca and P are respectively measured by a potassium permanganate method and a colorimetric method.
The results are shown in Table 6: compared with a control group, the apparent digestibility of the bacillus beilesensis for crude protein, crude fat and crude fiber is improved in the whole-stage irrigation, but the difference is not significant (P is more than 0.05); the apparent digestibility of calcium is reduced, but the difference is not significant (P > 0.05); total energy, ash and phosphorus apparent digestibility were significantly improved (P < 0.05).
TABLE 6 Effect of full-term drenching of Bacillus beleisis M2 on apparent digestibility of nutrients in SD rat diets
Note: the same row of data is marked with different letters to show significant difference (P <0.05), and no letter to show insignificant difference (P > 0.05).
Claims (7)
1. The Min pig source Bacillus beliezensis GX-1 for degrading cellulose is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2018841 and is named as Bacillus velezensis GX-1 in classification.
2. The cellulose degrading Bacillus belgii GX-1 of Min swine origin according to claim 1, wherein the biological characteristics comprise: white wax, white colony, irregular edge, dryness, and wrinkles.
3. The cellulose degrading Bacillus belgii GX-1 of Min swine origin according to claim 1, wherein the fermentation culture method comprises: taking OD6001-5% of the seed liquid with the inoculum size of 1.0 is inoculated into a fermentation medium, and the fermentation medium is cultured by shaking a table under the conditions of the temperature of 31-41 ℃, the rotation speed of 180-: 5g/L of yeast powder, 10g/L of trypsin vein, 10g/L of sodium chloride, 10g/L of CMC-Na and 6 +/-0.5 of pH.
4. The Bacillus belgii GX-1 of Min swine origin for degrading cellulose of claim 3, wherein the temperature is 35 ℃, the pH is 5.0, and the inoculation amount is 2%.
5. The Bacillus belgii GX-1 as a cellulose-degrading enzyme of claim 1, wherein the cellulase produced by the method is capable of degrading cellulose at a temperature of 40-70 ℃, a pH of 4.0-7.0, and a reaction time of 5-40 min.
6. The Bacillus belgii GX-1 as a cellulose degradation product of claim 5, wherein the cellulase produced by the method has a cellulose degradation temperature of 55 ℃, a pH of 4.5, and a reaction time of 25 min.
7. Use of the cellulose degrading Bacillus belgii GX-1 of Min swine origin according to claim 1 for the preparation of fibrous feed.
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| CN112094775B (en) * | 2020-09-22 | 2022-07-26 | 中国石油天然气集团有限公司 | Bacillus belgii and screening culture method and application thereof |
| CN112175877B (en) * | 2020-10-19 | 2021-04-27 | 山东碧蓝生物科技有限公司 | Cellulose degrading strain with sewage ammonia nitrogen degrading capability and application thereof |
| CN112662599B (en) * | 2021-01-27 | 2022-06-14 | 吉林省农业科学院 | Poultry source Bacillus belgii CL-4 and application thereof |
| CN113249277B (en) * | 2021-07-05 | 2021-09-24 | 广东海洋大学 | Application of Bacillus beiLeisi in extraction of heparin from aquatic products |
| CN114058532B (en) * | 2021-08-13 | 2023-06-06 | 黑龙江省农业科学院畜牧兽医分院 | Bacillus bailii for producing enzyme and separation and application thereof |
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