CN112239735B - Bacillus subtilis, microbial inoculum, screening method and application - Google Patents
Bacillus subtilis, microbial inoculum, screening method and application Download PDFInfo
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- CN112239735B CN112239735B CN202010985674.7A CN202010985674A CN112239735B CN 112239735 B CN112239735 B CN 112239735B CN 202010985674 A CN202010985674 A CN 202010985674A CN 112239735 B CN112239735 B CN 112239735B
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Abstract
The invention relates to a Bacillus subtilis strain which is named as Bacillus subtilis Ch9 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020380. The invention also discloses a microbial inoculum prepared from the strain, a screening method and application. The strain has strong capability of producing protease, amylase, lipase and cellulase. The strain can be applied to grass carp culture, and can obviously improve the specific growth rate of grass carps and reduce the feed coefficient in growth; reducing liver injury caused by high fat diet and aeromonas hydrophila infection, maintaining normal lipid metabolism function of liver, and reducing accumulation of lipid in liver. The strain can also inhibit the adhesion of Aeromonas hydrophila to grass carp intestinal epithelial cells, weaken the damage of Aeromonas hydrophila to cells, and protect the grass carp intestinal mucosa structure damage caused by Aeromonas hydrophila.
Description
Technical Field
The invention relates to the technical field of strain cultivation, in particular to bacillus subtilis, a microbial inoculum, a screening method and application.
Background
Aeromonas hydrophila is widely existed in the culture water body and is a main pathogenic bacterium in the culture process of freshwater fishes. Among them, the intestinal tract is an important infection route for fish, and aeromonas hydrophila invading the fish body can enter blood circulation through the intestinal tract, thereby causing systemic infection of the fish body. The aeromonas hydrophila can cause intestinal inflammation and structural damage of the grass carp; also can cause the reduction of the oxidation resistance of the fish and the occurrence of lipid peroxidation.
The use of antibiotics is effective in controlling the occurrence and spread of diseases, but interferes with the normal growth and reproduction of beneficial microbial flora in the aquaculture environment and in the intestinal tracts of aquatic animals, resulting in susceptibility of the aquatic animals to pathogenic microorganisms, and more seriously, in increased resistance to pathogenic bacteria. The probiotics is utilized to adjust the micro-ecological balance in the animal body, restore the normal physiological function of the organism, prevent and treat diseases and promote health, and is gradually becoming a hot tide in the world, and the probiotics product is nontoxic, has no residue and can not generate drug resistance, so the probiotics has good prospect for replacing antibiotics.
Disclosure of Invention
In view of the above, the invention provides a Bacillus subtilis strain, which is Bacillus subtilis Ch9 preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020380.
Specifically, the 16S rDNA sequence of the bacillus subtilis Ch9 is shown as SEQ ID No. 1.
The invention also provides a screening method of the bacillus subtilis, which comprises the following steps:
s1, separating and purifying the grass carp intestinal tract to obtain bacillus;
s2, screening the bacillus obtained in the S1 through extracellular enzyme production, and screening a strain with extracellular enzyme production capacity, namely the bacillus subtilis Ch 9.
The enzymes include proteases, lipases, amylases, and cellulases.
The invention also provides a bacillus subtilis microbial inoculum which is prepared by activating, fermenting and culturing the bacillus subtilis strain.
The invention also provides application of the bacillus subtilis or the microbial inoculum thereof in grass carp breeding.
The invention has at least the following beneficial effects:
the strain is separated from the healthy grass carp intestinal tract, and has stronger capacity of producing protease, amylase, lipase and cellulase. The strain is applied to grass carp culture, so that the specific growth rate of grass carps can be obviously improved and the bait coefficient can be reduced in growth; reducing liver injury caused by high fat diet and aeromonas hydrophila infection, maintaining normal lipid metabolism function of liver, and reducing accumulation of lipid in liver. In the aspect of immune protection, the preparation can inhibit the adhesion of the aeromonas hydrophila to grass carp intestinal epithelial cells, weaken the damage of the aeromonas hydrophila to the cells and have a certain protection effect on the damage of an intestinal mucosa structure caused by the aeromonas hydrophila.
Drawings
FIG. 1 is a graph showing the oil red O staining pattern of the liver of grass carp fed with Bacillus subtilis Ch9 according to the present invention; note: at 400X magnification, 28D (A-D diagram) and 56D (E-H diagram) of each group (control group, Ah + Bs group and Bs + Ah group).
FIG. 2 is a graph showing the results of lipid deposition in the liver of grass carp fed with Bacillus subtilis Ch9 according to the present invention; i, drawing: area results of stained areas were counted using Image-Pro Plus 6.0 software; j diagram: fat content of the liver; values in the figure are expressed as mean ± sem (n ═ 6). Different superscript letters indicate significant differences between different treatment groups (P < 0.05). Indicates a significant difference between 28d and 56d (P < 0.05).
FIG. 3 is a graph showing the effect of Bacillus subtilis on the intestinal mucosa structure of grass carp (200X magnification ratio) according to an embodiment of the present invention; note: A-B belong to the grouping of a control group, C-E belong to the grouping of an Ah group, and C '-E' belong to the grouping of an Ah + Bs group; 1. 3, 4, 5, 6, 7 represent days post infection, respectively; as shown in the figure, A-1 represents a day-1 micrograph of the A control group, and B-3 represents a day-3 micrograph of the B control group; in the figure, S represents epithelial degeneration, necrosis or exfoliation, I represents inflammatory cell infiltration, BI represents hemorrhage, and G represents goblet cell proliferation.
FIG. 4 shows the effect of Bacillus subtilis on the structure of grass carp intestinal mucosa (200 × magnification ratio) according to an embodiment of the present invention; note: F-H belong to the grouping of the Ah group, and F '-H' belong to the grouping of the Ah + Bs group; 1. 3, 4, 5, 6, 7 represent days post infection, respectively; for example, F-4 represents day 4 micrographs of the F group (subgroup of Ah group), F '-4 represents day 4 micrographs of the F' group (subgroup of Ah + Bs group); in the figure, S represents epithelial degeneration, necrosis or exfoliation, I represents inflammatory cell infiltration, BI represents hemorrhage, and G represents goblet cell proliferation.
FIG. 5 is a schematic diagram (5000 ×) showing the ultrastructure of the effect of Bacillus subtilis on the ultrastructure of grass carp intestinal epithelial cells according to an embodiment of the present invention; note: control (A, B), Ah (C, D), Ah + Bs (E, F); TJ stands for tight junction, ER for endoplasmic reticulum, and M for mitochondria.
FIG. 6 is a ultramicro structural diagram (200 × enlarged scale) of the effect of Bacillus subtilis Ch9 on the microfilament skeleton of grass carp intestinal epithelial cells according to an embodiment of the present invention; note: A-B: control, C-E: ah group, C '-E': ah + Bs group; 1. 3, 7 represent days post infection, respectively; v: the microfilaments at the striated edges appear green and cloudy.
FIG. 7 is a microscopic view (200X) showing the effect of Bacillus subtilis Ch9 on Caco-2 cell morphology according to the present invention; a: control, B: group Bs, C: ah group, D: ah + Bs group; 3. 6, 9 represent the time (h) after infection, respectively.
FIG. 8 is a microscopic view (10000 ×) of the effect of Bacillus subtilis Ch9 on the ultrastructure of Caco-2 cells according to the present invention; a: control group, B: group Bs, C: ah group, D: ah + Bs group; TJ: and (4) tightly connecting.
FIG. 9 is a microscopic view (400X) of the effect of Bacillus subtilis Ch9 on the Caco-2 cell microwire skeleton according to the present invention; a: control, B: group Bs, C: ah group, D: ah + Bs groups, 1, 3, 6 represent time post infection, respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Screening of Bacillus subtilis Ch9
In one implementation aspect of the invention, the Bacillus subtilis is named as Bacillus subtilis Ch9, is preserved in Wuhan Chinese type culture Collection in 7-30 months in 2020, and has a preservation number of CCTCC NO: M2020380, and has a preservation address of No. 299 of eight paths in Wuchang district in Wuhan, Hubei province. The 16S rDNA sequence of the bacillus subtilis Ch9 is shown in SEQ ID No. 1.
1. Screening of relevant materials
Screening sources: the average weight of grass carp is about 250g, the grass carp is taken from a Tangson lake fishery, and the grass carp is placed in an aquarium for temporary culture for 3d before sampling.
Culture medium: FWA medium;
the culture medium for detecting the protease of the strain: 10g of casein; na (Na)2HPO42g of the total weight of the mixture; 13g of agar; 1000mL of water; 12.5mL of 0.4% bromothymol blue solution; the pH value is 7.2-7.4.
Determination of lipase production by the strains culture medium: 10g of peptone; 5g of NaCl; CaCl20.1 g; tween-8010 mL; 13g of agar; 1000mL of water; the pH value is 7.2-7.4.
Determining the culture medium for producing amylase by the strain: 5g of peptone; 10g of yeast extract; 5g of NaCl; 10g of soluble starch; 13g of agar; 1000mL of water; the pH value is 7.2-7.4.
And (3) determining a culture medium for producing the cellulase by the strain: (NH)4)2SO4 2g；MgSO4 0.5g；K2HPO41g of a compound; NaCl 0.5 g; 2g of sodium carboxymethylcellulose (CMC-Na); congo red 0.4 g; 13g of agar; 1000mL of water; the natural pH value.
2. Screening method
S1, separation and purification of bacillus
Separating foregut, midgut and hindgut of each 3 fish according to aseptic technique, homogenizing, diluting by 10 times, and taking 10-4、10-5、10-6The bacteria were isolated by plating three dilutions, using fresh water agar medium (FWA medium). Selecting clear and well-dispersed colonies on a bacterial separation plate, purifying, inoculating on an FWA slant, culturing at 28 ℃ for 24-28h, and storing at 4 ℃ for later use.
S2 screening of extracellular enzyme-producing bacteria
The bacteria purified in S1 were streaked onto four selection plates (protease-producing medium, lipase-producing medium, amylase-producing medium, and cellulase-producing medium, provided above) to form single colonies. The diameter of the hydrolytic ring and the diameter of the colony were observed and measured after culturing at 28 ℃ for 48 hours. The extracellular enzyme secretion ability of the bacteria is judged by comparing the hydrolysis loop diameter/colony diameter (H/D) values, the larger the value is, the stronger the enzyme production ability of the strain is, each strain is measured for 5 colonies, and the repetition is carried out for 3 times, so as to determine the enzyme production stability.
And (3) selecting the strain which has the hydrolysis ring on each screening plate and has the largest difference between the diameter of the hydrolysis ring and the diameter of the bacterial colony, namely the bacillus subtilis Ch9 obtained by final screening. As a result, as shown in Table 1, the Bacillus subtilis Ch9 pair was able to simultaneously produce protease, lipase, amylase and cellulase.
TABLE 1 digestive enzyme-producing ability of Strain Ch9
The preparation of the fermentation liquid of the bacillus subtilis Ch9 provided by the embodiment of the invention comprises the following steps: inoculating bacillus subtilis Ch9 in a liquid fermentation culture medium, performing fermentation culture at 25-30 ℃, and shaking in a shaking table for 2-4 days to obtain a fermentation liquid of bacillus subtilis Ch 9.
Wherein the shaking table oscillates at a rotating speed of 180 r/min.
The liquid fermentation medium comprises the following components in concentration: 10g/L of tryptone, 5g/L of yeast extract, 20g/L of sucrose and 5g/L of NaCl. The pH value of the liquid fermentation medium during preparation is 7.2-7.4, and the liquid fermentation medium is sterilized for 20min at the temperature of 121 ℃.
Identification of strains
16S rDNA sequence analysis:
extracting Ch9 DNA with kit, and using primer
27F: 5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID No. 2;
1492R: 5 '-TACGGYTACCTTGTTACGACTT-3', SEQ ID No. 3;
performing PCR amplification, wherein a PCR reaction system comprises: 2 XTaq PCR Master Mix 12.5. mu.L, primers 27F and 1492R each 1. mu.L, template DNA 1. mu.L, and finally 25. mu.L was supplemented with sterile double distilled water. The reaction procedure is as follows: 5min at 94 ℃; at 94 ℃ for 40s, at 55 ℃ for 40s, and at 72 ℃ for 2.5min, for 35 cycles; finally, the reaction is finished at 72 ℃ for 10min and 4 ℃. And (3) carrying out 1% (w/v) agarose gel electrophoresis on the PCR amplification product, cutting and recovering the product, and then sending the product to Shanghai biological engineering technical service company Limited for sequencing. The 16S rDNA sequence of the bacillus subtilis Ch9 is shown in SEQ ID No. 1. The sequence is subjected to Blast comparison on Genbank, and the identification result shows that the strain is highly homologous with bacillus subtilis (B.subtilis), determined and named as bacillus subtilis Ch9, the 16S rDNA sequence of the bacillus subtilis Ch9 is shown as SEQ ID No.1, and is preserved in the Wuhan Chinese type culture preservation center in 7-30 th 2020, with the preservation number of CCTCC NO: M2020380, and the preservation address of eight-way 299 in the Wuhan city, Hubei province.
Applications of the invention
The application research of the bacillus subtilis Ch9 and the fermentation liquid thereof in grass carp culture is carried out in the embodiment of the invention, and the research content is as follows.
1. Influence on grass carp growth and liver lipid metabolism
1.1 test methods
Grouping tests: the experimental fish is hungry for 24h, the body length and the weight are measured, namely, a formal experiment is started, and grass carp with healthy appearance and consistent specification (the average weight is 50.53 +/-0.70 g) is selected and randomly distributed to 12 culture pots with the capacity of 300L (4 groups in total, three in each group are repeated, as shown in table 2), and 25 fish are in each pot.
TABLE 2 Experimental groups
TABLE 3 basic feed formulation and nutritional ingredients
Note:1vitamin premix (mg/kg): vitamin a, 6500 IU; vitamin D34500 IU; vitamin C, 120 mg; vitamin E, 25 mg; vitamin K 35 mg; vitamin B112.5 mg; vitamin B212.5 mg; vitamin B615.0 mg; vitamin B120.025 mg; nicotinamide, 50 mg; pantothenic acid, 40 mg; inositol, 75 mg; folic acid, 2.5 mg; biotin, 0.08 mg.2Mineral premix (mg/kg): sodium chloride, 1.0; magnesium sulfate, 15.0; sodium dihydrogen phosphate, 25.0; aluminum chloride hexahydrate, 0.06; potassium dihydrogen phosphate, 32.0; 20.0 parts of monocalcium phosphate; ferric citrate, 2.5; calcium lactate, 3.5; 0.353 parts of zinc sulfate heptahydrate; manganese sulfate tetrahydrate, 0.162; 0.031 parts of copper sulfate pentahydrate; cobalt chloride hexahydrate, 0.001; potassium iodate, 0.003; cellulose, 0.39.
Feeding program: feeding basal feed 28d for control group, Ah + Bs group, and feeding basal feed containing Bacillus subtilis Ch9 for Bs + Ah group (wherein the content of Ch9 is 10)7CFU/g)28d；
Injecting 0.1mL PBS into abdominal cavity of grass carp of control group, and continuously feeding basal feed for 56 days; ah groups the intraperitoneal injection of a suspension of Aeromonas hydrophila A.hydrophila (bacteria concentration of 10)5CFU/mL), and continuously feeding the basic feed to 56 d; intraperitoneal injection of A.hydrophila bacterial suspension (bacterial concentration is 10) to grass carp in Ah + Bs group5CFU/mL), and continuing to feed basic feed containing Bacillus subtilis Ch9 (wherein the content of Ch9 is 10)7CFU/g) to 56 d; injecting A.hydrophila bacterial suspension (with the bacterial concentration of 10) into abdominal cavity of Bs + Ah group grass carp5CFU/mL), continue feeding basal feed to 56 d.
The basic feed containing the bacillus subtilis Ch9 is prepared by mixing the bacterial powder of the bacillus subtilis Ch9 into the basic feed.
The preparation method of the bacillus subtilis Ch9 comprises the following steps: bacillus subtilis Ch9 was inoculated from a slant into the liquid medium provided in the above example, the liquid fermentation medium comprising the following components in concentrations: 10g/L of tryptone, 5g/L of yeast extract, 20g/L of sucrose and 5g/L of NaCl. The pH value of the liquid fermentation medium during preparation is 7.2-7.4, and the liquid fermentation medium is sterilized for 20min at the temperature of 121 ℃. Fermenting for 18-24h until the final concentration of thallus is not less than 10 9After CFU/ml, adding protective agent, etc., and freeze drying to obtain bacterial powder for later use.
Sampling: after 28d and 56d of cultivation, the grass carps are starved for 24 h. After anesthesia with MS-222, each fish in the jar was counted and weighed. Then, 24 fish samples were randomly selected for each group (i.e., 8 fish were taken per cylinder). Measuring the length and the weight of an individual by using 6 fish, taking blood from tail veins to obtain serum, analyzing biochemical indexes of the serum, dissecting on ice, separating out viscera and liver, and weighing the viscera weight and the liver weight; immediately separating liver of 6 fish with sterile scissors, rapidly freezing in liquid nitrogen, and storing at-80 deg.C (no more than 2w) for total RNA extraction and determination of antioxidase; 6, taking the liver of the tail fish for measuring the lipid content of the liver; and 6, taking the liver of the fish, and fixing the liver in paraformaldehyde fixing solution for subsequent frozen section.
TABLE 4 list of abbreviations
In the context of Table 4, the following examples are,
1) survival rate (SR,%) 100 × (final/initial)
2) Weight gain (Weight gain, WG, g) — end Weight-initial Weight
3) Weight gain ratio (Weight gain, WGR,%) of 100 × (final Weight-initial Weight)/initial Weight
4) Specific growth rate (SGR,%/d) of 100 × (Ln last weight-Ln initial weight)/number of days of rearing
5) Food intake (Food intake, FI, g/fish) feed consumption (g, dry weight)/fish number
6) Feed Factor (FCR) total Feed per cylinder/total fish body weight gain per cylinder
1.2 oil Red O staining analysis
Fixing the liver sample with paraformaldehyde for 48h, dehydrating with 15% and 30% sucrose solution, embedding with OCT embedding medium, slicing with a freezing microtome (thickness of 8 μm), and staining with oil red O; photographs were taken under a microscope as shown in FIG. 1.
Randomly selected 10 fields from each sample, the relative area of red-stained lipid droplets in liver tissue was calculated using Image-Pro Plus 6.0 software, and the graphs were counted using a double blind method to summarize the results, as shown in FIG. 2.
1.3 determination of fat content
The liver samples were freeze-dried at-50 ℃ for 24h and then the crude fat content of the liver was determined by soxhlet extraction. The mass of the air-dried sample is recorded as m, the mass of the filter paper after extraction with constant weight is recorded as m1, the mass of the filter paper after extraction with constant weight and the degreased sample is recorded as m2, and the formula is used as follows: fat content (%). 100% × (m + m1-m2)/m, and the liver crude fat content was calculated.
1.4 serum Biochemical index determination
Serum Cholesterol (CHO), Triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) content and serum glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) activities are measured by a Helanwei diagram full-automatic biochemical analyzer, and the kits are all purchased from Zhongsheng Bei accuse biotechnology GmbH. The results are shown in Table 4.
1.5 results
In FIG. 1, the liver tissue sections were stained red for lipids and blue for nuclei.
In fig. 2:
1. results of oil red O staining (a-H) and statistics of the relative area occupied by lipid droplets staining for oil red O (I) show: the lipid droplet area stained with oil red O at 28d increased significantly in the Ah group (P <0.05), while there was no statistical difference in the Bs + Ah and Ah + Bs groups (P > 0.05); there was no significant difference in lipid droplet area for each group at 56d (P > 0.05).
2. J diagram shows the results of liver fat content determination: at 28d, the fat content of the Ah group was significantly higher than the control group (P <0.05), and the fat content of the Ah + Bs and Bs + Ah groups was significantly reduced compared to the Ah group (P < 0.05). At 56d, there was no statistical difference in liver fat content for each group (P > 0.05). The liver fat content at 56d was significantly higher in the control, Bs + Ah and Ah + Bs groups than at 28d (P < 0.05).
The fat content of the liver and the oil red O staining result show that the aeromonas hydrophila can cause the lipid accumulation of the liver of the grass carp, and the condition can be improved by adding the bacillus subtilis into the feed, so that the lipid accumulation in the liver of the grass carp is reduced. Specifically, at the 28 th day, the liver fat content of grass carp in Ah + Bs and Bs + Ah groups is obviously lower than that of the Ah group, and is at the same level as that of the control group.
TABLE 5 influence of Bacillus subtilis Ch9 on growth performance and food intake of grass carp
Note: the values in the table are expressed as mean ± sem (n ═ 3 replicate cylinders). Different letters after each row of values indicate significant differences (P < 0.05).
Table 5 shows that, starting from the 28 th day, the grass carp fed with the probiotic feed exhibited better growth performance, and at the 56 th day, the growth performance was significantly better than that of the groups not fed with the probiotic (control group and Ah group) and the FCR (feed factor) of the Ah + Bs group was the smallest, both when the probiotic was fed before the injection of the aeromonas hydrophila (Bs + Ah group) and after the injection of the aeromonas hydrophila (Ah + Bs group), indicating that the addition of bacillus subtilis Ch9 to the feed promoted the growth of the grass carp and improved the feed utilization efficiency.
TABLE 6 influence of probiotic Bacillus subtilis on biochemical indexes of grass carp serum
Note: the values in the table are expressed as mean ± sem (n ═ 6). Different letters after each row of values indicate significant difference (P < 0.05).
The results of the serum biochemical indexes show that TG of the Ah group is remarkably reduced at 28 th day after the injection of the aeromonas hydrophila compared with the control group, CHO and LDL-C are remarkably reduced to 56 th day, and HDL-C also has a tendency to be reduced; the results of the Ah + Bs group and the Bs + Ah group showed that the blood lipid lowering condition was improved and the blood lipid was restored to the same level as the control group, regardless of whether the Bacillus subtilis feed was fed before the injection of Aeromonas hydrophila or after the injection. In addition, the results of the Ah + Bs group and the Bs + Ah group show that the AST and ALT activities in the serum of the two groups are obviously reduced compared with the Ah group after the bacillus subtilis is added, and the bacillus subtilis Ch9 probably reduces the damage of aeromonas hydrophila to the liver.
2. Protection for grass carp intestinal epithelial cell structure damage
2.1 in vivo experiments
2.1.1 Experimental methods
Healthy experimental grass carps with the average weight of (70.0 +/-5.0) g are randomly divided into 3 groups which are respectively a control group: feeding feed without probiotics for 7d after 0.3 mL/tail of sterile PBS is poured into the mouth; ah group: 0.3 mL/tail (1.0X 10) of aeromonas hydrophila liquid for oral irrigation7cfu/mL) followed by feeding the probiotic-free feed for 7 d; feed group of Ah + Bs (bacillus subtilis Ch 9): the bacteria solution of Aeromonas hydrophila is fed after 0.3 mL/tail of the bacteria solution is fed, and the concentration of the bacteria solution is 1.0 multiplied by 107Experimental feed of cfu/g Bs 7 d; each group was set with 3 replicates and there was no significant difference in weight between groups. The experimental fish is raised in a culture system, the volume of each tank is 300L, and 20 tail fishes are contained in each tank.
And observing the disease condition of the fish body at any time after infection, wherein each group starts to sample at the 1 st d after the mouth is filled with sterile PBS or aeromonas hydrophila bacterial liquid, and samples are continuously taken at the same time period of 7 d.
2.1.2 Paraffin sectioning and histological microscopic examination
1) Fixing: the fresh tissue mass in the middle of the midgut was washed with ice-cold PBS and quickly fixed with 4% paraformaldehyde for over 24h after impurities were removed. Taking out the intestinal tissue from the fixing liquid, flattening the intestinal tissue in a fume hood by using a scalpel, and putting the trimmed intestinal tissue and the corresponding label into a dehydration box.
2) And (3) dehydrating: the intestinal tissues are dehydrated by different gradient alcohols in sequence in the dehydration box. And (3) dehydrating procedure: 4 hours of 75% alcohol, 2 hours of 85% alcohol, 2 hours of 90% alcohol, 1 hour of 95% alcohol, 30 minutes of absolute ethyl alcohol I, 30 minutes of absolute ethyl alcohol II, 5-10 minutes of alcohol benzene, 5-10 minutes of xylene I, 5-10 minutes of xylene II, 1h of wax I, 1 hour of wax II and 1 hour of wax III.
3) Embedding: embedding the wax-soaked intestinal tissue in an embedding frame. Firstly, molten wax is put into an embedding frame, before the wax is solidified, intestinal tissues are taken out and put into the embedding frame according to the requirements of an embedding surface, corresponding labels are pasted on the intestinal tissues, and the intestinal tissues are frozen and cooled at the temperature of minus 20 ℃. And after the wax is solidified, taking the wax block out of the embedding frame and finishing the wax block.
4) Slicing: the trimmed wax block was sliced on a paraffin slicer to a thickness of 4 μm. The slices were floated on a spreader in warm water (40 ℃) to spread out, picked up with a glass slide and baked in a 60 ℃ oven. Taking out after the water is dried, and storing at normal temperature for later use.
5) Paraffin section dewaxing to water: placing the slices in xylene I20 min, xylene II 20min, anhydrous ethanol I10min, anhydrous ethanol II 10min, 95% ethanol 5min, 90% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min, and distilled water sequentially.
6) Hematoxylin staining nuclei: and (3) placing the slices into Harris hematoxylin for dyeing for 3-8 min, washing with tap water, differentiating for several seconds by 1% hydrochloric acid alcohol, washing with tap water, returning blue by 0.6% ammonia water, and washing with running water.
7) Eosin staining of cytoplasm: and putting the slices into an eosin dye solution for dyeing for 1-3 min.
8) Dewatering and sealing: and (3) putting the slices into 95% alcohol I for 5min, 95% alcohol II for 5min, absolute ethanol I for 5min, absolute ethanol II for 5min, xylene I for 5min and xylene II for 5min in sequence, dehydrating and transparent, taking out the slices from the xylene, slightly drying, and sealing with neutral gum.
9) And (5) microscopic examination and photographing: the tissue sections were observed and compared under an optical microscope (ZEISS, Axio Imager A2) for changes in the intestinal mucosal structure of grass carp in each group, and pictures were taken, as shown in FIGS. 3 and 4. 2.1.3 ultrathin sections and ultrastructural observations
1) Material taking: a fresh tissue mass (typically cut to about 1cm 3) from the middle of the midgut was quickly stored at 4 ℃ with 2.5% glutaraldehyde.
2) Double fixation: the fixed intestinal tissue was rinsed 3 times in PBS (0.1M) and the supernatant was centrifuged off for 20min each time. Intestinal tissue was fixed with pre-cooled (4 ℃) 1% osmic acid for 2-3h at 4 ℃ and then rinsed 3 times with PBS (0.1M) for 20min each.
3) And (3) dehydrating: dehydrating the fixed intestinal tissue sequentially with different gradient alcohol (50%, 70%, 80%, 85%, 90%, 95%, 100%) for 10-20min (generally 15min) each time, and thoroughly dehydrating with 100% alcohol for 2 times (10 min each time).
4) And (3) infiltration: the penetrant is acetone: epoxy resin (2:1), acetone: epoxy resin (1:1) and epoxy resin, and permeating overnight for 12h each time, and heating in an incubator at 37 ℃.
5) Embedding: putting the penetrated intestinal tissue into a small capsule, adding embedding agent epoxy resin, curing for 48h, and heating at 60 deg.C.
6) Slicing and staining: the solidified intestinal tissue was trimmed to size and shape, sectioned on a microtome (Leica, EM UC7) to a thickness of about 60-100nm, and double stained with lead and uranium.
7) And (5) microscopic examination and photographing: the tissue sections were observed under a transmission electron microscope (FEI Co., Tecnai G220 TWIN; acceleration voltage: 200kv) to compare the changes of the tight junction and ultrastructure of the intestinal epithelial cells of each group of grass carp, and a picture was taken, as shown in FIG. 5.
2.1.4 Observation of cytoskeleton
And (3) fixing, dehydrating, embedding, slicing and dewaxing the paraffin section to water according to the paraffin section and the steps 1) to 5) in the histological microscopic observation.
6) Antigen retrieval: the tissue slices are placed in a repair box filled with EDTA antigen repair buffer (pH9.0) and subjected to antigen repair in a microwave oven. After the medium fire is boiled, the power is cut off and the interval is 10min, then the low fire is boiled, and the buffer solution is prevented from being excessively evaporated in the process, so that dry tablets are not cut. After natural cooling, the slide was washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.
7) Staining with phalloidin fluorescent reagent: gently throwing off the confining liquid, dripping a phalloidin fluorescent reagent (the dilution ratio is 1:200) prepared by PBS according to a certain proportion on the section, and flatly placing the section in a wet box for incubation at 4 ℃ overnight.
8) DAPI counterstaining of nuclei: slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. After the section is slightly dried, DAPI dye liquor is dripped into the circle, and the section is incubated for 10min at room temperature in a dark place.
9) Sealing: slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. The slices were slightly spun dry and mounted with an anti-fluorescent quenching mounting agent.
10) And (5) microscopic examination and photographing: the tissue sections were observed and compared under an upright fluorescence microscope (ZEISS, Axio Imager A2) for changes in the fluorescence intensity of microfilaments in the intestinal epithelial cells of grass carp of each group, and a picture was taken, as shown in FIG. 6.
2.1.5 intestinal villus height determination
The grass carp is sampled continuously for 7 days at the same time according to the experimental method of 2.1.1, and then the intestinal tract is dissected and taken out, and then the grass carp intestinal tract tissue section is prepared by fixing, dehydrating, transparentizing, embedding, slicing, dewaxing, HE staining and sealing. The intestinal villus height was measured with a micrometer under an optical microscope and the data was recorded. The results are shown in Table 7.
2.1.6 results
As can be seen from fig. 3 and 4:
1. The grass carp intestinal villi in the control group are regularly arranged, the intestinal mucosa structure is complete, and no obvious pathological change appears (see a-1 and a B-3).
2. On day 1, the Ah group and the Ah + Bs group both showed a small amount of inflammatory cell infiltration (as indicated by "I" in FIG. C-1 and FIG. C ' -1) and an increase in goblet cell number (as indicated by "G" in FIG. C-1 and FIG. C ' -1), and the lamina propria of the Ah group was slightly bleeding (as indicated by "BI" in FIG. C-1), whereas the Ah + Bs group did not show this phenomenon (FIG. C ' -1).
3. On day 2, the Ah group and the Ah + Bs group both exhibited slight exfoliation of part of the epithelial cells (indicated by "S" in FIG. D-2 and FIG. D '-2) and bleeding of the lamina propria (indicated by "BI" in FIG. D-2 and FIG. D' -2), and the bleeding of the lamina propria was more severe in the Ah group than in the Ah + Bs group.
4. On day 3, the damage degree of intestinal villi of the Ah group is more serious than that of the Ah + Bs group, mucosal epithelial cells are exfoliated in a large area (shown as S in a figure E-3), more exfoliated cell fragments exist in the intestinal lumen, and the inherent layer edema is thickened; whereas only a part of the epithelial cells in the Ah + Bs group was exfoliated (indicated by "S" in FIG. E' -3).
5. On day 4, the damage degree of intestinal mucosa of Ah group and Ah + Bs group is improved to a certain extent compared with that of day 3. Both groups had varying degrees of epithelial cell detachment (indicated by "S" in FIGS. F-4 and F '-4) and inflammatory cell infiltration within the mucosal layer (indicated by "I" in FIGS. F-4 and F' -4). The Ah group had slight bleeding in the lamina propria (shown as "BI" in FIG. F-4), while the Ah + Bs group did not show this (shown in FIG. F' -4).
6. On days 5-6, the intestinal villus damage degree of the Ah group and the Ah + Bs group is obviously reduced compared with that of the 3 rd-4 th day, and the phenomena of mucosal epithelial cell shedding and inflammatory cell infiltration are obviously improved. In 2 experimental groups, the intestinal mucosal epithelial goblet cell increase ("G" in FIG. G-5 and FIG. G '-5) and bleeding of the lamina propria ("BI" in FIG. G-5 and FIG. G' -5) were observed.
7. On day 7, the damage degree of intestinal villi in Ah group was more severe than that in Ah + Bs group, and part of mucosal epithelial cells were exfoliated (shown as "S" in FIG. H-7), and intestinal villi was shortened and thickened; whereas mucosal epithelial cells of the Ah + Bs group showed only a small amount of necrosis and slight exfoliation (shown as "S" in FIGS. H' -7). The goblet cell increase of intestinal mucosa epithelium occurred in 2 experimental groups (shown by "G" in FIG. H-7, FIG. H' -7).
As can be seen from FIGS. 3 and 4, the mucosal epithelial cells of Ah group grass carp are degenerated, necrosed and even exfoliated; goblet cells increase, and more exfoliated epithelial cell fragments and mucus exist in the intestinal lumen; bleeding and edema of the lamina propria become thick; mucosal layer inflammatory cell infiltration. The lesions are serious in 3-4 days after infection, and the 3d intestinal mucosa has the greatest damage degree, which is specifically characterized in that the epithelial cells of the intestinal mucosa are largely shed, and more cell fragments can be seen in the intestinal cavity; edema and coarsening of the lamina propria; there is a massive infiltration of inflammatory cells within the mucosal layer. On days 5-6, the above lesions were improved, but on day 7, mucosal epithelial cells were exfoliated, and intestinal villi were shortened and thickened.
Similar symptoms appear in Ah + Bs grass carps, but compared with the Ah group, the mucosal epithelial cell shedding phenomenon is reduced in 3-4 days. The lesions are obviously improved on days 5-7, and compared with the Ah group, the damage of the intestinal mucosa structure is greatly improved.
In FIG. 5, it can be seen that:
1. the structure of the tight connection (shown as TJ in the picture A) of the control group is clear and regular, and no gap exists in the middle; the microvilli are dense and orderly arranged, so that filamentous tow is arranged in the microvilli, and the tow extends into cytoplasm; the cytoplasm is rich in mitochondria, endoplasmic reticulum, etc., and mitochondria are rod-shaped with clear ridges ("M" in FIG. B).
2. The Ah group (4 d) tight junction structure was severely damaged, and the gap was significantly widened (indicated by "TJ" in FIG. C); the microvilli atrophy is shortened, the number is reduced, and the distribution is disordered; the micro-filament bundles in the micro villi are not clear; marked swelling, reduction of cristae (shown as "M" in panel D), marked expansion of endoplasmic reticulum (shown as "ER" in panel D), and increased lysosomes were observed in mitochondria.
3. The damage degree of the Ah + Bs group (4 d) tight junction structure is improved, and gaps are obviously narrowed (shown as 'TJ' in a graph E); the number of the microvilli is increased and the microvilli are neatly arranged; mitochondria were abundant, structurally intact, and no significant swelling ("M" in panel F).
Fig. 6 shows that:
1. the microfilaments in the intestinal epithelial cells of the control group emit uniform green fluorescence, and the fluorescence intensity is relatively strong (see FIGS. A-1 and B-3).
2. The fluorescence intensity of the microfilaments in the intestinal epithelial cells of the Ah group on days 1-3 is gradually weakened, and the fluorescence intensity of the microfilaments of the 3D group is weakest and is obviously lower than that of a control group (shown in figures D-3), and the microfilaments at the striatal edge of the intestinal epithelial cells are particularly obvious in a green fog state; the fluorescence intensity of the 4 th to 7 th microfilaments (see FIG. E-7) is slightly increased.
3. The fluorescence intensity of the microfilament in the intestinal epithelial cells of the Ah + Bs group is gradually reduced from day 1 to day 3, the fluorescence intensity of the microfilament is gradually enhanced from day 4 to day 7 (see figure E ' -7), but the fluorescence intensity of the microfilament is higher than that of the Ah group (see figure C ' -1 and figure D ' -3).
TABLE 7 influence of Bacillus subtilis on the height of the villi in the intestine of grass carp
As can be seen from table 7, as the experiment proceeded, the intestinal villus height of the Ah group was significantly reduced, while the control group had no significant change; the intestinal villus height of the Ah + Bs group is obviously reduced compared with that of the control group in days 1-7, but is obviously improved in days 2-7 compared with the Ah group. This indicates that Bacillus subtilis Ch9 provided by the present invention was fed at the same time. The in vivo experiment shows that the bacillus subtilis Ch9 has an improvement effect on symptoms such as degeneration, shedding and bleeding of grass carp mucous epithelium cells caused by A.hydrophila when fed. The method has an effect of improving the damage conditions of the grass carp intestinal villi caused by A.hydrophila, such as micro-connection structure, micro-villi quantity, mitochondrion quantity and the like. Meanwhile, the antibacterial agent also has a protective effect on the damage of microfilament structures in grass carp intestinal epithelial cells caused by A.hydrophila, and can reduce the damage of aeromonas hydrophila to grass carp intestinal villi, so that the absorption function of grass carp intestines is adjusted, the secretion capacity of grass carp intestines is enhanced, and the small intestinal mucosa is kept in a good state.
2.2 in vitro experiments
2.2.1 grouping of Caco-2 cells
When Caco-2 cells grow to 80% -90% fusion, about 1mL of cells containing 0.3g/L EDTA +2.5g/L pancreatin are added to digest the cells at 37 ℃, centrifugation is carried out at 1000r/min for 5min, and then the cell suspension is inoculated into a 12-hole cell culture plate (1 mL of cell suspension in each hole) at 37 ℃ and 5% CO2And (3) growing for 3-4 days under the condition until the cells grow into a monolayer, discarding the old cell culture solution, washing the cells by using sterile PBS (pH7.4), adding 1mL of double-antibody-free DMEM cell culture solution, and performing the following experiment.
The experiments were divided into 4 groups:
control group: firstly adding 0.5mL of double-antibody-free DMEM cell culture solution for culture, and then adding 0.5mL of double-antibody-free DMEM cell culture solution for culture at 1 h;
② Bacillus group (Bs group): 0.5mL of Bacillus subtilis Ch9 bacterial liquid (1.0X 10) provided in the above example was added6cfu/mL), and then adding 0.5mL of double-antibody-free DMEM cell culture solution for culturing at 1 h;
③ Aeromonas hydrophila group (Ah group): 0.5mL of Aeromonas hydrophila bacterial liquid (1.0X 10) is added6cfu/mL), and then adding 0.5mL of double-antibody-free DMEM cell culture solution for culturing at 1 h;
(iv) Aeromonas hydrophila + Bacillus group (Ah + Bs group): 0.5mL of Aeromonas hydrophila bacterial liquid (1.0X 10) is added 6cfu/mL), and then 0.5mL of Bacillus subtilis Ch9 bacterial liquid (1.0X 10) was added at 1h6cfu/mL);
each treatment group was set to 3 replicates, cells were incubated at 37 ℃ and 5% CO throughout the experiment2Culturing under the condition.
2.2.2 Observation of Caco-2 cell morphology
Each group was sampled at 3h, 6h, and 9h after the start of the experiment. And taking out the 12-hole cell culture plate, observing the change of the morphological structure of each group of cells under an inverted microscope, and taking a picture. As shown in fig. 7.
2.2.3 Observation of Caco-2 cell Tight Junction (TJ) and ultrastructure
When the cells grow in a T25 cell culture bottle for 7-10 d to 80% -90% fusion, the old cell culture solution is discarded, the cells are washed with sterile PBS (pH7.4), and then 2mL of double-antibody-free DMEM cell culture solution is added for the following experiment.
Experiment Caco-2 cells were grouped according to 2.2.1, and the concentration of Aeromonas hydrophila liquid and Bacillus liquid added in each group was 1.0X 106cfu/mL, the dose was changed to 1mL, and accordingly the dose of the double-antibody-free DMEM cell culture solution added to each group was changed to 1 mL.
Material taking: sampling is carried out 6 hours after the experiment is started, culture solution is removed, 2.5% glutaraldehyde is quickly added and fixed for 2-4 hours at 4 ℃, 0.1M phosphate buffer PBS (pH7.4) is rinsed for 3 times, 15 minutes each time, a cell scraper is used for scraping, and cell sediment is centrifugally collected (cell blocks with the size of mung bean are generally required to be collected).
Referring to 2.1.3 ultra-thin section and step 2-7 in ultrastructural observation, fixing, dehydrating, infiltrating, embedding, sectioning and staining of cells, and taking pictures by microscopic examination are completed, as shown in FIG. 8.
2.2.4 Observation of the Caco-2 cytoskeleton
1) Grouping and sampling of cells: cells were seeded into 12-well cell culture plates (1 mL cell suspension per well) containing sterile coverslips at 37 ℃ with 5% CO2The cells were grown to a monolayer under the conditions, the old cell culture solution was discarded, the cells were washed with sterile PBS (pH7.4), and the following experiment was performed after adding 1mL of double-antibody-free DMEM cell culture solution. The experiment was divided into 4 groups as described above, according to the grouping of 2.2.1 Caco-2 cells. Samples were taken at 1h, 3h, and 6h after the start of the experiment, the medium was discarded, fixed with 4% paraformaldehyde for 30min, and washed with PBS 3 times for 5min each.
2) Staining with phalloidin fluorescent reagent: after the slide is slightly dried, a histochemical pen is used for drawing circles at positions where cells are uniformly distributed in the middle of a cover glass (preventing liquid from flowing away), 50-100 mu L of phalloidin working solution is added, the mixture is incubated for 2 hours at room temperature, and the mixture is washed for 3 times by PBS (5 min each time).
3) DAPI counterstained nuclei: the slide was washed 3 times with PBS (pH7.4), 5min each time. After PBS was removed, DAPI staining solution was added dropwise to the circle, and incubated for 10min at room temperature in the dark.
4) Sealing: the slide was washed 3 times with PBS (pH7.4), 5min each time. After the slide is slightly spun dry, the side with cells faces downwards, and the slide is mounted on a glass slide by an anti-fluorescence quenching mounting piece.
5) And (3) photographing: the sections were observed under an upright fluorescence microscope and images were collected as shown in FIG. 9.
2.2.5 results
In FIG. 7, it can be seen that:
1. caco-2 cells of the control group and the Bs group both have polygonal shapes, the monolayer growth is in a paving stone shape, and the cell morphology is basically normal at 3h, 6h and 9 h.
2. Caco-2 cells of the Ah group disappear in shape, become round and have small volume, and are more and more seriously dead and shed in 3h, 6h and 9h, and a large cavity is formed due to death and shedding of most Caco-2 cells in 9 h.
3. Caco-2 cells in the Ah + Bs group disappear in shape, become round and have small volume, the phenomena of more and more serious death and shedding appear in 3h, 6h and 9h, and cavities with different sizes are formed due to death and shedding of the cells. However, compared with the Ah group at the same time point, the phenomena of Caco-2 cell death and shedding are obviously reduced.
In FIG. 8, it can be seen that:
1. the tight connection structures of the control group and the Bs group are neat and clear, and no gap is formed in the middle (shown as 'TJ' in the images A and B); filamentous microfilament bundles can be seen in the microvilli; the cytoplasm is rich in mitochondria, endoplasmic reticulum and the like, and the mitochondrial cristae is clear.
2. The Ah group (6h) tight junction structure is damaged, the gap is widened, or the tight junction structure becomes blurred (shown as "TJ" in FIG. C); the micro-filament bundles in the micro-villus are not clear or disappear; obvious swelling, ridge reduction or rupture and even vacuolation of mitochondria can be seen, and the endoplasmic reticulum is obviously expanded; more autophagosomes appeared.
3. The damage degree of the tight connection structure of the Ah + Bs group (6h) is smaller, the gap widening is not obvious, and the tight connection structure is clearer than that of the Ah group (shown as 'TJ' in a graph D); the mitochondria structure is complete without obvious swelling; there were a small number of autophagosomes.
In FIG. 9, it can be seen that:
1. the nucleus of the Caco-2 cell is blue under the excitation of ultraviolet, and the microfilament is green. The microfilaments in the cells of the control group and the Bs group emit uniform green fluorescence, the fluorescence is mainly distributed at the cell membrane and the center of the cell, and the fluorescence intensity is larger. The microfilaments are mutually connected in a bundle shape at the contact parts between cells, the outline is clear and complete, no obvious gap exists, and the cell boundary is clear; irregular fiber filaments are also visible in the center of the cells, and are scattered in the form of cord (see fig. a and B).
2. In the Ah group, the intracellular fluorescence intensity slightly decreases at 1h, the cord-like distribution of a part of cytoskeleton disappears, and the contact part of microfilaments among cells becomes fuzzy and rough and irregular (see figure C-1). The microfilament is gathered in a small area around the cell nucleus in 3h and 6h, and the central fluorescence intensity is strong; the fluorescence intensity of the peripheral part of the cell is weak, and the microwires are fuzzy and foggy (FIG. C-3 and FIG. C-6).
3. The Ah + Bs group showed a slight decrease in intracellular fluorescence intensity at 1h, and the microfilaments became blurred and uneven at the contact sites between cells (see FIG. C-1). At 3h and 6h, the microfilaments are also gathered in small areas around the cell nucleus (figure C-3 and figure C-6), but compared with the Ah group at the same time point, the fluorescence intensity and the microfilament gathering phenomenon are improved to a certain extent, the microfilament gathering range is smaller than that of the Ah group, the microfilaments at the cell membrane are broken in a ring-point shape, and the microfilaments are fuzzy and foggy.
The in vitro experiments show that when the bacillus subtilis Ch9, the aeromonas hydrophila and the Caco-2 cells are co-cultured, the death and falling caused by the Caco-2 cells due to the aeromonas hydrophila can be reduced, the protective effect on organelles such as microvilli, mitochondria and endoplasmic reticulum of the Caco-2 cells can be achieved, and the damage situations such as microfilament breakage of the Caco-2 cell membranes caused by the aeromonas hydrophila can be improved.
Since Caco-2 cells are a human clonal colon adenocarcinoma cell, structurally and functionally similar to differentiated small intestine epithelial cells, with microvilli and like structures, cells growing on porous permeable polycarbonate membranes can fuse and differentiate into intestinal epithelial cells under cell culture conditions to form a continuous monolayer, unlike the case where normal mature small intestine epithelial cells show counter-differentiation during in vitro culture, which is a widely adopted in vitro model for small intestine absorption. Therefore, when the bacillus subtilis Ch9 is co-cultured with bacillus subtilis Ch9, aeromonas hydrophila and Caco-2 cells, the adhesion of the aeromonas hydrophila to the Caco-2 cells can be inhibited, the damage caused by the aeromonas hydrophila is further reduced, the protection of oxidative stress damage to the Caco-2 cells caused by aeromonas hydrophila infection is provided, and the protection effect and the antioxidant capacity of the bacillus subtilis Ch9 to intestinal epithelial cells are reflected.
In conclusion:
1. the bacillus subtilis Ch9 provided by the invention is fed to grass carp, so that liver damage and lipid accumulation in liver caused by high-fat daily ration and aeromonas hydrophila infection can be reduced, the specific growth rate of grass carp is obviously improved, and the feed coefficient is reduced.
2. The bacillus subtilis Ch9 provided by the invention is fed to grass carp to protect the integrity and close connection structure of intestinal villi of grass carp, inhibit inflammatory reaction caused by aeromonas hydrophila in grass carp body, and protect intestinal mucosa structure damage caused by aeromonas hydrophila.
3. The bacillus subtilis Ch9 provided by the invention is fed to grass carp, so that the oxidation resistance and the immunity of the grass carp can be effectively improved, and the grass carp is prevented from oxidative stress damage caused by aeromonas hydrophila infection.
4. The bacillus subtilis Ch9 provided by the invention can inhibit the adhesion of aeromonas hydrophila to fish intestinal epithelial cells and can weaken the damage of aeromonas hydrophila to the cells when fed to grass carps.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (1)
1. The application of the bacillus subtilis in preparing the product for improving the intestinal mucosa structure damage of the grass carp is characterized in that the bacillus subtilis strain is bacillus subtilis (Bacillus subtilis)Bacillus subtilis) Ch9, preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020380; the structural damage of grass carp intestinal mucosa comprises the symptoms of grass carp intestinal mucosal epithelial cell degeneration, shedding and bleeding caused by aeromonas hydrophila, and the damage of the micro-connection structure, the micro-villus quantity and the mitochondrion quantity of grass carp intestinal villus caused by aeromonas hydrophila.
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| CN104411182A (en) * | 2012-04-12 | 2015-03-11 | 杜邦营养生物科学有限公司 | Microbial strains and their use in animals |
| CN105368749A (en) * | 2015-12-04 | 2016-03-02 | 青岛玛斯特生物技术有限公司 | Bacillus subtilis and feed additive containing same |
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| CN105368749A (en) * | 2015-12-04 | 2016-03-02 | 青岛玛斯特生物技术有限公司 | Bacillus subtilis and feed additive containing same |
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