Solid state fermentation method of yak source bacillus pumilus TS2 and application of yak source bacillus pumilus TS2 in mutton sheep feeding

Abstract

The invention discloses a solid state fermentation method of yak source bacillus pumilus TS2 and application of the yak source bacillus pumilus TS2 in feeding mutton sheep. The preservation number of the yak source bacillus pumilus TS2 is CCTCC (China Center For Type Culture Collection) NO: M2023246; the initial mixture and the bacterial liquid are evenly mixed according to the proportion of 7: 3 and then fermented to obtain the fermented feed, and after the fermented feed is fed, the meat quality of mutton can be improved by remarkably increasing the eye muscle area, improving the cooked meat rate and reducing the water loss rate; the oxidation resistance and the immunocompetence of the mutton sheep can be improved, and the disease resistance of the mutton sheep is improved. Meanwhile, the fermented feed is fed, so that the length and the width of rumen nipples can be increased to a certain extent before and after toxic counteracting, the thickness of a base layer is increased, the relative abundance of conditioned pathogenic bacteria in rumen after toxic counteracting is reduced, the relative abundance of beneficial bacteria is improved, and digestion and absorption of mutton sheep are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of livestock feed additives, and particularly relates to a solid-state fermentation method of Bacillus pumilus TS2 from yaks and its application in feeding meat sheep. Background Art

[0002] To effectively ensure the safety of animal-derived food and achieve the sustainable development of the livestock and poultry breeding industry, in order to promote the requirements of "antibiotic ban" in feed and "antibiotic reduction" in breeding nationwide, in the feed production and animal breeding links, the development and application of antibiotic alternatives have become the key. Among them, probiotic feed additives can maintain the balance of gastrointestinal flora through the synergistic effects of various probiotics, inhibit the growth of harmful microorganisms, and provide beneficial metabolites, thereby promoting the growth and development of the body. Bacillus can secrete highly active enzymes such as protease, amylase, and cellulase, and can also secrete antibacterial peptides with broad-spectrum antibacterial effects. Therefore, Bacillus is currently used as a probiotic supplement in animal feed; the main active ingredients of wolfberry include wolfberry polysaccharides, flavonoids, carotenoids, etc. Among them, wolfberry polysaccharide (LBP) has the effects of anti-oxidative aging, anti-tumor, anti-inflammatory, enhancing immunity, reducing blood sugar and blood lipids, protecting the retina, liver, kidney, and nerves, and has great potential for the prevention and treatment of various diseases.

[0003] In the aspect of meat sheep breeding, it has been found that adding Bacillus licheniformis and Bacillus subtilis to the diet significantly increased the average daily feed intake and average daily weight gain of weaned goats, improved the serum biochemical indexes of weaned goats, promoted the digestion and absorption of goats, and increased the richness and diversity of the flora in feces. At the same time, some studies have shown that adding 5% wolfberry residue can significantly improve the apparent digestibility of nutrients in the total mixed ration of fattening Tan sheep and enhance its antioxidant capacity and immune function. Feeding weaned male lambs of Tan sheep with a total mixed granular diet containing wolfberry residue, it was found that the digestion and metabolism ability, meat quality, and slaughter performance of fattening Tan sheep were all improved, indicating that wolfberry residue can also promote the growth and development of fattening Tan sheep.

[0004] Adding different types of probiotics to the diet has different effects on the rumen microbial diversity and metabolites, but can significantly increase the content of volatile fatty acids in the rumen, thereby improving the growth performance of ruminants. Most of the strains used in the current market are Bacillus subtilis isolated from the soil or other commercially available strains. The development of probiotic Bacillus from ruminants is insufficient. Moreover, when different types and proportions of compound probiotics are added to the high-concentrate diet of ruminants, what changes will occur in the pH of rumen fluid and rumen microbial diversity? There is currently a lack of relevant research. And in the current related technical inventions, there is a lack of comprehensive evaluation of the biochemical indexes in the blood and the pH and diversity of rumen microorganisms after feeding probiotic fermented feed, and some fermentation processes are relatively cumbersome and costly. Summary of the Invention

[0005] The purpose of this application is to provide a solid-state fermentation method of Bacillus pumilus TS2 from yak and its application in feeding meat sheep to solve the deficiencies in the prior art.

[0006] To solve the above technical problems, this application provides a solid-state fermentation method of Bacillus pumilus TS2 from yak. The preservation unit name of Bacillus pumilus TS2 from yak is China Center for Type Culture Collection; the preservation unit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is March 6, 2023; the preservation number is CCTCC NO: M2023246; the taxonomic naming is Bacillus pumilus TS2, including:

[0007] Screen 20% - 25% of wolfberry pomace, 24% - 28% of corn distillers' grains, 20% - 25% of rice bran, 10% - 12% of corn, 5% - 8% of soybean meal, 3% - 5% of distillers' grains, 5% - 8% of cottonseed meal;

[0008] Crush the wolfberry pomace, the corn distillers' grains, the rice bran, the corn, the soybean meal, the distillers' grains and the cottonseed meal, and then conduct the first mixing to obtain an initial mixture;

[0009] Add the bacterial liquid containing Bacillus pumilus TS2 from yak to the initial mixture and conduct the second mixing. Among them, the initial mixture and the bacterial liquid are mixed evenly according to a ratio of 7:3;

[0010] Load the material containing the bacterial liquid into a unidirectional breathing membrane bag with a breathing valve, heat-seal the bag, and then push it into a fermentation chamber at a temperature of 28 - 30 °C for fermentation. The fermentation time is 7 days;

[0011] Take out the material, prepare an LB solid medium, perform serial dilution on the test sample, and evenly coat it on an agar plate for colony counting. When the colony reaches 1×10 8 CFU / kg, it meets the standard and can be removed from the fermentation chamber and stored at room temperature.

[0012] To solve the above technical problems, this application also provides an application of the fermented feed fermented according to the above fermentation method in feeding meat sheep.

[0013] As a preferred embodiment, the feed for feeding meat sheep includes a basal diet, and the mass percentage of the fermented feed in the basal diet is 0.2% - 0.4%.

[0014] As a preferred embodiment, feed twice a day, and the feeding times are 8:00 and 17:00 respectively.

[0015] As a preferred embodiment, the fermented feed can enhance the antioxidant capacity and immune capacity of meat sheep.

[0016] As a preferred embodiment, the fermented feed can promote the digestion and absorption of meat sheep.

[0017] Compared with the prior art, it has at least the following beneficial effects:

[0018] In the present invention, an initial mixture and a bacterial liquid are mixed evenly in a ratio of 7:3 and then fermented to obtain a fermented feed. After feeding the fermented feed, the quality of mutton can be improved by significantly increasing the eye muscle area, increasing the cooked meat rate, and decreasing the water loss rate; it can also enhance the antioxidant capacity and immune capacity of meat sheep, and improve the disease resistance of meat sheep. At the same time, feeding the fermented feed can, to a certain extent, increase the length and width of rumen papillae and the thickness of the basal layer before and after virus challenge, reduce the relative abundance of conditional pathogenic bacteria in the rumen after virus challenge, and increase the relative abundance of beneficial bacteria, thereby promoting the digestion and absorption of meat sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the present application, the following briefly introduces the drawings required to be used in the embodiments. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is an analysis diagram of the growth performance results of a meat sheep provided by an embodiment of the present application;

[0021] Figure 2 It is an analysis diagram of the mutton quality results provided by an embodiment of the present application;

[0022] Figure 3 It is an analysis diagram of the slaughter performance results of mutton provided by an embodiment of the present application;

[0023] Figure 4 It is an analysis diagram of the antioxidant index results of a meat sheep provided by an embodiment of the present application;

[0024] Figure 5 It is an analysis diagram of the immune index results of a meat sheep provided by an embodiment of the present application;

[0025] Figure 6 It is an analysis diagram of the inflammatory factor results of a meat sheep provided by an embodiment of the present application;

[0026] Figure 7a It is a characteristic venn diagram before virus challenge provided by an embodiment of the present application;

[0027] Figure 7b It is a characteristic venn diagram after virus challenge provided by an embodiment of the present application;

[0028] Figure 8 A species distribution map at the phylum level provided by an embodiment of the present application;

[0029] Figure 9 A species distribution map at the genus level provided by an embodiment of the present application;

[0030] Figure 10 A graph showing the results of α-diversity analysis for each group before and after virus challenge provided by an embodiment of the present application;

[0031] Figure 11 A graph showing the significant analysis of α-diversity for each group after virus challenge provided by an embodiment of the present application;

[0032] Figure 12 PCoA analysis graphs for each group provided by an embodiment of the present application;

[0033] Figure 13 A graph showing the morphological characteristics of the rumen for each group before and after virus challenge provided by an embodiment of the present application;

[0034] Figure 14 A graph showing the changes in rumen papilla height, papilla width, and muscular layer thickness before and after virus challenge provided by an embodiment of the present application. Detailed implementation manners

[0035] In order to enable those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0036] The core of the present application is to provide a solid-state fermentation method of Bacillus pumilus TS2 derived from yaks and its application in feeding meat sheep, solving the deficiencies in the prior art.

[0037] Figure 1 A graph showing the analysis results of the growth performance of meat sheep provided by an embodiment of the present application, Figure 2 A graph showing the analysis results of the meat quality of meat sheep provided by an embodiment of the present application, Figure 3 A graph showing the analysis results of the slaughter performance of meat sheep provided by an embodiment of the present application, Figure 4 A graph showing the analysis results of the antioxidant indexes of meat sheep provided by an embodiment of the present application, Figure 5 A graph showing the analysis results of the immune indexes of meat sheep provided by an embodiment of the present application, Figure 6 A graph showing the analysis results of the inflammatory factors of meat sheep provided by an embodiment of the present application, Figure 7a A venn diagram of the characteristics before virus challenge provided by an embodiment of the present application, Figure 7b A venn diagram of the characteristics after virus challenge provided by an embodiment of the present application, Figure 8A species distribution map at the phylum level provided by an embodiment of the present application, Figure 9 A species distribution map at the genus level provided by an embodiment of the present application, Figure 10 A graph showing the results of α-diversity analysis for each group before and after virus challenge provided by an embodiment of the present application, Figure 11 A graph showing the significant analysis of α-diversity for each group after virus challenge provided by an embodiment of the present application, Figure 12 A PCoA analysis graph for each group provided by an embodiment of the present application, Figure 13 A graph showing the rumen morphological characteristics of each group before and after virus challenge provided by an embodiment of the present application, Figure 14 A graph showing the changes in rumen papilla height, papilla width and muscular layer thickness before and after virus challenge provided by an embodiment of the present application, see Figures 1 to 14 shown. The Bacillus pumilus TS2 derived from yak used in the present invention was deposited at the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province on March 6, 2023, with the deposit number: CCTCC NO: M2023246.

[0038] A solid-state fermentation method for Bacillus pumilus TS2 derived from yak, comprising the following steps:

[0039] Screen 20% - 25% of wolfberry pomace, 24% - 28% of corn distillers grains, 20% - 25% of rice bran, 10% - 12% of corn, 5% - 8% of soybean meal, 3% - 5% of distillers grains, 5% - 8% of cottonseed meal;

[0040] Crush the wolfberry pomace, the corn distillers grains, the rice bran, the corn, the soybean meal, the distillers grains and the cottonseed meal, and then conduct a first mixing to obtain an initial mixture;

[0041] Add a bacterial liquid containing Bacillus pumilus TS2 derived from yak to the initial mixture, and conduct a second mixing, wherein the initial mixture and the bacterial liquid are mixed evenly at a ratio of 7:3;

[0042] Load the material containing the bacterial liquid into a unidirectional breathing membrane bag with a breathing valve, heat-seal the bag, and then push it into a fermentation chamber at a temperature of 28 - 30 °C for fermentation, and the fermentation time is 7 days;

[0043] Take out the material, prepare an LB solid medium, perform serial dilution on the test sample, and evenly coat it on an agar plate to perform colony counting. When the colony reaches 1×10 8 CFU / kg, it meets the standard and can be removed from the fermentation chamber and stored at room temperature.

[0044] To solve the above technical problems, the present application also provides an application of a fermented feed fermented according to the above fermentation method in feeding meat sheep.

[0045] Preferably, the feed for feeding meat sheep includes a basal diet, and the mass percentage of the fermented feed in the basal diet is 0.2% - 0.4%. The composition and nutritional level of the basal diet are shown in Table 1.

[0046] Table 1 Composition and nutritional level of the basal diet

[0047]

[0048] Preferably, the feeding method is to feed twice a day, and the feeding times are 8:00 and 17:00 respectively.

[0049] Preferably, the fermented feed can improve the antioxidant capacity and immune capacity of meat sheep.

[0050] Preferably, the fermented feed can promote the digestion and absorption of meat sheep.

[0051] To enable those skilled in the art to better understand this solution, the following uses specific experiments to elaborate on this solution in detail:

[0052] Experimental process

[0053] Grouping, administration and modeling

[0054] Select healthy meat sheep at 2 - 3 months old with similar genetic background and body weight, and randomly divide them into 4 groups. The control group (CON) is fed the basal diet (the ratio is shown in Table 1), the wolfberry residue group (GQ) is fed the basal diet + the feed fermented with wolfberry residue, the experimental group I (2%) is fed the basal diet + 2% fermented feed, and the experimental group II (4%) is fed the basal diet + 4% fermented feed. The experimental period is 70 days, including a 10 - day preliminary trial period and a 60 - day formal trial period. After feeding for a period of time, the same number of meat sheep in each group are selected for Salmonella challenge, and the dose is 1.0×10 9 CFU / kg, to observe whether Bacillus pumilus TS2 from yaks and the addition of wolfberry residue in the feed play a role in reducing the inflammation caused by Salmonella. The fermented feed is the feed solid - state fermented with Bacillus pumilus TS2 from yaks.

[0055] Feeding management

[0056] Before the start of the experiment, all experimental sheep houses are cleaned and disinfected, and all experimental sheep are numbered and weighed and grouped. Feed twice a day, the feeding times are 8:00 and 17:00. The fermented feed is added proportionally to the diet every morning, mixed evenly and then fed. Free access to feed and water is provided. The experimental condition is high - bed feeding.

[0057] Determination indexes and methods

[0058] (1) Effects on improving growth performance: ① Average daily gain: Fasting weights were measured on the 1st, 30th, and 60th days of the formal period, and the average daily gain was calculated; ② Slaughter performance: After the end of the formal trial period, the sheep had free access to water, and the pre-slaughter live weights of all experimental sheep were measured after fasting for 12 hours. Then, 2 sheep were randomly selected from each group and sacrificed by jugular vein exsanguination to measure carcass weight, eye muscle area, and backfat thickness; The heart, liver, spleen, kidney, and lung were collected and weighed, and the organ indices were calculated. ③ The longissimus dorsi muscle between the 5th and 12th ribs was intercepted for meat quality determination, including indicators such as pH, cooked meat rate, water loss rate, shear force, and meat color.

[0059] (2) Diversity analysis of intestinal microbial communities: Fecal samples from different groups were sent to Beijing Biomarker Technologies Co., Ltd. for microbial community diversity analysis, and the intestinal microbial communities were identified at the phylum level and genus level of species categories.

[0060] (3) Effects on anti-inflammatory and antioxidant effects: Determination of serum biochemical and immune indices: On the 30th and 60th days of the experiment, all sheep were fasting and blood was collected from the jugular vein. 5 mL of jugular vein blood was collected, allowed to stand for 2 hours, then centrifuged at 3500 r / min for 15 minutes, and the serum was collected and stored at -20 °C to measure serum biochemical indices and serum anti-inflammatory and antioxidant indices, such as TP (total protein), ALB (albumin), GLB (globulin), BUN (blood urea nitrogen), GLU, SOD, MDA, GSH-Px, and IgG, IgM, IgA, etc. that reflect the immune capacity of the body.

[0061] Experimental results

[0062] 1. Analysis of the determination results of the growth performance of meat sheep

[0063] Fasting weights were measured on the 1st, 30th, and 60th days of the formal period, and their weight changes and average daily gain were calculated. The slaughter performance was measured according to the method in "Sheep Production Science". After the end of the formal trial period, the sheep had free access to water, and the pre-slaughter live weights of all experimental sheep were measured after fasting for 12 hours. Then, 2 sheep were randomly selected from each group and sacrificed by jugular vein exsanguination to measure carcass weight, eye muscle area, and backfat thickness (at the back where the 12th and 13th thoracic vertebrae are combined, the thickness of the subcutaneous fat was measured with a ruler, not including the skin).

[0064] The results of the growth performance of meat sheep are as Figure 1 shown. Compared with the control group, the average daily gain of the other three groups increased, but there was no significant difference. After slaughter, compared with the control group, the eye muscle area of the wolfberry residue group and experimental group I decreased (P<0.05), and the eye muscle area of experimental group II increased significantly (P<0.01); The backfat thickness of the wolfberry residue group, experimental groups I and II decreased compared with the control group but not significantly; The carcass weight of experimental group II increased, and that of group I decreased significantly (P<0.05).

[0065] 2. Determination results of mutton quality

[0066] The pH of the meat sample was measured using a portable pH meter 45 minutes after slaughter, and the brightness (L*), redness (a*), and yellowness (b*) values of the meat sample were measured using a color difference meter. Each meat sample was measured 3 times and the average value was taken.

[0067] Figure 2 The results of mutton quality are shown. Compared with the control group, the pH value of the wolfberry residue group decreased significantly (P<0.05), and the pH value of experimental group II increased significantly (P<0.01); the brightness of the wolfberry residue group also increased but not significantly, and the yellowness increased significantly (P<0.01).

[0068] 3. Determination results of slaughter performance of meat sheep

[0069] The longissimus dorsi muscle between the 5th and 12th ribs was intercepted for meat quality determination, including cooked meat rate, water loss rate, slaughter rate, shear force, etc.

[0070] The determination results of the slaughter performance of meat sheep are as Figure 3 shown. Compared with the control group, the cooked meat rate of experimental group I increased significantly (P<0.05), and the cooked meat rate of experimental group II increased significantly (P<0.01); the water loss rate of experimental group II decreased significantly (P<0.05); compared with the control group, the slaughter rate of experimental group I decreased (P<0.05); the shear force of the wolfberry residue group, experimental group I, and experimental group II all decreased, and the shear force of the wolfberry residue group decreased significantly (P<0.01).

[0071] 4. Determination results of antioxidant indexes of meat sheep

[0072] On the 30th and 60th days of the experiment, all sheep were bled from the jugular vein on an empty stomach. 5 mL of jugular vein blood was collected, allowed to stand for 2 h, then centrifuged at 3500 r / min for 15 min, the serum was collected, stored at -20 °C, and the serum biochemical indexes and serum antioxidant indexes were measured.

[0073] Figure 4 It can be seen that after 60 days of feeding, compared with the control group, the content of GSH-Px in experimental group I, experimental group II, and the wolfberry residue group all increased, and the increase in experimental group II was more significant (P<0.05); the activity of SOD in experimental group I increased significantly (P<0.05).

[0074] 5. Determination results of immune indexes of meat sheep

[0075] From Figure 5It can be seen that after 60 days of feeding, the contents of IgA and IgG in the experimental group II increased extremely significantly compared with the blank control group (P<0.05), the content of IgM increased significantly (P<0.01), and the content in the wolfberry residue group increased but not significantly; after 30 days of feeding, the protein synthesis ability of the experimental group II also increased extremely significantly compared with the blank control group (P<0.01), and that of the experimental group I increased significantly after 60 days (P<0.05), while the ability of the wolfberry residue group decreased.

[0076] 6. Determination results of anti-inflammatory level of meat sheep

[0077] It is Figure 6 known that compared with before inoculation, the contents of three inflammatory factors, IL-1β, IL-6 and TNF-α, in the serum of meat sheep increased significantly after inoculation. However, compared with the control group after inoculation, the contents of TNF-α and IL-6 in the serum of the wolfberry group after inoculation decreased (P<0.05), the content of IL-6 in the experimental group I decreased extremely significantly (P<0.0001), the content of IL-1β decreased significantly (P<0.01), and the contents of TNF-α and IL-1β in the experimental group II also decreased extremely significantly (P<0.001).

[0078] 7. Analysis of rumen microbial diversity

[0079] After the end of the formal trial period, 2 sheep were randomly selected from each group (fasted for 12 h and allowed free drinking water), and sacrificed by jugular vein bleeding. Immediately after slaughter, the rumen was isolated, and the rumen contents were collected and aliquoted into 2 50-ml centrifuge tubes for determination of rumen flora abundance.

[0080] (1) Analysis of the number of OTU / ASVs

[0081] ASVs (amplicon sequence variants) represent the truly existing biological sequences, can distinguish the differences between sequences at the single nucleotide precision, and can achieve the identification of real species or even strain-level species in samples. The dada2 method in the QIIME2 2020.6 software was used to denoise the sequences to obtain ASVs. The following table and figure show the distribution of the number of ASVs in each sample obtained by clustering: the numbers above the bars in the figure are the ASV numbers of the corresponding samples.

[0082] The determination was divided into 8 groups, among which C, G, T2, and T4 were the control group, the wolfberry residue group, experimental group I, and experimental group II respectively; CS, GS, TS2, and TS4 were the control group, the wolfberry residue group, experimental group I, and experimental group II after challenge. Before challenge, the number of core OTUs common to the four experimental groups was 7, the number of unique OTUs in the control group was 4321, and the numbers of unique OTUs in the wolfberry residue group, experimental group I, and experimental group II were 4071, 4396, and 3913 respectively; after challenge, the number of core OTUs common to the four experimental groups was 124, the number of unique OTUs in the control group was 1207, and the numbers of unique OTUs in the wolfberry residue group, experimental group I, and experimental group II were 1403, 971, and 710 respectively. It can be seen that the number of OTUs of rumen bacteria decreased significantly after challenge.

[0083] (2) Species taxonomic analysis

[0084] Under the conditions of this experiment, the species distribution of each group at the phylum level is shown in Figure 8 . Before challenge, the phyla with relatively high relative abundances at the phylum level in the samples were Acidobacteria, Proteobacteria, Actinobacteria, Bacteroidota, Gemmatimonadota, Chloroflexi, etc.; after challenge, the phyla with relatively high relative abundances at the phylum level were Bacteroidota, Firmicutes, Proteobacteria, Spirochaetes, etc. It can be seen that there were obvious changes in the dominant microbial flora in the rumen of meat sheep before and after challenge.

[0085] The species distribution of each group at the genus level is shown in Figure 9Before virus challenge, the genera with relatively high relative abundances at the genus level in the samples were Sphingomonas, unclassified-Gemmatimonadaceae, Vicinamibacterales, etc. Among them, the relative abundance of Sphingomonas increased significantly in experimental groups I and II; after virus challenge, the phyla with relatively high relative abundances at the genus level were Succiniclasticum, Prevotella, Treponema, Rikenellaceae_RC9_gut_group, etc. As a conditional pathogen that can cause inflammation, the relative abundance of Prevotella decreased significantly in the wolfberry residue group and experimental groups I and II compared with the control group; while the Rikenellaceae_RC9_gut_group can promote the digestion and absorption of carbohydrates in the intestine, and the bacteria of the genus Succiniclasticum have the effects of clearing succinic acid, improving the body's glucose tolerance and inflammation, and the relative abundance increased in the wolfberry residue group and experimental group I.

[0086] (3) α-diversity analysis

[0087] α-diversity reflects the species richness and species diversity of a single sample. Among them, the Chao1 index and ACE index are related to the microbial community abundance, while the Shannon index and Simpson index are related to the diversity of the intestinal flora. As Figure 10 can be seen, compared with before virus challenge, the species abundance and colony diversity after virus challenge decreased significantly.

[0088] As Figure 11 can be seen, compared with the control group, the Chao1 index of the GQ group increased extremely significantly after virus challenge (P < 0.01); and the Shannon index of the GQ group and experimental group I increased significantly (P < 0.01).

[0089] It can be seen that adding wolfberry to the feed can significantly increase the abundance of the rumen microbial community and the diversity of the flora; adding Bacillus-fermented feed can reduce the abundance of the communities in experimental groups I and II and increase the diversity of the flora in experimental group I.

[0090] (4) β-diversity analysis

[0091] β-diversity is mainly used to compare the similarity in species diversity between different samples. Principal Coordinate Analysis (PCoA) can extract the most important elements and structures from multi-dimensional data. Through principal coordinate analysis, the classification of multiple samples can be achieved, and further show the species diversity differences between samples. The closer the samples are on the coordinate graph, the greater the similarity. As Figure 12It can be seen that the differences between groups are obvious while the within-group differences are small, and the clustering is good, indicating that the community structures are highly similar.

[0092] 8. Experimental section analysis

[0093] The rumen tissues were subjected to HE staining to observe the morphological changes of the rumen ( Figure 13 ). Among them, Group A was before challenge, Group B was after challenge, CON represented the control group, GQ represented the wolfberry residue group, 2% represented Experimental Group I fed with 2% fermented feed, and 4% represented Experimental Group II fed with 4% fermented feed. The length and width of rumen papillae and the thickness of muscular layer were recorded, and the changes before and after challenge were compared ( Figure 14 ).

[0094] Compared with the control group, before challenge, the length, width of rumen papillae and the thickness of muscular layer in the other three groups all decreased. Among them, the width of rumen papillae in the wolfberry group and Experimental Group I was higher than that of the control group (P < 0.05), the height of rumen papillae and the thickness of muscular layer in Experimental Group I were significantly higher than those of the control group (P < 0.01), the width of rumen papillae in Experimental Group II was significantly higher than that of the control group (P < 0.001), and the thickness of muscular layer was also significantly higher than that of the control group (P < 0.01). Compared with before challenge, after challenge, the length of rumen papillae and the thickness of muscular layer decreased overall, but compared with the control group, the length of rumen papillae in the wolfberry group and Experimental Group II increased significantly (P < 0.01), and the length of rumen papillae and the thickness of muscular layer in Experimental Group I increased extremely significantly (P < 0.001); the width of rumen papillae in Experimental Group II increased significantly (P < 0.01), and the thickness of muscular layer increased (P < 0.05).

[0095] From the experimental data of this feeding trial, it can be concluded that compared with the control group, feeding the fermented feed fermented by Bacillus pumilus TS2 from yaks can improve the meat quality of mutton by significantly increasing the eye muscle area, increasing the cooked meat rate and decreasing the water loss rate.

[0096] Meanwhile, the content of GSH-Px and the activity of SOD in the three experimental groups increased after 60 days of feeding (P < 0.05). After 60 days of feeding, the contents of IgA and IgG in Experimental Group II were significantly higher than those in the blank control group (P < 0.05), and the content of IgM increased significantly (P < 0.01), indicating that feeding the feed fermented by wolfberry residue and Bacillus pumilus (fermented feed) can improve the antioxidant capacity and immune capacity of mutton sheep; and after challenge, the contents of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of mutton sheep decreased significantly compared with the control group (P < 0.05).

[0097] According to the analysis of rumen microbial communities, after LPS-induced inflammatory response, the Chao1 index of rumen microbial diversity will decrease, and the shannon index of the GQ group and the LDG group will increase significantly after challenge, indicating that feeding Bacillus-fermented feed can reduce the abundance of the flora and increase the diversity of the flora. Such results may be related to the antibacterial properties of Bacillus pumilus used in this experiment, and its antibacterial properties will regulate the balance of the flora.

[0098] Other embodiments of the present application will be readily apparent to those skilled in the art upon consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present application, which follow the general principles of the present application and include the common general knowledge or conventional technical means in the technical field disclosed in the present application. The specification and examples are only illustrative, and the true scope of the present application is pointed out by the claims.

[0099] It should be understood that the present application is not limited to the exact structures described above and shown in the drawings, and various modifications and changes can be made without departing from its scope. The embodiments of the present application described above do not constitute a limitation on the protection scope of the present application.

Claims

1. A solid-state fermentation method of yak-derived Bacillus pumilus TS2, characterized in that: The depository name of the yak-derived Bacillus pumilus TS2 is: China Center for Type Culture Collection; the depository address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit date is: March 6, 2023; the deposit number is: CCTCC NO: M2023246; the classification name is: Bacillus pumilus TS2, including: Screening 20% ​​to 25% wolfberry residue, 24% to 28% corn distiller's grains, 20% to 25% rice bran, 10% to 12% corn, 5% to 8% soybean meal, 3% to 5% distiller's grains, and 5% to 8% cottonseed meal; The wolfberry residue, the corn distiller's grains, the rice bran, the corn, the soybean meal, the distiller's grains and the cottonseed meal are crushed and then mixed for the first time to obtain an initial mixture; Adding a bacterial solution containing yak-derived Bacillus pumilus TS2 to the initial mixture, and performing secondary mixing, wherein the initial mixture and the bacterial solution are evenly mixed in a ratio of 7:3; The material containing bacterial liquid is put into a one-way breathing film bag with a breathing valve, and after heat sealing, it is pushed into a fermentation tank with a temperature of 28-30°C for fermentation for 7 days; Take out the material, prepare LB solid medium, dilute the sample to be tested in multiple ratios, evenly spread it on an agar plate and count its colonies. When the colony count reaches 1×10 8 CFU / kg meets the standard and can be moved out of the fermentation warehouse and stored at room temperature.

2. Use of fermented feed obtained by the fermentation method according to claim 1 in feeding mutton sheep.

3. The use according to claim 2, characterized in that: The feed for feeding mutton sheep comprises a basic diet, and the mass percentage of the fermented feed in the basic diet is 0.2% to 0.4%.

4. The use according to claim 2, characterized in that: Feed twice a day, at 8:00 and 17:00 respectively.

5. The use according to claim 1, characterized in that: The fermented feed can enhance the antioxidant capacity and immune capacity of mutton sheep.

6. The use according to claim 1, characterized in that: The fermented feed can promote digestion and absorption of mutton sheep.

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