Buffalo rumen source lignin degrading bacterium composite microecological preparation and application thereof

Abstract

The invention discloses a buffalo rumen-derived lignin degrading bacterium composite microecological preparation and application thereof, and relates to the technical field of biology. Active components of the buffalo rumen-derived lignin degrading bacterium composite microecological preparation comprise a buffalo rumen-derived lignin degrading bacterium combination; the buffalo rumen source lignin degrading bacterium composition is prepared from bacillus sorola desert, bacillus cereus and ochrobactrum pseudointermedium. The buffalo rumen source lignin degrading bacterium composite microecological preparation can obviously improve the digestion and absorption capacity of beef cattle, and especially has an obvious degradation effect on wood fiber substances which are difficult to decompose, so that the feed utilization rate is increased, and the feeding cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, focusing on a compound microecological preparation of buffalo rumen-derived lignin-degrading bacteria and its application. Background Technology

[0002] The cell walls of roughage plants contain a high proportion of lignin, which has a complex and difficult-to-degrade structure. This lignin forms a natural physical barrier within the plant cell wall, preventing most enzymes that can degrade polysaccharides such as cellulose and hemicellulose from penetrating. Overcoming this lignin barrier and exposing cellulose, hemicellulose, and other polysaccharide nutrients can not only improve the utilization efficiency of crude fiber in ruminants but may also drive significant breakthroughs in the feed industry and biomass energy. In nature, microorganisms, including fungi and bacteria, can degrade lignin. In-depth research into the mechanisms of microbial lignin degradation is of significant scientific importance and application potential for developing new technologies and products to improve the utilization rate of roughage.

[0003] As an important livestock species, water buffalo, compared to other breeds, can not only survive and reproduce in extreme environments such as high temperature and humidity, but also effectively cope with various disease challenges, and have a high tolerance for extensive feeding methods. Numerous studies have shown that, compared to other breeds, water buffalo rely mainly on roughage during the fattening stage, especially exhibiting significant advantages in low-quality roughage such as corn stalks and rice straw, which have extremely low lignin degradation. Previous research by the inventors also found that water buffalo are significantly better than cattle in their ability to break down rice straw cell walls.

[0004] Water buffalo rely on the synergistic effect of their rumen microbial community to effectively degrade lignocellulose in plants. In contrast, although beef cattle are also ruminants, their lignocellulose degradation efficiency is lower than that of water buffalo due to differences in rumen microbial composition or different feeding and management methods, resulting in the incomplete degradation and absorption of various nutrients within plant cell walls. Since chemical and enzymatic agents have limited effectiveness in improving rumen degradation efficiency, rumen microbial transplantation (RMT) technology offers a new direction for improving the degradation efficiency of roughage fiber. For example, by transplanting rumen microorganisms from animals with high fiber-degrading capabilities into other animals, it can help other ruminants utilize low-quality roughage more efficiently, reducing feed costs and environmental pollution. It shows promising application prospects in regulating ruminant growth performance and digestive metabolism.

[0005] Currently, some microbial preparations and enzymes related to cellulose and hemicellulose degradation are used as feed additives to improve the digestibility and utilization of roughage by ruminants. These additives not only improve the digestibility of crude fiber in feed but also help improve the rumen environment, promote rumen fermentation, and thus enhance ruminant growth performance, contributing to the sustainable development of ruminant animal husbandry. However, research on the use of lignin-degrading microbial preparations as feed additives is limited. Therefore, the preparation of lignin-degrading microbial preparations for use as feed additives and their application in livestock production will create significant economic and social benefits. Summary of the Invention

[0006] The purpose of this invention is to provide a buffalo rumen-derived lignin-degrading bacteria compound microecological preparation and its application, in order to solve the aforementioned technical problems. This buffalo rumen-derived lignin-degrading bacteria compound microecological preparation can significantly improve the digestive and absorptive capacity of beef cattle, especially exhibiting a significant degradation effect on difficult-to-decompose lignocellulose substances, thereby improving feed utilization and reducing feeding costs.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a buffalo rumen-derived lignin-degrading bacterial combination, wherein the buffalo rumen-derived lignin-degrading bacterial combination includes Sonora desert Bacillus (… Bacillus sonorensis ), Bacillus cereus ( Bacillus cereus ) and pseudo-intermediate paleobacterium ( Ochrobactrum pseudintermedium ); The Sonora desert Bacillus was deposited at the China Center for Type Culture Collection on March 18, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025512. The Bacillus cereus was deposited at the China Center for Type Culture Collection on March 18, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025511. The *Pseudomonas intermedia* was deposited on March 18, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025513.

[0008] Furthermore, the ratio of the number of Sonora desert Bacillus, the number of Bacillus cereus, and the number of Pseudomonas intermedia is 20:20:1.

[0009] The present invention also provides the application of the above-mentioned combination of buffalo rumen-derived lignin-degrading bacteria in the degradation of lignin.

[0010] The present invention also provides the application of the above-mentioned buffalo rumen-derived lignin-degrading bacteria combination in the preparation of lignin-degrading bacteria compound microecological preparations.

[0011] The present invention also provides a lignin-degrading bacteria composite microecological preparation, the active ingredients of which include the above-mentioned buffalo rumen-derived lignin-degrading bacteria combination.

[0012] Furthermore, the lignin-degrading bacteria compound microecological preparation also includes excipients.

[0013] This invention also provides a method for preparing the above-mentioned lignin-degrading bacteria composite microecological preparation, comprising the following steps: The above-mentioned Sonora desert Bacillus and Bacillus cereus were fermented and cultured, and the bacterial cells were isolated and then spray-dried to obtain Sonora desert Bacillus spray-dried powder and Bacillus cereus spray-dried powder. The above-mentioned Pseudomonas intermedia was fermented and cultured, and the bacterial cells were isolated and then freeze-dried to obtain Pseudomonas intermedia freeze-dried powder. The lignin-degrading bacteria composite microecological preparation is obtained by uniformly mixing the spray-dried powder of Bacillus desertica, the spray-dried powder of Bacillus cereus, and the freeze-dried powder of Bacillus pseudointermedia.

[0014] The present invention also provides the application of the above-mentioned lignin-degrading bacteria compound microecological preparation in the degradation of lignin.

[0015] The present invention also provides the application of the above-mentioned lignin-degrading bacteria compound microecological preparation in the preparation of feed additives or feeds that improve the growth performance of beef cattle.

[0016] The present invention also provides a feed additive or feed to improve the growth performance of beef cattle, including the above-mentioned lignin-degrading bacteria compound microecological preparation.

[0017] The present invention discloses the following technical effects: This invention isolates and purifies three lignin-degrading bacteria from the rumen of buffalo: *Bacillus desertica* YB, *Bacillus cereus* AH7-7, and *Pseudomonas intermedius* CB-2. A compound microecological preparation using these three bacteria provides a novel solution for beef cattle production. This compound microecological preparation significantly improves the digestive and absorptive capacity of beef cattle, particularly exhibiting a significant degradation effect on difficult-to-decompose lignin fibers, thereby improving feed utilization and reducing feeding costs. Experiments show that after using this preparation, the daily weight gain of beef cattle is significantly increased, and feed conversion ratio is significantly optimized. This not only promotes the growth and development of beef cattle but also shortens the breeding cycle and improves economic benefits. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a plate colony diagram of strain YB; Figure 2 This is a plate colony diagram of strain AH7-7; Figure 3 This is a plate colony diagram of strain CB-2; Figure 4 Phylogenetic tree diagram of strain YB; Figure 5 Phylogenetic tree diagram of strain AH7-7; Figure 6 Phylogenetic tree diagram of strain CB-2; Figure 7 This is a statistical graph of the lignin degradation rate in Example 2. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The reagents and culture media used in the following examples are as follows: Lignin solid screening medium: Buffer A 165 mL / L, Buffer B 165 mL / L, cell-free rumen fluid 170 mL / L, NaHCO3 5.0 g / L, sodium lignin sulfonate 1.0 g / L and agar 18.0 g / L.

[0026] Lignin degradation medium: Buffer A 165 mL / L, Buffer B 165 mL / L, cell-free rumen fluid 170 mL / L, 0.1% resazurin 1.0 mL / L, NaHCO3 5.0 g / L, sodium lignin sulfonate 1.0 g / L, peptone 1.0 g / L and yeast extract 1.0 g / L.

[0027] Cellless rumen fluid: Fresh buffalo rumen fluid was filtered through four layers of gauze, centrifuged at 10,000 r / min for 10 min at 4°C, and the supernatant was collected and frozen in a freezer at -20°C for later use.

[0028] Buffer A: (NH4)2SO4 3g / L, NaCl 6g / L, KH2PO4 3g / L, CaCl2·2H2O 0.4g / L and MgSO4·7H2O 0.6g / L.

[0029] Buffer B: K2HPO4·3H2O 4g / L.

[0030] 0.1% Rhazinosa: Weigh 0.1g of Rhazinosa solid, dilute to 100mL with double-distilled water, and store at 4℃ for later use.

[0031] YB fermentation medium formula: corn starch 33.4 g / L, soybean meal powder 17 g / L, yeast powder 8.5 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 1 g / L, calcium carbonate 2.1 g / L and manganese sulfate 0.2 g / L.

[0032] The CB-2 fermentation medium formula is as follows: sucrose 30 g / L, soybean meal powder 20 g / L, tryptone 2 g / L, sodium chloride 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 1 g / L, and calcium carbonate 1 g / L.

[0033] AH7-7 fermentation medium formula: glucose 39.6g / L, soybean meal powder 14g / L, yeast powder 6.9g / L, sodium chloride 2.4g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 1g / L, calcium carbonate 1g / L and manganese sulfate 0.2g / L.

[0034] Example 1 1. Strains Isolation and Purification A rumen solid-liquid mixture sample was obtained from a buffalo rumen fistula and screened using lignin solid screening medium. After isolation and purification, three lignin-degrading bacteria were obtained and named YB, AH7-7 and CB-2, respectively.

[0035] 2. Strain identification The three bacterial strains were inoculated onto LB aerobic culture plates and cultured aerobically for 24 hours. The colony morphology is as follows: Figures 1-3 As shown.

[0036] Physiological and biochemical identification of the strains was performed according to the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification (9th Edition)".

[0037] The 16S rDNA sequence was amplified by PCR and the product was sent to Qingke Biotechnology Co., Ltd. for sequencing. Sequence comparison was performed using the 16S rDNA sequence at http: / / blast.nB-04i.nlm.nih.gov / Blast. Sequences of several bacteria with the highest sequence similarity were downloaded, and a phylogenetic tree was further constructed using MEGA 6.0 software. The results are shown in [Figure number missing]. Figures 4-6 .

[0038] Based on the above identification results, strain YB was identified as Sonora desert bacillus (Bacillus desertis). Bacillus sonorensis ); strain AH7-7 is Bacillus cereus ( Bacillus cereus ); strain CB-2 is *Pseudomonas intermedius* ( Ochrobactrum pseudintermedium ).

[0039] 3. Preservation of microbial strains Sonora desert bacillus ( Bacillus sonorensis YB was deposited on March 18, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025512.

[0040] Bacillus cereus ( Bacillus cereus AH7-7 was deposited on March 18, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025511.

[0041] Pseudomonas intermedia ( Ochrobactrum pseudintermedium CB-2 was deposited on March 18, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025513.

[0042] Example 2 The three activated strains (concentration 1×10⁻⁶) 7 (CFU / mL) was inoculated at an inoculum of 2.5% into 250mL serum bottles containing 200mL of lignin degradation medium. The culture was incubated at 39℃ in an anaerobic incubator. At 6h, 12h, 15h, 18h, 21h, and 24h, 2mL of fermentation broth was aseptically sampled using a syringe for lignin content determination. The experiment lasted for 24 hours. A composite strain group was also set up, consisting of bacterial suspensions of YB, AH7-7, and CB-2 (each at a concentration of 1×10⁻⁶). 7 After mixing the CFU / mL solution at a volume ratio of 20:20:1, the mixture was inoculated at a rate of 2.5% into a 250mL serum bottle containing 200mL of lignin degradation medium. The mixture was then incubated in an anaerobic incubator at 39℃. At 6h, 12h, 15h, 18h, 21h, and 24h, 2mL samples of the fermentation broth were aseptically taken using a syringe for lignin content determination. The experiment lasted for 24 hours. Based on the results, the degradation rate of sodium lignin sulfonate by the strain was calculated.

[0043] The method for determining lignin content is as follows: A sodium lignosulfonate solution with a concentration of 1000 mg / L was prepared, and then further diluted to prepare sodium lignosulfonate solutions with concentrations of 0, 100, 200, 300, 400, 500, and 600 mg / L. The absorbance (OD) of each concentration of sodium lignosulfonate was measured at a wavelength of 285 nm. A standard curve was plotted with the sodium lignosulfonate concentration on the x-axis and the OD value on the y-axis, and a linear regression equation was derived.

[0044] The degraded lignin degradation medium was centrifuged at 10000 rpm for 10 min, and the supernatant was collected as the test sample. The test sample was diluted 5 times with double-distilled water, and the OD value of the sample was measured at a wavelength of 285 nm. The concentration of lignin in the culture medium was calculated by substituting the values ​​into the linear regression equation of the standard curve. Finally, the degradation rate of sodium lignin sulfonate by the strain was calculated using the following formula: Lignin degradation rate (%) = (C1-C2) / C1 × 100%; Wherein, C1 is the concentration of sodium lignin sulfonate in the culture medium without the addition of the strain (i.e., the control group), and C2 is the concentration of sodium lignin sulfonate after degradation by the strain.

[0045] The statistical results of the degradation rate of sodium lignosulfonate by each strain are shown in the figure. Figure 7The results showed that after 24 hours of degradation culture, the degradation rate of sodium lignin sulfonate in the YB group reached 9.54%, that in the AH7-7 group reached 17.16%, that in the CB-2 group reached 11.99%, and that in the combined strain group reached 22.66%. This demonstrates that the combined use of these three strains can produce a synergistic effect, effectively improving the degradation of sodium lignin sulfonate.

[0046] Example 3 YB strain was fermented and cultured on YB fermentation medium to obtain bacterial cells, which were then spray-dried to obtain YB spray-dried bacterial powder.

[0047] After fermenting the AH7-7 strain in AH7-7 fermentation medium, the bacterial cells were obtained, and then spray-dried AH7-7 bacterial powder was obtained by spray drying.

[0048] After fermenting the CB-2 strain in CB-2 fermentation medium, the bacterial cells were obtained, and then CB-2 freeze-dried bacterial powder was obtained by freeze-drying.

[0049] YB spray-dried powder, AH7-7 spray-dried powder and CB-2 spray-dried powder were mixed evenly at a live bacteria ratio of 20:20:1 to obtain a compound microecological preparation.

[0050] Example 4 1. Test Methods Twenty-four beef cattle (half Charolais and half Simmental, with similar sex, age, and weight) were randomly divided into three groups: a blank control group, a low-dose group, and a high-dose group, with two breeds in each group and four replicates for each breed. The control group was fed the farm's TMR diet; the low-dose group was fed the farm's TMR diet plus 4 g / day / head of the compound microecological preparation prepared in Example 3; and the high-dose group was fed the farm's TMR diet plus 8 g / day / head of the compound microecological preparation prepared in Example 3. The nutritional and energy levels of the diets were kept consistent among the groups. The pre-trial period was 12 days, and the formal trial period was 167 days. Feeding was stopped at day 52 to investigate the persistence of the lignin-degrading bacteria preparation after discontinuation and the colonization of the strains in the rumen.

[0051] On days 1, 26, 52, 94, and 167 after the start of the formal experiment, fasting weights were measured in the control group, low-dose group, and high-dose group before morning feeding to calculate relative weight gain, average daily weight gain, and fattening index. Feed intake and uneaten feed were recorded daily to calculate average daily feed intake. The feed conversion ratio was calculated based on average daily weight gain and average daily feed intake. Body dimensions of the cattle were measured at the midpoint of the experiment (day 94) to evaluate the fattening effect.

[0052] Average daily feed intake = Feed intake of each group of beef cattle during the trial period / Number of trial days / 8 cattle; Average daily weight gain = (final weight - initial weight) / number of days in the trial; Relative weight gain = (final weight - initial weight) / initial weight; Fattening index = weight / height × 100.

[0053] 2. Test Results The growth performance indicators of beef cattle were measured during the experiment, and the results are shown in Table 1. The results showed that from day 1 to 26, the relative weight gain and average daily weight gain of the high-dose group were significantly higher than those of the control group, increasing by 54.41% and 53%, respectively. P <0.05. During the 1-52d and 1-167d periods, there were no significant differences in relative weight gain and average daily weight gain among the three groups of beef cattle. P >0.05), but from 1 to 94 days, the relative weight gain and average daily weight gain of beef cattle in the high-dose group were significantly higher than those in the control group, increasing by 21.73% and 20.24%, respectively. P <0.05, the fattening index of beef cattle in the high-dose group increased by 8.96% compared with the control group ( P <0.05), reaching 377.

[0054] Table 1. Effects of lignin-degrading bacterial preparations on the growth performance of beef cattle. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A combination of buffalo rumen-derived lignin-degrading bacteria, characterized in that, The buffalo rumen lignin-degrading bacterial combination comprises Bacillus sonorensis ( Bacillus sonorensis ), Bacillus cereus ( Bacillus cereus ), and Pseudintermedius ( Ochrobactrum pseudintermedium ). The Bacillus sonorensis was preserved in the China Center for Type Culture Collection on March 18, 2025, the address of the preservation center is China. Wuhan. Wuhan University, and the preservation number is CCTCC NO: M2025512; The Bacillus cereus was preserved in the China Center for Type Culture Collection on March 18, 2025, the address of the preservation center is China. Wuhan. Wuhan University, and the preservation number is CCTCC NO: M2025511; The Ochrobactrum pseudintermedium was preserved in the China Center for Type Culture Collection on March 18, 2025, the address of the preservation center is China. Wuhan. Wuhan University, and the preservation number is CCTCC NO: M2025513.

2. The buffalo rumen lignin-degrading bacterial consortium according to claim 1, characterized in that, The quantity ratio of the Bacillus sonorensis, the Bacillus cereus and the Ochrobactrum pseudintermedium is 20:20:

1.

3. Use of the buffalo rumen lignin-degrading bacterial combination of claim 1 or 2 in degrading lignin.

4. Use of the buffalo rumen lignin-degrading bacterial combination of claim 1 or 2 in preparing a lignin-degrading bacterial composite microecological preparation.

5. A lignin-degrading bacteria complex microecological preparation, characterized in that, The active ingredient comprises the buffalo rumen lignin-degrading bacterial combination of claim 1 or 2.

6. The lignin-degrading bacterial complex microecological preparation according to claim 5, characterized in that, The lignin-degrading bacterial composite microecological preparation further comprises adjuvants.

7. A method for preparing a complex microecological preparation of lignin-degrading bacteria according to claim 5 or 6, characterized in that, The method comprises the following steps: The Bacillus sonorensis and the Bacillus cereus of claim 1 are respectively subjected to fermentation culture, and the obtained bacterial bodies are subjected to spray drying treatment to obtain Bacillus sonorensis spray dried powder and Bacillus cereus spray dried powder; The Ochrobactrum pseudintermedium of claim 1 is subjected to fermentation culture, and the obtained bacterial bodies are subjected to freeze drying treatment to obtain Ochrobactrum pseudintermedium freeze dried powder; The Bacillus sonorensis spray dried powder, the Bacillus cereus spray dried powder and the Ochrobactrum pseudintermedium freeze dried powder are uniformly mixed to obtain the lignin-degrading bacterial composite microecological preparation.

8. Use of the lignin-degrading bacterial composite microecological preparation of claim 5 or 6 in degrading lignin.

9. Use of the lignin-degrading bacterial composite microecological preparation of claim 5 or 6 in preparing a feed additive or feed for improving the growth performance of beef cattle.

10. A feed additive or feed for improving growth performance of beef cattle, characterized by, The lignin-degrading bacterial composite microecological preparation of claim 5 or 6.

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