Application of bacillus altitudinis H18 and glucoside hydrolase gene of bacillus altitudinis H18 in improving rumen degradation of rice straws

Abstract

The invention provides application of bacillus altitudinis H18 and a glucoside hydrolase gene thereof in improving rumen degradation of rice straws, and belongs to the technical field of straw reutilization. The bacillus altitudinis H18 is separated from goat rumen fluid, the bacillus altitudinis H18 can promote degradation of rice straw cellulose, and reference is provided for feed application of rice straw. Sequencing analysis finds that a whole genome of the strain H18 contains a large number of genes related to glucoside hydrolase GH, carbohydrate hydrolase CE and the like, and the content of neutral detergent fibers and lignin in the rice straw can be remarkably reduced by performing fermentation treatment on the rice straw by using the strain H18 through an in-vitro fermentation method. The strain can be used as a bacterial strain for feed application of the rice straws so as to improve the conversion rate and the utilization rate of the rice straws.

Description

Technical Field

[0001] This invention belongs to the field of straw recycling technology, specifically relating to the application of Bacillus subtilis H18 and its glycoside hydrolase gene in improving the rumen degradation of rice straw. Background Technology

[0002] Rice straw is a key component of agricultural waste, not only produced in huge quantities but also widely distributed geographically. However, rice straw has a low crude protein content and a high crude fiber content, resulting in low nutritional value and making it an unsuitable feed ingredient. Furthermore, the neutral detergent fiber content of rice straw is as high as 60% or more, significantly impacting feed intake and production performance in herbivores.

[0003] Given the scarcity of roughage resources, the utilization of rice straw as animal feed holds immense potential. Studies have shown that chemical methods such as silage and ammoniation, along with biological treatment technologies, can further improve the utilization rate and digestibility of rice straw. However, the process of resource utilization of rice straw faces numerous challenges, particularly the extremely complex cell wall structure of rice straw, composed of multiple tightly packed cells, including a thick cuticle, a non-degradable waxy layer, and high levels of lignin and silicates—components that are difficult to degrade and hydrolyze in nature. It is precisely because of the inherent structural characteristics of rice straw that its exceptionally robust physical structure makes it difficult for animal digestive systems to effectively break it down. Therefore, feed researchers are actively exploring physical, chemical, or biological pretreatment methods to improve the digestibility and utilization of rice straw. Summary of the Invention

[0004] This invention provides the application of Bacillus hygroscopicus H18 and its glycoside hydrolase gene in improving the rumen degradation of rice straw. The strain can significantly reduce the content of neutral detergent fiber and promote fiber degradation. It can be used as a strain for the application of rice straw as feed to improve feed conversion rate and utilization rate.

[0005] This invention provides a highland Bacillus strain with straw degradation capabilities (… Bacillus altitudinis The preservation number of Bacillus hygroscopicus H18 is GDMCC No:67153.

[0006] The present invention also provides a method for culturing the above-mentioned Bacillus hygroscopicus H18, comprising inoculating the Bacillus hygroscopicus H18 onto LB medium for aerobic culture.

[0007] The present invention also provides a biological agent containing the above-mentioned Bacillus hygroscopicus H18.

[0008] In a preferred embodiment of the present invention, the effective viable count of Bacillus hygroscopicus H18 in the biological agent is not less than 1×10⁻⁶. 6 CFU / g.

[0009] The present invention also provides a method for preparing the above-mentioned biological agent, comprising inoculating the strain of Bacillus hygroscopicus H18 onto LB agar plates for aerobic culture, picking single colonies into LB broth culture medium for aerobic culture, and then expanding the culture to obtain the biological agent.

[0010] In a preferred embodiment of the present invention, the LB agar plate contains 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride and 15 g / L agar powder.

[0011] In a preferred embodiment of the present invention, the LB broth culture medium contains 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride.

[0012] In a preferred embodiment of the present invention, the temperature for aerobic culture is 37°C.

[0013] The present invention also provides the application of the above-mentioned Bacillus hygroscopicus H18 or the above-mentioned biological agent in the degradation of straw.

[0014] In a preferred embodiment of the present invention, the degraded straw includes degraded rice straw.

[0015] Beneficial Effects: This invention isolates Bacillus hygroscopicus H18 from goat rumen fluid. Bacillus hygroscopicus H18 can promote the degradation of cellulose in rice straw, providing a reference for the application of rice straw as feed. Sequencing analysis revealed that the whole genome of strain H18 contains a large number of genes related to glycoside hydrolases GH (GH5, GH9, GH48) and carbohydrate hydrolases CE (CE1, CE4). Using an in vitro fermentation method, strain H18 was used to simulate fermentation treatment of rice straw, significantly reducing the content of neutral detergent fiber and lignin in rice straw. Therefore, it can be used as a strain for the application of rice straw as feed, thereby improving the conversion rate and utilization rate of rice straw.

[0016] Biological Preservation Information Highland Bacillus ( Bacillus altitudinis H18 was deposited on October 23, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67153. Attached Figure Description

[0017] Figure 1 Image of decolorized Congo Red; Figure 2 Colony morphology; Figure 3 A phylogenetic tree diagram of the 16S rRNA gene; Figure 4 A genome loop diagram; Figure 5 A statistical graph of coding gene lengths; Figure 6 This is a statistical chart showing the percentage of different categories of carbohydrate-active enzymes. Detailed Implementation

[0018] This invention provides a highland Bacillus H18 with straw degradation capabilities, the preservation number of which is GDMCC NO:67153.

[0019] The strain H18 described in this invention has a good ability to degrade cellulose and lignin. After treating rice straw with strain H18, the concentrations of butyric acid and valeric acid can be significantly increased, proving that strain H18 promotes the decomposition of recalcitrant polysaccharides in straw and has a strong fiber degradation ability.

[0020] In this invention, gas production is used as an important indicator for evaluating the rumen fermentation efficiency of ruminants, directly reflecting the degradation rate of substrates and the activity of metabolic pathways by microorganisms. In one embodiment of this invention, the gas production performance of strain H18 at multiple time points was statistically analyzed. Compared with existing commercial inoculants, it has a higher gas production, especially showing a significant difference at 12 hours, demonstrating that strain H18 can more efficiently convert degradation products into gas through glycolysis, promoting fiber degradation and decomposition.

[0021] Furthermore, after treatment with strain H18 described in this invention, the nutritional composition of rice straw changes, for example, the content of acid detergent fiber and neutral detergent fiber can be significantly reduced, and the treated rice straw has the potential to be used as feed.

[0022] The present invention also provides a method for culturing the above-mentioned Bacillus hygroscopicus H18, comprising inoculating the Bacillus hygroscopicus H18 onto LB medium for aerobic culture.

[0023] The aerobic culture temperature described in this invention is 37°C, accompanied by shaking at a frequency of 220 rpm. During the aerobic culture described in this invention, the oxygen content of the natural air is sufficient.

[0024] The present invention also provides a biological agent containing the above-mentioned Bacillus hygroscopicus H18.

[0025] The effective viable count of Bacillus hygroscopicus H18 in the biological agent of this invention is not less than 1×10⁻⁶. 6CFU / g.

[0026] The present invention also provides a method for preparing the biological agent, comprising inoculating a strain of Bacillus hygroscopicus H18 onto an LB agar plate for aerobic culture, picking a single colony into LB broth for aerobic culture, and then expanding the culture to obtain the biological agent.

[0027] This invention involves streaking frozen bacterial cultures onto LB agar plates for aerobic culture. The LB agar plate comprises the following components at the following concentrations: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, and agar powder 15 g / L. The aerobic culture temperature described in this invention is 37°C.

[0028] In this invention, after the aerobic culture is completed, a single colony is picked and placed into 5 mL of LB broth for aerobic culture. The LB broth contains the following components at concentrations: yeast extract 5 g / L, tryptone 10 g / L, and sodium chloride 10 g / L. The aerobic culture temperature is 37°C, accompanied by shaking at a frequency of 220 rpm for a total of 6 hours to obtain the seed culture.

[0029] The present invention expands the culture of the seed liquid by inoculating the seed liquid at a ratio of 2% into 100mL LB broth and shaking it aerobically at 37°C for 12 hours to obtain the biological agent.

[0030] The present invention also provides the application of the above-mentioned Bacillus hygroscopicus H18 or the above-mentioned biological agent in the degradation of straw.

[0031] This invention is based on an in vitro fermentation method. By using the strain H18 to simulate fermentation treatment of rice straw, the content of neutral detergent fiber and lignin in rice straw can be significantly reduced. It can be used as a strain for the application of rice straw as feed to improve the conversion rate and utilization rate of rice straw.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, illustrates the application of Bacillus subtilis H18 and its glycoside hydrolase gene in enhancing the rumen degradation of rice straw, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] In the data processing methods used in this embodiment of the invention, fermentation characteristics and chemical composition data were first initially organized using Excel 2023, then analyzed using R (V4.1.3) and visualized using OrginPro 2023. Unless otherwise specified, the results in this embodiment of the invention are expressed as mean ± standard deviation (Mean ± SD). Analysis results are presented in... P <0.05 indicates a significant difference, 0.05≤ P<0.1 indicates a trend of change.

[0034] Example 1 1. Experimental materials Collect the contents of the goat's rumen and isolate the bacterial strain from the supernatant.

[0035] Prepare the culture medium: LB medium: 10 g tryptone, 5 g yeast extract, 1 g NaCl, 1000 mL distilled water; Sodium carboxymethyl cellulose (CMC-Na) medium: 15 g sodium carboxymethyl cellulose, 2 g tryptone, 0.2 g MgSO4, 1 g NaCl, 1 g KH2PO4, 1 g yeast extract and 1000 mL distilled water. All media were sterilized at 121℃ for 20 min.

[0036] 2. Separation and purification Take 1 mL of goat rumen contents supernatant, dilute it, and spread it on CMC-Na medium using the plate method. Place it in a 30℃ constant temperature incubator and invert it for 48 h. Observe the morphology of single colonies after culture, and complete the selection, purification and preservation.

[0037] 2.1 Strain screening The isolated and purified strain was inoculated onto CMC-Na medium using the spot inoculation method and incubated upside down in a 30℃ constant temperature incubator for 48 h. 20 mL of 1 mg / mL Congo red solution was added for staining for 30 min. After pouring out the staining solution, 20 mL of 1 mol / L NaCl solution was added for decolorization for 30 min. The colony diameter (d) and the decolorization zone diameter (D) were measured at this time, and the ratio between the two (D / d) was calculated.

[0038] Several bacterial strains were isolated and purified from sheep rumen fluid contents using the dilution plating method. Initial screening was performed using Congo red staining and selection medium. A strain with a diameter (D) of 7.5 mm, a diameter (d) of 0.9 mm, and a D / d ratio of 8.3 was selected, indicating its ability to degrade cellulose and lignin. This strain was named H18. The results of the isolated, purified, and stained strain H18 are shown below. Figure 1 As shown, the colony morphology is as follows Figure 2 As shown.

[0039] 2.2 Strain Identification DNA was extracted from strain H18, and primers were designed based on the bacterial sequence information for PCR amplification. The PCR products were detected by 1% agarose gel electrophoresis and then sequenced. The results were assembled using DNAMAN software, and species were identified through sequence similarity using NCBI's BLAST tool. A phylogenetic tree was constructed using MEGA6.

[0040] 27F (SEQ ID No. 1): AGAGTTTGATCMTGGCTCAG; 1492R (SEQ ID No. 2): GGTTACCTTGTTACGACTT; Sequence alignment of strain H18 was performed using the BLAST tool in NCBI, and the results are as follows: Figure 3 As shown, strain H18 and Bacillus virescens (Belasis alpineus) Bacillus altitudinis The homology of strain H18 with PQ285122.1 reached 98.53%. In the phylogenetic tree, strain H18 clustered with Bacillus vesicularis PQ285122.1, therefore the rice straw degrading bacterium H18 was named Bacillus vesicularis H18.

[0041] 3. Genome sequencing 3.1 DNA Extraction Activated H18 cells were inoculated at 1% in MRS liquid medium, cultured overnight, and then collected by centrifugation. Bacterial genomic DNA for Denovo sequencing was extracted using the cetyltrimethylammonium bromide (CTAB) method. The total DNA content was determined using the Quant-iT PicoGreen dsDNA Assay Kit, and DNA integrity was assessed using 1% agarose gel electrophoresis.

[0042] 3.2 Sequencing Data Assembly and Basic Analysis De novo sequencing uses a "second-generation + third-generation" sequencing approach to obtain zero-gap genomic data. The second-generation sequencing machine employs the standard Illumina TruSeq Nano DNA LT library preparation workflow (Illumina TruSeq DNA Sample Preparation Guide) to construct the required genomic libraries for sequencing. The kit used is TruSeq. TM The DNA Sample Prep Kit was used. For the third-generation machine, the standard PacBio Template Prep Kit 1.0 library preparation workflow (20kb Template Preparation Using BluePippin Size Selection) was used to construct the required genome library. The genome sequence was then aligned with the Nucleotide Sequence Database (NT).

[0043] The second-generation sequencing data are shown in Table 1. The total number of bases is 967,352,206. The percentages of bases with error rates less than 0.01 and 0.001 in the raw data are 98.6524% and 95.8242%, respectively. The third-generation sequencing data are shown in Table 2. The total number of reads is 48,953, the total base length is 508,598,784 bp, the longest read length is 30,245 bp, and the average read length is 10,389.53 bp.

[0044] Table 1. Second-generation sequencing data

[0045] Table 2. Third-generation sequencing data

[0046] The third-generation genome data was assembled using software, and then the second-generation data was used to correct errors in the third-generation results to obtain the zero-gap whole-genome information. The constructed genome is as follows: Figure 4 As shown in Table 3, the statistical results show that the genome length of H18 is 3,814,674 bp (approximately 3.81 Mb) and the GC content is 41.49%.

[0047] Table 3 Genomic Information

[0048] Of the H18 genes identified, the vast majority were over 1,000 bp in length. Figure 5 In addition, the H18 gene contains multiple copies of ribosomal RNA (rRNA), as well as transfer RNA (tRNA) and non-coading RNA (ncRNA) (Table 4).

[0049] Table 4. Encoding gene data

[0050] The Carbohydrate-Active Enzymes Database (CAZy) lists various information about enzyme molecular sequences, including family information and species origin. Based on enzyme type, they can be divided into five main categories: Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Polysaccharide Lyases (PLs), Carbohydrate Esterases (CEs), and Auxiliary Activities (AAs). Figure 6 The proportions of carbohydrate-active enzymes by category show that GH, CE, and GT account for the largest share. Among them, the GH5_7 family (gene1717) of this bacterium functions as an endoglucanase, exhibiting strong specificity for crystalline cellulose in plant cell walls, which can break down the crude fiber structure of corn stalks and provide a target site; the GH48 family (gene1715) acts as an exocellulase, continuously hydrolyzing the chain ends after endocellulase cleavage to produce cellobiose, providing core support for the "endocellulase + exocellulase" synergistic system; the GH9 family (gene1714) encodes a multifunctional enzyme with both endoglucanase activity and cellobiose hydrolysis function, adapting to the complex environment of cellulose and hemicellulose intertwined, improving degradation stability. The three work together to achieve efficient degradation of corn stalk cellulose.

[0051] The antiSMASH database was used to analyze the secondary metabolite gene clusters on the H18 genome, and the MiBIG database was used to predict the most similar compounds matched to the gene clusters. The results are shown in Table 5. A total of 6 regions were predicted to be secondary metabolite gene clusters, including NRPS, terpene, RRE-containing, beta-actone, T3PKS, and RiPP-like.

[0052] Table 5 Results of secondary metabolite gene clusters

[0053] 4. Fermentation Experiment Design Fresh rice straw was collected from the teaching and research base of South China Agricultural University in Guangdong Province. The stubble was left at 0.2-0.3 m. The straw was dried in an oven at 105℃ for two hours. The temperature was then adjusted to 65℃ and dried for another 48 hours before being crushed to obtain feed samples.

[0054] Rumen fluid was collected from Yuehai Food Slaughterhouse in Foshan City, Guangdong Province. The experiment consisted of two treatment groups and one control group, namely (1) adding 1×10 6Treatment group of CFU / g commercial Bacillus cereus, (2) with 1×10 6 The treatment group with CFU / g H18, (3) the blank control group without the addition of Bacillus hygroscopicus.

[0055] After filtering the rumen fluid through three layers of gauze, the mixture was mixed with buffer solution to prepare the artificial fermentation broth. 200 mL of the broth was then added to a soy milk bag (20 cm × 40 cm) with a cap. 1 g of feed sample was accurately weighed into a fiber analysis bag. Each group was treated in triplicate. After sealing, the bag was perforated, a glass bead was inserted, and the bag was placed in the fermentation broth. After the air was expelled, the bag was sealed with a rubber stopper and incubated in a 39°C water bath at 60 rpm for 48 hours. At the 2nd, 4th, 6th, 8th, 12th, 24th, 36th, and 48th hours, the gas in the soy milk bag was extracted with a disposable syringe, and the gas production was measured.

[0056] 5. Sampling and processing After fermentation, centrifuge 10 mL of the fermentation broth and collect the clear supernatant. Store the supernatant at 4°C for later use. Rinse the sample bag and bake it in a 105°C oven for two hours. Then, reduce the temperature to 65°C and continue baking for 48 hours. After baking, remove the sample and cool it in a desiccator. Weigh the sample. After weighing, cut open the fiber bag and remove the sample for nutrient content determination.

[0057] 6. Measurement Indicators and Methods 6.1 Nutritional composition determination The dry matter (DM) testing method is the 105℃ drying method.

[0058] The analysis of crude fat (EE) and crude ash (Ash) was performed according to the "Feed Analysis and Feed Quality Testing Technology".

[0059] Crude protein (CP) content in the sample was calculated using a Kjeltec 18 automated analyzer (FOSS Analytical AB, Schillerod, Denmark).

[0060] The content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) was assessed using the Van Soest method (ANKOMA-19I fiber analysis equipment, ANKOM Corporation, Macedonia, NY, USA).

[0061] The amount of water-soluble carbohydrates (WSC) was estimated using the sulfuric acid-anthrone colorimetric method.

[0062] Table 6. Results of nutrient composition determination of rice straw raw materials

[0063] 6.2 Fermentation quality assessment The pH of the fermentation broth was determined using a pB-7890 (Sartorius) pH meter, and the ammonia nitrogen (NH3-N) content was determined using the phenol-sodium hypochlorite colorimetric method. The contents of valeric acid (VA), butyric acid (BA), propionic acid (PA), and acetic acid (AA) were analyzed using a gas chromatograph (Agilent 7890B).

[0064] As shown in Table 7, the pH values ​​of the commercial group and the experimental group were slightly lower than those of the control group, and the pH value of the experimental group was lower than that of the commercial group, but there was no significant difference among the three groups. P <0.05. The ammonia nitrogen levels in both the commercial and experimental groups were slightly higher than those in the control group, with the experimental group showing a higher ammonia nitrogen level than the commercial group; however, there was no significant difference among the three groups. P <0.05. The concentrations of acetic acid and butyric acid in the commercial and experimental groups were slightly higher than those in the control group, and the concentrations in the experimental group were higher than those in the commercial group, but there was no significant difference among the three groups. P <0.05%. The propionic acid concentrations in the commercial and experimental groups were higher than those in the control group, and the concentration in the commercial group was slightly higher than that in the experimental group, but there was no significant difference among the three groups. P <0.05%. The valeric acid concentrations in both the commercial and experimental groups were higher than those in the control group, and there was a significant difference between the valeric acid concentrations in the experimental and control groups. P <0.05, while there was no significant difference between the commercial group and the experimental group, or the control group ( P >0.05). Compared to the commercial group and the blank control group, the experimental strain of this invention (experimental group) has the following advantages: the valeric acid (VA) content in the experimental group may be significantly higher (0.28±0.01 mmol / L), significantly higher than that in the commercial group and the control group (…). P =0.017<0.05), suggesting that its fermentation metabolic activity may be more vigorous and its degradation of rice straw may be more complete; the butyric acid (BA) content (2.16±0.07 mmol / L) is higher than that of the commercial group and the control group, which may indicate a stronger potential to promote the degradation of straw fiber.

[0065] Table 7. Effects of different treatments on the fermentation quality of rice straw.

[0066] In the table described in this invention, different lowercase letters in the superscript of data in the same row indicate differences ( P <0.05, identical letters or no letters indicate no significant difference ( P >0.05), the same below.

[0067] 6.2.1 Results of nutrient composition determination As shown in Table 8, the content of acidic and neutral detergent fibers in the commercial group was significantly higher than that in the control and experimental groups, while there was a significant difference in the content of neutral detergent fibers between the control and experimental groups. P <0.05); The contents of crude protein, crude fat, and soluble carbohydrates in the control group, commercial group, and experimental group were basically the same, with no significant differences. The lignin content in the experimental group was significantly lower than that in the control group ( P <0.05), while the commercial group had the lowest. The experimental group had slightly lower hemicellulose levels than the control and commercial groups, but the difference was not statistically significant. P >0.05%. Cellulose content was significantly higher in both the commercial and experimental groups than in the control group ( P <0.05%. Neutral detergent fiber (NDF), as a core component of straw structural fibers, directly reflects the overall degradation degree of straw. The NDF content in the experimental group was significantly lower than that in the commercial group. P =0.002) and the lowest level among the three groups, indicating that this strain has outstanding degradation ability for the main structural fibers of straw; lignin, as a key rate-limiting factor in the straw degradation process, has a decomposition efficiency that is an important indicator for evaluating the degradation efficacy of the strain. Although the lignin content in the experimental group was not the lowest among the three groups, it was significantly lower than that in the blank control group ( P The value of <0.001 indicates the strain's potential to overcome the straw lignin barrier, creating favorable conditions for further degradation of subsequent fiber components.

[0068] Table 8. Effects of different treatments on the nutrient composition of fermented rice straw.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of highland Bacillus with straw degradation capabilities ( Bacillus altitudinis H18, characterized in that, The preservation number of the Bacillus hygroscopicus H18 is GDMCC No:67153.

2. The method for culturing Bacillus hygroscopicus H18 according to claim 1, characterized in that, This includes inoculating the Bacillus hygroscopicus H18 onto LB medium for aerobic culture.

3. A biological agent comprising Bacillus hygroscopicus H18 as described in claim 1.

4. The biological agent according to claim 3, characterized in that, The effective viable count of Bacillus hygroscopicus H18 in the biological agent is not less than 1×10⁻⁶. 6 CFU / g.

5. The method for preparing the biological agent according to claim 3 or 4, characterized in that, The process involves inoculating a strain of Bacillus hygroscopicus H18 onto LB agar plates for aerobic culture, picking single colonies and culturing them aerobically in LB broth, followed by expansion culture to obtain the biological agent.

6. The preparation method according to claim 5, characterized in that, The LB agar plate contains 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and 15 g / L agar powder.

7. The preparation method according to claim 5, characterized in that, The LB broth culture medium contains 5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride.

8. The preparation method according to claim 5, characterized in that, The temperature for all aerobic cultures was 37°C.

9. The application of the Bacillus hygroscopicus H18 of claim 1 or the biological agent of claim 3 or 4 in the degradation of straw.

10. The application according to claim 9, characterized in that, The degraded straw includes degraded rice straw.

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