Bacillus capable of efficiently degrading fibers and application thereof

Abstract

The invention discloses a bacillus capable of efficiently degrading fibers and application of the bacillus. The name of the bacillus altitudinis DF34 is bacillus altitudinis, the preservation number of the bacillus altitudinis is GDMCC No: 65492, and the bacillus altitudinis is preserved in Guangdong Microbial Culture Collection Center on December 4, 2024. The bacillus has the characteristics of strong stress resistance and high growth speed; the strain has the capability of efficiently degrading fibers, and can degrade fiber components of forage grass, promote fermentation and improve the fermentation quality of the forage grass with low soluble sugar water content in the ensiling process. Therefore, the bacillus altitudinis DF34 can be widely applied to various regions in China, and especially has an outstanding effect on silage raw materials with high fiber content. The strain successfully solves the problem of poor silage fermentation caused by low water-soluble carbohydrate content, and provides a new solution for improving the quality of the silage.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacillus capable of efficiently degrading fiber and application thereof. Background Art

[0002] Silage has attracted much attention due to its sweet smell, soft and juicy texture, high nutrient retention rate and good palatability. As a key feed preservation method in the field of animal husbandry, silage technology can not only improve the quality of milk and meat, but also convert plant resources that cannot be directly used as feed into usable materials, while reducing the waste of agricultural product processing by-products. Silage is an effective means of storing green and juicy forage under anaerobic conditions through lactic acid fermentation. It mainly involves lactic acid bacteria attached to the surface of fresh grass to ferment soluble sugar compounds, produce a large amount of lactic acid, lower the pH value, and inhibit the growth of harmful microorganisms, thereby obtaining a juicy feed that can be preserved for a long time.

[0003] At present, most of the bacterial additives in the silage production process are lactic acid bacteria. It should be noted that for forage varieties with low soluble carbohydrate content and high cellulose content, since they cannot provide enough fermentation substrates for lactic acid bacteria, they often lead to insufficient fermentation, which in turn affects the storage effect and quality of the feed. Although there are measures to improve the fermentation quality by adding sugars, high-sugar byproducts or cellulases, these methods are not only complicated to operate, require more manpower and material resources, but also have high costs. Bacillus is a type of spore-forming bacteria that has the advantages of being able to produce a variety of enzymes and strong tolerance to extreme environments. It shows extremely high convenience and practicality in actual operations and industrial production. If Bacillus with efficient fiber degradation can be introduced into the silage process, they can effectively decompose the fiber components in the forage into monosaccharides, provide a rich source of nutrition for the growth of lactic acid bacteria, and reduce the fiber content of silage at the same time, which is of great significance to improving resource utilization efficiency and promoting the sustainable development of animal husbandry. Summary of the invention

[0004] The primary purpose of the present invention is to provide a bacillus that can efficiently degrade fiber. The bacillus is a high-tech bacillus that has a high fiber degradation ability and can efficiently degrade fiber in feed raw materials.

[0005] Another object of the present invention is to provide the use of the above-mentioned Bacillus capable of efficiently degrading fiber in silage preparation.

[0006] To achieve the above object, the present invention is implemented by the following technical solutions:

[0007] A strain of Bacillus that can efficiently degrade fiber, named Bacillus altitudinis DF34, with a deposit number of GDMCC No: 65492, was deposited on December 4, 2024 at the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0008] The above-mentioned Bacillus capable of efficiently degrading fiber is a Gram-positive bacillus, which grows grayish white, opaque colonies with wrinkles around them on LB culture medium. The strain grows relatively quickly, entering the logarithmic growth phase in 2 to 10 hours, growing rapidly, and reaching a peak in 10 hours.

[0009] The use of the above-mentioned Bacillus DF34 with high efficiency in degrading fiber in silage preparation preferably comprises the following steps:

[0010] (1) Cut the feed to be fermented into short pieces;

[0011] (2) evenly mixing the chopped feed to be fermented and the above-mentioned Bacillus capable of efficiently degrading fiber;

[0012] (3) Degas, seal and store the mixed feed.

[0013] The feed to be fermented in step (1) includes pasture grass and fodder grass.

[0014] The forage grass comprises at least one of corn straw, rice straw and wheat straw.

[0015] The forage grass is preferably elephant grass.

[0016] The degree of shortening described in step (1) is preferably shortening to 2 to 3 cm.

[0017] The amount of the Bacillus capable of efficiently degrading fiber in step (2) is preferably 1.0×10 5 ~1.0×10 8 CFU / g of fermented feed; more preferably 1.0×10 5 ~1.0×10 6 Calculation of CFU / g of feed to be fermented.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention screens out a strain of Bacillus subtilis DF34 from silage by primary screening through Congo red staining and secondary screening by measuring cellulase activity. The strain has extremely high cellulase activity and fiber degradation ability, can form spores under unfavorable conditions, and can adapt to high temperature and strong acid-base environments. The strain has a short culture cycle and has great industrial application prospects, and can overcome the problem of poor fermentation quality of silage caused by low water-soluble carbohydrate content.

[0020] (2) Compared with the patented high-yield cellulase-producing Bacillus subtilis GST-24, DF34 showed better performance in terms of cellulase activity and fiber degradation effect, and grew faster.

[0021] (3) The present invention utilizes Bacillus subtilis DF34 to improve the fermentation quality of silage, which has the advantages of low cost, safety, reliability and easy utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transparent circle diagram produced by Bacillus DF34 that efficiently degrades fiber during the initial screening of the examples of the present invention.

[0023] Figure 2 This is a graph showing the degradation rate of corn straw powder by different fiber-degrading strains.

[0024] Figure 3 This is the colony morphology of Bacillus subtilis DF34.

[0025] Figure 4 This is the phylogenetic tree of Bacillus subtilis DF34 constructed based on 16S rDNA sequence.

[0026] Figure 5 This is a growth curve diagram of Bacillus subtilis DF34 and Bacillus subtilis GST-24. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] Culture medium formulation and reagent preparation:

[0029] LB medium: The liquid medium is 10.0 g of peptone, 5.0 g of yeast extract, 1.0 g of glucose, and 5.0 g of sodium chloride. The solid medium is 15 g / L agar added to the liquid medium, and the volume is adjusted to 1 L with distilled water, pH 7.0, and sterilized at 121°C for 20 min.

[0030] Sodium carboxymethyl cellulose (CMC) medium: sodium carboxymethyl cellulose 10.0 g, ammonium sulfate 4.0 g, potassium dihydrogen phosphate 2.0 g, magnesium sulfate (MgSO4·7H2O) 0.5 g, peptone 10.0 g, agar 15.0 g, distilled water to 1 L, pH 7.0, sterilize at 121°C for 20 min.

[0031] Fermentation medium: sodium carboxymethyl cellulose 10.0 g, peptone 10.0 g, yeast extract 5.0 g, potassium dihydrogen phosphate 2.0 g, magnesium sulfate (MgSO4·7H2O) 1.0 g, sodium chloride 5.0 g, distilled water to 1 L, pH 7.0, sterilized at 121°C for 20 min.

[0032] Seed preservation medium: 10.0 g skim milk powder, 1.0 g L-proline, 1.0 g trehalose, 1.0 mL glycerol, distilled water to 100 mL, sterilize at 121°C for 20 min.

[0033] 0.2% Congo red staining solution: Weigh 0.2 g of Congo red, dissolve it in distilled water, and make up to 100.0 mL.

[0034] 1 mol / L NaCl solution: Weigh 29.2 g NaCl, dissolve it in distilled water, and make up to 500.0 mL.

[0035] DNS colorimetric reagent (3,5-dinitrosalicylic acid): Weigh 10.0 g of 3,5-dinitrosalicylic acid and dissolve it in 100.0 mL of 2.5 mol / L sodium hydroxide solution, add 500 mL of distilled water, stir magnetically in a 50°C water bath to dissolve, then add 200.0 g of potassium sodium tartrate, 2.0 g of redistilled phenol, and 5.0 g of anhydrous sodium sulfite in sequence. After all the solution is dissolved and clarified, cool to room temperature, dilute to 1 L with distilled water, filter, store in a brown reagent bottle, and place in a dark place for 7 days before use.

[0036] Example 1: Screening of cellulose-degrading bacteria

[0037] 1) Enrichment culture

[0038] Weigh 10 g of each sample collected from soil, litter, and silage, place in a conical flask containing 90 mL of sterile water, and shake in a shaker at 300 rpm for 30 min until the suspension is uniform. Dilute the suspension to 10 –2 ~10 –6 A series of concentration gradients were prepared, and 200 μL of the suspension was respectively applied to LB solid medium using a pipette. The plate was placed at 37°C and inverted for 24 to 48 hours, and colonies with good growth and different morphologies were selected for separation and purification until a pure strain was obtained, and the seed culture medium was used for seed preservation and placed at -80°C.

[0039] 2) Initial screening

[0040] A single bacterium was inoculated into a sodium carboxymethyl cellulose (CMC) solid culture medium, one strain was inoculated into each plate, and three points were inoculated into each strain. The plate was then incubated at 37°C for 2 days. After staining with 0.2% Congo red solution for 15 minutes, the solution was discarded, and a 1 mol / L NaCl solution was added for 15 minutes, and the waste liquid was discarded. Whether a transparent hydrolysis zone appears around the colony, if a hydrolysis zone is produced, it indicates that the strain has the ability to degrade fiber. The colonies with a larger ratio of the hydrolysis zone diameter to the colony diameter are marked and rescreened. The transparent zone produced by the Bacillus DF34 that can efficiently degrade fiber obtained by the present invention is as follows: Figure 1 shown.

[0041] 3) Rescreening

[0042] The DNS (3,5-dinitrosalicylic acid) colorimetric method was used to determine the cellulase activity of each strain. The strains with obvious degradation zones obtained in the initial screening were inoculated into LB liquid culture medium, cultured at 30°C for 24 hours to obtain bacterial liquid, and then inoculated into LB liquid culture medium, cultured at 37°C for 12 hours, and then inoculated into the fermentation medium at an inoculum of 2% v / v. 1 mL of the fermentation liquid was centrifuged and the supernatant was collected as the crude enzyme solution to be tested. Using sodium carboxymethyl cellulose as the substrate, the DNS method was used to determine the reducing sugar and calculate the cellulase activity.

[0043] From the data in Table 1, it can be seen that the cellulase activity of strain DF34 is the highest, significantly higher than that of other fiber-degrading strains, so this strain was selected for identification.

[0044] Table 1 Cellulase activity of rescreened strains

[0045]

[0046] Note: Different treatments with different lowercase letters in the same column have significant differences.

[0047] Example 2: Application of Bacillus to degrade corn straw powder

[0048] Freshly harvested corn stalk samples were placed in a 65°C oven for 48 hours, crushed with a powder machine, and filtered with an 18-mesh sieve for later use. Weigh 1g of corn stalk powder into a 100mL centrifuge tube, and add 10mL of sterile water to each tube. The fiber-degrading strains screened in Table 1 were inoculated into LB liquid culture medium and cultured at 30°C for 24 hours to obtain bacterial liquid. The number of bacteria inoculated in each sample was approximately 1×10 6cfu / g, mixed evenly. Each sample was repeated 3 times and cultured in a 37°C incubator for 24 hours. The cultured product was repeatedly rinsed 3 times with distilled water and filtered. The obtained solid was dried at 65°C to constant weight, and the straw powder degradation rate was calculated by weight loss method.

[0049] The results are as follows Figure 2 As shown, the results show that there are significant differences in the ability of different strains to degrade corn straw powder. Among them, the strain DF34 screened by the present invention showed the highest corn straw powder degradation rate, reaching 31.98%, which is significantly higher than the 26.34% of the strain GST-24.

[0050] Example 3: Silage application of Bacillus

[0051] According to the results of Example 2, five strains with the highest corn straw degradation rates were selected for silage addition experiments. The five strains were DF34, GST-24, NF117, KC24 and HT4.

[0052] Using elephant grass as the test material, an silage addition test was conducted. The characteristics of fresh elephant grass are shown in Table 2. The elephant grass was chopped into 2-3 cm pieces, mixed evenly, and 1.0×10 5 cfu / g strain. A control group was set up at the same time, and an equal amount of sterile water was added. The fermentation quality was analyzed after 14, 45, and 60 days of ensiling.

[0053] The results showed that after 14 days of ensiling, the pH of the silage with DF34 added dropped to 4.53, which was significantly lower than that of the control and the silage with GST-24 added (Table 3). The pH and ammonia nitrogen content of the silage with DF34 added were the lowest at different days of ensiling (Table 3). After 60 days of ensiling, the lactic acid content of the silage with DF34 added was the highest and the acetic acid content was the lowest (Table 4). This shows that the addition of DF34 significantly improved the silage fermentation quality of elephant grass.

[0054] Table 2 Chemical characteristics and microbial counts of elephant grass before ensiling

[0055]

[0056] Note: FM, fresh matter of silage; DM, dry matter of silage.

[0057] Table 3 Effects of adding Bacillus on pH and ammonia nitrogen content of grass silage

[0058]

[0059] Note: DM, dry matter of silage. The lowercase letters of different treatments in the same column are significantly different.

[0060] Table 4 Effect of adding Bacillus on the organic acid content of grass silage (g / kg DM)

[0061]

[0062] Note: DM, dry matter of silage. The treatments with different lowercase letters in the same row have significant differences.

[0063] Example 4: Identification of Bacillus

[0064] According to the results of Example 1, Example 2, and Example 3, DF34 has the highest cellulase activity, the strongest ability to degrade corn straw powder, and the best silage effect. Referring to the "Common Bacteria System Identification Manual" edited by Dong Xiuzhu, the Gram staining and shape observation of strain DF34 were performed, and its morphology was as follows: Figure 3 As shown, it is off-white, opaque, with wrinkles around it, positive in Gram staining, and produces spores. The physiological and biochemical characteristics of Bacillus DF34, which efficiently degrades fiber, are shown in Table 5. It is positive in hydrogen peroxide reaction, has strong stress resistance, can grow in the range of pH 4.5 to 9.0, is salt-tolerant (7% NaCl grows well), high temperature-tolerant (50°C grows well), and can also grow under anaerobic conditions.

[0065] Table 5 Physiological and biochemical characteristics of strain DF34

[0066]

[0067] +++: vigorous growth; ++: good growth; +: growth; -: no growth.

[0068] The full length of the 16S rDNA sequence of strain DF34 is 1446 bp, and the sequence is shown below. Using the BLAST function of NCBI, the sequence obtained was searched and compared with the GeneBank database for homology. The results showed that the homology with the 16S rDNA sequence of Bacillus saltitudinis was as high as 99.93%. The basic phylogenetic tree was constructed using MEGA13 software, as shown in Figure 4 As shown, strain DF34 was finally determined to be a strain of Bacillus altitudinis.

[0069] The sequence of 16S rDNA of Bacillus subtilis DF34 is as follows:

[0070] TTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTGCAAACTCTCGTG

[0071] GTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGC

[0072] GATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAA

[0073] CAGATTTGTGGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAG

[0074] CACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCC

[0075] GGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAG

[0076] GGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCAT

[0077] GCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGAT

[0078] GTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTG

[0079] TGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAG

[0080] TGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCAT

[0081] CGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCT

[0082] CAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC

[0083] GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTT

[0084] CCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGC

[0085] GAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGC

[0086] TGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTACTC

[0087] TTGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGC

[0088] GGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG

[0089] GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCA

[0090] TCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTA

[0091] AGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCGGTA

[0092] TTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCA

[0093] CCCGTCCGCCGCTAACATCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACTTGCATGTATT

[0094] AGGCACGCCGCCAGCGTTC。

[0095] The strain was biologically preserved and named Bacillus altitudinis DF34, with the preservation number GDMCC No: 65492. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 4, 2024.

[0096] Example 5: Growth curve determination of Bacillus

[0097] The high-producing cellulase-producing Bacillus subtilis GST-24 and the strain DF34 screened by the present invention were inoculated into LB liquid culture medium and cultured at 37°C and 180 r / min for 24 hours to obtain bacterial liquid. The bacterial liquid was inoculated into LB liquid culture medium at a 1% v / v inoculation amount and cultured for 24 hours. During this period, samples were taken out every 2 hours, and the OD was measured with sterile water as a blank control. 600 With the culture time as the horizontal axis, OD 600 The value is the vertical axis, and the growth curve is drawn. The result is as follows Figure 5 shown. Figure 5 The results showed that compared with the high-producing cellulase Bacillus GST-24, the growth rate of strain DF34 was faster, and its growth trend was first rising and then decreasing, reaching the highest peak around 10h. At this time, OD 600 The value is 1.63.

[0098] Determination methods of nutritional components and silage fermentation indicators:

[0099] 1) Nutrient composition determination and microbial analysis

[0100] The dry matter content was determined by drying at 70°C for 48 hours in Zhang Liying's feed analysis, cooling in a desiccator to room temperature and weighing. The buffering energy was determined by hydrochloric acid and sodium hydroxide titration. The crude ash content was determined by the burning method. The crude fiber, neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by the filter bag method (ANKOMA-200i, Beijing Ankoborui Technology Co., Ltd., Beijing); the water-soluble carbohydrate content was determined by the anthrone-sulfuric acid colorimetric method. Lactic acid bacteria were cultured and counted on MRS (de-Man Rogosa Sharpe, Guangdong Huankai Microbiology Co., Ltd.) agar medium, bacteria were cultured and counted on nutrient agar medium (Nutrient ager, Guangdong Huankai Microbiology Co., Ltd.), and yeast and mold counts were cultured and counted on potato dextrose agar (Potato-dextrose agar, Guangdong Huankai Microbiology Co., Ltd.). Lactic acid bacteria are cultured in an anaerobic chamber at 37°C for 1 to 2 days; bacteria, yeasts, and molds are cultured in a biochemical incubator at 30°C for 2 to 4 days.

[0101] 2) Fermentation quality analysis:

[0102] After the silage bag was opened, 20 g of the mixed silage was put into a ziplock bag, 80 mL of distilled water was added, and the mixture was placed in a 4 °C refrigerator for 18 h, then filtered and the pH was measured using a pH meter (Mettler Toledo FE28 pH meter). The organic acid content was measured using a Shimadzu LC-20AT high performance liquid chromatograph: Chromatographic conditions: chromatographic column (Eleven Organic Acids on TransgenomicCOREGel 87H3), detector: SPD-20A, mobile phase was 0.1 mmol / L phosphoric acid solution, flow rate was 1 mL / min, column temperature was 40 °C, detection wavelength was 210 nm, and injection volume was 20 μL. Ammonia nitrogen was measured using a colorimetric method.

[0103] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Bacillus strain capable of efficiently degrading fiber, characterized in that: The name of the Bacillus that can efficiently degrade fiber is Bacillus altitudinis DF34, the preservation number is GDMCC No: 65492, and it was deposited on December 4, 2024 at the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

2. Use of the Bacillus DF34 capable of efficiently degrading fiber as claimed in claim 1 in silage preparation.

3. The use of Bacillus DF34 capable of efficiently degrading fiber in silage preparation according to claim 2, characterized in that The steps include: (1) Cut the feed to be fermented into short pieces; (2) evenly mixing the chopped feed to be fermented and the above-mentioned Bacillus capable of efficiently degrading fiber; (3) Degas, seal and store the mixed feed.

4. The use of the Bacillus DF34 capable of efficiently degrading fiber in silage preparation according to claim 3, characterized in that: The feed to be fermented in step (1) includes pasture grass and fodder grass.

5. The use of the Bacillus DF34 capable of efficiently degrading fiber in silage preparation according to claim 4, characterized in that: The forage grass is at least one of corn straw, rice straw and wheat straw; The forage grass is elephant grass.

6. The use of the Bacillus DF34 capable of efficiently degrading fiber in silage preparation according to claim 3, characterized in that: The shortening described in step (1) is performed to a length of 2 to 3 cm.

7. The use of the Bacillus DF34 capable of efficiently degrading fiber in silage preparation according to claim 3, characterized in that: The amount of the Bacillus capable of efficiently degrading fiber in step (2) is 1.0×10 5 ~1.0×10 8 Calculation of CFU / g of feed to be fermented.

8. The use of the Bacillus DF34 capable of efficiently degrading fiber in silage preparation according to claim 7, characterized in that: The amount of the Bacillus capable of efficiently degrading fiber is 1.0×10 5 ~1.0×10 6 Calculation of CFU / g of feed to be fermented.

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