Bacillus licheniformis RDY21, bacterial agent containing the bacteria and application thereof

Abstract

The application discloses bacillus licheniformis RDY21, a bacterial agent containing the bacillus licheniformis and application of the bacillus licheniformis and belongs to the technical field of microorganisms. The application aims to provide bacillus licheniformis RDY21. The application provides bacillus licheniformis RDY21, the preservation number of the bacillus licheniformis is CCTCC NO: M 20221606, the bacillus licheniformis is preserved in the China Center for Type Culture Collection, and the preservation date is October 20, 2022. The bacillus licheniformis RDY21 has the effects of degrading zearalenone, improving bacterial diversity in a rumen of a cervidae, optimizing a flora structure and improving contents of crude protein and acid-soluble protein in soybean meal.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Bacillus licheniformis RDY21, bacterial agents containing this bacterium, and their applications. Background Technology

[0002] Zearalenone, a harmful mycotoxin produced by various Fusarium species, is widely found in grains, animal feed, and agricultural byproducts. According to a survey by Zhang Yong et al. on mycotoxin contamination in domestic feed and feed ingredients, the exceedance rate of zearalenone in corn byproducts was as high as 17.27%, followed by corn and compound feed at 13.30% and 12.41%, respectively. Zearalenone's chemical structure is similar to estradiol, and it can competitively bind to estrogen receptors, causing reproductive problems such as abortion, reducing animal production performance, and resulting in economic losses for the livestock industry. Due to its strong thermal stability, zearalenone can still remain in processed agricultural products such as vegetable oils, and meat, eggs, and milk from livestock and poultry, posing a threat to human health. In addition to reproductive toxicity, zearalenone has also been shown to have cytotoxicity and genotoxicity. Currently, the main detoxification methods for zearalenone both domestically and internationally include physical methods, chemical methods, and biodegradation methods. Physical and chemical methods have disadvantages such as high cost, loss of nutrients, and secondary pollution, thus limiting their application in practical production. Biodegradation is a method that uses enzymes adsorbed or secreted by microorganisms to degrade toxins. This method is carried out under mild conditions and has little impact on the physicochemical properties of feed and feed ingredients. It has the advantages of safety, high efficiency and low cost, and is currently the most promising measure to solve the problem of zearalenone contamination in feed and feed ingredients. Summary of the Invention

[0003] The purpose of this invention is to provide a Bacillus licheniformis RDY21 that can degrade zearalenone while simultaneously increasing the diversity of rumen bacteria in deer, optimizing the microbial community structure, and improving the content of crude protein and acid-soluble protein in soybean meal.

[0004] This invention provides a Bacillus licheniformis RDY21, which has the accession number CCTCC NO: M 20221606 and is deposited at the China Center for Type Culture Collection on October 20, 2022.

[0005] This invention provides a microbial preparation of the above-mentioned Bacillus licheniformis.

[0006] This invention provides the application of the above-mentioned Bacillus licheniformis RDY21 or the above-mentioned microbial preparation in the degradation of zearalenone.

[0007] This invention provides the application of the above-mentioned Bacillus licheniformis RDY21 or the above-mentioned microbial preparation in increasing the crude protein and acid-soluble protein content in soybean meal.

[0008] This invention provides the application of the above-mentioned Bacillus licheniformis RDY21 or the above-mentioned microbial preparation in improving rumen bacterial diversity and optimizing the microbial community structure.

[0009] To further define, improving rumen bacterial diversity and optimizing the microbial community structure means increasing the Chaol index of the rumen microbiota in deer, while simultaneously increasing the relative abundance of Bacteroidetes and decreasing the relative abundance of Proteobacteria.

[0010] The present invention provides a method for degrading zearalenone, wherein the above-mentioned Bacillus licheniformis RDY21 or the above-mentioned microbial preparation is added to a reaction solution containing zearalenone or soybean meal for reaction.

[0011] This invention provides a method for increasing the crude protein and acid-soluble protein content in soybean meal, wherein the above-mentioned Bacillus licheniformis RDY21 or the above-mentioned microbial preparation is added to soybean meal at 10% (v:w) for fermentation reaction.

[0012] This invention provides a method for improving rumen bacterial diversity and optimizing the microbial community structure by adding the above-mentioned Bacillus licheniformis RDY21 or the above-mentioned microbial preparation to feed sika deer.

[0013] Further specified, Bacillus licheniformis RDY21 was added to the feed at a rate of 10⁸ cfu / kg.

[0014] Beneficial Effects: The *Bacillus licheniformis* RDY21 strain involved in this invention possesses highly efficient ability to degrade zearalenone, achieving a degradation rate of up to 93.83% in liquid culture medium and 73.31% in fermented soybean meal. Solid-state fermentation with this strain can also increase the crude protein and acid-soluble protein content in soybean meal. In vivo experiments on sika deer demonstrate that *Bacillus licheniformis* RDY21 can improve rumen bacterial diversity and optimize the bacterial community structure.

[0015] [Biological Preservation Information]: Bacillus licheniformis RDY21, accession number: CCTCCNO: M 20221606, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on October 20, 2022. Attached Figure Description

[0016] Figure 1 The image shows the results of microscopic examination (1000X) of strain RDY21;

[0017] Figure 2 This is a photograph of the plate results for strain RDY21;

[0018] Figure 3 A heatmap of the whole genome of strain RDY21 was generated using OAT software.

[0019] Figure 4 The degradation rate of zearalenone by strain RDY21 in liquid culture medium;

[0020] Figure 5 The degradation rate of zearalenone by strain RDY21 in solid-state fermentation of soybean meal;

[0021] Figure 6 This is a graph showing the principal coordinate analysis results based on the Bray-Curtis distance;

[0022] Figure 7 A diagram showing the phylum-level composition of rumen fluid from sika deer;

[0023] Figure 8 This is a diagram showing the results of bacterial genus composition in the rumen fluid of sika deer. Detailed Implementation

[0024] Example 1: Isolation and Identification of Bacillus licheniformis RDY21

[0025] Fresh feces were collected from three-year-old male sika deer. Sterile phosphate-buffered saline (PBS) was added at a ratio of 1:5 (w:v), and the mixture was shaken for 30 min to mix thoroughly. The mixture was then incubated in an 80°C water bath for 20 min. After standing, 200 μL of the supernatant was inoculated into LB liquid medium and incubated at 37°C for 12–18 h. The medium was then serially diluted, and 10 μL of the supernatant was used as the final product. -3 10 -4 and 10 -5 Diluted bacterial suspensions were spread onto LB solid medium and incubated upside down at 37°C for 24–36 hours. Single colonies of different morphologies were picked and purified for 3–4 generations until *Bacillus licheniformis* and its biologically pure culture were obtained. Morphological observation of the purified *Bacillus licheniformis* revealed that the colonies were slightly yellow, flat, irregularly shaped, with a rough, wrinkled, and moist surface. Figure 1 The bacteria are short rod-shaped, arranged singly or in pairs, and are Gram-positive. Figure 2 Whole-genome sequencing results showed that, compared with the whole-genome data of the Bacillus licheniformis (ATCC_14580) type strain, the ANI value of the strain of this invention was 99.02%. Figure 3The results were greater than the ANI threshold of 96%; dDDH values ​​were 97.6%, 91.5%, and 98.1%, which were greater than the dDDH threshold of 70%; the bacteria were identified as Bacillus licheniformis.

[0026] Example 2: Inoculum containing Bacillus licheniformis RDY21

[0027] Bacillus licheniformis RDY21 fermentation broth: Inoculate Bacillus licheniformis RDY21 seed culture at 2% (v:v) onto LB liquid medium and incubate at 37°C for 24–48 hours. Collect the fermentation broth; the viable count should be 10⁻⁶. 10-11 cfu / mL.

[0028] Bacillus licheniformis RDY21 freeze-dried mycelial powder: Bacillus licheniformis RDY21 solution was inoculated at 2% (v:v) onto LB liquid medium and fermented at 37℃ for 24–48 h. After centrifugation at 4500 rpm / min for 15 min, the mycelial sludge was collected. A protectant (a mixture of 10% skim milk and trehalose) was added at 5 times its weight of the mycelial sludge. The mixture was thoroughly mixed and placed in a -80℃ freeze-dryer overnight. The resulting mycelial powder was prepared by vacuum freeze-drying, with a viable count of 10-1. 12-13 cfu / g.

[0029] Example 3: Degradation ability of Bacillus licheniformis RDY21 on zearalenone

[0030] Activated Bacillus licheniformis RDY21 bacterial culture was added at a 2% (v:v) inoculum to LB liquid basal medium containing 10 μg / mL zearalenone. The culture was incubated at 37°C with shaking. Samples were taken at different incubation times (0, 24 h, 48 h, 72 h), and the ability of this strain to degrade zearalenone was determined by HPLC. Figure 4 It can be seen that after 24 hours of cultivation, the degradation rate of zearalenone by Bacillus licheniformis RDY21 was 56.64%, and when the cultivation time reached 72 hours, the degradation rate of zearalenone by Bacillus licheniformis RDY21 was as high as 93.83%.

[0031] Zearalenone degradation rate (%) = [(m1-m2)÷m1]×100, where m1 is the zearalenone content in the initial culture medium and m2 is the zearalenone content in the fermentation broth at different culture times.

[0032] Example 4: Application of Bacillus licheniformis strain RDY21 in fermented feed

[0033] 200g of soybean meal containing 10.09μg / g zearalenone was weighed into a 1000mL Erlenmeyer flask. Activated Bacillus licheniformis RDY21 bacterial culture was inoculated into the soybean meal at a 10% (v:w) inoculation rate. An appropriate amount of sterile water was added to bring the final moisture content of the soybean meal to 50%. After thorough mixing, the flask was sealed with a bacterial breathable membrane and fermented at 37℃ for 5 days. The fermented sample was first dried at 105℃ for 30 minutes to inhibit fermentation, then dried at 65℃ for 24 hours, pulverized, and passed through a 40-mesh sieve for zearalenone and nutrient composition determination.

[0034] Depend on Figure 5 It was found that fermentation with Bacillus licheniformis RDY21 reduced the zearalenone content in soybean meal from 10.09 ug / g to 2.67 ug / g. Table 1 shows that compared to the original soybean meal, the crude protein and acid-soluble protein contents of the soybean meal fermented with Bacillus licheniformis RDY21 were significantly increased (P < 0.05). This indicates that adding Bacillus licheniformis RDY21 during soybean meal fermentation can significantly remove zearalenone content, increasing crude protein content by 9.18% and acid-soluble protein content by 78.64%.

[0035] Nutritional content improvement rate (%) = [(m1 - m2) ÷ m2] × 100, where m1 is the nutrient content after fermentation and m2 is the nutrient content before fermentation.

[0036] Table 1. Nutritional composition of soybean meal before and after fermentation

[0037] crude protein <![CDATA[45.08±1.26 b ]]> <![CDATA[49.22±2.84 a ]]> Coarse ash 6.82±0.06 7.18±0.05 Neutral detergent fibers 14.86±0.56 15.12±0.72 Acid detergent fibers 7.68±0.62 6.84±0.63 Acid-soluble protein <![CDATA[6.04±0.36 b ]]> <![CDATA[10.79±0.30 a ]]>

[0038] Example 5: Application of Bacillus licheniformis RDY21 in sika deer during antler growth period

[0039] Ten 3-year-old sika deer in the antler-growing stage were randomly divided into two groups of five each. The groups were fed the following diets: basal diet (control group) and basal diet plus microbial agent (experimental group), with Bacillus licheniformis RDY21 at a concentration of 10... 8 CFU / kg was added to the concentrate feed for a 30-day trial period.

[0040] The basic diet is made from corn flour, soybean meal, corn germ meal, peanut meal, and wheat bran as the main raw materials, with the addition of nutritional additives composed of minerals, vitamins, etc. Silage straw is used as roughage, and the composition of the concentrate is shown in Table 2. The nutritional level of the feed is shown in Table 3.

[0041] Table 2 Dietary Composition

[0042] cornmeal 26 soybean meal 19.5 Corn germ meal 11.7 wheat bran 3.9 Fish meal 1.3 Silage straw 35 salt 0.65 <![CDATA[Premix a > 1.95

[0043] The premix provides per kilogram of feed: MgO 0.083g; MnSO4·H2O 0.047g; FeSO4·H2O 0.058g; ZnSO4·H2O 0.039g; FeSO4·H2O 0.034g; Vitamin A 532.4IU; Vitamin D3 546.48IU; Vitamin E 0.911IU; Vitamin K3 0.253mg; Vitamin B1 0.101mg; Vitamin B2 0.759mg; Vitamin B... 12 0.015mg; Folic acid 0.025mg; Niacin 1.78mg; Calcium pantothenate 1.26mg; CaHPO4 5.68g; CaHPO4 5.02g.

[0044] Table 3 Nutritional levels of feed diets

[0045] dry matter 88.01 crude protein 19.89 Crude fat 3.92 Neutral detergent fibers 45.53 Acid detergent fibers 25.53 Total energy (MJ / kg) 18.15

[0046] Sample collection method:

[0047] On day 45 of the experiment, each sika deer was anesthetized by injecting 0.6–1.3 mg / kg body weight of thiazide hydrochloride before morning feeding. Rumen fluid was collected using a rumen fluid collector into 10 mL cryovials, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0048] Sample determination method:

[0049] Microbial genomic DNA extraction from rumen fluid samples was performed according to the Fast DNA RSPIN Kit for Feces instructions. The extracted genomic DNA was detected by 1% agarose gel electrophoresis at 120V for 23 min using a λ-HindⅢ DNA Maker. The DNA product was approximately 14,000 bp in length, with high band brightness, no significant degradation, and no impurities, indicating it was ready for further experiments. The target DNA was sent to Beijing Novogene Technology Co., Ltd., and sequencing was performed using Illumina Miseq. Microbial sequencing results were fused with paired-end sequences using Flash software (FLASH v1.2.7). Low-quality data were truncated using PRINSEQ software (PRINSEQ-lite 0.19.5), and sequences were clustered into Operational Taxonomic Units (OTUs) based on 97% similarity. OTU clustering was performed using Uclust software (Uclust v1.1.579), and species classification was performed using the RDP classifier software. The alpha diversity index of the microbial community was calculated using QIIME 1.9.0.

[0050] Data analysis methods:

[0051] The experimental data were statistically analyzed using the GLM program in SAS software. Multiple comparisons were performed using Ducan's method, where P < 0.01 was considered highly significant, P < 0.05 was considered significant, and P > 0.05 was considered not significant. The results are expressed as mean ± standard deviation.

[0052] Data Analysis and Conclusions:

[0053] Table 4 shows that the goods_coverage index of both groups was higher than 0.998, indicating sufficient sequencing depth and the results could present more than 99.8% of the bacterial community composition. Compared with the control group, the chao1 and shannon indices of the experimental group were significantly increased (P<0.05). In the alpha diversity analysis, the higher the Chaol index, the higher the species richness, indicating that the addition of Bacillus licheniformis RDY21 has a trend of improving the rumen bacterial diversity of sika deer during the antler-growing period.

[0054] Table 4. Alpha diversity of rumen flora in 3-year-old antler-growing sika deer after the addition of Candida aegypti DZ018.

[0055]

[0056] like Figure 6 As shown in the PCoA figure (PC1 vs PC2), the principal coordinate analysis (PCoA) based on the Bray-Curtis distance shows that the sum of the two coordinate axes explained 64.78% of the variable composition. The bacterial community composition of the samples in the two groups is basically similar but there are still some differences. It can be seen that the addition of Bacillus licheniformis RDY21 has a certain impact on the bacterial composition and structure of the rumen fluid of sika deer during the antler-growing period.

[0057] The results of the phylum-level abundance analysis of bacterial composition in the rumen fluid of sika deer are as follows: Figure 7 As shown, the three bacterial phyla with the highest relative abundance in both groups were Firmicutes, Bacteroides, and Proteobacteria. Compared with the control group, the relative abundance of Bacteroides was significantly increased in the experimental group (P < 0.05), while the relative abundance of Proteobacteria was significantly decreased (P < 0.05). Bacteroides are important degradative bacteria of dietary carbohydrates and proteins and can promote the development of the gastrointestinal immune system. Abnormal proliferation of Proteobacteria is often used as a microbial signal of intestinal microbial dysbiosis. Therefore, the addition of Bacillus licheniformis RDY21 has a positive effect on optimizing the rumen bacterial community structure of sika deer during the antler-growing period.

[0058] The results of the genus-level abundance analysis of bacterial composition in the rumen fluid of sika deer are as follows: Figure 8As shown, compared with the control group, the relative abundance of *Methanobrevibacter* and *Succiniclasticum* was significantly increased in the experimental group (P < 0.05), while the abundance of *Mycoplasma* was significantly decreased (P < 0.05). *Methanobrevibacter* can enhance cellulose degradation by regulating the metabolism of anaerobic fungi. *Succiniclasticum* can convert succinic acid to propionic acid and can break down starch and cellulose. This table shows that the addition of *Bacillus licheniformis* RDY21 can promote the digestion and utilization of nutrients in feed by altering the composition of the rumen bacterial flora.

Claims

1. A type of Bacillus licheniformis ( Bacillus licheniformis RDY21, characterized in that, The Bacillus licheniformis has the accession number CCTCC NO: M 20221606 and is deposited at the China Center for Type Culture Collection on October 20, 2022.

2. A microbial preparation containing Bacillus licheniformis as described in claim 1.

3. The use of Bacillus licheniformis RDY21 as described in claim 1 or the microbial preparation as described in claim 2 in the degradation of zearalenone.

4. The application of Bacillus licheniformis RDY21 as described in claim 1 or the microbial preparation as described in claim 2 in increasing the crude protein and acid-soluble protein content in soybean meal.

5. The use of Bacillus licheniformis RDY21 as described in claim 1 in the preparation of microbial preparations that improve rumen bacterial diversity and optimize microbial community structure.

6. The application according to claim 5, characterized in that, Improving rumen bacterial diversity and optimizing the microbial community structure refers to increasing the Chaol index of the rumen microbiota in deer, while simultaneously increasing the relative abundance of Bacteroidetes and decreasing the relative abundance of Proteobacteria.

7. A method for degrading zearalenone, characterized in that, The Bacillus licheniformis RDY21 of claim 1 or the microbial preparation of claim 2 is added to a reaction solution containing zearalenone or soybean meal for reaction.

8. A method for increasing the crude protein and acid-soluble protein content in soybean meal, characterized in that, The Bacillus licheniformis RDY21 of claim 1 or the microbial preparation of claim 2 is added to soybean meal at a ratio of 10% v:w for fermentation.

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