Bacillus licheniformis RDY21, microbial inoculum containing bacillus licheniformis RDY21 and application of bacillus licheniformis RDY21

Abstract

The invention discloses bacillus licheniformis RDY21, a microbial agent containing the bacillus licheniformis RDY21 and application of the bacillus licheniformis RDY21 and belongs to the technical field of microorganisms. The invention aims to provide the bacillus licheniformis RDY21 and the application thereof. The invention provides bacillus licheniformis RDY21, the preservation number of the bacillus licheniformis is CCTCC (China Center for Type Culture Collection) 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 degrades zearalenone, meanwhile, the bacillus licheniformis RDY21 has the effects of improving the diversity of rumen bacteria of cervidae animals, optimizing the flora structure and improving the content of crude protein and acid soluble protein in soybean meal.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Bacillus licheniformis RDY21, a bacterial agent containing the bacterium and application thereof. Background Art

[0002] Zearalenone is a harmful fungal toxin produced by a variety of Fusarium species and is widely found in grains, animal feed and agricultural and sideline products. According to a survey on the contamination of mycotoxins in domestic feed and feed raw materials by Zhang Yong et al., the excess rate of zearalenone in corn by-products was as high as 17.27%, followed by corn and compound feed, which were 13.30% and 12.41% respectively. The chemical structure of zearalenone is similar to that of estradiol, and it can competitively bind to estrogen receptors, causing reproductive problems such as miscarriage, reducing the production performance of animals and causing economic losses to the breeding industry. Because zearalenone has strong thermal stability, it will still remain in processed agricultural products such as vegetable oils, meat, eggs and milk of livestock and poultry, causing harm to human health. In addition to reproductive toxicity, zearalenone has also been shown to have cytotoxicity and genotoxicity. At present, the detoxification methods of zearalenone at home and abroad mainly include physical method, chemical method and biodegradation method. Physical and chemical methods have disadvantages such as high cost, loss of nutrients and secondary pollution, which limits the application of these two methods in actual production. The biodegradation method is to degrade toxins through enzymes adsorbed or secreted by microorganisms. This method is carried out under mild conditions and has little effect on the physical and chemical properties of feed and feed raw materials. It has the advantages of safety, high efficiency and low cost. It is currently the most promising measure to solve the problem of zearalenone contamination in feed and feed raw materials. Summary of the invention

[0003] The purpose of the present invention is to provide a Bacillus licheniformis RDY21, which can degrade zearalenone and at the same time improve the rumen bacterial diversity of deer animals, optimize the flora structure and increase the crude protein and acid-soluble protein content in soybean meal.

[0004] The present invention provides a Bacillus licheniformis RDY21, the Bacillus licheniformis has a preservation number of CCTCC NO: M 20221606, and is deposited in China Center for Type Culture Collection on October 20, 2022.

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

[0006] The present invention provides an application of the Bacillus licheniformis RDY21 or the microbial preparation in degrading zearalenone.

[0007] The present invention provides an application of the Bacillus licheniformis RDY21 or the microbial preparation in increasing the contents of crude protein and acid-soluble protein in soybean meal.

[0008] The present invention provides an application of the Bacillus licheniformis RDY21 or the microbial preparation in improving rumen bacterial diversity and optimizing bacterial flora structure.

[0009] It is further defined that improving rumen bacterial diversity and optimizing bacterial flora structure refers to improving the Chaol index of the rumen flora of Cervidae, while increasing the relative abundance of rumen Bacteroidetes and reducing 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 into a reaction solution containing zearalenone or soybean meal for reaction.

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

[0012] The present invention provides a method for improving rumen bacterial diversity and optimizing bacterial flora structure, wherein the above-mentioned Bacillus licheniformis RDY21 or the above-mentioned microbial preparation is added into feed to feed sika deer.

[0013] It is further defined that Bacillus licheniformis RDY21 is added to the feed at 108 cfu / kg.

[0014] Beneficial effects: The Bacillus licheniformis RDY21 of the present invention has the ability to efficiently degrade zearalenone. Its degradation rate of zearalenone in liquid culture medium is as high as 93.83%, and its degradation rate of zearalenone in fermented soybean meal is 73.31%. When the strain is used for solid-state fermentation, the crude protein and acid-soluble protein content in soybean meal can also be increased. In vivo experiments on sika deer show that Bacillus licheniformis RDY21 has the effect of increasing rumen bacterial diversity and optimizing bacterial community structure.

[0015] [Biological preservation information]: Bacillus licheniformis RDY21, preservation number: CCTCCNO: M 20221606, deposited in China Center for Type Culture Collection, the preservation address is: Wuhan University, Wuhan, China, and the preservation date is October 20, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the microscope (1000X) result of strain RDY21;

[0017] Figure 2 This is the result of the plate photo of strain RDY21;

[0018] Figure 3 A heat map was generated for the strain RDY21 using the strain-wide genome calculated using the OAT software;

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

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

[0021] Figure 6 This is the result of principal coordinate analysis based on Bray-Curtis distance;

[0022] Figure 7 This is the bacterial phylum composition result of rumen fluid of sika deer;

[0023] Figure 8 This is the composition result of bacteria at genus level in rumen fluid of sika deer. DETAILED DESCRIPTION

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

[0025] Fresh feces of three-year-old male sika deer were collected and added to sterile phosphate buffered saline (PBS) at a ratio of 1:5 (w:v). The mixture was shaken for 30 min and then placed in a water bath at 80°C for 20 min. After standing, 200 μL of the supernatant was inoculated into LB liquid culture medium and incubated at 37°C for 12-18 h. The supernatant was diluted in a gradient and 10 -3 , 10 -4 and 10 -5 The diluted bacterial solution was spread on LB solid medium and inverted for 24-36 hours at 37°C. Single colonies of different morphologies were picked out and purified for 3-4 generations until Bacillus licheniformis and its biological pure culture were obtained. Morphological observation of the purified Bacillus licheniformis revealed that the colonies were slightly yellow, flat, irregular in shape, 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 The whole genome sequencing results showed that the ANI value of the strain of the present invention compared with the whole genome data of the model strain of Bacillus licheniformis (ATCC_14580) was: 99.02% ( Figure 3), the result was greater than the ANI same-species judgment threshold of 96%; dDDH value: 97.6%, 91.5%, 98.1%, the result was greater than the dDDH same-species judgment threshold of 70%; it was identified as Bacillus licheniformis.

[0026] Example 2: Bacterial agent containing Bacillus licheniformis RDY21

[0027] Bacillus licheniformis RDY21 fermentation broth: 2% (v:v) of Bacillus licheniformis RDY21 seed liquid was inoculated into LB liquid medium and cultured at 37°C for 24-48 hours. The fermentation broth was collected and the number of viable cells was 10 10-11 cfu / mL.

[0028] Bacillus licheniformis RDY21 freeze-dried powder: Bacillus licheniformis RDY21 liquid was inoculated into LB liquid medium at 2% (v:v), fermented and cultured at 37°C for 24-48h, centrifuged at 4500rpm / min for 15min, and the bacterial sludge was taken. A protective agent (a mixture of 10% skim milk and trehalose) was added according to 5 times the weight of the bacterial sludge. After being fully mixed, it was placed in a -80°C refrigerator overnight and vacuum freeze-dried to obtain bacterial powder. The number of viable cells was 10 12-13 cfu / g.

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

[0030] Take the activated Bacillus licheniformis RDY21 bacterial solution, add it to the LB liquid basal medium containing 10 μg / mL zearalenone at a 2% (v:v) inoculation amount, and culture it at 37°C with shaking. Samples are taken at different culture times (0, 24h, 48h, 72h), and the ability of the strain to degrade zearalenone is 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%. When the cultivation time reached 72 hours, the degradation rate of zearalenone by Bacillus licheniformis RDY21 was as high as 93.83%.

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

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

[0033] Weigh 200g of soybean meal containing 10.09μg / g zearalenone in a 1000mL triangular flask, take the activated Bacillus licheniformis RDY21 bacterial solution, inoculate it into the soybean meal at a 10% (v:w) inoculation rate, add an appropriate amount of sterile water to make the final moisture content of the soybean meal 50%, stir evenly, seal the bottle mouth with a filter membrane, and ferment at 37°C for 5d. The fermented sample was first dried at 105°C for 30min to block fermentation, then dried at 65°C for 24h, crushed and passed through a 40-mesh sieve for the determination of zearalenone and nutritional components.

[0034] Depend on Figure 5 It can be seen that the zearalenone content in soybean meal decreased from 10.09ug / g to 2.67ug / g after fermentation with Bacillus licheniformis RDY21. As shown in Table 1, compared with the original soybean meal, the crude protein and acid-soluble protein content in soybean meal fermented with Bacillus licheniformis RDY21 were significantly increased (P < 0.05). This shows that during the fermentation of soybean meal, the addition of Bacillus licheniformis RDY21 can significantly remove the zearalenone content, increase the crude protein content by 9.18%, and increase the acid-soluble protein content by 78.64%.

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

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

[0037] project Original soybean meal (%) Fermented soybean meal (%) 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 fiber 14.86±0.56 15.12±0.72 Acid detergent fiber 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 on sika deer in the antler-growing period

[0039] Ten 3-year-old sika deer in the antler-growing stage were randomly divided into two groups, with 5 deer in each group. They were fed the following diets: basic diet (control group), basic diet + bacterial agent (experimental group), and Bacillus licheniformis RDY21 at 10 8 cfu / kg was added to the concentrated feed, and the experimental period was 30 days.

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

[0041] Table 2 Diet composition

[0042] project content(%) Corn flour 26 Soybean meal 19.5 Corn Germ Meal 11.7 Wheat bran 3.9 Fishmeal 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

[0045] Nutritional Information content(%) Dry matter 88.01 Crude Protein 19.89 Crude fat 3.92 Neutral detergent fiber 45.53 Acid detergent fiber 25.53 Total energy (MJ / kg) 18.15

[0046] Sample collection method:

[0047] On the 45th day of the experiment, each sika deer was anesthetized by injection of 0.6-1.3 mg / kg body weight of xylazine hydrochloride before morning feeding. The rumen fluid was collected by a rumen fluid collector into a 10 mL cryotube, quickly frozen in liquid nitrogen, and then stored in a -80℃ refrigerator.

[0048] Sample determination method:

[0049] The extraction of microbial genomic DNA from rumen fluid samples was carried out according to the instructions of Fast DNA RSPIN Kit for Feces. The extracted genomic DNA was detected by 1% agarose gel electrophoresis, the voltage was 120V, the electrophoresis was 23min, the marker was λ-HindⅢDNA Maker, the DNA product was about 14 000bp, and the DNA band brightness was high, there was no obvious degradation and no impurities, and the next step of the experiment could be carried out. The target DNA was sent to Beijing Novogene Technology Co., Ltd., and the sequencing platform was IlluminaMiseq. The microbial sequencing results were fused with double-end sequences using Flash software (FLASH v1.2.7). PRINSEQ software (PRINSEQ-lite 0.19.5) was used to cut off low-quality data, and the sequences were clustered into (OperationalTaxonomicUnits OTU) according to 97% similarity. The software used for OTU clustering was Uclust version (Uclust v1.1.579), and the software RDP classifier was used for species classification. The Alpha diversity index of the bacterial 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, and multiple comparisons were performed using Ducan's method, where P < 0.01 was considered extremely significant, P < 0.05 was considered significant, and P > 0.05 was considered insignificant. The results were expressed as mean ± standard deviation.

[0052] Data analysis and conclusion:

[0053] As shown in Table 4, the goods_coverage index of both groups was higher than 0.998, indicating that the sequencing depth was sufficient and the results could present more than 99.8% of the bacterial community composition. Compared with the control group, the Chao1 and Shannon indexes of the experimental group increased significantly (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 tendency to increase the diversity of rumen bacteria in sika deer during the antler growth period.

[0054] Table 4 Effect of adding Candida agastache DZ018 on the alpha diversity of rumen fluid flora of 3-year-old sika deer in the antler-growing stage

[0055]

[0056] like Figure 6 (PCoA PC1 vs PC2 diagram) shows that the principal coordinate analysis (PCoA) based on Bray-Curtis distance showed that the addition of the two coordinate axes explained 64.78% of the variable composition. The sample flora composition between the two groups was basically similar but there were still some differences. It can be seen that the addition of Bacillus licheniformis RDY21 has a certain effect on the bacterial composition and structure of the rumen fluid of sika deer in the antler-growing period.

[0057] The results of the phylum-level abundance analysis of rumen fluid bacteria in sika deer are as follows: Figure 7 As shown in the figure, the top three bacterial phyla with higher relative abundance in the two groups were Firmicutes, Bacteroidota, and Proteobacteria; compared with the control group, the relative abundance of Bacteroidetes in the experimental group was significantly increased (P < 0.05), while the relative abundance of Proteobacteria was significantly decreased (P < 0.05). Bacteroidetes are important degraders of dietary carbohydrates and proteins, which can promote the development of the gastrointestinal immune system. Abnormal expansion of Proteobacteria is often used as a microbial signal of intestinal microbial imbalance. It can be seen that the addition of Bacillus licheniformis RDY21 has a positive effect on optimizing the rumen bacterial flora structure of sika deer in the antler growth period.

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

Claims

1. A Bacillus licheniformis RDY21, characterized in that: The Bacillus licheniformis preservation number is: CCTCC NO: M 20221606, and it is deposited in the China Center for Type Culture Collection, and the preservation date is October 20, 2022.

2. A microbial preparation containing the Bacillus licheniformis according to claim 1.

3. Use of the Bacillus licheniformis RDY21 according to claim 1 or the microbial preparation according to claim 2 in degrading zearalenone.

4. Use of the Bacillus licheniformis RDY21 according to claim 1 or the microbial preparation according to claim 2 in increasing the content of crude protein and acid-soluble protein in soybean meal.

5. Use of the Bacillus licheniformis RDY21 according to claim 1 or the microbial preparation according to claim 2 in improving rumen bacterial diversity and optimizing bacterial flora structure.

6. The use according to claim 5, characterized in that: Improving rumen bacterial diversity and optimizing bacterial community structure means increasing the Chaol index of the rumen flora of deer, while increasing the relative abundance of rumen Bacteroidetes and reducing the relative abundance of Proteobacteria.

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

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

9. A method for increasing rumen bacterial diversity and optimizing bacterial flora structure, characterized in that: The Bacillus licheniformis RDY21 described in claim 1 or the microbial preparation described in claim 2 is added into feed to feed sika deer.

10. The method according to claim 9, characterized in that Bacillus licheniformis RDY21 press 10 8 cfu / kg added to feed.

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