Bacillus tequila QH1 producing α-tocopherol and its application

Abstract

The invention discloses alpha-tocopherol-producing Bacillus tequilensis QH1 and its application. The strain used is QH1, which is identified as Bacillus tequilensis and has a strain collection number of CGMCC NO.23715. The main biological characteristics are Gram-positive staining, bacillus, acid resistance, fast growth rate, and particularly outstanding ability to produce alpha-tocopherol, and has considerable economic value in the field of silage and pig fermented feed production.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application and feed preparation and processing, and in particular to alpha-tocopherol-producing Bacillus tequila QH1 and application thereof. Background Art

[0002] The hydrolysis product of α-tocopherol is vitamin E, which is also its main effector. It is highly safe and often used as an antioxidant. It has strong anti-stress ability and promotes reproduction. In recent years, α-tocopherol has been mainly used in the fields of medicine, cosmetics, food and health products, and feed. There are two types of α-tocopherol: natural and synthetic. Natural α-tocopherol is generally obtained from plants and animals, but there are no reports of obtaining it from bacteria. Compared with synthetic α-tocopherol, natural α-tocopherol is better in biological activity, optical rotation, safety, and economic benefits, and people tend to choose natural α-tocopherol.

[0003] my country is a major rice-growing country, and rice straw accounts for about 46% of the total rice grown. However, due to improper handling, more than 50% of the straw is wasted. According to analysis, rice straw contains a large amount of cellulose, hemicellulose and lignin, which is difficult to be decomposed by microorganisms, and has a strict epidermal keratin, low content of crude protein, crude fat, minerals, α-tocopherol, etc., resulting in poor palatability and low digestibility of rice straw. Silage is an effective method for long-term preservation of nutrients such as fresh forage and feed crops in an anaerobic and acidic environment, which can improve the palatability and digestibility of silage raw materials.

[0004] Piglets experience many cruel pressures from physiology, environment and society during the weaning process. Since piglets cannot adapt to these pressures quickly, these pressures often lead to a decrease in piglet feed intake and growth performance, disordered intestinal flora structure, decreased immunity and resistance, and increased incidence of various inflammations and disease infection rates. Therefore, vitamin E additives are often added to piglet feed to improve piglet anti-stress ability and intestinal health. However, simply adding vitamin E additives to piglet feed is costly, slow to rebuild beneficial microorganisms in the piglet intestine, and cannot quickly reduce stress.

[0005] In view of the fact that rice straw and piglet feed contain less α-tocopherol, the present invention uses the α-tocopherol-producing strain QH1 to be applied to rice straw silage alone, and the α-tocopherol-producing strain QH1 and Lactobacillus plantarum are combined to be applied to fermented piglet feed. The results show that the α-tocopherol-producing strain QH1 improves the fermentation quality of rice straw silage. Compared with the α-tocopherol content of rice straw and piglet feed, the α-tocopherol content of rice straw silage and piglet fermented feed is increased by about 700% and 166%, respectively, which has great application value in production. Summary of the invention

[0006] The present invention provides a strain QH1 capable of producing α-tocopherol, and a method for preparing high-quality rice straw silage and piglet fermented feed using the strain, in view of the fact that rice straw and piglet feed contain less α-tocopherol and livestock need vitamin E to maintain health and robust production performance. The strain can significantly improve the quality and α-tocopherol content of rice straw silage and piglet fermented feed, and has great application value in production.

[0007] The invention provides application of an alpha-tocopherol-producing strain QH1 in preparing rice straw silage and piglet fermented feed.

[0008] The invention achieves the above-mentioned purpose through the following technical solutions:

[0009] The present invention provides an α-tocopherol-producing strain QH1, which is identified as Bacillus equilensis, and was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on November 3, 2021, with the strain collection number of CGMCC NO.23715; its main biological characteristics are Gram-positive bacilli, fast growth rate [the pH of reinforced Clostridium (RCM) liquid culture medium can be reduced to below 4.6 after culturing at 37°C for 1 day], and its ability to produce α-tocopherol is particularly outstanding (50.72 mg / L).

[0010] The growth temperature of the strain is 20°C-50°C, and the growth temperature of the strain is preferably 37°C; the growth pH value is pH 4.5-pH 9.0.

[0011] The bacteria was obtained by enrichment culture and dilution plate screening: a conical flask containing 500 mL of distilled water, a test tube containing 9 mL of distilled water, RCM solid culture medium and a plate were sterilized, and then the RCM culture medium was poured into the plate. Under sterile conditions, 10 g of silage sample was placed in a plastic bag, and then 90 mL of sterile distilled water was added and shaken evenly, and then 1 mL of the diluted sample solution was added to a test tube containing 9 mL of sterile distilled water and shaken evenly, and finally the final concentration was diluted by 10 -3 ~10 -5. Take 0.03mL of the dilution and spread it on the plate. Incubate anaerobically at 37℃ for 36-72h until a single colony can be seen. Insert the single colony into a test tube containing Bars medium with an inoculation loop. After anaerobic incubation at 37℃ for 48 hours, take 1mL of the bacterial solution and store it in a -80℃ refrigerator for determination and evaluation of the strain's ability to produce α-tocopherol. The RCM medium is: 10.0g of peptone (Proteose peptone NO.3); 10.0g of beef extract (Beef extract); 3.0g of yeast extract (Yeast extract); 5.0g of glucose (Dextrose); 1.0g of soluble starch (soluble starch); 3.0g of sodium acetate (NaAc); 5.0g of sodium chloride (NaCl); 0.5g of L-cysteine ​​hydrochloride (L-Cysteine ​​hydrochloride). Dissolve the above reagents, make up to 1000mL with distilled water, add 15g / L agar to the solid culture medium, and sterilize at 121℃ for 15min. Bars culture medium is: K2HPO4·3H2O 0.66g, NH4Cl 1.0g, CaSO4 2.0g, sodium citrate dihydrate 5.0g, 60% sodium lactate 46mL, yeast extract 0.5g, (NH)2SO4·FeSO4·6H2O 0.5g, C2H3NaO2S 0.05g. Dissolve the above reagents, make up to 1000mL with distilled water, and dispense into 15mL test tubes, 10mL per test tube, and sterilize at 121℃ for 15min.

[0012] The present invention uses physiological and biochemical methods to screen out the strain QH1 that produces the most alpha-tocopherol and grows fast from 48 strains of bacteria. The full-length gene is extracted, and then the PCR amplification primers 25f (5`-AAC TGA AGA GTT TGA TCC TGG CTC-3`) and 1492r (5`-TAC GGC TAC CTT GTT ACG ACT-3`) are used to amplify the 16S rDNA gene, and the related sequences are compared on NCBI to determine the similar strains. Finally, the screened QH1 strain is inoculated into rice straw and piglet feed to improve the fermentation quality of the two and increase the alpha-tocopherol content.

[0013] A bacterial agent prepared by the Bacillus tequilensis QH1.

[0014] As a preferred embodiment of the present invention, the bacterial agent is prepared by the following method: 1 to 3 colonies of strain QH1 are picked to 5 mL of reinforced Clostridium liquid medium (RCM, sterilized at 121°C for 15 minutes), mixed and anaerobically cultured at 30 to 40°C for 36 to 72 hours to prepare a seed QH1 bacterial preparation, so that the bacterial count reaches at least 1.0×10 11 pcs / mL, which is convenient for subsequent production.

[0015] The invention discloses an application of Bacillus tequilensis QH1 in synthesizing alpha-tocopherol.

[0016] The invention discloses an application of Bacillus tequilensis QH1 in preparing silage and / or fermented piglet feed.

[0017] The bacterial agent of the present invention is used in preparing silage and / or fermented piglet feed.

[0018] A method for preparing silage using the strain QH1 comprises the following steps: ① cutting the rice straw to be silaged into 1-2 cm pieces and mixing them evenly; ② adding 1.0×10 strain QH1 per gram of rice straw. 6 or more; ③ the feed after adding strain QH1 is sealed and stored anaerobically at room temperature for more than 30 days, preferably 42 days.

[0019] A method for fermenting piglet feed comprises the following steps: ① taking piglet feed as fermentation raw material, mixing it with water at a ratio of 10:(3-3.5) by fresh weight, preferably 10:3.3; ② inoculating Lactobacillus plantarum and QH1 strains in an amount of 1×10 6 cfu / g FM, mix well and put into a fermentation tank; ③ ferment at 37°C for 5-10 days, preferably for 7 days.

[0020] As a preferred embodiment of the present invention, in step ②, the combined inoculation of Lactobacillus plantarum and the QH1 bacterial agent described in claim 2 is performed so that the two bacteria reach 1×10 6 cfu / g FM or more.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Using microorganisms to improve the fermentation quality of silage and increase the α-tocopherol content is low-cost, safe, reliable and easy to use.

[0023] (2) This strain can be used in various parts of my country, especially the QH1 strain, which has an outstanding ability to produce α-tocopherol. It is also the first strain that produces α-tocopherol isolated in my country.

[0024] (3) The Bacillus tergillosis QH1 used in the present invention improves the fermentation quality of rice straw and piglet feed silage and the content of α-tocopherol. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 α-Tocopherol production of QH1 strain, Enterococcus, Lactococcus lactis and Lactobacillus plantarum in Bars medium

[0026] Note: Different lowercase letters above the column indicate significant differences (P<0.05). QH1 strain, Bacillus tequilensis.

[0027] Figure 2 Phylogenetic diagram of QH1 strain and reference strains

[0028] Figure 3 Effect of inoculation with QH1 strain on the α-tocopherol content of rice straw silage

[0029] Note: Different lowercase letters above the column indicate significant differences, P<0.05.

[0030] Biomaterial Deposit Information

[0031] QH1, classified as Bacillus tequilensis, was deposited in the General Microbiology Center of China Culture Collection (CGMCC) on November 3, 2021. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing. The strain accession number is CGMCC NO.23715. DETAILED DESCRIPTION

[0032] Example 1: Screening of α-tocopherol producing strains

[0033] 1 Materials and methods

[0034] 1.1 Strain origin and characteristics

[0035] 48 strains were isolated from silage, and the α-tocopherol content was determined. According to the concentration of α-tocopherol produced, the tekila Bacillus QH1 with the highest α-tocopherol content and the fastest growth was screened. The results showed that the QH1 strain was a Gram-positive bacillus with a strong ability to produce α-tocopherol, could grow at 50°C and low pH = 4.5, had strong high temperature resistance, and could ferment most sugars (Table 1).

[0036] Table 1 Characteristics of strain QH1

[0037]

[0038]

[0039]

[0040] -: no growth; ±: weak growth; +: growth; ++: good growth.

[0041] 1.2 Physiological and biochemical characteristics of α-tocopherol-producing strain QH1 and medium preparation method:

[0042] 1) Gram staining, fermentation type and shape observation refer to the Manual of Identification of Common Bacterial Systems edited by Dong Xiuzhu.

[0043] 2) The pH of the growth of Bacillus tequila was adjusted using 0.1 mol / L NaOH and 0.1 mol / L HCl.

[0044] 3) Sugar fermentation was analyzed using API 50 CHL (bioMérieux, l'Etoile, France).

[0045] 4) Culture medium, RCM culture medium (for purification and culture of strain QH1): Proteose peptone NO.3 10.0 g; Beef extract 10.0 g; Yeast extract 3.0 g; Dextrose 5.0 g; Soluble starch 1.0 g; Sodium acetate (NaAc) 3.0 g; Sodium chloride (NaCl) 5.0 g; L-Cysteine ​​hydrochloride 0.5 g. Dissolve the above reagents and make up to 1000 mL with distilled water. Add 15 g / L agar to the solid culture medium and sterilize at 121°C for 15 min. Bars culture medium (for culture of strain QH1).

[0046] Dissolve the above reagents, make up to 1000 mL with distilled water, divide into 15 mL test tubes, 10 mL per test tube, and sterilize at 121°C for 15 min.

[0047] 1.3 Preparation of QH1 bacterial culture

[0048] Pick two colonies of Bacillus tergrillii QH1 and add them to 5 ml of sterilized fortified Clostridium liquid medium. Mix well and culture anaerobically at 37 °C for 48 h to prepare seed QH1 bacterial preparation. Make the bacterial count reach at least 1.0 × 10 11 Pieces / mL.

[0049] 1.4 Method for measuring α-tocopherol content in bacterial culture

[0050] Assay reagents: ① Color developer: phenanthroline (C29 H 13 ②FeCl3 ethanol solution: 400mg of FeCl3·6H2O is dissolved in 1000mL of anhydrous ethanol and diluted to 0.4mg / mL. ③α-Tocopherol standard solution: Weigh 10mg of α-tocopherol and dissolve it in 100mL of anhydrous ethanol as α-tocopherol mother solution, with a concentration of 100mg / L. The concentrations of the α-tocopherol standard curve are: 0, 2.5, 5.0, 10, 20, 40, 60mg / L (2.5-60mg / L is a linear relationship) Note: The above concentrations are obtained by diluting the mother solution, of which 0mg / L is the blank control group. ④Ethanol phosphoric acid solution: 6.23mL of concentrated phosphoric acid is dissolved in anhydrous ethanol to 100mL and stored in a brown bottle. ⑤Extracting solution: Anhydrous ethanol: n-heptane = 6:10 mixed and stored in a brown bottle. ⑥Lysozyme: 20 mg / mL lysozyme.

[0051] Determination steps: Take 0.4mL of Bars culture medium, centrifuge, transfer the supernatant to a new centrifuge tube for later use. Add 100uL of lysozyme to the bacteria at the bottom, shake well, and treat in a 37℃ metal bath for 60 minutes. Add the reserved supernatant to the centrifuge tube treated with lysozyme and containing bacteria, mix well, centrifuge at 10000 rpm for 2 minutes at 4℃, and keep the supernatant for later use. ⑧ Take 0.4mL of sample supernatant and place it in a centrifuge tube, add 1.6mL of extract. After mixing with a vortex mixer for 5 minutes, centrifuge at 1500 rpm for 10 minutes at room temperature, and take 0.7mL of supernatant to a new 2mL centrifuge tube. Add 0.1mL of color developer and 0.05mL of FeCl3 ethanol solution, and shake to mix. After color development at room temperature for 5 minutes, add 1.0 mL of ethanolic phosphoric acid to obtain a reaction solution, take 200 uL of the reaction solution and add it to the ELISA plate, colorimetric at a wavelength of 560 nm, and record the data. Deduct the blank control data, and make a standard curve with concentration as the horizontal axis and absorbance as the vertical axis. According to the standard curve and the absorbance of the sample, calculate the α-tocopherol concentration in the bacterial solution sample.

[0052] Excel was used to organize the data, and SPSS 21.0 statistical software was used for one-way analysis of variance. Tukey's multiple comparisons were used to analyze the differences between the mean values ​​of storage time and vacuum extraction time, P < 0.05.

[0053] 2 Results Analysis

[0054] The α-tocopherol production of the α-tocopherol-producing QH1 strain in Bars medium (56.61 mg / L) was significantly higher than that of Enterococcus gallinarum (12.55 mg / L), Lactococcus lactis (20.56 mg / L) and Lactobacillus plantarum (0 mg / L) ( Figure 1 ).

[0055] Example 2: Identification of α-tocopherol producing strains

[0056] 1 Materials and methods

[0057] The strain was cultured anaerobically at 37°C in RCM liquid medium for 48 hours. The bacterial solution was transferred to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm / min for 5 min to collect the bacteria, and then DNA was extracted using the TakaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver. 3.0 (Baori Physician Technology CO., LTD, Beijing, China). The DNA concentration of the extracted genomic DNA was measured by an ELISA instrument to determine whether it met the DNA extraction standard. Then, PCR amplification was performed, and the amplification primers of 16S rDNA were 25f (5`-AAC TGA AGA GTT TGA TCC TGG CTC-3`) and 1492r (5`-TAC GGC TAC CTT GTT ACG ACT-3`), and the PCR reaction was 95℃ (5min)-94℃ (30s)-55℃ (1min)-72℃ (1.5min)-72℃ (10min), wherein the reaction cycle of 94℃ (30s)-55℃ (1min)-72℃ (1.5min) was 30 times. The amplified product was sent to Boshang Biotechnology Co., Ltd. (China) for sequencing (Table 1), and the results were compared with the gene library of NCBI to find the standard strain Bacillus tequilensis that was closely related to this bacterium, and analyzed with DNAman software, and the partial sequence of 16S rDNA of the screened strain (SEQ ID NO.1) was analyzed for similarity with the standard bacteria.

[0058] 2. Results Analysis

[0059] In comparison with Genbank, the 16S rDNA sequence of QH1 was 100% similar to that of Bacillus tequilensis and was assigned to Bacillus tequilensis ( Figure 2 ).

[0060] Example 3: Effect of adding to silage

[0061] 1. Materials and Methods

[0062] The rice straw at the mature stage was used as the material for silage test. The rice straw was cut into 1-2 cm, 200 g of rice was weighed into a plastic basin, and the bacterial solution of strain QH1 cultured at 37°C for 48 hours was added. After mixing, it was filled into a polyethylene bag (20×25 cm), and vacuumed for 14 seconds with a vacuum sealer and then sealed. It was placed in a room temperature environment and kept away from light. On the 42nd day after silage, the pit was opened, and samples were taken to analyze the fermentation quality, and the fermentation quality and nutritional quality related indicators and α-tocopherol content were determined.

[0063] Nutritional composition and microbial analysis of fresh materials: Dry matter content was determined by 70℃ drying method, crude protein content was determined by Kjeldahl nitrogen determination method (nitrogen determination instrument KDN-103F, Shanghai Fiber Inspection Instrument Co., Ltd.), crude fiber and neutral detergent fiber content were determined by filter bag method. Soluble carbohydrate content was determined by anthrone-sulfuric acid method, buffering energy was determined by hydrochloric acid and sodium hydroxide titration method, lactic acid bacteria, bacteria and yeast were counted by MRS (de-Man Rogosa Sharpe) agar medium, nutrient agar medium and potato dextrose agar medium, respectively. Lactic acid bacteria were cultured in an anaerobic chamber at 37℃ for 48h; bacteria and yeast were cultured in a biochemical incubator at 30℃ for 48h.

[0064] Silage fermentation quality analysis: After the silage bag is opened, take 20g of mixed silage and put it into a polyethylene plastic sealable bag, add 80mL of distilled water, soak at 4℃ for 18h, filter, and measure the pH value of the extract with a pH meter (PHS-3B, Shanghai Pengshun Scientific Instrument Co., Ltd.). The number of lactic acid bacteria, bacteria and yeast was determined in the same way as the fresh sample, and the organic acid content was determined by Agilent high performance liquid chromatography: Chromatographic conditions: chromatographic column (KC-811), mobile phase was 3mmol / L HClO4 solution, flow rate was 1mL / min, column temperature was 60℃, and detection wavelength was 210nm.

[0065] α-Tocopherol content detection method: Weigh 3g of fresh sample, freeze it with liquid nitrogen and grind the sample. Place all samples in a conical flask, add 17.5mL of anhydrous ethanol, 7.5mL of methanol, 7.5mL of 10% vitamin C, and 5mL of 50% KOH solution in sequence, and then bathe in a water bath at 80°C in the dark for 30 minutes. After cooling to room temperature, pour all into a 50mL centrifuge tube, add 3mL of heptane and 5mL of distilled water, shake well, and centrifuge at room temperature for 5 minutes at 3500 rpm. Cover the lid tightly again and shake up and down, and centrifuge at room temperature for 5 minutes at 3500 rpm again. Pipette 1mL of supernatant into a brown sample bottle for analysis and detection. The α-tocopherol content is determined by high performance liquid chromatography. Chromatographic conditions: Agilent high performance liquid chromatograph; chromatographic column, InersilODS-4; mobile phase, 90% methanol + 10% acetonitrile; flow rate, 2 mL·min-1; column temperature, 45°C; detector, UV-visible detector; detection wavelength of α-tocopherol, 280 nm.

[0066] 1. Results Analysis

[0067] 2.1 Nutritional value of rice straw before ensiling

[0068] As shown in Table 2, rice straw has low contents of α-tocopherol and crude protein, but high contents of neutral detergent fiber and acid detergent fiber, and is a low-grade roughage.

[0069] Table 2 Chemical and microbial components of rice straw before ensiling

[0070]

[0071]

[0072] DM: dry matter; FM: fresh matter; lg: data logarithm to base 10; cfu / g: colony forming unit.

[0073] 2.2 Rice straw silage fermentation quality and α-tocopherol content

[0074] As shown in Table 3, the pH, butyric acid and ammonia nitrogen content of the rice straw silage with the addition of strain QH1 were significantly lower than those of the control group without addition (P<0.05), while the lactic acid content was significantly higher than that of the control group (P<0.05), indicating that the addition of α-tocopherol-producing strain QH1 can significantly improve the fermentation quality of rice straw silage. Compared with the rice straw raw material before ensiling (7.90 mg / kg DM), the α-tocopherol content in the rice straw silage inoculated with the QH1 strain increased by about 7 times (49.71 mg / kg DM) after 77 days of ensiling, which was about 2 times higher than the α-tocopherol content in the control group (25.09 mg / kg DM) ( Figure 3 ).

[0075] Table 3 Effect of inoculation with QH1 strain on the fermentation quality of rice straw silage

[0076]

[0077] Note: *, significant difference, P<0.05; **, extremely significant difference, P<0.01; N, nitrogen; NS, no significant difference, P>0.05. QH1 strain, Bacillus tequilensis QH1.

[0078] Example 4: Fermentation effect of adding to piglet feed

[0079] 1. Materials and Methods

[0080] Piglet feed was used as the fermentation raw material and mixed with water at a ratio of 10:3.3. Lactobacillus plantarum came from our laboratory and has been published in the literature (QH, Liu, T, et al. The effect of fibrolytic enzyme, Lactobacillus plantarum and two food antioxidants on the fermentation quality, alpha-tocopherol and beta-carotene of high moisture napier grasssilage ensiled at different temperatures [J]. Animal Feed Science & Technology, 2016.1-11). Weigh about 200 grams in a plastic basin and inoculate 1×10 6 cfu / g FM, or its combination with QH1 strain (the number of bacteria reached 1×10 6 cfu / g FM), loaded into a 250mL fermenter, fermented at 37℃ for 7 days, and then opened. Samples were taken to analyze the fermentation quality, and the fermentation quality and nutritional quality-related indicators as well as the α-tocopherol content were determined.

[0081] Analysis of nutritional components of piglet feed before fermentation: dry matter (DM) content was determined by 70℃ drying method, crude protein content was determined by Kjeldahl nitrogen determination method, crude fat content was determined by residual method, crude ash content was determined by burning method, calcium and phosphorus content were determined by EDTA complexometric titration and spectrophotometry, soluble carbohydrate content was determined by anthrone-sulfuric acid method, ammonia nitrogen (NH3-N) content was determined by direct distillation of Kjeldahl nitrogen analyzer (Hangzhou Top QSY-II), buffer energy was determined by hydrochloric acid and sodium hydroxide titration, crude fiber and neutral detergent fiber (NDF) content were determined by filter bag method, lactic acid bacteria, aerobic bacteria, yeast and mold were counted by MRS agar medium, nutrient agar medium and potato dextrose agar medium, respectively. Lactic acid bacteria were cultured in an anaerobic box (YQX-Ⅱ type) at 37℃ for 48h; aerobic bacteria, yeast and mold were cultured at 37℃ under aerobic conditions for 24h, 48h and 72h, respectively.

[0082] Silage fermentation quality analysis: After the silage bag was opened, 20 g of mixed silage was put into a polyethylene plastic sealed bag, 80 mL of distilled water was added, and the extract was filtered after soaking at 4 °C for 18 h. The pH value of the extract was measured with a pH meter (PHS-3B, Shanghai Pengshun Scientific Instrument Co., Ltd.). The number of lactic acid bacteria, bacteria and yeast was determined in the same way as the fresh sample. The organic acid content was determined by Agilent high performance liquid chromatography. The chromatographic conditions were: chromatographic column (KC-811), mobile phase was 3 mmol / L HClO4 solution, flow rate was 1 mL / min, column temperature was 60 °C, and detection wavelength was 210 nm.

[0083] α-Tocopherol content detection method: Weigh 3g of fresh sample, freeze it with liquid nitrogen and grind the sample. Place all the samples in a conical flask, add 17.5mL of anhydrous ethanol, 7.5mL of methanol, 7.5mL of 10% vitamin C, and 5mL of 50% KOH solution in sequence, and then water bath at 80℃ in the dark for 30 minutes. After cooling to room temperature, pour all into a 50mL centrifuge tube, add 3mL of heptane and 5mL of distilled water, shake well, and centrifuge at room temperature for 5 minutes at 3500 rpm. Cover the lid tightly again and shake up and down, and centrifuge at room temperature for 5 minutes at 3500 rpm again. Pipette 1mL of supernatant into a brown sample bottle for analysis and detection. The α-tocopherol content is determined by high performance liquid chromatography. Chromatographic conditions: Agilent high performance liquid chromatograph; chromatographic column, InersilODS-4; mobile phase, 90% methanol + 10% acetonitrile; flow rate, 2mL·min -1 ; Column temperature, 45°C; Detector, UV-visible detector; Detection wavelength of α-tocopherol, 280nm.

[0084] 2. Results Analysis

[0085] 2.1 Characteristics of piglet feed before fermentation

[0086] As shown in Table 4, a certain amount of premix was used in the piglet feed, which contained a certain amount of α-tocopherol (Table 5). The crude protein of the piglet feed before fermentation was moderate, the content of α-tocopherol was low, and the number of aerobic bacteria and fungi was greater than that of lactic acid bacteria.

[0087] Table 4 Piglet feed formula

[0088]

[0089] Table 5 Chemical and microbial composition of pig feed before fermentation

[0090]

[0091]

[0092] Note: FW, fresh weight; FM, fresh matter; DM, dry matter; cfu, colony forming unit.

[0093] 2.2 Piglet feed fermentation quality

[0094] The pH of all fermented piglet feeds was not lower than 4.8, indicating that the acidity was moderate. The lactic acid and acetic acid contents of the inoculated Lactobacillus plantarum and its combination with QH1 strain were lower than those of the control group (P<0.05), and the α-tocopherol content was significantly higher than that of the control group (P<0.05). The α-tocopherol content of the combination of Lactobacillus plantarum and QH1 strain was significantly higher than that of the Lactobacillus plantarum strain (P<0.05), so QH1 helps to further increase the tocopherol content of fermented piglet feed. Compared with the control group and the raw material before fermentation, the combination of Lactobacillus plantarum and QH1 strain increased α-tocopherol by about 229% and 166%, respectively. The ammonia nitrogen content of the combination of Lactobacillus plantarum and QH1 strain was lower than that of the control group (P<0.05), indicating less protein decomposition. In summary, the combination of Lactobacillus plantarum and QH1 strain has the effect of increasing α-tocopherol (Table 6).

[0095] Table 6. Effects of Lactobacillus plantarum and its combination with QH1 strain on fermentation quality and α-tocopherol content of pig feed 7 days after inoculation

[0096]

[0097]

[0098] Note: FW, fresh weight; FM, fresh matter; DM, dry matter; cfu, colony forming unit; different letters in the same row indicate significant differences, P<0.05; *, significant difference, P<0.05; **, extremely significant difference, P<0.01; N, nitrogen; NS, no significant difference, P>0.05. QH1 strain, Bacillus tequilensis QH1. Sequence Listing <110> Nanjing Agricultural University <120> Bacillus tequila QH1 producing α-tocopherol and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1420 <212> DNA <213> Bacillus tequilensis <400> 1 tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60 cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120 tggttgtttg aaccgcatgg ttcaaacata aaaggtggct tcggctacca cttacagatg 180 gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcaac gatgcgtagc 240 cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300 gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360 atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtacc gttcgaatag 420 ggcggtacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480 gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540 ttcttaagtc tgatgtgaaa gccccggct caaccgggga gggtcattgg aaactgggga 600 acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660 ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720 cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780 gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840 agtacggtg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900 atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960 cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080 cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140 gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200 aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260 cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320 ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380 ccgaagtcgg tgaggtaacc tttaggagcc agccgccgaa 1420

Claims

1. A strain of Bacillus tequila ( Bacillus tequilensis ) QH1, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 3, 2021, and the culture collection number is CGMCC NO. 23715.

2. The Bacillus tequila of claim 1 ( Bacillus tequilensis ) The bacterial agent prepared by QH1.

3. The bacterial agent according to claim 2, characterized in that The preparation method is as follows: 1 to 3 colonies of Bacillus tergrillii QH1 were picked and added to 5 ml of sterilized fortified Clostridium liquid culture medium, mixed and cultured anaerobically at 30 to 40 °C for 36 to 72 h to prepare a seed QH1 bacterial preparation, so that the bacterial count reached at least 1.0 × 10 11 Pieces / mL.

4. The Bacillus tergiornicotianae of claim 1 ( Bacillus tequilensis )Application of QH1 in the synthesis of α-tocopherol.

5. The Bacillus tergiornicotianae of claim 1 ( Bacillus tequilensis ) Application of QH1 in preparing silage and / or fermented piglet feed.

6. Use of the bacterial agent according to claim 2 in preparing silage and / or fermented piglet feed.

7. A method for preparing rice straw silage, characterized by comprising the following steps: ① Cut the rice straw to be ensiled into 1-2 cm pieces and mix them evenly; ② Add 1.0×10 Teguciella QH1 described in claim 1 to each gram of rice straw. 6 ③ The feed with strain QH1 added is sealed and stored anaerobically at room temperature for more than 30 days.

8. The method according to claim 7, characterized in that The feed to which strain QH1 was added was sealed and stored anaerobically at room temperature for 42 days.

9. The method according to claim 7, characterized in that Step ②: add the bacterial agent of claim 2, so that the concentration of Bacillus tergilacinus QH1 of claim 1 in each gram of rice straw is 1.0×10 6 indivual.

10. A method for fermenting piglet feed, characterized in that The method comprises the following steps: ① taking piglet feed as fermentation raw material, mixing it with water at a ratio of 10: (3-3.5) of fresh weight; ② inoculating Lactobacillus plantarum ( Lactobacillus plantarum ) and the QH1 strain described in claim 1, respectively, the number of which reaches 1×10 6 cfu / g FM, mix well and put into the fermentation tank; ③ Ferment at 37℃ for 5-10 days.

11. The method according to claim 10, characterized in that Step ② Combined inoculation of Lactobacillus plantarum and the QH1 bacterial agent of claim 2, so that both bacteria reach 1×10 6 cfu / g FM or more.

12. The method according to claim 10, characterized in that Step ③ Ferment for 7 days.

Images