A kind of Bacillus Velez and its application in wine lees fermentation and recycling

Abstract

The present invention discloses a Bacillus velezensis and its application in fermentation and recycling of winemaking byproducts-white wine lees. The classification name of the Bacillus velezensis is Bacillus velezensis, which has been sent to the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee for preservation, with a preservation date of June 27, 2022 and a preservation number of CGMCC No. 25202. The Bacillus velezensis strain provided by the present invention can efficiently grow in white wine lees, and can increase the crude protein content, reduce the crude fiber content, and improve the nutritional value of white wine lees.

Description

Technical Field

[0001] The invention belongs to the technical field of high-value utilization of liquor brewing waste, and specifically relates to a Bacillus velez and application thereof in the fermentation and reuse of vinasse. Background Art

[0002] China is a major producer and consumer of liquor. With the development of China's liquor industry, the output of liquor lees, a byproduct of liquor brewing, has also increased, reaching nearly 20 million tons. As the main byproduct of the brewing industry, lees have complex nutritional components and are difficult to store. At present, most companies deal with lees by simply drying them and then selling them directly as cattle feed or fertilizer, with extremely low added value.

[0003] At present, one of the effective ways to utilize distiller's grains is to use microbial fermentation to treat distiller's grains. However, there are few bacterial agents specifically used for distiller's grains at this stage, and distiller's grains have adverse conditions such as high ester, high alcohol, and high acid. Most of the bacteria that can be used as feed are not suitable for growing in distiller's grains. In addition, a certain proportion of husks are mixed in the process of liquor production and brewing, resulting in a high content of crude fiber in the distiller's grains. Due to its dense structure, it is difficult for animals to digest and has low nutritional value, which limits its application in monogastric animals (pigs).

[0004] To this end, the present invention provides a strain and a method for culturing the strain in white wine lees, which can grow well in white wine lees and has the effects of significantly increasing crude protein and reducing crude fiber. Summary of the invention

[0005] The present invention aims to solve the above-mentioned deficiencies in the prior art. The first purpose is to provide a Bacillus Velezii and its application in the fermentation and recycling of wine lees. The second purpose is to provide optimal fermentation conditions.

[0006] The present invention adopts a plate streaking method, uses an inoculation loop to dip different feed products, performs three-zone streaking on an LB solid culture medium, places the culture medium in a 30-40°C constant temperature incubator and cultures it for 36-48 hours; 40 strains separated are further streaked and cultured, and pure strains are separated and purified.

[0007] The strain was applied to distiller's grains fermentation. First, the growth performance of the strain was verified in distiller's grains fermentation. Then, the crude protein and crude fiber contents in the distiller's grains before and after fermentation were measured. It was found that Bacillus Velezii could grow efficiently in white wine distiller's grains and could reduce crude fiber and increase crude protein content.

[0008] The Bacillus velezensis provided by the present invention is classified and named Bacillus velezensis, and has been sent to the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration for preservation. The preservation date is June 27, 2022, and the preservation number is CGMCC No.25202.

[0009] The present invention also provides an application of Bacillus Velez, which is to use the strain for distiller's grains fermentation and reuse. Specifically, the following steps are included: the Bacillus Velez strain is cultured in a test tube slant (cultured at a constant temperature of 30°C for 3 days) and stored in a 4°C refrigerator; the culture solution of the strain obtained by the slant culture is placed in a 30-40°C constant temperature shaker and cultured at 180-300r / min for 24-36h; then inoculated into a sterilized distiller's grains culture medium and cultured and fermented in a 20-30°C constant temperature incubator.

[0010] The wine lees culture medium is composed of: 10-15 g of dry wine lees, 10-30 mL of pure water, and the pH is adjusted to 7.0-7.5. The wine lees culture medium is sterilized by high-pressure steam sterilization at 121° C. for 20 minutes.

[0011] Furthermore, the present invention optimizes the application of the screened and cultured Bacillus Velezii, and the optimal inoculation amount of the strain is 6%-10%, the optimal pH is 6-9, the best effect is 3-6 flips, and the optimal fermentation time is 48-72h.

[0012] The crude fiber and crude protein of the wine lees before and after fermentation were determined. It was found that the crude fiber content was further reduced and the crude protein content was increased before and after the optimization fermentation.

[0013] The beneficial effects of the present invention are embodied in:

[0014] 1. The combined application of this strain and the fermentation optimization process can significantly reduce the crude fiber of distiller's grains, increase the protein content, and improve the bioavailability;

[0015] 2. This fermentation strain is a strain allowed to be added to feed and is safe and non-toxic;

[0016] 3. Liquor lees is a by-product of winemaking. This fermentation method can increase its added value and improve its economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a photo of a plate of Bacillus Velez strain.

[0018] Figure 2 Data submitted to NCBI for Bacillus velez (strain accession number ON680836), as well as NCBI sequencing alignment results.

[0019] Figure 3The effect of different pH values ​​of the fermentation system on the crude fiber and crude protein content (%).

[0020] Figure 4 The effect of different turnover times during fermentation on crude fiber and crude protein content (%).

[0021] Figure 5 The effect of different inoculation amounts on crude protein and crude fiber content (%).

[0022] Figure 6 The effect of different fermentation time on crude protein and crude fiber content (%). DETAILED DESCRIPTION

[0023] The dry distiller's grains used in the examples were purchased from Anhui Gujing Gongjiu Co., Ltd.

[0024] Embodiment 1:

[0025] 1. By streaking on a plate, take a small amount of different feed products (Nanhua Qianmu straw fermentation agent, Huaxu fermentation source bacteria, fermented feed products of Anhui Zhengdayuan Feed Group, etc.) with an inoculation loop, and make three-zone streaking on LB solid culture medium (composed of: fish meal peptone 10g / L, yeast extract (Yeast extract) 5g / L, sodium chloride (NaCl) 10g / L, agar powder 25g / L, natural pH), and culture in a 30℃ constant temperature incubator for 36h. Different strains are streaked and cultured again using the same method, and the separated pure strains are cultured in test tube slant culture (constant temperature culture at 30℃ for 3 days), and stored in a 4℃ refrigerator.

[0026] 2. The 40 different strains purified in the previous step were cultured in LB liquid medium (fish meal peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, natural pH.) and inoculated. They were placed in a 30℃ constant temperature shaker and cultured at 180r / min for one day. 1mL of fermentation liquid was pipetted into lees medium (lees medium: dry lees 10g, pure water 20mL, ammonia water adjusted to pH 7.0, 121℃, 20min high pressure steam sterilization), and cultured at 30℃ for 3 days. Using the dilution coating method, take 1000μL of the original fermentation liquid and dilute it to 10 of the original -6 times, spread on LB medium plate; take 1000 mg of wine lees medium after 3 days of fermentation, dilute to 10% of the original -6 The above culture medium was placed at 30°C for one and a half days, and then the number of colonies was counted. The results showed that 11 strains (see Table 1, strain numbers 1-11) could grow normally in the wine lees medium.

[0027] Embodiment 2:

[0028] The 11 strains screened out were inoculated into LB liquid culture medium in sequence, cultured in a constant temperature shaker at 30°C and 180r / min for 24h, and then 1mL of fermentation liquid was inoculated into lees culture medium (lees culture medium: 10g dry lees, 20mL pure water, pH adjusted to 7.0 with ammonia water, sterilized by high pressure steam at 121°C and 20min), and then 1mL of bacterial fermentation liquid was inoculated into lees culture medium as a blank control. All the above culture media were cultured at a constant temperature of 30°C for 72h.

[0029] Dry and grind the wine lees culture medium, weigh 0.1g sample in a 250mL conical flask, pour 25mL water and petroleum ether respectively, let it stand for degreasing for 3 hours, filter it, and weigh it after drying. Put the sample into a 10mL centrifuge tube after drying, add 5mL mixed acid, and boil it in a boiling water bath for 25 minutes. Then centrifuge it at 3500r / min for 10 minutes and discard the supernatant. Then add 10mL distilled water to the precipitate and mix it. Centrifuge it for 10 minutes in the same way, discard the supernatant, and repeat this three times. Use a pipette to transfer 10mL 0.5mol sulfuric acid-potassium dichromate solution into the centrifuge tube of the precipitate. Hydrolyze it in a boiling water bath for 10 minutes. Cool it slightly after taking it out. Transfer the solution to a 150mL triangular flask. At the same time, wash the centrifuge tube with 15mL distilled water. Add 2 to 3 drops of phenanthroline indicator to the triangular flask with the washing solution, and titrate it with 0.1mol / L ferrous ammonium sulfate solution. The reaction end point is when the solution color changes from green to brownish red. This is used to calculate the crude fiber content.

[0030] Weigh 0.5g of sample, 0.2g of copper sulfate, 0.5g of potassium sulfate and 10ml of sulfuric acid, place in a digestion tube, heat with an electric furnace, keep the bottle slightly boiling, and continue heating for half an hour when the liquid in the bottle turns clear and transparent grass green. After cooling, add an appropriate amount of distilled water, shake well, cool to room temperature, transfer the solution to a 100ml volumetric flask, add pure water to the 100ml mark, shake well and set aside. Take 0.5ml of digestion solution, add 3.5ml of Nessler's reagent to a colorimetric tube, add water to the 25ml mark; take another colorimetric tube, add 0.2ml of 50% sulfuric acid solution, 3.5ml of Nessler's reagent, dilute to 25ml, and use this as a blank. Measure the absorbance at 400nm on a spectrophotometer. Calculate the crude protein content in this way.

[0031] After the determination, the results are shown in Table 2 and Table 3. After the determination, it was found that the crude fiber content of strain No. 10 (with higher crude protein and lower crude fiber, good overall performance) before and after fermentation was reduced by an average of 34.97%, and the crude protein content was increased by an average of 126.29%.

[0032] Embodiment 3:

[0033] After extracting the DNA of strain number 10, PCR amplification was performed: sequence amplification was performed using universal primers:

[0034] Gene amplification was performed using bacterial universal primers.

[0035] The primer sequences were upstream primer (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′);

[0036] Downstream primer (1492R: 5'-GGTTACCTTGTTACGACTT-3').

[0037] The 20 μl PCR reaction system includes: 2.0 μl 10×Ex Taq buffer, 0.2 μl 5U Ex Taq, 1.6 μl 2.5 mM dNTP Mix, 1 μl primer 1 / primer 2 (sequence 1 and sequence 2), 0.5 μl DNA template, and 13.7 μl ddH2O.

[0038] The amplification conditions were: 95°C pre-denaturation for 5 min, entering the cycle, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1.5 min, 35 cycles, and finally 72°C extension for 1.5 min. The obtained product was purified with a kit, cloned into a T vector, and positive colonies were identified for sequencing.

[0039] The amplified products were sequenced using the first-generation sequencing platform 3730. Generally, the sequence quality at both ends of the sequencing was poor. The low-quality sequences at both ends were removed by quality shearing, and the double-end sequencing results after quality control were assembled to obtain the 16SrRNA sequence.

[0040] NCBI database comparison to confirm species: Use the assembled sequence of the sample to compare with the database, and determine the species of the sample based on the coverage and similarity of the comparison results. In general, the one with the highest comparison score is selected and identified as Bacillus velezensis.

[0041] Embodiment 4:

[0042] Bacillus Velez was inoculated in LB medium and cultured in a shaking table at 36°C and 220r / min for 2 days. The dried distiller's grains were divided into 250ml triangular bottles, about 10g in each triangular bottle, water was added to keep it in a semi-wet and semi-dry state, the pH was adjusted to neutral with diluted ammonia water, and the bottle was sealed with sealing film and newspaper to prevent the volatilization of ammonia water, and sterilized at 121°C for 30 minutes. The seed liquid after shaking the bottle was inoculated into the sterilized distiller's grains culture medium, and cultured at 30°C for 2 days for distiller's grains fermentation. The fermented distiller's grains were dried, and about 0.1000-0.2000g of the sample that passed the 60-mesh sample sieve was weighed to make the nitrogen content between 30mg and 50mg, and transferred into the digestive tube, and 0.2g of copper sulfate, 0.5g of potassium sulfate and 10ml of sulfuric acid were added, and heated in an electric furnace for about 60 minutes until the boiling liquid turned into grass green clarification, and then heated for half an hour, the volume was fixed to 100ml, and cooled for use. The control and sample tubes were prepared in the same manner as above. 0.5 ml of digestion solution was added to the sample tubes and allowed to stand for 15 minutes. The absorbance of each tube was read at 400 nm to calculate the crude protein content.

[0043] Then, the crude fiber is determined. The sample is ground, passed through a 100-mesh sample sieve, degreased with petroleum ether, and dried. Weigh about 0.0500g of the sample, put it into a 10ml tube, and add about 5ml of the prepared acid. Place it in a boiling water bath for 25min, shake and stir continuously, and then centrifuge it at 3500-4000r / min for 10min, discard the supernatant, then add 10ml of distilled water, centrifuge it in the same way for 10min, and repeat this 3 times. Use a pipette to transfer 10ml of 0.5N sulfuric acid-potassium dichromate solution, heat it with warm water for 10min, let it stand for 10min, transfer the solution into a 250ml conical flask, wash it with 10ml of pure water, add 2-3 drops of indicator o-phenanthroline, and then titrate it with ammonium ferrous sulfate solution until the sudden change from green to brown-red is the end point, and calculate the crude fiber content.

[0044] This experiment used a single factor experiment to explore the fermentation time ( Figure 3 )、pH value( Figure 4 )、Number of flips( Figure 5 ) and inoculum size ( Figure 6 ) on the content of crude protein, crude fiber and other components in the fermentation products of white wine lees, and then the orthogonal experiment (Table 4, Table 5) and verification test (Table 6) were carried out to determine the optimal combination of fermentation conditions: fermentation time of 72h, initial pH value of 7-8, turnover number of 5 times, inoculation amount of 8%, that is, A3B3C1D2 combination. After the conditions were optimized, the crude fiber content of Bacillus Velez was reduced by 36.07% and the crude protein was increased by 126.60% compared with the white wine lees fermented before optimization.

[0045] Table 1 Colony counts of different strains before and after fermentation in liquor lees

[0046]

[0047]

[0048] Table 2 Crude fiber content of liquor lees before and after strain fermentation

[0049]

[0050] *The numbered bars in Table 2 are the same and are all determined according to the crude fiber method

[0051] Table 3 Crude protein content of white wine lees before and after strain fermentation

[0052]

[0053]

[0054] *The numbered bars in Table 3 are the same and are all determined according to the crude protein method

[0055] Table 4 Factors and levels design of orthogonal experiment

[0056]

[0057] Table 5 Orthogonal test results and analysis

[0058]

[0059] Table 6 Verification test

[0060]

Claims

1. A Bacillus Velezii ( Bacillus velezensis ), has been sent to the General Microbiology Center of China Microorganism Culture Collection Administration for preservation. The preservation date is June 27, 2022, and the preservation number is CGMCC No. 25202.

2. The use of the Bacillus Velezii according to claim 1, characterized in that: The Velez Bacillus is applied to wine lees fermentation. The strain can grow efficiently with white wine lees as the only carbon source and can reduce crude fiber and increase crude protein content. Specifically, the Velez Bacillus strain is cultured on a test tube slant, cultured at a constant temperature of 30°C for 3 days, and stored at 4°C; the strain culture solution obtained by the slant culture is placed in a constant temperature shaker at 30-40°C, and cultured at 180-300r / min for 24-36h; then inoculated into an inactivated wine lees culture medium, cultured and fermented in a constant temperature incubator at 20-30°C, the inoculation amount is 6%-10%, the system pH is 6-9, the culture and fermentation process is turned over 3-6 times, and the fermentation time is 48-72h; The wine lees culture medium is composed of 10-15 g of dry wine lees and 10-30 mL of pure water, and the pH is adjusted to 7.0-7.

5.

3. The use according to claim 2, characterized in that: The method of inactivating the wine lees culture medium is to sterilize it by high pressure steam at 121°C for 20 minutes.

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