Application of vinasse cellar bottom water-to-liquid feed

Abstract

The invention discloses application of a vinasse cellar bottom water-to-liquid feed. The vinasse cellar bottom water-to-liquid feed is used for culturing Wickerhamomyces anomalus FBKL2.00K8, and the preservation number of the Wickerhamomyces anomalus FBKL2.00K8 is CCTCC (China Center for Type Culture Collection) NO: M 2020249; the Wickerhamomyces anomalus has the functions of increasing the pH value of the vinasse cellar bottom water and reducing the total acid, and components in the vinasse cellar bottom water are converted. The method is used for research and development of liquid feed, a new thought is provided for cyclic utilization of the white spirit byproduct cellar bottom water, the water-free treatment cost of enterprises is expected to be reduced, and the method is of practical guiding significance for environmental protection and research and development of a novel feed technology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to screening out functional microorganisms that degrade lactic acid from wine lees bottom water, adjusting optimal fermentation parameters and applying the microorganisms to ferment in wine lees bottom water. Background Art

[0002] The water from the bottom of the wine lees cellar is a liquid by-product produced in the traditional liquor brewing process and is considered brewing wastewater.

[0003] The water from the bottom of the wine lees pit has the characteristics of high acidity, biochemical oxygen demand (BOD) and chemical oxygen demand (COD). In addition, the water from the bottom of the wine lees pit is rich in nutrients such as residual starch, protein, crude fat, amino acids and minerals. In the process of liquor fermentation and production, the output is large, the treatment cost is high and the process is cumbersome. If it is discharged directly, it will seriously pollute the water source and soil. In recent years, improving the utilization rate of organic matter in the bottom of the wine lees pit and increasing its added value have gradually become research hotspots. At the same time, the rational use of resource recycling is the general trend of "green development". This paper first analyzes the conventional physical and chemical indicators, nutritional components, toxic and harmful components, and microbial structure of the bottom of the wine lees pit to explore whether the bottom of the wine lees pit has nutritional value and safety as a liquid feed. Summary of the invention

[0004] Due to the high lactic acid content in the bottom water of the wine lees, the cellar mud, seven rounds of discarded lees and bottom water of the wine lees in the brewing environment of a certain Maotai-flavor liquor factory in Zunyi, Guizhou were selected as bacterial sources. Lactic acid was used as the only carbon source to screen functional microorganisms with the ability to degrade lactic acid. Through the cell morphology and molecular biology identification of the strains, combined with the liquid lactic acid fermentation of the strains and the co-fermentation test of lactic acid + glucose, the consumption rates of lactic acid and glucose were detected by high-performance liquid chromatography and microplate reader, respectively, to comprehensively screen functional microorganisms with the ability to degrade lactic acid. Finally, through the functional stress test of functional strains such as strain growth curve, pH, temperature, ethanol, etc., the selected functional microorganisms were inoculated into the bottom water of the wine lees for fermentation, and the optimal fermentation parameters of the strains were determined by fine-tuning.

[0005] We explored ways to adjust the distiller's grains bottom water to the intestinal pH range adapted to ruminants from the two aspects of microbial and edible alkali regulation. We observed the performance of the target strain and the degradation of lactic acid based on the pH, total acid and OD600 of the fermentation broth of the functional strain, in order to provide a reference plan for the research and development of distiller's grains bottom water into liquid feed.

[0006] The main findings are as follows:

[0007] 1. The microbial structure of the bottom water of the wine lees is complex, dominated by bacteria with many species, among which Firmicutes, Proteobacteria and Actinobacteriota are the dominant bacterial phyla, and the dominant genera are Limosilactobacillus, Wickerhamomyces and Streptococcus. There are only 4 phyla of fungi, 99% of which are Ascomcota. At the genus level, Zygosaccharomyces, Saccharomyces, Schizosaccharomyces and Kazachstania have the highest abundance.

[0008] 2. The bottom water and lees of a certain Maotai-flavor liquor enterprise in Guizhou were taken as samples. Using a screening medium with lactic acid as the carbon source, 16 single colonies that grew well and had the largest colonies on this medium were selected through separation and purification. Combined with liquid lactic acid fermentation and glucose-lactic acid co-fermentation experiments, a strain of efficient lactic acid degradation bacteria was screened out: abnormal Wickham yeast FBKL2.00K89, referred to as S9. Combined with the identification of cell morphology and molecular biology, it was preliminarily determined to be Wickerhamomyces anomalus.

[0009] 3. The strain function stress test showed that the bacteria grew well in lactic acid liquid culture medium, and could grow between 25 and 30℃, with the optimum growth temperature of 30℃, between pH3.5 and 6.5, with the optimum pH3.5, and could grow well below 6% ethanol concentration, with the optimum ethanol concentration of 1.5%. The application test of the strain in the bottom water of the wine lees showed that the functional microorganisms could not grow in the original solution, which was related to the high acidity and complex composition of the bottom water of the wine lees. Therefore, when the bottom water of the wine lees was diluted 8 times, the functional strain activation solution was inoculated into the bottom water of the wine lees and fermented for 7 days, and the pH could be adjusted from about 3.5 to 7-8.1. The pH of the bottom water of the wine lees was adjusted to different concentration gradients with edible alkali. The strain grew most vigorously at pH5.5-6.5, and the number of viable bacteria was as high as 1.55E+09-6.20E+09CFU / g. Lactic acid-degrading bacteria have the function of increasing the pH of the bottom water of the wine lees and reducing the total acid. Therefore, the selected lactic acid degradation functional strains are inoculated into the bottom water of the wine lees for fermentation, and the organic components in the bottom water of the wine lees are converted into nutrients required by ruminants. The fermentation liquid is added to the diet of ruminants, which not only realizes the recycling of the bottom water resources of the wine lees, but also reduces the proportion of concentrated feed and can increase the nutrient content of feed. Therefore, fermenting the bottom water of the wine lees with functional microorganisms and using the fermentation liquid as the raw material of liquid feed has application value and development prospects.

[0010] The bottom water and lees of a Maotai-flavor liquor company in Guizhou were selected as samples. Using a screening medium with lactic acid as the carbon source, 16 single colonies that grew well and had the largest colonies on this medium were selected through separation and purification. Combined with liquid lactic acid fermentation and glucose-lactic acid co-fermentation experiments, an efficient lactic acid degradation bacteria S9 was screened out, and combined with cell morphology and molecular biology identification, it was preliminarily determined to be Wickerhamomyces anomalus.

[0011] Its isolation and culture method and colony characteristics are as follows:

[0012] (1) Preliminary screening: Weigh 10.0 g of distiller's grains, 10.0 g of pit mud, and 10.0 mL of pit bottom water respectively into a 250 mL conical flask containing 90 mL of enrichment medium, place it in a shaker at 160 r / min and 30°C for 30 min, take it out and let it stand for 10 min to clarify, and prepare a bacterial suspension for use. Pipet 100 μL of the supernatant and transfer it into a centrifuge tube with 900 μL of sterile physiological saline for 10-fold concentration gradient dilution. -2 , 10 -3 , 10 -4 , 10 -5 , select 3 appropriate concentration gradients 10 -3 , 10 -4 , 10 -5 , set up 2 plates for each gradient, take 100 μL of dilution and inoculate it on Lu screening medium, spread it evenly with a glass coating rod, invert it in a 30℃ incubator and observe its growth after culturing for 3 days, select different single colonies that can grow on the screening medium, and carry out the next purification experiment;

[0013] (2) Rescreening: Observe the morphology of individual colonies on the screening culture medium plate with the naked eye, use an inoculation loop to pick different strains that grow well and continue to purify in the new screening culture medium Lu, streak the single colony at least three times, invert it in a 30°C constant temperature biochemical incubator and culture it for 3 days, and carefully observe whether the strain is purified. If two or more strains appear on the same plate, it means that the colony is not pure. It is necessary to purify repeatedly according to the above purification method until a single unknown strain appears. The purified single colony can be judged as a microorganism with the function of degrading lactic acid. Through purification, single colonies that grow well in the culture medium with lactic acid as the sole carbon source are screened out;

[0014] (3) Cultivation

[0015] ① Identification culture medium:

[0016] Lactic acid 4.2mL / L, peptone 10g / L, autoclave at 121℃ for 15min, sterilize at 115℃ for 20min;

[0017] ② Screening culture medium:

[0018] Lactic acid 4.2ml / L, Na2HPO 4 12H2O14.3 g / L, KH2PO 4 3g / L, (NH4)2SO 4 2 g / L, agar 20 g / L, autoclave at 121°C for 15 min. Sterilize at 115°C for 20 min;

[0019] (4) Preservation and subculture of bacterial strains:

[0020] ① Culture medium for bacterial strain preservation:

[0021] Glycerol and purified water were added into a triangular flask at a ratio of 1:1, mixed and shaken thoroughly, and then sealed with a breathable film. The flask was placed in a sterilizer and sterilized at 121°C for 15 minutes. The flask was taken out and cooled to room temperature for use. A single colony was picked up with an inoculation loop and placed in a liquid culture medium for amplification culture. When the growth of the microorganism entered the end of the logarithmic growth phase, 1 mL of activated bacterial solution and 1 mL of 25% glycerol sterile mixture were respectively drawn. Glycerol + water were added to the sterilized glycerol tube, mixed and shaken thoroughly, and placed in a -80°C ultra-low temperature refrigerator for freezing.

[0022] (5) Ecological characteristics of the strain:

[0023] The colonies of the six strains on the plates were small, all in milky white or grayish yellow, round or irregular circles. The cells were oval to rod-shaped, straight or slightly curved under a microscope, with a size of about 0.6-0.8μm×1.0-4.0μm. The cells could appear individually, in pairs or in chains. In addition, with the increase in strain passages or long storage time, the strains may also have degenerate bodies, which are in different rod-shaped, filamentous, curved and other forms. The cell morphology of a single bacterial colony was different.

[0024] (6) Lactic acid degradation and mixed carbon source fermentation characteristics

[0025] After conducting liquid fermentation tests on strains that grew well on the lactic acid screening medium, it was found that the lactic acid degradation rate was as high as 99%. In the presence of glucose, S9 was still able to degrade lactic acid better, with a degradation rate of 99.9%;

[0026] (7) Growth characteristics:

[0027] Temperature: 25-30℃, and the optimum growth temperature is 30℃

[0028] pH: pH 3.5 to 6.5, and the optimal pH for growth is 3.5

[0029] Ethanol: It can be grown at concentrations between 0% and 6%, and the optimum concentration for growth is 1.5%.

[0030] The results showed that when glucose coexists, S9 has a significant degradation effect, with a degradation rate of up to 99.9%. Molecular biological identification of this strain of microorganisms that degrade lactic acid well under aerobic conditions showed that the S9 strain is Wickerhamomyces anomalus FBKL2.00K8, with a homology of 100%. The use of microorganisms to transform the components of wine lees bottom water for the development of liquid feed provides a new idea for the recycling of wine lees bottom water, a byproduct of liquor, in order to reduce the cost of waterless treatment for enterprises, and has practical guiding significance for environmental protection and the development of new feed technology.

[0031] Wickerhamomyces anomalus FBKL2.00K8 has been deposited in the China Center for Type Culture Collection on June 30, 2020, with the accession number CCTCC No: M 2020249.

[0032] Instruction Manual

[0033] Figure 1 The screening result of the lactic acid liquid of the present invention;

[0034] Figure 2 It is the glucose utilization of the present invention;

[0035] Figure 3 The lactic acid utilization of the present invention;

[0036] Figure 4 This is the growth curve in the S9 nutrient broth of the present invention;

[0037] Figure 5 is the growth curve of S9 in Lu-Ye according to the present invention;

[0038] Figure 6 The cell morphology of the six bacterial strains described in the present invention;

[0039] Figure 7 It is the S9 temperature tolerance of the present invention;

[0040] Figure 8 It is the S9 pH tolerance of the present invention;

[0041] Fig. 9 This is the S9 ethanol tolerance of the present invention. DETAILED DESCRIPTION

[0042] Example 1

[0043] 1. Screening and identification of functional strains

[0044] This chapter will combine traditional isolation and culture methods with molecular biology techniques. Through morphological observation, physiological and biochemical characteristics, and metabolite detection of the strains, molecular biology techniques such as PCR and 18SrRNA gene sequencing will be used to quickly detect and determine the genus and species of the strains. The mechanism of lactic acid degradation will be further studied. Microorganisms with lactic acid degradation functions will be screened from the liquor brewing environment, and their application in the fermentation process of liquor cellar bottom water will be explored. This will provide a theoretical basis for the research and development of liquid feed conversion technology for distiller's grains cellar bottom water, and promote the further development and application of related technologies.

[0045] (1) Weigh 10.0 g of distiller's grains, 10.0 g of pit mud, and 10.0 mL of pit bottom water respectively into a 250 mL conical flask containing 90 mL of enrichment medium, place it in a shaker at 160 r / min and 30°C for 30 min, take it out and let it stand for 10 min to clarify, and prepare a bacterial suspension for use. Pipette 100 μL of the supernatant into a centrifuge tube containing 900 μL of sterile physiological saline and perform a 10-fold concentration gradient dilution for 10 -2 , 10 -3 , 10 -4 , 10 -5 , select 3 appropriate concentration gradients 10 -3 , 10 -4 , 10 -5 , set up 2 plates for each gradient, take 100 μL of dilution and inoculate it on Lu screening medium, spread it evenly with a glass coating rod, invert it in a 30℃ incubator and observe its growth after culturing for 3 days, select different single colonies that can grow on the screening medium, and carry out the next purification experiment;

[0046] (2) Visually observe the morphology of individual colonies on the screening medium plate, use an inoculation loop to pick out different strains that grow well and continue to purify in the new screening medium Lu, streak the single colony at least three times, invert it and culture it in a 30°C constant temperature biochemical incubator for 3 days, and carefully observe whether the strain is purified;

[0047] (3) Molecular biological identification of strains: DNA extraction: The S9 strain was inoculated into a conical flask containing 25 mL of lactic acid liquid culture medium, and cultured in a shaking incubator at 30°C and 160 r / min for 24 h. 1 mL of bacterial solution was taken and placed in a high-speed centrifuge for 1 min at 12,000 r / min. The supernatant was discarded and an appropriate amount of bacterial mud was taken for later use. The strain DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit, and then PCR amplification was performed. The PCR amplification product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for gene sequencing. The gene sequences of the obtained amplified fragments were spliced, and then the spliced ​​sequences were copied to the strain sequences on the NCBI website for BLAST homology comparison. The strain name with the highest homology in the gene library was selected as a reference.

[0048] 2. Lactic acid reduction test using the selected microorganisms

[0049] (1) Activation of bacterial strains: using lactic acid liquid medium Lu-Ye: lactic acid 4.2 ml / L, Na2 HPO4·12H2O14.3 g / L, KH2PO43 g / L, (NH4)2SO42 g / L, sterilized at 121℃ for 15 min;

[0050] (2) Seed solution preparation: Prepare a bottle of 200 ml lactic acid liquid culture medium, seal it, sterilize it at 121°C for 15 min, cool it, and then inoculate it with activated S9, and culture it in a shaking incubator at 30°C and 160 r / min for 24 h;

[0051] (3) Lactic acid degradation rate: 37 strains of bacteria separated and purified from wine lees, pit bottom water and pit mud were used to pick independent single colonies on solid screening culture medium with sterile inoculation loops, and then placed in a 30°C constant temperature biochemical incubator for inversion culture for 3 days. 16 strains of bacteria grew well in culture medium Lu with lactic acid as the only carbon source and had large colonies. Therefore, it can be further determined that they are functional microorganisms with lactic acid degradation. Therefore, these 16 strains of bacteria were selected for lactic acid liquid fermentation test. The lactic acid consumption of each strain fermentation broth on the 0th and 3rd day was detected by high performance liquid chromatography. The lactic acid degradation rates of the 16 strains of bacteria are shown in the attached figure;

[0052] (4) During the high-temperature sterilization process of the liquid culture medium, lactic acid may be decomposed and volatilized. Therefore, the sample should be sampled from 0 days after sterilization and inoculation. As shown in the attached figure, after culturing for 3 days under aerobic conditions, S8, S9, and S25 were screened from the bottom water of the cellar, and Z16, Z24, and Z27 were screened from the lees. The lactic acid consumption rates of S8, S9, S25 and Z16, Z24, and Z276 strains were all above 93%. Under anaerobic conditions, the lactic acid consumption rates of Z20, Z21, and Z24 were more advantageous than those of other strains. However, under aerobic conditions, their lactic acid consumption effects were not as good as those of S8, S9, and S25. The strains N3, N10, and N13 screened from the cellar mud had poor lactic acid degradation effects, and N19, N26, and N28 had almost no degradation effects.

[0053] (5) The above results show that the six strains of microorganisms, S8, S9, S25, Z24, and Z27, can utilize lactic acid well in both aerobic and anaerobic environments. Therefore, strains with good lactic acid degradation effects under aerobic conditions were selected to carry out fermentation experiments with the coexistence of glucose and lactic acid.

[0054] 3. Effect of glucose on lactic acid degradation

[0055] (1) Effect of glucose on lactic acid degradation: Under aerobic conditions, the glucose consumption rates of S8, S9, S25, Z16, Z24, and Z27 were 82.50%, 63.20%, 64.62%, 68.18%, 53.01%, and 39.07%, respectively, all of which were above 39%. Among them, the glucose consumption rate of S8 was higher than that of the other five strains. Under anaerobic conditions, the glucose consumption rates of the six strains were all above 47%.

[0056] (2) Among them, the glucose consumption rate of S9 was significantly higher than that of the other three strains. As shown in the attached figure, under aerobic conditions, the lactic acid consumption rates of S8, S9, S25, Z16, Z24, and Z27 were 99.37%, 99.98%, 99.98%, 99.98%, 88.92%, and 99.98%, respectively. Under the same culture conditions, the lactic acid consumption rate of Z24 was 11.06% lower than that of the other five strains. Under anaerobic conditions, only S8 and Z27 could utilize lactic acid, with consumption rates of 3.62% and 1.13%, respectively. The other four strains hardly utilized lactic acid. In summary, the six strains could also better degrade lactic acid when glucose and lactic acid were mixed. S9 was better than S8, Z26, and Z24 in the utilization of lactic acid in the presence of glucose.

[0057] (3) If the target strains have strong lactic acid utilization ability, they can convert glucose into lactic acid and further utilize other organic substances in the bottom water for metabolism. This can achieve the rational utilization of the bottom water resources of the wine lees and alleviate the pressure of environmental pollution. Therefore, when selecting lactic acid utilization strains, their metabolic ability for glucose and other organic substances can be considered. Strains with higher lactic acid production and good growth performance can be given priority. In addition, their effects on other by-products, such as ethanol and carbon dioxide, should also be considered to ensure that the fermentation process is stable and efficient. It should be noted that each strain has different metabolic characteristics and adapts to environmental conditions. Therefore, in actual production applications, screening and optimization of culture conditions should be carried out in combination with specific conditions to achieve the best fermentation effect.

[0058] Example 2

[0059] 1. Study on the functional characteristics of the four strains and the fermentation conditions in the bottom water of the wine lees

[0060] (1) Four strains of bacteria were inoculated at different temperatures, pH values, ethanol concentrations, and nutrients to explore the fermentation conditions of lactic acid-degrading bacteria in bottom water of the pit. These culture conditions are important factors affecting the microbial fermentation process. By adjusting these conditions, the growth and metabolic activity of the strains can be optimized, and the quality and yield of the fermentation products can be improved. This can improve the effect of microbial fermentation feed and meet the more refined regulation of the nutritional needs of mutton sheep.

[0061] (2) In order to improve the quality stability of microbial fermented feed, it is usually possible to control the fermentation temperature and optimize the selection of strains and inoculation amount. In addition, the growth of microorganisms in the bottom water of the cellar can be dynamically monitored through indicators such as the OD600 value and pH of the fermentation liquid using an ELISA instrument, so that the operation can be adjusted in time and the fermentation parameters of the liquid fermented feed can be ultimately determined.

[0062] 2. Growth trend of lactic acid degrading bacteria

[0063] (1) Growth of lactic acid-degrading bacteria in two culture media

[0064] A strain S9 with good lactic acid degradation effect screened out through lactic acid liquid fermentation was inoculated into lactic acid liquid culture medium for activation for 24 hours, and was inoculated into two culture mediums, namely lactic acid liquid culture medium Lu-Ye and nutrient broth culture medium, with an inoculation amount of 5%. Three parallels were set for each strain. In addition, in order to accurately observe the growth of the strain and whether there was contamination, blank groups of lactic acid liquid culture medium Lu-Ye and nutrient broth culture medium without inoculation of bacteria were set up. The groups were aerobically cultured on a shaker at 30°C and 160r / min. Samples were taken every 1 day to measure the OD600 value of the fermentation liquid to observe the growth status of the microorganisms.

[0065] 3. Stress test of lactic acid degrading bacteria

[0066] (1) Temperature tolerance test of lactic acid degrading bacteria

[0067] The activated liquid of S9 was inoculated into 25 mL of lactic acid liquid culture medium at a 5% inoculation rate, and placed in a constant temperature incubator set at 25°C, 30°C, 35°C, and 40°C in advance. Three parallels were set for each gradient, and aerobic culture was carried out at 160 r / min for 3 days. From day 0, 2 mL of samples were taken every day, and the OD600 of the fermentation liquid was measured with an enzyme marker to determine the growth status of the target strain.

[0068] (2) pH tolerance test of lactic acid degrading bacteria

[0069] Take the activated liquid of S9 strain and inoculate it into the lactic acid liquid culture medium of pH 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, and 9.5 at an inoculum volume of 5%, respectively. Incubate aerobically at 30°C and 160 r / min for 3 days in a constant temperature shaker. Set 3 blanks for each pH gradient. Take 2 mL of fermentation liquid from 0 day, detect the OD600 value of the culture liquid by a microplate reader, and observe the growth of the target strain.

[0070] (3) Ethanol tolerance test of lactic acid-degrading bacteria

[0071] The S9 lactic acid liquid activation liquid was inoculated with the activated seed liquid in lactic acid liquid culture medium containing 0%, 1.5%, 3%, 4.5%, 6%, and 7.5% ethanol at an inoculation rate of 5%, and aerobic culture was carried out in a constant temperature shaker at 30°C and 160r / min for 3 days. Three blanks were made for each gradient. Sampling began at 0d, and 2mL was sampled every day. The OD600 value of the culture medium was measured to observe the growth trend of the target strain.

[0072] 4. Application test of lactic acid degrading bacteria in bottom water of pit

[0073] (1) Effect of seed solution inoculation amount on fermentation

[0074] S9 was inoculated at 3%, 6%, 9%, 12% and 15% with 3 parallels for each gradient. Sampling was started from the 0th day, and 2 mL of fermentation liquid was taken every other day. The pH change of the bottom water of the wine lees was dynamically detected with a pH meter, and the fermentation liquid was diluted and spread to observe the growth of the strain.

[0075] (2) Effect of water concentration at the bottom of the lees on fermentation

[0076] Because the composition of the wine lees bottom water is complex, the concentration is high and the acidity is large, it will inhibit the growth of functional strains. Therefore, the wine lees bottom water was diluted to 2, 4, 6, 8 and 10 times, sealed with a breathable sealing film and placed in a sterilizer for high-temperature sterilization at 115℃ for 15min. After cooling, it was taken out and placed in an ultra-clean workbench to cool to room temperature. Three parallels were set for each gradient of S9 and cultured on a constant temperature shaker at 30℃ and 160r / min for 7 days. Sampling was started from the 0th day, and the absorbance of the fermentation liquid at a wavelength of 600nm was measured by a spectrophotometer, and the pH value was measured by a pH meter.

[0077] (3) pH adjustment method of wine lees bottom water

[0078] Microbial regulation: 25 mL of wine lees bottom water was placed in a 150 mL conical flask, sealed with a breathable sealing film, and placed in a sterilizer for sterilization at 115°C for 20 min. The flask was taken out and cooled to room temperature for later use. Three parallel cultures of each strain of S9 were set up in a sterile environment and placed in a constant temperature shaker at a speed of 160 r / min and 30°C for aerobic culture. 2 mL of fermentation liquid was taken every other day, and the pH value and OD600 value of the fermentation liquid were measured to observe the growth of microorganisms.

[0079] Adjustment with edible alkali: After adjusting the pH value of the distiller's grains bottom water to 3.5, 4.5, 5.5, and 6.5 with a saturated solution of edible alkali, take 25 mL of the distiller's grains bottom water of each pH concentration and put it into a 150 mL triangular flask, seal it with a breathable membrane, place it in a sterilizer and autoclave at 115°C for 20 min, remove and cool to room temperature, and inoculate S9 seed liquid into the bottom water of each pH concentration under a sterile environment, and ferment it in a constant temperature shaker at 30°C and 160 r / min for 7 days. After diluting and spreading the fermented liquid on the 1st, 3rd, 5th, and 7th days, place it in a 30°C incubator and culture it for 72 h, and then count the plates.

[0080] This patent selects efficient bacteria that can degrade the lactic acid content in the bottom water of distiller's grains, transforms the organic components in the bottom water of distiller's grains, and uses the fermented liquid of the bottom water of distiller's grains as liquid feed, which is the latest research in China. It optimizes the fermentation process, improves the absorption and utilization rate of nutrients such as energy and protein in the bottom water of distiller's grains, and converts the bottom water of distiller's grains into body fluid feed for ruminants, which is innovative and has application value. It can provide basic support for the subsequent research on the solution of feeding ruminants with yellow pulp water to reduce the amount of concentrated feed and realize the recycling of the bottom water resources of distiller's grains.

[0081] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, technicians familiar with the field may make various corresponding changes and deformations based on the present invention, but these corresponding changes and deformations should all fall within the scope of protection of the claims attached to the present invention.

[0082] Table 1 Colony and cell morphology of six bacterial strains

[0083]

Claims

1. An abnormal Wickerhamomyces anomalus FBKL2.00K8, characterized in that The accession number is CCTCC N O :M 2020249.

2. Use of the abnormal Wickham yeast as claimed in claim 1 in degrading lactic acid in wine lees bottom water.

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