Research method for degrading crude fibers by synergistically fermenting vinasse through artificially combined flora

Abstract

The invention relates to the technical field of modern breeding industry, and discloses a research method for degrading crude fibers by synergistically fermenting distillers' grains with artificial combined flora, which comprises the following steps: S1, activating single bacteria of the artificial combined flora; s2, preparing an artificial combined flora solution; s3, vinasse pretreatment and screening; s4, preparing a solid fermentation culture medium; s5, inoculation and solid fermentation; s6, monitoring the fermentation process; s7, measuring a product after fermentation; and S8, evaluating the in-vitro digestibility. The three strains are combined in proportion to form an artificial combined flora to cooperatively ferment the vinasse, so that the technical effects of efficiently degrading crude fibers in the vinasse in a targeted manner and cooperatively improving the activity of a key degrading enzyme so as to strengthen the overall degradation efficiency are achieved, and compared with the common technical scheme of single-strain fermentation or simple mixed flora fermentation without a specific proportion in the prior art, the technical scheme has the advantages that the cost is reduced, and the production efficiency is improved. The defects that the crude fiber degradation is not thorough, the enzyme activity secretion type is single, the synergy among strains is poor, and the anti-enzymolysis structure of the crude fiber in the vinasse is difficult to break through are overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of modern breeding, in particular to a research method for artificial combined microbial flora to synergistically ferment distiller's grains to degrade crude fiber. BACKGROUND

[0002] Distiller's grains is a large amount of by-product produced in the production process of liquor brewing industry, especially in the production process of strong-flavor liquor. The annual output is huge. As the processing residue of brewing raw materials, distiller's grains retains crude protein, minerals, vitamins and part of fermentable carbohydrates in the raw materials which are not completely utilized. It has the natural potential to be converted into livestock feed. If it can be efficiently utilized, it can not only alleviate the contradiction between supply and demand of feed raw materials, but also reduce solid waste pollution, which meets the demand of green circular economy development. However, the content of crude fiber in distiller's grains is high, and the lignin and hemicellulose in it form a tight cross-linked structure, which has strong anti-enzymatic properties. This characteristic leads to low digestibility and absorption rate after direct or simple treatment, and it is difficult to fully realize its feeding value, which becomes one of the core factors restricting its large-scale application in the feed field.

[0003] In the prior art, attempts have been made to degrade the crude fiber in distiller's grains by microbial fermentation. Single microbial strain or simple mixed microbial flora without specific proportion is mainly used for treatment. The enzyme system of single strain has limitations and can only efficiently decompose a certain component in cellulose or hemicellulose, and it is difficult to simultaneously produce effective degradation effect on the cross-linked structure of lignin and hemicellulose. And the simple mixed microbial flora without specific proportion is prone to problems such as nutritional competition between strains and mutual inhibition of enzyme activity due to the lack of consideration of metabolic synergy between strains, which leads to low overall degradation efficiency and cannot break through the anti-enzymatic barrier of crude fiber in distiller's grains. Ultimately, it is still difficult to achieve deep and complete degradation of crude fiber, and it cannot meet the requirement of low fiber content for high-quality feed. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a research method for artificial combined microbial flora to synergistically ferment distiller's grains to degrade crude fiber, which solves the problem of enzyme limitation or poor microbial flora synergy in the scheme of using single strain or simple mixed microbial flora to ferment distiller's grains to degrade crude fiber, and the problem of difficult complete degradation of crude fiber containing lignin and hemicellulose in distiller's grains.

[0005] To achieve the above purpose, the present application realizes the following technical scheme: a research method for artificial combined microbial flora to synergistically ferment distiller's grains to degrade crude fiber, comprising the following steps: S1, single-bacterium activation of artificial combined bacterial flora: Paenibacillus woosongensis, Bacillus thermoamylovorans, and Clostridium were selected and inoculated into PCS straw culture medium, and then incubated at corresponding temperatures for 3 days to obtain three single-bacterium activation liquids; S2, preparation of artificial combined bacterial flora liquid: the three single-bacterium activation liquids of S1 were mixed in a volume ratio of 2-4:1-3:1, and the bacterial concentration was adjusted to 1×10 8 CFU / mL to obtain an artificial combined bacterial flora liquid; 9 CFU / mL to obtain an artificial combined bacterial flora liquid; S3, pretreatment and selection of distiller's grains: after being dried, crushed, and sieved, the concentration of crude fiber and crude protein in the Luzhou-flavor liquor distiller's grains was determined, and the pretreated distiller's grains with a crude fiber content of ≥28% and a crude protein content of ≥22% were selected; S4, preparation of solid fermentation medium: a culture medium containing the pretreated distiller's grains of S3 was prepared, and the solid-liquid ratio was adjusted to 28%-32%. After sterilization, the medium was cooled; S5, inoculation and solid fermentation: the combined bacterial flora liquid of S2 was inoculated into the cooled culture medium of S4 at an inoculation amount of 8%-12%, and then uniformly stirred. After being incubated at 35-39℃ for 6-8 days, samples were taken every 24 hours; S6, monitoring of fermentation process: on the 3rd-5th day of fermentation, the removal rate of crude fiber components and enzyme activity were determined; S7, determination of post-fermentation product: after 6-8 days of fermentation, the total degradation rate, crude fiber removal rate, crude protein content, and dietary fiber proportion were determined. If the indicators do not meet the standards, the proportion of bacterial flora in S2 is adjusted, and the subsequent steps are repeated; S8, evaluation of in vitro digestion rate: the in vitro digestion rate of the product of S7 that meets the standards is determined. If the digestion rate does not meet the standards, the fermentation temperature in S5 is adjusted, and the subsequent steps are repeated.

[0006] Preferably, in S1, the formula of PCS straw culture medium is 8-12 g / L alkali-treated straw, 4-6 g / L peptone, 4-6 g / L NaCl, 0.5-2 g / L yeast powder, and 4-6 g / L CaCO3, and the medium is sterilized at 121℃ for 20 min; The culture temperature of Paenibacillus woosongensis is 30-38℃, the culture temperature of Bacillus thermoamylovorans is 45-55℃, and Clostridium is anaerobically cultured at 35-39℃. The bacterial concentration of the single-bacterium activation liquid is ≥1×10 8 CFU / mL.

[0007] Preferably, in S2, the concentration of the mixed bacteria liquid is adjusted with sterile physiological saline, and when the volume ratio of the bacterial population does not meet the requirements, the adjustment ratio is Paenibacillus woosongensis: Bacillus thermoamylovorans: Clostridium = 3-5: 1-3: 1.

[0008] Preferably, in S3, the dried distiller's grains are dried at 65-75°C under vacuum to a constant weight, crushed, and then sieved through a 30-50 mesh sieve.

[0009] Preferably, in S4, the culture medium is prepared using the pretreated distiller's grains screened in S3 as the core raw material, and the formula is 4-6 g / L peptone, 1.5-2.5 g / L yeast powder, 4-6 g / L NaCl, 18-22 g / L CaCO3, and 280-320 g / L of S3 pretreated distiller's grains. The culture medium is divided into 50 mL blue reagent bottles, sterilized at 121°C for 18-22 min, and then cooled to room temperature for standby.

[0010] Preferably, in S5, the inoculation is completed in a clean bench, and the culture medium is stirred with a sterile glass rod for not less than 1 min to ensure that the bacterial population liquid does not have local aggregation, and when the fermentation temperature does not meet the requirements, the temperature is adjusted to 36-40°C.

[0011] Preferably, in S6, the enzyme activity is determined by the DNS method, and when the hemicellulase activity is determined by the DNS method, the substrate is 0.4-0.6 g / mL xylan, and the water bath is 48-52°C for 28-32 min. When measuring ligninase activity, the substrate is 0.4-0.6 g / mL ABTS, and the water bath is 38-42°C for 28-32 min. When measuring cellulase activity, the substrate is 0.4-0.6 g / mL cellulose, and the water bath is 58-62°C for 28-32 min. The sample needs to pass through a 40 mesh sieve before detection.

[0012] Preferably, in S7, when measuring the total degradation rate, the fermented distiller's grains residue is filtered with a filter bag, and the residue is dried to a constant weight and weighed. When measuring the crude fiber removal rate, the mass and corresponding crude fiber content of the dry distiller's grains before fermentation, and the mass and corresponding crude fiber content of the residue after fermentation are weighed, and then the removal rate is calculated. During the measurement, a blank test is performed to deduct the reagent residue, and the residue obtained from the dietary fiber measurement is subjected to protein content measurement and ash content measurement. The actual dietary fiber content is calculated by deducting the protein mass and ash mass from the residue mass.

[0013] Preferably, in S8, when the in vitro digestion rate is determined, the reaction termination method is heating at 98-102 DEG C for 8-12 min, the centrifugation condition is centrifugation at 7800-8200 rpm for 8-12 min, and the residue drying condition is drying at 58-62 DEG C for 22-26 h to constant weight.

[0014] Preferably, in S8, when the in vitro digestion rate is determined, the simulated gastrointestinal enzyme solution is composed of 1.8-2.2 mg / mL trypsin and 3.8-4.2 mg / mL cellulase, and is dissolved in a hydrochloric acid buffer with pH=1.8-2.2. 28-32 g of the fermentation product is weighed, and a constant temperature water bath reaction is carried out at 36-38 DEG C for 1.8-2.2 h, and stirring is carried out every 18-22 min for 28-32 s. After the reaction is completed, the reaction is terminated by heating at 98-102 DEG C for 8-12 min, centrifugation is carried out at 7800-8200 rpm for 8-12 min, the solid residue is dried at 58-62 DEG C for 22-26 h to constant weight, and after drying, the residue is cooled in a desiccator for 30 min and then weighed.

[0015] The present application provides a research method for artificial combined flora to degrade crude fiber by synergistic fermentation of distiller's grains. 1. The present application forms an artificial combined flora by combining Paenibacillus woosongensis, Bacillus thermoamylovorans and Clostridium in a certain proportion, and the combined flora is used to synergistically ferment distiller's grains, so that the technical effect of efficiently and targetedly degrading crude fiber in the distiller's grains and synergistically improving the activity of key degradation enzymes to strengthen the overall degradation efficiency is achieved. Compared with the technical solutions of the prior art in which a single strain or a simple mixed flora without a specific proportion is used for fermentation, the present application solves the problems of incomplete degradation of crude fiber, single type of enzyme secretion and poor synergy between strains, and difficulty in breaking through the anti-enzymolysis structure of crude fiber in distiller's grains.

[0016] 2. The present application adopts a solid fermentation technical solution in which a Luzhou-flavor liquor distiller's grain is used as a core substrate, a culture medium formula is optimized, and a solid-liquid ratio is controlled, so that the technical effect of reducing the energy consumption and equipment cost of the fermentation system, improving the contact efficiency of the flora and the distiller's grain substrate, and reducing the loss or dilution of nutritional components during the fermentation process is achieved. Compared with the technical solutions of the prior art in which liquid fermentation is adopted, the present application solves the problems of insufficient contact between the substrate and the flora, high cost caused by additional concentration treatment of the product after fermentation, and loss of nutritional components such as crude protein.

[0017] 3、The technical scheme of the present application adopts the fermentation mode of crude fiber degradation, crude protein reservation and dietary fiber conversion, which degrades the crude fiber while maintaining and moderately increasing the crude protein content in the distiller's grains through the metabolic action of the microbial flora, and promotes the conversion of insoluble dietary fiber to soluble dietary fiber, achieving the technical effect of improving the digestibility and overall nutritional value of the distiller's grains for feeding. Compared with the technical scheme in the prior art which only focuses on crude fiber degradation and ignores nutrient reservation and dietary fiber optimization, the present application solves the problems of crude protein loss or insufficient proportion of soluble dietary fiber, and cannot meet the comprehensive requirements of high-quality feed with low fiber, high nutrition and high digestibility. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The present application is a process diagram of the research method of artificial combined microbial flora for synergistic fermentation of distiller's grains to degrade crude fiber. DETAILED DESCRIPTION

[0019] The technical scheme of the present application will be described clearly and completely below with reference to the drawings of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. Embodiment:

[0020] Please refer to the drawings of the present application Figure 1 The present application provides a research method of artificial combined microbial flora for synergistic fermentation of distiller's grains to degrade crude fiber, which comprises the following steps: S1, single-bacterium activation of artificial combined microbial flora: selecting Paenibacillus woosongensis, Bacillus thermoamylovorans and Clostridium, respectively, and inoculating them into PCS straw culture medium, and then incubating them at the corresponding temperature for 3 days to obtain three single-bacterium activation liquids; The PCS straw culture medium has the following formula: 8-12 g / L alkali-treated straw, 4-6 g / L peptone, 4-6 g / L NaCl, 0.5-2 g / L yeast powder and 4-6 g / L CaCO3, and is sterilized at 121°C for 20 min; The culture temperature of Paenibacillus woosongensis is 30-38°C, the culture temperature of Bacillus thermoamylovorans is 45-55°C, and Clostridium is anaerobically cultured at 35-39°C, and the bacterial concentration of the single-bacterium activation liquid is all ≥1×10 8 CFU / mL.

[0021] S2, preparation of artificial combined bacterial flora liquid: mix the three single bacterial activation liquids of S1 in a volume ratio of 2-4: 1-3: 1, and adjust the bacterial concentration to 1x10 8 -1x10 9 CFU / mL to obtain the artificial combined bacterial flora liquid; Wherein, the mixed bacterial liquid concentration is adjusted with sterile normal saline, and when the bacterial flora volume ratio does not meet the standard, the ratio is adjusted to Paenibacillus woosongensis: Bacillus thermoamylovorans: Clostridium = 3-5: 1-3: 1.

[0022] S3, distiller's grains pretreatment and screening: select Luzhou-flavor liquor distiller's grains, dry, crush, and sieve, then measure the crude fiber and crude protein content, and select pretreated distiller's grains with crude fiber ≥28% and crude protein ≥22%; Wherein, the distiller's grains drying conditions are vacuum drying at 65-75°C to constant weight, and crushing and sieving through a 30-50 mesh sieve.

[0023] S4, preparation of solid fermentation medium: prepare a medium containing S3 pretreated distiller's grains, adjust the solid-liquid ratio to 28%-32%, and sterilize and cool; Wherein, the medium uses S3 screened pretreated distiller's grains as the core raw material, and the formula is 4-6 g / L peptone, 1.5-2.5 g / L yeast powder, 4-6 g / L NaCl, 18-22 g / L CaCO3, and 280-320 g / L S3 pretreated distiller's grains. The medium is divided into 50 mL blue reagent bottles, sterilized at 121°C for 18-22 min, and cooled to room temperature for standby.

[0024] S5, inoculation and solid fermentation: inoculate S2 combined bacterial flora liquid into the cooled medium of S4 at an inoculation amount of 8%-12%, stir uniformly, and then place at 35-39°C for 6-8d of static fermentation, taking samples every 24h during the fermentation; Wherein, inoculation is completed in a super-clean bench, and the medium is stirred with a sterilized glass rod for not less than 1 min to ensure that the bacterial flora liquid does not have local aggregation, and when the fermentation temperature does not meet the standard, the temperature is adjusted to 36-40°C.

[0025] S6, fermentation process monitoring: on the 3rd-5th day of fermentation, measure the removal rate of crude fiber components and enzyme activity, which needs to meet the requirements of hemicellulose removal rate ≥30%, lignin removal rate ≥12%, cellulose removal rate ≥5%, and hemicellulase activity ≥0.6U, ligninase activity ≥0.55U; Wherein, the enzyme activity is determined by DNS method, and when the hemicellulase activity is determined by DNS method, the substrate is 0.4-0.6 g / mL xylan, and the water bath is 48-52°C for 28-32 min. When measuring the activity of ligninase, the substrate is 0.4-0.6 g / mL ABTS, 38-42℃ water bath for 28-32 min. When measuring the activity of cellulase, the substrate is 0.4-0.6 g / mL cellulose, 58-62℃ water bath for 28-32 min, and the sample needs to pass through a 40-mesh sieve before detection.

[0026] S7, product measurement after fermentation: after 6-8 days of fermentation, the total degradation rate, crude fiber removal rate, crude protein content and dietary fiber proportion are measured, and the total degradation rate ≥24%, the crude fiber removal rate ≥33%, the crude protein ≥23.5%, the soluble dietary fiber ≥60%, and the insoluble dietary fiber ≤40% are required. If the indicators do not meet the standards, adjust the proportion of the microbial community in S2, and repeat the subsequent steps. Among them, when measuring the total degradation rate, the filter bag is used to filter and retain the fermented residue, and the residue is dried to constant weight and weighed. When measuring the crude fiber removal rate, the mass and corresponding crude fiber content of the dry distiller's grains before fermentation, and the mass and corresponding crude fiber content of the residue after fermentation are weighed, and then the removal rate is calculated. During the measurement, a blank test is carried out to deduct the reagent residue, and the residue obtained by measuring the dietary fiber is subjected to protein content measurement and ash content measurement, and the actual dietary fiber content is calculated by deducting the protein mass and ash mass from the residue mass.

[0027] S8, in vitro digestion rate evaluation: the in vitro digestion rate of the product meeting the standards in S7 is measured, and the in vitro digestion rate ≥72% is required. If the digestion rate does not meet the standards, adjust the fermentation temperature in S5, and repeat the subsequent steps.

[0028] Among them, when measuring the in vitro digestion rate, the reaction termination method is 98-102℃ heating for 8-12 min, the centrifugation condition is 7800-8200 rpm centrifugation for 8-12 min, and the residue drying condition is 58-62℃ drying for 22-26 h to constant weight.

[0029] When measuring the in vitro digestion rate, the simulated gastrointestinal enzyme solution is composed of 1.8-2.2 mg / mL trypsin and 3.8-4.2 mg / mL cellulase, and is dissolved in a hydrochloric acid buffer with pH=1.8-2.2; 28-32 g of fermentation product is weighed, and the reaction is carried out in a 36-38℃ constant temperature water bath for 1.8-2.2 h, and the stirring time is 28-32 s every 18-22 min; After the reaction is completed, the reaction is terminated by heating at 98-102℃ for 8-12 min, centrifuged at 7800-8200 rpm for 8-12 min, and the solid residue is dried at 58-62℃ for 22-26 h to constant weight. After drying, it is cooled in a desiccator for 30 min and then weighed.

[0030] The following will be introduced in combination with specific examples: Example 1 Strain: Paenibacillus woosongensis Bacillus thermoamylovorans Clostridium.

[0031] Luzhou-flavor distiller's grains, purchased from Lude Bio-Environmental Technology Co., Ltd., with initial crude fiber 28.2% and crude protein 22.1%, 15 g dry weight for single experiment.

[0032] PCS straw medium raw materials: alkali-treated straw 8 g / L, peptone 4 g / L, NaCl 4 g / L, yeast powder 0.5 g / L, CaCO3 4 g / L, 50 mL for single preparation, and the corresponding raw material dosages are 0.4 g, 0.2 g, 0.2 g, 0.025 g, and 0.2 g, respectively.

[0033] Peptone 4 g / L, yeast powder 1.5 g / L, NaCl 4 g / L, CaCO3 18 g / L, and pretreated distiller's grains 280 g / L, 50 mL for single preparation.

[0034] Trypsin concentration 1.8 mg / mL, single experimental dosage 0.054 g, cellulase concentration 3.8 mg / mL, single experimental dosage 0.114 g, α-amylase activity 10,000 U / mL, protease activity 300 U / mL, and glucosidase activity 2,000 U / mL.

[0035] Xylan concentration 0.4 g / mL and ABTS concentration 0.4 g / mL, single dosage 0.6 g, and cellulose concentration 0.4 g / mL, single dosage 0.6 g.

[0036] Sterile physiological saline pH 7.0, single dosage 10 mL, hydrochloric acid concentration 1 mol / L, single dosage 2 mL, boric acid concentration 10%, single dosage 5 mL, HCl concentration 0.1 mol / L, single dosage 10 mL, all of which are analytical or biochemical pure.

[0037] The research method for artificial combined microbial flora to synergistically ferment distiller's grains to degrade crude fiber comprises the following steps: S1, single strain activation of artificial combined flora: after the 3 strains of -80℃ glycerol frozen tubes were thawed, 1% inoculation amount was inoculated into 0.5 mL strain to 50 mL PCS straw culture medium, which was prepared and sterilized at 121℃ for 20 min; wherein Paenibacillus woosongensis was incubated at 30℃ for 3 days, Bacillus thermoamylovorans was incubated at 45℃ for 3 days, and Clostridium was incubated at 35℃ for 3 days, 3 kinds of single strain activation liquid were obtained, and the bacterial concentration was 1.1×10 8 CFU / mL, meeting the requirement of ≥1×10 8 CFU / mL; S2, preparation of artificial combined flora liquid: the 3 kinds of single strain activation liquid obtained in S1 were mixed in a volume ratio of 2:1:1, i.e. 2 mL:1 mL:1 mL, and the concentration of mixed bacterial liquid was adjusted to 1×10 8 CFU / mL with sterile normal saline, the total volume was 5 mL, and the artificial combined flora liquid was obtained; S3, wine lees pretreatment and screening: 15 g of strong-flavor wine lees was dried in a vacuum drying oven at 65℃ to constant weight, the water content of the dried wine lees was 4.8%, and after crushing, it was passed through a 30 mesh sieve, the crude fiber content was 28.2%, and the crude protein content was 22.1%, meeting the requirements of crude fiber ≥28% and crude protein ≥22%, and serving as pretreated wine lees, with 14 g used for culture medium preparation; S4, preparation of solid fermentation medium: 0.02 g of peptone, 0.0075 g of yeast powder, 0.02 g of NaCl, 0.09 g of CaCO3, and 14 g of S3 pretreated wine lees were mixed, and deionized water was added to a total system of 50 mL, and the solid-liquid ratio was adjusted to 28%, and then it was divided into 50 mL blue reagent bottles, sterilized at 121℃ for 18 min, and then cooled to room temperature of 25℃, and then it was ready for use; S5, inoculation and solid fermentation: in a clean bench, 4 mL of S2 artificial combined flora liquid was inoculated into the cooled culture medium of S4 at an inoculation amount of 8%, and a sterile glass rod was used for stirring for 1 min to ensure that the flora liquid was not locally aggregated, and then the reagent bottle was placed in a 35℃ incubator for static fermentation for 6 days, and during the period, 2 g of sample was taken every 24 h; S6, monitoring of fermentation process: 2 g of sample was taken on the 3rd day of fermentation, and the sample was first passed through a 40 mesh sieve and then the removal rate of crude fiber components was measured, and the removal rates of hemicellulose, lignin and cellulose were 30.2%, 12.1% and 5.1% respectively, and the enzyme activity was measured by DNS method, 0.5 mL of supernatant bacterial liquid was taken, and 0.6 g of xylan, 0.6 g of ABTS and 0.6 g of cellulose substrate were added respectively, and the corresponding water bath was 48℃, 38℃ and 58℃ for 28 min, and finally the hemicellulase activity was 0.61 U and the ligninase activity was 0.56 U, both meeting the index requirements; S7, post-fermentation product determination: filter the entire post-fermentation vinasse with a filter bag, collect the residue and dry at 65°C to constant weight, the residue mass is 11.4g, the total degradation rate is calculated to be 24.1%, take 15g of dry vinasse before fermentation and the corresponding crude fiber content is 28.2%, take 11.4g of residue after fermentation and the corresponding crude fiber content is 19.5%, the crude fiber removal rate is calculated to be 33.2%, the crude protein content is determined to be 23.6%, the dietary fiber is determined, 1g of residue is taken for a blank test and the protein and ash are corrected, the soluble dietary fiber SDF is measured to be 60.1%, the insoluble dietary fiber IDF is measured to be 39.9%, which meets the index requirements; S8, in-vitro digestion rate evaluation: take 0.054g of trypsin and 0.114g of cellulase, dissolve in a pH=1.8 hydrochloric acid buffer, the buffer is adjusted with 1mol / L hydrochloric acid, and dilute to 100mL to prepare a simulated gastrointestinal enzyme solution, take 28g of fermentation product, add 100mL of the above enzyme solution, and react in a 36°C constant temperature water bath for 1.8h, stir every 18min for 28s, after the reaction is completed, heat at 98°C for 8min to terminate the reaction, centrifuge at 7800rpm for 8min, collect the solid residue and dry at 58°C for 22h to constant weight, weigh after cooling for 30min, and calculate the in-vitro digestion rate to be 72.1%.

[0038] Example 2 The strain is consistent with example 1.

[0039] Luzhou-flavor liquor vinasse, initial crude fiber 29.1%, crude protein 22.3%, single experiment dosage 15g dry weight.

[0040] PCS straw culture medium raw materials: alkali-treated straw 10g / L, proteose peptone 5g / L, NaCl 5g / L, yeast powder 1g / L, CaCO3 5g / L, single preparation 50mL, corresponding raw material dosage is 0.5g, 0.25g, 0.25g, 0.05g, 0.25g respectively.

[0041] Solid-state fermentation culture medium raw materials: proteose peptone 5g / L, yeast powder 2g / L, NaCl 5g / L, CaCO3 20g / L, pretreated vinasse 300g / L, single preparation 50mL, corresponding raw material dosage is 0.025g, 0.01g, 0.025g, 0.1g, 15g respectively.

[0042] Enzymatic reagents: trypsin concentration 2mg / mL, single experiment dosage 0.06g; cellulase concentration 4mg / mL, single experiment dosage 0.12g; α-amylase activity 10000U / mL, used for enzymolysis; protease activity 300U / mL, used for enzymolysis; glucosidase activity 2000U / mL, used for enzymolysis.

[0043] Substrate reagents: Xylan concentration 0.5 g / mL, used for enzyme activity assay, single dose 0.75 g; ABTS concentration 0.5 g / mL, used for enzyme activity assay, single dose 0.75 g; cellulose concentration 0.5 g / mL, used for enzyme activity assay, single dose 0.75 g.

[0044] Other reagents: sterile physiological saline pH 7.0, 10 mL per dose; hydrochloric acid 1 mol / L, 2 mL per dose; boric acid 10%, 5 mL per dose; HCl 0.1 mol / L, 10 mL per dose, all of which are analytical grade or biochemical grade.

[0045] The research method for the synergistic fermentation of distiller's grains by artificially combined microbial communities to degrade crude fiber includes the following steps: S1, Single-cell activation of artificially combined microbial communities: Three bacterial strains were inoculated at a 1% inoculum rate into 0.5 mL of 50 mL PCS straw medium and sterilized at 121℃ for 20 min. *Paenibacillus woosongensis* was cultured at 35℃ for 3 days, *Bacillus thermoamylovorans* at 50℃ for 3 days, and *Clostridium* was anaerobically cultured at 37℃ for 3 days. The resulting activated single-cell concentration was 1.3 × 10⁻⁶. 8 CFU / mL; S2, Preparation of artificially combined bacterial culture solution: Mix the three single-bacterial activation solutions from S1 at a volume ratio of 3:2:1, i.e., 3mL:2mL:1mL, and adjust the concentration to 1×10⁻⁶ with sterile physiological saline. 8 CFU / mL, total volume 5mL, to obtain artificial combined bacterial culture solution; S3, Pretreatment and screening of distiller's grains: 15g of distiller's grains were dried in a vacuum drying oven at 70℃ to constant weight, pulverized and passed through a 40-mesh sieve. The crude fiber content was measured to be 29.1% and the crude protein content was measured to be 22.3%, which met the requirements of crude fiber ≥28% and crude protein ≥22%. As pretreated distiller's grains, all of them were used for the preparation of culture medium. S4, Preparation of solid fermentation medium: Mix 0.025g peptone, 0.01g yeast powder, 0.025g NaCl, 0.1g CaCO3, and 15g pretreated distiller's grains, add deionized water to a total volume of 50mL, adjust the solid-liquid ratio to 30%, dispense into 50mL blue reagent bottles, sterilize at 121℃ for 20min, and cool to room temperature (25℃) for later use; S5, Inoculation and Solid Fermentation: In a clean bench, inoculate 5 mL of S2 artificial combination bacterial culture into the cooled S4 culture medium at a 10% inoculation rate. Stir with a sterile glass rod for 1 min to ensure that there is no local aggregation of the bacterial culture. Place the reagent bottle in a 37℃ incubator for static fermentation for 7 days. During this period, take a sample every 24 hours, and take 2 g of sample each time. S6, fermentation process monitoring: 2g of sample was taken on the 4th day of fermentation, the sample was first sieved through a 40-mesh sieve and then the crude fiber component removal rate was measured, the hemicellulose removal rate was 33.2%, the lignin removal rate was 13.5%, and the cellulose removal rate was 5.5%, the enzyme activity was measured by DNS method, 0.5mL of supernatant bacteria liquid was taken, 0.75g of xylan, 0.75g of ABTS, and 0.75g of cellulose substrate were added respectively, and the corresponding water bath was 50℃, 40℃, and 60℃ for 30min, finally the hemicellulase activity was 0.65U, and the ligninase activity was 0.59U; S7, product determination after fermentation: filter the whole fermented vinasse with a filter bag, collect the residue and dry at 65℃ until constant weight, the residue mass is 11.3g, the total degradation rate is 24.7%, take 15g of dry vinasse before fermentation and the corresponding crude fiber content is 29.1%, take 11.3g of residue after fermentation and the corresponding crude fiber content is 19.4%, the crude fiber removal rate is 33.5%, the crude protein content is 23.9%, and the dietary fiber is measured, 1g of residue is taken for a blank test and the protein and ash are corrected, the soluble dietary fiber SDF is 61.2%, and the insoluble dietary fiber IDF is 38.8%; S8, in vitro digestion rate evaluation: 0.06g of trypsin and 0.12g of cellulase are weighed and dissolved in a hydrochloric acid buffer solution with pH=2.0, the buffer solution is adjusted with 1mol / L hydrochloric acid, and the volume is made up to 100mL to prepare a simulated gastrointestinal enzyme solution, 30g of fermentation product is weighed and added into 100mL of the above enzyme solution, and the reaction is carried out in a constant temperature water bath at 37℃ for 2h, stirring for 30s every 20min, after the reaction is completed, the reaction is terminated by heating at 100℃ for 10min, centrifugation is carried out at 8000rpm for 10min, the solid residue is collected and dried at 60℃ for 24h until constant weight, and after cooling for 30min, the weight is weighed, and the in vitro digestion rate is 72.6%.

[0046] Example 3 The strain is consistent with example 1.

[0047] Vinasse specifications: Luzhou-flavor liquor vinasse, initial crude fiber 28.8%, crude protein 22.5%, single experiment dosage 15.5g dry weight.

[0048] PCS straw culture medium raw materials: alkali-treated straw 11g / L, peptone 5.5g / L, NaCl 5.5g / L, yeast powder 1.5g / L, CaCO3 5.5g / L, single preparation 50mL, corresponding raw material dosage 0.55g, 0.275g, 0.275g, 0.075g, 0.275g.

[0049] The solid fermentation medium raw materials are: 5.5 g / L of proteose peptone, 2.2 g / L of yeast powder, 5.5 g / L of NaCl, 21 g / L of CaCO3, and 310 g / L of pretreated vinasse; 50 mL is prepared at a time, and the corresponding raw material amounts are 0.0275 g, 0.011 g, 0.0275 g, 0.105 g, and 15.5 g, respectively.

[0050] The enzyme reagent is: 2.1 mg / mL of trypsin, 0.063 g of which is used at a time; 4.1 mg / mL of cellulase, 0.123 g of which is used at a time; and the specifications of α-amylase, protease, and glucosidase are the same as those in Example 1.

[0051] The substrate reagent is: 0.5 g / mL of xylan, 0.75 g of which is used at a time; 0.5 g / mL of ABTS, 0.75 g of which is used at a time; and 0.5 g / mL of cellulose, 0.75 g of which is used at a time.

[0052] Other reagents are: 10 mL of sterile normal saline with pH 7.0, which is used at a time; 2 mL of hydrochloric acid with a concentration of 1 mol / L, which is used at a time; 5 mL of boric acid with a concentration of 10%, which is used at a time; and 10 mL of hydrochloric acid with a concentration of 0.1 mol / L, which is used at a time; all of which are of analytical or biochemical purity.

[0053] The research method for synergistic fermentation of vinasse by artificially combined microbial flora to degrade crude fiber comprises the following steps: S1, single-strain activation of the artificially combined microbial flora: 3 strains are inoculated into 0.5 mL of the strain to 50 mL of PCS straw culture medium at an inoculation amount of 1%, and sterilized at 121°C for 20 min; Paenibacillus woosongensis is cultured at 36°C for 3 days, Bacillus thermoamylovorans is cultured at 52°C for 3 days, and Clostridium is anaerobically cultured at 38°C for 3 days; and the obtained single-strain activation liquid has a bacterial concentration of 1.4×10 8 CFU / mL; S2, preparation of the artificially combined microbial flora liquid: the 3 single-strain activation liquids in S1 are mixed in a volume ratio of 3.5:2.5:1, i.e., 3.5 mL:2.5 mL:1 mL; the concentration is adjusted to 5×10 8 CFU / mL with sterile normal saline, and the total volume is 5.5 mL, to obtain the artificially combined microbial flora liquid; S3, vinasse pretreatment and screening: 15.5 g of vinasse is dried in a vacuum drying oven at 72°C until the weight is constant, and the water content of the dried vinasse is 4.5%; after being crushed, the vinasse is sieved through a 45-mesh sieve; the crude fiber content is 28.8%, and the crude protein content is 22.5%, which meets the requirements of crude fiber ≥28% and crude protein ≥22%; and the pretreated vinasse is used as needed. S4, solid fermentation medium preparation: 0.0275 g of peptone, 0.011 g of yeast powder, 0.0275 g of NaCl, 0.105 g of CaCO3, 15.5 g of pretreated vinasse were mixed, and deionized water was added to a total system of 50 mL to adjust the solid-liquid ratio to 31%, and then 50 mL blue reagent bottles were divided and sterilized at 121°C for 21 min and then cooled to room temperature of 25°C for standby; S5, inoculation and solid fermentation: in the clean bench, 5.5 mL of S2 artificial combined microbial flora liquid was inoculated into the cooled medium of S4 at an inoculation amount of 11%, and a sterile glass rod was used for stirring for 1.5 min to ensure that the microbial flora liquid was not locally aggregated, and then the reagent bottle was placed in a 38°C incubator for static fermentation for 7 days, and 2 g of sample was taken every 24 h during the fermentation; S6, fermentation process monitoring: 2 g of sample was taken on the 4th day of fermentation, and the sample was first passed through a 40-mesh sieve and then the crude fiber component removal rate was measured. The measured hemicellulose removal rate was 31.8%, the lignin removal rate was 12.8%, and the cellulose removal rate was 5.3%. The enzyme activity was measured by the DNS method. 0.5 mL of supernatant microbial liquid was taken, and 0.75 g of xylan, 0.75 g of ABTS, and 0.75 g of cellulose substrate were added, respectively. The corresponding water bath was at 50°C, 40°C, and 60°C for 30 min. Finally, the hemicellulase activity was 0.63 U, and the ligninase activity was 0.57 U. S7, product determination after fermentation: all the fermented vinasse was filtered with a filter bag, the residue was collected and dried at 65°C to a constant weight. The residue mass was 11.4 g, and the total degradation rate was 24.4%. The dry vinasse before fermentation was weighed at 15.5 g, and the corresponding crude fiber content was 28.8%. The residue mass after fermentation was 11.4 g, and the corresponding crude fiber content was 19.5%. The crude fiber removal rate was calculated to be 33.3%. The crude protein content was measured to be 23.7%. The dietary fiber was measured. 1 g of residue was taken for a blank test and the protein and ash were corrected. The soluble dietary fiber SDF was 60.8%, and the insoluble dietary fiber IDF was 39.2%. S8, in vitro digestion rate evaluation: 0.063 g of trypsin and 0.123 g of cellulase were weighed and dissolved in a pH=2.1 hydrochloric acid buffer. The buffer was adjusted with 1 mol / L hydrochloric acid, and the volume was made to 100 mL to prepare a simulated gastrointestinal enzyme solution. 31 g of fermentation product was weighed and added to 100 mL of the above enzyme solution. The reaction was carried out in a constant temperature water bath at 37.5°C for 2.1 h. Every 21 min, the solution was stirred for 31 s. After the reaction was completed, the reaction was terminated by heating at 101°C for 11 min. The solution was centrifuged at 8100 rpm for 11 min. The solid residue was collected and dried at 61°C for 25 h to a constant weight. After cooling for 30 min, the weight was measured. The in vitro digestion rate was calculated to be 72.3%.

[0054] Example 4 The strain was consistent with example 1.

[0055] Distiller's grains: Luzhou-flavor distiller's grains, initial crude fiber 28.5%, crude protein 22.2%, single experiment dosage 16 g dry weight.

[0056] PCS straw medium raw materials: alkali-treated straw 12 g / L, peptone 6 g / L, NaCl 6 g / L, yeast powder 2 g / L, CaCO3 6 g / L, single preparation 50 mL, corresponding raw material dosage 0.6 g, 0.3 g, 0.3 g, 0.1 g, 0.3 g respectively.

[0057] Solid fermentation medium raw materials: peptone 6 g / L, yeast powder 2.5 g / L, NaCl 6 g / L, CaCO3 22 g / L, pretreated distiller's grains 320 g / L, single preparation 50 mL, corresponding raw material dosage 0.03 g, 0.0125 g, 0.03 g, 0.11 g, 16 g respectively.

[0058] Enzymatic reagents: trypsin concentration 2.2 mg / mL, single experiment dosage 0.066 g; cellulase concentration 4.2 mg / mL, single experiment dosage 0.126 g; α-amylase, protease, glucosidase specifications consistent with example 1.

[0059] Substrate reagents: xylan concentration 0.6 g / mL, single dosage 0.9 g; ABTS concentration 0.6 g / mL, single dosage 0.9 g; cellulose concentration 0.6 g / mL, single dosage 0.9 g.

[0060] Other reagents: sterile physiological saline pH 7.0, single dosage 10 mL; hydrochloric acid concentration 1 mol / L, single dosage 2 mL; boric acid concentration 10%, single dosage 5 mL; HCl concentration 0.1 mol / L, single dosage 10 mL, all are analytical pure or biochemical pure.

[0061] The research method for degrading crude fiber by artificial combined microbial flora synergistic fermentation of distiller's grains comprises the following steps: S1, single strain activation of artificial combined microbial flora: inoculate 3 strains into 0.5 mL of strain to 50 mL of PCS straw medium at an inoculation amount of 1%, sterilize at 121 ℃ for 20 min, cultivate Paenibacillus woosongensis at 38 ℃ for 3 days, cultivate Bacillus thermoamylovorans at 55 ℃ for 3 days, cultivate Clostridium anaerobically at 39 ℃ for 3 days, and obtain single strain activation liquid with a bacterial concentration of 1.6 × 10 8 CFU / mL; S2, preparation of artificial combined microbial flora liquid: mix the 3 kinds of single strain activation liquids of S1 according to the volume ratio of 4:3:1, that is, 4 mL:3 mL:1 mL, and adjust the concentration to 1 × 10 9CFU / mL, total volume 6 mL, to obtain artificial combined flora liquid; S3, distiller's grains pretreatment and screening: 16 g of distiller's grains was placed in a vacuum drying oven at 75℃ and dried to constant weight. After drying, the moisture content of the distiller's grains was 4.2%, and after crushing and passing through a 50 mesh sieve, the crude fiber content was measured to be 28.5% and the crude protein content was measured to be 22.2%, meeting the requirements of crude fiber ≥ 28% and crude protein ≥ 22%, and serving as pretreated distiller's grains for standby; S4, preparation of solid fermentation medium: according to the above-mentioned raw material proportions of the solid fermentation medium, 0.03 g of peptone, 0.0125 g of yeast powder, 0.03 g of NaCl, 0.11 g of CaCO3, and 16 g of pretreated distiller's grains were mixed, and deionized water was added to a total system of 50 mL, and the solid-liquid ratio was adjusted to 32%, and then it was divided into 50 mL blue reagent bottles, sterilized at 121℃ for 22 min, and then cooled to room temperature of 25℃ for standby; S5, inoculation and solid fermentation: in a clean bench, 6 mL of S2 artificial combined flora liquid was inoculated into the cooled medium of S4 at an inoculation amount of 12%, and a sterile glass rod was used to stir for 2 min to ensure that the flora liquid was not locally aggregated, and then the reagent bottle was placed in a 39℃ incubator for static fermentation for 8 days, and during the period, 2 g of sample was taken every 24 h; S6, monitoring of fermentation process: 2 g of sample was taken on the 5th day of fermentation, and the sample was first passed through a 40 mesh sieve and then the removal rate of crude fiber components was measured. The removal rate of hemicellulose was measured to be 32.5%, the removal rate of lignin was measured to be 13.2%, and the removal rate of cellulose was measured to be 5.4%. The enzyme activity was measured by DNS method. 0.5 mL of supernatant bacteria liquid was taken, and 0.9 g of xylan, 0.9 g of ABTS, and 0.9 g of cellulose substrate were added, respectively. The corresponding water bath was at 52℃, 42℃, and 62℃ for 32 min. Finally, the hemicellulase activity was measured to be 0.64 U, and the ligninase activity was measured to be 0.58 U; S7, determination of post-fermentation products: all the fermented distiller's grains were filtered with a filter bag, the residue was collected and dried at 65℃ to constant weight, the mass of the residue was 11.5 g, the total degradation rate was calculated to be 24.3%, the mass of the dry distiller's grains before fermentation was 16 g, and the corresponding crude fiber content was 28.5%, the mass of the residue after fermentation was 11.5 g, and the corresponding crude fiber content was 19.6%, the removal rate of crude fiber was calculated to be 33.4%, the crude protein content was measured to be 23.8%, and the dietary fiber was measured. 1 g of residue was taken for a blank test and the protein and ash were corrected. The soluble dietary fiber SDF was measured to be 60.5%, and the insoluble dietary fiber IDF was measured to be 39.5%; S8, in vitro digestion rate evaluation: 0.066 g trypsin, 0.126 g cellulase were weighed and dissolved in hydrochloric acid buffer solution with pH = 2.2, the buffer solution was adjusted with 1 mol / L hydrochloric acid, and the volume was made up to 100 mL to prepare simulated gastrointestinal enzyme solution. 32 g of the fermentation product was weighed and added to 100 mL of the above enzyme solution, and the reaction was carried out in a constant temperature water bath at 38°C for 2.2 h. Stirring was carried out every 22 min for 32 s. After the reaction was completed, the reaction was terminated by heating at 102°C for 12 min. Centrifugation was carried out at 8200 rpm for 12 min. The solid residue was collected and dried at 62°C for 26 h to constant weight. After cooling for 30 min, the weight was weighed, and the in vitro digestion rate was calculated to be 72.2%.

[0062] Comparative Example 1 The strain used was only Paenibacillus woosongensis.

[0063] S1, single strain activation: only Paenibacillus woosongensis was inoculated into 50 mL PCS straw culture medium at an inoculation amount of 1%, and incubated at 35°C for 3 days to obtain a single strain activation liquid, and the bacterial concentration was 1.3×10 8 CFU / mL; S2, fermentation broth preparation: the single strain activation liquid of Paenibacillus woosongensis obtained in S1 was directly used, and the concentration was adjusted to 1×10 8 CFU / mL with sterile normal saline as a fermentation broth; S3 to S8 steps, including distiller's grains pretreatment, solid fermentation medium preparation, inoculation amount, fermentation temperature, fermentation time, sampling monitoring, product determination and in vitro digestion rate evaluation, are all completely consistent with Example 2.

[0064] Comparative Example 2 The difference between this comparative example and Example 2 is that the solid fermentation medium is changed to a liquid medium; The formula is: peptone 5 g / L, yeast powder 2 g / L, NaCl 5 g / L, CaCO3 20 g / L. No distiller's grains are added.

[0065] S1 to S3 steps are consistent with Example 2; S4, liquid fermentation medium preparation: 0.025 g of peptone, 0.01 g of yeast powder, 0.025 g of NaCl and 0.1 g of CaCO3 were dissolved in 50 mL of deionized water according to the formula, and sterilized at 121°C for 20 min, and then cooled to room temperature 25°C; S5, inoculation and liquid fermentation: in the clean bench, 1.5 g of sterilized pretreated vinasse was added into 50 mL of cooled liquid medium to make the concentration 10% (w / v), then 5 mL of S2 artificial combined microbial flora liquid was inoculated at 10%, and stirred with a sterilized glass rod for 1 min, and the reagent bottle was placed in a 37℃ incubator shaker for culture for 7 days, the shaking speed was 180 rpm, and sampling was performed once every 24 h during the period; S6 to S8 steps, including sampling monitoring, product determination and in vitro digestion rate evaluation, are all completely consistent with example 2.

[0066] Table 1 Crude fiber removal rate (%) Total degradation rate (%) Crude protein content (%) Soluble dietary fiber SDF (%) Insoluble dietary fiber IDF (%) Hemicellulase activity (U) Ligninase activity (U) In vitro digestibility (%) Example 1 33.2 24.1 23.6 60.1 39.9 0.61 0.56 72.1 Example 2 33.5 24.7 23.9 61.2 38.8 0.65 0.59 72.6 Example 3 33.3 24.4 23.7 60.8 39.2 0.63 0.57 72.3 Example 4 33.4 24.3 23.8 60.5 39.5 0.64 0.58 72.2 Comparative Example 1 21.8 18.5 22.7 52.5 47.5 0.42 0.35 65.3 Comparative Example 2 25.2 20.3 23.1 54.8 45.2 0.51 0.48 67.5 Crude fiber removal rate detection Reference standard: GB / T6434-2006 "Determination of crude fiber in feed"; The mass (M0) and crude fiber content (C0) of dry vinasse before fermentation, the mass (M1) and crude fiber content (C1) of residue after fermentation were respectively weighed; Residue treatment after fermentation: the fermentation product was filtered with a F58 filter bag, vacuum dried at 65℃ to constant weight, and crushed through a 40 mesh sieve; Crude fiber determination: the acid digestion method was used, 0.13 mol / L sulfuric acid was boiled for 30 min, and the alkali digestion method was used, 0.23 mol / L potassium hydroxide was boiled for 30 min, and the crude fiber content was calculated after deducting the filter bag mass and ash content; Crude fiber removal rate (%) = (C0 x M0 - C1 x M1) / (C0 x M0) x 100%.

[0067] Total degradation rate detection The accurate mass (M0) of dry vinasse before fermentation was recorded; After the fermentation was completed, all the fermentation products were filtered with a F58 filter bag, and the residue was collected; The residue was placed in a vacuum drying oven at 65℃ and dried to constant weight, and the mass of the residue (M1) was recorded; Total degradation rate (%) = (M0 - M1) / M0 x 100%.

[0068] Crude protein content detection Reference standard: GB5009.5-2016 "Determination of protein in food (Kjeldahl method)" in food safety national standards; Operation steps: 1.0000 g of dried vinasse sample (passed through a 40 mesh sieve) was weighed in a digestion tube, 10 mL of concentrated sulfuric acid, 0.4 g of copper sulfate, and 6.0 g of potassium sulfate were added; Digestion: pre-digestion at 220℃ for 0.5 h, constant temperature digestion at 420℃ for 2.5 h until the digestion solution was transparent light green; Distillation: dilute with 20 mL of deionized water, add 40 mL of 40% NaOH solution, distill for 6 min, and absorb the distillate with 10% boric acid solution; Titration: titrate with 0.1 mol / L hydrochloric acid standard solution, record the consumption volume (V), and perform a blank control (V0) at the same time; Calculation: crude protein content (%) = (V-V0) x C x 14.007 x 6.25 / (M0 x 1000) x 100%; Wherein, C is the concentration of hydrochloric acid, M0 is the sample mass, and 6.25 is the protein conversion coefficient.

[0069] Dietary fiber (SDF / IDF) detection Reference standard: GB5009.88-2014 "Food safety national standard for determination of dietary fiber"; Sample pretreatment: vacuum drying of distiller's grains at 70°C to constant weight, crushing through a 0.4 mm sieve, and weighing 1 g of sample; Enzymatic hydrolysis: add 40 mL of MES-TRIS buffer, and sequentially add 50 μL of α-amylase, 100 μL of protease, and 50 μL of glucosidase; Insoluble dietary fiber (IDF) determination: vacuum filtration of the enzymatic hydrolysate through a pretreated diatomite crucible, washing with hot water at 70°C for 3 times, washing with acetone, and drying at 105°C to constant weight after washing with acetone; deduct the mass of blank, protein, and ash; Soluble dietary fiber (SDF) determination: add 4 times the volume of 95% ethanol to the enzymatic hydrolysate, stand at 4°C for 12 h, collect the precipitate by filtration, wash with 78% ethanol and acetone, and dry at 105°C to constant weight; similarly, deduct the mass of blank, protein, and ash; Calculation: SDF / IDF (%) = (residue mass - blank mass - protein mass - ash mass) / sample mass x 100%.

[0070] Enzyme activity (hemicellulase, ligninase) detection Detection method: DNS method; Hemicellulase activity determination: Substrate preparation: 0.4-0.6 g / mL xylan solution; Reaction system: 1.5 mL of substrate and 0.5 mL of fermentation supernatant bacteria solution, 48-52°C water bath for 28-32 min; Termination and determination: add 1.5 mL of DNS reagent, boil in a water bath for 5 min, and measure the absorbance at 540 nm wavelength after cooling; take 1 mg of reducing sugar released per minute as 1 enzyme activity unit (U); Ligninase activity determination: Substrate preparation: 0.4-0.6 g / mL ABTS solution; Reaction system: 1.5 mL substrate and 0.5 mL fermentation supernatant + 2 mL 1% hydrogen peroxide, 38-42℃ water bath for 28-32 min; Termination and determination: heat for 10 min at 100℃ to terminate the reaction, measure the absorbance at 470 nm wavelength, and take 1 mg of oxidized product released per minute as 1 enzyme activity unit (U).

[0071] In vitro digestion rate detection Operation steps: Preparation of simulated gastrointestinal enzyme solution: dissolve trypsin and cellulase in hydrochloric acid buffer with pH 1.8-2.2; Enzymatic reaction: weigh 28-32 g of fermentation product, add 100 mL of enzyme solution, and react in a 36-38℃ constant temperature water bath for 1.8-2.2 h, stir for 28-32 s every 18-22 min; Termination and separation: terminate the reaction by heating at 100℃ for 8-12 min, centrifuge at 7800-8200 rpm for 8-12 min, and collect the solid residue; Drying and weighing: dry the residue at 58-62℃ for 22-26 h until the weight is constant, and weigh after cooling for 30 min; Calculation: in vitro digestion rate (%) = (initial sample mass-residue mass) / initial sample mass × 100%.

[0072] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A research method for the synergistic fermentation of distiller's grains by artificially combined microbial communities to degrade crude fiber, characterized by: Includes the following steps: S1, Single-strain activation of artificially combined microbial communities: Paenibacillus_woosongensis, Bacillus_thermoamylovorans and Clostridium were selected and inoculated into PCS straw culture medium respectively. They were then statically cultured for 3 days under the corresponding temperature conditions to obtain three single-strain activation solutions. S2, Preparation of artificial combined bacterial culture solution: Mix the three single-strain activation solutions from S1 at a volume ratio of 2-4:1-3:1, and adjust the bacterial concentration to 1×10⁻⁶. 8 -1×10 9 CFU / mL was used to obtain an artificial bacterial culture solution; S3, Pretreatment and Screening of Distillers' Grains: Select strong-aroma baijiu lees, dry, crush and screen them, and determine the crude fiber and crude protein content. Screen out pretreated lees with crude fiber ≥28% and crude protein ≥22%. S4, Preparation of solid fermentation medium: Prepare a medium containing S3 pretreated distiller's grains, adjust the solid-liquid ratio to 28%-32%, sterilize and cool; S5, Inoculation and Solid Fermentation: Inoculate the combined bacterial culture of S2 into the cooled culture medium of S4 at an inoculation rate of 8%-12%, stir evenly, and let it ferment at 35-39℃ for 6-8 days, taking samples every 24 hours during the period. S6, Fermentation process monitoring: On days 3-5 of fermentation, the crude fiber component removal rate and enzyme activity were measured; S7, Determination of fermentation products: After 6-8 days of fermentation, determine the total degradation rate, crude fiber removal rate, crude protein content and dietary fiber ratio. If the indicators do not meet the standards, adjust the bacterial community ratio in S2 and repeat the subsequent steps. S8, In vitro digestibility assessment: The in vitro digestibility of the product that meets the S7 standard is measured. If the digestibility does not meet the standard, the fermentation temperature in S5 is adjusted and the subsequent steps are repeated.

2. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In S1, the PCS straw culture medium formula is 8-12 g / L alkali-treated straw, 4-6 g / L peptone, 4-6 g / L NaCl, 0.5-2 g / L yeast powder, and 4-6 g / L CaCO3, and is sterilized at 121℃ for 20 min; Paenibacillus woosongensis was cultured at 30-38℃, Bacillus thermoamylovorans at 45-55℃, and Clostridium at 35-39℃ using anaerobic culture. The concentration of single-cell activated cultures was ≥1×10⁻⁶. 8 CFU / mL.

3. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In step S2, sterile physiological saline is used to adjust the concentration of the mixed bacterial solution. If the bacterial volume ratio adjustment does not meet the standard, the adjustment ratio is Paenibacillus_woosongensis:Bacillus_thermoamylovorans:Clostridium=3-5:1-3:

1.

4. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In S3, the lees are dried under vacuum at 65-75℃ to constant weight, and then pulverized and passed through a 30-50 mesh sieve.

5. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In S4, the culture medium uses the pretreated distiller's grains that have passed the screening in S3 as the core raw material, and the formula is 4-6 g / L peptone, 1.5-2.5 g / L yeast powder, 4-6 g / L NaCl, 18-22 g / L CaCO3, and 280-320 g / L of S3 pretreated distiller's grains. Dispense the culture medium into 50mL blue reagent bottles, sterilize at 121℃ for 18-22min, and cool to room temperature before use.

6. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In step S5, inoculation is completed in a clean bench. The culture medium is stirred with a sterile glass rod for at least 1 minute to ensure that there is no local aggregation of the bacterial culture. If the fermentation temperature is not adjusted to the standard, the temperature is adjusted to 36-40℃.

7. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In S6, enzyme activity was determined using the DNS method. When determining hemicellulase activity using the DNS method, the substrate was 0.4-0.6 g / mL xylan, and the solution was in a water bath at 48-52℃ for 28-32 min. When determining ligninase activity, the substrate is 0.4-0.6 g / mL ABTS, and the solution is in a water bath at 38-42℃ for 28-32 min. When determining cellulase activity, the substrate is 0.4-0.6 g / mL cellulose, and the sample is placed in a water bath at 58-62℃ for 28-32 min. The sample must pass through a 40-mesh sieve before testing.

8. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In S7, when determining the total degradation rate, the fermented lees residue is filtered using a filter bag and then dried to constant weight before being weighed. When determining the crude fiber removal rate, first weigh the dry lees before fermentation and the corresponding crude fiber content, and weigh the residue after fermentation and the corresponding crude fiber content, and then calculate the removal rate. A blank test was performed simultaneously during the determination process to deduct reagent residues. The protein content and ash content of the residue obtained from the dietary fiber determination were determined separately. The actual dietary fiber content was calculated by subtracting the protein mass and ash mass from the residue mass.

9. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In S8, during the in vitro digestibility determination, the reaction termination method is heating at 98-102℃ for 8-12 minutes, centrifugation conditions are centrifugation at 7800-8200 rpm for 8-12 minutes, and residue drying conditions are drying at 58-62℃ for 22-26 hours until constant weight.

10. The research method for the synergistic fermentation of distiller's grains to degrade crude fiber according to claim 1, characterized in that: In S8, during in vitro digestibility determination, the simulated gastrointestinal enzyme solution consists of 1.8-2.2 mg / mL trypsin and 3.8-4.2 mg / mL cellulase, and is dissolved in hydrochloric acid buffer solution with pH=1.8-2.2; Weigh 28-32g of fermentation product and react it in a constant temperature water bath at 36-38℃ for 1.8-2.2h, stirring for 28-32s every 18-22min. After the reaction is complete, heat at 98-102℃ for 8-12 minutes to terminate the reaction, centrifuge at 7800-8200 rpm for 8-12 minutes, dry the solid residue at 58-62℃ for 22-26 hours until constant weight, cool in a desiccator for 30 minutes and weigh again.

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