Method for fermenting vinasse feed through mixed bacteria

Abstract

The invention discloses a method for fermenting vinasse feed through mixed bacteria, and belongs to the field of microbial fermentation. The invention firstly provides a microbial agent for increasing the content of crude protein in fermented vinasse. The microbial agent contains a bacillus subtilis liquid, a corynebacterium glutamicum liquid, a saccharomyces cerevisiae liquid, an enterococcus faecalis liquid and a bifidobacterium animalis liquid. The invention also provides an application of the microbial agent in preparation of a fermented vinasse feed. The contents of crude protein, crude fat and amino acid in the fermented vinasse feed are increased. Through mixed fermentation, the content of amino acid in the vinasse is increased, the content of lysine is increased by 108.33%, and the content of threonine is increased by 59.09%.

Description

Technical Field

[0001] The invention relates to a method for mixed-bacteria fermentation of distiller's grains feed, and belongs to the field of microbial fermentation. Background Art

[0002] Distillers' grains, a major byproduct of the brewing industry, are rich in nutrients such as protein, carbohydrates, and cellulose. However, improper handling can lead to resource waste and environmental problems. Currently, single-strain fermentation of distillers' grains has limitations in increasing product value and fully utilizing the grains. Mixed-strain fermentation technology can leverage the synergistic effects of different microorganisms to more effectively transform the components of distillers' grains, enrich their nutritional content, and improve fermentation efficiency and product quality. Summary of the Invention

[0003] In order to further improve the utilization rate of distiller's grains and improve the feeding value of distiller's grains, the present invention provides a method for fermenting distiller's grains with mixed bacteria.

[0004] The present invention first provides a method for preparing distiller's grains feed, which comprises: using distiller's grains as fermentation raw materials and fermenting with microorganisms; the microorganisms include Bacillus subtilis; the Bacillus subtilis includes Bacillus subtilis FMME ZK002, and the Bacillus subtilis ZK002 is Bacillus subtilis CCTCCM2021168. The microorganisms also include yeast. The yeast includes feed yeast or brewer's yeast. Optionally but not limiting, the brewer's yeast is brewer's yeast CCTCC NO: M 2023098, and the microorganisms also include Corynebacterium glutamicum, and the Corynebacterium glutamicum is Corynebacterium glutamicum CCTCC NO: M 2023742. The microorganisms also include Enterococcus faecalis and Bifidobacterium. The Enterococcus faecalis is Enterococcus faecalis 21869. The bifidobacterium is Bifidobacterium animalis subsp. lactis 24672.

[0005] In one embodiment, the method comprises: using wine lees as raw material and fermenting one strain of bacteria or a mixture of several strains of bacteria.

[0006] In one embodiment, the method comprises the following steps:

[0007] (1) Strain activation: Inoculate the strains into seed culture medium and culture to obtain bacterial culture fluid;

[0008] (2) Fermentation: Mix the lees with 1-3 mL / min. 100g风干酒糟 molasses, 1-2 mL / 100g风干酒糟 Corn starch, 40~60mL / 100g风干酒糟 Water, 4~8mL / 100g风干酒糟 of bacterial culture and ferment for 36 to 72 hours.

[0009] In one embodiment, the bacterial culture solution is at OD 600 Vaccination is between 6 and 10 o'clock.

[0010] In one embodiment, when the yeast, Bacillus subtilis, Corynebacterium glutamicum, Enterococcus faecalis and Bifidobacterium are subjected to mixed fermentation, the culture broth volume ratio of the bacterial liquid is 1:1:1:1:1.

[0011] In one embodiment, the temperature is 30-42°C.

[0012] The present invention also provides white wine lees or (and) yellow wine lees feed prepared by the method.

[0013] The present invention also provides a microbial agent for increasing the crude protein content in fermented vinasse. The microbial agent contains a bacterial solution of Bacillus subtilis, a bacterial solution of Corynebacterium glutamicum, a bacterial solution of Saccharomyces cerevisiae, a bacterial solution of Enterococcus faecalis, and a bacterial solution of Bifidobacterium animalis.

[0014] In one embodiment, in the microbial agent, the Bacillus subtilis solution, the Corynebacterium glutamicum solution, the Saccharomyces cerevisiae solution, the Enterococcus faecalis solution, and the Bifidobacterium animalis solution are mixed in a ratio of (1-3): (1-3): (1-4): (1-3): (1-3);

[0015] In one embodiment, the OD600 of the added Bacillus subtilis bacterial solution is 10-12; the OD600 of the added Corynebacterium glutamicum bacterial solution is 10-12; the OD600 of the added Saccharomyces cerevisiae bacterial solution is 10-12; the OD600 of the added Enterococcus faecalis bacterial solution is 10-12; and the OD600 of the added Bifidobacterium animalis bacterial solution is 10-12.

[0016] In one embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae CCTCC NO: M 2023098, the Bacillus subtilis is Bacillus subtilis CCTCC NO: M 2021168, the Corynebacterium glutamicum is Corynebacterium glutamicum CCTCC NO: M2023742, the Bifidobacterium animalis is Bifidobacterium animalis CICC 24672, and the Enterococcus faecalis is Enterococcus faecalis CICC 21869;

[0017] In one embodiment, the method for preparing the brewer's yeast liquid is as follows: inoculating brewer's yeast into a seed culture medium, culturing at 30-35° C. and 200-250 rpm for 10-12 hours to prepare the brewer's yeast liquid;

[0018] In one embodiment, the preparation method of the Bacillus subtilis bacterial liquid is as follows: inoculating Bacillus subtilis into a seed culture medium, culturing at 35-37° C. and 200-250 rpm for 10-12 hours to prepare the Bacillus subtilis bacterial liquid;

[0019] In one embodiment, the preparation method of the Corynebacterium glutamicum bacterial liquid is: inoculating Corynebacterium glutamicum into a seed culture medium, culturing at 30-35° C. and 200-250 rpm for 10-12 hours to prepare the Corynebacterium glutamicum bacterial liquid;

[0020] In one embodiment, the preparation method of the animal Bifidobacterium bacterial liquid and the Enterococcus faecalis bacterial liquid is: inoculate animal Bifidobacterium and Enterococcus faecalis into seed culture medium respectively, and culture them at 35-37°C and 200-250 rpm for 10-12 hours to prepare animal Bifidobacterium bacterial liquid and Enterococcus faecalis bacterial liquid respectively.

[0021] The present invention also provides a fermented distiller's grains feed, which is obtained by adding the above-mentioned microbial agent to distiller's grains and fermenting them.

[0022] In one embodiment, the vinasse further contains nutrients such as carbon source, nitrogen source, inorganic salts and water;

[0023] In one embodiment, the nutrients in the vinasse are: molasses: 2% (w / w), corn starch: 1% (w / w), dipotassium hydrogen phosphate: 0.5% (w / w), and water 50% (w / w).

[0024] In one embodiment, the carbon source is soluble starch, molasses or glucose; the nitrogen source is ammonium chloride, ammonium sulfate or corn starch; the inorganic salt is magnesium sulfate, sodium chloride or potassium dihydrogen phosphate;

[0025] In one embodiment, the added amount of the carbon source is: 1-3% (w / w), the added amount of the nitrogen source is: 1-3% (w / w), the added amount of the inorganic salt is: 0.1-1.0% (w / w), and the added amount of water is 40-60% (w / w).

[0026] In one embodiment, the amount of the microbial agent added is 8 to 16% by mass to volume ratio;

[0027] In one embodiment, the fermentation conditions are: temperature 20-35° C., and fermentation time at least 36 hours.

[0028] The present invention also provides a method for increasing the crude protein, crude fat and amino acid content in fermented distiller's grains feed, wherein the method comprises adding a microbial agent to the distiller's grains for fermentation.

[0029] In one embodiment, the vinasse further contains nutrients such as carbon source, nitrogen source, inorganic salts and water;

[0030] In one embodiment, the carbon source is soluble starch, molasses or glucose; the nitrogen source is ammonium chloride, ammonium sulfate or corn starch; the inorganic salt is magnesium sulfate, sodium chloride or potassium dihydrogen phosphate;

[0031] In one embodiment, the added amount of the carbon source is: 1-3% (w / w), the added amount of the nitrogen source is: 1-3% (w / w), the added amount of the inorganic salt is: 0.1-1.0% (w / w), and the added amount of water is 40-60% (w / w).

[0032] In one embodiment, the nutrients in the vinasse are: molasses: 2% (w / w), corn starch: 1% (w / w), dipotassium hydrogen phosphate: 0.5% (w / w), and water 50% (w / w).

[0033] In one embodiment, the amount of the microbial agent added is 8 to 16% by mass to volume ratio;

[0034] In one embodiment, the fermentation conditions are: temperature 20-35° C., and fermentation time at least 36 hours.

[0035] The present invention also provides the use of the above-mentioned microbial agent in preparing fermented distiller's grains feed or in increasing the crude protein, crude fat, and amino acid content in fermented distiller's grains feed, or in preparing products containing crude protein, crude fat, and amino acids in fermented distiller's grains feed.

[0036] Beneficial effects

[0037] (1) The feed yeast provided by the present invention can effectively increase the crude protein content in the vinasse by fermenting the vinasse at a temperature of 30°C, and the crude protein content is increased by about 2%.

[0038] (2) The present invention further improves the crude protein content in the fermented lees by inoculating yeast and Bacillus subtilis, Bifidobacterium, Enterococcus faecalis and Corynebacterium glutamicum. In addition, yeast is cultured separately with Bacillus subtilis, Bifidobacterium, Enterococcus faecalis and Corynebacterium glutamicum for mixed fermentation. The protein content after fermentation is the highest, which is 22.47%, and the crude protein content is increased by about 7%.

[0039] (3) The present invention effectively reduces the cellulose and hemicellulose in the lees through mixed fermentation. The cellulose content in the raw material is 21.45%, and the hemicellulose content is 16.84%. The cellulose content in the experimental group is reduced to 16.31%, a decrease of about 6%, and the hemicellulose content is reduced to 14.34%, a decrease of about 2.5%. The flavor and palatability of the feed are significantly improved.

[0040] (4) The present invention increases the amino acid content in the lees through mixed fermentation, with the lysine content increasing by 108.33% and the threonine content increasing by 59.09%. DETAILED DESCRIPTION

[0041] The molasses involved in the following examples were purchased from Sugarcane Molasses Source Company, corn starch was purchased from Shandong Tongsheng Co., Ltd., and dipotassium hydrogen phosphate was purchased from China National Pharmaceutical Co., Ltd.

[0042] The strain information involved in the following examples is as follows:

[0043] The yeast is Saccharomyces cerevisiae, with a deposit number of CCTCC NO: M 2023098, which is recorded in the Chinese invention patent application with publication number CN116144516A; the Bacillus subtilis has a deposit number of CCTCC NO: M2021168, which is recorded in the Chinese invention patent application with publication number CN 113061555A; and the Corynebacterium glutamicum has a deposit number of CCTCC NO: M 2023742, which is recorded in the Chinese invention patent application with publication number CN116656566A.

[0044] The bifidobacterium and enterococcus faecalis were purchased from China Industrial Microorganism Culture Collection Center; the bifidobacterium was animal bifidobacterium, specifically animal bifidobacterium lactis subsp. lactis, numbered: CICC 24672, and the enterococcus faecalis was numbered: CICC 21869.

[0045] The detection methods involved in the following embodiments are as follows:

[0046] Crude protein content detection:

[0047] Refer to GB T 6432-2018 Determination of crude protein in feed.

[0048] Detection of cellulose content

[0049] Refer to the acid and alkali washing method in GB T 6434-2022 Determination of crude cellulose.

[0050] The culture medium involved in the following examples is as follows:

[0051] YPD medium (1 L): 10 g yeast extract powder, 20 g peptone, 20 g glucose, 1000 mL distilled water, pH 6.6-7.0.

[0052] LB medium (1 L): yeast extract powder 5 g, peptone 10 g, sodium chloride 10 g.

[0053] LBGB medium (1 L): peptone 10 g, yeast extract 5 g, glucose 5 g, sodium chloride 10 g, brain heart infusion 18.5 g.

[0054] Beef extract peptone medium (1 L): 3 g beef extract, 10 g peptone, 15 g sodium chloride, 1000 mL distilled water, pH 7.0-7.2, autoclave at 121°C for 20 min.

[0055] Wine lees culture medium (1 L): crushed dry wine lees 200 g, glucose 10 g, distilled water 1000 mL, sterilize at 121 ° C for 10 min, and set aside.

[0056] MRS medium (1 L): peptone 10.0 g, beef extract 10.0 g, yeast powder 5.0 g, glucose 20.0 g, sodium acetate 5.0 g, diammonium citrate 2.0 g, Tween 80 1.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, distilled water 1000 mL, adjust pH to 6.8, and autoclave at 121°C for 30 min.

[0057] Secondary seed culture medium (1 L): 20 g brown sugar, 5 g corn starch, 5 g (NH4)2HPO4, 1 g K2HPO4·3H2O, 1 g KH2PO4; sterilize at 115°C for 15 min.

[0058] The preparation method of the bacterial solution of the strain involved in the following examples is as follows:

[0059] Preparation of brewer's yeast culture:

[0060] Take 100 mL of the glycerol tube bacterial solution and add it to 30 mL of YPD liquid medium. Incubate in a shaker at 30°C and 220 rpm for 12 h to obtain a bacterial concentration of: OD 600 =12 brewer's yeast liquid.

[0061] Preparation of Bacillus subtilis culture solution:

[0062] Take 100 mL of the glycerol tube bacterial solution and add it to 30 mL of LB liquid medium. Incubate in a shaker at 37°C and 220 rpm for 12 h to obtain a bacterial concentration of: OD 600 =12 of Bacillus subtilis liquid.

[0063] Preparation of Corynebacterium glutamicum bacterial solution:

[0064] Take 100 mL of the glycerol tube bacterial solution and add it to 30 mL of LBGB liquid medium. Incubate in a shaker at 30°C and 220 rpm for 12 h to obtain a bacterial concentration of: OD 600 =12 of Corynebacterium glutamicum bacterial liquid.

[0065] Preparation of Bifidobacterium and Enterococcus faecalis bacterial liquid:

[0066] Take 100 mL of glycerol tube bacterial solution and add it to 30 mL of MRS liquid medium, culture it in a shaker at 37°C and 220 rpm for 12 h, and get the bacterial concentration as follows: OD 600 =10 Bifidobacterium bacterial solution and bacterial concentration are: OD 600 =10 Enterococcus faecalis liquid.

[0067] The preparation method of the air-dried vinasse involved in the following examples:

[0068] The wet lees are dried under hot air at 65°C to evaporate moisture, and the moisture content of the dried lees is about 10%.

[0069] Example 1: Screening of vinasse fermentation strains

[0070] The specific steps are as follows:

[0071] 1. Preparation of different culture media

[0072] The formula of the culture medium (Table 1) is as follows:

[0073] Based on 1000 g of air-dried distiller's grains, carbon source: 1-3% (w / w), nitrogen source: 1-3% (w / w), inorganic salt: 0.1-1.0% (w / w), moisture 40-60% (w / w);

[0074] Table 1: Medium formulation for distiller's grains fermentation

[0075]

[0076] 2. Effects of different culture media on strains

[0077] 10 mL of saccharomyces cerevisiae solution, Bacillus subtilis solution, Corynebacterium glutamicum solution, Bifidobacterium solution, and Enterococcus faecalis solution were inoculated into the culture medium obtained in step 1 and cultured for 48 h (culture conditions: Saccharomyces cerevisiae: 30 ° C; Bacillus subtilis, Enterococcus faecalis, Bifidobacterium: 37 ° C; Corynebacterium glutamicum: 33 ° C) to prepare a culture solution. The bacterial concentration of the prepared culture solution was detected, and the results are shown in the following table:

[0078] Table 2: Effect of the formula of the culture medium for distiller's grains expansion on the growth of the tested strains (unit: ×10 9 CFU / g)

[0079]

[0080] The results show:

[0081] The optimal conditions are molasses: 2% (w / w), corn starch: 1% (w / w), dipotassium hydrogen phosphate: 0.5% (w / w), and water 50% (w / w);

[0082] 3. Effects of different strains on crude protein in distiller's grains under optimal conditions

[0083] The specific steps are as follows:

[0084] Based on 1000g of air-dried lees, add 20g of molasses, 10g of corn starch, and 5g of dipotassium hydrogen phosphate (control the pH to be 4.8-5.3), and keep the total moisture content (calculated according to the total weight of the culture medium) at 50%.

[0085] Saccharomyces cerevisiae, Bacillus subtilis, Corynebacterium glutamicum, Bifidobacterium, and Enterococcus faecalis were fermented at 100 ml of each culture under the conditions described in Table 3. After fermentation, the samples were dried at 65° C., crushed, and the nutrient content after fermentation was determined.

[0086] Table 3: Distillers grains feed fermented by different strains

[0087]

[0088]

[0089] The results show:

[0090] (1) The crude protein content of different strains after fermentation is shown in Table 4:

[0091] Table 4: Crude protein content before and after fermentation

[0092]

[0093] (2) Crude protein content, cellulose and hemicellulose content Table 4 shows that the protein content has been significantly improved. Among them, the fermentation effect of feed brewer's yeast is the best, with the protein content increased by about 1%, cellulose decreased by about 0.5%, and hemicellulose decreased by about 0.5%. However, in the fermentation of other strains, the protein content has increased, and the cellulose and hemicellulose contents have decreased. In the subsequent experiments, feed yeast was selected as one of the starting strains for mixed fermentation. Considering that the fermentation strains have their own advantages in increasing protein content, the next experiment was carried out through mixed fermentation.

[0094] Example 2: Double-bacteria fermentation of distiller's grains feed

[0095] The specific steps are as follows:

[0096] Taking 1000 g of air-dried distiller's grains as an example, add 20 g of molasses, 10 g of corn starch, and 5 g of dipotassium hydrogen phosphate (control the pH to be 4.8-5.3), maintain the total moisture content (calculated based on the total weight of the culture medium) at 50%, and ferment at a temperature of 30°C for 48 h.

[0097] The fermentation strains were mixed at a volume ratio of 1:1, and the amount of bacteria added was 100 ml (i.e., 50 ml of each strain was added). After mixing, they were inoculated into the above-mentioned culture medium and fermented under the conditions described in Table 5. After completion, the samples were dried at 65°C, crushed, and the nutrient content after fermentation was determined.

[0098] Table 5: Distillers grains feed fermented with two strains

[0099]

[0100]

[0101] The crude protein content of different strains after fermentation is shown in Table 6:

[0102] Table 6: Crude protein content before and after fermentation

[0103]

[0104] It can be seen that the crude protein content is optimal after fermentation using the combination of Bacillus subtilis and Saccharomyces cerevisiae. The nutrient content of this group before and after fermentation was tested, and the results are shown in Table 7:

[0105] Table 7: Nutrient content before and after fermentation

[0106]

[0107] The crude protein content of the distiller's grains raw material is 14.82%, the cellulose content is 21.45%, and the hemicellulose content is 16.84%. After 48 hours of mixed fermentation, the highest crude protein content in the distiller's grains is 16.84%, the cellulose content is 20.08%, and the hemicellulose content is 16.19%.

[0108] Example 3: Three-bacteria fermentation of distiller's grains feed

[0109] The specific steps are as follows:

[0110] Taking 1000 grams of air-dried distiller's grains as an example, add 20 grams of molasses, 10 grams of corn starch, and 5 grams of dipotassium hydrogen phosphate (control the pH to be: 5.4-6.0), the amount of culture added is 100 ml, the total moisture content is maintained at 50%, the fermentation temperature is: 30°C, and the time is 48 hours.

[0111] The fermentation strains were mixed at a volume ratio of 1:1:1 (i.e., 33.33 ml of each strain was added). After mixing, the mixture was inoculated into the above-mentioned culture medium and fermented under the conditions described in Table 8. After fermentation, the samples were dried at 65°C, pulverized, and the nutrient content after fermentation was determined.

[0112] Table 8: Distillers grains feed fermented by three strains

[0113]

[0114] The crude protein content of different strains after fermentation is shown in Table 9:

[0115] Table 9: Crude protein content before and after fermentation

[0116]

[0117] It can be seen that the crude protein content after fermentation with the combination of Bacillus subtilis, yeast, and Enterococcus faecalis is the best. The nutrient content of this group before and after fermentation was tested, and the results are shown in Table 10:

[0118] Table 10: Nutrient content before and after fermentation

[0119]

[0120] The crude protein content of the distiller's grains raw material is 14.82%, the cellulose content is 21.45%, and the hemicellulose content is 16.84%. After 48 hours of mixed fermentation, the highest crude protein content in the distiller's grains is 18.92%, the cellulose content is 18.37%, and the hemicellulose content is 15.47%.

[0121] Example 4: Four-bacteria fermentation of distiller's grains feed

[0122] The specific steps are as follows:

[0123] Taking 1000 grams of air-dried distiller's grains as an example, add 20 grams of molasses, 10 grams of corn starch, and 5 grams of dipotassium hydrogen phosphate (control the pH to be: 5.4-6.0), the amount of culture added is 100 ml, the total moisture content is maintained at 50%, the fermentation temperature is: 30°C, and the time is 48 hours.

[0124] The fermentation strain broths were mixed at a volume ratio of 1:1:1:1 (i.e., 25 ml of each strain was added), inoculated into the above-mentioned culture medium, and fermented under the conditions described in Table 11. After completion, the samples were dried at 65°C, crushed, and the nutrient content after fermentation was determined.

[0125] Table 11: Distillers grains feed fermented by four strains

[0126]

[0127] The crude protein content after fermentation of different strains is shown in Table 12:

[0128] Table 12: Crude protein content before and after fermentation

[0129]

[0130] It can be seen that the crude protein content after fermentation with the combination of Bacillus subtilis, yeast, Enterococcus faecalis, and Bifidobacterium is the best. The nutrient content of this group before and after fermentation was tested, and the results are shown in Table 13:

[0131] Table 13: Nutrient content before and after fermentation

[0132]

[0133] The crude protein content of the distiller's grains raw material is 14.82%, the cellulose content is 21.45%, and the hemicellulose content is 16.84%. After 48 hours of mixed fermentation, the highest crude protein content in the distiller's grains is 19.97%, the cellulose content is 17.42%, and the hemicellulose content is 14.79%.

[0134] Example 5: Five-bacteria fermentation of distiller's grains feed

[0135] 1. Screening of fermentation conditions:

[0136] (1) Set the inoculum volume of different bacterial strains. The total inoculum volume is: 8% (v / v), 10% (v / v), 12% (v / v), and 16% (v / v). The amount of each strain added is shown in the following table:

[0137] Table 14: Selection of strain inoculum volume (v / v)

[0138]

[0139] (2) Based on 1000 g of air-dried lees, add 20 g of molasses, 10 g of corn starch, and 5 g of dipotassium hydrogen phosphate (control the pH to be 4.8-5.3), and maintain the total moisture content (calculated based on the total weight of the culture medium) at 50%.

[0140] According to the addition amount of each bacterial species in Table 14, different bacterial liquids were weighed and mixed, and then inoculated into the above-mentioned culture medium. Fermentation was carried out according to the culture conditions in Table 15. After the fermentation, the samples were dried at 65°C, crushed, and the crude protein content after fermentation was determined; the results are shown in Table 15.

[0141] Table 15: Effects of different fermentation conditions on crude protein content after fermentation

[0142]

[0143] The results show:

[0144] The inoculum size of the strain was 10% (v / m, i.e., mL / 100 g), the inoculum volume ratio was 1:1:1:1:1, the moisture content was 50%, and the fermentation temperature was 30° C., which were the optimal conditions.

[0145] 2. Under optimal conditions, nutrient content before and after fermentation

[0146] Taking 1000 grams of air-dried distiller's grains as an example, 20 grams of molasses, 10 grams of corn starch, and 5 grams of dipotassium phosphate were added (controlling the pH at 5.4-6.0). The bacterial strain was added in an amount of 100 milliliters, the total moisture content was maintained at 50%, the fermentation temperature was 30°C, and the fermentation time was 48 hours. The fermentation strains (Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Enterococcus faecalis, and Bifidobacterium animalis) were mixed in a volume ratio of 1:1:1:1:1 (i.e., 20 milliliters of each strain was added), and fermentation was carried out according to the conditions in Table 16.

[0147] Table 16: Distillers grains feed fermented by five strains

[0148]

[0149] The results of fermentation of distiller's grains feed by composite strains are shown in the table below:

[0150] Table 17: Nutrient content before and after fermentation

[0151]

[0152] Table 18: Changes in amino acids in distillers grains feed before and after fermentation

[0153]

[0154] The crude protein content of the distiller's grains raw material is 14.82%, the cellulose content is 21.45%, and the hemicellulose content is 16.84%. After 48 hours of mixed fermentation, the highest crude protein content in the distiller's grains is 22.47%, the cellulose content is 16.31%, and the hemicellulose content is 14.34%.

[0155] Before and after fermentation, the amino acid content in the distiller's grains feed increased. As the limiting amino acid for animals, the lysine content increased from 0.36g / 100g to 0.75g / 100g, an increase of 108.33%, and the threonine content increased from 0.44g / 100g to 0.71g / 100g, an increase of 59.09%.

[0156] Example 6: Factory mass production test

[0157] 1. Taking 10,000 kg of air-dried distiller's grains as an example, add 200 kg of molasses, 100 kg of corn starch, and 50 kg of dipotassium hydrogen phosphate (maintaining pH at 5.4-6.0), maintain the total moisture content at 50%, and ferment at a temperature of 30°C for 48 hours.

[0158] Bacillus subtilis, Corynebacterium glutamicum, yeast, Enterococcus faecalis, and Bifidobacterium bacterial solutions were mixed at a volume ratio of 1:1:1:1:1 (i.e., each bacterial solution was added at 2%), and then inoculated into the above-mentioned culture medium for fermentation. The bacterial strain addition amount was 1000 ml. After fermentation, the samples were dried at 65°C, crushed, and the crude protein content after fermentation was determined; the results are shown in Tables 19 to 21.

[0159] The results show:

[0160] Table 19: Nutrient content before and after fermentation

[0161]

[0162] 2. Since the price of dipotassium hydrogen phosphate is relatively high, calcium carbonate is used instead of dipotassium hydrogen phosphate. The specific method is the same as step 1, except that the dipotassium hydrogen phosphate is replaced with calcium carbonate, and the addition amount is 80 kg.

[0163] The results show:

[0164] Table 20: Nutrient content before and after fermentation

[0165]

[0166] 3. Due to the high ash content, the adjustment plan is the same as step 1, except that the potassium hydrogen phosphate is replaced with calcium carbonate and potassium hydrogen phosphate, with the addition amounts being 10 kg and 40 kg respectively;

[0167] The results show:

[0168] Table 21: Nutrient content before and after fermentation

[0169]

[0170]

[0171] The results showed that adjusting dipotassium hydrogen phosphate to calcium carbonate and dipotassium hydrogen phosphate reduced the ash content and increased the crude protein content compared with using calcium carbonate alone.

[0172] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A microbial agent for increasing the crude protein content in fermented vinasse, characterized in that: The microbial agent contains Bacillus subtilis bacterial liquid, Corynebacterium glutamicum bacterial liquid, Saccharomyces cerevisiae bacterial liquid, Enterococcus faecalis bacterial liquid and Bifidobacterium animalis bacterial liquid.

2. The microbial agent according to claim 1, characterized in that In the microbial agent, the Bacillus subtilis solution, the Corynebacterium glutamicum solution, the Saccharomyces cerevisiae solution, the Enterococcus faecalis solution, and the Bifidobacterium animalis solution are mixed in a ratio of (1-3): (1-3): (1-4): (1-3): (1-3); Preferably, the OD600 of the added Bacillus subtilis bacterial solution is 10-12; the OD600 of the added Corynebacterium glutamicum bacterial solution is 10-12; the OD600 of the added Saccharomyces cerevisiae bacterial solution is 10-12; the OD600 of the added Enterococcus faecalis bacterial solution is 10-12; and the OD600 of the added Bifidobacterium animalis bacterial solution is 10-12.

3. The microbial agent according to claim 1 or 2, characterized in that The cerevisiae yeast is Saccharomyces cerevisiae CCTCC NO: M 2023098, the Bacillus subtilis is Bacillus subtilis CCTCC NO: M 2021168, the Corynebacterium glutamicum is Corynebacterium glutamicum CCTCC NO: M 2023742, the Bifidobacterium animalis is Bifidobacterium animalis CICC24672, and the Enterococcus faecalis is Enterococcus faecalis CICC 21869; Preferably, the method for preparing the brewer's yeast liquid is: inoculating brewer's yeast into a seed culture medium, culturing at 30-35° C. and 200-250 rpm for 10-12 hours to prepare the brewer's yeast liquid; The preparation method of the Bacillus subtilis bacterial liquid is as follows: inoculating Bacillus subtilis into a seed culture medium, culturing at 35-37° C. and 200-250 rpm for 10-12 hours to prepare the Bacillus subtilis bacterial liquid; The preparation method of the Corynebacterium glutamicum bacterial liquid is as follows: Corynebacterium glutamicum is inoculated into a seed culture medium, and cultured at 30-35° C. and 200-250 rpm for 10-12 hours to prepare the Corynebacterium glutamicum bacterial liquid; The preparation methods of the animal bifidobacterium bacterial liquid and the enterococcus faecalis bacterial liquid are as follows: animal bifidobacterium and enterococcus faecalis are inoculated into seed culture medium respectively, and cultured at 35-37° C. and 200-250 rpm for 10-12 hours to prepare animal bifidobacterium bacterial liquid and enterococcus faecalis bacterial liquid respectively.

4. A fermented distiller's grains feed, characterized in that: The fermented distiller's grains feed is obtained by adding the microbial agent according to any one of claims 1 to 3 to distiller's grains and fermenting them.

5. The fermented distiller's grains feed according to claim 4, characterized in that The vinasse also contains a carbon source, a nitrogen source, inorganic salts and water; Preferably, the carbon source is soluble starch, molasses or glucose; the nitrogen source is ammonium chloride, ammonium sulfate or corn starch; and the inorganic salt is magnesium sulfate, sodium chloride or potassium dihydrogen phosphate; Preferably, the amount of the carbon source added is 1-3% (w / w), the amount of the nitrogen source added is 1-3% (w / w), and the amount of the inorganic salt added is: 0.1~1.0% (w / w), the added amount of water is 40~60% (w / w).

6. The fermented distiller's grains feed according to claim 4 or 5, characterized in that The amount of the microbial agent added is 8 to 16% by mass to volume ratio; Preferably, the fermentation conditions are: temperature 20-35° C., and fermentation time at least 36 hours.

7. A method for increasing the crude protein, crude fat and amino acid content in fermented distiller's grains feed, characterized in that: The method comprises adding the microbial agent according to any one of claims 1 to 3 to wine lees for fermentation.

8. The method according to claim 7, characterized in that The vinasse also contains a carbon source, a nitrogen source, inorganic salts and water; Preferably, the carbon source is soluble starch, molasses or glucose; the nitrogen source is ammonium chloride, ammonium sulfate or corn starch; and the inorganic salt is magnesium sulfate, sodium chloride or potassium dihydrogen phosphate; Preferably, the amount of the carbon source added is 1-3% (w / w), the amount of the nitrogen source added is 1-3% (w / w), and the amount of the inorganic salt added is: 0.1~1.0% (w / w), the added amount of water is 40~60% (w / w).

9. The method according to claim 7 or 8, characterized in that The amount of the microbial agent added is 8 to 16% by mass to volume ratio; Preferably, the fermentation conditions are: temperature 20-35° C., and fermentation time at least 36 hours.

10. Use of the microbial agent according to any one of claims 1 to 3 in preparing fermented distiller's grains feed or in increasing the crude protein, crude fat, and amino acid content in fermented distiller's grains feed, or in preparing a product for increasing the crude protein, crude fat, and amino acid content in fermented distiller's grains feed.

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