Fermented distiller's grains, preparation method therefor, and use thereof

Abstract

The present invention provides fermented distiller's grains, a preparation method therefor, and a use thereof. In the method, encapsulated Rhodotorula glutinis powder and a bacterial liquid of Bacillus velezensis capable of efficiently degrading crude fiber are inoculated into distiller's grains for mixing and fermentation to obtain fermented distiller's grains. A solid-state fermentation process enables the encapsulated Rhodotorula glutinis powder to avoid oxidation of astaxanthin produced by same, thereby effectively improving the stability of the astaxanthin and ensuring that the fermented distiller's grains can be stored at room temperature for at least 60 days. Moreover, the content of anti-nutritional factors in the fermented distiller's grains is greatly reduced, and the nutritional content is significantly improved, thereby greatly improving the utilization rate of fermented distiller's grains. Adding the fermented distiller's grains to the non-breeding feed diet of pigs, cattle, and sheep and replacing 40-50% of the diet can effectively reduce the incidence of disease.

Description

Fermented distiller's grains, preparation method and application thereof Technical Field

[0001] The invention belongs to the technical field of animal feed, and particularly relates to fermented distiller's grains, a preparation method and application thereof. Background Art

[0002] my country has a significant production capacity of distiller's grains (DDGs). According to relevant data, national baijiu production reached 6.712 million kiloliters in 2022. Each ton of base liquor produces approximately 3 tons of DDGs, resulting in a DDG output exceeding 20 million tons in 2022. However, due to factors such as the high lignocellulose content in DDGs, direct feeding of DDGs to animals is nutritionally poor. Solid-state fermentation can effectively increase the nutritional content of DDGs, thereby promoting their use in animal feed and effectively avoiding environmental pollution caused by DDGs.

[0003] Astaxanthin is a non-provitamin A, fat-soluble carotenoid found primarily in certain microorganisms and marine animals. Its molecular structure primarily consists of a polyene chain and two ketone rings, located at either end of the astaxanthin molecule. This structural molecule can easily penetrate biological membranes and is effective in scavenging free radicals within animals. The unsaturated double bonds in the polyene chain also have excellent antioxidant properties. It also has excellent anti-cancer effects, reduces cardiovascular disease, and regulates the immune system, making it an important feed additive. However, astaxanthin has poor storage stability and is easily oxidized, leading to inactivation. However, existing encapsulation processes present issues with food safety, high costs, and poor stability.

[0004] Therefore, how to improve the stability of astaxanthin to avoid oxidation and apply it well in feed is a technical problem that scientific researchers urgently need to solve.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a fermented wine lees and a preparation method and application thereof in view of the above-mentioned deficiencies in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide a method for preparing fermented distiller's grains, comprising the following specific steps: S1, liquid fermentation of Rhodotorula glutinosus to obtain a first fermentation broth; liquid fermentation of Bacillus velezensis to obtain a second fermentation broth; the Rhodotorula glutinosus was purchased from the China Agricultural Microorganism Culture Collection Center with a strain catalog number of ACCC20030, and the Bacillus velezensis was obtained from the General Microorganism Center of the China Microorganism Culture Collection Committee with a collection number of CGMCC NO: 25202;

[0009] S2. Prepare a gelatin solution, add the first fermentation broth and olive oil thereto, stir evenly, then add sodium alginate, stir at a low temperature of 0-5°C for 15-20 minutes, add calcium chloride, raise the temperature to 55-60°C and continue stirring to obtain an embedded material, filter and wash, vacuum freeze-dry and crush to obtain embedded Rhodotorula glutinosus powder;

[0010] S3. Adding the second fermentation bacterial liquid and the embedded Rhodotorula glutinosus powder to a distiller's grains culture medium for solid-state fermentation to obtain a fermented product, and drying and crushing the fermented product at low temperature to obtain fermented distiller's grains.

[0011] Furthermore, in step S1, the Rhodotorula glutinosus is fermented and cultured in a first liquid culture medium at 28° C. and 180 rpm to 200 rpm for 4 to 5 days, wherein the first liquid culture medium comprises: 4 wt.% citric acid, 1.5 wt.% beef extract, 0.1 wt.% potassium dihydrogen phosphate, 0.04 wt.% magnesium sulfate heptahydrate, 0.04 wt.% sodium chloride, and the balance water; and the pH value of the first liquid culture medium is 6.5.

[0012] Furthermore, the Bacillus velezensis is cultured in LB liquid culture medium at 36° C. and 220 rpm to 250 rpm for 48-72 hours to obtain a first fermentation broth.

[0013] Furthermore, the gelatin is 1-4wt.%, sodium alginate is 1-4wt.%, olive oil is 0.5-7g, and calcium chloride is 1-1.2g.

[0014] Furthermore, the formula of the vinasse culture medium includes: 10-12 g of dry vinasse, 20-25 mL of deionized water, and adjusting the pH value to 7.0 with ammonia water.

[0015] Furthermore, in step S3, the mass ratio of the added amount of the second fermentation bacteria liquid to the said vinasse culture medium is (1.5-2 mL):10 g, and the mass ratio of the added amount of the embedded Rhodotorula glutinosus powder to the said vinasse culture medium is (0.1-0.2 g):10 g.

[0016] Furthermore, in step S3, the solid-state fermentation temperature is 30° C., and the culture is carried out on a shaking table at 180 rpm to 200 rpm for 7 to 8 days.

[0017] The second object of the present invention is to provide fermented wine grains prepared by the above method.

[0018] The third object of the present invention is to provide the use of the fermented distiller's grains in animal feed, wherein the fermented distiller's grains are added to a basic feed and fed as animal feed.

[0019] Furthermore, the fermented distiller's grains are added to the non-breeding feed rations of pigs, cattle and sheep, replacing 40-50% of the rations.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a fermented vinasse, a preparation method, and an application thereof. This method involves inoculating embedded Rhodotorula glutinosus powder and a bacterial solution of Bacillus velezensis, which is highly efficient in degrading crude fiber, into vinasse for mixed fermentation to obtain fermented vinasse. The solid-state fermentation process allows the embedded Rhodotorula glutinosus solution to prevent oxidation of the astaxanthin produced thereby, thereby effectively improving the stability of the astaxanthin and ensuring that the fermented vinasse can be stored at room temperature for at least 60 days. Furthermore, the content of anti-nutritional factors in the fermented vinasse is significantly reduced, and the nutritional content is significantly improved, thereby greatly improving the utilization rate of the fermented vinasse.

[0022] (2) The fermented distiller's grains provided by the present invention contain high-quality protein and plant functional factors such as polyphenols, flavonoids, and terpenoids. Through microbial fermentation, an organic combination of composite probiotics, flavonoids, terpenoids, astaxanthin, etc. is achieved, which can greatly enhance the antioxidant capacity of organisms. Adding the fermented distiller's grains provided by the present invention to the non-breeding feed diet of pigs, cattle, and sheep, replacing 40-50% of the diet, can effectively reduce the incidence of disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a comparison of the main component contents of lees before and after fermentation. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention are described in further detail below in conjunction with specific examples and accompanying drawings. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0025] The dry distiller's grains used in this embodiment refer to sorghum dry distiller's grains, which are provided by Anhui Gujing Gongjiu Co., Ltd.

[0026] It's important to note that the nutritional properties of sorghum distillers dried grains (DDGS) are primarily composed of insoluble starch polysaccharides such as low-fermentable arabinoxylan, cellulose, and lignin, along with high levels of fiber. The quality of DDGS depends on factors such as crop quality, fermentation process, and drying temperature and time. Compared to corn distillers dried grains, sorghum distillers dried grains contain higher levels of crude protein, neutral detergent fiber, and acid detergent fiber.

[0027] The Bacillus velezensis used in the present invention is classified as Bacillus velezensis and has been deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms. The deposit date is June 27, 2022, and the deposit number is CGMCC No. 25202.

[0028] The Rhodotorula glutinosus used in the present invention was purchased from the China Agricultural Microbial Culture Collection Center, with a strain catalog number of ACCC20030.

[0029] Example 1

[0030] This embodiment provides a method for preparing fermented distiller's grains.

[0031] The specific steps are as follows:

[0032] 1. Liquid fermentation of Rhodotorula glutinosus to obtain a first fermentation liquid.

[0033] (1) Preparation of fermentation medium: 4% citric acid, 1.5% beef extract, 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 0.04% sodium chloride and the balance water; the pH value of the liquid fermentation medium is 6.5.

[0034] (2) The Rhodotorula glutinosus ACCC20030 was cultured in a shaking incubator at 28°C and 180 rpm for 4 days.

[0035] 2. Liquid fermentation of Bacillus velezensis to obtain a second fermentation liquid.

[0036] (1) LB liquid culture medium: 10 g / L fish meal peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0037] (2) Bacillus velezensis CGMCC NO: 25202 was cultured in a shaking incubator at 36°C and 220 r / min for 48 h.

[0038] 3. Preparation of embedded Rhodotorula mucilaginosa powder

[0039] (1) Place a known volume of 70 mL of 4% gelatin solution in a beaker and stir thoroughly with a magnetic stirrer at approximately 60°C. This temperature remains constant throughout the experiment.

[0040] (2) Then, add 1 mL of olive oil and 0.1 g of the first fermentation broth. Add the bacterial agent to be embedded and stir evenly. Add 10 mL of 4% sodium alginate to achieve complete phase separation. In the experiment, the ratio of gelatin to sodium alginate was 3.5:1, and the pH was maintained at 3.75. Add ice cubes and lower the temperature to 0°C to 5°C and stir for 15 minutes. Add 1 g of calcium chloride, then raise the temperature to 60°C and continue stirring. Filter and wash to obtain the embedded material.

[0041] (3) The embedded material is freeze-dried in vacuum and then crushed to obtain embedded Rhodotorula glutinosus powder.

[0042] 4. Preparation of fermented lees

[0043] (1) Preparation of vinasse culture medium: 10 g dry vinasse, 20 mL deionized water, and pH adjusted to 7.0 with aqueous ammonia.

[0044] (2) Add 0.1 g of embedded Rhodotorula glutinosus powder and 2 mL of the second fermentation solution to the vinasse culture medium, and culture at 30° C. and 180 rpm for 6 days to obtain a fermentation product, which is then dried at low temperature and crushed to obtain fermented vinasse.

[0045] Example 2

[0046] This embodiment provides a method for preparing fermented distiller's grains.

[0047] The specific steps are as follows:

[0048] 1. Liquid fermentation of Rhodotorula glutinosus to obtain a first fermentation liquid.

[0049] (1) Preparation of fermentation medium: 4% citric acid, 1.5% beef extract, 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 0.04% sodium chloride, and the balance water; the pH value of the liquid fermentation medium is 6.5.

[0050] (2) The Rhodotorula glutinosus ACCC20030 was cultured in a shaking incubator at 28°C and 190 rpm for 4.5 days.

[0051] 2. Liquid fermentation of Bacillus velezensis to obtain a second fermentation liquid.

[0052] (1) LB liquid culture medium: 10 g / L fish meal peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0053] (2) Bacillus velezensis CGMCC NO: 25202 was cultured in a shaking incubator at 36°C and 230 rpm for 36 h.

[0054] 3. Preparation of embedded Rhodotorula mucilaginosa powder

[0055] (1) Place a known volume of 70 mL of 4% gelatin solution in a beaker and stir thoroughly with a magnetic stirrer at approximately 60°C. This temperature remains constant throughout the experiment.

[0056] (2) Then, add 1 mL of olive oil and 0.1 g of the first fermentation broth. Add the bacterial agent to be embedded and stir evenly. Add 10 mL of 4% sodium alginate to achieve complete phase separation. In the experiment, the ratio of gelatin to sodium alginate was 3.5:1, and the pH was maintained at 3.75. Add ice cubes and lower the temperature to 0°C to 5°C and stir for 15 minutes. Add 1 g of calcium chloride, then raise the temperature to 60°C and continue stirring. Filter and wash to obtain the embedded material.

[0057] (3) The embedded material is freeze-dried in vacuum and then crushed to obtain embedded Rhodotorula glutinosus powder.

[0058] 4. Preparation of fermented lees

[0059] (1) Preparation of vinasse culture medium: 11 g dry vinasse, 23 mL deionized water, and pH adjusted to 7.0 with aqueous ammonia.

[0060] (2) Add 0.15 g of embedded Rhodotorula glutinosus powder and 1.5 mL of the second fermentation solution to the vinasse culture medium, and culture at 30° C. and 190 rpm for 7 days to obtain a fermentation product, which is then dried at low temperature and crushed to obtain fermented vinasse.

[0061] Example 3

[0062] This embodiment provides a method for preparing fermented distiller's grains.

[0063] The specific steps are as follows:

[0064] 1. Liquid fermentation of Rhodotorula glutinosus to obtain a first fermentation liquid.

[0065] (1) Preparation of fermentation medium: 4% citric acid, 1.5% beef extract, 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 0.04% sodium chloride, and the balance water; the pH value of the liquid fermentation medium is 6.5.

[0066] (2) The Rhodotorula glutinosus ACCC20030 was cultured in a shaking incubator at 28°C and 200 r / min for 5 days.

[0067] 2. Liquid fermentation of Bacillus velezensis to obtain a second fermentation liquid.

[0068] (1) LB liquid culture medium: 10 g / L fish meal peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0069] (2) Bacillus velezensis CGMCC NO: 25202 was cultured in a shaking incubator at 36°C and 250 rpm for 72 h.

[0070] 3. Preparation of embedded Rhodotorula mucilaginosa powder

[0071] (1) Place a known volume of 70 mL of 4% gelatin solution in a beaker and stir thoroughly with a magnetic stirrer at approximately 60°C. This temperature remains constant throughout the experiment.

[0072] (2) Then, add 1 mL of olive oil and 0.1 g of the first fermentation broth. Add the bacterial agent to be embedded and stir evenly. Add 10 mL of 4% sodium alginate to achieve complete phase separation. In the experiment, the ratio of gelatin to sodium alginate was 3.5:1, and the pH was maintained at 3.75. Add ice cubes and lower the temperature to 0°C to 5°C and stir for 15 minutes. Add 1 g of calcium chloride, then raise the temperature to 60°C and continue stirring. Filter and wash to obtain the embedded material.

[0073] (3) The embedded material is freeze-dried in vacuum and then crushed to obtain embedded Rhodotorula glutinosus powder.

[0074] 4. Preparation of fermented lees

[0075] (1) Preparation of vinasse culture medium: 12 g dry vinasse, 25 mL deionized water, and pH adjusted to 7.0 with aqueous ammonia.

[0076] (2) Add 0.2 g of embedded Rhodotorula glutinosus powder and 1.8 mL of the second fermentation solution to the vinasse culture medium, culture at 30° C. and 200 r / min for 8 days to obtain a fermentation product, and dry the fermentation product at low temperature to obtain fermented vinasse.

[0077] In order to better illustrate the ability of the fermented vinasse prepared by the present invention to improve the stability and functional activity of astaxanthin, the applicant conducted the following research:

[0078] The embedded Rhodotorula mucilaginosa powders prepared in Examples 1-3 have similar structures and functions, and will be described in detail using Example 1 as an example.

[0079] 1. Determination of astaxanthin content in embedded Rhodotorula mucilaginosa powder.

[0080] Determination of astaxanthin embedding efficiency:

[0081] To prepare an astaxanthin standard curve: Accurately weigh 10 mg of astaxanthin standard and thoroughly dissolve it in a small amount of dichloromethane. Dissolve it in chromatographic and methanol solutions to prepare a 0.1 mg / mL astaxanthin stock solution. Take an appropriate amount of the stock solution and dilute it with chromatographically grade methanol to prepare astaxanthin standard solutions at concentrations of 1, 2, 3, 4, 6, 8, and 10 μg / mL. To determine the astaxanthin content in microencapsulated ...

[0082] Detection of astaxanthin content on the surface of embedded Rhodotorula glutinosus powder: Accurately weigh 1.0 g of embedded Rhodotorula glutinosus powder, add 40 mL of n-hexane, shake thoroughly to mix until the solution becomes colorless, centrifuge, discard the supernatant, quickly evaporate to dryness, take appropriate amount of acetone to dissolve, measure the absorbance at 480 nm, the average value of which is 0.945.

[0083] Embedding rate = (astaxanthin content in the embedded Rhodotorula glutinosus powder - astaxanthin content on the surface of the embedded Rhodotorula glutinosus powder) / astaxanthin content in the embedded Rhodotorula glutinosus powder × 100% = 51.6%.

[0084] 2. Study on the stability of astaxanthin in encapsulated Rhodotorula mucilaginosa powder.

[0085] Five 50ml test tubes were labeled 1#, 2#, 3#, 4#, and 5#. 1# contained an unembedded Rhodotorula glutinosus control tube; 2# contained an unembedded Rhodotorula glutinosus control tube after five days of room temperature storage; 3# contained embedded Rhodotorula glutinosus tube; 4# contained embedded Rhodotorula glutinosus tube after five days of room temperature storage; and 5# was a blank tube (no Rhodotorula glutinosus was added during embedding to offset the error introduced by the embedding material). The above method was used to test the embedded Rhodotorula glutinosus powder. The results are shown in Table 1.

[0086] Table 1. Astaxanthin stability experimental data

[0087] As shown in Table 1, astaxanthin is more stable after encapsulation, while unencapsulated astaxanthin loses almost all its activity after five days. The inactivation rate of unencapsulated astaxanthin is 92.9%, while that of encapsulated astaxanthin is only 30.2%. The effect is quite significant, while the stability of the encapsulated astaxanthin is 69.8%.

[0088] 3. Study on the changes of the main components in the lees before and after fermentation

[0089] 3.1 Study on the changes of total phenol content in wine lees before and after fermentation.

[0090] Preparation of total phenol test solution: Pulverize the pre- and post-fermentation lees. Weigh 1g of each lees, add 10mL of 70% ethanol, and centrifuge at 4000 rpm for 15 minutes in an ultrasonicator for 1 hour. Collect the supernatant, and extract the residue twice as described above. Finally, dilute to 30mL with distilled water. Total phenol content was determined using the Folin-phenol colorimetric (FC) method.

[0091] Referring to FIG2 , the total phenol content of the vinasse before fermentation was 1.228 μg / mL, and the total phenol content of the vinasse after fermentation was 1.477 μg / mL, and the total phenol content increased by 20.3%.

[0092] 3.2 Study on the changes in total triterpenoid content in wine grains before and after fermentation.

[0093] Preparation of total triterpene test solution: Pulverize the pre- and post-fermentation lees. Weigh 1g of each lees, add 10mL of 70% ethanol, and centrifuge at 4000 rpm for 15 minutes in an ultrasonicator for 1 hour. Collect the supernatant, and extract the residue twice as described above. Finally, dilute to 30mL with distilled water. Determine the intracellular triterpene content using the vanillin-glacial acetic acid method, using oleanolic acid as the standard.

[0094] Referring to FIG2 , the total triterpene content of the vinasse before fermentation was 2.7 μg / mL, and the total triterpene content of the vinasse after fermentation was 3.2 μg / mL, with the total triterpene content increased by 17.3%.

[0095] 3.3 Study on the changes in total sugar content in wine dregs before and after fermentation.

[0096] Preparation of polysaccharide test solution: Weigh 1g of pre-fermented and post-fermented lees, add 10mL of pure water, and extract at 95°C for 1 hour. Collect the supernatant and repeat the extraction process twice for the residue. Add pure water to 30mL to create the aqueous extract. Add 6mL of the aqueous extract to 24mL of 40% ethanol and incubate at 4°C for 24 hours. Determine total sugar content using the phenol-sulfuric acid method.

[0097] Referring to Figure 2 , the total sugar content of the vinasse before fermentation was 23.8 μg / mL, and the total sugar content of the vinasse after fermentation was 60.5 μg / mL, with the total sugar content increased by 154.2%.

[0098] 3.4 Study on the changes in crude fiber content in wine grains before and after fermentation.

[0099] The results showed that the crude fiber content of the lees before fermentation was 26.4%, while the crude fiber content of the lees after fermentation was 23.7%, with the crude fiber content decreasing by 2.7%.

[0100] 3.5 Study on the changes in crude protein content in distiller's grains before and after fermentation.

[0101] Referring to FIG2 , the crude protein content of the vinasse before fermentation was 14.5%, and the crude protein content of the vinasse after fermentation was 17.2%, an increase of 2.7%.

[0102] 3.6 Study on the changes in volatile basic nitrogen content in wine grains before and after fermentation.

[0103] 1) Sample Preparation: Weigh 1g each of pre- and post-fermentation distiller's grains, add 10mL of pure water, and extract at 95°C for 30min. Collect the supernatant and repeat the extraction process twice for the residue. Make up to 30mL with pure water, let stand, and then centrifuge to obtain the aqueous extract.

[0104] 2) Distillation: Place 5 mL of the sample to be tested in a distillation tube. Add 5 mL of a 10 g / mL magnesium oxide solution and place the sample in a Kjeldahl nitrogen analyzer. Add 10 mL of a 20 g / mL boric acid solution to a 250 mL Erlenmeyer flask and place it at the lower end of the condenser tube. Distill for 5-6 minutes.

[0105] 3) Titration: Use hydrochloric acid standard solution (0.01 mol / L) to titrate the blue-green solution in the conical flask until the solution turns blue-purple, which is the titration endpoint. At the same time, perform a blank test.

[0106] Calculate the volatile basic nitrogen content in the sample = ((v1-v2)xCx14) / (mx5 / 100)x100

[0107] V1: The volume of hydrochloric acid or sulfuric acid standard solution consumed by the sample solution for determination, in mL.

[0108] V2: Volume of blank hydrochloric acid standard solution consumed by the reagent. Unit: mL.

[0109] C: actual concentration of hydrochloric acid, in mol / mL.

[0110] m: mass of the sample, in g.

[0111] The final determination showed that the volatile basic nitrogen content in the lees before fermentation was 80.4 mg / kg, and the volatile basic nitrogen content in the lees after fermentation was 95.6 mg / kg.

[0112] 3.7 Study on the changes in pH value and total acid content in wine dregs before and after fermentation.

[0113] Weigh 1g each of the lees before and after fermentation, add 3-4mL of pure water, extract at 95℃ for 30min, collect the supernatant, and repeat the extraction of the residue twice according to the above method. Use pure water to make up to 10mL, let it stand and then centrifuge to obtain the test solution. Then use a pH meter to measure its pH value. Measure three times and take the average value. Use acid-base titration to determine the total acid content. Use a large-bellied pipette to draw 50.0mL of the sample into a 250mL conical flask, add 2 drops of phenolphthalein indicator (10g / L), and titrate with sodium hydroxide standard titration solution (0.1mol / L) until it turns slightly red, which is the end point.

[0114] Calculation formula: X = (C × V × 60) / 50.0 Where:

[0115] X is the mass concentration of total acid in the sample (calculated as acetic acid), in grams per liter (g / L);

[0116] C——actual concentration of sodium hydroxide standard titration solution, in moles per liter (mol / L);

[0117] V - the volume of sodium hydroxide standard titration solution consumed during titration, in milliliters (mL).

[0118] The pH of the vinasse liquid before fermentation was 4.2, and the total acid content was 11.2 mg / g. The pH of the vinasse liquid after fermentation was 6.0, and the total acid content was 10.9 mg / g.

[0119] 4. Study on the changes of functional activity of distiller's grains before and after fermentation

[0120] 4.1 Determination of antioxidant activity DPPH scavenging rate

[0121] Weigh 1g of pre- and post-fermentation lees, add 10mL of 70% ethanol, sonicate for 1 hour, and centrifuge at 4000 rpm for 15 minutes. Collect the supernatant and extract the residue twice as above. Finally, dilute to 20mL with distilled water to prepare the test solution.

[0122] Preparation of DPPH solution: Accurately weigh 4 mg and dissolve it in 100 mL of anhydrous ethanol to make a 0.04 mg / mL DDPH solution, which is then placed in a brown bottle (prepare for immediate use).

[0123] How to do it:

[0124] 1) Dilute all samples to 7%. Take 2 mL of each 7% pre-fermentation and post-fermentation sample solution and add 2 mL of DDPH solution. Mix well. After standing in the dark at room temperature for 10 minutes, measure the absorbance value A1 at 517 nm. Set up a blank group accordingly: 2 mL of 7% sample solution added with 2 mL of anhydrous ethanol.

[0125] 2) Add 2 mL of pure water to 2 mL of DDPH solution, mix well, and place in the dark at room temperature for 10 min. Measure the absorbance A2 at 517 nm. Set up a blank group with 2 mL of pure water and 2 mL of anhydrous ethanol.

[0126] 3) Use 0.1mg / mL V C As positive control, the samples before and after fermentation were numbered as 1# and 3#, and the blank controls were numbered as 2#, 4#, 0.1mg / mL V C The group number is 5# and the control number is 6#.

[0127] The DDPH clearance formula is: K% = (1-A1 / A2) × 100%

[0128] Table 2. DPPH clearance

[0129] As shown in Table 2, the DDPH clearance rate in the lees after fermentation was 66.7%, while the DDPH clearance rate in the lees before fermentation was only 30%, an increase of 36.7% compared to the DDPH clearance rate before fermentation. This shows that the content of antioxidant substances in the lees after fermentation is increased.

[0130] 4.2 Determination of antioxidant activity ABTS clearance rate

[0131] Preparation of ABTS mother solution: Accurately weigh 0.067g K2S2O8 and dissolve it in 100mL pure water, then weigh 0.384g ABTS and dissolve it in it to make 7mmol / L ABTS mother solution. Then let it stand at room temperature in the dark for 12-16h. The mother solution can be stable for 3-4d.

[0132] Preparation of ABTS working solution: Immediately before use, dilute the ABTS stock solution with anhydrous ethanol so that its absorbance at a wavelength of 734 nm reaches 0.700±0.020. Set up a blank group and use anhydrous ethanol instead.

[0133] How to do it:

[0134] Dilute the pre- and post-fermentation sample solutions to 7%. Take 1 mL of each 7% sample solution and add 2 mL of ABTS working solution. Shake well and measure the absorbance (A) at 734 nm after 20 seconds of reaction. Label the pre- and post-fermentation samples 1# and 2#, respectively, and the 0.1 mg / mL VC group 3#.

[0135] The ABTS clearance formula is: K% = (1-A / 0.700) × 100%

[0136] Table 3. ABTS clearance

[0137] As shown in Table 3, the ABTS clearance rate in the lees after fermentation was 91.7%, while the ABTS clearance rate in the lees before fermentation was only 85.9%, an increase of 5.8% compared to the ABTS clearance rate before fermentation. This shows that the antioxidant content in the lees after fermentation is increased.

[0138] 5. Study on the stability of astaxanthin in fermented distiller's grains.

[0139] Test group: fermented wine lees prepared in Example 1, i.e., sample 1;

[0140] Comparative group: The preparation method was basically the same as that of Example 1, except that the embedded Rhodotorula glutinosus powder was not added. Fermented vinasse was prepared, and then the embedded Rhodotorula glutinosus powder was added in a certain ratio (mass ratio of 10 g:0.1 g) to fermented vinasse. The mixture was physically mixed to obtain Sample 2.

[0141] Sample Preparation: Pulverize Sample 1 and Sample 2 separately, weigh 5g of each, add 10mL of 70% ethanol, sonicate for 1 hour, and centrifuge at 4000 rpm for 15 minutes. Collect the supernatant and extract the residue twice as described above. Finally, dilute to 30mL with distilled water for astaxanthin content determination using the same method as above.

[0142] Stability test: Sample 1 and Sample 2 were stored at room temperature, and the astaxanthin content in Sample 1 and Sample 2 was tested at 0, 15, 30, 45, and 60 days to examine the changes.

[0143] Table 4. Stability of astaxanthin in the experimental and control groups

[0144] As shown in Table 4, the data in the table are astaxanthin content values. It can be seen that the astaxanthin content in the fermented lees provided by the experimental group has a small change trend and is relatively stable, indicating that the astaxanthin in the fermented lees is not easily oxidized and can be stored for up to 60 days. However, the astaxanthin content in the physically mixed samples is significantly reduced, indicating that the physical mixing method is not conducive to the storage of embedded astaxanthin.

[0145] The fermented distiller's grains prepared based on the present invention have the above-mentioned functional activities and are rich in astaxanthin. In some embodiments, the fermented distiller's grains can be added to a basic feed and fed as animal feed.

[0146] Example 4

[0147] Ninety healthy pigs weighing approximately 100 kg were randomly divided into three groups and fed different diets at Hongshun Pig Farm in Huaibei City, Anhui Province. After 30 days of feeding on the three different diets, two healthy pigs of similar weight, condition, and age were selected from each group. Appropriate amounts of fresh blood were collected from the ear vein. The collected blood was placed into labeled tubes and centrifuged at 3000 rpm for 15 minutes. The supernatant was aliquoted and stored at 4°C until further use, preservatives (0.01% thimerosal or 0.02% sodium azide, final concentration) were added. Serum total protein, serum albumin, and immunoglobulins were measured using methods similar to those used in the study of the effects of fermented, antibiotic-free feed on growth performance, intestinal flora, blood biochemical parameters, and immune function in weaned piglets. Superoxide dismutase was measured using methods similar to those used in the study of the effects of fermented soybean meal on growth performance, serum biochemical parameters, and intestinal function in weaned piglets. Symptoms of diarrhea, colds, pica, and indigestion were reported. Morbidity rate = (number of affected animals × number of days of onset) / (total number of animals × number of experimental days) × 100%.

[0148] The results are shown in Table 5.

[0149] Table 5 Statistics of feeding results

[0150] Note: Among them, formula A is entirely soybean meal; formula B is soybean meal: unfermented distiller's grains feed weight ratio = 1:1; formula C is soybean meal: fermented distiller's grains weight ratio = 1:1.

[0151] As shown in Table 5, the biochemical indicators of feed formula C showed the highest serum total protein, albumin and superoxide dismutase activity, and the immune indicators and incidence rate were also the most appropriate. Therefore, the mixed fermented feed ratio of soybean meal: fermented distiller's grains = 1:1 was the best.

[0152] Any matters not mentioned above shall be subject to the existing technology.

[0153] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art of the present invention may make various modifications or additions to the described specific embodiments or replace them in similar ways, but they will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. A preparation method of fermented distillers' grains, characterized in that, it includes the following specific steps: S1. Rhodotorula glutinis is subjected to liquid fermentation to obtain a first fermentation broth; Bacillus velezensis is subjected to liquid fermentation to obtain a second fermentation broth; the Rhodotorula glutinis is purchased from the China Center for Agricultural Culture Collection, and the strain catalog number is ACCC20030. The Bacillus velezensis has a preservation number of CGMCC NO: 25202 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; S2. Prepare a gelatin solution, add the first fermentation broth and olive oil thereto, stir evenly, then add sodium alginate, stir at a low temperature of 0-5°C for 15-20 minutes, add calcium chloride, raise the temperature to 55-60°C and continue stirring, filter and wash to obtain an embedding material, and pulverize it after vacuum freeze-drying to obtain embedded Rhodotorula glutinis powder; S3. Add the second fermentation broth and the embedded Rhodotorula glutinis powder to the distillers' grains culture medium for solid-state fermentation to obtain a fermented product, and dry and pulverize the fermented product at a low temperature to obtain the fermented distillers' grains.

2. The preparation method according to claim 1, characterized in that, in step S1, the Rhodotorula glutinis is fermented and cultured on a shaker at 28°C and 180 r / min - 200 r / min in a first liquid medium for 4-5 days. The first liquid medium includes: 4 wt.% citric acid, 1.5 wt.% beef extract, 0.1 wt.% potassium dihydrogen phosphate, 0.04 wt.% magnesium sulfate heptahydrate, 0.04 wt.% sodium chloride, and the balance of water; the pH value of the first liquid medium is 6.

5.

3. The preparation method according to claim 1, characterized in that, in step S1, the Bacillus velezensis is cultured in an LB liquid medium at 36°C and 220 r / min - 250 r / min for 48-72 h to obtain the first fermentation broth.

4. The preparation method according to claim 1, characterized in that, in step S2, the gelatin is 1-4 wt.%, the sodium alginate is 1-4 wt.%, the olive oil is 0.5-7 g, and the calcium chloride is 1-1.2 g.

5. The preparation method according to claim 1, characterized in that, in step S3, the formula of the distillers' grains culture medium includes: 10-12 g of dry distillers' grains, 20-25 mL of deionized water, and the pH value is adjusted to 7.0 with ammonia water.

6. The preparation method according to claim 5, characterized in that, in step S3, the mass ratio of the addition amount of the second fermentation broth to the distillers' grains culture medium is (1.5-2 mL):10 g, and the mass ratio of the addition amount of the embedded Rhodotorula glutinis powder to the distillers' grains culture medium is (0.1-0.2 g):10 g.

7. The preparation method according to claim 6, characterized in that, in step S3, the solid-state fermentation temperature is 30°C, and it is cultured on a shaker at 180 r / min - 200 r / min for 6-8 days.

8. A fermented distillers' grains prepared by the preparation method according to any one of claims 1-7.

9. Use of the fermented distillers grains as described in claim 8 in animal feed, characterized in that, the fermented distillers grains are added to the basal feed and used as animal feed for feeding.

10. The use as described in claim 9, characterized in that, the fermented distillers grains are added to the feed rations for pigs, cattle and sheep during the non-breeding period to replace 40-50% of the rations.

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