CN117467711A - Fermented distiller's grains and preparation method and application thereof - Google Patents

Abstract

The invention provides fermented lees and a preparation method and application thereof. According to the method, the embedded rhodotorula glutinis powder and the bacterial liquid of bacillus bailii for efficiently degrading crude fibers are inoculated into the distillers ' grains for mixed fermentation, so that the fermented distillers ' grains are obtained, and the solid state fermentation technology enables the embedded rhodotorula glutinis powder to avoid the oxidation of astaxanthin produced by the embedded rhodotorula glutinis powder, so that the stability of astaxanthin is effectively improved, and the fermented distillers ' grains are ensured to be stored for at least 60 days at normal temperature. And the anti-nutritional factor content in the prepared fermented vinasse is greatly reduced, the nutritional ingredients are obviously improved, and the utilization rate of the fermented vinasse is greatly improved. The fermented distiller's grains are added into non-reproductive feed ration for pigs, cattle and sheep to replace 40-50% of ration, so that morbidity can be effectively reduced.

Description

Fermented distiller's grains and preparation method and application thereof

Technical field

The invention belongs to the technical field of animal feed, and specifically relates to fermented distiller's grains and its preparation method and application.

Background technique

The output of my country's distiller's grain resources is relatively large. According to relevant data, the national liquor production in 2022 will be 6.712 million kiloliters, and each ton of base liquor will produce about 3 tons of distiller's grain. Therefore, the output of my country's liquor grain in 2022 has exceeded 20 million tons. However, due to unfavorable factors such as the high lignocellulose content in white distiller's grains, it is poorly nutritious to feed animals directly. Solid-state fermentation can effectively improve the nutritional content of distiller's grains, thus promoting the promotion of animal feed and effectively avoiding environmental pollution caused by distillers' grains.

Astaxanthin is a non-provitamin A, fat-soluble carotenoid that mainly exists in certain microorganisms and marine animals. Its molecular structure is mainly composed of a polyene chain and two ketone rings. The two ketone rings are located at both ends of the astaxanthin molecule. This structural molecule can easily pass through biological membranes and has a good effect on scavenging free radicals inside animals. At the same time, the unsaturated double bonds on the polyene chain have good antioxidant properties. It also has good anti-cancer effects, reduces cardiovascular disease and regulates the immune system. It is an important feed additive. However, astaxanthin has poor storage stability and is extremely easily oxidized, leading to inactivation. However, the existing embedding process has problems of food safety, high cost, and poor stability.

Therefore, how to improve the stability of astaxanthin so as to avoid oxidation and be well used in feed is a technical problem that scientific researchers urgently need to solve.

Contents of the invention

The object of the present invention is to provide a fermented distiller's grain and its preparation method and application in view of the above-mentioned shortcomings of the prior art.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The first object of the present invention is to provide a preparation method for fermented distiller's grains, which includes the following specific steps: S1. Liquid fermentation of Rhodotorula to obtain the first fermentation liquid; Bacillus veleis liquid fermentation to obtain the second fermentation liquid ; The Rhodotorula glutinosa was purchased from the China Agricultural Microbial Culture Collection and Management Center, and the strain catalog number is ACCC20030; the Bacillus velezensis, the collection number of the General Microbiology Center of the China Microbial Culture Collection and Management Committee is CGMCC NO:25202;

S2. Prepare the gelatin solution, add the first fermentation bacteria liquid and olive oil to it, stir evenly, add sodium alginate, stir for 15-20 minutes at a low temperature of 0-5°C, add calcium chloride, and raise the temperature to 55-55°C. Continue stirring at 60°C to obtain the embedded substance, which is filtered and washed, vacuum freeze-dried and then pulverized to obtain embedded Rhodotorula yeast powder;

S3. Add the second fermentation bacteria liquid and the embedded Rhodotorula yeast powder to the distiller's grain culture medium for solid-state fermentation to obtain a fermented product. The fermented product is dried and pulverized at low temperature to obtain the fermented distiller's grain.

Further, in step S1, the Rhodotorula glutinosa is fermented and cultured in a first liquid culture medium at 28°C, 180r/min-200r/min on a shaking table for 4 to 5 days, and the first liquid culture medium includes: citric acid 4wt .%, beef extract 1.5wt.%, potassium dihydrogen phosphate 0.1wt.%, magnesium sulfate heptahydrate 0.04wt.%, sodium chloride 0.04wt.% and the balance water; the pH of the first liquid culture medium The value is 6.5.

Further, the Bacillus veleis was cultured in LB liquid culture medium at 36° C. and 220 r/min to 250 r/min for 4872 hours to obtain the first fermentation bacterial liquid.

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

Further, the formula of the distiller's grain culture medium includes: 10 to 12 g of dried distiller's grain, 20 to 25 mL of deionized water, and ammonia water to adjust the pH value to 7.0.

Further, in step S3, the mass ratio of the added amount of the second fermentation bacteria liquid to the distiller's grain culture medium is (1.5-2 mL): 10g, and the added amount of the embedded red yeast powder is equal to the mass ratio of the The mass ratio of distiller's grain culture medium is (0.1~0.2g):10g.

Further, in step S3, the solid-state fermentation temperature is 30°C, and the solid-state fermentation is cultured on a shaking table at 180r/min-200r/min for 7-8 days.

The second object of the present invention is to provide fermented distiller's grains prepared by the above method.

The third object of the present invention is to provide the application of the above-mentioned fermented distiller's grains in animal feed. The fermented distiller's grains are added to the basic feed and fed as animal feed.

Further, the fermented distiller's grains are added to the non-breeding period feed diets of pigs, cattle and sheep, replacing 40-50% of the diets.

Compared with the prior art, the beneficial effects of the present invention are:

(1) Fermented distiller's grains provided by the invention and its preparation method and application. This method inoculates the embedded Rhodotorula yeast powder and the bacterial liquid of Bacillus veleis that efficiently degrades crude fiber into the distiller's grains for mixed fermentation to obtain fermented distiller's grains. The solid-state fermentation process makes the embedded Rhodotorula yeast liquid It can avoid the oxidation of the astaxanthin produced by it, thereby effectively improving the stability of 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 prepared fermented distiller's grains is greatly reduced, and the nutritional components are significantly improved, which greatly improves the utilization rate of the fermented distiller's grains.

(2) The fermented distiller's grains provided by the present invention contain high-quality proteins and plant functional factors such as polyphenols, flavonoids and terpenoids. Through microbial fermentation, compound probiotics, flavonoids, terpenoids, shrimps and other ingredients are realized. The organic combination of cyanins, etc. can greatly improve the antioxidant capacity of organisms. Adding the fermented distiller's grains provided by the invention to the non-breeding period feed diet for pigs, cattle and sheep, replacing 40-50% of the diet, can effectively reduce the incidence rate.

Description of the drawings

Figure 1 is a comparison chart of the main component contents of distiller’s grains before and after fermentation.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the specific implementation modes of the present invention will be further described in detail below with reference to specific embodiments and drawings. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

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

It should be noted that the nutritional properties of sorghum dried distiller's grains (DDGS) are mainly composed of insoluble starch polysaccharides such as low-fermentation arabinoxylose, cellulose and lignin, and also contain high levels of fiber. The quality of DDGS often depends on the quality of the crop. , fermentation process, drying temperature and time and other factors. Compared with corn distillers dried grains, sorghum dried distillers grains have higher crude protein, neutral detergent fiber and acid detergent fiber.

The classification name of Bacillus velezensis used in the present invention is Bacillus velezensis, and it has been sent to the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Committee (CGMCC) for preservation. The preservation date is June 27, 2022. The deposit number is CGMCC No. 25202. The deposit number is CGMCC No. 25202.

The Rhodotorula glutinosa used in the present invention was purchased from the China Agricultural Microbial Culture Collection and Management Center, and the bacterial strain catalog number is ACCC20030.

Example 1

This embodiment provides a preparation method for fermented distiller's grains.

Specific steps are as follows:

1. Liquid fermentation of Rhodotorula to obtain the first fermentation liquid.

(1) Configuration of the 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 of water; the described The pH value of liquid fermentation medium is 6.5.

(2) The shaking culture conditions of Rhodotorula glutinosa ACCC20030 are 28°C and 180r/min for 4 days.

2. Liquid fermentation of Bacillus veleis to obtain the second fermentation bacterial liquid.

(1) The configuration of LB liquid culture medium: fish meal peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH value is 7.0.

(2) The shaking culture conditions of Bacillus veleis CGMCC NO: 25202 are 36°C and 220r/min for 48 hours.

3. Preparation of embedded red yeast powder

(1) Take a known volume of 70 mL of 4% gelatin solution in a beaker and stir it fully with a magnetic stirrer at about 60°C. This temperature remains unchanged during this experiment.

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

(3) The embedded material is vacuum freeze-dried and then pulverized to obtain embedded Rhodotorula yeast powder.

4. Preparation of fermented distiller’s grains

(1) Configuration of distiller’s grain culture medium: 10g of dried distiller’s grains, 20 mL of deionized water, and adjust the pH value to 7.0 with ammonia.

(2) Add 0.1g of embedded red yeast powder and 2mL of the second fermentation bacteria liquid to the distiller's grain culture medium, and culture it at 30°C and 180r/min for 6 days to obtain the fermentation product. The fermentation product is dried and powdered at low temperature. , that is, fermented distiller's grains are obtained.

Example 2

This embodiment provides a preparation method for fermented distiller's grains.

Specific steps are as follows:

1. Liquid fermentation of Rhodotorula to obtain the first fermentation liquid.

(1) Configuration of the 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 of water; the described The pH value of liquid fermentation medium is 6.5.

(2) The shaking culture conditions of Rhodotorula glutinosa ACCC20030 are 28°C and 190r/min for 4.5 days.

2. Liquid fermentation of Bacillus veleis to obtain the second fermentation bacterial liquid.

(1) The configuration of LB liquid culture medium: fish meal peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH value is 7.0.

(2) The shaking culture conditions of Bacillus veleis CGMCC NO:25202 are 36°C and 230r/min for 36h.

3. Preparation of embedded red yeast powder

(1) Take a known volume of 70 mL of 4% gelatin solution in a beaker and stir it fully with a magnetic stirrer at about 60°C. This temperature remains unchanged during this experiment.

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

(3) The embedded material is vacuum freeze-dried and then pulverized to obtain embedded Rhodotorula yeast powder.

4. Preparation of fermented distiller’s grains

(1) Configuration of distiller’s grain culture medium: 11g of dried distiller’s grains, 23 mL of deionized water, and adjust the pH value to 7.0 with ammonia.

(2) Add 0.15g of embedded red yeast powder and 1.5mL of the second fermentation liquid into the distiller's grain culture medium, and culture it at 30°C and 190r/min for 7 days to obtain the fermentation product. The fermentation product is dried at low temperature into powder. Puree, that is, fermented distiller's grains are obtained.

Example 3

This embodiment provides a preparation method for fermented distiller's grains.

Specific steps are as follows:

1. Liquid fermentation of Rhodotorula to obtain the first fermentation liquid.

(1) Configuration of the 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 of water; the described The pH value of liquid fermentation medium is 6.5.

(2) The shaking culture conditions of Rhodotorula glutinosa ACCC20030 are 28°C and 200r/min for 5 days.

2. Liquid fermentation of Bacillus veleis to obtain the second fermentation bacterial liquid.

(1) The configuration of LB liquid culture medium: fish meal peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH value is 7.0.

(2) The shaking culture conditions of Bacillus veleis CGMCC NO: 25202 are 36°C and 250r/min for 72h.

3. Preparation of embedded red yeast powder

(1) Take a known volume of 70 mL of 4% gelatin solution in a beaker and stir it fully with a magnetic stirrer at about 60°C. This temperature remains unchanged during this experiment.

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

(3) The embedded material is vacuum freeze-dried and then pulverized to obtain embedded Rhodotorula yeast powder.

4. Preparation of fermented distiller’s grains

(1) Configuration of distiller’s grain culture medium: 12g of dried distiller’s grains, 25mL of deionized water, and adjust the pH value to 7.0 with ammonia.

(2) Add 0.2g of embedded red yeast powder and 1.8mL of the second fermentation liquid to the distiller's grains culture medium, and culture it at 30°C and 200r/min for 8 days to obtain the fermentation product. The fermentation product is dried at low temperature into powder. Puree, that is, fermented distiller's grains are obtained.

In order to better explain the ability and functional activity of the fermented distiller's grains prepared by the present invention to improve the stability of astaxanthin, the applicant conducted the following research:

The embedded Rhodotorula yeast powder prepared in Examples 1-3 has similar structure and function. Example 1 is taken as an example for detailed description.

1. Determination of astaxanthin content in embedded red yeast powder.

Determination of astaxanthin entrapment rate:

Drawing of astaxanthin standard curve: Accurately weigh 10 mg of astaxanthin standard, first fully dissolve it with a very small amount of methylene chloride, and then use chromatography and methanol to dissolve it to prepare an astaxanthin mother solution with a concentration of 0.1 mg/mL. Take an appropriate amount of the mother liquor and dilute it with chromatographically pure methanol into astaxanthin standard solutions with concentrations of 1, 2, 3, 4, 6, 8 and 10 μg/mL. Test the content of microencapsulated astaxanthin: Accurately weigh 1.0g of embedded red yeast powder, first add 40mL of water and stir briefly with a glass rod, then add 40mL of acetone, shake repeatedly to make it colorless, add an appropriate amount Anhydrous magnesium sulfate dehydrates. Centrifuge and discard the supernatant, dry, dissolve the powder in acetone, and measure the absorbance at 480 nm. The average value is 1.953.

Detect the astaxanthin content on the surface of embedded Rhodotorula yeast powder: Accurately weigh 1.0g of embedded Rhodotorula yeast powder, add 40mL of n-hexane, shake and mix thoroughly until the solution becomes colorless, centrifuge, discard the supernatant, and evaporate quickly Dry, dissolve an appropriate amount of acetone, and measure the absorbance at 480 nm. The average value is 0.945.

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

2. Study on the stability of astaxanthin in embedded red yeast powder.

Take five 50ml test tubes and number them 1#, 2#, 3#, 4#, and 5# respectively. Among them, 1# is the control of Rhodotorula that was not embedded on the same day, 2# is the control of Rhodotorula that was not embedded after five days of storage at room temperature, 3# is the control of Rhodotorula that was embedded on the same day, and 4# is the control of Rhodotorula that was stored at room temperature and was embedded. Five days later, the red yeast and 5# are blank, that is, no red yeast is added during embedding to offset the error caused by the embedding material. Use the above method to detect the embedded red yeast powder, and the result is as follows As shown in Table 1.

Table 1. Stability experimental data of astaxanthin

As can be seen from Table 1, astaxanthin is more stable after being embedded, while the activity of astaxanthin without being embedded is almost completely lost after five days. The inactivation rate of unembedded astaxanthin was 92.9%, and the inactivation rate of astaxanthin after encapsulation was only 30.2%. The effect is very obvious, and the embedding stability is 69.8%.

3. Study on the changes in the content of main components in distiller’s grains before and after fermentation

3.1 Study on changes in total phenolic content in distiller’s grains before and after fermentation.

Preparation of the liquid to be tested for total phenols: crush the distiller's grains before and after fermentation, quantitatively weigh 1g of the distillers' grains before and after fermentation, add 10mL of 70% ethanol, and centrifuge at 4000r/min for 15min on an ultrasonic instrument for 1h. Collect the supernatant and extract the residue twice as described above. Finally, adjust the volume to 30mL with distilled water. The total phenolic content was tested using the Folin-phenol colorimetric method (FC).

Referring to Figure 1, the total phenolic content of the distiller's grains before fermentation was 1.228 μg/mL, and the total phenolic content of the distiller's grains after fermentation was 1.477 μg/mL. The total phenolic content increased by 20.3%.

3.2 Study on changes in total triterpene content in distiller’s grains before and after fermentation.

Preparation of the total triterpene test liquid: crush the distiller's grains before and after fermentation, quantitatively weigh 1g of the distillers' grains before and after fermentation, add 10mL of 70% ethanol, and centrifuge at 4000r/min for 15min on an ultrasonic instrument for 1h. , collect the supernatant, and extract the residue twice according to the above method. Finally, adjust the volume to 30mL with distilled water. Using oleanolic acid as the standard, the intracellular triterpene content was determined using the vanillin-glacial acetic acid method.

Referring to Figure 1, the total triterpene content of the distiller's grains before fermentation was 2.7 μg/mL, and the total triterpene content of the distiller's grains after fermentation was 3.2 μg/mL. The total triterpene content increased by 17.3%.

3.3 Study on changes in total sugar content in distiller’s grains before and after fermentation.

Preparation of polysaccharide test solution: Weigh 1g of distiller's grains before and after fermentation, add 10 mL of pure water, extract at 95°C for 1 hour, collect the supernatant, and repeat the extraction of the residue 2 times according to the above method. Use pure water to adjust the volume to 30mL, which is the water extract. Take 6 mL of water extract, add 24 mL of 40% ethanol solution, and let stand at 4°C for 24 hours. Total sugar content was determined using the phenol-sulfuric acid method.

Referring to Figure 1, the total sugar content of the distiller's grains before fermentation is 23.8 μg/mL, and the total sugar content of the distiller's grains after fermentation is 60.5 μg/mL. The total sugar content increased by 154.2%.

3.4 Study on the changes in crude fiber content in distiller’s grains before and after fermentation.

The results were as follows: the crude fiber content of the distiller's grains before fermentation was 26.4%, and the crude fiber content of the distiller's grains after fermentation was 23.7%. The crude fiber content decreased by 2.7%.

3.5 Study on the changes in crude protein content in distiller’s grains before and after fermentation.

Referring to Figure 1, the crude protein content of distiller's grains before fermentation is 14.5%, and the crude protein content of distiller's grains after fermentation is 17.2%, an increase of 2.7%.

3.6 Study on changes in volatile base nitrogen content in distiller’s grains before and after fermentation.

1) Sample preparation: Weigh 1g each of the distiller's grains before fermentation and after fermentation, add 10mL pure water, extract at 95°C for 30 minutes, collect the supernatant, and repeat the extraction of the residue 2 times according to the above method. Use pure water to adjust the volume to 30mL, let it stand and centrifuge to obtain the water extract.

2) Distillation: Put 5ml of the sample to be tested into a distillation tube, and at the same time add 5ml of magnesium oxide solution with a concentration of 10g/ml and put it into the Kjeldahl nitrogen analyzer. Put 10 mL of boric acid solution with a concentration of 20 g/ml into a 250 mL Erlenmeyer flask, and place it at the lower end of the condenser tube. Then carry out distillation, the distillation time is 5-6min.

3) Titration: Use hydrochloric acid standard solution (0.01mol/L) to titrate the blue-green solution in the Erlenmeyer flask. Titrate until the solution turns blue-violet as the titration end point, and do a blank experiment at the same time.

Calculate the volatile base nitrogen content of the sample = ((v1-v2)xCx14)/(mx5/100)x100

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

V2: The volume of blank hydrochloric acid standard solution consumed by the reagent. The unit is mL.

C: The actual concentration of hydrochloric acid, in mol/mL.

m: mass of the sample, unit is g.

The final determination of the volatile base nitrogen content in the lees before fermentation was 80.4 mg/kg, and the volatile base nitrogen content in the lees after fermentation was 95.6 mg/kg.

3.7 Study on changes in pH value and total acid content in lees before and after fermentation.

Weigh 1g each of the distiller's grains before and after fermentation, add 3-4 mL of pure water, extract at 95°C for 30 minutes, collect the supernatant, and repeat the extraction of the residue two times according to the above method. Use pure water to adjust the volume to 10mL, let it stand and then centrifuge, which is the liquid to be tested. Then use a pH meter to measure its pH. Measure three times and take the average value. Use the acid-base titration method to determine the total acid content. Use a big-belly pipette to draw 50.0mL of the sample into a 250mL Erlenmeyer flask, add 2 drops of phenolphthalein indicator (10g/L), and titrate with sodium hydroxide standard solution (0.1mol/L ) Titrate until it is slightly red, which is the end point.

Calculation formula X=(C×V×60)/50.0 where:

X——The mass concentration of total acid in the sample (calculated as acetic acid), in grams per liter (g/L);

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

V——The volume of sodium hydroxide standard titration solution consumed during titration, in milliliters (mL).

The pH of the distiller's grains liquid to be tested before fermentation was finally measured to be 4.2, and the total acid content was 11.2 mg/g. The pH value of the distiller's grains to be tested after fermentation is 6.0, and the total acid content is 10.9mg/g.

4. Research on changes in functional activity of distiller’s grains before and after fermentation

4.1 Determination of antioxidant activity DPPH clearance rate

Weigh 1g of distiller's grains before and after fermentation, add 10 mL of 70% ethanol, centrifuge on an ultrasonicator for 1 hour at 4000 r/min for 15 minutes, collect the supernatant, and extract the residue twice according to the above method. Finally, use distilled water to adjust the volume to 20mL. This is the sample liquid to be tested.

Preparation of DPPH solution: Accurately weigh 4 mg and dissolve it in 100 mL of absolute ethanol to make a 0.04 mg/mL DDPH solution, and place it in a brown bottle (prepare and use now).

How to operate:

1) Dilute all samples to 7%, take 2mL of each 7% pre-fermentation and post-fermentation sample solution, add 2mL of DDPH solution, mix evenly, leave it at room temperature in the dark for 10 minutes, measure the absorbance value A1 at 517nm, and set the blank group accordingly Add 2 mL of absolute ethanol to 2 mL of 7% sample solution.

2) Take 2 mL of pure water and add 2 mL of DDPH solution. Mix evenly. After leaving it at dark room temperature for 10 minutes, measure the absorbance value A ₂ at 517 nm. Correspondingly, set the blank group to 2 mL of pure water and add 2 mL of absolute ethanol.

3) Use 0.1mg/mL V _C as a positive control. The samples before and after fermentation were coded as 1# and 3#, the blank control was coded as 2#, 4#, the 0.1mg/mL V _C group was coded as 5#, and its control was coded as 6#.

The formula for DDPH clearance rate is: K%=(1-A ₁ /A ₂ )×100%

Table 2. DPPH clearance rate

It can be seen from Table 2 that the DDPH clearance rate in the distiller's grains after fermentation is 66.7%, and the DDPH clearance rate in the distiller's grains before fermentation is only 30%. Compared with the DDPH clearance rate before fermentation, the DDPH clearance rate increased by 36.7%. It can be seen that the antioxidant content in the lees after fermentation increases.

4.2 Determination of antioxidant activity ABTS clearance rate

Preparation of ABTS mother liquor: Accurately weigh 0.067g K ₂ S ₂ O ₈ and dissolve it in 100 mL pure water, then weigh 0.384g ABTS and dissolve it in it to prepare a 7mmol/L ABTS mother liquor, then let it stand for 12 seconds at room temperature and away from light. ~16h, the mother liquor can be stable for 3~4d.

Preparation of ABTS working solution: Just before use, dilute the ABTS mother solution with absolute ethanol so that the absorbance reaches 0.700±0.020 at a wavelength of 734nm, and replace the blank group with absolute ethanol.

How to operate:

Dilute the pre-fermentation and post-fermentation sample solutions to 7%, add 1mL of each 7% sample solution to 2mL of ABTS working solution, shake well, and measure the absorbance value A at 734nm wavelength at 20S of reaction. The samples before and after fermentation were coded as 1# and 2#, and the 0.1mg/mL VC group was coded as 3#.

The formula for ABTS clearance rate is: K%=(1-A/0.700)×100%

Table 3. ABTS clearance rate

It can be seen from Table 3 that the ABTS clearance rate in the distiller's grains after fermentation is 91.7%, and the ABTS clearance rate in the distiller's grains before fermentation is only 85.9%, which is an increase of 5.8% compared to the ABTS clearance rate before fermentation. It can be seen that the antioxidant content in the lees after fermentation increases.

5. Study on the stability of astaxanthin in fermented distiller’s grains.

Test group: fermented distiller's grains prepared in Example 1, which is sample 1;

Comparative group: The preparation method is basically the same as that of Example 1, with the difference: fermented distiller's grains are prepared without adding embedded red yeast powder, and then the embedded red yeast powder is added according to a certain proportion. The mass ratio of red yeast powder is 10g:0.1g, and the physical mixture is evenly mixed to obtain sample 2;

Sample preparation: Crush sample 1 and sample 2 separately, weigh 5g of each, add 10mL of 70% ethanol, centrifuge on an ultrasonicator for 1h at 4000r/min for 15min, collect the supernatant, and extract the residue twice according to the above method. Finally, use distilled water to adjust the volume to 30 mL for the determination of astaxanthin content. The determination method is the same as the above determination method.

Stability test: Store sample 1 and sample 2 at room temperature, detect the astaxanthin content in sample 1 and sample 2 on 0, 15, 30, 45, and 60 days, and observe their changes.

Table 4. Investigation on the stability of astaxanthin in the test group and control group

As shown in Table 4, the data in the table is the astaxanthin content value. It can be seen that the astaxanthin content in the fermented distiller's grains provided by the experimental group has a small change trend and is relatively stable, indicating that the astaxanthin in the fermented distiller's grains is not easily oxidized. Storage of up to 60 days can be achieved, but the astaxanthin content in physically mixed samples is significantly reduced, indicating that the physical mixing method is not conducive to the storage of embedded astaxanthin.

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

Example 4

90 healthy pigs weighing close to 100kg were selected and randomly divided into 3 groups. They were raised with different feed formulas at the Hongshun Pig Farm in Huaibei City, Anhui Province. After feeding with 3 different types of feed for 30 days, the pigs from the three groups were selected. Two healthy pigs each with similar weight, body condition and age were selected and an appropriate amount of fresh blood was collected from the ear vein. Put the collected blood into labeled test tubes, centrifuge at 3000r/min for 15min, take the supernatant and add preservative (0.01% thimerosal or 0.02% sodium azide, final concentration), aliquot and store in a 4°C refrigerator for later use. . Serum total protein, serum albumin, and immunoglobulin were measured respectively. The methods were based on the effects of anti-antibiotic fermented feed on the growth performance, intestinal flora, blood biochemical indicators and immune performance of weaned piglets. The determination of superoxide dismutase refers to the effects of fermented soybean meal on the growth performance, serum biochemical indicators and intestinal function of weaned piglets. The symptoms include diarrhea, cold, pica and food accumulation respectively. Incidence rate = (number of infected heads × number of days of onset)/(total number of heads × number of experimental days) × 100%.

The results are shown in Table 5.

Table 5 Feeding results statistics table

Note: Formula A is entirely soybean meal; Formula B is soybean meal: unfermented distiller's grain feed weight ratio = 1:1; Formula C soybean meal: fermented distiller's grain weight ratio = 1:1.

It can be seen from Table 5 that among the feed biochemical indicators of Formula C, the serum total protein, albumin and superoxide dismutase activity are the highest, and the immune indicators and incidence rates are also the most suitable. Therefore, the mixed fermented feed of soybean meal: fermented distiller's grains = 1:1 Best proportions.

Things not covered above are applicable to the existing technology.

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 will Various modifications, additions, or similar substitutions may be made to the described specific embodiments without departing from the direction of the present invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. The preparation method of the fermented lees is characterized by comprising the following specific steps of:

s1, performing liquid fermentation on rhodotorula glutinis to obtain a first fermentation bacterial liquid; liquid fermentation of bacillus belicus to obtain second fermentation bacteria liquid; the rhodotorula glutinis is purchased from China center for type culture collection of microorganisms, and the strain catalog number is as follows: ACCC20030, the bacillus belicus bacillus has a preservation number of CGMCC NO:25202;

s2, preparing gelatin solution, adding first fermentation bacteria liquid and olive oil into the gelatin solution, uniformly stirring, adding sodium alginate, stirring for 15-20 min at a low temperature of 0-5 ℃, adding calcium chloride, heating to 55-60 ℃, continuously stirring, filtering and washing to obtain an embedded substance, and crushing after vacuum freeze drying to obtain embedded rhodotorula glutinis powder;

s3, adding the second fermentation broth and the embedded rhodotorula glutinis powder into a vinasse culture medium for solid state fermentation to obtain a fermented product, and drying and crushing the fermented product at a low temperature to obtain the fermented vinasse.

2. The preparation method of claim 1, wherein in step S1, the rhodotorula glutinis is subjected to shake cultivation in a first liquid medium at 28 ℃ and 180-200 r/min for 4-5 days, and the first liquid medium comprises: 4wt.% citric acid, 1.5wt.% beef extract, 0.1wt.% monopotassium phosphate, 0.04wt.% magnesium sulfate heptahydrate, 0.04wt.% sodium chloride, and the balance water; the pH value of the first liquid culture medium is 6.5.

3. The preparation method of claim 1, wherein in the step S1, the bacillus belicus is cultured in LB liquid medium at 36 ℃ for 48-72 h at 220-250 r/min to obtain a first fermentation broth.

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

5. The method of claim 1, wherein in step S3, the distillers grains medium is formulated to include: 10-12 g of distiller's grains, 20-25 mL of deionized water, and adjusting the pH value to 7.0 by ammonia water.

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

7. The process according to claim 6, wherein in step S3, the solid state fermentation temperature is 30℃and the culture is carried out in a shaking bed at 180 to 200r/min for 6 to 8 days.

8. Fermented grain produced by the production method according to any one of claims 1 to 7.

9. Use of the fermented grain of claim 8 in animal feed, wherein the fermented grain is added to a basal feed for feeding the animal feed.

10. The use according to claim 9, wherein the fermented grain is added to the non-reproductive feed ration of pigs, cattle and sheep instead of 40-50% of the ration.