Production method of fungus, enzyme and adsorbent coupled solid-state fermentation detoxified white spirit vinasse and white spirit vinasse feed prepared by production method

Abstract

The invention discloses a production method of fungus, enzyme and adsorbent coupled solid-state fermentation detoxified white spirit vinasse and a white spirit vinasse feed prepared by the production method of fungus, enzyme and adsorbent coupled solid-state fermentation detoxified white spirit vinasse. The production method comprises the following steps: inoculating bacterial solutions of saccharomyces cerevisiae CJM26, bacillus subtilis YLZ7 and lactobacillus salivarius CRS23 obtained by screening into fresh distiller's grains, then adding compound enzyme, then adding plant carbon black, uniformly mixing, sealing, and fermenting at 30-37 DEG C for 3-4 days to obtain fermented distiller's grains. According to the fermented distiller's grains produced by the method, crude protein and true protein are remarkably improved, crude fiber and neutral detergent fiber are remarkably reduced, the content of zearalenone and the content of fumonisins are both lower than the feed hygienic standard, and the feeding safety and the nutritional value of the distiller's grains are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of solid-state fermentation, and more specifically to a production method of white wine lees by solid-state fermentation of bacteria, enzymes and adsorbents, and white wine lees feed prepared therefrom. Background Art

[0002] Liquor lees are one of the by-products in the process of liquor brewing. They are rich in nutrients, high in crude protein content, a full range of amino acids, and rich in bioactive substances. They can be developed and utilized as excellent non-conventional feed raw material resources. However, due to the high acidity, high crude fiber content, and high water content of fresh liquor lees, they are easy to mildew and have high mycotoxin content, which limits their application in animal feed production. At present, most technical studies use microbial solid-state fermentation methods to solve the above problems, but the strains used have poor specificity. Liquor lees are a material that has been fermented by microorganisms, and the content of nutrients suitable for microbial growth is low. When fermenting liquor lees, non-protein nitrogen and other auxiliary materials need to be added to the lees to increase the pH value and crude protein content of the liquor lees and ensure that the microorganisms can successfully grow in the lees to achieve the purpose of fermentation. However, the use of this fermentation process often leads to unsatisfactory fermentation effect of liquor lees, increased production costs, and cannot meet the requirements of livestock and poultry feed production. In addition, liquor lees are prone to excessive mycotoxin during storage, and there is currently no effective solution.

[0003] Therefore, there is an urgent need to develop a production method with simple process that can effectively improve the feeding value of white wine grains and reduce their toxin content, thereby greatly improving the feeding safety and utilization rate of white wine grains. Summary of the invention

[0004] In view of the shortcomings of the prior art, such as poor strain specificity, the need to add auxiliary materials to adjust the pH, moisture and protein content of distiller's grains, and the inability to solve the problems of excessive mycotoxins, the first object of the present invention is to provide a production method of solid-state fermentation of distiller's grains by coupling bacteria, enzymes and adsorbents. This method can effectively improve the above problems.

[0005] The second object of the present invention is to provide a white wine grains feed prepared by the above method, which can replace soybean meal and be used in livestock and poultry breeding.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for producing detoxified distiller's grains by solid-state fermentation coupled with bacteria, enzymes, and adsorbents, the production method comprising:

[0008] Inoculate the bacterial liquid of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7 and Lactobacillus salivarius CRS23 into fresh white wine lees at an inoculation rate of 1%-5%, then add complex enzyme and mix evenly, then add plant carbon black and mix evenly, seal, and ferment at 30-37°C for 3-4 days;

[0009] Among them, the Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7 and Lactobacillus salivarius CRS23 are deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit numbers are CGMCC No.31751, CGMCC No.31753 and CGMCC No.31752 respectively;

[0010] The complex enzyme consists of acid protease, cellulase, pectinase, xylanase and glucose oxidase.

[0011] The present invention targets the characteristics of fresh distiller's grains, uses acid resistance, cellulase production, mycotoxin removal and whether the bacteria can grow in nutrient-poor distiller's grains as indicators to selectively screen and obtain a bacterial strain combination suitable for fermentation of distiller's grains, targets mycotoxins zearalenone and fumonisin that are easily exceeded during storage of distiller's grains, screens microorganisms, enzymes and adsorbents that have significant removal effects on the two toxins, and combines the three to ferment distiller's grains without adding auxiliary materials or adjusting pH and water content, directly performs detoxification solid-state fermentation of bacteria, enzymes and adsorbents on the distiller's grains, and the crude protein and true protein of the fermented distiller's grains are significantly improved, the crude fiber and neutral detergent fiber are significantly reduced, the zearalenone and fumonisin contents are both lower than the feed hygiene standard, and the feeding safety and nutritional value of the distiller's grains are greatly improved.

[0012] Furthermore, the fresh distiller's grains in the present invention refer to grain distiller's grains produced by brewing liquor without any treatment. Generally, the fresh distiller's grains contain a large amount of water, illustratively including but not limited to 57%-60%.

[0013] Furthermore, the bacterial solution is prepared by the following method: a ring of colonies is picked from the preservation slopes of the saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7, and Lactobacillus salivarius CRS23, and they are respectively inoculated into YPD, LB and MRS liquid culture media for activation and enrichment, wherein the saccharomyces cerevisiae activation solution is oscillated and activated at 180 rpm and 28-30° C. for 45-48 hours, the Bacillus subtilis activation solution is oscillated and activated at 180 rpm and 35-37° C. for 20-24 hours, and the Lactobacillus salivarius activation solution is statically activated at 35-40° C. for 20-24 hours.

[0014] Furthermore, the YPD medium is prepared by mixing 10 g of peptone, 5 g of yeast extract powder, and 20 g of glucose, adding the mixture to 1000 mL of distilled water, heating and boiling to dissolve, adjusting the pH to natural, and sterilizing the mixture by high pressure sterilization at 121° C. for 20 min after packaging.

[0015] The LB medium is prepared as follows: 10 g of peptone, 5 g of yeast powder, and 10 g of sodium chloride are mixed and added into 1000 mL of distilled water, heated and boiled to dissolve, the pH value is adjusted to 7.2-7.4, and after packaging, the medium is sterilized at 121° C. for 20 min.

[0016] The MRS culture medium is prepared as follows: 10 g of peptone, 5 g of beef powder, 20 g of glucose, 4 g of yeast powder, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 2 g of triammonium citrate, 0.05 g of manganese sulfate, and 1 mL of Tween 80; the mixture is added into 1000 mL of distilled water, heated and boiled to dissolve, the pH value is adjusted to 6.2-6.4, and after packaging, the mixture is sterilized at 121° C. for 15 min.

[0017] Further, the addition amount of the acidic protease, cellulase, pectinase and xylanase is 100-200u / g dry white wine lees, and the addition amount of the glucose oxidase is 20-50u / g dry white wine lees. The dry white wine lees is prepared by weighing fresh white wine lees, measuring its water content, calculating the weight of the dry white wine lees, and then calculating the addition amount of the above enzymes according to the weight of the dry white wine lees.

[0018] Furthermore, the added amount of the plant carbon black is 1‰-5‰ of the mass of fresh white wine lees.

[0019] Furthermore, during the fermentation process, it is best to stir the mixture regularly; preferably, stir the mixture every 8-12 hours.

[0020] Furthermore, the production method also includes the steps of drying, crushing and packaging the fermented distiller's grains.

[0021] Furthermore, the drying temperature is 70-80°C and the drying time is 30-60 minutes.

[0022] In a second aspect, the present invention provides a white wine grains feed prepared by the above method.

[0023] In addition, unless otherwise specified, the raw materials used in the present invention can be obtained from commercial sources, and any range recorded in the present invention includes the end value and any value between the end values ​​and any sub-range formed by the end value or any value between the end values. The solutions are all aqueous solutions unless otherwise specified.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) The present invention provides a method for producing detoxified distiller's grains by solid-state fermentation coupled with bacteria, enzymes, and adsorbents. The fermentation process of distiller's grains is simple, easy to operate, and easy to realize industrial production.

[0026] (2) Unlike the prior art which uses air-dried distiller's grains as fermentation raw materials, the production method of the present invention does not require the fresh distiller's grains to be dehydrated and dried, and can be directly fermented. The reason why air-dried distiller's grains are used in the prior art is to prevent the fresh distiller's grains from becoming moldy and producing toxins during storage, which makes them unusable. However, the problem with this is that the drying cost is increased, and additional water must be added during fermentation, which complicates the process.

[0027] (3) The bacteria, enzymes, adsorbents and fermentation process provided by the present invention can effectively remove zearalenone and fumonisin, which are easily produced during long-term storage of white wine grains, with a detoxification rate of more than 95%. The pH of the white wine grains is increased from 3.3 to about 5.4-5.8, the crude protein content is increased by more than 25%, the acid-soluble protein is increased by more than 30%, the true protein is increased by more than 45%, the crude fiber is reduced by more than 20%, and the neutral detergent fiber is reduced by more than 30%.

[0028] Collection Instructions

[0029] Reference biomaterial strain: CJM26

[0030] Suggested taxonomic name: Saccharomyces cerevisiae

[0031] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0032] Abbreviation of depository institution: CGMCC

[0033] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0034] Storage date: August 27, 2024

[0035] The registration number of the Collection Center is: CGMCC No.31751.

[0036] Reference biomaterial (strain): YLZ7

[0037] Suggested taxonomic name: Bacillus subtilis

[0038] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0039] Abbreviation of depository institution: CGMCC

[0040] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0041] Storage date: August 27, 2024

[0042] The registration number of the Collection Center is: CGMCC No.31753.

[0043] Reference biomaterial strain: CRS23

[0044] Suggested taxonomic name: Lactobacillus salivarius

[0045] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0046] Abbreviation of depository institution: CGMCC

[0047] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0048] Storage date: August 27, 2024

[0049] The registration number of the Collection Center is: CGMCC No.31752. DETAILED DESCRIPTION

[0050] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the content specifically described below is illustrative and not restrictive, and should not be used to limit the scope of protection of the present invention. The enzymes and plant carbon black used in the embodiments are all commercially available products.

[0051] Example 1 Bacterial strain screening

[0052] The yeast, bacillus and lactic acid bacteria isolated from fresh distiller's grains, soil, naturally fermented yogurt, fermented feed and other samples were used as the starting strains, and acid resistance, cellulase production, mycotoxin removal and suitable growth using oligonutrients in distiller's grains were used as indicators to select one strain suitable for fermenting detoxified distiller's grains from each of the three types of bacteria. The specific screening results are shown in Tables 1 to 4.

[0053] Table 1 OD of different strains after culture under different pH conditions 600 value

[0054] Strain number Species pH=3.0 pH = 4.0 pH=5.0 CJM21 Saccharomyces cerevisiae <![CDATA[11.83±1.61 Aa ]]> <![CDATA[9.24±1.99 Bab ]]> <![CDATA[6.92±0.78 CDb <!-- 3 -->]]> CJM26 Saccharomyces cerevisiae <![CDATA[9.23±3.70 Aa ]]> <![CDATA[13.74±3.11 Aa ]]> <![CDATA[10.69±1.51 Ba ]]> CJM27 Saccharomyces cerevisiae <![CDATA[9.61±3.56 Aa ]]> <![CDATA[8.76±0.41 Ba ]]> <![CDATA[5.84±0.48 DEFa ]]> CJM29 Marine red yeast <![CDATA[11.01±5.29 Aa ]]> <![CDATA[14.51±2.84 Aa ]]> <![CDATA[16.3±1.54 Aa ]]> SC19-1 Saccharomyces cerevisiae <![CDATA[10.26±3.82 Aa ]]> <![CDATA[11.89±4.98 ABa ]]> <![CDATA[7.28±0.33 Ca ]]> ADM Candida utilis <![CDATA[3.18±0.02 Ba ]]> <![CDATA[3.09±0.04 CDa ]]> <![CDATA[3.10±0.05 Ia ]]> SC17-1 Saccharomyces cerevisiae <![CDATA[1.73±0.11 Bb ]]> <![CDATA[3.36±0.16 CDa ]]> <![CDATA[3.43±0.14 Ia ]]> PYB3 Bacillus licheniformis <![CDATA[0.19±0.00 b ]]> <![CDATA[0.17±0.00 b ]]> <![CDATA[1.10±0.05 a ]]> PYB5 Bacillus licheniformis <![CDATA[0.21±0.00 b ]]> <![CDATA[0.19±0.00 b ]]> <![CDATA[2.32±0.09 Aa ]]> CYB2-1 Bacillus amyloliquefaciens <![CDATA[0.13±0.00 b ]]> <![CDATA[0.12±0.01 b ]]> <![CDATA[1.17±0.01 a ]]> CYB2-2 Bacillus subtilis <![CDATA[0.20±0.00 b ]]> <![CDATA[0.19±0.00 b ]]> <![CDATA[2.36±0.04 Aa ]]> CYB5-2 Bacillus amyloliquefaciens <![CDATA[0.13±0.00 b ]]> <![CDATA[0.12±0.00 b ]]> <![CDATA[1.25±0.01 a ]]> CYB7-2 Bacillus licheniformis <![CDATA[0.09±0.00 b ]]> <![CDATA[0.08±0.00 b ]]> <![CDATA[0.63±0.10 a ]]> CYB11-1 Bacillus subtilis <![CDATA[0.20±0.00 b ]]> <![CDATA[0.19±0.00 b ]]> <![CDATA[2.31±0.05 Aa ]]> CYB12-1 Bacillus subtilis <![CDATA[0.19±0.00 b ]]> <![CDATA[0.18±0.00 b ]]> <![CDATA[2.29±0.07 Aa ]]> CYB20 Bacillus subtilis <![CDATA[0.14±0.00 b ]]> <![CDATA[0.12±0.00 b ]]> <![CDATA[0.96±0.02 a ]]> YLZ Bacillus subtilis <![CDATA[0.16±0.01 c ]]> <![CDATA[1.75±0.02 Aa ]]> <![CDATA[1.56±0.02 b ]]> CRS2-1 Lactobacillus salivarius <![CDATA[0.33±0.02 c ]]> <![CDATA[1.43±0.16 Ab ]]> <![CDATA[2.79±0.16 Aa ]]> CRS5-1 Lactobacillus salivarius <![CDATA[0.14±0.00 c ]]> <![CDATA[0.62±0.00 b ]]> <![CDATA[1.23±0.07 a ]]> CRS7-1 Lactobacillus salivarius <![CDATA[0.20±0.00 c ]]> <![CDATA[1.44±0.05 Aa ]]> <![CDATA[1.35±0.02 b ]]> CRS18 Enterococcus faecium <![CDATA[0.10±0.00 b ]]> <![CDATA[0.11±0.00 b ]]> <![CDATA[0.72±0.00 a ]]> CRS19 Lactobacillus salivarius <![CDATA[0.10±0.01 c ]]> <![CDATA[1.12±0.04 Ba ]]> <![CDATA[0.71±0.01 b ]]> CRS23 Lactobacillus salivarius <![CDATA[0.21±0.01 c ]]> <![CDATA[0.74±0.08 b ]]> <![CDATA[1.72±0.07 a ]]> FRS38 Lactobacillus plantarum <![CDATA[0.25±0.00 c ]]> <![CDATA[0.83±0.04 Cb ]]> <![CDATA[2.74±0.06 Aa ]]> CRS55 Lactobacillus salivarius <![CDATA[0.21±0.01 c ]]> <![CDATA[1.35±0.18 Ab ]]> <![CDATA[1.88±0.04 Ca ]]> CRS57 Enterococcus faecium <![CDATA[0.09±0.00 b ]]> <![CDATA[0.09±0.00 b ]]> <![CDATA[0.35±0.00 a ]]>

[0055] Note: In the table, data with different lowercase letters on the shoulders of the same row indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05); data with different uppercase letters on the shoulders of the same row indicate significant differences

[0056] (P<0.05), the same capital letters indicate no significant difference (P>0.05).

[0057] Table 2 Size of transparent zone produced by different strains growing on sodium carboxymethyl cellulose solid medium

[0058]

[0059]

[0060] Table 3 Detoxification rate of mycotoxins by different strains

[0061]

[0062]

[0063] The results of acid resistance screening showed that 5 out of 7 yeast strains (CJM21, CJM26, CJM27, CJM29, and SC19-1) had the best acid resistance, among which strain CJM21 had the strongest acid resistance; 5 out of 10 Bacillus strains (YLZ7, PYB5, CYB2-2, CYB11-1, and CYB12-1) had the best acid resistance, among which strain YLZ7 had the strongest acid resistance; 3 out of 9 lactic acid bacteria strains (CRS2-1, CRS7-1, and CRS55) had the best acid resistance, among which strain CRS2-1 had the strongest acid resistance.

[0064] The results of cellulase activity screening showed that among the 7 yeast strains, only strain SC17-1 had the strongest cellulase production ability; among the 10 Bacillus strains, strain YLZ7 had the highest cellulase production activity, followed by strains CYB2-1, CYB2-2 and CYB5-2; none of the 9 lactic acid bacteria had cellulase production activity.

[0065] The results of detoxification ability screening showed that among the 7 yeast strains, CJM26 had the strongest detoxification ability, followed by CJM29; among the 10 Bacillus strains, YLZ7 had the strongest ability to remove zearalenone, followed by PYB3; CYB7-2 had the strongest ability to remove fumonisin, followed by YLZ7; among the 9 lactic acid bacteria strains, CRS23 had the strongest detoxification ability.

[0066] Based on the screening results of the above three indicators, CJM21, CJM26, and SC17-1 are preferred among yeasts, PYB3, CYB2-2, and YLZ7 are preferred among Bacillus, and CRS2-1, CRS7-1, CRS55, and CRS23 are preferred among lactic acid bacteria as candidate strains for screening oligonutrients using white wine grains.

[0067] Table 4 Growth of different strains using oligonutrients from liquor lees

[0068] Strain number Species 0h 3d △Lg CJM21 Saccharomyces cerevisiae <![CDATA[1.3×10 6 ]]> <![CDATA[2.5×10 7 ]]> 1.28 CJM26 Saccharomyces cerevisiae <![CDATA[5.6×10 5 ]]> <![CDATA[6.3×10 7 ]]> 2.05 SC17-1 Saccharomyces cerevisiae <![CDATA[1.1×10 6 ]]> <![CDATA[9.3×10 7 ]]> 1.93 PYB3 Bacillus licheniformis <![CDATA[2.4×10 6 ]]> <![CDATA[8.4×10 7 ]]> 1.54 CYB2-2 Bacillus subtilis <![CDATA[6.6×10 6 ]]> <![CDATA[2.8×10 7 ]]> 0.63 YLZ Bacillus subtilis <![CDATA[5.5×10 6 ]]> <![CDATA[6.0×10 8 ]]> 2.03 CRS2-1 Lactobacillus salivarius <![CDATA[3.5×10 6 ]]> <![CDATA[1.0×10 5 ]]> <0 CRS7-1 Lactobacillus salivarius <![CDATA[1.8×10 6 ]]> <![CDATA[2.1×10 4 ]]> <0 CRS23 Lactobacillus salivarius <![CDATA[2.6×10 6 ]]> <![CDATA[1.9×10 4 ]]> <0 CRS55 Lactobacillus salivarius <![CDATA[1.9×10 6 ]]> <![CDATA[3.8×10 5 ]]> <0

[0069] Through screening by utilizing the oligonutrient capacity of white wine grains, Saccharomyces cerevisiae CJM26 is selected among yeasts as an alternative strain for fermenting white wine grains. Although the bacterium has no cellulase-producing activity, it has strong acid resistance, has a high removal rate for zearalenone and fumonisins, and can grow well in white wine grains; Bacillus subtilis YLZ7 is selected among Bacillus as an alternative strain for fermenting white wine grains. The bacterium has strong acid resistance and high cellulase-producing activity, especially outstanding removal performance for zearalenone, and grows well in white wine grains, so it is a strain very suitable for white wine grains fermentation; Lactobacillus salivarius CRS23 is selected among lactic acid bacteria as an alternative strain for fermenting white wine grains. The acid resistance, cellulase-producing activity and ability to utilize oligonutrients of white wine grains of the bacterium are not very good, but the efficiency of removing zearalenone and fumonisins of the bacterium is significantly higher than that of other candidate lactic acid bacteria, so the bacterium is also selected as an alternative strain for fermenting white wine grains.

[0070] Table 5 Nutrient composition and pH value of white wine lees fermented by different strain combinations

[0071]

[0072] Note: Different capital letters in the same column indicate significant differences (P < 0.05), and the same letters indicate no significant differences (P > 0.05)

[0073] The three strains obtained by screening were used to carry out solid-state fermentation of white wine lees with different combinations and ratios, and the strain combination that can significantly improve the nutritional value of white wine lees, reduce toxin concentration and increase pH value was selected. The specific process is as follows: a ring of colonies was picked from the storage slopes of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7, and Lactobacillus salivarius CRS23 and inoculated in YPD, LB and MRS liquid culture media for activation and enrichment, wherein the activation liquid of Saccharomyces cerevisiae CJM26 was activated at 180rpm and 30℃ for 48h, the activation liquid of Bacillus subtilis YLZ7 was activated at 180rpm and 37℃ for 24h, and the activation liquid of Lactobacillus salivarius CRS23 was activated at 37℃ for 24h. The enriched liquid of the three strains was inoculated with fresh white wine lees raw materials according to the combination of Table 5 according to the inoculation amount of 1%, placed in an open container, sealed, stirred every 8 hours, and fermented at 30℃ for 3 days. After fermentation, the nutrients, zearalenone, fumonisin and pH in the fermented white wine grains were tested.

[0074] Judging from the changes in pH, the pH of the white wine grains in all fermentation groups increased at the end of fermentation. Judging from the changes in nutritional components and toxin removal, the nutritional components of the white wine grains were best improved after mixed fermentation of the three strains of bacteria, with crude protein increased by 34.3%, true protein increased by 44.55%, crude fiber decreased by 14.54%, zearalenone degradation rate of 28.63%, and fumonisin degradation rate of 10.85%.

[0075] Based on the above strain screening results, the optimal strain combination for fermenting white wine lees was determined to be a mixture of CJM26, YLZ7 and CRS23. The three strains were deposited in the General Microbiology Center of China Microbiological Culture Collection on August 27, 2024, and the deposit numbers were CGMCC No.31751, CGMCC No.31753 and CGMCC No.31752 respectively.

[0076] Example 2 Mixed bacteria solid state fermentation of white wine lees

[0077] The above three strains of bacteria were inoculated in equal proportions in fresh white wine lees to produce fermented white wine lees products. The specific process was as follows: a ring of colonies was picked from the storage slopes of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7, and Lactobacillus salivarius CRS23 and inoculated in YPD, LB and MRS liquid culture media for activation and enrichment, wherein the activation solution of Saccharomyces cerevisiae CJM26 was activated by shaking at 180rpm and 28℃ for 45h, the activation solution of Bacillus subtilis YLZ7 was activated by shaking at 180rpm and 35℃ for 20h, and the activation solution of Lactobacillus salivarius CRS23 was activated by standing at 35℃ for 20h. The enriched solution of the three strains of bacteria was inoculated with fresh white wine lees raw materials according to the inoculation amount of 1%, loaded into an open container, sealed, stirred every 8 hours, and fermented at 30℃ for 3 days. After the fermentation, the fermented white wine lees were subjected to sensory evaluation, and the number of live bacteria, pH, nutrients and mycotoxins in the fermented white wine lees were detected. The fermented liquor lees are dried at 70°C for 60 minutes using a drum drying device, crushed and packaged.

[0078] Example 3: Solid-state fermentation of distiller's grains by bacterial enzyme synergy

[0079] A loop of colonies was picked from the storage slopes of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7, and Lactobacillus salivarius CRS23 and inoculated into YPD, LB, and MRS liquid culture media for activation and enrichment, wherein the activation solution of Saccharomyces cerevisiae CJM26 was activated by shaking at 180 rpm and 28°C for 45 hours, the activation solution of Bacillus subtilis YLZ7 was activated by shaking at 180 rpm and 35°C for 20 hours, and the activation solution of Lactobacillus salivarius CRS23 was activated by standing at 35°C for 20 hours. The enrichment solutions of the three strains were inoculated with fresh white wine lees according to a 1% inoculation amount, and then acid protease, cellulase, pectinase, xylanase, and glucose oxidase were added, mixed evenly, and then placed in an open container, sealed, stirred every 8 hours, and fermented at 30°C for 3 days. The addition amount of acid protease, cellulase, pectinase and xylanase is 100u / g dry white wine lees, and the addition amount of glucose oxidase is 20u / g dry white wine lees. After fermentation, the fermented white wine lees are subjected to sensory evaluation to detect the number of viable bacteria, pH, nutrients and mycotoxins in the fermented white wine lees. The fermented white wine lees are dried at 70°C for 60 minutes by a drum drying equipment, crushed and packaged.

[0080] Example 4: Solid-state fermentation of distiller's grains by coupling bacteria, enzymes and adsorbents

[0081] A loop of colonies was picked from the storage slopes of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7, and Lactobacillus salivarius CRS23 and inoculated in YPD, LB, and MRS liquid culture media for activation and enrichment, wherein the activation solution of Saccharomyces cerevisiae CJM26 was activated by shaking at 180 rpm and 28°C for 45 hours, the activation solution of Bacillus subtilis YLZ7 was activated by shaking at 180 rpm and 35°C for 20 hours, and the activation solution of Lactobacillus salivarius CRS23 was activated by standing at 35°C for 20 hours. The enrichment solutions of the three strains were inoculated with fresh white wine lees according to a 1% inoculation amount, and then acid protease, cellulase, pectinase, xylanase, and glucose oxidase were added and mixed evenly, and then plant carbon black was added, further mixed, and placed in an open container, sealed, stirred every 8 hours, and fermented at 30°C for 3 days. The addition amount of acid protease, cellulase, pectinase and xylanase is 100u / g dry white wine lees, the addition amount of glucose oxidase is 20u / g dry white wine lees, and the addition amount of plant carbon black is 1‰ of the mass of fresh white wine lees. After the fermentation, the fermented white wine lees are subjected to sensory evaluation to detect the number of viable bacteria, pH, nutrients and mycotoxins in the fermented white wine lees. The fermented white wine lees are dried at 70℃ for 60min by drum drying equipment, crushed and packaged.

[0082] Example 5: Solid-state fermentation of distiller's grains by coupling bacteria, enzymes and adsorbents

[0083] A loop of colonies was picked from the storage slopes of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7, and Lactobacillus salivarius CRS23 and inoculated in YPD, LB, and MRS liquid culture media for activation and enrichment, wherein the activation solution of Saccharomyces cerevisiae CJM26 was activated by shaking at 180 rpm and 30°C for 48 hours, the activation solution of Bacillus subtilis YLZ7 was activated by shaking at 180 rpm and 37°C for 24 hours, and the activation solution of Lactobacillus salivarius CRS23 was activated by standing at 37°C for 24 hours. The enrichment solutions of the three strains were inoculated with fresh white wine lees according to a 3% inoculation amount, and then acid protease, cellulase, pectinase, xylanase, and glucose oxidase were added and mixed evenly, and then plant carbon black was added, further mixed, and placed in an open container, sealed, stirred every 10 hours, and fermented at 35°C for 3 days. The addition amount of acid protease, cellulase, pectinase and xylanase is 150u / g dry white wine lees, the addition amount of glucose oxidase is 30u / g dry white wine lees, and the addition amount of plant carbon black is 3‰ of the mass of fresh white wine lees. After fermentation, the fermented white wine lees are subjected to sensory evaluation to detect the number of viable bacteria, pH, nutrients and mycotoxins in the fermented white wine lees. The fermented white wine lees are dried at 75℃ for 45min by drum drying equipment, crushed and packaged.

[0084] Example 6: Solid-state fermentation of distiller's grains by coupling bacteria, enzymes and adsorbents

[0085] A loop of colonies was picked from the storage slopes of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7, and Lactobacillus salivarius CRS23 and inoculated into YPD, LB, and MRS liquid culture media for activation and enrichment, wherein the activation solution of Saccharomyces cerevisiae CJM26 was activated by shaking at 180 rpm and 29°C for 46 hours, the activation solution of Bacillus subtilis YLZ7 was activated by shaking at 180 rpm and 36°C for 22 hours, and the activation solution of Lactobacillus salivarius CRS23 was activated by standing at 40°C for 22 hours. The enrichment solutions of the three strains were inoculated with fresh white wine lees according to a 5% inoculation amount, and then acid protease, cellulase, pectinase, xylanase, and glucose oxidase were added and mixed evenly, and then plant carbon black was added, further mixed, placed in an open container, sealed, stirred every 12 hours, and fermented at 37°C for 4 days. The addition amount of acid protease, cellulase, pectinase and xylanase is 200u / g dry white wine lees, the addition amount of glucose oxidase is 50u / g dry white wine lees, and the addition amount of plant carbon black is 5‰ of the mass of fresh white wine lees. After the fermentation, the fermented white wine lees are subjected to sensory evaluation, and the number of viable bacteria, pH, nutrients and mycotoxins in the fermented white wine lees are detected. The fermented white wine lees are dried at 80℃ for 30min by drum drying equipment, crushed and packaged.

[0086] Test Example 1 Index Detection

[0087] 1. Detection method:

[0088] (1) Sensory evaluation: After fermentation, samples of fermented liquor lees were randomly taken for color, odor, and texture evaluation.

[0089] (2) pH: Take 1 g of fermented liquor lees and place it in a 10 mL centrifuge tube. Add 9 mL of sterile water, shake and let stand. Use a pH meter to measure the pH of the supernatant.

[0090] (3) Number of viable bacteria: Take 10g of the white wine lees sample before and after fermentation in a 300mL Erlenmeyer flask, add 90mL of sterile water and an appropriate amount of glass beads, and shake in a shaking incubator (25℃, 180r / min) for 30min. After the shaking, perform a 10-fold gradient dilution, and make three parallels for each sample. (1) Determination of the number of viable bacteria of Bacillus subtilis: Use the dilution coating method to culture on an LB plate at 37℃ for 24h and then count; (2) Determination of the number of viable bacteria of Saccharomyces cerevisiae: Use the dilution coating method to culture on a Bengal red plate at 30℃ for 48h and then count; (3) Determination of the number of viable bacteria of lactic acid bacteria: Use the dilution pouring method to culture on an MRS solid medium containing 0.5% CaCO3 at 37℃ for 48h and then count the number of colonies that produce calcium-dissolving circles.

[0091] (4) Dry matter: refer to GB / T 6435-2014 “Determination of moisture in feeds” and use a constant temperature forced air drying oven for determination.

[0092] (5) Crude protein: refer to GB / T 6432-2018 “Determination of crude protein in feeds” and use FOSS fully automatic Kjeldahl nitrogen analyzer for determination.

[0093] (6) Acid-soluble protein: According to NY / T3801-2020 "Determination of acid-soluble protein in feed raw materials": weigh 2g of white wine lees, add 40mL, 150g / L trichloroacetic acid solution in a vortex shaker for half an hour, let it stand for 5 minutes, centrifuge at 4000r / min for 5 minutes, take 10mL and add 12mL concentrated sulfuric acid to the FOSS digestion tube, add a catalyst sheet, and determine according to the crude protein determination method. Acid-soluble protein % = mgN / mL × 6.25 × sample volume / trichloroacetic acid volume × sample mass.

[0094] (7) Determination of true protein: refer to DB13 / T1098-2009 "Determination of protein in feed" and use the copper sulfate precipitation method for determination.

[0095] (8) Determination of crude fiber content: refer to GB / T 6434-2022 and use the filter bag method.

[0096] (9) Determination of neutral detergent fiber: refer to GBT20806-2022 and use the filter bag method.

[0097] (10) Crude fat: Determined using a fully automatic Soxhlet extractor in accordance with GB / T 6433-2006 “Determination of crude fat content in feeds”.

[0098] (11) Crude ash: refer to GB / T 6438-2007 “Determination of crude ash content in feeds” and use a high-temperature muffle furnace for determination.

[0099] 2. The test results are shown in Table 6-Table 8:

[0100] Table 6 Sensory evaluation of liquor lees before and after fermentation

[0101] project texture color odor White wine lees Loose without lumps tan Wine aroma Embodiment 2, 3, 4, 5, 6 Loose, soft and no lumps Dark Brown Sour aroma

[0102] Table 7 Live bacteria count and pH before and after fermentation of white wine lees

[0103]

[0104] Table 8 Changes in nutritional components of liquor lees before and after fermentation

[0105]

[0106]

[0107] Compared with unfermented white wine lees, fermented white wine lees have a soft and loose texture, dark brown color and a sour and fragrant smell, and their palatability is significantly improved.

[0108] After the detoxification of liquor lees by coupled fermentation with bacteria, enzymes and adsorbents, the pH value was significantly increased from 3.3 to 5.4-5.8, reducing the stimulation of high acidity to livestock and poultry. After solid-state fermentation with only mixed strains, the crude protein content of liquor lees increased by 26.25%, the acid-soluble protein increased by 25.22%, the true protein increased by 40.39%, the crude fiber decreased by 15.15%, and the neutral detergent fiber decreased by 21.3%. The zearalenone of corn decreased by 30.97% and the fumonisin decreased by 12.67%. On the basis of mixed fermentation, after adding acid protease, cellulase, pectinase, xylanase and glucose oxidase at the same time, the crude protein content of liquor lees increased by 32.37%, the acid-soluble protein increased by 46.54%, the true protein increased by 50.87%, the crude fiber decreased by 28.11%, and the neutral detergent fiber decreased by 31.16%. The zearalenone of corn decreased by 42.09% and the fumonisin decreased by 44.87%. On the basis of the synergistic fermentation of bacteria and enzymes, plant carbon black was added at the same time to form a solid-state fermentation process coupled with bacteria, enzymes and adsorbents. The crude protein content of the fermented liquor lees increased by 31.03%, the acid-soluble protein increased by 44.05%, the true protein increased by 49.33%, the crude fiber decreased by 28.47%, and the neutral detergent fiber decreased by 31.1%. The zearalenone content of zearalenone decreased by 98.32%, and the fumonisin content decreased by 98.62%.

[0109] It can be seen that by adopting the bacteria, enzymes, adsorbents coupled solid-state fermentation detoxification white wine grains technology provided by this patent, it is possible to simultaneously solve the bottleneck problems that restrict the large-scale use of white wine grains as livestock and poultry feed raw materials, such as the high acidity, high fiber, and high water content of white wine grains, which lead to easy mildew and high mycotoxin content and are not suitable for microbial growth, thereby producing high-quality livestock and poultry feed raw material products.

[0110] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for producing detoxified distiller's grains by solid-state fermentation coupled with bacteria, enzymes and adsorbents, characterized in that: The production method comprises: Inoculate the bacterial liquid of Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7 and Lactobacillus salivarius CRS23 into fresh white wine lees at an inoculation rate of 1%-5%, then add complex enzyme and mix evenly, then add plant carbon black and mix evenly, seal, and ferment at 30-37°C for 3-4 days; Among them, the Saccharomyces cerevisiae CJM26, Bacillus subtilis YLZ7 and Lactobacillus salivarius CRS23 are deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit numbers are CGMCC No.31751, CGMCC No.31753 and CGMCC No.31752 respectively; The complex enzyme consists of acid protease, cellulase, pectinase, xylanase and glucose oxidase.

2. The production method according to claim 1, characterized in that The water content of the fresh distiller's grains is 57%-60%.

3. The production method according to claim 1, characterized in that The bacterial solution is prepared by the following method: a loop of colonies is picked from each of the preservation slants of the saccharomyces cerevisiae CJM26, the Bacillus subtilis YLZ7, and the Lactobacillus salivarius CRS23, and inoculated into YPD, LB and MRS liquid culture media for activation and enrichment, wherein the saccharomyces cerevisiae activation solution is oscillated and activated at 180 rpm and 28-30° C. for 45-48 hours, the Bacillus subtilis activation solution is oscillated and activated at 180 rpm and 35-37° C. for 20-24 hours, and the Lactobacillus salivarius activation solution is statically activated at 35-40° C. for 20-24 hours.

4. The production method according to claim 1, characterized in that The addition amount of the acidic protease, cellulase, pectinase and xylanase is 100-200u / g dry white wine grains, and the addition amount of the glucose oxidase is 20-50u / g dry white wine grains.

5. The production method according to claim 1, characterized in that: The added amount of the plant carbon black is 1‰-5‰ of the mass of the fresh white wine lees.

6. The production method according to claim 1, characterized in that: During the fermentation process, stir regularly.

7. The production method according to claim 6, characterized in that: Stir every 8-12 hours.

8. The production method according to claim 1, characterized in that: The production method also includes the steps of drying, crushing and packaging the fermented liquor lees.

9. The production method according to claim 8, characterized in that: The drying temperature is 70-80° C. and the drying time is 30-60 min.

10. A white wine lees feed, characterized in that: Prepared by the production method according to any one of claims 1 to 9.

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