Method for producing coarse feed through fermentation by fully utilizing two-step heterotopic white spirit brewing bottom pot water and waste distiller's grains

Abstract

The invention discloses a method for producing coarse feed through fermentation by fully utilizing pot-bottom water and waste distiller's grains in two-step ectopic liquor brewing, and belongs to the technical field of fermented feed. In order to realize white spirit vinasse and bottom pot water resource utilization and high-nutritional-value feed production, the invention provides a method for producing coarse feed by fermentation through two-step ectopic white spirit brewing bottom pot water vinasse full utilization, which comprises the following steps: carrying out activation culture on lactobacillus buchneri to obtain a lactobacillus buchneri activated seed solution; activating and culturing aspergillus oryzae to obtain an aspergillus oryzae activated strain; inoculating the lactobacillus buchneri activated seed solution into a pot-bottom water culture medium to obtain lactobacillus buchneri fermentation liquor; inoculating the aspergillus oryzae activated strain into the bottom pot water culture medium to obtain aspergillus oryzae fermentation liquor; uniformly mixing vinasse, calcium carbonate and sesame seed meal to obtain a fermentation substrate; and uniformly mixing the lactobacillus buchneri fermentation liquor, the aspergillus oryzae fermentation liquor and the fermentation substrate, and fermenting to obtain the vinasse roughage. The fermented products of the waste distiller's grains and the pot bottom water are all used as feed, and no waste is generated.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented feed, and in particular relates to a method for producing roughage by fermenting the whole waste grains of two-step ex situ liquor brewing bottom pot water. Background Art

[0002] Liquor lees are solid waste from liquor production, with an annual output of more than 20 million tons. Since the raw materials for liquor brewing usually include grains such as sorghum, wheat, rice and corn, most of the organic matter is utilized after microbial fermentation, and a small part of the organic matter remains in the lees. Therefore, the lees still have great utilization value, which contains a large amount of water and rich nutrients, including a large amount of cellulose, hemicellulose, protein, amino acids, vitamins (such as vitamin A, vitamin B, vitamin C and vitamin E, etc.), various organic acids and fats, etc. For the direct use of solid liquor lees as feed, small enterprises usually adopt this method, directly providing the lees to surrounding farmers as feed, but the price is relatively low, generally 80-100 yuan / ton. Liquor lees contain a lot of water, which is not convenient for storage and transportation. If it is not effectively treated and utilized, it is easy to mold, which will also cause pollution to the environment, and it is also a waste of resources.

[0003] Liquor lees is a high-quality feed raw material. The use of probiotics to ferment liquor lees to reduce fiber content and improve the nutritional content of feed is a hot topic in the current research on lees in feed. Liquor lees are processed into ruminant feed (such as roughage, concentrate feed, fermented feed, etc.), and the rich nutrients they contain are beneficial to the growth and development of animals. In the study of Yu Xingyu et al., it was found that adding 60% liquor lees to the roughage of Simmental beef cattle is beneficial to improving the production performance of beef cattle. Zhou Dongnian's research shows that the rumen utilization efficiency of crude protein and the fiber degradation rate of liquor lees are improved after fermentation by brewer's yeast. At the same time, the fermented lees feed can significantly improve the production performance and immunity of dairy cows. In the study of Ren Zhihua et al., adding an appropriate amount of liquor lees to beef cattle feed can increase the meat yield. Chen Guangji et al. found that in the process of yak feeding, adding 9.25% fermented liquor lees can improve the digestibility and production performance of feed, while the rumen fermentation type does not change and will not affect serum biochemical indicators. There are many ways to utilize the spent grains of liquor. The spent grains can also be used to produce organic fertilizer, cultivation medium and spent grain koji, etc.

[0004] Brewing wastewater is mainly composed of yellow water, site cleaning water, bottom pot water, etc. The bottom pot water of liquor is produced in the upper steamer cooking section of the liquor brewing process. It is the liquid that condenses and refluxes the steam in the pot and gathers at the bottom of the pot. It contains a large amount of alcohol, acid and ester. The liquor production process will produce a large amount of industrial wastewater, including bottom pot water, cellar bottom water and cooling water. According to statistics, in the liquor production process, every ton of liquor produced will produce about 17 tons of brewing wastewater, of which the bottom pot water of liquor accounts for the largest proportion of brewing wastewater. With the development of the liquor industry, the output of bottom pot water has been very huge. At the same time, the bottom pot water contains a large amount of organic matter (organic acid, starch and small molecule sugar, etc.), with a turbidity of about 1650NTU, chemical oxygen demand (COD) of about 11000mg / L, and suspended matter concentration of about 17500ms / L. If it is not effectively treated, it will cause serious pollution to the environment. It is also urgent to find a way to efficiently utilize the bottom pot water.

[0005] CN116769840A discloses a method for producing chlorella growth factor using liquor brewing bottom pot water, which uses liquor brewing bottom pot water to cultivate chlorella, reduce the COD, phosphorus and nitrogen of the bottom pot water, and generate chlorella growth factor at the same time. Deng Hansen et al. used yeast to treat the bottom pot water, reduce the organic matter in the bottom pot water, and obtain bacterial protein at the same time. Lin Jie et al. (Lin Jie, Zheng Jia, Wang Fengqing, et al. Research on the full utilization of liquor brewing by-products to produce fermented feed [J / OL]. China Feed, 1-7 [2024-04-13].) Use liquor brewing by-products (waste, bottom pot water) as the main raw materials, add food processing by-products, and use microbial fermentation to produce feed. After fermentation, the acid-soluble protein of the feed is increased by 125.46%.

[0006] CN1119495A discloses a method for producing active protein feed using distiller's grains, comprising: a. preparing bacterial strains, diluting and acidifying with water from a bottom pot to a pH value of about 3.0 as a culture medium, pouring the culture medium into an open basin, inoculating yeast strains, and intermittently stirring and culturing at a temperature of 30±1°C until the pH value rises to about 6.0, and the bacterial strain preparation is completed; b. when the fresh distiller's grains are dried to about 35°C, adding bacterial strains at an inoculation amount of 10%, mixing evenly, and fermenting in a pool or pile, and stopping fermentation when the initial pH value of the distiller's grains is about 3.0 and rises to about 6.0, and the distiller's grains are covered with white bacteria. However, this method uses fresh distiller's grains as a substrate to culture yeast to produce bacterial protein, and the formed product only considers bacterial proliferation, and does not involve metabolite enrichment; at the same time, it uses a single yeast fermentation, which is not conducive to the degradation of the fermentation substrate, nor is it conducive to the conversion of macromolecules into small molecules, and the animal's absorption and utilization rate of its feed is limited.

[0007] CN115074261A discloses a boulardii yeast and a fermentation medium thereof, a ruminant feed and a preparation method thereof, wherein the composite bacteria include boulardii yeast, Lactobacillus paracasei and Lactobacillus buchneri; the bacterial fermentation medium includes: waste grains, cottonseed meal, soybean meal, bran, and bottom pot water; the boulardii yeast or the composite bacteria is inoculated into the fermentation medium, and anaerobically fermented for 60 to 96 hours to obtain a ruminant feed. The method adopts one-step fermentation, and the crude fiber degradation rate in the obtained feed is low, the acid-soluble protein content is low, and after the feed is fed, the fiber utilization rate of the feed is low.

[0008] In summary, the use of bottom pot water to prepare probiotic fermentation liquid to inoculate the fermentation substrate with spent grains as the main matrix to produce ruminant roughage, improve the palatability of spent grains directly as feed, and increase the nutritional content and absorption rate of the feed; at the same time, it can effectively reduce the wastewater treatment cost in the operation of the liquor industry (can be reduced by about 50%), provide an effective and high-value comprehensive utilization method for the spent grains, a by-product of the liquor industry, and provide a local feed resource utilization plan for the feed industry. Summary of the invention

[0009] The present invention provides a two-step method for producing roughage by fermenting the waste dregs of liquor brewing bottom pot water and fully utilizing the waste dregs of liquor brewing bottom pot water to achieve high-value resource utilization of liquor and produce feed with high nutritional value, which comprises the following steps:

[0010] A. inoculating Lactobacillus buchneri into MRS liquid culture medium, and activating and culturing the medium to obtain Lactobacillus buchneri activated seed solution;

[0011] B. Aspergillus oryzae was streaked on the PDA medium and, after activation culture, an activated strain of Aspergillus oryzae was obtained;

[0012] C. Inoculate the activated Lactobacillus buchneri seed liquid obtained in step A into MRS bottom pot water culture medium, and culture at 34-38° C. for 20-30 h to obtain Lactobacillus buchneri fermentation liquid;

[0013] D, the Aspergillus oryzae activated strain obtained by step B is inoculated in the PDA bottom pot water culture medium, and cultivated for 30~48h at 28~32 ℃ to obtain Aspergillus oryzae fermentation liquid;

[0014] E. Evenly mixing the waste grains, calcium salt and sesame meal to obtain a fermentation matrix;

[0015] F. Evenly mix the Lactobacillus buchneri fermentation liquid obtained in step C, the Aspergillus oryzae fermentation liquid obtained in step D and the fermentation substrate obtained in step E, and ferment at 29-39° C. for 36-96 h to obtain fermented distiller's grains roughage.

[0016] Among them, in the method for producing roughage by fermenting the discarded grains from the bottom pot of the above-mentioned two-step ectopic liquor brewing, in step A, the formula of the MRS liquid culture medium is: 20.0g of glucose, 10.0g of beef extract, 10.0g of peptone, 5.0g of yeast extract powder, 5.0g of anhydrous sodium acetate, 2.0g of triammonium citrate, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate, 1.0g of Tween 80, 1.0L of distilled water, and natural pH.

[0017] Among them, in the above-mentioned two-step method for producing roughage by fermenting the discarded grains from the bottom pot of ex situ liquor brewing, in step A, the activation culture is cultured at 37° C. for 24 hours.

[0018] Among them, in the above-mentioned two-step method for producing roughage by fermenting the discarded grains from the bottom pot of ectopic liquor brewing, in step B, the formula of the PDA culture medium is: 6.0g potato extract powder, 20.0g glucose, 15.0g agar, 1.0L deionized water, and natural pH.

[0019] Among them, in the above two-step method for producing roughage by fermenting the discarded grains from the bottom pot of ex situ liquor brewing, in step B, the activation culture is cultured at 30° C. for 72 hours.

[0020] Wherein, in the above two-step method for producing roughage by fermenting the waste grains from the bottom pot of ectopic liquor brewing, in step C, the inoculation amount of the activated seed liquid of Lactobacillus buchneri is 1-5%.

[0021] The present invention adopts two-step ectopic fermentation (liquid fermentation first, then solid fermentation) to make the strain enriched metabolites more abundant. In step A of the present invention, the MRS liquid culture medium is a conventional culture medium in the art, and in step C, on the basis of the MRS liquid culture medium, the bottom pot water of liquor brewing is used to replace water, and the bottom pot water is used as one of the first step liquid fermentation matrices to prepare the Lactobacillus buchneri fermentation liquid. Among them, in the method for producing roughage by fermenting the discarded grains of the bottom pot water of the above-mentioned two-step ectopic liquor brewing, in step C, the formula of the MRS bottom pot water culture medium is: 20.0g of glucose, 10.0g of beef extract, 10.0g of peptone, 5.0g of yeast extract powder, 5.0g of anhydrous sodium acetate, 2.0g of triammonium citrate, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate, 1.0g of Tween 80, and 1.0L of liquor brewing bottom pot water with a pH of 3-7 (the pH of the liquor brewing bottom pot water can be adjusted by using an appropriate amount of sodium hydroxide solution).

[0022] In order to make the metabolites enriched by the strain more abundant, the present invention takes into account the difference in fermentation time and reflects the indicators of the fermentation broth. The biomass and total acid content of the fermentation broth of Lactobacillus buchneri are given. Among them, in the above two-step method of fermenting roughage using the waste dregs of the bottom pot water of the ectopic liquor brewing, in step C, the cell concentration OD of the fermentation broth of Lactobacillus buchneri is 600nm It is 1.2~1.5, and the total acid content is 14.50~17.50g / L.

[0023] Among them, in the above-mentioned two-step method for producing roughage by fermenting the discarded grains from the bottom pot water of ectopic liquor brewing, in step D, the inoculation amount of the activated Aspergillus oryzae strain is: a ring of the activated Aspergillus oryzae strain is dipped with an inoculation loop and inoculated into 30-70 mL of PDA bottom pot water culture medium.

[0024] The present invention adopts two-step ectopic (liquid fermentation first, solid fermentation later) to ferment, so that the metabolites enriched by the strain are more abundant. In step B of the present invention, the PDA culture medium is a conventional culture medium in the art, and in step D, on the basis of the PDA culture medium, the water in the bottom pot of liquor brewing is used to replace water, and the bottom pot water is used as one of the first liquid fermentation matrices, and the liquid culture medium does not need to add agar at the same time, for preparing Aspergillus oryzae fermentation liquid. Wherein, the above-mentioned two-step ectopic liquor brewing bottom pot water discarded dregs are fully utilized for fermentation production of roughage, and in step D, the formula of the PDA bottom pot water culture medium is: 6.0g potato extract powder, 20.0g glucose, 1.0L of liquor brewing bottom pot water with a pH of 3 to 7 (the pH of the liquor brewing bottom pot water can be adjusted with an appropriate amount of sodium hydroxide solution).

[0025] In order to make the metabolites enriched by the strain more abundant, the fermentation time is considered to reflect the index of the fermentation liquid, and the Aspergillus oryzae fermentation liquid gives its biomass. Among them, in the above-mentioned two-step method of fermenting roughage by fully utilizing the waste dregs of the bottom pot water of the ectopic liquor brewing, in step D, the cell content of the Aspergillus oryzae fermentation liquid is 0.3-0.5 g / 100 mL.

[0026] Among them, in the above-mentioned two-step method for producing roughage by fermenting the waste grains from the bottom pot of ex-situ liquor brewing with full utilization, in step E, the calcium salt is at least one of calcium carbonate, calcium hydroxide or calcium chloride.

[0027] Among them, in the method for producing roughage by fermenting the discarded dregs from the bottom pot of the two-step ex situ liquor brewing, in step E, the mass ratio of the discarded dregs, calcium salt and sesame meal is 200-210:15-16:24-26.

[0028] Among them, in the above-mentioned two-step method for producing roughage by fermenting the waste grains from the bottom pot of ectopic liquor brewing, in step F, the usage ratio of the Lactobacillus buchneri fermentation liquid and the Aspergillus oryzae fermentation liquid is 6-20 mL:19-6 mL.

[0029] Wherein, in the above two-step method for producing roughage by fermenting the waste grains from the bottom pot of ectopic liquor brewing, in step F, the total inoculation amount of the Lactobacillus buchneri fermentation liquid and the Aspergillus oryzae fermentation liquid is 8-16%.

[0030] Among them, in the method for producing roughage by fermenting the discarded grains from the bottom pot of the above-mentioned two-step ex situ liquor brewing, in step F, the acid detergent fiber content of the fermented grains roughage is 19-23%, the neutral detergent fiber content is 25-26.5%, the crude protein is 15-17g / 100g, the acid soluble protein content is 23-26%, and the total acid content is 14-15g / kg.

[0031] In the present invention, the water in the bottom pot of liquor brewing is a conventional material in the field of liquor brewing. When the grain is cooked in the steamer barrel and the liquor is distilled, the water vapor condenses and dissolves the soluble matter in the mash and then flows back to the liquid at the bottom of the steamer barrel. The mash is extracted with water, and its components contain high-boiling point nutrients such as fatty acids, reducing sugars, starch, crude protein, etc. At the same time, due to the continuous distillation of yellow water and wine tails, its nutritional components are richer, and it has great comprehensive recycling value.

[0032] Beneficial effects of the present invention:

[0033] The present invention selects composite strains to make the enzyme system of the starter richer and the degradation of the fermentation substrate more thorough, so as to obtain a fermented feed with a high acid-soluble protein ratio and a relatively low acid detergent fiber; at the same time, a two-step ectopic fermentation method is used, in which the first step is liquid fermentation, that is, bottom pot water is used as a solvent, and fermentation is performed after nutrition is supplemented, and metabolites are enriched to form a fermentation liquid; in the second step, the fermentation liquid of the first step is inoculated into a fermentation matrix with waste grains as a main component, and the second step of solid fermentation is performed. Under the condition of a richer enzyme system and more enriched metabolites, the degradation of the fermentation substrate is more thorough, the macromolecular substances in the raw materials are converted into small molecular substances, and the cellulose-related indicators are reduced, and finally the fermented feed with a high acid-soluble protein ratio and a relatively low acid detergent fiber is obtained, which is more suitable for animal absorption and utilization.

[0034] The present invention realizes the resource utilization of bottom pot water and can reduce the wastewater treatment cost by 50%; the consumption of wasted grains in the feed production process is relatively large, and the wasted grains account for more than 80% of the fermentation substrate, laying a foundation for its subsequent transformation of results; based on the wasted grains and bottom pot water, anaerobic breathing bag fermentation is adopted, and all the fermentation products are used as feed without any waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The figure is the effect of different fermentation bacteria on the ASP content of different fermentation substrates; among them, A1 is Saccharomyces cerevisiae CICC1398, A2 is Saccharomyces cerevisiae CICC 9080, A3 is Saccharomyces cerevisiae CICC 9763, A4 is Saccharomyces boulardii, B1 is Lactobacillus buchneri, B2 is Lactobacillus paracasei, B3 is Lactobacillus plantarum, C is Enterococcus faecium, D1 is Bacillus licheniformis, D2 is Bacillus subtilis, D3 is Bacillus subtilis xl, and E is Aspergillus oryzae; a~d represent significant differences among the groups, p<0.05.

[0036] Figure 2 The figure shows the degradation effect of different fermentation bacteria on the acidic and neutral detergent fibers of the fermentation substrates; among them, A1 is Saccharomyces cerevisiae CICC1398, A2 is Saccharomyces cerevisiae CICC 9080, A3 is Saccharomyces cerevisiae CICC 9763, A4 is Saccharomyces boulardii, B1 is Lactobacillus buchneri, B2 is Lactobacillus paracasei, B3 is Lactobacillus plantarum, C is Enterococcus faecium, D1 is Bacillus licheniformis, D2 is Bacillus subtilis, D3 is Bacillus subtilis xl, and E is Aspergillus oryzae; a~d represent significant differences among the groups, p<0.05.

[0037] Figure 3 It is a fermentation effect diagram under different volume ratios of composite bacterial fermentation liquid; among them, test group 1 is Saccharomyces cerevisiae 9763: Aspergillus oryzae = 1:1, test group 2 is Lactobacillus bukakii: Aspergillus oryzae = 1:1; test group 3 is Bacillus licheniformis: Aspergillus oryzae = 1:1; test group 4 is Saccharomyces cerevisiae 9763: Lactobacillus bukakii: Aspergillus oryzae = 1:1:1; test group 5 is Saccharomyces cerevisiae 9763: Bacillus licheniformis: Aspergillus oryzae = 1:1:1; test group 6 is Lactobacillus bukakii: Bacillus licheniformis: Aspergillus oryzae = 1:1:1; test group 7 is Saccharomyces cerevisiae 9763: Lactobacillus bukakii: Bacillus licheniformis: Aspergillus oryzae = 1:1:1:1. DETAILED DESCRIPTION

[0038] In the early stage of the present invention, the fermentation effect of different fermentation bacteria on different fermentation substrates was first tested. Figure 1 shown. Figure 1 This is a graph showing the effect of different fermentation bacteria on the ASP content of different fermentation substrates. Figure 1 Among them, A1 is Saccharomyces cerevisiae CICC1398, A2 is Saccharomyces cerevisiae CICC 9080, A3 is Saccharomyces cerevisiae CICC 9763, A4 is Saccharomyces boulardii, B1 is Lactobacillus buchneri, B2 is Lactobacillus paracasei, B3 is Lactobacillus plantarum, C is Enterococcus faecium, D1 is Bacillus licheniformis, D2 is Bacillus subtilis, D3 is Bacillus subtilis xl, and E is Aspergillus oryzae.

[0039] Depend on Figure 1It can be seen that with fresh waste grains as the main matrix and 6 kinds of grains such as sesame meal and peanut meal as auxiliary materials, after fermentation by 12 strains of bacteria, the sesame meal has the highest acid-soluble protein content and is most suitable for use as feed.

[0040] Then, the present invention uses sesame meal as auxiliary material, acid detergent fiber content as evaluation index, and combines fermentation mode (solid-state anaerobic fermentation) to screen strains suitable for fermentation. The results are as follows Figure 2 shown. Figure 2 This is a diagram showing the degradation effects of different fermentation bacteria on acidic and neutral detergent fibers as fermentation substrates. Figure 2 Among them, A1 is Saccharomyces cerevisiae CICC1398, A2 is Saccharomyces cerevisiae CICC9080, A3 is Saccharomyces cerevisiae CICC 9763, A4 is Saccharomyces boulardii, B1 is Lactobacillus buchneri, B2 is Lactobacillus paracasei, B3 is Lactobacillus plantarum, C is Enterococcus faecium, D1 is Bacillus licheniformis, D2 is Bacillus subtilis, D3 is Bacillus subtilis xl, and E is Aspergillus oryzae.

[0041] according to Figure 2 The present invention selects aerobic and facultative anaerobic strains of Saccharomyces cerevisiae A3, Aspergillus oryzae E, Lactobacillus buchneri B1, and Bacillus licheniformis D1 with low acid detergent fiber content as composite fermentation strains; subsequently, the strains are matched to obtain the best matching strains.

[0042] Subsequently, the present invention also tested the fermentation effect of different composite bacterial fermentation broths, and the results were as follows: Figure 3 shown. Figure 3 This is a diagram of the fermentation effect under different volume ratios of composite bacterial fermentation broth. Figure 3 In the experiments 1, 2, 3, 4, 5, 6, and 7, the ratios of Saccharomyces cerevisiae 9763 and Aspergillus oryzae were 1:1; Lactobacillus bukakii and Aspergillus oryzae were 1:1; Bacillus licheniformis and Aspergillus oryzae were 1:1; Saccharomyces cerevisiae 9763: Lactobacillus bukakii and Aspergillus oryzae were 1:1:1; Saccharomyces cerevisiae 9763: Bacillus bukakii and Aspergillus oryzae were 1:1:1; Lactobacillus bukakii and Bacillus licheniformis and Aspergillus oryzae were 1:1:1; Saccharomyces cerevisiae 9763: Lactobacillus bukakii and Bacillus licheniformis and Aspergillus oryzae were 1:1:1:1.

[0043] Depend on Figure 3 It can be seen that the acidic detergent fiber degradation effect of the fermented feed of group 2 (Lactobacillus buchneri fermentation liquid: Aspergillus oryzae fermentation liquid is 1:1) is the best, with an acidic detergent fiber content of 19.08%, and a relatively low neutral detergent fiber content of 26.23%. Therefore, the present invention selects Lactobacillus buchneri and Aspergillus oryzae for composite fermentation.

[0044] In the present invention, Saccharomyces cerevisiae CICC1398, Saccharomyces cerevisiae CICC 9080, Saccharomyces cerevisiae CICC 9763, Saccharomyces boulardii, Lactobacillus buchneri, Lactobacillus paracasei, Lactobacillus plantarum, Enterococcus faecium, Bacillus licheniformis, Bacillus subtilis, Bacillus subtilis XL and Aspergillus oryzae are all commercially available.

[0045] The present invention is further described in detail below by way of examples, but the protection scope of the present invention is not limited to the scope of the examples.

[0046] The Lactobacillus buchneri strain described in the embodiment of the present invention was purchased from the China Microorganism Culture Collection Center CGMCC, with the collection number of CGMCC: 1.15607.

[0047] The Aspergillus oryzae strain described in the embodiment of the present invention was purchased from China General Microbiological Culture Collection Center CGMCC, with a collection number of CGMCC: 3.13905.

[0048] The bottom pot water and discarded dregs described in the embodiments of the present invention are both bottom pot water and discarded dregs produced in the brewing production of Wuliang Luzhou-flavor liquor, and are sourced from a winery in Yibin City.

[0049] All culture media involved in the embodiments of the present invention were sterilized at 121° C. under high pressure for 30 minutes before use after preparation.

[0050] Example 1

[0051] 1. The Lactobacillus buchneri of laboratory preservation was inoculated in liquid culture medium (formula: glucose 20.0g, beef extract 10.0g, peptone 10.0g, yeast extract powder 5.0g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, Tween 80 1g, distilled water 1L, natural pH), the culture medium liquid amount was 50mL / 250mL triangular flask, and cultured at 37°C for 24h, as the Lactobacillus buchneri activated seed liquid.

[0052] 2. The Aspergillus oryzae preserved in the laboratory was streaked onto a solid culture medium (formula: 6 g potato extract powder, 20 g glucose, 1 L deionized water, natural pH (15.0 g agar was added when preparing the solid culture medium)), and cultured in a 30° C. constant temperature incubator for 72 h to serve as an activated strain of Aspergillus oryzae.

[0053] 3. The activated seed liquid of Lactobacillus buchneri was inoculated into a liquid culture medium (prescription: 20.0 g of glucose, 10.0 g of beef extract, 10.0 g of peptone, 5.0 g of yeast extract powder, 5.0 g of anhydrous sodium acetate, 2.0 g of triammonium citrate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 1 g of Tween 80, 1.0 L of pan water whose pH was adjusted to 6 with sodium hydroxide solution) at an inoculum size of 2%. The culture medium was loaded with liquid in a 50 mL / 250 mL triangular flask and cultured for 27 h at 37 ° C. As the fermented liquid of Lactobacillus buchneri, its cell concentration OD 600nm It is 1.43, and the total acid content is 17.24g / L.

[0054] 4. Dip a loop of the activated Aspergillus oryzae strain with an inoculating loop and inoculate it into a liquid culture medium (the formula is: 6 g potato extract powder, 20 g glucose, 1.0 L of pot water adjusted to 5 with sodium hydroxide solution), the culture medium liquid amount is 50 mL / 250 mL triangular flask, and cultured at 30 ° C for 42 h. As Aspergillus oryzae fermentation liquid, its cell content is 0.41 g / 100 mL.

[0055] 5. Mix 209.25 g of dregs, 15.75 g of calcium carbonate and 25 g of sesame meal evenly as the fermentation matrix.

[0056] 6. 12.5 mL of Lactobacillus buchneri fermented liquid and 12.5 mL of Aspergillus oryzae fermented liquid were mixed with 250 g of fermentation substrate, and 275 g of each anaerobic respiration bag was packaged and cultured at 37 ° C for 48 h to obtain a fermented distiller's grains roughage with an acid detergent fiber content of 21.47%, a neutral detergent fiber content of 25.39%, a crude protein content of 16.48 g / 100 g, an acid soluble protein content (ASP / CP) of 24.62%, and a total acid content of 14.27 g / kg.

[0057] Example 2

[0058] 1. The Lactobacillus buchneri of laboratory preservation was inoculated in liquid culture medium (formula: glucose 20.0g, beef extract 10.0g, peptone 10.0g, yeast extract powder 5.0g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, Tween 80 1g, distilled water 1L, natural pH), the culture medium liquid amount was 50mL / 250mL triangular flask, and cultured at 37°C for 24h, as the Lactobacillus buchneri activated seed liquid.

[0059] 2. The Aspergillus oryzae preserved in the laboratory was streaked onto a solid culture medium (formula: 6 g potato extract powder, 20 g glucose, 1 L deionized water, natural pH (15.0 g agar was added when preparing the solid culture medium)), and cultured in a 30° C. constant temperature incubator for 72 h to serve as an activated strain of Aspergillus oryzae.

[0060] 3. The activated seed liquid of Lactobacillus buchneri was inoculated into a liquid culture medium (prescription: 20.0 g of glucose, 10.0 g of beef extract, 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of anhydrous sodium acetate, 2.0 g of triammonium citrate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 1 g of Tween 80, 1 L of pot water whose pH was adjusted to 6 with sodium hydroxide solution) at an inoculum size of 2%. The culture medium was loaded with liquid in a 50 mL / 250 mL triangular flask and cultured at 37 ° C for 27 h. As the fermented liquid of Lactobacillus buchneri, its cell concentration OD 600nm It is 1.43, and the total acid content is 17.24g / L.

[0061] 4. Dip a loop of the activated Aspergillus oryzae strain with an inoculating loop and inoculate it into a liquid culture medium (the formula is: 6 g potato extract powder, 20 g glucose, 1.0 L of pot water adjusted to 5 with sodium hydroxide solution), the culture medium liquid amount is 50 mL / 250 mL triangular flask, and cultured at 30 ° C for 42 h. As Aspergillus oryzae fermentation liquid, its cell content is 0.41 g / 100 mL.

[0062] 5. Mix 209.25 g of dregs, 15.75 g of calcium carbonate and 25 g of sesame meal evenly as the fermentation matrix.

[0063] 6. 10mL of Lactobacillus buchneri fermented liquid and 15mL of Aspergillus oryzae fermented liquid were mixed with 250g of fermentation substrate, and 275g of each anaerobic respiration bag was packed, and fermented for 48h at 31°C to obtain a fermented distiller's grains roughage with an acid detergent fiber content of 22.38%, a neutral detergent fiber content of 25.51%, a crude protein of 16.01g / 100g, an acid soluble protein content (ASP / CP) of 25.43%, and a total acid content of 14.07g / kg.

[0064] Example 3

[0065] 1. The Lactobacillus buchneri of laboratory preservation was inoculated in liquid culture medium (formula: glucose 20.0g, beef extract 10.0g, peptone 10.0g, yeast extract powder 5.0g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, Tween 80 1g, distilled water 1L, natural pH), the culture medium liquid amount was 50mL / 250mL triangular flask, and cultured at 37°C for 24h, as the Lactobacillus buchneri activated seed liquid.

[0066] 2. The Aspergillus oryzae preserved in the laboratory was streaked onto a solid culture medium (formula: 6 g potato extract powder, 20 g glucose, 1 L deionized water, natural pH (15.0 g agar was added when preparing the solid culture medium)), and cultured in a 30° C. constant temperature incubator for 72 h to serve as an activated strain of Aspergillus oryzae.

[0067] 3. The activated seed liquid of Lactobacillus buchneri was inoculated into a liquid culture medium (prescription: 20.0 g of glucose, 10.0 g of beef extract, 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of anhydrous sodium acetate, 2.0 g of triammonium citrate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 1 g of Tween 80, 1 L of pot water whose pH was adjusted to 6 with sodium hydroxide solution) at an inoculum size of 2%. The culture medium was loaded with liquid in a 50 mL / 250 mL triangular flask and cultured at 37 ° C for 27 h. As the fermented liquid of Lactobacillus buchneri, its cell concentration OD 600nm It is 1.43, and the total acid content is 17.24g / L.

[0068] 4. Dip a loop of the activated Aspergillus oryzae strain with an inoculating loop and inoculate it into a liquid culture medium (the formula is: 6 g potato extract powder, 20 g glucose, 1.0 L of pot water adjusted to 5 with sodium hydroxide solution), the culture medium liquid amount is 50 mL / 250 mL triangular flask, and cultured at 30 ° C for 42 h. As Aspergillus oryzae fermentation liquid, its cell content is 0.41 g / 100 mL.

[0069] 5. Mix 209.25 g of dregs, 15.75 g of calcium carbonate and 25 g of sesame meal evenly as the fermentation medium.

[0070] 6. 10mL of Lactobacillus buchneri fermented liquid and 15mL of Aspergillus oryzae fermented liquid are mixed, mixed with 250g of fermentation substrate, packaged with the amount of 275g bagged for each anaerobic respiration, fermented at 37°C for 48h, and obtained a fermented distiller's grains roughage with an acid detergent fiber content of 22.01%, a neutral detergent fiber content of 25.87%, a crude protein of 16.04g / 100g, an acid soluble protein content (ASP / CP) of 23.61%, and a total acid content of 14.07g / kg.

[0071] Comparative Example 1

[0072] 1. The Lactobacillus buchneri of laboratory preservation was inoculated in liquid culture medium (formula: glucose 20.0g, beef extract 10.0g, peptone 10.0g, yeast extract powder 5.0g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, Tween 80 1g, distilled water 1L, natural pH), the culture medium liquid amount was 50mL / 250mL triangular flask, and cultured at 37°C for 24h, as the Lactobacillus buchneri activated seed liquid.

[0073] 2. The Aspergillus oryzae preserved in the laboratory was streaked onto a solid culture medium (formula: 6 g potato extract powder, 20 g glucose, 1 L deionized water, natural pH (15.0 g agar was added when preparing the solid culture medium)), and cultured in a 30° C. constant temperature incubator for 72 h to serve as an activated strain of Aspergillus oryzae.

[0074] 3. The activated seed liquid of Lactobacillus buchneri was inoculated into a liquid culture medium (prescription: 20.0 g of glucose, 10.0 g of beef extract, 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of anhydrous sodium acetate, 2.0 g of triammonium citrate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 1 g of Tween 80, 1 L of pot water whose pH was adjusted to 6 with sodium hydroxide solution) at an inoculum size of 2%. The culture medium was loaded with liquid in a 50 mL / 250 mL triangular flask and cultured at 37 ° C for 27 h. As the fermented liquid of Lactobacillus buchneri, its cell concentration OD 600nm It is 1.43, and the total acid content is 17.24g / L.

[0075] 4. Dip a loop of the activated Aspergillus oryzae strain with an inoculating loop and inoculate it into a liquid culture medium (the formula is: 6 g potato extract powder, 20 g glucose, 1.0 L of pot water adjusted to 5 with sodium hydroxide solution), the culture medium liquid amount is 50 mL / 250 mL triangular flask, and cultured at 30 ° C for 42 h. As Aspergillus oryzae fermentation liquid, its cell content is 0.41 g / 100 mL.

[0076] 5. Mix 209.25 g of dregs, 15.75 g of calcium carbonate and 25 g of peanut meal evenly as the fermentation medium.

[0077] 6. After 10mL Lactobacillus buchneri fermented liquid and 15mL Aspergillus oryzae fermented liquid were mixed, mixed with 250g fermentation substrate, bagged 275g of each anaerobic respiration and packaged, fermented at 37 ℃ for 72h, and obtained an acid detergent fiber content of 24.01%, a neutral detergent fiber content of 27.87%, a crude protein of 16.04g / 100g, an acid soluble protein content (ASP / CP) of 23.61%, and a total acid content of 11.07g / kg of fermented distiller's grains roughage.

[0078] Comparative Example 2

[0079] 1. The Lactobacillus buchneri of laboratory preservation was inoculated in liquid culture medium (formula: glucose 20.0g, beef extract 10.0g, peptone 10.0g, yeast extract powder 5.0g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, Tween 80 1g, distilled water 1L, natural pH), the culture medium liquid amount was 50mL / 250mL triangular flask, and cultured at 37°C for 24h, as the Lactobacillus buchneri activated seed liquid.

[0080] 2. The Aspergillus oryzae preserved in the laboratory was streaked onto a solid culture medium (formula: 6 g potato extract powder, 20 g glucose, 1 L deionized water, natural pH (15.0 g agar was added when preparing the solid culture medium)), and cultured in a 30° C. constant temperature incubator for 72 h to serve as an activated strain of Aspergillus oryzae.

[0081] 3. The activated seed liquid of Lactobacillus buchneri was inoculated into a liquid culture medium (prescription: 20.0 g of glucose, 10.0 g of beef extract, 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of anhydrous sodium acetate, 2.0 g of triammonium citrate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 1 g of Tween 80, 1 L of pot water whose pH was adjusted to 6 with sodium hydroxide solution) at an inoculum size of 2%. The culture medium was loaded with liquid in a 50 mL / 250 mL triangular flask and cultured at 37 ° C for 27 h. As the fermented liquid of Lactobacillus buchneri, its cell concentration OD 600nm It is 1.43, and the total acid content is 17.24g / L.

[0082] 4. Dip a loop of the activated Aspergillus oryzae strain with an inoculating loop and inoculate it into a liquid culture medium (the formula is: 6 g potato extract powder, 20 g glucose, 1.0 L of pot water adjusted to 5 with sodium hydroxide solution), the culture medium liquid amount is 50 mL / 250 mL triangular flask, and cultured at 30 ° C for 42 h. As Aspergillus oryzae fermentation liquid, its cell content is 0.41 g / 100 mL.

[0083] 5. Mix 209.25 g of dregs, 15.75 g of calcium carbonate and 25 g of rapeseed meal evenly as the fermentation matrix.

[0084] 6. After 10mL of Lactobacillus buchneri and 15mL of Aspergillus oryzae fermented liquid were mixed, they were mixed with 250g of fermentation substrate, and the amount of 275g was bagged for each anaerobic respiration and packaged, and fermented at 37°C for 72h to obtain a fermented distiller's grains roughage with an acid detergent fiber content of 25.02%, a neutral detergent fiber content of 27.41%, a crude protein of 16.13g / 100g, an acid soluble protein content (ASP / CP) of 23.89%, and a total acid content of 12.42g / kg.

Claims

1. A method for producing roughage by fermenting the waste dregs of two-step ex situ liquor brewing bottom pot water using full utilization, characterized in that: The following steps are involved: A. inoculating Lactobacillus buchneri into MRS liquid culture medium, and activating and culturing the medium to obtain Lactobacillus buchneri activated seed solution; B. Aspergillus oryzae was streaked on the PDA medium and, after activation culture, an activated strain of Aspergillus oryzae was obtained; C. Inoculate the activated Lactobacillus buchneri seed liquid obtained in step A into MRS bottom pot water culture medium, and culture at 34-38° C. for 20-30 h to obtain Lactobacillus buchneri fermentation liquid; D, the Aspergillus oryzae activated strain obtained by step B is inoculated in the PDA bottom pot water culture medium, and cultivated for 30~48h at 28~32 ℃ to obtain Aspergillus oryzae fermentation liquid; E. Evenly mixing the waste grains, calcium salt and sesame meal to obtain a fermentation matrix; F. Evenly mix the Lactobacillus buchneri fermentation liquid obtained in step C, the Aspergillus oryzae fermentation liquid obtained in step D and the fermentation substrate obtained in step E, and ferment at 29-39° C. for 36-96 h to obtain fermented distiller's grains roughage.

2. The method for producing roughage by fermentation of two-step ectopic liquor brewing bottom pot water discarded dregs according to claim 1, characterized in that: At least one of the following is met: In step A, the formula of the MRS liquid culture medium is: 20.0 g glucose, 10.0 g beef extract, 10.0 g peptone, 5.0 g yeast extract powder, 5.0 g anhydrous sodium acetate, 2.0 g triammonium citrate, 2.0 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate, 1.0 g Tween 80, 1.0 L distilled water, pH natural; In step A, the activation culture is carried out at 37° C. for 24 hours.

3. The method for producing roughage by fermentation of two-step ectopic liquor brewing bottom pot water discarded dregs according to claim 1, characterized in that: At least one of the following is met: In step B, the formula of the PDA medium is: 6.0 g potato extract powder, 20.0 g glucose, 15.0 g agar, 1.0 L deionized water, natural pH; In step B, the activation culture is carried out at 30° C. for 72 hours.

4. The method for producing roughage by fermentation of two-step ectopic liquor brewing bottom pot water discarded dregs according to claim 1, characterized in that: At least one of the following is met: In step C, the inoculation amount of the activated Lactobacillus buchneri seed solution is 1-5%; In step C, the formula of the MRS bottom pot water culture medium is: 20.0g glucose, 10.0g beef extract, 10.0g peptone, 5.0g yeast extract powder, 5.0g anhydrous sodium acetate, 2.0g triammonium citrate, 2.0g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate, 1.0g Tween 80, and 1.0L of liquor brewing bottom pot water with a pH of 3 to 7.

5. The method for producing roughage by fermentation of two-step ectopic liquor brewing bottom pot water discarded dregs according to claim 1, characterized in that: In step C, the cell concentration OD of the Lactobacillus buchneri fermentation broth is 600nm It is 1.2~1.5, and the total acid content is 14.50~17.50g / L.

6. The method for producing roughage by fermentation of two-step ex situ liquor brewing bottom pot water discarded dregs using full utilization according to claim 1, characterized in that: At least one of the following is met: In step D, the inoculation amount of the activated Aspergillus oryzae strain is as follows: a ring of the activated Aspergillus oryzae strain is dipped in an inoculation loop and inoculated into 30-70 mL of PDA bottom pot water culture medium; In step D, the formula of the PDA bottom pot water culture medium is: 6.0g potato extract powder, 20.0g glucose, and 1.0L of liquor brewing bottom pot water with a pH of 3-7.

7. The method for producing roughage by fermentation of two-step ex situ liquor brewing bottom pot water discarded dregs according to claim 1, characterized in that: In step D, the Aspergillus oryzae fermentation broth has a bacterial cell content of 0.3-0.5 g / 100 mL.

8. The method for producing roughage by fermentation of two-step ex situ liquor brewing bottom pot water discarded dregs using full utilization according to claim 1, characterized in that: At least one of the following is met: In step E, the calcium salt is at least one of calcium carbonate, calcium hydroxide or calcium chloride; In step E, the mass ratio of the waste grains, calcium salt and sesame meal is 200-210:15-16:24-26.

9. The method for producing roughage by fermentation of two-step ex situ liquor brewing bottom pot water discarded dregs according to claim 1, characterized in that: At least one of the following is met: In step F, the ratio of the Lactobacillus buchneri fermentation broth to the Aspergillus oryzae fermentation broth is 6-20 mL:19-6 mL; In step F, the total inoculation amount of the Lactobacillus buchneri fermentation broth and the Aspergillus oryzae fermentation broth is 8-16%.

10. The method for producing roughage by fermentation of two-step ex situ liquor brewing bottom pot water discarded lees according to any one of claims 1 to 9, characterized in that: In step F, the fermented distiller's grains roughage has an acid detergent fiber content of 19-23%, a neutral detergent fiber content of 25-26.5%, a crude protein content of 15-17 g / 100 g, an acid-soluble protein content of 23-26%, and a total acid content of 14-15 g / kg.

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