Aquatic feed fermentation method of white spirit vinasse
Through microbial fermentation of liquor lees by Bacillus subtilis, Candida prion and white dime, the quality problems of directly using fresh liquor lees as feed are solved, and the nutritional value and bioavailability of the feed are improved.
Patent Information
- Application Number
- CN202411663086.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-02-25
AI Technical Summary
In the prior art, fresh wine lees are directly used as feed, which have problems such as high water content, short storage time, low protein content and low animal utilization, and cannot effectively solve the problem of the final destination of wine lees.
The lees of liquor are fermented by microbial fermentation of Bacillus subtilis, Candida and White Dimethymium, controlled moisture content, autoclave and static fermentation, followed by drying, crushing and sieve the finished product.
It reduces the content of crude fiber and anti-nutrition factors, improves the abundance of nutrients such as proteins and amino acids, improves the bioavailability of nutrients, and improves the palatability and nutritional value of feed.
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Figure CN119498449A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of liquor lees, and in particular to a method for fermenting liquor lees into aquatic feed. Background Art
[0002] Fish meal is one of the important components of aquatic feed and is widely used in aquatic animal feed. However, the increase in demand for fish meal in the aquaculture industry and the sharp decline in fish catch have exacerbated the contradiction between supply and demand of fish meal, leading to an increase in the price of fish meal. General plant protein sources (such as cottonseed meal, soybean meal, rapeseed, etc.) have poor palatability, high content of anti-nutritional factors, and unbalanced amino acids, and the proportion that can replace fish meal is limited. Therefore, finding healthy, safe, and resource-rich new feed protein sources to replace fish meal, ensure the normal growth performance of aquatic animals, and reduce feed costs has become the focus of the aquaculture industry.
[0003] Liquor lees are the main by-product of the liquor brewing industry. Due to the large-scale production and consumption of liquor, China produces about 100 million tons of lees every year. However, lees are rich in organic acids, alcohols, water and nutrients, which are not only prone to mildew and rot, but also difficult to store. A large amount of lees are concentrated, and if not handled properly, it will cause serious environmental pollution. Although lees are regarded as waste, their cost as raw materials is relatively low, and their residual nutrients are rich, containing protein, fat, minerals, vitamins and a variety of growth factors that are beneficial to animal growth. They are rich in nutrients and low in price, making them a potential source of feed protein for aquatic feed. In addition, liquor undergoes complex biochemical changes during solid-state fermentation. Liquor lees contain trace beneficial components such as ribonucleic acid and purine produced by microbial cells, all of which are incomparable to grains. This is an important feature of the nutritional composition of lees feed that is different from that of grains.
[0004] The existing comprehensive utilization technology of distiller's grains is mainly reflected in the preparation of animal feed, edible fungus cultivation, biofertilizer, biomass fuel, fuel ethanol, etc. Among them, the most common way to use distiller's grains in China is as animal feed. Usually, distiller's grains are directly used as feed or fed after adding other feed additives; or the white distiller's grains are separated from the rice husk and dried, and then combined with other raw materials to produce different types of feed; or distiller's grain powder is combined with other raw materials to produce feed.
[0005] However, directly using fresh distiller's grains as feed has a high water content, a short storage time, a low protein content, and a low animal utilization rate, which cannot meet the industrial requirements for feed quality. Using distiller's grains for the cultivation of edible fungi can increase the utilization value of the distiller's grains and reduce production costs, but there are still residues that need to be processed; using distiller's grains to produce enzyme preparations, prepare glycerol, extract complex amino acids and trace elements, etc. cannot fundamentally solve the problem of the final destination of the distiller's grains, and may produce more residues. In addition, the distiller's grains have a high water content, are not easy to store, and contain materials such as rice husks that are difficult to utilize. Therefore, it does not meet the existing needs. In this regard, we propose a method for fermenting white wine distiller's grains into aquatic feed. Summary of the invention
[0006] The purpose of the present invention is to provide a method for fermenting white wine lees into aquatic feed, so as to solve the problem proposed in the above background technology that directly using fresh wine lees as feed has high water content, short storage time, low protein content, low animal utilization rate, and cannot meet the industrial requirements for feed quality. In addition, using wine lees for cultivating edible fungi can improve the utilization value of wine lees and reduce production costs, but there are still residues that need to be processed; using wine lees to produce enzyme preparations, prepare glycerol, extract complex amino acids and trace elements, etc. cannot fundamentally solve the problem of the final destination of wine lees, and it is possible to produce more lees residues, and the wine lees have high water content, are not easy to store, and contain materials such as rice husks that are difficult to utilize.
[0007] To achieve the above object, the present invention provides the following technical solution: a method for fermenting liquor lees into aquatic feed, comprising the following steps:
[0008] Step 1: Inoculate 0.1 g of Bacillus subtilis powder into LB liquid culture medium, place in a shaker at 37°C, and activate and culture at a speed of 150 r / min for 24 hours to obtain liquid seed A;
[0009] Step 2: Inoculate 0.1 g of Candida utilis powder into YPD liquid culture medium, place in a shaker at 30°C, and activate and culture at a speed of 150 r / min for 24 hours to obtain liquid seeds B;
[0010] Step 3: inoculate the freeze-dried powder of Geotrichum candidum into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours for activation, then take 5% of the activated bacterial solution and inoculate it into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours as the first generation culture, then take 5% of the first generation culture and inoculate it into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours to obtain a second generation bacterial solution, which is the liquid seed C;
[0011] Step 4: Using the white wine lees that have been washed to remove the rice husk to control the moisture content;
[0012] Step 5: 100 g of vinasse was placed in a 250 mL conical flask, the conical flask was sealed with a sealing film and covered with aluminum foil, and sterilized by high pressure at 120° C. for 18-22 min to obtain the original vinasse;
[0013] Step 6: After the white wine lees are sterilized, remove the aluminum foil and the sealing film, cool naturally, mix the liquid seed A, liquid seed B and liquid seed C to obtain a mixed bacterial solution, take 10% of the mixed bacterial solution and inoculate it into the wine lees, add ammonium sulfate and potassium dihydrogen phosphate, stir evenly at a natural pH, and seal with a sealing film;
[0014] Step 7: placing the sealed conical flask in a constant temperature incubator at different fermentation temperatures T for static fermentation for 7 days;
[0015] Step 8: Dry and crush the fermented lees, and then sieve them through a sieve to make the finished product.
[0016] Preferably, the moisture content of the distiller's grains after washing to remove the rice husk is controlled to be 60%.
[0017] Preferably, the mixing ratio of the liquid seed A, the liquid seed B and the liquid seed C is 1:1:1.
[0018] Preferably, the specific surface volume of the mixed bacterial liquid is 10 ml / 100 g.
[0019] Preferably, the concentration of ammonium sulfate is 2%, and the concentration of potassium dihydrogen phosphate is 0.5%.
[0020] Preferably, the fermentation temperature T includes 19°C (room temperature), 26°C, 28°C, 30°C, 32°C, 34°C and 36°C.
[0021] Preferably, the distiller's grains fermented at different fermentation temperatures T are divided into groups, including CT group, TA group, TB group, TC group, TD group, TE group and TF group, the CT group corresponds to 19°C (room temperature), the TA group corresponds to 26°C, the TB group corresponds to 28°C, the TC group corresponds to 30°C, the TD group corresponds to 32°C, the TE group corresponds to 34°C, and the TF group corresponds to 36°C, and the original distiller's grains are divided into the RT group.
[0022] Preferably, the particle size of the sieve is 40-60 mesh.
[0023] Preferably, the fermentation temperature condition T is 34°C.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] 1. The vinasse obtained by fermentation with microorganisms containing Bacillus subtilis, Candida utilis and Geotrichum candidum has reduced contents of crude fiber and anti-nutritional factors, and is richer in nutrients such as protein and amino acids, and has improved bioavailability of nutrients, which can effectively improve the palatability and nutritional value of feed;
[0026] 2. The present invention uses composite bacteria to ferment white wine lees under mixed bacterial strain conditions and selects the optimal temperature under multiple sets of different room temperature conditions to improve the nutritional value of the lees and effectively evaluate the feasibility of fermented lees as a substitute for plant protein sources such as fish meal. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a flow chart of the method for preparing protein feed by fermenting liquor lees of the present invention;
[0028] Figure 2 This is a schematic diagram of tannin content in fermented liquor lees;
[0029] Figure 3 This is a schematic diagram of phytic acid content in fermented liquor grains;
[0030] Figure 4 This is a schematic diagram of the general nutritional content of fermented liquor lees;
[0031] Figure 5 This is a schematic diagram of the essential amino acid content in fermented liquor lees;
[0032] Figure 6 It is the non-metric multidimensional scaling (NMDS) analysis diagram of the changes in microbial diversity during fermentation;
[0033] Figure 7 Genus-level diagram of microbial diversity during fermentation at different temperatures. DETAILED DESCRIPTION
[0034] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0035] See also Figures 1 to 7 The present invention provides a method for fermenting liquor lees into aquatic feed, comprising the following steps:
[0036] Step 1: Inoculate 0.1 g of Bacillus subtilis powder into LB liquid culture medium, place in a shaker at 37°C, and activate and culture at a speed of 150 r / min for 24 hours to obtain liquid seed A;
[0037] Step 2: Inoculate 0.1 g of Candida utilis powder into YPD liquid culture medium, place in a shaker at 30°C, and activate and culture at a speed of 150 r / min for 24 hours to obtain liquid seeds B;
[0038] Step 3: inoculate the freeze-dried powder of Geotrichum candidum into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours for activation, then take 5% of the activated bacterial solution and inoculate it into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours as the first generation culture, then take 5% of the first generation culture and inoculate it into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours to obtain a second generation bacterial solution, which is the liquid seed C;
[0039] Step 4: using the white wine lees that have been washed with water to remove the rice husks, and controlling the moisture content, wherein the moisture content of the white wine lees that have been washed with water to remove the rice husks is controlled to be 60%;
[0040] Step 5: 100 g of vinasse was placed in a 250 mL conical flask, the conical flask was sealed with a sealing film and covered with aluminum foil, and sterilized by high pressure at 120° C. for 18-22 min to obtain the original vinasse;
[0041] Step 6: After the white wine lees are sterilized, the aluminum foil and the sealing film are removed, and the mixture is cooled naturally. The liquid seed A, the liquid seed B and the liquid seed C are mixed to obtain a mixed bacterial solution, wherein the specific surface volume of the mixed bacterial solution is 10 ml / 100 g, 10% of the mixed bacterial solution is inoculated into the wine lees, and 2% ammonium sulfate and 0.5% potassium dihydrogen phosphate are added, and the mixture is stirred evenly at a natural pH, and sealed with a sealing film;
[0042] Step 7: placing the sealed conical flask in a constant temperature incubator at different fermentation temperatures T for static fermentation for 7 days;
[0043] Step 8: Dry and crush the fermented lees, and then sieve them through a 40-60 mesh screen to make the finished product.
[0044] The mixing ratio of the liquid seed A, liquid seed B and liquid seed C is 1:1:1.
[0045] The fermentation temperatures T include 19°C (room temperature), 26°C, 28°C, 30°C, 32°C, 34°C and 36°C. Room temperature 19°C is used as a control. The lees fermented at different fermentation temperatures T are divided into groups, including CT group, TA group, TB group, TC group, TD group, TE group and TF group. The CT group corresponds to 19°C (room temperature), the TA group corresponds to 26°C, the TB group corresponds to 28°C, the TC group corresponds to 30°C, the TD group corresponds to 32°C, the TE group corresponds to 34°C, and the TF group corresponds to 36°C. The original lees are divided into the RT group.
[0046] The changes in anti-nutritional factors of liquor lees before and after fermentation are as follows:
[0047] Phytic acid is a heat-resistant anti-nutritional factor that can react with divalent and trivalent mineral ions (such as Ca 2+ Mg 2+ 、Zn 2+ , Cu 3+ and Fe 3+ ) Chelation inhibits the utilization of these minerals by the animal gastrointestinal tract, and tannins inhibit digestive enzymes, which will also hinder the absorption of minerals and limit the bioavailability of nutrients, such as Figure 2 and Figure 3 As shown, the original liquor lees contained 1.76 mg / g tannin and 8.10 mg / g phytic acid, which were significantly improved by composite bacteria fermentation at different temperatures; the contents of tannin and phytic acid in all treatment groups were significantly reduced (P < 0.05).
[0048] As the temperature increased, the contents of tannin and phytic acid showed a trend of first increasing and then decreasing. At the fermentation temperature of 34°C (TE group), the phytic acid content reached the lowest value of 0.97 mg / g, and the degradation rate was as high as 88.05% compared with the original wine grains. The tannin content decreased to 1.22 mg / g, and the degradation rate reached 30.69%. In addition, when the fermentation temperature was 36°C (TF group), the tannin content reached the lowest value of 1.13 mg / g, and the degradation rate was 35.49%.
[0049] The nutritional components of liquor lees before and after fermentation change as follows:
[0050] like Figure 4 As shown in the data, except for crude fat, the crude fiber, crude protein, amino acids and starch contents of the conventional nutrients of liquor lees after solid-state fermentation all changed significantly (P < 0.05). After composite bacteria fermentation, the crude protein and total amino acid contents showed an overall upward trend under all temperature conditions, among which the protein content reached the maximum value at a fermentation temperature of 34°C (TE group), increasing by 15.5%. The total amino acids reached the maximum value in the TB group, followed by the TE group, increasing by 8.7% and 7.13%, respectively.
[0051] like Figure 5As shown, the content of essential amino acids in the treatment groups changed to varying degrees. Leucine and isoleucine increased in all treatment groups, and the content was the highest in the TB group, which increased by 17.57% and 18.18% respectively compared with the original distiller's grains (RT group). Threonine, phenylalanine, and arginine were the highest in the TE group, increasing by 14.29%, 15.15%, and 8.33% respectively. There were differences in crude fiber content at different fermentation temperatures. Compared with the original distiller's grains (RT), the crude fiber content decreased at a fermentation temperature of 26-34°C, and the crude fiber content reached the lowest at a temperature of 34°C (TE group), a decrease of 15.34%.
[0052] The increase in crude protein content may be the result of the reduction in starch and lipid content in microorganisms. Higher fermentation temperatures can provide optimal growth conditions for most microorganisms that produce amylase and glucoamylase and promote fermentation. These microorganisms can metabolize starch to produce energy, protein, organic acids and other substances, and lipids may be converted into protein. Crude fat is a high-energy nutrient in feed, but the presence of a large amount of crude fat may accelerate the oxidation and rancidity of raw materials.
[0053] Compared with the original distiller's grains (RT), the crude fat content was reduced by 8.77-36.84% (P>0.05) under the fermentation of composite bacteria at different temperatures, and the starch content in the TC, TD, TE, and TF groups was significantly lower than that in the original distiller's grains (RT).
[0054] The above results all show that composite bacteria fermentation at different temperatures improves the anti-nutritional factors and conventional nutrients of white wine grains, and when the fermentation temperature is 34°C, the anti-nutritional factors and crude fiber content in the fermented wine grains are greatly reduced, and the crude protein and amino acid contents are significantly increased. The optimal temperature for composite bacteria fermentation is 34°C.
[0055] The changes in microbial diversity during the fermentation process were analyzed by non-metric multidimensional scaling (NMDS) based on unweighted UniFrac distance to analyze the differences in the microbial community structure in different treatment groups. Figure 6 As shown in the data, during the solid-state fermentation of liquor lees, the bacterial community structure was constantly evolving. There was a significant difference in the microbial diversity at room temperature (CT group) and that in the TB-TE group. As the temperature increased, the CT and TE group samples were clearly separated, indicating that the bacterial communities in the CT and TE groups showed significant differences. When the fermentation temperature was 34°C (TE group), it had a greater impact on the microbial community in the fermentation matrix.
[0056] Some Weissella can produce biogenic amines during the fermentation process. These biogenic amines are common harmful substances in traditional fermented foods and can cause allergies, headaches and respiratory disorders, such as Figure 7As shown, compared with the control group (CT group), with the increase of temperature, the relative abundance of Weissella in the treated groups (except TA group) decreased, and reached a lower level in the TD (12.89%) and TE (13.12%) groups. The reduction in Weissella species indicates that appropriately increasing the temperature is beneficial to reducing the risks brought by the accumulation of biogenic amines and enhancing its feed safety.
[0057] Studies have shown that Stenotrophomonas, Bacillus, and Pseudomonas can produce lignocellulose degrading enzymes and have the characteristics of nitrogen fixation and cellulose degradation. Paenibacillus is considered to be the producer of various enzymes such as cellulase, amylase, protease, pectinase, and xylanase. The species it contains have strong hydrolysis ability and can effectively degrade cellulose and protein during solid-state fermentation. The relative abundance of Bacillus, Pseudomonas, and Paenibacillus all reached the maximum in the TE group. Although the proportion is small, their functions cannot be ignored. In addition, Stenotrophomonas has been greatly improved in the TD group (22.19%), followed by the TE group (18.60%). This shows that these microorganisms can utilize lignocellulose for bioconversion to a greater extent in the TE group and retain protein.
[0058] The specific nutritional components of fermented distiller's grains are shown in the following table:
[0059] Table 1 Detection results of anti-nutritional factors in fermented liquor lees
[0060]
[0061] Table 2 Detection results of nutritional components of fermented liquor lees
[0062]
[0063] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.
Claims
1. A method for fermenting liquor lees into aquatic feed, characterized in that: The steps include: Step 1: Inoculate 0.1 g of Bacillus subtilis powder into LB liquid culture medium, place in a shaker at 37°C, and activate and culture at a speed of 150 r / min for 24 hours to obtain liquid seed A; Step 2: Inoculate 0.1 g of Candida utilis powder into YPD liquid culture medium, place in a shaker at 30°C, and activate and culture at a speed of 150 r / min for 24 hours to obtain liquid seeds B; Step 3: inoculate the freeze-dried powder of Geotrichum candidum into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours for activation, then take 5% of the activated bacterial solution and inoculate it into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours as the first generation culture, then take 5% of the first generation culture and inoculate it into PDA liquid culture medium, place it in a shaker at a temperature of 30°C, and culture it at a constant temperature of 150 r / min for 24 hours to obtain a second generation bacterial solution, which is the liquid seed C; Step 4: Using the white wine lees that have been washed to remove the rice husk to control the moisture content; Step 5: 100 g of vinasse was placed in a 250 mL conical flask, the conical flask was sealed with a sealing film and covered with aluminum foil, and sterilized by high pressure at 120° C. for 18-22 min to obtain the original vinasse; Step 6: After the white wine lees are sterilized, remove the aluminum foil and the sealing film, cool naturally, mix the liquid seed A, liquid seed B and liquid seed C to obtain a mixed bacterial solution, take 10% of the mixed bacterial solution and inoculate it into the wine lees, add ammonium sulfate and potassium dihydrogen phosphate, stir evenly at a natural pH, and seal with a sealing film; Step 7: placing the sealed conical flask in a constant temperature incubator at different fermentation temperatures T for static fermentation for 7 days; Step 8: Dry and crush the fermented lees, and then sieve them through a sieve to make the finished product.
2. The method for fermenting liquor lees into aquatic feed according to claim 1, characterized in that: The moisture content of the white wine grains after washing to remove the rice husk is controlled to be 60%.
3. The method for fermenting liquor lees into aquatic feed according to claim 1, characterized in that: The mixing ratio of the liquid seed A, the liquid seed B and the liquid seed C is 1:1:
1.
4. The method for fermenting liquor lees into aquatic feed according to claim 1, characterized in that: The specific surface volume of the mixed bacterial solution is 10 ml / 100 g.
5. The method for fermenting liquor lees into aquatic feed according to claim 1, characterized in that: The concentration of the ammonium sulfate is 2%, and the concentration of potassium dihydrogen phosphate is 0.5%.
6. The method for fermenting liquor lees into aquatic feed according to claim 1, characterized in that: The fermentation temperature T includes 19°C (room temperature), 26°C, 28°C, 30°C, 32°C, 34°C and 36°C.
7. The method for fermenting liquor lees into aquatic feed according to claim 6, characterized in that: The distiller's grains fermented at different fermentation temperatures T were divided into groups, including CT group, TA group, TB group, TC group, TD group, TE group and TF group. The CT group corresponded to 19°C (room temperature), the TA group corresponded to 26°C, the TB group corresponded to 28°C, the TC group corresponded to 30°C, the TD group corresponded to 32°C, the TE group corresponded to 34°C, and the TF group corresponded to 36°C. The original distiller's grains were divided into the RT group.
8. The method for fermenting liquor lees into aquatic feed according to claim 1, characterized in that: The particle size of the sieve is 40-60 mesh.
9. The method for fermenting liquor lees into aquatic feed according to claim 6, characterized in that: The fermentation temperature T is 34°C.