Method for preparing high-protein-concentration feed protein from DDGS

Abstract

The invention discloses a method for preparing high-protein-concentration feed protein from DDGS (distillers dried grains with soluble). According to the method disclosed by the invention, the DDGS is used as a fermentation raw material, the high-protein-concentration feed protein is produced by adopting multi-bacteria mixing two-step fermentation, and the content of crude protein in the feed protein finally produced by 30L amplified fermentation reaches 70%-75%, so that the requirements of harmless treatment and utilization of the alcohol waste DDGS are met, and the utilization rate of the DDGS is increased.

Description

Technical Field

[0001] The invention relates to the fields of microbial fermentation and feed protein, and in particular to a method for preparing high-protein-concentration feed protein from DDGS. Background Art

[0002] Corn distillers dried grains with solubles (DDGS) are the residues left after industrial production of corn alcohol, which are dried and processed. In recent years, the production of industrial fuel ethanol in my country has increased significantly, and the amount of DDGS feed from corn ethanol production has also gradually increased. Therefore, DDGS has become a hot topic in the feed industry. However, DDGS has a low crude protein content and poor quality, and contains a large amount of crude fiber, which is difficult for livestock and poultry to digest. This has seriously affected the large-scale addition and large-scale application of corn DDGS in livestock and poultry feed.

[0003] At present, microbial feed protein has developed from fermentation of a single strain to the coordinated fermentation of mixed strains. Commonly used fermentation strains are mainly Bacillus subtilis, yeast, mold, etc. The protein content of microbial feed protein affects the quality and price of the product. Therefore, it is urgent to increase the protein content of feed protein.

[0004] The present invention uses a two-step microbial fermentation method for DDGS, and adds urea and ammonium sulfate utilizing strains to convert inorganic nitrogen into organic nitrogen, and utilizes the effects and synergistic effects of various strains to degrade the cellulose content of DDGS and increase the protein content, thereby establishing a method for preparing high-protein concentration feed protein from DDGS. The above invention has not been reported. Summary of the invention

[0005] The purpose of the present invention is to establish a method for preparing high-protein-concentration feed protein from DDGS.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows: A method for preparing high-protein concentration feed protein, characterized in that it uses a cheap nitrogen source and multiple bacteria mixed two-step fermentation to prepare the high-protein concentration feed protein, and the specific method steps are as follows: (1) After DDGS is sterilized and cooled, a mixed strain seed liquid is inoculated at an inoculation rate of 10%-15%, 1%-3% urea is added, and the solid-liquid ratio is adjusted to 1:1-1:1.5 (g:mL) with sterile water. After being fully stirred and mixed, the mixture is fermented at 30°C-35°C for 72-120 hours, and the fermentation is performed every 24 hours to obtain a fermentation product A; the mixed strains are Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacillus coagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Aspergillus oryzae ATCC11488, and the volume ratio thereof is 1:1.5:1:1:2:1:1; the Aspergillus oryzae and Candida utilis used in the present invention can utilize urea, and the urea utilization rate reaches 87-91% after mixed fermentation.

[0007] (2) After the solid-state fermentation is completed, it is converted to liquid fermentation. The fermentation product A is transferred to a 5 L fermentation tank, 0.5%-2% ammonium sulfate is added, and the solid-liquid ratio is adjusted to 1:3-1:7 (g:mL) with sterile water. Saccharomyces cerevisiae seed liquid is inoculated at a rate of 5-15%, and fermented at 30°C for 48-60 h to obtain a fermentation product B. The Saccharomyces cerevisiae CICC1562 is purchased from the China Industrial Microbiological Culture Collection Center, which uses ammonium sulfate and has a high bacterial protein content of 60.15%.

[0008] (3) The fermentation product B is separated into solid and liquid, and the solid part is dried and crushed at 60°C-65°C to obtain the high-protein concentration feed protein.

[0009] The present invention further discloses the application of the preparation method in improving the utilization value of DDGS raw materials; the experimental results show that the crude protein content of the feed protein produced by the final 30 L enlarged fermentation reaches 70%-75%, which meets the needs of harmless treatment and utilization of alcohol waste DDGS and improves the utilization rate of DDGS.

[0010] The more detailed preparation method of the present invention is as follows: A solid-state fermentation bacterial strain combination, specifically as follows: Preferably, solid-state fermentation is performed with Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacilluscoagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Aspergillus oryzae ATCC11488 to degrade the cellulose content in DDGS, and inorganic nitrogen urea is used to convert inorganic nitrogen into organic nitrogen to increase the protein content of the product; Preferably, the spore count of Aspergillus niger CICC41672, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Aspergillus oryzae ATCC11488 is ≥10 8 spores / mL, volume ratio was 1:2:1:1; Preferably, the number of viable bacteria in the bacterial solution of Candida utilis CICC1807, Bacillus subtilis CICC24713, and Bacillus coagulans CICC21736 is ≥ 10 9 CFU / mL, volume ratio was 1.5:1:1. Another preferred embodiment of the present invention is to prepare high-protein-concentration feed protein by two-step fermentation using a cheap nitrogen source and multiple bacteria mixture, as follows: The two-step fermentation comprises: inoculating the mixed strain seed liquid into the koji block, adding urea, adjusting the solid-liquid ratio to 1:1-1:1.5 (g:mL) with sterile water, stirring and mixing thoroughly, fermenting at 30°C-35°C for 72-120 h to obtain fermentation product A, transferring the fermentation product A to a 5 L fermentation tank, adding ammonium sulfate, adjusting the solid-liquid ratio to 1:3-1:7 (g:mL) with sterile water, inoculating the brewer's yeast CICC1562 seed liquid, and fermenting at 30°C for 48-60 h to obtain fermentation product B; the fermentation product B is separated into solid and liquid, and the solid part is dried and crushed at 60°C-65°C to obtain the obtained high-protein concentration feed protein; Preferably, the inoculation amount of the mixed strain seed solution is 10%-15%; Preferably, the amount of urea added is 1%-3%; Preferably, the inoculation amount of the Saccharomyces cerevisiae CICC1562 seed solution is 5%-15%; Preferably, the amount of ammonium sulfate added is 0.5%-2%.

[0011] The present invention mainly solves the problem of low protein content in feed protein prepared with DDGS as raw material, alleviates the tense situation of protein feed resources in my country, and focuses on the method of preparing feed protein by two-step fermentation of DDGS. The main difficulty lies in the combination of fermentation strains and fermentation process.

[0012] Compared with the prior art, the method for preparing high-protein-concentration feed protein from DDGS disclosed in the present invention has the following positive effects: (1) The present invention provides a method for preparing high-protein-concentration feed protein from DDGS, using DDGS as a fermentation raw material and adopting a two-step fermentation method with multiple bacteria to produce high-protein-concentration feed protein. The crude protein content of the feed protein produced by the final 30 L amplified fermentation reaches 70%-75%; (2) The present invention improves the utilization value of DDGS raw materials and improves the application value of DDGS in livestock and poultry feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Aspergillus oryzae ATCC11488 in medium with urea as the sole nitrogen source; Figure 2 Aspergillus oryzae CICC41461 in medium with urea as the sole nitrogen source; Figure 3 Figure 1. Candida utilis CICC1807 grown in a medium with urea as the sole nitrogen source. Figure 4 Figure 1. Saccharomyces cerevisiae CICC 1562 grown on medium with ammonium sulfate as the sole nitrogen source.

[0014] The biological materials involved in the present invention include Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacillus coagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Saccharomyces cerevisiae CICC1562 purchased from the China Industrial Microbiological Culture Collection Center. The biological materials involved in the present invention also include Aspergillus oryzae ATCC11488 purchased from the American Type Culture Collection Center, and the above strains are all available for purchase by the public.

[0015] The main experimental methods involved in the present invention are as follows: (1) Urea-decomposing bacteria Aspergillus oryzae ATCC11488 and Aspergillus oryzae CICC41461 were streaked from glycerol tubes onto PDA plates (1L potato extract, 20 g / L glucose, 15 g / L agar), and inverted in a 30-degree incubator for 48 hours for activation. Candida utilis CICC1807 was streaked from a glycerol tube onto MEA plates (0.78 g / L peptone, 12.75 g / L maltose, 2.75 g / L dextrin, 2.35 g / L glycerol, 15 g / L agar), and inverted in a 30-degree incubator for 16 hours for activation. The activated strain was streaked onto a medium with urea as the sole nitrogen source (5 g / L sodium chloride, 2 g / L potassium dihydrogen phosphate, 1 g / L glucose, 0.2 g / L peptone, 0.012 g / L phenol red, 20 g / L urea, pH -6.8) and inverted for 3 days. Urea-decomposing bacteria can produce urease, which catalyzes the decomposition of urea to produce CO3 2+ and NH 4+ , NH 4+ The pH around the colony increases. The phenol red indicator turns yellow when the pH is lower than 6.6 and turns red when the pH is higher than 8.4. Therefore, the strain that changes the culture medium from yellow to red is the urea-decomposing bacteria.

[0016] (2) Culture medium with ammonium sulfate as the only nitrogen source Ammonium sulfate 30%, magnesium sulfate 0.025%, dipotassium hydrogen phosphate 0.0655%, sodium chloride 0.1%, glucose 0.5%.

[0017] (3) Determination of bacterial protein content Saccharomyces cerevisiae CICC1562 was streaked from the glycerol tube onto a MEA plate (peptone 0.78 g / L, maltose 12.75 g / L, dextrin 2.75 g / L, glycerol 2.35 g / L, agar 15 g / L), and placed in an incubator at 30 degrees for 16 hours to complete activation. After activation, it was inoculated into liquid culture medium and cultured on a shaker until OD 600 =0.8 to obtain seed liquid, and the seed liquid was inoculated into 300 mL liquid culture medium at an inoculum of 1%, and cultured under shaking at 30°C and 180 rpm for 48 h to obtain bacterial suspension. The bacterial suspension was centrifuged at 4500 rpm for 10 min, the supernatant was discarded, and the suspension was washed with distilled water. After washing was repeated 3 times, it was placed in a 105°C oven and dried to constant weight or freeze-dried to constant weight. After cooling, the bacterial protein content was determined using a Kjeldahl nitrogen analyzer.

[0018] (4) Preparation of high-protein feed protein by two-step fermentation of multiple bacteria The mixed strain seed liquid of Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacilluscoagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461 and Aspergillus oryzae ATCC11488 was inoculated into the koji block at a volume ratio of 1:1.5:1:1:2:1:1, urea was added, and the solid-liquid ratio was adjusted to 1:1-1:1.5 (g:mL) with sterile water. After fully stirring and mixing, the mixture was fermented at 30°C-35°C for 72-120 h to obtain a fermentation product A, and the fermentation product A was transferred to 5 Ammonium sulfate is added to the L fermentation tank, and the solid-liquid ratio is adjusted to 1:3-1:7 (g:mL) with sterile water, and the seed liquid of Saccharomyces cerevisiae CICC1562 is added for liquid fermentation to obtain a fermentation product B; the fermentation product B is separated into solid and liquid, and the solid part is dried and crushed at 60°C-65°C to obtain a high-protein concentration feed protein; The inoculation amount of mixed strain seed solution is 10%-15%; the amount of urea added is 1%-3%; The inoculation amount of brewer's yeast seed liquid is 5%-15%; the amount of ammonium sulfate added is 0.5%-2%; the brewer's yeast seed culture medium is 1% yeast powder, 2% peptone, and 2% glucose; The spore count of Aspergillus niger CICC41672, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Aspergillus oryzae ATCC11488 is ≥10 8 spores / mL; The number of live bacteria in the culture medium of Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacillus coagulans CICC21736, and Saccharomyces cerevisiae CICC1562 is ≥10 9 CFU / mL; Liquid fermentation is carried out at 30°C, air flux 0.2-1.0 vvm, stirring 200-500 r / min, and the fermentation time is 48-60 h. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not used to limit the present invention. Unless otherwise specified, the content of the present invention is further described in combination with examples. Example

[0020] Determination of crude protein content in DDGS raw materials The DDGS raw material was dried and crushed at 60°C, and the crude protein content was determined using the national standard method GB / T6432-2018. The crude protein content of DDGS was measured to be 30.50%. Example

[0021] DDGS multi-bacteria mixed solid-state fermentation 90 g of DDGS raw material was loaded into each koji plate. After sterilization and cooling at 115°C for 20 min, a mixed strain seed liquid of Aspergillus niger CICC41672, Bacillus Subtilis CICC24713, Bacillus coagulans CICC21736, and Trichoderma reesei CICC2626 was inoculated into the koji block at a volume ratio of 1:1:1:1.5 at an inoculation rate of 13%. The solid-liquid ratio was adjusted to 1:1.5 (g:mL) with sterile water. After thorough stirring and mixing, the mixture was fermented at 30°C for 96 h, and the koji was turned every 24 h to obtain the fermentation product.

[0022] Under these fermentation conditions, the crude protein content of the fermentation product was 38.51%. Example

[0023] Solid-state fermentation of DDGS with multiple bacteria and urea and urea-utilizing strains 90 g of DDGS raw material was loaded into each koji plate. After sterilization and cooling at 115℃ for 20 min, 13% inoculation amount of mixed strain seed liquid of Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacillus coagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Aspergillus oryzae ATCC11488 was inoculated into the koji block at a volume ratio of 1:1.5:1:1:2:1:1, 2% urea was added, and the solid-liquid ratio was adjusted to 1:1.5 (g:mL) with sterile water. After thorough stirring and mixing, the koji was fermented at 30℃ for 96 h, and the mixture was stirred every 24 hours. h to turn over the koji and obtain the fermentation product.

[0024] Under these fermentation conditions, the crude protein content of the fermentation product was 55.92%. Example

[0025] Preparation of feed protein by two-step fermentation of DDGS with multiple bacteria 90 g of DDGS raw material was loaded into each koji plate. After sterilization and cooling at 115℃ for 20 min, 13% inoculation amount of mixed strain seed liquid of Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacillus coagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Aspergillus oryzae ATCC11488 was inoculated into the koji block at a volume ratio of 1:1.5:1:1:2:1:1, 2% urea was added, and the solid-liquid ratio was adjusted to 1:1.5 (g:mL) with sterile water. After thorough stirring and mixing, the koji was fermented at 30℃ for 96 h, and the mixture was stirred every 24 hours. h to turn over the koji and obtain fermentation product A. Fermentation product A was transferred to a 5 L fermentation tank, 1.25% ammonium sulfate was added, the solid-liquid ratio was adjusted to 1:5 (g:mL) with sterile water, and the seed liquid of Saccharomyces cerevisiae CICC1562 was inoculated at a 10% inoculation rate. The initial air flux was 0.2 vvm, the initial speed was 200 r / min, and the fermentation was maintained at 30°C for 50 h to obtain fermentation product B. The solid-liquid separation of fermentation product B was carried out, and the solid part was dried and crushed at 60°C to obtain feed protein. Under this fermentation condition, the crude protein content of feed protein was 67.31%. Example

[0026] 30 L scale-up to produce high protein concentration feed protein The DDGS raw material was charged into a solid fermentation tank at a charging ratio of 60%, sterilized at 115℃ for 20 min, cooled to 30℃, and inoculated with 13% of the mixed strain seed liquid of Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacilluscoagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, and Aspergillus oryzae ATCC11488 at a volume ratio of 1:1.5:1:1:2:1:1, 2% urea was added, and the solid-liquid ratio was adjusted to 1:1.5 (g:mL) with sterile water. After sufficient stirring and mixing, the mixture was fermented at 30℃ for 96 min. h, and fermentation product A was obtained. Fermentation product A was transferred to a 30 L fermentation tank, 1.25% ammonium sulfate was added, the solid-liquid ratio was adjusted to 1:5 (g:mL) with sterile water, and the 10% inoculation amount of Saccharomyces cerevisiae CICC1562 seed liquid was inoculated, the initial air flux was 0.2 vvm, the initial speed was 200 r / min, and the fermentation was maintained at 30°C for 50 h to obtain fermentation product B. The solid-liquid separation of fermentation product B was carried out, and the solid part was dried and crushed to obtain high-protein concentration feed protein.

[0027] Under this fermentation condition, the fermentation was repeated for 5 batches, and the crude protein content of the fermented feed protein was 73.01%, 70.87%, 73.02%, 74.69% and 71.33%, and the crude protein content was all between 70% and 75%.

[0028] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A method for preparing high protein concentration feed protein, characterized in that It uses cheap nitrogen source and multi-bacteria mixed two-step fermentation to prepare high-protein concentration feed protein. The specific method and steps are as follows: After DDGS is sterilized and cooled, a mixed strain seed liquid is inoculated at an inoculation rate of 10%-15%, 1%-3% urea is added, and the solid-liquid ratio is adjusted to 1:1-1:1.5 (g:mL) with sterile water. After being fully stirred and mixed, the mixture is fermented at 30°C-35°C for 72-120 h, and the fermentation is performed every 24 h to obtain a fermentation product A; the mixed strains are Aspergillus niger CICC41672, Candida utilis CICC1807, Bacillus Subtilis CICC24713, Bacillus coagulans CICC21736, Trichoderma reesei CICC2626, Aspergillus oryzae CICC41461, Aspergillus oryzae ATCC11488, the volume ratio is 1:1.5:1:1:2:1:1; (2) After the solid-state fermentation is completed, it is converted to liquid fermentation. The fermentation product A is transferred to a 5 L fermentation tank, 0.5%-2% ammonium sulfate is added, and the solid-liquid ratio is adjusted to 1:3-1:7 (g:mL) with sterile water. The brewer's yeast seed liquid that uses ammonium sulfate and has a bacterial protein content of 60.15% is inoculated at a rate of 5-15%, and fermented at 30°C for 48-60 h to obtain the fermentation product B; (3) The fermentation product B is separated into solid and liquid, and the solid part is dried and crushed at 60°C-65°C to obtain the high-protein concentration feed protein.

2. The method according to claim 2, characterized in that: Aspergillus oryzae CICC41461, Aspergillus oryzae ATCC11488 and Candida utilis CICC1807 in the mixed strains in the above step are urea-utilizing strains, and the urea utilization rate reaches 87-91% after multi-strain mixed solid-state fermentation.

3. Application of the preparation method of claim 1 in improving the utilization value of DDGS raw materials; the improved utilization value of DDGS raw materials refers to: the crude protein content of the feed protein produced by the final 30 L enlarged fermentation reaches 70%-75%.

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