Method for extracting rice bran protein through cooperation of aspergillus oryzae seed starter fermentation and enzymolysis
Aspergillus oryzae seed culture was prepared by fermentation with Aspergillus oryzae YY-21. The cellulase and protease in the seed culture were used to decompose the cellulose and disulfide bonds in rice bran, which enabled the efficient extraction of rice bran protein. This solved the problems of low extraction rate and poor solubility of rice bran protein and improved the utilization efficiency of rice bran protein.
Patent Information
- Application Number
- CN202511000215.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-18
- Publication Date
- 2025-10-28
Smart Images
Figure BDA0005508701970000061 
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of rice bran protein preparation technology, specifically relating to a method for extracting rice bran protein by fermentation with Aspergillus oryzae koji and synergistic enzymatic hydrolysis. Background Technology
[0002] Rice bran is the part of rice that is removed during the milling process to produce polished rice. It is a major byproduct of rice processing and mainly consists of the rice pericarp, germ, and aleurone layer. Rice bran contains the essence of rice's nutrition, rich in dietary fiber, high-quality protein, vitamins, and minerals. However, currently, rice bran is primarily used directly as an animal feed ingredient, resulting in a waste of resources.
[0003] Rice bran has a high protein content, approximately 10%–18%. Rice bran protein has a balanced amino acid composition, high bioavailability, and low allergenicity, making it an ideal high-quality protein source. However, the high fiber content in rice bran makes protein extraction very difficult. The dense network structure formed by the fiber in rice bran encapsulates the protein, creating a poorly soluble, dense complex that hinders protein dissolution and diffusion during extraction, resulting in a low extraction rate. Furthermore, rice bran protein contains numerous disulfide bonds, causing the protein molecules to aggregate in aqueous solutions, leading to poor solubility and limiting its effective utilization. Summary of the Invention
[0004] In view of this, one of the objectives of the present invention is to provide a method for preparing Aspergillus oryzae starter that can improve the extraction rate of rice bran protein. The Aspergillus oryzae starter prepared according to the method of the present invention has high cellulase activity.
[0005] The second objective of this invention is to provide a method for extracting rice bran protein by fermentation with Aspergillus oryzae koji and synergistic enzymatic hydrolysis. By using the Aspergillus oryzae koji prepared by this invention to ferment rice bran, high-efficiency extraction of rice bran protein can be achieved, thereby realizing the efficient utilization of rice bran protein.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] This invention provides a method for preparing Aspergillus oryzae koji, comprising the following steps:
[0008] Aspergillus oryzae YY-21 was inoculated into a culture medium and fermented to obtain Aspergillus oryzae seed culture; the culture medium consisted of rice bran, soybean meal and water, and the weight ratio of rice bran to soybean meal was (6:4) to (7:3); the preservation number of Aspergillus oryzae YY-21 was CGMCC No. 6381.
[0009] This invention uses rice bran as the main culture medium to prepare Aspergillus oryzae koji. Because rice bran has a high fiber content, it can induce Aspergillus oryzae to secrete more cellulase, and also increase the specificity of the secreted cellulase for decomposing cellulose in rice bran, thus enhancing the decomposition effect of the cellulase in the Aspergillus oryzae koji on rice bran. In this invention, the water content of the culture medium is preferably 30%–40%, more preferably 32%–38%, and even more preferably 35%. In this invention, the amount of Aspergillus oryzae YY-21 inoculated is based on the weight of the culture medium; preferably, 6 mL–7 mL of Aspergillus oryzae YY-21 spore suspension is inoculated per 100 g of culture medium, and preferably each mL of Aspergillus oryzae YY-21 spore suspension contains 10 8 One Aspergillus oryzae YY-21 spore. In this invention, the fermentation temperature is preferably 20℃~23℃, more preferably 21℃~22℃; the fermentation time is preferably 36h~60h, more preferably 54h~56h. This invention does not have specific limitations on the specific sources of rice bran, soybean meal, and water; conventional commercially available products in the art can be used.
[0010] The present invention also provides an Aspergillus oryzae seed culture, which is prepared by the above preparation method.
[0011] The present invention also provides the above-mentioned preparation method or the application of the above-mentioned Aspergillus oryzae seed in the extraction of rice bran protein.
[0012] The present invention also provides a method for extracting rice bran protein, comprising the following steps: mixing rice bran, water and the above-mentioned Aspergillus oryzae koji for fermentation, inactivating enzymes after fermentation, alkali extraction, taking the supernatant, and freeze-drying to obtain rice bran protein.
[0013] In this invention, the preferred weight-to-volume ratio of rice bran to water is 1g:6-16mL, more preferably 1g:8-14mL; the amount of Aspergillus oryzae starter added is based on the weight of protein in the rice bran, preferably 20g-30g of Aspergillus oryzae starter per 100g of protein, more preferably 23g-27g of Aspergillus oryzae starter per 100g of protein, and even more preferably 25g of Aspergillus oryzae starter per 100g of protein. In this invention, the preferred fermentation temperature is 45℃-55℃, more preferably 48℃-52℃, and even more preferably 50℃; the preferred fermentation time is 2h-6h, more preferably 3h-5h, and even more preferably 4h; the preferred fermentation pH is 7-9, more preferably 7.5. In this invention, the preferred method of enzyme inactivation is boiling water bath inactivation, and the preferred inactivation time is 10min. In this invention, after enzyme inactivation, the pH is adjusted to 9-10 for alkali extraction. The preferred pH for alkali extraction is 9.5, and the preferred extraction time is 2h. After alkali extraction, centrifugation is preferably performed, and the supernatant is collected. The centrifugation speed is preferably 8000 r / min, and the centrifugation time is preferably 20 min. This invention does not specifically limit the freeze-drying method; conventional freeze-drying methods in the art can be used.
[0014] In the method for preparing rice bran protein of this invention, the cellulase in the Aspergillus oryzae koji prepared by this method can better decompose the cellulose in rice bran, disrupt the tight structure between cellulose and protein, and improve the release rate of rice bran protein; the protease in the Aspergillus oryzae koji can break the disulfide bonds in rice bran protein, increase the solubility of rice bran protein, and thus improve the extraction rate of rice bran protein. Furthermore, the Aspergillus oryzae koji is rich in a large number of hyphae and spores (approximately 10...). 10 (Spores / g Aspergillus oryzae seed koji), during the reaction, the fermentation of Aspergillus oryzae also breaks down the tight structure between cellulose and protein, enhancing the extraction rate of rice bran protein.
[0015] The beneficial effects of this invention are:
[0016] This invention is the first to use rice bran as the main raw material and use Aspergillus oryzae YY-21 fermentation to prepare Aspergillus oryzae starter with high cellulase activity. The Aspergillus oryzae starter prepared by the method of this invention is rich in cellulase and Aspergillus oryzae mycelium, and has high enzyme activity, with the cellulase activity increasing from the initial 25.73 U / g to 204.04 U / g.
[0017] The Aspergillus oryzae inoculum prepared by this invention can be applied to the preparation of rice bran protein, enabling high-efficiency extraction and utilization of rice bran protein. Using the method for preparing rice bran protein solution provided by this invention, the protein extraction rate from rice bran raw material can reach as high as 84.8%.
[0018] Preservation Instructions
[0019] The Aspergillus oryzae strain YY-21 of this invention is classified as Aspergillus oryzae and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 6381 and deposit date of July 19, 2012. Attached Figure Description
[0020] Figure 1 The effect of inoculum size of Aspergillus oryzae YY-21 spore suspension on the activities of neutral cellulase and protease;
[0021] Figure 2 The results show the effect of the mass ratio of rice bran to soybean meal in the culture medium on the activities of neutral cellulase and protease.
[0022] Figure 3 The results show the effect of culture medium moisture content on the activities of neutral cellulase and protease.
[0023] Figure 4 The results show the effect of fermentation temperature on the activities of neutral cellulase and protease.
[0024] Figure 5 The results show the effect of fermentation time on the activities of neutral cellulase and protease.
[0025] Figure 6 The effect of fermentation time for inoculating Aspergillus oryzae starter on rice bran protein extraction rate;
[0026] Figure 7 The effect of pH on the extraction rate of rice bran protein during alkaline extraction;
[0027] Figure 8 The effect of Aspergillus oryzae inoculum addition amount on rice bran protein extraction rate;
[0028] Figure 9 The effect of the weight-to-volume ratio of rice bran and water (substrate-to-liquid ratio) on the extraction rate of rice bran protein was investigated.
[0029] Figure 10 The effect of fermentation temperature on rice bran protein extraction rate when inoculated with Aspergillus oryzae seed koji;
[0030] Figure 11 The effect of pH on rice bran protein extraction rate during fermentation reaction inoculated with Aspergillus oryzae seed koji;
[0031] In the above figures, different lowercase letters on the bars represent significant differences between groups, P<0.05. Detailed Implementation
[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0033] Unless otherwise specified, the following embodiments are all conventional methods.
[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0035] The preparation method of Aspergillus oryzae YY-21 spore suspension in the following examples is as follows:
[0036] (1) Cultivation of Aspergillus oryzae YY-21: Potato agar (PDA) was sterilized in an autoclave at 121℃ for 20 min and then prepared into a slant culture medium in a sterile room. After cooling, Aspergillus oryzae YY-21 was scraped with an inoculation loop and spread in a "Z" pattern. Finally, it was placed in an incubator at 30℃ and cultured for 2 days.
[0037] (2) Preparation of Aspergillus oryzae YY-21 spore suspension: 40 mL of sterile water was injected eight times into a subcultured Aspergillus oryzae slant. After each injection, the bacteria were scraped off with an inoculation loop (scraping until only the culture medium was exposed). The scraped liquid was poured into a 250 mL Erlenmeyer flask containing glass beads. The flask was placed in a shaker and incubated at 180 r / min and 30 °C for 1 h. After incubation, the mixture was filtered through four layers of gauze. 1 mL of the filtered liquid was serially diluted with deionized water. The spores were observed and counted using a hemocytometer under a 20x microscope, yielding a spore count of 10. 8 Aspergillus oryzae YY-21 spore suspension per mL.
[0038] Example 1
[0039] A type of Aspergillus oryzae koji is prepared by the following method:
[0040] Add 20g of rice bran and soybean meal (passed through a 50-mesh sieve) to a 250mL Erlenmeyer flask, with a weight ratio of 7:3. Add tap water to the flask and stir well to make the moisture content of the culture medium 35%. Sterilize in an autoclave at 121℃ for 20min to obtain the culture medium.
[0041] In a sterile room, the spore count was 10. 8 Aspergillus oryzae YY-21 spore suspension was inoculated into the culture medium at a rate of 6 mL per 100 g of culture medium (inoculation amount of 6.0% (v / w)). The culture medium was then fermented in a constant temperature incubator at 21℃ for 56 h to obtain Aspergillus oryzae inoculum. The preservation number of Aspergillus oryzae YY-21 was CGMCC No. 6381.
[0042] Test Example 1
[0043] A type of Aspergillus oryzae koji is prepared by the following method:
[0044] Add 20g of rice bran and soybean meal (passed through a 50-mesh sieve) to a 250mL Erlenmeyer flask, with a weight ratio of 7:3. Add tap water to the flask and stir well to make the moisture content of the culture medium 50%. Sterilize in an autoclave at 121℃ for 20min to obtain the culture medium.
[0045] In a sterile room, the spore count was 10. 8 Aspergillus oryzae YY-21 spore suspension was inoculated into the culture medium at amounts of 4 mL, 5 mL, 6 mL, 7 mL, or 8 mL per 100 g of culture medium (inoculation amounts of 4.0%, 5.0%, 6.0%, 7.0%, and 8.0% (v / w), respectively), and fermented in a constant temperature incubator at 30°C for 48 h to obtain Aspergillus oryzae inoculum; the preservation number of Aspergillus oryzae YY-21 is CGMCC No. 6381.
[0046] After the fermentation culture period is over, the activities of cellulase and protease in the Aspergillus oryzae inoculum are determined. The specific determination method is as follows:
[0047] Preparation of crude enzyme solution: Add 110 mL of solution (pH 7.0) to the fermented culture medium, extract at room temperature for 1 h, and then filter using eight layers of gauze. The liquid obtained after filtration is the crude enzyme solution.
[0048] Neutral cellulase activity assay: Place sodium carboxymethyl cellulose solution in a 50℃ intelligent digital display constant temperature water bath and preheat for 5 minutes. Take four identical 25mL stoppered tubes (one blank tube and three parallel tubes). Accurately add 2.00mL of sodium carboxymethyl cellulose solution (pH 7) to each stoppered tube (2.50mL for the blank). Accurately add 0.50mL of the crude enzyme solution to be tested to each of the three parallel tubes, mix thoroughly by repeated pipetting, and stopper. Place all four tubes simultaneously in a 50℃ water bath for hydrolysis for 30 minutes, then remove. Quickly and accurately add 3.0mL of the prepared 3,5-dinitrosalicylic acid reagent to each of the four tubes, stopper, and shake well. Place them simultaneously in a boiling water bath for 10 minutes, quickly remove, cool to room temperature, and dilute to volume with deionized water. Measure the absorbance at a spectrophotometer wavelength of 540nm, using the blank tube as the instrument zero point, and then calculate the cellulase activity.
[0049] Method for determining protease activity: The Folin-phenol method was used to determine the protease activity in Aspergillus oryzae koji.
[0050] The effect of inoculum size of Aspergillus oryzae YY-21 spore suspension on the activities of neutral cellulase and protease, using cellulase activity as an indicator, is as follows: Figure 1 As shown, the optimal inoculum size for Aspergillus oryzae YY-21 spore suspension is 6%.
[0051] Test Example 2
[0052] Following the preparation method of Experimental Example 1, the inoculum amount of Aspergillus oryzae YY-21 spore suspension was fixed at 6.0% (v / w), and the mass ratio of rice bran to soybean meal in the culture medium was set to 5:5, 6:4, 7:3, 8:2 or 9:1, respectively, with the rest being the same as in Experimental Example 1.
[0053] The effect of the mass ratio of rice bran to soybean meal in the culture medium on the activities of neutral cellulase and protease is shown in the following results. Figure 2 As shown, the optimal mass ratio of rice bran to soybean meal in the culture medium is 7:3.
[0054] Experimental Example 3
[0055] Following the preparation method of Example 1, the inoculum amount of Aspergillus oryzae YY-21 spore suspension was fixed at 6.0% (v / w), and the water content of the culture medium was set at 10%, 20%, 30%, 40%, 50% and 60% (v / w), respectively, with the rest being the same as in Example 1.
[0056] The effect of culture medium water content on the activities of neutral cellulase and protease is as follows: Figure 3 As shown, the optimal water content in the culture medium is 40%.
[0057] Test Example 4
[0058] Following the preparation method of Experimental Example 1, the inoculum amount of Aspergillus oryzae YY-21 spore suspension was fixed at 6.0% (v / w), the water content of the culture medium was fixed at 40% (v / w), and the fermentation temperature was set at 20℃, 25℃, 30℃, 35℃ and 40℃, respectively. All other conditions were the same as in Experimental Example 1.
[0059] The effects of fermentation temperature on the activities of neutral cellulase and protease are as follows: Figure 4 As shown, the optimal fermentation temperature is 20℃.
[0060] Experimental Example 5
[0061] Following the preparation method of Example 1, the inoculum amount of Aspergillus oryzae YY-21 spore suspension was fixed at 6.0% (v / w), the water content of the culture medium was fixed at 40% (v / w), the fermentation temperature was fixed at 20℃, and the fermentation time was set to 24h, 30h, 36h, 42h, 48h, 54h, 60h and 66h, respectively. All other settings were the same as in Example 1.
[0062] The effects of fermentation time on the activities of neutral cellulase and protease are as follows: Figure 5 As shown, the optimal fermentation time is 54 hours.
[0063] Experimental Example 6
[0064] Based on the results of the single-factor experiments in Examples 1-5, L9(3) was used. 4 An orthogonal experimental design was used, with neutral cellulase activity as the indicator. The orthogonal experimental analysis was conducted considering all factors. Three factors that significantly affected neutral cellulase and protease activity—culture medium moisture content, fermentation temperature, and fermentation time—were optimized. The levels of these factors in the orthogonal experiment are shown in Table 1, and the results are shown in Table 2.
[0065] Based on the above results, the optimal preparation conditions for the *Aspergillus oryzae* koji culture of this invention are as follows: a rice bran:soybean meal weight ratio of 7:3 in the culture medium, an inoculum of *Aspergillus oryzae* YY-21 spore suspension of 6%, a culture medium moisture content of 35%, a fermentation temperature of 21℃, and a fermentation time of 56 h. Under these optimal conditions, the cellulase activity of the *Aspergillus oryzae* koji culture prepared was 204.04 U / g. The protease activity in the *Aspergillus oryzae* koji culture prepared under the above optimal conditions was determined (using the same method as in Experiment 1) to be 797.12 U / g.
[0066] Table 1L9(3) 4 Orthogonal Experiment Factors and Levels
[0067] level Water content of culture medium A (%) B. Fermentation temperature / °C C Fermentation time / h 1 35 19 52 2 40 20 54 3 45 21 56
[0068] Table 2 Results of the orthogonal experiment
[0069]
[0070]
[0071] Example 2
[0072] A method for extracting rice bran protein using Aspergillus oryzae starter culture obtained in Example 1 via fermentation and synergistic enzymatic hydrolysis comprises the following steps:
[0073] Rice bran and water were mixed at a weight-to-volume ratio of 1g:10mL and stirred thoroughly to obtain a rice bran suspension. The pH of the rice bran suspension was adjusted to 7.5. Based on the weight of protein in the rice bran, 25g of the Aspergillus oryzae inoculum obtained in Example 1 was added to every 100g of protein (the addition amount was 25%). The Aspergillus oryzae inoculum obtained in Example 1 was added to the rice bran suspension and mixed. Fermentation was carried out at 50℃ for 4h. After fermentation, the mixture was placed in a boiling water bath for 10min to inactivate the enzymes. The system was then adjusted to pH 9.5 with 0.5mol / L NaOH solution and alkali extracted at 55℃ for 2h. After cooling to room temperature, the mixture was centrifuged at 8000r / min for 20min. The supernatant was collected to obtain a rice bran protein solution. The rice bran protein solution was freeze-dried to obtain rice bran protein.
[0074] Example 3
[0075] Following the method of Example 2, the fermentation reaction time was set to 1 h, 2 h, 4 h, 6 h, and 8 h, respectively, with all other parameters remaining the same as in Example 2. After obtaining rice bran protein solutions of different groups, the total nitrogen content in the supernatant was determined using the Dumas combustion method, and the extraction rate of rice bran protein was calculated. The formula for calculating the extraction rate of rice bran protein is:
[0076]
[0077] The results are as follows Figure 6 As shown, the rice bran protein extraction rate is highest when the fermentation reaction time is 4 hours.
[0078] Example 4
[0079] Following the method of Example 2, the fermentation reaction time was set to 4 hours, and the pH of the alkali extraction was set to 7, 7.5, 8, 8.5, 9, 9.5 and 10, respectively, with the rest being the same as in Example 2.
[0080] After obtaining rice bran protein solutions of different groups, the extraction rates of rice bran protein in different groups were obtained according to the determination and calculation method in Example 3. The results are as follows. Figure 7 As shown, the highest extraction rate of rice bran protein was achieved when the pH of the alkaline extraction was 9.5.
[0081] Example 5
[0082] Following the method of Example 2, the fermentation reaction time was set to 4 hours, the pH of the alkali extraction was set to 9.5, and the amount of Aspergillus oryzae seed koji added was set to 10%, 15%, 20%, 25%, 30%, and 35%, respectively. The rest were the same as in Example 2.
[0083] After obtaining rice bran protein solutions of different groups, the extraction rates of rice bran protein in different groups were obtained according to the determination and calculation method in Example 3. The results are as follows. Figure 8As shown, the highest extraction rate of rice bran protein was achieved when the amount of Aspergillus oryzae seed culture added was 25%.
[0084] Example 6
[0085] Following the method of Example 2, the fermentation reaction time was set to 4 hours, the pH of the alkali extraction was set to 9.5, the amount of Aspergillus oryzae starter added was set to 25%, and the weight-to-volume ratio (material-to-liquid ratio) of rice bran and water was set to 1:6, 1:8, 1:10, 1:12, 1:14 and 1:16 (g / mL), respectively. All other aspects were the same as in Example 2.
[0086] After obtaining rice bran protein solutions of different groups, the extraction rates of rice bran protein in different groups were obtained according to the determination and calculation method in Example 3. The results are as follows. Figure 9 As shown, the highest extraction rate of rice bran protein is achieved when the weight-to-volume ratio of rice bran to water is 1g:14mL.
[0087] Example 7
[0088] Following the method of Example 2, the fermentation time was set to 4 hours, the pH of the alkali extraction was set to 9.5, the amount of Aspergillus oryzae starter added was set to 25%, the weight-to-volume ratio of rice bran and water (material-to-liquid ratio) was set to 1:14 (g / mL), and the fermentation temperature was set to 40℃, 45℃, 50℃, 55℃ and 60℃ respectively. All other settings were the same as in Example 2.
[0089] After obtaining rice bran protein solutions of different groups, the extraction rates of rice bran protein in different groups were obtained according to the determination and calculation method in Example 3. The results are as follows. Figure 10 As shown, the highest extraction rate of rice bran protein is achieved when the fermentation temperature is 50℃.
[0090] Example 8
[0091] Following the method of Example 2, the fermentation time was set to 4 hours, the pH of the alkali extraction was set to 9.5, the amount of Aspergillus oryzae starter added was set to 25%, the weight-to-volume ratio of rice bran and water (material-to-liquid ratio) was set to 1:14 (g / mL), the fermentation temperature was set to 50°C, and the pH values of the fermentation were set to 5, 6, 6.5, 7, 7.5, 8, 8.5 and 9, respectively. All other settings were the same as in Example 2.
[0092] After obtaining rice bran protein solutions of different groups, the extraction rates of rice bran protein in different groups were obtained according to the determination and calculation method in Example 3. The results are as follows. Figure 11 As shown, the highest extraction rate of rice bran protein was achieved when the pH value of the fermentation reaction was 7.5.
[0093] Example 9
[0094] A method for extracting rice bran protein, comprising the following steps:
[0095] Rice bran and water were mixed at a weight-to-volume ratio of 1g:14mL and stirred thoroughly to obtain a rice bran suspension. The pH of the rice bran suspension was adjusted to 7.5. Based on the weight of protein in the rice bran, 25g of the Aspergillus oryzae inoculum obtained in Example 1 was added to every 100g of protein (the addition amount was 25%). The Aspergillus oryzae inoculum obtained in Example 1 was added to the rice bran suspension and mixed. Fermentation was carried out at 50℃ for 4h. After fermentation, the mixture was placed in a boiling water bath for 10min to inactivate the enzymes. The system was then adjusted to pH 9.5 with 0.5mol / L NaOH solution and alkali extracted at 55℃ for 2h. After cooling to room temperature, the mixture was centrifuged at 8000r / min for 20min. The supernatant was collected to obtain a rice bran protein solution. The rice bran protein solution was freeze-dried to obtain rice bran protein.
[0096] The total nitrogen content in the rice bran protein solution was determined, and the extraction rate of rice bran protein was calculated using the same method as in Example 3. The results showed that the extraction rate of rice bran protein was 84.8%.
[0097] Comparative Example 1
[0098] The difference from Example 9 is that Aspergillus oryzae inoculum was not added; otherwise, it was the same as Example 9. The rice bran protein extraction rate of this comparative example 1 was determined to be 57.00%, significantly lower than that of Example 9.
[0099] Comparative Example 2
[0100] The difference from Example 9 is that no Aspergillus oryzae starter was added, but cellulase and neutral protease with activities equivalent to those in the Aspergillus oryzae starter were added. The cellulase was 5101U per 100g of protein (Shanghai Sangon Biotech Co., Ltd., catalog number A002598), and the neutral protease was 19928U per 100g of protein (Novozymes Biotechnology Ltd., Neutrase 5.0). All other aspects were the same as in Example 9. The results showed that the rice bran protein extraction rate of this comparative example 2 was 68.62%, which was significantly lower than that of Example 9.
[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing Aspergillus oryzae koji, characterized in that, The steps include: Aspergillus oryzae YY-21 was inoculated into a culture medium and fermented to obtain Aspergillus oryzae seed culture; the culture medium consisted of rice bran, soybean meal and water, and the weight ratio of rice bran to soybean meal was (6:4) to (7:3); the preservation number of Aspergillus oryzae YY-21 was CGMCC No. 6381.
2. The preparation method according to claim 1, characterized in that, The water content of the culture medium is 30% to 40%.
3. The preparation method according to claim 1, characterized in that, The amount of Aspergillus oryzae YY-21 inoculated is based on the weight of the culture medium, with 6-7 mL of Aspergillus oryzae YY-21 spore suspension inoculated per 100 g of culture medium. Each mL of Aspergillus oryzae YY-21 spore suspension contains 10 8 One Aspergillus oryzae YY-21 spore.
4. The preparation method according to claim 1, characterized in that, The fermentation temperature is 20℃~23℃, and the fermentation time is 36h~60h.
5. A type of Aspergillus oryzae starter culture, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 4.
6. The preparation method according to any one of claims 1 to 4 or the Aspergillus oryzae seed culture according to claim 5 in the extraction of rice bran protein.
7. A method for extracting rice bran protein, characterized in that, The process includes the following steps: mixing rice bran, water, and the Aspergillus oryzae koji strain described in claim 5 for fermentation; after fermentation, inactivating the enzymes; extracting with alkali; taking the supernatant; and freeze-drying to obtain rice bran protein.
8. The method according to claim 7, characterized in that, The weight-to-volume ratio of rice bran to water is 1g:6-16mL; the amount of Aspergillus oryzae starter added is based on the weight of protein in the rice bran, with 20g-30g of Aspergillus oryzae starter added per 100g of protein.
9. The method according to claim 7, characterized in that, The fermentation temperature is 45℃~55℃, the fermentation time is 2h~6h, and the fermentation pH is 7~9.
10. The method according to claim 7, characterized in that, The pH of the alkaline extraction is 9-10.