Method for increasing protein content of DDGS (distillers dried grains with soluble)
By enzymatically hydrolyzing corn DDGS using cellulase and xylanase and optimizing the hydrolysis conditions, the protein content of DDGS was increased, solving the problem of insufficient protein content in aquatic feed and realizing the efficient application and toxin removal of DDGS in aquatic feed.
Patent Information
- Application Number
- CN202510972737.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-11-21
AI Technical Summary
DDGS has a low protein content in aquatic feed, which limits its addition ratio in aquatic feed formulations and leads to dependence on imported protein raw materials such as fishmeal and soybean meal. Existing technologies are not able to effectively increase the protein content of DDGS.
Maize DDGS was enzymatically hydrolyzed using cellulase and xylanase, and then concentrated maize DDGS protein was prepared by combining enzyme inactivation, centrifugation and drying. The enzymatic hydrolysis conditions, including enzyme ratio, temperature, pH and time, were optimized.
The protein content of DDGS was increased to over 39%, and the mycotoxin content was reduced, enabling the efficient application of DDGS in aquatic feed and reducing dependence on imported protein raw materials.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of aquatic feed protein sources, and particularly relates to a method for increasing the protein content of DDGS. BACKGROUND
[0002] China is a large country in aquaculture, and the aquaculture output in 2023 exceeds 5800 tons, ranking first in the world. However, as the main protein sources of aquatic feed, fish meal and soybean meal are highly dependent on imports. Reducing the dependence on imported fish meal and soybean meal has become a problem that needs to be solved in the field of aquatic feed. Corn is one of the main crops in China, and has the advantages of large yield and wide source. It has great potential to solve the problem of dependence on imported feed protein raw materials. In recent years, corn has been used to produce ethanol in China, and the production is increasing, which leads to an increasing production of corn processing by-products dry distiller's grains and their solubles (DDGS). Therefore, DDGS has become a stable and low-cost feed protein source.
[0003] The protein content of DDGS is relatively low, generally between 20-30%, and it is widely used in livestock and poultry feed at present. However, as a protein source for aquatic feed, there is a problem of low protein content. Because the demand for feed protein of aquatic animals is generally higher than that of livestock and poultry animals, which leads to a higher protein level in aquatic feed than in livestock and poultry feed. The low protein content of DDGS limits its addition amount in aquatic feed formulations. Therefore, increasing the protein content of DDGS can increase its addition proportion in aquatic feed formulations, thereby increasing the application scale of DDGS in aquatic feed, and further helping to solve the problem of excessive dependence on imported protein raw materials such as fish meal and soybean meal in aquatic feed. SUMMARY
[0004] The purpose of the present application is to provide a method for increasing the protein content of DDGS, which has a simple preparation process and low cost, and can be used for large-scale industrial production.
[0005] To achieve the above-mentioned purpose, the present application provides a method for increasing the protein content of DDGS, comprising: using a hydrolytic enzyme to hydrolyze corn DDGS, and then performing enzyme inactivation, centrifugal separation and drying to obtain corn DDGS concentrated protein.
[0006] The hydrolytic enzyme comprises one or more of cellulase, xylanase, beta-glucanase, beta-glucosidase and cellobiase.
[0007] Further, the hydrolytic enzyme comprises cellulase and xylanase.
[0008] Further, the mass ratio of the cellulase and the xylanase is 1:(0.8-1.2).
[0009] Further, the mass ratio of the hydrolytic enzyme and the corn DDGS is (0.01-0.2):1.
[0010] Further, the enzymolysis temperature is 50-54℃, and the time is 16-24h.
[0011] Further, the enzymolysis pH value is 5-6.
[0012] Further, the enzyme inactivation is kept at 85-95℃ for 15-30min.
[0013] Further, the centrifugal separation comprises: centrifuging the inactivated reaction liquid at 3500rpm for 10min to remove the enzymolysis liquid and part of the oil, taking out and discarding the supernatant after the centrifugation, adding distilled water and stirring uniformly, then putting into the centrifuge again at 3000rpm for 5min, taking out and discarding the supernatant after the centrifugation, and repeating 4 times.
[0014] Further, the corn DDGS is crushed before hydrolysis.
[0015] Further, the protein mass content in the corn DDGS is less than or equal to 30%, and the protein mass content in the corn DDGS concentrated protein is greater than or equal to 39%.
[0016] Overall, compared with the prior art, the above technical scheme conceived by the present application mainly has the following technical advantages:
[0017] (1) Compared with the method for improving the protein content of DDGS by using microbial fermentation, the present application can avoid the adverse effects of microorganisms and their own metabolites on the health of aquatic animals.
[0018] (2) The preparation method of the present application is simple, the process is easy to control, and the cost is low. The cellulase and xylanase used have commercialized products, so that the concentration and preparation process of DDGS can be standardized and scaled up, ensuring that the obtained DDGS concentrated protein with stable nutritional ingredients can be applied on a large scale in aquatic feed.
[0019] (3) By using the cellulase and xylanase in combination under suitable pH and temperature conditions, the present application can increase the protein level of DDGS to more than 39%, thereby improving the application value of DDGS in aquatic feed.
[0020] (4) The present application can also reduce the mycotoxin content of DDGS, further reducing the vomitoxin content of DDGS from 2mg / kg to an undetectable level. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1The change of protein growth rate of DDGS concentrated protein with time. DETAILED DESCRIPTION
[0022] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.
[0023] The present application provides a method for increasing the protein content of DDGS, which uses a cheap and stable composite commercial enzyme that can hydrolyze cellulose, and realizes the concentration of DDGS protein in combination with a water washing process. The specific method steps are as follows:
[0024] 1. Raw material crushing: The obtained commercial corn DDGS is crushed.
[0025] 2. Enzyme dissolution: Dissolve the commercial cellulase used for enzyme hydrolysis with distilled water.
[0026] 3. Mixing of corn DDGS and enzyme: Add the dissolved enzyme to the corn DDGS, and mix according to the ratio of corn DDGS to water of 1:4.
[0027] 4. pH adjustment: Adjust the pH of the reaction solution with 1 mol / L hydrochloric acid solution and 3 mol / L sodium hydroxide solution.
[0028] 5. Shaking enzyme hydrolysis: After reaching the enzyme hydrolysis temperature, shake during enzyme hydrolysis to ensure sufficient reaction.
[0029] 6. Enzyme inactivation: Increase the temperature of the reaction system to 90℃ for 20 minutes to inactivate the enzyme.
[0030] 7. Centrifugal water washing: Centrifuge the reaction solution at 3500 rpm for 10 min to remove the enzyme hydrolysis liquid and part of the oil, etc. After centrifugation, pour out the supernatant, add distilled water and stir evenly, then put it into a low-speed centrifuge at 3000 rpm for 5 min. After centrifugation, pour out the supernatant. This process is repeated 4 times.
[0031] 8. Drying: Dry the dry matter after centrifugal water washing in an oven.
[0032] 9. Crushing: After drying, use a crusher to crush it to obtain corn DDGS concentrated protein.
[0033] Optimization of enzyme hydrolysis conditions:
[0034] 1. Select two enzymes that can efficiently hydrolyze DDGS cellulose, which are cellulase and xylanase.
[0035] 2. The cellulase and xylanase are added directly mixed.
[0036] 3. The reaction time of the cellulase and xylanase with the DDGS is 16-24h.
[0037] 4. The optimal pH for the mixed addition of the cellulase and xylanase is 5.5.
[0038] 5. The optimal addition ratio of the DDGS to the cellulase and xylanase is (1:0.05), and the optimal enzymolysis temperature is 52℃.
[0039] The application is further described below through specific examples.
[0040] Example 1 Comparison of the effects of five cellulases on DDGS protein concentration
[0041] (1) Experimental materials
[0042] The experimental materials include commercial DDGS and five representative cellulases (cellulase, xylanase, β-glucanase, β-glucosidase, cellobiase) selected according to the type of cellulase.
[0043] (2) Enzymolysis condition setting
[0044] The initial pH of the five cellulases β-glucanase, xylanase, cellobiase, β-glucosidase and cellulase is set to be 4.0-5.5, 5.0-6.0, 3.5-6.0, 4-7, 4.0-5.5, respectively; the enzymolysis temperature is set to be 50℃; the addition ratio of the enzyme and the substrate is 1:10 (w:w); and the enzymolysis time is set to be 6h.
[0045] (3) Experimental design
[0046] Five experimental groups (β-glucanase, xylanase, β-glucosidase, cellobiase, cellulase) and one blank control group are set up. No cellulase is added in the blank control group, but the rest of the experimental process is still carried out. There are three repetitions in each group. After the enzymolysis, the supernatant is removed by centrifugation, the DDGS concentrated protein is dried at 105℃, the crude protein content of the DDGS concentrated protein is determined, and the cellulase enzymolysis capacity of each enzyme is compared.
[0047] (4) Experimental results and conclusions
[0048] The results of Example 1 are shown in Table 1. The initial protein content of DDGS was (29.61 ± 0.07)%, and the protein content of the blank control group, which was simply washed with water, was increased to (34.82 ± 0.16)%. The protein contents after the use of β-glucanase, β-glucosidase, cellobiase, xylanase and cellulase were (35.19 ± 0.12)%, (35.87 ± 0.16)%, (35.39 ± 0.17)%, (37.74 ± 0.13)% and (38.66 ± 0.30)%, respectively. These results show that among the five cellulases, cellulase and xylanase have the most obvious effect on protein concentration after enzymolysis.
[0049] Table 1 Comparison of the effects of different types of cellulases on protein concentration
[0050]
[0051] Example 2 Comparison of the effects of cellulase and xylanase addition methods on DDGS protein concentration
[0052] (1) Experimental materials
[0053] The experimental materials include commercial DDGS and cellulase and xylanase.
[0054] (2) Enzymolysis condition setting
[0055] The initial pH of the mixed xylanase and cellulase is set to 5.0-5.5; the enzymolysis temperature is set to 50°C; the addition ratio of the mixed enzyme and the substrate is 1:1:10 (w:w:w), and the reaction ratio of the single xylanase or cellulase and the substrate is 1:10 (w:w); and the enzymolysis time is set to 6 h.
[0056] (3) Experimental design
[0057] According to the results of Example 1, efficient enzymes are selected, and single cellulase and xylanase are used as the control group. The experimental group includes five treatment groups, i.e., simultaneous addition of cellulase and xylanase, addition of cellulase first and then xylanase after 3 h of enzymolysis, and addition of xylanase first and then cellulase after 3 h of enzymolysis, with three replicates for each group. After enzymolysis, the supernatant is removed by centrifugation, the DDGS concentrated protein is dried at 105°C, the crude protein content of the DDGS concentrated protein is determined, and the effects of cellulase and xylanase addition methods on cellulose hydrolysis are compared.
[0058] (4) Experimental results and conclusions
[0059] The results of Example 2 are shown in Table 2. The protein content of the xylanase group was (37.74±0.13)%, the protein content of the cellulase group was (38.66±0.30)%, and the protein content of the mixed cellulase and xylanase group was (39.61±0.21)%. The protein content of the cellulase and xylanase group added in sequence was (39.26±0.13)%, and the protein content of the xylanase and cellulase group added in sequence was (39.4±0.37)%. The results of the experiment show that the mixed cellulase and xylanase group has no significant difference compared with the cellulase and xylanase group added in sequence, and is higher than the single enzyme group. According to the simplicity of operation, it is recommended to choose single mixed addition.
[0060] Table 2 Comparison of the effects of different mixed addition methods on DDGS protein concentration
[0061]
[0062] Example 3 Influence of different enzyme hydrolysis time on the effect of DDGS protein concentration
[0063] (1) Experimental materials
[0064] The experimental materials include commercial DDGS and cellulase and xylanase.
[0065] (2) Enzymolysis condition setting
[0066] The initial pH of the mixed xylanase and cellulase is set to 5.0-5.5; the enzyme hydrolysis temperature is set to 50°C; and the addition ratio of the mixed enzyme and the substrate is 0.025:0.025:1 (w:w:w).
[0067] (3) Experimental design
[0068] According to the results of Examples 1 and 2, the cellulase and xylanase mixed addition method is selected, and the enzyme hydrolysis reaction time is set to 4h, 8h, 12h, 16h, 20h, 24h and 48h, with 3 repeats in each group. After enzyme hydrolysis, the supernatant is removed by centrifugation, the DDGS concentrated protein is dried at 105°C, the crude protein content of the DDGS concentrated protein is determined, and the cellulase hydrolysis ability of each enzyme is compared.
[0069] (4) Experimental results
[0070] The influence of the enzyme hydrolysis time of the cellulase and xylanase complex enzyme on the protein content of the DDGS concentrated protein is shown in Table 3. From 4h to 48h of enzyme hydrolysis time, the protein content increased from (37.53±0.31)% to (39.66±0.27)%, and especially from 24h to 48h, the protein content not only did not increase, but also decreased slightly. The growth rate of the protein content is shown in Table 4. Figure 1The enzymatic hydrolysis time from 16h to 24h is relatively slow, therefore, it is suggested that the enzymatic hydrolysis time is 16-24h.
[0071] Table 3 Effect of enzymatic hydrolysis time on protein concentration of DDGS
[0072]
[0073] Example 4 Effect of different enzyme addition amount on protein concentration of corn DDGS
[0074] (1) Experimental materials
[0075] The experimental materials include commercial corn DDGS and cellulase and xylanase.
[0076] (2) Enzymatic hydrolysis condition setting
[0077] The initial pH of mixed xylanase and cellulase is set at 5.0-5.5, the enzymatic hydrolysis temperature is set at 50℃, the addition ratio of mixed enzyme and substrate is 0.025:0.025:1(w:w:w), 0.1:0.1:1(w:w:w), and the enzymatic hydrolysis time is 16h, 20h, 24h, 48h.
[0078] (3) Experimental design
[0079] According to the results of examples 1-3, the mixed enzyme is used as the experimental group, and the blank with only synchronous water washing is used as the control group. The blank control group does not add any fiber hydrolytic enzyme, but still carries out the rest of the experimental process. Each group has 3 repeats. After the enzymatic hydrolysis, the supernatant is removed by centrifugation, the corn DDGS concentrated protein is dried at 105℃, the crude protein content of the corn DDGS concentrated protein is determined, and the cellulose enzymatic hydrolysis ability of each enzyme is compared.
[0080] (4) Experimental results and conclusions
[0081] The results of the effect of different enzyme addition amounts on the corn DDGS protein concentration are shown in Table 4. When the ratio of mixed enzymes to substrate is 0.025:0.025:1 (w:w:w), the protein content after 24 h is the highest, reaching (39.96±0.05)%, and when the ratio of mixed enzymes to substrate is 0.1:0.1:1 (w:w:w), the protein content after 48 h is the highest, reaching (41.36±0.78)%. Although 0.1:0.1:1 (w:w:w) can obtain higher DDGS concentrated protein, 0.025:0.025:1 (w:w:w) enzyme hydrolysis for 24 h has reached 96.62% thereof, and enzyme hydrolysis for 16 h has reached 93.54% thereof. Therefore, considering the enzyme addition amount, resource cost such as power consumption, and time factors, the enzyme hydrolysis can be selectively carried out for 16-24 h, and the ratio of mixed enzymes to substrate is 0.025:0.025:1 (w:w:w).
[0082] Table 4 Effect of enzyme addition amount on corn DDGS protein concentration (% dry matter)
[0083]
[0084] Example 5 Effect of different initial pH on DDGS protein concentration
[0085] (1) Experimental materials
[0086] The experimental materials include commercial DDGS and cellulase and xylanase.
[0087] (2) Enzymolysis condition setting
[0088] The enzyme hydrolysis time of mixed xylanase and cellulase is set to 16 h according to the results of Example 3; the enzyme hydrolysis temperature is set to 50°C; the addition ratio of mixed enzymes to substrate is 0.025:0.025:1 (w:w:w); and the addition ratio of single enzyme to substrate is 0.05:1.
[0089] (3) Experimental design
[0090] According to the results of Examples 1 and 2, the cellulase and xylanase are mixed and added, and the pH is selected with reference to the pH provided in the instruction manual of each cellulase and xylanase. The recommended pH of cellulase is 4.0-5.5, and the recommended pH of xylanase is 5.0-6.0. Therefore, three pH gradients, 4.0, 5.5 and 6.0, are designed in this experiment, and the specific design process is shown in Table 5. Each group has three repeats. After enzyme hydrolysis, the supernatant is removed by centrifugation, the DDGS concentrated protein is dried at 105°C, the crude protein content of the DDGS concentrated protein is determined, and the cellulase hydrolysis capacity of each enzyme is compared.
[0091] Table 5 pH design gradient for enzymatic hydrolysis
[0092]
[0093] Note: √ represents addition, and × represents no addition
[0094] (4) Experimental results and conclusions
[0095] The results of different pH on DDGS protein concentration are shown in Table 6. When the initial pH is 4, the protein content of mixed addition of cellulase and xylanase is only (37.53±0.15)%, which is even lower than that of single addition of cellulase (37.65±0.22)%. When the initial pH is 5.5 and 6.0, the protein content of mixed addition of cellulase and xylanase is higher than that of single addition. When the initial pH is 5.5, the protein content of mixed addition is (37.91±0.03)%, which is the highest among all treatment groups. Therefore, it is recommended that the pH of enzymatic hydrolysis reaction is 5.5. At the same time, it is shown that under suitable pH conditions, when the total amount of hydrolytic enzyme is the same, the mixed use of cellulase and xylanase is more conducive to improving the protein content, which indicates that the enzymatic hydrolysis effect is better.
[0096] Table 6 Effect of different pH on DDGS protein concentration
[0097]
[0098] Example 6 Effect of different enzymatic hydrolysis temperatures on DDGS protein concentration
[0099] (1) Experimental materials
[0100] The experimental materials include commercial DDGS and cellulase and xylanase.
[0101] (2) Enzymatic hydrolysis condition setting
[0102] The enzymatic hydrolysis time of mixed xylanase and cellulase is set to 16h according to the results of Example 3; the pH of enzymatic hydrolysis is set to 5.5; the addition ratio of mixed enzymes and substrate is 0.025:0.025:1 (w:w:w); and the addition ratio of single enzyme and substrate is 0.05:1.
[0103] (3) Experimental design
[0104] According to the results of Examples 1 and 2, the cellulase and xylanase are mixedly added, the temperature for enzyme hydrolysis is selected according to the temperature provided in the instruction manual of the cellulase and xylanase, the recommended temperature for cellulase is 50-60℃, and the recommended temperature for xylanase is 48-52℃; therefore, three temperature gradients are designed, i.e. 48, 52 and 60℃, and the specific design process is shown in Table 7, each group has three repeats, after the enzyme hydrolysis, the supernatant is removed by centrifugation, the DDGS protein concentrate is dried at 105℃, the crude protein content of the DDGS protein concentrate is determined, and the cellulase hydrolysis capacity of each enzyme is compared.
[0105] Table 7: Design of enzyme hydrolysis temperature gradient
[0106]
[0107] (4) Experimental results and conclusions
[0108] The results of DDGS protein concentration at different temperatures are shown in Table 8. With the increase of enzyme hydrolysis temperature, the protein content of DDGS protein concentrate is increased after the addition of cellulase or xylanase alone or after the mixed addition, and the protein content is the highest after the mixed addition of cellulase and xylanase at 52℃, reaching (39.77±0.09)%, therefore, the recommended enzyme hydrolysis temperature is 52℃.
[0109] Table 8: Effect of different enzyme hydrolysis temperatures on DDGS protein concentration
[0110]
[0111] In summary, according to the results of Examples 1-6, it can be found that the DDGS can be hydrolyzed by the complex cellulase under suitable conditions to increase the protein content, the specific conditions are as follows: the cellulase and xylanase are mixedly added, the addition ratio is 0.025:0.025:1 (W:W:W), the optimum temperature is 52℃, the optimum pH is 5.5, and the enzyme hydrolysis time is recommended to be 16-24h. Finally, the mycotoxin zearalenone, emetic toxin and aflatoxin B1 in the DDGS protein concentrate of the corn DDGS raw material sample and the sample treated by enzyme hydrolysis under the optimum conditions are detected, and only about 2mg / kg of emetic toxin is detected in the corn DDGS raw material sample, while no emetic toxin is detected in the DDGS protein concentrate treated by the method, which proves that the method can also reduce the emetic toxin in the DDGS. The reason may be that the emetic toxin is dissolved in water and removed by centrifugation during the enzyme hydrolysis process, and the enzyme hydrolysis process helps to dissolve the emetic toxin, further improving the removal effect of the emetic toxin.
[0112] Those skilled in the art can easily understand that the above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method of increasing the protein content of DDGS, characterized by, The application relates to a method for preparing corn DDGS concentrated protein. The hydrolytic enzyme comprises one or more of cellulase, xylanase, beta-glucanase, beta-glucosidase and cellobiase. The hydrolytic enzyme comprises cellulase and xylanase.
2. The method of increasing the protein content of DDGS of claim 1, wherein, The mass ratio of the cellulase to the xylanase is 1:(0.8-1.2).
3. The method of increasing the protein content of DDGS of claim 2, wherein, The mass ratio of the hydrolytic enzyme to the corn DDGS is (0.01-0.2):
1.
4. The method of increasing the protein content of DDGS of claim 1, wherein, The enzymolysis temperature is 50-54 DEG C, and the time is 16-24 h.
5. The method of increasing the protein content of DDGS of claim 1, wherein, The enzymolysis pH value is 5-6.
6. The method of increasing the protein content of DDGS of claim 1, wherein, The enzyme inactivation is carried out at 85-95 DEG C for 15-30 min.
7. The method of increasing the protein content of DDGS of claim 1, wherein, The centrifugal separation comprises the following steps: centrifuging the inactivated reaction liquid at 3500 rpm for 10 min to remove the enzymolysis liquid and part of oil, taking out and discarding the supernatant after the centrifugation, adding distilled water and stirring uniformly, then putting into the centrifuge again and centrifuging at 3000 rpm for 5 min, taking out and discarding the supernatant after the centrifugation, and repeating the above steps for 4 times.
8. The method of increasing the protein content of DDGS of claim 1, wherein, The corn DDGS is crushed before being hydrolyzed.
9. The method of increasing the protein content of DDGS of claim 1, wherein, The protein mass content in the corn DDGS is less than or equal to 30%, and the protein mass content in the corn DDGS concentrated protein is greater than or equal to 39%.
10. The method of increasing the protein content of DDGS of claim 1, wherein,