A method for processing enzymatic corn gluten meal

Abstract

The application discloses a processing method of zymolyzed corn gluten meal, and belongs to the technical field of biology. The processing method comprises the following steps: (1) after corn gluten meal is treated with alkali, composite enzyme I is added, and I-stage enzymolysis is carried out to obtain an enzymolysis liquid; (2) composite enzyme II and lactic acid bacteria liquid are added into the enzymolysis liquid, II-stage synchronous enzymolysis and fermentation are carried out, filtration and drying are carried out, and the zymolyzed corn gluten meal is obtained. The zymolyzed corn gluten meal is prepared by using the deep bacteria-enzyme synergistic fermentation method, the protein enzymolysis efficiency is improved, the number of lactic acid bacteria is increased, and the digestive utilization efficiency of the corn gluten meal and the product taste are greatly improved. The application can be applied to high-end aquatic products, egg-laying poultry and various young animals, can improve the animal production performance, and can reduce the feed cost and the breeding cost.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a processing method of enzymatically hydrolyzed corn gluten powder. Background Art

[0002] Corn gluten meal is a byproduct of corn kernels after starch production in the food industry or purification in the brewing industry. It has a high protein content, approximately 62% to 74% protein, accounting for about 65% of the dry matter. It is also rich in various amino acids, such as methionine, cystine, and leucine. Furthermore, the carotene and lutein in corn gluten meal can promote lipid metabolism in broiler chickens. As a high-protein feed ingredient, corn gluten meal has potential for application and development in animal production.

[0003] Although corn gluten meal has a high protein content, its main components are alcohol-soluble proteins (68%) and gluten (22%). Alcohol-soluble proteins contain large regions of α-helical structure, making them highly hydrophobic. Gluten is only soluble in alkaline solutions; the two have poor water solubility. Traditional corn gluten meal is made by wet-milling corn kernels to produce corn starch, which is then concentrated, dehydrated, separated, and dried. Due to differences in processing equipment and production processes, the protein content of corn gluten meal varies greatly. The high alcohol-soluble protein content in corn gluten meal is not conducive to animal digestion and absorption, which significantly limits the amount of corn gluten meal used in feed and the types of people who can use it.

[0004] Corn gluten meal can be modified and processed through fermentation and enzymatic hydrolysis, improving its feed value. During the fermentation process, microbial proteases break down the macromolecular proteins in corn gluten meal into small peptides and free amino acids, making them more readily absorbed and utilized by animals. Fermentation improves the amino acid profile of corn gluten meal, replenishing essential amino acids that are previously deficient and better meeting the nutritional needs of animals. However, solid-state fermentation of corn gluten meal also has certain drawbacks. Due to the complexity and uncertainty of the solid-state fermentation process, the quality of fermented corn gluten meal can vary significantly between batches. Solid-state fermentation cycles are typically relatively long, increasing production costs and posing potential contamination risks. Enzymatically hydrolyzed corn gluten meal has a shorter production cycle, more stable quality, a higher degree of protein hydrolysis than fermented corn gluten meal, and a higher product value. However, these are limited by the high cost of enzyme preparations, stringent reaction conditions, and significant equipment investment. Furthermore, enzymatic hydrolysis does not alter the amino acid composition of corn gluten meal, nor does it improve its amino acid balance. Summary of the Invention

[0005] The present invention aims to provide a method for processing enzymatically hydrolyzed corn gluten meal to address the aforementioned problems of the prior art. This method utilizes a deep bacterial-enzyme synergistic fermentation method to prepare enzymatically hydrolyzed corn gluten meal, improving protein hydrolysis efficiency, increasing the number of lactic acid bacteria, and significantly enhancing the digestibility and utilization efficiency of the corn gluten meal and the product's taste. This method can be applied to high-end aquatic products, laying poultry, and various young animals, improving animal production performance and reducing feed and breeding costs.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for processing enzymatically hydrolyzed corn gluten meal, comprising the following steps:

[0008] (1) treating corn gluten meal with alkali, adding complex enzyme I, and performing I-stage enzymolysis to obtain an enzymolysis solution;

[0009] (2) adding compound enzyme II and compound bacterial solution to the enzymatic hydrolysis solution, performing stage II simultaneous enzymatic hydrolysis and fermentation, filtering, and drying to obtain the enzymatic hydrolyzed corn gluten powder;

[0010] The complex enzyme I includes alkaline protease and neutral protease; the complex enzyme II includes neutral protease, flavor protease, acid protease and saccharifying enzyme;

[0011] The composite bacterial liquid includes Bacillus coagulans and Streptococcus thermophilus.

[0012] Furthermore, the alkali treatment process is to mix corn gluten meal, alkali and 60-70° C. water; the mass ratio of the corn gluten meal, alkali and water is 1: (0.02-0.03): (2-4).

[0013] Optionally, the base includes but is not limited to sodium hydroxide.

[0014] Furthermore, in the complex enzyme I, the inoculation amount of the alkaline protease is 1000-2000 U / g, and the inoculation amount of the neutral protease is 500-1000 U / g.

[0015] Furthermore, the temperature of the stage I enzymatic hydrolysis is 53° C.-60° C., the time is 6-10 h, and the pH is 8.0±0.2.

[0016] Furthermore, in the complex enzyme II, the inoculation amount of the neutral protease is 800-1000 U / g, the inoculation amount of the flavor protease is 300-600 U / g, the inoculation amount of the acid protease is 800-1000 U / g, and the inoculation amount of the saccharifying enzyme is 100-300 U / g.

[0017] Furthermore, in the composite bacterial solution, the ratio of live bacteria of Bacillus coagulans and Streptococcus thermophilus is 1:1, and the inoculation amount of the composite bacterial solution is (0.1-5)×10 7 CFU / g.

[0018] Furthermore, the temperature of the stage II simultaneous enzymatic hydrolysis and fermentation is 40° C.-50° C., and the time is 18-24 hours.

[0019] The present invention also provides enzymatically hydrolyzed corn gluten powder obtained by the processing method.

[0020] The present invention also provides a use of the enzymatically hydrolyzed corn gluten meal in preparing animal feed.

[0021] The present invention also provides an animal feed, which contains the enzymatically hydrolyzed corn gluten meal.

[0022] The present invention discloses the following technical effects:

[0023] The present invention uses a deep bacterial-enzyme synergistic fermentation method to prepare enzymatically hydrolyzed corn gluten meal. Compared with traditional microbial fermentation, deep bacterial-enzyme synergistic fermentation can not only break down corn gluten meal into smaller molecules, providing a better substrate for microbial fermentation, but also promoting microbial growth and metabolism, thereby improving fermentation efficiency and effectiveness. Simultaneously, the microbial enzymes produced during the fermentation process can further promote the enzymatic hydrolysis of corn gluten meal. The two methods work together to improve the nutritional value and functional properties of corn gluten meal.

[0024] The present invention improves protein enzymatic hydrolysis efficiency (increasing small peptide content and conversion rate) by optimizing the core enzymatic hydrolysis process. By adding a lactic acid bacteria fermentation process, the lactic acid bacteria count is increased, significantly increasing the digestion and utilization efficiency of corn gluten meal and improving the product taste. The present invention has a simple and low-cost processing process. The resulting enzymatically hydrolyzed corn gluten meal can be used as a high-quality protein feed raw material, replacing high-cost protein raw materials. It can be applied to high-end aquatic products, laying poultry, and various young animals, improving animal production performance and reducing feed and breeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The present invention is a flow chart of the preparation process of enzymatically hydrolyzed corn gluten meal;

[0027] Figure 2This is the UV absorption chromatogram of enzymatically hydrolyzed corn gluten meal. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1 Enzymatic Hydrolysis Process Optimization

[0034] This example investigates the effects of different enzymatic hydrolysis processes on the treatment of corn gluten meal. The enzyme preparations were selected from alkaline protease, neutral protease, acidic protease, flavor protease, or saccharifying enzymes; and the fermentation strain was selected from Bacillus coagulans. The enzymatic hydrolysis scheme is shown in Table 1. The specific process is as follows:

[0035] Take 20 Erlenmeyer flasks and divide them into 5 groups, with 4 bottles in each group. Add 50g corn gluten meal, 1g sodium hydroxide, and 150mL water to each bottle. Add the corresponding protease according to the scheme in Table 1, and then place the Erlenmeyer flasks in a 45℃ shaker at 150rpm for enzymatic hydrolysis. Among them, Groups C, D, and E were enzymatically hydrolyzed for 6 hours, and the pH of the material was adjusted to 8.0 and the corresponding protease was added. Group E was also added with saccharifying enzyme and inoculated with 10mL of LMRS cultured Bacillus coagulans. After 30 hours of enzymatic hydrolysis, the corn gluten meal was placed in a 90℃ water bath for 15 minutes, and then samples were taken to determine the moisture, pH, acid-soluble protein, and crude protein content of the corn gluten meal in each group after enzymatic hydrolysis.

[0036] Crude protein was determined by Kjeldahl method. For acid-soluble protein determination, 15% trichloroacetic acid was used as the extract, shaken well, and centrifuged for 5 minutes. The supernatant was taken and the protein content was determined by Kjeldahl method.

[0037] Table 1 Enzymatic hydrolysis scheme of corn gluten meal

[0038]

[0039] The test results are shown in Table 2. As can be seen, the crude protein content of each sample group after enzymatic hydrolysis was approximately 62%, with similar differences. However, the acid-soluble protein content and pH values ​​of the enzymatic hydrolysis samples varied significantly. Group E had the highest acid-soluble protein content after enzymatic hydrolysis, reaching 37.38%, and an acid-soluble protein conversion rate of 60%. Its pH value was 4.14, the lowest among all groups. A comparison of the results from Groups B and A shows that the addition of acid protease and flavor protease during enzymatic hydrolysis increased the acid-soluble protein content. A comparison of the results from Groups C and B shows that pH adjustment during enzymatic hydrolysis can increase the acid-soluble protein content. A comparison of the results from Groups D and C shows that the addition of neutral protease during stage II also increased the acid-soluble protein content. In contrast to Group D, the addition of saccharifying enzymes and lactic acid bacteria during stage II increased the acid-soluble protein content of the enzymatic hydrolysis. Therefore, Group E achieved the highest acid-soluble protein conversion rate and served as the basis for subsequent process optimization.

[0040] Table 2 Effects of different enzymatic hydrolysis schemes on crude protein, acid-soluble protein and pH of enzymatic hydrolysis corn gluten meal (dry basis)

[0041]

[0042]

[0043] Example 2 Screening of fermentation strains

[0044] This example explores the effect of fermentation bacteria on the treatment effect of corn gluten meal. The fermentation bacteria are selected from Bacillus coagulans, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecalis, or Pediococcus acidilactici. The specific process is as follows:

[0045] (1) Mix 1000g corn gluten powder, 20g sodium hydroxide, and 2000mL water (60℃) in a ratio of 1:0.02:2.

[0046] (2) Stage I enzymatic hydrolysis

[0047] The premix was added to a 5 L fermenter, and composite protease (alkaline protease 1000 U / g, neutral protease 1000 U / g) was added to start timed enzymolysis. The enzymolysis process was maintained at a stirring rate of 150 rpm, and the temperature of the material was controlled at 53-55 ° C. 20% sodium hydroxide solution was added every 2 hours to adjust the pH of the material to 8.0. The total enzymolysis time was 6 h.

[0048] (3) Stage II enzymatic hydrolysis and fermentation

[0049] After the completion of stage I enzymatic hydrolysis, compound protease (flavor protease 300 U / g, acid protease 800 U / g) and saccharifying enzyme (300 U / g) were added and divided into 500 mL triangular flasks, 200 g of enzymatic hydrolysate was added to each bottle, and 5 mL of MRS cultured lactic acid bacteria liquid was respectively inoculated. One group was added with 5 mL of MRS culture medium as a control, and fermented at 45 ° C in a shaker with a speed of 150 rpm for 24 h.

[0050] (4) Sampling and testing

[0051] Determine the acid-soluble protein, crude protein, and lactic acid content of raw corn gluten meal and enzymatically hydrolyzed corn gluten meal. Samples were taken at the end of fermentation to count lactic acid bacteria. Crude protein was determined using the Kjeldahl method. For the acid-soluble protein determination, extract the mixture using 15% trichloroacetic acid. After shaking, centrifuge for 5 minutes, and measure protein content in the supernatant using the Kjeldahl method.

[0052] The test results are shown in Table 3. As can be seen, after enzymatic hydrolysis and fermentation in stage II, the acid-soluble protein, lactic acid content, and viable lactic acid bacteria counts varied significantly across the groups inoculated with different lactic acid bacteria. Bacillus coagulans and Streptococcus thermophilus exhibited higher lactic acid bacteria counts, lactic acid content, and acid-soluble protein content after fermentation compared to the other lactic acid bacteria. However, there was little difference in product indices between Bacillus coagulans and Streptococcus thermophilus after fermentation and enzymatic hydrolysis. Therefore, using Bacillus coagulans and Streptococcus thermophilus as fermentation strains significantly improved the acid-soluble protein yield, the fermentation bacteria count, and the lactic acid content of the product compared to the other four lactic acid bacteria and the group without lactic acid bacteria inoculation.

[0053] Table 3 Effect of inoculation with different lactic acid bacteria on enzymatic hydrolysis of corn gluten meal (dry basis)

[0054]

[0055]

[0056] Example 3 Optimization of inoculum amount of lactic acid bacteria

[0057] This example investigates the effect of lactic acid bacteria inoculation on the treatment of corn gluten meal. The fermentation strains used were Bacillus coagulans and Streptococcus thermophilus. The specific process is as follows:

[0058] (1) Mix 1000g corn gluten powder, 20g sodium hydroxide, and 2000mL water (60℃) in a ratio of 1:0.02:2.

[0059] (2) Stage I enzymatic hydrolysis

[0060] The premix was added to a 5 L fermenter, and composite protease (alkaline protease 1000 U / g, neutral protease 1000 U / g) was added to start timed enzymolysis. The enzymolysis process was maintained at a stirring rate of 150 rpm, and the temperature of the material was controlled at 53-55 ° C. 20% sodium hydroxide solution was added every 2 hours to adjust the pH of the material to 8.0. The total enzymolysis time was 6 h.

[0061] (3) Stage II enzymatic hydrolysis and fermentation

[0062] After the completion of stage I enzymatic hydrolysis, compound protease (neutral protease 800 U / g, flavor protease 600 U / g, acid protease 800 U / g) and saccharifying enzyme (300 U / g) were added and divided into 500 mL triangular flasks, with 200 g of enzymatic hydrolysate in each bottle. Lactic acid bacteria were inoculated according to the proportions and addition amounts in Table 4, and fermented at 50°C in a shaker at 150 rpm for 24 h.

[0063] Table 4 Lactic acid bacteria addition amount and inoculation ratio scheme

[0064]

[0065]

[0066] (4) Sampling and testing

[0067] Determine the acid-soluble protein, crude protein, and lactic acid content of raw corn gluten meal and enzymatically hydrolyzed corn gluten meal. Samples were taken at the end of fermentation to count lactic acid bacteria. Crude protein was determined using the Kjeldahl method. For acid-soluble protein determination, extract the mixture using 15% trichloroacetic acid. After shaking, centrifuge for 5 minutes, and measure protein content in the supernatant using the Kjeldahl method.

[0068] The test results are shown in Table 5. It can be seen that when the inoculation ratio is (0.1-1): (1-0.1), after fermentation and enzymatic hydrolysis, the number of lactic acid bacteria produced in each group is (0.7-1.2)×10 10 CFU / g, which is much greater than the 6.94×10 4CFU / g content; acid-soluble protein content in each group was greater than 38%, which was also higher than the 36.77% of the uninoculated group; lactic acid content in each group was between 7.5% and 9.0%, much higher than the 2.12% of the uninoculated group; crude protein content in each group was similar, all around 62%. The results showed that the ratio of inoculated Bacillus coagulans to thermophilic Streptococcus was within the range of (0.1-1): (1-0.1), and the inoculation size was 1×10 6 -1×10 7 CFU / g range, each treatment group can obtain a relatively ideal fermentation effect. When the inoculation ratio of Bacillus coagulans to Streptococcus thermophilus is 1:1, the fermentation and enzymatic hydrolysis effect is the best, and the inoculation amount range is (0.1-5)×10 7 CFU / g have similar excellent effects.

[0069] Table 5 Effects of different ratios and inoculation amounts of lactic acid bacteria on the indices of enzymatically hydrolyzed corn gluten meal (dry basis)

[0070] Serial number Crude protein% Acid-soluble protein% Lactic acid% Lactic acid bacteria count CFU / g comparison 62.98 36.77 2.12 <![CDATA[6.94×10 4 ]]> 1 63.26 38.33 7.51 7.69 x 10 9 <!-- 6 -->]]> 2 62.86 39.06 7.93 8.01 x 10 9 ]] 3 62.85 40.30 8.24 <![CDATA[8.35×10 9 ]]> 4 62.36 40.16 8.43 1.03 x 10 10 ]]> 5 61.45 41.47 8.98 <![CDATA[1.21×10 10 ]]> 6 61.61 41.43 8.14 <![CDATA[1.10×10 10 ]]> 7 63.12 39.88 8.25 <![CDATA[9.85×10 9 ]]> 8 62.95 40.27 7.98 <![CDATA[9.39×10 9 ]]> 9 62.88 40.38 7.69 8.94 x 10 9 ]]

[0071] Example 4

[0072] 1. Enzymatic hydrolysis of corn gluten powder pilot experiment 1

[0073] 1. Processing method

[0074] (1) Premix dry corn gluten meal, alkali and a small amount of water

[0075] Take 10 kg of corn gluten powder, 0.25 kg of calcium hydroxide powder, and 2.5 kg of water, mix them in a mixer.

[0076] (2) Stage I enzymatic hydrolysis

[0077] Add 20kg of 70°C hot water to a 50L fermenter, start stirring, and slowly add the premixed corn gluten meal. After stirring, the material temperature is 53°C and the pH is 8.9. Then add a composite protease (2000U / g alkaline protease and 500U / g neutral protease) and mix well. Start the timed enzymatic hydrolysis. The temperature during the enzymatic hydrolysis process is controlled at 53-55°C. 20% sodium hydroxide solution is added every 2 hours to adjust the material pH to 8.0±0.2. The total enzymatic hydrolysis time is 6 hours.

[0078] (3) Stage II enzymatic hydrolysis and fermentation

[0079] After the completion of the first stage of enzymatic hydrolysis, the temperature was lowered to 45°C, and a composite protease was added for the second stage of enzymatic hydrolysis and fermentation. The addition amount of acidic protease and neutral protease was 1000U / g, and the addition amount of flavor protease was 400U / g. A mixed strain of Bacillus coagulans and Streptococcus thermophilus was added, with a viable cell count ratio of 1:1 and a total inoculation amount of 1×10 7CFU / g. 200 U / g of saccharifying enzyme was also added. The fermentation and enzymatic hydrolysis were maintained at 45°C for 18 h, with no pH control during the fermentation and enzymatic hydrolysis.

[0080] (4) Spray drying

[0081] After the enzymatic hydrolysis was completed, the temperature was raised to 80°C and maintained for 10 min. The enzymatic hydrolysate was then passed through a 100-mesh sieve and spray-dried.

[0082] 2. Detection indicators

[0083] The acid-soluble protein, crude protein and lactic acid contents in the raw corn gluten meal and the enzymatically hydrolyzed corn gluten meal were determined, and samples were taken to count the number of lactic acid bacteria at the end of fermentation.

[0084] 2. Enzymatic hydrolysis of corn gluten powder pilot experiment 2

[0085] 1. Processing method

[0086] (1) Premix dry corn gluten meal, alkali and a small amount of water

[0087] Take 10 kg of corn gluten powder, 0.30 kg of calcium hydroxide powder, and 4.0 kg of water, mix them in a mixer.

[0088] (2) Stage I enzymatic hydrolysis

[0089] Add 20 kg of 70°C hot water to a 50L fermenter, add the premixed material to the fermenter, stir and mix until the material temperature is 53°C and the pH is 9.1, then add composite protease (alkaline protease 1000 U / g, neutral protease 500 U / g) and mix well before starting the enzymatic hydrolysis. The temperature during the enzymatic hydrolysis process is controlled at 57-60°C. 20% sodium hydroxide solution is added every 2 hours to adjust the material pH to 8.0±0.2. The total enzymatic hydrolysis time is 10 hours.

[0090] (3) Stage II enzymatic hydrolysis and fermentation

[0091] After the completion of the first stage of enzymatic hydrolysis, the temperature was lowered to 45 °C, and compound protease was added for the second stage of enzymatic hydrolysis and fermentation. The addition amount of acid protease and neutral protease was 800 U / g, and the addition amount of flavor protease was 300 U / g. A mixed strain of Bacillus coagulans and Streptococcus thermophilus was added, with a live cell count ratio of 1:1 and a total inoculation amount of 1×10 7 CFU / g, and glucoamylase was added at the same time at a dosage of 200 U / g. The time was started from the addition of lactic acid bacteria, compound protease, and glucoamylase, and fermentation and enzymolysis were maintained at 45°C for 24 hours. The pH of the fermentation and enzymolysis process was not controlled.

[0092] (4) Spray drying

[0093] After the enzymatic hydrolysis was completed, the temperature was raised to 80°C and maintained for 10 min. The enzymatic hydrolysate was then passed through a 100-mesh sieve and spray-dried.

[0094] 2. Detection indicators

[0095] The acid-soluble protein, crude protein and lactic acid contents in the raw corn gluten meal and the enzymatically hydrolyzed corn gluten meal were determined, and samples were taken to count the number of lactic acid bacteria at the end of fermentation.

[0096] 3. Experimental Results

[0097] The test results are shown in Table 6. As can be seen, in small test 1, after the corn gluten meal raw material was processed by fermentation and enzymatic hydrolysis of this process, acid-soluble protein (small peptide) was increased from 2.07% to 41.47%, which was increased by about 20 times, lactic acid content was increased from 1.28% to 7.84%, starch content was reduced from 17.47% to 8.23%, crude protein content was 62.85%, and small peptide conversion rate reached 66.00%; small test 2 results were similar to small test 1. After fermentation and enzymatic hydrolysis, acid-soluble protein (small peptide) was increased from 2.05% to 41.79%, which was also increased by about 20 times, lactic acid content was increased from 1.31% to 8.03%, starch content was reduced from 17.83% to 8.49%, crude protein content was 63.14%, and small peptide conversion rate reached 66.19%. The results of the two batches were similar, indicating that the product obtained by the process of the present invention is more stable.

[0098] Table 6 Comparison of corn gluten meal before and after enzymatic fermentation in small scale test (dry basis)

[0099]

[0100] Example 5

[0101] Pilot experiment of enzymatic hydrolysis of corn gluten meal

[0102] 1. Processing method

[0103] (1) Premix dry corn gluten meal, alkali and a small amount of water

[0104] Take 500 kg of corn gluten powder, 12.5 kg of calcium hydroxide powder, and 125 kg of water, mix them in a mixer.

[0105] (2) Stage I enzymatic hydrolysis

[0106] Add 1000 kg of 70°C hot water to a 5-ton fermenter, add the premixed material, and stir until the material temperature reaches 55°C and the pH is 9.0. Add a composite protease (1000 U / g alkaline protease and 700 U / g neutral protease) and mix thoroughly before starting the timed enzymatic hydrolysis. The temperature during the hydrolysis process is controlled at 55°C ± 2°C. Every two hours, add 20% sodium hydroxide solution to adjust the pH to 8.0 ± 0.1. The total enzymatic hydrolysis time is 10 hours.

[0107] (3) Stage II enzymatic hydrolysis and fermentation

[0108] After the completion of the first stage of enzymatic hydrolysis, the fermentation tank was cooled to 45 ° C, and the compound protease was added to carry out the second stage of enzymatic hydrolysis and fermentation. The amount of acid protease and neutral protease added was 1000 U / g, and the amount of flavor protease added was 300 U / g. At the same time, a mixed strain of Bacillus coagulans and Streptococcus thermophilus was added, with a viable cell count ratio of 1:1 and a total inoculum size of 1×10 7 CFU / g, and glucoamylase was added at the same time at a dosage of 100 U / g. The time was started from the addition of lactic acid bacteria, compound protease, and glucoamylase, and fermentation and enzymolysis were maintained at 45°C for 24 hours. The pH of the fermentation and enzymolysis process was not controlled.

[0109] (4) Spray drying

[0110] After the enzymatic hydrolysis was completed, the temperature was raised to 80°C and maintained for 10 min. The enzymatic hydrolysate was then passed through a 100-mesh sieve and spray-dried.

[0111] 2. Detection indicators

[0112] The acid-soluble protein, crude protein and lactic acid contents in corn gluten meal raw material and enzymatically hydrolyzed corn gluten were determined, and samples were taken to count the number of lactic acid bacteria at the end of fermentation.

[0113] 3. Experimental results

[0114] The test results are shown in Table 7. It can be seen that in the pilot experiment, after the corn gluten meal raw material was fermented and enzymatically processed by this process, the acid-soluble protein (small peptide) increased from 2.07% to 42.03%, an increase of about 20 times, the lactic acid content increased from 1.32% to 7.85%, the crude protein content was 62.45%, the small peptide conversion rate reached 67.30%, and the number of lactic acid bacteria after fermentation reached 1.19×10 10 The results were similar to those in Example 4, indicating that the process of the present invention is stable.

[0115] Table 7 Comparison of corn gluten meal before and after pilot enzymatic fermentation (dry basis)

[0116] sample corn gluten meal Enzymatic corn egg powder Acid-soluble protein (small peptide)% 2.07 42.03 Crude protein% 66.47 62.45 Lactic acid% 1.32 7.85 Small peptide conversion rate % / 67.30 Lactic acid bacteria count CFU / g / <![CDATA[1.19×10 10 ]]>

[0117] Example 6

[0118] In order to explore the effect of bacterial enzyme synergistic fermentation treatment on the amino acid content in corn gluten meal, the enzymatic hydrolyzed corn gluten meal prepared in Example 5 was used as a sample to test the amino acid content. The test results are shown in Table 8. It can be seen that the total amino acid content in the enzymatic hydrolyzed corn gluten meal prepared by the method of the present invention was slightly reduced from 63.89% to 59.67%, a decrease of 4.22%. This is because the addition of alkali during the fermentation process "diluted" the crude protein content. In addition, due to the growth and metabolism of lactic acid bacteria during the fermentation process, the amino acid composition changed significantly compared with the raw material, and the proportion of the total essential amino acid composition to the total amino acid composition decreased slightly, but the overall difference was not large.

[0119] Table 8 Effect of enzymatic fermentation on the amino acid content in corn gluten meal

[0120]

[0121]

[0122] Example 7

[0123] To explore the effect of bacterial enzyme synergistic fermentation on the amino acid content of corn gluten meal, the enzymatic hydrolyzed corn gluten meal prepared in Example 5 was used as a sample to analyze the protein molecular weight. The specific process is as follows:

[0124] (1) Sample preparation: Take an appropriate amount of enzymatically hydrolyzed corn gluten powder sample, dissolve it in water to a final concentration of 5 μg / μL, centrifuge it at 4°C, 12000 rcf for 10 min, and prepare the supernatant for loading onto the instrument.

[0125] (2) Liquid chromatography conditions

[0126] 1) Column temperature is 25°C;

[0127] 2) Flow rate of 0.7 mL / min;

[0128] 3) The injection volume is 10 μL;

[0129] 4) Chromatographic column: BioCore SEC-1205 μm, 7.8 × 300 mm;

[0130] 5) Mobile phase: 150 mmol / L phosphate mobile phase (8.99 g anhydrous sodium dihydrogen phosphate, 10.65 g anhydrous disodium hydrogen phosphate in 1 L of water);

[0131] 6) Detection wavelength: UV 214nm.

[0132] Test results such as Figure 2As shown in Table 9, it can be seen that the protein molecular weight distribution of the sample is mainly concentrated between 100-1000 Da, accounting for about 63% of the soluble protein, and 25.63% of the soluble protein is hydrolyzed into amino acids with a molecular weight of less than 100 Da, and only a small amount of protein has a molecular weight greater than 3000 Da, indicating that after fermentation and enzymatic hydrolysis by the method of the present invention, the macromolecular protein in the corn gluten meal is mainly decomposed into small peptides with a molecular weight of less than 1000 Da.

[0133] Table 9 Molecular weight distribution of enzymatically hydrolyzed corn gluten meal

[0134] Components Molecular weight range Area% 1 ≥10kDa 0.00 2 5-10 kDa 0.08 3 3-5 kDa 0.44 4 1-3 kDa 10.15 5 0.5-1 kDa 25.98 6 0.5-0.1 kDa 37.73 7 0.1kDa 25.62 total - 100

[0135] Example 8

[0136] In order to explore the effect of the enzymatically hydrolyzed corn gluten meal prepared by the method of the present invention on the growth performance of juvenile sea bass by replacing fish meal, an experiment was conducted using the enzymatically hydrolyzed corn gluten meal prepared in Example 5 as a sample instead of fish meal.

[0137] Healthy, similarly sized juvenile striped seabass (Japonica seabass) were selected and divided equally into three treatment groups (CK, D1, and D2). Three rearing tanks were set up in each treatment group, with 30 Japonica seabass juveniles per tank. The Japonica seabass juveniles used in the experiment were from the same batch of fry from the same company. Initial weights of 12.20 ± 1.20 g were selected and the experiment began after one week of acclimatization. The experiment lasted 56 days, with feeding twice daily at 8:30 AM and 4:00 PM. Water quality was monitored daily during the rearing period, with water temperature around 28°C, salinity between 16‰ and 22‰, dissolved oxygen concentrations >5.0 mg / L, and ammonia nitrogen concentrations <0.05 mg / L. The experimental feed formulas and ingredients are shown in Table 10. CK was a conventional feed; D1 was supplemented with 3% enzymatically hydrolyzed corn gluten meal in place of an equal amount of fish meal; and D2 was supplemented with 6% enzymatically hydrolyzed corn gluten meal in place of an equal amount of fish meal.

[0138] Table 10 Feed formula and ingredients (%)

[0139]

[0140]

[0141] Measurement indicators and methods: Record and clean the feed feeding amount, feed residue and the number of dead sea bass in each breeding box every day, calculate the survival rate, measure the weight of each sea bass, and calculate the weight gain rate and feed coefficient.

[0142] Calculation method:

[0143] Weight gain rate (%) = (m1-m2) / m2(1)

[0144] Among them, m1 is the mass after the test, and m2 is the mass before the test.

[0145] Feed coefficient = (R1-R2) / (m3+m4-m5) (2)

[0146] Among them, R1 and R2 are the feeding amount and residual feed amount, respectively, m3 is the total mass of fish at the end, m4 is the total mass of dead fish, and m5 is the total mass of initial fish.

[0147] Statistical analysis and variance analysis of each element index were performed using Microsoft EXCEL 2010 and IBM SPSS Statistics 26 software.

[0148] The test results are shown in Table 11. It can be seen that replacing 3% or 6% of fish meal with the enzymatically hydrolyzed corn gluten meal of the present invention in a conventional sea bass feed formula had no significant effect on the survival rate, weight gain rate, specific growth rate, and feed conversion rate of the sea bass (p>0.05). This indicates that the product of the present invention can partially replace fish meal in sea bass feed, reducing feed costs, and has no significant effect on sea bass growth indicators.

[0149] Table 11 Effects of enzymatic hydrolyzed corn gluten meal on growth performance indicators of Lateolabrax

[0150]

[0151]

[0152] Example 9

[0153] In order to explore the effect of replacing fish meal with different proportions of enzymatically hydrolyzed corn gluten meal on the growth performance of Penaeus monodon, an experiment was conducted using the enzymatically hydrolyzed corn gluten meal prepared in Example 5 as a sample.

[0154] 900 healthy, vigorous Penaeus monodon (Penaeus monodon) weighing approximately 13.20 ± 0.10 g were selected and divided equally into three treatments (CK, T1, and T2). Three cages were set up in each treatment, with 100 P. monodon shrimp per cage. The amounts of fish meal and enzymatically hydrolyzed corn gluten meal added to each treatment were as follows. The experimental feed formula and composition are shown in Table 12. CK used fish meal as the primary protein ingredient; T1 replaced 3% of the fish meal with enzymatically hydrolyzed corn gluten meal; and T2 replaced 6% of the fish meal with enzymatically hydrolyzed corn gluten meal.

[0155] Table 12 Formula and composition of basic feed for Penaeus monodon (%)

[0156]

[0157]

[0158] Measurement indicators and methods: Clean and record the number of dead Penaeus monodon shrimp in each cage daily, and calculate the survival rate. Measure the weight of each shrimp and calculate the weight gain rate, specific growth rate, and feed efficiency.

[0159] The test results are shown in Table 13. As can be seen, the survival rates of Penaeus monodon in the CK, T1, and T2 treatment groups were 99.0%, 98.5%, and 98.0%, respectively, with no significant differences in survival rates between the groups. The weight gain rate and specific growth rate of the T2 treatment group were slightly lower than those of the CK group, but there were no significant differences. There were no significant differences in feed conversion rates between the T1 and T2 treatment groups compared to the CK group (p>0.05). This indicates that replacing 3% or 6% fish meal with the enzymatically hydrolyzed corn gluten meal of the present invention has no significant effect on the growth parameters of Penaeus monodon. This product can be used to partially replace fish meal in feeding to reduce feed costs.

[0160] Table 13 Survival rate and growth index of each group of Penaeus monodon

[0161] Group CK T1 T2 Initial body weight (g) 13.21±0.04 13.25±0.06 13.40±0.11 Final body weight (g) 31.74±1.62 31.92±2.21 31.37±3.22 Survival rate (%) 99.00±1.00 98.50±1.50 98.00±1.00 Weight gain rate (%) 140.21±4.85 141.73±3.59 133.62±7.41 Specific growth rate (%) 1.50±0.04 1.53±0.02 1.47±0.03 Feed coefficient 1.74±0.03 1.74±0.05 1.79±0.10

[0162] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for processing enzymatically hydrolyzed corn gluten meal, characterized in that: The steps include: (1) After treating corn gluten meal with alkali, adding complex enzyme I and performing I-stage enzymatic hydrolysis to obtain enzymatic hydrolyzate; (2) adding compound enzyme II and compound bacterial solution to the enzymatic hydrolysis solution to perform stage II simultaneous enzymatic hydrolysis and fermentation, filtering, and drying to obtain the enzymatic hydrolyzed corn gluten powder; The complex enzyme I is composed of alkaline protease and neutral protease; the complex enzyme II is composed of neutral protease, flavor protease, acidic protease and saccharifying enzyme; The composite bacterial solution consists of Bacillus coagulans and Streptococcus thermophilus; In the composite bacterial solution, the ratio of live bacteria of Bacillus coagulans and Streptococcus thermophilus is 1:1; the inoculation amount of the composite bacterial solution is (0.1-5)×10 7 CFU / g.

2. The processing method according to claim 1, characterized in that: The alkali treatment process comprises mixing corn gluten meal, alkali and water at 60-70° C.; the mass ratio of the corn gluten meal, alkali and water is 1: (0.02-0.03): (2-4).

3. The processing method according to claim 1, characterized in that: In the complex enzyme I, the inoculation amount of the alkaline protease is 1000-2000 U / g, and the inoculation amount of the neutral protease is 500-1000 U / g.

4. The processing method according to claim 1, characterized in that: The temperature of the stage I enzymatic hydrolysis is 53° C.-60° C., the time is 6-10 h, and the pH is 8.0±0.

2.

5. The processing method according to claim 1, characterized in that: In the complex enzyme II, the inoculation amount of the neutral protease is 800-1000 U / g, the inoculation amount of the flavor protease is 300-600 U / g, the inoculation amount of the acid protease is 800-1000 U / g, and the inoculation amount of the saccharifying enzyme is 100-300 U / g.

6. The processing method according to claim 1, characterized in that: The temperature of the stage II synchronous enzymatic hydrolysis and fermentation is 40° C.-50° C., and the time is 18-24 hours.

7. An enzymatically hydrolyzed corn gluten meal obtained by the processing method according to any one of claims 1 to 6.

8. Use of the enzymatically hydrolyzed corn gluten meal according to claim 7 in preparing animal feed.

9. An animal feed, characterized in that The animal feed contains the enzymatically hydrolyzed corn gluten meal according to claim 7.

Images