Method for preparing high-F-value oligopeptide based on whey protein

Abstract

The invention discloses a method for preparing high-F-value oligopeptide from whey protein, and belongs to the technical field of food biology. According to the method, whey protein is taken as a raw material, step-by-step synergistic enzymolysis of alkaline protease-chymotrypsin-flavourzyme is performed, and an activated carbon selective adsorption dearomatization process is combined, so that the F value and the hydrolysis degree of oligopeptide are remarkably improved. The enzymolysis efficiency is high, and the final hydrolysis degree is gt; 30%; the dearomatization effect is excellent, and the product F value is gt; 26; the method is simple in process and low in cost, high-value conversion of whey protein can be achieved, and the prepared high-F-value oligopeptide can be applied to the fields of sports nourishment, food for special medical purposes and the like and has remarkable industrial value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food science and bioengineering, and specifically relates to a method for preparing high Fischer ratio oligopeptide (HFO) from whey protein by multi-step enzymatic hydrolysis combined with activated carbon adsorption technology. BACKGROUND

[0002] High Fischer ratio oligopeptide is a short-chain polypeptide composed of 2-9 amino acid residues, and its core feature is high content of branched-chain amino acids (BCAA) and low content of aromatic amino acids (AAA), with a Fischer ratio (molar ratio of BCAA to AAA) usually greater than 20. Such oligopeptides have significant physiological functions, such as anti-fatigue, metabolic regulation, free radical scavenging, and antioxidant effects, and have broad application prospects in the fields of sports nutrition and special medical use formula foods. Whey protein is a natural high-quality complete protein that contains all 9 essential amino acids required by the human body, with BCAA accounting for 26% and the molar ratio of BCAA to AAA being 2.7:1, making it an ideal raw material for preparing high Fischer ratio oligopeptides. Currently, the preparation of high Fischer ratio oligopeptides mainly relies on enzymatic hydrolysis technology, but existing processes have the following key problems: 1. Low enzymatic hydrolysis efficiency: the specificity of single enzyme species for whey protein hydrolysis is limited, which easily leads to insufficient protein degradation and low yield of target oligopeptides; the parameters of multi-enzyme synergistic hydrolysis (such as enzyme species combination, temperature, pH, and enzyme-substrate ratio) have not been systematically optimized, and the degree of hydrolysis is difficult to break through 30%. 2. Difficulty in achieving a Fischer ratio of 20 or more: free aromatic amino acids (such as phenylalanine and tyrosine) are easily produced during enzymatic hydrolysis, resulting in an initial Fischer ratio of 3-5 in the hydrolysate, which needs to be improved by de-aromatic treatment. However, existing de-aromatic processes are not optimized for whey protein hydrolysates, and there are problems such as incomplete removal of AAA, high loss rate of BCAA, or F value that cannot be stably achieved above 20. 3. Poor process economics: some processes use complex separation and purification techniques (such as ultrafiltration-chromatography combination), which have high equipment costs and are difficult to operate, making industrialization difficult; at the same time, whey protein is a byproduct of dairy products, and its high-value transformation path has not yet formed an efficient technical system. Therefore, developing a method for preparing high Fischer ratio oligopeptides from whey protein with high enzymatic hydrolysis efficiency, stable Fischer ratio, simple process, and controllable cost is of great significance for promoting the industrial application of functional peptide products and the green upgrading of the dairy industry chain. SUMMARY

[0003] In view of the defects of low enzymatic hydrolysis efficiency, difficulty in achieving a Fischer ratio, and poor process economics in existing high Fischer ratio oligopeptide preparation processes, the purpose of the present application is to provide a method for efficiently preparing high Fischer ratio oligopeptides from whey protein, which significantly improves the yield and Fischer ratio of HFO by optimizing the multi-step enzymatic hydrolysis process and activated carbon de-aromatic conditions, while adapting to the needs of industrial production.

[0004] The technical solution of the present application is as follows: To achieve the above-mentioned purpose, the application adopts an integrated process of "three-step synergistic enzymatic hydrolysis + activated carbon directional defragmentation", and the specific steps are as follows: 1. Step-by-step synergistic enzymatic hydrolysis (1) First step, alkaline protease hydrolysis: adjust the pH of the whey protein solution (mass-volume concentration 5%-7%) to 8.0-11.0 (preferably 9.0) with 1 mol / L NaOH solution, add alkaline protease according to the enzyme-substrate mass ratio of 1:12.5-1:50 (preferably 1:25), and place it in a constant temperature water bath at 45-55°C (preferably 50°C) for enzymatic hydrolysis for 2.0-3.0 h (preferably 3.0 h); after the enzymatic hydrolysis is completed, high-temperature treatment at 85-95°C for 10-15 min is used to inactivate the enzyme, and then it is cooled to room temperature. This step utilizes the specificity of alkaline protease for the cleavage of basic amino acid residues in whey protein, preliminarily destroys the three-dimensional structure of the protein, and releases short peptide fragments containing BCAAs.

[0005] To determine the optimal parameters of alkaline protease hydrolysis, the application conducts a plurality of single-factor optimization experiments: Whey protein concentration optimization: set mass-volume concentrations of 3%, 4%, 5%, 6%, and 7% for a total of 5 gradients, and fix the remaining parameters at pH 9.0, enzyme-substrate mass ratio of 1:25, and 50°C enzymatic hydrolysis for 3.0 h. The results show that (Fig. 1): Figure 1 ): When the whey protein concentration is increased from 3% to 6%, the F value after the first step of enzymatic hydrolysis is increased from 2.86 to 3.79, and the degree of hydrolysis is increased from 5.43% to 12.76%; when the concentration exceeds 6%, the enzyme-substrate contact is not sufficient due to the increased viscosity of the solution, and the F value and the degree of hydrolysis are slightly reduced (F=3.70, degree of hydrolysis=11.52%), so the optimal range of whey protein mass-volume concentration is determined to be 5%-7%.

[0006] Enzymatic hydrolysis time optimization: set 2.0, 2.5, 3.0, 3.5, and 4.0 h for a total of 5 gradients, and fix the remaining parameters at whey protein mass-volume concentration of 6%, pH 9.0, enzyme-substrate mass ratio of 1:25, and 50°C. The results show that (Fig. 2): Figure 1 ): When the time is extended from 2.0 h to 3.0 h, the F value is increased from 3.47 to 3.66, and the degree of hydrolysis is increased from 7.38% to 9.62%; when the time exceeds 3.0 h, the F value is significantly reduced, and the energy consumption is increased, so the optimal time range is determined to be 2.0-3.0 h, and the preferred time is 3.0 h.

[0007] Temperature optimization: set 40, 45, 50, 55, and 60°C for a total of 5 gradients, and fix the remaining parameters at whey protein mass concentration of 6%, pH 9.0, enzyme-substrate mass ratio of 1:25, and enzymatic hydrolysis for 3.0 h. The results show that (Fig. 3): Figure 1): the F value and the degree of hydrolysis reach the peak value when the temperature is 45-60℃; when the temperature is lower than 45℃, the enzyme activity is insufficient; when the temperature is 60℃, the enzyme is partially inactivated, and the degree of hydrolysis is slightly reduced, so the optimal temperature range is determined to be 45-55℃, and preferably 50℃.

[0008] pH optimization: 8.0, 9.0, 10.0, 11.0 and 12.0 are set as 5 gradients, and the remaining parameters are fixed as whey protein mass concentration 6%, enzyme and substrate mass ratio 1:25, enzyme hydrolysis at 50℃ for 3.0 h. The results show that (Fig. 2): Figure 1 ): the F value reaches 4.25 (the highest) at pH 12.0, but the degree of hydrolysis is 9.21%, and the hydrolysis efficiency is obviously reduced; considering the reduction of alkali dosage, the improvement of hydrolysis efficiency and the cost saving, the optimal pH range is determined to be 8.0-11.0, and preferably 9.0.

[0009] Enzyme and substrate mass ratio optimization: 1:12.5, 1:25, 1:50, 1:100 and 1:150 are set as 5 gradients, and the remaining parameters are fixed as whey protein mass concentration 6%, pH 9.0, enzyme hydrolysis at 50℃ for 3.0 h. The results show that (Fig. 3): Figure 1 ): when the enzyme and substrate mass ratio is 1:12.5-1:50, the F value and the degree of hydrolysis are optimal; when the enzyme and substrate mass ratio is 1:12.5, the enzyme dosage is too high, and the cost is increased; when the enzyme and substrate mass ratio is 1:100, the enzyme dosage is insufficient (F=3.91, degree of hydrolysis=12.52%); therefore, the optimal enzyme and substrate mass ratio range is determined to be 1:12.5-1:50, and preferably 1:25.

[0010] After the enzyme hydrolysis is completed, high-temperature enzyme inactivation is carried out at 85-95℃ for 10-15 min, and then the temperature is cooled to room temperature.

[0011] (2) The second step, chymotrypsin hydrolysis: in the enzyme hydrolysis solution after the enzyme inactivation in the first step, the pH is adjusted to 8.0-9.0 (preferably 8.5) by using 1 mol / L NaOH solution, chymotrypsin is added according to the enzyme and substrate mass ratio of 1:50-1:100 (preferably 1:50), and then the enzyme hydrolysis is carried out in a constant-temperature water bath at 35-45℃ (preferably 40℃) for 4.0-5.0 h (preferably 4.5 h); after the enzyme hydrolysis is completed, high-temperature enzyme inactivation is repeated at 85-95℃ for 10-15 min, and then the temperature is cooled to room temperature. In this step, the selective cleavage of the carboxyl terminal peptide bond of aromatic amino acids by chymotrypsin reduces the content of AAA in the system, and further degrades the macromolecular peptides, so that the degree of hydrolysis is increased to 27%-28%.

[0012] In order to determine the optimal hydrolysis parameters of chymotrypsin, the present application carries out a plurality of single-factor optimization experiments: Time optimization: 5 gradients of 3.0, 3.5, 4.0, 4.5 and 5.0 h were set, and the remaining parameters were fixed as pH 8.5, 40℃, enzyme to substrate mass ratio 1:50. The results showed that (Table 1): Figure 2 ): F value reached 4.42 and the degree of hydrolysis reached 22.79% at 4.5 h, both of which were the highest; F value and the degree of hydrolysis were reduced after more than 4.5 h, so the time range was determined as 4.0-5.0 h, and 4.5 h was preferred.

[0013] Temperature optimization: 5 gradients of 30, 35, 40, 45 and 50℃ were set, and the remaining parameters were fixed as pH 8.5, enzyme to substrate mass ratio 1:50, and enzymolysis for 4.5 h. The results showed that (Table 2): Figure 2 ): F value (4.89) and the degree of hydrolysis (23.39%) reached the peak at 40℃; the enzymolysis efficiency was low at 30℃ (F=4.73, degree of hydrolysis 13.28%), and F value was significantly reduced at 50℃, so the temperature range was determined as 35-45℃, and 40℃ was preferred.

[0014] pH optimization: 5 gradients of 7.5, 8.0, 8.5, 9.0 and 9.5 were set, and the remaining parameters were fixed as enzyme to substrate mass ratio 1:50, 40℃, and enzymolysis for 4.5 h. The results showed that (Table 3): Figure 2 ): F value reached 4.09 (the highest) and the degree of hydrolysis was 20.30% at pH 8.5; enzyme activity was inhibited when pH was lower than 8.0, and AAA cutting specificity decreased when pH was higher than 9.0, and F value was significantly reduced, so the optimal pH range was determined as 8.0-9.0, and 8.5 was preferred.

[0015] Enzyme to substrate mass ratio optimization: 5 gradients of 1:25, 1:50, 1:75, 1:100 and 1:125 were set, and the remaining parameters were fixed as pH 8.5, 40℃, and enzymolysis for 4.5 h. The results showed that (Table 4): Figure 2 ): F value was 4.83 and the degree of hydrolysis was 27.72% when enzyme to substrate mass ratio was 1:50; F value and the degree of hydrolysis were significantly reduced when enzyme dosage was increased at 1:25; F value was reduced to 4.05 when enzyme dosage was insufficient at 1:125, so the enzyme to substrate mass ratio range was determined as 1:50-1:100, and 1:50 was preferred.

[0016] After enzymolysis, high-temperature enzyme inactivation at 85-95℃ for 10-15 min was repeated, and then cooled to room temperature.

[0017] (3) Third step, flavor protease hydrolysis: Adjust the pH of the enzymatic hydrolysate after enzyme inactivation in the second step to 6.0-7.0 (preferably 6.0) with 6 mol / L HCl solution, add flavor protease at an enzyme-to-substrate mass ratio of 1:12.5-1:50 (preferably 1:25), and incubate in a constant temperature water bath at 50-60℃ (preferably 55℃) for 3.0-5.0 h (preferably 4.0 h); after enzymatic hydrolysis, inactivate the enzyme again at 85-95℃ for 10-15 min, and cool to room temperature. This step utilizes the exopeptidase activity of flavor protease to remove the free amino acids (especially some AAA) at the ends of the peptide chain, and further degrades the peptide chain into oligopeptides of 2-9 amino acid residues, with a final degree of hydrolysis of over 32%.

[0018] To determine the optimal parameters for flavor protease, this invention conducted multiple sets of single-factor optimization experiments: Time optimization: Five time gradients were set: 1.0, 2.0, 3.0, 4.0, and 5.0 h. Other parameters were fixed at pH 6.0, 55℃, and an enzyme-to-substrate mass ratio of 1:25. Results showed ( Figure 3 At 4.0 h, the F value reached 4.22 and the degree of hydrolysis reached 27.86%, both reaching their highest levels. At 1.0 and 2.0 h, the hydrolysis time was insufficient, resulting in lower F values ​​and degrees of hydrolysis. After 4.0 h, the F value decreased slightly. Therefore, the time range was determined to be 3.0-5.0 h, with 4.0 h being the preferred time.

[0019] Temperature optimization: Five temperature gradients were set: 45, 50, 55, 60, and 65℃. Other parameters were fixed at pH 6.0, enzyme to substrate mass ratio of 1:25, and hydrolysis time of 4.0 h. Results showed ( Figure 3 At 55℃, the F value reaches 4.58 and the degree of hydrolysis is 27.83%; at 45℃, the hydrolysis efficiency is low (F=4.01, degree of hydrolysis=15.29%), and at 65℃, the peptide chain is excessively degraded (F=4.62, degree of hydrolysis=19.65%). Therefore, the temperature range is determined to be 50-60℃, with 55℃ being the preferred temperature.

[0020] pH optimization: Five pH gradients were set: 5.5, 6.0, 6.5, 7.0, and 7.5. Other parameters were fixed at an enzyme-to-substrate mass ratio of 1:25, 55℃, and hydrolysis time of 4.0 h. Results showed ( Figure 3 At pH 6.0, the F value is 5.09 and the degree of hydrolysis is 31.00%; at pH 5.5, the enzymatic hydrolysis is insufficient (F=4.60, degree of hydrolysis=26.38%); at pH 7.5, the exopeptidase activity decreases and the degree of hydrolysis decreases. Therefore, the pH range is determined to be 6.0-7.0, with 6.0 being the preferred value.

[0021] Enzyme to substrate mass ratio optimization: 5 gradients of 1:12.5, 1:25, 1:50, 1:75 and 1:100 were set, and the remaining parameters were fixed at pH 6.0, 55°C, enzymolysis for 4.0 h. The results showed that (Figs. 1 and 2): Figure 3 ): when the enzyme to substrate mass ratio was 1:25, the F value and the degree of hydrolysis were the highest, being 4.76 and 31.59%, respectively; when the enzyme to substrate mass ratio was 1:75 and 1:100, the F value and the degree of hydrolysis were significantly reduced, so the enzyme to substrate mass ratio range was determined to be 1:12.5-1:50, and 1:25 was preferred.

[0022] After the enzymolysis was completed, the enzyme was inactivated again at a high temperature of 85-95°C for 10-15 min, and then cooled to room temperature.

[0023] 2. Activated carbon de-aromatization process (1) Pretreatment: the enzyme inactivation solution after the third step was adjusted to pH 2.5-3.5 (preferably 3.0) with 6 mol / L HCl solution, and was ready for use; (2) Adsorption de-aromatization: 200 mesh powdered activated carbon was added to the above enzyme solution at a carbon to liquid mass ratio of 1:5-1:15 (preferably 1:10), and the mixture was placed in a constant temperature water bath at 40-50°C (preferably 50°C) under magnetic stirring for adsorption for 55-65 min (preferably 60 min); (3) Separation and purification: after the adsorption was completed, the mixture was centrifuged in a centrifuge at a speed of 8000 r / min for 10 min, and the supernatant was collected; after filtration through qualitative filter paper, a high F value oligopeptide solution was obtained.

[0024] 3. Key index determination Degree of hydrolysis (DH) determination: O-phthalaldehyde (OPA) method was used to determine the degree of hydrolysis of the enzyme solution, with L-serine as the standard. 0.4 mL of the sample to be analyzed was mixed with 3 mL of OPA reagent (containing 3.81 g of disodium tetraborate decahydrate, 100 mg of SDS, 80 mg of DTT, 80 mg of OPA and 4 mL of ethanol, and then made up to 100 mL with distilled water) and uniformly mixed, and then placed at room temperature for 2 min, and then the absorbance was measured at 340 nm. The absorbance of a 0.1 mg / mL serine standard solution was also measured in the same way. The calculation formula of the degree of hydrolysis is as follows: Degree of hydrolysis (DH) = h / htot x 100% h = (serine-NH2 - 1) / 0.4 serine-NH2 = 0.951 x 0.3 x 100 x (OD 样 - OD 空 ) / XP (OD 标 - OD 空 ) In the formula, h: the number of free amino groups released by enzymatic hydrolysis; htot: the total free amino groups of the raw material measured by acid hydrolysis, which is 8.8 mmol / g; X: the mass (g) of the sample; P: the protein content (%) in the sample.

[0025] F value determination: UV spectrophotometry was used to determine the absorbance at 220 nm (BCAA characteristic absorption) and 280 nm (AAA characteristic absorption), and the F value was obtained by calculating the absorbance ratio (OD 220 / OD 280 ). Beneficial effects

[0026] 1. Significant improvement in enzymatic efficiency: Through step-by-step synergistic enzymatic hydrolysis of alkaline protease-chymotrypsin-flavor protease, the substrate specificity advantages of each enzyme are fully utilized, and the final degree of hydrolysis reaches 32.22%, which is more than 40% higher than single enzyme hydrolysis, and the yield of target oligopeptide (2-9 amino acid residues) is significantly improved. 2. Stable F value meets standards: The optimized activated carbon de-aromatization process (50°C, pH 3.0, carbon liquid ratio 1:10, adsorption for 60 min) can efficiently remove AAA, making the product F value reach 26.46, which is much higher than the industry standard (F>20) for high F value oligopeptide. 3. Excellent process economy: Conventional enzyme preparations and powdered activated carbon are used, without the need for complex chromatography equipment, and the operation steps are simple; at the same time, whey protein (dairy by-product) is converted into high-value, reducing raw material costs, and suitable for large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a single-factor parameter optimization diagram for the first step (alkaline protease) enzymatic hydrolysis of whey protein; Figure 2 is a single-factor parameter optimization diagram for the second step (chymotrypsin) enzymatic hydrolysis of whey protein; Figure 3 is a single-factor parameter optimization diagram for the third step (flavor protease) enzymatic hydrolysis of whey protein. DETAILED DESCRIPTION Example 1

[0028] This example provides a method for preparing a high F value oligopeptide from whey protein, the specific operation is as follows: 1. Preparation of whey protein solution: weigh 12.0 g of whey protein and dissolve it in 200 mL of ultrapure water to prepare a whey protein solution with a mass concentration of 6%; 2. First step of enzymolysis (alkaline protease): adjust the pH of the above solution to 9.0 with 1 mol / L NaOH solution, add 0.48 g of alkaline protease according to the mass ratio of enzyme to substrate 1:25, and place in a 50°C constant temperature water bath for enzymolysis for 3.0 h; after the enzymolysis is completed, high-temperature enzyme inactivation at 90°C for 12 min, and cool to room temperature; 3. Second step of enzymolysis (chymotrypsin): adjust the pH of the first step of enzymolysis solution to 8.5 with 1 mol / L NaOH solution, add 0.24 g of chymotrypsin according to the mass ratio of enzyme to substrate 1:50, and place in a 40°C constant temperature water bath for enzymolysis for 4.5 h; after the enzymolysis is completed, high-temperature enzyme inactivation at 90°C for 12 min, and cool to room temperature; 4. Third step of enzymolysis (flavor protease): adjust the pH of the second step of enzymolysis solution to 6.0 with 6 mol / L HCl solution, add 0.48 g of flavor protease according to the mass ratio of enzyme to substrate 1:25, and place in a 55°C constant temperature water bath for enzymolysis for 4.0 h; after the enzymolysis is completed, high-temperature enzyme inactivation at 90°C for 12 min, and cool to room temperature; 5. Activated carbon de-aromatization: adjust the pH of the third step of enzymolysis solution to 3.0 with 6 mol / L HCl solution, add 7.5 g of 200 mesh powdered activated carbon according to the mass ratio of carbon solution 1:10, and place in a 50°C constant temperature water bath for magnetic stirring adsorption for 60 min; after the adsorption is completed, centrifuge at 8000 r / min for 10 min, collect the upper clear liquid and filter; 6. Index determination: the degree of hydrolysis is 32.20% determined by OPA method; the F value of the enzymolysis solution is 26.46 determined by ultraviolet spectrophotometry, meeting the requirement of high F value oligopeptide. It is especially suitable for high-end special medical use food (such as hepatic encephalopathy auxiliary nutritional agent) scene, and such products usually require F value > 25. Example 2

[0029] The embodiment provides a preparation method of high F value oligopeptide from whey protein source, and the specific operation is as follows: 1. Preparation of whey protein solution: weigh 10.0 g of whey protein, dissolve in 200 mL of ultrapure water, and prepare a whey protein solution with a mass-volume concentration of 5%.

[0030] 2. First step of enzymolysis (alkaline protease): adjust the pH to 9.0, add 0.32 g of alkaline protease according to the mass ratio of enzyme to substrate 1:25, and perform enzymolysis at 50°C for 3.0 h, and inactivate the enzyme; 3. Second step of enzymolysis (chymotrypsin): adjust the pH to 8.5, add 0.16 g of chymotrypsin according to the mass ratio of enzyme to substrate 1:50, and perform enzymolysis at 40°C for 4.5 h, and inactivate the enzyme; 4. Third step of enzymolysis (flavour protease): adjust pH to 6.0, add 0.32 g of flavour protease with a mass ratio of enzyme to substrate of 1:25, and perform enzymolysis at 55°C for 4.0 h, and then inactivate the enzyme; 5. Activated carbon de-aromatization: the same de-aromatization parameters as in Example 1 (200-mesh activated carbon, pH 3.0, 50°C, mass ratio of carbon to liquid 1:10, adsorption for 60 min) were adopted, and the F value was measured to be 24.83 and the degree of hydrolysis was 30.12% after centrifugal filtration.

[0031] 6. Result analysis: this process is suitable for low-concentration whey protein raw materials, and although the final degree of hydrolysis is slightly lower than that in Example 1, the F value is still stably higher than 20, the viscosity of the enzymatic hydrolysate is low, the subsequent filtration efficiency is improved, and it is suitable for high-flowability functional beverage scenarios. Example 3

[0032] This example provides a preparation method of whey protein source high-F-value oligopeptide, and the specific operation is as follows: 1. First step of enzymolysis: refer to step 2 of Example 1; 2. Second step of enzymolysis: adjust pH to 8.5, add 0.16 g of chymotrypsin (0.24 g in Example 1) with a mass ratio of enzyme to substrate of 1:75, and perform enzymolysis at 40°C for 4.5 h, and then inactivate the enzyme; 3. Third step of enzymolysis: refer to step 4 of Example 1, add 0.48 g of flavour protease with a mass ratio of enzyme to substrate of 1:25, and perform enzymolysis at 55°C for 4.0 h, and then inactivate the enzyme; 4. Activated carbon de-aromatization: refer to step 5 of Example 1, and the F value is measured to be 24.12 after centrifugal filtration.

[0033] 5. Result analysis: the dosage of chymotrypsin is reduced to a mass ratio of enzyme to substrate of 1:75, and the product F value is still as high as 24.12, which not only ensures the efficiency of aromatic amino acid peptide bond cleavage and high F value of the product, but also directly reduces the cost of enzymes and indirectly reduces the cost of subsequent processes, thereby achieving cost reduction and efficiency increase. Example 4

[0034] This example provides a preparation method of whey protein source high-F-value oligopeptide, and the specific operation is as follows: 1. Enzymolysis stage: refer to steps 1-4 of Example 1 completely to prepare the third step of enzymolysis (degree of hydrolysis 32.20%, F value 5.13).

[0035] 2. Activated carbon de-aromatization: adjust the pH of the enzymolysis liquid to 3.0, add 7.5 g of 200-mesh powdered activated carbon with a mass ratio of carbon to liquid of 1:10, set the adsorption temperature to 45°C, the adsorption time to 60 min, and the other conditions to be consistent with those in Example 1.

[0036] 3. Index determination: F value = 23.15 after 45°C adsorption.

[0037] 4. Result analysis: under the condition of adsorption temperature reduced to 45℃, the F value of the product treated by the above activated carbon still reached 23.15, which was significantly higher than the benchmark requirement of 20. This showed that the activated carbon still had good de-aromatic amino acid efficiency under a relatively milder adsorption temperature, and the process operation window was wide, which was beneficial to reduce energy consumption and control production cost. Example 5

[0038] This embodiment provides a method for preparing high F value oligopeptide from whey protein source, the specific operation is as follows: 1. Enzymolysis stage: completely refer to steps 1-4 of example 1 to prepare the third step enzyme solution (hydrolysis degree 32.20%, F value 5.13).

[0039] 2. Activated carbon de-aromatic: adjust pH to 3.0, add 200 mesh activated carbon (carbon liquid mass ratio 1:10), set adsorption time for 50 min, 60 min and 70 min respectively, adsorption temperature 40℃ (lower than 50℃ of example 1), other conditions remain unchanged.

[0040] 3. Index determination: 50 min adsorption, F value = 21.37; 60 min adsorption, F value = 22.89; 70 min adsorption, F value = 23.05.

[0041] 4. Result analysis: although the F value is lower at 40℃ low temperature adsorption (22.89 vs 26.46), it still meets the standard of F>20, and can reduce the oxidation loss of oligopeptide at high temperature; extend the adsorption time to 70 min, the F value only increases by 0.16, so the optimal low temperature process is 40℃, 60 min, which is suitable for products with high requirements for the retention of heat-sensitive components (such as lactoferrin remaining in whey protein). Example 6

[0042] This embodiment provides a method for preparing high F value oligopeptide from whey protein source, the specific operation is as follows: 1. Preparation of whey protein solution: weigh 600 g of whey protein, dissolve in 10 L of ultrapure water, use an industrial grade stirring tank (rotating speed 200 r / min) to stir for 45 min until dissolved, mass volume concentration 6%.

[0043] 2. Stepwise enzymolysis: First step: adjust pH to 9.0, add 24 g of alkaline protease (enzyme to substrate mass ratio 1:25), 50℃ stirring enzymolysis for 3.0 h, 90℃ pump into plate heat exchanger to inactivate enzyme; Second step: After cooling, adjust pH to 8.5, add 12 g chymotrypsin (enzyme to substrate mass ratio 1:50), stir for 4.5 h at 40℃, and use a plate heat exchanger to inactivate the enzyme; Third step: adjust pH to 6.0, add 24 g flavor protease (enzyme to substrate mass ratio 1:25), stir for 4.0 h at 55℃, and use a plate heat exchanger to inactivate the enzyme; Take a sample to determine the degree of hydrolysis of the enzyme solution, which is 32.15%, and the F value is 5.10.

[0044] 3. Activated carbon de-aromatization: adjust pH to 3.0, add 375 g Guangdong Shunye activated carbon (carbon liquid mass ratio 1:10), stir for 60 min at 50℃, separate using an industrial disc centrifuge (8000 r / min), and then filter using a plate and frame filter (filter cloth pore size 1 μm).

[0045] 4. Index verification: the F value of the final product is 26.32, and the degree of hydrolysis is 31.98%, which is less than 0.6% different from the laboratory test (Example 1), proving that the process can be stably scaled up to industrial production scale, and the equipment adaptability is good. Comparative Example 1

[0046] This comparative example uses a single enzyme hydrolysis process, and only alkaline protease is used in the entire enzyme hydrolysis process.

[0047] 1. Process steps: (1) Prepare a whey protein solution with a mass-volume concentration of 6%; (2) Single enzyme hydrolysis using alkaline protease: pH 9.0, enzyme to substrate mass ratio 1:25, enzyme hydrolysis for 10.5 h at 50℃ (total enzyme hydrolysis time is the same as Example 1); (3) Inactivate the enzyme at 85℃ for 15 min, and then cool and treat according to the de-aromatization parameters of Example 1 (200 mesh activated carbon, pH 3.0, 50℃, carbon liquid mass ratio 1:10, 60 min).

[0048] 2. Results: (1) Degree of hydrolysis: 18.76% (only 58.2% of Example 1); (2) F value: 12.35 (not reaching the high F value oligopeptide standard).

[0049] 3. Comparative analysis: single enzyme hydrolysis cannot fully destroy the three-dimensional structure of whey protein, resulting in low degree of hydrolysis and insufficient release of aromatic amino acids, even if the enzyme hydrolysis time is extended, it is difficult to reach the target F value, proving the necessity of three-step synergistic enzyme hydrolysis. Comparative Example 2

[0050] This comparative example uses a two-step enzyme hydrolysis process, which omits the chymotrypsin hydrolysis process compared to Example 1.

[0051] 1. Process steps: (1) Preparation of a whey protein solution with a mass / volume concentration of 6%; (2) Stepwise enzymatic hydrolysis: First step: same as the first step of Example 1 (alkaline protease, 3.0 h); Second step: directly proceed to the third step of Example 1 (flavourzyme, 4.0 h), total enzymatic hydrolysis time 7.0 h; (3) De- aromatic process same as Example 1.

[0052] 2. Result indicators: (1) Degree of hydrolysis: 25.32% (lower than 32.22% of Example 1); (2) F value: 18.79 (close to but not reaching the standard of F>20).

[0053] 3. Comparative analysis: The specific cleavage of the peptide bond at the carboxyl end of aromatic amino acids by chymotrypsin is the key to improving the F value. After omitting this step, even if the enzymatic hydrolysis time is extended, it is still not possible to effectively reduce the content of aromatic amino acids, proving that the synergistic effect of three-step enzymatic hydrolysis cannot be replaced. Comparative Example 3

[0054] This comparative example is compared with Example 1, and the order of enzymatic hydrolysis is reversed, first using flavourzyme for enzymatic hydrolysis, and finally using alkaline protease for enzymatic hydrolysis.

[0055] 1. Process steps: Preparation of a whey protein solution with a mass / volume concentration of 6%, the order of enzymatic hydrolysis is adjusted to: flavourzyme→ chymotrypsin→ alkaline protease; the enzymatic hydrolysis parameters (pH, temperature, enzyme / substrate ratio, time) remain unchanged, and the de-aromatic process is the same as Example 1.

[0056] 2. Result indicators: (1) Degree of hydrolysis: 22.15% (significant decrease in enzymatic efficiency); (2) F value: 15.87 (not reaching the standard).

[0057] 3. Comparative analysis: The order of enzymatic hydrolysis directly affects the efficiency of the gradual deconstruction of protein structure, reversing the order will result in the suppression of the activity of subsequent enzyme preparations, and the formation of an effective peptide chain cleavage gradient, proving the scientificity of the order of "alkaline protease→ chymotrypsin→ flavourzyme" in the present application. Comparative Example 4

[0058] This comparative example omits the de-aromatic treatment compared with Example 1.

[0059] 1. Process steps: (1) The enzymatic hydrolysis stage is completely the same as Example 1 (three-step enzymatic hydrolysis, degree of hydrolysis 32.20%); (2) omitting the activated carbon de-aromatization step, directly centrifugal filtration.

[0060] 2. Result index: F value: 5.13 (only 19.4% of Example 1).

[0061] 3. Comparative analysis: de-aromatization treatment is the core step to achieve high F value, and only through enzymolysis cannot increase F value to more than 20, and the product without de-aromatization has bitter taste problem, which is not suitable for food application. Summary of comparative examples

[0062] The above comparative examples compare the process of the present application from the aspects of enzymolysis mode (single / two-step vs. three-step), enzymolysis order, necessity of de-aromatization, etc. The results show that: 1. Three-step synergistic enzymolysis is the basis to ensure high degree of hydrolysis; 2. Specific enzymolysis order (alkaline protease -> chymotrypsin -> flavor protease) is the key to improve F value; 3. Omitting any step of key process (such as de-aromatization, specific enzyme species) cannot prepare high F value oligopeptide meeting the requirements.

[0063] These comparative data further verify the innovativeness and irreplaceability of the process of the present application, and highlight the decisive influence of synergistic effect of each step on product quality.

[0064] The applicant declares that the present application is illustrated by the above examples to explain the detailed method of the present application, but the present application is not limited to the above detailed method, i.e. it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific mode, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for preparing high-F-value oligopeptides from whey protein, characterized in that, Includes the following steps: 1) Prepare a whey protein solution with a mass-volume concentration of 5%-7%; 2) The whey protein solution was subjected to a first-step enzymatic hydrolysis using alkaline protease. The conditions for the first-step enzymatic hydrolysis were: pH 8.0-11.0, enzyme to substrate mass ratio 1:12.5-1:50, temperature 45-55℃, and hydrolysis time 2.0-3.0 h. 3) Use chymotrypsin to perform a second enzymatic hydrolysis on the first step hydrolysate. The conditions for the second enzymatic hydrolysis are: pH 8.0-9.0, enzyme to substrate mass ratio 1:50-1:100, temperature 35-45℃, and hydrolysis time 4.0-5.0 h. 4) The second-step enzymatic hydrolysate was subjected to a third enzymatic hydrolysis using flavor protease. The conditions for the third enzymatic hydrolysis were: pH 6.0-7.0, enzyme to substrate mass ratio 1:12.5-1:50, temperature 50-60℃, and hydrolysis time 3.0-5.0 h. 5) The enzymatic hydrolysate from the third step is subjected to dearomatization treatment using activated carbon. The dearomatization treatment conditions are: pH 2.5-3.5, carbon-liquid mass ratio 1:5-1:15, temperature 40-50℃, and adsorption time 55-65 min.

2. The method according to claim 1, characterized in that, In step 2), the pH of the first enzymatic hydrolysis is 9.0, the enzyme to substrate mass ratio is 1:25, the temperature is 50℃, and the hydrolysis time is 3.0 h.

3. The method according to claim 1, characterized in that, In step 3), the pH of the second enzymatic hydrolysis is 8.5, the enzyme to substrate mass ratio is 1:50, the temperature is 40℃, and the hydrolysis time is 4.5 h.

4. The method according to claim 1, characterized in that, In step 4), the pH of the third enzymatic hydrolysis is 6.0, the enzyme to substrate mass ratio is 1:25, the temperature is 55℃, and the hydrolysis time is 4.0 h.

5. The method according to claim 1, characterized in that, In step 5), the activated carbon is 200-mesh powdered activated carbon, the pH of the dearomatization treatment is 3.0, the carbon-liquid mass ratio is 1:10, the temperature is 50℃, and the adsorption time is 60 min.

6. The method according to claim 1, characterized in that, In steps 2), 3), and 4), after enzymatic hydrolysis, enzymes are inactivated by high-temperature treatment at 85-95℃ for 10-15 minutes.

7. The method according to claim 1, characterized in that, In step 5), after the aromatic removal treatment, there are also centrifugation and filtration steps, wherein the centrifugation speed is 8000 r / min and the time is 10 min.

8. The method according to claim 1, characterized in that, The prepared high-F-value oligopeptides have a degree of hydrolysis ≥30% and an F-value ≥26.

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