Preparation method of highland barley protein peptide with hypoglycemic activity

Abstract

The invention discloses a preparation method of highland barley protein peptide with hypoglycemic activity, and belongs to the field of biotechnology and functional food. According to the preparation method, highland barley protein is extracted from highland barley powder by adopting an alkali-solution and acid-isolation method, then the highland barley protein peptide is prepared by sequentially adopting alkaline protease and bromelain to carry out step-by-step enzymolysis, and the complementary and synergistic effects of different restriction enzyme cutting sites are realized through a step-by-step enzymolysis strategy of'alkaline protease-bromelain 'in a specific sequence. Aromatic and hydrophobic amino acids are preferentially cut by the alkaline protease, the space structure of the protein is opened, and more effective sites are provided for bromelain, so that small molecular peptide fragments with hypoglycemic activity are more efficiently released, and in-vitro experiments show that the inhibition rate of the bromelain on alpha-amylase is up to 52.49%, and the inhibition rate of the bromelain on the bromelain is up to 52.49%. The inhibition rate of alpha-glucosidase reaches up to 73.35%, which is far better than that of a single enzymolysis product.

Description

Technical Field

[0001] This invention relates to a method for preparing barley protein peptides with hypoglycemic activity, belonging to the field of biotechnology and functional foods. Background Technology

[0002] Diabetes is a major global chronic metabolic disease, and existing drugs suffer from side effects and poor patient adherence. Natural bioactive peptides, due to their wide availability, high safety profile, and diverse bioactivities, have become a hot topic in diabetes intervention research. Highland barley, a specialty crop of the Qinghai-Tibet Plateau, is rich in protein resources, especially essential amino acids, making it an excellent raw material for preparing bioactive peptides.

[0003] Currently, there are reports on the preparation of bioactive peptides from various plant proteins using enzymatic hydrolysis. However, research on barley protein is still in its early stages. Existing techniques mostly employ single proteases for hydrolysis, resulting in low hydrolysis efficiency, high product heterogeneity, and unsatisfactory yields of target bioactive peptides. In particular, effective technical solutions are still lacking for how to efficiently release and enrich barley protein peptides with synergistic inhibitory activities against multiple key hypoglycemic enzymes such as α-glucosidase and α-amylase through enzymatic hydrolysis.

[0004] Therefore, developing a method for the efficient and targeted preparation of highly active barley protein peptides has significant application value and market prospects. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for preparing barley protein peptides with hypoglycemic activity. The method involves extracting barley protein from barley flour using an alkali-soluble acid precipitation method, followed by stepwise enzymatic hydrolysis with alkaline protease and bromelain to obtain the barley protein peptides. The barley protein peptides prepared by this invention exhibit excellent comprehensive hypoglycemic activity.

[0006] A method for preparing barley protein peptides with hypoglycemic activity includes the following steps: (1) Preparation of barley protein raw material: Barley protein was extracted from barley flour using the alkali dissolution and acid precipitation method. Specifically, barley flour was first mixed with water, and 3000 mL of deionized water was added to the barley flour at a material-to-liquid ratio of 1:10 (w / v, g / mL). The pH of the system was adjusted to 11.0 with alkali solution to fully dissolve the barley protein. Then, insoluble impurities were removed by centrifugation, and the supernatant was collected. Subsequently, the pH of the supernatant was adjusted to the isoelectric point of barley protein (pH 4.5) with acid solution to cause protein aggregation and precipitation. Finally, after centrifugation, washing, and drying, high-purity barley protein was obtained. (2) Stepwise enzymatic hydrolysis: Barley protein is mixed with deionized water to prepare a solution, and then stepwise enzymatic hydrolysis is carried out using alkaline protease and bromelain in sequence; Step 1 Enzymatic hydrolysis: Under the conditions of pH 9.5-10.5 and temperature 40-50℃, add alkaline protease at an enzyme dosage of 1500-2500 U / g protein and react for 60-120 minutes. The second step of enzymatic hydrolysis: Adjust the pH of the first step enzymatic hydrolysate to 6.5-7.5, add bromelain at a rate of 1500-2500 U / g protein at a temperature of 40-50℃, and react for 90-150 minutes. (3) Post-processing: After the enzymatic hydrolysis is completed, the enzyme inactivation solution is performed, centrifuged, the supernatant is collected, and the barley protein peptide is obtained after drying.

[0007] In one embodiment of the present invention, in step (2), the conditions for the first enzymatic hydrolysis are: pH 10.0, temperature 45°C, enzyme dosage 2000 U / g protein, and reaction time 90 minutes.

[0008] In one embodiment of the present invention, in step (2), the conditions for the second enzymatic hydrolysis are: pH 7.0, temperature 45°C, enzyme dosage 2000 U / g protein, and reaction time 120 minutes.

[0009] This invention provides barley protein peptides prepared by the method described above.

[0010] This invention provides the application of the above-described highland barley protein peptide in the preparation of functional foods, special medical purpose formula foods, or nutritional supplements with the function of assisting in lowering blood sugar.

[0011] Compared with the prior art, the present invention has the following significant advantages: 1. Synergistic Effect: Through a stepwise enzymatic digestion strategy of "alkaline protease → bromelain" in a specific sequence, complementary and synergistic effects are achieved at different cleavage sites. The alkaline protease preferentially cleaves aromatic and hydrophobic amino acids, opening up the protein's spatial structure and providing more actionable sites for bromelain, thereby releasing small molecule peptides with hypoglycemic activity more efficiently.

[0012] 2. Significantly Enhanced Activity: The barley protein peptides prepared in this invention exhibit excellent comprehensive hypoglycemic activity. In vitro experiments show that their inhibition rate against α-amylase is as high as 52.49%, and their inhibition rate against α-glucosidase is as high as 73.35%, which is far superior to single enzymatic hydrolysis products.

[0013] 3. Targeted and controllable process: By optimizing and combining key parameters such as enzyme type, sequence, pH, temperature, and time, the hydrolysis process can be precisely controlled, enabling the targeted enrichment of target active peptides, avoiding ineffective and excessive hydrolysis, and resulting in products with high activity and stability.

[0014] 4. Natural raw materials and broad application prospects: This invention uses natural barley protein as raw material, and the process is green and safe. The resulting product can be used to develop functional foods, special medical purpose formula foods, or nutritional supplements with auxiliary blood sugar lowering function. Attached Figure Description

[0015] Figure 1 This is a process flow diagram of the preparation method of the present invention.

[0016] Figure 2 A comparison of the hypoglycemic activities of hydrolysates obtained by different enzymatic hydrolysis methods.

[0017] Figure 3 The figure shows the results of single-factor optimization of alkaline protease hydrolysis conditions.

[0018] Figure 4 The figure shows the results of single-factor optimization of bromelain hydrolysis conditions. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0020] The sources of the raw materials used in this invention are as follows: The method for extracting barley protein described in this invention uses commercially available varieties of barley as raw material. As a specific embodiment, the barley used in this experiment was provided by Qinghai Tianyoude Technology Investment Management Group Co., Ltd., and the variety is "Kunlun 14". The scope of protection of this invention is not limited to this specific variety.

[0021] In the following examples, the enzyme preparation information is as follows: 1. The alkaline protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No. 9014-01-1, with an enzyme activity of ≥200 U / g.

[0022] 2. Bromelain was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No. 37189-34-7, with an enzyme activity of ≥300 U / g.

[0023] In the following examples, the hypoglycemic activity of barley protein peptides was evaluated by measuring their α-amylase inhibition rate and α-glucosidase inhibition rate. The specific measurement methods are as follows: Assay for α-amylase inhibitory activity: Mix 100 μL of sample solution (2.0 mg / mL) with 250 μL of α-amylase solution (10 U / mL), shake, add 50 μL of phosphate buffer (pH 7.0), and incubate at 37℃ for 10 min; then add 1 mL of 5% soluble starch solution, continue the reaction for 10 min, add 500 μL of DNS colorimetric solution, boil in a water bath for 5 min, cool, and measure the absorbance at 540 nm and calculate the inhibition rate.

[0024] The α-amylase inhibition rate is calculated according to Formula 1: α-Amylase inhibition rate (%) = [1 - (S - S0) / (C - C0)] × 100 (Formula 1) Formula 1: Sample group (S), blank sample group (S0, with buffer solution instead of enzyme solution), control group (C, with buffer solution instead of sample solution) and blank control group (C0, with buffer solution instead of sample solution and enzyme solution).

[0025] Assay for α-glucosidase inhibitory activity: Mix 100 μL of sample solution (2.0 mg / mL) with 250 μL of PNPG solution (3 mmol / L), shake, add 375 μL of phosphate buffer (pH 6.8), and incubate at 37℃ for 10 min; then add 125 μL of α-glucosidase solution (1 U / mL), continue the reaction for 10 min, measure the absorbance at 405 nm and calculate the inhibition rate.

[0026] The α-glucosidase inhibition rate is calculated according to Formula 2: α-glucosidase inhibition rate (%) = [1 - (S - S0) / (C - C0)] × 100 (Formula 2) Formula 2: Sample group (S), blank sample group (S0, with buffer solution instead of enzyme solution), control group (C, with buffer solution instead of sample solution) and blank control group (C0, with buffer solution instead of sample solution and enzyme solution).

[0027] Example 1 Extraction of barley protein Weigh 300 g of dried highland barley and intermittently grind it for 20 min using a small high-speed grinder. Pass the powder through an 80-mesh sieve to obtain highland barley flour. Add the highland barley flour to 3000 mL of deionized water at a material-to-liquid ratio of 1:10 (w / v). Adjust the pH of the system to 11.0 using 1.0 mol / L NaOH solution. Extract the powder by stirring at 40℃ for 2 h, followed by centrifugation at 4000 rpm for 15 min. Collect the supernatant, adjust the pH to 4.5 (isoelectric point) with 1.0 mol / L HCl, allow it to settle, centrifuge again, collect the precipitate, and freeze-dry it to obtain highland barley protein powder. Store the powder at -20℃ for later use.

[0028] Example 2 Preparation of barley protein peptides (1) Take 1 g of the barley protein prepared in Example 1 and add 10 mL of deionized water and mix well.

[0029] (2) Step 1: Alkaline protease hydrolysis: Adjust the pH of the protein solution to 10.0 with NaOH, preheat in a 45°C water bath, add alkaline protease at a rate of 2000 U / g protein, and react at a constant temperature of 45°C for 90 minutes, maintaining the pH at 10.0 with NaOH during the reaction.

[0030] (3) Second step: bromelain hydrolysis: After the first step reaction is completed, the pH of the hydrolysate is adjusted to 7.0 with HCl, and bromelain is added at a rate of 2000 U / g protein. The reaction is carried out at a constant temperature of 45℃ for 120 minutes, during which the pH is maintained at 7.0 with HCl.

[0031] (4) After the enzymatic hydrolysis is completed, place the reaction system in a boiling water bath for 10 minutes to inactivate the enzyme, then cool to room temperature, centrifuge at 4000 rpm for 15 minutes, and collect the supernatant.

[0032] (5) The supernatant was freeze-dried to obtain the white powder product of the present invention, which is barley protein peptide.

[0033] Example 3 Optimization of alkaline protease hydrolysis conditions The enzymatic hydrolysis conditions of alkaline protease were optimized using α-amylase inhibition rate and α-glucosidase inhibition rate as evaluation indicators.

[0034] (1) pH: The effect of pH 9.0-11.0 was investigated at a fixed temperature of 45℃, an enzyme dosage of 2000 U / g, and a time of 90 min. The results are as follows. Figure 3 (A) indicates that both inhibition rates reached their peak at pH 10.0.

[0035] (2) Temperature: With pH fixed at 10.0, enzyme dosage at 2000 U / g, and time at 90 min, the effect of temperature (30-50℃) was investigated. The results are as follows: Figure 3 (B) indicates that both inhibition rates reached their highest values ​​at 45°C.

[0036] (3) Enzyme dosage: With pH fixed at 10.0, temperature at 45℃, and time at 90 min, the effect of enzyme dosages ranging from 500 to 3000 U / g was investigated. Results are as follows... Figure 3 (C) indicates that the inhibition rate of α-amylase was the highest at 2000 U / g, and the inhibition rate of α-glucosidase also remained at a high level.

[0037] (4) Time: The effects of time (30-150 min) on pH 10.0, temperature 45℃, and enzyme dosage of 2000 U / g were investigated. Results are as follows: Figure 3 (D) indicates that both inhibition rates reached their peak at 90 min.

[0038] In summary, the optimal conditions for the first step of alkaline protease are determined to be: pH 10.0, temperature 45℃, enzyme dosage 2000 U / g, and reaction time 90 min.

[0039] Example 4 Optimization of enzymatic hydrolysis conditions for bromelain Based on the alkaline protease hydrolysis product from the first step, without intermediate purification, the system conditions were directly adjusted for the second step of enzymatic hydrolysis. The hydrolysis conditions of bromelain were optimized using single-factor optimization with α-amylase inhibition rate and α-glucosidase inhibition rate as evaluation indicators.

[0040] (1) pH: The effect of pH 5.0-8.0 was investigated at a fixed temperature of 45℃, an enzyme dosage of 2000 U / g, and a time of 120 min. The results are as follows. Figure 4 (A) indicates that both inhibition rates reached their peak at pH 7.0.

[0041] (2) Temperature: With pH fixed at 7.0, enzyme dosage at 2000 U / g, and time at 120 min, the effect of temperature (35-60℃) was investigated. The results are as follows: Figure 4 (B) indicates that both inhibition rates reached their highest values ​​at 45°C.

[0042] (3) Enzyme dosage: With pH 7.0, temperature 45℃, and time 120 min fixed, the effect of enzyme dosage (500-3000 U / g) was investigated. Results are as follows: Figure 4 (C) indicates that at 2000 U / g, both inhibition rates can be balanced and the effect is optimal.

[0043] (4) Time: The effects of pH 7.0, temperature 45℃, and enzyme dosage of 2000 U / g on the time of 30-180 min were investigated. Results are as follows: Figure 4 (D) indicates that the α-amylase inhibition rate peaked at 120 min, and the α-glucosidase inhibition rate showed a decreasing trend after 150 min. Therefore, 120 min was selected as the optimal reaction time.

[0044] In summary, the optimal conditions for the second step of bromelain production were determined to be: pH 7.0, temperature 45℃, enzyme dosage 2000 U / g, and reaction time 120 min.

[0045] Comparative Example 1 The order of stepwise enzymatic hydrolysis is changed (bromelain → alkaline protease). The preparation method is the same as in Example 2, except that: first, bromelain (pH 7.0, 45℃, 2h) is used for enzymatic hydrolysis, and then the pH is adjusted to 10.0 before enzymatic hydrolysis with alkaline protease (45℃, 2h).

[0046] Comparative Example 2 The preparation method is the same as in Example 2, except that the stepwise enzymatic hydrolysis in steps (2) and (3) is replaced by co-enzymatic hydrolysis of alkaline protease and bromelain (both at a dose of 2000 U / g) at pH 6.5 and 45°C for 4 h.

[0047] Comparative Example 3 The preparation method is the same as in Example 2, except that the stepwise enzymatic hydrolysis in steps (2) and (3) is replaced by co-enzymatic hydrolysis of alkaline protease and bromelain (both at a dose of 2000 U / g) at pH 8.5 and 45°C for 4 h.

[0048] Comparative Example 4 Single bromelain hydrolysis The preparation method is the same as in Example 2, except that step (2) is omitted and only bromelain is used to hydrolyze the enzyme at pH 7.0 and 45°C for 4 hours.

[0049] Comparative Example 5 Single alkaline protease hydrolysis The preparation method is the same as in Example 2, except that step (3) is omitted and only alkaline protease is used to hydrolyze the enzyme at pH 10.0 and 45°C for 4 hours.

[0050] Experimental Example: Assay for Hypoglycemic Activity The barley protein peptide samples prepared in Example 2 and Comparative Examples 1-5 were respectively prepared into 2 mg / mL solutions, and their α-amylase inhibitory activities and α-glucosidase inhibitory activities were measured. The results are shown in the table below: Table 1. α-Amylase inhibitory activity and α-glucosidase inhibitory activity of Examples 2 and Comparative Examples 1-5

[0051] Conclusion: As shown in the table above, the barley protein peptides prepared by the stepwise enzymatic hydrolysis method of "alkaline protease → bromelain" specific to this invention have significantly higher hypoglycemic activity than any product prepared by single enzymatic hydrolysis or by changing the enzymatic hydrolysis order, which fully demonstrates the synergistic effect and inventiveness of the technical solution of this invention.

[0052] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing barley protein peptides, characterized in that, Includes the following steps: (1) Preparation of barley protein raw materials: Barley protein was extracted from barley flour by alkaline dissolution and acid precipitation method; (2) Stepwise enzymatic hydrolysis: Barley protein is mixed with deionized water to prepare a solution, and then stepwise enzymatic hydrolysis is carried out using alkaline protease and bromelain in sequence; (3) Post-processing: After the enzymatic hydrolysis is completed, the enzyme inactivation solution is performed, centrifuged, and the supernatant is collected and dried to obtain the barley protein peptide.

2. The method according to claim 1, characterized in that, In step (1), the preparation method of barley protein includes: first, mixing barley flour with water, adjusting the pH of the system to 11.0 with alkaline solution to fully dissolve the barley protein; then removing insoluble impurities by centrifugation and collecting the supernatant; subsequently adjusting the pH of the supernatant to the isoelectric point of barley protein, pH 4.5, with acid solution to cause protein aggregation and precipitation; finally, after centrifugation, washing, and drying, barley protein is obtained.

3. The method according to claim 2, characterized in that, Add barley flour to deionized water at a ratio of 1:10, g / mL.

4. The method according to claim 1, characterized in that, In step (2), the stepwise enzymatic hydrolysis specifically includes: Step 1 enzymatic hydrolysis: Under the conditions of pH 9.5-10.5 and temperature 40-50℃, add alkaline protease at an enzyme dosage of 1500-2500 U / g protein and react for 60-120 minutes; Step 2 enzymatic hydrolysis: Adjust the pH of the first step enzymatic hydrolysate to 6.5-7.5, and under the conditions of temperature 40-50℃, add bromelain at an enzyme dosage of 1500-2500 U / g protein and react for 90-150 minutes.

5. The method according to claim 4, characterized in that, The conditions for the first step of enzymatic hydrolysis are: pH 10.0, temperature 45℃, enzyme dosage 2000 U / g protein, and reaction time 90 minutes.

6. The method according to claim 4, characterized in that, The conditions for the second step of enzymatic hydrolysis are: pH 7.0, temperature 45℃, enzyme dosage 2000 U / g protein, and reaction time 120 minutes.

7. The method according to claim 1, characterized in that, In step (2), the enzyme activity of alkaline protease is ≥ 200 U / g.

8. The method according to claim 1, characterized in that, In step (2), the enzyme activity of bromelain is ≥ 300 U / g.

9. Barley protein peptides prepared by any one of claims 1-8.

10. The use of the barley protein peptide according to claim 9 in the preparation of functional foods, special medical purpose formula foods or nutritional supplements with auxiliary hypoglycemic function.

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