A rye pollen peptide with antioxidant, anti-glycation, and α-amylase inhibitory activities, its preparation method, and functional products thereof.

Abstract

This invention relates to the technical field of deep processing of rye pollen, and particularly to a rye pollen peptide with antioxidant, anti-glycation, and α-amylase inhibitory activities, its preparation method, and functional products. The preparation method includes the following steps: mixing rye pollen with water, then sequentially adding cellulase and pectinase for pretreatment; after enzyme inactivation in the pretreatment solution, sequentially adding Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease for three hydrolysis cycles; and obtaining powdered rye pollen peptides after solid-liquid separation, filtration, and drying. The rye pollen peptides prepared by the method of this invention possess antioxidant, α-amylase inhibitory, and anti-glycation activities, and also have prebiotic effects that promote the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum*.

Description

Technical Field

[0001] This invention relates to the technical field of deep processing of rye pollen, and in particular to a rye pollen peptide with antioxidant, anti-glycation and α-amylase inhibitory activities, its preparation method, and functional products. Background Technology

[0002] Rye pollen, derived from rye (Secale Cereale L.), a plant belonging to the genus Secale of the Poaceae family, is processed through pollen harvesting, drying, and separation, and has become another new food resource based on pollen. Rye pollen is rich in various vitamins, such as B vitamins (e.g., vitamin B6 and vitamin B12), vitamin C, and vitamin E. It also provides several important minerals, such as zinc, copper, and selenium. Rye pollen contains antioxidants, such as bioflavonoids, carotenoids, and vitamin E, which help fight free radical damage and maintain cell health.

[0003] There is currently a lot of research on rye pollen extract, mainly focusing on its pharmacological functions. For example, the article "Pollenextract improves pain in men with CP / CPPS" cites a study showing that rye pollen extract has a good effect on men with chronic prostatitis / chronic pelvic pain syndrome.

[0004] Meanwhile, pollen peptides have become a research hotspot in recent years. As the reproductive cells of plants, pollen contains a large amount of vitamins, antioxidants and proteins. Without destroying the original nutritional value of pollen, pollen proteins can be broken down into peptides and amino acids through enzymatic hydrolysis technology, which facilitates human absorption and digestion while improving its antioxidant properties and other functional activities.

[0005] Current research on rye pollen mainly focuses on its direct preparation via drying, with few reports on its protein processing. Consequently, there are few reports on the preparation of rye pollen peptides with high antioxidant activity using multiple enzymes in combination. It is clear that existing literature and patents do not report on the preparation of rye pollen peptides with antioxidant properties, α-amylase inhibitory activity, anti-glycation activity, and prebiotic effects that promote the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum*. Therefore, the preparation of multifunctional rye pollen peptides from rye pollen has significant theoretical and practical value. Summary of the Invention

[0006] To address the shortcomings of the prior art mentioned in the background section, this invention provides a method for preparing rye pollen peptides, the technical solution of which is as follows:

[0007] The preparation method of this rye pollen peptide includes the following steps:

[0008] Rye pollen was mixed evenly with water, and cellulase was added for the first pretreatment, followed by the first enzyme inactivation treatment to obtain the first pretreated solution.

[0009] Pectinase was added to the first pretreatment solution for a second pretreatment, followed by a second enzyme inactivation treatment to obtain the second pretreatment solution.

[0010] In the second pretreatment solution, Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease were added sequentially for three-step hydrolysis, followed by enzyme inactivation treatment to obtain the hydrolysate.

[0011] The hydrolysate was subjected to solid-liquid separation to obtain a filtrate containing rye pollen peptides;

[0012] The filtrate is filtered to retain rye pollen peptides, thus obtaining the rye pollen peptides.

[0013] In the first pretreatment process, the mass ratio of rye pollen to water is 1:10 to 1:20; the amount of cellulase added is 1% to 1.5% of the mass of the rye pollen.

[0014] The temperature of the first pretreatment is 40℃~45℃, the pH of the reaction system is 4.5~5.0, and the reaction time is 0.5~1.5h; the temperature of the first enzyme inactivation treatment is 85℃~90℃, and the enzyme inactivation treatment time is 15~20min.

[0015] In the second pretreatment process, the amount of pectinase added is 1% to 1.5% of the mass of the rye pollen; the temperature of the second pretreatment is 50℃ to 55℃, the pH of the reaction system is 3.5 to 4.5, and the reaction time is 0.5 to 1.5 h; the temperature of the second enzyme inactivation treatment is 85℃ to 90℃, and the enzyme inactivation treatment time is 15 to 20 min.

[0016] The amount of Bacillus licheniformis alkaline protease added is 1% to 1.5% of the mass of the rye pollen; the amount of papain added is 0.5% to 0.8% of the mass of the rye pollen; and the amount of Bacillus subtilis neutral protease added is 1% to 1.5% of the mass of the rye pollen.

[0017] A first hydrolysis was performed by adding Bacillus licheniformis alkaline protease to the second pretreatment solution. The temperature of the first hydrolysis was 50℃~55℃, the pH of the reaction system was 8.5~9.0, and the hydrolysis time was 1~3h. Then, the enzyme was inactivated at 85℃~90℃ for 15~20min to obtain the first hydrolysate. A second hydrolysis was performed by adding papain to the first hydrolysate. The temperature of the second hydrolysis was 50℃~55℃, the pH of the reaction system was 6.5~7.0, and the hydrolysis time was 0.5~1.5h. Then, the enzyme was inactivated at 85℃~90℃ for 15~20min to obtain the second hydrolysate. A third hydrolysis was performed by adding Bacillus subtilis neutral protease to the second hydrolysate. The temperature of the third hydrolysis was 50℃~55℃, the pH of the reaction system was 6.5~7.0, and the hydrolysis time was 0.5~1.5h. Then, the enzyme was inactivated at 85℃~90℃ for 15~20min to obtain the third hydrolysate.

[0018] The hydrolysate obtained from the three hydrolysis processes is centrifuged to separate solids and liquids, resulting in a filtrate containing rye pollen peptides. The filtrate is then subjected to ultrafiltration to retain rye pollen peptides with a molecular weight of less than 5000 Da, followed by drying to obtain the rye pollen peptides.

[0019] In one embodiment, spray drying is used to obtain the rye pollen peptide in powder form.

[0020] The present invention also provides a rye pollen peptide, which is prepared by the rye pollen peptide preparation method described above.

[0021] The present invention also provides a functional product, the components of which include rye pollen peptides prepared by the preparation method described above.

[0022] Based on the above, compared with the prior art, the method for preparing rye pollen peptides of the present invention has the following beneficial effects:

[0023] The method of this invention can be used to prepare rye pollen peptides, which have antioxidant properties, α-amylase inhibitory activity, anti-glycation activity, and prebiotic effects that promote the proliferation of Lactobacillus plantarum and Lactobacillus fermentum.

[0024] The rye pollen peptides prepared by this invention have no obvious bitterness or astringency, and have a good taste and flavor, which helps to improve the user experience.

[0025] The present invention uses a simple combination of operations such as mixing, pretreatment, enzymatic hydrolysis, and filtration to obtain the desired rye pollen peptides. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0028] This invention provides an operational example of a method for preparing rye pollen peptides with antioxidant, anti-glycation, and α-amylase inhibitory activities. The specific steps are as follows:

[0029] Step 1

[0030] (1) Mix rye pollen with water evenly, wherein the mass ratio of rye pollen to pure water is 1:(10-20).

[0031] (2) Add cellulase to the rye pollen solution, adjust the pH of the system to 4.5-5.0, hydrolyze at 40℃-45℃ for 0.5-1.5h, and then inactivate the enzyme at 85℃-90℃ for 15-20min to obtain the first pretreated solution; wherein, the amount of cellulase added is (1-1.5)% of the mass of rye pollen.

[0032] (3) Add pectinase to the first pretreatment solution, adjust the pH of the system to 3.5-4.5, hydrolyze at 50℃-55℃ for 0.5-1.5h, and then inactivate the enzyme at 85℃-90℃ for 15-20min to obtain the second pretreatment solution; wherein, the amount of pectinase added is (1-1.5)% of the mass of rye pollen.

[0033] Step Two

[0034] Add Bacillus licheniformis alkaline protease (enzyme activity 200,000 U / g) to the second pretreatment solution, adjust the pH of the system to 8.5–9.0, hydrolyze at 50–55°C for 1–3 h, and then inactivate the enzyme at 85–90°C for 15–20 min to obtain the first hydrolysate; wherein, the amount of Bacillus licheniformis alkaline protease added is (1–1.5)% of the mass of rye pollen.

[0035] Step 3

[0036] Papain (enzyme activity 500,000 U / g) was added to the first hydrolysate, the pH of the system was adjusted to 6.5–7.0, and hydrolysis was carried out at 50–55°C for 0.5–1.5 h, followed by enzyme inactivation treatment at 85–90°C for 15–20 min to obtain the second hydrolysate; wherein the amount of papain added was (0.5–0.8)% of the mass of rye pollen.

[0037] Step Four

[0038] Add Bacillus subtilis neutral protease (enzyme activity 200,000 U / g) to the second hydrolysate, adjust the pH of the system to 6.5–7.0, hydrolyze at 50–55°C for 0.5–1.5 h, and then inactivate the enzyme at 85–90°C for 15–20 min to obtain the third hydrolysate; wherein, the amount of Bacillus subtilis neutral protease added is (1–1.5)% of the mass of rye pollen.

[0039] Step 5

[0040] The third hydrolysate was cooled to room temperature and centrifuged at 4000–6000 rpm for 5–10 min. After centrifugation, the solution was ultrafiltered to obtain a filtrate with a molecular weight below 5000 Da, which was then spray-dried to obtain the rye pollen peptide.

[0041] Step Six

[0042] The activity of rye pollen peptides was tested, and they showed antioxidant activity, α-amylase inhibitory activity, anti-glycation activity, and also prebiotic effects that promote the proliferation of Lactobacillus plantarum and Lactobacillus fermentum.

[0043] The present invention provides the following embodiments and comparative examples.

[0044] The Bacillus licheniformis alkaline protease used in the examples and comparative examples had an enzyme activity of 200,000 U / g, papain had an enzyme activity of 500,000 U / g, and Bacillus subtilis neutral protease had an enzyme activity of 200,000 U / g.

[0045] Example 1

[0046] 1. Take 500g of rye pollen (protein content 50%), mix it evenly with 5000g of pure water, heat it to 40℃, adjust the pH to 4.8, add 5g of cellulase to hydrolyze for 1 hour, and heat it to 85℃ to inactivate the enzyme for 20 minutes.

[0047] 2. Cool to 55℃, adjust the pH to 4.0 with hydrochloric acid, add 5g of pectinase to hydrolyze for 1 hour, and heat to 85℃ to inactivate the enzyme for 20 minutes;

[0048] 3. Cool to 55℃, adjust the pH to 8.5 with sodium hydroxide, add 5g of Bacillus licheniformis alkaline protease to hydrolyze for 2h, and heat to 85℃ to inactivate the enzyme for 20min;

[0049] 4. Cool to 55℃, adjust the pH to 6.5 with hydrochloric acid, add 2.5g of papain to hydrolyze for 1 hour, and heat to 85℃ to inactivate the enzyme for 20 minutes.

[0050] 5. After cooling to 55℃, adjust the pH to 7.0 with sodium hydroxide, add 5g of Bacillus subtilis neutral protease to hydrolyze for 1.5h, and heat to 85℃ to inactivate the enzyme for 20min.

[0051] 6. The solution was centrifuged and filtered (centrifugation speed of 5000 rpm for 10 min), and then ultrafiltered to obtain a rye pollen peptide solution with a molecular weight of less than 5000 Da.

[0052] 7. Rye pollen peptide powder is obtained by spray drying.

[0053] Example 2

[0054] 1. Take 500g of rye pollen (protein content 50%), mix it evenly with 7500g of pure water, heat it to 45℃, adjust the pH to 4.5, add 5g of cellulase to hydrolyze for 0.5h, and heat it to 90℃ to inactivate the enzyme for 15min.

[0055] 2. Cool to 50℃, adjust the pH to 3.5 with hydrochloric acid, add 5g of pectinase to hydrolyze for 0.5h, and heat to 90℃ to inactivate the enzyme for 15min;

[0056] 3. Cool to 55℃, adjust pH to 9.0 with sodium hydroxide, add 5g of Bacillus licheniformis alkaline protease to hydrolyze for 1 hour, and heat to 90℃ to inactivate enzyme for 15 minutes;

[0057] 4. Cool to 50℃, adjust the pH to 7.0 with hydrochloric acid, add 4g of papain to hydrolyze for 1 hour, and heat to 90℃ to inactivate the enzyme for 15 minutes.

[0058] 5. After cooling to 50℃, adjust the pH to 6.5 with hydrochloric acid, add 5g of Bacillus subtilis neutral protease to hydrolyze for 1h, and heat to 90℃ to inactivate the enzyme for 15min.

[0059] 6. The solution was centrifuged and filtered (centrifugation speed of 5000 rpm for 10 min), and then ultrafiltered to obtain a rye pollen peptide solution with a molecular weight of less than 5000 Da.

[0060] 7. Rye pollen peptide powder is obtained by spray drying.

[0061] Example 3

[0062] 1. Take 500g of rye pollen (protein content 50%), mix it evenly with 10000g of pure water, heat it to 40℃, adjust the pH to 5.0, add 7.5g of cellulase to hydrolyze for 1.5h, and heat it to 90℃ to inactivate the enzyme for 15min.

[0063] 2. Cool to 55℃, adjust the pH to 4.5 with hydrochloric acid, add 7.5g of pectinase to hydrolyze for 1.5h, and heat to 90℃ to inactivate the enzyme for 15min;

[0064] 3. Cool to 50℃, adjust pH to 9.0 with sodium hydroxide, add 7.5g of Bacillus licheniformis alkaline protease to hydrolyze for 3h, and heat to 90℃ to inactivate enzyme for 15min;

[0065] 4. Cool to 55℃, adjust the pH to 6.5 with hydrochloric acid, add 3g of papain (by weight of rye pollen) to hydrolyze for 1 hour, and heat to 90℃ to inactivate the enzyme for 15 minutes.

[0066] 5. Cool to 55℃, adjust the pH to 7.0 with sodium hydroxide, add 7.5g of Bacillus subtilis neutral protease to hydrolyze for 0.5h, and heat to 90℃ to inactivate the enzyme for 15min;

[0067] 6. The solution was centrifuged and filtered (centrifugation speed of 5000 rpm for 10 min), and then ultrafiltered to obtain a rye pollen peptide solution with a molecular weight of less than 5000 Da.

[0068] 7. Rye pollen peptide powder is obtained by spray drying.

[0069] Comparative Example 1 (The hydrolysis process used only Bacillus licheniformis alkaline protease, without Bacillus subtilis neutral protease and papain)

[0070] The amount of Bacillus licheniformis alkaline protease added in step 3 was changed to 16.25g, steps 4 and 5 were removed, and the remaining operations and processes were the same as in Example 1.

[0071] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 5g. Based on the protein content in rye pollen, the enzyme addition amount of Bacillus licheniformis alkaline protease was 4000 U / g. The specific calculation process is as follows:

[0072] The enzyme activity of 5g of Bacillus licheniformis alkaline protease is: 200,000 U / g * 5 = 1,000,000 U. Converted to the enzyme addition amount based on the protein content per gram of rye pollen, it is: 1,000,000 U / (500g * 50%)g = 4,000 U / g.

[0073] The enzyme activities of Bacillus licheniformis alkaline protease (200,000 U / g), papain (500,000 U / g), and Bacillus subtilis neutral protease (200,000 U / g) used in the examples and comparative examples described herein refer to the enzyme activity per gram of enzyme (used to characterize the enzyme's ability to catalyze a specific chemical reaction). This differs in meaning from the enzyme addition unit U / g calculated per gram of rye pollen based on protein content. The conversion method for the addition amount of other enzymes described below is the same as the calculation method described above.

[0074] The amount of papain added was 2.5g, which, based on the protein content of rye pollen, represents an enzyme addition of 5000 U / g; the amount of Bacillus subtilis neutral protease added was 5g, which, based on the protein content of rye pollen, represents an enzyme addition of 4000 U / g; therefore, the 16.25g of Bacillus licheniformis alkaline protease in this comparative example, based on the protein content of rye pollen, represents 13000 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease used in Example 1.

[0075] Comparative Example 2 (the hydrolysis process used only papain, without Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease)

[0076] The amount of papain added in step 4 was changed to 6.5g, and steps 3 and 5 were removed. The remaining operations and processes were the same as in Example 1.

[0077] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 5g, which, based on the protein content of rye pollen, represents 4000 U / g of Bacillus licheniformis alkaline protease. The amount of papain added was 2.5g, which, based on the protein content of rye pollen, represents 5000 U / g of papain. The amount of Bacillus subtilis neutral protease added was 5g, which, based on the protein content of rye pollen, represents 4000 U / g of Bacillus subtilis neutral protease. Therefore, the 6.5g of papain in this comparative example, based on the protein content of rye pollen, represents 13000 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease used in Example 1.

[0078] Comparative Example 3 (the hydrolysis process used only Bacillus subtilis neutral protease, without Bacillus licheniformis alkaline protease and papain)

[0079] The amount of Bacillus subtilis neutral protease added in step 5 was changed to 16.25g, steps 3 and 4 were removed, and the remaining operations and processes were the same as in Example 1.

[0080] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 5g, which, based on the protein content of rye pollen, represents 4000 U / g of Bacillus licheniformis alkaline protease. The amount of papain added was 2.5g, which, based on the protein content of rye pollen, represents 5000 U / g of papain. The amount of Bacillus subtilis neutral protease added was 5g, which, based on the protein content of rye pollen, represents 4000 U / g of Bacillus subtilis neutral protease. Therefore, the 16.25g of Bacillus subtilis neutral protease in this comparative example, based on the protein content of rye pollen, represents 13000 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease used in Example 1.

[0081] Comparative Example 4 (The hydrolysis process only used Bacillus licheniformis alkaline protease and papain, without Bacillus subtilis neutral protease)

[0082] The amount of Bacillus licheniformis alkaline protease added in step 3 of Example 1 was changed to 8.75g, the amount of papain added in step 4 was changed to 3g, step 5 was removed, and the remaining operations and processes were the same as in Example 1.

[0083] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 5g (enzyme activity of 200,000 U / g per unit mass), which is 4,000 U / g based on the protein content in rye pollen; the amount of papain added was 2.5g, which is 5,000 U / g based on the protein content in rye pollen; and the amount of Bacillus subtilis neutral protease added was 5g, which is 4,000 U / g based on the protein content in rye pollen.

[0084] Therefore, the amount of Bacillus licheniformis alkaline protease added in this comparative example is 7000 U / g based on the protein content in rye pollen, and the amount of papain added is 6000 U / g based on the protein content in rye pollen. The enzymes in the comparative example are equal in amount to the total amount of Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease used in Example 1.

[0085] Comparative Example 5 (The hydrolysis process used only Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease, without papain)

[0086] The amount of Bacillus licheniformis alkaline protease added in step 3 was changed to 8.125g, the amount of Bacillus subtilis neutral protease added in step 5 was changed to 8.125g, step 4 was removed, and the remaining operations and processes were the same as in Example 1.

[0087] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 5g, which, based on the protein content in rye pollen, was 4000 U / g; the amount of papain added was 2.5g, which, based on the protein content in rye pollen, was 5000 U / g; and the amount of Bacillus subtilis neutral protease added was 5g, which, based on the protein content in rye pollen, was 4000 U / g.

[0088] Therefore, the enzyme addition amount of 8.125g of Bacillus licheniformis alkaline protease in this comparative example, based on the protein content in rye pollen, is 6500 U / g, and the enzyme addition amount of 8.125g of Bacillus subtilis neutral protease, based on the protein content in rye pollen, is also 6500 U / g. The enzyme amount in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease used in Example 1.

[0089] Comparative Example 6 (without cellulase and pectinase pretreatment)

[0090] Step one is omitted, and the other operations and conditions are the same as in Example 1.

[0091] It should be noted that the enzymes with the same name used in the above embodiments and comparative examples are from the same commercially available company and brand, that is, the enzyme activity of the enzymes with the same name used in each embodiment and comparative example is consistent.

[0092] The performance of the rye pollen peptide products obtained in the above examples and comparative examples was tested:

[0093] 1. Antioxidant activity of rye pollen peptides

[0094] The rye pollen peptides from the examples and comparative examples were prepared into a 20 mg / ml solution (using water as the solvent), and their hydroxyl radical scavenging rate, DPPH radical scavenging rate, ABTS radical scavenging rate, and ferrous ion chelating ability were tested. The test results are shown in Table 1.

[0095] The method for detecting the superoxide anion scavenging rate of rye pollen peptides was based on the study of "Extraction Process and Antioxidant Activity of Gleditsia sinensis Leaf Polysaccharide"; the methods for detecting the DPPH and ABTS free radical scavenging rates were based on the DPPH and ABTS methods for peptide antioxidant determination in GB / T 39100~2020; the method for detecting the hydroxyl free radical scavenging rate was based on the study of "Antioxidant Activity of Panax notoginseng Polysaccharide"; and the method for detecting the ferrous ion chelating ability was based on the study of "Preparation of Angelica sinensis Peptides and Study on Antioxidant Activity Mechanism and Structure-Activity Relationship".

[0096] Table 1. Antioxidant capacity of rye pollen peptides processed by different methods

[0097]

[0098] Note: - indicates no activity detected.

[0099] As can be seen from Table 1:

[0100] Both the examples and comparative examples exhibited high hydroxyl radical scavenging, ABTS scavenging, and DPPH scavenging rates, demonstrating high free radical scavenging efficiency. However, the DPPH scavenging rates of the comparative examples were lower than those of the examples. No ferrous ion chelating ability was detected in Comparative Examples 2, 3, and 6, indicating weak chelating ability for metal ions, while the examples showed strong ferrous ion chelating ability.

[0101] 2. α-Amylase inhibitory activity of rye pollen peptides

[0102] The rye pollen peptides from the examples and comparative examples were prepared into a 20 mg / ml solution (using water as the solvent), and their α-amylase inhibition rate was detected. The test results are shown in Table 2.

[0103] The determination of the inhibitory activity of rye pollen peptide α-amylase was performed in accordance with GB / T 24401~2009—Appendix B, Visual colorimetric method for determination of α-amylase activity at medium temperature.

[0104] Table 2. α-Amylase inhibition rate of rye pollen peptides from different processing methods

[0105]

[0106] Note: - indicates no inhibitory activity.

[0107] As shown in Table 2:

[0108] The α-amylase inhibition rates of the examples were all higher than those of the comparative examples, and no α-amylase inhibitory activity was detected in comparative examples 3 and 6.

[0109] The α-amylase inhibitory activity of rye pollen peptides obtained in the examples using a three-step hydrolysis method involving Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease was higher than that obtained using the comparative methods employing single protease and dual-enzyme hydrolysis. This indicates that the enzymatic combination of Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease can improve the α-amylase inhibition rate of rye pollen peptides, thus facilitating the release of active peptide fragments.

[0110] The α-amylase inhibitory activity of rye pollen peptides obtained by enzymatic hydrolysis using Bacillus licheniformis alkaline protease and papain in Comparative Example 4 and by enzymatic hydrolysis using Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease in Comparative Example 5 was higher than that obtained by enzymatic hydrolysis using a single protease in Comparative Examples 1-3.

[0111] 3. AGEs inhibitory (anti-glycation) activity of rye pollen peptides

[0112] The rye pollen peptides from the examples and comparative examples were prepared into 5 mg / ml solutions (using water as the solvent), and their AGEs inhibition rates were detected. The test results are shown in Table 3.

[0113] The determination of the AGEs inhibitory (anti-glycation) activity of rye pollen peptides was based on the study of "Multi-model evaluation of the anti-glycation effect and active components of Sophora japonica flower water extract".

[0114] Table 3. AGEs inhibition (anti-glycation) rate of rye pollen peptides from different processes

[0115]

[0116] As can be seen from Table 3:

[0117] The AGEs inhibition rates of Comparative Examples 4 and 5 (dual-enzyme hydrolysis) were higher than those of Comparative Examples 1, 2, and 3 (single-enzyme hydrolysis), indicating that dual-enzyme hydrolysis by Bacillus licheniformis alkaline protease and papain, and by Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease can have a synergistic effect on the AGEs inhibition rate. The AGEs inhibition rate of rye pollen peptides obtained by dual-enzyme hydrolysis is higher than that obtained by single-enzyme hydrolysis.

[0118] The AGEs inhibition rate of the rye pollen peptides obtained in the examples was higher than that of all comparative examples, indicating that the enzymatic hydrolysis combination of Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease can improve the AGEs inhibition rate of rye pollen peptides compared with enzymatic hydrolysis of equal enzyme activity using two or one enzyme.

[0119] 4. Probiotic activity of rye pollen peptides

[0120] The probiotic promotion experiment was conducted by measuring the OD of probiotics cultured in MRS medium for 20 hours. 600As a preliminary screening indicator, the promotion rate of probiotics was calculated, and the test results are shown in Table 4. MRS liquid culture medium was prepared and dispensed into 10 mL tubes, with 20 mg / mL of rye pollen peptide (water as solvent) added. A blank control group (without added rye pollen peptide) was also required. The probiotic inoculum was 3% (w / v), and the culture was incubated at 37°C. After the culture was completed, the bacterial suspension was mixed and diluted 20 times before the OD of the bacteria was measured. 600 The calculation is as follows:

[0121] Probiotic promotion rate % = (BA) / A x 100%

[0122] Note: A: OD of the blank group 600 B: OD of the experimental group 600 .

[0123] The formula for MRS liquid culture medium is as follows: 10g casein peptone, 10g beef extract, 5g yeast extract, 5g glucose, 5g sodium acetate, 2g K2HPO4, 2g diammonium citrate, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 1g Tween 80; pH 6.2. The solid culture medium is prepared by adding 2% agar and 2% CaCO3 to the above formula and sterilizing at 121℃ for 15min.

[0124] Lactobacillus plantarum BXM2 was deposited on September 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.16436.

[0125] The fermenting *Lactobacillus fermentum* B153 was deposited on September 10, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16454. Technicians can obtain the sample from the collection center using this accession number.

[0126] Table 4. Probiotic promotion rate of rye pollen peptides from different processes

[0127]

[0128] Note: - indicates not detected, which means that it has no effect on promoting the growth of beneficial bacteria. In other words, compared with the control group, it does not promote the growth of beneficial bacteria, that is, its growth rate is less than or equal to 0.

[0129] As can be seen from the data in Table 4:

[0130] As shown in Table 4, the *Lactobacillus plantarum* promotion rates of Comparative Examples 1, 2, 4, and 5 were all low, while no promotion effect on *Lactobacillus plantarum* was detected in Comparative Examples 3 and 6. This indicates that the rye pollen peptides obtained by single-enzyme hydrolysis with *Bacillus subtilis* neutral protease in Comparative Example 3 and without pretreatment with cellulase and pectinase in Comparative Example 6 did not have the activity of promoting *Lactobacillus plantarum*. Examples 1, 2, and 3 all showed a higher *Lactobacillus plantarum* promotion effect than the comparative examples, indicating that the enzymatic hydrolysis combination of *Bacillus licheniformis* alkaline protease, papain, and *Bacillus subtilis* neutral protease can produce rye pollen peptides that promote the growth of *Lactobacillus plantarum* compared to the same amount of dual-enzyme or single-enzyme hydrolysis in the comparative examples.

[0131] No growth rate of *Lactobacillus fermentans* was detected when hydrolyzed with neutral enzymes only (Comparative Example 3). The growth rate of rye pollen peptides obtained by hydrolysis with alkaline protease of *Bacillus licheniformis* (Comparative Example 1) and papain (Comparative Example 2) was relatively low. The growth rate of rye pollen peptides obtained in the examples was higher than that of all comparative examples. Compared with the same amount of dual-enzyme or single-enzyme hydrolysis in the comparative examples, the combination of three enzymes, alkaline protease of *Bacillus licheniformis*, papain, and neutral protease of *Bacillus subtilis*, can produce rye pollen peptides with higher activity that promote the growth of *Lactobacillus fermentans*.

[0132] 5. Sensory characteristics of rye pollen peptides

[0133] (1) The sample polypeptide powders of the examples and comparative examples were prepared into a 20 mg / ml solution (solvent was water). The sensory characteristics were evaluated in terms of bitterness, astringency, and sweetness. The sensory evaluation results are shown in Table 5 (1-4 represent obvious taste; 5-7 represent present taste; 8-10 represent indistinct taste):

[0134] Table 5 Sensory Analysis and Evaluation Table

[0135]

[0136] (2) From the data in Table 5, we can see that:

[0137] In this embodiment of the invention, rye pollen peptides are prepared by a combination of various enzymatic hydrolysis methods. These rye pollen peptides have no obvious bitterness or astringency and have a distinct sweetness, resulting in a good taste and flavor that enhances the user experience.

[0138] In summary, the method for preparing rye pollen peptides provided by this invention has at least the following mechanisms of action and technical effects:

[0139] (1) This invention is the first to discover that rye pollen peptides prepared by pretreatment with cellulase and pectinase followed by enzymatic hydrolysis with a combination of alkaline protease from Bacillus licheniformis, papain, and neutral protease from Bacillus subtilis have antioxidant properties, α-amylase inhibitory activity, anti-saccharification activity, and prebiotic effects that promote the proliferation of Lactobacillus plantarum and Lactobacillus fermentum.

[0140] In this invention, specific cellulase and pectinase are used for pretreatment, and a specific combination of Bacillus licheniformis alkaline protease, specific papain, and specific Bacillus subtilis neutral protease is used for enzymatic hydrolysis to achieve the desired effect. It is not that single-enzyme hydrolysis, dual-enzyme combination hydrolysis, or any other combination of neutral and alkaline protease hydrolysis can achieve the effect of this application.

[0141] (2) Based on the above-mentioned effects and functions of rye pollen peptides, the rye pollen peptides prepared by this invention can be used as a functional factor in functional foods, cosmetics and health products.

[0142] Free radicals, being highly reactive atoms or groups with unpaired electrons, pose various health risks to the human body when present in excess, including attacking cells and breaking down tissues. Based on the high antioxidant properties of this rye pollen peptide, its application in functional products (such as food, health supplements, and skincare products) can promote health, alleviate aging, and prevent skin aging.

[0143] Because inhibiting α-amylase in the human body can effectively suppress carbohydrate absorption, it not only reduces obesity but also lowers blood sugar levels. Based on the high α-amylase inhibitory activity of this rye pollen peptide, its application in functional products (such as food and health supplements) can achieve functions such as weight loss and blood sugar reduction.

[0144] As the body's sugar intake accumulates and metabolism gradually slows down, the ingested sugar easily accumulates, combines with proteins, oxidizes, and eventually forms AGEs (Advanced Glycation End Products). Rye pollen peptides have a high ability to inhibit AGEs and can play an anti-glycation role. Based on its anti-glycation effect, it can be used in anti-glycation functional products.

[0145] Since the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum* is beneficial to intestinal digestion and defecation, based on the prebiotic effect of this rye pollen peptide in promoting the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum*, its application in functional products (such as food) can play a role in aiding digestion and defecation.

[0146] (2) The rye pollen peptides obtained by this invention are prepared by a combination of various enzymatic hydrolysis methods. They have no obvious bitterness or astringency, and have a good taste and flavor, which is conducive to improving the user experience.

[0147] (3) The method of the present invention can obtain the desired polypeptide by simply combining operations such as mixing, enzymatic hydrolysis and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0148] In summary, this invention studies the extraction and purification of active polypeptides from rye pollen to obtain rye pollen peptides with prebiotic effects, including antioxidant activity, α-amylase inhibitory activity, anti-glycation activity, and promotion of the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum*. These polypeptides can be used as raw material components in functional foods and applied in products with effects such as weight loss, blood sugar reduction, antioxidant activity, anti-glycation activity, and regulation of intestinal health.

[0149] It should be noted that:

[0150] (1) Definition:

[0151] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0152] The term "food" as used herein is used in a broad sense, encompassing both human food and drink. In some embodiments, the food product is suitable for and designed for human consumption.

[0153] In the text, "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazine (free radical).

[0154] In the text, "ABTS" refers to the free radical 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0155] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0156] The terms "ultrafiltration," "centrifugation," and "spray drying" used in this article are conventional names for processing steps in the field, and their names accurately describe the processing procedures, so they will not be repeated here.

[0157] (2) Raw materials used in implementation:

[0158] The cellulase, pectinase, Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease used are all commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0159] (3) Application of rye pollen peptides:

[0160] Rye pollen peptides possess the following characteristics: (1) antioxidant activity; (2) α-amylase inhibitory activity; (3) anti-glycation activity; and (4) prebiotic effect, promoting the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum*. Based on these characteristics (1)-4), rye pollen peptides can also be applied to functional products (which can be foods, health products, cosmetics, skin care products, etc.) that have significant effects such as weight loss, blood sugar reduction, antioxidant activity, anti-glycation, and gut health regulation.

[0161] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of the present invention, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of the present invention.

[0162] In addition, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this embodiment do not specify the manufacturer or other information, and are all conventional products that can be purchased from the market.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing rye pollen peptides, characterized in that, Includes the following steps: Rye pollen was mixed evenly with water, and cellulase was added for the first pretreatment, followed by the first enzyme inactivation treatment to obtain the first pretreated solution. Pectinase was added to the first pretreatment solution for a second pretreatment, followed by a second enzyme inactivation treatment to obtain the second pretreatment solution. In the second pretreatment solution, Bacillus licheniformis alkaline protease, papain, and Bacillus subtilis neutral protease were added sequentially for three-step hydrolysis, followed by enzyme inactivation treatment to obtain the hydrolysate. The hydrolysate was subjected to solid-liquid separation to obtain a filtrate containing rye pollen peptides; The filtrate is filtered to retain rye pollen peptides, thus obtaining the rye pollen peptides. In the first pretreatment process, the mass ratio of rye pollen to water is 1:10 to 1:20; the amount of cellulase added is 1% to 1.5% of the mass of the rye pollen. The temperature of the first pretreatment is 40℃~45℃, the pH of the reaction system is 4.5~5.0, and the reaction time is 0.5~1.5h; the temperature of the first enzyme inactivation treatment is 85℃~90℃, and the enzyme inactivation treatment time is 15~20min. In the second pretreatment process, the amount of pectinase added is 1% to 1.5% of the mass of the rye pollen; The second pretreatment is performed at a temperature of 50℃ to 55℃, with a pH of 3.5 to 4.5 and a reaction time of 0.5 to 1.5 h; the second enzyme inactivation treatment is performed at a temperature of 85℃ to 90℃ and a treatment time of 15 to 20 min. The amount of Bacillus licheniformis alkaline protease added is 1% to 1.5% of the mass of the rye pollen; The amount of papain added is 0.5% to 0.8% of the mass of the rye pollen; The amount of Bacillus subtilis neutral protease added is 1% to 1.5% of the mass of the rye pollen; Bacillus licheniformis alkaline protease was added to the second pretreatment solution for the first hydrolysis. The temperature of the first hydrolysis was 50℃~55℃, the pH of the reaction system was 8.5~9.0, and the hydrolysis time was 1~3h. Then, the enzyme was inactivated at 85℃~90℃ for 15~20min to obtain the first hydrolysate. Papain was added to the first hydrolysate for a second hydrolysis at a temperature of 50℃ to 55℃, a pH of 6.5 to 7.0, and a hydrolysis time of 0.5 to 1.5 h. The enzyme was then inactivated at 85℃ to 90℃ for 15 to 20 min to obtain the second hydrolysate. Bacillus subtilis neutral protease was added to the second hydrolysate for a third hydrolysis. The temperature of the third hydrolysis was 50℃~55℃, the pH of the reaction system was 6.5~7.0, and the hydrolysis time was 0.5~1.5h. Then, the enzyme was inactivated at 85℃~90℃ for 15~20 min to obtain the third hydrolysate; The hydrolysate obtained from the three hydrolysis processes was centrifuged to separate the solid and liquid components, resulting in a filtrate containing rye pollen peptides. The filtrate was subjected to ultrafiltration to retain rye pollen peptides with a molecular weight of less than 5000 Da, and then dried to obtain the rye pollen peptides.

2. The method for preparing rye pollen peptides according to claim 1, characterized in that: The rye pollen peptides were obtained in powder form by spray drying.

3. A rye pollen peptide, characterized in that: It was prepared using the method for preparing rye pollen peptide as described in claim 1 or 2.

4. A functional product, characterized in that: Its components include rye pollen peptides prepared by the preparation method as described in claim 1 or 2.

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