Processing method of flaxseed active polypeptide powder

Abstract

The invention provides a processing method of flaxseed active polypeptide powder. The processing method comprises the following steps: adding water into flaxseed meal which is peeled and is subjected to grease extraction and is used as a raw material, grinding into pulp, adding neutral protease for first-time hydrolysis, performing enzyme deactivation on hydrolysate, adding proline protease for second-time hydrolysis, performing enzyme deactivation, centrifugation and membrane separation on the hydrolysate for the second time, and concentrating and drying the obtained polypeptide liquid to obtain the flaxseed polypeptide. The flaxseed polypeptide powder capable of inhibiting tyrosinase activity is obtained, and the polypeptide powder can be applied to beauty cosmetics or functional food.

Description

Technical Field

[0001] The present invention relates to the comprehensive utilization and deep processing of flaxseed meal, specifically using defatted and dehulled flaxseed meal as raw material

[0002] A method for processing active polypeptides. The polypeptide powder obtained by the method can be used in cosmetics or functional foods. Background Art

[0003] Flaxseed is the raw material for linseed oil and sesame oil in my country. The protein content of the cake obtained after linseed oil extraction is 30-40%. The amino acid ratio of linseed protein is balanced, making it a high-quality protein source. Linseed meal is a by-product of oil extraction using linseed as raw material by machine pressing or leaching. Although linseed cake contains a lot of protein, the linseed skin contains 7-10% water-soluble linseed gum and a large amount of insoluble dietary fiber, which makes the extraction and processing of linseed protein and polypeptides more difficult. At present, linseed meal is mainly used as animal feed or fertilizer.

[0004] Existing literature and patents reveal limited research on the production of functional peptides from flaxseed meal. Most studies primarily involve extracting flaxseed protein using alkaline-acid precipitation, followed by protease hydrolysis. Zhou Haochun et al., in their paper "Optimization of Flaxseed Cake Protein Extraction Process and Study on the Inhibition of α-Amylase Activity by Its Hydrolyzate," describe extracting flaxseed meal protein using alkaline extraction and acid precipitation, followed by enzymatic hydrolysis with alkaline protease to yield a product with α-amylase inhibitory activity. Wu Feng, in his master's thesis "Preparation of Flaxseed Polypeptides and Study on the Inhibition of α-Glucosidase Activity by Natural Trypsin Inhibitors," reported on the enzymatic hydrolysis of flaxseed meal protein isolate using 3.350 koji black acid protease. The resulting enzymatically hydrolyzed flaxseed peptides exhibited excellent antioxidant activity. In patent CN202110355086.X, the inventors disclose the anti-inflammatory effects of flaxseed active peptides extracted using alkaline extraction and acid precipitation, followed by hydrolysis with papain. The resulting hydrolyzate was then filtered through a 10 kDa ultrafiltration membrane and subjected to anti-inflammatory activity in mice with DSS-induced colitis. Only patent CN201610247829 discloses the use of enzymes such as papain to hydrolyze flaxseed meal to produce cholesterol-lowering flaxseed peptides. This method does not involve alkali dissolution and acid precipitation to extract protein from the raw material. However, to improve the activity and purity of the peptides, a pretreatment process using pectin lyase to remove flaxseed gum was employed. Even so, the resulting flaxseed peptide product only contained over 60% peptide. This suggests that some pectin hydrolysate or other polysaccharides remain unremoved in the resulting peptide hydrolysate. There are few reports of directly hydrolyzing dehulled and defatted flaxseed to produce functionally active peptides.

[0005] A search for flaxseed's ability to inhibit tyrosinase activity revealed only Zhao Yingying's article, "Optimization of Microwave-Assisted Deep Eutectic Solvent Extraction of Flaxseed Cake Polysaccharides and Study of Their Biological Activity," which stated that polysaccharides in flaxseed cake have a high tyrosinase inhibition rate at certain concentrations. No other literature or patents have reported flaxseed protein or peptides possessing tyrosinase inhibitory activity.

[0006] Existing literature and technical reports indicate that proline protease is an auxiliary enzyme used to eliminate precipitation and turbidity in beer. Proline protease works by breaking down proline in hordein, thereby reducing turbidity and precipitation caused by the binding of proline in the protein to polyphenols. However, there are currently no reports of using proline protease to process flaxseed or flaxseed meal polypeptides. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for processing flaxseed active polypeptide powder, wherein the polypeptide powder obtained by the method has the activity of inhibiting tyrosinase.

[0008] The present invention provides a flaxseed polypeptide powder processing method, comprising the following steps: using peeled and defatted flaxseed meal as a raw material, adding clean water at a material-liquid ratio of 1:9-1:19, grinding with a colloid mill, adjusting the pH of the liquid to 4.0-6.0, adding 2000-3500 u / g of Aspergillus oryzae protease activity units based on the protein mass in the raw material, and performing a first hydrolysis; after inactivating the enzyme in the first hydrolysis liquid, adjusting the pH value of the seasoning liquid to 4.5-6.5, adding 500-1000 u / g of proline protease activity units based on the protein mass in the raw material, and performing a second hydrolysis; after inactivating the enzyme in the second hydrolysis liquid, centrifuging at 4000-5000 rpm for 10-15 minutes to remove the precipitate, separating the obtained supernatant with a membrane to obtain a polypeptide liquid with a molecular weight of 200-3000 Da, and concentrating and drying to obtain flaxseed active polypeptide powder having tyrosinase inhibitory activity.

[0009] The first hydrolysis temperature is 35-45° C., and the time is 100-150 min; the second hydrolysis temperature is 40-60° C., and the time is 40-60 min.

[0010] The dehulled and defatted flaxseed meal refers to flaxseed meal that has been dehulled by a dry method and then de-fatted to less than 3% by pressing or leaching.

[0011] The first hydrolysis solution inactivation refers to inactivation of enzymes at 98°C for 5 minutes; the second hydrolysis solution inactivation refers to inactivation of enzymes at 90-98°C for 4-7 minutes.

[0012] The Aspergillus oryzae protease is the M-SD type Aspergillus oryzae protease produced by Amano Amano Enzyme Preparation Co., Ltd.; the proline protease is the proline protease produced by Ningxia Xiasheng Industrial Group Co., Ltd.

[0013] The membrane separation refers to that the hydrolyzate is first ultrafiltered through an ultrafiltration membrane with a molecular cutoff of 3000Da, and the filtrate is then nanofiltered through a 200Da nanofiltration membrane.

[0014] The flaxseed active polypeptide powder processed in this invention exhibits tyrosinase inhibition activity, meaning that when the purified polypeptide solution after membrane separation is at a concentration of 10 mg / ml, the tyrosinase inhibition rate is greater than 70%. Therefore, the flaxseed active polypeptide powder processed in this invention has melanin inhibition and whitening properties, and can be used in cosmetics or functional foods. The tyrosinase inhibition rate was determined according to T / SHRH 015-2018 Cosmetics - Tyrosinase Activity Inhibition Test Method.

[0015] Compared with the prior art, the present invention has the following advantages: 1. Direct enzymatic hydrolysis of polypeptides using defatted and peeled flaxseed meal as raw material eliminates the need for alkali dissolution and acid precipitation to extract the protein and then process the polypeptides, thus simplifying the process and reducing the discharge of acidic and alkaline wastewater. 2. Utilizing industrial waste to process functional polypeptides with potential whitening activity has low raw material costs and good application prospects. 3. Utilizing peeled and defatted flaxseed meal removes flaxseed gum, resulting in a hydrolyzate of the flaxseed meal from the enzymatic hydrolysis of the flaxseed meal with high polypeptide purity and low impurities, making it less likely for the ultrafiltration membrane to clog. DETAILED DESCRIPTION

[0016] Example 1

[0017] 1. The first enzymatic hydrolysis: 100 g of dehulled and defatted linseed meal containing 1.85% oil and 34% protein was added with 1200 g of water. After grinding with a colloid mill, the pH of the slurry was adjusted to 4.5. 70,000 u (activity units) of Aspergillus oryzae protease M-SD was added and hydrolyzed at 45 ° C for 110 min. After the hydrolysis, the enzyme was inactivated at 98 ° C for 5 min.

[0018] 2. Second hydrolysis and precipitation removal: The pH of the first inactivation solution was adjusted to 5.0, 20,000 units of proline protease were added, and hydrolysis was carried out at 60°C for 45 minutes. After the second hydrolysis solution was inactivated at 95°C for 6 minutes, the precipitate was removed by centrifugation at 4000 rpm for 15 minutes.

[0019] 3. Membrane separation: The centrifugal supernatant is first ultrafiltered through an ultrafiltration membrane with a molecular cutoff of 3000Da, and the filtrate is then nanofiltered through a 200Da nanofiltration membrane to obtain a polypeptide retentate with a molecular weight of 200-3000Da.

[0020] 4. Concentration and drying: The polypeptide retentate is vacuum concentrated and spray dried to obtain a powdered polypeptide with a polypeptide content of 93.15%.

[0021] The polypeptide was prepared into a solution with a concentration of 10 mg / ml, and the tyrosinase inhibition rate of the polypeptide solution was measured to be 83.65% (see Table 1). It can be used as a beauty ingredient or functional food.

[0022] Comparative Example 1

[0023] 100 g of the same raw material as in Example 1 was hydrolyzed using the same procedures and conditions as in Example 1, with the addition of protease M-SD. Following hydrolysis, the enzyme was inactivated at 98°C for 5 minutes, and a second hydrolysis was omitted. Subsequent membrane separation, concentration, and drying procedures were identical to those in Example 1. The resulting dried polypeptide powder was prepared into a 10 mg / ml solution, and the tyrosinase inhibition rate of the polypeptide was determined (see Table 1).

[0024] Comparative Example 2

[0025] 100 g of the same raw materials as in Example 1 were added to 1200 g of water and refined. The pH of the slurry was adjusted to 5.0, and 34,000 units of proline protease were added. Hydrolysis was performed at 60°C for 60 min (M-SD protease was not added for hydrolysis). The hydrolyzate was inactivated at 95°C for 6 min. All subsequent steps and conditions were the same as in Example 1. The dried polypeptide powder was prepared into a 10 mg / ml solution, and the tyrosinase inhibition rate of the polypeptide was determined (see Table 1).

[0026] Comparative Example 3

[0027] 100 g of the same raw materials as in Example 1 were added to 1200 g of water, and after refining, the pH was adjusted to 3.0. 3.350 g of Aspergillus niger acid protease (replacing M-SD protease) and 70,000 u were added, and the mixture was hydrolyzed at 45°C for 110 min. The remaining steps and conditions were the same as in Example 1.

[0028] Comparative Example 4

[0029] 100 g of the same raw materials as in Example 1 were added to 1200 g of water, refined, and adjusted to pH 3.0. 70,000 units of Aspergillus zomeprazole acid protease (replacing M-SD protease) were added, and hydrolysis was carried out at 45°C for 110 min. The remaining steps and conditions were the same as in Example 1.

[0030] Comparative Example 5

[0031] 100 g of the same raw material as in Example 1 was added with 1200 g of water, the pulp was refined, the pH was adjusted to 6.0, 70,000 u of papain (replacing M-SD protease) was added, and the mixture was hydrolyzed at 50° C. for 110 min. The remaining steps and conditions were the same as in Example 1.

[0032] Comparative Example 6

[0033] Table 1 Tyrosinase inhibition rate of peptide samples hydrolyzed by different enzymatic hydrolysis combinations (10 mg / ml)

[0034] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Tyrosinase inhibition rate% 83.65 -- -- 6.88 -- -- --

[0035] Note: -- indicates an inhibition rate of 0 or a negative value; there are also several groups of control groups with no inhibitory activity, which are not listed in this table.

[0036] 100 g of the same raw material as in Example 1 was added to 1200 g of water, and after refining, the pH was adjusted to 7.5. 70,000 units of Bacillus subtilis neutral protease (replacing M-SD protease) was added, and the mixture was hydrolyzed at 50°C for 110 min. The remaining steps and conditions were the same as in Example 1.

[0037] The test data from Comparative Examples 1 and 2 show that hydrolysis of sea buckthorn seed meal with either Aspergillus oryzae protease M-SD or proline protease alone does not produce polypeptides with tyrosinase inhibitory activity. Substituting Aspergillus oryzae protease M-SD for hydrolysis of sea buckthorn seed meal with other neutral proteases, acidic proteases, or sulfhydryl proteases, followed by continued hydrolysis with proline protease, also produces no polypeptides with tyrosinase inhibitory activity. In Comparative Example 3, the hydrolysis of the sea buckthorn seed meal with 3.350 g of Aspergillus niger acidic protease instead of M-SD protease showed very weak tyrosinase inhibitory activity, making it unsuitable for extraction and utilization. Only in Example 1, where the hydrolysis was first performed with Aspergillus oryzae protease M-SD followed by proline protease, could a polypeptide with strong tyrosinase inhibitory activity be produced.

[0038] This active peptide was actually discovered unexpectedly during other experiments. To develop a flaxseed peptide beverage, researchers were using different proteases to hydrolyze flaxseed meal to screen for a peptide solution with a good taste. They found that the peptide solution hydrolyzed with Aspergillus oryzae protease M-SD had a pleasant taste and no bitterness, but it tended to produce turbidity and precipitation upon refrigeration. Considering that flaxseed also contains phenolic compounds and proline, they sought to incorporate a small amount of proline protease into the hydrolyzate to reduce turbidity, similar to methods used to reduce beer turbidity. However, this method was ineffective. Later, when the enzymatic hydrolyzate was incubated with zebrafish, it was found to reduce melanin in the fish. Subsequent experiments demonstrated that flaxseed meal peptides hydrolyzed with Aspergillus oryzae protease M-SD, followed by moderate hydrolysis with proline protease (presumably by removing some proline residues from the peptides), could produce active peptides that inhibit tyrosinase. Although this serendipitous discovery led to the development of an active peptide product and a method for further processing flaxseed meal.

[0039] Example 2

[0040] 1. The first enzymatic hydrolysis: 1000 g of dehulled and defatted flaxseed meal containing 1.85% fat and 34% protein was added to 15000 g of water. After grinding with a colloid mill, the pH of the slurry was adjusted to 5.0, and 1.02 million u of Aspergillus oryzae protease M-SD was added. The slurry was hydrolyzed at 40°C for 130 min. After the hydrolysis, the enzyme was inactivated at 98°C for 5 min.

[0041] 2. Second enzymatic hydrolysis and precipitation removal: The pH of the first enzyme inactivation solution was adjusted to 6.0, 270,000 units of proline protease were added, and hydrolysis was carried out at 50°C for 60 minutes. After the second hydrolysis solution was inactivated at 98°C for 5 minutes, the precipitate was removed by centrifugation at 5000 rpm for 10 minutes.

[0042] 3. Polypeptide membrane separation and fractionation. The centrifuged supernatant was ultrafiltered and fractionated using ultrafiltration membranes with molecular cutoffs of 10,000 Da, 5,000 Da, and 3,000 Da, respectively, to obtain polypeptide A with an average molecular weight Mw > 10,000 Da, polypeptide B with an Mw of 5,000-10,000 Da, polypeptide C with an Mw of 3,000-5,000 Da, and polypeptide molecular segment D with an Mw < 3,000. The polypeptide with an Mw < 3,000 Da was then subjected to a 200 Da nanofiltration membrane to remove salt and free amino acids, yielding a molecular segment E of 200-3,000 Da.

[0043] 4. Concentration, Drying, and Testing. Vacuum concentrate and freeze-dry the peptide ultrafiltrate fractions A, B, C, D, and E. Prepare each sample to a peptide concentration of 10 mg / ml. Test the tyrosinase inhibition rate for each sample. The results are shown in Table 2.

[0044] Table 2 Tyrosinase inhibition rate (10 mg / ml) of peptides with different molecular segments

[0045] Centrifuge fluid before ultrafiltration A segment>10000Da B segment 10000-5000Da C segment 3000-5000Da D segment <3000Da E segment 200-3000Da Tyrosinase inhibition rate% 57.44 -- -- 6.80 78.95 82.71 Peptide content% 79.80 87.64 90.35 94.87

[0046] Analysis of the tyrosinase inhibition rates of peptides in various molecular segments by ultrafiltration and nanofiltration revealed that peptides with inhibitory activity were present in the ultrafiltrate with a molecular weight of less than 3000 Da. Nanofiltration of the ultrafiltrate in segment E further increased the inhibitory activity to over 82%, making it the optimal active peptide segment identified by the present invention. Peptides with molecular weights greater than 10,000 Da and those between 5,000 and 10,000 Da showed no tyrosinase inhibitory activity. Although the 3,000-5,000 Da segment exhibited 6.8% inhibitory activity, the inhibition rate was very low. The small amount of tyrosinase inhibitory activity in this segment is likely due to the activity of residual active peptides less than 3,000 Da that were not filtered out of the 3,000-5,000 Da ultrafiltrate retentate.

[0047] Even without ultrafiltration and hydrolysis, the centrifuge liquid has a 57% tyrosinase inhibition rate and a peptide content of 79.8%. If the focus is on increasing peptide yield and economic efficiency, the hydrolyzate can be directly concentrated and dried without ultrafiltration and used as the primary raw material for beauty active peptides.

[0048] Example 3

[0049] 1. The first enzymatic hydrolysis: 1000 g of dehulled and defatted linseed meal containing 1.85% oil and 34% protein was added with 18,000 g of water. After grinding with a colloid mill, the pH of the slurry was adjusted to 6.0, and 850,000 u of Aspergillus oryzae protease M-SD was added. The slurry was hydrolyzed at 35°C for 150 min. After the hydrolysis, the enzyme was inactivated at 98°C for 5 min.

[0050] 2. Second hydrolysis and precipitation removal: The pH of the first enzyme inactivation solution was adjusted to 6.5, and 340,000 U of proline protease was added. After stirring evenly, the solution was divided into 6 equal portions and hydrolyzed at 40°C for 30, 40, 50, 60, 70, and 80 min respectively. After the second hydrolysis, the 6 samples were placed in a 98°C water bath to inactivate the enzyme for 4 min, and then centrifuged at 5000 rpm for 10 min to remove the precipitate.

[0051] 3. Membrane separation The centrifugal supernatant of the six samples was first ultrafiltered through an ultrafiltration membrane with a molecular cutoff of 3000 Da, and the filtrate was then nanofiltered through a 200 Da nanofiltration membrane to obtain six polypeptide retentates of 200-3000 Da.

[0052] 4. Concentrate and dry. The 6 parts of polypeptide retentate are vacuum concentrated and freeze-dried to obtain 6 parts of powdered polypeptide.

[0053] The 6 portions of polypeptide were prepared into solutions with a concentration of 10 mg / ml. The inhibition rates of the 6 portions of polypeptide solutions on tyrosinase were measured and shown in Table 3.

[0054] Table 3 Tyrosinase inhibition rate (10 mg / ml) of proline protease hydrolyzates at different times

[0055] Hydrolysis 30min Hydrolysis 40min Hydrolysis 50min Hydrolysis for 60 minutes Hydrolysis 70min Hydrolysis 80min Tyrosinase inhibition rate% 27.26 76.25 86.08 77.89 26.40 0

[0056] As can be seen from Example 3, when proline protease was added after the first protease hydrolysis and a second hydrolysis was performed, the tyrosinase inhibition rate of the resulting second polypeptide hydrolyzate was less than 30% when the hydrolysis time was less than 30 minutes or greater than 70 minutes. This indicates that the hydrolyzed polypeptides of Aspergillus oryzae protease M-SD can only produce tyrosinase inhibitory activity after being moderately hydrolyzed by proline protease for a period of time. Short hydrolysis times result in insufficient removal of proline from the polypeptide or protein chain, resulting in insufficient tyrosinase inhibitory polypeptides. Excessive hydrolysis times result in excessive cleavage of polypeptides with tyrosinase inhibitory activity, leading to a rapid decrease in inhibition rate. Therefore, the present invention determines that the appropriate hydrolysis time is 40-60 minutes.

[0057] Corresponding enzyme addition experiments also confirmed that the tyrosinase inhibitory activity of the second hydrolyzate can only be achieved at a high level when the proline protease addition level is within the range of 500-1000 u / g (this experiment is also presented in this example).

Claims

1. A method for processing flaxseed active polypeptide powder, characterized in that The following steps are involved: The method uses dehulled and defatted flaxseed meal as raw material, adds clean water at a material-liquid ratio of 1:9-1:19, and grinds the slurry with a colloid mill. The pH of the slurry is adjusted to 4.0-6.0, and 2000-3500 u / g of Aspergillus oryzae protease is added based on the protein content of the raw material for a first hydrolysis. After the enzyme is inactivated in the first hydrolysis solution, the pH value of the slurry is adjusted to 4.5-6.5, and 500-1000 u / g of proline protease is added based on the protein content of the raw material for a second hydrolysis. After the enzyme is inactivated in the second hydrolysis solution, the solution is centrifuged at 4000-5000 rpm for 10-15 minutes to remove the precipitate. The obtained supernatant is separated by a membrane to obtain a polypeptide solution with a molecular weight of 200-3000 Da. The flaxseed active polypeptide powder is concentrated and dried to obtain the polypeptide powder. The polypeptide powder has tyrosinase inhibitory activity.

2. The method for processing flaxseed active polypeptide powder according to claim 1, characterized in that: The first hydrolysis temperature is 35-45° C., and the time is 100-150 min; the second hydrolysis temperature is 40-60° C., and the time is 40-60 min.

3. The method for processing flaxseed active polypeptide powder according to claim 1, characterized in that: The dehulled and defatted flaxseed meal refers to flaxseed meal that has been dehulled by a dry method and then de-fatted to less than 3% by pressing or leaching.

4. The method for processing flaxseed active polypeptide powder according to claim 1, characterized in that: The first hydrolysis solution inactivation refers to inactivation of enzymes at 98°C for 5 minutes; the second hydrolysis solution inactivation refers to inactivation of enzymes at 90-98°C for 4-7 minutes.

5. The method for processing flaxseed active polypeptide powder according to claim 1, characterized in that: The Aspergillus oryzae protease is the M-SD type Aspergillus oryzae protease produced by Amano Amano Enzyme Preparation Co., Ltd.; the proline protease is the proline protease produced by Ningxia Xiasheng Industrial Group Co., Ltd.

6. The method for processing flaxseed active polypeptide powder according to claim 1, characterized in that: The membrane separation refers to the hydrolyzate first being ultrafiltered through an ultrafiltration membrane with a molecular cutoff of 3000Da, and the filtrate then being nanofiltered through a 200Da nanofiltration membrane to obtain a polypeptide retentate.

7. Use of the flaxseed active polypeptide powder obtained by the processing method according to any one of claims 1 to 7 in cosmetics or functional foods.