A tea tree flower peptide with weight loss and anti-glycation activities, its preparation method, and functional products.

Abstract

The present invention relates to the technical field of deep processing of tea flowers, and in particular to a tea flower peptide with weight loss and anti-glycation activity, a preparation method thereof, and a functional product. The tea flower peptide preparation method comprises the following steps: extracting protein from tea flowers to obtain a tea flower protein extract; adding a composite enzyme to the tea flower protein extract for a first hydrolysis to obtain a first hydrolyzate; adding a protease to the first hydrolyzate for a second hydrolysis to obtain a second hydrolyzate; filtering the second hydrolyzate to intercept polypeptides to obtain tea flower peptides; wherein the composite enzyme includes pectinase, hemicellulase, and cellulase. The tea flower peptide prepared by the method of the present invention has anti-glycation, α-glucosidase inhibitory activity, pancreatic lipase inhibitory activity, tyrosinase inhibitory activity, antioxidant activity, and the role of a prebiotic that promotes the proliferation of fermented mucus lactobacillus.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology of tea flower, and in particular to a tea flower peptide with weight loss and anti-glycation activities, its preparation method, and functional products. Background Technology

[0002] Tea, as one of China's important crops, has a cultivation history of over 5,000 years. Tea flowers are a byproduct of the tea plantation. For a long time, people have focused on using tea leaves to make tea, paying little attention to the flowers and resulting in a waste of this abundant resource. In recent years, researchers have discovered that the polysaccharides and polyphenols in tea flowers possess anti-inflammatory, anti-tumor, anti-cancer, and immune-stimulating biological activities.

[0003] Compared to the polysaccharides and polyphenols in tea flowers, research on proteins in tea flowers is relatively limited. In fact, proteins account for 30-50% of the dry weight of tea flowers, indicating that tea flowers are an excellent raw material for protein extraction. Peptides are the active building blocks of proteins; currently, there are almost no reports on the deep processing of tea flowers to prepare active peptides, nor on the bioactivity of tea flower peptides. Regarding tea flower proteins, there are only reports on the preparation of enzymes or low-purity protein extracts from tea flowers, for example:

[0004] Chinese invention patent application CN201610067080.1, entitled "A tea flower protease and its preparation method and application," discloses a method for obtaining tea flower protease by treating tea flowers using a salting-out method. However, this method only states that the obtained tea flower protease can significantly increase the amino acid content in tea beverages, without studying the bioactivity of the obtained tea flower protease.

[0005] Chinese invention patent application number CN201410429372.6, entitled "A Tea Tree Flower Protein Extract and Its Application", discloses a method for obtaining tea tree flower protein extract by enzymatic or alkaline treatment of tea tree flowers. This method only describes that the obtained tea tree flower protein extract has good protein functional properties, such as foaming and gelling properties, but does not study the bioactivity of the obtained tea tree flower protein extract. Summary of the Invention

[0006] To address the gaps in the prior art mentioned above, this invention provides a method for preparing tea tree flower peptides with weight-loss and anti-glycation activities, the technical solution of which is as follows:

[0007] The preparation method of this tea tree flower peptide includes the following steps:

[0008] Tea flower powder was extracted using an alkaline method. The extract was then separated into solid and liquid components, and the supernatant was collected to obtain a tea flower protein extract.

[0009] A complex enzyme is added to the tea flower protein extract for the first hydrolysis to obtain a first hydrolysate; wherein the complex enzyme includes pectinase, hemicellulase and cellulase;

[0010] A second hydrolysis is performed by adding a protease to the first hydrolysate to obtain a second hydrolysate; wherein the protease includes Aspergillus oryzae aminopeptidase and Bacillus subtilis neutral protease.

[0011] The second hydrolysate was filtered to retain the polypeptide, thus obtaining the tea flower peptide.

[0012] In some embodiments, the process of extracting tea tree flower powder using the alkaline method is as follows: the tea tree flower powder is mixed evenly with an aqueous calcium hydroxide solution, and the extraction reaction is carried out at 80-100°C for 0.5-6 hours. Then, the extract is centrifuged to separate the solid and liquid, and the supernatant is collected to obtain a tea tree flower protein extract. The concentration of the aqueous calcium hydroxide solution is 0.1-0.4 mol / L, and the ratio of tea tree flower powder to the aqueous calcium hydroxide solution is 1 g: 10-40 kg.

[0013] In some embodiments, in the first hydrolysis step, the pH of the tea flower protein extract is adjusted to 5.0–6.0, the compound enzyme is added for the first hydrolysis, the first hydrolysis temperature is 40–50°C, and the first hydrolysis time is 100–240 min; in the second hydrolysis step, the pH of the first hydrolysate is adjusted to 6.0–7.0, the protease is added for the first hydrolysis, the second hydrolysis temperature is 45–60°C, and the second hydrolysis time is 60–180 min.

[0014] In some embodiments, in the first hydrolysis step, the amount of the compound enzyme added is 1-5% of the mass of the tea flower powder, and the mass ratio of the pectinase, the hemicellulase and the cellulase is 1:(0.8-1.2):(0.8-1.2); in the second hydrolysis step, the amount of Aspergillus oryzae aminopeptidase added is 0.1-1.0% of the mass of the tea flower, and the amount of Bacillus subtilis neutral protease added is 0.5-1.5% of the mass of the tea flower powder.

[0015] In some embodiments, the tea flower protein extract is subjected to a first hydrolysis treatment, followed by enzyme inactivation at 85-90°C for 20-30 minutes to obtain a first hydrolysate; the first hydrolysate is subjected to a second hydrolysis treatment, followed by enzyme inactivation at 85-90°C for 20-30 minutes to obtain a second hydrolysate.

[0016] In some embodiments, during the filtration step, the second hydrolysate is filtered to retain polypeptides with a molecular weight cutoff of 200 to 5000 Da.

[0017] In some embodiments, during the filtration step, the second hydrolysate is filtered to obtain a polypeptide filtrate with a polypeptide molecular weight of 200 to 5000 Da; the polypeptide filtrate is then concentrated to a polypeptide mass content of 12% to 22%, and the concentrate is dried to form a powder.

[0018] In some embodiments, spray drying or freeze drying is used for the drying into powder process.

[0019] This invention provides a tea tree flower peptide prepared using the method described above.

[0020] This invention provides a functional product whose components include tea tree flower peptides prepared by the method described above.

[0021] In some embodiments, the functional products include functional foods, health products, cosmetics, and skincare products.

[0022] In some embodiments, the functional product includes at least one of the following functions:

[0023] (1) It has anti-glycation effects;

[0024] (2) Possesses α-glucosidase,

[0025] (3) It possesses anti-pancreatic lipase inhibitory activity.

[0026] (4) It possesses tyrosinase inhibitory activity;

[0027] (5) Possesses antioxidant properties;

[0028] (6) It has the activity of promoting fermentation of Lactobacillus mucinus.

[0029] Based on the above, compared with the prior art, the method for preparing tea tree flower peptides of the present invention has the following beneficial effects:

[0030] The method of this invention can produce tea tree flower peptides with high anti-glycation activity. These tea tree flower peptides also have α-glucosidase inhibitory activity, pancreatic lipase inhibitory activity, tyrosinase inhibitory activity, antioxidant activity, and prebiotic effects that promote the proliferation of fermenting Lactobacillus mucin. These tea tree flower peptides can be used as raw material components of functional foods and applied in products with effects such as weight loss, anti-oxidation, anti-glycation, whitening, regulating intestinal health, and preventing skin aging.

[0031] This tea tree flower peptide is prepared by a combination of enzymatic hydrolysis. It has a rich tea tree flower aroma and when applied to food, it has a good taste and flavor, which helps to improve the user experience. In addition, it is resistant to acid and high temperature, so it will not cause the problem of easy precipitation of plant peptides at high temperatures.

[0032] The method of this invention can produce the desired peptides 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.

[0033] Other features and beneficial effects of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other beneficial effects of the invention can be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Unless otherwise specified, the positional relationships shown in the drawings in the following description are based on the direction in which the components are drawn in the figure.

[0035] Figure 1 The images show the phenomena of peptide samples prepared under different preparation process conditions in the examples and comparative examples under acidic and high-temperature treatment.

[0036] Figure 2 The graph shows the anti-glycation ability data of peptide samples prepared under different preparation process conditions for the examples and comparative examples;

[0037] Figure 3 The graph shows the inhibitory activity data of α-glucosidase and pancreatic lipase in peptide samples prepared under different preparation process conditions for the examples and comparative examples.

[0038] Figure 4 The graph shows the tyrosinase inhibitory activity data of peptide samples prepared under different preparation process conditions in the examples and comparative examples.

[0039] Figure 5 The graph shows the antioxidant capacity data of peptide samples prepared under different preparation process conditions for the examples and comparative examples;

[0040] Figure 6 The figures show the activity data of *Lactobacillus mucinus* promoting fermentation of peptide samples prepared under different preparation process conditions for the examples and comparative examples. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, 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. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] 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.

[0043] This invention provides an operational example of a method for preparing tea tree flower peptides, the specific steps of which are as follows:

[0044] (1) After drying the tea flowers, pulverize them using a pulverizer to obtain tea flower powder;

[0045] (2) Mix tea tree flower powder with calcium hydroxide solution with a concentration of 0.1-0.4 mol / L evenly, with a mass ratio of 1 g: (10-40) kg, and extract at 80-100℃ for 0.5-6 h. Then centrifuge the extract mixture and collect the supernatant to obtain tea tree flower protein extract.

[0046] (3) After cooling the tea tree flower protein extract to 40-50℃, adjust the pH of the tea tree flower protein extract to 5.0-6.0 with citric acid, add compound enzyme at 1-5% of the raw material mass, and hydrolyze at 40-50℃ for 100-240 min. After hydrolysis, heat to 85-90℃ for 20-30 min to inactivate the enzyme and obtain the first hydrolysate.

[0047] The complex enzyme is composed of pectinase, hemicellulase and cellulase, and the mass ratio of pectinase, hemicellulase and cellulase is 1: (0.8-1.2): (0.8-1.2).

[0048] (4) Cool the first hydrolysate to 45-60°C, adjust the pH of the first hydrolysate to 6.0-7.0 with calcium hydroxide, add 0.1-1.0% of Aspergillus oryzae aminopeptidase and 0.5-1.5% of Bacillus subtilis neutral protease according to the raw material mass, hydrolyze at 45-60°C for 60-180 min, heat to 85-90°C to inactivate enzyme for 20-30 min, and cool to room temperature to obtain the second hydrolysate.

[0049] (5) The second hydrolysate was filtered using a plate and frame filter to remove insoluble substances and obtain a filtrate; the filtrate was ultrafiltered through a 5000 Da ultrafiltration membrane to obtain an ultrafiltrate with peptide molecular weight less than 5000 Da; the ultrafiltrate was then nanofiltered through a 200 Da nanofiltration membrane to obtain tea tree flower peptides with molecular weight of 200-5000 Da.

[0050] (6) The obtained tea tree flower peptide liquid with a molecular weight of 200 to 5000 Da is concentrated to a polypeptide mass content of 12% to 22% and dried into powder; spray drying or freeze drying can be used for the drying into powder process.

[0051] (7) The acid and high temperature resistance tests on tea tree flower peptides do not produce the problem of easy precipitation of plant peptides at high temperatures.

[0052] (8) The activity of tea tree flower peptides was tested and found that they have anti-glycation, α-glucosidase inhibitory activity, pancreatic lipase inhibitory activity, tyrosinase inhibitory activity, antioxidant activity, and prebiotic effect of promoting the proliferation of fermenting Lactobacillus mucinus.

[0053] The present invention also provides the following embodiments and comparative examples to verify the effectiveness of the present invention:

[0054] The pectinase used in the examples and comparative examples had an enzyme activity of 30,000 U / g; hemicellulase had an enzyme activity of 100,000 U / g; cellulase had an enzyme activity of 20,000 U / g; Aspergillus oryzae aminopeptidase had an enzyme activity of 50,000 U / g; Bacillus subtilis neutral protease had an enzyme activity of 80,000 U / mL and a density of 1.26 g / mL, resulting in an enzyme activity of 6.35 U / g per unit mass; papain had an enzyme activity of 800,000 U / g; and Bacillus licheniformis alkaline protease had an enzyme activity of 200,000 U / g.

[0055] Example 1

[0056] 1. After drying the tea flowers, grind them using a grinder.

[0057] 2. Mix 1 kg of tea tree flower powder with 40 kg of 0.1 mol / L calcium hydroxide solution until homogeneous, react at 80℃ for 0.5 h, then centrifuge the mixture and collect the supernatant to obtain tea tree flower protein extract.

[0058] 3. After cooling to 40℃, adjust the pH of the tea flower protein extract to 5.0 with citric acid, add 10g of compound enzyme, and enzymatically hydrolyze at 40℃ for 100min. After hydrolysis, heat to 85℃ for 20min to inactivate the enzyme and obtain the first hydrolysate. The mass ratio of pectinase, hemicellulase and cellulase in the compound enzyme is 1:0.8:0.8.

[0059] 4. After cooling to 45℃, adjust the pH of the first hydrolysate to 6.0 with calcium hydroxide, add 1g of Aspergillus oryzae aminopeptidase and 5g of Bacillus subtilis neutral protease, and hydrolyze at 45℃ for 60min. After hydrolysis, heat to 85℃ for 20min to inactivate the enzyme, and then cool to room temperature to obtain the second hydrolysate.

[0060] 5. Filter the second hydrolysate using a plate and frame filter to remove insoluble substances and obtain a filtrate; pass the filtrate through a 5000 Da ultrafiltration membrane to obtain an ultrafiltrate with a molecular weight of less than 5000 Da; pass the ultrafiltrate through a 200 Da nanofiltration membrane to obtain tea tree flower peptides with a molecular weight of 200–5000 Da.

[0061] 6. Concentrate the obtained tea tree flower peptide liquid with a molecular weight of 200-5000 Da to a polypeptide content of 18%, and then spray dry it into powder for storage.

[0062] Example 2

[0063] 1. After drying the tea flowers, grind them using a grinder.

[0064] 2. Mix 1 kg of tea tree flower powder with 10 kg of 0.4 mol / L calcium hydroxide solution until homogeneous, and react at 100℃ for 6 h. Then centrifuge the mixture and collect the supernatant to obtain tea tree flower protein extract.

[0065] 3. After cooling to 50℃, adjust the pH of the tea flower protein extract to 6.0 with citric acid, add 50g of compound enzyme, and enzymatically hydrolyze at 50℃ for 240min. After hydrolysis, heat to 90℃ for 30min to inactivate the enzyme, obtaining the first hydrolysate. The mass ratio of pectinase, hemicellulase, and cellulase in the compound enzyme is 1:1.2:1.2.

[0066] 4. After cooling to 60℃, adjust the pH of the first hydrolysate to 7.0 with calcium hydroxide, add 10g of Aspergillus oryzae aminopeptidase and 15g of Bacillus subtilis neutral protease, and hydrolyze at 60℃ for 180min. After hydrolysis, heat to 90℃ for 30min to inactivate the enzyme, and then cool to room temperature to obtain the second hydrolysate.

[0067] 5. Filter the second hydrolysate using a plate and frame filter to remove insoluble substances and obtain a filtrate; pass the filtrate through a 5000 Da ultrafiltration membrane to obtain an ultrafiltrate with a molecular weight of less than 5000 Da; pass the ultrafiltrate through a 200 Da nanofiltration membrane to obtain tea tree flower peptides with a molecular weight of 200–5000 Da.

[0068] 6. Concentrate the obtained tea tree flower peptide liquid with a molecular weight of 200-5000 Da to a polypeptide content of 12%, and then spray dry it into powder for storage.

[0069] Example 3

[0070] 1. After drying the tea flowers, grind them using a grinder.

[0071] 2. Mix 1 kg of tea tree flower powder with 20 kg of 0.2 mol / L calcium hydroxide solution until homogeneous, react at 90℃ for 3 h, then centrifuge the mixture and collect the supernatant to obtain tea tree flower protein extract.

[0072] 3. After cooling to 45℃, adjust the pH of the tea flower protein extract to 5.5 with citric acid, add 30g of compound enzyme, and enzymatically hydrolyze at 45℃ for 180min. After hydrolysis, heat to 85℃ for 25min to inactivate the enzyme, obtaining the first hydrolysate. The mass ratio of pectinase, hemicellulase, and cellulase in the compound enzyme is 1:1:1.

[0073] 4. After cooling to 50℃, adjust the pH of the first hydrolysate to 6.5 with calcium hydroxide, add 5g of Aspergillus oryzae aminopeptidase and 10g of Bacillus subtilis neutral protease, and hydrolyze at 50℃ for 120min. After hydrolysis, heat to 85℃ for 25min to inactivate the enzyme, and then cool to room temperature to obtain the second hydrolysate.

[0074] 5. Filter the second hydrolysate using a plate and frame filter to remove insoluble substances and obtain a filtrate; pass the filtrate through a 5000 Da ultrafiltration membrane to obtain an ultrafiltrate with a molecular weight of less than 5000 Da; pass the ultrafiltrate through a 200 Da nanofiltration membrane to obtain tea tree flower peptides with a molecular weight of 200–5000 Da.

[0075] 6. Concentrate the obtained tea tree flower peptide liquid with a molecular weight of 200-5000 Da to a polypeptide content of 15%, and then spray dry it into powder for storage.

[0076] Example 4

[0077] 1. After drying the tea flowers, grind them using a grinder.

[0078] 2. Mix 1 kg of tea tree flower powder with 20 kg of 0.2 mol / L calcium hydroxide solution until homogeneous, react at 90℃ for 3 h, then centrifuge the mixture and collect the supernatant to obtain tea tree flower protein extract.

[0079] 3. After cooling to 45℃, adjust the pH of the tea flower protein extract to 5.5 with citric acid, add 30g of compound enzyme, and enzymatically hydrolyze at 45℃ for 180min. After hydrolysis, heat to 85℃ for 25min to inactivate the enzyme, obtaining the first hydrolysate. The mass ratio of pectinase, hemicellulase, and cellulase in the compound enzyme is 1:1:1.

[0080] 4. After cooling to 50℃, adjust the pH of the first hydrolysate to 6.5 with calcium hydroxide, add 5g of Aspergillus oryzae aminopeptidase and 10g of Bacillus subtilis neutral protease, and hydrolyze at 50℃ for 120min. After hydrolysis, heat to 85℃ for 25min to inactivate the enzyme, and then cool to room temperature to obtain the second hydrolysate.

[0081] 5. Filter the second hydrolysate using a plate and frame filter to remove insoluble substances and obtain a filtrate; pass the filtrate through a 5000 Da ultrafiltration membrane to obtain an ultrafiltrate with a molecular weight of less than 5000 Da; pass the ultrafiltrate through a 200 Da nanofiltration membrane to obtain tea tree flower peptides with a molecular weight of 200–5000 Da.

[0082] 6. Concentrate the obtained tea tree flower peptide liquid with a molecular weight of 200-5000 Da to a polypeptide content of 20%, and freeze-dry it into powder for preservation.

[0083] Comparative Example 1

[0084] The difference between this comparative example and Example 1 is that step 3 in Example 1 (i.e., the treatment of the first hydrolysate with a compound enzyme is omitted) is omitted, while the remaining steps and processes are the same as in Example 1.

[0085] Comparative Example 2

[0086] The difference between this comparative example and Example 1 is that in step 3, the compound enzyme was replaced with an enzyme of pectinase activity or other similar amounts for post-treatment (i.e., the amount of pectinase used in Comparative Example 2 is equal to the amount of compound enzyme used in Example 1). Everything else is the same as in Example 1.

[0087] The specific calculation process for replacing the compound enzyme with the activity of enzymes such as pectinase is as follows:

[0088] In Example 1, the total enzyme activity of the added pectinase was 30,000 U / g * 10g * 1 / 2.6 = 115,400 U; the total enzyme activity of the added hemicellulase was 100,000 U / g * 10 * 0.8 / 2.6 = 307,700 U; and the total enzyme activity of the added cellulase was 20,000 U / g * 10 * 0.8 / 2.6 = 61,540 U. Therefore, the total enzyme activity of the complex enzyme was 484,640 U.

[0089] The total enzyme activity of the pectinase and the complex enzyme in this comparative example is equal, and the converted pectinase mass is: 484,640 U / 30,000 U / g = 16.154g.

[0090] The conversion method for the amount of enzyme added in the other comparative examples below is the same as the calculation method above.

[0091] Comparative Example 3

[0092] The difference between this comparative example and Example 1 is that in step 3, the activity of enzymes such as hemicellulase is used to replace the complex enzyme for post-treatment (i.e., the amount of hemicellulase used in Comparative Example 3 is equal to the amount of complex enzyme used in Example 1). Everything else is the same as in Example 1.

[0093] Comparative Example 4

[0094] The difference between this comparative example and Example 1 is that in step 3, the complex enzyme was replaced with an enzyme of cellulase activity or other similar amounts for post-treatment (i.e., the amount of cellulase used in Comparative Example 3 is equal to the amount of complex enzyme used in Example 1). Everything else is the same as in Example 1.

[0095] Comparative Example 5

[0096] The difference between this comparative example and Example 1 is that in step 4, the activity of Aspergillus oryzae aminopeptidase and other enzymes is used to replace the neutral protease of Bacillus subtilis for post-treatment (that is, the amount of Aspergillus oryzae aminopeptidase in Comparative Example 3 is equal to the sum of its amount and the amount of neutral protease of Bacillus subtilis in Example 1); everything else is the same as in Example 1.

[0097] Comparative Example 6

[0098] The difference between this comparative example and Example 1 is that in step 4, the activity of Bacillus subtilis neutral protease and other enzymes is used to replace Aspergillus oryzae aminopeptidase for post-treatment (i.e., the amount of Bacillus subtilis neutral protease in Comparative Example 3 is equal to the sum of its amount and the amount of Aspergillus oryzae aminopeptidase used in Example 1). Everything else is the same as in Example 1.

[0099] Comparative Example 7

[0100] In step 4, the pH was adjusted to 6.0, and Aspergillus oryzae aminopeptidase and Bacillus subtilis neutral protease were replaced with enzymes such as papain. Everything else was the same as in Example 1.

[0101] Comparative Example 8

[0102] In step 4, the pH was adjusted to 7.0, and the aminopeptidase of Aspergillus oryzae and the neutral protease of Bacillus subtilis were replaced with enzymes such as Bacillus licheniformis alkaline protease. Everything else was the same as in Example 1.

[0103] The performance of the peptide products obtained in the above examples and comparative examples was tested:

[0104] 1. Effects of different processing conditions on the acid and heat resistance of peptide products

[0105] (1) The peptide powder samples from the examples and comparative examples were prepared into solutions of 50 mg / mL (using water as the solvent). The pH of the solutions was adjusted to 4.0 and 5.0 respectively with citric acid, and sterilized at 121°C for 30 min. The acid and heat resistance properties of the peptide samples from the examples and comparative examples were tested, and the results are as follows: Figure 1 As shown in (a)-(d).

[0106] in, Figure 1 The treatment conditions in (a) are pH 4.0 and no sterilization. Figure 1 (b) The treatment conditions were pH 4.0 and sterilization at 121°C for 30 min; Figure 1 (c) The treatment conditions were pH 5.0 and non-sterilized; Figure 1 (d) The treatment conditions were pH 5.0 and sterilization at 121°C for 30 min. Figure 1 In (a)-(d), from left to right are Examples 1-4 and Comparative Examples 1-8, respectively.

[0107] (2) By Figure 1 From (a) to (d), we can conclude that:

[0108] Examples 1-4 all exhibit good acid and heat resistance. Figure 1 No precipitate was formed in any of (a)-(d).

[0109] In Comparative Examples 1-8, Comparative Example 7 (using papain to replace Aspergillus oryzae aminopeptidase and Bacillus subtilis neutral protease by the same mass) showed severe turbidity and whitening at pH 4.0 without sterilization, and turbidity and slight whitening at pH 5.0 without sterilization; however, the solution became clear after sterilization at 121℃ for 30 min. This may be because papain cannot effectively hydrolyze the hydrophobic proteins in tea flower peptides, leading to turbidity in the solution before sterilization; after high-temperature treatment, the hydrophobic protein structure expands due to the high temperature, resulting in increased solubility and the disappearance of turbidity.

[0110] Peptide products are susceptible to precipitation due to their insensitivity to acid and heat. This can cause the beverage solution to become cloudy or develop flocculent matter after sterilization when used in acidic liquid beverages, which are considered defective products. Similarly, when used in solid beverages, the powdered peptides may agglomerate or precipitate after being brewed with hot water, which are also considered defective products. Therefore, the insensitivity of peptide products to acid and heat and their tendency to precipitate limit their application.

[0111] 2. Effects of different processing conditions on the anti-glycation properties of peptide products

[0112] (1) The peptide powder samples from the examples and comparative examples were prepared into a 5 mg / mL solution (using water as the solvent). The anti-glycation performance of the peptide samples from the examples and comparative examples was tested, and the results are as follows: Figure 2 As shown in Table 1. The method for detecting anti-glycation performance was carried out in accordance with the "Multi-model Evaluation of the Anti-glycation Effect and Active Components of Sophora japonica Water Extract", with aminoguanidine 40 mg / mL as the positive control.

[0113] Table 1

[0114]

[0115] In Table 1, "-" indicates that it was not detected.

[0116] (2) Through detection data ( Figure 2 As can be seen from Table 1:

[0117] Examples 1-4 all exhibited good anti-glycation properties, all exceeding 80%; Example 1 showed relatively good anti-glycation properties, at 86.44%.

[0118] A comparison of the examples with Comparative Examples 1-8 shows that the anti-glycation performance of Comparative Examples 1-8 is reduced compared to the examples. Specifically, the data indicate that the use of specific complex enzymes (pectinase, hemicellulase, and cellulase) has a relatively small impact on the anti-glycation performance of tea tree flower peptides, while the use of specific proteases has a significant impact. Among them, Comparative Example 6 showed the highest AGEs inhibition rate of tea tree flower peptides, at 70.32%.

[0119] The reason for this may be that the complex enzymes (pectinase, hemicellulase, and cellulase) are more conducive to hydrolyzing tea flower proteins, thus exposing more anti-glycation protein structures. The Aspergillus oryzae aminopeptidase, papain (used in Comparative Example 7), and Bacillus licheniformis alkaline protease (used in Comparative Example 8) in the proteases can cleave the anti-glycation protein structures, resulting in the tea flower proteins having no anti-glycation effect after treatment. Therefore, Comparative Example 5 used an excessive amount of Aspergillus oryzae aminopeptidase to replace the complex protease in the embodiments of this invention, leading to a significant reduction in the anti-glycation performance of the peptides.

[0120] 3. Effects of different process conditions on α-glucosidase inhibition rate and pancreatic lipase inhibition rate

[0121] (1) The polypeptide powders from the examples and comparative examples were prepared into a 20 mg / ml solution (using water as the solvent), and the α-glucosidase inhibition rate and pancreatic lipase inhibition rate were detected. For detailed data, please refer to [link to relevant documentation]. Figure 3 And Table 2; wherein, the method for detecting α-glucosidase inhibition rate was in accordance with "Inhibitory effect of chestnut (Castanea mollissima Blume) inner skin extract on the activity of α-amylase, α-glucosidase, dipeptidyl peptidase IV and in vitro digestibility of starches", and the method for detecting pancreatic lipase inhibition rate was in accordance with "Study on the inhibitory effect of lotus leaf flavonoids on pancreatic lipase".

[0122] Table 2

[0123]

[0124] (2) By Figure 3 From the data in Table 2, we can see that:

[0125] Examples 1-4 all showed good α-glucosidase inhibition rates and pancreatic lipase inhibition rates, all above 70%; Example 1 showed relatively good α-glucosidase inhibition rates and pancreatic lipase inhibition rates, at 96.86% and 77.52%, respectively.

[0126] The α-glucosidase inhibition rate and pancreatic lipase inhibition rate of Comparative Examples 1-8 were all lower than those of Examples 1-8. A comparison between Examples 1-8 and Comparative Examples 1-8 shows that different combinations of protease digestion and complex enzyme digestion can significantly affect the inhibitory activities of the obtained peptides on α-glucosidase and pancreatic lipase.

[0127] Compared with Examples 1-4, Comparative Examples 1-4 did not use a compound enzyme (pectinase, hemicellulase, cellulase) or only used one of the compound enzymes for the first hydrolysis, which had a greater impact on the inhibition rate of α-glucosidase of tea flower peptides and a smaller impact on the inhibition rate of pancreatic lipase. This indicates that the structural changes of pectin, hemicellulose and cellulose present in tea flower peptides have a greater impact on the inhibition rate of α-glucosidase of tea flower peptides. It is speculated that this may be because sugars such as pectin have a certain encapsulating effect on peptides, preventing them from effectively inhibiting α-glucosidase.

[0128] Compared to the examples, the α-glucosidase inhibition rate and pancreatic lipase inhibition rate of the peptides obtained in Comparative Examples 5-8 were both reduced. In Comparative Examples 7-8, the second hydrolysis using only one of papain or Bacillus licheniformis alkaline protease to hydrolyze the tea flower protein significantly reduced the pancreatic lipase inhibition rate of the tea flower peptide, and the α-glucosidase inhibition rate was also reduced. The possible reason is that both papain and Bacillus licheniformis alkaline protease have the characteristic of excessively hydrolyzing proteins, indicating that the sites where these enzymes cleave the tea flower peptide cannot effectively bind to pancreatic lipase, thereby reducing the pancreatic lipase inhibition rate of the tea flower peptide.

[0129] In Comparative Examples 5-6, the second hydrolysis was performed using only Aspergillus oryzae aminopeptidase to hydrolyze tea flower protein and Bacillus subtilis neutral protease. Compared with the examples, the inhibition rates of pancreatic lipase and α-glucosidase of tea flower peptides were both reduced.

[0130] 4. Effects of different process conditions on tyrosinase inhibition rate

[0131] (1) The polypeptide powders from the examples and comparative examples were prepared into a 20 mg / ml solution (using water as the solvent), and the tyrosinase inhibition rate was detected. For detailed data, please refer to [link to relevant data]. Figure 4 See Table 3. The method for detecting tyrosinase inhibition rate was performed in accordance with "T / SHRH015-2018 Shanghai Daily Chemical Industry Association Group Standard: Cosmetics - Test Method for Tyrosinase Activity Inhibition".

[0132] Table 3

[0133]

[0134] In the table, "-" indicates that it was not detected.

[0135] (2) Through detection data ( Figure 4 As can be seen from Table 3:

[0136] Examples 1-4 showed good tyrosinase inhibition rates, all above 65%, with Example 1 showing the highest tyrosinase inhibition rate at 72.59%.

[0137] Compared with Examples 1-4, the tyrosinase inhibition rates of the peptides obtained in Comparative Examples 1-8 were all reduced. The second hydrolysis in Comparative Example 7, which used only papain, and Comparative Example 8, which used only Bacillus licheniformis alkaline protease, resulted in lower tyrosinase inhibition rates for the tea flower protein, at 31.28% and 0%, respectively. This may be because both papain and Bacillus licheniformis alkaline protease have the characteristic of excessively hydrolyzing proteins. This indicates that the sites where both enzymes cleave tea flower peptides cannot effectively bind to tyrosinase, thus reducing the tyrosinase inhibition rate of the tea flower peptides.

[0138] 5. Effects on antioxidant activity

[0139] (1) The sample peptide powders from the examples and comparative examples were prepared into a 20 mg / ml solution (using water as the solvent), and their DPPH radical scavenging rate, ABTS radical scavenging rate, and hydroxyl radical scavenging rate were detected. Detailed detection data can be found in [link to relevant documentation]. Figure 5 And Table 4.

[0140] The detection methods for DPPH free radical scavenging rate, ABTS free radical scavenging rate and hydroxyl free radical scavenging rate are in accordance with "Isolation and characterization of three antioxidant peptides from protein hydrolysate of bluefin leatherjacket".

[0141] Table 4

[0142]

[0143] (2) From Figure 5 As can be seen from the data in Table 4:

[0144] Examples 1-4 exhibit extremely high scavenging rates for DPPH radicals, ABTS radicals, and hydroxyl radicals, demonstrating extremely high antioxidant activity, which is higher than that of the comparative examples. In particular, the scavenging rates of DPPH radicals and hydroxyl radicals in Examples 1-4 are all higher than 90%, and the scavenging rate of ABTS radicals is 100%.

[0145] In Comparative Examples 1-8, the ABTS radical scavenging rate of all comparative examples was higher than 90%. Except for Comparative Example 5, where the DPPH radical scavenging rate was 75.81%, the DPPH and hydroxyl radical scavenging rates of the other comparative examples were higher than 80%. However, the DPPH and hydroxyl radical scavenging rates were lower than those of the examples, indicating that the antioxidant activity of the peptides prepared in the comparative examples was relatively reduced compared to the examples.

[0146] The above results indicate that the enzymatic combination of specific complex enzymes (pectinase, hemicellulase, cellulase), specific Aspergillus oryzae aminopeptidase, and Bacillus subtilis neutral protease in the embodiments of the present invention can produce the best antioxidant activity.

[0147] 6. Effects on promoting the activity of beneficial bacteria

[0148] (1) The obtained tea tree flower peptide powder was used to detect the promotion rate of fermented Lactobacillus mucin. The specific results are shown in [the table below]. Figure 6 See Table 5. The detection method was based on "Study on the In Vitro Promotion of Probiotic Growth by Chinese Yam Polypeptide", with slight modifications.

[0149] The specific testing method is as follows:

[0150] MRS liquid culture medium was prepared and dispensed into 10 mL tubes. 300 μL of probiotic strain (Lactobacillus fermentum) was inoculated into each tube. The experimental group received 500 μL of 20 mg / mL tea tree flower peptide aqueous solution, while the control group received 500 μL of sterile water. The cultures were incubated at 37°C for 24 h. The OD was then measured after diluting the culture medium 20-fold. 600 Calculate the probiotic promotion rate.

[0151] Probiotic promotion rate % = (BA) / A x 100%;

[0152] Note: A: OD of the blank group 600 B: OD of the experimental group 600 .

[0153] The MRS liquid culture medium formula is as follows: 10.0g casein digest, 10.0g beef extract, 5.0g yeast extract, 2.0g triammonium citrate, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 5.0g sodium acetate, 1.08g Tween-80, 0.2g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·4H2O), and distilled water to a final volume of 1000mL, pH 5.7–5.9.

[0154] 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.

[0155] Table 5

[0156]

[0157] In the table, "-" indicates no proliferation-promoting effect.

[0158] (2) Through detection data ( Figure 6 As can be seen from Table 5:

[0159] The examples demonstrated good beneficial bacteria-promoting activity, with a proliferation promotion rate of over 9% for fermented *Lactobacillus mucinus*. In Example 3, the proliferation promotion rate of *Lactobacillus mucinus* reached 10.6%.

[0160] Comparative Example 8 did not show any promoting activity against beneficial bacteria; Comparative Examples 1-7 showed lower promoting activity than Examples 1-4. This indicates that the enzymatic combination of specific complex enzymes (pectinase, hemicellulase, cellulase), specific Aspergillus oryzae aminopeptidase, and Bacillus subtilis neutral protease in the embodiments of the present invention can produce polypeptides with better probiotic activity.

[0161] 7. Sensory characteristics of tea tree flower peptides

[0162] (1) The sample peptide powders of the examples and comparative examples were prepared into solutions of 20 mg / ml (solvent was water), and their sensory characteristics were evaluated from aspects such as taste and odor. The sensory evaluation results are shown in Table 6:

[0163] Table 6 Sensory Analysis and Evaluation Table

[0164]

[0165] The sensory levels in the table are divided into "heavy, medium, light, slight, and none".

[0166] (2) From the data in Table 6, we can see that:

[0167] Examples 1-4 all exhibited good taste and floral aroma. Comparative Examples 2-4 all had similar taste to the examples, while the floral aroma of Comparative Example 1 was weaker than that of the examples, and the taste of Comparative Examples 5-8 was more bitter than that of the examples.

[0168] This invention employs a combination of various enzymatic hydrolysis methods to prepare tea tree flower peptides. The resulting tea tree flower peptides have a rich tea tree floral aroma and, when applied to food, offer a superior taste and flavor, thus enhancing the user experience.

[0169] In summary, the method for preparing tea tree flower peptides provided by this invention has at least the following mechanisms of action and technical effects:

[0170] (1) This invention is the first to discover that tea flower peptides prepared by enzymatic hydrolysis of tea flower by pectinase, hemicellulase and cellulase complex enzymes, followed by enzymatic hydrolysis by Aspergillus oryzae aminopeptidase and Bacillus subtilis neutral protease complex protease have high anti-glycation ability, high α-glucosidase inhibition rate, high pancreatic lipase and tyrosinase inhibition activity, high antioxidant activity (high DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, ABTS free radical scavenging rate), and promote the activity of beneficial bacteria (fermenting Lactobacillus mucinus). It can be used as a functional factor in functional foods, cosmetics and health products.

[0171] 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). Tea tree flower 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.

[0172] By inhibiting the activity of α-glucosidase secreted by the small intestine, it can prevent the breakdown of oligosaccharides into monosaccharides such as glucose in the small intestine; by inhibiting the activity of lipase secreted by the intestine, it can prevent the breakdown of lipids in the intestine. Based on its high pancreatic lipase inhibitory activity, this tea tree flower peptide can be used in functional products (such as food and health products) to achieve functions such as weight loss; by inhibiting the secretion of tyrosinase by human melanocytes, it can prevent the synthesis of melanin from tyrosine. Based on its high α-glucosidase inhibition rate and high pancreatic lipase and tyrosinase inhibitory activity, this active peptide can be used in functional products (such as food and health products) to achieve functions such as lowering blood sugar, weight loss, and skin whitening.

[0173] Since the proliferation of fermented Lactobacillus mucin is beneficial to intestinal digestion and defecation, based on the prebiotic effect of this tea tree flower peptide in promoting the proliferation of fermented Lactobacillus mucin, its application in functional products (such as food) can play a role in aiding digestion and defecation.

[0174] Because free radicals are atoms or groups with highly reactive unpaired electrons, excessive free radicals and oxidants in the human body can cause various harms, including attacking body cells and breaking down body tissues. Based on the high antioxidant properties of this tea tree flower peptide, its application in functional products (such as food, health supplements, and skincare products) can provide functions such as health maintenance, alleviating aging, and preventing skin aging.

[0175] (2) The tea tree flower peptide is prepared by a combination of various enzymatic hydrolysis. The tea tree flower has a rich aroma. When the prepared tea tree flower peptide is used in food, it has a good taste and flavor, which is conducive to improving the user experience. Moreover, it is acid-resistant and heat-resistant, so it will not cause the problem of easy precipitation of plant peptides at high temperatures.

[0176] (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.

[0177] In summary, this invention extracts and purifies active polypeptides from tea tree flowers to obtain tea tree flower peptides with prebiotic effects, including anti-glycation, α-glucosidase inhibitory activity, pancreatic lipase inhibitory activity, tyrosinase inhibitory activity, antioxidant activity, and promotion of the proliferation of *Lactobacillus fermentum*. These peptides can be used as raw material components in functional foods, applied to products with effects such as weight loss, anti-oxidation, anti-glycation, whitening, regulating intestinal health, and preventing skin aging. This invention not only fills a gap in tea tree flower peptide research but also provides a theoretical basis and practical solution for the deep processing of tea tree flowers and the industrialization of tea tree flower peptides.

[0178] It should be noted that:

[0179] (1) Definition:

[0180] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0181] 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.

[0182] In this article, "promoting activity of fermenting Lactobacillus mucinus" refers to the ability to promote the proliferation of fermenting Lactobacillus mucinus.

[0183] In the text, "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazine (free radical).

[0184] In the text, "ABTS" refers to the free radical 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0185] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0186] The terms "ultrafiltration" and "nanofiltration" 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.

[0187] (2) Raw materials used in implementation:

[0188] The enzymes used, such as Aspergillus oryzae aminopeptidase, Bacillus subtilis neutral protease, pectinase, hemicellulase, cellulase, Bacillus licheniformis alkaline protease, and papain, are all commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0189] (3) Application of tea tree flower peptides:

[0190] Tea tree flower peptides possess the following characteristics: 1) anti-glycation activity; 2) inhibitory activity against α-glucosidase, pancreatic lipase, and tyrosinase; 3) antioxidant properties; and 4) beneficial bacteria-promoting activity. Based on these characteristics (1)-4), tea tree flower peptides can also be applied to functional products (including food, health supplements, cosmetics, skincare products, bath products, and cleaning products) that offer skincare, whitening, and antioxidant effects, among others.

[0191] 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.

[0192] 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.

[0193] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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 tea tree flower peptides, characterized in that... This includes the following steps: Tea tree flowers are dried and then pulverized to obtain tea tree flower powder. The tea tree flower powder is mixed evenly with a calcium hydroxide aqueous solution and extracted at 80–100°C for 0.5–6 hours. The extract is then centrifuged to separate the solid and liquid components, and the supernatant is collected to obtain a tea tree flower protein extract. The concentration of the calcium hydroxide aqueous solution is 0.1–0.4 mol / L, and the ratio of tea tree flower powder to calcium hydroxide aqueous solution is 1 kg: 10–40 kg. The pH of the tea flower protein extract was adjusted to 5.0–6.0, and a compound enzyme was added for the first hydrolysis. The first hydrolysis temperature was 40–50°C, and the first hydrolysis time was 100–240 min. Then, the enzyme was inactivated at 85–90°C for 20–30 min to obtain the first hydrolysate. The compound enzyme included pectinase, hemicellulase, and cellulase. The amount of the compound enzyme added was 1–5% of the mass of the tea flower powder, and the mass ratio of the pectinase, hemicellulase, and cellulase was 1:(0.8–1.2):(0.8–1.2). The pH of the first hydrolysate is adjusted to 6.0–7.0, and a second hydrolysis is performed by adding protease at a temperature of 45–60°C for 60–180 min. The enzyme is then inactivated at 85–90°C for 20–30 min to obtain the second hydrolysate. The protease comprises Aspergillus oryzae aminopeptidase and Bacillus subtilis neutral protease. The amount of Aspergillus oryzae aminopeptidase added is 0.1–1.0% of the mass of the tea flower, and the amount of Bacillus subtilis neutral protease added is 0.5–1.5% of the mass of the tea flower powder. The second hydrolysate is filtered to retain polypeptides with a molecular weight cutoff of 200-5000 Da, thus obtaining the tea tree flower peptide.

2. The method for preparing tea tree flower peptides according to claim 1, characterized in that: In the filtration step, the second hydrolysate is filtered to obtain a polypeptide filtrate with a polypeptide molecular weight of 200-5000 Da; the polypeptide filtrate is then concentrated to a polypeptide mass content of 12%-22%, and the concentrate is dried to form a powder.

3. A tea tree flower peptide, characterized in that: The tea tree flower peptide is prepared using the method described in any one of claims 1-2.

4. A functional product, characterized in that: Its components include tea flower peptides prepared by the preparation method according to any one of claims 1-2.

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