Preparation Method and Application of Polypeptides from Morus atropurpurea Roxb

Abstract

The present application provides a method for preparing protein mulberry polypeptide, which includes the steps of washing, pulverizing and enzymatically extracting protein mulberry leaves. After suitable glycosylation modification, the α-glucosidase inhibitory activity and antioxidant activity of the protein mulberry polypeptide are further improved, and it can better tolerate the digestive tract environment, and is suitable for preparing health products for assisting in reducing blood sugar.

Description

Technical Field

[0001] This application belongs to the field of health products. Specifically, this application provides a preparation method and application of protein mulberry polypeptide. Background Art

[0002] Mulberry leaves belong to the category of Chinese medicinal materials that can be used both medicinally and as food. Traditional Chinese medicine believes that it has the effects of calming the liver and improving eyesight, clearing the lungs and moistening dryness, dispersing wind-heat, etc. Modern medical research has also proven that many of its components have the effects of lowering blood sugar, lowering blood lipids, anti-inflammatory, antioxidant, etc.

[0003] Protein mulberry is a mulberry variety cultivated in the late 1990s of the last century. Its main feature is its high protein content. The protein content can account for more than 30% of the dry weight of mulberry leaves and can also account for more than 20% of the dry weight in tender branches. In the prior art, there are already a large number of methods for extracting protein mulberry protein, such as enzymolysis-assisted extraction and ultrasonic-assisted extraction.

[0004] How to further enhance the effect and stability of protein mulberry protein to make it have more significant effects in practical applications is one of the potential research directions in this field. Summary of the Invention

[0005] Based on the existing mature preparation process of protein mulberry polypeptide, the applicant introduced the glycosylation modification method, which effectively improved the α-glucosidase inhibitory activity and antioxidant activity of the product and could provide better stability during the simulation process.

[0006] On the one hand, this application provides a preparation method of protein mulberry polypeptide, and the method includes the steps of washing, pulverizing, and enzymatically extracting protein mulberry leaves.

[0007] Further, the method includes:

[0008] (1) Remove the petiole part of protein mulberry leaves, wash and dry them;

[0009] (2) Pulverize the dried protein mulberry leaves and sieve them to obtain protein mulberry leaf powder;

[0010] (3) Add the protein mulberry leaf powder to water with a mass of 6 - 8 times, add papain 400 - 600U / mL and cellulase 700 - 1000U / mL to the water, and adjust the pH to 6.5 - 7.5;

[0011] (4) Enzymatically hydrolyze at 50 - 60°C for 4 - 5h, inactivate the enzyme at 80 - 95°C for 5 - 15min to obtain an enzymolysis solution;

[0012] (5) Filter and concentrate the enzymolysis solution to obtain an enzymolysis extract;

[0013] (6) Dry the enzymolysis extract at 50 - 70°C, pulverize and sieve it to obtain enzymolysis powder.

[0014] Further, the method further includes a glycosylation modification step.

[0015] Further, in the glycosylation modification step, Gracilaria lemaneiformis gum is used for glycosylation modification.

[0016] Further, the glycosylation modification step is as follows:

[0017] (7) Prepare the hydrolysate powder obtained in step (6) into an aqueous solution with a concentration of 20 g / L, and prepare Gracilaria lemaneiformis gum into an aqueous solution with a concentration of 10 g / L;

[0018] (8) Mix the Gracilaria lemaneiformis gum aqueous solution and the hydrolysate powder aqueous solution obtained in step (7) in equal volume, place them in a water bath at 60 °C and react for 120 minutes, and then place them in a water bath at 85 °C and react for 60 minutes;

[0019] (9) Concentrate and freeze-dry the product of step (8).

[0020] On the other hand, the present application provides the use of the protein mulberry polypeptide prepared by the above method in the preparation of a health product with the efficacy of assisting in reducing blood sugar.

[0021] Further, the health product is a solid beverage.

[0022] Further, the solid beverage also contains cassia seed extract, dendrobium officinale extract, and Rana temporaria chensinensis extract.

[0023] Further, the solid beverage also contains maltulose, corn starch, malic acid, and sucralose.

[0024] Further, the preparation method of the Rana temporaria chensinensis extract is as follows: Add dry Rana temporaria chensinensis bodies to an acetic acid solution with a mass of 10 - 20 times that of the dry Rana temporaria chensinensis bodies and a concentration of 4 - 8% w / v, and soak for 0.5 - 1 hour; Ultrasonically assist in extracting the acetic acid solution and the dry Rana temporaria chensinensis bodies soaked therein 1 - 2 times, each time for 30 - 40 minutes; Filter and centrifuge to obtain an extract; Concentrate and freeze-dry the extract to obtain the Rana temporaria chensinensis extract.

[0025] In addition to the solid beverage dosage form, the health product of the present application can also be prepared into dosage forms such as tablets, capsules, and oral liquids, and excipients in these dosage forms can be conventionally selected by those skilled in the art.

[0026] In addition to mulberry leaves, the young petioles of mulberry leaves can also be used. When using raw materials without removing the petioles, the dosage of the enzyme needs to be appropriately increased and the enzymolysis time needs to be extended during enzymolysis.

[0027] The cassia seed extract and dendrobium officinale extract can be water extracts or alcohol extracts purchased commercially or prepared by conventional methods.

[0028] The Rana temporaria chensinensis extract is an extract obtained by using dried Rana temporaria chensinensis as raw material and through steps of soaking with acetic acid, ultrasonic-assisted extraction, concentration, and freeze-drying. Non-limiting examples are as follows: adding dried Rana temporaria chensinensis into an acetic acid solution with a mass 10 - 20 times that of the dried Rana temporaria chensinensis and with a concentration of 4 - 8% w / v, and soaking for 0.5 - 1 hour; performing ultrasonic-assisted extraction on the acetic acid solution and the soaked dried Rana temporaria chensinensis for 1 - 2 times, with each time lasting for 30 - 40 minutes; filtering and centrifuging to obtain an extract solution; concentrating and freeze-drying the extract solution to obtain the Rana temporaria chensinensis extract. Description of the Drawings

[0029] Figure 1 It is a flow chart for the preparation of the enzymolysis extract of Morus atropurpurea Roxb.

[0030] Figure 2 It is the α-glucosidase activity inhibition rate and DPPH free radical scavenging rate of the Morus atropurpurea Roxb. polypeptide with monosaccharide to trisaccharide glycosylation modification.

[0031] Figure 3 It is the α-glucosidase activity inhibition rate and DPPH free radical scavenging rate of the Morus atropurpurea Roxb. polypeptide with polysaccharide modification.

[0032] Figure 4 It is the change of the α-glucosidase activity inhibition rate of the glycosylated Morus atropurpurea Roxb. polypeptide after being treated in artificial gastric juice. Detailed Description of the Invention

[0033] Unless otherwise specified, the reagents and raw materials in the following examples are all domestic conventional food grades.

[0034] Example 1 Preparation of Morus atropurpurea Roxb. Polypeptide

[0035] According to the operation instruction manual for the extraction of Morus atropurpurea Roxb. enzymolysis powder formulated by the applicant, the basic preparation process of the Morus atropurpurea Roxb. enzymolysis powder is as follows, and refer to Figure 1 :

[0036] (1) Remove the petiole part of the Morus atropurpurea Roxb. leaves, wash and dry them.

[0037] (2) Crush and sieve the dried Morus atropurpurea Roxb. leaves to obtain Morus atropurpurea Roxb. leaf powder.

[0038] (3) Add the Morus atropurpurea Roxb. leaf powder into water with a mass 6.5 times that of the powder, add papain at 500 U / mL and cellulase at 800 U / mL to the water, and adjust the pH to 7.0.

[0039] (4) Perform enzymolysis at 55 °C for 4 h, inactivate the enzyme at 90 °C for 15 min to obtain an enzymolysis solution.

[0040] (5) Filter and concentrate the enzymolysis solution to obtain an enzymolysis extract paste.

[0041] (6) Dry the enzymolysis extract paste at 55 - 65 °C, crush and sieve it.

[0042] Every 100 kg of fresh Morus alba L. leaves can produce 30 kg of Morus alba L. polypeptide powder. Calculated by the Kjeldahl method, the protein extraction rate is about 32 - 35%. The Morus alba L. polypeptide powder is a brownish powder, which can be sealed and stored for later use. A homogeneous product of the same batch is used in the glycosylation experiment.

[0043] Example 2 Glycosylated Modified Morus alba L. Polypeptide

[0044] Similar to the research results of previous papers, the Morus alba L. extract has antioxidant and α-glucosidase activities. To further improve its antioxidant and α-glucosidase inhibitory activities, provide a better adjuvant hypoglycemic effect, and at the same time solve its stability problem during digestion (as described in Example 3 below), the applicant tried to carry out glycosylation modification on it.

[0045] Basic method:

[0046] The Morus alba L. polypeptide obtained by the process of Example 1 was formulated into an aqueous solution of 20 g / L and left standing at 4°C for 12 h to fully hydrate the protein and sugar components therein; various sugars were formulated into an aqueous solution of 10 g / L (dissolved with hot water if necessary), and the sugar aqueous solution was mixed with an equal volume of the Morus alba L. polypeptide aqueous solution and placed in a water bath at 60°C for 120 minutes, and then placed in a water bath at 85°C for 60 minutes; a solution product with a slightly darker color was obtained (the Morus alba L. enzymolysis extract solution itself has a slightly brownish color), which can be stored for later use in a liquid state at low temperature or concentrated and made into a freeze-dried powder for storage.

[0047] The α-glucosidase activity inhibition rate of the product was detected according to the following method:

[0048] The sample was formulated into a solution of 20 mg / mL; 25 μL of the sample solution and the distilled water control were respectively incubated with 50 μL of 1 U / mL α-glucosidase solution (prepared with 0.2 M PBS, pH 7.0) at 37°C for 10 min; 25 μL of 5 mM PNPG solution was added and incubated at 37°C for 5 min; the absorbance was measured at 405 nm.

[0049] α-glucosidase activity inhibition rate = (1 - (A sample - A sample blank / A control - A control blank)) × 100%

[0050] The DPPH free radical scavenging rate of the product was detected according to the following method:

[0051] Prepare a 5 mg / mL sample solution for the sample to be tested; take 2.5 mL of the liquid sample to be tested, mix it with 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution (sample) or 2.5 mL of anhydrous ethanol (control), and react in the dark at 25 °C for 30 min; measure the absorbance of the sample reaction solution, the control reaction solution, and the reference reaction solution (2.5 mL of anhydrous ethanol plus 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution) at 517 nm with a spectrophotometer; repeat for three groups.

[0052] DPPH radical scavenging rate % = (1 - (A_sample - A_control) / A_reference) × 100%.

[0053] The glycosylation modification effects of various sugars were tested, and the results are as Figure 2 and Figure 3 shown. Among monosaccharides to trisaccharides, only lactose and raffinose had relatively obvious positive effects on both indicators, while xanthan gum, locust bean gum, guar gum, and gloiopeltis furcata in polysaccharides all had relatively obvious improvement effects.

[0054] Further optimize the ratio of sugar to the enzymolysis extract of protein mulberry. It was found that when mixed with an equal volume of 20 g / L aqueous solution of protein mulberry polypeptide, the optimal concentrations of lactose and raffinose were 20 g / L, the optimal concentrations of xanthan gum, locust bean gum, and guar gum were 15 g / L, and the optimal concentration of gloiopeltis furcata was 10 g / L. The α-glucosidase activity inhibition rate and DPPH radical scavenging rate of various glycosylated modified protein mulberry enzymolysis extracts are shown in Table 1 at this time.

[0055] Table 1 Performance of glycosylated modified protein mulberry polypeptide after optimizing the ratio

[0056] Sugar α-Glucosidase activity inhibition rate DPPH free radical scavenging rate Lactose 57.2±0.8 63.5±0.7 Raffinose 55.3±0.1 61.7±1.2 Xanthan gum 56.8±0.1 67.9±0.8 Locust bean gum 55.9±0.6 64.9±0.1 Guar gum 58.7±0.4 68.2±0.3 Gloiopeltis furcata polysaccharide 57.1±0.5 66.3±1.1

[0057] Example 3 Stability of different glycosylated modified products during digestion

[0058] To investigate the possible effect changes of protein mulberry polypeptide during digestion, use simulated digestive fluid (modified from the pharmacopoeia, slightly adjusting the pH: 15 mL of 10% dilute hydrochloric acid plus 800 mL of water, adjusting the pH to 2.5 with 1 M NaOH) to prepare the product into the concentration described in the detection method of Example 2, and sample and detect the α-glucosidase activity inhibition rate after standing at room temperature for a certain time.

[0059] As Figure 4As shown, the inhibition rate of α-glucosidase activity of the protein mulberry polypeptide without glycosylation modification decreased rapidly in the simulated digestive fluid, and the activity could not be restored when the pH returned to neutral. This indicates that an irreversible change occurred in the spatial structure of the protein mulberry polypeptide at low pH. If solid beverages and oral liquid preparations are directly prepared without using treatment methods such as capsules, coatings, and inclusion complexes, their effects after passing through the stomach will be greatly reduced, and the introduction of methods such as capsules, coatings, and inclusion complexes has adverse effects on the sensory properties, taste, and potential safety of health products.

[0060] Monosaccharide glycosylation modification has no obvious effect on stability. In contrast, the protein mulberry polypeptide modified with polysaccharides has obvious improvements in this regard, especially the product modified with gloiopeltis tenax polysaccharide, such as Figure 4 As shown, within 15 minutes, the inhibition rate of its α-glucosidase activity did not decrease significantly. At 20 minutes, about 60% of the activity was still retained.

[0061] Example 4 Example of a protein mulberry polypeptide solid beverage health product

[0062] 52 parts by mass of glycosylated modified protein mulberry polypeptide, 7 parts by mass of cassia seed extract, 5 parts by mass of dendrobium officinale extract, 1.8 parts by mass of Rana temporaria chensinensis extract, 30 parts by mass of isomaltulose, 4 parts by mass of corn starch, 0.1 part by mass of malic acid, 0.1 part by mass of sucralose.

Claims

1. A method for preparing protein mulberry polypeptide, characterized in that: The method comprises: (1) removing the petiole of the mulberry leaves, washing and drying; (2) crushing and sieving the dried protein mulberry leaves to obtain protein mulberry leaf powder; (3) Add 6.5 times the mass of protein mulberry leaf powder to water, add 500 U / mL of papain and 800 U / mL of cellulase to the water, and adjust the pH to 7.0; (4) enzymolysis at 55°C for 4 h, and then inactivate the enzyme at 90°C for 15 min to obtain an enzymolysis solution; (5) filtering and concentrating the enzymolyzate to obtain an enzymolyzate extract; (6) drying the enzymolyte extract at 55-65° C., crushing and sieving to obtain enzymolyte powder; (7) preparing a 20 g / L aqueous solution of the enzymatic hydrolysate powder obtained in step (6), and preparing a 10 g / L aqueous solution of the seaweed gum; (8) mixing the sea urchin gum aqueous solution obtained in step (7) and the enzymatic hydrolysate powder aqueous solution in equal volumes, placing in a 60° C. water bath to react for 120 minutes, and then placing in a 85° C. water bath to react for 60 minutes; (9) Concentrating and freeze-drying the product of step (8) to obtain protein mulberry polypeptide.

2. Use of the protein mulberry polypeptide prepared by the method according to claim 1 in the preparation of a health product with auxiliary blood sugar lowering effect.

3. The use according to claim 2, wherein the health product is a solid beverage.

4. The use according to claim 3, wherein the solid beverage further comprises one or more of radix strychnifoliae extract, cassia seed extract, dendrobium officinale extract, rana extract, hawthorn extract, maltulose, corn starch, malic acid, and sucralose.

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