Preparation method of loquat active peptide

Abstract

The invention relates to the technical field of deep processing of loquats, and particularly discloses a preparation method of a loquat active peptide. The preparation method comprises the following steps: denucleating fresh loquats, pulping, preparing water, degrading cellulose and pectin, performing enzyme digestion modification, performing solid-liquid separation, performing enzyme deactivation, adding monomethoxy polyethylene glycol, performing combined modification, performing centrifugal purification, performing nanofiltration to remove polyethylene glycol, and performing concentration until the diopter is 50-55% so as to obtain a loquat active peptide liquid product. According to the combined modification technology, monomethoxy polyethylene glycol is adopted as a modifier, protein drugs are well modified, the in-vivo half-life period of the product is prolonged, immunogenicity is reduced, and meanwhile the water solubility of the product can be improved. The loquat active peptide prepared by the method is high in yield, the obtained active peptide is high in activity, the active peptide is mainly 2-6 peptides, and the glycopeptide yield of the active peptide reaches 90% or above.

Description

Technical Field

[0001] The invention relates to the technical field of loquat deep processing, and in particular to a method for preparing loquat active peptide. Background Art

[0002] Loquat (Eriobotrya japonica), commonly known as Luju, Luju, and Jinwan, belongs to the genus Eriobotrya in the Rosaceae family. As a plant with both medicinal and edible properties, loquat has significant development potential in functional foods, health supplements, and pharmaceuticals. Loquat has numerous beneficial effects and benefits for the human body. For example, loquat is rich in vitamins such as B and C, as well as nutrients such as protein and citric acid. The amygdalin contained in loquat has the effects of moistening the lungs, relieving coughs, and reducing phlegm. Furthermore, the large amount of organic acids contained in loquat can stimulate the digestive glands, promote digestion and absorption of food, and help alleviate various symptoms caused by indigestion. Loquat is also rich in carotene, which can be converted into vitamin A in the body. Furthermore, loquat can be used to prevent influenza, lung heat cough, wind-heat cough, and chronic cough due to lung deficiency.

[0003] The crude protein content in loquat leaves can reach 8-12%, and it contains a variety of functional amino acids (such as glutamic acid and aspartic acid), which have antioxidant and anti-inflammatory potential. At present, research on the chemical composition of loquat is mostly focused on amygdalin and trace elements, and the development of loquat protein resources is relatively lagging. Traditional extraction methods have problems such as high temperature, low efficiency and high cost. Specifically, the decoction method is performed under high temperature conditions (80-100°C), which will destroy heat-sensitive proteins (peptide chain breakage and loss of function), and the extraction rate is <30%. Organic solvent extraction will cause protein denaturation, and the subsequent desolvation cost is high. The alcohol precipitation method can only separate large molecular polysaccharides (>10kDa), and oligosaccharides and small molecular peptides are seriously lost. At present, there is no enzymatic hydrolysis-directed preparation technology for loquat, so the application of loquat is limited.

[0004] Traditional proteins (such as whey protein) require gastric acid and enzymatic hydrolysis before they can be absorbed, with a bioavailability of only 40-60%. Peptides with a molecular weight of less than 1 kDa can be directly absorbed through the intestinal mucosa, with an utilization rate of over 90%. With the development of modern society, people's pace of life is constantly accelerating. Under the influence of various life and work pressures, people are often more prone to fatigue, causing varying degrees of chronic damage to the body. Based on this, the demand for health foods is gradually increasing. Although health products containing nutrients such as protein, vitamins, polysaccharides, or trace elements are currently available on the market, the absorption and metabolism of these nutrients in the human body are limited by various factors, resulting in low absorption rates. Therefore, it is of great significance to develop a small molecule loquat active peptide that is more easily absorbed and utilized by the human body and can be widely used in nutritional formula foods, health products, special medical foods, and pharmaceuticals.

[0005] In addition, on the one hand, due to differences in understanding among those skilled in the art; on the other hand, because the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] In view of the problems that the existing loquat production processing technology is backward, the active substances are difficult to control, and the product nutrition and efficacy are low, the present invention provides a method for preparing loquat active peptides, which comprises the following steps:

[0007] (1) removing the core of the loquats and beating them into pulp, adding 8 to 9 times of purified water to obtain a diluted loquat pulp;

[0008] (2) Enzymatic hydrolysis: The temperature of the diluted loquat slurry was raised to 56-58°C, and cellulase, pectinase, neutral protease and aminopeptidase were added in sequence for enzymatic hydrolysis. During the enzymatic hydrolysis, the stirrer speed was controlled at 39-41 r / min, and the enzymatic hydrolysis time was 3-4 h.

[0009] (3) enzyme inactivation;

[0010] (4) after inactivating the enzyme, filtering the mixture using a plate filter press to obtain a loquat filtrate;

[0011] (5) controlling the temperature of the loquat filtrate at 35-37° C., adding 0.1-0.2% of monomethoxy polyethylene glycol by weight of the raw material, maintaining the stirrer speed at 35-45 r / min, and combining and modifying for 5-5.5 hours to obtain a synthetic liquid;

[0012] (6) centrifuging the synthetic solution using a three-phase centrifuge to obtain a centrifuge;

[0013] (7) The centrifuged liquid is filtered to remove polyethylene glycol, and the nanofiltrate is retained;

[0014] (8) The nanofiltrate is concentrated by a double-effect concentrator, and the refractive index of the concentrate reaches 50-55%, thereby obtaining a liquid product of loquat active peptide.

[0015] Optionally, in step (2), the amount of cellulase added is 1 to 1.2% of the loquat raw material.

[0016] Specifically, in step (2), the amount of cellulase added is 1% of the loquat raw material.

[0017] Specifically, in step (2), the amount of cellulase added is 1.1% of the loquat raw material.

[0018] Specifically, in step (2), the amount of cellulase added is 1.2% of the loquat raw material.

[0019] Optionally, in step (2), the amount of pectinase added is 0.8-1% of the loquat raw material.

[0020] Specifically, in step (2), the amount of pectinase added is 0.8% of the loquat raw material.

[0021] Specifically, in step (2), the amount of pectinase added is 0.9% of the loquat raw material.

[0022] Specifically, in step (2), the amount of pectinase added is 1% of the loquat raw material.

[0023] Optionally, in step (2), the amount of neutral protease added is 1 to 1.2% of the loquat raw material.

[0024] Specifically, in step (2), the amount of neutral protease added is 1% of the loquat raw material.

[0025] Specifically, in step (2), the amount of neutral protease added is 1.1% of the loquat raw material.

[0026] Specifically, in step (2), the amount of neutral protease added is 1.2% of the loquat raw material.

[0027] Optionally, in step (2), the amount of aminopeptidase added is 0.3-0.5% of the loquat raw material.

[0028] Specifically, in step (2), the amount of aminopeptidase added is 0.3% of the loquat raw material.

[0029] Specifically, in step (2), the amount of aminopeptidase added is 0.4% of the loquat raw material.

[0030] Specifically, in step (2), the amount of aminopeptidase added is 0.5% of the loquat raw material.

[0031] Optionally, in step (3), the temperature of the enzymatically hydrolyzed loquat slurry is raised to 92-95° C., and the enzyme is inactivated for 10-13 minutes.

[0032] Optionally, in step (5), the molecular weight of the monomethoxypolyethylene glycol used is 2000 to 3000 Daltons.

[0033] Optionally, in step (6), the rotation speed of the three-phase centrifuge is maintained at 16000-18000 r / min.

[0034] Optionally, in step (7), when removing polyethylene glycol by nanofiltration, the pore size of the nanofiltration membrane used is 0.001 to 0.002 microns.

[0035] A loquat active peptide liquid product prepared by the above-mentioned loquat active peptide preparation method has a glycopeptide rate of ≥90%. The glycopeptide rate of the present invention is the ratio of glycopeptide to the obtained product.

[0036] The aforementioned preparation method of loquat active peptide and the use of the loquat active peptide liquid product prepared by the aforementioned preparation method of loquat active peptide in the preparation of nutritional formula foods, health products, special medical foods or medicines.

[0037] The technical solution of the present invention has the following advantages:

[0038] 1. Loquat fruit is rich in cellulose and pectin. The present invention first adds cellulose and pectinase, and utilizes these two enzymes to degrade the loquat fruit, which is beneficial to reducing the viscosity of the liquid and separating the trace elements in the loquat fruit. This operation is beneficial to subsequent processing steps and maintains the clarity of the product.

[0039] 2. On the other hand, the preparation method disclosed in the present invention performs enzymatic hydrolysis and peptide bond modification on loquat protein by adding neutral protease and aminopeptidase, so that the protein degradation is more thorough, the product yield is higher, the activity of active peptides is high, and they are mainly 2 to 6 peptides, the glycopeptide rate is more than 90%, the taste is better, and the human body absorption effect is better.

[0040] 3. The present invention uses monomethoxy polyethylene glycol combined modification to significantly increase the effective concentration and duration of active peptides in vivo through mechanisms such as extended circulation, targeted enrichment, anti-enzymatic hydrolysis, and conformational optimization, further improving the activity of loquat active peptides and meeting the different requirements of pharmaceutical grade. Combination modification has the advantages of improving their solubility and stability, reducing immunogenicity, improving biodistribution and targeting, simple reaction mechanism, versatility, and adjustable molecular weight. In addition, in the combination modification technology used in the present invention, the molecular weight of monomethoxy polyethylene glycol is 2000-3000 Daltons, and the hydroxyl groups on its molecular chain can be close to each other, resulting in a greater effect of hydrogen bonding, promoting the combination modification of loquat peptides and the synthesis of other active substances in loquat. The combination modification technology used in the present invention achieves flexible restriction of cyclized active peptides, stabilizes the secondary structure of the polypeptide, and improves the affinity of the modified peptides with receptors. In addition, the combination modification technology uses monomethoxy polyethylene glycol as a modifying agent, which is better for modifying protein drugs, increasing the half-life of the product in vivo, reducing immunogenicity, and also increasing the water solubility of the product. Specifically, the loquat active peptides prepared by the present invention have a high absorption rate, a short peak time, and a high peak concentration; in terms of specific component content, the synthesis rate of small peptides and amygdalin is high; in terms of active ingredient purity, the content of composite peptides is high, reaching more than 96%; cell growth and metabolism are significantly affected; and cell signaling pathways are obvious.

[0041] 4. The nanofiltration technology of the present invention uses nanofiltration to remove polyethylene glycol, and the pore size of the nanofiltration membrane is 0.001 to 0.002 microns. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present disclosure. In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, it is obvious that one or more embodiments can also be implemented without these specific details. In addition, in the following description, the description of the known technology is omitted to avoid unnecessary confusion of the concept of the present disclosure. In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially. All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used here should be interpreted as having meanings consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0043] In the present invention, "inactivating an enzyme" means terminating an enzyme reaction or terminating enzyme activity.

[0044] In the present invention, "small molecule peptide" refers to a short peptide chain with a small molecular weight (eg, <1000 Da), usually composed of 2 to 10 amino acids.

[0045] Example 1

[0046] This embodiment provides a method for preparing loquat active peptide. The preparation method comprises the following steps:

[0047] (1) 1000 g of loquats were pitted and pulped, and 8000 ml of purified water were added to obtain a diluted loquat pulp;

[0048] (2) raising the temperature of the diluted loquat slurry to 56° C., adding 10 g of cellulase, 8 g of pectinase, 10 g of neutral protease, and 3 g of aminopeptidase in sequence for enzymatic hydrolysis, controlling the stirrer speed at 39 r / min, and performing enzymatic hydrolysis for 3 h to obtain enzymatically hydrolyzed loquat slurry;

[0049] (3) heating the enzymatically hydrolyzed loquat slurry to 95°C and inactivating the enzyme for 10 min;

[0050] (4) after inactivating the enzyme, filtering the mixture using a plate filter press to obtain a loquat filtrate;

[0051] (5) The temperature of the loquat filtrate was controlled at 35°C, 1 g of monomethoxy polyethylene glycol was added to the loquat filtrate, the stirrer speed was maintained at 35 r / min, the temperature was maintained at 35°C, and the combination modification was performed for 5 hours to obtain a synthetic solution;

[0052] (6) Purifying the synthetic liquid by centrifugation in a three-phase centrifuge at a speed of 16,000 r / min to obtain a centrifuge;

[0053] (7) The centrifuged liquid is filtered to remove polyethylene glycol, and the nanofiltrate is retained;

[0054] (8) The nanofiltrate is further concentrated by a double-effect concentrator until the refractive index of the concentrate reaches 50%, thereby obtaining a liquid product of loquat active peptide.

[0055] According to this example, the activity of the neutral protease is 1.2×10 5 μ / g, aminopeptidase activity was 4.0×10 5 μ / mL.

[0056] Example 2

[0057] The present invention provides another method for preparing loquat active peptide, which comprises the following steps:

[0058] (1) 100 kg of loquats were pitted and pulped, and 850 L of purified water were added to obtain a diluted loquat slurry;

[0059] (2) raising the temperature of the diluted loquat slurry to 57° C., and sequentially adding 1.1 kg of cellulase, 0.9 kg of pectinase, 1.1 kg of neutral protease, and 0.4 kg of aminopeptidase thereto for enzymatic hydrolysis. The stirrer speed was controlled at 40 r / min, and the enzymatic hydrolysis time was 3.5 h to obtain the enzymatically hydrolyzed loquat slurry;

[0060] (3) The enzymatically hydrolyzed loquat slurry was heated to 92°C and the enzyme was inactivated for 13 min;

[0061] (4) after inactivating the enzyme, filtering the mixture using a plate filter press to obtain a loquat filtrate;

[0062] (5) The temperature of the loquat filtrate was controlled at 36°C, 110 g of monomethoxy polyethylene glycol was added to the loquat filtrate, the stirrer speed was maintained at 36 r / min, the temperature was maintained at 36°C, and the combination modification was performed for 5 hours to obtain a synthetic solution;

[0063] (6) Purifying the synthetic liquid by centrifugation in a three-phase centrifuge at a speed of 17,000 r / min to obtain a centrifuge;

[0064] (7) The centrifuged liquid is filtered to remove polyethylene glycol, and the nanofiltrate is retained;

[0065] (8) The nanofiltrate is further concentrated by a double-effect concentrator, and the refractive index of the concentrate reaches 52%, thereby obtaining a liquid product of loquat active peptide.

[0066] Example 3

[0067] The present invention provides another method for preparing loquat active peptide, which comprises the following steps:

[0068] (1) 500 kg of loquats were pitted and pulped, and 4500 L of purified water were added to obtain a diluted loquat slurry;

[0069] (2) raising the temperature of the diluted loquat slurry to 58° C., adding 6 kg of cellulase, 5 kg of pectinase, 6 kg of neutral protease, and 2.5 kg of aminopeptidase in sequence for enzymatic hydrolysis, controlling the stirrer speed at 45 r / min, and performing enzymatic hydrolysis for 4 h to obtain enzymatically hydrolyzed loquat slurry;

[0070] (3) heating the enzymatically hydrolyzed loquat slurry to 95°C and inactivating the enzyme for 10 min;

[0071] (4) after inactivating the enzyme, filtering the mixture using a plate filter press to obtain a loquat filtrate;

[0072] (5) The temperature of the loquat filtrate was controlled at 37°C, 1 kg of monomethoxy polyethylene glycol was added to the loquat filtrate, the stirrer speed was maintained at 45 r / min, the temperature was maintained at 37°C, and the combination modification was carried out for 5.5 hours to obtain a synthetic solution;

[0073] (6) The synthetic liquid was centrifuged and purified using a three-phase centrifuge at a speed of 18,000 r / min to obtain a centrifuge;

[0074] (7) The centrifuged liquid is then filtered to remove polyethylene glycol, and the nanofiltrate is retained;

[0075] (8) The nanofiltrate is further concentrated by a double-effect concentrator, and the refractive index of the concentrate reaches 55%, thereby obtaining a liquid product of loquat active peptide.

[0076] Product molecular weight distribution Proportion Body absorption rate Less than 1000 Daltons 95％ 98％ 2-3 peptide 49％ 99.99％ amino acids 7％ 30％

[0077] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the description of the present invention is illustrative and does not constitute a limitation on the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts, such as "optionally" indicating that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the feature guided by "optionally" is only an optional method and should not be understood as a must-have setting. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A method for preparing loquat active peptide, characterized in that: The steps include: (1) removing the core of the loquats and beating them into pulp, adding 8 to 9 times of purified water to obtain a diluted loquat pulp; (2) Enzymatic hydrolysis: The temperature of the diluted loquat slurry was raised to 56-58°C, and cellulase, pectinase, neutral protease, and aminopeptidase were added respectively for enzymatic hydrolysis. During the enzymatic hydrolysis, the stirrer speed was controlled at 39-41 r / min, and the enzymatic hydrolysis time was 3-4 h. (3) enzyme inactivation; (4) after inactivating the enzyme, filtering the mixture using a plate filter press to obtain a loquat filtrate; (5) controlling the temperature of the loquat filtrate at 35-37° C., adding 0.1-0.2% of monomethoxy polyethylene glycol by weight of the raw material, maintaining the stirrer speed at 35-45 r / min, and combining and modifying for 5-5.5 hours to obtain a synthetic liquid; (6) centrifuging the synthetic liquid in a three-phase centrifuge at a speed of 16,000 to 18,000 r / min to obtain a centrifuge; (7) The centrifuged liquid is then filtered to remove polyethylene glycol, and the nanofiltrate is retained; (8) The nanofiltrate is concentrated by a double-effect concentrator, and the refractive index of the concentrate reaches 50-55%, thereby obtaining a liquid product of loquat active peptide.

2. The method for preparing loquat active peptide according to claim 1, characterized in that: In step (2), the amount of cellulase added is 1 to 1.2% of the loquat raw material.

3. The method for preparing loquat active peptide according to claim 1, characterized in that: In step (2), the amount of pectinase added is 0.8-1% of the loquat raw material.

4. The method for preparing loquat active peptide according to claim 1, characterized in that: In step (2), the amount of the neutral protease added is 1 to 1.2% of the loquat raw material.

5. The method for preparing loquat active peptide according to claim 1, characterized in that: In step (2), the amount of aminopeptidase added is 0.3-0.5% of the loquat raw material.

6. The method for preparing loquat active peptide according to claim 1, characterized in that: In step (3), the temperature of the enzymatically hydrolyzed loquat slurry is raised to 92-95° C., and the enzyme is inactivated for 10-13 minutes.

7. The method for preparing loquat active peptide according to claim 1, characterized in that: In step (5), the molecular weight of the monomethoxy polyethylene glycol used is 2000 to 3000 Daltons.

8. The method for preparing loquat active peptide according to claim 1, characterized in that: In step (7), when removing polyethylene glycol by nanofiltration, the pore size of the nanofiltration membrane used is 0.001 to 0.002 microns.

9. The loquat active peptide liquid product prepared by the preparation method of loquat active peptide according to any one of claims 1 to 8, characterized in that: The glycopeptide rate in the loquat active peptide liquid product is ≥90%.

10. The preparation method of the loquat active peptide according to any one of claims 1 to 8 and use of the loquat active peptide liquid product according to claim 9 in the preparation of nutritional formula foods, health products, special medical foods or medicines.