阅读说明：本技术 一种除湿的发酵型小分子肽及其制备方法和应用 (Dehumidifying fermented small molecular peptide and preparation method and application thereof ) 是由 赵海峰 赵国安 康明 葛振龙 皮子凤 刘志强 杨茂君 高波 韩葳葳 李婉南 韩璐 于 2020-12-22 设计创作，主要内容包括：本发明涉及一种除湿的发酵型小分子肽及其制备方法和应用,所述发酵型小分子肽包括ADFW和HADF。本发明以红色食品为原料,通过采用合适的水解条件,制备得到了富含本发明小分子肽的水解物,本发明的小分子肽具有较高的抗氧化活性,由此所得到的多肽水解物对于除湿,特别是治疗丘疹样荨麻疹、湿疹、皮肤瘙痒具有重要意义。有望在抗衰老、美容方面取得功效。(The invention relates to a dehumidifying fermentation type small molecular peptide and a preparation method and application thereof, wherein the fermentation type small molecular peptide comprises ADFW and HADF. The hydrolysate rich in the small molecular peptide is prepared by taking red food as a raw material and adopting proper hydrolysis conditions, the small molecular peptide has higher antioxidant activity, and the obtained polypeptide hydrolysate has important significance for dehumidifying, particularly treating papular urticaria, eczema and skin pruritus. Is expected to have the effects of resisting aging and beautifying.)

1. A dehumidified, fermentable small molecule peptide comprising ADFW and HADF.

2. The small molecule peptide according to claim 1, wherein the content of ADFW is 0.9 wt% or more and the content of HADF is 0.7 wt% or more.

3. Use of the small molecule peptide of claim 1 or 2 as an antioxidant and/or for dehumidification.

4. Hydrolysate containing fermentable small-molecule peptides according to claim 1 or 2, wherein the short peptides are contained in an amount of 60% by weight or more.

5. The method for preparing the hydrolysate of claim 4, wherein the hydrolysate is prepared by the following steps:

(1) 200-300 parts by weight of red bean; red riceRed riceRed pumpkinPeanutHawBrown sugar50-100 of Chinese dates; red quinoaChinese wolfberry fruit Hericium erinaceus powder10-30 parts of poria cocos; roselle flowerRed ginsengBreaking cell wall, pulverizing, and dissolving in water;

(2) adding mixture of cellulase and pectinase, adjusting pH to 4-5, heating the reaction solution to 50-60 deg.C, placing into a stirrer, and performing enzymatic hydrolysis for 1-3 hr;

(3) adding trypsin and bromelain after hydrolysis, uniformly stirring to obtain a reaction solution, adjusting the pH of the reaction solution to 7-8, heating the reaction solution to 50-60 ℃, putting the reaction solution into a stirrer, and carrying out enzymatic hydrolysis for 2-4 hours;

(4) adding serine protease after hydrolysis, and performing enzymatic hydrolysis at pH6.5-7.5 and 50-60 deg.C for 3-5 hr;

(5) denaturing the enzyme in a heat treatment;

(6) centrifuging and recovering the liquid phase;

(7) ultrafiltering and percolating the liquid phase through a 4-6kDa membrane and recovering the permeate;

(8) then the permeate from the ultrafiltration is subjected to nanofiltration through a 150-300Da membrane and the retentate is recovered;

(9) freeze drying to obtain hydrolysate.

6. The method according to claim 5, wherein the hydrolysate is obtained.

7. Use of the hydrolysate of claim 4 or 6 for dehumidification.

8. Use of the hydrolysate of claim 4 or 6 for the preparation of a medicament or food for the treatment of papular urticaria, eczema and/or cutaneous pruritus.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a dehumidifying fermented small molecular peptide, and a preparation method and application thereof.

Background

Red food refers to food in red, orange red or red brown. The food is rich in natural active plant pigment (such as lycopene, anthocyanin, capsorubin, betacyanin and the like), carotene, iron, protein and partial amino acid, is an important source of high-quality protein, carbohydrate, dietary fiber, B vitamins and various inorganic salts, and can make up the nutritional deficiency in polished round-grained rice and white noodles. Scientific research shows that the red food has the functions of treating iron-deficiency anemia and relieving fatigue, preventing and treating tumor diseases such as breast cancer, etc., gives people excitement, increases appetite, smoothens skin, enhances epidermal cell regeneration, prevents skin aging, prevents cold, etc. The red food can further improve the utilization rate of the nutrition in the staple food after being eaten frequently, and the hawthorn, red ginseng and other foods have the effect of treating cancers. It is called "red Shengli Jun". "nutritionists think that the most typical advantage of red vegetables and fruits is that they are all foods rich in natural iron, for example, peanuts, Chinese dates and the like which are frequently eaten are all natural good medicines for anemia patients, and are also suitable for nourishing after blood loss in female menstrual period. Therefore, the women can eat the red vegetables and fruits as much as possible. The red food also contains 'killers' of pathogenic microorganisms, namely macrophages, so that the red food can effectively resist microorganisms such as cold viruses and the like, and the cold resistance of a human body is enhanced.

However, the conventional application methods of the red food are mostly developed and utilized as common food or active natural plant pigment extracted from the common food, and the protein components in the red food are rarely concerned. Scientific research shows that protein hydrolysates (small molecular peptides) of various foods have strong antioxidant capacity, and common antioxidants such as vitamin E and the like in the market can remove active oxygen, but have weak effect of repairing the caused damage and generate side effect. The invention develops and utilizes the composite small molecular peptide in the red food, makes up for the defect that the common antioxidant does not have the repair function after damage, and more comprehensively develops and utilizes the nutritional value of the red food.

The preparation of antioxidant peptides by enzymatic hydrolysis of proteins is the mainstream method, but because many proteases have specific cleavage sites and the uniqueness of protein amino acid sequences, the selection of different proteins and enzyme combinations has important significance for the generation of peptide compounds with unique functionality. In the case of the existing enzymatic methods, some of them use non-specific or widely specific proteases, and such methods have low production reproducibility and produce free amino acids, or the peptide chain is too small to be hydrolyzed to cause reduced functionality, and the yield of the final product is also low, and generally, there is no method for stably and efficiently preparing antioxidant peptides.

The inventor of the application previously applies CN111357865A to disclose a red food composite small molecule peptide, a preparation method and application in cell repair, which comprises the following raw materials: red bean, red rice, red pumpkin, peanut, Chinese date, hawthorn, quinoa, wolfberry fruit, roselle and red ginseng; the preparation method of the composite small molecule peptide based on the red food raw material comprises the following steps of preparing raw material powder; preparing a fermentation product; preparing an enzymolysis product; and (3) preparing a finished product of the composite small molecular peptide. The composite small molecular peptide provided by the invention has the characteristics of pure nature, no additive, bright red color, attractive appearance, strong appetite, wide range of people for eating, good taste and high nutritional value, and has the health-care effects of repairing damaged cells, delaying senescence, removing oxygen free radicals, tonifying qi and blood and enhancing immunity. However, since the content of the active ingredient in the polypeptide hydrolysate is low, particularly, the antioxidative property of ADF as an active ingredient is yet to be further improved, and thus the dehumidifying effect of the resulting polypeptide hydrolysate is yet to be further improved.

Disclosure of Invention

The invention aims to provide a dehumidifying fermented small molecular peptide and a preparation method and application thereof. Specifically, in order to achieve the purpose of the present invention, the following technical solutions are proposed:

the invention relates to a dehumidifying fermentation type small molecule peptide, which comprises ADFW and HADF.

In a preferred embodiment of the present invention, the content of ADFW is 0.9 wt% or more and the content of HADF is 0.7 wt% or more.

The invention also relates to a hydrolysate containing the fermentation type small molecule peptide, which contains more than 60 wt% of short peptide, wherein the short peptide refers to peptide with the peptide chain length of less than 10 and more than 3.

In another aspect, the present invention relates to a method for preparing the hydrolysate, which is characterized by comprising the following steps:

(1) 200-300 parts by weight of red bean; red riceRed riceRed pumpkinPeanutHawBrown sugar50-100 of Chinese dates; red quinoaChinese wolfberry fruit Hericium erinaceus powder10-30 parts of poria cocos; roselle flowerRed ginsengBreaking cell wall, pulverizing, and dissolving in water;

(2) adding mixture of cellulase and pectinase, adjusting pH to 4-5, heating the reaction solution to 50-60 deg.C, placing into a stirrer, and performing enzymatic hydrolysis for 1-3 hr;

(3) adding trypsin and bromelain after hydrolysis, uniformly stirring to obtain a reaction solution, adjusting the pH of the reaction solution to 7-8, heating the reaction solution to 50-60 ℃, putting the reaction solution into a stirrer, and carrying out enzymatic hydrolysis for 2-4 hours;

(4) adding serine protease after hydrolysis, and performing enzymatic hydrolysis at pH6.5-7.5 and 50-60 deg.C for 3-5 hr;

(5) denaturing the enzyme in a heat treatment;

(6) centrifuging and recovering the liquid phase;

(7) ultrafiltering and percolating the liquid phase through a 4-6kDa membrane and recovering the permeate;

(8) then the permeate from the ultrafiltration is subjected to nanofiltration through a 150-300Da membrane and the retentate is recovered;

(9) freeze drying to obtain hydrolysate.

The invention further relates to the application of the small molecule peptide as an antioxidant and/or a dehumidifying agent.

In another aspect, the invention also relates to the application of the hydrolysate in dehumidification and the application in preparing medicines or foods for treating papular urticaria, eczema and/or cutaneous pruritus.

Advantageous effects

The hydrolysate rich in the small molecular peptide is prepared by adopting proper hydrolysis conditions, the small molecular peptide has high antioxidant activity, and the obtained polypeptide hydrolysate has important significance for dehumidifying, particularly treating papular urticaria, eczema and skin pruritus. Is expected to have the effects of resisting aging and beautifying.

Detailed Description

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1:

(1) weighing 300g of phaseolus calcaratus, 300g of red rice, 300g of red comedy rice, 300g of red pumpkin, 200g of peanut, 150g of hawthorn, 150g of brown sugar, 100g of Chinese date, 50g of quinoa wheat, 30g of medlar, 30g of hericium erinaceus powder, 30g of poria cocos, 30g of roselle and 30g of red ginseng; cleaning with water, oven drying to remove water, breaking cell wall with cell wall breaking machine, and pulverizing;

(2) adding mixture of cellulase (2.0%/DM) and pectase (1.0%/DM), adjusting pH to 4.5, heating reaction solution to 55 deg.C, placing into a stirrer, reacting at 40rpm, and performing enzymatic hydrolysis for 2 hr;

(3) hydrolyzing for 2 hours, adding trypsin (1.5%/DM) and bromelain (2.0%/DM), stirring uniformly to obtain a reaction solution, adjusting the pH of the reaction solution to 7.5, heating the reaction solution to 55 ℃, putting the reaction solution into a stirrer, and reacting at 40rpm for 3 hours;

(4) after hydrolysis, serine protease (1.5% DM) was added and enzymatic hydrolysis was carried out at pH 7.0 and a temperature of 55 ℃ for 4 hours;

(5) heat treatment at 85 ℃ for 45 minutes to denature the enzyme;

(6) centrifuging and recovering the liquid phase;

(7) the liquid phase is first ultrafiltered and diafiltered through a 5kDa membrane and the permeate is recovered;

(8) then nano-filtering the permeate from the ultrafiltration through a 200Da membrane and recovering the retentate;

(9) freeze drying to obtain hydrolysate. The hydrolysate was tested to contain 62% by weight of short peptides, 27% polysaccharides and 11% other materials.

Comparative example 1:

reference is made to example 1, with the difference that no trypsin is added in step (3). The hydrolysate thus obtained, tested, contained 47% by weight of short peptides, 35% of polysaccharides and 18% of other substances.

Comparative example 2:

reference is made to example 1, with the difference that no bromelain is added in step (3). The hydrolysate thus obtained, tested, contained 37% by weight of short peptides, 32% of polysaccharides and 31% of other substances.

Comparative example 3:

referring to example 1, the difference is that step (4) is performed first, and then step (3) is performed. The hydrolysate thus obtained, tested, contained 53% by weight of short peptides, 28% of polysaccharides and 19% of other substances.

The antioxidant activity of the above hydrolysate was compared by measuring ORAC value (. mu. mol TE/mmol) and DPPH value (. mu. mol TE/mmol), and the results are shown in Table 1.

Table 1: test results of antioxidant Activity of hydrolysates

From the above comparison results, the antioxidant activity of example 1 was higher than that of comparative examples 1 to 3, thereby illustrating that the hydrolysate of example 1 has a higher antioxidant activity.

With respect to the hydrolysates obtained in example 1 and comparative examples 1 to 3, the contents of short peptides contained therein were analyzed, and it was found by examination that the most abundant short peptides in example 1 were ADFW and HADF in amounts of 0.92% and 0.76% of the hydrolysate, while the most abundant short peptides in comparative example were ADFW and HADF in amounts of only 0.57% and 0.53% of the hydrolysate in comparative example 3.

To further determine whether the antioxidant activity of the hydrolysate was high due to the presence of ADFW and HADF, the detected short peptides with high abundance were synthesized by Fmoc solid phase synthesis, which is a common peptide chemical synthesis method, and the most abundant ADF obtained in CN111357865A was used as a control for comparison as a sample for measuring antioxidant activity. For the synthesized short peptides, ORAC (. mu.mol TE/mmol) and DPPH (. mu.mol TE/mmol) were determined, and the results of analysis of the peptide sequences are summarized in Table 2.

Table 2: test results of antioxidant activity of short peptides

Based on the above table, it can be seen that by simultaneously containing ADFW and HADF showing high ORAC value and high DPPH value, the antioxidant property of the hydrolysate was overall excellent, and in particular, the peptide of the present invention had higher antioxidant activity as compared to ADF disclosed in CN 111357865A.

Moisture removal effect of hydrolysate:

first, clinical data

The test method comprises the following steps: randomized, positive drug control clinical study.

Safety evaluation index: general physical examination, blood, urine and stool routine, liver and kidney functions, electrocardiogram, adverse reaction and the like.

The number of cases: 40 cases of treatment groups, 40 cases of negative control groups and 40 cases of positive control groups, and 120 cases in total, clinically manifested by papuloid urticaria, eczema and skin pruritus.

Second, test the medicine

Treatment groups: the hydrolysate prepared in the embodiment 1 of the invention is taken with warm boiled water 10g each time, 3 times a day.

Negative control group: no treatment is performed.

Positive control group: CN111357865A the small molecule peptide solution prepared in example 1 was administered 3 times a day, 40g (dry matter: 10g) per time, and was heated.

Third, treatment of the subject

The course of treatment is as follows: 1 week, follow-up 1 time per day in the observation period, except for patients with serious adverse reaction, follow-up is generally not carried out after 1 week of observation.

Fourthly, counting the results

As can be seen from the table above, the hydrolysate of the invention can effectively relieve papular urticaria, eczema and skin pruritus. In addition, no obvious adverse reaction is seen during observation, and no damage to liver, kidney function and hematopoietic system is seen, which indicates that the small molecular peptide is safe to use.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Sequence listing

<110> Jilin province specialist food Biotechnology Co., Ltd

Changchun Institute of Applied Chemistry, Chinese Academy of Sciences

<120> dehumidifying fermentation type small molecular peptide and preparation method and application thereof

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<141> 2020-12-22

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