阅读说明：本技术 一种减轻体重的发酵型小分子肽及其制备方法和应用 (Weight-reducing fermented small-molecule peptide and preparation method and application thereof ) 是由 赵海峰 赵国安 康明 葛振龙 韩璐 李婉南 韩葳葳 高波 皮子凤 常浩 周喜鹏 于 2020-12-22 设计创作，主要内容包括：本发明涉及一种减轻体重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RKVP和EQFL。本发明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,对于减轻体重具有重要意义。(The invention relates to a fermented small molecule peptide for reducing weight, and a preparation method and application thereof. The hydrolysate rich in the small molecular peptide is prepared by adopting proper hydrolysis conditions, and the small molecular peptide has high antioxidant activity and has important significance for reducing weight.)

1. A small molecule, fermentable peptide for weight loss comprising RKVP and EQFL.

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

3. The hydrolysate containing the fermentable small-molecule peptide according to claim 1, wherein the hydrolysate comprises 50 wt% or more of the short peptide, 20 wt% or more of the polysaccharide and 2.5 wt% or more of the anthocyanin.

4. The hydrolyzate of claim 3, wherein RKVP is present in an amount of 0.8 wt% or more and EQFL is present in an amount of 0.6 wt% or more.

5. A process for the preparation of a hydrolysate according to claim 3 or 4, which is obtainable by the steps of:

(1) 50-100 parts of black sesame, 50-100 parts of black rice, 50-100 parts of black beans, 50-100 parts of black medlar, 50-100 parts of black peanuts, 50-100 parts of black fungus and 50-100 parts of black mulberries by weight are crushed and dissolved 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 papain and bromelain after hydrolysis, stirring uniformly 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 hydrolyzate produced by the production process according to claim 5.

7. A meal replacement powder, which is characterized by comprising 30-50 parts of hydrolysate as described in claim 3, 4 or 6, 60-100 parts of concentrated milk protein, 60-100 parts of crystalline fructose, 60-100 parts of mixed fruit and vegetable powder, 6-10 parts of compound vitamin, 6-10 parts of compound mineral, 10-50 parts of chicken powder, 10-50 parts of beef powder and 100-200 parts of konjac powder.

8. Use of the meal replacement powder of claim 7 for weight loss.

Technical Field

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

Background

Aerobic organisms including humans take in oxygen through respiration for survival, and a part of the oxygen taken in the body is converted into active oxygen species such as peroxide radicals, hydrogen peroxide, hydroxyl radicals and the like during energy metabolism. Such active oxygen species are originally involved in organism defense and the like represented by a mechanism of removing a pathogen from macrophages when infected with bacteria or viruses, and play an important role in maintaining health. However, it is considered that environmental factors such as air pollution and ultraviolet rays, living habits such as smoking, stress, and the like destroy the balance in the living body, and that excessive reactive oxygen species react with proteins, lipids, DNA, and the like in the living body to cause protein denaturation, lipid peroxide production, genetic damage, and the like, thereby causing living habit disorders and aging. Therefore, it is gradually recognized that the intake of antioxidant active ingredients from the diet is very important for maintaining health in addition to the mechanism possessed by the organism, and it is recommended to actively take antioxidant substances as the 7 th nutrient.

The synthetic antioxidant has the advantages of low price, high yield and the like, and is widely applied to the industries of food, skin care products and cosmetics. However, with the economic development, people pay more and more attention to health, so that people prefer safe, pure natural and non-toxic and side-effect natural antioxidants, such as tea polyphenol, ascorbic acid, protein peptide and the like. There are many kinds of food-derived antioxidant peptides, and they are classified into antioxidant peptides of plant origin, animal origin, milk origin, egg origin, and the like according to their source. The plant source mainly comprises corn, soybean, wheat grain, rice bran, rapeseed, peanut, bean olecranon, rice residue, pricklyash seed, walnut, black bean, wheat germ, etc.

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 CN110024950A to disclose a black plant small molecular peptide total nutrient meal replacement powder, which achieves the effects of slowing down protein consumption rate, controlling diet, reducing weight, improving sleep, relieving eye dryness, resisting fatigue and the like through the optimal combination of protein proportion on the basis of meeting daily nutrient requirements of a human body, and the preparation method effectively simplifies the production flow, maintains the nutrient value of the product and increases the stability of a protein system. However, the weight-reducing effect of the meal replacement powder is still to be further improved due to the low content of the active ingredient in the meal replacement powder.

Disclosure of Invention

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

one aspect of the invention relates to a small molecule, fermentable peptide for weight loss comprising RKVP and EQFL.

In a preferred embodiment of the present invention, the RKVP content is 0.8 wt% or more and the EQFL content is 0.6 wt% or more.

The invention also relates to a hydrolysate containing the fermented small molecule peptide, wherein the hydrolysate contains more than 50 wt% of short peptide, more than 20 wt% of polysaccharide and more than 2.5 wt% of anthocyanin.

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

(1) 50-100 parts of black sesame, 50-100 parts of black rice, 50-100 parts of black beans, 50-100 parts of black medlar, 50-100 parts of black peanuts, 50-100 parts of black fungus and 50-100 parts of black mulberries by weight are crushed and dissolved 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 papain and bromelain after hydrolysis, stirring uniformly 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 also relates to meal replacement powder which is characterized by comprising 30-50 parts of hydrolysate, 60-100 parts of concentrated milk protein, 60-100 parts of crystalline fructose, 60-100 parts of mixed fruit and vegetable powder, 6-10 parts of compound vitamin, 6-10 parts of compound mineral, 10-50 parts of chicken meal, 10-50 parts of beef powder and 100-200 parts of konjac powder.

The invention further relates to the application of the small molecule peptide as an antioxidant and/or weight loss.

The invention also relates to application of the meal replacement powder in weight reduction.

Advantageous effects

The hydrolysate rich in the small molecular peptide is prepared by adopting proper hydrolysis conditions, and the small molecular peptide has high antioxidant activity and has important significance for reducing weight.

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) crushing 100 parts by weight of black sesame, 100 parts by weight of black rice, 100 parts by weight of black beans, 100 parts by weight of black medlar, 100 parts by weight of black peanuts, 100 parts by weight of black fungus and 100 parts by weight of black mulberries, and dissolving 10% of Dry Matter (DM) in water to form paste;

(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 papain (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) the permeate from the ultrafiltration was then subjected to nanofiltration through a 200Da membrane and the retentate was recovered.

(9) Lyophilization was performed to give a hydrolysate which was tested to contain 57% by weight short peptides, 25% polysaccharides, 3% anthocyanins and 15% other materials.

Comparative example 1:

reference is made to example 1, with the difference that no papain is added in step (3). The hydrolysate thus obtained, tested, contained 37% by weight of short peptides, 35% of polysaccharides, 2% of anthocyanins and 26% 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 32% by weight of short peptides, 32% of polysaccharides, 1% of anthocyanins and 35% 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 43% by weight of short peptides, 37% of polysaccharides, 2% of anthocyanins and 18% 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 from example 1 and comparative examples 1-3, the content of short peptides contained therein was analyzed, and it was found by detection that the most abundant short peptides in example 1 were RKVP and EQFL, and the content thereof reached 0.83% and 0.67% of the hydrolysate, while the most abundant short peptides in comparative example 1 were RKVP and EQFL, and only 0.45% and 0.36% in comparative example, and ERRQ and FTSE, respectively, in comparative example 3, and the higher content thereof was ERRQ and FTSE, and 0.69% and 0.59% in comparative example.

In order to further determine whether the antioxidant activity of the hydrolysate was high due to the presence of RKVP and EQFL, the short peptides with high abundance were synthesized as samples for measuring antioxidant activity by Fmoc solid phase synthesis, which is a common peptide chemical synthesis method. 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 the antioxidant properties of the hydrolysate are comprehensively excellent by simultaneously including RKVP and EQFL exhibiting high ORAC values and exhibiting high DPPH values.

The effect of the meal replacement weight loss implementation is as follows:

taking 40 parts of hydrolysate in example 1, mixing with 60 parts of concentrated milk protein, 60 parts of crystalline fructose, 60 parts of mixed fruit and vegetable powder, 6 parts of vitamin complex, 6 parts of compound mineral substance, 10 parts of chicken powder, 10 parts of beef powder and 200 parts of konjac powder, uniformly mixing, setting the rotating speed of a dry mixer to be 1500r/min, uniformly mixing for 10min, sieving the mixed dry powder, and controlling the particle diameter of the solid powder to be below 600 mu m so as to ensure that the product has a better dissolving effect. And (3) placing the sieved dry powder into an aluminum foil composite bag, filling nitrogen gas into the aluminum foil composite bag, and sealing the aluminum foil composite bag to obtain the total-nutrient meal replacement powder.

The brewing method comprises the following steps: and (3) taking 20g of the total nutrient meal replacement powder, adding 300ml of 100-80 ℃ water, and uniformly stirring for eating, wherein the time is 3 times per day.

For comparison, meal replacement powder prepared according to example 2 of CN110024950a was used as a comparison.

Subject: selecting 80 adults of 20-50 years old with equivalent weight as subjects to be investigated (half of both men and women), and randomly dividing the subjects into 2 groups of 40 adults (half of both men and women);

the weight measurements of the meal replacement population for one week were counted and the weight changes before and after meal replacement were recorded, with the results shown in table 3. It can be seen in table 3 that the weight-reducing effect of the total nutrient meal replacement powder of the present invention is significantly better than that of the meal replacement powder of the prior art, and the weight-reducing effect is improved by reducing excessive intake of starch and fat during meal.

Table 3 results of the weight loss test

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> fermentation type small molecular peptide for reducing weight and preparation method and application thereof

<130> CP20787

<141> 2020-12-22

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