Fruiting body of a basidiomycete and method for producing the same

Abstract

The present application belongs to the technical field of resource utilization of liquor lees, and provides a sugar-reducing polypeptide of Qing-flavor liquor lees, a preparation method and application thereof. The sequence of the sugar-reducing polypeptide of Qing-flavor liquor lees is FFGPG. The present application uses Qing-flavor liquor lees as a starting material, and prepares a lees polypeptide by using enzymatic hydrolysis technology. The lees polypeptide is further subjected to fractionation by using ultrafiltration membranes with different molecular weight cut-off, and the inhibitory activity of each component on alpha-glucosidase is determined. The component with the best inhibitory effect is subjected to mass spectrometry identification. Cell experiment research finds that the lees hydrolysis polypeptide FFGPG prepared by the method of the present application can significantly improve the glucose consumption of insulin-resistant HepG2 cells. Therefore, the lees polypeptide FFGPG can be used as an effective ingredient to prepare a medicine or health care product with a sugar-reducing effect, and has important application value, and lays a foundation for high-value utilization of Qing-flavor liquor lees.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of resource utilization of liquor lees, and particularly relates to a sugar-reducing polypeptide for a Qing-flavor liquor lees and a preparation method and application thereof. BACKGROUND

[0002] China is a big country in liquor production and consumption, and liquor lees is the largest by-product in the process of liquor production. It is a mixed solid waste produced by fermentation and distillation of cereal crops. The liquor lees contains crude fiber, crude starch, fat, amino acids, minerals, enzymes and various vitamins. The composition and nutritional level of liquor lees are different in different liquor factories. Fresh liquor lees is rich in nutrition and has high water content, high acidity and is prone to mold. If such a large amount of liquor lees is not properly treated, it will cause resource waste and environmental pollution. Therefore, actively carrying out research on the reuse of liquor lees and improving the comprehensive utilization efficiency to produce high value-added products are of great significance to the resource utilization and environmental protection of the liquor industry.

[0003] Protein is an important component for the normal growth of all cells and tissues of the human body, and is an important carrier of life activities. Most physiological reactions of the human body require the participation of protein. However, protein generally has a large molecular weight and a complex structure, and its important functions cannot be fully played. However, when it is converted into polypeptide, the structure is relatively simple, so it can play an important role in various life activities of the body. The nutrient substances in liquor lees, especially protein, are rich, mainly composed of cereal protein and microbial protein, and are a protein resource with application potential in industry. Compared with Maotai-flavor and Nongxiang-flavor liquor lees, the protein content in Qing-flavor liquor lees is the highest, which can reach about 20.96%. Therefore, how to extract these liquor lees proteins and use their enzymatic products to produce functional polypeptides has become a hot spot of current research.

[0004] Enzymatic hydrolysis has been the preferred method for polypeptide preparation for a long time because of its low price, mild reaction conditions, and easy access to polypeptide products similar to natural active polypeptides, and is widely used in the production of functional polypeptides from different protein sources. Such polypeptides often have good stability, easy absorption, and less toxicity and side effects compared with general small molecule drugs, and have certain research and development value. Recently, Jiang et al. used alkaline protease to isolate a tetrapeptide Asp-Arg-Glu-Leu (DREL) with antioxidant activity from liquor lees protein hydrolysate, and confirmed it through in vitro biochemical experiments and in vivo cell and animal experiments, and revealed its molecular mechanism of participating in in vivo antioxidant activity (Yunsong J, Jinyuan S, Zhongtian Y, et al.Evaluation of antioxidant peptides generated from Jiuzao (residueafter Baijiu distillation) protein hydrolysates and their effect of enhancing healthy value of Chinese Baijiu [J]. Journal of the science of food and agriculture, 2020, 100(1):59-73). It can be seen from this that the antioxidant effect of enzymatically hydrolyzed peptides from baijiu lees has been widely recognized, but there are few reports on their other effects such as hypoglycemic effects. It can be said that our current research on enzymatically hydrolyzed peptides in baijiu lees is still in its infancy, especially in the research on light-fragrant baijiu lees represented by Fenjiu. Summary of the Invention

[0005] To overcome the above problems, the present invention aims to provide a light-fragrance distiller's grains hypoglycemic polypeptide, its preparation method, and application. Research has found that light-fragrance distiller's grains enzymatic hydrolysis polypeptides exhibit in vitro hypoglycemic activity. Furthermore, a polypeptide identified from these polypeptides, FFGPG, can significantly enhance glucose consumption in insulin-resistant HepG2 cells. Therefore, this polypeptide has significant application value as an active ingredient in the preparation of hypoglycemic drugs or health supplements.

[0006] To achieve the above object, the present invention provides the following solution: a light-fragrant distiller's grains hypoglycemic polypeptide, wherein the sequence of the polypeptide is FFGPG.

[0007] The method for preparing the light-fragrant distiller's grains hypoglycemic polypeptide comprises the following steps:

[0008] (1) Dried lees powder obtained by drying light-fragrant lees;

[0009] (2) The vinasse powder is defatted with an organic solvent, n-hexane, and then dissolved in distilled water in a certain proportion and mixed evenly. The vinasse protein is precipitated by an alkali dissolution and acid precipitation method, and then freeze-dried to obtain vinasse protein powder;

[0010] (3) enzymatically hydrolyzing the dissolved vinasse protein powder with alkaline protease to obtain vinasse enzymatic hydrolyzate;

[0011] (4) The lees hydrolyzed liquid was ultrafiltered using ultrafiltration tubes with different molecular weight cutoffs to obtain three fractions with molecular weights > 10 kD, 3 kD < molecular weight < 10 kD, and molecular weight < 3 kD. After concentration and drying, the light-fragrance lees hydrolyzed polypeptides of different fractions were obtained.

[0012] (5) the components less than 3 kD are separated into four components F1-F4 by Superdex 30 gel column chromatography, and in vitro alpha-glucosidase inhibitory activity determination is carried out; the component with the strongest alpha-glucosidase inhibitory effect is subjected to mass spectrometric identification;

[0013] (6) the polypeptide after mass spectrometric identification is subjected to BIOPEP database search by selecting the average local confidence (ALC) > 80% peptide segment, and a sugar-reducing polypeptide of a light-flavor wine lees is screened and obtained;

[0014] (7) the screened polypeptide is synthesized according to its sequence by a solid-phase synthesis method, and the purity is greater than 95%.

[0015] The drying method in step (1) is that the fresh wine lees is dried at 20-30℃ for ≥10 hours to sufficiently remove water.

[0016] In step (2), the mass / volume ratio of the wine lees powder and n-hexane is 1:5 for defatting; the specific method for defatting is that the wine lees is dried, crushed and separated through an 80-mesh sieve; defatting is carried out by mixing n-hexane reagent at a ratio of 1:5, sufficient extraction, 13000r centrifugation for 30 min, removal of the solvent, and then the wine lees is placed in a ventilated place to volatilize the remaining solvent; the dried wine lees is dissolved in 20 times the volume of distilled water, the pH value is adjusted to 9.5-10.0 by 1 mol / L sodium hydroxide, and then the mixture is placed in a 56℃ water bath for 2h of heat preservation and stirring, and then centrifuged to take the supernatant; the precipitate is extracted and centrifuged twice; the combined supernatant is adjusted to pH 4.0-4.5 by dilute hydrochloric acid to precipitate the protein, and the protein is washed twice with water, the pH value of the water-washed protein is adjusted to 7.0 by 1 mol / L sodium hydroxide, and then freeze-dried, the freeze-drying temperature is-76℃, and the freeze-drying time is 12h to obtain the finished wine lees protein powder.

[0017] In step (3), the enzymolysis conditions are that the temperature is 40℃, the pH is 10.0, the time is 3h, and the enzyme amount is 5500U / g.

[0018] The specific method of the solid-phase synthesis method is that the first amino acid is fixed on a solid support, other amino acids are gradually added through chemical reactions, and finally a polypeptide chain is obtained.

[0019] The application further provides application of the sugar-reducing polypeptide of a light-flavor wine lees in preparation of a sugar-reducing product.

[0020] Further, the application of the sugar-reducing polypeptide of a light-flavor wine lees in preparation of a product for inhibiting alpha-glucosidase.

[0021] The present application finds in research that the component less than 3 kD has better in-vitro inhibitory activity on alpha-glucosidase than the other two components; therefore, it is used as an effective component for preparing medicine or health care product with hypoglycemic effect, which has important application value. The enzymatic polypeptide FFGPG of the light-flavor distiller's grains can significantly improve the glucose consumption of insulin-resistant HepG2 cells.

[0022] The present application provides a method for preparing the enzymatic polypeptide of the light-flavor distiller's grains; and a distiller's grains hypoglycemic polypeptide FFGPG is obtained; research shows that the distiller's grains polypeptide prepared by the method has good hypoglycemic activity, and the preparation method is simple, low in cost, easy to operate and implement, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only represent some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0024] Figure 1 Inhibition activity of different components of the enzymatic polypeptide extract of the distiller's grains on alpha-glucosidase after ultrafiltration;

[0025] Figure 2 Effect of different components of the enzymatic polypeptide extract of the distiller's grains on IR-HepG2 cell activity and glucose consumption after ultrafiltration. C represents the control group; IR represents the insulin resistance model group; M represents the 2mM metformin treatment group; and the different concentrations of the distiller's grains enzymatic polypeptide treatment groups are shown in the figure.

[0026] Figure 3 Gel chromatography analysis of the component less than 3 kD and determination of the inhibitory activity of the corresponding component on alpha-glucosidase;

[0027] Figure 4 Effect of FFGPG on IR-HepG2 cell activity and glucose consumption. C represents the control group; IR represents the insulin resistance model group; M represents the 2mM metformin treatment group; and the different concentrations of the FFGPG polypeptide treatment groups are shown in the figure. DETAILED DESCRIPTION

[0028] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an individual value from the range can be expressly disclosed herein and is expressly incorporated into its range. For example, a range of 2.0 to 4.0 can be disclosed, and the values 2.0, 3.0, and 4.0 can be expressly disclosed as well as the values 2.0-3.0, 2.0-4.0, 3.0-4.0, and 3.0-4.0. Unless otherwise stated, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the application. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art in the field of the application. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application.

[0031] Various modifications and variations to the specific implementation of the application can be practiced from the teachings of the present application without departing from the spirit and scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given herein are exemplary and are not intended to be limiting.

[0032] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended and do not limit the object to which the terms are applied to only consist of the recited elements.

[0033] Example 1: Preparation of saccharide-reducing polypeptide by enzymatic hydrolysis of light-flavor distiller's grains: The light-flavor distiller's grains were dried at 20-30°C for more than 10 hours to remove water, and then separated by a 80-mesh sieve. The sieved distiller's grains were mixed with n-hexane at a ratio of 1:5, stirred, and extracted. After removing the solvent by centrifugation, the distiller's grains were placed in a ventilated kitchen to volatilize. Then, a certain amount of the distiller's grains powder was dissolved in distilled water at a ratio of 1:10, and stirred in a water bath at 57°C for 2 hours. During the process, the pH value was maintained at 9.5-10.0 to ensure that the proteins were fully dissolved. The supernatant was collected by centrifugation, and the process was repeated twice. The pH value was adjusted to 4.0-4.5 by dilute hydrochloric acid, and the proteins were precipitated and washed with water. The protein powder was obtained by freeze-drying.

[0034] A certain amount of the dried distiller's grains protein powder was dissolved in 10 times the volume of distilled water, and the dissolved distiller's grains protein powder was added to alkaline protease for enzymatic hydrolysis. The enzymatic hydrolysis conditions were set as follows: temperature 40°C, pH 10.0, time 3 hours, and enzyme dosage 5500 U / g. After the enzymatic hydrolysis, the enzyme was inactivated to obtain a distiller's grains enzymatic hydrolysate.

[0035] Further ultrafiltration of the enzymatic hydrolysate of distiller's grains was performed using 3 kD and 10 kD ultrafiltration tubes. The results showed that the distiller's grains polypeptides less than 3 kD had good α-glucosidase inhibitory effect. The less than 3 kD component was further separated and purified using a Superdex 30 gel column. First, the tubing, column wall and chromatographic column were flushed with ultrapure water and PBS buffer at pH 7.3 until no absorption peak was detected at 220 nm by the ultraviolet detector. After adjusting the flow rate to 1 mL / min for loading, the polypeptide component was eluted with the same buffer and the same flow rate. The polypeptide component was mainly separated by different molecular weights. During the whole process, the eluate was collected every 2 mL, and then the samples were collected according to the peaks. Multiple injections were used to enrich the concentration. The obtained samples were freeze-dried and stored at low temperature for standby use (e.g. Figure 3 A).

[0036] Example 2: Determination of α-glucosidase inhibitory activity of distiller's grains enzymatic polypeptide

[0037] Different concentrations of distiller's grains enzymatic polypeptide solution and 0.5 U / mL α-glucosidase solution were prepared. 0.2 M 4-nitrophenyl-β-D-galactopyranoside (pNPG) was used as the substrate, and pH 10.95 sodium carbonate was used as the termination solution. The absorbance value was measured at 410 nm. The specific operation is shown in Table 1.

[0038] Table 1: Operation process of determination of α-glucosidase inhibitory activity of distiller's grains enzymatic polypeptide

[0039]

[0040] The α-glucosidase activity unit is defined as: the amount of enzyme required to hydrolyze pNPG to generate 1 μmol / L pNP per minute in a buffer system at 37°C and pH 6.8 is one activity unit. The specific calculation formula is as follows:

[0041] The inhibition rate (%) of hypoglycemic polypeptide on α-glucosidase activity = [△A control group - (△A experimental group - △A background group)] / △A control group x 100% (in the formula: △A control group represents the reaction without distiller's grains polypeptide; △A experimental group represents the reaction with distiller's grains polypeptide; △A background group represents the reaction without α-glucosidase).

[0042] According to the above-mentioned determination steps, different components of the distiller's grains polypeptide were determined respectively. According to the inhibition rate of the hypoglycemic polypeptide with different concentrations on the activity of α-glucosidase, it can be seen that when the concentration of 3-10 kD and >10 kD distiller's grains polypeptide is 1.6 mg / mL and 1.5 mg / mL respectively, the inhibition rate of the activity of α-glucosidase is maintained at about 15% and 8%. The component less than 3 kD has the best inhibition effect on the activity of α-glucosidase, and when the concentration of the distiller's grains polypeptide is 1.2 mg / mL, the polypeptide yield is about 25% (for example Figure 1 ). Further, the F4 component has a strong α-glucosidase inhibitory effect, and the inhibition rate is about 24%, so the F4 component is identified by mass spectrometry (for example Figure 3 B).

[0043] Example 3: Determination of the toxicity of distiller's grains polypeptide on IR-HepG2 cells and glucose consumption

[0044] HepG2 cells in the logarithmic phase were inoculated into a 96-well cell culture plate, and the cells were treated with 30 mM glucose and 0.2 mM palmitic acid for 24 hours to induce the formation of insulin-resistant cells. Different concentrations of each component of the distiller's grains polypeptide and the hypoglycemic peptide FFGPG were added, and the culture was carried out for 48 hours. After the culture, the subsequent operation was carried out according to the experimental steps described by Lu Ke, and the absorbance was finally detected to calculate the cell survival rate. The cells were treated with 2 mM metformin as a positive control. In the glucose consumption experiment of IR-HepG2 cells, the culture solution was first collected, and the operation was carried out according to the operation manual of the glucose content determination kit to calculate the residual glucose content. The results of the IR-HepG2 cell glucose consumption experiment showed that after the diabetes modeling treatment, the cell activity was not significantly affected, but the glucose consumption was significantly reduced. It shows that the modeling is successful. The addition of metformin and 0.8-1.2 mg / ml JZP-3 can improve the glucose consumption of IR-HepG2 cells, which is increased by 16% compared with the control group; among them, when the concentration of JZ-1 is 1.91 μM, the effect of promoting glucose consumption is extremely significant, which is increased by 15.9% compared with the IR group. (for example Figure 2 and Figure 4 )

[0045] In summary, the distiller's grains polypeptide mixture and the active polypeptide FFGPG in the present application have the activity of inhibiting α-glucosidase in vitro, can significantly improve the glucose consumption of insulin-resistant HepG2 cells, and can be used as effective components for preparing hypoglycemic drugs or health products.

[0046] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall all fall within the protection scope of the present application as defined by the claims.

[0047] Finally, it should be noted that the above embodiments are merely used to illustrate the technical solutions of the present application, and are not intended to limit the scope of the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still make modifications to the technical solutions recorded in the foregoing embodiments, or make equivalent replacements to some or all of the technical features; and these modifications or replacements 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 application.

Claims

1. A light-fragrant distiller's grains hypoglycemic polypeptide, characterized by: The sequence of the polypeptide is FFGPG.

2. Use of the light-fragrant vinasse hypoglycemic polypeptide according to claim 1 in the preparation of hypoglycemic products.

3. The use according to claim 2, characterized in that: The application of the light-fragrant vinasse hypoglycemic polypeptide in the preparation of a hypoglycemic product that inhibits α-glucosidase.

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