阅读说明：本技术 一种提高酿酒酵母增殖和乙醇耐受性的蛋白活性肽及其制备方法与应用 (Protein active peptide for improving saccharomyces cerevisiae proliferation and ethanol tolerance as well as preparation method and application thereof ) 是由 赵海锋 阳辉蓉 于 2019-10-22 设计创作，主要内容包括：本发明公开了一种提高酿酒酵母增殖和乙醇耐受性的蛋白活性肽及其制备方法及应用。本发明以小麦面筋蛋白为原料,通过酶法水解,制备得到小麦面筋蛋白酶解液,再利用乙醇沉淀和反相高效液相色谱对其进行分离纯化得到七种新的能促进酵母增殖和提高酵母乙醇耐受性的活性肽,该方法制备得到的目标活性肽包括：Glu-Pro、Leu-Leu、Leu-Trp、Leu-Met-Leu、Leu-Leu-Leu、Leu-Leu-Trp、Glu-Phe-Pro-Leu。本发明所制得的小麦面筋蛋白肽能显著提高酿酒酵母的乙醇耐受性,将其应用于高浓度乙醇条件下,可有效促进酿酒酵母增殖以及提高细胞活性。(The invention discloses a protein active peptide for improving saccharomyces cerevisiae proliferation and ethanol tolerance, and a preparation method and application thereof. The invention takes wheat gluten protein as raw material, prepares wheat gluten enzymolysis liquid through enzymatic hydrolysis, and then separates and purifies the wheat gluten enzymolysis liquid by utilizing ethanol precipitation and reversed-phase high performance liquid chromatography to obtain seven new active peptides capable of promoting yeast proliferation and improving yeast ethanol tolerance, and the target active peptides prepared by the method comprise: Glu-Pro, Leu-Leu, Leu-Trp, Leu-Met-Leu, Leu-Leu-Trp, Glu-Phe-Pro-Leu. The wheat gluten protein peptide prepared by the invention can obviously improve the ethanol tolerance of the saccharomyces cerevisiae, and can effectively promote the proliferation of the saccharomyces cerevisiae and improve the cell activity when being applied to the condition of high-concentration ethanol.)

1. A preparation method of wheat gluten protein active peptide for improving saccharomyces cerevisiae proliferation and ethanol tolerance is characterized by comprising the following steps:

(1) enzymolysis of wheat gluten protein: mixing wheat gluten protein with water, adding protease, and performing enzymolysis under constant temperature stirring; after enzymolysis, inactivating enzyme, cooling to room temperature, centrifuging, and removing precipitate to obtain wheat gluten protein enzymolysis clear liquid;

(2) carrying out alcohol precipitation treatment on the wheat gluten protein enzymolysis clear liquid: concentrating the wheat gluten protein enzymolysis clear liquid under reduced pressure to obtain a concentrated solution, adding absolute ethyl alcohol for alcohol precipitation to remove macromolecular protein and polypeptide, and freeze-drying the supernatant to obtain wheat gluten protein alcohol precipitation clear liquid freeze-dried powder WP;

(3) reversed-phase high-performance liquid phase separation: dissolving the freeze-dried powder WP in water, separating and purifying by using a reversed phase high performance liquid chromatography, and freeze-drying the obtained separated component to obtain the wheat gluten protein active peptide for improving the saccharomyces cerevisiae proliferation and the ethanol tolerance.

2. The method according to claim 1, wherein in the step (1), the ratio of the wheat gluten protein to the water is 1: 8-1: 10; adding protease accounting for 1-2 wt% of the wheat gluten protein.

3. The preparation method according to claim 1, wherein in the step (1), the enzymolysis is carried out at 45-55 ℃ for 18-22 h; inactivating the enzyme in boiling water for 10-30 min.

4. The method according to claim 1, wherein in the step (2), the absolute ethanol is added in an amount of: so that the concentration of the ethanol in the mixed solution of the absolute ethanol and the concentrated solution reaches 70-90 vol.%.

5. The method according to claim 1, wherein in the step (2), the concentration under reduced pressure is 0.2 to 0.6 times of the original enzymolysis solution at a temperature of 40 to 50 ℃ and a vacuum degree of 0.08 to 0.1 MPa.

6. The production method according to claim 1, wherein in the step (3), in the reversed-phase high-performance liquid phase separation method: dissolving the freeze-dried powder WP in water, and purifying by using a reversed phase high performance liquid chromatography, wherein a chromatographic column is a BEH-C18 preparative column, gradient elution is carried out at a flow rate of 1-3 ml/min, and the conditions are as follows: 0-5 min, 10vol.% acetonitrile; 5-35 min, 10-60vol.% acetonitrile; 35-36 min, 60vol.% acetonitrile; 36-40 min, 60-10vol.% acetonitrile; 41-42 min, 10vol.% acetonitrile.

7. The production method according to claim 6, wherein in the step (3), the mobile phases are phase A and phase B, the phase A is ultrapure water containing 0.1 vol.% trifluoroacetic acid, and the phase B is acetonitrile.

8. The method according to claim 1, wherein in the step (3), the elution rate during the further separation by the reversed-phase high-performance liquid phase is 1 ~ 3 mL/min.

9. A wheat-gluten-active peptide obtained by the production process as claimed in any one of claims 1 to 8, characterized in that the wheat-gluten-active peptide comprises active peptide fragments having the amino acid sequence Glu-Pro, Leu-Leu, Leu-Trp, Leu-Met-Leu, Leu-Leu-Trp and Glu-Phe-Pro-Leu.

10. Use of the wheat gluten peptide of claim 9 for increasing the cell proliferation and cell activity of saccharomyces cerevisiae under high ethanol stress conditions.

Technical Field

The invention relates to the technical field of protein peptides, in particular to a wheat gluten protein active peptide for improving yeast proliferation and ethanol tolerance, and a preparation method and application thereof.

Background

Saccharomyces cerevisiae is a very important industrial microorganism, has the characteristics of high fermentation speed, high ethanol yield and the like, and is mainly applied to the ethanol and brewing industries. However, as fermentation proceeds, the accumulation of ethanol can cause deleterious effects on yeast cells, which in turn can affect cell proliferation, survival rates, and ethanol fermentation, especially ultra-high concentration fermentation. In ultra-high concentration fermentations, ethanol stress is the most dominant stress experienced by yeast. Therefore, in order to improve the fermentation efficiency of yeast in ultra-high concentration fermentation, it becomes important and urgent to improve the proliferation and ethanol stress tolerance of yeast under high concentration ethanol conditions.

Wheat gluten protein is a high-quality plant protein which is rich, economic and potential, and contains a plurality of amino acids. Wheat gluten proteins contain a number of biologically active amino acid sequences. However, due to the limitations of certain functional properties of wheat gluten, especially its poor solubility, the exertion of other functionalities is hindered, limiting the use of gluten in the food and non-food industries. The application of wheat gluten protein in food and food biotechnology industry is expanded through enzymolysis. Because the specific protease hydrolyzes the wheat gluten protein, the peptide segment with biological activity is possibly released, thereby improving the economic value of the wheat gluten protein.

CN108342439A discloses that a wheat gluten protein peptide separation component capable of improving ethanol tolerance of saccharomyces cerevisiae is prepared by taking wheat gluten protein as a raw material through ultrasonic treatment, homogenization pretreatment, enzymolysis, alcohol precipitation and macroporous resin column chromatography, but the mixture is obtained by the method, and specific and definite polypeptides are not obtained; meanwhile, CN108342439A adopts ultrasonic and homogenization to increase the treatment cost of wheat gluten protein.

Disclosure of Invention

In order to overcome the problem of poor yeast proliferation and metabolic activity in high-concentration fermentation, the invention aims to provide a wheat gluten protein active peptide for improving yeast proliferation and ethanol tolerance, and a preparation method and application thereof.

The invention takes wheat gluten protein as a raw material, provides seven wheat gluten protein peptides which can promote the cell proliferation of saccharomyces cerevisiae and improve the ethanol tolerance of yeast in the presence of high-concentration ethanol by utilizing food-grade commercial protease to carry out enzymolysis on the wheat gluten protein and separating and purifying zymolyte.

The object of the present invention is achieved by at least one of the following means.

A preparation method of wheat gluten protein active peptide for improving saccharomyces cerevisiae proliferation and ethanol tolerance comprises the following steps:

(1) enzymolysis of wheat gluten protein: mixing wheat gluten protein with water, adding protease, and performing enzymolysis under constant temperature stirring; after enzymolysis, inactivating enzyme, cooling to room temperature, centrifuging, and removing precipitate to obtain wheat gluten protein enzymolysis clear liquid;

(2) carrying out alcohol precipitation treatment on the wheat gluten protein enzymolysis clear liquid: concentrating the wheat gluten protein enzymolysis clear liquid under reduced pressure to obtain a concentrated solution, adding absolute ethyl alcohol for alcohol precipitation to remove macromolecular protein and polypeptide, and freeze-drying the supernatant to obtain wheat gluten protein alcohol precipitation clear liquid freeze-dried powder WP;

(3) reversed-phase high-performance liquid phase separation: dissolving the freeze-dried powder WP in water, separating and purifying by using a reversed phase high performance liquid chromatography, and freeze-drying the obtained separated component to obtain the wheat gluten protein active peptide for improving the saccharomyces cerevisiae proliferation and the ethanol tolerance.

Further, in the step (1), the mass ratio of the wheat gluten protein to the water is 1: 8-1: 10; adding protease accounting for 1-2 wt% of the wheat gluten protein.

Further, in the step (1), the enzymolysis is carried out for 18-22 hours at 45-55 ℃; inactivating the enzyme in boiling water for 10-30 min.

Further, in the step (2), the addition amount of the absolute ethyl alcohol is as follows: so that the concentration of the ethanol in the mixed solution of the absolute ethanol and the concentrated solution reaches 70-90 vol.%.

Further, in the step (2), the reduced pressure concentration is 0.2-0.6 times of that of the original enzymolysis solution under the conditions of the temperature of 40-50 ℃ and the vacuum degree of 0.08 MPa-0.1 MPa.

Further, in the step (3), in the reversed-phase high-performance liquid phase separation method: dissolving the freeze-dried powder WP in water, purifying by using a reversed phase high performance liquid chromatography, and performing gradient elution at a flow rate of 1-3 ml/min under the conditions of: 0-5 min, 10vol.% acetonitrile; 5-35 min, 10-60vol.% acetonitrile; 35-36 min, 60vol.% acetonitrile; 36-40 min, 60-10vol.% acetonitrile; 41-42 min, 10vol.% acetonitrile.

Further, in step (3), the reversed-phase high-performance liquid separation column was a BEH-C18 preparative column (10 mm x 150 mm, 5 μm).

Further, in the step (3), the mobile phases are phase a and phase B, the phase a is ultrapure water containing 0.1 vol.% trifluoroacetic acid, and the phase B is acetonitrile.

Further, in the step (3), during the further separation process through the reversed-phase high-performance liquid phase, the elution speed is 1 ~ 3 mL/min.

The wheat gluten active peptide obtained by the preparation method comprises active peptide segments of Glu-Pro (245.1127), Leu-Leu (245.1849), Leu-Trp (318.1816), Leu-Met-Leu (376.2277), Leu-Leu-Leu (358.2702), Leu-Leu-Trp (431.267) and Glu-Phe-Pro-Leu (505.2678).

The wheat gluten protein peptide is applied to increasing the cell proliferation and cell activity of saccharomyces cerevisiae under the condition of high-concentration ethanol stress.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the preparation method takes the wheat gluten protein as a raw material, adopts a biological enzyme preparation to degrade the wheat gluten protein, fully releases active peptide segments in the protein, reduces macromolecular substances in zymolyte through ethanol precipitation, and finally combines a reversed-phase high-performance liquid separation technology to further separate and enrich the wheat gluten protein peptide, thereby achieving the purpose of efficiently enriching the active ingredients of the wheat gluten protein peptide;

(2) the preparation method has the characteristics of simple operation, low cost, high safety, good enzymolysis and separation effect and high extraction rate;

(3) the wheat gluten protein peptide prepared by the preparation method has the effect of improving ethanol tolerance of saccharomyces cerevisiae, and can be applied to improving ethanol tolerance of saccharomyces cerevisiae in a high-concentration or high-density fermentation process, and cell proliferation and cell vitality under high ethanol concentration.

Drawings

FIG. 1 is a graph of the reversed-phase high performance liquid chromatography separation and purification of wheat gluten protein active peptide in step 3 in example 2;

FIG. 2 is a graph of growth of cells of Saccharomyces cerevisiae in 10vol.% ethanol YPD medium supplemented with the wheat gluten peptide and without any reagents of the semi-preparative reverse phase high performance liquid chromatography fractions of example 2;

FIG. 3 is a graph of the effect of cell viability of Saccharomyces cerevisiae in 10vol.% ethanol YPD medium supplemented with wheat gluten peptide and without any reagents for each fraction isolated by semi-preparative reverse phase high performance liquid chromatography of example 2;

FIG. 4 is a graph showing the growth of cells of Saccharomyces cerevisiae in 10vol.% ethanol YPD medium supplemented with each of the wheat gluten target peptides (Glu-Pro, Leu-Leu, Leu-Trp, Leu-Met-Leu, Leu-Leu-Trp, and Glu-Phe-Pro-Leu) and without any reagents in example 2;

FIG. 5 is a graph showing the effect of the cell activity of Saccharomyces cerevisiae in 10vol.% ethanol YPD medium to which each of the wheat gluten target peptides (Glu-Pro, Leu-Leu, Leu-Trp, Leu-Met-Leu, Leu-Leu-Trp, and Glu-Phe-Pro-Leu) was added and no reagent was added in example 2.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, but the scope and implementation of the present invention are not limited thereto.

In the following examples, experiments were conducted under conventional conditions, under conditions described in a laboratory manual, or under conditions recommended by the manufacturer, unless specific conditions are specified.