阅读说明：本技术 富锌酵母多肽及其制备方法、应用 (Zinc-rich yeast polypeptide and preparation method and application thereof ) 是由 孙怀庆 裴运林 张伟强 郭朝万 胡露 于 2021-09-02 设计创作，主要内容包括：本申请涉及微生物发酵领域,具体而言,涉及一种富锌酵母多肽及其制备方法、应用。富锌酵母多肽的制备方法,包括：采用含有ZnSO-(4)的培养基培养酵母菌株ATCC204508；分离并取酵母细胞,对酵母细胞破壁后采用蛋白酶酶解,过滤酶解后的产物并取滤液。经过发明人的多次实验后发现,采用含有ZnSO-(4)的培养基酵母菌株ATCC204508,可以使ATCC204508酵母细胞吸收结合锌离子,培养成富锌酵母,再对富锌酵母进行酶解,得到含锌的酵母源多肽。最终得到的富锌酵母多肽在抗氧化和酪氨酸酶抑制活性等方面有显著的提升。(The application relates to the field of microbial fermentation, in particular to a zinc-rich yeast polypeptide and a preparation method and application thereof. The preparation method of the zinc-rich yeast polypeptide comprises the following steps: by using a composition containing ZnSO 4 Culturing yeast strain ATCC 204508; separating and taking yeast cells, breaking the walls of the yeast cells, performing enzymolysis by adopting protease, filtering the enzymolysis product and taking filtrate. Warp beamAfter a plurality of experiments of the inventor, the ZnSO-containing material is adopted 4 The culture medium yeast strain ATCC204508 can enable ATCC204508 yeast cells to absorb and combine zinc ions, the zinc-rich yeast is cultured, and then the zinc-rich yeast is subjected to enzymolysis to obtain the zinc-containing yeast source polypeptide. The finally obtained zinc-rich yeast polypeptide has remarkable improvement on the aspects of antioxidation, tyrosinase inhibitory activity and the like.)

1. A preparation method of zinc-rich yeast polypeptide is characterized by comprising the following steps:

by using a composition containing ZnSO₄Culturing yeast strain ATCC 204508;

separating and taking yeast cells, breaking the walls of the yeast cells, performing enzymolysis by adopting protease, filtering the enzymolysis product and taking filtrate.

2. The method of claim 1, wherein the polypeptide comprises ZnSO₄In the culture medium of (1), ZnSO₄The concentration of (A) is 100-300 mg/L.

3. The method for producing a zinc-rich yeast polypeptide according to claim 1, wherein the yeast is a yeastStrain ATCC204508 and said ZnSO-containing₄The volume ratio of the medium of (a) is 1: (50-200);

alternatively, said yeast strain ATCC204508 and said ZnSO-containing strain₄The volume ratio of the medium of (a) is 1: (90-110).

4. The preparation method of the zinc-rich yeast polypeptide according to claim 1, wherein the culture temperature of the cultured yeast strain ATCC204508 is 27-30 ℃, and the culture time is 24-72 h;

optionally, the culture temperature is 28-29 ℃, and the culture time is 46-50 h.

5. The method for preparing a zinc-rich yeast polypeptide according to any one of claims 1-4, wherein the protease comprises at least one of bromelain, papain, neutral protease and complex protease;

optionally, the protease is papain.

6. The method for preparing a zinc-rich yeast polypeptide according to any one of claims 1-4, wherein the step of separating and collecting the yeast cells comprises centrifugation at 8000-12000 r/min.

7. The method of any one of claims 1-4, wherein the zinc-rich yeast polypeptide comprises ZnSO₄The culture medium comprises ZnSO₄And YPD medium.

8. The method for preparing zinc-rich yeast polypeptide according to any one of claims 1-4, wherein the step of breaking the yeast cell wall comprises:

drying the yeast cells and then crushing to obtain powder, mixing the powder with water, and ultrasonically breaking the walls, wherein the ratio of the powder to the water is 1: (10-100).

9. A zinc-rich yeast polypeptide prepared by the method of any one of claims 1-8.

10. Use of a zinc-rich yeast polypeptide according to claim 9 in the preparation of a skin care product.

Technical Field

The application relates to the field of microbial fermentation, in particular to a zinc-rich yeast polypeptide and a preparation method and application thereof.

Background

The yeast polypeptide is a yeast fermentation product, contains active polypeptide with antioxidant activity, can be used for feed and food, and has good effect when being applied to the field of cosmetics. Is beneficial to the absorption of the skin and plays a role.

At present, the performance of yeast polypeptides is in a constantly discovered stage; how to further enhance certain properties of yeast polypeptides is also an ongoing endeavor of researchers.

The present application provides a novel yeast polypeptide.

Disclosure of Invention

The embodiment of the application aims to provide a zinc-rich yeast polypeptide, a preparation method and application thereof, which aim to improve the antioxidation of the yeast polypeptide.

In a first aspect, the present application provides a method for preparing a zinc-rich yeast polypeptide, comprising:

by using a composition containing ZnSO₄Culturing yeast strain ATCC 204508;

separating and taking yeast cells, breaking the walls of the yeast cells, performing enzymolysis by adopting protease, filtering the enzymolysis product and taking filtrate.

After a plurality of experiments of the inventor, the ZnSO-containing material is adopted₄The culture medium yeast strain ATCC204508 can enable ATCC204508 yeast cells to absorb and combine zinc ions, the zinc-rich yeast is cultured, and then the zinc-rich yeast is subjected to enzymolysis to obtain the zinc-containing yeast source polypeptide. The finally obtained zinc-rich yeast polypeptide has remarkable improvement on the aspects of antioxidation, tyrosinase inhibitory activity and the like.

In some embodiments of the first aspect of the present application, the ZnSO-containing compound₄In the culture medium of (1), ZnSO₄The concentration of (A) is 100-300 mg/L.

In some embodiments of the first aspect of the present application, yeast strain ATCC204508 and said ZnSO-containing strain₄The volume ratio of the medium of (a) is 1: (50-200);

alternatively, said yeast strain ATCC204508 and said ZnSO-containing strain₄The volume ratio of the medium of (a) is 1: (90-110).

In some embodiments of the first aspect of the present application, yeast strain ATCC204508 is cultured at a temperature of 27 to 30 ℃ for 24 to 72 hours;

optionally, the culture temperature is 28-29 ℃, and the culture time is 46-50 h.

In some embodiments of the first aspect of the present application, the protease comprises at least one of bromelain, papain, neutral protease, and complex protease;

optionally, the protease is papain.

In some embodiments of the first aspect of the present application, the step of separating and removing the yeast cells comprises centrifugation at 8000r/min to 12000 r/min.

In some embodiments of the first aspect of the present application, ZnSO is contained₄The culture medium comprises ZnSO₄And YPD medium.

In some embodiments of the first aspect of the present application, the step of performing enzymolysis with protease after breaking the yeast cell wall comprises:

drying the yeast cells and then crushing to obtain powder, mixing the powder with water, and ultrasonically breaking the walls, wherein the ratio of the powder to the water is 1: (10-100).

In a second aspect, the present application provides a zinc-rich yeast polypeptide, which is prepared by the above-mentioned preparation method of the zinc-rich yeast polypeptide.

The zinc-rich yeast polypeptide obtained by the application has excellent antioxidant activity and tyrosinase inhibitory activity, and can play a role in resisting aging and whitening if the zinc-rich yeast polypeptide is used in a skin care product.

In a third aspect, the application provides an application of the zinc-rich yeast polypeptide provided in the second aspect of the application in preparing a skin care product.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 shows DPPH free radical scavenging rates for various concentrations of the zinc-rich yeast polypeptide of example 1.

Figure 2 shows ABTS free radical clearance by various concentrations of the zinc-rich yeast polypeptide of example 1.

Figure 3 shows hydroxyl radical scavenging rates for various concentrations of the zinc-rich yeast polypeptide of example 1.

FIG. 4 shows tyrosinase inhibition by various concentrations of the zinc-rich yeast polypeptide of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The zinc-rich yeast polypeptide of the embodiments of the present application, and the preparation method and application thereof are specifically described below.

The application provides a preparation method of zinc-rich yeast polypeptide, which mainly comprises the steps of culturing a yeast strain, separating yeast cells, breaking the walls of the yeast cells, performing enzymolysis and finally collecting enzymolysis products.

Specifically, the culture of yeast strains is carried out first, and in the present application, a culture medium containing ZnSO is used₄The culture medium of (2) culturing yeastStrain ATCC 204508.

After a plurality of experiments of the inventor, the ZnSO-containing material is adopted₄The zinc-rich yeast polypeptide finally obtained by the culture medium yeast strain ATCC204508 has better effects on the aspects of antioxidation, tyrosinase inhibitory activity and the like. It is clearly superior to the culture of yeast strain ATCC9763, yeast strain ATCC9080, yeast strain ATCC9987, yeast strain ATCC4021258, yeast strain ATCC22023 and the like under the same conditions.

Furthermore, ZnSO in the culture Medium₄The addition of (b) significantly increases the antioxidant activity and tyrosinase inhibitory activity of the resulting polypeptide.

Illustratively, containing ZnSO₄The culture medium comprises ZnSO₄And YPD medium.

YPD: yeast Extract Peptone Dextrose Medium, a Yeast Extract Dextrose Medium, agar Medium called Yeast Extract Dextrose (YPD) agar Medium.

By way of illustration, ZnSO₄The concentration of (b) is 100 to 300mg/L, and may be, for example, 100mg/L, 120mg/L, 130mg/L, 150mg/L, 160mg/L, 190mg/L, 200mg/L, 210mg/L, 240mg/L, 250mg/L, 280mg/L, 290mg/L, 300mg/L, or the like.

Illustratively, seed solutions of yeast strain ATCC204508 are mixed with a mixture containing ZnSO₄The volume ratio of the medium of (a) is 1: (50-200); for example, the volume ratio of the two may be 1: 50. 1: 60. 1: 80. 1: 90. 1: 100. 1: 110. 1: 130. 1: 150. 1: 180. 1: 190. 1: 200, etc.

As an example, the culture conditions are: the culture temperature is 27-30 ℃, and the culture time is 24-72 h; for example, the culture temperature may be 27 ℃, 28 ℃, 29 ℃, 30 ℃ and the like, and the culture time may be 24h, 28h, 30h, 32h, 34h, 40h, 46h, 50h, 60h, 70h, 72h and the like.

It should be noted that, in other embodiments of the present application, ZnSO is contained₄The medium of (A) may have other compositions, and is not limited to the above-mentioned ZnSO₄And YPD media, which can be configured according to the growth and metabolism of the yeast strain. Accordingly, the raw material benefit is not consideredThe inoculation amount of the yeast strain ATCC204508 can be other values under the conditions of the rate, the yield and the like, and the culture conditions can be adaptively adjusted according to the growth habit of the yeast strain ATCC 204508.

And (3) separating the yeast cells after the culture of the yeast strains is finished, and separating the yeast cells from the culture medium and the metabolite to obtain the yeast cells.

Illustratively, the separation is carried out by adopting a centrifugal mode, and the centrifugation is carried out for 3-8min under the condition of 8000 r/min-12000 r/min; the rotation speed can be 8000r/min, 9000r/min, 10000r/min, 11000r/min, 12000r/min and the like.

It should be noted that in other embodiments, filtration and the like may be used for separation without considering the separation efficiency and yield.

After yeast cells are obtained by separation, yeast cell wall breaking and enzymolysis are carried out, and finally, enzymolysis products are collected.

Illustratively, the yeast cells are dried and then crushed to obtain a powder, the wall breaking is performed by ultrasonic wave on the powder and water, and the ratio of the powder to the water is 1: (10-100); for example, the ratio of 1: 10. 1: 20. 1: 30. 1: 40. 1: 50. 1: 60. 1: 70. 1: 80. 1: 90. 1: 100, etc.

The ultrasonic wall breaking is carried out in ice bath, and the time for ultrasonic wall breaking can be 40-80min, such as 60min, 80min, etc.

And adding protease for enzymolysis after the ultrasonic wall breaking is finished.

Illustratively, the protease includes at least one of bromelain, papain, neutral protease, and complex protease;

for example, the protease is papain.

The temperature of the enzymatic hydrolysis is 50 ℃ to 70 ℃, and may be, for example, 50 ℃, 55 ℃, 60 ℃, 67 ℃, 70 ℃ or the like. The enzymolysis time is 100-120min, for example, 100min, 110min, 120min, etc.; the pH is natural.

As mentioned above, the enzymolysis conditions and time in the enzymolysis process can be set according to the reaction activity of the protease.

And collecting an enzymolysis product after enzymolysis is finished. In some embodiments, the enzymatic hydrolysate can be lyophilized to a powder; it should be noted that, in other embodiments of the present application, the enzymatic hydrolysate can also be in a liquid form.

The preparation method of the zinc-rich yeast polypeptide provided by the application at least has the following advantages:

in the presence of ZnSO₄The yeast strain ATCC204508 is cultured by the culture medium, so that the yeast absorbs and combines zinc ions to culture the zinc-rich yeast, and then the zinc-rich yeast is subjected to enzymolysis to obtain the zinc-containing yeast source polypeptide extract. The method has simple preparation process and mild process conditions, and can obtain zinc-rich yeast polypeptide.

The application also provides a zinc-rich yeast polypeptide, and the zinc-rich yeast polypeptide is prepared by the preparation method of the zinc-rich yeast polypeptide.

The zinc-rich yeast polypeptide obtained by the application has better antioxidant activity and tyrosinase inhibitory activity, and can play a role in resisting aging and whitening if the zinc-rich yeast polypeptide is used in a skin care product.

Based on the aforementioned advantages of the zinc-rich yeast polypeptide provided by the application, the application also provides an application of the zinc-rich yeast polypeptide in preparation of skin care products.

For example, a skin care product comprising an adjuvant and the zinc-rich yeast polypeptide; or, a skin care product, the effective component of which comprises the zinc-rich yeast polypeptide.

It should be noted that, in the embodiments of the present application, the form of the skin care product is not limited, and for example, it may be oil, cream, emulsion, solution, and the like. Accordingly, the present application is not limited to materials other than zinc-rich yeast polypeptides in skin care products.

The features and properties of the present application are described in further detail below with reference to examples.

Yeast strain ATCC204508, yeast strain ATCC9763, yeast strain ATCC9080, yeast strain ATCC9987, yeast strain ATCC4021258 and yeast strain ATCC22023 are all purchased from ATCC strain preservation center;

example 1

The embodiment provides a zinc-rich yeast polypeptide, which is mainly prepared by the following steps:

(1) using a solution containing ZnSO₄Culturing ATCC204508 strain in YPD medium; ZnSO₄The concentration of (A) is 300 mg/L; strains and ZnSO-containing strains₄The YPD medium of (1): 100, respectively; the culture temperature is 28 ℃, and the culture time is 48 h.

(2) After fermentation, the fermentation liquor and thalli are separated by centrifugation at 10000r/min for 5min, the fermentation liquor is discarded, and yeast cells are dried to constant weight at 60 ℃.

(3) And (3) crushing the dry yeast obtained by the treatment in the step (2), and then, mixing the crushed dry yeast with the powder according to the volume ratio of water to the powder of 20: 1 dissolving yeast powder, breaking cell wall in ice bath with 600w ultrasonic wave for 60min, adding wt1 ‰ papain after ultrasonic wave, performing enzymolysis at 60 deg.C for 120min, and keeping pH natural.

(4) And (4) freeze-drying the enzymolysis liquid obtained in the step (3) to obtain the zinc-rich yeast polypeptide.

Example 1 Experimental data for live zinc-rich yeast polypeptides are shown in Table 1, where the polypeptide content in Table 1 is measured using a BCA protein concentration assay kit and the zinc content is measured using a zinc (Zn) assay kit.

TABLE 1

Adding amount of yeast powder	10g
		Zinc-rich yeast polypeptide extract yield	2.945g
Yield of	29.45％
		Polypeptide content	20.18％
Zinc content	1.65‰

Experimental example 1

Taking the zinc-rich yeast polypeptide obtained in the example 1, and carrying out DPPH (dual-ph) free radical clearance experiments on the zinc-rich yeast polypeptides with different concentrations;

the experimental procedure was as follows:

preparing 0.2mmol/L DPPH-methanol (or absolute ethyl alcohol) solution; preparing a sample (zinc-rich yeast polypeptide) solution to be detected with the concentration of 200 mg/mL; VC was used as a positive control sample.

Respectively sucking 2mL of sample solution and 2mL of LDPPH solution into a test tube with a plug, uniformly mixing, reacting for 30min in a dark place, and measuring the light absorption value A1 at the wavelength of 517 nm; respectively sucking 2mL of sample solution and 2mL of methanol into a test tube with a plug, uniformly mixing, reacting for 30min in a dark place, and measuring the light absorption value A2 at the wavelength of 517 nm; respectively sucking 2mL of the solution of the PPH and 2mL of methanol into a test tube with a plug, uniformly mixing, reacting for 30min in a dark place, and measuring the light absorption value A0 at the wavelength of 517 nm; 3 replicates were made for each sample.

The results of the experiment are shown in FIG. 1.

As can be seen from FIG. 1, the IC of DPPH radical scavenging ratio of the zinc-rich yeast polypeptide obtained in example 1₅₀It was 1.854 mg/mL.

Experimental example 2

Taking the zinc-rich yeast polypeptide obtained in the example 1, and carrying out ABTS free radical clearance experiments on the zinc-rich yeast polypeptides with different concentrations;

the experimental procedure was as follows:

the following reagents were prepared: 1, 2.45mmol/L potassium persulfate solution; (ii) 10mmol/L PBS phosphate buffer (pH 7.4); ③ 7mmol/L ABTS stock solution (prepared by using prepared solvent).

Taking ABTS stock solution, diluting the ABTS stock solution by 60-70 times with 10mmol/L PBS (pH 7.4) by using absolute ethyl alcohol to ensure that the light absorption value of the ABTS stock solution at 734nm is 0.7 +/-0.02 to obtain ABTS working solution; absorbing 6mLABTS working solution, adding 60 μ L sample solution, oscillating for 10s, standing at 30 deg.C for 6min, and measuring light absorption value A at 734nm₁(ii) a Absorbing 6mL of absolute ethyl alcohol or PBS buffer solution, adding 60 μ L of sample solution, oscillating for 10s, standing at 30 deg.C for 6min, and measuring light absorption value A at 734nm₂(ii) a Absorbing 6ml of LaBTS working solution, adding 60 μ L of absolute ethanol or PBS buffer solution, oscillating for 10s, standing at 30 deg.C for 6min, and measuring light absorption value A at 734nm₀. VC was used as a positive control for the samples, and triplicates were made for each sample.

The results of the experiment are shown in FIG. 2.

As can be seen from FIG. 2, the IC of the radical scavenging efficiency of the zinc-rich yeast polypeptide ABTS obtained in example 1₅₀It was 0.6727 mg/mL.

Experimental example 3

Taking the zinc-rich yeast polypeptide obtained in the example 1, and carrying out hydroxyl radical scavenging rate experiments on the zinc-rich yeast polypeptides with different concentrations;

the experimental procedure was as follows:

taking 25mL test tubes, and adding 2mmol/L FeSO respectively₄6mmol/L salicylic acid 3mL each, and 3mL 1mmol/L H₂O₂Starting the reaction, heating in 37 deg.C water bath for 15min, and measuring absorbance (A) at 510nm₀). Diluting 0.2, 0.4, 0.6, 0.8, 1.0mL of the solution to be tested and 0.8, 0.6, 0.4, 0.2, 0mL of distilled water to 1mL, adding 2mmol/L FeSO₄6mmol/L salicylic acid 3mL each, and 3mL 1mmol/L H₂O₂Starting the reaction, heating in 37 deg.C water bath for 15min, and measuring absorbance (A) at 510nm₁) And absorbance (A) of the liquid to be measured₂). Each sample was done in 3 replicates.

The results of the experiment are shown in FIG. 3.

As can be seen from FIG. 3, the IC of hydroxyl radical scavenging ratio of the zinc-rich yeast polypeptide obtained in example 1₅₀It was 4.516 mg/mL.

Experimental example 4

Taking the zinc-rich yeast polypeptide obtained in the example 1, and carrying out tyrosinase inhibition rate experiments on the zinc-rich yeast polypeptides with different concentrations;

the detection method is shown in the standard implementation guide [ M ] of the ChengTree army cosmetic evaluation alternative method: the method for detecting the tyrosinase inhibition rate in Chinese quality inspection press and Chinese Standard press, 2017.

The results of the experiment are shown in FIG. 4.

As can be seen from FIG. 4, the IC of the tyrosinase inhibition rate of the zinc-rich yeast polypeptide obtained in example 1₅₀It was 6.347 mg/mL.

Comparative example 1

Please refer to example 1 and experimental examples 1-4. This comparative example was conducted by replacing ATCC204508 with yeast strain ATCC9763 and carrying out the experiments of example 1 and Experimental examples 1-4, the results of which are shown in Table 2.

TABLE 2

Comparative example 2

Please refer to example 1 and experimental examples 1-4. In this comparative example, yeast strain ATCC9080 was used in place of ATCC204508 and the experiments of example 1 and Experimental examples 1-4 were carried out, the results of which are shown in Table 3.

TABLE 3

Comparative example 3

Please refer to example 1 and experimental examples 1-4. This comparative example used yeast strain ATCC9987 in place of ATCC204508 strain and conducted the experiments of example 1 and Experimental examples 1-4, and the results are shown in Table 4.

TABLE 4

Comparative example 4

Please refer to example 1 and experimental examples 1-4. This comparative example used the yeast strain ATCC4021258 in place of ATCC204508 and conducted the experiments of example 1 and Experimental examples 1-4, the results of which are shown in Table 5.

TABLE 5

Comparative example 5

Please refer to example 1 and experimental examples 1-4. This comparative example used yeast strain ATCC22023 in place of ATCC204508 strain and conducted the experiments of example 1 and Experimental examples 1-4, the results of which are shown in Table 6.

TABLE 6

Comparative example 6

Please refer to example 1 and experimental examples 1-4. This comparative example differs from example 1 in that the medium does not contain ZnSO₄(ii) a The results of the experiment are shown in Table 7.

TABLE 7

From example 1, experimental examples 1 to 4, and comparative examples 1 to 6, it can be seen that:

DPPH free radical clearance rate IC of zinc-rich yeast polypeptides obtained in comparative examples 1-6₅₀Much larger than example 1; the zinc-rich yeast polypeptide ABTS free radical clearance rate IC obtained in comparative examples 1-6₅₀Much larger than example 1; comparative examples 1-6 obtained zinc-rich yeast polypeptide hydroxyl radical clearance rate IC₅₀Much larger than example 1; IC of the inhibition ratios of the zinc-rich yeast polypeptide tyrosinase obtained in comparative examples 1-6₅₀Much larger than example 1; description of the addition of ZnSO to the Medium₄Can improve the oxidation resistance and the tyrosinase inhibition rate of the yeast polypeptide; it is also shown that the performance of the yeast polypeptide obtained by culturing yeast strain ATCC9763, yeast strain ATCC9080, yeast strain ATCC9987, yeast strain ATCC4021258 and yeast strain ATCC22023 under the same conditions is lower than that of yeast strain ATCC204508 in terms of antioxidant capacity and tyrosinase inhibition rate.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.