阅读说明：本技术 一种含花色苷果酒的护色发酵的方法 (Color-protecting fermentation method of anthocyanin-containing fruit wine ) 是由 白卫滨 滕昭军 孙建霞 李旭升 王超 曾颖钰 于 2021-07-23 设计创作，主要内容包括：本发明公开了一种生成Methyl-Vitisin B的含花色苷果酒的护色发酵的方法,酿酒酵母通过保留花色苷和促进矢车菊素进一步反应生成Methyl-Vitisin B,起到了对发酵液的颜色保护作用。本发明提供的酵母CICC 31905可显著保留花色苷含量,增加Methyl-Vitisin B含量,使果酒颜色转变为陈酿成熟稳定的橙红色泽,提升果酒的色泽稳定性。(The invention discloses a method for color-protecting fermentation of anthocyanin-containing fruit wine for generating Methyl-vitasin B. The yeast CICC 31905 provided by the invention can obviously retain the content of anthocyanin, increase the content of Methyl-vitasin B, change the color of fruit wine into the orange red color with mature and stable aging, and improve the color stability of the fruit wine.)

1. A method for color-protecting fermentation of anthocyanin-containing fruit wine for generating Methyl-vitasin B is characterized in that fermentation is carried out by using one or more of Saccharomyces cerevisiae CICC 31898, Saccharomyces cerevisiae CICC 31895, Saccharomyces cerevisiae CICC 31905 and/or Saccharomyces cerevisiae CICC 31966 which are preserved in China center for industrial microorganism strain preservation management, wherein Methyl-vitasin B is anthocyanin derivative, and the chemical structural formula of the derivative is shown as the formula (I):

2. the method according to claim 1, wherein the fermentation is carried out using Saccharomyces cerevisiae CICC 31905, deposited at the China center for Industrial culture Collection of microorganisms.

3. The method of claim 1, wherein the anthocyanin is cyanidin-3-O-glucoside.

4. The method according to claim 1, wherein the initial pH of the color-protected fermentation broth is in the range of 3.0 to 3.5.

5. The method according to claim 1, wherein the sugar degree of the fermentation broth of the color-protecting fermentation is measured by a Brix meter and is in the range of 19 to 22.

6. A anthocyanin derivative Methyl-Vitisin B, wherein the chemical structural formula is shown as formula (I):

Technical Field

The invention relates to the technical field of fruit wine processing, in particular to a color-protecting fermentation method of anthocyanin-containing fruit wine for generating Methyl-vitasin B.

Background

The fruit wine is prepared from fresh fruit or fruit juice by crushing, fermenting or soaking. The brewing process of the fermented fruit wine comprises raw material pretreatment, fermentation, clarification, aging and the like. Wherein fermentation is the most important step in the fruit wine brewing process and is influenced by various factors, and the fermentation microorganisms are the key of the fruit wine brewing and influence the color of the fruit wine. The color is one of the main parameters for measuring the quality of the fruit wine, and can visually reflect the brewing process, the flavor, the wine age and the like of the fruit wine, thereby bringing the most obvious visual feeling to consumers.

The color of the fruit wine is mainly derived from anthocyanin, and the form and the content of the anthocyanin have great influence on the color tone, the color and the stability of the fruit wine. C3G is anthocyanin with the most abundant content in nature, is widely distributed in various fruits such as mulberries, waxberries, blueberries and the like, and plays an important role in the color of the fruit wine. The change of anthocyanin mainly occurs in the early stage of fruit wine fermentation, and the components and contents of anthocyanin and derivatives thereof directly influence the change of fruit wine color and stability in the later fermentation process. At present, in the prior art, the color of the fruit wine is generally regulated and maintained by adding a pigment or a chemical color fixative in the brewing process, so that great food safety hazards exist.

Saccharomyces cerevisiae is an important microorganism for producing fermented fruit wine, and has important influence on the quality and sensory quality of red wine. In the process of fermenting the fruit wine, on one hand, the cell wall of the yeast can adsorb anthocyanin in the solution; on the other hand, beta-glucosidase secreted by yeast cells also causes anthocyanin to be degraded, thereby causing the content of anthocyanin in the solution to be reduced, and finally causing the color of the wine body to be reduced. Due to the difference of adsorption effect of different saccharomyces cerevisiae on anthocyanin and capability of degrading anthocyanin, proper saccharomyces cerevisiae strains are selected for fruit wine fermentation, color loss in the fermentation process can be effectively reduced, and the color protection effect is achieved to a certain extent. The method provides theoretical guidance for keeping the content of anthocyanin in the fruit wine, stabilizing the wine body color of the fruit wine, increasing the nutritional functionality and producing high-quality fruit wine.

There are studies reporting that "metabolites (acetaldehyde and pyruvic acid) produced by yeast at glycolysis stage are condensed with anthocyanin in grape to form the more stable anthocyanins vitasin a and vitasin B" (helnosia. influence of brewing factor on anthocyanin derivative vitasins and color quality in dry red wine [ D ]. university of gansu agriculture, 2016.), and vitasins contribute to the stability of wine color.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides a method for color-protecting fermentation of anthocyanin-containing fruit wine for generating Methyl-vitasin B, which reserves the anthocyanin content in a solution, increases the Methyl-vitasin B content and improves the color quality of the fruit wine.

The invention aims to provide a method for color-protecting fermentation of anthocyanin-containing fruit wine for generating Methyl-vitasin B.

Another object of the present invention is to provide a Methyl-vitasin B derivative of anthocyanin, which has a chemical structural formula represented by the following formula (I):

in order to achieve the purpose, the invention is realized by the following scheme:

according to the condition of early fermentation of fruit wine in actual production, various auxiliary factors suitable for yeast growth are added, the sugar degree and the pH value are adjusted, and simulated fermentation liquor for early fermentation of fruit wine is constructed, so that the method lays a foundation for subsequent research on the mechanism of anthocyanin in the yeast fermentation process. Inoculating single strain or composite strain of Saccharomyces cerevisiae into simulated fermentation liquid containing anthocyanin, and observing color change during fermentation. And (3) taking fermentation samples at different times, and carrying out qualitative and quantitative analysis on color development substances (anthocyanin and anthocyanin products) in the fermentation liquor by using UPLC-MS. Thereby determining the retention condition of anthocyanin and the generation condition of the derivative Methyl-vitasin B in the fermentation process of the yeast in the fruit wine.

A method for color-protecting fermentation of anthocyanin-containing fruit wine for generating Methyl-vitasin B is characterized in that fermentation is carried out by using one or more of Saccharomyces cerevisiae CICC 31898, Saccharomyces cerevisiae CICC 31895, Saccharomyces cerevisiae CICC 31905 and/or Saccharomyces cerevisiae CICC 31966 which are preserved in China center for industrial microorganism strain preservation management, wherein Methyl-vitasin B is anthocyanin derivative, and the chemical structural formula of the derivative is shown as the formula (I):

preferably, the fermentation is carried out using Saccharomyces cerevisiae CICC 31905, deposited at the China center for Industrial culture Collection of microorganisms.

Preferably, the anthocyanin is one or more of cyanidin-3-O-glucoside (C3G).

More preferably, the anthocyanin is cyanidin-3-O-glucoside (C3G).

Preferably, the initial pH value of the fermentation liquor of the color-protecting fermentation is within the range of 3.0-3.5.

Preferably, the sugar degree of the fermentation liquor of the color-protecting fermentation is measured by a Brix meter, and the value of the sugar degree is within the range of 19-22.

A anthocyanin derivative Methyl-Vitisin B, wherein the chemical structural formula is shown as formula (I):

compared with the prior art, the invention has the following beneficial effects:

the invention utilizes Saccharomyces cerevisiae D254 yeast, 2323 yeast, RC212 yeast and VL1 yeast to brew the fruit wine, reserves more C3G, promotes cyanidin to generate Methyl-vitasin B, wherein the RC212 yeast reserves C3G the most and generates Methyl-vitasin B the most. Methyl-vitasin B is a product obtained by further methylating an anthocyanin derivative, and the substance is a novel anthocyanin derivative. The invention provides an effective color protection method in fruit wine brewing, and provides an effective tool and process for improving the color quality of the fruit wine.

Drawings

FIG. 1 is a graph of color change of fermentation broth at different times;

FIG. 2 is a liquid chromatogram of each group at 520 nm;

FIG. 3 is a chart of the UV-VIS absorption spectrum of C3G;

FIG. 4 is a secondary mass spectrum of C3G;

FIG. 5 is a diagram of the UV-VIS absorption spectrum of cyanidin;

FIG. 6 is a secondary mass spectrum of cyanidin;

FIG. 7 is a graph of the UV-VIS absorption spectrum of Methyl-vitasin B;

FIG. 8 is a secondary mass spectrum of Methyl-Vitisin B;

FIG. 9 shows the content of C3G in four yeasts at different fermentation times, wherein 3 samples are selected from each group, and labeled with different lower case letters to show significant inter-group variability (p <0.05), and labeled with the same lower case letters to show insignificant inter-group variability (p > 0.05);

FIG. 10 shows the cyanidin content of four yeasts at different fermentation times, wherein 3 samples are selected from each group, and are marked with different lower case letters to show that the difference between the groups is significant (p <0.05), and the same lower case letters to show that the difference between the groups is not significant (p > 0.05);

FIG. 11 shows the content of Methyl-vitasin B in four yeasts at different fermentation times, wherein 3 samples are selected from each group, and the samples are marked with different lower case letters to show significant difference between groups (p <0.05), and the samples are marked with the same lower case letters to show insignificant difference between groups (p > 0.05).

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The materials used in the following examples are as follows:

the Saccharomyces cerevisiae is obtained from China center for Industrial culture Collection of microorganisms (CICC), CICC 31898 (original number D254), CICC 31895 (original number 2323), CICC 31905 (original number RC212) and CICC 31966 (original number VL 1).

Experimental reagent: cyanidin-3-O-glucoside, obtained by separation in the laboratory, with a purity of more than 94%; methanol, Shanghai Tantake Technique, Inc.; formic acid (chromatographic grade), tianjin, komi europe chemicals ltd; acetonitrile (chromatographic grade), merck group ltd, germany.

Experimental apparatus equipment: LE104E/02 electronic balance, mettler-toledo instruments ltd; KS15 biosafety cabinet, Thermo Scientific, usa; electrothermal blowing drying ovens, shanghai-chang scientific instruments ltd; BXM-30R sterilization pot, xiamen micro instruments ltd; UPLC/MS-8045 triple quadrupole LC Mass spectrometer, Shimadzu (SHIMADZU); c18 (2.1X 100mm,1.8 μm) column, Aigel-Feinumet; waters liquid chromatograph, Waters technologies ltd; c18 (4.6X 250mm, 5.0 μm) column, BornaeIjer technologies, Inc.

Example 1 Effect of four yeasts on anthocyanin during fermentation of fruit wine

1. Experimental methods

(1) Preparation phase

Cleaning a 60mL special fermentation bottle, placing the cleaned fermentation bottle in a beaker, packaging the fermentation bottle, placing the packaged fermentation bottle in a high-pressure steam sterilization pot, sterilizing the fermentation bottle for 20min at a high temperature of 121 ℃, placing the fermentation bottle in a drying oven at a temperature of 65 ℃ for drying, and placing the fermentation bottle in a super clean workbench for later use.

(2) Simulation architecture construction

Accurately weighing 110.0g of glucose, 110.0g of fructose, 2.0g of tartaric acid, 2.0g of monopotassium phosphate, 0.5g of ammonium chloride and 0.2g of magnesium sulfate heptahydrate, using a volumetric flask to fix the volume to 1000mL to prepare a fermentation liquor for later use, and adding a monomer anthocyanin cyanidin-3-glucoside (C3G) into the fermentation liquor to prepare a simulated fermentation liquor containing anthocyanin. The pH of the solution was adjusted to 3.5 using saturated sodium hydroxide solution, and then 50mL of the solution was taken through a 0.22 μm filter using a syringe and transferred to a 60mL fermentation flask.

(3) Conditions of yeast inoculation and fermentation

The activated agar medium was dipped with yeast by using an inoculating loop and transferred to YPD medium, and cultured at 30 ℃ for 24 hours at 150 r/min. And (3) respectively sucking 20 mu L of the bacterial suspension from the liquid culture medium into a 1.5mL EP tube filled with 200 mu L of distilled water, performing vortex mixing, dripping 20 mu L of the mixed solution onto a cell counting plate, covering the cell counting plate with a cover glass, counting the cell counting plate on a microscope, and recording the corresponding yeast number. The volume of liquid medium inoculated was determined according to the amount of yeast, the insufficient volume was made up with sterilized blank medium so that the amount of yeast in each bottle of fermentation broth was equal and the total volume of medium added was 5 mL. And (3) fermenting the inoculated fermentation liquor in a biochemical incubator at 25 ℃, and keeping the bottle cap in an incompletely screwed state so as to discharge carbon dioxide, wherein the fermentation process is strictly protected from light.

(4) Qualitative analysis of anthocyanin and its derivatives

Analyzing anthocyanin derivative components in the fermentation liquor by using UPLC-MS.

Sample pretreatment: after centrifuging 1.0mL of the fermentation broth, the filtrate was filtered through a 0.22 μm pore size aqueous membrane and placed in a brown liquid vial for use.

Liquid phase conditions: a C18 reverse chromatographic analysis column (2.1 multiplied by 100mm,1.8 mu m) is used, the temperature of the chromatographic column is 35 ℃, the mobile phase A is 2% formic acid water, the mobile phase B is 100% acetonitrile, the single sample injection volume is 5.0 mu L, the flow rate is 0.3mL/min, the detection wavelength is 520nm, the gradient elution program is 0-3 min, and the B content is 6% -6%; 3-8 min, 6% -13% of B; 8-11 min, 13% -25% of B; 11-15 min, 25% -95% B; 15-16 min, 95% -95% B; 16-19 min, 95% -30% of B; 19-21 min, 30% -13% B; 21-23 min, 13% -6% of B; 23-25 min, 6% -6% of B.

Mass spectrum conditions: performing mass spectrometry by using a MS-8045 triple quadrupole mass spectrometer of Shimadzu, ESI interface, interface voltage of 4.0kV, interface temperature of 300 ℃, DL tube temperature of 250 ℃, heating block temperature of 400 ℃, and performing analysis by using positive and negative ion scanning modes; nitrogen gas is used as atomizing gas, drying gas and heating gas, argon gas is used as collision gas, wherein the flow rate of atomizing gas is 3.0L/min, the flow rate of drying gas is 10.0L/min, the flow rate of heating gas is 10.0L/min, and the pressure of a collision chamber is 230 kPa.

(5) Quantitative analysis of anthocyanins and their derivatives

The anthocyanin derivative has absorption at 520nm wavelength and the content is in direct proportion to the corresponding peak area, so the content change condition can be calculated by analyzing the peak area of a liquid chromatogram and referring to a standard curve of C3G.

2. Results of the experiment

(1) Color change of fermentation broth

Four strains of Saccharomyces cerevisiae (2323, D254, RC212 and VL1) were used for fermentation experiments, and color change of each strain was observed during fermentation.

As is clear from the experimental results (FIG. 1), the color of the yeast group and that of the yeast-free group did not differ significantly at 0 day of fermentation, and the solutions of the respective groups exhibited bright red colors. When the fermentation is carried out for 2 days, the solution without the yeast is bright red; the fermentation action of the yeast will significantly reduce the color in solution; the color difference of the fermentation liquor under the fermentation action of different yeasts is not obvious. When the fermentation is carried out for 5 days, the color of the solution without the yeast is not obviously attenuated; the color of the solution is obviously reduced by the fermentation effect of the yeast, wherein the color of the solution after the yeast VL1 is fermented is reddish, the color of the solution after the fermentation of other yeast is yellowish, and the color of the fermentation liquor RC212 is higher than that of the fermentation liquor of other three groups of yeast.

(2) Liquid phase and mass spectrometry results

The fermentation liquor without the yeast fermentation control group, the yeast 2323, the yeast D254, the yeast RC212 and the yeast VL1 all has three absorption peaks (figure 2) at the wavelength of 520nm, wherein the retention time of the No. 1 peak is 13.6min, the maximum absorption wavelength (figure 3) is 517nm, and the secondary mass spectrum result (figure 4) shows that the substance is C3G. The retention time of peak 2 was 15.5min, the maximum absorption wavelength (FIG. 5) was 527nm, and the secondary mass spectrometry result (FIG. 6) indicated that the substance was cyanidin. The retention time of peak 3 was 16.1min, the maximum absorption wavelength (FIG. 7) was 505nm, and the secondary mass spectrometry results (FIG. 8) indicated that the substance was presumed to be Methyl-Vitisin B produced by methylation and derivatization of cyanidin.

And (4) analyzing the contents of the anthocyanin and anthocyanin-related products in the fermentation liquor by using HPLC. The standard curve of C3G is obtained by external standard analysis, and the contents of cyanidin and Methyl-vitasin B are calculated by using the standard curve of C3G. Experimental results show that the content of C3G in the RC212 yeast fermentation liquid (figure 9) is obviously higher than that of other three yeasts when the fermentation is carried out for 1-5 days. When the fermentation is carried out for 1-5 days, the cyanidin aglycone content (shown in figure 10) in the four yeast fermentation liquids has no significant change. On day 5 of fermentation, the content of Methyl-vitasin B in the RC212 yeast broth (fig. 11) was significantly higher than that of the other three yeasts.

(3) Analysis of results

The color change in the fermentation liquor is mainly related to the content of the color-developing substances, and only one color-developing substance, namely anthocyanin, is added in the experiment for carrying out the experiment. The color change in the solution can be explained by comparing the fermentation phenomenon with the change in the contents of the main coloring substances (anthocyanins and their products).

The color of the RC212 fermentation broth is higher than that of the other three groups of yeast fermentation broths, mainly because the contents of C3G and Methyl-Vitisin B in the fermentation broth are significantly higher than that of the other three groups of yeast fermentation broths.

Research shows that different saccharomyces cerevisiae cell walls have different adsorption effects on anthocyanin, beta-glucosidase activities of different yeast cells also influence the content change of anthocyanin, and the retention effect of RC212 yeast on anthocyanin may be related to the adsorption effect of yeast cell walls and the beta-glucosidase activity.

Research shows that active small molecular substances in the fruit wine fermentation process, such as pyruvic acid, acetaldehyde and the like, can chemically react with anthocyanin to generate anthocyanin derivatives with more stable structures than the anthocyanin. The Methyl-vitasin B obtained in the experiment is a product obtained after further methylation of the anthocyanin derivative, and the substance is a novel anthocyanin derivative and has not been reported by others. The production of the yeast strain is greatly related to the formula of fermentation liquor and the fermentation and metabolism of yeast, which is also the reason for the difference of the final content of Methyl-vitasin B of different yeasts, and RC212 yeast can generate higher content of Methyl-vitasin B under the fermentation condition, thereby playing a role in protecting the color of fruit wine.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.