Co-fermentation method for improving color and luster of fruit wine and application

Abstract

The invention belongs to the technical field of food fermentation, and particularly relates to a co-fermentation method for improving the color of fruit wine. According to the method, lactic acid bacteria and saccharomycetes are adopted to cooperatively ferment and enzymatically hydrolyze fruit juice, and the fruit wine is prepared through a constant-temperature dark fermentation process. According to the brewing method provided by the invention, the degradation of anthocyanin is effectively inhibited and the color quality of a fruit wine product is remarkably improved by utilizing the pH regulation effect generated by double-bacterium co-fermentation and the synergistic effect of the anthocyanin derivative. Compared with the prior art, the method breaks through the technical limitation of a traditional co-fermentation process in the aspect of color improvement, and the obtained fruit wine is brighter and more stable in color compared with fruit juice primary pulp.

Description

Technical Field

[0001] The present application relates to the field of food fermentation technology, and in particular to a co-fermentation method and application for improving the color stability of fruit wine. Background Art

[0002] Mulberries, a classic berry with both medicinal and edible properties, are rich in vitamins (such as vitamin C and B vitamins), organic acids (malic acid, citric acid), and phenolic compounds (anthocyanidins, resveratrol, etc.). Their bioactive components impart multiple health benefits, including lowering blood pressure, regulating glucose and lipid metabolism, protecting the liver, and providing antioxidant benefits, making them a research hotspot for functional food development. Currently, mulberry processed products primarily include juice, jam, vinegar, and wine. Mulberry wine, in particular, enjoys growing global demand due to its enhanced flavor and sensory quality during the brewing process, combining nutritional value with drinkability.

[0003] Innovation in fruit wine brewing technology is key to increasing product added value. While traditional single-yeast fermentation can achieve sugar conversion, it suffers from issues such as a monotonous aroma structure and unstable color. In recent years, the co-fermentation of yeast and lactic acid bacteria has garnered widespread attention due to its synergistic metabolic properties. Lactic acid bacteria degrade organic acids and produce ester precursors, forming a metabolic network with the yeast's alcohol fermentation process, significantly enriching the aroma profile of fruit wine (e.g., increasing the diversity of esters and higher alcohols).

[0004] However, existing research mainly focuses on the regulatory mechanism of co-fermentation on flavor substances, while the impact on color, an important sensory indicator, has not yet been clarified; and color is not only an intuitive reflection of the quality of fruit wine, but the retention and conversion of its core component anthocyanin is more directly related to the functional activity of the product. Therefore, it is imperative to seek a fermentation technology that can improve the color of fruit wine in the long term. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a co-fermentation method and application for improving the color of fruit wine, which is used to solve the technical bottleneck that the existing co-fermentation technology cannot improve the color of fruit wine in the long term.

[0006] To achieve the above technical objectives, the present invention provides a co-fermentation method for improving the color of fruit wine. The fermentation steps are: inoculating lactic acid bacteria and yeast into the enzymatically hydrolyzed juice, and fermenting it at a constant temperature and in the dark to obtain fruit wine.

[0007] Furthermore, a step-by-step inoculation method is adopted: lactic acid bacteria are first inoculated, and then yeast is inoculated after the lactic acid bacteria ferment for a preset time.

[0008] Furthermore, a step-by-step inoculation method is adopted: lactic acid bacteria are inoculated into the juice after enzymatic hydrolysis, and fermented at a constant temperature and in the dark for 3 to 5 days, and then yeast is inoculated and fermented at a constant temperature and in the dark to obtain fruit wine.

[0009] Furthermore, the pH of the juice is 3-5.

[0010] Furthermore, the sweetness of the juice is 25~35 °Bx.

[0011] Furthermore, the cell inoculation density ratio of yeast and lactic acid bacteria is 1:(1~1000).

[0012] Furthermore, the temperature range of constant temperature fermentation is 20~30℃.

[0013] The present invention provides an application of a co-fermentation method for improving the color of fruit wine, which is used for brewing fruit wine.

[0014] Furthermore, the fruit wine is fruit wine containing anthocyanins.

[0015] The invention provides a fruit wine which is prepared by adopting a co-fermentation method and has a pH value of less than 3.5.

[0016] In summary, the present invention provides a co-fermentation method for improving the color of fruit wine. The fermentation steps are: inoculating lactic acid bacteria and yeast into the enzymatically hydrolyzed juice, and fermenting at a constant temperature and in the dark to obtain fruit wine. The brewing method provided in this application uses the co-fermentation technology of lactic acid bacteria and yeast to significantly improve the stability of cyanidin-3-O-glucoside (C3G); and also by strengthening the color parameter a * The pH regulation effect of the dual-strain co-fermentation compensates for the color loss caused by anthocyanin degradation during single-strain fermentation. Therefore, the present invention significantly improves the color of fruit wine through the pH regulation effect of dual-strain co-fermentation and the synergistic effect of anthocyanin derivatives. Compared with existing technologies, the fruit wine obtained by the brewing method provided in this application has a color that is superior to both single-strain fermentation and puree. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0018] Figure 1 Schematic diagram of the change of viable bacteria count under different inoculation time sequences;

[0019] Figure 2 Schematic diagram of the changes in anthocyanin and total phenol content at different inoculation time sequences;

[0020] Figure 3 Schematic diagram of the changes in fermentation kinetics, residual sugar content, ethanol content, L-lactic acid and L-malic acid content at different inoculation time sequences;

[0021] Figure 4 Schematic diagram of the change of viable bacteria count under different inoculation ratios;

[0022] Figure 5 Schematic diagram of the changes in fermentation kinetics, residual sugar content, ethanol content, L-lactic acid and L-malic acid content at different inoculation ratios;

[0023] Figure 6 Schematic diagram of the changes in anthocyanin and total phenol content under different inoculation ratios;

[0024] Figure 7 Schematic diagram of the change of viable bacterial count at different fermentation temperatures;

[0025] Figure 8 Schematic diagram of the changes in fermentation kinetics, residual sugar content, ethanol content, L-lactic acid and L-malic acid content at different fermentation temperatures;

[0026] Figure 9 Schematic diagram of the changes in anthocyanin and total phenol content at different fermentation temperatures;

[0027] Figure 10 A schematic diagram and liquid phase diagram showing changes in anthocyanin retention during the brewing process of the mulberry wine provided in Example 14 and Comparative Example 1;

[0028] Figure 11 Schematic diagram of changes in pH and titratable acidity during the brewing process of mulberry wine provided in Example 14 and Comparative Example 1;

[0029] Figure 12 Schematic diagram and color block diagram of CIELAB changes during the brewing process of mulberry wine provided in Example 14 and Comparative Example 1;

[0030] Figure 13 Schematic diagram of changes in color characteristics (CD, T, WCP, WC, CDR SO2, CAW) during the brewing process of mulberry wine provided in Example 14 and Comparative Example 1;

[0031] Figure 14 Pearson correlation analysis provided for Example 14 and Comparative Example 1. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions of the embodiments of this application in conjunction with the accompanying drawings. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this specification, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection requested by this application.

[0033] The sources of all raw materials in the present invention are not particularly limited and can be purchased on the market or prepared according to conventional methods known to those skilled in the art.

[0034] The embodiment of the present invention provides a co-fermentation method for improving the color of fruit wine. The fermentation steps are: inoculating lactic acid bacteria and yeast into enzymatically hydrolyzed juice, and fermenting at a constant temperature and in the dark to obtain fruit wine.

[0035] It should be noted that the lactic acid produced by lactic acid bacteria fermentation can quickly lower the pH of the system. This change has multiple effects: the low pH system not only helps maintain the stability of anthocyanins, but also inhibits the alcohol fermentation process of brewer's yeast. At the same time, the lactic acid produced by lactic acid bacteria metabolism can provide a carbon source for brewer's yeast, reducing the brewer's yeast's consumption of anthocyanins. In addition, the organic acids produced by lactic acid bacteria form a co-coloring effect with anthocyanins, thereby improving the color stability of the product. Therefore, the co-fermentation system formed by lactic acid bacteria and yeast compensates for the color loss caused by anthocyanin degradation during single-strain fermentation. It also significantly improves the color of the fruit wine through the pH regulation effect of the dual-bacteria co-fermentation and the synergistic effect of anthocyanin derivatives.

[0036] In some specific embodiments, the yeast is wine yeast MST (Maestoso), purchased from Angel Yeast Co., Ltd.; the lactic acid bacteria is Lactobacillus plantarum SCLN1, deposited in Guangdong Provincial Microbiological Culture Collection Center with the accession number GDMCC No. 60944.

[0037] In some embodiments, a step-by-step inoculation method is adopted: lactic acid bacteria are first inoculated, and then yeast is inoculated after the lactic acid bacteria ferment for a preset time.

[0038] In some preferred embodiments, a step-by-step inoculation method is adopted: lactic acid bacteria are inoculated into the enzymatically hydrolyzed juice, and fermented at a constant temperature and in the dark for 3 to 5 days, and then yeast is inoculated and fermented at a constant temperature and in the dark to obtain fruit wine.

[0039] It should be noted that in a co-fermentation system, brewer's yeast exhibits a stronger competitive advantage than lactic acid bacteria. Therefore, the strategy of inoculating lactic acid bacteria first and then delaying the inoculation of brewer's yeast can create more favorable conditions for the growth of lactic acid bacteria and promote their proliferation. Secondly, the inoculation time of brewer's yeast is significantly correlated with the inhibitory effect of lactic acid bacteria. The later the inoculation time, the more obvious the inhibitory effect of lactic acid bacteria on subsequent alcohol fermentation. However, if brewer's yeast is inoculated too late, alcohol fermentation may not proceed fully, and the residual sugar concentration in the system may be too high, increasing the risk of spoilage bacteria growth.

[0040] In some embodiments, the pH of the juice is 3-5.

[0041] In some embodiments, the sweetness of the juice is 25-35 °Bx.

[0042] In some embodiments, the cell inoculation density ratio of yeast and lactic acid bacteria is 1:(1-1000).

[0043] In some preferred embodiments, the cell inoculation density ratio of yeast and lactic acid bacteria is 1:(1-10).

[0044] It's important to note that fermentable sugars in juice serve as a carbon source for yeast growth and reproduction, effectively boosting their metabolic activity and accelerating fermentation initiation. Especially when yeast is inoculated later, an appropriate sugar concentration can maintain the yeast's competitive advantage and reduce the inhibitory effects of lactic acid bacteria metabolites on the yeast.

[0045] In some embodiments, the temperature range of constant temperature fermentation is 20-30°C.

[0046] In some preferred embodiments, the temperature range of constant temperature fermentation is 20-25°C.

[0047] In some more preferred embodiments, the temperature range of the constant temperature fermentation is 25°C.

[0048] It is important to note that mulberry wines brewed at 20°C and 25°C significantly outperformed those brewed at 30°C in key indicators such as viable cell count, residual sugar content, and antioxidant retention. Furthermore, the 25°C group had slightly lower total phenol content than the 20°C group, but also lower residual sugar levels, indicating more thorough alcohol fermentation. This characteristic can effectively reduce the risk of microbial spoilage, improve wine stability, and provide a better foundation for subsequent aging processes. Furthermore, 25°C, being close to industrial production ambient temperatures, can significantly reduce energy costs for temperature control.

[0049] The embodiment of the present invention provides an application of a co-fermentation method for improving the color of fruit wine, which is used for brewing fruit wine.

[0050] Furthermore, the fruit wine includes fruit wine containing anthocyanins.

[0051] Furthermore, fruit wines containing anthocyanins include mulberry wine and grape wine.

[0052] An embodiment of the present invention provides a fruit wine prepared by a co-fermentation method, wherein the pH value of the fruit wine is less than 3.5.

[0053] The applicant further provides the following reference specific embodiments to describe the present invention. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0054] Example 1

[0055] This embodiment provides a co-fermentation method for improving the color of fruit wine, comprising the following steps:

[0056] Step S1, strain activation: Lactic acid bacteria (Lactic Acid Bacteria) and Saccharomyces cerevisiae (Yeast) stored at -80°C in a 25% mass fraction glycerol solution were inoculated into MRS medium (Deborah's medium) and YPD liquid medium (yeast extract peptone dextrose medium), respectively, and cultured at 30°C for 12-14 hours to obtain first-activated lactic acid bacteria strains and Saccharomyces cerevisiae strains; the first-activated strains were inoculated into MRS medium and YPD medium, respectively, and cultured at 30°C for 12-14 hours to obtain second-activated lactic acid bacteria strains and Saccharomyces cerevisiae strains; the second-activated strains were inoculated into MRS and YPD liquid media, respectively, and cultured at 30°C for 12-14 hours to obtain third-activated lactic acid bacteria strains and Saccharomyces cerevisiae strains;

[0057] Step S2, fermentation substrate preparation:

[0058] Fresh mulberries and deionized water were crushed and squeezed in a mass ratio of 1:1 to obtain mulberry pulp. Pectinase (0.05% by mass of the mulberry pulp) was added to the mulberry pulp, and the mulberry pulp with pectinase was placed in a constant temperature incubator at 50°C for enzymatic hydrolysis for 150 min. After the enzymatic hydrolysis, the mulberry juice was filtered with gauze to obtain the mulberry juice. White sugar was added to the mulberry juice to adjust the sugar content to 28 °Bx, and 0.009% potassium metabisulfite was added for preservation. Finally, the pH value of the mulberry juice was adjusted to 4.00 using food-grade citric acid and food-grade sodium bicarbonate. The mulberry juice was then heated at 85°C for 10 min for pasteurization, and then cooled to room temperature for later use.

[0059] Step S3, strain inoculation:

[0060] The activated lactic acid bacteria strain and the saccharomyces cerevisiae strain were inoculated into the reserved mulberry juice at the same time (the inoculation method of this embodiment is abbreviated as Y+L in the accompanying drawings) so that the cell density of lactic acid bacteria and saccharomyces cerevisiae in the mulberry juice was 10 6 CFU / mL, and the inoculated mulberry juice was placed in a constant temperature incubator at 30 ℃ in the dark and fermented for 14 days to obtain mulberry wine.

[0061] Example 2

[0062] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 1 is that the inoculation sequence of the lactic acid bacteria strain and the cerevisiae yeast strain is different. Specifically, the activated cerevisiae yeast strain is inoculated into mulberry juice, and the activated lactic acid bacteria is inoculated after culturing for 1 day (the inoculation method of this embodiment is abbreviated as Y+1dL in the accompanying drawings). The other preparation steps, raw material ratios, and process parameters are the same as those in Example 1.

[0063] Example 3

[0064] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 1 is that the inoculation sequence of the lactic acid bacteria strain and the cerevisiae yeast strain is different. Specifically, the activated cerevisiae yeast strain is inoculated into mulberry juice, and the activated lactic acid bacteria is inoculated after culturing for 2 days (the inoculation method of this embodiment is abbreviated as Y+2dL in the accompanying drawings). The other preparation steps, raw material ratios, and process parameters are the same as those in Example 1.

[0065] Example 4

[0066] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 1 is that the inoculation sequence of the lactic acid bacteria strain and the cerevisiae yeast strain is different. Specifically, the activated lactic acid bacteria strain is inoculated into mulberry juice, and the activated cerevisiae yeast is inoculated after culturing for 1 day (the inoculation method of this embodiment is abbreviated as L+1dY in the accompanying drawings). The other preparation steps, raw material ratios, and process parameters are the same as those in Example 1.

[0067] Example 5

[0068] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 1 is that the inoculation sequence of the lactic acid bacteria strain and the cerevisiae yeast strain is different. Specifically, the activated lactic acid bacteria strain is inoculated into mulberry juice, and the activated cerevisiae yeast is inoculated after culturing for 2 days (the inoculation method of this embodiment is abbreviated as L+2dY in the accompanying drawings). The other preparation steps, raw material ratios, and process parameters are the same as those in Example 1.

[0069] Example 6

[0070] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 1 is that the inoculation sequence of the lactic acid bacteria strain and the cerevisiae yeast strain is different. Specifically, the activated lactic acid bacteria strain is inoculated into mulberry juice, and the activated cerevisiae yeast is inoculated after culturing for 3 days (the inoculation method of this embodiment is abbreviated as L+3dY in the accompanying drawings). The other preparation steps, raw material ratios, and process parameters are the same as those in Example 1.

[0071] Example 7

[0072] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 1 is that the inoculation sequence of the lactic acid bacteria strain and the cerevisiae yeast strain is different. Specifically, the activated lactic acid bacteria strain is inoculated into mulberry juice, and the activated cerevisiae yeast is inoculated after culturing for 5 days (the inoculation method of this embodiment is abbreviated as L+5dY in the accompanying drawings). The other preparation steps, raw material ratios, and process parameters are the same as those in Example 1.

[0073] Example 8

[0074] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 1 is that the inoculation sequence of the lactic acid bacteria strain and the cerevisiae yeast strain is different. Specifically, the activated lactic acid bacteria strain is inoculated into mulberry juice, and the activated cerevisiae yeast is inoculated after culturing for 7 days (the inoculation method of this embodiment is abbreviated as L+7dY in the accompanying drawings). The other preparation steps, raw material ratios, and process parameters are the same as those in Example 1.

[0075] Example 9

[0076] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 7 is that the cell densities of the saccharomyces cerevisiae strain and the lactic acid bacteria strain after inoculation are different. Specifically, the cell densities of the saccharomyces cerevisiae strain and the lactic acid bacteria strain after inoculation are 10 6 CFU / mL and 10 7 CFU / mL, at this time the cell density ratio of Saccharomyces cerevisiae and lactic acid bacteria is 1:10.

[0077] Example 10

[0078] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 7 is that the cell densities of the lactic acid bacteria strain and the saccharomyces cerevisiae strain after inoculation are different. Specifically, the cell densities of saccharomyces cerevisiae and lactic acid bacteria after inoculation are 10 6 CFU / mL and 10 8 CFU / mL, at this time the cell density ratio of Saccharomyces cerevisiae and lactic acid bacteria is 1:100.

[0079] Example 11

[0080] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 7 is that the cell densities of the lactic acid bacteria strain and the saccharomyces cerevisiae strain after inoculation are different. Specifically, the cell densities of saccharomyces cerevisiae and lactic acid bacteria after inoculation are 10 6 CFU / mL and 5*10 8 CFU / mL, at this time the cell density ratio of Saccharomyces cerevisiae and lactic acid bacteria is 1:500.

[0081] Example 12

[0082] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 7 is that the cell densities of the lactic acid bacteria strain and the saccharomyces cerevisiae strain after inoculation are different. Specifically, the cell densities of saccharomyces cerevisiae and lactic acid bacteria after inoculation are 10 6 CFU / mL and 10 9 CFU / mL, at this time the cell density ratio of brewer's yeast and lactic acid bacteria is 1:1000.

[0083] Example 13

[0084] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 7 is that the fermentation temperature is different; the fermentation temperature is 20°C.

[0085] Example 14

[0086] This embodiment provides a co-fermentation method for improving the color of fruit wine. The difference from Example 7 is that the fermentation temperature is different; the fermentation temperature is 25°C.

[0087] Comparative Example 1

[0088] This comparative example provides a fermentation method for improving the color of fruit wine. The difference from Example 1 is that a single strain inoculation method is adopted, and the inoculated strain is Saccharomyces cerevisiae (the inoculation method of this comparative example is abbreviated as CKY in the accompanying drawings), and the cell density after inoculation is 10 6 CFU / mL.

[0089] Comparative Example 2

[0090] This comparative example provides a fermentation method for improving the color of fruit wine. The difference from Example 1 is that a single strain inoculation method is adopted, and the inoculated strain is lactic acid bacteria (the inoculation method of this comparative example is abbreviated as CKL in the accompanying drawings), and the cell density after inoculation is 10 6 CFU / mL.

[0091] Comparative Example 3

[0092] This comparative example provides a co-fermentation method for improving the color of fruit wine. The difference from Example 7 is that the fermentation temperature is different; the fermentation temperature is 37°C.

[0093] In order to evaluate and verify the fermentation methods provided in the above embodiments and comparative examples, the physical and chemical indexes of the fermented fruit wine were tested. The specific test items and test methods are as follows:

[0094] Test 1: Fermentation kinetics test

[0095] Test method: Fermentation dynamics is measured by monitoring CO2 emissions. In this test, mulberry juice was weighed every 24 hours during the fermentation phase.

[0096] Test 2: Determination of reducing sugars, organic acids, and ethanol

[0097] Reducing sugar test method: 3,5-dinitrosalicylic acid colorimetric method (DNS);

[0098] L-lactic acid and L-malic acid assay methods: Mulberry juice from the fermentation stage was assayed by high-performance liquid chromatography (HPLC). Organic acids were separated using a Venusil ASB C18 column (4.6 mm × 250 mm, 5 μm particle size, Tianjin, China). Detection was performed at a wavelength of 210 nm using a diode array detector. Peaks were identified based on retention times and quantified using an external standard curve. The mobile phase consisted of 0.1% H3PO4 solution at a flow rate of 0.8 mL / min, the column temperature was 30°C, and the injection volume was 10.0 μL.

[0099] Ethanol content in fermented mulberry juice was determined using a Shimadzu GC-2010 Pro gas chromatograph equipped with an SH-5 capillary column (30 m × 0.25 mm × 0.25 μm) and an FID detector. Instrument parameters were as follows: FID detector and injection port temperatures were set at 250°C and 225°C, respectively; hydrogen and nitrogen flow rates were 40 mL / min and 400 mL / min, respectively; the equilibrium temperature was 75°C for 1 min; the column temperature was 60°C, the sample tube temperature was 80°C, the transfer tube temperature was 90°C, the cycle time was 7.50 min, the purge flow rate was 3 mL / min, and the injection volume was 10 μL.

[0100] Test 3: Test of viable bacteria count

[0101] Test Method: Microbial counts were performed using the standard plate count method. The test method employed in this invention is as follows: Serial decimal dilutions were prepared using sterile physiological saline (0.9% NaCl solution by mass). 0.1 mL of each dilution was evenly spread onto a fresh plate and incubated at 30°C for 48 hours. After incubation, dilutions were selected that resulted in 30 to 300 CFU (colony-forming units) with clear boundaries between colonies. The viable count in the original solution was calculated and expressed as 1 g CFU / mL. Note: For counting Saccharomyces cerevisiae, incubation should be performed on Red Bengal agar plates; for counting lactic acid bacteria, incubation should be performed on MRS agar plates containing 0.1 g / L natamycin.

[0102] Test 4: Determination of total anthocyanin content and total phenolic content

[0103] Total Anthocyanin Content Test Method: This method makes a simple improvement based on the pH differential method. The specific method is as follows: 0.025 mol / L KCl buffer at a pH of 1.0 and 0.4 mol / L CH3COONa buffer at a pH of 4.5 are uniformly mixed with fermented mulberry juice (or mulberry wine). After mixing, the mixture is incubated in the dark for 30 minutes. The absorbance of the incubated mulberry juice (or mulberry wine) is measured at 520 nm and 700 nm. The total anthocyanin content is expressed as the equivalent of cyanidin-3-O-glucoside, calculated as follows:

[0104] Anthocyanin content (mg / L) = (A × MW × DF × 10 3 ) / (ε×1) Formula (1);

[0105] In formula (1),

[0106] A represents absorbance, A=(A 520nm -A 700nm ) pH1.0 -(A 520nm -A 700nm ) pH4.5 ;

[0107] MW indicates the molecular mass of cyanidin-3-O-glucoside, 449.2;

[0108] ε represents the molar extinction coefficient of cyanidin-O-glucoside, 26900;

[0109] 1 means the optical path of the cuvette is 1 cm;

[0110] DF indicates the dilution factor.

[0111] Method for determining total phenol content: The present invention is slightly improved on the basis of the Folin-Ciocalteu method. The specific test method is as follows: dilute the mulberry juice (or mulberry wine) in the fermentation stage, take 1 mL of a 1:10 diluted Folin~Ciocalteu reagent and mix it with 200 μL of the diluted mulberry juice (or mulberry wine) for 4 minutes. After the reaction is completed, add 800 μL of sodium carbonate with a mass concentration of 75 g / L, continue to incubate at room temperature in the dark for 2 hours, and finally test the absorbance at 765 nm. The calculated result is expressed as grams of gallic acid per liter of fermented mulberry juice (mulberry wine).

[0112] Test 5: Anthocyanin monomer content test

[0113] Test method: Mulberry juice (or mulberry wine) in the fermentation stage was filtered through a 0.22 μm filter and collected in a brown liquid sample bottle. The sample bottle was placed in the injection area of ​​a Wukong instrument K2025 (Shanghai, China). Anthocyanins were separated using a Venusil ASB C18 column (4.6 mm × 250 mm, 5 μm particle size, Tianjin, China) and detected using a diode array detector at 520 nm. The mobile phases A and B were methanol and 2% formic acid water, respectively, with a gradient program of: 0.0–30.0 min, 6–90% A; 30.0–31.0 min, 90–95% A; 31.0–34.0 min, 95–95% A; 34.0–37.0 min, 95–6% A; 37.0–40.0 min, 6–6% A; the injection volume was 10 μL, the flow rate was 1 mL / min, and the column temperature was 30 °C.

[0114] Test 6: Determination of pH and titratable acidity

[0115] Test method: Titratable acidity is tested according to GB / T12456~2021 "Determination of total acid in foods"; pH value is measured using a pH meter.

[0116] Test 7: Color Evaluation

[0117] CIELAB determination: The color of fermented mulberry juice (or mulberry wine) was measured using a benchtop spectrophotometer (Hangzhou Caipu Technology Co., Ltd., Zhejiang, China). The specific test method is as follows: the diluted fermented mulberry juice (or mulberry wine) was placed in a 12.5 × 12.5 × 45 mm quartz cuvette and the color characteristics of the sample were measured using a spectrophotometer. Distilled water was used as a control. The parameters were set to illuminant D65 and the observation angle was 10°. In transmission mode, the CIELAB color parameter brightness (L) was calculated using the following formula: * )、Red and Green Value(a * )、yellow-blue value(b * ), chroma (C * ), hue angle (h ab ), total color difference (ΔE):

[0118] C * =(a *2 +b *2 ) 1 / 2 Formula (2);

[0119] h ab =arctan(b * / a * ) Formula (3);

[0120] ΔE=[(ΔL * )2 + (Δa * ) 2 + (Δb * ) 2 ] 1 / 2 Formula (4).

[0121] Test 8: Determination of color density and hue

[0122] Test method: The color characteristics of fermented mulberry juice (or mulberry wine) are further characterized by measuring color density (CD) and hue (Tint, T) using a microplate reader. The specific test method is: measure the absorbance of diluted fermented mulberry juice (or mulberry wine) at 420 nm, 520 nm, and 620 nm, and calculate CD and T according to the following formula:

[0123] CD=A 420 +A 520 +A 620 Formula (5);

[0124] T=A 420 / A 520 Formula (6).

[0125] Test 9: Determination of mulberry wine color (WCP) at pH 1.0

[0126] Test method: Take 0.3 mL of mulberry wine and dissolve it in 2.7 mL of 1 mol / L hydrochloric acid solution, mix well, and then place it in the dark for 30 minutes. Use a microplate reader to measure the absorbance of the sample at 520 nm, and calculate the chromaticity of the mulberry wine according to the coefficient formula:

[0127] WCP=A 520 ×10 Formula (7).

[0128] Test 10: Determination of the color of anthocyanins resistant to SO2 bleaching (CDR SO2), determination of the color of mulberry wine (WC), and calculation of the chemical age of wine (CAW)

[0129] CDR SO2 test method: Add 45 μL of 20% sodium metabisulfite solution to 3.0 mL of fermentation-stage mulberry juice (or mulberry wine), place in the dark for 10 minutes, and measure the absorbance of the mulberry juice (or mulberry wine) at 520 nm using a microplate reader.

[0130] WC test method: Add 30 μL of 10% acetaldehyde solution to 3.0 mL of fermentation-stage mulberry juice (or mulberry wine), place the mixture in the dark for 45 min, and measure the absorbance of the mulberry juice (or mulberry wine) at 520 nm using a microplate reader.

[0131] The calculation method of Chemical Alcohol Age (CAW) is as follows:

[0132] CAW=(CDR SO2 / WC)×100% Equation (8).

[0133] Data processing method 11: All experiments were repeated three times, and the final results were expressed as mean ± standard deviation. At the same time, one-way analysis of variance was performed, and Duncan's multiple range test was used to compare the significance between the data. When P < 0.05, it indicated that there was a significant difference between the samples; and the Pearson correlation coefficient was used to examine the correlation of the color characteristics of the mulberry juice fermentation process.

[0134] In order to systematically evaluate the effects of different inoculation timings or methods on the quality of mulberry wine, the present invention monitored the fermentation dynamics of Examples 1 to 8 and Comparative Examples 1 to 2. The specific results are as follows:

[0135] (1) Viable bacteria count test results:

[0136] The growth and reproduction of cells is one of the key indicators for evaluating fermentation efficiency and competition between strains. Figure 1 The changes in the bacterial counts of saccharomyces cerevisiae and lactic acid bacteria after different inoculation methods in Examples 1 to 8 and Comparative Examples 1 to 2 are shown (Note: The viable bacterial counts of the inoculated lactic acid bacteria and / or saccharomyces cerevisiae were not tested on day 0, and the preset inoculation density was used at this time; and Figure 1 G and Figure 1 J, the initial inoculation of Saccharomyces cerevisiae was on the 2nd and 7th day, respectively, and the viable count of Saccharomyces cerevisiae on the day of inoculation was not tested. The determination of the viable count began on the 3rd and 8th day, respectively. Specific details are as follows:

[0137] In separate fermentation systems, Saccharomyces cerevisiae and lactic acid bacteria showed significantly different growth trends (e.g. Figure 1 A and 1B). Saccharomyces cerevisiae has a short lag phase. After inoculation with mulberry juice at an initial inoculum of 6.0 lg CFU / mL, it quickly enters the growth phase. On the first day of fermentation, the bacterial count reaches a peak of 8.2 lg CFU / mL. For the next four days, the viable bacterial count remains above 8.0 lg CFU / mL. From the fifth day of fermentation, the growth activity of Saccharomyces cerevisiae begins to weaken and gradually decreases over the subsequent period until it drops to 2.3 lg CFU / mL (e.g. Figure 1 A). The growth of lactic acid bacteria showed a one-day lag phase, followed by an exponential growth phase, with the bacterial count rapidly increasing to a peak of 8.4 lg CFU / mL on the fifth day. Subsequently, its growth activity decreased, and the bacterial count slowly decreased, eventually stabilizing at 7.3 lg CFU / mL (e.g. Figure 1B). This shows that Saccharomyces cerevisiae can reach its peak bacterial count faster than lactic acid bacteria, demonstrating its rapid cell growth and strong adaptability.

[0138] In the dynamic changes of microbial populations in the co-fermentation system, the growth of lactic acid bacteria is not ideal (e.g. Figure 1 C-1J). Regardless of the inoculation method, the viable lactic acid bacteria count rapidly decreased after the addition of Saccharomyces cerevisiae, significantly inhibiting their growth. This phenomenon may be due to a variety of factors. Analysis of strain characteristics revealed that Saccharomyces cerevisiae and lactic acid bacteria fermentations alone showed that Saccharomyces cerevisiae adapted more quickly to the mulberry juice system, potentially suggesting greater competitiveness and a greater advantage in nutrient competition. Furthermore, the rapid proliferation and fermentation of Saccharomyces cerevisiae continuously produce metabolites such as ethanol. In fact, during co-fermentation, Saccharomyces cerevisiae and lactic acid bacteria interact at numerous chemical and physical levels. Chemical interactions primarily manifest as the effects of metabolites on each other's growth, while physical interactions involve direct cell-to-cell contact. These interactions influence the compatibility of the two and the success rate of fermentation. Generally, the presence of Saccharomyces cerevisiae inhibits the growth of co-fermented lactic acid bacteria, and the degree of inhibition is closely related to the inoculation time of Saccharomyces cerevisiae and lactic acid bacteria. The longer the alcoholic fermentation lasts and the later the lactic acid bacteria are inoculated, the greater the inhibitory effect. This test also fully confirmed this point. The inoculation method of inoculating brewer's yeast first is more detrimental to the growth of lactic acid bacteria (such as Figure 1 D-1E). The saccharomyces cerevisiae used in the present invention and the lactic acid bacteria mainly exhibit an inhibitory effect. Therefore, in the present invention, the method of first inoculating lactic acid bacteria and delaying the inoculation time of saccharomyces cerevisiae can promote the growth of lactic acid bacteria to a certain extent (such as Figure 1 F-1J).

[0139] (2) Anthocyanin test results:

[0140] During the fermentation process, the anthocyanin content continued to decline, and the decline in the early stage of fermentation was significantly greater than that in the later stage (e.g. Figure 2 A). This is primarily due to the presence of yeast during wine fermentation, which causes a significant amount of anthocyanin degradation. Furthermore, yeast cell walls also adsorb anthocyanins, causing further anthocyanin loss. However, anthocyanins adsorbed by yeast cell walls only account for 1.60–5.80% of the total anthocyanins in wine.

[0141] In the early stage of fermentation, the brewer's yeast is in the period of vigorous reproduction (such as Figure 1 A), at which point it plays a leading role in the degradation of anthocyanins; in the late fermentation stage, the brewer's yeast enters the decline stage (e.g. Figure 1 A), the loss of anthocyanins is mainly due to adsorption on yeast cell walls. During the entire fermentation process, the anthocyanin content of CKL is always much higher than that of CKY (e.g. Figure 2 A). This may be because lactic acid bacteria fermentation causes a rapid decrease in pH. Under low pH conditions, anthocyanins exist as stable cations called chlorophyll. Therefore, the anthocyanin content of whole mulberry pulp changes little when lactic acid bacteria ferment it, while yeast fermentation, whether aerobic or anaerobic, results in a decrease in anthocyanin content.

[0142] Under the inoculation method of first inoculating brewer's yeast (Y+L, Y+1dL, Y+2dL), the trend of anthocyanin content change was basically consistent with that of CKY, which was consistent with the results of the viable cell count test (e.g. Figure 1 C-1E), indicating that lactic acid bacteria are difficult to grow under these inoculation methods, and fermentation mainly relies on Saccharomyces cerevisiae.

[0143] The fermentation strategy of inoculating lactic acid bacteria first (L+5dY, L+7dY) seemed to effectively exert the effect of lactic acid bacteria, and the anthocyanin content after fermentation was significantly higher than that of CKY (such as Figure 2 B). This may be because lactic acid fermentation by lactic acid bacteria causes a rapid drop in pH, which, on the one hand, enhances the stability of anthocyanins and, on the other hand, inhibits the subsequent alcoholic fermentation by brewer's yeast. In addition, the lactic acid produced by lactic acid fermentation can serve as a carbon source for brewer's yeast, reducing the yeast's metabolism of anthocyanins. Moreover, the organic acids produced by lactic acid bacteria metabolism form a co-pigmenting effect with anthocyanins, further enhancing the stability of anthocyanins.

[0144] (3) Total phenol test results:

[0145] Total phenols are one of the key indicators for measuring the quality of fruit wine. They are not only closely related to the color of the wine, but also have the effects of scavenging free radicals in the human body, anti-oxidation and anti-tumor. Therefore, it is of great significance to measure the total phenol content of co-fermented mulberry wine. The trend of total phenol content is similar to that of anthocyanins, showing a downward trend throughout the fermentation process, and CKL is significantly higher than CKY (such as Figure 2 C-2D). However, the total phenol content fluctuates, with an upward trend in the early stages of fermentation. This may be because the present invention utilizes fermentation with mulberry pomace, which releases phenolic compounds during the fermentation and maceration process. In the later stages of fermentation, phenolic compounds may undergo chemical reactions such as enzymatic oxidation and polymerization with anthocyanins, leading to a decrease in phenolic compounds. Specifically, anthocyanins can react with various phenolic compounds through enzymatic reactions, forming quinones via coupled oxidation or condensation between quinones. Alternatively, anthocyanin monomers can form polymers through non-enzymatic reactions.

[0146] (4) Fermentation kinetics test results:

[0147] As a key parameter in the fermentation process, the reaction rate can indirectly reflect the status of substrate consumption, microbial growth and product formation in the fermentation system. The present invention explores the fermentation rate and degree of alcohol fermentation by monitoring the mass loss during the fermentation process. Specifically, the greater the mass loss per unit time, the faster the fermentation rate; the greater the mass loss, the more thorough the alcohol fermentation.

[0148] The weight loss of CKY and CKL was in line with expectations (e.g. Figure 3 A). CKY’s alcohol fermentation process is smooth, producing a large amount of The mass decreased rapidly and tended to be constant in the later stage of fermentation, indicating that the alcohol fermentation was basically completed; CKL also showed a slight weight loss, which may be due to the heterolactic fermentation of lactic acid bacteria.

[0149] The final weight loss of Y+L, Y+1dL, Y+2dL, L+1dY and L+2dY was basically the same as that of CKY, which means that the addition of lactic acid bacteria did not affect the alcohol fermentation of brewer's yeast. This result is consistent with Figure 1 This finding supports the conclusion that Saccharomyces cerevisiae strongly inhibits lactic acid bacteria, supporting the conclusion that fermentation is primarily driven by Saccharomyces cerevisiae. L+3dY, L+5dY, and L+7dY showed smaller weight losses compared to CKY, and the later the Saccharomyces cerevisiae yeast was inoculated, the smaller the weight loss. This suggests that a period of initial fermentation by lactic acid bacteria inhibits subsequent alcohol fermentation by Saccharomyces cerevisiae. However, in the case of L+7dY, the fact that it did not maintain a constant weight at the end of fermentation suggests that introducing Saccharomyces cerevisiae yeast too late can lead to incomplete alcohol fermentation.

[0150] (5) Residual sugar test results:

[0151] A specific amount of residual sugar can neutralize the organic acids (such as malic acid and citric acid) in the fruit wine, reduce the sourness and astringency, make the taste softer and mellower, and interact with flavor substances such as fruit aroma and esters to enhance the aroma complexity of the fruit wine. Figure 3 B) showed significant correlation with fermentation kinetics data.

[0152] In the Y+L, Y+1dL, Y+2dL, L+1dY, and L+2dY treatment groups, the residual sugar content was not significantly different from the control group CKY, while the residual sugar content of the L+3dY, L+5dY, and L+7dY treatment groups was significantly higher than that of CKY. However, from the perspective of microbial stability, excessively high residual sugar concentrations increase the risk of spoilage bacteria growth. According to QB / T5476-2020 "General Technical Requirements for Fruit Wine," the residual sugar limit for fruit wine is 150 g / L. Comprehensively judging, the residual sugar content of the L+5dY inoculation method is most in line with industry standards. It is worth noting that the residual sugar content of the CKL group is approximately 9 times that of the CKY group, which may be related to the metabolic pattern of lactic acid bacteria—lactic acid bacteria can obtain energy by breaking down organic acids such as malic acid and citric acid, and their dependence on sugar metabolism is significantly lower than that of brewer's yeast.

[0153] (6) Ethanol test results:

[0154] Ethanol is a characteristic product of alcoholic fermentation, and its content is closely related to fermentation kinetics and residual sugar levels. In the co-fermentation system, the ethanol content of mulberry wine is lower than that of the single yeast fermentation group (CKY) (e.g. Figure 3 C) This phenomenon is driven by two mechanisms: first, nutrient competition between lactic acid bacteria and yeast inhibits yeast metabolic activity; second, organic acids produced by lactic acid bacteria undergo esterification with ethanol, consuming some of the ethanol. The ethanol content in the L+5dY treatment group was 13% (volume fraction), fully meeting wine quality standards.

[0155] (7) Organic acid test results:

[0156] The progress of malic acid-lactic acid fermentation (MLF) was verified by monitoring the changes in organic acid content. The L+5dY treatment group completely converted the initial malic acid into lactic acid and accumulated additional lactic acid through sugar metabolism (e.g. Figure 3 D), a phenomenon directly attributable to the preferential inoculation strategy of lactic acid bacteria. The CKL group (fermentation with pure lactic acid bacteria) showed no detectable malic acid and had the highest lactic acid content, further demonstrating the ability of lactic acid bacteria to metabolize malic acid and their dominant role in the acid metabolism network.

[0157] Based on the above test results (1) to (7), the following conclusions were drawn: 1) In the co-fermentation system, brewer's yeast showed a stronger competitive advantage than lactic acid bacteria. Therefore, the strategy of inoculating lactic acid bacteria first and delaying the inoculation time of brewer's yeast can create more favorable conditions for the growth of lactic acid bacteria and promote their proliferation. 2) The lactic acid produced by lactic acid bacteria fermentation can quickly reduce the pH value of the system. This change has multiple effects: it not only helps to maintain the stability of anthocyanins, but also has an inhibitory effect on the alcohol fermentation process of brewer's yeast; at the same time, the lactic acid produced by lactic acid bacteria metabolism can provide a carbon source for brewer's yeast, reducing the consumption of anthocyanins by brewer's yeast; in addition, the organic acids produced by lactic acid bacteria form a co-color effect with anthocyanins, which improves the color stability of the product. 3) The inoculation time of brewer's yeast is significantly correlated with the inhibitory effect of lactic acid bacteria. The later the inoculation time, the more obvious the inhibitory effect of lactic acid bacteria on subsequent alcohol fermentation. However, if the brewer's yeast is inoculated too late, the alcohol fermentation may not proceed fully. 4) Inoculating brewer's yeast too early can result in low residual sugar levels, while inoculating too late can lead to high residual sugar concentrations, increasing the risk of spoilage bacteria growth. Considering factors such as fermentation efficiency, product quality, and microbiological safety, the L+3dY and L+5dY inoculation sequences offer superior performance.

[0158] In order to explore the effect of different inoculation ratios on the brewing effect of mulberry wine, the present invention monitored the changes in various indicators during the brewing process of Examples 7 and 9 to 12, and the specific results are as follows:

[0159] (1) Population dynamics test results

[0160] like Figure 4 The results show that the population changes of co-fermented mulberry wine under different inoculation ratios did not improve the coexistence of lactic acid bacteria with brewer's yeast, but accelerated the decline of the number of live lactic acid bacteria (such as Figure 4 C-4E). This may be due to the increased lactic acid bacteria inoculation increasing competition with Saccharomyces cerevisiae. Commercial yeast, through artificial breeding and improvement, has the characteristics of rapid fermentation initiation, strong fermentation capacity, and high stability, which has given it a consistent advantage over the competition.

[0161] (2) Fermentation kinetics and residual sugar test results

[0162] Although the results of viable bacteria count were not ideal, increasing the inoculum of lactic acid bacteria did enhance the inhibitory effect on subsequent alcohol fermentation. Figure 5 As shown in A-5B, as the inoculum of lactic acid bacteria increased, the final weight loss decreased and the residual sugar content increased. There was no significant difference in the ethanol content between the treatment groups with different inoculum ratios. This may be because the heterolactic fermentation of lactic acid bacteria also contributed to the ethanol content (e.g. Figure 5 C). All treatment groups were able to achieve complete conversion of malic acid (e.g. Figure 5 D), which is consistent with the results of the L+5dY inoculation method mentioned above, with a malic acid conversion rate of 100%. In addition, increasing the lactic acid bacteria inoculation level promoted the conversion of more sugars into lactic acid.

[0163] (3) Anthocyanin and total phenol test results

[0164] There was little difference in the anthocyanin and total phenol content of mulberry wine in different inoculation ratio treatment groups (e.g. Figure 6 A-6D). Although increasing the lactic acid bacteria inoculation rate inhibits the subsequent alcohol fermentation of brewer’s yeast and is beneficial to the retention of anthocyanins and total phenols, it should be noted that lactic acid bacteria can also degrade anthocyanins and phenolic substances (such as Figure 2 As the lactic acid bacteria inoculation rate increased, its degradation of anthocyanins and total phenols intensified, which offset the reduced consumption of brewer's yeast. As a result, there was no significant difference in the anthocyanin and total phenol content of mulberry wines brewed with different inoculation ratios.

[0165] Based on the above (1) to (3), it can be seen that increasing the inoculum of lactic acid bacteria did not improve the coexistence of lactic acid bacteria with brewer's yeast, nor did it have a significant effect on the anthocyanin and total phenol content. Instead, it led to an increase in the residual sugar content after fermentation. From the perspective of industrial production, increasing the inoculum of lactic acid bacteria will also lead to an increase in production costs. Therefore, the inoculum density ratio of yeast to brewer's yeast is 1: (1-10) which is a more economical and practical inoculation method.

[0166] In order to further explore the effect of fermentation temperature on the brewing effect of mulberry wine, the present invention monitored the changes in various indicators of the brewing process of Example 7, Examples 13-14, and Comparative Example 3, and the monitoring results are as follows:

[0167] (1) Viable bacteria count test results:

[0168] Fermentation temperature has a direct and critical impact on the growth, metabolism and fermentation activity of the strain. Experimental data show that (e.g. Figure 7 D) When the fermentation temperature rose to 37°C, the bacterial counts of both Saccharomyces cerevisiae and lactic acid bacteria in the late fermentation period were <10 CFU / mL, below the detection limit. This phenomenon was attributed to the fact that 37°C exceeded the optimal growth temperature range for both strains, and the high temperature destroyed the integrity of the cell membrane, triggering bacterial autolysis.

[0169] Lowering the fermentation temperature can significantly improve the coexistence of brewer's yeast and lactic acid bacteria. The rate of decline of lactic acid bacteria after inoculation with brewer's yeast is significantly slowed down, especially under the fermentation condition of 20 ℃ (such as Figure 7 A-7B), the two strains achieved excellent synergistic growth. It is speculated that the low temperature effectively inhibited the metabolic activity of Saccharomyces cerevisiae, creating a more suitable habitat for lactic acid bacteria. Furthermore, compared to 30°C and 37°C, lactic acid bacteria exhibited greater tolerance to environmental stress factors at 20°C and 25°C.

[0170] It is worth noting that at fermentation temperatures of 20°C and 25°C, the time for S. cerevisiae to maintain a high viable count was significantly prolonged (e.g. Figure 7 A-7B), which is Figure 8 The residual sugar content in B corresponds to the stable growth and metabolism of yeast at lower temperatures, which directly affects the consumption rate of residual sugar during fermentation and the final residual amount.

[0171] (2) Test results of fermentation kinetics, residual sugar content, and ethanol content:

[0172] There is a close correlation between fermentation kinetics, residual sugar content, ethanol content and viable cell count results. Figure 8 A), the weight loss of mulberry wine fermented at 25 ℃ was the largest, which was consistent with the good coexistence of brewer's yeast and lactic acid bacteria at this temperature (such as Figure 7 B), the lowest residual sugar content (such as Figure 8 B) and the highest ethanol content (e.g. Figure 8 C) form a corresponding relationship. On the other hand, at 37 ℃, the weight loss of mulberry wine is the smallest (e.g. Figure 8 A), which is consistent with the rapid death of both strains at this temperature (e.g. Figure 7 D), the highest residual sugar content (such as Figure 8 B) and the lowest ethanol content (e.g. Figure 8C) The results are consistent.

[0173] From the comparison of temperature gradients, the ethanol content of mulberry wine at 20 ℃ and 25 ℃ is higher than that at 30 ℃ (e.g. Figure 8 C), and the viable bacterial count was also better than that at 30 ℃ (such as Figure 7 This result not only confirms that high temperatures increase microbial sensitivity to environmental stressors but also suggests that the primary inhibitory metabolite on lactic acid bacteria may not be ethanol, but rather other substances such as fatty acids and SO₂. Notably, although the viable count of mulberry wine at 20°C was higher than that at 25°C, its residual sugar content did not decrease further. This may be because, while low temperatures promote the coexistence of bacterial strains, they inhibit the metabolic activity of both Saccharomyces cerevisiae and lactic acid bacteria, preventing the fermentation process from proceeding fully.

[0174] like Figure 8 Organic acid analysis of D showed that L-malic acid was completely converted at all fermentation temperatures. Within the 20-30°C range, L-lactic acid content decreased as the fermentation temperature decreased, consistent with the conclusion that low temperatures inhibit microbial metabolic activity.

[0175] (3) Anthocyanin test results:

[0176] Temperature is a key environmental variable affecting the stability of anthocyanins and total phenols. Its regulatory mechanism on the quality of co-fermented mulberry wine is as follows: Figure 9 As shown. Studies have shown that low temperatures (20°C and 25°C) significantly increase anthocyanin retention, a phenomenon driven by dual mechanisms: first, low temperatures inhibit the metabolic activity of Saccharomyces cerevisiae, reducing its biodegradation of anthocyanins; second, low temperatures effectively reduce the catalytic efficiency of polyphenol oxidase (PPO) and β-glucosidase, enzymes that have been shown to be the primary biocatalysts responsible for anthocyanin structural degradation. In stark contrast, the significant loss of anthocyanins at 37°C is directly attributed to their heat sensitivity—high temperatures accelerate the oxidative degradation of anthocyanin molecules.

[0177] (4) Total phenol test results:

[0178] The trend in total phenolic content showed some synergy with that of anthocyanins, but there were significant differences: the total phenolic content reached its peak in the fermentation group at 20°C, while no statistically significant difference was found between the 25°C and 30°C groups (P>0.05). This phenomenon suggests that polyphenols other than anthocyanins (such as flavonoids and tannins) may have different temperature response characteristics, with their degradation and release processes reaching a dynamic equilibrium in the medium temperature range (25-30°C).

[0179] From the comprehensive performance evaluation of the fermentation system, mulberry wine at 20°C and 25°C conditions was significantly better than that at 30°C and 37°C in key indicators such as viable bacteria count, residual sugar content, and antioxidant retention. Although the total phenol content of the 25°C group was slightly lower than that of the 20°C group, its residual sugar level was lower, indicating that the alcohol fermentation was more thorough. This characteristic can effectively reduce the risk of microbial spoilage, improve the stability of the wine body, and provide a better foundation for subsequent aging processes. In addition, 25°C is close to the industrial production environment temperature, which can significantly reduce the energy consumption cost of temperature control, and has both quality assurance and economic feasibility.

[0180] Based on the experimental data of Examples 1 to 14 and Comparative Examples 1 to 3, Example 14 (see the attached Figures 10-14 The brewing parameters (abbreviated as Best in this example) performed optimally in key indicators such as fermentation efficiency, microbial coexistence, and product quality. The following is an in-depth mechanistic analysis of the brewing process of Example 14 from three perspectives: microbial interaction mechanisms, metabolic pathway regulation, and the synergistic effects of environmental factors.

[0181] (1) Anthocyanin test results:

[0182] Anthocyanin is the core pigment that gives fruit wine its attractive color, and its retention rate is a key indicator for quality evaluation in this invention. Figure 10 The dynamic patterns of anthocyanin retention during mulberry wine fermentation under optimal process conditions were revealed. Compared to fermentation with Saccharomyces cerevisiae alone, the co-fermentation system significantly improved anthocyanin retention: the anthocyanin retention rate in the co-fermentation group (48.99%) was 31.23% higher than that in the yeast control group (37.33%). This demonstrates that the co-fermentation strategy significantly protects anthocyanins without significantly affecting residual sugar and ethanol content.

[0183] High performance liquid chromatography analysis (e.g. Figure 10 B-10C) demonstrates that anthocyanin degradation during mulberry wine fermentation exhibits significant structural selectivity. The primary anthocyanin components in mulberry are cyanidin-3-O-glucoside (C3G) and cyanidin-3-O-rutinoside (C3R), corresponding to the left and right peaks in the chromatogram. In the single-yeast fermentation system, the loss rate of C3G reached 97.25%, while the loss rate of C3R was 32.32%, indicating that S. cerevisiae has a stronger tendency to degrade C3G. The protective effect of the co-fermentation system on anthocyanins is primarily reflected in the retention of C3G: compared to the yeast control, the peak area of ​​C3G increased by 742.80%, while that of C3R only increased by 7.28%.

[0184] These differences are closely related to the chemical properties of the anthocyanin glycosyl structure: C3G is linked to a monoglucose group at the C3 position of anthocyanidins, while C3R is linked to a rutose disaccharide structure composed of glucose and rhamnose via an α-1,6 glycosidic bond. This difference in glycosyl structure leads to significant differences in their stability: 1) Enzymatic susceptibility: β-glucosidase is more efficient at catalyzing the monosaccharide structure of C3G, while the rutose structure of C3R, due to steric hindrance, requires the removal of rhamnose to form C3G before further degradation. This mechanism was validated in human intestinal microbial metabolism experiments: C3G was completely hydrolyzed within 1 hour of incubation, while intact C3R molecules were still detected after 2 hours. 2) pH stability: Pre-acidification by lactic acid bacteria in the co-fermentation system causes a rapid drop in the ambient pH. Under low pH conditions, C3G exists as a stable cation, and its degradation rate is significantly lower than in neutral or alkaline environments.

[0185] In summary, the co-fermentation system selectively improved the stability of the simpler C3G through pH regulation driven by lactic acid bacteria metabolism, thereby achieving an increase in the overall retention rate of anthocyanins.

[0186] (2) CIELAB analysis results

[0187] pH is a key environmental factor influencing the stability and coloration of anthocyanins. Under strongly acidic conditions (pH < 2), anthocyanins primarily exist as yellow cations with a characteristic red color. When the pH is in the weakly acidic range, their molecular structure isomerizes into colorless methanol pseudobases or hemiacetals. As the pH approaches neutrality, anthocyanins undergo deprotonation to form quinone bases or, through ring opening, convert to chalcone structures. The latter, due to their poor chemical stability, further cleave into phenolic aldehydes or phenolic acids. As the pH continues to rise, anthocyanins gradually transform into purple neutral quinone bases and blue ionized quinone bases. Therefore, systematically measuring the dynamic changes in pH and titratable acidity during mulberry wine fermentation is of great scientific significance for revealing the evolution of its color.

[0188] Experimental results (such as Figure 11) showed that the pH value of the single-bacteria fermentation system of Saccharomyces cerevisiae mainly decreased during the first five days of fermentation, followed by a slight upward trend; while the pH value of the mixed fermentation system continued to decrease until the end of fermentation. The trend of titratable acidity was significantly negatively correlated with the pH value, that is, the stage of pH decrease corresponded to the stage of titratable acidity increase. The decrease in pH value in the early stage of single-bacteria fermentation of Saccharomyces cerevisiae was mainly due to the large-scale production of organic acids during the fermentation process, while the recovery of pH value in the later stage may be related to the utilization of nitrogen sources by the bacteria and the release of alkaline metabolites during cell autolysis. Compared with single-bacteria fermentation, the mixed fermentation system not only contains organic acids produced by the metabolism of Saccharomyces cerevisiae, but also includes lactic acid produced by lactic acid bacteria fermentation, which makes the mixed fermentation mulberry wine have a lower pH value and higher titratable acidity.

[0189] The present invention uses the CIELAB color space system to quantitatively analyze the color value of mulberry wine. This method uses the three visual perception dimensions of hue, lightness and saturation to achieve an objective evaluation of the color of wine and its dynamic changes, and can effectively track the color evolution during the fermentation process. The CIELAB three-dimensional space coordinate system includes the lightness axis (L * ;L * =0 means black, L * =100 represents ideal white), red / green axis (a * ;a * >0 corresponds to red tone, a * <0 corresponds to green hue) and the yellow / blue axis (b * ; b * >0 corresponds to yellow tone, b * <0 corresponds to blue hue), and derives chroma (C * ) and hue angle (h ab ) and other color vision related parameters. Figure 12 The CIELAB parameter changes and corresponding color block diagrams of mixed fermentation mulberry wine during the fermentation process are shown. Figure 12 B) The color changes of the fermentation system are intuitively presented. The color change of the single-strain fermentation system of Saccharomyces cerevisiae is relatively small throughout the fermentation cycle, while the mixed fermentation system shows a significant color improvement on the 5th day, and the color at the end of the fermentation is better than that at the beginning of the fermentation. This result is of great research value because both the single-strain and mixed fermentation systems lose more than 50% of anthocyanins at the end of the fermentation (such as Figure 10 ), but its color did not show the expected attenuation trend, and the mixed fermentation system even achieved color improvement, which shows that the color of mulberry wine is not entirely determined by the single factor of anthocyanin content, and the effect of pH change on the color of mulberry wine is greater than that of anthocyanin content.

[0190] CIELAB parameter analysis (e.g. Figure 12A) provides further data support for the above phenomenon: L * 、a * 、b * and C * The parameter change trend is gentle; it is worth noting that during the storage of fruit wine, anthocyanin degradation usually leads to a * The value gradually decreases (a * The pH value is positively correlated with the red intensity), but this phenomenon was not observed in the present invention. This difference can be attributed to the regulatory effect of pH on the stability of anthocyanins. In the early stage of single-bacteria fermentation, the brewer's yeast is in the logarithmic growth phase. With the rapid decline of anthocyanin content, the pH value of the system decreases sharply, allowing low-concentration anthocyanins to still maintain good color performance. Specifically, the pH drop causes a * The effect of increasing the value and the decrease of anthocyanin content caused a * The decreasing effect of the value forms a dynamic balance, and finally the fermentation end point a * The value was slightly higher than the initial value. This result shows that the influence of pH value on the color of mulberry wine is more important than that of anthocyanin content. The experimental data of the late stage of single-bacteria fermentation further verified the above mechanism: when the pH value of the fermentation system tends to be stable (such as Figure 11 A) When the anthocyanin content continues to decrease slowly (e.g. Figure 10 A), a * The value showed a downward trend with the decrease of anthocyanin content. The mixed fermentation system showed significant changes in CIELAB parameters, which was closely related to the continuous and significant decrease in its pH value. Under the synergistic effect of low pH environment and high anthocyanin retention rate, the a of mixed fermentation mulberry wine was * value, b * Value and C * The value was significantly higher than that of the single-bacteria fermentation system, while L * The value is significantly lower than the latter, exhibiting a deeper chroma and more vivid color characteristics. ΔE, a comprehensive indicator of color space shift (this invention uses day 0 as the reference point), is perceptible to human vision when its value is greater than 2.8. The final ΔE value of the mixed-fermentation mulberry wine reached 16.31, significantly above the visual perception threshold, fully consistent with the significant color improvement shown in the color block diagram.

[0191] (3) Anthocyanin color index analysis results

[0192] This paper focuses on the color indexes related to anthocyanins, aiming to deeply analyze the color evolution mechanism of co-fermented mulberry wine during fermentation. Specifically, the color density (CD), hue (T), mulberry wine chroma at pH 1.0 (WCP), mulberry wine chroma (WC), and anti- Bleached anthocyanin color (CDR ) and chemical alcohol age (CAW) and other parameters were systematically analyzed.

[0193] Color density (CD), the sum of absorbance at wavelengths of 420 nm (yellow), 520 nm (red), and 620 nm (blue), reflects the dynamic changes in anthocyanin structural isomerization with pH. The CD values ​​for both single-strain and mixed fermentation systems of Saccharomyces cerevisiae initially increased rapidly, followed by a fluctuating decline. The sharp increase in CD values ​​during the initial fermentation phase is directly attributed to the decrease in pH, which leads to the predominance of anthocyanins in the red-yellow cationic form, significantly increasing absorbance at 520 nm. As fermentation progresses, the pH stabilizes while the anthocyanin content continues to decrease, leading to a downward trend in CD values ​​due to their dependence on anthocyanin concentration. Notably, the volatility of the CD value during this decline may be related to the dynamic equilibrium between the reduction in free anthocyanin concentration and the formation of anthocyanin derivatives in the wine.

[0194] The hue (T value), which represents the relative ratio of yellow to red absorbance, decreases after fermentation, a phenomenon also driven by a significant increase in red hue due to a decrease in pH.

[0195] Mulberry wine color (WCP) at pH 1.0 is determined by converting colorless hemiacetal anthocyanins and bisulfite adducts into sulfite ions and measuring their absorbance. It is a key indicator of the total amount of free and polymerized anthocyanins in wine. Typically, degradation of free anthocyanins causes the WCP value to decrease during fermentation. In the present invention, the decrease in WCP values ​​in both single-strain and mixed fermentation systems using Saccharomyces cerevisiae is related to the degradation and adsorption of anthocyanins by microorganisms. However, the mixed fermentation system exhibits significantly higher WCP values ​​than the single-strain fermentation system due to its higher anthocyanin retention rate.

[0196] Mulberry wine chromaticity (WC), defined as the wine's chromaticity at 520 nm, is positively correlated with the co-pigmentation effect between anthocyanins, phenolic acids, and flavanols. Its variation mechanism has dual attributes: on the one hand, it is regulated by pH, and on the other hand, it may be related to the dynamic changes in the co-pigmentation intensity of the anthocyanin-phenolic acid complex system.

[0197] anti- Bleached anthocyanin color (CDR ) is the core indicator that indirectly reflects the content of anthocyanin derivatives, characterizing the generation of stable coloring substances in wine and the color's ability to resist oxidation. During the fermentation and aging process, The released sulfite ions easily combine with free anthocyanins to form colorless and unstable bisulfite complexes, which make them easily bleached. However, anthocyanin derivatives substituted at the 4th carbon position have higher structural stability and are not very effective. In the present invention, the CDRs of the two types of fermentation systems The values ​​all show an upward trend, indicating resistance The enhanced bleaching ability suggests the transformation of anthocyanins into stable derivatives. The values ​​were significantly higher, indicating that more structurally stable anthocyanin derivatives were generated.

[0198] Chemical alcohol age (CAW) is defined as CDR The CAW value of the single-strain fermentation system of Saccharomyces cerevisiae shows a continuous upward trend, which is in line with the expected aging law; while the CAW value of the mixed fermentation system shows a fluctuating characteristic of first decreasing and then increasing, and the difference between the values ​​before and after fermentation is not significant. This phenomenon seems to be consistent with the CDR. The changing trend of CAW is contradictory. In-depth analysis shows that CAW value is also affected by CDR Synergistic effect with WC: The lower pH value of the mixed fermentation system significantly improved the WC value by enhancing the coloring ability of anthocyanins, while CDR The synchronous growth of the WC value ultimately resulted in no significant change in the CAW value.

[0199] The above research results reveal that the mixed fermentation system optimizes the color of mulberry wine by regulating pH value and promoting the production of stable anthocyanin derivatives through a dual mechanism, providing an important theoretical basis for the development of fruit wine color regulation technology.

[0200] (4) Pearson correlation analysis results

[0201] In order to further reveal the potential correlation between pH value, anthocyanin content and different color parameters, the present invention conducted Pearson correlation analysis on the relevant variables. The results are as follows Figure 14 The analysis shows that whether it is a single fermentation of Saccharomyces cerevisiae or a mixed fermentation system, the pH value and a * The correlation between the pH value and anthocyanin content was significantly higher than that of anthocyanin content, which further confirmed the effect of pH value on a * The regulatory effect of the value is stronger than that of the anthocyanin concentration. * The value was strongly positively correlated with ΔE (single-bacteria fermentation: 0.98; mixed fermentation: 1.00), indicating that the color improvement of mulberry wine after fermentation was mainly driven by the enhancement of red hue. * The values ​​were significantly positively correlated with the CD values ​​and significantly negatively correlated with the T values, indicating that the changes in the CD values ​​and T values ​​were mainly caused by the fluctuation of the absorbance at 520 nm (red band), which was consistent with the conclusion of the previous analysis.

[0202] The WCP value is highly positively correlated with the anthocyanin content (single-bacteria fermentation: 0.99; mixed fermentation: 0.98), which is consistent with the general rule that the degradation of free anthocyanins during fermentation leads to a decrease in the WCP value. The CAW value was significantly negatively correlated with the anthocyanin content (single-bacteria fermentation: -0.7; mixed fermentation: -0.94), suggesting that the reduction of anthocyanin content may involve both degradation reactions and transformation to stable derivatives. In the mixed fermentation system, the CAW value was significantly negatively correlated with the WC value and CDR value. The correlation coefficients of the values ​​were -0.76 and 0.21, indicating that the change of CAW value was mainly dominated by WC value, and CDR The changes in CAW values ​​were masked by WC values, a mechanism that explains why CAW values ​​in mixed fermentations did not show the expected trend. In addition, WC values ​​showed a significant negative correlation of -0.83 with pH, ​​confirming the speculation that pH indirectly affects CAW values ​​by regulating WC values.

[0203] Compared with single-bacteria fermentation, the introduction of lactic acid bacteria significantly enhanced the pH value and L * 、a * 、b * 、C * 、h ab 、CDR The correlation between color parameters such as pH and anthocyanin highlights the core regulatory role of pH in the color formation of mixed fermentation mulberry wine. This study constructed a multivariate interaction network of pH, anthocyanin, and color parameters through correlation analysis, providing a key scientific basis for the precise control of fruit wine color.

[0204] Based on the test results of the above embodiments and comparative examples, the present application has the following beneficial effects:

[0205] (1) The present invention synergistically improves the color and flavor of fruit wine by optimizing the inoculation timing, inoculation ratio and fermentation temperature.

[0206] (II) The present invention uses a co-fermentation method to achieve color optimization of mulberry wine through a dual path: 1) Lactic acid bacteria metabolism drives the pH value of the fermentation system to decrease rapidly, thereby improving the stability of cyanidin-3-O-glucoside (C3G); (2) The reduced pH value in the co-fermentation system has a significant effect on the color parameter a * The regulatory effect of the value is significantly stronger than the change in anthocyanin content, which compensates for the color loss caused by anthocyanin degradation and achieves a systematic improvement in the color of fruit wine.

[0207] The above are only preferred embodiments of the present application and are not intended to limit the present invention. Although the present application has been described in detail with reference to examples, those skilled in the art can still modify the technical solutions described in the aforementioned examples or make equivalent replacements for some of the technical features therein. However, any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A co-fermentation method for improving the color of fruit wine, characterized in that: The fermentation steps are: Lactic acid bacteria and yeast are inoculated into the juice after enzymatic hydrolysis, and the fruit wine is obtained by fermenting it at a constant temperature and in the dark.

2. The co-fermentation method for improving the color of fruit wine according to claim 1, characterized in that: Use a step-by-step vaccination approach: First inoculate lactic acid bacteria, and then inoculate yeast after the lactic acid bacteria ferment for a preset time.

3. The co-fermentation method for improving the color of fruit wine according to claim 2, characterized in that: Use a step-by-step vaccination approach: Lactic acid bacteria are inoculated into the juice after enzymatic hydrolysis, and fermented at a constant temperature and in the dark for 3 to 5 days. Yeast is then inoculated and fermented at a constant temperature and in the dark to obtain fruit wine.

4. The co-fermentation method for improving the color of fruit wine according to claim 1, characterized in that: The pH of the juice is 3-5.

5. The co-fermentation method for improving the color of fruit wine according to claim 1, characterized in that: The sweetness of the juice is 25-35°Bx.

6. The co-fermentation method for improving the color of fruit wine according to claim 1, characterized in that: The cell inoculation density ratio of the yeast and the lactic acid bacteria is 1: (1-1000).

7. The co-fermentation method for improving the color of fruit wine according to claim 1, characterized in that: The temperature range of the constant temperature fermentation is 20-30°C.

8. An application of the co-fermentation method for improving the color of fruit wine according to any one of claims 1 to 7, characterized in that: Used to make fruit wine.

9. The use according to claim 8, characterized in that The fruit wine is fruit wine containing anthocyanin.

10. A fruit wine, characterized in that: The fruit wine is prepared by the co-fermentation method according to any one of claims 1 to 7, and the pH value of the fruit wine is less than 3.5.

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