Method for fermenting grape juice and use thereof

Abstract

The application provides a grape juice enzyme fermentation method and application, and relates to the technical field of fermentation.The grape juice enzyme fermentation method provided by the application comprises the following steps: inoculating fermentation bacteria in a culture medium containing grape juice to perform fermentation, and preparing grape juice enzyme; the fermentation bacteria comprise lactobacillus plantarum Zhang-LL and kloeckera apiculata M3; the preservation number of the lactobacillus plantarum Zhang-LL is CGMCC No.6936; and the preservation number of the kloeckera apiculata M3 is CGMCC No.1811.The fermentation method uses the lactobacillus plantarum Zhang-LL and the kloeckera apiculata M3 fermentation bacteria, and through the synergistic cooperation of the two, the fermentation time is shortened, and a high viable cell count can be achieved; the grape juice enzyme can be significantly improved in terms of antioxidant property and total protease activity; and the application provides a theoretical basis for industrialized preparation of grape enzyme and subsequent research and development of multifunctional products.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a fermentation method and application of grape juice enzyme. Background Technology

[0002] As people's living standards continue to improve, they are paying more attention to their own health, and therefore the demand for enzymes is growing stronger.

[0003] Grapes are a type of vine belonging to the Vitaceae family, and their fruiting season is typically in August and September. They are popular due to their rich nutritional content and refreshing taste. Consuming grapes may have positive effects on human health; current research suggests that grape consumption can prevent or delay fatty liver disease, extend lifespan, and that grapes have the potential to regulate the human gut microbiota. Grapes are widely cultivated in my country, with 10% used for winemaking, 10% for deep processing, and nearly 80% for fresh consumption. However, the short shelf life of fresh grapes and their susceptibility to mechanical damage during transportation limit the development of the fresh grape market.

[0004] Enzymes are fermented products made from fruits, plants, and traditional Chinese medicines through microbial fermentation, resulting in a wealth of nutrients. Enzymes are rich in various bioactive substances and possess anti-aging, antioxidant, and immune-boosting effects. The nutrients in fruit and vegetable juices provide excellent conditions for the growth of lactic acid bacteria. Fermentation helps maintain the characteristics of the raw materials, adds new active ingredients, and extends shelf life to some extent.

[0005] However, current enzyme fermentation methods are not efficient enough, and the antioxidant properties and total protease activity of enzymes are low. It is necessary to find a new fermentation method.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a method for fermenting grape juice enzymes to solve the aforementioned problems.

[0008] The second objective of this invention is to provide the application of the above-mentioned fermentation method for grape juice enzymes in improving the antioxidant properties of enzyme products.

[0009] A third objective of this invention is to provide the application of the above-mentioned fermentation method for grape juice enzymes in improving the total protease activity of enzyme products.

[0010] The fourth objective of this invention is to provide a grape juice enzyme.

[0011] To achieve the above objectives, the following technical solution is proposed:

[0012] In a first aspect, the present invention provides a method for fermenting grape juice enzymes, comprising the following steps:

[0013] Grape juice enzyme is prepared by inoculating a culture medium containing grape juice with fermenting bacteria and fermenting it.

[0014] The fermentation bacteria include Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3;

[0015] The preservation number of the *Lactobacillus plantarum* Zhang-LL is CGMCC No. 6936;

[0016] The preservation number of the Kluyveromyces martensii M3 is CGMCC No. 1811.

[0017] As a further technical solution, the inoculation ratio of Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3 is 1:2 to 2:1, preferably 1:1;

[0018] The inoculation amount of the fermentation bacteria is 4-61 g CFU / mL, preferably 51 g CFU / mL.

[0019] As a further technical solution, the fermentation temperature is 31-37℃, preferably 34℃;

[0020] The fermentation time is 15 to 21 hours, preferably 18 hours.

[0021] As a further technical solution, the culture medium mainly consists of grape juice and a nitrogen source;

[0022] In the culture medium, the mass percentage of nitrogen source is 0.25% to 0.75%, preferably 0.5%.

[0023] As a further technical solution, the method for preparing grape juice includes: mixing grapes with water and then pulping the mixture, followed by solid-liquid separation, and obtaining grape juice by taking the clear liquid;

[0024] The mass ratio of grapes to water is 1:2 to 2:1, preferably 1:1.

[0025] As a further technical solution, the nitrogen source includes at least one of tryptone, soy peptone, casein peptone or gelatin peptone, preferably soy peptone.

[0026] Secondly, the present invention provides the application of the above-mentioned fermentation method of grape juice enzyme in improving the antioxidant properties of enzyme products.

[0027] Thirdly, the present invention provides the application of the above-mentioned fermentation method of grape juice enzyme in improving the total protease activity of enzyme products.

[0028] Fourthly, the present invention provides a grape juice enzyme, which is obtained by fermentation using the above-described fermentation method.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The fermentation method for grape juice enzyme provided by this invention uses Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3 fermentation strains. Through the synergistic effect of the two, the fermentation time can be shortened while achieving a high number of viable cells. It can significantly improve the antioxidant capacity and total protease activity of grape juice enzyme. It provides a theoretical basis for the industrial preparation of grape enzyme and the subsequent development of multifunctional products. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 The growth curve of Lactobacillus plantarum Zhang-LL;

[0033] Figure 2 The growth curve of Kluyveromyces martensii M3;

[0034] Figure 3 The viable counts of lactic acid bacteria and yeasts in different fermentation methods;

[0035] Figure 4 DPPH scavenging rate of enzymes in different fermentation methods;

[0036] Figure 5 Total protease activity of enzymes produced by different fermentation methods;

[0037] Figure 6 The effect of initial inoculum size on the viable count of the two strains;

[0038] Figure 7 The effect of different nitrogen sources on the viable count of the two bacteria;

[0039] Figure 8 The effect of nitrogen source addition on the viable count of the two strains;

[0040] Figure 9 The effect of fermentation temperature on the viable cell count of the two strains;

[0041] Figure 10 The effect of fermentation time on the viable cell count of the two strains;

[0042] Figure 11aThe response surface and contour lines of the effect of the interaction between fermentation temperature and inoculum size on the viable count of grape juice fermentation broth;

[0043] Figure 11b The response surface and contour lines of the interaction between inoculum size and nitrogen source addition on the viable count of grape juice fermentation broth;

[0044] Figure 11c The response surface and contour lines of the effect of the interaction between fermentation temperature and nitrogen source addition on the viable count of grape juice fermentation broth;

[0045] Figure 11d The response surface and contour lines of the effect of the interaction between fermentation temperature and inoculum size on the viable count of grape juice fermentation broth;

[0046] Figure 11e The response surface and contour lines of the interaction between nitrogen source addition and inoculum amount on the viable count of grape juice fermentation broth;

[0047] Figure 11f The response surface and contour lines show the effect of the interaction between nitrogen source addition and fermentation temperature on the viable count of grape juice fermentation broth. Detailed Implementation

[0048] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0049] In a first aspect, the present invention provides a method for fermenting grape juice enzymes, comprising the following steps:

[0050] Grape juice enzyme is prepared by inoculating a culture medium containing grape juice with fermenting bacteria and fermenting it.

[0051] The fermentation bacteria include Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3;

[0052] The Lactobacillus plantarum Zhang-LL has the accession number CGMCC No. 6936 (this strain has been disclosed in the applicant's prior patent application, and the fermentation study in this invention uses a strain preserved in the laboratory).

[0053] The Kluyveromyces martensii M3 strain has the accession number CGMCC No. 1811 (this strain has been disclosed in the applicant's prior patent application; the fermentation study in this invention uses a laboratory-preserved strain).

[0054] *Lactobacillus plantarum* Zhang-LL, as a probiotic, can survive in the intestines, has a hypoglycemic effect, and can also alleviate ulcerative colitis. Furthermore, the plantar lactobacillus toxin produced by this bacterium has a strong inhibitory effect on Gram-positive bacteria. *Kluyveromyces* contains killer yeasts that can inhibit contamination by putrefactive yeasts, ensuring a good fermentation system and thus improving the quality of fermented products. *Kluyveromyces martensii* M3, derived from *Gnaphalium affine*, exhibits superior performance in enzyme, ethanol, and aroma production, and has cholesterol-lowering effects and can regulate the balance of intestinal flora.

[0055] The fermentation method for grape juice enzyme provided by this invention uses Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3 fermentation strains. Through the synergistic effect of the two, the fermentation time can be shortened while achieving a high number of viable cells. It can significantly improve the antioxidant capacity and total protease activity of grape juice enzyme. It provides a theoretical basis for the industrial preparation of grape enzyme and the subsequent development of multifunctional products.

[0056] In some preferred embodiments, the inoculation ratio of Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3 can be, for example, but not limited to, 1:2, 1:1 or 2:1, preferably 1:1;

[0057] The inoculation amount of the fermentation bacteria can be, for example, but not limited to, 41g CFU / mL, 51g CFU / mL or 61g CFU / mL, preferably 51g CFU / mL.

[0058] In some preferred embodiments, the fermentation temperature can be, for example, but not limited to, 31°C, 33°C, 35°C or 37°C, preferably 34°C;

[0059] The fermentation time can be, for example, but not limited to, 15h, 17h, 19h or 21h, preferably 18h.

[0060] In some preferred embodiments, the culture medium mainly consists of grape juice and a nitrogen source;

[0061] In the culture medium, the mass percentage of the nitrogen source can be, for example, but not limited to, 0.25%, 0.5%, or 0.75%, preferably 0.5%.

[0062] In some preferred embodiments, the method for preparing the grape juice includes: mixing grapes with water and then pulping the mixture, followed by solid-liquid separation, and obtaining grape juice by taking the clear liquid;

[0063] The mass ratio of grapes to water can be, for example, but not limited to, 1:2, 1:1 or 2:1, but preferably 1:1.

[0064] In some preferred embodiments, the nitrogen source includes, but is not limited to, at least one of tryptone, soy peptone, casein peptone or gelatin peptone, or other nitrogen sources known to those skilled in the art. In this invention, soy peptone is preferred as the nitrogen source.

[0065] Through further optimization and adjustment of the fermentation process, the viable count of Lactobacillus plantarum Zhang-LL during fermentation reached as high as 6.60 × 10⁻⁶. 8 The viable count of Kluyveromyces martensii M3 was as high as 8.20 × 10⁻¹¹ CFU / mL. 7 CFU / mL significantly improved the antioxidant properties and total protease activity of grape juice enzymes.

[0066] Secondly, the present invention provides the application of the above-mentioned fermentation method of grape juice enzyme in improving the antioxidant properties of enzyme products.

[0067] The inventors have discovered that the fermentation method provided by this invention can significantly improve the antioxidant properties of grape juice enzymes.

[0068] Thirdly, the present invention provides the application of the above-mentioned fermentation method of grape juice enzyme in improving the total protease activity of enzyme products.

[0069] The inventors have discovered that the fermentation method provided by this invention can significantly increase the total protease activity of grape juice enzymes.

[0070] Fourthly, the present invention provides a grape juice enzyme, which is obtained by fermentation using the above-described fermentation method.

[0071] This grape juice enzyme has high antioxidant and total protease activity.

[0072] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0073] Example 1

[0074] 1. Materials and Methods

[0075] 1.1 Experimental Reagents and Materials

[0076] Table 1 Main Reagents and Materials

[0077]

[0078] 1.2 Experimental Methods

[0079] 1.2.1 Activation of strains: Glycerol preservation tubes of Lactobacillus plantarum Zhang-LL and yeast M3 strains were used. Lactobacillus plantarum was inoculated into MRS medium and cultured on a shaker at 37℃ and 180 r / min for 12 h, while yeast was inoculated into YPD medium and cultured on a shaker at 28℃ and 150 r / min for 24 h. After two subcultures, activated strains were obtained and used for later fermentation after plate counting.

[0080] 1.2.2 Grape Juice Enzyme Preparation

[0081] (1) Grape processing: Select fresh Summer Black grapes, wash and dry them, then pulp them according to a material-to-water ratio of 1:1 (g:g), centrifuge at 4000r / min for 10min, and remove impurities from the supernatant through filter paper to obtain clear grape juice.

[0082] (2) Preparation of fermentation substrate: Add nitrogen source to the filtered grape juice, adjust pH to 6.5, and filter and sterilize using a 0.22μm hollow fiber microfiltration membrane.

[0083] (3) Inoculation and fermentation: The activated lactic acid bacteria and yeast are inoculated into the grape juice at a ratio of 1:1 according to the required amount for fermentation.

[0084] 1.2.3 Colony Counting Method: Samples were serially diluted 10-fold. The viable count of lactic acid bacteria and yeast was determined using the pour plate method. Three appropriate dilution gradients were selected, and samples were poured into suitable culture plates using the pour plate method. The plates were inverted for colony counting. A selective medium was prepared by adding 1% potassium sorbate to MRS medium. Bengal red agar was used as the selective medium for yeast.

[0085] 1.2.4 Screening Grape Juice Fermentation Methods

[0086] (1) Growth curve determination. To estimate the growth status of Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3 in grape juice, the two bacteria were inoculated into grape juice of the same system. The lactic acid bacteria were cultured for 24 h, and the viable cell count was measured every 6 h. The yeast was cultured for 48 h, and the viable cell count was measured every 12 h. The growth curves of the two bacteria in grape juice were then constructed.

[0087] (2) Effect of different inoculation methods on viable cell count. Five inoculation methods were used: single-strain lactic acid bacteria fermentation, single-strain yeast fermentation, inoculation with lactic acid bacteria followed by yeast, inoculation with yeast followed by lactic acid bacteria, and simultaneous inoculation of both. The fixed fermentation conditions were: initial inoculation amount 2.00 × 10⁻⁶. 6 The optimal fermentation method was selected by measuring the number of live lactic acid bacteria and yeast, using CFU / mL, fermentation temperature of 34℃, and the addition of 1% tryptone as a nitrogen source.

[0088] (3) Effects of different inoculation methods on antioxidant capacity. Five inoculation methods were used: single-strain lactic acid bacteria fermentation, single-strain yeast fermentation, inoculation with lactic acid bacteria followed by yeast, inoculation with yeast followed by lactic acid bacteria, and simultaneous inoculation of both. The fixed fermentation conditions were: initial inoculation amount 2.00 × 10⁻⁶. 6 The fermentation method was determined by setting the total antioxidant activity (CFU / mL), fermentation temperature (34℃), and adding 1% tryptone as a nitrogen source. The total antioxidant activity of the fermentation broth was measured using a total antioxidant activity kit to select the optimal fermentation method.

[0089] (4) Effects of different inoculation methods on protease activity. Five inoculation methods were used: single-strain lactic acid bacteria fermentation, single-strain yeast fermentation, inoculation with lactic acid bacteria followed by yeast, inoculation with yeast followed by lactic acid bacteria, and simultaneous inoculation of both. The fermentation conditions were fixed as follows: initial inoculation amount 2.00 × 10⁻⁶. 6 The fermentation method was determined by setting the concentration of CFU / mL, the fermentation temperature to 34℃, and adding 1% tryptone as a nitrogen source. The total protease activity of the fermentation broth was measured using the Folin-Ciocalteu method to select the optimal fermentation method.

[0090] 1.2.5 Optimization of Grape Juice Fermentation Process

[0091] (1) Effect of initial inoculum quantity on viable count of grape juice fermentation broth. The initial inoculum quantity and final concentration were set at 2.00, 3.00, 4.00, 5.00, and 6.00 lg CFU / mL. 1% tryptone was added as a nitrogen source. The fermentation time was 12 h and the fermentation temperature was 34 ℃. The viable counts of lactic acid bacteria and yeast were used to evaluate the fermentation broth and determine the optimal inoculum quantity for both bacteria.

[0092] (2) Effect of different nitrogen sources on viable cell count in grape juice fermentation broth. Different nitrogen sources were set up, namely tryptone, soybean peptone, casein peptone, peptone, and gelatin peptone, with an addition amount of 1%. The initial inoculum concentration was 5.001g CFU / mL, the fermentation time was 12h, and the fermentation temperature was 34℃. The evaluation index of the fermentation broth was the viable cell count of lactic acid bacteria and yeast, and the optimal nitrogen source was determined.

[0093] (3) Effect of nitrogen source addition on viable count of grape juice fermentation broth. The addition amounts of soybean peptone were set at 0.25%, 0.5%, 0.75%, 1%, and 1.25%, the initial inoculum concentration was 5.00 lg CFU / mL, the fermentation time was 12 h, and the fermentation temperature was 34 ℃. The evaluation index of the fermentation broth was the viable count of lactic acid bacteria and yeast, and the optimal nitrogen source addition amount was determined.

[0094] (4) Effect of fermentation temperature on viable count of grape juice fermentation broth. Fermentation temperatures were set at 28℃, 31℃, 34℃, 37℃, and 40℃, with an initial inoculum concentration of 5.001g CFU / mL, 0.5% soybean peptone added, and fermentation time of 12h. The viable counts of lactic acid bacteria and yeast were used to evaluate the fermentation broth and determine the optimal fermentation temperature.

[0095] (5) Effect of fermentation time on viable count of grape juice fermentation broth. Fermentation times were set at 12h, 15h, 18h, 21h, and 24h, with an initial inoculum concentration of 5.001g CFU / mL, 0.5% soybean peptone added, and fermentation temperature at 34℃. The fermentation broth was evaluated based on the viable counts of lactic acid bacteria and yeast to determine the optimal fermentation time.

[0096] 1.2.6 Plackett-Burman Experimental Design

[0097] The significance of the initial inoculum size (A), nitrogen source addition (B), fermentation time (C), and fermentation temperature (D) was investigated using Plackett-Burman design experiments. The low (-1) and high (+1) levels of these four single factors were used to conduct 12 experiments. The levels of each factor in the PB design are shown in Table 2.

[0098] Table 2. Factor Levels in the Plackett-Burman Experiment

[0099]

[0100] 1.2.7 Box-Behnken Central Composite Experiment

[0101] Based on the Plackett-Burman screening of factors with main effects, a three-factor, three-level response surface methodology was designed using the Box-Behnken principle. The number of viable cells of the two strains was used as the response value. The optimal fermentation process conditions were obtained through data analysis. Three parallel experiments were conducted for verification. The factors and levels are shown in Table 3.

[0102] Table 3. Box-Behnken Experiment Factor Levels

[0103]

[0104] 1.3 Data Processing

[0105] Microsoft Excel 2019 was used for data processing, GraphPad Prism 8.0.1 software was used for chart creation, and Design-Expert 10 software was used to design Plackett-Burman and Box-Behnken central combination experiments. The results were then analyzed and plotted.

[0106] 2 Results Analysis

[0107] 2.1 Results of growth curve determination of two bacteria in grape juice

[0108] Because the nutrients in grape juice differ significantly from those in the culture medium, to better understand the growth status of the bacteria in the grape juice system and to select an appropriate fermentation time for subsequent screening of fermentation methods, two bacterial strains were inoculated separately into the same grape juice system. Figure 1 It can be seen that the growth trend of *Lactobacillus plantarum* Zhang-LL in grape juice reaches a plateau after 12 hours of culture. Figure 2 It was found that *Kluyveromyces martensii* M3 remained in a stationary phase in grape juice from 12 to 24 hours. Both strains can reproduce rapidly within a short period. Rapid growth is essential for industrial applications, therefore *Lactobacillus plantarum* Zhang-LL and *Kluyveromyces martensii* M3 can be considered excellent fermentation strains. The growth curve measurements provide a reasonable fermentation time for subsequent screening of fermentation methods.

[0109] 2.2 Screening of Grape Juice Fermentation Methods

[0110] Comparing three fermentation methods—inoculating with lactic acid bacteria first followed by yeast (Z+M), inoculating with yeast first followed by lactic acid bacteria (M+Z), and inoculating both simultaneously (G)—based on the growth curves measured in section 2.1, the fermentation time for lactic acid bacteria was fixed at 12 hours, and the fermentation time for yeast was fixed at 24 hours to ensure a consistent total fermentation time. Therefore, the co-culture fermentation time was 36 hours. Lactic acid bacteria and yeast were used as evaluation indicators. Figure 3 It is evident that the yeast strain with the highest number of live cells is G, followed by M+Z, while the lactic acid bacteria strain with the highest number of live cells is Z+M, followed by G. Based on the combined live cell counts of the two strains, mixed-culture fermentation and co-cultivation fermentation are the most effective.

[0111] The antioxidant activity of single-strain fermentation and co-culture fermentation broths was analyzed and compared, and the results of their hydroxyl radical scavenging rates were as follows: Figure 4 The DPPH scavenging rates of Lactobacillus plantarum (Z) and Kluyveromyces martensii (M) fermentation broths were 53.42% and 31.44%, respectively. The DPPH scavenging rates of the fermentation broths inoculated with lactic acid bacteria first and then yeast (Z+M) and inoculated with yeast first and then lactic acid bacteria (M+Z) were 42.18% and 50.36%, respectively, both significantly lower than the DPPH scavenging rate of 78.95% in the co-fermentation group (G). These results indicate that co-fermentation of Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3 can significantly improve the antioxidant properties of enzyme products.

[0112] Furthermore, the total protease activity of single-strain fermentation and co-culture fermentation broths was analyzed and compared, and the results are as follows: Figure 5The total protease activity of the fermentation broth containing *Lactobacillus plantarum* Zhang-LL (Z) was the lowest at 30.22 U / ml, lower than that of the fermentation broths inoculated with lactic acid bacteria followed by yeast (Z+M) and those inoculated with yeast followed by lactic acid bacteria (M+Z), and also significantly lower than the total protease activity (258.38 U / ml) of the grape enzyme fermentation broth inoculated solely with *Kluyveromyces martensii* M3 (M). Among all groups, the total protease activity in grape juice co-fermented with *Lactobacillus plantarum* Zhang-LL and *Kluyveromyces martensii* M3 (G) was the highest, reaching 385.69 U / ml, significantly higher than other fermentation groups. These results indicate that co-fermentation with *Lactobacillus plantarum* Zhang-LL and *Kluyveromyces martensii* M3 can significantly improve the total protease activity of enzyme products.

[0113] In summary, this study shows that, compared with using Lactobacillus plantarum Zhang-LL and Kluyveromyces martensii M3 alone to ferment grape juice, co-fermentation of the two strains can significantly improve the number of viable yeast cells, the DPPH scavenging rate, and the total protease activity.

[0114] 2.3 Optimization of Grape Juice Fermentation Process

[0115] 2.3.1 Effect of initial inoculum size on viable cell count in grape juice fermentation broth. Experimental results are as follows: Figure 6 As shown, nutrients in the fermentation system cannot be fully utilized when the inoculum amount is too low, while nutrients are rapidly consumed when the inoculum amount is too high, which not only reduces the fermentation rate but also wastes resources. Under the premise of industrial production and considering cost savings, the highest viable count of *Lactobacillus plantarum* Zhang-LL in the grape juice fermentation broth reached 2.67 × 10⁻⁶ when the inoculum amount was 5.00 lg CFU / mL. 8 The viable count of Max Kluyveromyces M3 yeast was the second highest at 3.47 × 10⁻⁶ CFU / mL. 7 CFU / mL, therefore, an inoculum of 4.001g CFU / mL was considered the low level for the PB experiment, and 6.001g CFU / mL was considered the high level.

[0116] 2.3.2 Effects of Different Nitrogen Sources on Viable Bacterial Count in Grape Juice Fermentation Broth. Grape juice contains nutrients such as sugars, amino acids, and vitamins, but only trace amounts of protein. Nitrogen sources are essential for the growth of lactic acid bacteria and yeast. To promote better bacterial growth and reproduction, different nitrogen sources were added to the grape juice fermentation system to investigate their effects on viable bacterial count. The experimental results are as follows: Figure 7 (In the figure, 1: tryptone; 2: soybean peptone; 3: casein peptone; 4: peptone; 5: gelatin peptone) As shown, adding soybean peptone as a nitrogen source can achieve a high level of viable cell count for both bacteria, with the viable cell count of *Lactobacillus plantarum* Zhang-LL reaching 2.33 × 10⁻⁶.8 The viable count of Kluyveromyces martensii M3 reached 5.40 × 10⁻¹¹ CFU / mL. 7 CFU / mL. Since different nitrogen sources and their amounts are closely related, the amount of nitrogen source added will change when the type of nitrogen source changes. Therefore, this factor is not considered in the PB experiment.

[0117] 2.3.3 Effect of Nitrogen Source Addition on the Viable Count of Grape Juice Fermentation Broth. The amount of nitrogen source added affects the viable count of lactic acid bacteria and yeast. Insufficient nitrogen source cannot meet the nutritional needs of bacterial growth, while excessive nitrogen source will cause excessively vigorous bacterial growth, leading to premature cell death. Figure 8 It can be seen that when the amount of soybean peptone added is 0.5%, the viable count of *Lactobacillus plantarum* Zhang-LL reaches the highest level of 2.57 × 10⁻⁶. 8 The viable count of Kluyveromyces martensii M3 reached 3.60 × 10⁻¹¹ CFU / mL. 7 CFU / mL, therefore, 0.25% nitrogen source was added as the low level for the PB experiment, and 0.75% as the high level.

[0118] 2.3.4 Effect of Fermentation Temperature on the Viable Cell Count of Grape Juice Fermentation Broth. Too low a fermentation temperature results in slow cell growth and metabolism, while too high a temperature also affects cell growth and can affect enzyme activity, even causing enzyme inactivation. Figure 9 It was found that the viable count of *Lactobacillus plantarum* Zhang-LL during co-culture fermentation remained at a high level between 28 and 34°C, began to decrease slightly at 37°C, and dropped rapidly at 40°C. The viable count of *Kluyveromyces martensii* M3 during co-culture fermentation also increased with increasing temperature between 28 and 40°C. However, higher temperatures would increase production costs. Considering all factors, 34°C was chosen as the optimal fermentation temperature, at which the viable count of *Lactobacillus plantarum* Zhang-LL was measured to be 3.18 × 10⁻⁶. 8 The viable count of Kluyveromyces martensii M3 was 2.60 × 10⁻⁶ CFU / mL. 7 CFU / mL, therefore the fermentation temperature was designed as 31℃ as the low level of the PB experiment and 37℃ as the high level.

[0119] 2.3.5 Effect of fermentation time on viable cell count in grape juice fermentation broth. With prolonged fermentation time, the viable cell counts of both strains showed a trend of first increasing and then decreasing, as shown in the experimental results. Figure 10 When the fermentation time reached 18 hours, the viable count of *Lactobacillus plantarum* Zhang-LL was 6.30 × 10⁻⁶. 8 The viable count of Kluyveromyces martensii M3 was 1.01 × 10⁻¹¹ CFU / mL. 8 CFU / mL, therefore, a fermentation time of 15h was chosen as the low level for the PB experiment, and 21h as the high level.

[0120] 2.4 Plackett-Burman test results

[0121] Based on the results of the single-factor experiment, the Design-Expert 10 was used to design the experiment, with the number of viable bacteria of the two strains as the response value, and the factors with significant influence were screened. The design and results of the PB experiment are shown in Table 4.

[0122] Table 4. Plackett-Burman test results

[0123]

[0124] Table 5. Results of Analysis of Variance (Zhang-LL)

[0125]

[0126] Note: ** indicates highly significant (P < 0.01), * indicates significant (0.01 < P < 0.05) (the same applies below). Table 6: Results of ANOVA (M3)

[0127]

[0128] Table 5 shows that for *Lactobacillus plantarum* Zhang-LL, the model has F>1 and P<0.01, indicating extremely significant differences and a reasonable model design. The inoculum size (A) has a highly significant effect on the viable cell count, while the amount of nitrogen source added (B) and fermentation temperature (C) have significant effects. Fermentation time (D) has no significant effect on the viable cell count; the order of their influence is: inoculum size (A) > fermentation temperature (C) > nitrogen source added (B). Since the P-value for fermentation time (D) is 0.0793 > 0.05, this factor has no significant effect on the viable cell count.

[0129] Table 6 shows that for Kluyveromyces martensii M3, the model has F>1 and P<0.05, indicating a significant difference and suggesting a reasonable model design. The inoculum size (A) has a highly significant effect on the viable cell count, while the amount of nitrogen source added (B), fermentation temperature (C), and fermentation time (D) have no significant effect on the viable cell count. The order of influence of the four factors on the yeast viable cell count is: inoculum size (A) > nitrogen source added (B) > fermentation time (D) > fermentation temperature (C).

[0130] Although only the inoculum size can affect the viable yeast count, the fermentation method in this study was co-culture, which needs to be considered in conjunction with factors affecting Lactobacillus plantarum Zhang-LL. Therefore, three factors were selected for the next step of response surface optimization: inoculum size, nitrogen source addition amount, and fermentation temperature.

[0131] 2.5 Results of the Box-Behnken Central Composite Experiment

[0132] 2.5.1 Experimental Design Results and Analysis. Based on the single-factor experiments and Plackett-Burman experiments, the Box-Behnken central composite experimental design and results processing were performed using Design-Expert 10 software, as shown in Table 7. Regression analysis was then performed on the data from 17 BB experiments. The ANOVA of the Zhang-LL viable cell count regression model is shown in Table 8. The regression model F = 51.66, P < 0.0001, indicating the model is highly significant. The P-value for the lack-of-fit term is 0.0633 > 0.05, indicating no significant difference in the lack-of-fit test. The correlation coefficient R² is 0.9852, demonstrating a relatively good fit between the results and the model. R²adj is 0.9661, indicating good reliability and feasibility of the model. The ANOVA of the M3 viable cell count regression model is shown in Table 9. The regression model F = 47.18, P < 0.0001. The P-value for the lack-of-fit term is 0.1275 > 0.05, indicating no significant difference in the lack-of-fit test. The correlation coefficient R² is 0.9838, showing a relatively good fit. R²adj is 0.9629, indicating high reliability. Therefore, this model can be used to analyze and predict fermentation conditions.

[0133] Table 7. Experimental Results of the Box-Behnken Design

[0134]

[0135]

[0136] Table 8. Analysis of variance of the Zhang-LL viable cell count regression model.

[0137]

[0138] Table 9. Analysis of variance of the M3 viable cell count regression model.

[0139]

[0140]

[0141] Table 8 shows that inoculum size (A) and fermentation temperature (B) have a highly significant effect on the number of Zhang-LL viable cells in the grape juice fermentation broth, while the effect of nitrogen source addition (C) is not significant. The effect of the three factors on the number of viable yeast cells is: inoculum size (A) = nitrogen source addition (C) > fermentation temperature (B). Table 9 shows that both the linear and quadratic terms of the factor variables have a highly significant effect on the number of M3 viable cells in the grape juice fermentation broth. The effect of the three factors on the number of viable yeast cells is: inoculum size (A) = nitrogen source addition (C) > fermentation temperature (B).

[0142] 2.5.1 Response Surface Plot and Contour Plot Analysis. The slope of the three-dimensional surface plot formed by the response value and each factor indicates the degree of interaction between the two factors. The greater the influence of the dependent variable on the response value, the steeper the surface, and vice versa. Contour plots also provide a clear visual analysis of the influence of factors on the response value. The more elliptical the contour lines, the greater the interaction between the two factors; denser contour lines also indicate a significant influence of the factors on the response value. Figures 11a to 11f ( Figures 11a to 11f The top figure (response surface plot) and the bottom figure (contour plot) are both response surface plots opening downwards, indicating that the response value increases with the increase of variable factors. After reaching the maximum value, the response value decreases with the increase of variable factors. The contour plots also mostly show an elliptical shape. Although the analysis in Table 8 shows that the pairwise interactions of groups a, b, and c are not significant, the analysis in Table 9 shows that the pairwise interactions of groups d, e, and f are extremely significant and can affect the experimental results. After comprehensive analysis, the influence of the interaction between the two factors on the grape juice fermentation broth is in the order AB > BC > AC.

[0143] Further fitting and analysis of the existing data, using the viable cell count in the grape juice fermentation broth as an indicator, and employing Design-Expert 10 software, the optimal parameters for the grape juice fermentation broth were predicted to be: inoculum size 5.25 lg CFU / mL, fermentation temperature 33.74℃, and nitrogen source addition 0.457%. It was predicted that with these process parameters, the viable cell count of *Lactobacillus plantarum* Zhang-LL would reach 6.80 × 10⁻⁶. 8 CFU / mL, the viable count of Max Kluwer M3 reached 8.41 × 10⁻⁶ CFU / mL. 7 CFU / mL. Considering operational feasibility, the fermentation conditions were modified to: inoculum size 5.001 g CFU / mL, fermentation temperature 34℃, and nitrogen source addition 0.46%. Three parallel experiments were conducted to verify the feasibility and reliability of response surface methodology, following the adjusted optimal process parameters. The final viable count of *Lactobacillus plantarum* Zhang-LL was determined to be 6.50 × 10⁻⁶ CFU / mL. 8 CFU / mL, viable count of Saccharomyces martensii M3 was 8.20 × 10⁻⁶. 7 At this point, the DPPH clearance rate of grape enzyme was 85.25%, and the total protease activity was 472.6 U / mL.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermentation method of grape juice enzyme, characterized by, The method comprises the following steps: The grape juice enzyme is prepared by simultaneously inoculating Lactobacillus plantarum Zhang-LL and Kluyveromyces marxianus M3 in a culture medium comprising grape juice and then performing fermentation; The Lactobacillus plantarum Zhang-LL has a preservation number of CGMCC No.6936; The Kluyveromyces marxianus M3 has a preservation number of CGMCC No.1811; The inoculation ratio of the Lactobacillus plantarum Zhang-LL and the Kluyveromyces marxianus M3 is 1:

1.

2. The fermentation process according to claim 1, characterized in that, The inoculation amount of the fermentation bacteria is 4-6 lg CFU / mL.

3. The fermentation process according to claim 2, characterized in that, The inoculation amount of the fermentation bacteria is 5 lg CFU / mL.

4. The fermentation process of claim 1, wherein, The fermentation temperature is 31-37℃. The fermentation time is 15-21 h.

5. The fermentation process of claim 4, wherein, The fermentation temperature is 34℃. The fermentation time is 18 h.

6. The fermentation process of claim 1, wherein, The culture medium mainly comprises grape juice and a nitrogen source. In the culture medium, the mass percentage of the nitrogen source is 0.25%-0.75%.

7. The fermentation process of claim 6, wherein, In the culture medium, the mass percentage of the nitrogen source is 0.5%.

8. The fermentation process of claim 6, wherein, The grape juice is prepared by the following method: mixing grapes with water, beating the mixture to pulp, then performing solid-liquid separation, and obtaining grape juice from the clear liquid. The mass ratio of the grapes to water is 1:2-2:

1.

9. The fermentation process of claim 8, wherein, The mass ratio of the grapes to water is 1:

1.

10. The fermentation process of claim 6, wherein, The nitrogen source comprises at least one of tryptone, soybean peptone, casein peptone or gelatin peptone.

11. The fermentation process of claim 10, wherein, The nitrogen source is soybean peptone.

12. Use of the fermentation method of the grape juice enzyme according to any one of claims 1-11 in improving the antioxidant property of the enzyme product.

13. Use of the fermentation method of the grape juice enzyme according to any one of claims 1-11 in improving the total protease activity of the enzyme product.

14. A grape juice ferment, characterized in that, The grape juice enzyme is prepared by the fermentation method according to any one of claims 1-11.

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