Fermentation process of malus micromalus takahashi and hippophae rhamnoides composite fruit wine

Abstract

This invention discloses a fermentation process for a prickly pear and sea buckthorn compound fruit wine, belonging to the field of fruit wine fermentation technology. The invention includes the following steps: selecting prickly pear juice and sea buckthorn juice, mixing them in a 2:1 volume ratio as fermentation raw materials; adjusting the pH of the juice to a suitable range, adding sugar and preservatives, and adjusting the initial sugar content; activating yeast and adding it to the juice in a specific ratio, stirring evenly; placing the inoculated juice into a fermentation container and fermenting for 9 days under constant temperature conditions; filtering to remove sediment after fermentation to obtain primary fruit wine; clarifying the wine by low-temperature settling to promote impurity sedimentation; using moderate heating for sterilization to ensure product safety and stability; bottling after all indicators pass testing, and sealing and storing the finished product. This invention, by optimizing the compound ratio of prickly pear and sea buckthorn and the synergistic fermentation process parameters, can achieve an effective fusion and balance of the two raw materials in terms of flavor, nutrition, and fermentation kinetics.

Description

Technical Field

[0001] This invention belongs to the field of fruit wine fermentation technology, and in particular relates to a fermentation process for a compound fruit wine made from prickly pear and sea buckthorn. Background Technology

[0002] In the field of fruit wine fermentation, developing compound fruit wines has become an important trend for enriching product variety and enhancing nutritional functions. Both prickly pear and sea buckthorn are fruit raw materials with outstanding nutritional value; the former has a unique flavor and high vitamin C content, while the latter is rich in various bioactive substances. Combining the two for compound fermentation theoretically allows for the fusion of their advantages, producing beverages with superior taste and health benefits, demonstrating clear market potential.

[0003] However, achieving effective compound fermentation of prickly pear and sea buckthorn is not a simple matter of mixing. The two fruits have inherent differences in sugar-acid composition, flavor precursors, and fermentation characteristics. Improper handling can easily lead to flavor incoordination and imbalanced taste during fermentation, failing to create a stable and unified complex wine style. Furthermore, asynchronous fermentation processes may even affect the physicochemical stability and sensory quality of the final product. Therefore, the following solutions are proposed to address these issues. Summary of the Invention

[0004] The purpose of this invention is to provide a fermentation process for prickly pear and sea buckthorn compound fruit wine. By optimizing the compound ratio of prickly pear and sea buckthorn and the entire set of fermentation process parameters that work together with them, the two raw materials can be effectively integrated and balanced in terms of flavor, nutrition and fermentation kinetics. This solves the problem of flavor incoordination and asynchronous fermentation process that easily occurs when prickly pear and sea buckthorn are simply fermented together in the prior art, resulting in poor sensory quality and physicochemical stability of the product.

[0005] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: This invention relates to a fermentation process for a prickly pear and sea buckthorn compound fruit wine, comprising the following steps: Raw material compounding: prickly pear juice and sea buckthorn juice are provided, and the prickly pear juice and sea buckthorn juice are mixed in a predetermined ratio to obtain compound fruit juice; Pre-fermentation treatment: The compound fruit juice is subjected to pH adjustment, initial sugar content adjustment and preservative addition treatment in sequence to obtain fermentation substrate; Yeast activation and inoculation: Saccharomyces cerevisiae is provided, the Saccharomyces cerevisiae is activated, and then the activated yeast is inoculated into the fermentation substrate; Primary fermentation: The inoculated fermentation substrate is placed in a sealed fermentation container and fermented under controlled temperature conditions until the primary fermentation stage is completed; Post-processing: The wine after primary fermentation is subjected to solid-liquid separation, clarification, and sterilization to obtain the prickly pear and sea buckthorn compound fruit wine.

[0006] Furthermore, in the raw material compound, the mixing ratio of prickly pear juice and sea buckthorn juice is an optimized ratio determined by a comprehensive evaluation of the sensory quality and physicochemical indicators of the finished wine.

[0007] Furthermore, in the pre-fermentation treatment, pH adjustment is performed by using a food-grade alkaline regulator to adjust the pH of the compound fruit juice to a weakly acidic range suitable for the growth and fermentation of the brewing yeast.

[0008] Furthermore, in the pre-fermentation treatment, the initial sugar content adjustment is achieved by adding a sugar source to the compound fruit juice to adjust its sugar content to a level that can support the fermentation process to reach the target alcohol content.

[0009] Furthermore, in the yeast activation and inoculation process, the activation treatment includes placing the brewing yeast in a warm sugar solution for static culture until the yeast regains its activity; the inoculation refers to adding the activated yeast liquid to the fermentation substrate at a predetermined inoculation amount.

[0010] Furthermore, in the primary fermentation, temperature control refers to maintaining the fermentation temperature within a constant and suitable range, and the duration of fermentation is determined according to the fermentation kinetics characteristics until the alcoholic fermentation is essentially completed.

[0011] Furthermore, in the post-processing, solid-liquid separation is achieved by physical filtration to remove yeast residues and coarse suspended particles from the wine; the clarification process is achieved by low-temperature settling or assisted clarification to further stabilize and clarify the wine.

[0012] Furthermore, in the post-processing, the sterilization treatment is performed using low-temperature, long-time or medium-temperature, short-time pasteurization to achieve microbial stability and preserve the flavor of the wine.

[0013] Furthermore, in the post-processing, the clarification process is carried out before the sterilization process, or after the sterilization process and before packaging, it also includes a period of aging and stabilization.

[0014] An experimental method for evaluating the quality of a prickly pear and sea buckthorn compound fruit wine includes: comprehensively scoring the color, aroma, taste, and style of the fruit wine using a specific sensory evaluation system; determining the alcohol content, total acid content, and total ester content of the fruit wine using standard physicochemical analysis methods; and determining the antioxidant activity of the fruit wine using a free radical scavenging method, in order to comprehensively evaluate the quality and functional characteristics of the compound fruit wine.

[0015] The present invention has the following beneficial effects: This invention optimizes the compound ratio of prickly pear and sea buckthorn, enabling their flavor compounds and nutrients to synergistically create a unique style characterized by harmonious taste and a fusion of fruity and wine aromas. The entire fermentation parameter system has been optimized and matched to ensure smooth fermentation and a balanced and stable final product with stable physicochemical indicators such as alcohol content and acid-ester ratios. The integrated process flow design is clear and the operation is seamless, facilitating production control and large-scale implementation. The final product retains the typical characteristics of fruit wine while exhibiting enhanced antioxidant activity and superior overall quality.

[0016] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the fermentation process of a prickly pear and sea buckthorn compound fruit wine according to the present invention. Figure 2 This is an experimental flow chart of a prickly pear and sea buckthorn compound fruit wine fermentation process according to the present invention; Figure 3 This is a three-dimensional schematic diagram illustrating the effect of the interaction of factors in this invention on the alcohol content of the prickly pear and sea buckthorn compound fruit wine. Figure 4 This is a three-dimensional schematic diagram illustrating the effect of the interaction of factors in this invention on the sensory score of the prickly pear and sea buckthorn compound fruit wine. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Please see Figure 1 As shown, this invention is a fermentation process for a prickly pear and sea buckthorn compound fruit wine, comprising the following steps: Step S1: Raw material preparation and pretreatment Step S11: Raw material selection and processing Prickly pear juice: Fresh or commercially available prickly pear juice is selected, with a pH value of approximately 3.4, and free from spoilage and off-odors.

[0021] Sea buckthorn juice: Made from fresh or commercially available sea buckthorn juice, free of impurities and mold.

[0022] Sugar source: Food-grade white sugar is used to adjust the initial sugar content during fermentation.

[0023] Yeast: Use Angel BV818 brewing yeast, which needs to be activated in advance.

[0024] Additives: Food-grade sodium bicarbonate (for pH adjustment), potassium metabisulfite (as a preservative and antioxidant).

[0025] Step S12, Raw material ratio: The volume ratio of prickly pear juice to sea buckthorn juice is 2:1. This ratio has been optimized and determined to effectively balance flavor and nutrients.

[0026] Step S2, Pre-fermentation treatment Step S21, pH adjustment: Measure the initial pH of the prickly pear juice (approximately 3.4); slowly adjust the pH of the prickly pear juice to 4.7±0.1 using edible sodium bicarbonate solution. This pH range is conducive to yeast growth and stable fermentation process.

[0027] Step S22, Initial sugar content adjustment: Mix the pH-adjusted prickly pear juice and sea buckthorn juice in proportion; calculate and weigh the corresponding mass of white sugar according to the target initial sugar content of 25°BX, add it to the mixed juice, and stir until completely dissolved.

[0028] Step S23: Add preservative: Add 100 mg / L of potassium metabisulfite to the mixed juice and stir well to inhibit the growth of miscellaneous bacteria and prevent oxidation.

[0029] Step S3, Yeast Activation and Inoculation Step S31, Yeast activation: Weigh the required amount of BV818 brewing yeast (0.56% of the total fermentation liquid mass); add the yeast to a 5% sugar solution and activate it at 36±1℃ for 20 minutes until the yeast is completely suspended and there is no obvious sediment.

[0030] Step S32, Inoculation: Add the activated yeast liquid to the mixed fruit juice with adjusted sugar content and pH at a ratio of 10 mL / L; stir gently to distribute the yeast evenly.

[0031] Step S4, Primary Fermentation Process Step S41: Preparation of fermentation container: Use a 250mL glass fermentation bottle, sterilize it by high pressure steam (121℃, 20 minutes) or alcohol, and then let it dry for later use.

[0032] Step S42, Filling and Sealing: Fill each bottle with 180mL of the inoculated mixed fruit juice, leaving an appropriate headspace (approximately 20-30mL); install the fermentation plug or venting membrane to allow for... To drain and prevent the entry of other bacteria.

[0033] Step S43, Fermentation Condition Control: Place the fermentation bottle in a constant temperature incubator and control the fermentation temperature at 25±1℃; the fermentation time is 9 days, during which vibration and strong light should be avoided; observe the fermentation status daily and record the bubble production.

[0034] Step S5: Determination and handling of fermentation endpoint Step S51, Fermentation endpoint: On the 9th day of fermentation, the bubbles in the wine will decrease significantly and the wine will become clearer, indicating that the primary fermentation is basically complete.

[0035] Step S52, Preliminary filtration: Use double-layered degreased cotton gauze to filter the fermentation liquid under normal pressure to remove most of the yeast mud and fruit residue; collect the filtrate, which is the primary fruit wine.

[0036] Step S6, Post-processing and Clarification Step S61, Clarification by Stagnation: Place the filtered fruit wine in a refrigerated environment at 4-8℃ and let it stand for 7 days to further clarify and stabilize the wine.

[0037] Step S62, Secondary Filtration: If fine suspended matter still exists in the wine, diatomaceous earth or plate and frame filters can be used for fine filtration to improve the transparency of the wine.

[0038] Step S7, Sterilization and Stabilization Step S71, pasteurization: Place the clarified fruit wine in a water bath at 65-70℃ for 15-20 minutes to kill residual microorganisms and terminate enzyme activity; after pasteurization, quickly cool to room temperature.

[0039] Step S72, Quality Stability: Appropriate amounts of food-grade antioxidants (such as vitamin C) or stabilizers may be added as needed to prevent oxidation and precipitation during storage.

[0040] Step S8, Finished Product Inspection and Packaging Step S81, Physicochemical index testing: Test indicators such as alcohol content, total acid, total esters, and sugar content to ensure compliance with fruit wine product standards.

[0041] Step S82, Sensory evaluation: The wine is orange-yellow to orange-red in color, clear and transparent; it has a harmonious fruity and wine aroma of prickly pear and sea buckthorn; the taste is moderately sweet and sour, with a unique flavor and no off-flavors.

[0042] Step S83, Filling and Storage: Use aseptic filling technology to fill the glass bottles and seal them for storage; the finished product should be stored in a cool, dark place, and the recommended storage temperature is 10-15℃.

[0043] experiment: The process flow used in this embodiment is shown in the figure. Figure 2 The specific process is as follows: pH adjustment: The original prickly pear juice was measured to be 3.4. Edible sodium bicarbonate was used to adjust the pH of the prickly pear juice to 4.7. Adjusting the initial sugar content and adding potassium metabisulfite: After adjusting the pH value of the prickly pear juice and sea buckthorn juice, weigh out a certain amount of white sugar according to the target initial sugar content and add it to dissolve. At the same time, add 100 mg / L potassium metabisulfite.

[0044] Inoculation: Activate BV818 brewing yeast in 5% sugar water at 36℃ for 20 min according to the required amount. Measure 10 ml of the activated yeast using a graduated cylinder and add it to the prepared prickly pear and sea buckthorn juice.

[0045] Fermentation: Add 180 mL of prickly pear and sea buckthorn juice to a sterilized 250 mL fermentation container and place it in an incubator at a preset temperature for fermentation.

[0046] Filtration: Filtration is carried out under normal pressure using double-layer degreased cotton gauze.

[0047] Sterilization: The mixed fermentation liquid is heat-treated by pasteurization and maintained in a constant temperature water bath environment of 65-70 ℃ for 15-20 minutes. This temperature range can effectively inactivate the microbial community while retaining the heat-sensitive active ingredients in the raw materials, and finally produce a prickly pear and sea buckthorn compound fruit wine that meets the safety standards for beverages.

[0048] Single-factor experiment on prickly pear and sea buckthorn compound fruit wine: 90 ml each of prickly pear juice and sea buckthorn juice were taken and mixed in a 1:1 volume ratio. This mixture was used as the base raw material for fermenting prickly pear and sea buckthorn compound fruit wine. Under the baseline conditions of an initial sugar content of 24°Bx, a yeast inoculation amount of 0.6%, a stable fermentation temperature of 25℃, and a fermentation period of 8 days, the effects of different factors on the quality of the prickly pear and sea buckthorn fruit wine were investigated by changing a single variable. These factors included: the ratio of prickly pear to sea buckthorn (e.g., 1:1, 2:1, 1:2, 3:1, 1:3, etc.), the initial sugar content (including different values ​​such as 18°Bx, 20°Bx, 22°Bx, 24°Bx, 26°Bx, etc.), the amount of yeast added (0.2%, 0.4%, 0.6%, 0.8%, 1.0%, etc.), and the fermentation period (4 days, 6 days, 8 days, 10 days, 12 days, etc.). Sensory test of prickly pear and sea buckthorn compound fruit wine: According to the "General Analytical Methods for Wines and Fruit Wines," the sensory evaluation criteria for prickly pear and sea buckthorn compound fruit wine include appearance, color, aroma, taste, and style. Specifically, an evaluation team of 10 food science students was formed to conduct the sensory evaluation.

[0049] Table 1 Sensory Evaluation Form

[0050] Response surface methodology for prickly pear and sea buckthorn compound fruit wine: Based on a thorough understanding of the results of single-factor experiments, key factors significantly influencing the fermentation process of prickly pear and sea buckthorn compound fruit wine were selected, and response surface methodology was conducted. Subsequently, the fermented fruit wine underwent rigorous alcohol content testing and sensory evaluation analysis. Using BOX-Behken software, an optimized experimental process was designed, and the optimal process parameters derived by the software were verified to ensure their effectiveness in practical applications.

[0051] Physicochemical index determination: Determination of total acidity: The total acidity was determined by acid-base titration in accordance with the national standard GB 123456-2021 "National Food Safety Standard - Determination of Total Acidity in Food". This acid-base titration method is a commonly used quantitative analysis method in the field of acidity analysis of wine and fruit wine.

[0052] Total esters: Referring to GB / T 10345—2022 "Analytical Methods for Baijiu", acid-base titration was used as the core detection method in the experiment. Bromocresol green-methyl red composite indicator was prepared. By controlling the reflux condensation time, the full saponification reaction of esters was ensured. The endpoint was determined by controlling the color change range within the pH range of 3.8-5.4.

[0053] Alcohol content: The alcohol content was determined by alcohol meter method according to Method II of GB 5009.225—2023 "National Food Safety Standard - Determination of Ethanol Concentration in Wine and Edible Alcohol"; Antioxidant activity: The two-system free radical scavenging method was used for determination. The DPPH free radical scavenging effect was detected according to the national standard document GB / T 39100—2020 on the evaluation of antioxidant properties of peptides. The specific operation was based on the improved ultraviolet spectrophotometric method of Yang Bingxin et al., and the free radical scavenging rate was calculated by the change of characteristic absorption peak shift. The ABTS+ free radical scavenging effect was determined by the spectrophotometric detection process established by Qin Jinying's team. The change of absorbance of the reaction system was measured at a wavelength of 734 nm, and the neutralization efficiency of cationic free radicals was quantitatively analyzed by the standard curve method.

[0054] Discussion and analysis of results: Univariate Results and Analysis: The effect of yeast inoculation amount on prickly pear and sea buckthorn compound fruit wine: Table 2 Effect of yeast inoculum size on prickly pear and sea buckthorn compound fruit wine

[0055] Note: The numbers represent the mean and standard deviation. Different lowercase letters in the same column indicate significant differences (P<0.05). Table 2 shows that the yeast inoculation amount has a significant impact on the alcohol content of the prickly pear and sea buckthorn compound fruit wine (P<0.05). When the yeast inoculation amount is too low during the fermentation process, the fermentation process is slow, the sugar conversion rate is low, resulting in a low alcohol content. When the yeast inoculation amount is too high, the yeast grows too vigorously in the early stages of fermentation, the fermentation speed is fast, a large amount of nutrients are consumed, and the yeast may undergo premature autolysis, resulting in a yeasty and bitter taste in the fruit wine, affecting its taste and flavor, and reducing its quality. Therefore, choosing an appropriate amount of yeast is crucial to the fruit wine's quality. Table 2 also shows that when the yeast inoculation amount is in the range of 0.2%~0.6%, the alcohol content and sensory score increase with increasing yeast addition. At this point, the nutrients in the fruit wine are gradually utilized, there is no off-flavor, and the taste is normal. When the inoculation amount is in the range of 0.6%~1.0%, the alcohol content and sensory score decrease with increasing yeast addition. At this point, the taste of the fruit wine deteriorates, the wine becomes cloudy, the yeasty taste is strong, the overall sourness and astringency are significantly increased, and a small amount of sediment is present. When the yeast inoculation amount was 0.6%, the alcohol content of the prickly pear and sea buckthorn compound fruit wine reached 14.53% vol, and the sensory score was as high as 72.33 points. At this point, the fruit wine had a suitable taste, no significant sedimentation, and a harmonious balance between the fruit aroma and the wine aroma. Therefore, the optimal yeast addition amount for prickly pear and sea buckthorn fruit wine was determined to be 0.6%.

[0056] The effect of fermentation time on prickly pear and sea buckthorn compound fruit wine: Table 3. Effect of fermentation time on prickly pear and sea buckthorn compound fruit wine

[0057] Note: Values ​​are mean ± standard deviation. Different lowercase letters in the same column indicate significant differences (P<0.05). Table 3 shows that the alcohol content and sensory score initially increased and then decreased with prolonged fermentation time. During the first 4-8 days of fermentation, both alcohol content and sensory score increased. This is because in the early stages of fermentation, yeast utilizes the sugars in the fruit juice for alcoholic fermentation, producing alcohol. Under suitable fermentation conditions, the flavor compounds and aroma components of the fruit wine are relatively rich, resulting in a harmonious taste, and the color and appearance are also at a good level. The longer the fermentation time, the more complete the fermentation, leading to higher alcohol content and sensory scores. After 8 days of fermentation, both alcohol content and sensory scores began to decrease. This may be because as fermentation time further extends, the yeast's nutrients are gradually depleted, and the alcohol and other substances produced during fermentation inhibit yeast activity, causing a decrease in the fermentation rate. Consequently, the alcohol content not only stops increasing but actually decreases. Lower sensory scores may be due to prolonged fermentation altering some flavor compounds in the fruit wine, producing unpleasant flavors; or over-fermentation may have made the fruit wine's taste unbalanced, thus affecting the overall sensory quality. Fermentation time has a significant impact on the alcohol content and sensory score of prickly pear and sea buckthorn compound fruit wine. On the 8th day, the alcohol content of 11.43% vol and the sensory score of 78.66 reached their highest levels. Taking into account both alcohol content and sensory score, 8 days of fermentation is selected as the optimal fermentation time.

[0058] The effect of initial sugar content on prickly pear and sea buckthorn compound fruit wine: Table 4 Effect of initial sugar content on prickly pear and sea buckthorn compound fruit wine

[0059] Note: Values ​​are mean ± standard deviation. Different lowercase letters in the same column indicate significant differences (P<0.05). Table 4 shows that initial sugar content is a crucial factor affecting alcoholic fermentation in fruit wine. When the initial sugar content is low, the amount of sugar available for yeast fermentation is limited, resulting in lower alcohol yield and alcohol content. Simultaneously, due to the relatively small amount of fermentation products, the flavor compounds in the fruit wine are insufficiently generated, leading to a lower sensory score. As the initial sugar content increases, the yeast has more sugar available for fermentation and aging, producing more alcohol. The alcohol content gradually increases, the flavor of the fruit wine gradually becomes richer, and the sensory score rises accordingly. When the initial sugar content is in the range of 18–24°BX, both alcohol content and sensory score increase with increasing initial sugar content. At this point, the taste and flavor of the fruit wine gradually improve, and the balance between alcohol content and flavor compounds is relatively good. However, when the initial sugar content reaches 26°BX, the alcohol content shows a downward trend, and the sensory score also decreases. This may be because excessively high sugar content leads to excessively high osmotic pressure in the fermentation environment, inhibiting yeast growth and fermentation activity, resulting in incomplete fermentation and some sugar failing to be converted into alcohol. Simultaneously, excessively high sugar content may make the fruit wine overly sweet, affecting the overall flavor and causing problems such as a lack of crispness and uncoordinated aromas, leading to a decrease in sensory scores. When the initial sugar content is 24°BX, the prickly pear and sea buckthorn compound fruit wine reaches its highest alcohol content of 11.9% vol, and its sensory score of 74.33 is also at a relatively high level. At this point, the fruit wine has a moderate taste, a reasonable balance between sweetness and alcohol, and a harmonious combination of fruit and wine aromas. Therefore, considering both alcohol content and sensory scores, 24°BX is chosen as a more suitable initial sugar content.

[0060] The effect of different proportions of prickly pear and sea buckthorn on prickly pear and sea buckthorn compound fruit wine: Table 5. Effects of different proportions on prickly pear and sea buckthorn compound fruit wine

[0061] Note: Values ​​are mean ± standard deviation. Different lowercase letters in the same column indicate significant differences (P<0.05). Table 5 shows that the ratio of prickly pear to sea buckthorn affects the substrate composition and flavor formation of fruit wine fermentation. When the ratio of prickly pear to sea buckthorn changes from 1:2 to 1:1 and then to 3:1, the alcohol content gradually increases, and the sensory score also improves. This is because prickly pear and sea buckthorn each contain different nutrients and flavor compounds. Increasing the proportion of prickly pear may provide a more suitable nutritional environment for yeast fermentation, promoting yeast growth and fermentation, thus increasing the alcohol content. It also makes the flavor of the fruit wine richer and more harmonious, thereby improving the sensory score. When the ratio reaches 2:1, the alcohol content reaches its highest value of 12.26% vol, and the sensory score reaches 74.0 points. At this point, the taste, aroma, and overall quality of the fruit wine are at their best. At this ratio, the flavor compounds of prickly pear and sea buckthorn blend ideally, forming a unique and harmonious flavor, while also providing optimal substrate conditions for yeast fermentation. However, when the ratio becomes 1:3, both the alcohol content and the sensory score decrease. The overly prominent flavor of sea buckthorn masks the flavor of prickly pear, leading to an imbalance in the fruit wine's flavor. Furthermore, it may inhibit yeast fermentation, resulting in lower alcohol content and ultimately affecting the overall quality of the fruit wine. Considering all factors, a 2:1 ratio is the optimal proportion for a prickly pear and sea buckthorn blended fruit wine.

[0062] Analysis of variance for one-way experiments: Analysis of variance on alcohol content and sensory evaluation as single variables yielded the results shown in Table 6. Four factors that significantly influenced the quality of the prickly pear and sea buckthorn compound fruit wine were identified, as shown in Table 7.

[0063] Table 6. Analysis of variance of different factors on prickly pear and sea buckthorn compound fruit wine

[0064] Note: ** indicates extremely significant effect (P<0.01); * indicates significant effect (P<0.05); Table 7. Response Surface Design Factor Level Coding Table

[0065] Response surface methodology: Based on the analysis results of the single-factor experiments, four key control points were identified through analysis of variance: yeast inoculum size (A), fermentation time (B), initial sugar concentration (C), and the ratio of prickly pear to sea buckthorn (D). A four-factor, three-level orthogonal experimental design was constructed based on the principle of response surface methodology, using alcohol content and sensory evaluation scores as target parameters for process optimization. Detailed experimental data regarding the relevant experimental design and response values ​​are shown in Table 8.

[0066] Table 8 Response Surface Experimental Design and Results

[0067] Response surface methodology: Analysis of variance (ANOVA) was performed on the response surface methodology using Design-Expert 13 software. Table 9 presents the detailed results of the response surface analysis, which include the degree of influence of each factor on the response variable and the significance of interactions. Focusing on these data allows for precise optimization of the experimental design, ultimately aiming to achieve optimal experimental results.

[0068] Table 9. Analysis of variance of the alcohol content regression equation

[0069] Note: ** indicates extremely significant effect (P<0.01); * indicates significant effect (P<0.05); Table 9 shows that the model sum of squares for alcohol content is 13.46, degrees of freedom is 14, standard deviation is 0.9617, F-value is 69.45, and P-value is <0.0001, indicating that the established model is highly significant and can well reflect the relationship between various factors and response values. The model's coefficient of determination R² = 0.9858 and Adj R² = 0.9716, both of which are very close to 1, indicating that the model fits the experimental data very well, and there is a strong correlation between the experimental values ​​and the predicted values ​​obtained using this equation. The model can be well used to predict and analyze relevant indicators of prickly pear and sea buckthorn wine. The quadratic multiple regression equation is Y1 = +11.74 + 0.2250A + 0.0958B + 0.1250C - 0.3125D - 0.1875AB - 0.0250AC - 0.2375AD - 0.0250BC - 0.1250BD - 0.1250CD - 0.7408A² - 0.6596B² - 0.6158C² - 1.07D. The effects of each factor and interaction term on the response value: For the linear terms: the p-values ​​for yeast inoculum size (A), fermentation time (B), initial sugar content (C), and the ratio of prickly pear to sea buckthorn (D) are all <0.01, indicating a highly significant effect on the response value. For the interaction terms: the p-values ​​for AB and AD are <0.01, indicating a highly significant effect on the response value, suggesting that the interaction between yeast inoculum size and fermentation time, and between yeast inoculum size and the ratio of prickly pear to sea buckthorn, has a significant impact on the relevant indicators of prickly pear and sea buckthorn wine. The p-values ​​for AC and BC are >0.05, indicating no significant effect on the response value. This suggests that the interaction between yeast inoculation amount and initial sugar content, and fermentation time and initial sugar content, has a relatively small impact on the results. The p-values ​​for BD and CD are 0.0519, close to the significance level, indicating that the interaction between fermentation time and the ratio of prickly pear to sea buckthorn, and the interaction between initial sugar content and the ratio of prickly pear to sea buckthorn, has some influence on the response value, but the significance is slightly weak. The p-values ​​for quadratic terms A², B², C², and D² are all <0.01, indicating a highly significant effect on the response value. The quadratic terms of yeast inoculation amount, fermentation time, initial sugar content, and the ratio of prickly pear to sea buckthorn have a significant non-linear effect on the relevant indicators of prickly pear and sea buckthorn wine. Based on the F-values, the degree of influence of these four factors on prickly pear and sea buckthorn compound fruit wine is as follows: the ratio of prickly pear to sea buckthorn (D) plays a decisive role, followed by yeast inoculation amount (A), then initial sugar content (C), while the influence of fermentation time (B) is relatively small.

[0070] Table 10. Analysis of variance of the regression equation for sensory ratings

[0071] Note: ** indicates extremely significant effect (P<0.01); * indicates significant effect (P<0.05); Table 10 shows that the model's sum of squares is 643.38, degrees of freedom is 14, standard deviation is 45.96, F-value is 42.71, and p-value < 0.0001, indicating that the established model is highly significant and can well reflect the relationship between each factor and the response value. The model's coefficient of determination R² = 0.9771 and Adj R² = 0.9452, both of which are very close to 1, indicating that the model fits the experimental data very well, and there is a strong correlation between the experimental values ​​and the predicted values ​​obtained by using this equation. The model can be well used to predict and analyze the relevant indicators of prickly pear and sea buckthorn fruit wine. The quadratic multiple regression equation is Y2=82.18+1.58A+0.5250B+0.8750C-2.04D-1.31AB-0.1750AC-1.66AD-0.1750BC-1.31BD-0.8750CD-5.26A²-4.47B²-4.38C²-7.36D². Interaction term analysis: The p-value for BD (interaction term between time and the ratio of prickly pear to sea buckthorn) is <0.01, clearly indicating that the interaction between these two factors has a significant impact on prickly pear and sea buckthorn wine; they synergistically shape the unique quality of the wine. The p-values ​​for AC (interaction term between yeast inoculation amount and initial sugar content) and CD (interaction term between initial sugar content and the ratio of prickly pear to sea buckthorn) are both <0.05, indicating that their interaction has a significant impact on prickly pear and sea buckthorn fruit wine. The p-values ​​of the interaction terms AB, AD, and BC are greater than 0.05, indicating that the interaction between yeast inoculation amount and time, the ratio of yeast inoculation amount to prickly pear and sea buckthorn, and time and initial sugar content have no significant impact on the quality of prickly pear and sea buckthorn wine. Therefore, these factors will not be considered in the process optimization for the time being.

[0072] Quadratic effect investigation: The p-values ​​for A², B², C², and D² were all <0.01, which fully demonstrates that the quadratic terms of yeast inoculation amount, time, initial sugar content, and the ratio of prickly pear to sea buckthorn have a highly significant nonlinear effect on prickly pear and sea buckthorn wine. According to the F-value, the influence of these four factors on prickly pear and sea buckthorn compound fruit wine is as follows: the ratio of prickly pear to sea buckthorn (D) is the most important factor, followed by fermentation time (B), then yeast inoculation amount (A), while the effect of initial sugar content (C) is relatively small.

[0073] Response surface methodology interaction analysis: A three-dimensional response surface plot is a visual description of a regression equation, which can intuitively reflect the interaction between various parameters and the maximum response value.

[0074] When alcohol content is used as the evaluation index, a three-dimensional surface plot of the effects of pairwise interactions between four factors—yeast inoculum size (A), fermentation time (B), initial sugar content (C), and the ratio of prickly pear to sea buckthorn (D)—on alcohol content was plotted using Design-expert13 based on the regression equation. Figure 3As shown. The inclination of the surface reflects the degree of influence of the experimental factors on the response value. The greater the inclination, the more significant the influence; conversely, the less significant. The shape of the contour lines represents the strength of the interaction between two factors. The closer the contour lines are to circles, the less significant the interaction between the two factors; conversely, the more they develop towards an ellipse, the more significant the interaction between the two factors. The center point inside the smallest ellipse in the contour map is the highest point of the response surface. From this point, it can be seen that there are extreme values ​​of the response value of the influencing factor within the experimental range. It can be seen that the contour lines between the inoculation amount and the ratio of prickly pear and sea buckthorn (D) are elliptical, indicating that the interaction between the yeast inoculation amount (A) and the ratio of prickly pear and sea buckthorn (D) is significant. When the initial sugar content (C) and fermentation time (B) remain constant, at a low yeast inoculum level (A), the alcohol content initially increases and then decreases with increasing prickly pear and sea buckthorn ratio (D), while the alcohol content increases with increasing yeast inoculum concentration (A). At a high prickly pear and sea buckthorn ratio (D), the alcohol content initially increases and then decreases with increasing yeast inoculum concentration (A). The contour lines between the initial sugar content (C) and the prickly pear and sea buckthorn ratio (D) are elliptical, indicating a significant interaction between the initial sugar content (C) and the prickly pear and sea buckthorn ratio (D). When the initial yeast inoculum level (A) and fermentation time (B) remain constant, at a low initial sugar content (C), the alcohol content initially increases and then decreases with increasing prickly pear and sea buckthorn ratio (D). At a high initial sugar content (C), the alcohol content generally decreases with increasing prickly pear and sea buckthorn ratio. At a low prickly pear and sea buckthorn ratio (D), the alcohol content increases with increasing initial sugar content (C). When the ratio of prickly pear to sea buckthorn (D) is high, the alcohol content first increases and then decreases with the increase of the initial sugar content (C).

[0075] Using the Design-expert13 software platform, extreme point analysis was performed on the multiple regression equation to obtain the optimal parameter combination: A=0.1287, B=0.416, C=-0.306, D=0.1426, with a theoretical predicted value R reaching 10.79% vol. Experimental data show that when the process parameters are set as follows: yeast inoculum amount 0.534%, fermentation cycle 8.49 days, initial sugar concentration 25.4°BX, and the ratio of prickly pear to sea buckthorn 1:2.57, the obtained alcohol content of 11.20% vol is in good agreement with the model prediction.

[0076] When sensory evaluation is used as the evaluation index, the contour lines between the fermentation time and the proportion of prickly pear and sea buckthorn are elliptical, indicating a significant interaction between fermentation time (B) and the proportion of prickly pear and sea buckthorn (D). When the yeast inoculum amount (A) and initial sugar content (C) remain constant, at low fermentation time levels, sensory evaluation initially increases and then decreases with increasing prickly pear and sea buckthorn proportion. At high fermentation time levels, sensory evaluation generally decreases with increasing prickly pear and sea buckthorn proportion (D). At low prickly pear and sea buckthorn proportion (D), sensory evaluation increases with increasing fermentation time (B) concentration. At high fermentation time levels, sensory evaluation initially increases and then decreases with increasing fermentation time (B) concentration. The elliptical contour lines between initial sugar content (C) and the proportion of prickly pear and sea buckthorn (D) indicate a significant interaction between these two factors. When the yeast inoculum (A) and fermentation time (B) remain constant, and the initial sugar content (C) is low, the sensory evaluation initially increases and then decreases with increasing prickly pear and sea buckthorn ratio (D). Conversely, when the initial sugar content (C) is high, the sensory evaluation generally decreases with increasing prickly pear and sea buckthorn ratio (D). When the prickly pear and sea buckthorn ratio (D) is low, the sensory evaluation increases with increasing initial sugar concentration. Conversely, when the prickly pear and sea buckthorn ratio is high, the sensory evaluation initially increases and then decreases with increasing initial sugar content (C).

[0077] Using Design-Expert 13, equation analysis was conducted, and the extreme points of the regression model were obtained as follows: A = -0.5159, C = 0.8087, B = -0.3251, and D = 0.271. Based on these extreme points, the predicted response value R reached 71.03. Under these conditions, with a fermentation time of 8.65 days, an initial sugar content of 24.31 °BX, a yeast inoculum of 0.558%, and a prickly pear to sea buckthorn ratio of 1:2.2, the sensory evaluation score of 72.15 points is in good agreement with the model.

[0078] Validation experiment of the optimal formulation: Through Design-Expert 13 analysis of the equations, the optimal process for alcohol content and sensory evaluation was obtained. Comparison revealed that the sensory evaluation showed superior performance across all indicators. Specifically, under the conditions of a fermentation time of 8.65 days, an initial sugar content of 24.31°BX, a yeast inoculation rate of 0.558%, and a prickly pear to sea buckthorn ratio of 1:2.2, the sensory evaluation score was 72.15. Considering the need for smooth experimental implementation in actual operation, the above optimal process parameters were adjusted. The fermentation time was modified to 9 days, the yeast inoculation rate to 0.56%, the initial sugar content to 24°BX, and the prickly pear to sea buckthorn ratio to 2:1. Under these optimized conditions, the actual measured average values ​​of the alcohol content and sensory evaluation score for the prickly pear and sea buckthorn blended fruit wine were 11.20±0.13% vol and 72.12±0.35 points, respectively. The two actual measured values ​​are quite close to the predicted values, which indicates that the optimal process conditions obtained by the software simulation are feasible.

[0079] Sensory scores and physicochemical properties of the samples: The sensory evaluation and physicochemical index test results of the prickly pear and sea buckthorn compound fruit wine fermented using the optimal process, compared with single prickly pear and sea buckthorn fruit wines, are shown in Table 11. As shown in Table 11, the test data indicate that the key parameters of the compound fruit wine, such as total acid content and total ester content, all meet the technical requirements for fermented wine categories in the T / GZSX prickly pear series product standard. Regarding the stability of the wine, the compound process exhibits better index balance than the single-raw-ingredient wine samples.

[0080] Table 11. Results of the test on the control of prickly pear and sea buckthorn fruit wine.

[0081] Table 12 Results of Antioxidant Activity Detection

[0082] As shown in Table 12, the prickly pear and sea buckthorn compound fruit wine exhibits superior antioxidant activity compared to single-fruit wines. Its DPPH free radical scavenging rate reached 62.14%, higher than the 56.73% of prickly pear wine and 57.11% of sea buckthorn wine; its ABTS cation scavenging rate was 59.40%, also exceeding the 54.20% of prickly pear wine and 53.62% of sea buckthorn wine, indicating that the combination of the two enhances antioxidant performance.

[0083] Conclusion: Based on the single-factor experiments, sensory evaluation was ultimately selected as the observation index. The process parameters were optimized by combining single-factor experiments with response surface methodology, and the effects of different factors on prickly pear and sea buckthorn compound fruit wine were studied. A Box-Behken design was used, yielding the quadratic multiple regression equation Y = 82.18 + 1.58A + 0.5250B + 0.8750C - 2.04D - 1.31AB - 0.1750AC - 1.66AD - 0.1750BC - 1.31BD - 0.8750CD - 5.26A² - 4.47B² - 4.38C² - 7.36D². The influence of the linear terms A, B, C, and D on sensory evaluation was ranked as follows: the proportion of prickly pear and sea buckthorn (D) had the greatest impact, followed by yeast inoculation amount (A), initial sugar content (C), and fermentation time (B) had the least impact. After experimental optimization, the optimal fermentation process parameters were determined as follows: yeast inoculation amount set at 0.56%, prickly pear to sea buckthorn ratio at 2:1, fermentation time at 9 days, and initial sugar content at 25°BX. Under the optimal process conditions, the prickly pear and sea buckthorn fruit wine had an alcohol content of 11.20% vol, a sensory score of 72.12 points, a total acid content of 10.32 g / L, a total ester content of 0.94 g / L, and antioxidant activity with a DPPH free radical scavenging rate of 62.10% and an ABTS cation scavenging rate of 59.40%. Therefore, it can be concluded that the prickly pear and sea buckthorn compound fruit wine has certain development potential.

[0084] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0085] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A fermentation process for a prickly pear and sea buckthorn compound fruit wine, characterized in that, The fermentation process includes the following steps: Raw material compounding: prickly pear juice and sea buckthorn juice are provided, and the prickly pear juice and sea buckthorn juice are mixed in a predetermined ratio to obtain compound fruit juice; Pre-fermentation treatment: The compound fruit juice is subjected to pH adjustment, initial sugar content adjustment and preservative addition treatment in sequence to obtain fermentation substrate; Yeast activation and inoculation: Saccharomyces cerevisiae is provided, the Saccharomyces cerevisiae is activated, and then the activated yeast is inoculated into the fermentation substrate; Primary fermentation: The inoculated fermentation substrate is placed in a sealed fermentation container and fermented under controlled temperature conditions until the primary fermentation stage is completed; Post-processing: The wine after primary fermentation is subjected to solid-liquid separation, clarification, and sterilization to obtain the prickly pear and sea buckthorn compound fruit wine.

2. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the aforementioned raw material compound, the mixing ratio of prickly pear juice and sea buckthorn juice is an optimized ratio determined through a comprehensive evaluation of the sensory quality and physicochemical indicators of the finished wine.

3. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the pre-fermentation treatment, pH adjustment is performed by using a food-grade alkaline regulator to adjust the pH of the compound fruit juice to a slightly acidic range suitable for the growth and fermentation of the brewing yeast.

4. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the pre-fermentation treatment, the initial sugar content adjustment is achieved by adding a sugar source to the compound fruit juice to adjust its sugar content to a level that can support the fermentation process to reach the target alcohol content.

5. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the yeast activation and inoculation process, the activation treatment includes placing the brewing yeast in a warm sugar solution for static culture until the yeast regains its activity; the inoculation refers to adding the activated yeast liquid to the fermentation substrate at a predetermined inoculation amount.

6. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the primary fermentation, temperature control means maintaining the fermentation temperature within a constant and suitable range, and the duration of fermentation is determined according to the fermentation kinetics characteristics until the alcoholic fermentation is essentially completed.

7. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the post-processing, solid-liquid separation is achieved by physical filtration to remove yeast residues and large suspended particles from the wine; the clarification process is achieved by low-temperature settling or assisted clarification to further stabilize and clarify the wine.

8. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the post-processing, the sterilization treatment is carried out by low-temperature long-time or medium-temperature short-time pasteurization to achieve microbial stability and preserve the flavor of the wine.

9. The fermentation process for a prickly pear and sea buckthorn compound fruit wine according to claim 1, characterized in that, In the post-processing, the clarification process is carried out before the sterilization process, or after the sterilization process and before packaging, it includes a period of aging and stabilization.

10. An experimental method for evaluating the quality of prickly pear and sea buckthorn compound fruit wine, characterized in that, The experimental methods include: using a specific sensory evaluation system to comprehensively score the color, aroma, taste, and style of the fruit wine; using standard physicochemical analysis methods to determine the alcohol content, total acid content, and total ester content of the fruit wine; and using a free radical scavenging method to determine the antioxidant activity of the fruit wine, so as to comprehensively evaluate the quality and functional characteristics of the compound fruit wine.

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