Method for preparing jujube and hawthorn enzyme by multi-strain two-stage fermentation

Abstract

The present application belongs to the field of food biotechnology, and specifically discloses a method for preparing jujube and hawthorn enzyme through multi-strain two-stage fermentation, which comprises the following steps: anaerobic fermentation of jujube and hawthorn pulp by lactic acid bacteria, followed by aerobic fermentation by yeast, centrifugation after fermentation to obtain supernatant, and obtaining jujube and hawthorn enzyme. The present application uses jujube and hawthorn as main raw materials, connects preferred composite probiotics after scientific compounding, greatly shortens the fermentation period compared with traditional natural fermentation, and determines the process parameters of each stage of fermentation through multiple rounds of optimization, so that the jujube and hawthorn enzyme is ruby red, sweet and sour, jujube aroma and hawthorn fruit aroma are coordinated, and there is no bitter aftertaste; the total phenol and total flavonoid retention rate is greater than or equal to 92%, the free radical scavenging h activity is greater than or equal to 95%, and the antioxidant activity is significantly better than that of commercially available similar enzymes, and the present application is rich in functional factors such as short-chain fatty acids, active peptides and organic acids. The present application has good repeatability and is suitable for industrial continuous production.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology, specifically relating to a method for preparing jujube and hawthorn enzyme through a two-stage fermentation process using multiple strains. Background Technology

[0002] red dates( Ziziphus jujuba (Mill.) is a plant belonging to the genus Ziziphus of the family Rhamnaceae. Ripe jujubes have a high sugar content, a sweet and mellow taste, and are rich in essential polysaccharides, polyphenols, dietary fiber, vitamins, and other bioactive components, making them an important plant with both medicinal and edible uses. However, the high sugar and water content of fresh jujubes leads to their poor storage properties; they are highly susceptible to mold and fermentation after harvest, with a shelf life of less than ten days at room temperature. To alleviate sales difficulties and reduce post-harvest losses, primary processing methods in major producing areas generally involve sun-drying and baking. Over 90% of jujube products are limited to the two traditional forms of dried jujubes (unwashed jujubes) and candied fruit. Low technological barriers and severe homogenization have become the core bottlenecks restricting the upgrading of the jujube industry.

[0003] Probiotic fermentation is the greenest, most economical, and most effective technology for preserving and amplifying functional components among various biotransformation and processing methods. Lactic acid bacteria, yeast, acetic acid bacteria, and other microbial resources have been widely used in the food fermentation industry, such as in the production of traditional fermented foods like yogurt, alcoholic beverages, kimchi, and soy sauce and vinegar. In recent years, mixed-strain symbiotic fermentation has rapidly gained popularity in the fruit and vegetable sector. Its main advantage lies in the complementary metabolic networks of the same strains, which can convert bound polyphenols into free aglycones, cleave large polysaccharides into easily absorbed oligomeric fragments, and simultaneously produce synergistic active ingredients such as B vitamins, γ-aminobutyric acid (GABA), and extracellular polysaccharides. Overall, its antioxidant and hypoglycemic potential is significantly superior to single-strain fermentation, providing a new leverage point for the functional premium of jujubes.

[0004] The concept of enzyme products originated in Japan. In its 2006 Health Food Regulations, Japan defined plant-based enzymes as "concentrated liquids containing active enzymes, organic acids, and secondary metabolites obtained through the combined fermentation of various fruits and vegetables using yeast, lactic acid bacteria, acetic acid bacteria, etc." As an emerging food product using mixed-culture fermentation technology, the nutritional and functional value of enzymes has been verified in preliminary studies. However, the overall scientific evidence remains fragmented, and systematic research has yet to reach a consensus. The jujube industry still has a significant gap in enzyme products, with fragmented data and a lack of in-depth mechanistic explanations and clinical evidence. Summary of the Invention

[0005] This invention uses dried hawthorn slices mixed with Xinjiang red dates (Junzao) in a certain proportion as a substrate to construct a two-stage fermentation symbiotic system of "three lactic acid bacteria and one yeast". This system fully releases polyphenols, folic acid, and triterpenoid bioactive components, while simultaneously enriching short-chain fatty acids, extracellular polysaccharides, and active enzymes. It not only breaks through the limitations of single-strain fermentation, but also leverages the new consumer label of "enzyme" to achieve product category upgrading, providing a high-value-added, green, low-carbon, and replicable breakthrough path for the red date processing industry.

[0006] This invention first provides a method for preparing jujube and hawthorn enzyme through a two-stage fermentation process using multiple bacteria, including anaerobic fermentation of jujube and hawthorn pulp with lactic acid bacteria, followed by aerobic fermentation with yeast, and centrifugation to collect the supernatant after fermentation to obtain jujube and hawthorn enzyme.

[0007] In the above method, the lactic acid bacteria are three types: Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus thermophilus.

[0008] In the above method, the yeast is yeast BV818.

[0009] In the above method, the jujube and hawthorn pulp is obtained by removing the pits from the jujubes, taking dried jujubes and hawthorn slices at a mass ratio of 5:1 as raw materials, adding water four times the mass of the raw materials, mixing and pulping, and then sterilizing at 73°C for 30 minutes.

[0010] In the above method, during anaerobic fermentation, the inoculum amount of *Lactobacillus plantarum* is 0.05% by volume, the inoculum amount of *Lactobacillus acidophilus* is 0.06% by volume, and the inoculum amount of *Streptococcus thermophilus* is 0.05% by volume.

[0011] In the above method, the anaerobic fermentation is carried out at 33°C for 36 hours.

[0012] In the above method, the aerobic fermentation involves an inoculation amount of yeast of 0.02% by volume.

[0013] In the above method, the aerobic fermentation is carried out at 28°C for 12 hours.

[0014] The present invention discloses a method for preparing jujube and hawthorn enzyme through a two-stage fermentation process using multiple microorganisms, the specific steps of which are as follows: 1) Remove the pits from the dates in advance, take the dates and dried hawthorn slices in a mass ratio of 5:1 as raw materials, add distilled water at a material-to-liquid ratio of 1:4 to 4 times the mass of the raw materials and mix and pulp. Sterilize in a constant temperature water bath at 73℃ for 30 minutes and then cool to room temperature to obtain jujube and hawthorn pulp. 2) The inoculum of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus acidophilus were weighed at volume percentages of 0.05%, 0.05%, and 0.06%, respectively, and dissolved in a 5% glucose solution. After activation at 37°C for 30 minutes, the solution was inoculated into the jujube and hawthorn pulp for anaerobic fermentation. Fermentation was carried out at 33°C for 36 hours to obtain a fruit pulp fermented with lactic acid bacteria for 36 hours. 3) Weigh out BV818 bacterial powder at a volume percentage of 0.02% and dissolve it in a glucose solution at a mass percentage of 5%. After activation at 37°C for 30 minutes, inoculate it into the fruit pulp fermented by the lactic acid bacteria for 36 hours for aerobic fermentation. Continue fermentation at 28°C for 12 hours, centrifuge and collect the supernatant to obtain jujube and hawthorn enzyme.

[0015] This invention relates to a high-antioxidant-activity jujube and hawthorn enzyme, using jujube and hawthorn as the main raw materials. After scientific compounding, a selected blend of probiotics is inoculated and the enzyme undergoes biotransformation in a controlled fermentation tank at a constant temperature of 33°C for 36 hours followed by a constant temperature of 28°C for 12 hours. This significantly shortens the fermentation cycle compared to traditional natural fermentation. Multiple rounds of laboratory optimization determined the optimal sugar-acid ratio, bacterial age, and inoculum size, resulting in a total phenol and flavonoid retention rate ≥92%, free radical scavenging activity ≥95%, and antioxidant activity significantly superior to commercially available enzymes. The resulting enzyme is ruby ​​red, with a pleasantly sweet and sour taste, a harmonious blend of jujube and hawthorn aromas, and no bitter aftertaste. It is also rich in short-chain fatty acids, active peptides, and organic acids. This invention features a standardized and reproducible process, suitable for continuous industrial production, providing a highly efficient, green, and controllable new technological pathway for functional fruit and vegetable enzymes.

[0016] The beneficial effects of this invention are as follows: 1. This invention adopts a two-stage sequential fermentation, first anaerobic fermentation with lactic acid bacteria for 36 h, and then aerobic fermentation with yeast for 12 h. The roles of the strains are clear, and the metabolic windows of the two types of strains are staggered. 2. Under the temperature gradient fermentation of 33 ℃ / 28 ℃, the finished product can be obtained in 48 hours. Compared with the long natural fermentation of traditional enzymes, the fermentation time has been greatly shortened. 3. The formula contains zero exogenous sugar and zero additives, using only the reducing sugar of jujubes as the carbon source. The yeast is lightly fermented, and the alcohol content at the end of fermentation is <0.5% (v / v), avoiding an overpowering alcohol flavor that masks the fruit aroma. 4. All process parameters are completed within food-grade containers, node parameters are determined, production scale can be linearly scaled up, significantly reducing industrial conversion risks. Attached Figure Description

[0017] Figure 1 This is the optimized process for the jujube and hawthorn enzyme in Example 1 of the present invention.

[0018] Figure 2The growth dynamic curves of eight lactic acid bacteria in jujube and hawthorn juice over 36 hours are shown in Example 2 of this invention.

[0019] Figure 3 This is a dynamic change curve of the total phenolic content and total flavonoid content of eight lactic acid bacteria in jujube and hawthorn pulp over 36 hours in Example 1 of this invention. Figure 3 A represents the total phenol content. Figure 3 B represents the total flavonoid content.

[0020] Figure 4 The results of the sequential gradient locking experiment for Lactobacillus plantarum inoculation amount in Example 1 of the present invention are shown.

[0021] Figure 5 This is the result of the sequential gradient locking experiment of Lactobacillus acidophilus inoculation amount in Example 1 of the present invention.

[0022] Figure 6 This is the result of the sequential gradient locking test of the inoculum amount of Streptococcus thermophilus in Example 1 of the present invention.

[0023] Figure 7 This invention illustrates the effect of the raw material compounding ratio on the active ingredients and antioxidant properties of lactic acid bacteria fermentation products in Example 1.

[0024] Figure 8 This illustrates the effect of fermentation temperature on the active components and antioxidant properties of lactic acid bacteria fermentation products in Example 1 of the present invention.

[0025] Figure 9 This describes the effect of fermentation time on the active ingredients and antioxidant properties of lactic acid bacteria fermentation products in Example 1 of the present invention.

[0026] Figure 10 This describes the effect of the material-to-liquid ratio on the active ingredients and antioxidant properties of lactic acid bacteria fermentation products in Example 1 of the present invention.

[0027] Figure 11 The three-dimensional response surface plot (AC) and corresponding contour plot (ac) in Embodiment 1 of the present invention illustrate the influence of the interaction of various factors on the total phenol content. Factors: raw material compound ratio and fermentation time (A, a), raw material compound ratio and material-liquid ratio (B, b), fermentation time and material-liquid ratio (C, c).

[0028] Figure 12 The three-dimensional response surface plot (AC) and corresponding contour plot (ac) in Embodiment 1 of the present invention illustrate the influence of the interaction of various factors on the total flavonoid content. Factors: raw material compound ratio and fermentation temperature (A, a), raw material compound ratio and fermentation time (B, b), fermentation temperature and fermentation time (C, c).

[0029] Figure 13 The changes in total flavonoid content during the fermentation process of different yeasts in Example 1 of the present invention are shown.

[0030] Figure 14 This illustrates the changes in total phenol content during the fermentation process of different yeast strains in Example 1 of the present invention.

[0031] Figure 15 This refers to the changes in DPPH free radical scavenging activity during the fermentation process of different yeast strains in Example 1 of the present invention.

[0032] Figure 16 This describes the changes in SOD activity during the fermentation process of different yeast strains in Example 1 of the present invention.

[0033] Figure 17 Sensory scores were obtained for different yeasts during fermentation in Example 1 of this invention.

[0034] Figure 18 This describes the effect of fermentation temperature on the active ingredients, antioxidant activity, and sensory scores in Example 1 of the present invention. Figure 18 A represents the active ingredient and antioxidant activity. Figure 18 B represents the sensory rating.

[0035] Figure 19 This describes the effect of yeast fermentation time on active ingredients, antioxidant activity, and sensory scores in Example 1 of the present invention. Figure 19 A represents the active ingredient and antioxidant activity. Figure 19 B represents the sensory rating.

[0036] Figure 20 This describes the effect of yeast inoculum amount on active ingredients, antioxidant activity, and sensory score in Example 1 of the present invention. Figure 20 A represents the active ingredient and antioxidant activity. Figure 20 B represents the sensory rating.

[0037] Figure 21 The three-dimensional response surface plot (AC) and corresponding contour plot (ac) in Embodiment 1 of the present invention illustrate the influence of the interaction of various factors on total phenols. Factors: inoculum size and fermentation time (A, a), inoculum size and fermentation temperature (B, b), fermentation time and fermentation temperature (C, c).

[0038] Figure 22 The three-dimensional response surface plot (AC) and corresponding contour plot (ac) in Embodiment 1 of the present invention illustrate the influence of the interaction of various factors on the DPPH free radical scavenging activity. Factors: inoculum size and fermentation time (A, a), inoculum size and fermentation temperature (B, b).

[0039] Figure 23The three-dimensional response surface plot (AC) and corresponding contour plot (ac) in Embodiment 1 of the present invention illustrate the influence of the interaction of various factors on SOD activity. Factors: inoculum size and fermentation time (A, a), inoculum size and fermentation temperature (B, b), fermentation time and fermentation temperature (C, c).

[0040] Figure 24 The three-dimensional response surface plot (AC) and corresponding contour plot (ac) in Embodiment 1 of the present invention illustrate the influence of the interaction of various factors on sensory scores. Factors: inoculum size and fermentation time (A, a), inoculum size and fermentation temperature (B, b), fermentation time and fermentation temperature (C, c). Figure 25 This is a flowchart of the production process of jujube and hawthorn enzyme in Embodiment 2 of the present invention. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0044] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0045] Example 1 like Figure 1 As shown, the applicant adopted a fermentation process of first inoculating with lactic acid bacteria and then with yeast. Taking the selection of fermentation strains as the starting point for optimization, the applicant conducted single-factor and response surface experiments to optimize the process of the two inoculation stages of lactic acid bacteria and yeast in sequence, and detected relevant antioxidant and bioactive component indicators.

[0046] Experimental materials: Jujubes were purchased from Aksu, Xinjiang, China, and hawthorn slices were purchased from the local market in Shihezi, Xinjiang, China.

[0047] Test strains: Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus thermophilus were purchased from Xianong Biotechnology (Shanghai, China) Co., Ltd., and active dry wine yeast (BV818) was purchased from Angel Yeast Co., Ltd. (Hubei, China).

[0048] 1. Lactic acid bacteria fermentation stage 1.1 Determination of Lactic Acid Bacteria Strains for Fermentation Objective: To screen three types of commercial lactic acid bacteria (see Table 1 for specific manufacturers and product codes) for use in fermenting jujube and hawthorn enzymes.

[0049] Table 1 Information on Eight Types of Lactic Acid Bacteria Products

[0050] Methods: The strains were screened and determined based on their growth curves and total phenol and total flavonoid curves within 36 hours in jujube and hawthorn pulp (the material-to-liquid ratio and raw material compounding ratio were tentatively set at 1:4 and 1:4).

[0051] Specifically, the dates are pitted in advance, and the dates and hawthorns are washed and mixed in a mass ratio of hawthorn:date = 1:4. Distilled water is added in a material-liquid ratio of 1:4 and the mixture is then pulped. The resulting pulp is sterilized in a constant temperature water bath at 73℃ for 30 minutes and then cooled to room temperature to obtain date and hawthorn pulp.

[0052] Eight commercial lactic acid bacteria were inoculated at a volume ratio of 0.03% in a fermenter with a volume of 500 mL, an initial pH of 5.3, and a fermentation temperature of 37°C for anaerobic fermentation.

[0053] Three replicates were set up to measure the growth curve and total phenol and total flavonoid curve within 36 hours.

[0054] See results Figure 2 and Figure 3 Based on the growth curves and total phenol and flavonoid curves within 36 hours, three lactic acid bacteria strains—Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus thermophilus—were selected as the optimal fermentation lactic acid bacteria strains.

[0055] 1.2 Optimization of inoculum amounts for three types of lactic acid bacteria Objective: To determine the optimal inoculation percentage of three lactic acid bacteria in jujube and hawthorn pulp.

[0056] Methods: For each type of lactic acid bacteria, the inoculation amounts of the other two types of lactic acid bacteria were kept constant. The changes in total phenols, total flavonoids, and DPPH free radical scavenging activities with inoculation amount at a fixed fermentation time and temperature were used to determine the optimal inoculation amount.

[0057] Specifically, using the jujube and hawthorn pulp from section 1.1 as the material, the inoculation amounts of three lactic acid bacteria—Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus thermophilus—were determined as follows: 1) The inoculum amounts of *Lactobacillus plantarum* were set at 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, and 0.09%, respectively, while the inoculum amounts of *Lactobacillus acidophilus* and *Streptococcus thermophilus* were fixed at 0.01%. The fermenter volume was 500 mL, the initial pH was 5.3, and the fermentation temperature was 37℃. Anaerobic fermentation was carried out, and three replicates were set up.

[0058] See results Figure 4 This indicates that the fermentation effect is optimal when the inoculum amount of Lactobacillus plantarum is 0.05% by volume.

[0059] 2) The inoculum amounts of Lactobacillus acidophilus were set at 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, and 0.09%, respectively, while the inoculum amounts of fixed Lactobacillus plantarum and Streptococcus thermophilus were 0.01%. The fermentation tank volume was 500 mL, the initial pH was 5.3, and the fermentation temperature was 37℃. Anaerobic fermentation was carried out, and 3 replicates were set up.

[0060] See results Figure 5 This indicates that the fermentation effect is optimal when the inoculum amount of Lactobacillus acidophilus is 0.06% by volume.

[0061] 3) The inoculum amounts of Streptococcus thermophilus were set at 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, and 0.09%, respectively; the inoculum amounts of Lactobacillus plantarum and Lactobacillus acidophilus were fixed at 0.01% and 0.01%, respectively; the fermentation tank volume was 500 mL; the initial pH was 5.3; the fermentation temperature was 37℃; and anaerobic fermentation was carried out with 3 replicates.

[0062] See results Figure 6 This indicates that the fermentation effect is optimal when the inoculum amount of Streptococcus thermophilus is 0.05% by volume.

[0063] Therefore, the inoculation amounts of Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus thermophilus were determined to be 0.05%, 0.06%, and 0.05% (volume percentage), respectively.

[0064] 1.3 Optimization of fermentation process during lactic acid bacteria inoculation stage Objective: To determine the remaining optimal process parameters for the lactic acid bacteria inoculation and fermentation stage based on the optimal fermentation strain and inoculation amount.

[0065] Methods: Using total phenolic and total flavonoid content and DPPH free radical scavenging rate as evaluation indicators, single-factor experiments were first conducted to investigate five factors in sequence: the ratio of jujube and hawthorn, fermentation temperature, fermentation time, material-to-liquid ratio, and initial pH. By analyzing the influence trends of changes in the levels of each factor on the evaluation indicators, the optimal level ranges for each factor were preliminarily determined.

[0066] Based on the results of single-factor experiments, a four-factor, three-level Box-Behnken design was used for response surface methodology optimization. The four factors—the ratio of jujube to hawthorn (A), fermentation temperature (B), fermentation time (C), and the solid-liquid ratio (D)—were used as independent variables. Based on the single-factor results, three levels—low (-1), medium (0), and high (+1)—were set for each factor. The response values ​​(dependent variables) were total phenolic content, total flavonoid content, and DPPH free radical scavenging rate. This design required a total of N=29 experiments (including 24 factorial sites and 5 central point replicates) to establish a quadratic polynomial regression model between each response value and the factors, analyze the interactions between factors, and ultimately predict and verify the optimal parameter combination for the fermentation process through model analysis.

[0067] The specific single-factor experiments were as follows: Fermentation was carried out at the optimal inoculum size of lactic acid bacteria. Under the conditions of fermentation temperature of 37℃, fermentation time of 36h, and material-to-liquid ratio of 1:4, the raw material compound ratio (hawthorn:jujube, w:w) was set to 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7. The volume of the fermentation tank was 500mL, and the initial pH was 5.3. The optimal raw material compound ratio was determined using TPC, TFC, and DPPH free radical scavenging activities as optimization indicators. The results are shown below. Figure 7 Preliminary results indicate that the fermentation effect is best when the raw material compound ratio is 1:4 by mass.

[0068] Single-factor experiment on fermentation temperature: Fermentation was conducted with the optimal inoculum size of lactic acid bacteria. Under the conditions of a raw material ratio of 1:4, a fermentation time of 36 h, and a material-to-liquid ratio of 1:4, the fermentation temperatures were set at 21℃, 25℃, 29℃, 33℃, 37℃, and 41℃. The fermenter volume was 500 mL, and the initial pH was 5.3. The optimal fermentation temperature was determined using TPC, TFC, and DPPH free radical scavenging activities as optimization indicators. Results are shown below. Figure 8 Preliminary results indicate that the fermentation effect is best when the fermentation temperature is 33℃.

[0069] Single-factor experiment on fermentation time: Fermentation was conducted with the optimal inoculum size of lactic acid bacteria. Under the conditions of a raw material ratio of 1:4, a fermentation temperature of 33℃, and a material-to-liquid ratio of 1:4, fermentation times were set at 24h, 28h, 32h, 36h, 40h, and 44h. The fermentation tank volume was 500mL, and the initial pH was 5.3. The optimal fermentation time was determined using TPC, TFC, and DPPH free radical scavenging activities as optimization indicators. Results are shown below. Figure 9 Preliminary results indicate that the fermentation effect is best when the fermentation time is 36 hours.

[0070] Single-factor experiment on the material-to-liquid ratio: Fermentation was carried out with the optimal inoculum size of lactic acid bacteria. Under the conditions of a raw material ratio (hawthorn:jujube) of 1:4, a fermentation temperature of 33℃, and a fermentation time of 36h, the material-to-liquid ratios were set to 1:4, 1:5, 1:6, 1:7, 1:8, and 1:9. The fermentation tank volume was 500mL, and the initial pH was 5.3. The optimal material-to-liquid ratio was determined using TPC, TFC, and DPPH free radical scavenging activities as optimization indicators. The results are shown below. Figure 10 Preliminary results indicate that the fermentation effect is best when the material-to-liquid ratio is 1:5.

[0071] Based on the results of the single-factor experiments above, three levels were set for the ratio of jujube to hawthorn: 1:3 (-1), 1:4 (0), and 1:5 (+1); three levels for fermentation temperature: 29℃ (-1), 33℃ (0), and 37℃ (+1); three levels for fermentation time: 32h (-1), 36h (0), and 40h (+1); and three levels for material-to-liquid ratio: 1:4 (-1), 1:5 (0), and 1:6 (+1). A Box-Behnken principle-based four-factor, three-level response surface methodology was used to optimize the fermentation conditions during the lactic acid bacteria inoculation stage. The fermenter volume was 500mL, with three replicates, and anaerobic fermentation was performed. Specific experimental designs and results are shown in Tables 2, 3, and 4. Figure 11 , Figure 12 .

[0072] Table 2 Response Surface Experimental Design and Results

[0073] Table 3. Analysis of variance of the regression model for total phenol content

[0074] Table 4. Analysis of variance of regression model for total flavonoid content

[0075] Therefore, the optimal process for the lactic acid bacteria inoculation stage was determined to be a material-to-liquid ratio of 1:4 (volume percentage) and a compound ratio of jujube and hawthorn of 5:1 (mass ratio), followed by constant temperature anaerobic fermentation at 33℃ for 36 hours.

[0076] 2. Yeast fermentation stage Objective: Based on the optimal process of lactic acid bacteria fermentation, to select one yeast strain from three commercial yeast strains (product information is shown in Table 5) for fermenting jujube and hawthorn enzyme.

[0077] Table 5 Information on three yeast products

[0078] Methods: The selection of strains was based on a comprehensive screening of SOD enzyme activity, sensory evaluation, total phenols, and total flavonoids within 12 hours in lactic acid bacteria fermented jujube and hawthorn pulp (based on the optimal lactic acid bacteria fermentation process in 1).

[0079] Specifically, the optimal process in step 1 was used to ferment jujube and hawthorn pulp with lactic acid bacteria. Three commercial yeast strains were inoculated at a volume percentage of 0.03%. The fermentation tank had a volume of 500 mL, an initial pH of 4.5, and a fermentation temperature of 25°C for aerobic fermentation.

[0080] Set 3 repetitions.

[0081] See results Figures 13-17 Based on comprehensive evaluation, wine active dry yeast BV8181 was determined to be the best fermentation yeast strain.

[0082] 2.2 Optimization of fermentation process during yeast inoculation stage Objective: Based on the optimal yeast strain for fermentation, determine the remaining optimal process parameters for the yeast inoculation and fermentation stage, and determine the final processing technology for jujube and hawthorn enzyme.

[0083] Methods: This study optimized the yeast fermentation process using key evaluation indicators such as total phenol content, total flavonoid content, DPPH free radical scavenging rate, SOD activity, and sensory evaluation. First, single-factor experiments were conducted to examine fermentation temperature, fermentation time, and yeast inoculum size, analyzing the influence trends of each factor's level changes on the evaluation indicators to preliminarily determine the optimal value range for each factor.

[0084] Based on the results of single-factor experiments, a three-factor, three-level Box-Behnken design was used for response surface methodology optimization. Fermentation temperature (A), fermentation time (B), and yeast inoculum size (C) were used as independent variables. Based on the single-factor results, three levels—low (-1), medium (0), and high (+1)—were set for each factor. The response values ​​(dependent variables) were total phenol content, SOD activity, sensory score, and DPPH free radical scavenging rate. This design required a total of N=17 experiments (including 12 factorial and 5 central point replicates) to establish a quadratic polynomial regression model between each response value and the factors, analyze the main effects and interactions among the factors, and predict and verify the optimal combination of process parameters for the yeast fermentation stage through model analysis.

[0085] The specific single-factor experiment is as follows: Single-factor experiment on fermentation temperature: Based on the optimal process of lactic acid bacteria inoculation and fermentation, fermentation was carried out at 19℃, 22℃, 25℃, 28℃, and 31℃ under the conditions of 0.02% yeast inoculation and 24h fermentation time. The fermentation tank volume was 500mL, and the initial pH was 4.5. The optimal fermentation temperature was determined using TPC, TFC, DPPH free radical scavenging activity, SOD activity, and sensory evaluation as optimization indicators. Results are shown below. Figure 18 Preliminary results indicate that the fermentation effect is best when the fermentation temperature is 28℃.

[0086] Single-factor experiment on fermentation time: Based on the optimal process of lactic acid bacteria inoculation and fermentation, fermentation was carried out at a yeast inoculation rate of 0.02% and a fermentation temperature of 25℃. Fermentation times were set at 0h, 4h, 8h, 12h, 16h, 20h, and 24h. The fermenter volume was 500mL, and the initial pH was 4.5. The optimal fermentation time was determined using TPC, TFC, DPPH free radical scavenging activity, SOD activity, and sensory evaluation as optimization indicators. Results are shown below. Figure 19 Preliminary results indicate that the fermentation effect is best when the fermentation time is 12 hours.

[0087] Single-factor experiment on yeast inoculum size: Based on the optimal fermentation process of lactic acid bacteria inoculation, fermentation was carried out at a fermentation temperature of 25℃ and a fermentation time of 24h. The yeast inoculum size was set at 0.01%, 0.015%, 0.02%, 0.025%, and 0.03%, the fermenter volume was 500mL, and the initial pH was 4.5. The optimal yeast inoculum size was determined using TPC, TFC, DPPH free radical scavenging activity, SOD activity, and sensory evaluation as optimization indicators. The results are shown below. Figure 20 Preliminary results indicate that the fermentation effect is optimal when the yeast inoculum is 0.02%.

[0088] Based on the results of the single-factor experiments above, three fermentation temperature levels were set: 25℃ (-1), 28℃ (0), and 31℃ (+1); three fermentation time levels were set: 8h (-1), 12h (0), and 16h (+1); and three yeast inoculum levels were set: 0.015% (-1), 0.02% (0), and 0.025% (+1). A three-factor, three-level response surface methodology was used to optimize the fermentation conditions during the yeast inoculation stage. The fermenter volume was 500mL, and 33 replicates were set up for aerobic fermentation. The experimental design and results are shown in Tables 6-10. Figures 21-24 .

[0089] Table 6 Response Surface Experimental Design and Results

[0090] Table 7. Analysis of Variance of Regression Model for Total Phenolic Content

[0091] Table 8. Analysis of variance of the regression model for DPPH free radical scavenging activity

[0092] Table 9. Analysis of Variance for the SOD Activity Regression Model

[0093] Table 10. Analysis of Variance for Sensory Rating Regression Model

[0094] Results: The optimal fermentation process for the yeast inoculation stage was determined to be 0.02% BV818 by volume, with constant temperature aerobic fermentation at 28℃ for 12 hours.

[0095] Example 2 Based on the optimization results of Example 1, the production process flow for jujube and hawthorn enzyme was set up, see [link / reference]. Figure 25 : 1) Remove the pits from the dates in advance. Take dates and dried hawthorn slices in a mass ratio of 5:1, wash them repeatedly with distilled water, add distilled water at a material-liquid ratio of 1:4 (four times the mass of the raw materials) and mix and pulp. Sterilize the resulting pulp in a constant temperature water bath at 73℃ for 30 minutes, and cool it to room temperature to obtain jujube and hawthorn pulp. 2) Weigh out *Lactobacillus plantarum*, *Streptococcus thermophilus*, and *Lactobacillus acidophilus* at inoculation rates of 0.05%, 0.05%, and 0.06% by volume, respectively. Add the inoculation powder to a 5% glucose solution (mass percentage) (starting at 5 mL) and shake thoroughly under aseptic conditions to obtain an unactivated lactic acid bacteria solution. Activate the unactivated lactic acid bacteria solution in a 37°C water bath for 30 minutes before inoculation. Perform water-sealed anaerobic fermentation on jujube and hawthorn pulp in a 500 mL fermentation tank at 33°C for 36 hours to obtain a fruit pulp fermented with lactic acid bacteria for 36 hours. 3) Weigh out BV818 yeast powder at a volume percentage of 0.02%, add it to a 5% glucose solution (starting from 5 mL), and shake well to obtain an unactivated yeast culture. Activate the unactivated yeast culture in a 37℃ constant temperature water bath for 30 minutes, then inoculate it into fruit pulp fermented with lactic acid bacteria for 36 hours. Switch the fermentation conditions to aerobic fermentation and continue fermentation at 28℃ for 12 hours. After fermentation, centrifuge the entire sample and collect the supernatant to obtain jujube and hawthorn enzyme.

[0096] The obtained jujube and hawthorn enzyme exhibited a total phenol and flavonoid retention rate of ≥92%, a free radical scavenging activity of ≥95%, and antioxidant activity significantly superior to commercially available similar enzymes. The final alcohol content at the fermentation endpoint was <0.5% (v / v). The resulting jujube and hawthorn enzyme was ruby ​​red in color, had a pleasantly sweet and sour taste, a harmonious blend of jujube and hawthorn aromas, and no bitter aftertaste.

[0097] This invention employs a two-stage sequential fermentation process: first, anaerobic fermentation with lactic acid bacteria for 36 hours, followed by aerobic fermentation with yeast for 12 hours. The roles of each microorganism are clearly defined, and their metabolic windows are staggered. Under a temperature gradient controlled fermentation at 33℃ / 28℃, the finished product can be obtained in 48 hours, significantly shortening the fermentation time compared to the long natural fermentation of traditional enzymes. The formula of this invention contains zero exogenous sugar and zero additives, using only the reducing sugars from the jujube itself as the carbon source. Yeast fermentation is light, and the final alcohol content is <0.5% (v / v), avoiding an overpowering alcohol flavor that masks the fruit aroma. All process parameters are completed in food-grade containers, and the node parameters are predetermined, allowing for linear scaling up of production and significantly reducing the risks associated with industrial conversion.

[0098] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing jujube and hawthorn enzyme through a two-stage fermentation process using multiple strains, characterized in that: The method includes the steps of anaerobic fermentation of jujube and hawthorn pulp with lactic acid bacteria, followed by aerobic fermentation with yeast, centrifugation after fermentation to obtain jujube and hawthorn enzyme.

2. The method according to claim 1, characterized in that: The lactic acid bacteria are three types: Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus thermophilus.

3. The method according to claim 1, characterized in that: The yeast strain is yeast BV818.

4. The method according to claim 1, characterized in that: The jujube and hawthorn pulp is made by removing the pits from the jujubes, taking dried jujubes and hawthorn slices at a mass ratio of 5:1 as raw materials, adding four times the mass of water to the raw materials, mixing and pulping, and then sterilizing at 73°C for 30 minutes.

5. The method according to claim 2, characterized in that: In the anaerobic fermentation, the inoculum amount of *Lactobacillus plantarum* is 0.05% by volume, the inoculum amount of *Lactobacillus acidophilus* is 0.06% by volume, and the inoculum amount of *Streptococcus thermophilus* is 0.05% by volume.

6. The method according to claim 5, characterized in that: The anaerobic fermentation was carried out at 33°C for 36 hours.

7. The method according to claim 3, characterized in that: In the aerobic fermentation, the inoculum amount of yeast is 0.02% by volume.

8. The method according to claim 7, characterized in that: The aerobic fermentation is carried out at 28°C for 12 hours.

9. The method according to any one of claims 1-8, characterized in that: The specific steps of the method are as follows: 1) Remove the pits from the dates in advance, take the dates and dried hawthorn slices in a mass ratio of 5:1 as raw materials, add distilled water at a material-to-liquid ratio of 1:4 to 4 times the mass of the raw materials and mix and pulp. Sterilize in a constant temperature water bath at 73℃ for 30 minutes and then cool to room temperature to obtain jujube and hawthorn pulp. 2) The inoculum of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus acidophilus were weighed at volume percentages of 0.05%, 0.05%, and 0.06%, respectively, and dissolved in a 5% glucose solution. After activation at 37°C for 30 minutes, the solution was inoculated into the jujube and hawthorn pulp for anaerobic fermentation. Fermentation was carried out at 33°C for 36 hours to obtain a fruit pulp fermented with lactic acid bacteria for 36 hours. 3) Weigh out BV818 bacterial powder at a volume percentage of 0.02% and dissolve it in a glucose solution at a mass percentage of 5%. After activation at 37°C for 30 minutes, inoculate it into the fruit pulp fermented by the lactic acid bacteria for 36 hours for aerobic fermentation. Continue fermentation at 28°C for 12 hours, centrifuge and collect the supernatant to obtain jujube and hawthorn enzyme.

10. The application of the method for preparing jujube and hawthorn enzyme by two-stage fermentation of multiple strains as described in any one of claims 1-9 in food processing.

Images