Process for the preparation of a fermented must of the wine nag grape
By screening efficient lactic acid bacteria strains and optimizing the fermentation process, the problems of insufficient strain screening and process parameters in the processing of Munage grapes have been solved, resulting in the production of high-quality fermented Munage grape juice, which enhances the nutritional and flavor characteristics of the product.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG UNIVERSITY
- Filing Date
- 2026-03-10
- Publication Date
- 2026-05-29
AI Technical Summary
Existing lactic acid bacteria fermentation technology for processing Munage grapes suffers from problems such as insufficient targeted strain selection, inadequate optimization of fermentation process parameters, and insufficient preservation of product flavor and nutrients, making it difficult to produce high-quality functional fermented beverages.
A combination of lactic acid bacteria strains that produce high levels of extracellular polysaccharides and also possess antioxidant and cholesterol-lowering functions was used. Fermentation process parameters were optimized using single-factor experiments and response surface methodology. The preparation method included pulp enzymatic hydrolysis, fermentation, and sterilization steps to ensure product stability and efficient production.
It significantly increased the yield of extracellular polysaccharides and antioxidant capacity, enhanced the nutritional value and flavor of Munage grape juice, ensured product quality stability, and realized the deep processing advantages of Munage grapes.
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Figure CN122096360A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial engineering and fermentation, and particularly relates to a method for preparing fermented Munage grape juice. Background Technology
[0002] Munage grapes ( Vitis vinifera L.cv.Munage Munage grapes are a high-quality, late-maturing fresh-eating variety with plump berries, a sweet taste, and are rich in glucose, fructose, phenolic compounds, vitamins, and other nutrients and bioactive substances, possessing extremely high edible and medicinal value. However, the skin of Munage grapes is relatively thin, making them highly susceptible to pathogens after harvest, leading to dehydration, browning, and spoilage. Furthermore, their ripening period is concentrated in August to October, limiting cold chain storage and transportation capacity and processing capacity, often resulting in seasonal and regional oversupply, severely impacting industry development and economic benefits. Currently, research on Munage grapes focuses primarily on disease control and preservation techniques, with limited research on deep processing. Existing grape processed products mainly consist of wine and raisins, with few varieties of grape juice beverages, and a lack of high-value-added products that combine nutritional value, probiotic functions, and excellent flavor.
[0003] Lactic acid bacteria fermentation technology is a new technology in fruit and vegetable beverage processing. It can maintain and improve the safety, nutritional value, taste, and shelf life of fruit and vegetable products, and has become a new trend in the production of plant-based functional beverages. The extracellular polysaccharides produced by lactic acid bacteria possess antioxidant, cholesterol-lowering, and antibacterial activities, which can enhance the functional properties of products. However, existing lactic acid bacteria fermentation technology for fruit and vegetable juices, when applied to Munage grapes, suffers from problems such as insufficient specificity in strain selection, inadequate optimization of fermentation process parameters, unreasonable flavor blending, and poor storage stability. This makes it difficult to fully utilize the resource advantages of Munage grapes and produce high-quality functional fermented beverages. Further research is needed. Summary of the Invention
[0004] In view of this, the purpose of this invention is to address the problems of high post-harvest loss, difficult storage and transportation, lack of deep-processed products, and the limited functionality, flavor, and nutritional retention of existing grape processed products. The invention provides a method for preparing Munage grape juice fermented with high-yield extracellular polysaccharide lactic acid bacteria, which can fully retain the nutritional components of Munage grapes, while endowing the product with probiotic functions and excellent flavor, and with a stable and controllable production process.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing fermented Munage grape juice, comprising the following steps: 1) Pulp the Munage grapes to obtain fruit pulp; 2) Mix the fruit pulp with the compound enzyme, enzymatically hydrolyze at 40-50℃ for 3-5 hours, then centrifuge and sterilize to obtain supernatant 1; 3) Mix the compound bacterial agent with supernatant 1, ferment at 35~40℃ for 28~36h, then centrifuge to obtain supernatant 2; 4) Sterilize the supernatant 2 to obtain fermented Munage grape juice; Step 2) The mass ratio of fruit pulp to compound enzyme is 80~120:1; The complex enzyme comprises the following components in the following mass ratio: Pectinase: Cellulase = 1~3:1; Step 3) The mass ratio of the supernatant 1 to the compound bacterial agent is 100:2~3; The bacterial concentration of the compound microbial agent is 10. 8 ~10 10 CFU / mL, comprising the following components in the following mass ratios: Lactobacillus plantarum: Lactobacillus acidophilus = 1~3: 1~3.
[0006] Preferably, the fruit pulp in step 2) is mixed with water before use, and the mass ratio of fruit pulp to water is 2~5:1.
[0007] Preferably, step 2) further includes an enzyme inactivation step after enzymatic hydrolysis, wherein the enzyme inactivation temperature is 70~90℃ and the enzyme inactivation time is 5~15 min.
[0008] Preferably, the centrifugation speed in step 2) or step 3) is 3000~8000 rpm; Centrifugation time is 10-20 min.
[0009] Preferably, the pH of the supernatant 1 in step 2) is 5.5 to 6.5.
[0010] Preferably, the sterilization temperature in step 2) or step 4) is 90~110℃ and the sterilization time is 8~12 min.
[0011] The present invention provides fermented Munage grape juice obtained by the preparation method described above.
[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention, targeting the characteristics and functional requirements of Munage grapes, screens a combination of lactic acid bacteria strains that produce high levels of extracellular polysaccharides (EPS) and possess strong antioxidant and cholesterol-lowering functions. This overcomes the problem of insufficient targeting in existing strain screening technologies, significantly increasing the yield of EPS in the fermented product (EPS content reaches 2618.43 mg / L after fermentation), enhancing antioxidant capacity, and increasing the content of bioactive substances (total phenols and total flavonoids). This fully leverages the synergistic nutritional advantages of Munage grapes and lactic acid bacteria. Furthermore, this invention employs single-factor experiments combined with response surface methodology to systematically investigate the impact of multi-factor interactions on fermentation effects, accurately determining the optimal combination of process parameters. This solves the problem of imprecise process optimization in existing technologies, significantly improving fermentation efficiency and product quality stability, ensuring that the yield of EPS and the number of viable bacteria reach ideal levels. Attached Figure Description
[0013] Figure 1 The figure shows the results of a single-factor experiment for the enzymatic hydrolysis conditions; Figure 2 Figure 1 shows the results of testing extracellular polysaccharide production, antioxidant capacity, and cholesterol-lowering capacity of different bacterial strains. Figure 3 The results of the strain ratio test; Figure 4 Results of single-factor experiments for inoculating the strain; Figure 5 For predicting regression models. Detailed Implementation
[0014] This invention provides a method for preparing fermented Munage grape juice, comprising the following steps: 1) Pulp the Munage grapes to obtain fruit pulp; 2) Mix the fruit pulp with the compound enzyme, enzymatically hydrolyze at 40-50℃ for 3-5 hours, then centrifuge and sterilize to obtain supernatant 1; 3) Mix the compound bacterial agent with supernatant 1, ferment at 35~40℃ for 28~36h, then centrifuge to obtain supernatant 2; 4) Sterilize the supernatant 2 to obtain fermented Munage grape juice; In this invention, Munage grapes are pulped to obtain fruit pulp; the Munage grapes are preferably fresh Munage grapes that are plump and free from mold infection; the pulping method is preferably to put the cleaned Munage grapes into a pulping machine to pulp them, and the sieve mesh size is preferably 80~120 mesh, and more preferably 100 mesh.
[0015] In this invention, fruit pulp is mixed with a compound enzyme, enzymatically hydrolyzed, centrifuged, and sterilized to obtain supernatant 1. The mass ratio of fruit pulp to compound enzyme is preferably 80-120:1, more preferably 90-110:1, and even more preferably 100:1. Before use, the fruit pulp is preferably diluted with water, and the mass ratio of fruit pulp to water is preferably 2-5:1, more preferably 2.5-4:1, and even more preferably 3:1. The compound enzyme preferably comprises components in the following mass ratio: pectinase: cellulase = 1-3:1, more preferably 1.5-2.5:1, and even more preferably 2:1. The enzymatic hydrolysis temperature is 40-50℃, preferably 42-48℃, and even more preferably 45℃. The enzymatic hydrolysis time is 3-5 hours, preferably 3.5-4.5 hours, and even more preferably 4 hours. After enzymatic hydrolysis, an enzyme inactivation step is preferably included, and the enzyme inactivation temperature is preferably 70-90℃, more preferably 75-85℃, and even more preferably 80℃. The enzyme inactivation time is preferably 5-15 minutes. The centrifugation time is preferably 7-12 min, and even more preferably 10 min; the centrifugation speed is preferably 3000-8000 rpm, more preferably 4000-6000 rpm, and even more preferably 5000 rpm; the centrifugation time is preferably 10-20 min, more preferably 12-18 min, and even more preferably 15 min; the sterilization temperature is preferably 90-110℃, more preferably 95-105℃, and even more preferably 100℃; the sterilization time is preferably 8-12 min, more preferably 9-11 min, and even more preferably 10 min; the pH of the supernatant 1 is preferably 5.5-6.5, more preferably 5.7-6.2, and even more preferably 5.9.
[0016] In this invention, the compound microbial agent is mixed with supernatant 1, fermented, and then centrifuged to obtain supernatant 2; the compound microbial agent comprises *Lactobacillus plantarum* and *Lactobacillus acidophilus*, and the mass ratio of *Lactobacillus plantarum* to *Lactobacillus acidophilus* is preferably 1~3:1~3, more preferably 1:1; the bacterial concentration of the compound microbial agent is 10. 8 ~10 10 CFU / mL, preferably 5×10⁻⁶ 8 ~5×10 9 CFU / mL, further preferably 1×10⁻⁶ 9CFU / mL; the mass ratio of the supernatant 1 to the compound bacterial agent is 100:2~3, preferably 100:2.2~2.8, more preferably 100:2.5; the fermentation temperature is 35~40℃, preferably 36~38℃, more preferably 37℃; the fermentation time is 28~36h, preferably 30~34h, more preferably 32h; the centrifugation speed is preferably 3000~8000 rpm, more preferably 4000~6000 rpm, even more preferably 5000 rpm; the centrifugation time is preferably 10~20 min, more preferably 12~18 min, even more preferably 15 min.
[0017] In this invention, the supernatant 2 is sterilized to obtain fermented Munage grape juice; the sterilization temperature is preferably 90~110℃, more preferably 95~105℃, even more preferably 100℃, and the sterilization time is preferably 8~12 min, more preferably 9~11 min, and even more preferably 10 min.
[0018] The present invention provides fermented Munage grape juice obtained by the preparation method described above.
[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0020] Source of materials:
[0021] Example 1
[0022] Enzymatic hydrolysis process optimization
[0023] (1.1) Single-factor experiment
[0024] The enzymatic hydrolysis method was as follows: Fresh, plump Munage grapes free from mold infection were selected, washed, and then pulped in a pulper to form grape pulp (hereinafter referred to as pulp). This pulp was then hydrolyzed using a compound enzyme. The effects of the following factors on the increase rates of total phenolic content and soluble solids content were investigated as single-factor studies: the material-to-liquid ratio (the pulp needs to be diluted with water before use; here, the material-to-liquid ratio refers to the mass of pulp to water), the compound enzyme ratio (mass ratio of pectinase to cellulase), the amount of compound enzyme added, the hydrolysis time, and the hydrolysis temperature. The optimal range of each factor was determined. See the results below. Figure 1 .
[0025] Figure 1 The results show that the best performance is achieved when the material-to-liquid ratio (grape pulp: water) is 4:1, the compound enzyme ratio is 1:1, the enzyme addition is 0.8%, the enzymatic hydrolysis time is 4 hours, and the enzymatic hydrolysis temperature is 45℃.
[0026] To further determine the optimal enzymatic hydrolysis process, orthogonal experiments were conducted for further research.
[0027] The factor level table is shown in Table 1, the factor orthogonal table is shown in Table 2, the orthogonal experimental results of the enzymatic hydrolysis conditions are shown in Table 3, the orthogonal experimental results of the enzymatic hydrolysis conditions are shown in Table 4, and the orthogonal experimental variance analysis is shown in Table 5.
[0028] Table 1 Factor Level Table
[0029] Table 2 Factor Orthogonal Table
[0030] Table 3. Results of the orthogonal experiment for enzymatic hydrolysis conditions
[0031] Table 4. Results of the orthogonal experiment for enzymatic hydrolysis conditions
[0032] Table 5. Variance Analysis of Orthogonal Experiments
[0033] Total phenol content increase rate: R 2 =0.929, soluble solids content increase rate: R 2 =0.963
[0034] Note: , P < 0.05 and P < 0.01, respectively, indicate significant differences.
[0035] Optimal enzymatic hydrolysis conditions: material-to-liquid ratio: 3:1, compound enzyme ratio: 2:1, enzyme addition: 1%, hydrolysis time: 4h, hydrolysis temperature: 45℃.
[0036] Prepare the enzymatic hydrolysis solution using the above-mentioned optimal enzymatic hydrolysis conditions, then inactivate the enzyme (80℃, 10 min), centrifuge (5000 rpm, 15 min), collect the supernatant, sterilize the supernatant (100℃, 10 min), and obtain supernatant 1 for later use.
[0037] Example 2
[0038] (2.1) Strain screening
[0039] Candidate strains: Ten common EPS-producing lactic acid bacteria were selected as candidate strains, including Lactobacillus acidophilus (CICC 6086), Lactobacillus casei (CICC6114), Lactobacillus paracasei (bio-53142), Lactobacillus rhamnosus (CICC6164), Lactobacillus fermentum (CICC25124), Lactobacillus reuteri (CICC6123), Lactobacillus plantarum (CICC 25125), Pediococcus lactis (bio-097553), Bifidobacterium thermophilum (bio-04950), and Pediococcus pentosus (CICC21865).
[0040] Initial screening: Candidate strains were fermented in MRS medium, and EPS yield was determined by phenol-sulfuric acid method to screen out strains with high EPS yield.
[0041] Secondary screening: The strains screened in the primary screening were inoculated into Munage grape juice. The total antioxidant capacity was determined by the FRAP method, and the cholesterol-lowering capacity was determined by the o-phthalaldehyde (OPA) method. The EPS yield, antioxidant capacity and cholesterol-lowering capacity were combined.
[0042] The results of the initial screening and secondary screening are shown in Figure 1 The extracellular polysaccharide production of ten bacterial strains was determined by phenol and sulfuric acid testing, and *Bifidobacterium thermophilum* (SRS) and *Lactobacillus paracasei* (LGL) were screened out. *Lactobacillus rhamnosus* (SLT), *Pediococcus lactis* (RSP), *Pediococcus pentosus* (WT), and *Lactobacillus reuteri* (LYS) were screened out by total antioxidant capacity testing. Finally, *Lactobacillus casei* (GL) was screened out by cholesterol-lowering capacity testing, and *Lactobacillus plantarum* (ZW), *Lactobacillus acidophilus* (SS), and *Lactobacillus fermentum* (FJ) were determined to be the optimal strains.
[0043] (2.2) Strains ratio experiment
[0044] The optimal inoculation combination was determined using extracellular polysaccharide production and viable cell count as indicators. See details for the results. Figure 3 (The mass ratio of the two bacteria is 1:1).
[0045] The results showed that *Lactobacillus plantarum* (ZW) and *Lactobacillus acidophilus* (SS) were the optimal inoculation combination (*Lactobacillus plantarum* (ZW): *Lactobacillus acidophilus* (SS) = 1:1). The physicochemical indicators of the two strains during the fermentation process were monitored, and both strains reached the stable growth period at 32 h.
[0046] (2.3) Single-factor test of strain inoculation
[0047] EPS yield was determined using the phenol-sulfuric acid method. Subsequent experiments were conducted with inoculum levels of 2%, 3%, and 4%, initial pH values of 5, 5.5, and 6, fermentation times of 24 h, 30 h, and 36 h, and fermentation temperatures of 34℃, 37℃, and 40℃. See details for the results. Figure 4 .
[0048] Figure 4 The results showed that the best results were achieved when the inoculum content was 3%, the initial pH was 5.5, the fermentation time was 30 h, and the fermentation temperature was 37 °C. To further determine the optimal fermentation process, response surface methodology was used for further research.
[0049] The results of the response surface methodology experiment are detailed in Table 6. Analysis of variance (ANOVA) of the response surface regression model is also shown. 2 =0.9212, adjust R 2 =0.8425) See Table 7, and the predictive regression model is shown in [Table 7]. Figure 5 .
[0050] Table 6 Results of Response Surface Experiment
[0051] Table 7. Analysis of Variance for Response Surface Regression Model
[0052] The results showed that, based on the predictive regression model, the optimal fermentation conditions for Munage grape juice using lactic acid bacteria were as follows: inoculum concentration of 2.534% (10 9 The initial pH was 5.908, the fermentation temperature was 37.606℃, and the fermentation time was 31.832 h. Under these conditions, the predicted EPS yield of Lactic Acid Bacteria fermented Munage grape juice was 1314.26 mg / L. Considering the practicality of the experiment, the inoculum concentration was set at 2.5% (10 CFU / mL). 9 The initial pH was set to 5.9, the fermentation time to 32 h, and the fermentation temperature to 37 °C. The supernatant 1 obtained from Example 1 was fermented using the final determined actual fermentation parameters. After fermentation, the fermentation broth was centrifuged at 5000 rpm for 15 min, the supernatant was collected, and bacterial cells and impurities were removed.
[0053] Sterilization of finished product: Boil the supernatant at 100℃ for 10 minutes to sterilize, then cool to room temperature to obtain the finished product.
[0054] The results of comparing the finished product with the supernatant 1 prepared in Example 1 are detailed in Table 8.
[0055] Table 8. Physicochemical Indicators Detection
[0056] Table 8 shows that the titratable acid content of the fermentation broth increased significantly, while the pH value and soluble solids content decreased. The content of extracellular polysaccharides and total flavonoids increased significantly, but the total phenol content did not change significantly. In addition, single-strain fermentation did not significantly improve the total antioxidant capacity of Munage grape juice (the highest was only 3.45%), but mixed-strain fermentation improved it by 20.21%.
[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing fermented Munage grape juice, characterized in that, Includes the following steps: 1) Pulp the Munage grapes to obtain fruit pulp; 2) Mix the fruit pulp with the compound enzyme, enzymatically hydrolyze at 40-50℃ for 3-5 hours, then centrifuge and sterilize to obtain supernatant 1; 3) Mix the compound bacterial agent with supernatant 1, ferment at 35~40℃ for 28~36h, then centrifuge to obtain supernatant 2; 4) Sterilize the supernatant 2 to obtain fermented Munage grape juice; Step 2) The mass ratio of fruit pulp to compound enzyme is 80~120:1; The complex enzyme comprises the following components in the following mass ratio: Pectinase: Cellulase = 1~3:1; Step 3) The mass ratio of the supernatant 1 to the compound bacterial agent is 100:2~3; The bacterial concentration of the compound microbial agent is 10. 8 ~10 10 CFU / mL, comprising the following components in the following mass ratios: Lactobacillus plantarum: Lactobacillus acidophilus = 1~3: 1~3.
2. The preparation method according to claim 1, characterized in that, Step 2) The fruit pulp is mixed with water before use, and the mass ratio of fruit pulp to water is 2~5:
1.
3. The preparation method according to claim 2, characterized in that, Step 2) The enzymatic hydrolysis is followed by an enzyme inactivation step, with the enzyme inactivation temperature being 70~90℃ and the inactivation time being 5~15 min.
4. The preparation method according to claim 1, characterized in that, The centrifugation speed in step 2) or step 3) is 3000~8000 rpm; Centrifugation time is 10-20 min.
5. The preparation method according to claim 1, characterized in that, Step 2) The pH of the supernatant 1 is 5.5~6.
5.
6. The preparation method according to claim 1, characterized in that, The sterilization temperature in step 2) or step 4) is 90~110℃, and the sterilization time is 8~12 min.
7. Fermented Munage grape juice obtained by the preparation method according to any one of claims 1 to 6.