Bryopsis taxifolia juice with high sod activity, bryopsis taxifolia juice probiotic fermentation product and preparation method thereof

Abstract

This invention discloses a high SOD activity *Gnaphalium affine* juice, a *Gnaphalium affine* juice probiotic fermented product, and its preparation method. Using *Gnaphalium affine* as raw material, this invention obtains a high SOD activity *Gnaphalium affine* juice through a combination of repeated low-temperature treatment, pulping, ultrasonic crushing pretreatment, and heat treatment. Fermentation is carried out using *Lactobacillus mucilaginosus* Grx07, with the addition of a commercial fermenting agent to regulate the fermentation process. This achieves a synergistic effect between probiotics and *Gnaphalium affine* juice in terms of SOD activity, improving the SOD activity, viable cell count, and sensory quality of the fermented product. It has broad application prospects in the preparation of probiotic fermented foods with high antioxidant activity.

Description

Technical Field

[0001] This invention belongs to the technical field of probiotic fermentation products, specifically relating to a rice juice with high SOD activity, a rice juice probiotic fermentation product, and its preparation method. Background Technology

[0002] Nostoc sphaeroides, belonging to the phylum Cyanobacteria, family Nostocaceae, and genus Nostoc, is a type of edible cyanobacteria with high nutritional and functional development value. It is rich in polysaccharides, proteins, phycobiliproteins, amino acids, and minerals, exhibiting strong antioxidant, immunomodulatory, and metabolic regulatory potential. Superoxide dismutase (SOD) is a particularly important activity indicator in the study of Nostoc sphaeroides' functional properties. SOD can catalyze the dismutation reaction of superoxide anion free radicals, generating hydrogen peroxide and oxygen, thereby mitigating the oxidative damage caused by excessive accumulation of reactive oxygen species.

[0003] However, the outer layer of Ge Xian Mi is coated with a relatively dense extracellular matrix of colloidal polysaccharides. Under conventional processing and direct consumption conditions, the release and utilization of its internal active ingredients such as polysaccharides, proteins, and SOD may be limited, which also restricts the high-value utilization of Ge Xian Mi. Summary of the Invention

[0004] The purpose of this invention is to provide a high SOD activity rice juice from *Gnaphalium affine*, a probiotic fermented product from rice juice, and a method for preparing the same. This invention uses *Gnaphalium affine* as raw material, and obtains a rice juice with high SOD activity through a combination of repeated low-temperature treatment, pulping, ultrasonic crushing pretreatment, and heat treatment. Fermentation is carried out using *Limosilactobacillus fermentum* Grx07, with the addition of a commercial starter culture to regulate the fermentation process. This achieves a synergistic effect between the probiotics and the rice juice in terms of SOD activity, improving the SOD activity, viable cell count, and sensory quality of the fermented product.

[0005] The technical solution for achieving the objective of this invention is as follows:

[0006] The preparation method of Ge Xian Mi juice with high SOD activity involves first pretreating Ge Xian Mi using a physical cell wall disruption method combining low-temperature treatment, pulping, and ultrasonic crushing, followed by heat treatment of the pretreated Ge Xian Mi liquid. The specific steps include:

[0007] (1) Pretreatment: Fresh Nostoc commune is first treated at low temperature in an ice bath at 0-4℃ for 30-40 min. Then, deionized water is added at 1 / 2 of the mass of Nostoc commune, and the mixture is pulped for 2-5 min. The resulting Nostoc commune pulp is then placed in an ultrasonic cell disruptor and ultrasonically disrupted in an ice bath for 25-30 min. The above process of low temperature treatment-pulping-ultrasonic disruption is repeated twice to obtain Nostoc commune pretreatment solution.

[0008] (2) Heat treatment: The pretreated liquid of Ge Xian Mi was sterilized at 115℃ for 15 minutes to obtain Ge Xian Mi juice with high SOD activity.

[0009] This invention provides a rice juice with high SOD activity prepared by the above preparation method.

[0010] This invention provides a probiotic-fermented rice juice made from *Lactobacillus mucinus* Grx07, which is the above-mentioned rice juice fermented by *Lactobacillus mucinus* Grx07, with the preservation number of *Lactobacillus mucinus* Grx07 being CGMCC NO.8874.

[0011] Furthermore, the above-mentioned probiotic-fermented Ge Xian rice juice also contained 0.025 g / L of commercial starter culture MO 094 BR.

[0012] The above-mentioned method for preparing probiotic-fermented Ge Xian rice juice is as follows: the activated fermented Lactobacillus graminearum seed liquid is inoculated into Ge Xian rice juice at an inoculation amount of 3%, and fermented at 39~43℃ for 18~24h. After fermentation, probiotic-fermented Ge Xian rice juice is obtained.

[0013] This invention provides a fermented product of Ge Xian rice juice probiotics with high SOD activity, which contains the above-mentioned probiotic fermented Ge Xian rice juice.

[0014] Furthermore, the Ge Xian rice juice probiotic fermentation product is a lactic acid bacteria solid beverage, which is prepared through the following steps: a protectant of fermenting Lactobacillus mucinus Grx07 is added to the probiotic fermented Ge Xian rice juice, and after vacuum freeze-drying, a freeze-dried powder with a water content of no more than 3% is obtained. Then, the freeze-dried powder is mixed with maltodextrin and xylooligosaccharide in proportion under sterile conditions to obtain a fermenting Lactobacillus mucinus Grx07 solid beverage with high SOD activity.

[0015] Furthermore, the Ge Xian rice juice probiotic fermentation product is a postbiotic product, which is prepared through the following steps: after pasteurizing the fermented Ge Xian rice juice, the probiotics are homogenized and crushed under aseptic high pressure to further release the active substances in the probiotics, thus obtaining a postbiotic product with high SOD activity.

[0016] This invention also provides a method for preparing a fermented milk with high SOD activity from Ge Xian rice juice probiotics. The specific steps are as follows: whole milk powder and water are mixed in a certain proportion to obtain a whole milk emulsion. Then, sucrose is added, homogenized, pasteurized, cooled to room temperature, and then Ge Xian rice juice is added. Fermented Lactobacillus fermentum Grx07 seed liquid is inoculated, and commercial starter culture MO094 BR is added. Fermentation is carried out at 41-43℃ until curdled to obtain Ge Xian rice juice probiotic fermented milk. The amount of each component added per 150mL of whole milk emulsion is: 15-19.5g whole milk powder, 7.5-10.5g sucrose, 15-30mL Ge Xian rice juice, 60-360μL commercial starter culture MO094 BR, and 4.5-7.5mL fermented Lactobacillus fermentum Grx07 seed liquid.

[0017] Furthermore, the homogenization pressure is 20 MPa.

[0018] Furthermore, the amount of each component added per 150 mL of whole milk emulsion is as follows: 17.25 g whole milk powder, 10.5 g sucrose, 22.5 mL of Ge Xian Mi juice, 240 μL of commercial starter culture MO 094 BR, and 4.5 mL of fermented Lactobacillus mucilaginosus Grx07 seed culture.

[0019] The present invention further provides a fermented milk with high SOD activity from rice juice containing Ge Xian rice juice probiotics prepared by the above preparation method.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) The present invention combines low temperature treatment-pulping-ultrasonic crushing pretreatment with high pressure treatment at 115℃ for 15 min heat treatment, which can effectively release the SOD active ingredients of Ge Xian Mi and obtain Ge Xian Mi juice with high SOD activity.

[0022] (2) In this invention, probiotics are involved in the fermentation of whole milk, probiotics that can further improve the SOD activity of Ge Xian rice juice are screened, and commercial fermentation agents are screened for co-fermentation to further improve the SOD activity, probiotic live bacteria count and sensory evaluation of Ge Xian rice juice.

[0023] (3) This invention prepares the active ingredient of the ginseng into a juice with high SOD activity, and then prepares it into lactic acid bacteria food products with high SOD activity, such as lactic acid bacteria solid beverages and postbiotics, providing theoretical support for the high-value utilization of ginseng and other cyanobacterial functional resources. Attached Figure Description

[0024] Figure 1 shows the effect of different heat treatment methods on the SOD activity of Ge Xian rice juice, where different letters indicate significant differences between different treatment groups for the same index (P<0.05).

[0025] Figure 2 shows the effects of different additives on the SOD activity, acidity, and sensory score of Grx07 fermented rice juice; where A represents the SOD activity of the sample, B represents the acidity of the sample, and C represents the sensory score of the sample. Different letters indicate significant differences between different treatment groups for the same indicator (P<0.05).

[0026] Figure 3 shows the effects of MO 094 BR addition on SOD activity, viable cell count, and sensory score of Ge Xian rice juice probiotic fermented milk; where A represents the SOD activity of the sample, B represents the viable cell count of the sample, and C represents the sensory score of the sample. Different letters indicate significant differences between different treatment groups for the same indicator (P<0.05).

[0027] Figure 4 shows the effects of the amount of *Ge Xian* rice juice added on the SOD activity, viable bacteria count, and sensory score of *Ge Xian* rice juice probiotic fermented milk; where A represents the SOD activity of the sample, B represents the viable bacteria count of the sample, and C represents the sensory score of the sample. Different letters indicate significant differences between different treatment groups for the same indicator (P<0.05).

[0028] Figure 5 shows the effects of the inoculum size of *Lactobacillus fermentum* Grx07 on the SOD activity, viable cell count, and sensory score of fermented milk containing *Gnaphalium affine* rice juice probiotics; where A represents the SOD activity of the sample, B represents the viable cell count of the sample, and C represents the sensory score of the sample. Different letters indicate significant differences between different treatment groups for the same indicator (P<0.05).

[0029] Figure 6 shows the effects of fermentation temperature on SOD activity, viable cell count, and sensory score of Ge Xian Rice Juice Probiotic Fermented Milk; where A represents the SOD activity of the sample, B represents the viable cell count of the sample, and C represents the sensory score of the sample. Different letters indicate significant differences between different treatment groups for the same indicator (P<0.05).

[0030] Figure 7 shows the effects of sucrose addition on SOD activity, viable cell count, and sensory score of Ge Xian rice juice probiotic fermented milk; where A represents the SOD activity of the sample, B represents the viable cell count of the sample, and C represents the sensory score of the sample. Different letters indicate significant differences between different treatment groups for the same indicator (P<0.05).

[0031] Figure 8 shows the effects of storage period on SOD activity, viable cell count, and sensory score of fermented milk containing Ge Xian rice juice probiotics; where A represents the SOD activity of the sample, B represents the viable cell count of the sample, and C represents the sensory score of the sample. Different letters indicate significant differences between different treatment groups for the same indicator (P<0.05). Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but this does not limit the invention to the scope of the embodiments described. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0033] In the following examples, the *Ge Xian Mi* (a type of rice) used was provided by Wuhan Zao Nongye Ecological Agriculture Technology Co., Ltd.; the whole milk powder was provided by Fonterra Trading Co., Ltd. of New Zealand; the white sugar was provided by Angel Yeast Co., Ltd.; the commercial starter cultures MO 094 BR and YO 15Z (10U) were provided by Shanghai Haoyue Biotechnology Co., Ltd.; and the resistant dextrin was provided by Shandong Bailong Chuangyuan Biotechnology Co., Ltd.

[0034] In the following embodiments, the accession number of *Lactobacillus fermentum* Grx07 is CGMCC NO. 8874, which has been fully disclosed in Chinese Patent ZL201410156237.9; the accession number of *Lactobacillus plantarum* Grx401 is CGMCC NO. 23544, which has been fully disclosed in Chinese Patent ZL 202111496871.3; and the accession number of *Lactobacillus plantarum* Grx402 is CGMCC NO. 28585, which has been fully disclosed in Chinese Patent ZL202311475848.5. All of the above-mentioned *Lactobacillus fermentum* Grx07, *Lactobacillus plantarum* Grx401, and *Lactobacillus plantarum* Grx402 were provided by the Provincial Key Laboratory of Probiotics and Deep Processing of Dairy Products (Yangzhou University).

[0035] In the following examples, the SOD activity of each sample was determined strictly in accordance with the product instructions of the SOD activity assay kit from Beijing Solarbio Science & Technology Co., Ltd.

[0036] In the following embodiments, the sensory evaluation team consisted of eight evaluators (four men and four women) who had received specialized training in dairy sensory evaluation. The evaluation was conducted in a standardized, independent sensory chamber. Fermented milk samples were stored at 4°C in transparent plastic cups labeled with a three-digit random code. Evaluators were required to rinse their mouths with water between tasting different samples.

[0037] The sensory evaluation has a total score of 10 points. The scoring dimensions are set based on the characteristics of Ge Xian Mi (a type of rice) such as flavor and color, and refer to GB 19302-2025 "National Food Safety Standard for Fermented Milk". The scoring is mainly based on five dimensions: color, flavor, taste, texture and overall acceptability. The specific scoring rules are shown in Table 1.

[0038] Table 1 Sensory Evaluation Criteria for Fermented Milk with Ge Xian Rice Juice

[0039]

[0040] Note: The final sensory score is the weighted average of the five indicators or the total score after direct conversion.

[0041] Example 1

[0042] This embodiment investigates the effects of different heat treatment methods on the SOD activity of Ge Xian rice juice. The specific experiments are as follows:

[0043] (1) Pretreatment: Fresh kudzu rice was first placed at 0℃ for 30 min, then deionized water was added at 1 / 2 of the mass of kudzu rice, and pulped for 2 min to obtain kudzu rice crude pulp; the crude pulp was then placed in an ultrasonic cell disruptor and ultrasonically disrupted for 25 min under ice bath conditions. The above low temperature treatment-pulping-ultrasonic disruption process was repeated twice to obtain kudzu rice pretreatment solution.

[0044] (2) The pretreated kudzu root extract was subjected to three heat treatments: autoclaving at 115℃ for 15 min, boiling in a water bath at 100℃ for 10 min, and heating in a water bath at 95℃ for 5 min. After each heat treatment, the samples were cooled to room temperature to obtain kudzu root juice treated by different heat treatment methods.

[0045] Before preparing fermented milk base, the raw materials need to undergo appropriate heat treatment to reduce the risk of contamination by other microorganisms and ensure the stability of the fermentation process. SOD activity is closely related to the spatial structure of enzyme proteins and their metal active sites; excessive heat treatment may lead to conformational changes in enzyme proteins or damage to active sites, thereby causing a decrease in SOD activity. However, due to… Figure 1 It was found that the untreated *Gnaphalium affine* pretreatment solution, serving as the Control group, exhibited low SOD activity. With increasing heat treatment intensity, SOD activity did not decrease as continuously as with conventional heat-sensitive proteins, but instead showed a significant trend towards the highest SOD activity (3.83 U / mL) under high pressure conditions (115℃, 15 min), which was significantly different from other treatment groups (P<0.05). Compared to water bath treatments at 95℃ and 100℃, the 115℃ high-temperature and high-pressure conditions provided stronger thermal and pressure effects, which was beneficial for disrupting the outer layer of the *Gnaphalium affine*'s gelatinous polysaccharide matrix and cell wall structure.

[0046] In summary, high-pressure treatment at 115℃ for 15 minutes is more effective in improving the SOD activity of *Gynostemma pentaphyllum* juice, while also meeting the sterilization requirements during raw material pretreatment. Therefore, this invention employs a heat treatment method that involves first treating *Gynostemma pentaphyllum* at low temperature, then pulping it, followed by ultrasonic crushing under ice bath conditions. This process of low-temperature treatment-pulping-ultrasonic crushing is repeated twice, followed by high-pressure sterilization at 115℃ for 15 minutes. This method yields *Gynostemma pentaphyllum* juice with high SOD activity, which is beneficial for the subsequent preparation of fermented *Gynostemma pentaphyllum* juice products. In subsequent experiments, *Gynostemma pentaphyllum* juice sterilized at 115℃ for 15 minutes was used as the raw material.

[0047] Example 2

[0048] This embodiment investigates the effects of different probiotic fermentation strains on the SOD activity of *Ge Xian Mi* (a type of herb). The specific experiments are as follows:

[0049] The activated fermented Lactobacillus mucinus Grx07, Lactobacillus plantarum Grx401, and Grx402 seed cultures were inoculated into the Ge Xian rice juice prepared in Example 1 at an inoculation rate of 3%, and fermented at 37°C for 18 hours. After fermentation, the Ge Xian rice juice fermented with different probiotics was taken and its SOD activity was measured. The results are shown in Table 2.

[0050] Table 2. SOD activity of rice juice fermented with different probiotics

[0051]

[0052] Table 2 shows that the SOD activity of *Lactobacillus mucilaginosus* fermented with *Lactobacillus mucilaginosus* Grx07 was 5.22 U / mL, an increase of 36.3% compared to the unfermented *Lactobacillus mucilaginosus* juice; the SOD activity of *Lactobacillus plantarum* fermented with *Lactobacillus plantarum* Grx401 was 4.62 U / mL, an increase of 20.6% compared to the unfermented *Lactobacillus plantarum* juice; and the SOD activity of *Lactobacillus plantarum* fermented with *Lactobacillus plantarum* Grx402 was 4.85 U / mL, an increase of 26.6% compared to the unfermented *Lactobacillus mucilaginosus* juice. These results indicate that fermentation with *Lactobacillus mucilaginosus* Grx07 significantly improves the SOD activity of *Lactobacillus mucilaginosus* juice compared to *Lactobacillus plantarum* Grx401 and Grx402 (P<0.05).

[0053] Example 3

[0054] To improve the fermentation activity of *Lactobacillus mucinus* Grx07 in a milk-based system and to screen commercial starter cultures suitable for fermented milk systems based on *Ge Xian* rice juice, this example investigates the effects of commercial starter cultures MO 094 BR, YO 15Z, and resistant dextrin at different fermentation temperatures on the acid production capacity, SOD activity, and sensory quality of Grx07 fermented milk. Specific experiments are as follows:

[0055] 17.25g of whole milk powder was added to deionized water to prepare 150mL of whole milk emulsion with a milk solids content of 11.5%. Then, 10.5g of sucrose was added to obtain an emulsion base containing 7% sucrose. Activated Lactobacillus fermentum Grx07 seed culture was inoculated into the emulsion base containing 7% sucrose at a 3% inoculation rate. A control group was established without the addition of commercial starter culture and resistant dextrin. The experimental groups were supplemented with resistant dextrin (1.5% w / w), commercial starter culture MO 094 BR (0.025 g / L), and YO 15Z (0.040 g / L), respectively. Each group was fermented in a constant temperature incubator at 37℃ and 42℃ until curdling. The SOD activity, titratable acidity, and sensory quality of the fermented milk in each group were measured. The results are as follows: Figure 2 As shown.

[0056] like Figure 2 As shown in Figure A, regarding SOD activity, the MO 094 BR group exhibited the highest SOD activity at 42℃, reaching 9.917 U / mL, significantly higher than the Control group, YO 15Z group, and resistant dextrin group (P<0.05). At 37℃, the overall SOD activity of all treatment groups was lower than that at 42℃, with the MO 094 BR group still showing superior performance. Appropriately increasing the fermentation temperature and adding MO 094 BR may enhance the growth and metabolic activity of Grx07, promote the release or accumulation of antioxidant-related active substances, and thus improve the antioxidant enzyme activity of the fermentation system.

[0057] like Figure 2 As shown in Figure B, the MO 094 BR group exhibited the highest acidity under both temperature conditions. Furthermore, at 42°C, the MO 094 BR group showed the highest acidity at 110.20 °T, significantly higher than the YO 15Z group's 95.21 °T, and also significantly higher than the resistant dextrin group and the Control group (P<0.05). These results indicate that at higher fermentation temperatures, MO 094 BR is more beneficial for increasing the system's acidity.

[0058] like Figure 2 As shown in C, the MO 094 BR group achieved high scores under both temperature conditions, with the highest score of 7.33 at 37℃ and 7.00 at 42℃, both higher than the Control group, YO 15Z group, and resistant dextrin group.

[0059] In summary, MO 094 BR demonstrated excellent performance in promoting Grx07 fermentation, enhancing the system's functional activity, and maintaining the product's sensory quality. Therefore, this invention selected the commercial starter MO 094 BR for subsequent optimization of the Ge Xian rice juice probiotic fermented milk process.

[0060] Example 4: Optimization of fermentation conditions for Ge Xian Rice Juice Probiotic Fermented Milk

[0061] 1. Single-factor optimization experiment of fermentation conditions for Ge Xian Rice Juice Probiotic Fermented Milk

[0062] To determine the optimal preparation parameters for Ge Xian rice juice fermented milk, this experiment used a 150 mL whole milk emulsion system (17.25 g of whole milk powder added to deionized water to prepare 150 mL of whole milk emulsion with a milk solids content of 11.5%) as the basis. Single-factor optimization experiments were conducted on the following factors: MO 094BR addition amount (μL), Ge Xian rice juice addition amount (mL), Grx07 inoculum amount (mL), fermentation temperature (°C), and sucrose addition amount (g). The experiment used SOD activity, viable lactic acid bacteria count, and sensory evaluation scores as evaluation indicators to compare the effects of different fermentation conditions on the antioxidant activity, fermentation characteristics, textural quality, and sensory acceptability of Ge Xian rice juice probiotic fermented milk, providing a basis for subsequent orthogonal experimental optimization.

[0063] (1) MO 094 BR addition amount

[0064] 10.5 g of sucrose was added to 150 mL of whole milk emulsion and homogenized. After pasteurization and cooling, 22.5 mL of *Ge Xian Mi* juice was added, and 4.5 mL of Grx07 seed culture was inoculated. 15, 60, 240, 960, and 3840 µL of MO 094 BR were added respectively, and fermentation was carried out at 37℃ until curdled. The results are shown in the figure. Figure 3 .

[0065] Depend on Figure 3 As shown in A and 3B, with the increase of MO 094 BR addition from 15 μL to 60 μL, the SOD activity in the fermentation system showed a significant increasing trend, reaching its highest value of 7.83 U / mL in the 240 μL treatment group, significantly higher than the 15 μL and 960 μL groups (P<0.05). When the MO 094 BR addition was further increased to 3840 μL, the SOD activity decreased significantly. Simultaneously, the increase in viable lactic acid bacteria count gradually leveled off with increasing addition. The viable bacteria count was low in the 15 μL group, and after further increasing the addition to 960 and 3840 μL, the change in viable bacteria count was not significant, remaining at 1.27 × 10⁻⁶ compared to the 240 μL group. 6 Compared with other groups, there was no significant difference in CFU / mL among the three groups (P>0.05). When the amount added reached 240 μL, further increasing MO 094 BR had a limited effect on increasing the number of viable bacteria.

[0066] Depend on Figure 3 As can be seen from C, the sensory scores of the 240 μL treatment group were at a high level, indicating that the product performed well in terms of overall flavor, taste, or acceptability at this dosage.

[0067] Based on the SOD activity, viable lactic acid bacteria count, and sensory evaluation of fermented milk, 60, 240, and 960 μL were selected as the addition amounts of MO 094 BR in fermented milk for further optimization.

[0068] (2) Amount of Ge Xian Mi Juice Added

[0069] 10.5 g of sucrose was added to 150 mL of whole milk emulsion and homogenized. After pasteurization and cooling, 7.5, 15, 22.5, 30, and 37.5 mL of *Ge Xian Mi* juice were added respectively. 4.5 mL of Grx07 and 60 µL of MO 094 BR were inoculated, and fermentation was carried out at 37°C until curdled. The results are shown in the figure. Figure 4 .

[0070] Depend on Figure 4 As shown in Figure A, the SOD activity of the fermented milk significantly increased with the increase of the amount of *Ge Xian Mi Zi* juice added from 7.5 mL to 15 mL; the SOD activity reached its highest value of 7.31 U / mL when the amount added was 30 mL. There were no significant differences among the 15, 22.5, and 30 mL treatment groups (P>0.05), but all were significantly higher than the 7.5 and 37.5 mL treatment groups (P<0.05). When the amount added was further increased to 37.5 mL, the SOD activity decreased to 4.17 U / mL.

[0071] Depend on Figure 4 As shown in B, increasing the added amount from 7.5 mL to 22.5 mL resulted in an increase in the viable bacteria count from 1.07 × 10⁻⁶. 6 CFU / mL increased to 1.25 × 10⁻⁶ 6 CFU / mL, appropriately increasing the amount of *Ge Xian Mi* juice may be beneficial for bacterial proliferation. The 22.5 and 30 mL treatment groups had higher viable cell counts, with no significant difference between the two groups (P>0.05), both significantly higher than other treatment groups (P<0.05). When the amount added was increased to 37.5 mL, the viable cell count decreased to 1.09 × 10⁻⁶. 6 CFU / mL, excessive addition of Ge Xian rice juice may hinder the continued proliferation of bacteria.

[0072] Depend on Figure 4 As shown in C, the 7.5 mL treatment group had a lower overall evaluation score. With the addition amount increasing to 15 mL, the overall evaluation score rose to 7.80, the highest among all groups; the scores of the 22.5 mL and 30 mL treatment groups also remained at a high level, with no significant difference from the 15 mL treatment group (P>0.05). When the addition amount increased to 37.5 mL, the overall evaluation score decreased, indicating that the excessively high proportion of *Ge Xian Mi Juice* (a type of herb) affected the product's flavor harmony and texture, leading to a decrease in overall acceptability.

[0073] Based on the combined SOD activity, viable lactic acid bacteria count, and sensory evaluation of the fermented milk, 10, 15, and 20 mL were selected as the addition amounts of Ge Xian Mi Juice to the fermented milk for further optimization.

[0074] (3) Inoculation amount of fermenting Lactobacillus mucin Grx07

[0075] 10.5 g of sucrose was added to 150 mL of whole milk emulsion and homogenized. After pasteurization and cooling, 22.5 mL of *Ge Xian Mi* juice was added. 1.5, 4.5, 7.5, 10.5, and 13.5 mL of Grx07 seed culture and 60 µL of MO 094 BR were inoculated respectively, and fermented at 37℃ until curdled. The results are shown in the figure. Figure 5 .

[0076] Depend on Figure 5 As shown in Figure A, the Grx07 inoculum amount significantly affects the SOD activity of fermented rice juice. The 4.5 mL inoculum group showed the highest SOD activity at 7.18 U / mL. When the inoculum amount increased to 7.5 mL, SOD activity decreased significantly. The enzyme activity further decreased in the 10.5 mL and 13.5 mL groups. A higher Grx07 inoculum amount is not necessarily more beneficial for increasing SOD activity; excessively high inoculum amounts may affect the stability of SOD enzyme activity.

[0077] Depend on Figure 5 As shown in B, the viable bacterial count was low in the 1.5 mL inoculum group; when the inoculum was increased to 4.5 mL, the viable bacterial count significantly increased to 1.23 × 10⁻⁶. 6 The CFU / mL concentration was significantly higher than that of the 1.5 mL treatment group (P<0.05). When the inoculum volume was further increased to 7.5 mL, 10.5 mL, and 13.5 mL, there were no significant differences among the groups (P>0.05). After the inoculum volume reached 4.5 mL, further increasing the Grx07 inoculum volume had no significant effect on increasing the viable bacterial count.

[0078] Depend on Figure 5 As shown in Figure C, the 1.5 mL inoculum group had a lower sensory score, indicating insufficient fermentation and poor product flavor and texture due to the low inoculum size. The 4.5 mL inoculum group had the highest sensory score (7.23 points), showing better performance in terms of acidity, aroma, taste harmony, and overall acceptability. The sensory scores of the 7.5 mL and 10.5 mL inoculum groups were both lower than those of the 4.5 mL group, and the sensory score of the 13.5 mL inoculum group decreased further.

[0079] Based on the combined SOD activity, viable lactic acid bacteria count, and sensory evaluation of the fermented milk, 4.5 mL was selected as the amount of Grx07 seed liquid to be added to the fermented milk.

[0080] (4) Fermentation temperature

[0081] 10.5 g of sucrose was added to 150 mL of whole milk emulsion and homogenized. After pasteurization and cooling, 22.5 mL of *Ge Xian Mi* juice was added, and 4.5 mL of Grx07 seed culture and 60 µL of MO 094 BR were inoculated. Fermentation was carried out at 37℃, 39℃, 41℃, 43℃, and 45℃ until curdling. The results are shown in the figure. Figure 6 .

[0082] Depend on Figure 6 As shown in Figure A, the SOD activity in the 37℃ treatment group was low, at 7.18 U / mL; when the temperature increased to 39℃, the SOD activity increased to 7.52 U / mL. The SOD activities in the 41℃ and 43℃ treatment groups further increased, with no significant difference between the two groups (P>0.05), but both were significantly higher than those in the 39℃ and 45℃ treatment groups (P<0.05). When the temperature increased to 45℃, the SOD activity decreased significantly to 3.61 U / mL, indicating that excessively high temperatures are not conducive to maintaining SOD activity.

[0083] Depend on Figure 6 As shown in B, the viable bacterial count was lower in the 37℃ treatment group and highest in the 43℃ treatment group, at 1.35 × 10⁻⁶. 6 The CFU / mL level was also high in the 41℃ treatment group, with no significant difference between the two groups (P>0.05). When the temperature increased to 45℃, the viable bacterial count decreased to 1.20×10⁻⁶. 6 The CFU / mL reading indicates that excessively high temperatures may affect bacterial growth and are not conducive to maintaining the viable cell count in the fermentation system.

[0084] Depend on Figure 6 As shown in Figure C, the sensory score of the 37℃ treatment group was low, indicating insufficient fermentation under low-temperature conditions, resulting in mediocre product flavor and texture. As the temperature increased, the sensory score gradually improved, reaching its highest at 8.87 points for the 43℃ treatment group; the 41℃ treatment group also scored relatively high at 8.37 points. The sensory score of the 45℃ treatment group decreased, as the excessively high temperature led to an unbalanced acidity and poor curd consistency, reducing sensory acceptance.

[0085] Based on the combined SOD activity, viable lactic acid bacteria count, and sensory evaluation of fermented milk, 39℃, 41℃, and 43℃ were selected as the fermentation temperatures for further optimization.

[0086] (5) Amount of sucrose added

[0087] 4.5, 7.5, 10.5, 13.5, and 16.5 g of sucrose were weighed and added to 150 mL of whole milk emulsion, homogenized, pasteurized, and cooled. 22.5 mL of *Gnaphalium affine* juice was added, followed by inoculation with 4.5 mL of Grx07 seed culture and 60 µL of MO 094 BR. Fermentation was carried out at 37℃ until curdled. Results are shown in […]. Figure 7 .

[0088] Depend on Figure 7 As shown in Figure A, when the sucrose addition amount was 4.5, 7.5, and 10.5 g, the SOD activity gradually increased, reaching its highest value at 10.5 g. Increasing the sucrose addition amount is beneficial for maintaining the antioxidant enzyme activity of the system. When the sucrose addition amount increased to 13.5 and 16.5 g, the SOD activity decreased significantly and was significantly lower than that of the 4.5, 7.5, and 10.5 g treatment groups (P<0.05). Excessively high sucrose concentration may be detrimental to maintaining SOD activity.

[0089] Depend on Figure 7 As shown in B, the viable cell count was low in the 4.5 g treatment group. When the sucrose addition increased to 7.5 and 10.5 g, the viable cell count gradually increased. The viable cell counts in the 13.5 and 16.5 g treatment groups further increased and remained at a high level, indicating that appropriately increasing the sucrose addition was beneficial for promoting cell proliferation. However, as the addition amount continued to increase, the increase in viable cell count tended to level off, and the differences among the latter three groups were not significant (P>0.05), suggesting that excessively high sucrose concentration had a limited effect on promoting cell growth.

[0090] Depend on Figure 7 As shown in C, the 4.5 g treatment group had the lowest sensory score. At lower sucrose levels, the product lacked sweetness, had a noticeable astringency, poor sweet-sour balance, and low overall acceptability. The highest sensory score was obtained in the 10.5 g treatment group, where the product had a balanced sweet-sour taste, harmonious flavor of the rice wine, and good overall sensory quality. When the sucrose level continued to increase to 13.5 and 16.5 g, the sensory scores decreased. Excessive sucrose addition resulted in poor curd formation, affecting the product's flavor balance and overall acceptability.

[0091] Overall, the 10.5 g sucrose addition group showed a high level of SOD activity, a high number of viable bacteria, and the highest sensory score, which can be used as a reference level for subsequent optimization of the Ge Xian rice juice fermented milk process.

[0092] 2. Orthogonal experimental optimization of fermentation conditions for Ge Xian Rice Juice Probiotic Fermented Milk

[0093] Single-factor experiments showed that the SOD activity of the fermented milk was highest when the inoculum size of *Lactobacillus mucinus* Grx07 seed culture was 4.5 mL, and there was no significant difference when the inoculum size was further increased (P>0.05). Similarly, the SOD activity of the fermented milk was highest when the sucrose addition was 10.5 g, and there was no significant difference when the addition amount was further increased (P>0.05). Therefore, 4.5 mL of Grx07 inoculum and 10.5 g of sucrose addition were selected as the fermentation conditions for the fermented milk. Based on this, three factors that significantly affected the quality of the fermented milk were selected for orthogonal optimization, with three levels for each factor, using an L9(3) quadratic algorithm.4 The orthogonal experimental design is shown in Tables 3 and 4 for specific parameters, and Table 5 shows the orthogonal experimental results.

[0094] Table 3. Factor Level Table for Orthogonal Experiment of Ge Xian Rice Juice Fermented Milk Process Optimization

[0095]

[0096] Table 4 L9(3) 4 Orthogonal experimental design scheme

[0097]

[0098] Table 5. Results of Orthogonal Experiments and Range Analysis

[0099]

[0100] Table 5 shows that the SOD activity of fermented milk containing *Gynostemma pentaphyllum* juice differed significantly under different combinations of factors. Analysis using k-values ​​and the range R-squared revealed that the order of influence of the three factors on SOD activity was: MO 094 BR addition amount (C) > fermentation temperature (B) > *Gynostemma pentaphyllum* juice addition amount (A). Therefore, A2B3C2 was determined as the preparation conditions for the subsequent product, namely, 22.5 mL of *Gynostemma pentaphyllum* juice, a fermentation temperature of 43℃, and 240 μL of MO 094 BR.

[0101] To further determine the statistical significance of the effects of each factor on SOD activity, an analysis of variance was performed on the orthogonal experimental results, as shown in Table 6.

[0102] Table 6. Results of Analysis of Variance (ANOVA) for SOD Activity Indicators

[0103]

[0104] Note: "*" indicates a significant difference (P<0.05), and "**" indicates an extremely significant difference (P<0.01).

[0105] Table 6 shows that the p-value of the modified model was 0.002, reaching a highly significant level, indicating that the orthogonal experimental model is statistically significant and can reflect the influence of various factors on the SOD activity of fermented milk containing *Gynostemma pentaphyllum* juice. Among the factors, the p-value of MO 094 BR addition (C) was 0.001, reaching a highly significant level (P<0.01); the p-value of fermentation temperature (B) was 0.026, reaching a significant level (P<0.05). The p-value of *Gynostemma pentaphyllum* juice addition (A) was 0.210, not reaching a significant level (P>0.05), indicating that the effect of *Gynostemma pentaphyllum* juice addition on SOD activity was relatively weak. Based on the F-values, the order of influence of the three factors on SOD activity was: MO 094 BR addition (C) > fermentation temperature (B) > *Gynostemma pentaphyllum* juice addition (A). This result is basically consistent with the range analysis results.

[0106] The combined results showed that the amount of MO 094 BR added and the fermentation temperature were the main factors affecting the SOD activity of fermented milk made from *Lactobacillus thunbergii* rice juice, while the amount of *Lactobacillus thunbergii* rice juice added had no significant effect within the experimental range. Therefore, the optimal process parameters for fermented milk made from *Lactobacillus thunbergii* rice juice were finally determined. A verification experiment was conducted according to combination A2B3C2, namely, 10.5 g sucrose was added to 150 mL of whole milk emulsion (containing 17.25 g whole milk powder), the amount of *Lactobacillus thunbergii* rice juice added was 22.5 mL, 240 μL of MO 094 BR and 4.5 mL of *Lactobacillus mucilaginosus* Grx07 seed culture were inoculated, and fermentation was carried out at 43℃ until curdling. The SOD activity of the fermented milk was measured to be 13.17 U / mL, and the viable cell count and sensory quality remained at a good level, which can be used as the preparation conditions for subsequent storage stability experiments.

[0107] Example 5: Study on the stability of fermented milk made from Ge Xian rice juice during storage

[0108] For functionally active fermented milk made from *Ge Xian* rice juice, maintaining good sensory quality during refrigeration is crucial, as is maintaining certain SOD activity and viable bacteria levels to ensure its antioxidant function and probiotic properties. Therefore, this example investigates the quality changes of fermented milk made from *Ge Xian* rice juice under 4°C refrigeration conditions and analyzes its suitable functional shelf life accordingly. The results are as follows: Figure 8 As shown.

[0109] Depend on Figure 8 As shown in Figure A, the antioxidant activity of the product remained relatively good during the initial storage period. The SOD activity gradually decreased from 13.17 U / mL on day 0 to 8.18 U / mL on day 11. Although it decreased, it still remained at a relatively high level. After day 13, the decrease in SOD activity further increased, dropping to 3.54 U / mL on day 15. This indicates that the stability of antioxidant enzyme activity in the fermented milk system decreased significantly with prolonged storage time (P<0.05).

[0110] Depend on Figure 8 According to B, the viable bacterial count increased from 1.41 × 10⁻¹⁵ within days 0 to 11. 6 CFU / mL decreased to 1.28 × 10⁻⁶ 6 The product maintained good probiotic activity during the early and middle stages of storage, with a CFU / mL count. However, by day 13, the viable count had decreased to 0.91 × 10⁻⁶. 6 The CFU / mL count was lower than the requirement for live lactic acid bacteria in the national standard GB 19302-2025 "National Food Safety Standard for Fermented Milk". The number of lactic acid bacteria decreased significantly in the later stages of storage (P<0.05).

[0111] Depend on Figure 8 As shown in C, the sensory scores of Ge Xian rice juice fermented milk generally declined with prolonged storage. On day 0, the sensory score was 8.13, indicating a relatively balanced sweet and sour taste, good texture, and high overall acceptability. From day 7 to 11, the sensory score remained above 7. After day 13, the sensory score continued to decline, dropping to 6.77 on day 15. At this point, the product had a poor flavor, a noticeable sour taste, and a decreased mouthfeel, indicating a gradual deterioration in sensory quality.

[0112] Based on the above changes, considering SOD activity, viable bacteria count, and sensory acceptance, the suitable functional shelf life of the fermented rice juice prepared by this invention at 4°C should be controlled at 11 days.

[0113] Example 6: Preparation of a lactic acid bacteria solid beverage with high SOD activity

[0114] The preparation of Ge Xian rice juice is the same as in Example 1. 3% *Lactobacillus fermentum* Grx07 is inoculated into the sterilized Ge Xian rice juice and fermented at 43°C for 18 h. A protectant for *Lactobacillus fermentum* Grx07 is added, and the mixture is freeze-dried under vacuum to obtain a lyophilized powder with a water content not exceeding 3%. The lyophilized powder is then mixed with maltodextrin and xylooligosaccharides in a certain proportion under aseptic conditions to obtain a *Lactobacillus fermentum* Grx07 solid beverage.

[0115] Example 7 Preparation of postbiotic products with high SOD activity

[0116] The preparation of Ge Xian rice juice is the same as in Example 1. 3% of *Lactobacillus fermentum* Grx07 is inoculated into the sterilized Ge Xian rice juice, and fermented at 43°C for 18 h. After pasteurization, the mixture is homogenized under aseptic high pressure to further release the active substances in the probiotics, resulting in a postbiotic product with high SOD activity.

Claims

1. A method for preparing Nostoc commune juice having high SOD activity, characterized by, Specifically, the following steps are included: (1) Pretreatment: Fresh Nostoc commune is first treated at low temperature in an ice bath at 0-4℃ for 30-40 min. Then, deionized water is added at 1 / 2 of the mass of Nostoc commune, and the mixture is pulped for 2-5 min. The resulting Nostoc commune pulp is then placed in an ultrasonic cell disruptor and ultrasonically disrupted in an ice bath for 25-30 min. The above process of low temperature treatment-pulping-ultrasonic disruption is repeated twice to obtain Nostoc commune pretreatment solution. (2) Heat treatment: The pretreated liquid of Ge Xian Mi was sterilized at 115℃ for 15 minutes to obtain Ge Xian Mi juice with high SOD activity.

2. The *Ge Xian Mi Juice* with high SOD activity prepared by the preparation method according to claim 1.

3. A probiotic fermented Nostoc commune juice, characterized by, The rice juice of claim 2 is fermented with Lactobacillus mucinus Grx07, wherein the Lactobacillus mucinus Grx07 has the accession number CGMCC NO.8874.

4. The probiotic fermented Nostoc juice according to claim 3, characterized in that, Commercial starter culture MO 094 BR was also added at a concentration of 0.025 g / L.

5. The method of claim 3 or 4, wherein the fermented Nostoc juice is prepared by culturing the Nostoc cell in a medium containing a carbon source, a nitrogen source, and a phosphorus source, and then fermenting the Nostoc cell in a medium containing a carbon source, a nitrogen source, and a phosphorus source. Specifically, the activated fermented Lactobacillus mucinus Grx07 seed liquid was inoculated into Ge Xian rice juice at an inoculation rate of 3%, and fermented at 39~43℃ for 18~24h. After fermentation, probiotic fermented Ge Xian rice juice was obtained.

6. A Nostoc sp. juice probiotic fermented product having high SOD activity, characterized by, It contains fermented rice juice with probiotics as described in claim 3 or 4.

7. The Nostoc sp. juice probiotic fermented product according to claim 6, characterized in that, The product is a lactic acid bacteria solid beverage, prepared through the following steps: A protectant of *Lactobacillus mucinus* Grx07 is added to probiotic-fermented *Ge Xian* rice juice. After vacuum freeze-drying, a freeze-dried powder with a water content not exceeding 3% is obtained. The freeze-dried powder is then mixed with maltodextrin and xylooligosaccharides in a specific ratio under aseptic conditions to obtain a *Lactobacillus mucinus* Grx07 solid beverage with high SOD activity. Alternatively, the product is a postbiotic product, prepared through the following steps: After pasteurizing probiotic-fermented *Ge Xian* rice juice, it is homogenized under aseptic high pressure to further release the active substances in the probiotics, thus obtaining a postbiotic product with high SOD activity.

8. A method for preparing a Nostoc commune juice probiotic fermented milk having high SOD activity, characterized by, The specific steps are as follows: whole milk powder and water are mixed in a certain proportion to prepare whole milk emulsion, then sucrose is added, homogenized and pasteurized, cooled to room temperature, and then the *Gynostemma pentaphyllum* rice juice as described in claim 2 is added. *Lactobacillus mucilaginosus* seed liquid is inoculated, and commercial starter culture MO 094 BR is added. Fermentation is carried out at 41-43℃ until curdled to obtain *Gynostemma pentaphyllum* rice juice probiotic fermented milk. The amount of each component added per 150mL of whole milk emulsion is: 15-19.5g whole milk powder, 7.5-10.5g sucrose, 15-30mL *Gynostemma pentaphyllum* rice juice, 60-360μL commercial starter culture MO 094 BR, and 4.5-7.5mL *Lactobacillus mucilaginosus* seed liquid.

9. The production method according to claim 8, characterized by, The homogenization pressure was 20 MPa; the amount of each component added per 150 mL of whole milk emulsion was: 17.25 g whole milk powder, 10.5 g sucrose, 22.5 mL of Ge Xian Mi juice, 240 μL of commercial starter culture MO 094 BR, and 4.5 mL of fermented Lactobacillus mucilaginosus Grx07 seed culture.

10. A fermented milk containing *Gnaphalium affine* rice juice probiotics with high SOD activity prepared by the preparation method according to claim 8 or 9.

Images