A fermentation seabuckthorn bacterial agent and a preparation method and application of the bacterial agent for improving functional characteristics of seabuckthorn juice

Abstract

The present application provides a kind of fermenting seabuckthorn bacterial agent and the preparation method and application for improving the functional characteristics of seabuckthorn juice, and the present application belongs to the field of food processing technology.The preparation method of the present application includes the following steps: (1) take seabuckthorn homogenate and mix with water, then add complex enzyme preparation for enzymolysis treatment, to obtain enzymolysis seabuckthorn juice; (2) take the enzymolysis seabuckthorn juice and add mixed strains for fermentation treatment, to obtain fermented seabuckthorn juice.The present application optimizes the enzyme-assisted extraction conditions by response surface method, and combines with dietary fiber synergistic fermentation, significantly improves the inhibitory activity of alpha-glucosidase and alpha-glucosidase (alpha-glucoamylase) in seabuckthorn juice, and enhances the inhibitory effect on staphylococcus aureus and escherichia coli.The fermented seabuckthorn juice of the present application can be used as a functional food raw material for preparing products that inhibit carbohydrate digestive enzymes, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, and in particular to a microbial agent for fermenting sea buckthorn and a preparation method and application for improving the functional properties of sea buckthorn juice. Background Technology

[0002] The application of sea buckthorn (Hippophae rhamnoides L, SB) as a functional fruit is increasingly attracting attention from the academic community due to its rich content of polysaccharides, flavonoids, phenolic compounds, and organic acids. These molecules possess antioxidant, anti-inflammatory, and metabolic regulatory functions. Furthermore, sea buckthorn is rich in various micronutrients, such as vitamins C, A, E, and K, carotenoids, unsaturated fatty acids, phytosterols, and amino acids. These components work synergistically to lay the foundation for its application in functional foods and nutritional supplements. Recent studies have highlighted the therapeutic potential of sea buckthorn while also indicating the need to develop enhanced processes to overcome matrix limitations—which hinder the efficient extraction of target bioactive compounds.

[0003] Traditional hot water extraction techniques suffer from low extraction rates of sea buckthorn polysaccharide (SBP) due to polysaccharide entrapment within cellulose-pectin cell wall complexes, hindering dissolution. While techniques such as ultrasound-assisted enzymatic extraction (UAEE) have been explored to address this deficiency, significant challenges remain regarding their industrial-scale application and operational complexity. Existing technologies also include enzymatic hydrolysis-assisted extraction (EAE), which uses enzymatic hydrolysis to disrupt cell wall structure and enhance polysaccharide release. Targeted cell wall enzymatic extraction can significantly improve recovery efficiency while maintaining structure-activity relationships. However, most studies only investigate the extraction or fermentation processes separately, failing to integrate extraction optimization with cellulose-mediated fermentation technology or assess its impact on the inhibition of dual carbohydrate-digestive enzymes. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing sea buckthorn juice with improved functional properties, aiming to enhance the functionality and nutritional and health benefits of sea buckthorn juice.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing sea buckthorn juice with enhanced functional properties, comprising the following steps: (1) Take sea buckthorn homogenate and mix it with water, then add a compound enzyme preparation for enzymatic hydrolysis to obtain enzymatically hydrolyzed sea buckthorn juice; (2) Take the enzymatically hydrolyzed sea buckthorn juice and add it to the mixed strain for fermentation treatment to obtain fermented sea buckthorn juice.

[0006] Preferably, the mass-to-volume ratio of sea buckthorn homogenate to water in step (1) is 1:10~30; the compound enzyme preparation is composed of pectinase powder and cellulase powder in a mass ratio of 1~2:1~2; the mass concentration of the compound enzyme preparation is 0.2~0.6%; the enzymatic hydrolysis conditions are: temperature of 40~60℃ and enzymatic hydrolysis time of 0.5~2.5 h.

[0007] Preferably, the mixed strains in step (2) are Lactobacillus plantarum LZU-J-LZ1-2 and Lactobacillus plantarum LZU-J-QA5-2 mixed in a 1:3 ratio, with an effective viable count >2×10⁻⁶. 8 CFU / mL; the fermentation treatment is as follows: fermentation temperature 35~39℃; fermentation time 20~28 hours; the inoculation amount of mixed strains is 1.5%~2.5% of the volume of enzymatically hydrolyzed sea buckthorn juice.

[0008] Preferably, dietary fiber is added before or after the fermentation process in step (2), and the amount added is 0.5% to 2.0% of the mass of the enzymatically hydrolyzed sea buckthorn juice.

[0009] Preferably, the dietary fiber is dietary fiber derived from traditional Chinese medicine, herbal ingredients, or a mixture of both.

[0010] Preferably, the medicinal fiber is selected from one or more of mulberry leaves, mulberry fruit, polygonatum, white kidney bean, and chicory; the herbal fiber is selected from one or more of bitter melon, lettuce, cabbage, celery, and white hyacinth bean.

[0011] Preferably, the amount of dietary fiber added is 0.5% to 2.0% of the mass of the enzymatically hydrolyzed sea buckthorn juice, and more preferably 1%.

[0012] Preferably, when the dietary fiber is a mixed dietary fiber, it is a mixture of dietary fiber from traditional Chinese medicine and dietary fiber from herbs in a mass ratio of 1~2:1~2.

[0013] The present invention also provides an application of the sea buckthorn juice prepared by the above preparation method in the preparation of functional foods.

[0014] The present invention also provides a mixed strain in the preparation method described above, characterized in that the mixed strain is *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum LZU-J-LZ1-2 and Lactobacillus plantarum ( Lactiplantibacillus plantarum LZU-J-QA5-2; The Lactobacillus plantarum LZU-J-LZ1-2 is deposited at Guangdong Microbial Culture Collection Center, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, on July 1, 2024, with accession number GDMCC No:64799. The Lactobacillus plantarum LZU-J-QA5-2 is deposited at Guangdong Microbial Culture Collection Center, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, on July 1, 2024, with accession number GDMCC No:64798.

[0015] The present invention also provides an application of the aforementioned mixed strain in the preparation of sea buckthorn juice.

[0016] The present invention provides a method for preparing sea buckthorn juice with enhanced functional properties. By optimizing enzyme-assisted extraction conditions using response surface methodology, the extraction rate of sea buckthorn polysaccharides is significantly improved. For the first time, enzyme-assisted extraction is combined with synergistic lactic acid bacteria fermentation of dietary fiber, resulting in fermented sea buckthorn juice with significantly enhanced inhibitory activity against α-glucosidase and α-glucoamylase. The fermented sea buckthorn juice prepared by this invention exhibits significant growth inhibition effects against Staphylococcus aureus and Escherichia coli. The modified sea buckthorn juice prepared by this invention possesses triple activities: antioxidant, carbohydrate digestive enzyme inhibition, and antibacterial activity. It can be directly used as a raw material to prepare functional foods such as beverages, powders, and gels, and can also be used as a natural food additive for food preservation. Simultaneously, the prepared sea buckthorn compound ingredients have synergistic activity and can be directly used in the development of functional food formulations, significantly reducing the R&D costs of food production and showing broad application prospects in the food processing field. Attached Figure Description

[0017] Figure 1 The results of the single-factor optimization experiment for enzymatic extraction in Example 1; Figure 2 The three-dimensional response surface plot and contour plot are shown for the effect of other enzymatic extraction conditions on polysaccharide yield when the extraction temperature is at the center level in Example 1. Figure 3 The graph shows the results of optimizing the fermentation conditions for sea buckthorn juice in Example 1. Figure 4 The graph shows the effect of fermentation on the physicochemical properties and antibacterial activity of sea buckthorn juice in Example 1. Figure 5 This is a comparative analysis of the bioactive metabolite profiles of fresh and fermented sea buckthorn in Example 1. Figure 6 A comparative analysis of the bioactive metabolite profiles of fresh and fermented sea buckthorn; Figure 7 This is a flowchart of the preparation of sea buckthorn juice in Example 1.

[0018] Preservation Certificate

[0019] The plant lactobacillus ( Lactiplantibacillus plantarumLZU-J-LZ1-2, deposited at Guangdong Microbial Culture Collection Center, address: Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, deposited on July 1, 2024, accession number: GDMCC No:64799; The plant lactobacillus ( Lactiplantibacillus plantarum LZU-J-QA5-2 is deposited at Guangdong Microbial Culture Collection Center, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. The deposit date is July 1, 2024, and the accession number is GDMCC No: 64798. Detailed Implementation

[0020] 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.

[0021] Example 1

[0022] 1. Materials and Reagents

[0023] DeMan-Rogosa-Sharpe (MRS) and Luria-Bertani (LB) media, DPPH, and ABTS reagents were all purchased from domestic suppliers. Pectinase, cellulase, α-glucosidase, and α-glucosidase were purchased from Nanning Bangbo Biotechnology Co., Ltd. SB berries were purchased from Gansu Fuxinghou Biomedical Technology Co., Ltd. (Gansu, China). Two Lactobacillus strains (LZU-J-LZ1-2 and LZU-J-QA5-2) [2×10⁻⁶] were used. 8 Lactobacillus strains LZU-J-LZ1-2 and LZU-J-QA5-2, with a concentration of [CFU / mL], were isolated from river water, a specialty fermented food from Northwest China. The strains were identified by 16S rRNA gene sequence analysis and deposited at the Guangdong Microbial Culture Collection Center (GDMCC) in Guangzhou, China, with accession numbers GDMCC No: 64799 and GDMCC No: 64798, respectively. All fiber raw materials were food-grade dried fine powders provided by Shaanxi Huike Plant Development Co., Ltd. Detailed botanical names and raw material descriptions are shown in Table 1. In this article, "dietary fiber from traditional Chinese medicine" and "dietary fiber from herbal sources" both refer to plant-derived dietary fiber.

[0024] Table 1. Dietary fiber raw materials used for co-fermentation

[0025] 2. Polysaccharide content determination

[0026] A calibration curve was constructed using glucose as a standard, with a concentration range of 0.02 to 0.20 mg / mL. The procedure was as follows: Take 0.2–1.2 mL of the standard solution or appropriately diluted sample solution and bring the volume to 2.0 mL with distilled water. Then, quickly add 1.0 mL of 5% (w / v) phenol solution and 5.0 mL of concentrated sulfuric acid, and mix thoroughly. After allowing the reaction mixture to stand at room temperature for 10 minutes, heat it in a boiling water bath for 15 minutes. After cooling, measure the absorbance at 490 nm using a UV-Vis spectrophotometer. We also calculated the polysaccharide content based on the glucose standard curve and expressed it as milligram glucose equivalents (GE) per milliliter of sample.

[0027] 3. Enzymatic extraction of SB polysaccharide

[0028] 3.1 Evaluation of RSM Optimization Extraction Conditions Based on Univariate Analysis

[0029] SB homogenate was mixed with distilled water, and a compound enzyme preparation consisting of pectinase and cellulase powders in equal proportions (1:1, w / w; food grade) was added. Enzymatic hydrolysis was carried out under controlled temperature and time conditions, followed by enzyme inactivation at 90°C for 10 minutes. The enzyme concentration was calculated as the ratio of the total mass of the enzyme powder to the dry weight of the pea raw material. Preliminary single-factor experiments evaluated the effects of hydrolysis temperature (40–60°C), extraction time (0.5–2.5 h), enzyme concentration (0.2–0.6%, w / w), and solid-liquid ratio (1:10–1:30, w / v) on polysaccharide yield (Table 2). The results are as follows: Figure 1 As shown.

[0030] Under optimized extraction conditions, the highest polysaccharide yield was obtained when the extraction time was 2.5 hours, the temperature was 50℃, the solid-liquid ratio was 1:25 (w / v), and the enzyme concentration was 0.3% (w / w). This result indicates that the combination of moderate thermal conditions and enzyme activity effectively promotes the release of polysaccharides from the SB matrix. The initial polysaccharide yield began to decrease after 2.5 hours of extraction, indicating that the polysaccharides underwent degradation or decomposition into smaller structures during the extended treatment process. Figure 1 a). Similarly, the extraction temperature exhibits a unimodal effect, with the highest yield at 50℃. Figure 1 b). Enzyme concentration also affects polysaccharide recovery rate; when the concentration increases to 0.3%, the yield tends to saturate. Figure 1 c) indicates the presence of enzyme saturation. Furthermore, the solid-liquid ratio is crucial; an ideal 1:25 (w / v) ratio provides favorable mass transfer conditions. Figure 1 d). These results laid the foundation for subsequent optimization of extraction conditions using the RSM-Box-Behnken design. The optimal parameters were determined to be: extraction time 2.5 hours, temperature 50°C, enzyme concentration 0.3% (w / w), and solid-liquid ratio 1:25 (w / v). All subsequent experiments used this parameter combination.

[0031] Table 2. Single-factor experimental design for enzymatic extraction of SB polysaccharides

[0032] 3.2 Response Surface Methodology Analysis and Optimization of Extraction Conditions

[0033] The polysaccharide yield was optimized using response surface methodology. The Box-Behnken experimental design and response values ​​are shown in Table 3, with yield being the most critical response variable in the analysis. The quadratic model used in the study showed high significance (p<0.0001) and good fit (R²=0.9844). The adjusted good fit (Adj R²=0.9687) and predicted good fit (Pred R²=0.9277, see Table 4) were both excellent. These values ​​indicate a strong correlation between the predicted values ​​and the experimental data. The missing fit test showed no significant value (p=0.5142), supporting the model's applicability. Based on the regression analysis results and optimal extraction conditions, the predicted polysaccharide yield was approximately 4.42%. The triple-replication validation experiment yielded an average polysaccharide content of 4.19%, which highly matched the predicted value. Key factors for optimizing polysaccharide yield included extraction time and enzyme concentration, while temperature had a relatively minor impact. Furthermore, the high sufficient precision value (28.40) indicates a significant signal-to-noise ratio, while the low coefficient of variation (CV=7.85) reflects the model's accuracy and reproducibility. Overall, these results demonstrate that the Box-Behnken random response model method can provide a reliable optimization scheme and accurate prediction for the polysaccharide extraction efficiency of this system.

[0034] Table 3 Box-Behnken Experimental Design and Response Values

[0035] Table 4. Analysis of variance (ANOVA) of the quadratic model

[0036] Analysis of variance showed that enzyme concentration (C), solid-liquid ratio (D), and extraction time (B) had the greatest impact on polysaccharide yield (F=562.40; p=0.0001). Conversely, extraction temperature (A) had no significant effect (Table 4). Quadratic terms (B², C², D²; p<0.05) were all statistically significant, indicating a highly curved response surface, meaning that the maximum polysaccharide recovery rate could be achieved within the tested parameter range. Most interaction terms were insignificant, indicating that each factor had an independent effect on the yield. These studies also found very few interactions between extraction parameters. The overall regression model (after coding) can be expressed as: Y=3.8406-0.0066A+1.5065B-0.3583C-0.3676D+0.0095AB-0.2045AC-0.0798 AD-0.3680BC-0.3080BD-0.0515CD-0.2031A²-1.0228B²-0.2925C²-0.9884D² Where Y represents the polysaccharide yield (percentage). A positive B coefficient indicates that extending the hydrolysis time increases the yield, while negative C and D coefficients indicate that reducing the enzyme dosage and reducing solvent ratio decreases the extraction efficiency. The model's high predictive accuracy demonstrates its applicability in process optimization and its ability to accurately predict polysaccharide yield within the scope of the study. To visualize the interactions between extraction variables, three-dimensional response surface plots and contour plots were generated. Figure 2 ).

[0037] 4. Preparation of SB Juice

[0038] The preparation of SB juice can be briefly described as follows: Wash fresh SB berries, remove seeds, filter to obtain juice, add a compound enzyme preparation composed of pectinase and cellulase for enzymatic hydrolysis, pasteurize at 80℃ for 15 minutes, cool to room temperature and then ferment.

[0039] 5. Lactic acid bacteria activation and fermentation

[0040] Lactobacillus plantarum strains (LZU-J-LZ1-2 and LZU-J-QA5-2) were activated in MRS medium at 37°C for 24 hours. The optimal fermentation process for preparing the solid extract under specific conditions was determined through single-factor fermentation experiments. Fermentation time, extraction temperature, strain ratio, and inoculum volume all significantly affected polysaccharide yield. Figure 3 Extended fermentation time can increase yield, reaching a peak and then stabilizing after 24 hours of fermentation. Figure 3 a). Fermentation temperature is also crucial; polysaccharide content reaches its peak at 37℃. Figure 3 (b) This temperature is the optimal temperature for the strongest microbial activity. Mixed-strain fermentation is superior to single-strain fermentation, with the highest polysaccharide concentration obtained when LZU-J-LZ1-2 and LZU-J-QA5-2 were co-existed in a 1:3 ratio. Figure 3 c) indicates a synergistic effect between the two strains. Inoculum volume also affects polysaccharide yield; an inoculum volume of 2% (volume ratio) yielded the optimal response. Figure 3 d). The increase in polysaccharide content induced by fermentation may be due to the depolymerization of complex carbohydrates by microorganisms and the increased solubility of bioactive components. The final selection criteria were: fermentation time of 24 h, extraction temperature of 37℃, a 1:3 mixture of LZU-J-LZ1-2 and LZU-J-QA5-2, and an inoculum volume of 2% for all subsequent fermentation experiments.

[0041] 6. Preparation of experimental samples

[0042] Three formulations were prepared for comparative evaluation: virgin SB juice, FSB juice (fermented sea buckthorn juice), and FSB+DF (dietary fiber-assisted fermentation of sea buckthorn, prepared via water-powder dispersion). SB was fresh juice without fermentation or added extracts; FSB was prepared by fermenting SB juice using strains (LZU-J-LZ1-2 and LZU-J-QA5-2) under the optimized fermentation conditions described above. To evaluate the effect of enrichment timing, the FSB+DF formulation was prepared using two methods: (i) pre-fermentation enrichment—adding water-soluble dietary fiber to the SB juice before lactic acid bacteria inoculation; and (ii) post-fermentation enrichment—adding the same water-soluble dietary fiber after fermentation. Table 1 details the components of the traditional Chinese medicine and herbal extracts. The enrichment and fermentation procedures were adjusted according to previously reported methods. The physicochemical properties of all formulations, including pH, °Brix value, and titratable acidity, were determined.

[0043] In this embodiment, comparative experiments were conducted using traditional Chinese medicine fiber, herbal fiber, and mixed fiber, respectively. The mixed fiber is a mixture of mulberry leaf powder and bitter melon powder in a 1:1 mass ratio.

[0044] The amount of fiber added is 1% of the weight of sea buckthorn juice.

[0045] The method of adding dietary fiber before fermentation is to add it before inoculating the microbial culture and then ferment it together; the method of adding it after fermentation is to add dietary fiber directly after fermentation is completed, without further fermentation. In this embodiment, the dietary fiber from traditional Chinese medicine sources is selected from mulberry leaves, and the dietary fiber from herbal sources is selected from bitter melon.

[0046] 7. Antioxidant activity

[0047] 7.1 DPPH free radical scavenging test

[0048] The extracts of SB, FSB, and FSB+DF samples were diluted 6 times with ethanol. 0.1 mL of each diluted SB, FSB, and FSB+DF sample was mixed with 3.9 mL of DPPH solution, for a total volume of 4.0 mL. After incubation at room temperature in the dark for 30 minutes, the absorbance was measured at 517 nm using a UV-Vis spectrophotometer. Ethanol was used as a blank control, and the DPPH solution without the sample was used as a negative control. The DPPH free radical scavenging rate was calculated according to formula (1): DPPH removal ability ×100% (1) A standard curve was plotted using Trolox as the standard (concentration range 0–200 mL). -¹), the regression equation was obtained as y = 0.0041 × + 0.0013 (R² = 0.9970), where y is the DPPH scavenging rate (%) and x is the Trolox concentration (μg / mL). Subsequently, the DPPH free radical scavenging capacity of SB, FSB, and FSB+DF was expressed as milligrams of Trolox per gram dry weight equivalent (mg TE / g DW).

[0049] 7.2 ABTS Free Radical Scavenging Test

[0050] ABTS solution was prepared by adding 7 mM ABTS and 2.45 mM potassium persulfate, and incubated at room temperature in the dark for 24 hours before use. The absorbance of the ABTS solution at 734 nm was adjusted to 0.7 ± 0.02 by dilution with ethanol. For detection, 0.1 mL of diluted extract (10% of the original extract) was mixed with 3.9 mL of diluted ABTS solution, and the mixture was reacted in the dark for 10 minutes. The absorbance was measured at 734 nm using a UV-Vis spectrophotometer. Ethanol was used as a blank control, and ABTS solution without the sample was used as a control group. The ABTS free radical scavenging rate (%) was calculated according to formula (2): ABTS clearance rate (%) ×100% (2) Calibration curves for terolox (0–200 μg / mL) were prepared under identical conditions, yielding the regression equation y = 1.4000x + 0.0002 (R² = 0.9982), where y represents ABTS scavenging activity (%) and x represents terolox concentration (μg / mL). ABTS free radical scavenging activity was expressed as milligrams of terolox equivalent per gram of dry weight (mg TE / g DW).

[0051] 7.3 Ferrous Reduction Antioxidant Capacity (FRAP)

[0052] The FRAP working solution was freshly prepared by mixing sodium acetate buffer (300 mM, pH 3.6), TPTZ solution (40 mM hydrochloric acid containing 10 mM TPTZ), and ferric chloride solution (20 mM) in a 10:1:1 (volume ratio). To determine FRAP activity, 0.1 mL of diluted SB, FSB, or FSB+DF extract (10% concentration of the original extract) was mixed with 2.4 mL of the FRAP working solution, and diluted with water to a final volume of 5 mL. After incubation in the dark for 30 minutes, the absorbance was measured at 593 nm.

[0053] Calibration curves were plotted using ferrous sulfate (0–600 μg / mL), yielding the regression equation y = 0.00065x + 0.0049 (R² = 0.9985), where y represents the absorbance at 593 nm and x represents the ferrous sulfate concentration (μg / mL). FRAP values ​​are expressed as milligrams of ferrous sulfate per gram of dry weight equivalent (mg FeSO4E / g DW). The results are shown in Table 5. Table 5. Antioxidant potential of sea buckthorn juice, fermented sea buckthorn, and fiber-rich fermented sea buckthorn in DPPH, ABTS, and FRAP assays.

[0054] Values ​​are expressed as mean ± standard deviation (n=3). Different superscript letters within the same column indicate significant differences (p<0.05).

[0055] 8. Antibacterial activity

[0056] The antibacterial activity against Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922) was determined using a 96-well microplate growth inhibition assay. The final concentrations of the sample solutions were 0.625, 1.25, 2.5, 5, 10, and 20 mg / mL, respectively. The bacterial strains were cultured overnight in nutrient broth at 37°C and adjusted to an initial concentration of approximately 1 × 10⁻⁶ mg / mL. 6 CFU / mL. In a sterile 96-well microplate, 180 μL of bacterial suspension was mixed with 20 μL of each sample solution. Wells containing only culture medium served as blank control (B), wells containing bacterial suspension but no sample served as negative control (NC), and wells containing ampicillin served as positive control (PC). The microplate was incubated at 37°C, and the absorbance at 600 nm was measured at set time points over 24 hours using a microplate reader. 600 Bacterial growth was monitored. Antimicrobial activity was expressed as the growth inhibition rate relative to the untreated control group. Results are as follows: Figure 4 As shown.

[0057] The fermentation process significantly increased the total solids content and titratable acidity, while decreasing the pH (p<0.05), which is consistent with the accumulation of organic acids during lactic acid bacteria metabolism. Figure 4 ac). Based on OD 600 Growth inhibition experiment of value ( Figure 4 The results (dg) indicated that the fermented samples had a stronger growth-inhibiting effect compared to the unfermented control group. The control samples showed negligible inhibitory effects on Staphylococcus aureus and Escherichia coli within the tested concentration range, while the fermented samples, through OD... 600 Measurements showed a significant concentration-dependent inhibitory effect on bacterial growth. The optimal concentration was 1.25 mg / mL, at which FSB exhibited significantly stronger inhibitory effects against Staphylococcus aureus and Escherichia coli than the unfermented control group. Figure 4 (e,g) Among these, fiber-assisted fermentation exhibited the strongest inhibitory effect. These results indicate that fermentation-induced physicochemical changes are associated with enhanced bacterial growth inhibition. Studies of fermented fruit matrices also showed a similar trend, with increased acidity and phenolic compound transformation jointly promoting improved antimicrobial properties. These results suggest that fermentation not only improves the physicochemical quality of the samples but also enhances their antimicrobial potential against representative Gram-positive (S. aureus) and Gram-negative (E. coli) pathogens.

[0058] 9. Biochemical and Functional Analysis

[0059] Solid content of SB, FSB, and FSB+DF samples was determined by gravimetric method: A quantitative sample was dried to constant weight at 105℃, and the result was expressed as total solids percentage (w / w). Brief procedure: 100 μL of sample extract was mixed with 500 μL of Folin-Ciocalt reagent, followed by the addition of 1 mL of sodium carbonate solution. After incubation at room temperature for 30 minutes, the absorbance was measured at 765 nm. Total tannin content was determined using a gallic acid calibration curve and expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE / g DW). Total titratable acidity was determined by titration with 0.1 N sodium hydroxide (NaOH) and expressed as the percentage of citric acid equivalent per 100 mL of juice. α-glucosidase and α-glucoamylase activities were determined using a standard enzyme inhibition method. The α-glucosidase assay used a reaction system consisting of 50 μL sample extract, 100 μL of 1 mM p-nitrophenyl-α-D-glucopyranoside (pNPG), and 50 μL of α-glucosidase solution. The reaction was incubated at 37°C for 30 minutes, and then terminated with 200 μL of 0.1 M NaOH. The absorbance of the released p-nitrophenol was measured at 405 nm. The α-glucanase inhibition assay was performed under similar conditions, using 4-nitrophenyl-α-D-glucopyranoside as a substrate. The absorbance was measured at 405 nm. The results are as follows: Figure 5 As shown.

[0060] Depend on Figure 5 It can be seen that (B is the blank control, C is the control group with only culture medium added, Pre is added before fermentation, Fer is the fermentation stage, Veg is dietary fiber from herbal sources, Her is dietary fiber from traditional Chinese medicine sources, Post is added after fermentation, and Mix is ​​mixed fiber), fermentation significantly enhanced the inhibitory effects of α-glucosidase and α-glucoamylase, compared to starch paste ( Figure 5 a, c). Fiber-assisted co-fermentation further enhanced inhibitory activity, with the mixed fiber pre-fermentation formulation exhibiting the strongest effect. Figure 5 b, d).

[0061] Fermentation significantly enhanced the inhibitory effects of α-glucosidase and α-glucoamylase. For example... Figure 5 As shown in Figure a, fermentation significantly enhanced the inhibitory effect of α-glucosidase compared to SB. Fiber-assisted co-fermentation further improved α-glucosidase inhibitory activity: dietary fiber from traditional Chinese medicine sources reached 68-71%, dietary fiber from herbs reached 58-61%, and mixed fiber reached 82-88% (…). Figure 5 b).

[0062] Regarding α-glucanase inhibition, the inhibition rate in unfermented SB was 15-20%, while in FSB this value rose to 45-50%. Figure 5 c). Fiber-assisted co-fermentation resulted in inhibition levels of: 37-61% dietary fiber from traditional Chinese medicine sources, 44-47% dietary fiber from herbal sources, and 53-61% mixed fiber. Figure 5 d). Higher inhibition rates may be related to increased organic acid production, the conversion of bound phenols to free states, and the formation of low-molecular-weight polysaccharides. The mixed fibers produced the strongest inhibitory effect, indicating a synergistic supportive effect on microbial metabolism.

[0063] Furthermore, we compared pre-fermentation and post-fermentation fiber addition strategies, termed co-fermentation. The results showed that adding herbal extracts, vegetable extracts, and mixed extracts (a blend of herbal and vegetable extracts) before fermentation had a stronger inhibitory potential compared to adding them after fermentation. Figure 5 b, 5d). Among all groups, the mixed pre-fermentation group exhibited the highest inhibitory activities of α-glucosidase (84%) and α-glucosidase (58%), while the inhibitory activities of the post-fermentation addition group were 64.5% and 33%, respectively. Figure 5 (b, d) Adding cellulose before fermentation may enhance the efficiency of microorganisms in utilizing the fermentable substrate, thereby promoting the accumulation of metabolites and enhancing enzyme inhibition.

[0064] 10. LC-MS analysis of SB and FSB samples

[0065] To characterize and compare the metabolite profiles of SB and FSB samples, liquid chromatography-mass spectrometry (LC-MS) was used for analysis. Fruit juice samples (n=6) were thawed at 4°C and vortexed. 100 μL of sample was added to 400 μL of pre-cooled methanol (-20°C), vortexed, and centrifuged at 12,000 rpm at 4°C. The supernatant was collected and gently dried under a nitrogen stream. The dried residue was dissolved in 150 μL of 80% methanol (containing 4 ppm of 2-chloro-L-phenylalanine) as an internal standard, vortexed, filtered through a 0.22 μm polytetrafluoroethylene membrane filter, and finally transferred to a 2 mL LC vial for analysis.

[0066] High-performance liquid chromatography (HPLC) analysis was performed using a Vanquish UHPLC system (Thermo, USA) equipped with an ACQUITY UPLC® HSS T3 column (2.1 × 100 mm, 1.8 μm; Waters, USA). The column was kept at a constant temperature of 40 °C, the injection volume was 2 μL, and the mobile phase flow rate was 0.3 mL / min. Metabolite identification was performed using a QExactive Focus mass spectrometer (Thermo, USA) equipped with an electrospray ionization source. Mass spectrometry analysis was performed in both positive and negative ion modes. Instrument parameters were set as follows: sheath gas pressure 40 arg, auxiliary gas flow rate 10 arg, injection voltage ESI(+) 3.50 kV, ESI(-) -2.50 kV, and capillary temperature 325 °C.

[0067] The raw LC-MS data were analyzed using Compound Discoverer software (Thermo Scientific), enabling detection, retention time alignment, peak area integration, and internal standard normalization of characteristic intensities. Metabolite annotation and speculative identification were achieved at MSI2 level by comparing precise quality, retention time, and MS / MS fragmentation spectra with existing open-source databases. To ensure analytical stability and data reliability, a mixed quality control sample was prepared by mixing equal volumes of all samples and injected periodically during the analytical process. Base peak chromatograms and principal component analysis (PCA) were used to evaluate the quality control performance. For highly reproducible characteristics, a filtering mechanism based on relative standard deviation (RSD) was employed, retaining only characteristics with a QC RSD ≤ 30%. Characteristic intensities were statistically analyzed after internal standard normalization, logarithmic transformation, and scaling to eliminate systematic variation. To enhance data robustness, characteristics with excessive missing values ​​were removed. By combining multivariate analysis (VIP scoring of the supervised model) with univariate statistical tests, the differences in metabolic characteristics leading to intergroup separation were revealed. To control for multiple comparisons, the Benjamini-Hochberg false discovery rate (FDR) was used to correct the p-value. Features with VIP > 1.0 and FDR-corrected p < 0.05 were considered significant changes.

[0068] The metabolites detected by LC-MS analysis, their classification, and relative distribution are summarized in Table 6 and presented in the table below. Figure 6 The study found that the metabolite profiles exhibited highly specific fermentation-related changes, mainly reflected in the relative abundance changes of key compound groups in the comparison between SB and FSB. Figure 6(a represents SB, b represents FSB). After fermentation, the characteristic flavonoid content increased slightly, accompanied by a trace increase in terpenoids. The relative percentages of phenylpropanoids, alkaloids, organic acids, and phenolic compounds did not differ significantly between SB and FSB. Amino acids, sugars, and other low-molecular-weight metabolites showed minimal changes, with sugars increasing slightly after fermentation. Principal component analysis (PCA) successfully separated the FSB and SB samples. Figure 6 (c) This indicates that metabolic differences are regulated by the fermentation process. The average distance between the centers of the FSB samples is smaller than that between the centers of the SB samples, indicating that metabolism is more homogeneous after fermentation, which may be due to the uniformity of lactic acid bacteria activity.

[0069] Table 6 LC-MS Analysis Results

[0070] The most abundant substances in both the unfermented and fermented groups were flavonoids, with a slightly higher relative abundance in the FSB group (128) than in the SB group (122). Figure 6 The data for metabolite categories shown in d are as follows. Terpenes also showed a slight increase, with their relative abundance rising from 13.39 to 14.34 after fermentation. In contrast, the distributions of phenylpropanoids, alkaloids, organic acids, and phenols did not change significantly. Similarly, the absolute numbers of phenolic compounds (53) and organic acids (30) identified during fermentation in the SB and FSB groups were similar, indicating that fermentation does not have a general effect by radically remodeling the metabolome, but rather acts on specific metabolite categories.

[0071] Beyond the categorical changes, multivariate analysis further identified subgroups of labeled metabolites with significant discriminative power, supported by their high variable importance projection values ​​(VIPs). These identified metabolites were primarily concentrated in flavonoid and terpene-related subclasses, consistent with the post-fermentation changes in compound category distribution. In summary, liquid chromatography-mass spectrometry analysis revealed that fermentation induced subtle but persistent metabolic changes in SB juice. These changes were mainly characterized by enhanced flavonoid signatures and slight shifts in terpene-related metabolites, while other metabolite categories remained largely unchanged. Although annotation based on untargeted LC-MS provides useful information on metabolic alterations during fermentation, targeted studies are still needed to identify specific metabolites and their biological significance.

[0072] 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 sea buckthorn juice with enhanced functional properties, characterized in that, Includes the following steps: (1) Take sea buckthorn homogenate and mix it with water, then add a compound enzyme preparation for enzymatic hydrolysis to obtain enzymatically hydrolyzed sea buckthorn juice; (2) Take the enzymatically hydrolyzed sea buckthorn juice and add it to the mixed strain for fermentation to obtain fermented sea buckthorn juice; The mixed strain is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum LZU-J-LZ1-2 and Lactobacillus plantarum ( Lactiplantibacillus plantarum )LZU-J-QA5-2.

2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of sea buckthorn homogenate to water in step (1) is 1:10~30; the compound enzyme preparation is composed of pectinase powder and cellulase powder in a mass ratio of 1~2:1~2; the mass concentration of the compound enzyme preparation is 0.2~0.6%; the enzymatic hydrolysis conditions are: temperature of 40~60℃ and enzymatic hydrolysis time of 0.5~2.5h.

3. The preparation method according to claim 1, characterized in that, The mixed strain mentioned in step (2) is a mixture of Lactobacillus plantarum LZU-J-LZ1-2 and Lactobacillus plantarum LZU-J-QA5-2 in a 1:3 ratio, with an effective viable count >2×10⁻⁶. 8 CFU / mL; the fermentation treatment is as follows: fermentation temperature 35~39℃; fermentation time 20~28 hours; the inoculation amount of mixed strains is 1.5%~2.5% of the volume of enzymatically hydrolyzed sea buckthorn juice.

4. The preparation method according to claim 1, characterized in that, In step (2), dietary fiber is added before or after the fermentation process, and the amount added is 0.5% to 2.0% of the mass of the enzymatically hydrolyzed sea buckthorn juice.

5. The preparation method according to claim 4, characterized in that, The dietary fiber is dietary fiber derived from traditional Chinese medicine, herbal ingredients, or a mixture of both.

6. The preparation method according to claim 4, characterized in that, When the dietary fiber is a mixed dietary fiber, it is a mixture of dietary fiber from traditional Chinese medicine and dietary fiber from herbs in a mass ratio of 1~2:1~2.

7. The application of sea buckthorn juice prepared by the preparation method according to any one of claims 1 to 6 in the preparation of functional foods.

8. A mixed bacterial strain in the preparation method according to claim 1, characterized in that, The mixed strains were Lactobacillus plantarum LZU-J-LZ1-2 and Lactobacillus plantarum LZU-J-QA5-2; The Lactobacillus plantarum LZU-J-LZ1-2 is deposited at Guangdong Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, on July 1, 2024, with accession number GDMCC No:64799. The Lactobacillus plantarum LZU-J-QA5-2 is deposited at Guangdong Microbial Culture Collection Center, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, on July 1, 2024, with accession number GDMCC No: 64798.

9. The use of the mixed strain of claim 8 in the preparation of sea buckthorn juice.

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