Phytobacterium plantarum NXU-F1 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to a plant lactobacillus NXU-F1 strain and application thereof. The classification name of the bacterial strain is lactobacillus plantarum, the bacterial strain is preserved in Guangdong Microbial Culture Collection Center on June 20, 2025, and the preservation number of the bacterial strain is GDMCC No.66563. The invention also discloses a preparation method of the bacterial strain. The plant lactobacillus is strong in growth capability, has good adaptive capacity to environment, and can obviously increase the content of antioxidant substances when being applied to plant matrix fermentation. The lycium barbarum and grape compound fermented beverage is prepared by fermenting the lycium barbarum and grape compound fermented beverage, the content of polyphenol, flavone, polysaccharide and procyanidine in the beverage is obviously increased, and the increase of resveratrol and ferulic acid is particularly obvious; meanwhile, ester compounds are increased and aldehyde substances are reduced, so that the beverage is endowed with strong longan fruity flavor, and the flavor of harmonious sour and sweet taste and lingering aftertaste is formed.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Lactobacillus plantarum NXU-F1 and its applications. Background Technology

[0002] Phenolic compounds in plant-based foods (such as catechins, quercetin, anthocyanins, and proanthocyanidins) are a class of aromatic secondary metabolites with hydroxyl groups. They have been proven to possess antioxidant, anti-inflammatory, cardiovascular disease risk-reducing, and antimicrobial activities. Polyphenolic beverages, rich in flavonoids, tannins, and phenolic acids, can synergistically enhance free radical scavenging capabilities with vitamins C and E, making them a popular choice for health-conscious consumers. However, natural polyphenols mostly exist in bound forms, and conventional processing techniques struggle to effectively release the free active ingredients, resulting in low polyphenol content and insufficient bioavailability in products, severely limiting their health benefits.

[0003] Goji berries and grapes are two representative raw materials rich in polyphenols. However, grape wines, due to their alcohol content and limited applicability, often suffer from bitterness and unbalanced flavors in non-alcoholic beverages developed using goji berries and grapes, severely impacting consumer acceptance. Furthermore, current processes have low efficiency in enriching key active ingredients such as free polyphenols and flavonoids, resulting in low levels of functional components. Existing technologies cannot simultaneously address the conversion of bound polyphenols and flavor regulation in polyphenol beverage development. There is an urgent need to develop a new technology that can effectively solve these problems, increasing the content and bioavailability of antioxidants in beverages while reconstructing their flavor, thereby meeting consumers' demand for healthy drinks. Summary of the Invention

[0004] To address the above technical problems, this invention provides a strain of Lactobacillus plantarum NXU-F1 and its applications. This strain has a fast growth rate, can increase the content of antioxidants in plant-based foods, significantly improve the antioxidant activity of food, and simultaneously optimize flavor and texture.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Lactobacillus plantarum* NXU-F1, whose taxonomic name is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The sample was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No. 66563; the deposit address is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0006] The *Lactobacillus plantarum* NXU-F1 provided by this invention was isolated from slurry in Guyuan, Ningxia. It is a typical probiotic with strong growth ability and strong environmental adaptability. When applied to plant substrate fermentation, it can increase the content of antioxidant substances.

[0007] Its 16S-rDNA sequence (as shown in SEQ ID No. 1) is as follows: Secondly, the present invention also provides a microbial agent comprising the aforementioned *Lactobacillus plantarum* NXU-F1.

[0008] The microbial agent may also include biologically acceptable microbial carriers and excipients. The *Lactobacillus plantarum* NXU-F1 provided by this invention can be prepared into different dosage forms by selecting suitable carriers and / or excipients to improve the stability, activity, and efficacy of the microbial agent, thereby meeting different production needs. The dosage forms of the microbial agent include, but are not limited to, liquids, powders, granules, tablets, and capsules; the excipients include, but are not limited to, starch, glucose, maltodextrin, sucrose, lactose, skim milk powder, trehalose, and chitosan; the carriers include, but are not limited to, culture media, water, starch, cellulose, and gelatin.

[0009] The bacterial agent may also include other probiotics, including but not limited to Bifidobacterium spp. Bifidobacterium ) strains, Lactobacillus genus ( Lactobacillus ), Yeast ( Saccharomyces ) strain.

[0010] Preferably, the microbial agent is a microbial strain preparation for food processing.

[0011] The food processing microbial preparations described in this invention are food raw material preparations made by fermentation, enrichment, emulsification or non-emulsification, drying or non-drying, mixing or non-mixing, and packaging of one or more live microorganisms (including bacteria, filamentous fungi, and yeast) that can be used in food.

[0012] Thirdly, the present invention also provides the application of the above-mentioned Lactobacillus plantarum NXU-F1 or the above-mentioned microbial agent in the preparation of plant-based fermented beverages.

[0013] The plant-based fermented beverages described in this invention are beverages made by using plant raw materials (such as grains, beans, fruits, vegetables, tea, etc.) as a base and then fermenting them with probiotics. These include, but are not limited to, plant-based beverages and fruit and vegetable juices and their beverages.

[0014] Fourthly, the present invention also provides the application of the above-mentioned Lactobacillus plantarum NXU-F1 or the above-mentioned bacterial agent in the preparation of health foods that help with antioxidation.

[0015] The *Lactobacillus plantarum* NXU-F1 provided by this invention, when applied to plant substrate fermentation, can increase the content of antioxidant active ingredients in plant-based foods. Simultaneously, it can efficiently hydrolyze bound polyphenols and flavonoids, significantly increasing the content of free antioxidants. In vitro experiments have demonstrated that a wolfberry and grape compound fermented beverage prepared using this bacterium has antioxidant effects. Therefore, the *Lactobacillus plantarum* NXU-F1 or the aforementioned bacterial agent provided by this invention can be used to prepare health foods that contribute to antioxidant effects.

[0016] Fifthly, the present invention also provides a wolfberry and grape compound fermented beverage, wherein the raw materials of the compound fermented beverage are wolfberry juice and grape juice, and the fermentation strain includes the above-mentioned Lactobacillus plantarum NXU-F1.

[0017] The *Lactobacillus plantarum* NXU-F1 provided by this invention can efficiently transform substrates in a wolfberry and grape compound fermentation substrate, significantly increasing the content of antioxidant active ingredients such as polyphenols, flavonoids, polysaccharides and proanthocyanidins. It also imparts higher antioxidant activity and a unique fruity flavor to the beverage by secreting extracellular proteases to degrade macromolecular proteins.

[0018] The wolfberry and grape compound fermented beverage provided by this invention has a high content of polyphenols and flavonoids, strong antioxidant activity, and a rich longan fruit aroma. The overall aroma is harmonious, the taste is sweet and sour, and the aftertaste is lingering.

[0019] The wolfberry juice / grape juice of this invention is a juice or slurry product made from wolfberry (fruit part) / grape using physical methods (mechanical methods, water extraction, etc.). Optionally, the preparation method involves rehydrating dried wolfberry / grape fruit with water and then juicing, followed by solid-liquid separation to obtain wolfberry juice / grape juice; or extracting dried wolfberry fruit with hot water and then separating the solid and liquid to obtain wolfberry juice; or directly juicing fresh wolfberry / fresh grapes or juicing with water followed by solid-liquid separation to obtain wolfberry juice / grape juice; or directly juicing fresh wolfberry / fresh grapes or juicing with water followed by solid-liquid separation to obtain wolfberry juice / grape juice.

[0020] Preferably, the goji berry juice is black goji berry juice, and the grape juice is red grape juice.

[0021] Sixthly, the present invention also provides a method for preparing a wolfberry and grape compound fermented beverage, comprising the following steps: S1: After soaking dried goji berries in water, the solid and liquid are separated to obtain goji berry juice; fresh grapes are pulped to prepare grape juice; the goji berry juice and the grape juice are mixed at a mass ratio of 1:1-2, and after solid and liquid separation, the fermentation stock solution is obtained. S2: Inoculate the above-mentioned *Lactobacillus plantarum* NXU-F1 or the above-mentioned bacterial agent into the fermentation stock solution at an inoculation amount of 1×10⁻⁶. 5 -1×10 6 The mixture of CFU / mL was cultured at 33-37℃ for 20-24 h to obtain a compound fermented beverage of wolfberry and grape.

[0022] Preferably, the preparation method further includes an activation step of the *Lactobacillus plantarum* NXU-F1 or the bacterial agent.

[0023] Activated Lactobacillus plantarum NXU-F1 or its inoculum can be inoculated into the fermentation stock solution either as an activation solution or as centrifuged cells. For activation, a common culture medium suitable for activating Lactobacillus plantarum strains can be selected, such as MRS medium, or the fermentation stock solution can be used.

[0024] Preferably, in step S2, the activated microbial cells can be centrifuged, resuspended in sterile water, and then inoculated.

[0025] This inoculation method avoids introducing the culture medium components used during bacterial activation into the fermentation broth, thus reducing impurities in the final beverage.

[0026] Preferably, in step S1, the mass-to-volume ratio (g / mL) of the dried wolfberry fruit and water is 1:40, the extraction temperature is 60°C, and the extraction time is 30 min.

[0027] Preferably, the goji berry juice is black goji berry juice, and the grape juice is red grape juice; the black goji berry juice and the red grape juice are mixed in a mass ratio of 1:1.5.

[0028] More preferably, in step S2, the culture is carried out at 37°C for 24 h. Attached Figure Description

[0029] Figure 1 The colony morphology (A) and Gram staining results (B) of *Lactobacillus plantarum* NXU-F1 strain in Example 1 are shown. Figure 2 This is a phylogenetic tree of the *Lactobacillus plantarum* NXU-F1 strain in Example 1; Figure 3 To verify the fingerprint spectrum of the black goji berry and grape compound fermented beverage before and after fermentation in Example 1 (U1, U2, and U3 are all fermented beverage treatment groups, and F1, F2, and F3 are all unfermented beverage treatment groups). Figure 4 To verify the liquid chromatogram of ferulic acid in Example 2; Figure 5 To verify the liquid chromatogram of resveratrol in Example 2; Figure 6 To verify the liquid chromatogram of proanthocyanidins in Example 2; Figure 7 To verify the protein concentration results of the black goji berry and grape compound fermented beverage before and after fermentation in Example 2; Figure 8 To verify the DPPH scavenging rate results of the black goji berry and grape compound fermented beverage before and after fermentation in Example 3; Figure 9 To verify the ABTS scavenging rate results of the black goji berry and grape compound fermented beverage before and after fermentation in Example 3; Figure 10To verify the hydroxyl radical scavenging rate results of the black goji berry and grape compound fermented beverage before and after fermentation in Example 3; Figure 11 To verify the DNA oxidative damage before and after fermentation of the black goji berry and grape compound fermented beverage in Example 3; (lane 1 is the DL5000 marker, lanes 2-5 are 1×10⁻⁶ markers respectively) -2 1×10 -3 1×10 -4 1×10 -5 The experimental group of mg / mL fermentation extract, lanes 6-9, was 1×10 mg / mL. -2 1×10 -3 1×10 -4 1×10 -5 The experimental group was composed of mg / mL unfermented extract; lane 10 was the PBS buffer control group; and lane 11 was the 1×10 mg / mL unfermented extract group. -3 (mg / mL ascorbic acid control group) Detailed Implementation To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] The strains used in the following embodiments of the present invention are from the following sources: Lactobacillus plantarum NXU-F1 was deposited on June 20, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 66563. The deposit address is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0031] The culture medium used in the following embodiments of the present invention is as follows: MRS medium: Take 10 g peptone, 5 g yeast extract, 20 g glucose, 10 g beef extract, 5 g anhydrous sodium acetate, 2 g triammonium citrate, 2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate and 1 mL Tween-80 and dissolve in 900 mL of water. Add 15 g agar powder to the solid medium and bring the volume to 1000 mL. Autoclave at 121℃ for 15-20 min.

[0032] Unless otherwise specified, all raw materials, reagents, and kits used in the following embodiments of the present invention are commercially available. The black goji berries used were produced in Haiyuan County, Zhongwei City, Ningxia, and the red grapes used were Red Globe grapes produced in Yinchuan, Ningxia.

[0033] Example 1 This embodiment provides a strain of Lactobacillus plantarum NXU-F1, which was isolated from slurry water in Guyuan, Ningxia.

[0034] 1. Isolation and screening of Lactobacillus plantarum NXU-F1 Take 1-2 g of solidified slurry and inoculate it into sterile 0.9% physiological saline. Mix thoroughly. Dilute the sample 10-fold serially with sterile physiological saline to form different concentration gradients from 10⁻¹ to 10⁻⁸. Take 200 μL of each concentration and spread it evenly on MRS agar plates, with three replicates for each concentration. Incubate the spread culture medium upside down in a 37℃ incubator for 48 h. Identify the *Lactobacillus plantarum* colonies based on their morphological and color characteristics, and perform the calcium dissolution zone test. The calcium dissolution zone specific medium is: add 1% CaCO₃ to the MRS solid medium. Select single colonies that produce the morphological characteristics of a clear zone on the plate and further isolate and purify them using the streak plate method. After streak purification 2-3 times, obtain a single strain. One strain with acid-producing ability was isolated and named NXU-F1. Prepare a glycerol strain by mixing bacterial culture with 50% glycerol at a ratio of 1:1 and store it at -80℃.

[0035] 2. Identification of Lactobacillus plantarum NXU-F1 strain 2.1 Colony and cell morphology characteristics of NXU-F1 strain Colony morphology observation: The colonies are milky white, round, with neat edges, smooth surface, and slightly raised. See details... Figure 1 -A.

[0036] Microscopic morphology of the bacteria: obligate aerobic Gram-negative bacteria, short rod-shaped, spore-forming; see details. Figure 1 -B.

[0037] 2.2 Sequencing analysis of 16S rDNA of NXU-F1 strain The selected strains were streaked onto MRS medium and cultured at 37°C for 2 days. The bacterial cells were collected and sent to Beijing Liuhe BGI Genomics Co., Ltd. for 16S rDNA sequencing. The sequencing results are shown in SEQ ID NO.1.

[0038] Alignment of the 16S rDNA sequence of strain NXU-F1 with EzBioCloud showed that this strain is derived from *Lactobacillus plantarum* (Lactobacillus). Lactiplantibacillus plantarum The homology was 99.72%. A phylogenetic tree was constructed using MEGA software, based on the 16S rDNA sequence, using the Neighbour-Joining method with a Btoostrap value of 1000. The phylogenetic tree is shown below. Figure 2 It can be seen that this strain is related to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum It has the highest affinity.

[0039] In summary, based on morphological characteristics and molecular biological analysis, strain NXU-F1 was identified as *Lactobacillus plantarum* (Lactobacillus plantarum). Lactiplantibacillus plantarum The strain was named *Lactobacillus plantarum* NXU-F1. It was deposited on June 20, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 66563); the address is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0040] Example 2 This embodiment provides a liquid inoculum of Lactobacillus plantarum NXU-F1, which is composed of Lactobacillus plantarum NXU-F1 and MRS liquid culture medium, and is prepared by the following steps: (1) Streak Lactobacillus plantarum NXU-F1 onto an MRS plate and incubate at 37°C for 1 day.

[0041] (2) Select a single colony of Lactobacillus plantarum NXU-F1 from the MRS plate and inoculate it onto the MRS liquid medium for seed culture. Culture at 37℃ and 200 rpm for 12 h.

[0042] (3) After the seed culture is completed, the inoculum is transferred to MRS liquid medium at 37℃ and 200 rpm for 12 h to obtain the liquid bacterial agent of Lactobacillus plantarum NXU-F1.

[0043] The test results showed that the viable count of the bacterial agent was 1×10⁻⁶. 8 -4×10 8 CFU / mL.

[0044] Example 3 This embodiment provides a *Lactobacillus plantarum* NXU-F1 powder, which is prepared by the following steps: S1: Inoculate *Lactobacillus plantarum* NXU-F1 onto MRS liquid medium for seed culture at 37°C and 200 rpm for 24 h. Re-inoculate into MRS liquid medium at a 2% inoculum for activation culture, repeating twice.

[0045] S2: Activated *Lactobacillus plantarum* NXU-F1 was inoculated into MRS liquid medium at a 2% inoculum size and cultured statically at 37°C for 20 h. The bacterial sludge was then collected by centrifugation at 6000 rpm / min for 10 min at 4°C and washed twice with 0.85% physiological saline. 10 g of the bacterial sludge was added to 10 mL of a 10 wt% trehalose solution and thoroughly vortexed to prepare a bacterial suspension.

[0046] S3: The obtained bacterial suspension was spray-dried to obtain *Lactobacillus plantarum* NXU-F1 powder. The spray-drying conditions were: inlet temperature 110℃, outlet temperature 55℃, fan frequency 60 Hz, impact pin interval 5 s, execution time 0.3 s, and feed rate 250 mL / h; or the obtained bacterial suspension was pre-frozen at -80℃ for 8 h and then transferred to a vacuum freeze dryer for freeze-drying. The freeze-drying parameters were: -80℃, vacuum degree 1.0 Pa, and freezing time 24 h.

[0047] The powder was tested and found to have a viable bacteria count of 1×10⁻⁶. 8 -3×10 8 CFU / g.

[0048] Example 4 This embodiment provides a black goji berry and grape compound fermented beverage, which is prepared by the following steps: S1: Weigh 5 g of black goji berries, add 200 mL of distilled water, and soak in a 60℃ water bath for half an hour to prepare black goji berry juice; accurately weigh 200 g of red grapes, put them into a pulper and pulp them to prepare grape juice.

[0049] S2: Mix black goji berry juice and grape juice at a mass ratio of 1:1.5, and filter the mixture using a vacuum filter to obtain the fermentation stock solution.

[0050] S3: After the frozen Lactobacillus plantarum NXU-F1 glycerol tubes were brought to room temperature, the inoculum was inoculated onto an MRS plate for culture. After two passages, a single colony was selected and inoculated into 150 mL of the original fermentation broth. The culture was activated at 37°C for 12 h to obtain the seed culture.

[0051] S4: Inoculate the seed culture into the fermentation stock solution, controlling the inoculation amount of *Lactobacillus plantarum* NXU-F1 to be 1×10⁻⁶. 5 The black goji berry and grape compound fermented beverage was obtained by culturing at 37℃ for 20 h with CFU / mL.

[0052] Example 6 This embodiment provides a black goji berry and grape compound fermented beverage, which is prepared by the following steps: S1: Weigh 5 g of black goji berries, add 150 mL of distilled water, and soak in a 40℃ water bath for half an hour to prepare black goji berry juice; accurately weigh 200 g of red grapes, put them into a pulper and pulp them to prepare grape juice.

[0053] S2: Mix black goji berry juice and grape juice in a 1:1 mass ratio, filter out solid insoluble matter to obtain fermentation stock solution.

[0054] S3: After the frozen Lactobacillus plantarum NXU-F1 glycerol tubes were brought to room temperature, the bacterial strain was inoculated onto MRS plates for culture. After two passages, a single colony was selected and inoculated into 150 mL of MRS liquid culture. After activation culture at 37°C for 12 h, the bacterial cells were obtained by centrifugation and resuspended in sterile water to obtain the resuspension.

[0055] S4: Inoculate the resuspended solution into the fermentation stock broth, controlling the inoculum size of *Lactobacillus plantarum* NXU-F1 to be 1×10⁻⁶. 6 The black goji berry and grape compound fermented beverage was obtained by culturing at 35℃ for 24 h with CFU / mL.

[0056] Example 6 This embodiment provides a black goji berry and grape compound fermented beverage, which is prepared by the following steps: S1: Weigh 5 g of black goji berries, add 250 mL of distilled water, and soak in a 60℃ water bath for half an hour to prepare black goji berry juice; accurately weigh 200 g of red grapes, put them into a pulper and pulp them to prepare grape juice.

[0057] S2: Mix black goji berry juice and grape juice in a mass ratio of 1:2 to obtain the fermentation stock solution.

[0058] S3: Add *Lactobacillus plantarum* NXU-F1 powder (provided in Example 3) to the fermentation stock solution, controlling the inoculum size of *Lactobacillus plantarum* NXU-F1 to be 1×10⁻⁶. 6 The black goji berry and grape compound fermented beverage was obtained by culturing at 33℃ for 24 h with CFU / mL.

[0059] Verification Example 1 The flavor composition changes and sensory evaluation of the black goji berry and grape compound fermented beverage prepared in Example 4 were determined.

[0060] 1. Detection of changes in flavor compounds A 10 mL sample of the black goji berry and grape compound fermented beverage was placed in a 20 mL headspace vial and incubated at 80℃ for 15 min. Volatile flavor components were then determined using a FlavorSpec® flavor analyzer, with three replicates. Chromatographic conditions: db-wax capillary column (15 m × 0.53 mm × 1.0 μm), carrier gas (drift gas) nitrogen (purity ≥99.999%), flow rate 150 mL / min, drift gas temperature 45℃; IMS temperature 45℃. Injector flow program: 2 mL / min for 10 min, increased to 10 mL / min for 10 min, analysis time 20 min.

[0061] By comparing the volatile components of unfermented and fermented beverages using GC-IMS technology, a total of 32 substances were identified (such as...). Figure 3 As shown in the figure, the compounds cover nine classes, including esters, aldehydes, and alcohols. The concentration of esters (such as ethyl acetate and ethyl hexanoate) increased significantly after fermentation, possibly due to the esterification of acid-alcohol precursors during fermentation, which imparts fruity aroma characteristics to the beverage. The unfermented sample showed higher levels of some aldehydes (such as hexanal), which decreased after fermentation due to the inhibition of lipid oxidation in a reducing environment. Simultaneously, sulfur-containing compounds and pyrazines showed localized enhancement after fermentation, possibly related to yeast metabolism, amino acid degradation, or Maillard reactions. These dynamic changes reveal the mechanism by which fermentation processes synergistically influence beverage flavor formation by regulating redox states and microbial metabolism. The brightness of sulfur-containing compounds and pyrazines showed localized enhancement after fermentation, and their formation may depend on specific metabolic pathways. These brightness changes not only reflect the synergistic effects of complex chemical reactions during fermentation but may also provide a material basis for the unique flavor of the beverage.

[0062] 2. Sensory evaluation Sensory evaluation was conducted on the black goji berry and grape compound fermented beverage prepared in Example 4. The evaluation team members (15 people) were asked to score the beverage according to four indicators: color, aroma, taste and state. For details, please refer to the sensory evaluation table (as shown in Table 1).

[0063] Table 1 Sensory Evaluation Form

[0064] The compound fermented beverage has a purple color, is clear and bright with a harmonious luster, scoring 19 points; the beverage has a rich longan fruit aroma, with a harmonious fragrance, scoring 38 points; the beverage has a sweet and sour taste, is mellow and harmonious, with a lingering aftertaste, scoring 29 points; the beverage has a uniform texture, is clear and free of sediment, scoring 10 points; the overall sensory score of the black goji berry and grape compound fermented beverage is 96 points.

[0065] Based on the aforementioned changes in flavor compounds, the beverage's rich longan aroma is likely due to the metabolic activity of probiotics, which significantly increases the content of esters [such as ethyl heptaate, hexyl butyrate, ethyl acetate, ethyl 2-oxopropionate, methyl anthranilate, 1-methoxy-2-propylacetate, methyl acetic acid, (4-methylphenyl) ester, etc.] and alcohols [such as 2-furanol, (E)-2-hexen-1-ol, butanol, etc.], resulting in the beverage's rich longan aroma. Simultaneously, the content of aldehydes [(E)-2-heptenal, 3-methylbutanal, etc.] and ketones [3-methyl-2(5H)-furanone, 4,5-dihydro-3(2H)-thiophenone, etc.] is significantly reduced, resulting in a marked decrease in the original astringency of the unfermented beverage after fermentation.

[0066] Verification Example 2 The content of antioxidant active substances and protein concentration of the black goji berry and grape compound fermented beverage prepared in Example 4 were detected.

[0067] 1. Detection of the content of antioxidant active substances such as polyphenols, flavonoids, and polysaccharides in black goji berry and grape compound fermented beverage. Total phenol content: determined using gallic acid as standard and the Folin-Ciocalteu method; Flavonoid content: determined using rutin as standard and the sodium nitrite-aluminum nitrate-sodium hydroxide method; Flavonoid content: determined using rutin as standard and the aluminum salt complexation method; Polysaccharide content: determined using glucose as standard and the phenol-sulfuric acid method.

[0068] Table 2. Types and concentrations of antioxidant active substances in fermented beverages

[0069] Note: Different letters in the numbers in the table represent significant differences between groups. P <0.05) The results showed that fermentation with *Lactobacillus plantarum* NXU-F1 significantly increased the content of polyphenols, flavonoids, and polysaccharides in the beverage (p<0.05), indicating that fermentation can increase the content of antioxidant active substances. The increase in flavonoids was the most significant (20.19%), followed by polysaccharides (8.49%). Their strong antioxidant properties and physiological regulatory functions significantly enhanced the health value of the beverage. The simultaneous increase in polyphenols and flavonoids (+6.78% and +6.62%, respectively) further synergistically improved the overall antioxidant and anti-inflammatory activities. This may be due to the biotransformation of the plant matrix by microbial metabolism during fermentation—releasing bound active ingredients (such as converting flavonoid glycosides to aglycones and dissociating polysaccharide complexes) through enzymatic hydrolysis of cell walls, and promoting the dissolution of macromolecules, thereby systematically improving the bioavailability of functional factors.

[0070] 2. Detection of ferulic acid, resveratrol, and proanthocyanidins in black goji berry and grape compound fermented beverage. A suitable amount of beverage sample was taken, filtered through a 0.45 μm aqueous filter membrane, and the contents of ferulic acid, resveratrol, and proanthocyanidins were determined by high-performance liquid chromatography (HPLC). The results are as follows: Figure 4-6 As shown.

[0071] Chromatographic conditions for ferulic acid determination: Column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (A) - 0.1% phosphoric acid (B) with gradient elution (0–8 min, 22% A; 9–19 min, 26% A; 20–34 min, 80% A; 35–40 min, 22% A); column temperature: 20 °C; flow rate: 1.0 mL / min; detection wavelength: 286 nm; injection volume: 10 μL.

[0072] Chromatographic conditions for measuring resveratrol: Agilent XDB-C18 column (4.6 mm × 250 mm, 5 μm), gradient elution with acetonitrile (A)-water (B) as mobile phase (0-30 min, A 22%-40%; 30-40 min, A 40%-22%); detection wavelength, 306 nm; flow rate, 1.0 mL / min; injection volume, 10 μL.

[0073] Chromatographic conditions for measuring proanthocyanidins: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); column temperature, 35℃; mobile phase, 1% formic acid aqueous solution: 1% formic acid acetonitrile solution, gradient elution (see table below); flow rate, 0.8 mL / min; wavelength, 530 nm; injection volume, 20.0 μL.

[0074] Table 3 Gradient elution conditions of mobile phase

[0075] The contents of ferulic acid, resveratrol, and proanthocyanidins in the black goji berry and grape fermented beverage before and after fermentation were calculated based on the standard curve and peak area. After fermentation, the ferulic acid content increased from 0.13 μg / mL to 4.16 μg / mL, resveratrol from 4.28 μg / mL to 11.81 μg / mL, and proanthocyanidins from 40.70 μg / mL to 56.18 μg / mL. This significant increase in the content of these substances after fermentation may be attributed to the biotransformation of bound phenolic acids in the substrate by microorganisms. Black goji berries and grapes are rich in phenolic esters (such as chlorogenic acid and caffeic acid ferulic acid esters). These esters exist mostly in bound form in their natural state and have low bioavailability. These ester compounds are hydrolyzed by esterases secreted by lactic acid bacteria, releasing free substances, thereby significantly increasing the content of these components.

[0076] 3. Measurement of protein concentration before and after fermentation of black goji berry and grape compound fermented beverage The protein concentration of the black goji berry and grape compound fermented beverage before and after fermentation was determined using the BCA protein concentration assay kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.). The results are shown in the table below. Figure 7 .

[0077] As shown in the figure, the protein concentration in the beverage after fermentation (21.67 mg / mL) was significantly higher than that before fermentation (17.49 mg / mL), with an increase of approximately 23.9%. This change is likely closely related to the metabolic activities of microorganisms during fermentation. In the early stages of fermentation, microorganisms may secrete extracellular proteases to degrade macromolecular proteins in the culture medium, generating soluble peptides or amino acids, thereby increasing the total protein concentration in the solution.

[0078] Verification Example 3 The in vitro antioxidant activity of the black goji berry and grape compound fermented beverage prepared in Example 4 was tested.

[0079] 1. DPPH scavenging rate determination After preparing samples of black goji berry and grape compound fermented beverage with different concentrations before and after fermentation, 1 mL of each sample was placed in stoppered test tubes and labeled as "unfermented sample" and "fermented sample," respectively. 2 mL of 0.1 mM DPPH anhydrous ethanol solution was added to each test tube, and the mixture was immediately shaken. The two test tubes were placed at room temperature in the dark for 30 min. After the reaction, the absorbance of the solution in each test tube at 517 nm was measured using a spectrophotometer. Simultaneously, the absorbance of the blank control group (sterile water instead of the sample) and the background group (solvent instead of DPPH anhydrous ethanol solution) were measured. The DPPH scavenging rate was calculated according to formula (1), and the results are shown in [the table below]. Figure 8 .

[0080] Clearance rate (%) = [1 - (A)] sample -A blank ) / (A control Formula (1) × 100; Among them: A sample A represents the absorbance value of the sample. control The absorbance value of the blank control group; A blank This represents the absorbance value of the background group.

[0081] 2. ABTS Scavenging Rate Determination Mix 0.89 mL of 140 mmol / L ammonium persulfate solution with 50 mL of 7 mmol / L ABTS solution, let stand overnight at room temperature in the dark, and then store at 4℃. Dilute the ABTS stock solution with deionized water to make the absorbance of the mixture at 734 nm between 0.68 and 0.72, thus obtaining the ABTS working solution. Take 0.1 mL of each of the black goji berry and grape compound fermented beverage samples before and after fermentation, place them in a 96-well microplate, and label them as "unfermented sample" and "fermented sample," respectively. Add 1.9 mL of ABTS working solution to each of the two wells and mix immediately. After reacting in the dark for 6 min, use a microplate reader to measure the absorbance of the solutions in the two wells at a wavelength of 734 nm. At the same time, measure the absorbance of the blank control group (sterile water instead of the sample) and the background group (solvent instead of ABTS solution). Calculate the DPPH scavenging rate according to formula (2), and the results are shown in [the table below]. Figure 9 .

[0082] Sweep rate (%) = [1 - (A1 - A2) / A0] × 100 Equation (2); Where: A1 is the absorbance value of the sample; A2 is the absorbance value of the background group; A0 is the absorbance value of the blank control group.

[0083] 3. Determination of hydroxyl radical scavenging rate Add 1.0 mL of 9.0 mmol / L FeSO4 solution to centrifuge tubes and label them as "unfermented sample" and "fermented sample," respectively. Add 1.0 mL of black goji berry and grape compound fermented beverage samples before and after fermentation to the two centrifuge tubes. Add 1.0 mL of 9.0 mmol / L H2O2 solution to the two centrifuge tubes, shake well, and let stand for 11 min. After standing, add 1.0 mL of 9.0 mmol / L salicylic acid-ethanol solution to the two centrifuge tubes and shake well. Place the two centrifuge tubes in a 37℃ constant temperature water bath for 30 min and then remove and cool. Centrifuge the cooled mixture at 3600 r / min for 11 min, and take the supernatant to measure the absorbance at 510 nm. At the same time, measure the absorbance of the sample control group (using 1 mL of distilled water instead of salicylic acid solution) and the blank control group (using 1 mL of distilled water instead of sample solution). Calculate the hydroxyl radical scavenging rate according to formula (3), and the results are shown in [the table below]. Figure 10 .

[0084] Sweep rate (%) = [A0 - (A1 - A2) / A0] × 100 (Equation 3); Where: A1 is the absorbance value of the sample; A0 is the absorbance value of the blank control group; A2 is the absorbance value of the sample control group.

[0085] The results show that, at the same dilution factor, the clearance rate of the fermented samples was significantly higher than that of the unfermented samples, and showed a clear dose-dependent effect, indicating that fermentation significantly enhances the antioxidant capacity of the beverage.

[0086] 4. DNA oxidative damage assay For the DNA damage protection assay, there were four groups: a sample group, an ascorbic acid control group, a PBS buffer control group, and a plasmid group. In the sample extract experimental group, FeSO4 solution, H2O2 solution, and extract solutions of different concentrations (1×10⁻⁶) were used. -2 1×10 -3 1×10 -4 and 1×10 -5 Add mg / mL of the solution to a reaction vessel, then add pBR322 plasmid, incubate at 37°C for 30 min, and then add loading buffer for electrophoresis. The optimized electrophoresis conditions are as follows: electrophoresis at 72 V for 15 min, followed by electrophoresis at 122 V for 40 min. After electrophoresis, images are taken using a gel imaging system. In the ascorbic acid control group, PBS buffer control group, and plasmid group, in addition to using 1×10 mg / mL of the solution... -3Except for replacing the extract solution with mg / mL ascorbic acid solution, 0.01 mol / L PBS buffer (pH 7.4), and distilled water, the other procedures were the same as those for the sample treatment group.

[0087] Electrophoresis images of the experimental group, ascorbic acid control group, PBS buffer control group, and plasmid group were obtained according to the experimental method. Figure 11 As can be seen, the brightness of bands 2-5 gradually decreased with decreasing concentration of the fermented beverage, indicating that the protective effect of the fermented beverage against DNA damage is dose-dependent. Simultaneously, the brightness of bands 6-9 in the unfermented beverage also gradually decreased with decreasing concentration. The bands of the fermented beverage extract at the same concentration were brighter than those of the unfermented beverage extract at the same concentration, indicating that the protective effect of the fermented beverage against DNA damage is significantly higher than that of the unfermented beverage.

[0088] In summary, the *Lactobacillus plantarum* NXU-F1 strain provided by this invention exhibits strong growth ability, can efficiently hydrolyze bound polyphenols and flavonoids, significantly increases the content of free antioxidants, and improves the bioavailability of polyphenols. It is suitable for preparing plant-based fermented beverages and health foods that contribute to antioxidant effects. Furthermore, the wolfberry and grape compound juice prepared using the *Lactobacillus plantarum* NXU-F1 strain provided by this invention is rich in polyphenols and flavonoids, exhibits a pleasant longan aroma, a harmonious sweet and sour taste, and excellent nutritional value and flavor.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* NXU-F1, characterized in that, Its classification name is *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The sample was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No. 66563; the deposit address is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

2. A microbial agent, characterized in that, It contains Lactobacillus plantarum NXU-F1 as described in claim 1.

3. The application of the *Lactobacillus plantarum* NXU-F1 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of plant-based fermented beverages.

4. The use of *Lactobacillus plantarum* NXU-F1 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of health foods that help with antioxidation.

5. A compound fermented beverage of wolfberry and grape, characterized in that, The raw materials for the compound fermented beverage are wolfberry juice and grape juice, and the fermentation strain includes *Lactobacillus plantarum* NXU-F1 as described in claim 1.

6. The wolfberry and grape compound fermented beverage according to claim 5, characterized in that, The goji berry juice is black goji berry juice, and the grape juice is red grape juice.

7. A method for preparing a wolfberry and grape compound fermented beverage, characterized in that, Includes the following steps: S1: After soaking dried goji berries in water, the solid and liquid are separated to obtain goji berry juice; fresh grapes are pulped to prepare grape juice; the goji berry juice and the grape juice are mixed at a mass ratio of 1:1-2, and after solid and liquid separation, the fermentation stock solution is obtained. S2: Inoculate the *Lactobacillus plantarum* NXU-F1 according to claim 1 or the bacterial agent according to claim 2 into the fermentation stock solution, with an inoculation amount of 1×10⁻⁶. 5 -1×10 6 The mixture of CFU / mL was cultured at 33-37℃ for 20-24 h to obtain a compound fermented beverage of wolfberry and grape.

8. The preparation method according to claim 7, characterized in that, The preparation method further includes an activation step of the *Lactobacillus plantarum* NXU-F1 or the bacterial agent.

9. The preparation method according to claim 7, characterized in that, In step S1, the mass-to-volume ratio (g / mL) of dried goji berries to water is 1:40, the extraction temperature is 60℃, and the time is 30 min; and / or The goji berry juice is black goji berry juice, and the grape juice is red grape juice; the black goji berry juice and the red grape juice are mixed in a mass ratio of 1:1.

5.

10. The preparation method according to claim 9, characterized in that, In step S2, the culture is carried out at 37°C for 24 h.

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