A strain of fermenting *Lactobacillus mucilaginosus* and its application in the preparation of fermented wolfberry juice to enhance immunity.

Abstract

This invention belongs to the field of food fermentation and functional beverage technology, specifically relating to a strain of *Lactobacillus fermentans* and its application in the preparation of fermented goji berry juice that enhances immunity. The *Lactobacillus fermentans* strain LFY21 was deposited on November 17, 2025, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20252569; the deposit address is Wuhan University. This strain grows vigorously in goji berry juice, but exhibits weak acid production during fermentation, resulting in a high sugar-acid ratio, good flavor and palatability, and high free radical scavenging activity and total phenol content. Furthermore, animal experiments have demonstrated that fermented goji berry juice prepared with this strain can effectively enhance immune function, showing potential as an immune enhancer.

Description

Technical Field

[0001] This invention belongs to the field of food fermentation and functional beverage technology, specifically relating to a strain of fermenting Lactobacillus mucilaginosus and its application in the preparation of fermented wolfberry juice that enhances immunity. Background Technology

[0002] Goji berries ( Lycium spp. Goji berries are rich in active ingredients such as polysaccharides, flavonoids, anthocyanins, and polyphenols, possessing health benefits such as antioxidant, anti-inflammatory, and immune-regulating effects, making them an important raw material for developing functional beverages. However, traditional goji berry juice products suffer from problems such as a monotonous flavor, excessive sourness, and low utilization rate of active ingredients.

[0003] In recent years, lactic acid bacteria fermentation technology has been widely used in the development of plant-based beverages, improving flavor and enhancing nutritional value. However, conventional lactic acid bacteria (such as...) Lactobacillus plantarum , Lactobacillus rhamnosus When fermenting high-sugar, high-acid substrates, wolfberries typically produce large amounts of lactic acid, resulting in excessive acidity and a sharp taste, which limits the sensory appeal of the product. Furthermore, traditional fermentation often directs carbon flow solely towards lactic acid production, failing to fully stimulate the release of functional substances such as polyphenols and anthocyanins. Therefore, developing a novel wolfberry fermentation process that exhibits significant low acidification characteristics, balanced flavor, and enhanced antioxidant activity has important industrial application value. Summary of the Invention

[0004] To address the above-mentioned technical problems, this invention provides a strain of *Lactobacillus fermentatus* and its application in the preparation of fermented wolfberry juice that enhances immunity. Fermenting wolfberry juice with this *Lactobacillus fermentatus* strain LFY21 yields a low-acidity, highly oxidizing fermented wolfberry juice, which also enhances the body's immunity.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides a strain of *Lactobacillus fermentatus* LFY21, whose taxonomic name is... Limosilactobacillus fermentum LFY21 was deposited at the China Center for Type Culture Collection (CCTCC) on November 17, 2025, with accession number CCTCC NO: M 20252569; the deposit address is Wuhan University.

[0007] The *Lactobacillus mucinus* strain LFY21 provided in this invention was isolated and screened from traditionally naturally fermented milk samples purchased from a local market in Kashgar, Xinjiang. Experiments show that this strain grows vigorously in wolfberry juice, but exhibits weak acid production during fermentation, resulting in a wolfberry juice with a high sugar-acid ratio, good flavor and palatability, and high free radical scavenging activity and total phenol content. Furthermore, animal experiments have demonstrated that fermented wolfberry juice prepared with this strain can induce DTH in mice, enhance the transformation ability of mouse spleen lymphocytes, increase the number of antibody-producing cells in mice, significantly increase serum hemolysin levels in mice, increase macrophage phagocytic rate and phagocytic index, and enhance NK cell activity, thereby effectively improving immune function. Moreover, the comprehensive effects of fermenting wolfberry juice with this strain are superior to those of other lactic acid bacteria.

[0008] A second aspect of the present invention provides the application of the above-mentioned fermented Lactobacillus mucinus LFY21 in fermented wolfberry juice.

[0009] Preferably, the goji berry juice is black goji berry juice.

[0010] A third aspect of this invention provides a method for preparing fermented wolfberry juice that enhances immunity using the above-mentioned *Lactobacillus fermentans* LFY21 strain, specifically including the following operations:

[0011] After removing the stems from the goji berries, wash them with water, and then soak them in a mixture of 0.01%-0.02% sodium isoascorbate solution for 8-15 hours to rehydrate them.

[0012] After rehydration, the goji berries are mashed into a pulp, and pectinase is added at 0.1%-0.15% of the weight of the goji berries before rehydration for 2-4 hours. After filtration, goji berry juice is obtained.

[0013] Filter the goji berry juice, pasteurize it at 60-65℃ for 30-40 minutes, cool it to room temperature, inoculate it with a 2%-3% v / v suspension of Lactobacillus fermentum strain LFY21, and ferment it at 30-37℃ for 24-48 hours.

[0014] Preferably, the goji berry fruit is black goji berry fruit.

[0015] Preferably, the pectinase has an enzyme activity of 30,000 U / g.

[0016] Preferably, the viable cell concentration of the *Lactobacillus fermentans* LFY21 strain suspension is 6 × 10⁻⁶. 6 CFU / m.

[0017] A fourth aspect of the present invention provides fermented wolfberry juice prepared by the above method.

[0018] The fifth aspect of the present invention provides the use of the above-mentioned fermented wolfberry juice in the preparation of health foods that help enhance immunity and / or help with antioxidation.

[0019] The beneficial effects of this invention are as follows:

[0020] The *Lactobacillus mucinus* LFY21 provided by this invention has the characteristic of low acid production. Using it as a fermentation bacterium to ferment goji berry juice can reduce the acidity of the resulting goji berry juice and increase the sugar-acid ratio, thereby improving the flavor and taste. Furthermore, the fermented goji berry juice also has high antioxidant capacity and high total phenolic content, solving the problems of monotonous flavor, excessive sourness, and low utilization rate of active ingredients in goji berry juice, as well as the problems of excessively high acidity and low release of polyphenols and other active substances when fermenting plant-based beverages with lactic acid bacteria. In addition, experiments conducted by this invention have shown that the fermented goji berry juice prepared using *Lactobacillus mucinus* LFY21 can induce DTH in mice, enhance the transformation ability of mouse spleen lymphocytes, increase the number of antibody-producing cells in mice, significantly increase the serum hemolysin level in mice, increase the phagocytic rate and phagocytic index of macrophages, and increase NK cell activity. Therefore, it has the effect of enhancing the body's immune function and has the potential to be used as an immune enhancer. Attached Figure Description

[0021] Figure 1 These are photographs of the plate colony morphology and microscopic morphology (oil immersion 100×) of Lactobacillus fermentans LFY21 in Example 1 of the present invention.

[0022] Figure 2 The viable cell counts of wolfberry juice fermented by different strains at different fermentation times in Example 2 of the present invention;

[0023] Figure 3 The pH values ​​of unfermented wolfberry juice and wolfberry juice fermented with different strains at different fermentation times in Example 2 of this invention;

[0024] Figure 4 The sugar-acid ratio of unfermented wolfberry juice and wolfberry juice fermented with different strains in Example 2 of the present invention;

[0025] Figure 5 The sensory characteristics of unfermented wolfberry juice and wolfberry juice fermented with different strains in Example 2 of the present invention;

[0026] Figure 6 The antioxidant properties of unfermented wolfberry juice and wolfberry juice fermented with different strains in Example 2 of the present invention;

[0027] Figure 7 The total phenol content of unfermented wolfberry juice and wolfberry juice fermented by different strains in Example 2 of the present invention;

[0028] Figure 8The content of volatile flavor compounds in unfermented wolfberry juice and wolfberry juice fermented with different strains in Example 2 of the present invention;

[0029] Figure 9 The effects of unfermented wolfberry juice and wolfberry juice fermented with different strains on delayed-type hypersensitivity in mice, as shown in Example 3 of this invention; * indicates comparison with the negative control group. p <0.05;

[0030] Figure 10 This describes the effect of unfermented wolfberry juice and wolfberry juice fermented with different strains on the transformation capacity of ConA-induced mouse spleen lymphocytes in Example 3 of this invention; * indicates comparison with the negative control group. p <0.05;

[0031] Figure 11 This describes the effect of unfermented wolfberry juice and wolfberry juice fermented with different strains on hemolysin levels in mice in Example 4 of this invention; * indicates comparison with the negative control group. p <0.05;

[0032] Figure 12 This describes the effects of unfermented wolfberry juice and wolfberry juice fermented with different strains on the function of antibody-producing cells in mice, as shown in Example 4 of this invention; * indicates comparison with the negative control group. p <0.05. Detailed Implementation

[0033] 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 and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0034] Traditional goji berry juice products suffer from problems such as monotonous flavor, excessive sourness, and low utilization of active ingredients. Lactic acid bacteria fermentation technology can be used to develop plant-based beverages, but it has drawbacks such as excessive acidity, a sharp taste, and failure to fully stimulate the release of functional substances such as polyphenols and anthocyanins in plant raw materials.

[0035] This invention provides a fermenting strain of *Lactobacillus mucinus* LFY21. Experiments have shown that fermenting goji berry juice with this strain results in moderate acidification, balanced flavor, and excellent palatability, and significantly enhances the antioxidant capacity and total phenolic content of the goji berry juice. Furthermore, animal experiments have demonstrated that goji berry juice fermented with this strain can effectively improve the immune function of mice.

[0036] This invention also provides the application of the fermented Lactobacillus mucinus LFY21 in fermented wolfberry juice.

[0037] The present invention will be described below through specific embodiments.

[0038] The red and black goji berries used in the following examples were harvested from Golmud City, Qinghai Province.

[0039] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, culture media, etc. used in the following examples are all commercially available.

[0040] Example 1

[0041] This embodiment provides the screening, isolation process and identification of the fermenting Lactobacillus mucinus LFY21 strain.

[0042] 1. Screening and isolation process of Lactobacillus fermentans strain LFY21

[0043] Traditional naturally fermented milk samples purchased from the local market in Kashgar, Xinjiang, were used as the source for screening lactic acid bacteria. This type of fermented dairy product involves the natural fermentation of fresh milk without the addition of any commercial starter culture, resulting in a rich and diverse microbial composition. After serial dilution with sterile water, the fermented milk samples were isolated and purified on MRS plates, yielding a fast-growing lactic acid bacterium, LFY21, which can tolerate high concentrations of acid and bile salts.

[0044] 2. Identification of LFY21 strain

[0045] 2.1 Colony morphology and physiological and biochemical characteristics

[0046] The plate colony morphology and microscopic morphology (oil immersion 100×) of strain LFY21 are as follows: Figure 1 As shown.

[0047] The physiological and biochemical characteristics of strain LFY21 are as follows: Gram-positive facultative anaerobic bacillus; capable of metabolizing glucose, galactose, lactose, mannose, fructose, and sucrose; heterofermentation, producing lactic acid and carbon dioxide; good acid tolerance, able to grow normally under culture conditions of pH=4-7.

[0048] 2.2 16S rRNA Sequencing

[0049] The 16S rRNA sequence of strain LFY21 was obtained by gene sequencing (as shown in SEQ ID No. 1):

[0050]

[0051] Identification was performed based on colony morphology, physiological and biochemical characteristics, and 16S rRNA gene sequencing results. The results showed that the LFY21 strain is *Lactobacillus fermentatus* (…). Limosilactobacillus fermentum It was named *Lactobacillus fermentatus* LFY21 and deposited on November 17, 2025, at the China Center for Type Culture Collection (CCTCC, address: Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC NO: M 20252569.

[0052] Example 2

[0053] This embodiment provides a method for preparing fermented goji berry juice that enhances immunity using fermented Lactobacillus mucinus LFY21, and also compares the effects of fermented goji berry juice produced with other lactic acid bacteria as well as unfermented red goji berry juice and black goji berry juice.

[0054] 1. Sample Preparation

[0055] 1.1 Preparation method of fermented wolfberry juice

[0056] Using red or black goji berries as raw materials, fermentation is carried out as follows: After removing the stems from the goji berries, wash them with water and then rehydrate them by soaking them in a 0.01% sodium isoascorbate solution for 10 hours. After rehydration, the goji berries are mashed into a pulp, and pectinase (enzyme activity 30000 U / g) is added at 0.15% of the weight of the goji berries before rehydration and treated for 2 hours. The pulp is then filtered to obtain goji berry juice. The filtered goji berry juice is pasteurized at 65℃ for 30 minutes, cooled to room temperature, and inoculated with a 3% (v / v) suspension of the fermentation strain (live bacteria concentration 6×10⁻⁶). 6 The culture was carried out at 37°C (CFU / ml) to initiate fermentation. The selected fermentation strains were *Lactobacillus fermentatus* LFY21, *Lactobacillus plantarum* TXZ 2-35, *Lactobacillus rhamnosus* XQ3, and *Lactobacillus paracasei* CZ6.

[0057] 1.2 Preparation methods of unfermented red and black goji berry juice

[0058] Using red or black goji berries as raw materials, the berries are destalked and washed with water, then rehydrated by soaking in a 0.01% sodium isoascorbate solution for 10 hours. After rehydration, the berries are mashed into a pulp, and pectinase (30,000 U / g enzyme activity) is added at 0.15% of the original weight of the berries for 2 hours. The pulp is then filtered to obtain goji berry juice. The filtered goji berry juice is pasteurized at 65°C for 30 minutes and cooled to room temperature.

[0059] 2. Testing Items

[0060] 2.1 Viable bacteria count and pH

[0061] The colony count in the fermentation broth was determined using the plate count method at 0, 6, 18, 24, and 28 hours after inoculation with the fermentation strain suspension. The viable count of lactic acid bacteria was expressed as the logarithm of colony forming units (CFU / mL).

[0062] The pH values ​​of red and black goji berry juice during fermentation were measured at each sampling time point using a calibrated pH meter, and the pH values ​​of unfermented red and black goji berry juice were also measured simultaneously.

[0063] 2.2 Sugar-acid ratio

[0064] At 24 and 48 hours after inoculation with the fermentation strain suspension, the fermentation broth and unfermented wolfberry juice were centrifuged at 9000 rpm for 10 min, respectively. The total soluble sugar content of the supernatant was determined using the phenol-sulfuric acid colorimetric method. 1.0 mL of proportionally diluted supernatant from fermented and unfermented wolfberry juice samples were mixed with 5.0 mL of freshly prepared phenol-sulfuric acid reagent in an ice bath, and then heated at 95 °C for 10 min. After cooling to room temperature, the absorbance was measured at 620 nm using a UV-Vis spectrophotometer. A standard curve was plotted using glucose as a standard, and the results are expressed as glucose equivalents (g / L).

[0065] The titratable acidity (TA) of fermented and unfermented wolfberry juice supernatants was determined using the AOAC 2005 method. 10.0 mL of each sample was diluted with 10 mL of distilled water and then titrated to pH 8.1 with 0.1 mol / L NaOH solution (using a pH meter). The total acidity (TA) was calculated based on the volume of sodium hydroxide solution consumed and expressed as lactic acid equivalents (g / L).

[0066] Titratable sugar ratio (SAR) = Total soluble sugar (g / L) / Titratable acidity (g / L).

[0067] 2.3 Sensory characteristics

[0068] Twenty trained evaluators (10 men and 10 women, aged 20-30 years) conducted sensory evaluations of the supernatants from centrifuged (9000 rpm for 10 min) fermented and unfermented wolfberry juices 24 hours after inoculation with the fermentation strain suspension using a 9-point hedonic scale (1 = very dislike, 9 = very like). Evaluators were trained on basic taste identification and calibration using reference samples (unfermented wolfberry juice and commercially available beverages) to ensure consistency of evaluations (Kappa coefficient > 0.75). Before evaluation, samples were centrifuged, filtered, and equilibrated in brown glasses at 25 ± 1 °C. No more than four samples were evaluated at a time, and evaluators cleaned their mouths with unsalted biscuits and deionized water between evaluations. This study complied with ISO 8589:2007 and was approved by the institution's ethics committee. All participants signed informed consent forms and fully understood the experimental procedures. Sensory evaluation and instrumental analysis were conducted in a standardized laboratory with a constant temperature (23 ± 1 ℃).

[0069] 2.4 Antioxidant properties

[0070] At 24 and 48 hours after inoculation with the fermentation strain suspension, the fermentation broth and unfermented wolfberry juice were centrifuged at 9000 rpm for 10 min, respectively. The antioxidant activity of the supernatant of fermented wolfberry juice and unfermented wolfberry juice was evaluated using the DPPH free radical scavenging method. The DPPH free radical scavenging activity was determined according to the method of Liu et al. (2019): the diluted wolfberry juice sample was mixed with 0.2 mmol / L DPPH reagent, incubated in the dark for 30 min, and then the absorbance was measured at 517 nm to determine its scavenging ability. DPPH free radical scavenging activity (%) = 1 - (A1 - A2) / A0 × 100%, where A0 represents the absorbance of the DPPH solution mixed with anhydrous ethanol, A1 represents the absorbance of the DPPH solution mixed with the sample, and A2 represents the absorbance of the sample solution mixed with anhydrous ethanol.

[0071] 2.5 Total phenol content

[0072] At 24 and 48 hours after inoculation with the fermentation strain suspension, the fermentation broth and unfermented wolfberry juice were centrifuged at 9000 rpm for 10 min, respectively. 100 µL L Lolin-Ciocalteu reagent was added to the supernatant of both the fermented and unfermented wolfberry juices, followed by the addition of 10% sodium carbonate solution, and incubation for 60 min. The absorbance was measured at 510 nm, and the results were expressed as gallic acid equivalents (g GA·kg). -1 DW indicates the total phenol content.

[0073] 2.6 Analysis of Changes in Volatile Flavor Compounds

[0074] Twenty-four hours after inoculation with the fermentation strain suspension, the fermentation broth and unfermented wolfberry juice were centrifuged at 9000 rpm for 10 min, respectively. The volatile compounds in the supernatant were analyzed using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS, Agilent Technologies, USA). 5 mL of sample was transferred to a 15 mL headspace vial, and 2 μL of cyclohexanone (0.25 g / g) was added as an internal standard. After equilibration for 15 min, a 50 / 30 μm DVB / CAR / PDMS fiber was placed in the headspace and extracted at 60 °C for 30 min. Then, desorption was performed at 240 °C in splitless mode at the gas chromatograph injection port for 3 min. The gas chromatographic temperature program was as follows: initial temperature 50 °C, hold for 2 min; increase to 180 °C at a rate of 5 °C / min, hold for 10 min; then increase to 260 °C at a rate of 10 °C / min. High-purity helium (99.99%) was used as the carrier gas at a flow rate of 1.2 mL / min. Volatile compounds were initially identified by comparison with the NIST 14 mass spectrometry library, and then further confirmed by literature and standard compounds. Relative contents were calculated using the internal standard method (cyclohexanone) and peak area normalization. Odor characteristic data of the compounds were obtained from FlavorDB, Flavornet, and relevant literature.

[0075] 3. Results

[0076] 3.1 Viable bacterial count and pH

[0077] Results of viable cell count examination as follows Figure 2 As shown, all strains exhibited rapid growth during the first 24 hours of fermentation, reaching a peak viable count around 24 hours, followed by a decline. Among them, *Lactobacillus mucinus* LFY21 showed the most viable growth in black goji berry juice, while *Lactobacillus paracasei* CZ6 had the highest viable count in red goji berry juice.

[0078] pH test results are as follows Figure 3 As shown, the pH gradually decreased with the extension of fermentation time. The overall pH of black goji berry juice was lower than that of red goji berry juice. Compared with other lactic acid bacteria, the fermentation of Lactobacillus mucilaginosus LFY21 had a weaker acid production capacity and a higher final pH.

[0079] 3.2 Sugar-acid ratio

[0080] The results are as follows Figure 4 As shown, fermentation significantly reduced the sugar-acid ratio of the two types of goji berry juice, but the goji berry juice fermented with Lactobacillus mucilaginosus LFY21 still maintained a high level, indicating that its acidification was moderate and its flavor was balanced; while Lactobacillus paracasei CZ6 had the strongest acidification.

[0081] 3.3 Sensory characteristics

[0082] The results are as follows Figure 5 As shown, the goji berry juice fermented with Lactobacillus myxobolus LFY21 scored the highest in terms of color, aroma, taste, and overall acceptability, indicating that Lactobacillus myxobolus LFY21 has a significant advantage in improving the flavor and palatability of goji berry juice.

[0083] 3.4 Antioxidant properties

[0084] The results are as follows Figure 6 As shown, fermentation significantly improved the free radical scavenging activity of both red and black goji berry juices. p <0.05). DPPH scavenging activity increased significantly after 24 h and 48 h of fermentation. The highest scavenging rate (99%) was observed in black goji berry juice after 48 h of fermentation with Lactobacillus mucilaginosus LFY21, followed by the Lactobacillus rhamnosus XQ3 fermentation group.

[0085] 3.5 Total phenol content

[0086] The results are as follows Figure 7 As shown, fermentation also significantly increased the total phenolic content (TPC) of both red and black goji berry juices. p <0.05), and the fermented Lactobacillus mucinus LFY21 had the best effect on increasing the total phenol content of red and black goji berry juice.

[0087] 3.6 Analysis of Changes in Volatile Flavor Compounds

[0088] The results are as follows Figure 8 As shown, there were significant differences in the number of volatile compounds detected in fermentation samples from different strains. In fermented red goji berries, the *Lactobacillus myxobin* LFY21 group had higher levels of 2,3-butanediol and 1-butanol (2-methyl and 3-methyl isomers), resulting in a smoother texture. In contrast, the *Lactobacillus myxobin* LFY21 sample group had the highest levels of acetic acid and 2,3-butanediol in black goji berries. These results indicate that *Lactobacillus myxobin* LFY21 has a unique influence on the flavor of fermented goji berries.

[0089] Example 3

[0090] This example investigated the effects of fermented wolfberry juice (fermented for 24 hours after inoculation with the bacterial suspension of the fermentation strain) prepared in Example 2 and unfermented wolfberry juice on the cellular immune function of mice.

[0091] 1. Experimental animals and dosage design

[0092] Healthy female Kunming mice (6 weeks old) were selected as experimental animals and randomly divided into a negative control group and 10 experimental groups (each group was given unfermented red goji berry juice, fermented red goji berry juice prepared with Lactobacillus plantarum TXZ 2-35, fermented red goji berry juice prepared with Lactobacillus rhamnosus XQ3, fermented red goji berry juice prepared with Lactobacillus paracasei CZ6, fermented red goji berry juice prepared with Lactobacillus fermentum LFY21, and unfermented black goji berry juice, fermented black goji berry juice prepared with Lactobacillus plantarum TXZ 2-35, fermented black goji berry juice prepared with Lactobacillus rhamnosus XQ3, fermented black goji berry juice prepared with Lactobacillus paracasei CZ6, and fermented black goji berry juice prepared with Lactobacillus fermentum LFY21), with 10 mice in each group. Referring to the procedures for immune function evaluation in the "Technical Specifications for Inspection and Evaluation of Health Foods," a dose conversion was performed using the body surface area normalization method, with a dosage 10 times the recommended human dose (50 mL / 60 kg body weight) as the benchmark. Fermented and unfermented wolfberry juice were concentrated 200 times and used as test samples. Mice in the experimental group were administered the test samples orally via gavage at a dose of 0.4 mL / (kg·bw), equivalent to 10 times the recommended human dose. The negative control group was administered an equal volume of distilled water via gavage. Mice in each group underwent intervention once daily for 30 consecutive days via gavage.

[0093] 2. Experimental Methods

[0094] 2.1 Delayed-type hypersensitivity (DTH) assay

[0095] On day 26 after gavage administration of the test sample, mice were sensitized by intraperitoneal injection of 0.20 mL of 2.0% (v / v) sheep red blood cell (SRBC) suspension per mouse. On day 4 post-sensitization, each mouse was challenged by subcutaneous injection of 20 μL of 20.0% (v / v) SRBC suspension into the left hind paw. The thickness of the same location on the left hind paw of the same mouse was measured using a precision pachymeter before and 24 h after challenge. The difference in paw thickness before and after challenge was calculated to represent the intensity of the DTH response. Experimental data were compared between each dose group and the negative control group.

[0096] 2.2 ConA-induced spleen lymphocyte transformation assay

[0097] After the DTH assay was completed, the mice were euthanized, and the spleens were aseptically harvested and placed in Hank's solution to prepare a spleen cell suspension. The cell concentration was then adjusted to 3 × 10⁻⁶ cells / mL. 6Spleen cell suspension was seeded into 24-well plates at a density of 1 mL per well. 75 μL of ConA solution was added to each experimental well, while none was added to the control wells. After incubation at 37 ℃ in a 5.0% CO2 incubator for 68 h, 0.70 mL of supernatant was discarded, and 0.70 mL of RPMI 1640 medium and 50 μL of MTT solution (5 mg / mL) were added to each well. Incubation continued for another 4 h. After incubation, 1 mL of acidic isopropanol was added to each well to dissolve formazan crystals, and the optical density of each well was measured at 570 nm using a UV spectrophotometer.

[0098] 3. Results

[0099] 3.1 Results of Delayed-Type Hypersensitivity (DTH) Measurement

[0100] like Figure 9 As shown, the difference in left hind paw thickness induced by SRBC-induced DTH in red and black goji berry juice fermented with Lactobacillus myxoid LFY21 was significantly different from that in the negative control group, and the effect was stronger than that in other strain fermentation groups. This indicates that goji berry juice fermented with Lactobacillus myxoid LFY21 can induce DTH in mice and enhance their immune function.

[0101] 3.2 Results of ConA-induced spleen lymphocyte transformation assay

[0102] like Figure 10 As shown, the lymphocyte proliferation OD difference in the group fermented with *Lactobacillus myxobin* LFY21 was significantly different from that in the control group, and the effect was more pronounced than that of other strains. This indicates that *Lactobacillus myxobin* LFY21-fermented goji berry juice can enhance the transformation capacity of splenic lymphocytes in mice and enhance their immune function.

[0103] Example 4

[0104] This example investigated the effects of fermented wolfberry juice (fermented for 24 hours after inoculation with the bacterial suspension of the fermentation strain) prepared in Example 2 and unfermented wolfberry juice on the humoral immune function of mice.

[0105] 1. Experimental animals and dosage design: Same as in Example 3.

[0106] 2. Experimental Methods

[0107] 2.1 Serum hemolysin assay

[0108] On day 26 after gavage, each mouse was intraperitoneally injected with 0.20 mL of 2.0% SRBC suspension. On day 4 post-immunization, blood was collected via ocular sampling and serum was separated. Serum was serially diluted with physiological saline, and 100 μL of each dilution was added to a microplate. 100 μL of 0.5% SRBC suspension was then added to each well, mixed thoroughly, and incubated at 37 ℃ for 3 h. The degree of hemagglutination was observed, and antibody volume was calculated. Experimental data were compared between each dose group and the negative control group, and statistical analysis was performed using ANOVA.

[0109] 2.2. Detection of antibody-producing cells

[0110] Mice that underwent blood collection as described in section "2.1" were sacrificed, and spleens were harvested to prepare spleen cell suspensions. The cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 6 / mL. Preheated and melted 1% agarose was mixed with an equal volume of double-concentration Hank's solution and dispensed into small test tubes, 0.5 mL per tube. Immediately, 50 μL of 10% SRBC and 20 μL of spleen cell suspension were added to each tube, quickly mixed, and poured onto a glass slide pre-coated with a thin layer of agarose. After the agarose solidified, the slide was placed in a slide holder and incubated in a CO2 incubator for 1.5 h. Subsequently, complement diluted with SA buffer (1:8) was added to the grooves of the slide holder, and incubation continued for another 1.5 h. The number of hemolytic plaques was then counted.

[0111] 3. Results

[0112] 3.1 Serum hemolysin assay results

[0113] like Figure 11 As shown, compared with the negative control group, the serum hemolysin level of mice in the group fermented with red and black goji berry juice fermented with Lactobacillus mucin LFY21 was significantly increased. p <0.05). This indicates that wolfberry juice fermented with *Lactobacillus myxobolus* LFY21 significantly increased serum hemolysin levels in mice, enhancing humoral immunity. Furthermore, compared to other fermentation groups, the effect of wolfberry juice fermented with *Lactobacillus myxobolus* LFY21 was more significant.

[0114] 3.2 Results of antibody-producing cell detection

[0115] like Figure 12 As shown, compared with the negative control group, the number of hemolytic plaques in the red and black goji berry juice group fermented with Lactobacillus fermentum LFY21 was significantly increased ( p <0.05). This indicates that wolfberry juice fermented with *Lactobacillus myxobin* LFY21 can increase the number of antibody-producing cells in mice and enhance immunity. Compared with other strains, wolfberry juice fermented with *Lactobacillus myxobin* LFY21 has a more significant effect.

[0116] Example 5

[0117] This example investigated the effects of fermented wolfberry juice (fermented for 24 hours after inoculation with the bacterial suspension of the fermentation strain) and unfermented wolfberry juice prepared in Example 2 on the function of mouse mononuclear-macrophages and the activity of NK cells.

[0118] 1. Experimental animals and dosage design: Same as in Example 3.

[0119] 2. Experimental Methods

[0120] 2.1 Peritoneal macrophage phagocytosis assay of chicken red blood cells

[0121] On day 30 after gavage, 1 mL of 20% chicken erythrocyte suspension was injected intraperitoneally into each mouse. Thirty minutes later, the mice were euthanized by cervical dislocation, and 2 mL of physiological saline was immediately injected into the peritoneal cavity. After gentle abdominal compression, 1 mL of peritoneal lavage fluid was aspirated and evenly dripped onto a glass slide. The slide was incubated at 37 °C for 30 min, rinsed with physiological saline, air-dried, fixed with acetone:methanol (1:1, v / v) solution, stained with 4% Giemsa-phosphate buffer, rinsed with distilled water, and air-dried. Microscopic observation was performed, and the number of phagocytic macrophages and the total number of phagocytosed chicken erythrocytes were counted. The phagocytic rate and phagocytic index were calculated using the following formulas: Phagocytic rate (%) = (Number of macrophages phagocytosed erythrocytes / Total number of macrophages) × 100%; Phagocytic index = Total number of phagocytosed erythrocytes / Total number of macrophages.

[0122] 2.2 NK cell activity assay (lactate dehydrogenase (LDH) method)

[0123] After completing the peritoneal cell collection in section 2.1, spleen cells were immediately harvested from the same mouse to prepare a spleen cell suspension for NK cell activity assay. 100 μL each of target cell (YAC-1 cells) and effector cell (spleen cells) suspensions (effector-target ratio of 50:1) were added to a U-shaped 96-well plate. Simultaneously, two wells were set up: one for natural target cell release (100 μL each of target cells and culture medium) and one for maximum target cell release (100 μL each of target cells and 1% NP40). All wells were tripled. The plate was incubated at 37 ℃ in a 5.0% CO2 incubator for 4 h. After incubation, the plate was centrifuged at 1500 r / min for 5 min, and 100 μL of supernatant was aspirated from each well and transferred to the corresponding well in a flat-bottomed 96-well plate. Immediately, 100 μL of LDH matrix solution was added to each well, and the reaction was carried out at room temperature in the dark for 8 min. Finally, 30 μL of 1 mol / L HCl solution was added to each well to terminate the reaction. The optical density of each well was measured at 490 nm using a microplate reader. NK cell activity was calculated based on the measured optical density values. Experimental data were compared between each dose group and the negative control group, and statistical analysis was performed using ANOVA.

[0124]

[0125] 3. Results

[0126] As shown in Table 1, compared with the negative control group, goji berry juice fermented with specific strains, especially fermented with *Lactobacillus mucinus* LFY21, significantly enhanced the body's immune function. Specifically, the black goji berry juice group fermented with *Lactobacillus mucinus* LFY21 showed the best immune-enhancing effect, with its macrophage phagocytic rate (31.10% ± 1.37%) and phagocytic index (0.83 ± 0.12) being significantly higher than those of the negative control group. p The concentration of *Lactobacillus mucinus* was <0.05, comparable to that of high-dose immune enhancers. Furthermore, the NK cell activity in this group (33.90% ± 2.81%) also reached its highest level. The red goji berry juice fermented with *Lactobacillus mucinus* LFY21 showed the second-best effect. In addition, compared with other tested strains, the fermentation effect of *Lactobacillus mucinus* LFY21 was more prominent, and its fermentation effect on black goji berries was generally better than that on red goji berries.

[0127] Table 1. Effects of different strains of fermented wolfberry juice on the function of mouse monocytes-macrophages and the activity of NK cells ( ±s)

[0128]

[0129] Note: P represents the test result compared with the negative control.

[0130] As can be seen from the above experiments, the black goji berry juice fermented with Lactobacillus mucinus LFY21 of this invention can serve as a highly effective immune enhancer and has broad prospects for development and application.

[0131] 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 fermentatus* LFY21, characterized in that, Its category name is Limosilactobacillus fermentum LFY21 was deposited at the China Center for Type Culture Collection (CCTCC) on November 17, 2025, with accession number CCTCC NO: M 20252569; the deposit address is Wuhan University.

2. The application of the fermented Lactobacillus mucinus LFY21 as described in claim 1 in fermented wolfberry juice.

3. The application according to claim 2, characterized in that, The goji berry juice is black goji berry juice.

4. A method for preparing immune-enhancing fermented wolfberry juice using the *Lactobacillus fermentans* LFY21 strain as described in claim 1, characterized in that... Specifically, the following operations are included: After removing the stems from the goji berries, wash them with water, and then soak them in a mixture of 0.01%-0.02% sodium isoascorbate solution for 8-15 hours to rehydrate them. After rehydration, the goji berries are mashed into a pulp, and pectinase is added at 0.1%-0.15% of the weight of the goji berries before rehydration for 2-4 hours. After filtration, goji berry juice is obtained. Filter the wolfberry juice, pasteurize it at 60-65℃ for 30-40 minutes, cool it to room temperature, inoculate it with a 2%-3% v / v suspension of Lactobacillus fermentans strain LFY21, and ferment it at 30-37℃ for 24-48 hours; the preservation number of the Lactobacillus fermentans strain LFY21 is CCTCC NO: M 20252569.

5. The method according to claim 4, characterized in that, The goji berry mentioned is the black goji berry.

6. The method according to claim 4, characterized in that, The pectinase activity is 30,000 U / g.

7. The method according to claim 4, characterized in that, The viable cell concentration of the *Lactobacillus fermentans* LFY21 strain suspension was 6 × 10⁻⁶. 6 CFU / m.

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