Lactobacillus plantarum p2 and application thereof

Abstract

This invention discloses a *Lactobacillus plantarum* P2 and its applications, belonging to the fields of food microbiology technology and agricultural product processing technology. The *Lactobacillus plantarum* P2 is classified and named... Lactiplantibacillus plantarum P2, with preservation number CCTCC NO: M 20261182. Application experiments show that the fermentation broth of *Lactobacillus plantarum* P2 can significantly improve the brightness of fruit and vegetable juices, while effectively inhibiting browning in freeze-dried fruits and fresh-cut fruits during processing and storage; its color-protecting activity is unaffected by storage time; and it has no impact on the natural nutrients in fruits and vegetables. Therefore, applying it to the pretreatment of fruit and vegetable juices, freeze-dried fruits, and fresh-cut fruits to construct a natural color-protecting technology for fruit and vegetable products can effectively improve the turbidity of fruit and vegetable juices and the browning problem of freeze-dried fruits and fresh-cut fruits.

Description

Technical Field

[0001] This invention relates to the fields of food microbiology technology and agricultural product processing technology, specifically to a plant lactobacillus P2 and its application in color protection of fruit and vegetable products. Background Technology

[0002] Fruit and vegetable products (including fruit and vegetable juices, freeze-dried fruits, fresh-cut fruits, etc.) are rich in vitamins, minerals, polyphenols and dietary fiber, and have both nutrition and flavor, making them highly popular health foods. However, during the processing of fruit and vegetable products, peeling and cutting will expose the flesh to the environment, which will easily cause browning (including enzymatic browning and non-enzymatic browning). This will manifest as dark color, decreased brightness and increased browning index, which will seriously affect the sensory quality, shelf life and market competitiveness of the products (the color-protecting effect of lactic acid bacteria fermentation on pear juice [J]. Modern Food Science and Technology, 2019, 35(03):148-153.).

[0003] Currently, industrial methods for improving the color of fruit and vegetable products mainly rely on chemical color-protecting agents such as ascorbic acid, citric acid, and sulfites, or physical methods such as blanching, light protection, nitrogen filling, and ultrafiltration (Research on the Formulation of Seabuckthorn Compound Fruit and Vegetable Juice Beverage [J]. Food Research and Development, 2015, 36(8):43-45.). Physical methods are costly and have limited color-protecting effects; although chemical color-protecting agents can inhibit browning in the short term, they have drawbacks such as safety controversies, flavor interference, residue risks, and non-compliance with clean labeling and the trend of natural and healthy consumption, making it difficult to meet the development needs of the modern food industry for green, safe, and natural products.

[0004] As a food-grade GRAS lactic acid bacterium, *Lactobacillus plantarum* produces organic acids, extracellular polysaccharides, reduced metabolites, and antioxidants during its metabolism. These substances can regulate pH, scavenge free radicals, stabilize pigments, and improve color, making it an ideal natural biological color-protecting resource. Existing research mainly focuses on the preservative, antibacterial, and antioxidant functions of *Lactobacillus plantarum* in fruit and vegetable products (Research progress on the antibacterial properties and applications of *Lactobacillus plantarum* extract [J]. Chinese Condiments, 2024, 49(12): 215-220.), while there are relatively few studies on its application in inhibiting browning and improving the color stability of fruit and vegetable products.

[0005] Studies show that the processing by-products and fruit surfaces of apples and strawberries are rich in natural lactic acid bacteria. Therefore, apples and strawberries are ideal natural materials for screening functional lactic acid bacteria for fruit and vegetable products. Isolating and screening lactic acid bacteria that can improve brightness, delay browning, and have antioxidant capabilities, and establishing their natural brightening and color-protecting technology in fruit and vegetable products, has important theoretical value and application prospects for promoting the green development of the fruit and vegetable industry and realizing the high-value utilization of by-products. Summary of the Invention

[0006] The purpose of this invention is to address the problems of color deterioration, unnatural color protection methods, and lack of specialized functional strains in existing fruit and vegetable products (including fruit and vegetable juices, freeze-dried fruits, fresh-cut fruits, etc.). This invention provides a beneficial strain that can enhance the brightness of processed fruit and vegetable products, delay browning, and has antioxidant capabilities. Applying this strain to the color protection and brightening of fruit and vegetable products provides a new biological strain and technical solution for the production of high-quality natural fruit and vegetable products.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention screens bacterial strains from apples and strawberries, belonging to the endophytic lactic acid bacteria of fruits and vegetables. The screened strains are inoculated into different types of fruit and vegetable juices for fermentation. By measuring indicators such as juice transmittance, color difference (L, a, b values), and browning index, a strain that improves the brightness of fruit and vegetable juices and reduces the browning index is identified. Based on morphological characteristics, culture properties, and physiological and biochemical characteristics, this strain is identified as belonging to the genus *Lactobacillus* (…). Lactiplantibacillus Lactobacillus plantarum ( Lactiplantibacillus plantarum Therefore, it was named *Lactobacillus plantarum*. Lactiplantibacillus plantarum P2.

[0008] Lactobacillus plantarum P2 was deposited on June 4, 2026 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261182.

[0009] Furthermore, the culture conditions for *Lactobacillus plantarum* P2 are as follows: cultured in MRS medium at 37±0.5℃ under anaerobic conditions.

[0010] This invention demonstrates that *Lactobacillus plantarum* P2 exhibits excellent brightness enhancement effects on apple juice, pear juice, and strawberry juice. It significantly improves the light transmittance of the juice, mitigates oxidative browning, optimizes color uniformity and visual clarity, and does not affect the polyphenol and flavonoid content or other nutritional components. In freeze-dried fruit applications, apple slices, strawberry slices, and freeze-dried pear slices pretreated with *Lactobacillus plantarum* P2 showed significantly higher brightness values ​​and a significantly lower browning index than the untreated control group, resulting in a significant improvement in sensory quality. In fresh-cut fruit applications, pretreatment with *Lactobacillus plantarum* P2 not only enhanced the brightness of fresh-cut pear slices but also effectively delayed browning.

[0011] Another object of the present invention is to provide the application of *Lactobacillus plantarum* P2 in the preparation of color-protecting agents for fruit and vegetable products. The application includes: using *Lactobacillus plantarum* P2 or its metabolites to prepare specific color-protecting agents for fruit and vegetable products.

[0012] The color-protecting agent for fruit and vegetable products prepared by this invention mainly consists of *Lactobacillus plantarum* P2 and its metabolites. By directly using the fermentation products of this strain as raw materials, there is no need for cumbersome purification and extraction processes, resulting in low production costs and a long-lasting and stable color-protecting effect, making it highly practical.

[0013] The color-protecting agent is used for at least one of the following purposes: inhibiting browning and enhancing color.

[0014] Specifically, the application of *Lactobacillus plantarum* P2 or its metabolites provided by this invention can be as follows: In the fruit and vegetable juice processing process, the fruit and vegetable juice is first subjected to high-temperature pasteurization, and then inoculated with *Lactobacillus plantarum* P2 for constant-temperature fermentation; In the freeze-drying fruit processing process, before freeze-drying, the fruit and vegetable raw materials are soaked in *Lactobacillus plantarum* P2 fermentation liquid (fermented fruit and vegetable liquid) or sprayed to make the fermentation liquid (fermented fruit and vegetable liquid) evenly adhere to the surface of the fruit and vegetables, which can also be used for ice coating treatment, and then freeze-drying is performed; In the fresh-cut fruit processing process, the fruit and vegetable raw materials are soaked in *Lactobacillus plantarum* P2 fermentation liquid (fermented fruit and vegetable liquid) or sprayed to make the fermentation liquid (fermented fruit and vegetable liquid) evenly adhere to the surface of the fruit and vegetables.

[0015] Furthermore, for fruit and vegetable juice processing, the effective concentration of *Lactobacillus plantarum* P2 is 1×10⁻⁶. 9 ~3×10 9 CFU / mL. Fermentation conditions: 37±0.5℃ for 20-24 h.

[0016] Furthermore, the method for preparing the *Lactobacillus plantarum* P2 seed culture includes: inoculating the *Lactobacillus plantarum* P2 into MRS solid medium for activation culture, then transferring it to MRS broth for culture until the exponential growth phase, collecting the strain by centrifugation, washing the bacterial cells with sterile sodium chloride solution, and resuspending to obtain a bacterial suspension of the required concentration.

[0017] Furthermore, the preparation method of the *Lactobacillus plantarum* P2 fermentation broth (fermented fruit and vegetable broth) includes: mixing *Lactobacillus plantarum* P2 in the exponential growth phase at a concentration of 1×10⁻⁶. 9 ~ 3×10 9 Inoculate sterilized fruit and vegetable juice with an inoculum concentration of CFU / mL and ferment at 37±0.5℃ for 20-24 h to obtain fermented fruit and vegetable juice. This invention uses fruit and vegetable juice as the fermentation substrate because, on the one hand, the juice contains natural carbon and nitrogen nutrients, which can induce the strain to efficiently synthesize color-protecting active ingredients such as lactic acid and antioxidant metabolites; on the other hand, the substrate is homologous to the components of the fruits and vegetables to be treated, resulting in good compatibility of the fermentation broth, no exogenous culture medium residue, and the endogenous antioxidants in the fruits and vegetables, after being activated by fermentation, can synergistically inhibit browning with the strain's metabolites, without destroying the original nutrients of the fruits and vegetables throughout the process.

[0018] This invention utilizes *Lactobacillus plantarum* P2 fermentation broth as a natural color-protecting agent specifically for fruit and vegetable products. This strain does not interact negatively with the natural nutrients in fruits and vegetables and will not destroy vitamins, minerals, or other nutrients. Therefore, *Lactobacillus plantarum* P2 fermentation broth can be used alone or in combination with other food additives in the processing of various fruit and vegetable products to achieve a stable improvement in their color.

[0019] Furthermore, in terms of scope of application, the *Lactobacillus plantarum* P2 or its metabolites provided by this invention can be applied to a variety of fruit and vegetable products, covering pome fruits, stone fruits, berries, tropical fruits and some vegetables, specifically including fruits such as apples, pears, strawberries, blueberries, mangoes, bananas, and kiwis, as well as their fruit and vegetable juices, freeze-dried fruits, and fresh-cut fruits, and can also be used in compound fruit and vegetable products.

[0020] The *Lactobacillus plantarum* P2 metabolites provided by this invention exhibit strong color-protecting activity and stability. The duration of storage does not affect their efficacy, and they maintain good color-protecting effects even after long-term storage, perfectly meeting the requirements of industrial processing and long-term storage of fruit and vegetable products. Furthermore, *Lactobacillus plantarum* P2 can be safely used in the preparation of food additives, demonstrating high biosafety. No harmful substances are generated during fermentation and metabolism, complying with food additive safety regulations.

[0021] Another object of this invention is to provide the application of *Lactobacillus plantarum* P2 in the preparation of antioxidants. Studies of this invention have shown that the metabolites of *Lactobacillus plantarum* P2 exhibit excellent antioxidant activity, effectively scavenging DPPH free radicals and ABTS. + Free radicals, with their strong reducing power, are one of the important mechanisms by which they exert their color-protecting effect.

[0022] The present invention also provides a method for delaying browning of fruit and vegetable juice, the method comprising: activating the aforementioned Lactobacillus plantarum P2 and inoculating it into sterilized fruit and vegetable juice, fermenting it at 37±0.5℃ for 20-24 h, and then storing it at low temperature.

[0023] In this invention, in addition to using *Lactobacillus plantarum* P2 as the core active ingredient, an auxiliary color-protecting step can be added before juicing. Specifically, the method for preparing the fruit and vegetable juice includes: washing and cutting fresh fruits and vegetables, soaking them in a vitamin C solution or a sodium D-isoascorbate solution for color protection pretreatment, then removing them, juicing them, filtering them, and sterilizing them to obtain the fruit and vegetable juice.

[0024] Furthermore, the color-protecting pretreatment method is as follows: soak the fruit and vegetable pieces in a vitamin C solution or sodium D-isoascorbate solution with a mass-volume percentage concentration of 0.5%-1% for 5-10 minutes to inhibit the enzymatic browning of the fruit and vegetable pieces in the early stage of juicing. After soaking, drain off the residual vitamin C solution or sodium D-isoascorbate solution on the surface.

[0025] The present invention also provides a method for delaying browning of freeze-dried fruit or fresh-cut fruit, the method comprising the following steps: (1) The plant lactobacillus P2 was activated and inoculated into sterilized fruit and vegetable juice, and fermented at 37±0.5℃ for 20-24 h to obtain fermented fruit and vegetable juice. (2) After washing and cutting the fresh fruits and vegetables, soak them in the fermented fruit and vegetable juice or spray the fermented fruit and vegetable juice evenly onto the surface of the fruits and vegetables, and then freeze-dry them.

[0026] Furthermore, in step (1), the activated *Lactobacillus plantarum* P2 is added at a final concentration of 1×10⁻⁶. 9 ~ 3×10 9 CFU / ml was inoculated into fruit and vegetable juice.

[0027] Furthermore, in step (2), for freeze-dried fruit, fresh fruits and vegetables are cut and soaked in the fermented fruit and vegetable juice for 5-10 minutes; for fresh-cut fruit, fresh fruits and vegetables are cut and soaked in the fermented fruit and vegetable juice for 15-30 minutes.

[0028] In this invention, in addition to using *Lactobacillus plantarum* P2 as the core active ingredient, auxiliary color-protecting steps such as blanching or vitamin C soaking can be added before soaking or spraying the fermented fruit juice to synergistically enhance the color-protecting effect.

[0029] The beneficial effects of this invention are as follows: (1) This invention provides a new strain of plant lactobacillus P2. Application experiments show that its fermentation liquid can significantly improve the brightness of fruit and vegetable juices, and can effectively inhibit browning of freeze-dried fruit and fresh-cut fruit during processing and storage, and improve the color of fruit and vegetable juices, freeze-dried fruit and fresh-cut fruit.

[0030] (2) The plant lactobacillus P2 provided by the present invention has no effect on the natural nutrients in fruits and vegetables. When applied to the pretreatment of fruit and vegetable juices, freeze-dried fruits and fresh-cut fruits, it can construct a natural color protection technology for fruit and vegetable products, effectively improve the turbidity of fruit and vegetable juices and the browning problem of freeze-dried fruits and fresh-cut fruits, improve the visual quality of fruit and vegetable products, and reduce economic losses caused by color deterioration.

[0031] (3) The plant lactobacillus P2 provided by the present invention has excellent stability, and the color-protecting activity of its metabolites is not affected by storage time. It is suitable for the processing and storage of fruit and vegetable products and can be used without additional adjustment of the processing flow. Attached Figure Description

[0032] Figure 1 The images show a color comparison of apple juice and strawberry juice after fermentation with four different strains. (A) is apple juice; (B) is strawberry juice.

[0033] Figure 2 The growth curve of strain P2 is shown.

[0034] Figure 3 The images show the plate growth diagram and Gram staining microscopic image of strain P2, where: (A) is the plate growth diagram; (B) is the Gram staining image.

[0035] Figure 4 Homology analysis for strain P2.

[0036] Figure 5 The color changes of apple juice after fermentation with strain P2 and storage for two weeks are shown in the figures. (A) represents brightness; (B) represents red saturation; (C) represents yellow saturation; and (D) represents browning index.

[0037] Figure 6 Comparison of freeze-dried pears prepared by soaking in pear juice fermented with P2 strain in different batches, where: (A) is the treatment in 2025, (B) is the treatment in April 2026, and (C) is the treatment in May 2026.

[0038] Figure 7 The images show a comparison of the color changes of fresh-cut pear slices of Qiuyue pear, Korla pear, and Crown pear after different treatments. Among them: (A) No treatment and left for 0 min; (B) No treatment and left for 20 min; (C) Soaked in P2 fermented pear juice for 20 min. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. For example, in the following examples, MRS broth and agar powder are conventional culture media / liquids in the art.

[0041] Example 1: Screening and identification of endogenous lactic acid bacteria that delay browning in fruits and vegetables 1. Experimental Materials The apples and strawberries used were samples from Qingdao, Shandong, and the freeze-dried strawberry blocks were selected from commercially available freeze-dried strawberries.

[0042] 2. Experimental Methods 2.1 Isolation of strains Weigh 10g from the sample and place it in a homogenizing bag. Add 90mL of sterile physiological saline and homogenize thoroughly. Then prepare a 10-fold serial dilution series of sample homogenates. The dilution was selected as 10. -5 10 -6 10 -7 Three gradients were spread onto MRS solid medium, three in parallel, and incubated in an anaerobic incubator at 37°C for 24-48 hours. After single colonies grew, colonies with significant differences were selected and streaked onto MRS solid medium for isolation, and incubated in an anaerobic incubator at 37°C for 24-48 hours. Four strains were obtained, numbered C2, C3, P2, and P3, where C2 and C3 were derived from fresh strawberries, and P2 and P3 were derived from fresh apples.

[0043] 2.2 Preparation of inoculum The selected bacterial strains were inoculated into MRS solid medium and anaerobically cultured at 37°C for 48 h. Single colonies were then picked from the solid medium and inoculated into MRS broth, and cultured at 37°C for 24 h to obtain a bacterial suspension. The reactivated bacterial suspension was centrifuged at 8000 r / min for 5 min, and the bacterial cells were washed twice with physiological saline and then resuspended in sterile physiological saline in an equal volume for later use.

[0044] 2.3 Preparation of Fermented Apple Juice Select fresh Fuji apples, wash them under running water, peel them, cut them into chunks, and soak them in a VC solution (1% by volume) for color protection for 5 minutes. Remove them, juice them, filter them through a 300-mesh filter cloth, bottle them, seal them, and pasteurize them in a 90℃ water bath for 5 minutes. After cooling, inoculate the bacterial suspension at a 2% (v / v) inoculation rate. The initial concentration of the bacterial strain in the fermentation system is 1×10⁻⁶. 9 ~ 3×10 9 The concentration of CFU / mL was increased and fermented in a 37°C incubator for 24 hours. After fermentation, the product was removed and stored in a 4°C refrigerator.

[0045] 2.4 Preparation of Fermented Strawberry Juice Grind freeze-dried strawberry blocks into powder, add distilled water at a ratio of 1:4 to make strawberry juice, bottle it, seal it, pasteurize it in a 90℃ water bath for 5 minutes, cool it, inoculate it with seed liquid at a 2% inoculation rate, and ferment it in a 37℃ incubator for 24 hours. After fermentation, remove it and store it in a 4℃ refrigerator.

[0046] 2.5 Determination of color of fermented fruit juice After fermentation, a certain amount of juice was taken and the color of the juice was measured using a portable colorimeter. The red saturation (a*), yellow saturation (b*), and brightness (L*) of different juice samples were measured, and the browning index BI value was calculated according to the following formula.

[0047] BI = 100 × (x - 0.31 / 0.172); x=(a + 1.75×L) / (5.645×L + a - 3.012×b); In the formula, a is the red saturation (a*), b is the yellow saturation (b*), and L is the brightness (L*).

[0048] Experimental results are as follows Figure 1 As shown in Table 1, the apple juice fermented by strain P2 had a higher brightness than the juice fermented by the other three strains, and its BI (browning index) was significantly lower than that of the juice fermented by the other three strains. This indicates that in the apple juice system, strain P2 has a better effect on inhibiting browning of fruits and vegetables and improving the brightness of apple juice.

[0049] Table 2 shows that the strawberry juice fermented by strain P2 had the highest brightness, but its browning index was higher than that of strains C3 and P3. Considering that the strawberry juice itself has a bright color, the browning index results will be affected by the color of the raw material. Based on the results of apple juice, strain P2 was selected for further research on inhibiting browning and improving the brightness of fruit and vegetable products.

[0050] Table 1. Color values ​​of apple juice fermented by four different bacterial strains Note: Different lowercase letters in the same column indicate significant differences between groups. p <0.05).

[0051] Table 2. Color values ​​of strawberry juice fermented by four different strains Note: Different lowercase letters in the same column indicate significant differences between groups. p <0.05).

[0052] 2.6 Drawing the growth curve Experimental Methods: The strain P2 isolated in section 2.1 was inoculated into MRS broth and anaerobically cultured at 37°C for 24 h. Then, it was inoculated into MRS solid medium using an inoculation loop and cultured for 48 h. A single colony was then picked and inoculated into MRS broth, and incubated at 37°C for 24 h. The absorbance of the cultured bacterial solution at 600 nm was adjusted to 0.1 using fresh MRS broth. 2 mL of the bacterial solution was taken and placed in a microbial growth analyzer to determine its growth curve. Three replicates were performed.

[0053] Experimental results: such as Figure 2 As shown, the absorbance of strain P2 initially increased rapidly and then stabilized during the measurement period. Around 4 hours, P2 entered the logarithmic growth phase and began to multiply rapidly, while the absorbance at 600 nm increased. Around 12 hours, P2 entered the stationary phase, and the absorbance remained stable from 12 to 48 hours.

[0054] 2.7 Strain Identification 2.7.1 Morphological identification Experimental methods: The strain was streaked and single bacteria were picked and cultured in MRS broth at 37°C for 24 hours. Morphological observation and Gram staining were then performed on the plates.

[0055] Experimental results: such as Figure 3 As shown, the colonies of strain P2 on MRS culture dishes are round, raised, and slightly whitish. After Gram staining, they turn blue-purple, are rod-shaped, and uniform in morphology, thus being identified as Gram-positive bacteria.

[0056] 2.7.2 16S rRNA gene sequence analysis Experimental methods: Single colonies were picked from strain P2 plates and inoculated into MRS broth. The culture was then anaerobic at 37°C for 24 h. The bacterial culture was then transferred to MRS solid medium and streaked for 48 h at 37°C. After two streaking purifications, single colonies were picked and anaerobically cultured in MRS broth at 37°C for 24 h. DNA was extracted from the bacterial culture using a DNA extraction kit, and the DNA concentration was determined before amplification.

[0057] Reaction system: 7.0 μL ultrapure water, 1.0 μL DNA template, 10.0 μL 2×M5 Hiper plus Taq HiFi PCR mix, 1 μL each of upstream and downstream primers.

[0058] Upstream primer sequence: 5'-AGAGTTTGATCMTGGCTCAG-3'; Downstream primer sequence: 5'-GGTTACCTTGTTACGACTT-3'.

[0059] Reaction procedure: pre-denaturation at 95℃ for 2 min, 95℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, 36 cycles; finally, hold at 72℃ for 10 min.

[0060] The qualified PCR amplification products were sent to Sangon Biotech for sequencing to obtain the amplified gene sequence of strain P2, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0061] BLAST homology analysis was performed on the processed gene sequences in the NCBI database. The analysis yielded known strains with the highest homology to those in the database, such as... Figure 4 As shown, compared with known strains Lactiplantibacillus plantarum The homology reached over 99%.

[0062] Based on the above analysis, P2 was identified as belonging to the Bacteria kingdom (Bacteria). Bacteria Firmicutes ( Firmicutes ), Bacillus class ( Bacilli Lactobacilli ( ) Lactobacillales Lactobacillus family ( Lactobacillaceae Lactobacillus ( ) Lactiplantibacillus Lactobacillus plantarum ( Lactiplantibacillus plantarum Therefore, it was named *Lactobacillus plantarum*. Lactiplantibacillus plantarum P2.

[0063] Lactobacillus plantarum P2 ( Lactiplantibacillus plantarum The sample was deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20261182. Testing was completed on June 11, 2026, and the sample was found to be viable.

[0064] Example 2: Detection of antioxidant capacity and active substance content in fruit juice fermented by Lactobacillus plantarum P2 1. Experimental strains Example 1: Isolation and identification of Lactobacillus plantarum P2.

[0065] 2. Experimental Methods 2.1 Fruit Juice Fermentation 2.1.1 Apple Juice Preparation Wash and cut the apples into chunks. Soak them in a color-protecting solution (1% sodium D-isoascorbate aqueous solution) for 5 minutes, then put them into a screw press to extract the juice. Filter the juice using a 300-mesh filter cloth, then pasteurize (90℃ for 5 minutes). Cool the juice to room temperature before use.

[0066] 2.1.2 Apple Juice Fermentation On MRS solid medium, pick single colonies of P2 and place them in MRS broth. Incubate at 37°C for 24 h to obtain a bacterial suspension. Inoculate the strain into MRS broth at a 2% (v / v) inoculum and incubate at 37°C for 24 h. Centrifuge the reactivated bacterial suspension at 8000 r / min for 5 min, wash the bacterial cells twice with physiological saline, resuspend in an equal volume, and then inoculate into pasteurized apple juice at a 2% (v / v) inoculum. Incubate at 37°C for 24 h and store at 4°C for later use.

[0067] 2.2 Scavenging ability of DPPH (2,2-diphenyl-1-picrylhydrazine) radicals Experimental method: Add 10 mL of 70% ethanol to 1 mL of P2 fermented fruit juice and use ultrasonic extraction method. Sonicate for 20 min to fully extract the antioxidant components. Then centrifuge (8000 rpm, 10 min) and take the supernatant as the "apple juice antioxidant extract" to be tested.

[0068] Accurately weigh 2.5 mg of DPPH and dissolve it in 100 mL of anhydrous ethanol to make the concentration of the mixture 6.5 × 10⁻⁶. -5 Prepare a DPPH-ethanol solution using mol / L. Add 2 mL of anhydrous ethanol and DPPH-ethanol solution to centrifuge tubes, mix well, and react at room temperature for 30 min. Measure the absorbance at 517 nm. Add 2 mL of the extract and anhydrous ethanol to centrifuge tubes, mix well, and react at room temperature for 30 min. Measure the absorbance at 517 nm. Add 2 mL of DPPH-ethanol solution and extract to centrifuge tubes, mix well, and react at room temperature for 30 min. Measure the absorbance at 517 nm. The formula for calculating the DPPH scavenging rate is: DPPH clearance rate = [1 - (A2 - A1) / A0] × 100%; In the formula: A0 is the absorbance of DPPH-ethanol solution and anhydrous ethanol; A1 is the absorbance of extract and anhydrous ethanol; A2 is the absorbance of extract and DPPH-ethanol solution.

[0069] 2.3 Total antioxidant activity Experimental Methods: The total antioxidant capacity was determined using the Nanjing Jiancheng Total Antioxidant Capacity Assay Kit (ABTS method). ABTS is oxidized to green ABTS under the action of a suitable oxidant. The production of ABTS is inhibited in the presence of antioxidants. The absorbance of ABTS was measured at 405 nm. The total antioxidant capacity of the sample was calculated using the standard curve equation y = -1.1214x + 1.1262, where y is the total antioxidant capacity (mM) and x is the absorbance.

[0070] 2.4 Determination of polyphenol content in fruit juice Experimental method: The polyphenol content was determined by the Folin-Ciocalteu method and expressed as gallic acid equivalent. 0.5 mL of a sample diluted 10 times was taken, 2.5 mL of distilled water and 0.5 mL of Folin-Ciocalteu reagent were added, and the mixture was reacted at room temperature for 3 min. Then, 1.5 mL of Na2CO3 was added and the mixture was reacted for 2 h. The absorbance was measured at 765 nm. The polyphenol content of the sample was calculated using the standard curve equation y = 0.00807x + 0.0023, where y is the absorbance and x is the polyphenol concentration (mg / L) in the reaction solution.

[0071] 2.5 Determination of Flavonoid Content Experimental method: Rutin was used as the equivalent. Take 1 mL of a sample diluted appropriately, add 1 mL of 5% (w / v) NaNO2, let stand at room temperature for 5 min, add 1 mL of 10% (w / v) AlCl3 solution, let stand at room temperature for 6 min, add 4 mL of 2 mol / L NaOH solution, react for 15 min, and measure the absorbance at 510 nm. Calculate the flavonoid content of the sample using the standard curve equation y = 0.00827x + 0.0003, where y is the absorbance and x is the flavonoid concentration (mg / L) in the reaction solution.

[0072] 3. Experimental Results Table 3 shows that P2 fermented apple juice has high DPPH, total antioxidant capacity, and flavonoid polyphenol content. The results indicate that P2 fermentation products have certain antioxidant capacity and can promote the production of polyphenol flavonoid bioactive substances in the juice.

[0073] Table 3. Antioxidant capacity and bioactive content of P2 fermented apple juice Example 3: Application of Lactobacillus plantarum P2 in delaying browning of apple juice 1. Experimental strains Example 1: Isolation and identification of Lactobacillus plantarum P2.

[0074] 2. Experimental Methods 2.1 Preparation of Fermented Apple Juice Select fresh Fuji apples, wash them under running water, peel them, cut them into pieces, and soak them in a 1% VC solution for color protection for 5 minutes. Remove them, juice them, filter them through a 300-mesh filter cloth, bottle them, seal them, pasteurize them in a 90℃ water bath for 5 minutes, cool them, inoculate them with Bacillus plantarum P2 seed liquid at a 2% inoculation rate, and ferment them in a 37℃ incubator for 24 hours. After fermentation, remove them and store them in a 4℃ refrigerator. Take samples of the fruits and vegetables during the first and second weeks of storage to measure their color and calculate the browning index.

[0075] 2.2 Juice color determination A certain amount of juice was taken, and the color of the juice was measured using a portable colorimeter. The red saturation (a*), yellow saturation (b*), and brightness (L*) of different juice samples were measured.

[0076] 3. Experimental Results like Figure 5 As shown, apple juice fermented with Lactobacillus plantarum P2 strain did not show significant differences in brightness (L value), red saturation (a value), and yellow saturation (b value) during the two weeks of storage, maintaining a stable overall level. Meanwhile, although the browning index of the juice increased slightly during storage, the increase was small, rising from 4.2 to 5.7. The browning index after 2 weeks of storage was still lower than that of the control on day 0 (6.71 in Table 1), indicating that the treatment can effectively maintain the color stability of the juice.

[0077] Example 4: Application of *Lactobacillus plantarum* P2 in delaying browning of freeze-dried pear slices 1. Experimental strains Example 1: Isolation and identification of Lactobacillus plantarum P2.

[0078] 2. Experimental Methods 2.1 Preparation of Fermented Pear Juice Fresh Akizuki pears were selected, washed under running water, peeled, cut into chunks, and soaked in a 1% VC solution for color protection for 5 minutes. The juice was then extracted, filtered through a 300-mesh filter cloth, bottled, sealed, and pasteurized in a 90℃ water bath for 5 minutes. After cooling, *Lactobacillus plantarum* P2 seed culture was inoculated at a 2% inoculation rate and fermented in a 37℃ incubator for 24 hours. After fermentation, the inoculated pears were stored in a 4℃ refrigerator for later use. *Lactobacillus plantarum* Q17 (patent application number CN202311409402.2) was used as a control.

[0079] 2.2 Preparation of freeze-dried pear slices Select fresh Akizuki pears, wash them under running water, peel them, cut them into pieces, and soak them in four solutions: VC solution (1%), sugar-salt solution (5% sugar (w / v), 0.5% salt (w / v)), Q17 fermented pear juice, and P2 fermented pear juice. After 5 minutes, take them out, spread them flat in a glass dish, and pre-freeze them in a -80℃ refrigerator for more than 4 hours. Then freeze-dry them in a vacuum freeze dryer. After taking them out, seal them in a bag and store them in a drying oven.

[0080] 2.3 Determination of color of freeze-dried pears After the samples were freeze-dried, three pieces were taken from each of the untreated and four types of freeze-dried pear samples soaked in solutions. The color of the freeze-dried pears was measured using a portable colorimeter. The red saturation (a*), yellow saturation (b*), and brightness (L*) of different freeze-dried pear samples were measured.

[0081] 3. Experimental Results Table 4 and Figure 6 A represents the color difference values ​​(2025) of freeze-dried pears corresponding to different treatment methods. The freeze-dried pears treated with P2 fermented pear juice had the highest brightness and the lowest browning index, showing significant differences from the other groups.

[0082] Table 5 and Figure 6 B represents the color difference values ​​of freeze-dried pears corresponding to three different treatment methods (April 2026). The freeze-dried pears treated with P2 fermented pear juice had the highest brightness among all groups, and their brightness and browning index were significantly different from the untreated group and the Q17 group.

[0083] Table 6 and Figure 6 C represents the color difference values ​​of freeze-dried pears corresponding to three different treatment methods (May 2026). The freeze-dried pears treated with P2 fermented pear juice from Lactobacillus plantarum showed the best results.

[0084] The results in summary indicate that Lactobacillus plantarum P2 has the ability to enhance the brightness of freeze-dried pears, while also exhibiting a certain browning inhibition effect.

[0085] Table 4. Color difference values ​​of freeze-dried pears corresponding to three different treatment methods, including pear juice fermented with strain P2 (2025) Note: Different lowercase letters in the same column indicate significant differences between groups. p <0.05).

[0086] Table 5. Color difference values ​​of freeze-dried pears corresponding to three different treatment methods, including pear juice fermented with strain P2 (April 2026) Note: Different lowercase letters in the same column indicate significant differences between groups. p <0.05).

[0087] Table 6. Color difference values ​​of freeze-dried pears corresponding to three different treatment methods, including pear juice fermented with strain P2 (May 2026) Note: Different lowercase letters in the same column indicate significant differences between groups. p <0.05).

[0088] Example 5: Application of Lactobacillus plantarum P2 in delaying browning of fresh-cut pear slices 1. Experimental strains Example 1: Isolation and identification of Lactobacillus plantarum P2.

[0089] 2. Experimental Methods 2.1 Preparation of fresh-cut pear slices Select fresh Qiuyue pears, Korla fragrant pears and Crown pears, wash them under running water, peel them, and slice them. Group A did not treat the three types of pear slices in any way and let them sit for 20 minutes. Group B soaked the three types of pear slices in P2 fermented pear juice for 20 minutes and then took them out.

[0090] 2.2 Color determination of freshly cut pear slices A portable colorimeter was used to measure the color of fresh-cut pear slices after 0 minutes of storage, as well as the colors of group A and group B. The red saturation (a*), yellow saturation (b*), and brightness (L*) of different fresh-cut pear slice samples were also measured.

[0091] 3. Experimental Results Tables 7 and 8 show that untreated fresh-cut pear slices all exhibited varying degrees of browning after 20 minutes. Table 9, however, shows that the browning phenomenon was effectively inhibited in fresh-cut pear slices treated with pear juice fermented by *Lactobacillus plantarum* P2 for 20 minutes. Specific results for fresh-cut pear slices are shown in Table 9. Figure 7 After 20 minutes, the browning index of the three types of fresh-cut pear slices in the control group increased by 66.77%, 46.19%, and 35.69% respectively compared with the group treated with P2 fermented pear juice.

[0092] Table 7. Color of three types of freshly cut pear slices after 0 min. Table 8. Color of three types of freshly cut pear slices after 20 minutes Table 9. Color of three types of fresh-cut pear slices after soaking in P2 fermented pear juice for 20 minutes In summary, *Lactobacillus plantarum* P2 can not only significantly improve the brightness of fresh-cut pear slices, but also effectively delay the browning process of fresh-cut pear slices.

[0093] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum P2, characterized in that, The classification name of *Lactobacillus plantarum* P2 is... Lactiplantibacillus plantarum P2 is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20261182.

2. The *Lactobacillus plantarum* P2 as described in claim 1, characterized in that, The culture conditions for *Lactobacillus plantarum* P2 were as follows: cultured in MRS medium at 37±0.5℃ under anaerobic conditions.

3. The application of *Lactobacillus plantarum* P2 as described in claim 1 or 2 in the preparation of color-protecting agents for fruit and vegetable products, characterized in that... The color-protecting agent is used for at least one of the following purposes: inhibiting browning and enhancing color.

4. The application as described in claim 3, characterized in that, The fruit and vegetable products include fruit and vegetable juices, freeze-dried fruits, and fresh-cut fruits.

5. The application as described in claim 4, characterized in that, The fruits and vegetables mentioned are at least one of apples, pears, strawberries, blueberries, mangoes, bananas, and kiwis.

6. The use of *Lactobacillus plantarum* P2 as described in claim 1 or 2 in the preparation of antioxidants.

7. A method for delaying browning of fruit and vegetable juices, characterized in that, The method includes: activating *Lactobacillus plantarum* P2 as described in claim 1 or 2, inoculating it into sterilized fruit and vegetable juice, fermenting it at 37±0.5℃ for 20-24 h, and then storing it at low temperature.

8. The method as described in claim 7, characterized in that, The method for preparing the fruit and vegetable juice includes: washing and cutting fresh fruits and vegetables, soaking them in a vitamin C solution or a sodium D-isoascorbate solution for color protection pretreatment, then taking them out to extract the juice, filtering them and sterilizing them to obtain the fruit and vegetable juice.

9. A method for delaying browning in freeze-dried fruit or fresh-cut fruit, characterized in that, The method includes the following steps: (1) The plant lactobacillus P2 as described in claim 1 or 2 is activated and inoculated into fruit and vegetable juice, and fermented at 37±0.5℃ for 20-24 h to obtain fermented fruit and vegetable juice. (2) After washing and cutting the fresh fruits and vegetables, soak them in the fermented fruit and vegetable juice or spray the fermented fruit and vegetable juice evenly onto the surface of the fruits and vegetables, and then freeze-dry or store them at low temperature.

10. The method for delaying browning of freeze-dried fruit or fresh-cut fruit as described in claim 9, characterized in that, For freeze-dried fruits, fresh fruits and vegetables are cut and then soaked in the fermented fruit and vegetable juice for 5-10 minutes; for fresh-cut fruits, fresh fruits and vegetables are cut and then soaked in the fermented fruit and vegetable juice for 15-30 minutes.

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