Lactobacillus degrading hippophae rhamnoides malic acid and application thereof in preparation of hippophae rhamnoides juice

Abstract

The application discloses lactobacillus degrading hippophae rhamnoides malic acid and application thereof in preparation of hippophae rhamnoides juice, wherein one strain is classified and named as lactobacillus acidophilus (Lactobacillus acidophilus) Lactobacillus acidipiscis ) with a preservation number of CGMCC No. 37351, and one strain is classified and named as lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus plantarum ) with a preservation number of CGMCC No. 37352. Fresh hippophae rhamnoides juice is mixed with sterile water according to a proportion, pasteurized after sugar degree is adjusted, cooled to room temperature, and pretreated; the lactobacillus is inoculated into the pretreated hippophae rhamnoides juice after being cultured to a logarithmic growth phase, and anaerobically cultured at 35±1 DEG C for 90-96 h. The lactobacillus screened in the application has a remarkable degradation effect on hippophae rhamnoides malic acid, can efficiently degrade hippophae rhamnoides malic acid, and meanwhile retains nutrition function and natural flavor, and can be used for preparation of hippophae rhamnoides juice.

Description

Technical Field

[0001] This application belongs to the field of microbial fermentation technology, specifically relating to a lactobacillus that degrades malic acid in sea buckthorn and its application in the preparation of sea buckthorn juice. Background Technology

[0002] Sea buckthorn juice is rich in various functional components such as total phenols, flavonoids, and vitamins, possessing extremely high nutritional value and antioxidant activity. However, the malic acid content in natural sea buckthorn juice is as high as 1.2~1.8g / 100mL, resulting in a pH value as low as 2.5~3.0, strong acidity, and a pungent taste, severely impacting consumer acceptance. Furthermore, the high concentration of malic acid masks the natural fruity aroma of sea buckthorn juice, limiting its application in the food industry.

[0003] Existing methods for reducing the acidity of sea buckthorn juice mainly include chemical neutralization, physical adsorption, and microbial fermentation. While chemical neutralization can rapidly reduce acidity, it destroys the natural flavor of sea buckthorn juice and leads to nutrient loss. Physical adsorption is complex, costly, and prone to secondary pollution. In microbial fermentation, commonly used lactic acid bacteria strains (such as Lactobacillus paracasei) have low degradation efficiency for malic acid (usually ≤60%), and fermentation easily leads to a decrease in the total phenol content and antioxidant activity of sea buckthorn juice, making it difficult to balance acid reduction and product quality.

[0004] Therefore, screening a lactic acid bacteria strain that can efficiently degrade malic acid in sea buckthorn while preserving its nutritional functions and natural flavor, and establishing an optimized fermentation process, is of great significance for promoting the industrialization of sea buckthorn juice products. Summary of the Invention

[0005] This application provides a lactobacillus that degrades malic acid in sea buckthorn and its application in the preparation of sea buckthorn juice, which can efficiently degrade malic acid in sea buckthorn while retaining its nutritional functions and natural flavor.

[0006] A strain of *Lactobacillus acidophilus* was classified and named *Lactobacillus acidophilus* ( Lactobacillus acidipiscis The strain number S3 was deposited on January 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37351.

[0007] Morphological characteristics of Lactobacillus acidophilus: Gram-positive, rod-shaped cells, measuring (0.6-0.9) μm × (2.5-5.0) μm, existing singly, in pairs, or forming short chains; non-spore-forming, non-capsulated, non-flagellated, and non-motile; under scanning electron microscopy, the cell surface is smooth and without special appendages.

[0008] Culture characteristics of Lactobacillus acidophilus: When anaerobically cultured on MRS solid medium at 30-37℃ for 48h, it forms round colonies with a diameter of 1.5-2.5mm, raised, with neat edges, smooth and dense surface, milky white color, uniform texture, and translucent to opaque.

[0009] After anaerobic culture at 30-37℃ for 24 hours in MRS liquid medium, the culture medium becomes uniformly turbid and the pH drops significantly (down to below 4.0). After 48 hours of culture, a small amount of bacterial precipitate can be seen.

[0010] The 16S RNA nucleotide sequence of *Lactobacillus acidophilus* is shown in SEQ ID NO: 1.

[0011] This application also provides the application of the aforementioned *Lactobacillus acidophilus* in the degradation of malic acid in sea buckthorn.

[0012] This application also provides the application of the aforementioned *Lactobacillus acidophilus* in the preparation of sea buckthorn juice.

[0013] This application also provides a strain of *Lactobacillus plantarum*, classified and named *Lactobacillus plantarum* (… Lactobacillus plantarum The strain number S4 was deposited on January 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37352.

[0014] Morphological characteristics of Lactobacillus plantarum: Gram-positive, cells are straight or slightly curved rods, measuring (0.7-1.0) μm × (3.0-8.0) μm, existing singly, in pairs, or forming short chains; non-spore-forming, non-flagellated, and non-motile; non-capsulated, with a smooth cell surface, and uniform cell morphology under scanning electron microscopy, without any special appendages.

[0015] Culture characteristics of Lactobacillus plantarum: When cultured anaerobically at 30-35℃ for 48h on MRS solid medium, it forms round colonies with a diameter of 2-3mm, raised, with neat edges, smooth and dense surface, milky white color, uniform texture, translucent to opaque, and viscous when picked up. In MRS liquid medium, after anaerobic culture at 30-35℃ for 24 hours, the culture medium becomes uniformly turbid and the pH can drop below 4.0. After 48 hours of culture, a small amount of bacterial precipitate can be seen.

[0016] The 16S RNA nucleotide sequence of Lactobacillus plantarum strain is shown in SEQ ID NO: 2.

[0017] This application also provides the application of the aforementioned *Lactobacillus plantarum* in the degradation of malic acid in sea buckthorn.

[0018] This application also provides the application of the aforementioned *Lactobacillus plantarum* in the preparation of sea buckthorn juice.

[0019] This application also provides a fermentation agent for preparing sea buckthorn juice, using the Lactobacillus acidophilus or Lactobacillus plantarum as the active ingredient.

[0020] This application also provides a method for preparing sea buckthorn juice, comprising: Take fresh sea buckthorn juice concentrate and mix it with sterile water according to the ratio, adjust the sugar content, pasteurize, and cool to room temperature to complete the pretreatment; The *Lactobacillus acidophilus* or the *Lactobacillus plantarum* was cultured to the logarithmic growth phase to obtain a bacterial suspension. The bacterial suspension was then inoculated into pretreated sea buckthorn juice concentrate and cultured anaerobically at 35±1℃ for 90-96 hours.

[0021] Optionally, the viable count of the bacterial suspension is 10-1. 8 CFU / mL ~10 9 CFU / mL; the bacterial suspension was inoculated into the pretreated sea buckthorn juice at a volume ratio of 1-3%. Further, the bacterial suspension was inoculated into the pretreated sea buckthorn juice at a volume ratio of 2%. The strains screened in this application achieved a malic acid degradation rate of over 80% at a 2% inoculation amount.

[0022] Optionally, fresh sea buckthorn juice concentrate can be mixed with sterile water at a mass ratio of 1:1.

[0023] Optionally, when adjusting the sugar content, add 6% (w / w) glucose to the mixture of fresh sea buckthorn juice concentrate and sterile water.

[0024] Optionally, the fresh sea buckthorn juice concentrate can be a commercially available product or prepared by conventional methods.

[0025] Compared with the prior art, this application has the following beneficial effects: (1) The strain of this application only needs 96 hours of anaerobic fermentation at 35°C in the sea buckthorn juice system. With an inoculum of 2%, the malic acid degradation rate is as high as 87.79% and 82.45%, respectively. It achieves a degradation effect far exceeding the existing technology in a shorter fermentation cycle, which greatly shortens the production cycle of sea buckthorn juice and reduces the industrial production cost.

[0026] (2) Existing technologies mostly focus on the single effect of malic acid degradation, failing to simultaneously optimize flavor and enhance functional activity: conventional lactic acid bacteria fermentation can easily lead to the loss of fruity aroma in sea buckthorn juice and the generation of unpleasant fermentation odors. However, after fermentation by the strain of this application, the astringency of sea buckthorn juice is significantly reduced, the acidity is mild, and it is highly coordinated with the natural fruity aroma of sea buckthorn. The overall sensory acceptance is significantly improved compared to the unfermented group, and it is significantly better than the fermentation effect of conventional Lactobacillus paracasei. At the same time, the DPPH free radical scavenging rate of sea buckthorn juice is increased from 20.38% in the unfermented group to a maximum of 56.82%. While efficiently degrading malic acid, it simultaneously activates the bioactivity of antioxidants in sea buckthorn juice, achieving simultaneous optimization of taste, flavor, and nutritional function, and solving the technical problem of not being able to simultaneously reduce acidity and enhance function in existing technologies. Attached Figure Description

[0027] Figure 1 The graph shows the degradation rate of malic acid in the fermented sea buckthorn juice of the 12 strains listed in Table 1.

[0028] Figure 2A The graph shows the L-malic acid content in sea buckthorn juice after pretreatment of raw material B1 and fermentation of sea buckthorn juice by strains S3, S4, YJ24, YJ2 and YSHM3.

[0029] Figure 2B The graph shows the malic acid degradation rate in sea buckthorn juice after fermentation by strains S3, S4, YJ24, YJ2, and YSHM3.

[0030] Figure 3A The graph shows the L-malic acid content in sea buckthorn juice after pretreatment of raw material B1 and fermentation by strains S3 and YJ2.

[0031] Figure 3B The graph shows the malic acid degradation rate in sea buckthorn juice after fermentation by strains S3 and YJ2.

[0032] Figure 4A The graph shows the L-malic acid content in sea buckthorn juice raw material B1 after pretreatment and in sea buckthorn juice fermented by strains YJ24 and YJ2.

[0033] Figure 4B The graph shows the malic acid degradation rate in sea buckthorn juice after fermentation by strains YJ24 and YJ2.

[0034] Figure 5A The graph shows the L-malic acid content in sea buckthorn juice raw material B1 after pretreatment and in sea buckthorn juice fermented by strains YJ2 and YSHM3.

[0035] Figure 5B The graph shows the malic acid degradation rate in sea buckthorn juice after fermentation by strains YJ2 and YSHM3.

[0036] Figure 6AThe graph shows the L-malic acid content in sea buckthorn juice after pretreatment of raw material B1 and fermentation by strains S4 and YJ2.

[0037] Figure 6B The graph shows the malic acid degradation rate in sea buckthorn juice after fermentation by strains S4 and YJ2.

[0038] Figure 7 The graph shows the polyphenol content in sea buckthorn juice after pretreatment of raw material B1 and fermentation by strains S3, S4, YJ24, YJ2 and YSHM3.

[0039] Figure 8 The graph shows the DPPH free radical scavenging rate of sea buckthorn juice after fermentation of pretreated sea buckthorn juice raw material B1 and strains S3, S4, YJ24 and YSHM3.

[0040] Figures 9A-9E The image shows the sensory evaluation results of sea buckthorn juice after fermentation by different strains.

[0041] Figure 10 Cluster heatmaps of sea buckthorn juice metabolites after fermentation of YJ2 and S4, respectively. Detailed Implementation

[0042] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0044] Example 1 The strains used in this application were all isolated from pig farm feed in Fuyang area. The isolation and identification process was carried out in accordance with Bergey's Manual of Bacterial Identification and the Classification, Identification and Experimental Methods of Lactic Acid Bacteria. The physiological and biochemical characteristics of the strains, such as contact enzymes, oxidases, and sugar fermentation, were measured.

[0045] Strain isolation: Feed samples were collected from pig farms in the Fuyang area, placed in sterile sampling bags, and transported to the laboratory at low temperature for immediate separation. 10.0 g of sample was weighed, added to 90 mL of sterile physiological saline, and thoroughly shaken to prepare 10... -1 The sample was homogenized and then serially diluted 10-fold to 10. -6 .

[0046] Take 100 μL of each graded dilution and spread it evenly on MRS solid medium plates containing calcium carbonate. Incubate anaerobically at 35 °C for 48 h. Select single colonies with a clear calcium dissolution zone and different morphologies from the plates for streak isolation and purification. Repeat the purification process three times to obtain pure cultures.

[0047] The purified strains were initially screened for physiological and biochemical characteristics such as Gram staining, catalase test, oxidase test, and sugar fermentation. Gram-positive, catalase-negative, and oxidase-negative lactic acid bacteria candidate strains were obtained, as shown in Table 1.

[0048] Table 1

[0049] Strain identification: The morphological characteristics of Lactobacillus acidophilus S3 are as follows: Gram-positive, rod-shaped cells, measuring (0.6-0.9) μm × (2.5-5.0) μm, existing singly, in pairs, or forming short chains; without spores, capsules, flagella, or motility; and with a smooth cell surface and no special appendages under scanning electron microscopy.

[0050] Culture characteristics of Lactobacillus acidophilus S3: When anaerobically cultured on MRS solid medium at 30-37℃ for 48h, it forms round colonies with a diameter of 1.5-2.5mm, raised, with neat edges, smooth and dense surface, milky white color, uniform texture, and translucent to opaque. After anaerobic culture at 30-37℃ for 24 hours in MRS liquid medium, the culture medium becomes uniformly turbid and the pH drops significantly (down to below 4.0). After 48 hours of culture, a small amount of bacterial precipitate can be seen.

[0051] Genomic DNA was extracted from strain S3 and amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO: 3) and 1492R (5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO: 4). The amplified products were purified and bidirectionally sequenced to obtain the full-length sequence of the 16S rRNA gene of this strain.

[0052] The full-length 16S rRNA gene sequence of S3 (SEQ ID NO: 1):

[0053] The sequenced samples were compared with those obtained by BLAST in the NCBI database. The results showed that this strain was similar to *Lactobacillus acidophilus* (Lactobacillus acidophilus). Lactobacillus acidipiscis The 16S rRNA gene sequence of the model strain showed ≥99.5% homology, and in the phylogenetic tree constructed based on the 16S rRNA gene sequence, it was consistent with... Lactobacillus acidipiscis Clustered within the same evolutionary branch, and combining morphological and cultural characteristics, the taxonomic position of this strain was ultimately determined to be *Lactobacillus acidophilus*. Lactobacillus acidipiscis ).

[0054] Morphological characteristics of Lactobacillus plantarum S4: Gram-positive, cells are straight or slightly curved rods, measuring (0.7-1.0) μm × (3.0-8.0) μm, existing singly, in pairs, or forming short chains; non-spore-forming, non-flagellated, and non-motile; non-capsulated, with a smooth cell surface, and uniform cell morphology under scanning electron microscopy, without any special appendages.

[0055] Culture characteristics of Lactobacillus plantarum S4: When cultured anaerobically at 30-35℃ for 48h on MRS solid medium, it forms round colonies with a diameter of 2-3mm, raised, with neat edges, smooth and dense surface, milky white color, uniform texture, translucent to opaque, and viscous when picked up. In MRS liquid medium, after anaerobic culture at 30-35℃ for 24 hours, the culture medium becomes uniformly turbid and the pH can drop below 4.0. After 48 hours of culture, a small amount of bacterial precipitate can be seen.

[0056] Genomic DNA was extracted from strain S4 and amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO: 5) and 1492R (5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO: 6). The amplified products were purified and bidirectionally sequenced to obtain the full-length sequence of the 16S rRNA gene of this strain.

[0057] Full-length 16S rRNA gene sequence of strain S4 (SEQ ID NO: 2):

[0058] The sequence obtained from sequencing was compared with the NCBI database using BLAST homology. The results showed that this strain is similar to *Lactobacillus plantarum*. (Lactobacillus plantarum) The 16S rRNA gene sequence of the model strain showed ≥99.8% homology, and in the phylogenetic tree constructed based on the 16S rRNA gene sequence, it was consistent with... Lactobacillus plantarum Clustered within the same evolutionary branch, and combining morphological and cultural characteristics, the taxonomic position of this strain was ultimately determined to be *Lactobacillus plantarum*. Lactobacillus plantarum ).

[0059] Strain S3 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, with accession number CGMCC No. 37351 and deposit date of January 12, 2026.

[0060] Strain S4 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, with accession number CGMCC No. 37352 and deposit date of January 12, 2026.

[0061] Example 2 Lactic acid bacteria fermentation: Pretreatment of sea buckthorn juice raw materials: Take fresh sea buckthorn juice concentrate purchased from the market (the one used in this example was purchased from Xinjiang Yuanmai Trading Co., Ltd.) and mix it with sterile water in a 1:1 ratio to obtain a mixture. Add 6% (w / w) glucose to the mixture to adjust the sugar content of the sea buckthorn juice, and then pasteurize at 65℃ for 30 minutes and cool to room temperature.

[0062] Activation of bacterial strains: Twelve lactic acid bacteria strains, as shown in Table 1, preserved in the laboratory, were inoculated into MRS liquid medium and anaerobically cultured at 35°C for 10 hours. The cultures were subcultured twice (5% inoculation each time, v / v) to induce the bacteria into the logarithmic growth phase. The bacterial concentration was adjusted to OD600 = 1 (10⁻⁶ viable cells) using PBS buffer. 8 CFU / mL ~10 9 (CFU / mL), for later use.

[0063] Fermentation treatment: Take sea buckthorn juice cooled to room temperature, inoculate it with 2% (v / v) activated lactic acid bacteria solution, seal it and place it in anaerobic culture at 35℃ for 96h. After fermentation, take 2mL of fermentation juice, centrifuge at 10000 r / min and 4℃ for 10 min, take the supernatant, filter it through a 0.22μm aqueous filter membrane and set it aside.

[0064] Performance testing: (1) Screening of dominant strains: The L-malic acid detection kit (WST-8 method) developed by Beyotime was used to detect the change in malic acid content before and after fermentation. Based on the degradation rate of malic acid content, the strain with the best degradation effect was selected to comprehensively measure and evaluate the total phenols, sugar content, antioxidant capacity, viable lactic acid bacteria count and sensory evaluation of the fermented sea buckthorn juice.

[0065] (2) Determination of total phenols: The polyphenol content was determined by the Folin-Ciocalteu colorimetric method. 10 μL of treated sea buckthorn juice was added to a 96-well plate, along with 100 μL of Folin-Ciocalteu reagent and 90 μL of 7.5% sodium carbonate solution. The mixture was allowed to react in the dark for 30 min, and the absorbance was measured at a wavelength of 765 nm. The final total phenol content was calculated as gallic acid equivalent.

[0066] (3) Determination of antioxidant capacity: Determination of DPPH free radical scavenging rate: Take 80 μL of the test solution and mix it thoroughly with 120 μL of 0.1 mmol / L DPPH ethanol solution. After reacting at room temperature in the dark for 30 min, measure the absorbance at a wavelength of 515 nm (recorded as sample); at the same time, set up a blank control group (80 μL of 70% ethanol to replace the test solution, the rest of the operation is the same, and the absorbance is recorded as blank) and a sample control group (80 μL of the test solution and 120 μL of anhydrous ethanol are mixed, and the absorbance is recorded as control). Calculate the scavenging rate according to the formula: DPPH free radical scavenging rate (%) = [1 - (sample - control) / blank] × 100%.

[0067] (4) Sensory evaluation: After the sea buckthorn juice fermentation was completed, a sensory evaluation team was formed by 10 professionals with sensitive sense of smell and taste and sensory evaluation training background. According to the scoring criteria in Table 2, the blind sample evaluation method was used to score the fermented sea buckthorn juice from 6 dimensions: color, aroma, sweetness, acidity, astringency, and overall acceptability. The final score was the sum of the scores of the 6 indicators.

[0068] Table 2 Sensory Evaluation Indicators

[0069] (5) Lactic acid bacteria count: The number of live lactic acid bacteria in sea buckthorn juice was determined according to the national standard GB 4789.35-2016 "National Food Safety Standard for Microbiological Examination of Food - Lactic Acid Bacteria Examination".

[0070] Take the fermented sea buckthorn juice sample and perform serial dilutions with sterile physiological saline. Select 0.1 mL of each of the three suitable dilutions and spread them onto 90 mm diameter circular MRS solid agar plates. Incubate under anaerobic conditions at 36℃±1℃ for 48 h. Select plates with colony counts between 30 and 300 CFU for counting. Count typical lactic acid bacteria colonies (milky white, round, with neat edges and smooth surface). Calculate the viable lactic acid bacteria count (CFU / mL) in the sample using the formula: Viable lactic acid bacteria count (CFU / mL) = (Average colony count on plate × Total dilution factor) / Spread volume (mL).

[0071] Experimental results: (1) Degradation rate of L-malic acid The changes in malic acid content before and after fermentation were detected using the L-malic acid assay kit (WST-8 method) developed by Beyotime. L-malic acid is oxidized to oxaloacetic acid (OAA) by malate dehydrogenase (MDH), during which NAD+ is reduced to NADH. The generated NADH is then reduced to WST-8 by the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium methyl sulfate) to form orange-yellow formazan, which has a maximum absorption peak at around 450 nm. The amount of formazan generated in the reaction system is directly proportional to the L-malic acid content in the sample.

[0072] The overall degradation rate of L-malic acid by the 12 strains in Table 1 is shown in the figure below. Figure 1 As shown, the changes in L-malic acid content after fermentation of strains S3, S4, YJ2, YJ24, and YSHM3 are as follows: Figure 2A As shown, the malic acid degradation rate is as follows: Figure 2B As shown, the effects of fermentation by strains S3 and YJ2 on the L-malic acid content of sea buckthorn juice are as follows: Figure 3A As shown, the malic acid degradation rate is as follows: Figure 3B As shown, the effects of fermentation by strains YJ24 and YJ2 on the L-malic acid content of sea buckthorn juice are as follows: Figure 4A As shown, the malic acid degradation rate is as follows: Figure 4B As shown, the effects of fermentation by strains YSHM3 and YJ2 on the L-malic acid content of sea buckthorn juice are as follows: Figure 5A As shown, the malic acid degradation rate is as follows: Figure 5B As shown, the effects of fermentation by strains S4 and YJ2 on the L-malic acid content of sea buckthorn juice are as follows: Figure 6A As shown, the malic acid degradation rate is as follows: Figure 6B As shown in the figure, B1 is the pretreated sea buckthorn juice raw material.

[0073] Experimental results showed that the L-malic acid content of the pretreated sea buckthorn juice reached 98.62 μg / mL. Lactobacillus acidipiscis After fermentation at 35℃ for 96 hours, the content of S3 decreased to 17.31 μg / mL, and the degradation rate reached 82.45%. Lactobacillus plantarum After fermentation at 35℃ for 96 hours, the content of S4 decreased to 12.04 μg / mL, and the degradation rate reached 87.79%. Lactobacillus plantarum strain After fermentation at 35℃ for 96 hours, the content of KF9 YJ24 decreased to 18.07 μg / mL, and the degradation rate reached 81.68%. Lactobacillus casei After fermentation at 35℃ for 96 hours, the content of YSHM3 decreased to 20.25 μg / mL, with a degradation rate of 79.47%. Similarly... Lactobacillus paracasei strain After fermentation at 35℃ for 96 hours, the content of KL1 and YJ2 decreased to 57.84 μg / mL, with a degradation rate of only 41.35%. Among the 12 strains in Table 1, the sea buckthorn juice fermented by four strains, S3, S4, YJ24 and YSHM3, showed a significant reduction in the sourness caused by excessive malic acid content. This application selected S3 and S4, which showed the best results, for preservation.

[0074] Cluster heatmaps of sea buckthorn juice metabolites after fermentation by YJ2 and S4 respectively are shown below. Figure 10 As shown, the results indicate that citric acid, isocitric acid, D-(+)-malic acid, trans-aconitic acid, and fumaric acid, which significantly decreased after fermentation, are the core sources of the original sourness and astringency of sea buckthorn. The organic acids enriched after fermentation are mainly mild carboxylic acids with secondary flavors, including succinic acid, hydroxy acids, and phenyllactic acid, which further soften the sourness. The significantly decreased levels of salvianolic acid, 3-O-p-coumaryl quinic acid, sinapic acid, and 2,3-dihydroxybenzoic acid after fermentation are the core contributors to the astringency and bitterness of sea buckthorn. (This information was obtained from Lactobacillus plantarum S4...) Lactobacillus plantarum After fermentation, the sour and astringent taste of sea buckthorn juice is reduced. The enriched ethoxyacetic acid brings a light fruity and sweet aroma, phenyllactic acid brings a floral and honey-sweet taste, long-chain fatty acid derivatives bring a light nutty and fatty aroma, and trans-orthocoumaric acid contributes vanilla and floral aroma. These substances together construct a complex aroma system, which significantly improves the richness and acceptability of the flavor of sea buckthorn juice.

[0075] (2) Changes in total phenol content Figure 7 The graph shows the changes in polyphenol content after fermentation of sea buckthorn juice by strains S3, S4, YJ24, YJ2, and YSHM3. The experimental results indicate that the polyphenol content of the pretreated sea buckthorn juice (B1) reached 0.6438 mg / mL. Lactobacillus acidipiscis The polyphenol content of S3 after fermentation was 0.6858 mg / mL; Lactobacillus plantarumThe polyphenol content of S4 after fermentation is 0.6857 mg / mL; Lactobacillus plantarum strain The polyphenol content of KF9YJ24 after fermentation is 0.6860 mg / mL; Lactobacillus casei The polyphenol content of YSHM3 after fermentation was 0.6702 mg / mL. Similarly... Lactobacillus paracasei strain After fermentation, the polyphenol content of KL1 and YJ2 was 0.6621 mg / mL. The total phenol content of sea buckthorn juice did not decrease after lactic acid bacteria fermentation; in fact, it increased in some areas, resulting in richer health-promoting properties.

[0076] (3) Changes in antioxidant capacity Figure 8 The total antioxidant capacity of sea buckthorn juice after fermentation with strains S3, S4, YJ24, YJ2, and YSHM3 was tested. The results showed that the DPPH free radical scavenging rate of unfermented sea buckthorn juice (B1) was only 20.38%. Lactobacillus acidipiscis The DPPH free radical scavenging rate of S3 after fermentation was 56.82%; Lactobacillus plantarum The DPPH free radical scavenging rate of S4 fermentation was 45.99%; Lactobacillus plantarum strain The DPPH free radical scavenging rate of KF9YJ24 after fermentation was 56.63%. Lactobacillus casei YSHM3 exhibited a DPPH free radical scavenging rate of 55.15% after fermentation. This indicates that the fermentation process of the strain in this application activated the activity of antioxidants in sea buckthorn juice, thereby enhancing the functional value of the product.

[0077] (4) Sensory evaluation The sensory evaluation results are shown in Table 1 and Figures 9A-9E As shown, Figure 9A The sensory evaluation results of sea buckthorn juice after fermentation by strains S3 and YJ2 are as follows. Figure 9B The sensory evaluation results of sea buckthorn juice after fermentation by strains S3, S4, YJ24 and YSHM3 are as follows. Figure 9C The sensory evaluation results of sea buckthorn juice after fermentation by strains YJ24 and YJ2 are as follows. Figure 9D The sensory evaluation results of sea buckthorn juice after fermentation by strains S4 and YJ2 are as follows. Figure 9E The sensory evaluation results are for sea buckthorn juice fermented by strains YJ2 and YSHM3.

[0078] Table 3: Sensory Evaluation Results

[0079] Photosensitive evaluation showed that the astringency of sea buckthorn juice fermented by S3 and S4 was significantly improved, and the overall acceptability was increased. Compared with the control group, the astringency was improved most significantly, which was positively correlated with the malic acid content.

[0080] (5) Lactic acid bacteria viable count For live-culture lactic acid bacteria beverages, the national standard requires a live lactic acid bacteria count of ≥1×10⁻⁶. 6 CFU / mL. The viable count of lactic acid bacteria in this application shows that the viable count of sea buckthorn juice reached 1.37 × 10⁻⁶ after 96 hours of fermentation. 8 CFU / mL (8.14 log(cfu / mL)) meets the national standard requirements.

[0081] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, The classification name is Lactobacillus plantarum ( Lactobacillus plantarum (The plant number is S4, and the preservation number is CGMCC No.37352).

2. The application of *Lactobacillus plantarum* as described in claim 1 in the degradation of malic acid in sea buckthorn.

3. The application of Lactobacillus plantarum as described in claim 1 in the preparation of sea buckthorn juice.

4. A strain of *Lactobacillus acidophilus*, characterized in that, The classification name is Lactobacillus acidophilus ( Lactobacillus acidipiscis (The plant number is S3, and the preservation number is CGMCC No.37351).

5. The application of *Lactobacillus acidophilus* as described in claim 4 in the degradation of malic acid in sea buckthorn.

6. The application of Lactobacillus acidophilus as described in claim 4 in the preparation of sea buckthorn juice.

7. A fermentation agent for preparing sea buckthorn juice, characterized in that, The active ingredient is either Lactobacillus plantarum as described in claim 1 or Lactobacillus acidophilus as described in claim 4.

8. A method for preparing sea buckthorn juice, characterized in that, include: Take fresh sea buckthorn juice concentrate and mix it with sterile water according to the ratio, adjust the sugar content and pasteurize it, cool it to room temperature to complete the pretreatment; The *Lactobacillus plantarum* as described in claim 1 or the *Lactobacillus acidophilus* as described in claim 4 is cultured to the logarithmic growth phase to obtain a bacterial suspension. The bacterial suspension is then inoculated into pretreated sea buckthorn juice concentrate and cultured anaerobically at 35±1℃ for 90-96 hours.

9. The preparation method according to claim 8, characterized in that, The viable count of the bacterial suspension is 10. 8 CFU / mL ~10 9 CFU / mL; the bacterial suspension was inoculated into the pretreated sea buckthorn juice at a volume ratio of 1-3%.

10. The preparation method according to claim 8, characterized in that, Fresh sea buckthorn juice concentrate and sterile water are mixed at a mass ratio of 1:1.

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