Method for fermenting blueberry juice by using lactobacillus plantarum

Abstract

The invention discloses a method for fermenting blueberry juice by using lactobacillus plantarum, and particularly, the lactobacillus plantarum S0045 is used for realizing blueberry juice fermentation, the strain is preserved in the China Center for Type Culture Collection, the preservation number of the strain is CCTCC M NO.20252288, the preservation date is October 22, 2025, and the preservation address is Wuhan University, Wuchang District, Wuhan City, Hubei Province. Through the synergistic effect of'enzymatic conversion of anthocyanin precursors and metabolism optimization of flavor substances' of the strain, the anthocyanin content is increased by 45% or above and the flavor quality is quantified and optimized synchronously, and the three bottlenecks that'anthocyanin increase is insufficient, flavor improvement has no data support and functions and sense are difficult to consider 'in the existing fermentation technology are solved; and the method is suitable for industrial production of high-added-value functional drinks.

Description

Technical Field

[0001] This invention belongs to the field of functional fruit juice processing technology, and provides a method for fermenting blueberry juice using Lactobacillus plantarum. Background Technology

[0002] Fresh blueberries, due to their thin skin and high water content, are susceptible to microbial contamination and rotting after harvest. At room temperature, the rot rate reaches 25%-30% within 3-5 days, and even low-temperature storage (4℃) only extends the shelf life to 15-20 days. This highly seasonal nature and short shelf life result in significant economic losses after harvest each year. Therefore, blueberry juice, as a processing method that efficiently transports the active substances (anthocyanins, polyphenols, etc.) of blueberries to diverse markets, has become the mainstream direction for blueberry deep processing due to its appealing color, convenience, and long shelf life. Blueberry juice not only has a sweet and sour flavor but is also rich in polyphenols and vitamin C, with significantly higher antioxidant capacity than other fruit and vegetable juices, making it widely popular among consumers.

[0003] Anthocyanins, the core functional component of blueberry juice (accounting for 60%-70% of the total polyphenols), belong to the flavonoid polyphenol class of compounds. They are mainly composed of cyanidin-3-glucoside and delphinidin-3-galactoside, possessing well-defined physiological functions such as delaying nerve aging, preventing cardiovascular disease, regulating gut microbiota, enhancing vision, and inhibiting microbial growth. However, anthocyanins are extremely sensitive to light, temperature, oxygen, and pH: light exposure (12 hours of natural light) leads to a 25%-30% degradation rate, high temperature (30 minutes at 60℃) results in a 35%-40% degradation rate, and neutral to alkaline environments (pH≥7.0) result in over 50% degradation. Traditional processing methods (such as fresh juicing and heat sterilization) not only fail to increase anthocyanin content but also lead to a 15%-20% loss. Therefore, overcoming the technical bottleneck of "easy degradation and difficulty in increasing anthocyanin content" to produce high-value-added blueberry juice products has become a critical issue that the industry urgently needs to address. Fermented blueberry juice is made through lactic acid fermentation by microorganisms (mainly lactic acid bacteria). It combines the nutritional properties of blueberry juice with the probiotic functions of probiotics (such as improving intestinal barrier function and enhancing immunity). In recent years, with the increasing consumer demand for "low-fat, low-sugar, and cholesterol-free" healthy diets (annual growth rate of 12%-15%), the market attention on fermented blueberry juice has significantly increased. Utilizing microbial fermentation technology to increase the content of active ingredients is an innovative research direction in the fermentation field. Blueberry juice fermented with lactic acid bacteria can retain original nutrients (such as vitamin C retention rate ≥85%) and antioxidants (total phenol retention rate ≥90%), while increasing anthocyanin content through the metabolic activity of the bacterial strains and generating flavor substances such as ethyl acetate and phenylethanol, giving the product a unique fermented flavor. However, the types and strains of lactic acid bacteria significantly affect fermentation results: currently, only 15%-20% of known lactic acid bacteria can be used for fruit and vegetable juice fermentation, and different genera have diametrically opposed effects on anthocyanin content. (Li Haikun) [1] Fermentation of blueberry juice using *Lactobacillus plantarum* and *Lactobacillus casei* showed a 24.4% and 32.9% reduction in anthocyanin content, respectively, compared to freshly squeezed juice. The core reason was the lack of anthocyanin-converting enzymes in the strains, and the accelerated oxidation of anthocyanins by hydrogen peroxide produced during metabolism. Even different strains of the same genus (*Lactobacillus plantarum*) showed significant differences in results. (Li Sujin) [2] It was found that fermentation with *Lactobacillus plantarum* Lp T15 resulted in a 30.28% decrease in anthocyanin content in blueberry juice (due to the strain's rapid acid production leading to a pH < 3.0, which exacerbated anthocyanin degradation); Li Hongfu [3] Although the reported Lactobacillus plantarum J26 can increase anthocyanin content, the increase rate is only 15.38%, and the fermentation cycle is as long as 36 hours, resulting in low efficiency for industrial production.

[0004] In summary, existing technologies either cause anthocyanin degradation by the strains or the strains that can improve anthocyanin content suffer from drawbacks such as "low amplitude, long cycle, and high cost." There is a lack of lactic acid bacteria strains specifically designed to improve the anthocyanin content of blueberry juice that are "low-cost (no need for exogenous enzyme addition), highly active (anthocyanin improvement rate ≥30%), and have a short cycle (≤24h)". Therefore, screening for Lactobacillus plantarum with the above characteristics is the key to achieving efficient enhancement of anthocyanins in blueberry juice, and also makes Lactobacillus plantarum the preferred strain for fermenting blueberry juice. Currently, none of the publicly disclosed patents related to lactic acid bacteria fermented blueberry juice in China have addressed the core need for "targeted enhancement of anthocyanins." Specific shortcomings are as follows: Patent CN105581218B discloses a method for improving the flavor quality of wild blueberry juice by fermentation with Lactobacillus plantarum. Its core objective is to "improve flavor quality". It reduces the astringency of the juice by optimizing the fermentation temperature (28-32℃). However, it does not use anthocyanins as an evaluation indicator. The actual test showed that the anthocyanin content did not change significantly after fermentation (the improvement rate was <5%), and it did not involve strain-specific screening. Patent application CN119174472A discloses a method for preparing lactic acid bacteria fermented blueberry juice, focusing on "extending shelf life". It achieves commercial sterility through compound sterilization (fermentation + pasteurization), but the strain is a conventional Lactobacillus plantarum (the specific strain number is not disclosed). After fermentation, the anthocyanin content only increases by 8%-10%, and the flavor substances are not optimized (ethyl acetate content <0.8mg / L).

[0005] Patent CN116569999B discloses a fermentation method for blueberry juice, which focuses on "increasing the content of total phenols and total flavonoids" and adopts an "ultrasound-assisted fermentation" process. The total phenol content is increased by 22%-25%, but the anthocyanin content is only increased by 12%-14%. Moreover, the ultrasonic treatment increases energy consumption (100-130 kWh / t), resulting in high industrialization costs. In summary, existing patents either focus on flavor, total phenols, or shelf life, but none of them have designed technical solutions for "highly efficient enhancement of anthocyanins," and none of the strains involved have the dual ability to "significantly enhance anthocyanins and optimize flavor." Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a method for fermenting blueberry juice using Lactobacillus plantarum.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. Lactobacillus plantarum, classified as Lacticacid bacteria S0045, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M NO. 20252288, deposited on October 22, 2025, at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Preferably, the 16S rDNA sequence of the *Lactobacillus plantarum* is shown in SEQ ID NO.1.

[0009] 2. The aforementioned application of Lactobacillus plantarum in the fermentation and preparation of blueberry juice.

[0010] 3. A method for fermenting blueberry juice using Lactobacillus plantarum, the specific steps of which are as follows: S1. Blueberry pretreatment: Take fresh blueberries, wash, crush, filter, sterilize, cool, and obtain juice; S2. Inoculate the juice with the aforementioned *Lactobacillus plantarum* and allow it to undergo anaerobic fermentation; S3. Post-ripening optimization yields blueberry juice.

[0011] Preferably, in step S1, the ripeness of the fresh blueberries is 85-90%, and the judgment criteria are: uniform purplish-black appearance with intact bloom; soft and elastic texture, without being hard or rotten; sweet with a slight sour taste; and soluble solids of 12-15%.

[0012] Preferably, in step S1, water is used for rinsing to remove surface impurities.

[0013] Preferably, in step S1, a juicer is used for crushing, and the crushing conditions are: rotation speed 1500 r / min, time 2 min.

[0014] Preferably, in step S1, 300-mesh gauze is used for filtration to remove fruit residue, with a fruit residue removal rate of ≥98%.

[0015] Preferably, in step S1, the sterilization conditions are: high-temperature sterilization at 95°C for 15 minutes to kill miscellaneous bacteria and endogenous enzymes.

[0016] Preferably, in step S1, the temperature is cooled to 30°C to avoid killing the bacterial strain with high temperature.

[0017] Preferably, in step S2, after activation, *Lactobacillus plantarum* is inoculated into the juice at a volume inoculation rate of 1.0%, specifically as follows: Take *Lactobacillus plantarum* freeze-dried powder (viable count ≥10) preserved at -80℃. 9 The bacterial culture was aseptically inoculated into MRS liquid medium (purchased from Beijing Luqiao Technology Co., Ltd.) at 37°C for 24 hours to obtain the first-generation bacterial culture. 1 mL of the first-generation culture was inoculated into fresh MRS liquid medium and aseptically incubated at 30°C for 12 hours to obtain the second-generation bacterial culture. The second-generation culture was centrifuged at 8000 rpm for 2 minutes at 4°C, the supernatant was discarded, and the cells were washed twice with sterile 0.85% (w / v) saline. Finally, the cells were resuspended in sterile 0.85% saline and the bacterial concentration was adjusted to 10⁻⁶ CFU / mL. 7 CFU / mL.

[0018] Preferably, in step S2, the anaerobic fermentation conditions are: anaerobic fermentation at 30℃ for 22 hours, with an anaerobic degree ≥95% in the fermenter.

[0019] Preferably, in step S3, the post-ripening optimization method is: refrigeration at 4℃ for 10 hours.

[0020] The beneficial effects of this invention are: This invention provides a method for fermenting blueberry juice using Lactobacillus plantarum. Specifically, the fermentation of blueberry juice is achieved using Lactobacillus plantarum S0045, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M NO., deposit date of October 22, 2025, and address at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0021] The plant lactobacillus of the present invention can increase anthocyanin content by more than 45% through its own metabolism without the need for exogenous assistance, and at the same time generate 30 key flavor substances, to obtain a fermented blueberry juice product with "high function + excellent sensory qualities", thus meeting consumers' demand for functional beverages.

[0022] This invention achieves a simultaneous increase of over 45% in anthocyanin content and quantitative optimization of flavor quality through the synergistic effect of strains “enzymatic conversion of anthocyanin precursors + metabolic optimization of flavor substances”. It solves the triple bottleneck of existing fermentation technology, namely “insufficient anthocyanin enhancement, lack of data support for flavor improvement, and difficulty in balancing function and sensory benefits”, and is suitable for the industrial production of high-value-added functional beverages.

[0023] The core advantage of this invention is "synergistic metabolism of three enzymes": β-Glucosidase: Enzyme activity 15.2 U / mL, it selectively hydrolyzes bound anthocyanin precursors (such as anthocyanin-3-glucosidase) in the cell wall, converting them into free active anthocyanins, thus achieving a leap in their content; Esterase: Activity 8.6 U / mL, catalyzes the synthesis of characteristic flavor compounds such as ethyl acetate (fruity aroma) and phenylethanol (floral aroma) from fatty acids and alcohols; Protease: Activity 3.2 U / mL, degrades bitter peptides in fruit juice (such as hydrophobic peptides with a molecular weight of 1000-2000 Da), reduces astringency, and optimizes taste.

[0024] This invention has the following advantages: 1. Function-Flavor Synergistic Breakthrough: Without the need for exogenous enzymes or physical assistance, it achieves an anthocyanin increase of over 45% (absolute content 27.04 mg / L) and simultaneous optimization of flavor quality solely through the endogenous enzyme system of the strain, resolving the contradiction of "one thing at the expense of another" in existing technologies. The sensory score reaches 8.7 points (close to the full score), meeting the needs of high-end functional beverages. 2. Technical cost advantage: The fermentation cycle is only 32 hours (22 hours of fermentation + 10 hours of post-ripening), which is 33.3% shorter than the existing patented process (48 hours), and no exogenous enzymes need to be added, which can reduce the cost of industrial production; 3. Strong market competitiveness: The product has an anthocyanin content of ≥27 mg / L (can be labeled with the "high anthocyanin" functional claim), and contains 30 kinds of key flavor substances, which have better flavor quality, thus making the product more competitive in the market.

[0025] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 Morphological characteristics of Lactobacillus plantarum S0045; Figure 2 Morphological identification of Lactobacillus plantarum S0045; Figure 3 Blueberry juice fermented with Lactobacillus plantarum S0045.

[0027] Biological Preservation Information Classification and nomenclature: Lacticacid bacteria S0045; Accession number: CCTCC M NO. 20252288; Preservation period: October 22, 2025; Depository: China Center for Type Culture Collection; Address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, inside Wuhan University. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments.

[0029] Example 1 Screening and identification of Lactobacillus plantarum S0045 1. Experimental Materials 1.1 Sample Source: Fermented vegetable samples from the Fruit and Vegetable Processing Laboratory, College of Food Science, Southwest University (fermentation time 60 days, pH 3.8, lactic acid bacteria content 10). 8 CFU / g); 1.2 Culture media: MRS liquid medium (tryptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, sodium acetate 5g / L, diammonium citrate 2g / L, Tween-80 1mL / L, MgSO4·7H2O 0.58g / L, MnSO4·4H2O 0.25g / L, pH 6.2±0.2, sterilized at 121℃ for 20min); MRS solid medium (added with 1.5% agar, the rest is the same as the liquid medium); 1.3 Reagents: β-glucosidase assay kit (Shanghai Enzyme-Linked Biotechnology, catalog number ELK-B0256), esterase assay kit (catalog number ELK-B0258), protease assay kit (catalog number ELK-B0260); 16S rDNA amplification primers (forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', synthesized by Beijing Qingke Biotechnology).

[0030] 2 Experimental Methods 2.1 Strain Isolation: Take 10g of fermented vegetable sample, add 90mL of sterile physiological saline (0.85% NaCl), shake for 30min (180r / min) to prepare 10⁻ 1 Diluent, serially diluted to 10⁻ 6 Take 0.1 mL of each dilution and spread it on MRS solid medium. Incubate anaerobically at 37℃ for 48 h. Pick single colonies that are "round, creamy white, and 0.5-2 mm in diameter" and inoculate them onto MRS liquid medium. Incubate at 37℃ for 24 h to obtain 32 pure culture strains. 2.2 Initial screening (anthocyanin enhancement rate): 32 strains were inoculated into blueberry juice at a volume of 1.0% and fermented at 30℃ for 24 hours. The anthocyanin content was measured by pH differential method. Eight strains with enhancement rates ≥10% were screened and numbered S0020, S0025, S0030, S0035, S0040, S0045, S0050, and S0053, respectively. 2.3 Secondary screening (enzyme activity and flavor): The activities of β-glucosidase, esterase and protease of 8 strains were determined, and the strain with the highest activities of all three enzymes (number S0045) was selected, with β-glucosidase activity of 15.2 U / mL, esterase activity of 8.6 U / mL and protease activity of 3.2 U / mL; 2.4 Molecular Identification: DNA from strain S0045 was extracted using a bacterial genomic DNA extraction kit (Tiangen Biotech, catalog number DP302) and amplified by 16S rDNA PCR (25μL system: 2μL DNA template, 12.5μL 2×Taq PCRMasterMix, 1μL each of forward and reverse primers, 8.5μL sterile water; amplification program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles, 72℃ final extension for 10 min). The amplified product was sent to Beijing Qingke Biotechnology for sequencing. The sequencing results were compared with NCBI BLAST and showed 99.2% homology with the Lacticacid bacteria S0045 standard strain (GenBank accession number MN813456.1), confirming its classification as Lactobacillus plantarum.

[0031] 3 Results and Analysis 3.1 Screening results: S0045 was selected from 32 strains, with an anthocyanin enhancement rate of 42.51% (initial screening). During the secondary screening, the activity of the three enzymes was significantly higher than that of other strains, showing dual potential for "anthocyanin conversion + flavor optimization". 3.2 Morphological characteristics: S0045 colonies were round and raised, creamy white, moist and smooth, and without hemolysis when cultured on MRS solid medium at 37℃ for 48 h (Figure 1); Gram staining was positive (purple short bacillus, Figure 2), which is consistent with the morphological characteristics of Lactobacillus plantarum. 3.3 16S rDNA sequence analysis of the strain The 16S rDNA sequence is shown in SEQ ID NO.1, and the results show that the strain is Lactobacillus plantarum.

[0032] Example 2 Application of Lactobacillus plantarum S0045 fermentation to enhance anthocyanin content in blueberry juice 1. Experimental Materials 1.1 Fresh blueberries: provided by Chongqing Ruihang Biotechnology Co., Ltd., variety "Mist", soluble solids 12.5%, initial anthocyanin content 15.20 mg / L.

[0033] 1.2 Experimental strains: Lactobacillus plantarum S0030 (laboratory collection), Lactobacillus plantarum S0035 (laboratory collection), Lactobacillus plantarum S0045 (the strain of this invention), and Lactobacillus plantarum S0053 (laboratory collection).

[0034] 1.3 Instruments: pH meter (Shanghai Leici, PHS-3C), PAL-1 portable saccharimeter (Japan Aituo), UV-Vis spectrophotometer (Shimadzu UV-2600), high-speed centrifuge (Xiangyi H1650-W).

[0035] 2 Experimental Methods 2.1 Activation of bacterial strains: Take each strain's cryopreservation tube (-80℃), and aseptically inoculate one loop into MRS liquid medium. Incubate anaerobically at 37℃ for 24 h (activation of the first generation). Take 1 mL of the first generation bacterial culture and inoculate into fresh MRS liquid medium. Incubate anaerobically at 30℃ for 12 h (activation of the second generation). Centrifuge the second generation bacterial culture at 8000 rpm for 2 min at 4℃, discard the supernatant, wash the cells twice with sterile 0.85% (w / v) saline, and finally resuspend in sterile 0.85% saline to adjust the bacterial concentration to 10. 7 CFU / mL (verified by plate counting method); 2.2 Preparation of Fermented Blueberry Juice: Fresh blueberries, with a ripeness of 85-90%, judged by the following criteria: uniform purplish-black appearance with intact bloom; soft and elastic texture, without being hard or rotten; sweet with a slight tartness; and soluble solids of 12-15%. Rinse with water to remove surface impurities. Crush the blueberries in a juicer at 1500 rpm for 2 minutes. Filter through 300-mesh gauze to remove pulp, achieving a pulp removal rate of ≥98%. Sterilize at 95℃ for 15 minutes and cool to 30℃.

[0036] (A) *Lactobacillus plantarum* S0030, (B) *Lactobacillus plantarum* S0035, (C) *Lactobacillus plantarum* S0045, and (D) *Lactobacillus plantarum* S0053 were inoculated at 1.0% (v / v) and fermented in a constant-temperature anaerobic incubator at 30℃ and ≥95% anaerobic concentration for 22 hours. After fermentation, post-ripening optimization was performed, and the viable cell count in the finished product was determined using the plate count method to be 1.0-1.2 × 10⁻⁶. 7 CFU / mL; 2.3 pH test: Take 10 mL of fermented blueberry juice sample and measure the pH value directly with a pH meter. Each sample is measured in parallel 3 times and the average value is taken. 2.4 Detection of soluble solids: Take 2 drops of blueberry juice sample with a dropper and drop it onto the detection prism of the PAL-1 portable saccharimeter. Wipe it clean before measuring. The result is expressed as "%". Perform 3 parallel measurements and take the average value. 2.5 Anthocyanin content detection: Take two 1 mL blueberry juice samples and add 5 mL of buffer solutions at pH 1.0 (KCl-HCl buffer, 0.2 mol / L KCl and 0.2 mol / L HCl mixed at a volume ratio of 25:67) and pH 4.5 (NaAc-HAc buffer, 0.4 mol / L NaAc and 0.2 mol / L HAc mixed at a volume ratio of 40:60). After standing in the dark for 1 h, measure the absorbance at wavelengths of 520 nm and 700 nm using a UV-Vis spectrophotometer (with the corresponding buffer solution as a blank control). Calculate the anthocyanin content by substituting the absorbance into the formula. The total anthocyanins in the sample are expressed as cyanidin-3-O-glucoside (C3G) equivalents.

[0037] Calculation formula: TAC = [(A 520 - A 700 (pH=1.0) - (A 520 - A 700 )(pH=4.5)] × Mw × DF × 1000 / (ε × L) Where: TAC represents the anthocyanin content of fermented blueberry juice (mg / L); A 520 A 700 The absorbance values ​​are 520 nm and 700 nm, respectively; Mw is the relative molecular mass of cyanidin-3-O-glucose (449 g / mol); DF is the dilution factor (DF=1 in this experiment); ε is the molar extinction coefficient of cyanidin-3-O-glucose (26900 L / (mol・cm)); L is the optical path length (1 cm).

[0038] 3 Results and Analysis 3.1 pH changes in fermented blueberry juice Figure 3 shows the actual product of blueberry juice fermented by Lactobacillus plantarum S0045. It is purplish-red, without layering, and has the typical appearance and color of blueberry juice. The pH of the blueberry juice before and after fermentation was 4.2±0.05 before fermentation, and decreased to 3.15±0.04 after 22 h of fermentation.

[0039] 3.2 Changes in soluble solids in fermented blueberry juice Table 1 shows the soluble solids content in blueberry juice after fermentation with four different strains of Lactobacillus plantarum. The soluble solids content in the unfermented group was 12.5±0.2%. After 22 hours of fermentation: S0030 group 11.8±0.2%, S0035 group 12.2±0.1%, S0045 group 12.0±0.1%, and S0053 group 11.6±0.2%, all showing a slight decreasing trend. The slight decrease in soluble solids is due to the strain metabolizing some sugars (such as glucose and fructose) to produce organic acids and energy, but the decrease is small (≤0.9%), indicating that strain S0045 has moderate sugar utilization efficiency and will not cause the juice to taste too bland.

[0040] Table 1. Comparison of soluble solids content in blueberry juice before and after fermentation for four different strains (n=3) Group Soluble solids content (%) Unfermented group 12.5±0.2 S0030 Fermentation group (22h) 11.8±0.2 S0035 Fermentation group (22h) 12.2±0.1 S0045 Fermentation group (22h) 12.0±0.1 S0053 Fermentation group (22h) 11.6±0.2 3.3 Changes in anthocyanins in fermented blueberry juice To investigate the effects of different *Lactobacillus plantarum* strains on the anthocyanin content of blueberry juice, the anthocyanin content was measured after 22 h of fermentation. The results are shown in Table 2. S0035 was the only non-inventory strain to show a slight positive increase (+5.23%), but the increase was only 11.6% of that of S0045. S0030 (-18.76%) and S0053 (-9.45%) showed varying degrees of decrease, further confirming the uniqueness and superiority of S0045 in enhancing anthocyanin content. This eliminates the inevitable factor that "all lactic acid bacteria strains can enhance anthocyanin content," highlighting the specific advantages of the strains of this invention.

[0041] Table 2 Comparison of anthocyanin content in blueberry juice before and after fermentation for four strains (n=3) Fermentation strains Anthocyanin content before fermentation (mg / L) Anthocyanin content (mg / L) after 22 hours of fermentation rate of change Blank group (unfermented) 15.20±0.12 15.20±0.12 0% Lactobacillus plantarum S0030 15.20±0.12 12.38±0.13 -18.76% Lactobacillus plantarum S0035 15.20±0.12 16.00±0.10 +5.23% Lactobacillus plantarum S0045 15.20±0.12 27.04±0.15 +45.0% Lactobacillus plantarum S0053 15.20±0.12 13.76±0.11 -9.45% To verify the industry-leading performance of *Lactobacillus plantarum* S0045, it was compared with literature strains (including patented strains), and the results are shown in Table 3. Significant core advantages: *Lactobacillus plantarum* S0045 achieved a 45.0% increase in anthocyanin content in fermented blueberry juice, 2.92 times higher than the best strain in existing literature, J26 (15.38%), completely breaking through the technical bottleneck of "increase rate ≤20%"; more importantly, S0045 achieved an absolute increase of 11.84 mg / L even with low initial anthocyanin content (15.20±0.12 mg / L), 1.65 times that of J26 (7.17 mg / L). It can efficiently convert blueberry raw materials from non-core production areas and with slightly lower ripeness, significantly reducing the cost of industrial production dependent on raw material quality. Existing strains (such as J26 and LP) rely solely on a passive mechanism of "acid production to lower pH and reduce anthocyanin degradation," resulting in limited enhancement effects. In contrast, S0045 constructs a technological barrier through a unique synergistic mechanism of "active enzymatic transformation + passive environmental stabilization." On one hand, its β-glucosidase activity reaches 15.2 U / mL, converting over 70% of bound anthocyanin precursors into free forms. On the other hand, the post-fermentation pH remains stable at 3.2±0.04, with a polyphenol oxidase inhibition rate of 78.5%, reducing oxidative losses. In summary, S0045 is currently the only dedicated strain capable of achieving "low initial content → high enhancement rate → high final content" of anthocyanins in blueberry juice, providing crucial support for the industrial production of high-anthocyanin functional blueberry juice.

[0042] Table 3. Comparison of anthocyanin content in blueberry juice fermented by the strains of this invention and those in the literature. Fermentation strains Anthocyanin content before fermentation (mg / L) Anthocyanin content after fermentation (mg / L) Improvement rate References Lactobacillus plantarum S0045 15.20±0.12 27.04±0.15 45.0% This invention Lactobacillus plantarum J26 46.62 53.79 15.38% [3] Lactobacillus plantarum SH-470 197.88±3.28 206.40±0.87 4.31% [4] Lactobacillus plantarum LP 5.19 6.04 14.07% [5] Example 3: Application of Lactobacillus plantarum S0045 in enhancing the flavor and quality of blueberry juice 1. Experimental Materials 1.1 Samples: Fermented blueberry juice S0030, Fermented blueberry juice S0035, Fermented blueberry juice S0045, Fermented blueberry juice S0053, and unfermented blueberry juice from Example 2; 1.2 Instruments: Electronic tongue (ASTREE II, Alpha MOS, France, sensor array: ZZ, AB, GA, BB, CA, DA, JE), gas chromatography-mass spectrometry (GC-MS, Agilent 7890A-5975C), solid phase microextraction fiber head (50 / 30μm DVB / CAR / PDMS, Supelco); 1.3 Reagents: Ethyl acetate, phenylethanol, ethyl hexanoate, and other standards (purity ≥98%, Sigma-Aldrich).

[0043] 2 Experimental Methods 2.1 Sensory Evaluation: A professional judging panel of 10 people (5 men and 5 women, aged 25-45, who have passed sensory training) was formed. A 9-point scoring method was used to score the samples from three dimensions: “sweetness and acidity balance (0-9 points, 9 points for complete sweetness and acidity balance), aroma richness (0-9 points, 9 points for rich and layered aroma), and no astringency (0-9 points, 9 points for no astringency). The total score is calculated as follows: sweetness and acidity balance × 0.4 + aroma richness × 0.3 + no astringency × 0.3. Each sample was scored 3 times and the average score was taken. 2.2 Electronic tongue detection: Take 10mL of juice sample, equilibrate at 4℃ for 30min, and use an electronic tongue to detect taste parameters (sweetness value, astringency value). The detection time is 120s, the cleaning time is 60s, and each sample is tested 3 times and the average value is taken. 2.3 Flavor compound detection (GC-MS): Take 5 mL of juice sample and place it in a 20 mL headspace vial. Add 10 μL of internal standard (2-octanol, 100 μg / mL), seal the vial, and equilibrate at 40 °C for 30 min. Insert the solid phase microextraction fiber head and allow it to adsorb for 30 min. Then, insert the GC injection port (250 °C) for desorption for 5 min. GC conditions: HP-5MS capillary column (30m×0.25mm×0.25μm); column temperature program: 40℃ for 3 min, then ramped up to 230℃ at 5℃ / min and held for 5 min; carrier gas: He (purity ≥99.999%), flow rate 1.0 mL / min; split ratio 10:1; MS conditions: Electron impact ionization source (EI), ionization energy 70 eV; ion source temperature 230℃; quadrupole temperature 150℃; scan range m / z 35-450; external standard method for quantitative analysis (gradual dilution of standards, plotting standard curves).

[0044] 3 Results and Analysis 3.1 Sensory rating results The sensory scores of different samples are shown in Table 4. The unfermented group had the lowest total score (5.2±0.3), followed by groups S0030 and S0053 (5.8±0.2 and 5.6±0.2, respectively), group S0035 had a moderate score (6.9±0.1), and group S0045 had the highest score (8.7±0.1), with statistically significant differences compared to other groups (P<0.05).

[0045] Table 4 Comparison of sensory scores of different samples (n = 3, 9-point scale) Sample type Sweet and sour balance Aroma richness No astringency Total Score Unfermented blueberry juice 4.5±0.2 3.8±0.3 2.2±0.2 5.2±0.3 S0030 Fermented Blueberry Juice 5.1±0.1 4.3±0.2 3.1±0.1 5.8±0.2 S0053 Fermented Blueberry Juice 4.9±0.1 4.1±0.2 2.9±0.1 5.6±0.2 S0035 Fermented Blueberry Juice 6.3±0.1 5.9±0.1 4.8±0.1 6.9±0.1 S0045 Fermented Blueberry Juice 8.9±0.1 8.5±0.1 8.7±0.1 8.7±0.1 3.2 Results of electronic tongue taste parameters The sweetness values and astringency values detected by the electronic tongue are shown in Table 5, presenting an obvious gradient: sweetness value: group S0045 (6.2 ± 0.1) > group S0035 (4.7 ± 0.1) > group S0030 (3.8 ± 0.1) > group S0053 (3.6 ± 0.1) > unfermented group (3.1 ± 0.2); the astringency value is opposite, group S0045 (2.1 ± 0.1) < group S0035 (4.1 ± 0.1) < group S0030 (5.7 ± 0.1) < group S0053 (5.9 ± 0.1) < unfermented group (6.8 ± 0.2).

[0046] Table 5 Comparison of electronic tongue taste parameters of different samples (n = 3) Sample type Sweetness level Astringency value Unfermented blueberry juice 3.1±0.2 6.8±0.2 S0030 Fermented Blueberry Juice 3.8±0.1 5.7±0.1 S0053 Fermented Blueberry Juice 3.6±0.1 5.9±0.1 S0035 Fermented Blueberry Juice 4.7±0.1 4.1±0.1 S0045 Fermented Blueberry Juice 6.2±0.1 2.1±0.1 3.3 Results of GC-MS flavor substance analysis The contents and total numbers of key flavor substances detected by GC-MS are shown in Table 6. A variety of juice characteristic flavor substances are detected in all samples, presenting a gradient increasing trend of "unfermented group → S0053 / S0030 group → S0035 group → S0045 group", and group S0045 is significantly leading in both "quantity + content": total number of flavor substances: group S0045 reaches 30 kinds, increasing by 100% compared with the unfermented group (15 kinds), increasing by 87.5% compared with group S0030 (16 kinds), increasing by 76.5% compared with group S0053 (17 kinds), and increasing by 50% compared with group S0035 (20 kinds); contents of core aroma substances: for key flavor substances such as ethyl acetate (fruity aroma), phenethyl alcohol (floral aroma), ethyl hexanoate (sweet aroma), etc., the contents of group S0045 are 1.6 times, 1.5 times, and 4.4 times that of group S0035 respectively, and are 2.9 - 3.1 times, 3.0 - 3.4 times that of group S0030 / S0053 group, and a breakthrough from not detected to detected; characteristic flavor substances: eugenol (spicy aroma) and γ-decalactone (milky aroma) are only detected in group S0045 with relatively high contents (0.22 mg / L, 0.18 mg / L), although detected in group S0035 but with extremely low contents (0.05 mg / L, 0.04 mg / L), and not detected in the other groups; geraniol (floral aroma) is only detected in group S0035 and group S0045, and the content of group S0045 is 2.1 times that of group S0035.

[0047] Table 6 Comparison of main flavor substance contents of different samples (n = 3, mg / L) Key flavor compounds Unfermented blueberry juice S0030 Fermented Blueberry Juice S0053 Fermented Blueberry Juice S0035 Fermented Blueberry Juice S0045 Fermented Blueberry Juice Ethyl acetate (fruity aroma) 0.32±0.02 0.55±0.02 0.51±0.02 0.98±0.03 1.58±0.04 Phenylacetyl alcohol (floral scent) 0.18±0.01 0.25±0.01 0.23±0.01 0.52±0.02 0.78±0.03 Ethyl hexanoate (sweet flavor) Not detected 0.05±0.01 Not detected 0.08±0.01 0.35±0.02 Eugenol (Spicy) Not detected Not detected Not detected 0.05±0.01 0.22±0.01 γ-Decanolactone (Boswellia carterii) Not detected Not detected Not detected 0.04±0.01 0.18±0.01 Ethyl caprylate (fruity flavor) 0.06±0.01 0.09±0.01 0.08±0.01 0.15±0.01 0.30±0.02 Geraniol (floral scent) Not detected Not detected Not detected 0.12±0.01 0.25±0.01 2-Heptanone (fruity aroma) 0.04±0.01 0.07±0.01 0.06±0.01 0.10±0.01 0.21±0.01 Isoamyl alcohol (aromatic aroma) Not detected 0.08±0.01 0.07±0.01 0.11±0.01 0.19±0.01 Limonene (citrus scent) Not detected Not detected 0.05±0.01 0.09±0.01 0.17±0.01 Total number of flavor compounds 15 kinds 16 kinds 17 kinds 20 kinds 30 kinds Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0048] References: [1] Li Haikun. Study on quality changes and gastric mucosal protection of probiotic fermented blueberry juice [D]. Shenyang Agricultural University, 2023. [2] Li Sujin. Screening of endophytic lactic acid bacteria from fruits and their functional characteristics in fermented blueberry juice [D]. Nanjing Agricultural University, 2021. [3] Li Hongfu. Study on the fermentation process and functional properties of blueberry juice by Lactobacillus plantarum [D]. Northeast Agricultural University, 2019. [4] Zhang Chenyan, Zhang Lixia, Wei Zhaohui, et al. Optimization of fermentation process and study on functional properties of blueberry juice by lactic acid bacteria [J]. Journal of Henan University of Technology (Natural Science Edition), 2022, 43(05): 77-85. [5] Ding Mingke, Chen Huizhi, Wu Weijie, et al. Analysis on nutritional quality and flavor changes of blueberry and mulberry pulp fermented with different lactic acid bacteria [J]. Journal of Nuclear Agricultural Sciences, 2025, 39(02):297-306.

Claims

1. Lactobacillus plantarum, characterized in that, Its classification name is Lacticacid bacteria (S0045) S0045. It has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M NO. 20252288, deposit date October 22, 2025, and deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The application of the *Lactobacillus plantarum* as described in claim 1 in the fermentation preparation of blueberry juice.

3. A method for fermenting blueberry juice using *Lactobacillus plantarum* as described in claim 1, characterized in that, The specific steps are as follows: S1. Blueberry pretreatment: Take fresh blueberries, wash, crush, filter, sterilize, cool, and obtain juice; S2. Inoculate the juice with the aforementioned *Lactobacillus plantarum* and allow it to undergo anaerobic fermentation; S3. Post-ripening optimization yields blueberry juice.

4. The method according to claim 3, characterized in that, In step S1, the fresh blueberries are 85-90% ripe.

5. The method according to claim 3, characterized in that, In step S1, a juicer is used for crushing, with the following crushing conditions: speed 1500 r / min, time 2 min.

6. The method according to claim 3, characterized in that, In step S1, filtration is achieved using 300-mesh gauze.

7. The method according to claim 3, characterized in that, In step S1, the sterilization conditions are: high-temperature sterilization at 95℃ for 15 minutes.

8. The method according to claim 3, characterized in that, In step S2, after activation, Lactobacillus plantarum is inoculated into the juice at a volume inoculation rate of 1.0%.

9. The method according to claim 3, characterized in that, In step S2, the anaerobic fermentation conditions are: anaerobic fermentation at 30℃ for 22 hours, with an anaerobic degree ≥95% in the fermenter.

10. The method according to claim 3, characterized in that, In step S3, the post-ripening optimization method is: refrigeration at 4℃ for 10 hours.

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