Aroma-enhancing lactobacillus plantarum with high beta-glucosidase activity and application of aroma-enhancing lactobacillus plantarum

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to aroma-enhancing lactobacillus plantarum with high beta-glucosidase activity and application of the aroma-enhancing lactobacillus plantarum. The lactobacillus plantarum ES-25 is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 24, 2023, the preservation number is CGMCC No.28003, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the strain is separated from dry red wine which is subjected to spontaneous malic acid-lactic acid fermentation in a Xinjiang producing area, and the lactobacillus plantarum ES-25 has the advantages that the lactobacillus plantarum ES-25 has the advantages that the lactobacillus plantarum ES-25 can be used for producing the dry red wine which is subjected to spontaneous malic acid-lactic acid fermentation in the Xinjiang producing area; the strain is recognized as a safe strain which can be used for food. The lactobacillus plantarum can efficiently complete malic acid-lactic acid fermentation, has high beta-glucosidase activity, and can significantly increase the content of esters, aldehydes, ketones and terpenes aroma substances in wine in the fermentation stage; wherein the content of the linalool and the content of the citronellol are respectively increased by 77.34 times and 50.66 times compared with the wine which is not subjected to malic acid-lactic acid fermentation, so that the aroma sensory characteristics of the wine are optimized.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a flavor-enhancing Lactobacillus strain with high β-glucosidase activity and its applications. Background Technology

[0002] Malolactic fermentation (MLF) is a crucial biotransformation process in winemaking, its core function being to enhance the sensory stability and flavor complexity of wine. This process involves lactic acid bacteria converting sharp malic acid into softer lactic acid, accompanied by the release of carbon dioxide. This naturally reduces the acidity of the wine, making it more rounded and mellow, a role particularly important for overly acidic red wines and high-acidity white wines.

[0003] Traditional malolactic fermentation in wine is carried out by *Cytococcus vinifera* (…). Oenococcus oeni Lactobacillus plantarum (Lactobacillus) is dominant due to its excellent tolerance to high-acid, high-ethanol environments. However, as winemakers increasingly demand flavor diversity and fermentation efficiency, Lactobacillus plantarum (Lactobacillus) has become a more important factor in the fermentation process. Lactiplantibacillus plantarum It has gradually become a research hotspot and is regarded as a highly promising alternative fermentation agent, providing a new way to optimize the sensory quality of wine.

[0004] Despite the significant advantages of *Lactobacillus plantarum* in multi-stage fermentation (MLF), its commercial application still faces multi-dimensional challenges, urgently requiring breakthroughs through collaboration between industry, academia, and research. Currently, the application of *Lactobacillus plantarum* is moving from the laboratory to commercialization, but its performance exhibits high strain specificity. Successful commercial application depends on the precise screening of specific strains and the optimization of fermentation processes to ensure that it can efficiently and stably complete MLF and impart ideal flavor characteristics to the wine, ultimately becoming a sophisticated tool for winemakers to shape specific wine styles.

[0005] Patent No. 202311139412.9, entitled "A Strain of Lactobacillus plantarum that Can Degrade Malic Acid and Its Application," Lactobacillus plantarum ZG-3, accession number CGMCC No. 27170. This strain, when simultaneously inoculated and fermented with Saccharomyces cerevisiae, can reduce malic acid in wine from 8.10 g / L to 0.39 g / L, achieving a reduction rate of 95.19%; when sequentially inoculated and fermented with Saccharomyces cerevisiae, it can reduce malic acid in wine from 8.30 g / L to 0.49 g / L, achieving a reduction rate of 94.10%. Patent No. 202211215250.8, entitled "Lactobacillus plantarum Strain and Its Application," discloses a strain of Lactobacillus plantarum and its application, screening out a native Lactobacillus plantarum P520 and simultaneously / sequentially inoculating and fermenting this strain with Saccharomyces cerevisiae in dry red wine. The problem with this patent is: 1. Stability and reliability deficiencies The fermentation process is unpredictable, and the start time and completion efficiency of malolactic fermentation vary greatly between different batches, resulting in unstable product quality and failing to meet the strict requirements of modern brewing for production planning and brand consistency.

[0006] 2. Flavor and safety risks Its metabolite profile is unclear, and there is a significant possibility of introducing sensory defects (such as excessive volatile acids and diacetyl) or safety risks (such as biogenic amines), posing a potential threat to the sensory quality and food safety of the final wine.

[0007] 3. Bottlenecks in process controllability Due to the lack of clear strain characteristics data and standardized process guidance, its application is highly dependent on experience, making it difficult to carry out precise quality control and technology replication, thus hindering the effective accumulation of brewing knowledge and the continuous optimization of the process.

[0008] In summary, these shortcomings collectively point to the fundamental deficiencies in the stability, safety, and controllability of domestically produced fermentation agents. Summary of the Invention: In view of this, the present invention provides a strain of aroma-enhancing Lactobacillus plantarum with high β-glucosidase activity and its application to effectively solve the three core problems in the wine industry: difficulty in initiating malolactic fermentation, insufficient aroma enhancement, and safety risks of biogenic amines.

[0009] To solve the above problems, the technical solution provided by the present invention is as follows: Flavor-enhancing Lactobacillus plantarum with high β-glucosidase activity Lactiplantibacillus plantarum ES-25, accession number: CGMCCNo.28003.

[0010] The plant lactobacillus ( Lactiplantibacillus plantarum ES-25 was cultured in MRS medium at 37°C for 11 hours, and the OD... 600 It is 1.7.

[0011] The plant lactobacillus ( Lactiplantibacillus plantarum The β-glucosidase activity of ES-25 in MRS medium was 10.7 U during mid-log growth and 5.9 U during the early stationary phase.

[0012] Application of aroma-enhancing Lactobacillus plantarum with high β-glucosidase activity in wine production.

[0013] The plant lactobacillus is used in malic-lactic acid fermentation during wine production.

[0014] The *Lactobacillus plantarum* completed malic-lactic fermentation within 5 days in a simulated wine culture medium with a pH of 3.5 and an ethanol concentration of 13% (v / v).

[0015] The application of Lactobacillus plantarum ES-25 in wine production to enhance the content of aroma substances.

[0016] The application of Lactobacillus plantarum ES-25 in improving sensory quality in wine production.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The Lactobacillus plantarum ES-25 of this invention has high β-glucosidase activity. This enzyme can hydrolyze aromaless glycoside precursors in wine, releasing free terpenes, phenols and other volatile aroma components, significantly enhancing the complexity of the fruit and floral aromas of wine.

[0018] 2. The Lactobacillus plantarum ES-25 of this invention has stronger environmental resistance and faster fermentation start-up ability, ensuring that malic acid-lactic acid fermentation can be completed efficiently and reliably.

[0019] 3. The Lactobacillus plantarum ES-25 of this invention can not only efficiently degrade malic acid, but also has the characteristics of stable stress environment tolerance, low production of diacetyl and other off-flavor byproducts, while avoiding the synthesis of histamine and other biogenic amines to ensure drinking safety.

[0020] 4. The Lactobacillus plantarum ES-25 of this invention has excellent tolerance to acid, ethanol and SO2, and can quickly complete malolactic fermentation in wine without any safety risks.

[0021] 5. The Lactobacillus plantarum ES-25 of this invention has high β-glucosidase activity, which can significantly increase the content of esters, aldehydes, ketones and terpenes in wine during the fermentation stage. Among them, the content of linalool and citronellol is increased by 77.34 times and 50.66 times respectively compared with wines that have not undergone malolactic fermentation, thereby optimizing the aroma and sensory characteristics of wine.

[0022] 6. The Lactobacillus plantarum ES-25 of this invention does not pose any safety risks related to arginine deimine metabolism and biogenic amine synthesis during fermentation, which meets the high food safety requirements of modern brewing. Attached Figure Description

[0023] Figure 1 The colony morphology (A), Gram staining (B), and transmission electron microscopy (C) of Lactobacillus plantarum ES-25 in this invention are shown. Figure 2 Growth curve of Lactobacillus plantarum cultured in MRS medium at 37°C; Figure 3Cell count and malic acid metabolism rate of Lactobacillus plantarum in simulated wine; Figure 4 β-glucosidase activity of Lactobacillus plantarum at different growth stages; Figure 5 The rate at which *Lactobacillus plantarum* metabolizes malic acid and produces lactic acid in Gansu Cabernet Sauvignon (A), Hebei Marselan (B), Shandong Marselan (C), and Shanxi Cabernet Sauvignon (D); Figure 6 Content of aroma compounds in Gansu Cabernet Sauvignon wine (A) and Hebei Marselan wine (B, C, D) before and after Lactobacillus plantarum malic-lactic fermentation; Figure 7 Sensory radar charts and aroma series radar charts (EH) of Cabernet Sauvignon (A), Cabernet Gernischt (B), Syrah (C) and Merlot (D) wines from the Helan Mountain East Foothills region of Ningxia before and after Lactobacillus plantarum malic-lactic fermentation. Detailed Implementation

[0024] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0025] This invention relates to Lactobacillus plantarum ( Lactiplantibacillus plantarum ES-25 was deposited on July 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28003, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] This strain was isolated from dry red wine produced in Xinjiang that underwent spontaneous malolactic fermentation and is a recognized safe strain for use in food.

[0027] This strain has high β-glucosidase activity, which can significantly increase the content of esters, aldehydes, ketones and terpenes in wine during the fermentation stage, thereby optimizing the aroma and sensory characteristics of wine.

[0028] The *Lactobacillus plantarum* ES-25 of this invention is a Gram-positive rod-shaped bacterium that does not produce spores. The colony and cell characteristics of this strain are as follows: after culturing on MRS solid medium for 36 h, the colonies are milky white, opaque, raised, moist, with smooth edges, and glossy. Under an optical microscope, the cell morphology shows Gram-positive staining, and the cells are short rod-shaped. Under a transmission electron microscope, the cell morphology shows that individual cells are approximately 1.0-1.5 μm long and 0.3-0.8 μm wide, with a capsule forming on the outer layer. Figure 1 As shown.

[0029] The strain does not pose any safety risks related to arginine deimine metabolism or biogenic amine synthesis.

[0030] I. Determination of strain characteristics 1. Growth characteristics The tested *Lactobacillus plantarum* glycerol tubes were removed from a -80℃ freezer and inoculated into MRS liquid medium at a 2% inoculum rate. The medium was then incubated at 37℃ for 12 h. Finally, the inoculum was transferred to fresh MRS liquid medium at a 2% inoculum rate. OD was monitored during the 37℃ incubation period. 600 .

[0031] The MRS culture medium formula is as follows: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, with the remainder being water, pH 5.4.

[0032] 2. Stress tolerance test After two activation cycles to the logarithmic growth phase (OD600 between 1.1 and 1.4), *Lactobacillus plantarum* was centrifuged at 10,000 r / min for 5 min. The cells were washed twice with 0.85% physiological saline and then resuspended in simulated wine. 250 mL of the simulated wine was transferred to a 330 mL anaerobic fermentation flask, and *Lactobacillus plantarum* was inoculated at a 1% inoculum, resulting in an initial cell concentration of 10⁻⁶. 7 The samples were collected at CFU / mL and placed in a 20℃ incubator. Samples were taken every 24 hours for plate plating for colony counting, and the L-malic acid content was determined at the same time.

[0033] The simulated wine formulation was as follows: tartaric acid 5.0 g / L, L-malic acid 3.5 g / L, glucose 2.0 g / L, D-fructose 2.0 g / L, sodium chloride 0.2 g / L, ammonium sulfate 1.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate 0.05 g / L, and yeast extract 2.0 g / L. The alcohol content of the simulated wine was adjusted to 13% (v / v) with anhydrous ethanol, and the pH was adjusted to 3.5 using HCl / NaOH.

[0034] The simulated wine was filtered through a 0.22 μm organic filter membrane in a clean bench before use. The L-malic acid content was detected using an Agilent 1260 Infinity II HPLC system. The chromatographic column used was a BioRadAminex HPX 87-H (300 mm × 7.8 mm), the mobile phase was 5 mmol / L sulfuric acid solution, the flow rate was 0.5 mL / min, the column temperature was set to 60℃, the injection volume was 20 μL, and the detection wavelength was set to 210 nm.

[0035] 3. Detection of β-glucosidase activity in strains Take 1 mL each of *Lactobacillus plantarum* suspension during the mid-logarithmic growth phase and the early stationary phase, centrifuge at 10000 r / min for 10 min to collect the cells, wash the cells twice with 0.85% NaCl solution, and finally suspend them in 0.5 mL of 0.85% NaCl solution. Add 0.5 mL of citrate-phosphate buffer-p-nitrophenol glucoside mixture (pH 5.0, p-nitrophenol glucoside concentration 5 mmol / L) to the bacterial suspension, mix, and react at 37℃ for 1 h. Immediately add 2 mL of 1000 mmol / L sodium carbonate solution to terminate the reaction. Centrifuge at 10000 r / min for 15 min, and transfer the supernatant to another test tube. Measure the absorbance at 400 nm using a full-wavelength microplate reader. The control sample was prepared using 0.85% NaCl buffer instead of the bacterial suspension, and other treatments were the same as the sample.

[0036] The standard curve used for β-glucosidase activity assay was obtained by measuring the absorbance of p-nitrophenol solutions at different concentrations (0–60 μmol / L) at a wavelength of 400 nm. The regression equation was y = 0.0188x + 0.0274, R0. 2 =0.9995, indicating a good linear relationship that meets experimental requirements. β-glucosidase activity is defined as the amount of p-nitrophenol produced per gram of bacterial cell (dry weight) per minute (μmol / (g·min)).

[0037] 4. Strain safety testing The safety of *Lactobacillus plantarum* was tested by PCR amplification of the functional genes for biogenic amines and ethyl carbamates. (Biogenic amine functional genes) arc A , arc B , arc C PCR amplification program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s, 32 cycles; 72℃ extension for 5 min. Ethyl carbamate functional gene. hdc , odcPCR amplification reaction program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 60 s, 32 cycles; 72℃ extension for 5 min. tdc Reaction procedure: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 48℃ annealing for 30 s, 72℃ extension for 60 s, 32 cycles; 72℃ extension for 5 min. After the reaction, the PCR products were detected by 1% agarose gel electrophoresis, and the results were observed and recorded using a gel electrophoresis system. Primers for detecting the functional genes of bioamines and ethyl carbamate produced by *Lactobacillus plantarum* are shown in Table 1 below: Growth characteristics monitored in strain-specific studies, such as Figure 2 As shown, similar to most Lactobacillus plantarum strains, SE-25 reached the stationary phase after 11 hours of culture in MRS liquid medium, with an OD600 of 1.7. Figure 3 As shown, *Lactobacillus plantarum* SE-25 under simulated wine conditions with combined stress underwent malolactic fermentation, completely degrading malic acid by day 5, and the viable cell count exceeded 10 at the end of fermentation. 7 CFU / mL. For example... Figure 4 As shown, among the 20 *Lactobacillus plantarum* strains tested, SE-25 exhibited the highest β-D-glucosidase activity, with a mid-log phase activity of 10.7 U and a pre-stationary phase activity of 5.9 U. Safety assessment of *Lactobacillus plantarum* SE-25 was performed using primers listed in Table 1, and the results showed that no β-D-glucosidase was detected. arcA , arcB , arcC , hdc , odc and tdc The gene indicates that the strain does not pose any safety risks related to arginine deimine metabolism and biogenic amine synthesis.

[0038] II. Application of bacterial strains in wine 1. Malic acid-lactic acid fermentation capacity The wine used to test the malic-lactic acid fermentation capacity of the strain had undergone prior alcoholic fermentation and was filtered through a 0.22 μm organic filter membrane in a clean bench, and then allowed to stand at 20℃ for 12 h. A *Lactobacillus plantarum* culture in the mid-logarithmic growth phase was centrifuged at 10000 rpm for 5 min and washed twice with 45 mL of 0.85% physiological saline. 250 mL of wine was transferred to a 330 mL anaerobic fermentation flask, and the culture was inoculated with bacteria at a 1% inoculum (10... 7 The malic acid-lactic acid fermentation was carried out in a 20 ℃ constant temperature incubator (CFU / mL). Changes in malic acid and lactic acid levels were monitored during the fermentation process.

[0039] 2. Aroma compounds The concentration of volatile compounds was determined using headspace solid-phase extraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). 1.0 g NaCl and 5 mL of wine sample were added to the headspace vial of the HS-SPME sample, followed by 10 μL of 4-methyl-2-pentanol (1.0083 g / L). The mixture was stirred at 40 °C for 1 h. GC-MS conditions: He was used as the carrier gas at a flow rate of 1 mL / min. The temperature was maintained at 40 °C for 3 min, then increased to 160 °C, and then further increased to 230 °C at a rate of 7 °C / min, and held for 8 min. The scan range was m / z between 33 and 450, with positive ion mode as the electron power source. The ion source temperature was 230 °C. Qualitative and quantitative analysis was performed by comparing the retention times of standard aroma components with those of the detected positional compounds.

[0040] 3. Sensory quality A panel of 15 people was selected for evaluation. The sensory evaluation team received standardized training. A sensory scoring sheet was designed to rate the color, aroma, taste, and overall quality of the wines, with a maximum score of 100 points. The average score was used. The scoring order was: visual inspection, aroma, and taste, followed by an overall evaluation. (Note: Before evaluating each wine sample, the panel members rinsed their mouths with purified water to avoid lingering aftertastes from the previous wine, which could lead to inaccurate sensory evaluation results.) like Figure 5 As shown, the malolactic fermentation capacity of *Lactobacillus plantarum* SE-25 in wine is as follows: In Gansu wine, SE-25 reduced the malic acid content to 0.16 g / L by day 9 of malolactic fermentation. In Hebei wine, SE-25 exhibited the fastest malic acid degradation rate, completing malic acid consumption in 5 days. In Shandong wine, all tested strains were able to complete malolactic fermentation, with SE-25 reducing the malic acid content from 2.80 g / L to below 0.2 g / L by day 9. In Shanxi wine, SE-25 completed malolactic fermentation on day 7. SE-25 demonstrated rapid malolactic fermentation in all tested wine samples and showed outstanding performance among the tested strains.

[0041] like Figure 6 As shown in Table 2, *Lactobacillus plantarum* SE-25 exhibits remarkable comprehensive aroma-producing capabilities after completing malolactic fermentation. The significant increase in ester content (ethyl decanoate, ethyl hexanoate, ethyl acetate) infuses the wine with rich fruit aromas; simultaneously, it effectively converts aldehydes and ketones, optimizing aroma quality; its fatty acid metabolism adds aging potential to the wine; and its effective preservation of isoprene compounds (linalool and citronellol) perfectly maintains the typical aroma characteristics of the Marselan varietal.

[0042] like Figure 7 As shown in the sensory radar chart, SE-25 enhances the sensory performance of Cabernet Sauvignon, Cabernet Gernischt, and Syrah wines in terms of finish, aroma intensity, and hue.

[0043] like Figure 7 As shown in the aroma radar chart, compared to wines without malolactic fermentation, SE-25 fermentation increased the expression of fruit and floral aromas in the tested wines. Syrah wines exhibited strong spicy and toasty aromas, and the intensity of spicy aromas significantly increased after SE-25 fermentation.

[0044] In summary, among the tested strains, SE-25 has a more positive impact on the aroma of wine.

[0045] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A flavor-enhancing Lactobacillus plantarum with high β-glucosidase activity, characterized in that: The plant lactobacillus ( Lactiplantibacillus plantarum ES-25, accession number: CGMCCNo.28003.

2. The flavor-enhancing *Lactobacillus plantarum* with high β-glucosidase activity according to claim 1, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum ES-25 was cultured in MRS medium at 37°C for 11 hours, and the OD... 600 It is 1.

7.

3. The flavor-enhancing *Lactobacillus plantarum* with high β-glucosidase activity according to claim 1, characterized in that, The plant lactobacillus ( Lactiplantibacillus plantarum The β-glucosidase activity of ES-25 in MRS medium was 10.7 U during mid-log growth and 5.9 U during the early stationary phase.

4. The application of the aroma-enhancing Lactobacillus plantarum with high β-glucosidase activity as described in claim 1 in wine production.

5. The application of the aroma-enhancing *Lactobacillus plantarum* with high β-glucosidase activity as described in claim 4 in wine production, characterized in that... The plant lactobacillus is used in malic-lactic acid fermentation during wine production.

6. The application of the flavor-enhancing *Lactobacillus plantarum* with high β-glucosidase activity as described in claim 5, characterized in that, The *Lactobacillus plantarum* completed malic-lactic fermentation within 5 days in a simulated wine culture medium with a pH of 3.5 and an ethanol concentration of 13% (v / v).

7. The application of Lactobacillus plantarum ES-25 as described in any one of claims 1 to 3 in enhancing the content of aroma substances in wine production.

8. The use of Lactobacillus plantarum ES-25 as described in any one of claims 1 to 3 in improving sensory quality in wine production.

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