Lactobacillus LS.1 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus LS.1 and application thereof. The invention provides the lactobacillus subsp. Sp. LS.1 which is resistant to acid, fast in acid production and high in acid accumulation amount, can inhibit growth of putrefying bacteria, is used for fermenting crystal allium chinense, and shows excellent effects of protecting color, keeping crispness and improving flavor. The crystal allium chinensis product fermented by adding the lactobacillus LS.1 has uniform jade-white inner and outer scales, is uniform in color and luster, does not become black or dark, and does not generate biological membranes; the sour allium chinense is pure and rich in flavor, free of spicy pungent smell and peculiar smell, mainly sweet and harmonious in sour and salty taste; in addition, the crispness of the product can be well ensured, the shelf life quality of the product can be ensured to be stable without swelling under the condition that no preservative is used, the product quality is comprehensively improved, the fermentation process is convenient and simple to control, and large-scale industrial technology popularization can be realized.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Lactobacillus LS.1 and its applications. Background Technology

[0002] Pickled vegetables are a traditional food made by naturally fermenting vegetables. However, because the vegetable raw materials are not sterilized during the production process, the presence of various microorganisms in the raw materials makes the fermentation process of pickled vegetables uncontrollable, resulting in prolonged fermentation time and unstable product quality, and even spoilage, thus becoming a bottleneck in industrial fermentation.

[0003] The main microbial groups involved in the natural fermentation of kimchi are lactic acid bacteria, yeast, and Bacillus. Most lactic acid bacteria provide the unique tangy flavor of kimchi. However, some yeasts and Bacillus produce biofilms during their reproduction and metabolism, which adhere to the product surface and are identified as visible foreign matter during packaging, increasing the difficulty of quality control during production and processing. Removing these biofilms by washing will result in the loss of fermented flavor. Some yeasts produce yeast-like or fermented alcoholic flavors during their metabolism. When yeasts convert acids into alcohols, the acid content in the fermentation system decreases significantly, subsequently leading to abnormal putrid or even foul odors. Simultaneously, the fermented product turns dark and loses its commercial value. Especially in the mid-temperature environment of summer, yeasts and Bacillus can proliferate rapidly in the early stages of fermentation, resulting in slow and insufficient acid production, insufficient sensory acidity, and abnormal aroma.

[0004] Shallots, hailed as the "Ganoderma lucidum of vegetables," are renowned not only for their delicious taste but also for their rich botanical history, boasting a long and enduring culinary tradition in southern China. Crystal shallots, with their crisp texture, unique flavor, and sweet taste, are widely loved. However, their natural fermentation is susceptible to contamination by yeast, spores, and other microorganisms. Furthermore, limitations imposed by refrigeration, cold chain conditions, and consumer spending habits lead to inconsistent quality in naturally fermented shallots during room temperature storage, distribution, and sales. Issues such as browning, softening, bloating, and flavor degradation severely impact product quality. Currently, industrial production often employs pasteurization and the addition of preservatives to extend shelf life, but this can result in significant nutrient and flavor loss, and long-term preservative intake poses health risks. Therefore, developing high-quality shallot fermentation agents to achieve zero-addition of preservatives such as sodium benzoate and sodium metabisulfite, thereby extending the shelf life of additive-free products and improving product safety, is an inevitable trend. Summary of the Invention

[0005] Based on the above technical problems, the main objective of this invention is to overcome the shortcomings of the aforementioned background technology. A strain of Lactobacillus sunkii LS.1 was screened from traditional naturally fermented crystal shallots. This strain produces acid rapidly, accumulates a large amount of acid, has excellent sensory flavor properties, and inhibits spoilage microorganisms. It can solve the problems of slow acid production, insufficient acid production, insufficient fermented acid aroma, and easy occurrence of putrid odors during the fermentation process of crystal shallots. It achieves the technical effect of producing fermented shallots with a rich and pure acid aroma, a mellow and harmonious acid taste, a lingering mellow and sweet flavor, good retention, unique flavor, and preventing spoilage during shelf life.

[0006] To achieve the above objectives, the inventors conducted in-depth research and, through repeated studies and demonstrations, obtained the solution of this invention, as detailed below:

[0007] In the first aspect, this application provides a strain of Lactobacillus sunkii subspecies LS.1, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2025, with accession number GDMCC No. 66350.

[0008] Lactobacillus LS.1 colonies on MRS medium are milky white, round, with regular edges, smooth surfaces, and a moist, glossy appearance. The bacteria are typically rod-shaped with varying lengths, some exhibiting a curved, coiled, or spiral form. The bacterial cells are 0.3–1.1 μm long and 0.2–0.4 μm wide.

[0009] Secondly, the present invention also provides a fermentation agent comprising the aforementioned Lactobacillus LS.1.

[0010] Thirdly, the present invention also provides the application of the above-mentioned fermentation agent in the fermentation of shallots, preferably crystal shallots, especially to improve the problems of insufficient sour aroma, uneven color, and easy occurrence of putrid odor in fermented crystal shallots, thereby improving the color, aroma, taste, and crispness of fermented crystal shallots and comprehensively improving the quality of fermented crystal shallots.

[0011] Fourthly, the present invention also provides a fermentation method for crystal shallots, the method comprising the step of fermentation using the aforementioned Lactobacillus LS.1 or fermentation agent.

[0012] Furthermore, the amount of Lactobacillus LS.1 or the fermentation agent added is 10. 6 -10 7 CFU / mL.

[0013] Furthermore, the Lactobacillus LS.1 or the fermentation agent is added at the initial stage of fermentation.

[0014] Furthermore, the fermentation time is 15-30 days.

[0015] Fifthly, the present invention provides a fermented crystal scallion, which is prepared by the method described in this application.

[0016] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0017] 1. The Lactobacillus LS.1 provided by this invention is acid-resistant, produces acid rapidly, and has a high acid accumulation, which can inhibit the growth of spoilage bacteria. When used to ferment crystal shallots, it exhibits excellent effects in protecting color, maintaining crispness, enhancing flavor, and extending shelf life.

[0018] 2. The Lactobacillus LS.1 strain provided by this invention has strong hydrophobicity and rapid self-aggregation, which is a prerequisite for adhering to intestinal epithelial cells and colonizing the gastrointestinal tract of animals to exert the beneficial effects of probiotics.

[0019] 3. The crystal shallots fermented with Lactobacillus LS.1 provided by this invention are crystal clear, with a uniform white skin and a uniform translucent jade white skin and pulp. They have a pure sour aroma, a pure and refreshing sour taste, a mellow and harmonious flavor, good retention, no off-flavors, and no biofilm formation. They can better preserve the texture of the shallots, ensure the crispness of the product, and comprehensively improve the product quality.

[0020] 4. The crystal shallot product fermented with Lactobacillus LS.1 provided by this invention has a uniform and stable color, and can ensure stable quality during the shelf life of the product without bloating when vacuum-packed without the use of preservatives. Moreover, its fermentation process is convenient and simple to control, and can be promoted for large-scale industrialization. Attached Figure Description

[0021] Figure 1 Figure 1 shows the test results of hydrophobicity and self-cohesion of different strains;

[0022] Figure 2 Figures showing the results of acid production tests for different bacterial strains during fermentation.

[0023] Figure 3 Figures showing the results of fermentation flavor and color difference analysis for different strains;

[0024] Figure 4 This is a colony morphology diagram of Lactobacillus LS.1.

[0025] Figure 5 This is a microscopic image of the cell morphology of Lactobacillus LS.1.

[0026] Figure 6 OD test of acid resistance of Lactobacillus LS.1 600 Stacked diagram;

[0027] The Lactobacillus sunkii subspecies LS.1 provided by this invention was deposited on May 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No. 66350. This strain was received and registered by the collection center on May 19, 2025, and was confirmed to be a viable strain by the collection center on May 19, 2025. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] 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 invention pertains.

[0030] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional food-grade reagents, methods and equipment in the art.

[0031] The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Example 1: Screening of Lactobacillus LS.1

[0033] 1. Raw material processing:

[0034] Under aseptic conditions, samples of naturally fermented crystal shallots with excellent flavor were examined microscopically. Numerous rod-shaped microorganisms were observed under the microscope, preliminarily identified as lactic acid bacteria, with very few yeasts. MRS medium was used for the isolation and purification of the target microorganisms. Under aseptic conditions, 25g of naturally fermented crystal shallots with good flavor were weighed and placed in 225mL of sterile water. The mixture was homogenized for 30 minutes and then serially diluted 10-fold with sterile water to a final concentration of 10. -1 -10 -6 Take 10 -4 10 -5 10 -6 Three dilution gradients of bacterial suspension were inoculated onto MRS solid medium using the pour culture method, with 1 mL inoculated per dish for each dilution. The dishes were then placed in a 30°C incubator and incubated anaerobically upside down for 48 h.

[0035] Culture dishes with a growth rate of 30-300 CFU / plate were selected. Colony morphology was labeled and morphological differences were described. Colonies with different morphologies were examined and described under a microscope. Typical lactic acid bacteria were selected for purification. After purification three times, Gram staining was used for microscopic examination to preliminarily determine the purified strains. Glycerol cryovials were prepared and stored at -80℃ in a medical refrigerator for later use. Ten rod-shaped microorganisms with excellent flavor were obtained after isolation and purification and numbered as LS.1, BDH-5, SC, HX, YY-2, CH-2, BDH-3, BDH-1, YY-1, and BDH-2.

[0036] 2. Preliminary screening of lactic acid bacteria

[0037] Prepare 100 mL / bottle of MRS liquid culture medium, sterilize at 121 °C for 20 mins, cool, and inoculate with 1 loop / bottle of the 10 isolated and purified microorganisms. Incubate at 30 °C for 48 h, and adjust the effective viable count of the microorganisms to 1 × 10⁻⁶. 8 ~5×10 8 CFU / mL is the test bacterial suspension.

[0038] (1) Cell hydrophobicity test

[0039] Centrifuge the bacterial suspension at 10000 r / min for 15 min, discard the supernatant, wash with an equal volume of sterile PBS solution, centrifuge again, resuspend with an equal volume of sterile PBS solution, and adjust the absorbance at 600 nm to about 0.8 Abs, which is A0.

[0040] Take 5 mL each of the hydrophobic agent n-hexane and chloroform (trichloromethane), and add them separately to two blank test tubes. Add 15 mL of PBS-adjusted bacterial suspension to each of the two test tubes, mix well, and place in a fume hood for 1 hour. Discard the organic phase and retain the aqueous phase for data measurement. Zero the test tube using the PBS solution as a control, and measure the absorbance of the aqueous phase; this is A1. Calculate the hydrophobicity using the following formula, and record the results. Figure 1 middle.

[0041]

[0042] analyze Figure 1Data shows that when chloroform is used as a hydrophobic agent, BDH-3 exhibits the best hydrophobicity at 42.5%, followed by LS.1 at 32.37%. The order of hydrophobicity from highest to lowest is BDH-3 > LS.1 > YY-1 > YY-2 > BDH-2 > BDH-5 > CH-2 > BDH-1 > SC > HX. When n-hexane is used as a hydrophobic agent, LS.1 exhibits the best hydrophobicity at 31.09%, followed by YY-2 at 30.55%. The order of hydrophobicity from highest to lowest is LS.1 > YY-2 > YY-1 > BDH-3 > BDH-5 > CH-2 > BDH-2 > SC > HX > BDH-1. LS.1 and YY-1 possess strong hydrophobic properties, which are prerequisites for adhering to intestinal epithelial cells and colonizing the gastrointestinal tract to exert the beneficial effects of probiotics.

[0043] (2) Cell self-cohesion test

[0044] The adjusted bacterial suspension was placed in a 25°C incubator for 5 hours and 20 hours without shaking. The supernatant was gently collected, and the absorbance was measured at 5 hours and 20 hours using PBS solution as a control. This value is A2. The cohesive force was calculated using the following formula, and the results were recorded. Figure 1 middle.

[0045]

[0046] analyze Figure 1 Data shows that at 5 hours, YY-1 had the strongest self-cohesion at 44.24%, CH-2 had the weakest at 17.38%, and LS.1 had 32.69%. At 20 hours, YY-2 had the best self-cohesion at 91.96%, CH-2 had the weakest at 38.41%, and LS.1 had 39.1%. Comprehensive analysis indicates significant differences in self-cohesion among different strains. Most strains showed an increase in self-cohesion over time, while the increase in self-cohesion for LS.1 was relatively small, suggesting a rapid sedimentation rate. Self-cohesion is a prerequisite for bacterial sedimentation and attachment in the gastrointestinal tract.

[0047] (3) Optimal selection of fermentation strains for crystal shallots

[0048] Take crystal shallots, wash and drain them, slice them, and place them in a 300mL Erlenmeyer flask (150g / flask). Add 10% white sugar solution (150mL / flask), incubate at 95℃ for 30 minutes, and cool before use. Perform aseptic inoculation of the test microorganisms, with an initial inoculation amount of 1×10⁻⁶. 6 ~5×10 6 To avoid cross-contamination, the CK treatment was the same as the experimental group except that no microorganisms were inoculated. After inoculation, the cells were incubated at 30°C. Samples were taken every 3 days to measure lactic acid titration. Results were recorded. Figure 2On day 15, microbial species were counted, and the results are recorded in Table 1. Leaf samples were taken for color difference testing, and the sample test values ​​L*, a*, and b* were recorded. The average color difference of all samples was calculated. The sum of the color differences between the sample and the average value is denoted as S. A positive S indicates that the color is better than the overall level; the larger the value, the better the color. Flavor analysis was performed according to GB / T 12315-2008 "Sensory Analysis Methodology - Ranking Method". The color and flavor results were recorded. Figure 3 middle.

[0049]

[0050] Table 1. Microbial community analysis of fermented crystal shallots by different strains after 14 days.

[0051]

[0052] Note: "-" indicates that no such bacterial community was observed on the counting plate. "+" indicates the presence of biofilm, and " / " indicates the absence of biofilm.

[0053] Depend on Figure 2 The results of pure scallion fermentation tests after pasteurization of different strains showed that the CK treatment produced almost no acid, with an initial titratable lactic acid content of 0.02% and a titratable lactic acid content of 0.05% after 14 days of fermentation. LS.1 produced the fastest acid, with a titratable lactic acid content of 0.39% on day 3, and the highest acid production, reaching 0.91% on day 14. BDH-5 produced the second highest acid, with a titratable lactic acid content of 0.62% after 14 days of fermentation. From the perspective of 14 days of fermentation, the acid production from highest to lowest was: LS.1 > BDH-5 > BDH-2 > BDH-3 > HX=CH-2 > SC > BDH-1 > YY-1 > YY-2 > CK.

[0054] Table 1 shows that after pasteurization, the viable cell counts of scallions fermented for 14 days on MRS medium were measured. The results showed that the six treatments (SC, CH-2, BDH-3, BDH-5, HX, and LS.1) only produced lactic acid bacteria and no biofilm growth was observed. The viable cell counts were 7.28 lg CFU / g, 7.94 lg CFU / g, 7.10 lg CFU / g, 7.70 lg CFU / g, 7.68 lg CFU / g, and 8.72 lg CFU / g, respectively. Among them, LS.1 had the highest viable cell count, indicating that inoculating LS.1 strain into crystal scallions can rapidly grow and reproduce, playing a dominant role and inhibiting the growth of putrefactive bacteria such as biofilm-producing yeasts and Bacillus.

[0055] Depend on Figure 3The results of pure fermentation tests of scallions after pasteurization showed that LS.1 had the best color (S = 10.67 NBS), the highest rank sum for taste and aroma, and the best overall sensory evaluation. BDH-5 was the next best, followed by HX and SC. CH-2 had average color protection, fermentation aroma, and taste. BDH-3 showed excellent color protection but poor fermentation flavor. BDH-2 showed excellent fermentation flavor but poor color. The sensory analysis ranking method (POP test) showed that when p = 11, j = 12, and α = 0.05, the critical value was 4949.62. Since L aroma (5907 > 4949.62) and L flavor (5986 > 4949.62) showed differences between samples when α = 0.05, and the ranking was consistent with the pre-set order. The aroma order is: BDH-5 > LS.1 > HX > SC > CH-2 > BDH-2 > YY-2 > CK > BDH-3 > YY-1 > BDH-1; the flavor order is: LS.1 > BDH-5 > BDH-2 > HX > SC > CH-2 > CK > BDH-3 > YY-2 > YY-1 > BDH-1. LS.1 and BDH-5 impart excellent color and flavor to fermented crystal shallots.

[0056] 3. Lactic acid bacteria rescreening

[0057] The above-mentioned CK putrefactive microorganisms were mixed and enriched. Since they contained Bacillus and yeast, LB medium and malt extract medium were prepared. The two mediums were mixed evenly, sterilized, and inoculated into the CK-treated mixed bacterial suspension under aseptic conditions. The inoculation amount was 1%, and the suspension was incubated at 30°C for 48 hours to obtain the putrefactive microorganism suspension.

[0058] The above-mentioned SC, CH-2, BDH-3, BDH-5, HX, and LS.1 strains were enriched using MRS medium. The precipitates and supernatants were separated by aseptic centrifugation at 10,000 rpm for 15 minutes. The precipitates were resuspended in different volumes of sterile physiological saline to prepare five concentrations: 81-fold, 27-fold, 9-fold, and 3-fold concentrated suspensions, as well as the original concentration suspension. These, along with the supernatant, resulted in six gradient solutions, which were used as the test solutions for each lactic acid bacteria.

[0059] Prepare sterile semi-solid agar (10 g / L). Accurately transfer 10 mL into a sterile empty petri dish, place it horizontally, and allow the agar to cool and solidify. Gently place six Oxford cups at equal intervals on the agar plate without pressing them down. Adjust the concentration of the prepared putrefactive microbial suspension to 1 × 10⁻⁶. 8 ~5×10 8 CFU / mL was added to a mixed LB and malt extract medium cooled to 45–50°C, and thoroughly mixed to achieve a putrefactive microbial concentration of 1 × 10⁻⁶. 6 ~5×106 CFU / mL. Measure 10 mL and pour it into a semi-solid agar plate. Gently shake the plate to evenly spread the bacterial suspension. Place it horizontally and allow it to solidify before use. Accurately measure 100 μL of the concentrated lactic acid bacteria sample and slowly add it to an Oxford cup with distilled water as a control. Perform three replicates for each treatment. Place the plates in a 4℃ refrigerator for diffusion for 4–10 hours. Remove the plates and place them in a 30℃±1℃ incubator, incubating upright until the inhibition zone is clear. Measure the diameter of the inhibition zone using a vernier caliper with a measurement accuracy of 0.02 mm–0.1 mm. The inhibition zone should range from 8 mm to 25 mm. Measure each inhibition zone three times in different directions and record the average value. Establish a linear equation (R²) with the logarithm of the reciprocal of the additive dilution factor as the x-axis and the diameter of the inhibition zone as the y-axis. 2 Additives with high antibacterial potency (≥0.9900) were selected from the pool. The results are recorded in Table 2.

[0060] Table 2 Evaluation of the antibacterial effect of lactic acid bacteria test solution on putrefactive microorganisms

[0061]

[0062]

[0063] Analysis of Table 2 shows that, based on the bacterial suspension test results, LS.1 exhibited the highest inhibitory titer against putrefactive bacteria at 81.15 AU / mg, followed by strain SC at 35.63 AU / mg. In the supernatant test, LS.1 showed the largest inhibition zone against putrefactive bacteria at 16.3 mm, followed by strain SC at 10.5 mm. Other strains did not show significant inhibitory effects against putrefactive bacteria. The reason they did not grow Bacillus or yeast in the previous test is attributed to their nutrient contention during growth and reproduction, becoming the dominant microorganism in the early stages of crystal shallot fermentation, thus preventing the reproduction of putrefactive bacteria. In conclusion, LS.1 has a significant inhibitory effect on putrefactive bacteria during crystal shallot fermentation, and its effect is superior to other lactic acid bacteria. Combined with control of the initial inoculum size, it can effectively control the outbreak of putrefactive bacteria in the fermentation system.

[0064] Based on the fermentation test indicators of acid content, color difference, sensory evaluation, and antibacterial ability, it can be seen that strain LS.1 can rapidly grow and reproduce in fermented crystal shallots, produce acid quickly and accumulate in large quantities, significantly improving the color and flavor of the product, effectively inhibiting the growth and reproduction of spoilage bacteria in crystal shallots, and improving the fermentation quality of vegetables. Therefore, this strain was selected as the target strain for fermenting crystal shallots.

[0065] The selected Lactobacillus LS.1 test strain suspension for fermentation testing was streaked and Gram-stained. Colony morphology and cell morphology were observed and described using both visual inspection and microscopy. The colony morphology of LS.1 is shown below. Figure 4As shown, the colonies are milky white, round, with neat edges, smooth surfaces, and a moist, glossy appearance. After Gram staining, the morphology of the obtained strain was observed under a 100x oil immersion microscope as follows. Figure 5 As shown, the bacteria are typically rod-shaped with varying lengths, some exhibiting a curved, coiled, or spiral form. The bacteria are 0.3–1.1 μm long and 0.2–0.4 μm wide.

[0066] The pure culture of the tested LS.1 strain was sent for sequencing and identified as Lactobacillus sunkii. It was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 19, 2025, with the accession number GDMCC No. 66350.

[0067] Example 2: Performance testing of Lactobacillus LS.1

[0068] (1) Acid resistance test

[0069] Lactobacillus LS.1 suspension was prepared according to the method described in Example 1.

[0070] MRS liquid culture media with citric acid concentrations of natural, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 1.75% were prepared, sterilized, and cooled before use. LS.1 bacterial suspensions were inoculated at 1% into the media with different acid concentrations and incubated at 30°C on a shaker at 150 rpm. OD values ​​were measured at the initial incubation, 24 h, 48 h, and 72 h. 600 The acid resistance of the strain was tested, and the results were recorded in [the relevant document / document]. Figure 6 middle.

[0071] analyze Figure 6 Data shows that Lactobacillus LS.1 strain has extremely strong acid resistance and can still grow and reproduce at an acid concentration of 1.75%.

[0072] (2) Aerobic demand characteristics test

[0073] Prepare MRS solid medium. Aseptically dilute the Lactobacillus LS.1 bacterial suspension to 1000 CFU / mL to 5000 CFU / mL. Spread the diluted bacterial suspension onto the solid medium at a rate of 100 μL / plate, for a total of 6 plates. Place 2 plates unsealed in an anaerobic jar for anaerobic culture, seal 2 plates with sealing film for culture, and leave 2 plates unsealed and place them in a 30°C constant temperature incubator for 72 hours before counting.

[0074] Table 3. Colony Count Results of Aerobic Characteristics Test

[0075]

[0076] Analysis of the data in Table 3 shows that Lactobacillus LS.1 has the highest growth rate of 116 CFU / plate under anaerobic conditions and 95 CFU / plate under aerobic conditions. This indicates that LS.1 can grow and reproduce normally in an anaerobic environment, and its growth is better than that in an aerobic environment. This growth characteristic meets the fermentation requirements of crystal shallots, which is conducive to inhibiting the growth and reproduction of putrefactive bacteria during the fermentation process and improving the fermentation quality of vegetables.

[0077] Example 3: Fermentation Test of Crystal Shallots

[0078] Lactobacillus LS.1 and BDH-5 bacterial suspensions were prepared according to the method described in Example 1.

[0079] Prepare six clean 25kg fermentation jars. Take fresh crystal shallots, remove inedible parts, wash, and drain excess water. Arrange the shallots neatly into the fermentation jars, filling them to the top, with each jar containing 13kg of shallots. Add 4% edible salt and 5% white sugar solution to the top of the jar, 12kg / jar. Group 1 is inoculated with LS.1 strain at 1% of the total system; Group 2 is inoculated with BDH-5 strain at 1% of the total system; the control group (CK) is left uninoculated and ferments naturally. After standing overnight, turn the jars the next day to ensure thorough mixing of the inoculum and the material. Natural fermentation serves as the control group; except for the absence of inoculation, all other treatments are identical to the tested groups. Fermentation is complete after 30 days. Samples are taken to determine the total acid content and perform sensory evaluation. The results are recorded in Tables 4-5.

[0080] After fermentation, the samples were packaged and their shelf life was tested. To eliminate the influence of acidity on the test results, citric acid was used to adjust the total acidity of the fermented shallots and broth to 1.0% (calculated as titratable lactic acid) at the end of fermentation. The samples after fermentation were packaged according to three different schemes, with 80% solids and the broth being the original broth from each fermentation scheme. Packaging scheme 1 was preservative-free, non-heat-treated vacuum packaging; scheme 2 was vacuum packaging with 1‰ sodium benzoate and 100ppm sodium metabisulfite as preservatives; and scheme 3 was preservative-free vacuum packaging and pasteurization at 85℃ for 30 minutes. Each treatment package contained 13 bags. Initial samples were taken for TPA texture analysis, and the results are recorded in Table 6. The remaining samples underwent accelerated evaluation at room temperature, 30℃, and 37℃, and the swelling of the packages was recorded every 15 days. When recording the swelling condition, the sample was placed vertically, and a liquid level line was drawn. The height inside the packaging was marked as h1, and the height of the liquid level line was marked as h2. The difference between h1 and h2 is the true reflection of the swelling condition, which is recorded as Q. The larger the Q value, the more severe the swelling condition. The average value of repeated data was statistically processed, and the results are recorded in Table 7.

[0081] Table 4. Statistics on total acidity and film formation at the end point of crystal shallot fermentation.

[0082]

[0083] Analysis of Table 4 shows that the LS.1 treatment had the highest acid accumulation at 0.96%, which is 2.34 times that of the CK treatment and 1.52 times that of BDH-5 lactic acid bacteria. Biofilm formation occurred in the scallions fermented by CK and BDH-5 strains, adhering to the surface and affecting the product's appearance. No biofilm formation occurred in the LS.1 treatment. This demonstrates that exogenously added strains can promote acid production in scallion fermentation, and different lactic acid bacteria exhibit significant differences in their acid-producing capabilities. LS.1 possesses the superior advantages of rapidly promoting acid production and accumulating a large amount of acid in scallion fermentation. This excellent characteristic can quickly inhibit the growth of biofilm-producing strains such as yeast and Bacillus, ensuring uniform product appearance, absence of visible foreign matter, strong adaptability to production and processing, and eliminating the need for washing to prevent flavor loss during fermentation, thus improving product quality stability.

[0084] Table 5. Sensory Evaluation of the Final Fermentation End of Crystal Shallots

[0085]

[0086] Analysis of Table 5 shows that the scallions fermented by LS.1 are crystal clear in color, with uniform color on both the inner and outer layers of scales. They have a sour scallion flavor, without any pungent or irritating odors, a balanced sour and salty taste, a pure, mellow, and harmonious sourness, a fresh and sweet flavor, a long-lasting and mellow aroma, and good crispness. The scallions fermented by BDH-5 have uneven color on the inner layers of scales, exhibiting alternating white and translucent white hues. They have a slightly fermented sour scallion flavor, a pungent and irritating odor, a weak sour aroma, a spicy taste that irritates the mouth, insufficient fermented sourness, and poor crispness. Although the scallions fermented by BDH-5 have a fermented sour aroma compared to the CK treatment, their overall sensory evaluation is inferior to that of the LS.1 treatment. The LS.1 treatment has a uniform color across the entire fermentation system, with no blackening or darkening, and no biofilm formation. CK-processed scallions have uneven coloring on both the inner and outer layers of scales, appearing dark and dull. They have a strong, pungent, and irritating odor, a weak sour scallion flavor, a yeasty taste, a spicy and irritating taste in the mouth, a prominent salty taste, a very weak fermented sour aroma, and poor crispness.

[0087] Table 6. Texture Analysis of Crystal Shallots under Different Packaging Processes

[0088]

[0089] Note: In the sensory crispness ranking, "1" indicates the best crispness, and the crispness decreases in the following order. The same number indicates that the difference in crispness is not significant.

[0090] Analysis of Table 6 shows that TPA testing revealed a significant decrease in the crispness of the crystal shallots after sterilization at 85℃ for 30 minutes. Therefore, heat treatment is not recommended for maintaining shelf-life quality stability. For the same fermentation process, there was no significant difference in crispness between non-heat-treated packaging with and without preservatives. The crispness of processed shallots was comprehensively evaluated using local maximum normal force, hardness, adhesiveness, and chewiness. Crispness (unique to samples with a crisp outer skin), resilience, and cohesiveness contributed relatively little to the sensory crispness of the product. The shallots fermented with LS.1 exhibited excellent hardness, adhesiveness, crispness, and chewiness. Their crispness-preserving principle stems from inhibiting the growth of spoilage microorganisms while producing acid, thus better retaining the texture of the shallots.

[0091] Table 7. Statistics on Packaging Expansion During Shelf Life of Crystal Shallots

[0092]

[0093] Table 7 shows that under the non-heat-treated preservative-added packaging process, groups LS.1, BDH-5, and CK did not exhibit bulging after 45 days at room temperature, 30℃, and 37℃. Under the zero-preservative heat-treated packaging storage process, groups BDH-5 and CK showed slight bulging after 15 days, with the CK group showing more significant bulging, which worsened with prolonged storage. Group LS.1, however, showed no bulging after 45 days at room temperature, 30℃, and 37℃. Under the zero-preservative non-heat-treated packaging process, groups BDH-5 and CK showed significant bulging after 15 days, with the CK group showing more significant bulging, which worsened with prolonged storage. Group LS.1, on the other hand, showed no bulging after 45 days at room temperature, 30℃, and 37℃. Comprehensive analysis shows that both lactic acid bacteria BDH-5 and naturally fermented crystal shallots, even after sterilization at 85℃ for 30 minutes, still exhibit swelling during their shelf life. Chemical preservatives are required to ensure product shelf-life quality stability and prevent swelling. Swelling can prevent the shallots from fully contacting the broth, leading to browning, abnormal flavor, and increased susceptibility to leakage during transport. This leakage can then rot due to contact with environmental bacteria, resulting in a foul odor, mushy appearance, and contamination of the entire batch, rendering it commercially worthless. LS.1 fermented shallots, however, can maintain shelf-life quality stability through 1.0% vacuum packaging, non-heat treatment, and no preservatives. This zero-preservative packaging and storage process promotes the green and healthy development of Yunnan's plateau-characteristic shallot industry.

[0094] In summary, Lactobacillus LS.1 cells are highly hydrophobic and possess rapid self-aggregation, giving them the potential to colonize the animal gastrointestinal tract. Their rapid acid production and high acid accumulation inhibit the growth of putrefactive bacteria. LS.1 fermented crystal shallots offer color protection, crispness retention, and flavor enhancement. The fermented crystal shallots are crystal clear, with uniform, translucent white scales inside and out. They have a pure and rich aroma, free of pungent or irritating odors, and a strong shallot flavor with a balanced acidity and saltiness, a pure, mellow, and harmonious sourness, a sweet and savory taste, and a long-lasting, mellow aftertaste. The entire fermentation system exhibits uniform color across all layers, without blackening or darkening, and no biofilm formation. It effectively preserves the shallot texture, ensuring product crispness. The packaged product has a uniform and stable color, and even in vacuum packaging without preservatives, it maintains stable shelf-life quality without bulging. Furthermore, its fermentation process is convenient and simple to control, enabling large-scale industrial-scale technology promotion.

[0095] The embodiments described above are merely illustrative of several implementations of the present invention, 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 the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A strain of lactobacillus ( Lentilactobacillus sunkii LS.1, characterized in that, The preservation number of the Lactobacillus LS.1 is GDMCC No. 66350.

2. A fermentation agent, characterized in that, The fermentation agent comprises Lactobacillus LS.1 as described in claim 1.

3. The application of Lactobacillus LS.1 as described in claim 1 or the fermentation agent as described in claim 2 in scallion fermentation.

4. The application as described in claim 3, characterized in that, The scallions in question are crystal scallions.

5. A fermentation method for crystal shallots, characterized in that, The method includes the step of fermentation using Lactobacillus LS.1 as described in claim 1 or the fermentation agent as described in claim 2.

6. The method as described in claim 5, characterized in that, The amount of Lactobacillus LS.1 or fermentation agent added is 10. 6 -10 7 CFU / mL.

7. The method as described in any one of claims 5 or 6, characterized in that, The Lactobacillus LS.1 or fermentation agent is added at the initial stage of fermentation.

8. The method as described in any one of claims 5 or 6, characterized in that, The fermentation time is 15-30 days.

9. A fermented crystal scallion, characterized in that, The fermented crystal shallots are prepared by the method described in any one of claims 5-8.

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