Lactobacillus strain ls.1 and its use

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a lactobacillus LS.1 and application thereof. Lentilactobacillus sunkii The lactobacillus sunkii subsp. LS.1 is acid-resistant, can produce acid quickly, has high acid accumulation, and can inhibit the growth of spoilage bacteria, and is used for fermenting crystal leek, and shows excellent color protection, crispness preservation and flavor improvement effects. The crystal leek product fermented by adding the lactobacillus LS.1 has uniform and transparent white scales on the inside and outside, uniform color without blackening and darkening, and no biological membrane is produced; the pickled leek has pure and mellow flavor, no pungent and irritating smell, no peculiar smell, sweet taste is dominant, and sour and salty tastes are coordinated; the product crispness can be better ensured, the product shelf life quality can be ensured to be stable without swelling under the condition of not using preservatives, the product quality is comprehensively improved, and the fermentation process control is convenient and simple, and the scale industrialization technology popularization can be realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a lactobacillus LS.1 and application thereof. BACKGROUND

[0002] Pickle is a kind of traditional food produced by natural fermentation of vegetables. Since the raw materials are not sterilized during the production process, various microorganisms in the raw materials make the fermentation process of pickles uncontrollable, resulting in prolonged fermentation time and unstable product quality, and even spoilage, which has become a bottleneck for industrial fermentation.

[0003] During the natural fermentation process of pickles, the main microbial groups are lactic acid bacteria, yeast and spore bacteria. Most lactic acid bacteria can provide a unique sour taste to pickles. Some yeast and spore bacteria produce biofilm during reproduction and metabolism, which adheres to the surface of the product and is defined as visible foreign matter during packaging, increasing the difficulty of quality control during the production and processing process. If cleaning is used to remove it, the fermentation flavor will be lost. Some yeast metabolizes to produce a yeast extract flavor and a fermented wine flavor. When yeast converts acid into alcohol, the acid content in the fermentation system is significantly reduced, followed by abnormal spoilage flavor and even foul odor. At the same time, the color of the fermented product becomes dark and loses commercial value. Especially in the summer, yeast and spore bacteria can proliferate rapidly in the early stage of fermentation, resulting in slow acid production, insufficient acid production, insufficient sensory acid, and abnormal aroma.

[0004] Allium fistulosum is known as "the Lingzhi of vegetables" and is famous for its delicious taste and deep historical background in botany. It has a long history of consumption in southern China. Water crystal allium fistulosum is loved by people because of its fresh and crisp taste, unique flavor, and sweet and delicious taste. However, its natural fermentation is also easily contaminated by yeast and spore bacteria. In addition, due to the limitations of refrigeration, cold chain conditions, and consumption level, natural fermentation of allium fistulosum has the problems of unstable quality, easy browning, soft texture, bag swelling, and flavor decline during normal temperature storage, circulation, and sales, which seriously affect product quality. Currently, pasteurization and the addition of preservatives are used to extend the shelf life in industrial production, but this may result in serious loss of nutrients and flavor, and long-term consumption of preservatives may pose a health risk. Therefore, it is an inevitable trend to develop high-quality allium fistulosum fermentation inoculant, achieve zero addition of preservatives such as sodium benzoate and sodium metabisulfite, extend the shelf life of zero-addition products, and improve product safety. SUMMARY

[0005] Based on the above technical problems, the main purpose of the present application is to overcome the shortcomings of the prior art. A Lentilactobacillus sunkii LS.1 strain is screened from traditional natural fermented water crystal leeks. The strain produces acid quickly, accumulates a large amount of acid, has excellent sensory flavor performance, and inhibits spoilage microorganisms. It can solve the problems of slow acid production, insufficient acid production, insufficient fermented acid aroma, and easy occurrence of spoilage odor during the fermentation of water crystal leeks, achieving the technical effects of fermented leeks with rich and pure acid aroma, soft and coordinated sour taste, lingering and sweet alcohol aroma, good retention, unique flavor, and prevention of spoilage during the shelf life.

[0006] To achieve the above-mentioned purpose, the inventors have conducted in-depth research and completed the present application scheme after repeated research and demonstration, as follows:

[0007] In a first aspect, the present application provides a Lentilactobacillus sunkii LS.1 strain, which has been deposited with the Guangdong Microbial Culture Collection Center on May 19, 2025, and has the accession number GDMCC No.66350.

[0008] The colony of the Lentilactobacillus sunkii LS.1 strain in the MRS culture medium is milky white, with round colonies, neat edges, smooth surface, and luster. The bacterial body is a typical long and short rod shape, and part of the morphology is curved and coiled into a cluster or a curved spiral shape. The length of the bacterial body is 0.3-1.1 μm, and the width is 0.2-0.4 μm.

[0009] In a second aspect, the present application further provides a fermentation inoculant, which comprises the above-mentioned Lentilactobacillus sunkii LS.1.

[0010] In a third aspect, the present application further provides the use of the above-mentioned fermentation inoculant in leek fermentation, preferably water crystal leek, especially for improving the problems of insufficient fermented acid aroma, uneven color, easy occurrence of spoilage odor, etc. of fermented water crystal leek, and improving the color, aroma, taste, and crispness of fermented water crystal leek, and comprehensively improving the quality of fermented water crystal leek.

[0011] In a fourth aspect, the present application further provides a fermentation method of water crystal leek, which comprises the step of using the above-mentioned Lentilactobacillus sunkii LS.1 or fermentation inoculant for fermentation.

[0012] Further, the addition amount of the Lentilactobacillus sunkii LS.1 or fermentation inoculant is 10 6 -10 7 CFU / mL.

[0013] Further, the addition time of the Lentilactobacillus sunkii LS.1 or fermentation inoculant is the initial stage of fermentation.

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

[0015] In a fifth aspect, the present application provides a fermented Chinese allium fistulosum, which is prepared by the method described in the present application.

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

[0017] 1. The lactobacillus LS.1 provided by the present application is acid-resistant, fast acid-producing, and high in acid accumulation, and can inhibit the growth of spoilage bacteria. It is used to ferment Chinese allium fistulosum, and shows excellent effects of color protection, crispness preservation, flavor improvement, and shelf life extension.

[0018] 2. The lactobacillus LS.1 strain provided by the present application has strong cell hydrophobicity and the performance of rapid self-aggregation, which can adhere to intestinal epithelial cells and colonize the gastrointestinal tract of animals to play the beneficial role of probiotics.

[0019] 3. The Chinese allium fistulosum fermented by the lactobacillus LS.1 provided by the present application is crystal clear, the skin is uniform white, the skin and the allium fistulosum flesh are uniform and translucent white, the acid aroma is pure, the acid taste is pure and refreshing, soft and harmonious, the retention degree is good, there is no odor, no biofilm is produced, the texture of the allium fistulosum can be well preserved, the crispness of the product is ensured, and the overall product quality is improved.

[0020] 4. The Chinese allium fistulosum fermented by the lactobacillus LS.1 provided by the present application has uniform and stable color, can ensure the quality stability of the product shelf life without using preservatives under vacuum packaging, is not inflated, and the fermentation process is easy to control, which can realize scale industrialization technology popularization. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a test result graph of hydrophobicity and self-aggregation of different strains;

[0022] Figure 2 It is a test result graph of acid production of different strains;

[0023] Figure 3 It is a flavor and color difference analysis result graph of different strains;

[0024] Figure 4 It is a lactobacillus LS.1 colony morphology graph;

[0025] Figure 5 It is a lactobacillus LS.1 cell morphology graph under a microscope;

[0026] Figure 6 It is an acid resistance performance test OD 600 Stacked chart;

[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] Select the growth of 30-300 CFU / dish of petri dish, mark the colony morphology and describe the morphological differences, and the colonies with different colony morphology are subjected to microscopic morphological test and description. Select typical lactic acid bacteria for purification, purify 3 times, and use gram staining technique for microscopic examination to preliminarily judge the purified strains, prepare glycerol freezing tube, and store in -80℃ medical refrigerator for standby. 10 strains of rod-shaped microorganisms numbered LS.1, BDH-5, SC, HX, YY-2, CH-2, BDH-3, BDH-1, YY-1, BDH-2 are obtained by separating and purifying samples with excellent flavor.

[0036] 2. Primary screening of lactic acid bacteria

[0037] Configure MRS liquid medium 100mL / bottle, 121℃, sterilize for 20mins, cool down, inoculate the above-mentioned 10 strains of microorganisms, 1 ring / bottle, place in 30℃ constant temperature incubator, and stand for culture for 48h, adjust the effective viable count of microorganisms to 1×10 8 ~5×10 8 CFU / mL, which is the test bacterial suspension.

[0038] (1) Cell hydrophobicity test

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

[0040] Take 5mL of hydrophobic agent n-hexane and chloroform (trichloromethane) respectively, add them into 2 empty test tubes, add 15mL of PBS adjusted bacterial suspension into each of the 2 test tubes, mix well, place in the fume hood for 1h, discard the organic phase, and retain the aqueous phase to measure the data. Take PBS solution as the absorbance control to adjust zero, measure the absorbance of the aqueous phase, which is A1. Calculate the hydrophobicity according to the following formula, and record the results in Figure 1 .

[0041]

[0042] Analysis 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 3It can be seen that the test results of pure strain fermentation of different strains after pasteurization show that LS.1 has the best color with S of 10.67NBS, the largest rank sum of taste and smell, and the best comprehensive sensory evaluation, followed by BDH-5, then HX and SC. CH-2, color protection and fermentation aroma and taste are general. Although BDH-3 shows excellent color protection effect, the fermentation flavor is poor. Although BDH-2 shows excellent fermentation flavor, the color is poor. Sensory analysis ranking method page test results show that when p = 11, j = 12, and α = 0.05, the critical value is 4949.62, because L aroma is 5907 > 4949.62, and L flavor is 5986 > 4949.62, so when α = 0.05, there is difference between samples, and the order is consistent with the pre-set order. The aroma rank sum order is: BDH-5 > LS.1 > HX > SC > CH-2 > BDH-2 > YY-2 > CK > BDH-3 > YY-1 > BDH-1; the flavor rank sum 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 can give fermented water crystal leek excellent color and flavor.

[0056] 3. Lactic acid bacteria rescreening

[0057] The above CK spoilage microorganisms are mixed and enriched. Because they contain Bacillus and yeast, LB medium and wort medium are prepared and mixed uniformly, sterilized, and inoculated with CK treatment mixed bacteria liquid in a sterile manner. The inoculation amount is 1%, and the culture is incubated at 30°C for 48h to obtain a spoilage microorganism suspension.

[0058] The above SC, CH-2, BDH-3, BDH-5, HX, and LS.1 strains are enriched using MRS medium, and the precipitate and supernatant are separated by centrifugation at 10000r / min for 15mins for standby use. The precipitate is resuspended with different volumes of sterile normal saline to prepare 81-fold concentrated liquid, 27-fold concentrated liquid, 9-fold concentrated liquid, 3-fold concentrated liquid, and original concentration bacteria suspension, a total of 5 concentrations of bacteria suspension, plus supernatant, a total of 6 gradient solutions, which are the test liquids of each lactic acid bacteria.

[0059] Sterile semi-solid MRS agar (10g / L) is prepared, 10mL is accurately transferred to a sterile empty culture dish, and placed horizontally. The semi-solid MRS agar plate is cooled and solidified. Six Oxford cups are placed at equal distances on the semi-solid MRS agar plate without pressing. The prepared spoilage microorganism suspension is adjusted to a concentration of 1×10 8 ~ 5×10 8 CFU / mL, and inoculated into the mixed LB and wort medium cooled to 45~50°C, mixed uniformly, so that the concentration of the mixed spoilage microorganism bacteria is 1×10 6 ~ 5×106 CFU / mL. 10 mL was pipetted into a semi-solid agar plate, the plate was gently shaken to evenly spread the bacterial suspension on the plate, and then placed horizontally. After solidification, 100 μL of the test lactic acid bacterial concentrate sample was accurately measured and slowly added into the Oxford cup with distilled water as the control, with 3 replicates for each treatment. The plate was moved into a 4°C refrigerator for 4-10 h, then taken out and placed in a 30°C±1°C incubator for upright culture until the inhibition zone was clear. The diameter of the inhibition zone was measured using a vernier caliper with a measurement accuracy of 0.02-0.1 mm, and the range of the inhibition zone was 8-25 mm. Each inhibition zone was measured in three different directions, and the average value was recorded. The logarithmic value of the reciprocal of the dilution factor of the additive was taken as the abscissa, and the diameter of the inhibition zone was taken as the ordinate to establish a linear equation (R 2 ≥0.9900), and the additive with high antibacterial titer was preferably selected. The results are shown in Table 2.

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

[0061]

[0062]

[0063] From the analysis of the data in Table 2, it can be seen that, in terms of the test effect of the bacterial suspension, LS.1 had the largest antibacterial titer against spoilage bacteria, which was 81.15 AU / mg, followed by SC strain, which was 35.63 AU / mg. In terms of the test effect of the supernatant, LS.1 had the largest inhibition zone against spoilage bacteria, which was 16.3 mm, followed by SC, which was 10.5 mm. Other strains did not show obvious inhibition effect on spoilage bacteria. The reason for the evaluation of the non-growth of Bacillus and yeast in the previous test was that the lactic acid bacteria occupied the nutrients in the growth and reproduction, and became the dominant flora in the early fermentation of crystal leek, so that the spoilage bacteria could not reproduce. In summary, LS.1 had obvious inhibition effect on spoilage bacteria in the fermentation process of crystal leek, and the effect was better than that of other lactic acid bacteria. Combined with the control of the initial inoculation amount, the outbreak of spoilage bacteria in the fermentation system could be effectively controlled.

[0064] Based on the test indicators of strain fermentation, such as acid content, color difference, sensory evaluation and antibacterial ability, it can be seen that the LS.1 strain can quickly grow and reproduce, produce acid quickly and accumulate a large amount, significantly improve the color and flavor of the product, effectively inhibit the growth and reproduction of spoilage bacteria in crystal leek, and improve the quality of the fermented vegetables. Therefore, the strain is selected as the target strain for the fermentation of crystal leek.

[0065] The test bacterial suspension of the lactic acid bacteria LS.1 selected by fermentation test was subjected to plate streaking and Gram staining, and the colony morphology and cell morphology of the strain were observed and described by naked eye and microscope. The colony morphology of LS.1 was as follows: Figure 4As shown, the colony morphology is milky white, round colony, with neat edges, smooth surface, moist and shiny. After Gram staining of the obtained strain, the bacterial morphology was observed under 100x oil immersion lens as shown in Figure 1. Figure 5 As shown, the bacterial morphology is typical long and short rod-shaped, part of which is curved and coiled into a cluster or curved spiral shape. The length of the bacterial body is 0.3-1.1 pm, and the width is 0.2-0.4 pm.

[0066] The pure culture of the test LS.1 strain was sent for sequencing and identified as Lentilactobacillus sunkii, and was preserved in the Guangdong Microbial Culture Collection Center on May 19, 2025, with the strain preservation number GDMCC No.66350.

[0067] Example 2 Performance test of lactobacillus LS.1

[0068] (1) Acid resistance performance test

[0069] Prepare a lactobacillus LS.1 bacterial suspension according to the method described in Example 1.

[0070] Prepare MRS liquid medium with natural, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 1.75% acid concentrations using citric acid, and sterilize and cool for standby. Inoculate the LS.1 bacterial suspension into the medium with different acid concentrations at an inoculation amount of 1%, and incubate in a 30°C constant temperature shaker at 150 r / min. Measure the OD 600 at the initial, 24h, 48h, and 72h, respectively, to test the acid resistance performance of the strain, and the results are recorded in Table 1. Figure 6

[0071] Analysis Figure 6 The data shows that the lactobacillus LS.1 strain has strong acid resistance, and can still grow and reproduce under an acid concentration of 1.75%.

[0072] (2) Aerobic characteristic test

[0073] Prepare MRS solid medium, and aseptically dilute the lactobacillus LS.1 bacterial suspension to 1000 CFU / mL-5000 CFU / mL. Spread the diluted bacterial suspension on the solid medium at 100 pL / dish, and spread 6 dishes. Incubate 2 dishes in an anaerobic tank under anaerobic conditions, seal 2 dishes with a sealing film, and place 2 dishes without sealing in a 30°C constant temperature incubator. Incubate for 72h and count.

[0074] Table 3 Aerobic characteristic test colony count results

[0075]

[0076] ​From the data in Table 3, it can be seen that the growth of Lactobacillus LS.1 was the largest under anaerobic conditions, reaching 116 CFU / dish, and the growth under aerobic conditions was 95 CFU / dish, indicating that LS.1 can grow and reproduce normally in an anaerobic environment, and the growth is better than in an aerobic environment. This growth characteristic meets the fermentation requirements of water crystal leeks and is conducive to inhibiting the growth and reproduction of spoilage bacteria during fermentation and improving the quality of fermented vegetables.

[0077] Example 3 Water Crystal Leek Fermentation Test

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

[0079] Six clean 25 kg fermentation jars were prepared, fresh water crystal leeks were taken, inedible parts were removed, washed, and drained. The leeks were neatly packed into the fermentation jars to the brim, with a leek mass of 13 kg per jar, 4% edible salt was added, 5% white sugar solution was added to the brim of the jar, and 12 kg per jar. Group 1 was inoculated with LS.1 strain, and the inoculation amount was 1% of the total system; group 2 was inoculated with BDH-5, and the inoculation amount was 1% of the total system; CK was not inoculated and naturally fermented. After placing overnight, the next day the jars were turned to mix the bacteria and materials evenly. The natural fermentation was the control group, and except for not inoculating the strain, the other treatments and test groups were exactly the same. After 30 days of fermentation, the total acid results were determined and sensory evaluation was performed, and the results are recorded in Tables 4-5.

[0080] After fermentation, the samples were packaged and the shelf life was tested. To exclude the influence of acid indicators on the test results, at the end of fermentation, the total acid of the leek and soup in the fermentation jar was adjusted to 1.0% (calculated as titratable lactic acid) using citric acid. The samples at the end of fermentation were packaged using three schemes, with 80% solids and soup being the original soup of each fermentation scheme. Scheme 1 was non-antiseptic non-heat treated vacuum packaging, scheme 2 was vacuum packaging with the addition of 1‰ sodium benzoate and 100 ppm sodium metabisulfite antiseptic, and scheme 3 was vacuum packaging without antiseptic at 85°C for 30 minutes. Each treatment was packaged in 13 bags, and the initial samples were sampled for TPA texture analysis, and the results are recorded in Table 6. The remaining samples were evaluated at room temperature, 30°C, and 37°C, and the swelling was counted every 15 days. When recording the swelling, the samples were placed vertically, a liquid level line was drawn, the height of the package was h1, the height of the liquid level line was h2, and h1-h2 was the true reflection of the swelling, which was Q. The larger the Q value, the more serious the swelling. The average value of the repeated data was calculated, and the results are recorded in Table 7.

[0081] Table 4 Total acid and film production at the end of water crystal leek fermentation

[0082]

[0083] From the data in Table 4, it can be seen that the acid accumulation of LS.1 treatment was the largest, 0.96%, which was 2.34 times of CK treatment and 1.52 times of BDH-5 lactic acid bacteria. The leeks fermented by CK and BDH-5 strains appeared biofilm production, adhered to the surface of leeks, affected the appearance of the product, and LS.1 strain treatment had no biofilm production. Therefore, the exogenous added strain can promote leek fermentation to produce acid, and there is a big difference in the acid production ability of different lactic acid bacteria. LS.1 has the advantages of excellent promotion of leek fermentation to rapidly produce acid and large acid accumulation, and this excellent feature can quickly inhibit the breeding of strains such as yeast and bacillus that produce biofilm, ensure the uniformity of product appearance, no visual foreign matter, strong production and processing adaptability, no need to clean to cause the loss of fermentation flavor, and improve the stability of product quality.

[0084] Table 5 Flavor sensory evaluation of water crystal leek at the end of fermentation

[0085]

[0086] From the data in Table 5, it can be seen that the leek fermented by LS.1 has a color that is crystal clear, uniform color of the inner and outer scales, sour leek flavor, no spicy irritating eye and nose odor, sour and salty coordination, pure and soft sour taste, fresh and sweet, long-lasting sweet and mellow, and good brittleness. The leek fermented by BDH-5 has uneven color of the inner and outer scales, slightly fermented sour leek flavor, spicy irritating eye and nose odor, weak sour aroma, spicy taste stimulating the oral cavity, insufficient fermentation sour taste, and poor brittleness. Although the leek fermented by BDH-5 has fermented sour aroma compared with CK treatment, the comprehensive sensory evaluation is not as good as LS.1 treatment. The leek treated by LS.1 has uniform color of the whole fermentation system, no blackening and darkening phenomenon, and no biofilm production. The leek treated by CK has uneven color of the inner and outer scales, blackening and darkening, strong spicy irritating eye and nose odor, weak sour leek flavor, yeast flavor, spicy taste stimulating the oral cavity, prominent salty taste, very weak fermented sour aroma, and poor brittleness.

[0087] Table 6 Texture analysis of water crystal leek with different packaging processes

[0088]

[0089] Note: The sensory brittleness ranking "1" indicates the best brittleness, and the brittleness decreases in turn. The same serial number indicates that the brittleness has no significant difference.

[0090] From the analysis of Table 6 data, it can be seen from the TPA detection analysis that the treatment of 85℃ sterilization for 30mins makes the crispness of water crystal leek decrease significantly, so it is not recommended to use heat treatment scheme to maintain shelf life quality stability. Under the same fermentation scheme, the crispness difference between the non-heat treatment packaging process with preservative and zero preservative is not significant. The crispness of leek processed by the process is evaluated comprehensively by local maximum positive force, hardness, gumminess and chewiness. Crispness (unique to samples with crispy skin), resilience and cohesiveness have less contribution to the sensory crispness of the product. LS.1 fermented leek shows excellent hardness, gumminess, crispness and chewiness. Its crispness protection principle comes from inhibiting the growth of spoilage microorganisms while producing acid, which better preserves the texture of leek.

[0091] Table 7 Water crystal leek shelf life bulging situation statistics

[0092]

[0093] From Table 7, under the non-heat treatment packaging process with preservative, LS.1, BDH-5 and CK groups did not appear bulging at room temperature, 30℃ and 37℃ for 45d. Under the zero preservative heat treatment packaging storage process, BDH-5 and CK groups appeared slight bulging after 15d, and the bulging of CK group was more significant, which intensified with the extension of storage time. LS.1 group did not appear bulging at room temperature, 30℃ and 37℃ for 45d. Under the zero preservative non-heat treatment packaging, BDH-5 and CK groups appeared obvious bulging after 15d, and the bulging of CK group was more significant, which intensified with the extension of storage time. LS.1 group did not appear bulging at room temperature, 30℃ and 37℃ for 45d. Comprehensive analysis shows that lactic acid bacteria BDH-5 and naturally fermented water crystal leek will still appear bulging even after 85℃ sterilization for 30mins, and chemical preservative needs to be used to realize the shelf life quality stability and prevent bulging. The occurrence of bulging makes leek unable to fully contact with soup, which easily leads to browning of color, abnormal flavor of product, breakage of bag during transportation, and contamination of the same batch of products, resulting in loss of commercial value of the same batch of products. LS.1 fermented leek can ensure the shelf life quality stability under the condition of 1.0% vacuum packaging, non-heat treatment and no preservative addition, realize the packaging storage process with zero preservative, and promote the green and healthy development of Yunnan plateau characteristic leek industry.

[0094] In summary, Lactobacillus LS.1 cells have strong hydrophobicity and the ability of rapid self-aggregation, which has the potential to colonize the gastrointestinal tract of animals. The characteristics of fast acid production and high acid accumulation can inhibit the growth of spoilage bacteria. LS.1 fermented crystal leek has the functions of color protection, crispness preservation and flavor improvement. The fermented crystal leek product has a crystal clear color, uniform and jade white outer and inner scales, fermented leek flavor, pure and rich aroma, no spicy and irritating smell, rich leek flavor, harmonious sour and salty taste, pure and soft sour taste, fresh and sweet, long-lasting sweet and salty taste, good crispness. The whole fermentation system has uniform color without darkening and biological membrane production. It can better preserve the texture of leek and ensure the crispness of the product. The packaged product has uniform and stable color, which can ensure the quality stability of the product during the shelf life without using preservatives under vacuum packaging. The product does not expand and the fermentation process control is simple and convenient, which can realize the popularization of large-scale industrialization technology.

[0095] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to 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, 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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