Lactobacillus mucilaginosus and application thereof

Abstract

The invention provides lactobacillus mucilaginosus and application thereof, and belongs to the technical field of microorganisms. According to the present invention, the Latin of the Lactobacillus mucus fermentation is Limosilactobacillusfermentum, the preservation name is Lactobacillus mucus fermentation MQ10, the preservation place is China Center for Type Culture Collection (CCTCC), the preservation date is July 22, 2024, and the preservation number is CCTCC NO: M 20241616. The strain has a broad-spectrum antibacterial effect and can inhibit growth and reproduction of various microorganisms. The biological fresh-keeping product prepared from the fresh-keeping agent can reduce the decay rate of food and the browning rate of fruit stems of fruits and vegetables, maintain the content and hardness of soluble solids, slow down the aging decay speed of the food in the storage period and prolong the storage period. Due to the characteristics, the lactobacillus mucilaginosus is expected to become a green and environment-friendly biological fresh-keeping agent, and the quality and the safety of foods are improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a fermented mucus lactobacillus and an application thereof. Background Art

[0002] Cherry belongs to the genus Prunus in the Rosaceae family, and there are many varieties. Fresh cherries are easily damaged mechanically during transportation. Since cherries are prone to cracking, the wounds are more susceptible to infection by pathogenic microorganisms. The post-harvest loss caused by microbial infection is as high as 50% each year. Among them, the common fungal pathogens that cause post-harvest corruption are mainly: Penicillium expansum, Botrytis cinerea, Alternaria alternata, Aspergillus niger, etc. These pathogenic fungi cause post-harvest diseases such as penicillium, brown rot, and gray mold. It can be seen that improving its storage quality and preventing fungal infection are the main problems that need to be solved after cherry is harvested. It is urgent to find an effective preservation method to reduce the loss of cherries after harvest.

[0003] At present, there are two main ways to preserve cherries after harvest: physical and chemical. Physical preservation mainly includes low-temperature refrigeration and controlled atmosphere preservation, which mainly delay fruit aging by inhibiting respiration and reducing the activity of related enzymes; however, physical preservation technology generally has high requirements for machinery and equipment, and the investment cost is relatively large. Chemical preservation technology mainly relies on chemical preservatives to be sprayed, soaked or fumigated, which can usually reduce the microorganisms on the surface of cherries or reduce their respiration rate to maintain quality. It has the advantages of low cost, simple process and energy saving; however, chemical treatment is extremely strict in considering the amount of chemical residues. With people's attention to safety issues and environmental pollution, the widespread use of chemical preservatives still faces huge challenges and controversies. It can be seen that the existing preservation methods have problems such as high cost and residue. Therefore, there is an urgent need for a natural, safe, residue-free and low-cost preservation method. Summary of the invention

[0004] The purpose of the present invention is to provide a fermented mucus lactobacillus and an application thereof, screen out a fermented mucus lactobacillus with high antibacterial activity, and apply the screened fermented mucus lactobacillus MQ10 to the biological preservation of food, delay spoilage, extend the shelf life, maintain its quality, and improve its antioxidant capacity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a fermented mucus lactobacillus. The Latin name of the fermented mucus lactobacillus is Limosilactobacillusfermentum, the preservation name is fermented mucus lactobacillus MQ10, the preservation place is China Center for Type Culture Collection, the preservation date is July 22, 2024, and the preservation number is CCTCC NO: M 20241616.

[0007] The invention also provides application of the fermented mucus lactobacillus in inhibiting the growth of microorganisms.

[0008] Preferably, the microorganisms include one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Penicillium expansum, Botrytis cinerea, and Aspergillus carbonarius.

[0009] The invention also provides application of the fermented mucus lactobacillus in preparing a product for inhibiting microbial growth.

[0010] Preferably, the microorganisms include one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Penicillium expansum, Botrytis cinerea, and Aspergillus carbonarius.

[0011] Preferably, the product is a biological preservative.

[0012] The invention also provides a biological preservative, which contains fermented mucus lactobacillus.

[0013] The present invention also provides a method for preparing the biological preservative, comprising the following steps:

[0014] (1) activating the fermented mucus lactobacillus to obtain an activated solution;

[0015] (2) inoculating the activation solution into the culture medium, culturing and centrifuging to obtain bacterial cells;

[0016] (3) Wash the cells and resuspend them to adjust the final concentration to 0.8×10 9 ~1.2×10 9 CFU / mL to obtain the biological preservative.

[0017] Preferably, the number of activations in step (1) is 1 to 3 generations;

[0018] The inoculation amount of the activation solution in step (2) is 2% to 6% of the volume of the culture medium;

[0019] The culture medium is MRS liquid culture medium;

[0020] The culture time is 12 to 16 hours, and the culture temperature is 35 to 39° C.;

[0021] The centrifugal temperature is 2-6°C, the centrifugal speed is 8000-12000rpm, and the centrifugal time is 10-20min;

[0022] The washing method in step (3) is to wash the bacteria with sterile saline for 1 to 3 times;

[0023] The resuspension method is: resuspending the washed bacteria in physiological saline.

[0024] The present invention also provides application of the biological preservative in preserving fruits, wherein the fruits include cherries.

[0025] The present invention provides a fermented mucus lactobacillus and an application thereof. The fermented mucus lactobacillus is named Limosilactobacillusfermentum in Latin, is deposited as fermented mucus lactobacillus MQ10, is deposited at China Center for Type Culture Collection, is deposited on July 22, 2024, and has a deposit number of CCTCC NO: M 20241616.

[0026] The beneficial effects of the present invention are:

[0027] 1. Fermented Lactobacillus mucus MQ10 has a broad-spectrum antibacterial effect and can inhibit the growth and reproduction of many common pathogenic bacteria and pathogenic fungi. At the same time, the safety and excellent probiotic properties of this strain indicate its great potential value in industrial applications, especially in the development of new biological preservatives. These characteristics make fermented Lactobacillus mucus MQ10 a green and environmentally friendly solution for post-harvest preservation of fruits and vegetables, which helps to reduce the use of chemical preservatives and improve the quality and safety of food.

[0028] 2. As a biological preservative, fermented Lactobacillus mucilaginosus MQ10 can effectively reduce the spoilage rate of food and the browning rate of fruit and vegetable stems, maintain the content and hardness of soluble solids, slow down the aging and spoilage rate of food during storage, and extend the storage period. At the same time, fermented Lactobacillus mucilaginosus MQ10 can enhance the nutritional value of food, improve the market value of food and the health benefits of consumers.

[0029] Collection Instructions

[0030] Fermented mucus lactobacillus, the preservation name is fermented mucus lactobacillus MQ10, the Latin name is Limosilactobacillusfermentum, the preservation place is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is July 22, 2024, and the preservation number is CCTCC NO: M 20241616. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The inhibitory effect of fermented Lactobacillus mucilaginosus MQ10 on Penicillium expansum;

[0032] Figure 2 The inhibitory effect of fermented Lactobacillus mucilaginosus MQ10 on Botrytis cinerea;

[0033] Figure 3 The inhibitory effect of fermented Lactobacillus mucilaginosus MQ10 on Aspergillus carbonarius;

[0034] Figure 4 For the hemolytic test of fermented Lactobacillus mucilaginosus MQ10;

[0035] Figure 5 To evaluate the hydrophobicity of fermentative Lactobacillus mucilaginosus MQ10;

[0036] Figure 6 To evaluate the self-aggregation ability of fermentative Lactobacillus mucilaginosus MQ10;

[0037] Figure 7 To evaluate the coaggregation ability of fermentative Lactobacillus mucilaginosus MQ10 with pathogenic bacteria;

[0038] Figure 8 The acid tolerance of fermenting Lactobacillus mucilaginosus MQ10;

[0039] Fig. 9 The bile salt tolerance of Lactobacillus mucilaginosus MQ10;

[0040] Fig.10 The sodium chloride tolerance of fermenting Lactobacillus mucilaginosus MQ10;

[0041] Fig.11 The hydrogen peroxide tolerance of fermenting Lactobacillus mucilaginosus MQ10;

[0042] Fig.12 The temperature tolerance of fermentation Lactobacillus mucilaginosus MQ10;

[0043] Fig.13 The changes of weight loss rate of cherry fruits in different treatment groups with the extension of storage time;

[0044] Fig.14 The changes of the spoilage rate of cherry fruits in different treatment groups with the extension of storage time;

[0045] Fig.15 The changes of stalk browning rate of cherry fruits in different treatment groups with the extension of storage time;

[0046] Fig.16 The changes of cherry fruit firmness in different treatment groups with the extension of storage time;

[0047] Fig.17 The changes of TSS of cherry fruits in different treatment groups with the extension of storage time;

[0048] Fig.18 The changes of titratable acid in cherry fruits of different treatment groups with the extension of storage time;

[0049] Fig.19 The changes of glutathione content in cherry fruits of different treatment groups with prolonged storage time;

[0050] Fig. 20 The changes of reduced ascorbic acid content in cherry fruits of different treatment groups with prolonged storage time;

[0051] Fig.21 The changes of total phenolic content in cherry fruits of different treatment groups with the extension of storage time;

[0052] Fig. 22 The changes of flavonoid content in cherry fruits of different treatment groups with the extension of storage time;

[0053] Fig.23 The changes of total anthocyanin content in cherry fruits of different treatment groups with the extension of storage time;

[0054] Fig.24 The changes of DPPH free radical scavenging ability of cherry fruits in different treatment groups with the extension of storage time;

[0055] Fig.25 The changes of total antioxidant capacity of cherry fruits in different treatment groups with prolonged storage time;

[0056] Fig.26 The changes of lignin content in cherry fruits of different treatment groups with extended storage time. DETAILED DESCRIPTION

[0057] The invention provides a fermented mucus lactobacillus. The Latin name of the fermented mucus lactobacillus is Limosilactobacillusfermentum, the preservation name is fermented mucus lactobacillus MQ10, the preservation place is China Center for Type Culture Collection, the preservation date is July 22, 2024, and the preservation number is CCTCC NO: M 20241616.

[0058] The invention also provides application of the fermented mucus lactobacillus in inhibiting the growth of microorganisms.

[0059] In the present invention, the microorganisms include one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Penicillium expansum, Botrytis cinerea, and Aspergillus carbonarius.

[0060] The invention also provides application of the fermented mucus lactobacillus in preparing a product for inhibiting microbial growth.

[0061] In the present invention, the microorganisms include one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Penicillium expansum, Botrytis cinerea, and Aspergillus carbonarius.

[0062] In the present invention, the product is a biological preservative.

[0063] The invention also provides a biological preservative, which contains fermented mucus lactobacillus.

[0064] The present invention also provides a method for preparing the biological preservative, comprising the following steps:

[0065] (1) activating the fermented mucus lactobacillus to obtain an activated solution;

[0066] (2) inoculating the activation solution into the culture medium, culturing and centrifuging to obtain bacterial cells;

[0067] (3) Wash the cells and resuspend them to adjust the final concentration to 0.8×10 9 ~1.2×10 9 CFU / mL, and obtain the biological preservative.

[0068] In the present invention, the number of activation in step (1) is 1 to 3 generations, more preferably 2 generations.

[0069] In the present invention, the inoculation amount of the activation solution in step (2) is preferably 2% to 6% of the volume of the culture medium, and more preferably 3 to 5%;

[0070] The culture medium is preferably MRS liquid culture medium;

[0071] The culturing time is preferably 12 to 16 hours, more preferably 13 to 15 hours;

[0072] The culture temperature is preferably 35-39°C, more preferably 36-38°C;

[0073] The centrifugal temperature is preferably 2 to 6°C, more preferably 3 to 5°C;

[0074] The centrifugal speed is preferably 8000-12000 rpm, more preferably 9000-110000 rpm;

[0075] The centrifugation time is preferably 10 to 20 minutes, more preferably 12 to 18 minutes;

[0076] The raw materials of the MRS liquid culture medium are preferably: glucose (20.00 g), peptone (10.00 g), beef extract (10.00 g), yeast extract (5.00 g), anhydrous sodium acetate (5.00 g), dipotassium hydrogen phosphate (2.00 g), diammonium hydrogen citrate (2.00 g), magnesium sulfate heptahydrate (0.58 g), manganese sulfate monohydrate (0.17 g) and Tween 80 (1.00 mL), distilled water (1000 mL);

[0077] The method for preparing the MRS liquid culture medium is preferably as follows: the above raw materials are mixed, heated and stirred to dissolve, and then dispensed into 100 mL reagent bottles, sterilized by high pressure at 121° C. for 15 min, and cooled and stored for later use.

[0078] In the present invention, the washing method in step (3) is preferably to wash the bacterial cells with sterile saline for 1 to 3 times, more preferably 2 times;

[0079] The resuspension method is preferably: resuspending the washed bacteria in physiological saline;

[0080] The final concentration of the bacteria is preferably 0.9×10 9 ~1.1×10 9 CFU / mL.

[0081] The present invention also provides application of the biological preservative in preserving fruits, wherein the fruits include cherries.

[0082] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0083] The kits used in the experimental examples of the present invention include: a plant flavonoid content detection kit, a plant total anthocyanin (total anthocyanin) content detection kit, a lignin content detection kit, a plant total phenol (Total Phenols, TP) content detection kit, a DPPH free radical scavenging ability detection kit, a total antioxidant capacity (T-AOC) detection kit, a reduced glutathione content detection kit, and a reduced ascorbic acid content detection kit, all of which were purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0084] The indicator bacteria used in the experimental examples of the present invention include: enteroaggregative Escherichia coli (Escherichiacoli EAEC CICC 25044), Staphylococcus aureus (Staphylococcus aureus CICC 23926), and Salmonelia enterica BNCC 292614. The indicator bacteria in the mold inhibition test include: Penicillium expansum (bio-25700), Botrytis cinerea (bio-81601), and Aspergillus carbonarius (bio-52150). The above strains are all preserved by this laboratory.

[0085] Example 1 Isolation, purification and identification of lactic acid bacteria

[0086] Isolation, purification and identification of lactic acid bacteria

[0087] According to the standard of "GB4789.35-2023 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test", a traditional fermented yak yogurt sample from a herdsman in Maqu County, Gannan Tibetan Autonomous Prefecture, Gansu Province was diluted and cultured with lactic acid bacteria. The diluted and coated MRS solid medium was cultured in a 37°C incubator for 60 hours. Pick the colonies with obvious characteristics on the plate and repeatedly streak them until colonies with consistent morphology and size are isolated. The isolated and purified lactic acid bacteria were identified by MALDI-TOF and confirmed as fermented mucus Lactobacillus, and then named fermented mucus Lactobacillus MQ10.

[0088] The MRS agar medium is prepared by adding 15 g of agar powder to the MRS liquid formula, and the subsequent steps are the same as those of the MRS liquid culture medium.

[0089] Example 2 Antibacterial effect verification test of fermented Lactobacillus mucilaginosus MQ10

[0090] a Common pathogenic bacteria inhibition test

[0091] A single colony of the indicator bacteria was inoculated into LB liquid culture medium and cultured at 37°C for 14 hours. The indicator bacteria was inoculated into LB semi-solid culture medium at 55°C at an inoculum amount of 1% of the volume of the LB semi-solid culture medium, mixed and introduced into a culture dish, and after the culture medium solidified, a sterile Oxford cup was placed, and overnight cultured Lactobacillus mucosa MQ10 culture medium was added to the cup, 200 μL per well, and cultured in a 37°C incubator for 16 hours. The size of the inhibition zone was measured using a vernier caliper.

[0092] The raw material composition of the LB liquid culture medium is: tryptone (10.00 g), yeast extract (5.00 g), sodium chloride (10.00 g). The preparation method is: after mixing the raw materials, heating and stirring to dissolve, divide them into 100 mL reagent bottles, sterilize them at 121° C. for 15 min, and cool and store them for later use.

[0093] The raw materials used in the LB solid culture medium are based on the LB liquid formula, with 15g of agar powder added additionally, and the subsequent steps are the same as those of the LB liquid culture medium.

[0094] The inhibition zones of fermented Lactobacillus mucilaginosus MQ10 against enteroaggregative Escherichia coli (Escherichia coli EAEC CICC25044), Staphylococcus aureus (Staphylococcus aureus CICC 23926), and Salmonella enterica BNCC 292614 are shown in Table 1. Fermented Lactobacillus mucilaginosus MQ10 has the strongest inhibitory ability against Staphylococcus aureus, with an inhibition zone of 16.88±1.52mm; it has a stronger effect on Salmonella enterica BNCC 292614, with an inhibition zone diameter of 12.91±1.65mm; and has a weaker inhibitory effect on enteroaggregative Escherichia coli, with an inhibition zone diameter of 10.55±0.96mm.

[0095] Table 1 Inhibitory ability of lactic acid bacteria against common pathogenic bacteria

[0096]

[0097] Note: The inner diameter of the Oxford cup is 6mm.

[0098] b. Fungus inhibition test

[0099] The fermented Lactobacillus mucilaginosus MQ10 after activation for 2 generations was inoculated into MRS solid medium (two lines located in the center of the medium, two parallel lines 2 cm long, 1.5 cm apart), cultured in a 37°C incubator for 48 hours, and then 15 mL of PDA medium mixed with mold spore suspension was poured onto the MRS medium after 48 hours of culture. The culture dish containing the double-layer medium was cultured at 25°C for 5 days.

[0100] The preparation method of the PDA solid culture medium is as follows: potatoes (200 g / L) are washed, peeled and cut into small pieces. The potato pieces are placed in a pot, 1000 ml of distilled water is added, and the potatoes are boiled for 25 minutes until the potatoes become soft. The filtrate is filtered through gauze, and the filtrate is collected and supplemented with distilled water to 1000 ml, and glucose (20 g / L) and agar powder (15 g / L) are added.

[0101] The inhibitory effects of fermented Lactobacillus mucilaginosus MQ10 on Penicillium expansum (bio-25700), Botrytis cinerea (bio-81601), and Aspergillus carbonarius (bio-52150) are as follows Figure 1 , Figure 2 , Figure 3 As shown in the figure, the fermented mucus Lactobacillus MQ10 has excellent inhibitory ability against Penicillium expansum, Botrytis cinerea, and Aspergillus carbonarius, and has a broad-spectrum antibacterial activity, and has an inhibitory effect on a variety of pathogenic fungi.

[0102] Example 3 Safety evaluation of fermented Lactobacillus mucilaginosus MQ10

[0103] aHemolytic activity test

[0104] After activating the fermented mucus Lactobacillus MQ10, use an inoculation loop to dip a small amount of culture solution and draw four lines on the Columbia blood agar plate. Place the blood plate at 37°C for incubation, and observe whether there is a lytic ring around the colony after 48 hours. If there is no hemolytic ring, it indicates that the strain has γ hemolytic activity, that is, it is non-hemolytic and safe for the human body. Staphylococcus aureus is used as the positive control strain.

[0105] The blood plate streaked with fermented Lactobacillus mucilaginosus MQ10 showed no hemolytic ring, indicating that it was non-hemolytic. Figure 4 express.

[0106] b Antibiotic sensitivity test

[0107] The sensitivity of the lactic acid bacteria to different antibiotics was determined on MRS solid medium by the disc diffusion method. 100 μL of bacterial solution was evenly spread on sterile MRS solid medium, and the solution was absorbed for 30 minutes and then cultured at 37°C for 48 hours. After culture, the size of the inhibition zone (mm) was measured, and the resistance of the lactic acid bacteria to be tested was classified according to the CLSI2021 parameters. Lactobacillus rhamnosus CICC6001 was used as the positive control strain.

[0108] The antibiotic sensitivity test (Table 2) showed that Lactobacillus mucosa fermenter MQ10 had a certain resistance to sulfamethoxazole and ciprofloxacin, but was sensitive to tetracycline, chloramphenicol, ceftriaxone, gentamicin, lincomycin, ampicillin, penicillin and erythromycin.

[0109] Table 2 Antibiotic susceptibility of Lactobacillus mucilaginosus MQ10

[0110]

[0111]

[0112] Note: R-resistant; I-intermediately sensitive; S-sensitive.

[0113] Example 4 Evaluation of the probiotic properties of fermented Lactobacillus mucilaginosus MQ10

[0114] a Evaluation of hydrophobicity, self-aggregation ability and co-aggregation ability with pathogenic bacteria

[0115] (1) Hydrophobicity evaluation:

[0116] After the fermented Lactobacillus mucilaginosus MQ10 was activated for 2 generations, the culture solution was centrifuged at 10000r / min and 4°C for 15 minutes, and the bacterial cells were collected. The concentration of the bacterial suspension was adjusted to 1×10 9 CFU / mL, and measure its absorbance at 600nm, which is recorded as A0. Take 2mL of bacterial suspension and add the same volume of xylene / ethyl acetate / chloroform, mix for 5min, let it stand at room temperature for 1 hour, then take the lower aqueous phase and measure its absorbance at 600nm, which is recorded as A t The positive control strain was (Lacticaseibacillus rhamnosus) CICC6001, and the surface hydrophobicity was calculated (the calculation method is shown in Formula 1).

[0117]

[0118] (2) Self-aggregation evaluation:

[0119] Take 5 ml of the resuspended bacterial solution, and place it at 37°C for 2 hours, 4 hours, 6 hours, and 24 hours, then take 200 μL of the bacterial solution and add it to a 96-well plate. Use an ELISA reader to measure the OD600nm value at different times, and record it as A2. Substitute it into the formula to calculate the self-aggregation (the calculation method is shown in Formula 2).

[0120]

[0121] (3) Evaluation of co-aggregation ability:

[0122] After the fermented Lactobacillus mucilaginosus MQ10 was activated for 2 generations, the culture solution was centrifuged at 10000r / min and 4°C for 15 minutes, and the bacterial cells were collected. The concentration of the bacterial suspension was adjusted to 1×10 9 CFU / mL, and its absorbance at 600nm was measured and recorded as A1. After the Escherichia coli and Staphylococcus aureus single colonies were activated for 2 generations, the culture medium was centrifuged at 10000r / min and 4℃ for 15 minutes, and the bacteria were collected. The concentration of the bacterial suspension was adjusted to 1×10 with sterile saline. 9CFU / mL, and measure its absorbance at 600nm, which is recorded as A2. Take 2mL of bacterial suspension and 2mL of Escherichia coli suspension, mix for 5min, and culture in a 37℃ incubator for 5 hours. Take the supernatant and measure its absorbance at 600nm, which is recorded as A mix , substitute into the formula to calculate the copolymerization of lactic acid bacteria strains (the calculation method is shown in Formula 3).

[0123]

[0124] The results of the hydrophobicity evaluation, self-aggregation ability evaluation, and co-aggregation ability evaluation with pathogenic bacteria of fermented Lactobacillus mucilaginosus MQ10 are shown in the figure below. Figure 5 , Figure 6 , Figure 7 As shown. The test results show that under the action of ethyl acetate and xylene, the hydrophobicity of MA10 can reach 28.36% and 16.70% respectively, which is significantly stronger than CICC6001. When the aggregation time is 2h, 4h and 6h, the self-aggregation ability of MQ10 is 12.32%, 9.84% and 14.99% respectively, which is stronger than CICC6001, and the difference in self-aggregation at 6h is statistically significant. Compared with CICC6001, MQ10 has a stronger co-aggregation ability against enteroaggregative Escherichia coli, which is 17.99%.

[0125] bTolerance evaluation

[0126] (1) Acid tolerance evaluation:

[0127] HCl (1 mol / L) and NaOH (1 mol / L) were used to adjust the pH value of the MRS liquid culture medium to 2.5, 3.5, 4.5, and 9.0. The activated fermented mucus lactobacillus MQ10 was inoculated in culture media with different pH values ​​according to an inoculation amount of 4% of the volume of the MRS liquid culture medium, and the MRS liquid culture medium without pH adjustment was used as a control. After culturing at 37°C for 0, 6, 12, 18, and 24 hours, the optical density of the bacterial solution at a wavelength of 600 nm was measured.

[0128] (2) Bile salt tolerance evaluation:

[0129] The activated fermented Lactobacillus mucosa MQ10 was inoculated in MRS liquid medium containing 0.3% and 0.5% ox bile salts at an inoculum volume of 4% of the MRS liquid medium, and the MRS liquid medium without bile salts was used as a control. After culturing at 37°C for 0, 6, 12, 18, and 24 hours, the optical density of the bacterial solution at a wavelength of 600 nm was measured.

[0130] (3) Sodium chloride tolerance evaluation:

[0131] The activated fermented mucus Lactobacillus MQ10 was inoculated into MRS liquid culture medium containing 3%, 5%, 8%, and 10% NaCl at an inoculation amount of 4% of the volume of the MRS liquid culture medium. The MRS liquid culture medium without sodium chloride was used as a control. After culturing at 37°C for 0, 6, 12, 18, and 24 hours, the optical density value of the bacterial solution at a wavelength of 600 nm was measured.

[0132] (4) Hydrogen peroxide tolerance evaluation:

[0133] After sterilization by filtration through a filter membrane, a 30% hydrogen peroxide solution was added to an MRS liquid culture medium, so that the final concentration of the hydrogen peroxide solution in the culture medium was 1.0 mmol / L, 2.0 mmol / L, and 3.0 mmol / L, respectively. The activated fermented mucus lactobacillus MQ10 was inoculated in culture mediums with different hydrogen peroxide concentrations according to an inoculation amount of 4% of the volume of the MRS liquid culture medium, and an MRS liquid culture medium without hydrogen peroxide was used as a control. After culturing at 37°C for 0, 6, 12, 18, and 24 hours, the optical density of the bacterial solution at a wavelength of 600 nm was measured.

[0134] (5) Temperature tolerance evaluation:

[0135] The activated fermented mucus Lactobacillus MQ10 was inoculated into MRS liquid culture medium at an inoculation amount of 4% of the volume of the MRS liquid culture medium and cultured in incubators at 25° C., 37° C., and 40° C., respectively. The optical density of the bacterial solution at a wavelength of 600 nm was measured 0, 6, 12, 18, and 24 hours after inoculation.

[0136] The acid tolerance evaluation, bile salt tolerance evaluation, sodium chloride tolerance evaluation, hydrogen peroxide tolerance evaluation, and temperature tolerance evaluation of fermented Lactobacillus mucilaginosus MQ10 were as follows: Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 As shown. The test results show that the fermented mucus Lactobacillus MQ10 grows faster and more in acidic conditions, and has a certain tolerance to bile salts and sodium chloride. MQ10 has good tolerance to hydrogen peroxide, and grows well at various hydrogen peroxide concentrations after 24 hours, and can tolerate 3.0mmol / L hydrogen peroxide, with good antioxidant capacity; MQ10 grows vigorously at 45℃, indicating that it has good tolerance to temperature, which is conducive to its application in real industry.

[0137] Example 5 Application of fermented mucus Lactobacillus MQ10 in the preservation of cherry fruits at room temperature

[0138] 1. Preparation of bacterial suspension

[0139] The fermented Lactobacillus mucilaginosus MQ10 was activated for 2 generations, and then inoculated into MRS liquid medium at an inoculum volume of 4% of the volume of the MRS liquid medium, cultured overnight at 37°C, and then centrifuged at 4°C, 10,000 rpm, and 15 min. The bacteria were washed twice with sterile saline and resuspended in sterile saline to adjust the concentration to 10 9 CFU / mL, stored at 4℃ for future use.

[0140] 2. Sample processing

[0141] Cherries with uniform size, maturity, and no disease or mechanical damage were selected, rinsed with distilled water, and then soaked in 1% (v / v) sodium hypochlorite for 2 minutes, picked up, and air-dried on the workbench. The experiment set up three groups: CK group (cherries were only disinfected); NaCl group (the disinfected and air-dried cherries were soaked in sterile saline for 2 minutes); LAB group (the disinfected and air-dried cherries were soaked in MQ10 bacterial suspension for 2 minutes). Cherries in each treatment group were soaked and air-dried until there was no moisture on the surface. Each group of 54 cherries was packed in 3 perforated and sterilized polyethylene lunch boxes and stored at room temperature for 8 days. Samples were taken on the 0th, 2nd, 4th, 6th and 8th days of storage for relevant tests.

[0142] 3. Post-harvest index testing of cherries

[0143] aDetermination of fruit weight loss rate and corruption rate

[0144] Weight loss rate: The cherries were weighed immediately after being air-dried with different treatments and recorded as W0. They were weighed at other storage time points and recorded as W. t , the weight loss rate is calculated based on (the calculation method is shown in Formula 4).

[0145] Weight loss rate (%) = (W0-W t ) / W0×100% Formula 4

[0146] Corruption rate: Observe all cherries at each sampling time point (calculation method is shown in Formula 5).

[0147] Decay rate = number of fruits showing rot symptoms / total number of fruits × 100% Formula 5

[0148] b Determination of fruit stem browning rate

[0149] Pedicel browning rate: All cherries at each sampling time point were observed (calculation method is shown in formula 6). Pedicels were graded from 0 to 3 according to the browning area covered on the pedicel surface, where 0 was no browning, 1 was slight (≤25% browning); 2 was moderate (26-50% browning); 3 was severe (≥50% browning).

[0150] Browning index calculation formula: Browning index = ∑ (browning grade × number of stems of each grade) / 3 × total number of stems in each treatment.

[0151] Formula 6

[0152] c Determination of fruit firmness

[0153] Hardness: Use the GY-1 fruit hardness tester to measure the hardness of cherries. Pierce the cherry once with a uniform and constant force, the puncture depth is 1 cm, and the probe diameter is 5 mm. The maximum force (N) measured is the hardness.

[0154] dTotal soluble solids (TSS) content and titratable acid determination

[0155] 30g of cherry juice was squeezed from each group and centrifuged to obtain clarified cherry juice for the determination of total soluble solids content. Total soluble solids (TSS) content: TSS content was determined using a handheld refractometer, expressed in °Birx. Calibrate it according to the instructions before use.

[0156] Titratable acid: homogenize 10 g of cherry pulp, dilute to 100 mL with CO2-free distilled water, filter, and titrate the filtrate with 0.01 mol / L NaOH standard solution. Use phenolphthalein as an indicator and titrate until the solution turns slightly red and does not fade within 30 seconds. Record the volume of NaOH consumed and use a pH meter to measure the pH of the mixed solution to determine the titration endpoint. The titration endpoint is a pH between 8.1 and 8.2 (the calculation method is shown in Formula 8).

[0157] Titratable acid (%) = V × c × (V1-V0) × f / (V s ×m)×100% Formula 8

[0158] Note: V is the total volume of sample extract (mL), V s is the volume of filtrate used for titration (mL), c is the sodium hydroxide concentration (mol / L), V1 is the volume of sodium hydroxide solution consumed in titrating the filtrate (mL), V0 is the volume of sodium hydroxide solution consumed in titrating distilled water (mL), m is the sample mass (g), and f is the conversion factor (g / mmol) = 0.067

[0159] eDetermination of antioxidant capacity index and lignin content in cherry fruit

[0160] The kit was used to detect the glutathione content, ascorbic acid content, total phenol content, flavonoid content, total anthocyanin content, DPPH free radical scavenging ability, total antioxidant capacity and lignin content in each treated cherry at each sampling time point.

[0161] 4. Experimental conclusion

[0162] Changes in quality indicators of cherry fruits during storage: The weight loss rate of cherries treated with fermented Lactobacillus mucilaginosus MQ10 suspension was significantly reduced ( Fig.13 ), MQ10 reduced the loss of fruit better. The corruption rate and stalk browning rate of cherries treated with bacterial suspension on the 8th day were 5.56% and 86.42% respectively ( Fig.14 , Fig.15 ), while the fruit corruption rate and stalk browning rate of the CK group on the 8th day were 11.11% and 89.82%, respectively, and the fruit corruption rate and stalk browning rate of the NaCl group on the 8th day were 11.11% and 98.15%, respectively, indicating that the treatment with bacterial suspension significantly reduced the fruit corruption rate and stalk browning rate, which helped to delay the fruit corruption process and reduce the corruption of cherries after harvest.

[0163] The fermented Lactobacillus mucilaginosus MQ10 suspension can significantly improve the firmness of cherries after the storage period and maintain a low level of total soluble solids content ( Fig.16 , Fig.17 ), which is beneficial to maintain the texture of the fruit, effectively slow down the aging of cherries during storage, significantly improve the quality of cherry fruits, delay the ripening process, and effectively extend the shelf life. During storage, the titratable acidity of cherry fruits showed a downward trend, which was due to the decrease in acidity caused by ripening. During the entire storage period, the cherry fruits treated with MQ10 maintained a high level of titratable acidity, indicating that it better maintained the quality of cherry fruits and extended the storage period ( Fig.18 ).

[0164] Cherry fruits treated with fermented Lactobacillus mucilaginosus MQ10 maintained high levels of glutathione during storage ( Fig.19 ) and reduced ascorbic acid content ( Fig. 20 ). Glutathione is an important antioxidant that works inside cells to protect them from oxidative damage, while reduced ascorbic acid is another powerful antioxidant that is essential for maintaining the freshness and nutritional value of the fruit.

[0165] The total phenolic content of cherry fruits treated with fermented Lactobacillus mucilaginosus MQ10 suspension was significantly higher than that of the two control groups ( Fig.21 In the later storage period (6 days and 8 days), the flavonoid content and total anthocyanin content of cherry fruits treated with MQ10 bacterial suspension were ( Fig. 22 , Fig.23 ) were significantly higher than those of the two control groups. During storage, the DPPH free radical scavenging capacity and total antioxidant capacity of the LAB group ( Fig.24 , Fig.25) were significantly higher than those in the two control groups. Total phenols, flavonoids, and total anthocyanins are all important phytochemicals with multiple biological activities such as antioxidant, anti-inflammatory, and anti-cancer. Flavonoids are a large class of polyphenolic compounds with powerful antioxidant and anti-inflammatory effects; anthocyanins are natural pigments that give fruits their color and also have antioxidant and health-promoting properties. These antioxidant indicators indicate that MQ10 treatment can provide antioxidant capacity for cherry fruits during storage, thereby helping to maintain human health.

[0166] The lignin content in cherry fruits treated with fermented Lactobacillus mucilaginosus MQ10 was significantly higher than that in the control group ( Fig.26 ), lignin is one of the main components of plant cell walls and does play an important role in maintaining the structure and hardness of fruits. It forms a strong cell wall structure by interweaving with cellulose and hemicellulose, providing mechanical support and protection for plants. At the same time, lignin also participates in the self-protection mechanism of plants in the plant body.

[0167] It can be seen from the above embodiments that the present invention provides a fermented mucus lactobacillus and its application. The Latin name of the fermented mucus lactobacillus is Limosilactobacillusfermentum, the preservation name is fermented mucus lactobacillus MQ10, the preservation location is China Center for Type Culture Collection, the preservation date is July 22, 2024, and the preservation number is CCTCC NO: M20241616. The strain has a broad-spectrum antibacterial effect and can inhibit the growth and reproduction of various microorganisms. It is made into a biological preservation product, which can reduce the corruption rate of food and the browning rate of fruit and vegetable stalks, maintain the content and hardness of soluble solids, slow down the aging and corruption rate of food during storage, and extend the storage period. This characteristic makes fermented mucus lactobacillus promising to become a green and environmentally friendly biological preservative, improving the quality and safety of food.

[0168] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fermented mucus lactobacillus, characterized in that The Latin name of the fermented mucus lactobacillus is Limosilactobacillusfermentum, the preservation name is fermented mucus lactobacillus MQ10, the preservation location is China Center for Type Culture Collection, the preservation date is July 22, 2024, and the preservation number is CCTCC NO: M 20241616.

2. Application of the fermented mucus lactobacillus according to claim 1 in inhibiting microbial growth.

3. The use according to claim 2, characterized in that: The microorganisms include one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Penicillium expansum, Botrytis cinerea, and Aspergillus carbonarius.

4. Use of the fermented mucus lactobacillus according to claim 1 in preparing a product that inhibits the growth of microorganisms.

5. The use according to claim 4, characterized in that: The microorganisms include one or more of Escherichia coli, Staphylococcus aureus, Salmonella, Penicillium expansum, Botrytis cinerea, and Aspergillus carbonarius.

6. The use according to claim 4, characterized in that: The product is a biological preservative.

7. A biological preservative, characterized in that: The biological preservative contains the fermented mucus lactobacillus according to claim 1.

8. The method for preparing the biological preservative according to claim 7, characterized in that: The steps include: (1) activating the fermented mucus lactobacillus to obtain an activated solution; (2) inoculating the activation solution into the culture medium, culturing and centrifuging to obtain bacterial cells; (3) Wash the cells and resuspend them to adjust the final concentration to 0.8×10 9 ~1.2×10 9 CFU / mL, and obtain the biological preservative.

9. The preparation method according to claim 8, characterized in that: The number of activations in step (1) is 1 to 3 generations; The inoculation amount of the activation solution in step (2) is 2% to 6% of the volume of the culture medium; The culture medium is MRS liquid culture medium; The culture time is 12 to 16 hours, and the culture temperature is 35 to 39° C.; The centrifugal temperature is 2-6°C, the centrifugal speed is 8000-12000rpm, and the centrifugal time is 10-20min; The washing method in step (3) is: washing the bacterial cells with sterile saline for 1 to 3 times; The resuspension method is: resuspending the washed bacteria in physiological saline.

10. The use of the biological preservative as claimed in claim 7 in fruit preservation, characterized in that: The fruit includes cherries.

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