TWI810868B - Use of leuconostoc fallax for mamufacturing anti-candida albicans and anti-staphylococcus aureus composition - Google Patents

Abstract

The present invention relates to a use of Leuconostoc fallaxfor manufacturing anti- Candida albicansand anti- Staphylococcus aureuscomposition, comprising applying a cultured medium of Leuconostoc fallaxto a subject needed to inhibit mycelial morphology transformation of Candida albicansand biofilm formation of Candida albicansand Staphylococcus aureus. The cultured medium of Leuconostoc fallaxis obtained by culturing Leuconostoc fallaxin a medium for 24-48 hours.

Description

Use of Leuconostoc sp. for the preparation of compositions for inhibiting Candida albicans and Staphylococcus aureus

The invention relates to the use of Leuconostoc sp. for the preparation of a composition for inhibiting Candida albicans and Staphylococcus aureus, which can effectively inhibit the mycelium transformation of Candida albicans and inhibit the formation of Candida albicans and Staphylococcus aureus biofilm.

Candida albicans ( Candida albicans ) belongs to polymorphic fungi, and its growth status can form hyphae or pseudohyphae, or exist in the form of yeast. Candida albicans can grow in a wide range of pH values and temperatures. It is one of the opportunistic pathogenic fungi. It usually lives in the mouth, skin, mucous membranes, digestive tract, vagina and other organs of the human body. When the human body is immune When the force is reduced, Candida albicans can reproduce and develop in large numbers, and transform into hyphae to invade the human body and cause clinical symptoms, including oral infection, esophageal or mucous membrane infection, and in severe cases, it will cause invasive or systemic infection, and even cause death . In addition, Candida albicans can form biofilm (biofilm) and attach to the surface of solid media, thereby increasing the resistance to drugs, so it is quite difficult to treat.

Staphylococcus aureus ( Staphylococcus aureus ) is also an opportunistic pathogen. It is a Gram-positive bacterium that usually exists in human skin, hair, nasal cavity, throat and other mucous membranes and feces, and can be found on purulent wounds Large numbers of Staphylococcus aureus; Staphylococcus aureus is currently the leading cause of hospital and community infections, and drug-resistant Staphylococcus aureus has posed a huge threat to the healthcare system.

Recent studies have found that when Candida albicans forms a biofilm, Staphylococcus aureus can adhere to the surface of the Candida albicans biofilm and form colonies, and then form a special biofilm structure, and at the same time increase Candida albicans and Staphylococcus aureus Tolerance to drugs, that is, during co-infection, Candida albicans will increase the chance of Staphylococcus aureus invading mucosal tissue and causing systemic infection in patients; therefore, how to simultaneously inhibit the infection of two opportunistic pathogens, important research direction.

Today, the inventor is in view of the fact that there is no effective method for simultaneously inhibiting Candida albicans and Staphylococcus aureus at the same time, so it is a tireless spirit, assisted by its rich professional knowledge and many years of practical experience, and researches accordingly Created the present invention.

The main purpose of the present invention is to provide a use of Leuconostoc fallax for the preparation of compositions for inhibiting Candida albicans and Staphylococcus aureus subsp.aureus , which is to use Leuconostoc fallax Bacteria culture solution is administered to a desired individual to inhibit Candida albicans mycelium morphological transformation and biofilm formation, as well as inhibit Staphylococcus aureus growth and biofilm formation; wherein the bacterium culture solution is selected from ATCC 700006 strain Leuconostoc was cultured in YSB medium at pH 6 to obtain a mixed bacterial solution, and after culturing the mixed bacterial solution at 30°C for 24 hours, the supernatant was collected by centrifugation, and then the supernatant was filtered to obtain the Leuconostoc culture medium.

In one embodiment of the present invention, the Leuconostoc culture solution contains organic acid and hydrogen peroxide (H ₂ O ₂ ).

In one embodiment of the present invention, the Leuconostoc culture solution inhibits the production of Aspartic Proteinase by Candida albicans.

In one embodiment of the present invention, the Leuconostoc culture solution inhibits the growth of Bacillus cactus, Escherichia coli, Salmonella and Pseudomonas aeruginosa.

Thereby, the Leuconostoc culture solution of the present invention can inhibit the hyphae transformation of Candida albicans and simultaneously inhibit the biofilm formation of Candida albicans and Staphylococcus aureus, and can be applied to Candida albicans and Staphylococcus aureus. Symptoms of bacterial co-infection, and Leuconostoc is a probiotic with extremely low toxicity to organisms, and is less likely to produce side effects or drug resistance.

The first picture: the optimal growth temperature and pH value test analysis chart of Leuconostoc.

The second picture: the analysis chart of the inhibition of the growth of Candida albicans and Staphylococcus aureus by Leuconostoc.

The third picture: the analysis chart of the inhibition of Candida albicans to produce SAP by Leuconostoc.

Figure 4: Analytical diagram of hyphal transformation of Candida albicans inhibited by Leuconostoc (1).

Figure 5: Analytical diagram of hyphal transformation of Candida albicans inhibited by Leuconostoc (2).

Figure 6: Analysis of the inhibition of Candida albicans and Staphylococcus aureus biofilm formation by Leuconostoc.

Figure 7: Analysis of bile-salt tolerance and salt-tolerance of Leuconostoc.

Figure 8: Electrophoresis analysis chart of extracellular protein expression of Leuconostoc.

The purpose of the present invention and the advantages of its structure and function will be described based on the structure shown in the following drawings with specific examples, so that the review committee can have a deeper and more specific understanding of the present invention.

In the present invention, a kind of leuconostoc fallax ( Leuconostoc fallax ) is used to prepare the purposes of inhibiting candida albicans and staphylococcus aureus composition, and it is to apply a leuconostoc fallax culture solution to a desired individual, to inhibit candida albicans Candida hyphae morphological transformation and biofilm formation; Leuconostoc culture solution is obtained by culturing Leuconostoc in a culture medium for 24 to 48 hours; Leuconostoc culture solution can be used for culturing The supernatant liquid or the culture solution containing Leuconostoc cells, in addition, the Leuconostoc culture solution contains organic acid and hydrogen peroxide, and the pH value is between pH3~pH5; thus, the present invention Leuconostoc or its culture solution can inhibit the mycelial transformation of Candida albicans, and can also inhibit the formation of biofilms between Candida albicans and Staphylococcus aureus at the same time; and Leuconostoc is a kind of probiotics, with biological toxicity Low, less likely to produce side effects or cause drug resistance.

In addition, the scope of practical application of the present invention can be further proved by the following specific examples, but it is not intended to limit the scope of the present invention in any form.

Leuconostoc fallax used in this test was purchased from the American Type Culture Collection (America Type Culture Collection), and the Leuconostoc fallax purchased was numbered ATCC 700006; again, the following examples used Candida albicans , Staphylococcus aureus subsp.aureus , Escherichia coli , Bacillus cereus , Pseudomonas aeruginosa , and Salmonella choleraesuis strains were purchased From the Bioresource Conservation Center (BCRC) of the Food Industry Development Research Institute, the strain numbers are listed in Table 1 below.

In addition, the following experiments were repeated more than 2 times, and the differences between groups were analyzed using unpaired student t-test (student uhpaired t-test). Or <0.01 and <0.001, it means that there is a statistical difference from the control group.

1. The culture condition test of Leuconostoc

(1), cultivation temperature

Take 0.01~0.1mL Leuconostoc (hereinafter referred to as L.fallax ) seed broth to MRS medium (De Mah, Rogosa and Sharpe broth) to obtain a culture broth, and make L.fallax in the culture broth The final concentration was 1 X 10 ⁶ CFU/mL, and then cultured at 25°C, 30°C or 37°C respectively, at the beginning of the culture (0 hour) and 24 hours after the culture, the measured The absorbance value at a wavelength of 595nm (abbreviated as OD ₅₉₅ ) was used to evaluate the growth of L.fallax .

Please refer to the first picture (A), the OD ₅₉₅ of the L.fallax culture cultured at 25°C is 0.0989±0.00551, the OD ₅₉₅ of the 30°C group is 0.3356±0.0005, and the 37°C group The OD ₅₉₅ was 0.0804±0.00473, that is, the growth condition of L.falla x cultured at 30°C was the best.

(2) The pH value of the culture medium

Mix a 24-hour-cultured L.fallax seed solution evenly, distribute it evenly among three centrifuge tubes, and then centrifuge at 8000rpm for 10 minutes, remove the supernatant and keep the bacteria; add 0.1M phosphate ( Phosphate) buffer solution (pH 7.0, hereinafter referred to as PBS buffer) to wash the bacteria twice, and evenly back-lyse the bacteria with MRS medium of pH 4.0, pH 6.0 and pH 8.0 respectively to obtain a lysate, and measure each OD ₅₉₅ , and then take 0.01~0.1mL of back-lysed bacteria solution and cultivate them in three kinds of MRS medium with the above three pH values to obtain a mixed bacteria solution. The concentration of L.fallax in the mixed bacteria solution is 1 X 10 ⁶ CFU/ mL, and then measure the OD ₅₉₅ of the mixed bacterial solution of each group at 30°C and aerobic culture for 24 hours.

Please refer to the first figure (B), the OD ₅₉₅ of L.fallax cultured at pH 6.0 is 0.669±0.011, and the OD ₅₉₅ of L.fallax cultured at pH 8.0 is 0.674±0.050. There are obvious differences; according to the results in the first figure, L.fallax has the best growth condition at 30°C, and the optimum pH value for cultivation is between pH 6.0-8.0.

2. Bacteriostasis test of Concretomonocera

(1) Preparation of Leuconostoc culture supernatant

First measure the OD ₅₉₅ of the L.fallax seed liquid cultured for 24 hours, then take 0.01~0.1mL of the seed liquid and culture it in 5.9~5.99mL of YSB medium with a pH of 6.0 to obtain a mixed bacterial liquid. The concentration of L.fallax is 1 X 10 ⁶ CFU/mL, and the final volume of the mixed bacterial solution is 6 mL. After the mixed bacterial solution is incubated at 30°C for 24 hours, the bacterial solution is centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected , and then filter the supernatant with a 0.45 μm filter membrane to obtain a culture solution of L. fallax (hereinafter referred to as LF culture solution), and store the filtrate at 4°C.

(2), inhibition of Candida albicans and Staphylococcus aureus growth test

In the experiment of testing the inhibitory effect of Candida albicans, first add 0.1mL seed solution of Candida albicans or Staphylococcus aureus cultured overnight at a concentration of 1 X 10 ⁶ CFU/mL to a 96-well culture plate, and then add 0.1mL of YSB medium or LF culture solution prepared above, cultured at 37°C for 24 hours, the group added with YSB medium was used as the control group, and the group added with LF culture solution was used as the experimental group; after 24 hours, take 100 μL of the above The cultured bacterial solution is serially diluted and then mixed evenly with the heated liquid TSA medium, then poured into the culture plate (culture plate) and allowed to stand to solidify into a culture plate, and then culture the culture plate at 37°C After 16-24 hours, calculate the number of colonies on the culture plate and multiply the dilution factor of the seed liquid to represent the total number of bacteria in the cultured liquid, compare the results of the experimental group with the control group, and calculate the LF culture The survival rate of Candida albicans or Staphylococcus aureus in the liquid group. The calculation formula of the survival rate is as follows: Survival rate (%)=(total number of bacteria in the experimental group/total number of bacteria in the control group) X 100%

Please refer to the second picture (A) and the second picture (B). Compared with the control group, the survival rate of Candida albicans in the group (LFs) cultured with LF medium decreased to 12.4±0.0038%, while the golden yellow The survival rate of Staphylococcus decreased to 1.9±0.00591%; see the second figure (C) again, in the co-cultivation group of Candida albicans and Staphylococcus aureus, the LF medium group (LFs), the survival rate of the two bacteria also decreased to 15.67±0.00471%; that is, the LF culture solution in this case can indeed effectively inhibit the growth of Candida albicans and Staphylococcus aureus.

(3), inhibition of Candida albicans and Staphylococcus aureus adhesion test

First add 10 mg/mL mucin to a 96-well culture plate, place it at 4°C for 24 hours, and absorb the supernatant that does not adhere to the culture plate; take 0.1 mL of 1 X 10 ⁶ CFU/mL for overnight culture Candida albicans or Staphylococcus aureus seed bacteria solution was added to the culture plate coated with mucin, and then 0.1 mL of YSB medium and ^0.1 mL of L. Bacteria-containing culture solution of fallax or 0.1mL LF culture solution, and cultivated at 37°C for 3 hours; remove the culture supernatant, and wash away the non-adhered bacteria with 0.1M PBS buffer (pH7.0), Then take the bacteria on the mucin, serially dilute it, mix it evenly with the heated liquid TSA medium, pour it into a culture plate and let it solidify into a culture plate, and then culture the culture plate at 37°C for 16- 24 hours; after culturing, calculate the number of colonies on the culture plate, and multiply the dilution factor to represent the total number of bacteria on the mucin, and calculate the adhesion inhibition rate; the group added with YSB medium is the control group, and the group added with L. The fallax -containing bacteria culture medium group is called LFc group, and the LF culture medium group is called LFs group. The formula for calculating the adhesion inhibition rate is as follows: adhesion inhibition rate (%)=[(total bacterial count in the control group/total bacterial count in the experimental group)/total bacterial count in the control group]X 100%

Please refer to Table 1. In the LFc group, the adhesion inhibition rate of Candida albicans was 81.28±4.61%, while the adhesion inhibition rate of Staphylococcus aureus was 63.27±5.43%, both of which had statistically significant differences compared with the control group ( p <0.01); and in the LFs group, the adhesion inhibition rate of Candida albicans was 50.47±4.08% ( p <0.01), and the adhesion inhibition rate of Staphylococcus aureus was 81.28±0.72% ( p <0.001). significantly different from the control group.

(4) Test of inhibiting the production of Aspartic Proteinase by Candida albicans

Candida albicans will produce secreted aspartic proteinase (Secreted Aspartic Proteinase, hereinafter referred to as SAP) to help it invade the organism and colonize the host's tissue, and avoid the host's immune response; SAP will also decompose extracellular As the source of its nitrogen source, it is believed that the ability of Candida albicans to produce SAP has a positive correlation with its pathogenicity.

The test method for the secretion of SAP by Candida albicans is briefly described as follows: MRS medium (hereinafter referred to as the control group) and 3.0 mL of 1 X 10 ⁶ CFU/mL or 1 X 10 ⁷ CFU/mL overnight culture L.fallax bacteria Albicans with 3.0mL concentration of 1 X 10 ⁷ CFU/mL were co-cultured in a 96-well culture plate and reacted at 25°C for 1 hour. The group added with MRS medium was the control group, and the added concentration was 1 X 10 ⁶ CFU/mL L.fallax bacterial solution is called 10 ⁶ group, and the addition of 1 X 10 ⁷ CFU/mL L.fallax bacterial solution is called 10 ⁷ group; Volume, PBS buffer solution containing 2 (v/v)% bovine serum albumin (BSA) and 0.1% (w/w) glucose (Glucose); after acting at 37°C for 6 hours, draw the supernatant to SAP enzyme-linked immunosorbent assay kit (Mouse SAP enzyme-linked immunosorbent assay kit, MyBioSource) was used to measure the SAP content in the supernatant.

Please refer to the third figure, when Candida albicans was co-cultured with L.fallax , compared with the control group, the SAP production rate of Candida albicans decreased significantly. In the ¹⁰⁶ group, the SAP production rate of Candida albicans decreased to 77.19±8.51%, which was significantly different from the control group ( p <0.01); in the ¹⁰⁷ group, the SAP production rate of Candida albicans decreased to 50.72±6.89%, which was also significantly different from the control group ( p <0.001) .

(5), inhibition of Candida albicans morphological transformation

Candida albicans has yeast form and hyphae form. At present, it is believed that the hyphae conversion ability of Candida albicans is directly related to its ability to cause disease, and the growth of its mycelium is also regarded as the main basis for judging its pathogenicity. one. In this experiment, the effect of LF culture medium on the transformation of Candida albicans hyphae was tested. Take 0.01mL of Candida albicans cultured overnight and add it to 0.99mL of 0.1M PBS buffer (pH 7.0) to obtain a mixture. The final concentration of Candida albicans in the mixture is 1 X 10 ⁶ CFU /mL, then centrifuge the mixture at 4°C at a speed of 4000rpm; remove the supernatant and keep the cells, then add 1.0mL of YSB medium or LF culture medium to mix the cells evenly to obtain a culture solution, and Place the cultured bacteria solution in a 96-well culture plate and act at 37°C for 1 hour. The group added with YSB medium is called the control group, and the group added with LF culture medium is called the LFs group; then add 0.1 mL of RPMI-1640 medium was mixed evenly and then cultured at 37°C; 24 hours and 48 hours after culture, use an optical microscope to observe, and calculate the ratio of the yeast-type thallus and hyphae of Candida albicans.

In another experiment, the Candida albicans cultured overnight was mixed with the Staphylococcus aureus cultured overnight to obtain a mixed seed culture, and then mixed with 0.1M PBS buffer (pH 7.0) The final volume of the seed bacterial liquid was adjusted to 1 mL to obtain a mixed bacterial liquid in which the concentration of Candida albicans was 1 X 10 ⁶ CFU/mL, and the concentration of Staphylococcus aureus was also 1 X 10 ⁶ CFU/mL. mL; centrifuge the mixed bacterial solution at 4°C at a speed of 4000rpm; remove the supernatant and keep the bacterial cells, then add 1mL of YSB medium or LF culture medium to mix the bacterial cells evenly to obtain a lysate, and then The back-lysed bacteria solution was added to a 96-well culture plate and reacted at 37°C for 1 hour. The group added with YSB medium was called the control group, and the group added with LF culture medium was called the LFs group; then 0.1 mL of RPMI was added to the back-lysed bacteria solution. -1640 medium, mixed evenly and cultured at 37°C for 24 hours; finally observed with an optical microscope to calculate the proportion of yeast-like cells and hyphae of Candida albicans.

The fourth picture (A) is an optical microscope observation photo. Compared with the control group, the mycelium production of Candida albicans in the LFs group was significantly reduced; please refer to the fourth picture (B) again, which is the statistics of the number of mycelium Figure, in the control group, the hyphae number was 785±29.70 hyphae no/mL 24 hours after culture, and 48 hours after culture The number of hyphae increased to 1410±72.71 hyphae no/mL; however, in the LFs group, the number of mycelia was 2.5±0.28 hyphae no/mL at 24 hours after culture, and 32.5±1.34 hyphae no/mL at 48 hours after culture, not only The total number of hyphae is much lower than that of the control group, and the range of mycelium increase is also much lower than that of the control group.

Please refer to the fifth figure again, in the group of Candida albicans cultured alone (Candida albicans group), the proportion of its hyphae is 1.68±0.0104%, while the proportion of yeast-type thalli is 98.32%; Candida albicans In the co-cultivation group with Staphylococcus aureus, the mycelial proportion of Candida albicans increased to 7.43±0.0305%, suggesting that Staphylococcus aureus had a beneficial effect on the mycelium conversion of Candida albicans; however, the two strains Co-cultivated in LF culture medium, the proportion of Candida albicans hyphae in this group is close to 0%, that is, LF culture medium can effectively inhibit the growth of Candida albicans from yeast form in the environment where the two strains grow together. The cells transformed into mycelial form.

(6), inhibition of Candida albicans biofilm formation

Take 0.002mL of Candida albicans cultured overnight and mix it with 0.098mL of YSB medium or LF culture medium to obtain a mixed bacterial solution. The concentration of Candida albicans in the mixed bacterial solution is 1 X 10 ⁶ CFU/mL, The mixed bacterial solution was reacted at 37°C for 1 hour, and the group added with YSB medium was called the control group, and the group added with LF medium was called the LFs group; then 0.1 mL of RPMI-1640 medium was added to the mixed bacterial solution, mixed evenly, and then incubated at 37°C Incubate for 24 hours; carefully remove the medium, wash the cells with 0.1M PBS buffer (pH 7.0), remove the PBS buffer and stain with 0.05% crystal violet stain for 10 minutes at room temperature to make crystal violet The dye stains the formed biofilm; remove the crystal violet dye, then add 95% ethanol to completely dissolve the stained biofilm, and then measure the OD ₅₉₅ value of the dissolved solution to calculate the biofilm formation rate.

In another experiment, the overnight culture of Candida albicans and the overnight culture of Staphylococcus aureus were mixed with YSB medium or LF culture medium to obtain a mixed bacterial solution with a volume of 0.098mL , the concentration of Candida albicans in the mixed bacterial solution is 1 X 10 ⁶ CFU/mL, and the concentration of Staphylococcus aureus is also 1 X 10 ⁶ CFU/mL. The mixed bacterial solution is reacted at 37°C for 1 hour, and YSB medium is added to it The group is the control group, and the group added with LF culture medium is called the LFs group; then add 0.1mL RPMI-1640 medium to the mixed bacterial solution, mix well and incubate at 37°C for 24 hours; carefully remove the medium, and then use 0.1M PBS Wash the cells with buffer (pH 7.0), remove the PBS buffer, and then stain with 0.05% crystal violet dye at room temperature for 10 minutes, so that the crystal violet dye will stain the formed biofilm; remove the crystal violet stain Then add 95% ethanol to completely dissolve the stained biofilm, then measure the OD ₅₉₅ of the dissolved solution to calculate the biofilm formation rate. The calculation formula of biofilm formation rate is as follows: formation rate (%)=(OD ₅₉₅ of LFs group / OD ₅₉₅ of control group) X 100%

Please refer to the sixth figure (A). In the test of culturing Candida albicans alone, when the biofilm formation rate of Candida albicans in the control group is defined as 100%, the biofilm formation rate of Candida albicans in the LFs group will drop to 17.00±0.0513% ; Also, see the sixth figure (B), in the test of culturing Staphylococcus aureus alone, compared with the control group, the biofilm formation rate of Staphylococcus aureus in the LFs group also decreased to 26.34±0.0246%; see also the sixth Figure (C), in the co-cultivation test of Candida albicans and Staphylococcus aureus, compared with the control group, the biofilm formation rate of the LFs group also decreased to 15.60±0.0506%; In the test of strains cultured alone or co-cultivated, LF medium can effectively inhibit the formation of biofilm.

(7) Analysis of the ability to inhibit opportunistic pathogens

Take 0.1mL of Bacillus cectus, Escherichia coli, Salmonella or Pseudomonas aeruginosa cultured overnight at a concentration of 1 X 10 ⁶ CFU/mL, culture them in 96-well culture plates, and then add 0.1mL of YSB medium Or LF culture solution, cultivated at 37°C for 24 hours, the group added with YSB medium was called the control group, and the group added with LF culture solution was called the LFs group; the cultured bacterial solution was serially diluted and mixed with the heated liquid Mix the TSA medium evenly, then put the mixed bacterial solution into a culture plate and let it solidify into a culture plate, then culture the culture plate at 37°C for 16-24 hours, then count the number of colonies on the culture plate, and return Multiply the dilution factor to calculate the total number of bacteria in each group, and then calculate the growth inhibition rate of the LF culture solution to the strains in each group. The calculation formula of growth inhibition rate is as follows: Growth inhibition rate (%)=[(total number of bacteria in control group-total number of bacteria in LFs group)/total number of bacteria in control group] X 100%

Please refer to Table 2 for the calculation results of the total number of bacteria in each group. In the test of four kinds of bacteria, the number of bacteria in the LFs group was far lower than that of the control group, and the growth inhibition rate was calculated. The growth of Escherichia coli in LF culture solution The inhibition rate was 99.77±0.0051%, 99.83±0.0011% for Bacillus cactus, 99.04±0.0010% for Salmonella, and 99.99±0.0003% for Pseudomonas aeruginosa.

3. Analysis of characteristics of Leuconostoc

(1) Bile salt resistance test

Take 0.003mL of L.fallax seed culture cultured overnight, and culture with 0.997mL containing 3% bile salts at 37°C for 1 hour and 2 hours respectively, then serially dilute the bacteria liquid, and culture the culture plate at 30°C for 16-24 hours , and then calculate the number of colonies to calculate the total number of bacteria L.fallax , and calculate its survival rate. The formula for calculating the survival rate is as follows: Survival rate (%) = (total number of bacteria after culture/total number of bacteria after 0 hours of culture) X 100%

Please refer to the seventh figure (A), after L.fallax was cultured in the environment of 3% bile salts for 1 hour, there was still a high survival rate of 95.00±4.30%; while cultured in the environment of 3% bile salts, L.fallax still remained The survival rate was 60.00±3.75%, which indicated that L.fallax has the characteristic of bile salt tolerance.

(2) Salt tolerance test

Inoculate mL of L.fallax bacteria solution into MRS medium without adding sodium chloride (NaCl) (control group), MRS medium containing 3wt% sodium chloride, or MRS medium containing 6wt% sodium chloride, Incubate at 37°C for 24 hours. Serially dilute the culture plate and incubate at 37°C for 16-24 hours. Count the colonies on the culture plate to calculate the total number of bacteria in each group to evaluate and calculate the amount of salt added (sodium chloride) environment, the survival rate of L.fallax . The formula for calculating the survival rate is as follows: Survival rate (%)=(total number of bacteria cultured in the group containing salt medium/total number of bacteria in the control group) X 100%

Please refer to the seventh figure (B). Compared with the control group, the survival rate of L.fallax cultured in 3% saline environment was 92.58±7.21%, which was not much different from the control group; % saline environment, the survival rate of L.fallax was 2.45±0.47%, significantly lower than that of the control group.

(3), analysis of LF culture medium

(1), pH value

Take mL of activated L. fallax cultured overnight, culture it in mL YSB medium with pH 6.0, and incubate at 30°C for 24 hours; centrifuge the bacterial liquid at 8000rpm for 10 minutes, and the supernatant, and then The supernatant was filtered with a 0.45 μm filter membrane to obtain the culture solution of L. fallax (hereinafter referred to as the LF culture solution), and the pH value of the LF culture solution was measured; the measurement results showed that the pH value of the LF culture solution was 3.81 ±0.19, indicating that L.fallax can produce a large amount of organic acids after cultivation.

(2), hydrogen peroxide (H ₂ O ₂ )

Take the above LF culture solution, mix with 5 μL of horseradish peroxidase (Horseradish Peroxidase) and 5 μL of 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine ) and mix well, then measure the absorbance value (OD650) of the mixed solution at 650nm wavelength. In this test, 30 v/v% hydrogen peroxide is used as a standard to calculate the concentration of hydrogen peroxide in the LF culture solution. The test results showed that the concentration of hydrogen peroxide in the LF culture medium was 0.075%.

(3), extracellular protein expression

The above LF culture solution was taken, and the protein concentration was measured by Bradford protein assay. The measurement result showed that the extracellular protein concentration of the LF culture solution was 9±0.5 μg/mL.

Again, the above-mentioned LF culture solution was concentrated with Vivaspin 20 protein concentration tube (GE Healthcare), and after the concentration was measured, the concentrated protein solution was analyzed by gel electrophoresis with 18% SDS-polyacrylamide colloid (SDS-PAGE ), and finally the gel piece was stained with 0.125wt% Coomassie Blue R250 (Coomassie Blue R250) dye for 30 minutes. If the colloid contains protein, it will be stained by the dye to show a blue band.

Please refer to the eighth picture, the stained SDS-PAGE colloid, between the protein molecular weight of 5-20kDa, blue bands are observed, especially between 5-11kDa and 17-22kDa, the color of the blue bands It is darker, indicating that there are indeed small molecular weight proteins in the LF medium.

(4), antioxidant capacity

Take 0.1-3mL of overnight cultured L.fallax bacteria solution and culture it in YSB medium with pH 6.0 to obtain a mixed bacteria solution with a final volume of 6mL, and make the concentration of L.fallax in the mixed bacteria solution 4 X 10 ⁸ CFU/mL, 2 X 10 ⁸ CFU/mL, 1 X 10 ⁸ CFU/mL and 8 X 10 ⁷ CFU/mL, incubate the mixed bacterial solution at 30°C for 24 hours, and then rotate the mixed bacterial solution at 8000rpm Centrifuge for 10 minutes, collect the supernatant after centrifugation, and then filter the supernatant with a 0.45 μm filter membrane to obtain different L.fallax culture fluids (hereinafter referred to as LF culture fluids); MRS medium or the above-mentioned LF The culture solution was mixed with 0.1M 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution, and then measured its absorbance at a wavelength of 520nm (OD ₅₂₀ ) to obtain the DPPH of the LF culture solution Free radical scavenging ability, wherein the group added with MRS medium is the control group. The calculation formula of free radical scavenging rate is: DPPH free radical scavenging rate (%)=[(control group OD ₅₂₀ -experimental group OD ₅₂₀ )/control group OD ₅₂₀ ]X 100%

The experimental results showed that the DPPH free radical scavenging rate of LF culture medium (4 X 10 ⁸ CFU/mL group) was 57.60±0.34%, and the DPPH free radical scavenging rate of LF culture medium (2 X 10 ⁸ CFU/mL group) was 50 %, and the DPPH free radical scavenging rate of LF culture medium (1 X 10 ⁸ CFU/mL group) was 30%.

As can be seen from the above description, compared with the prior art, the present invention has the following advantages:

1. The culture supernatant obtained from Leuconostoc in the present invention has the effect of inhibiting Candida albicans hyphae morphological transformation and inhibiting the formation of Candida albicans and Staphylococcus aureus biofilm, and the inhibitory effect is not inferior to existing medications.

2. Concrete of the present invention can secrete organic acids to make the environment slightly acidic, and also secrete hydrogen peroxide and other antibacterial substances, so it has the ability to inhibit the transformation of Candida albicans hyphae and inhibit Candida albicans and Efficacy of Staphylococcus aureus in forming biofilms.

3. The Leuconostoc of the present invention belongs to a kind of probiotics, has low toxicity to organisms, and is not easy to produce side effects or drug resistance.

To sum up, the application of Contomonococcus spp. of the present invention for the preparation of compositions for inhibiting Candida albicans and Staphylococcus aureus can indeed achieve the expected use effect through the above-disclosed embodiments, and this The invention has not been disclosed before the application, and it has fully complied with the provisions and requirements of the Patent Law. ￠It is really convenient to file an application for a patent for invention according to the law, and ask for the review and approval of the patent.

However, the illustrations and descriptions disclosed above are only preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present invention; those who are familiar with the art generally do other things based on the characteristics and scope of the present invention. Equivalent changes or modifications shall be regarded as not departing from the design scope of the present invention

Claims (5)

A use of Leuconostoc fallax for preparing a composition for inhibiting Candida albicans and Staphylococcus aureus subsp.aureus , which is to give a Leuconostoc fallax culture solution A required individual, to inhibit Candida albicans hyphae morphological transformation and biofilm formation and inhibit the growth of Staphylococcus aureus and biofilm formation; wherein the Leuconostoc culture fluid is selected from ATCC 700006 strains of Leuconostoc Cultivate in the YSB medium of pH 6 to obtain a mixed bacterial solution, then culture the mixed bacterial solution at 30°C for 24 hours, collect the supernatant by centrifugation, and then filter the supernatant to obtain the Leuconostoc culture solution . The use as described in claim 1, wherein the culture solution of Concrete spp. contains organic acid and hydrogen peroxide (H ₂ O ₂ ). The use as described in claim 1, wherein the culture solution of Contolomonococcus detritus inhibits the production of Aspartic Proteinase by Candida albicans. The purposes as described in claim item 1, wherein the concretomonocera culture fluid inhibits the growth of cactus bacteria, escherichia coli, salmonella and pseudomonas aeruginosa. The use as described in Claim 1, wherein the culture solution of Concrete spp. is further scavenging free radicals.