WO2021180250A1

WO2021180250A1 - Preparation for inhibiting candida albicans

Info

Publication number: WO2021180250A1
Application number: PCT/CN2021/088996
Authority: WO
Inventors: 李铮; 王艳; 王喜龙; 马恬然; 于汉杰
Original assignee: 西北大学
Priority date: 2020-03-10
Filing date: 2021-04-22
Publication date: 2021-09-16
Also published as: CN111249444A; CN111249444B

Abstract

Disclosed is a preparation for inhibiting Candida albicans. The active ingredient of the preparation contains an SA α 2-3 Gal sugar chain structure, in particular, a glycoprotein rich in an SA α 2-3 Gal sugar chain structure. The active ingredient of the preparation is preferably a glycoprotein rich in an SA α 2-3 Gal sugar chain structure separated from cows' milk and purified.

Description

A preparation for inhibiting Candida albicans

Technical field

This application relates to a preparation for inhibiting Candida albicans.

Background technique

Candida albicans, also known as Candida albicans, is the most common conditional pathogenic fungus. It mainly exists in the human respiratory tract, oral cavity and epidermis. It will not cause disease under normal conditions, but when the body’s immune function is low, it will cause disease, which will threaten the body’s mucous membranes, tissues, skin and other organs, causing different Candida albicans disease can cause infections of organs such as the oral cavity in mild cases, and systemic candida disease in humans in severe cases.

Candida albicans mainly grows as a single cell, and Gram staining is positive, but the staining is not very uniform. Candida albicans causes disease due to its ability to adapt to the environment and many virulence factors that cause different degrees of infection to the host. Its ability to adapt to the environment includes the ability to take in nutrients from the surrounding environment, flexible anti-stress response, and rapid adaptation to changes in the environment. Its virulence factors mainly include extracellular hydrolase, morphological transformation and surface adhesin. After Candida albicans adhere to host cells, hyphae grow and secrete a variety of enzymes, such as hydrolases, proteases, and lipases. The hydrolases secreted can help Candida albicans extract nutrients from the external environment. The most studied extracellular hydrolase at this stage is extracellular aspartase (SAP). SAP plays a very important role in the process of infection, such as destroying the cell membrane of the host to promote adhesion and digesting molecules for self-uptake Nourishment, destroy the host's immune system and evade the host's attack, etc.

Candida albicans is a single-celled fungus that grows in an elliptical and oval shape and can form bud tubes. Under normal circumstances, Candida albicans exists in three forms: spores, pseudohyphae and fungal filaments. The fungal filaments are parallel and straight hyphae, and pseudohyphae are a long string of yeast cells, and the cells are not separated from each other. , Pseudohyphae easily form a branched state, so it is easy to absorb nutrients from the outside world. The hyphae of Candida albicans can cause tissue infections. Its phenotypic transformation means that its pathogenicity is gradually increasing. The phenotype transformation speed is closely related to the pathogenic ability, and the formed hyphae can help It escapes the attack of immune cells and is therefore more pathogenic. Studies have shown that when Candida albicans turns from white to gray (Opaque), it can avoid host cell invasion and cause infection. Experiments have shown that the hyphae of Candida albicans are more pathogenic than normal spores. . Some researchers have also proved that the morphological transformation of Candida albicans can avoid the phagocytosis of host cells, which in turn causes host cell infection. It can be seen that Candida albicans flexibly changes between multiple forms, which not only helps it quickly adapt to changes in the surrounding environment, but also lays the foundation for infecting host cells and coexisting with the host. The surface of eukaryotic yeast cells has the characteristics of adhesion, mainly because the surface of yeast cells carries different kinds of adhesin, which can adhere to different substrates, and produce different adhesin according to the substrate, which provides survival for fungi to adapt to the environment. Base. The adhesion of Candida albicans to the host means the occurrence of infection. After the host cell is adhered, the bacteria will form hyphae, thereby exerting a pathogenic effect. If Candida albicans cannot form hyphae, its pathogenicity will be Significantly weakened.

According to the "IDF Global Diabetes Overview" survey, diabetes is one of the top ten causes of death in the world. The incidence of type 2 diabetes is increasing year by year. It is estimated that by 2030, the total number of diabetes patients in the world will reach 578 million. Among patients, type 2 diabetes (T2DM) patients account for up to 90%. In recent years, the number of fungal infections caused by Candida albicans has increased significantly. T2DM patients have a significant increase in oral Candida albicans due to the increase in their salivary glucose levels, which seriously threatens human health. The oral mucosal epithelium in the oral defense system is the primary site of Candida albicans invasion. It has adhesion and destruction effects on oral epithelial cells. After adhering to host cells, the bacteria sprout and grow hyphae, which in turn cause pathogenic effects. Currently, there is a lack of effective treatment methods for the treatment of oral Candida albicans infection in patients with T2DM, and most of them are treated with oral hypoglycemic drugs or antibiotics.

With the development of molecular biology and cell biology, many biological functions of sugar chains are constantly being recognized, such as glycoprotein sugar chains, proteoglycans, glycolipid sugar chains and sugar binding proteins, which participate in many important life activities, and are also related to Many diseases, such as cancer, bacterial and viral infections, are closely related.

Summary of the invention

The main purpose of this application is to study a preparation that can effectively inhibit Candida albicans.

Through research, the applicant found and clarified the following conclusions: the active ingredient with sialic acid (SA) α2-3Gal sugar chain structure has an inhibitory effect on Candida albicans. As the concentration of sialic acid glycoprotein increases, it has The inhibitory effect of Candida growth was significantly enhanced.

Specifically, the following schemes can be drawn:

In the first aspect, a preparation for inhibiting Candida albicans, the active ingredient of which contains SAα2-3Gal sugar chain structure (especially a glycoprotein rich in SAα2-3Gal sugar chain structure). The preparation can directly act on the skin surface, oral cavity and other parts with potential for the growth of Candida albicans.

Preferably, the type of formulation is spray or film coating.

Preferably, the SAα2-3Gal sugar chain structure is derived from milk.

Further preferably, the active ingredient to which the SAα2-3Gal sugar chain structure belongs is a glycoprotein enriched in the SAα2-3Gal sugar chain structure separated and purified from milk.

Further preferably, the glycoprotein rich in SAα2-3Gal sugar chain structure is obtained by separating and purifying from milk based on the lectin MAL-II-magnetic particle complex or serotonin-magnetic particle complex.

In the second aspect, the use of the active ingredient having the sugar chain structure of SAα2-3Gal in the preparation of a preparation for inhibiting Candida albicans.

Preferably, the active ingredient with SAα2-3Gal sugar chain structure is a glycoprotein enriched in SAα2-3Gal sugar chain structure separated and purified from milk.

Further preferably, the active ingredient with the SAα2-3Gal sugar chain structure is a sugar chain structure rich in SAα2-3Gal based on the lectin MAL-II-magnetic particle complex or serotonin-magnetic particle complex separated and purified from milk. Glycoprotein.

In the third aspect, the milk extract is used in the preparation of a preparation for inhibiting Candida albicans. The milk extract is a glycoprotein rich in SAα2-3Gal sugar chain structure obtained by separation and purification.

Description of the drawings

Figure 1 shows the results of Candida albicans culture. Among them, (a) culture until a milky white round colony grows, (b) transfer to an agar plate, and after the streaked bacteria liquid has dried out, continue to cultivate until a milky white colony grows.

Figure 2 shows the results of CAL-27 cell culture (40×).

Figure 3 shows the effects of different substances on the growth curve of Candida albicans. Among them, (a) the influence of different concentrations of sialoglycoprotein A on the growth curve of Candida albicans; (b) the influence of different control proteins on the growth curve of Candida albicans: the control group and the same concentration (400μg/mL) saliva Acid monomer, BSA, asialoprotein B1 (NaIO ₄ oxidation), asialoprotein (sialidase) C1, sialoglycoprotein A.

Figure 4 shows the experimental results of the effect of sialylated glycoprotein on the adhesion of Candida albicans. Among them, (a): Control group; (b): 50μg/mL sialoprotein A; (c): 100μg/mL sialoprotein A; (d): 200μg/mL sialoprotein A; (e): 200μg /mL Asialoprotein B1; (f): 200μg/mL Asialoprotein C1. Merge stands for superimposed field, DAPI stands for fluorescence field, and Bright stands for bright field. The blue ones are DAPI channels.

Figure 5 shows the statistical results of the effect of sialylated glycoprotein on the adhesion of Candida albicans.

Detailed ways

The following describes the experimental analysis related to the present invention in detail. Of course, the applicant's research and development work on the present invention is not limited to this.

1. Experimental part

(1) Extracting substances rich in galactose and sialic acid sugar chain structure (conventional method)

Extraction method 1: Based on serotonin-magnetic particle complex, separate and purify the glycoprotein rich in sialylation from the material rich in sialic acid sugar chain structure. This sialylated glycoprotein contains SAα2-3Gal and SAα2-6Gal sugar chain structures. For details, please refer to the Chinese patent document (Serotonin-magnetic particle complex and method for enriching sialylated glycoprotein, application number: 201711206127.9).

Extraction method 2: Lectin MAL-II and SNA-magnetic particle complexes were separated and purified to separate and purify glycoproteins rich in SAα2-3Gal sugar chain structures and glycoproteins rich in SAα2-6Gal sugar chain structures. For details, please refer to the Chinese patent document (Lozenges for the prevention of influenza virus, application number: 201610654636.7).

Using the above-mentioned extraction method one, one extract is obtained from the milk, divided into 3 parts, and denoted as A, B, and C respectively. Oxidize extract B with 1mmol/mL sodium periodate solution, remove the SA at the end of its sugar chain, and then record it as B1; treat extract C with α2-3 Neuraminidase to remove its SAα2- The terminal SA of the 3Gal sugar chain structure retains its SAα2-6Gal sugar chain structure, and is then denoted as C1.

Using the above-mentioned extraction method two, two extracts of glycoprotein rich in SAα2-3Gal sugar chain structure and glycoprotein rich in SAα2-6Gal sugar chain structure were separated and purified from milk, respectively, denoted as D and E.

(2) Test strain

Candida albicans (Candida albicans ATCC 10231), purchased from China Medical Culture Collection.

(3) Cell line

The CAL-27 (human tongue squamous cell carcinoma) cell line was cultured in DMEM containing 10% fetal bovine serum, 1% penicillin-streptomycin double antibody solution, and placed in a 5% CO ₂ , 37°C constant temperature incubator. Cultivate until the cells grow to the logarithmic phase.

(4) Preparation of culture medium and experimental solution

1) Preparation of medium: YMA medium and liquid Sabouraud medium were prepared according to standard formulas and sterilized at 115°C for 20 minutes, and stored at 4°C for later use.

2) 10×PBS buffer: 0.1mol/L Na ₂ HPO ₄ , 1.37mol/L NaCl, 0.027mol/L KCl, 0.0176mol/L KH ₂ PO ₄ dissolved in ultrapure water, adjust the pH to 7.4, Store at room temperature, dilute to 1×PBS when used.

3) BSA (0.8mg/mL): Weigh 0.8mg of BSA and dissolve it in 1mL of ultrapure water. After it is completely dissolved, store it at -20°C for use.

4) DAPI: Dissolve 2μl DAPI in 10mL sterile 1×PBS buffer (1:5000) and store at 4°C in the dark.

(5) Preservation, activation and inoculation of bacteria

1) Preservation of strains

Take a sterile 50mL centrifuge tube, add 10mL of liquid Sabouraud medium, use an inoculating loop to draw a small amount of Candida albicans standard strains into the centrifuge tube added with liquid Sabouraud medium, constant temperature shaker 37℃, 160rpm Cultivate for 24h. Take 1 mL of Candida albicans bacteria liquid into a 1.5 mL sterile centrifuge tube, collect the bacteria cells after low-temperature centrifugation, add sterile liquid Sabouraud medium to resuspend, then add 80% glycerol solution, mix well and place in- Store in a refrigerator at 80°C for later use.

2) Activation and inoculation of strains

Pour the YMA medium plate on the ultra-clean workbench, turn on the UV lamp and blow air to promote its solidification, then take out the preserved Candida albicans strain from the refrigerator at -80℃, and transfer a small amount of bacterial liquid with a pipette To the medium plate, spread evenly with a spreading rod, cover the petri dish after the bacterial liquid is dry, place it upside down in a constant temperature incubator at 37°C and cultivate until it grows milky white round colonies (Figure 1a). Then use an inoculating loop to pick up a little, then quickly transfer to the agar plate and streak. During this process, do not use too much force to prevent the culture medium from being scratched. After the streaking bacteria liquid on the culture medium is dry, the same conditions as above Continue to culture until milky white colonies grow (Figure 1b).

Take a 50mL sterile centrifuge tube and add 10mL of liquid Sabouraud's medium. Use an inoculating loop to pick colonies on the activated medium plate and transfer to the liquid Sabouraud's medium. Place it on a constant temperature shaker at 37°C and rotate at 160rpm. Cultivate for 24h. After the cultivation is completed, the concentration of the Candida albicans suspension is adjusted to 1×10 ⁵ CFU/mL with liquid Sabouraud medium for later use.

(6) Cultivation and digestion of CAL-27 cells

1) Cell recovery

CAL-27 cells are human oral squamous cell carcinoma cells. Under the microscope, the cells are flat and polygonal, arranged relatively tightly, and are paving stones (Figure 2). It was cultured in high-sugar DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution, and placed in a constant temperature cell incubator with _{5% CO 2 and 37°C.}

Remove the frozen CAL-27 cells from the liquid nitrogen tank and transfer them to a 37°C constant temperature water bath to thaw them quickly. After centrifugation at a low speed in a centrifuge, the supernatant was discarded, and the high-sugar DMEM medium was added and pipetted evenly, and then the liquid was transferred to a culture flask and cultured under the above-mentioned culture conditions.

2) Cell exchange and passage

Observe the growth of the cells and the color of the culture medium under a microscope, and change the medium in time for the cells. Change the medium to the cells for passage every 2 days.

Change the medium: firstly pour out the old culture medium in the culture flask gently, rinse 2-3 times with sterile 1×PBS to wash off the excess medium, then add fresh culture medium, gently shake the culture flask, and put it in Incubator, take log phase cells for follow-up experiments.

Passaging: When the cells grow to about 80% of the adherent area, they have reached the logarithmic growth phase, and then the cells will be subcultured. First, pour out the old culture medium gently, rinse with sterile 1×PBS for 2-3 times, then digest the cells with 0.25% trypsin, add trypsin, shake the culture flask gently, and put it in the incubator for 5 minutes , Then add culture medium to terminate the reaction, use pipette tip to gently pipette, transfer all the mixed cell culture solution to a 15mL centrifuge tube, centrifuge to discard the supernatant, add fresh culture medium and pipette evenly to resuspend the cells, then Distribute them evenly into each culture flask, add a little fresh medium, and continue to culture in _{a constant temperature incubator with 5% CO 2} and 37° C. to logarithmic phase.

3) Cell starvation culture

Before the adhesion experiment, the cells must be hungry culture. Serum-free culture can make the cells hungry. After adding the sialic acid protein, it can be better absorbed, which will help the protein to play its role.

The cells used in this experiment need to be cultured in a 3.5cm cell culture dish. When the cells in the culture flask grow to the logarithmic phase, use the same method as above to digest the cells, add fresh medium and blow evenly to ensure When the cells are in suspension, add 200μl to the cell culture dish, make up the volume to 990μl with high-sugar DMEM medium, and culture for 24h under the same conditions as above. After the cells adhere to the wall, slowly aspirate the old medium and replace it with Serum-free DMEM medium, shake gently, continue to _{culture at 37°C in a 5% CO 2} incubator, and take log phase cells for subsequent experiments.

(7) The inhibitory effect of sialylated glycoprotein on Candida albicans

The concentration of sialylated glycoprotein was determined by Nano-drop to be 0.8 mg/mL, and the sterile glycoprotein solution was obtained after filtration with a 0.22 μm filter membrane, which was stored at -20°C for later use. Adjust the concentration of the BSA and asialoprotein solution to 0.8 mg/mL, and set aside at -20°C.

Place the 96-well plate in the ultra-clean workbench, add 100μl of the prepared bacterial suspension (10 ⁵ CFU/mL) to wells 1-8, and add 100μl, 50μl, 25μl to wells 1-4, respectively , 12.5μl of sialylated glycoprotein solution to make the final protein concentration of 400, 200, 100, 50μg/mL, respectively. Add 100μl of BSA solution to well 5, add 100μl of asialoprotein (NaIO ₄ oxidation) solution to well 6 and add 100μl of neuraminidase-treated sialoglycoprotein (0.8mg/mL) to well 7 ) Solution, the final concentration of protein in wells 5-7 is 400μg/mL. Add 100μl of sterile water to well 8 and fill the total volume of the solution with sterile water to 200μl in the remaining wells. The final concentration of the bacterial solution in each well is 10 ³ CFU/mL～10 ⁴ CFU/mL. Take each well with sialoprotein as the experimental group, and the remaining wells as the control group. The whole experiment was repeated three times. Subsequently, the solution in each well was mixed and sucked evenly, and then incubated at 37°C in a shaker at a constant temperature with a rotating speed of 120 rpm. The OD value of each well was measured and recorded at 595 nm with a microplate reader every 12 hours.

Because the 96-well plate has good airtightness, the CO ₂ produced by Candida albicans is discharged through the space around the plate during the culture process. At 37°C, the water vapor in the plate will also be taken out of the plate. This will cause the volume in the edge of the 96-well plate to decrease. Therefore, we should pay attention to several points during the above experiment: first add 200μl of sterile water or PBS to the wells near the edge of the 96-well plate to avoid the rapid evaporation of the liquid in the experimental wells resulting in protein concentration during the shaking of the 96-well plate. Or the high concentration of the culture medium will affect the measurement results. In this way, it can not only reduce the loss of water in the internal wells, but also analyze the cause of the infection; secondly, wrap the 96-well plate and the lid with a sealing film to prevent the temperature from getting too high. High medium concentration will affect the results; in addition, after adding each group of solutions to a 96-well plate, the shaking speed should not be too fast during the culture process on a shaker, generally between 100-130rpm, to avoid spraying liquid between each well Affect the experimental results; finally, before each determination of the OD value, use a pipette to blow the solution in each well evenly, because as the incubation time increases, the bacterial solution precipitation increases. The accuracy of the measurement results.

(8) Sialyl glycoprotein inhibits the adhesion of Candida albicans to CAL-27 cells

For the growth curve of Candida albicans, various characteristics are generally selected at the end of the logarithmic growth phase and the beginning of the stable phase, because the bacteria at this stage are the most typical in terms of biological characteristics and metabolic activity. Therefore, in this experiment, Candida albicans was cultured to the end of the logarithmic stage, and subsequent adhesion experiments were carried out.

Under the ultra-clean workbench, take a 15mL sterile centrifuge tube, add 2mL of sterile liquid Sabouraud medium and bacterial solution, and cultivate to the end of logarithmic growth on a constant temperature shaker at 37°C and shaking at 160rpm. Centrifuge at low temperature for 20 minutes at 4000 rpm, discard the supernatant, add 1 mL of sterile 1×PBS buffer to the pellet, vortex to mix, and centrifuge again under the above conditions, repeat three times, the purpose is to wash off the excess medium. After centrifugation, the supernatant was discarded, and 1 mL of sterile 1×PBS buffer was added to resuspend the suspension. The concentration of the Candida albicans suspension was adjusted to 1×10 ⁶ CFU/mL, and it was used at 4°C for use.

Take out the CAL-27 cells that have grown to the logarithmic phase from the incubator, and add 10 μl of the Candida albicans solution (10 ⁶ CFU/mL) of the above known concentration to the culture dish. Then add 500μl, 250μl, 125μl of sialylated glycoprotein respectively, and make up the volume to 2mL with sterile water, so that the final protein concentration is 200μg/mL, 100μg/mL, 50μg/mL. Then add 500 μl of BSA and asialoprotein ( _{oxidized by NaIO 4} ) to other dishes, and make up the volume to 2 mL with sterile water so that the final concentration of the bacterial solution is 10 ³ CFU/mL～10 ⁴ CFU/mL. The petri dishes added with sialic acid protein were used as the experimental group, and the other dishes were used as the control group. Slowly shake the above culture dish and incubate it in a 5% CO ₂ , 37°C incubator for 4.5 hours. After the reaction is over, take the cell culture dish out of the incubator for fixation and staining. The detailed steps are as follows:

1) Fixation: Use a pipette to slowly suck up the excess culture solution from the petri dish along the wall, add 1mL sterile PBS to rinse the excess culture solution and pour it away, then quickly add 1mL 4% paraformaldehyde solution, room temperature Reaction for 30min. In order to prevent the culture dish from drying out during this process, it must be washed quickly to prevent the cells from leaving the culture solution and deforming.

2) Washing: After the above reaction is over, use a pipette to absorb the paraformaldehyde, wash the excess paraformaldehyde and unfixed cells with sterile 1×PBS buffer, shake and wash 3min×3 times.

3) DAPI staining: After washing is finished, aspirate the excess 1×PBS buffer, add 200μl of DAPI staining solution, and react for 20min in the dark at 4°C.

4) Cleaning: After the dyeing is finished, perform cleaning, and the cleaning procedure is the same as (2). After washing, add 200μl of sterile 1×PBS buffer to the cell culture dish. The purpose is to keep the bottom of the culture dish moist and avoid cell deformation.

After completing the above steps, take photos in the dark under an inverted fluorescence microscope. Each petri dish is taken 3 times under different viewing angles, and finally the average value is calculated.

(9) Data analysis

_{The average value and SD value of OD 595} measured in the experiment were calculated and graphed by Graphpad Prism8 software. The image taken by the fluorescence microscope was adjusted with PhotoShop software to adjust the brightness and gray level, and then the Image J software was used to count the cells and bacteria. The SPSS software was used for statistical analysis. The inhibitory effect of sialoglycoprotein on the growth and adhesion of Candida albicans was analyzed by one-way analysis of variance (t = 0.05 as the significance test level), and the least significant difference method (LSD) was used for pairwise comparison. ) Method, the obtained p<0.05 means that the difference is significant. (P<0.05 is represented by "*", p<0.01 is represented by "**", p<0.001 is represented by "***").

2. Research results

(1) Extraction method 1 Extracted sialylated glycoprotein

1) Effect on the growth curve of Candida albicans

This experiment uses mixed culture of Candida albicans and different concentrations of sialoglycoprotein A in a 96-well plate. The absorption peak at 595nm is measured with a microplate reader every 12h. The experiment is repeated three times, and Graphpad is used _{according to the OD 595 value of each group.} Prism8 software draws a line graph of the effect of sialic acid protein on the growth of Candida albicans (Figure 3a). It can be seen from the figure that within 12h, the OD _{595 of} Candida albicans treated with different concentrations of sialoprotein did not change significantly; when it grew to 16h, except for the increase _{in the OD 595 of the blank control group, the OD of each experimental group} no significant change in the value of _595; in the range of 18h-24h, the OD ₅₉₅ value of the control group continued to increase, gradually and the OD ₅₉₅ reaches the end value of the number of cell growth, 50μg / mL and 100μg / mL of the experimental group also Continue to increase, but both are smaller than the control group. The OD ₅₉₅ values of the 200μg/mL and 400μg/mL experimental groups are almost unchanged; when the incubation time is longer than 24h, only the number of bacteria in the 400μg/mL experimental group remains unchanged. All grow to a stable period. _{During the whole process, the OD 595} value of Candida albicans treated with 400μg/mL sialic acid protein was almost unchanged. It may be that the protein concentration is too high, which has a bactericidal effect on the bacteria.

_{The OD 595} value of each group after the stable period was analyzed by variance analysis. The experimental group and the control group were compared, and it was found that 50μg/mL, 100μg/mL, 200μg/mL, 400μg/mL sialoglycoprotein A was effective against Candida albicans. The growth of the plant has a significant inhibitory effect (p<0.001). From the above results, within the same time frame, as the concentration of sialylated glycoprotein increased, the OD ₅₉₅ value decreased significantly, and the number of Candida albicans growth decreased, indicating that sialylated glycoprotein A has a significant effect on the growth of Candida albicans The inhibitory effect, and was concentration-dose-dependent. According to the same determination method described above, the influence of each control group on the growth of Candida albicans was detected, and the _{following curve was drawn according to the OD 595} value (Figure 3b). It can be seen from the figure that the growth curve of Candida albicans after the addition of 400μg/mL BSA and sialic acid monomer is almost the same as the normal growth curve of Candida albicans, and the addition of 400μg/mL asialoprotein B1 and sialidase treatment The growth curve of Candida albicans after glycoprotein C1 is significantly different from the normal growth curve of Candida albicans (p<0.001), but at the same concentration of each protein (400μg/mL), sialic acid protein is significantly different from other controls Difference (p<0.001).

In summary, sialoglycoprotein A within the concentration range (400μg/mL) has a significant inhibitory effect on the growth of Candida albicans, and when the sialoglycoprotein concentration reaches 400μg/mL, the bacterial cells hardly grow. . However, the same concentration of BSA, asialoprotein B1, glycoprotein C1 and sialic acid monomer at the end SA of the sugar chain structure of SAα2-3Gal have weaker inhibitory effects on Candida albicans than that of sialic acid glycoprotein A, indicating that sugar The SAα2-3 sugar chain structure of the protein plays a major role in the inhibition of Candida albicans.

2) Effect on the adhesion of Candida albicans

In order to detect the effect of sialylated glycoprotein on the adhesion of Candida albicans, the cells that have grown to the logarithmic phase were first co-cultured with sialylated protein and Candida albicans for 4.5 hours, and after a series of steps such as washing, fixing, and staining, Take pictures under a fluorescence microscope to get the experimental results shown in Figure 4. Cells treated with different concentrations of sialic acid protein and bacterial cells were used as the experimental group, and the remaining groups were used as the control group. Use Image J software to count cells and bacteria, calculate the adhesion rate (adhesion rate = the number of cells with bacteria on the surface/total number of cells), and use the Graphpad Prism8 software to draw a histogram (Figure 5). It can be seen from Figure 4 that as the concentration of sialic acid protein increases, the number of Candida albicans decreases, and the aggregation phenomenon between the bacteria gradually weakens. Correspondingly, the number of bacteria attached to the cell surface also decreases, while the remaining controls The group of bacteria aggregation is not obvious and the number has not changed significantly. The adhesion rates of the experimental group and the control group were statistically analyzed with the blank control group, and it was found that the adhesion effects of 50μg/mL sialoglycoprotein A and 200μg/mL asialoprotein (B1 and C1) on Candida albicans There was no significant effect, but 200μg/mL, 400μg/mL sialoglycoprotein A had a significant inhibitory effect on the adhesion of Candida albicans compared with the control group, and the difference was statistically significant (p<0.001). At the same time, the sialoprotein A inhibition group was significantly larger than the blank control group and other protein groups.

In summary, when the concentration of sialoprotein A is in the range of 100μg/mL～200μg/mL, it has a significant inhibitory effect on the adhesion of Candida albicans to CAL-27 cells. The inhibitory effect of Candida albicans adhesion is more obvious, which is concentration-dose dependent. At the same time, the aggregation phenomenon of Candida albicans is also continuously weakened.

(2) Using the above-mentioned extraction method two, glycoprotein D rich in SAα2-3Gal sugar chain structure and glycoprotein E rich in SAα2-6Gal sugar chain structure are separately purified from milk.

1) Effect on the growth curve of Candida albicans

With the same method as above, the Candida albicans and glycoprotein D rich in SAα2-3Gal sugar chain structure were mixed cultured in 96-well plates, and the absorption peak at 595nm was measured with a microplate reader every 12h, and the experiment was repeated. three times. _{Within 12h, the OD 595} value of Candida albicans treated with different concentrations of sialoprotein did not change significantly; when it grew to 16h, except for the increase _{in the OD 595} value of the blank control group, there was no significant change in the _{OD 595 value of each experimental group;} In the range of 18h-24h, the OD ₅₉₅ value of the blank control group continued to rise, and gradually reached the end of the logarithm of bacterial growth. The OD ₅₉₅ value of the 25μg/mL and 50μg/mL experimental groups also continued to increase, but both were lower than the control. _{The OD 595} values of the 100μg/mL and 200μg/mL experimental groups were almost unchanged; when the culture time was longer than 24h, only the number of bacteria in the 200μg/mL experimental group remained unchanged, and the rest of the groups grew to a stable phase. _{During the whole process, the OD 595} value of Candida albicans treated with 200μg/mL sialoprotein D was almost unchanged. It may be that the protein concentration is too high, which has a bactericidal effect on the bacteria.

After the stable period, the OD ₅₉₅ values of each group were analyzed by variance analysis. Comparing the experimental group with the control group, it can be seen that 25μg/mL, 100μg/mL, 200μg/mL sialoglycoprotein D has an effect on the growth of Candida albicans. Significant inhibition (p<0.001). From the above results, in the same time frame, as the concentration of sialylated glycoprotein increased, the OD ₅₉₅ value decreased significantly, and the number of Candida albicans growth decreased, indicating that the glycoprotein D pair rich in SAα2-3Gal sugar chain structure The growth of Candida albicans has a significant inhibitory effect, and it is concentration-dose-dependent. According to the same measurement method as above, the influence of each control group on the growth of Candida albicans was tested. After adding 200μg/mL BSA and sialic acid monomer, the growth curve of Candida albicans was almost the same as the normal growth curve of Candida albicans. The 200μg/mL glycoprotein E, which is rich in SAα2-6Gal sugar chain structure, has an effect on the normal growth curve of Candida albicans, but there is no significant difference.

In summary, glycoprotein D, which is rich in SAα2-3Gal sugar chain structure, has a significant inhibitory effect on the growth of Candida albicans within the concentration range (200μg/mL), and when its concentration reaches 200μg/mL, the bacteria The body hardly grows. The same concentration of glycoprotein E rich in SAα2-6Gal sugar chain structure has an effect on the normal growth curve of Candida albicans, but there is no significant difference, indicating that the SAα2-3 sugar chain structure of glycoprotein inhibits Candida albicans in is a main factor.

2) Effect on the adhesion of Candida albicans

In the same way as above, to detect the influence of glycoprotein D, which is rich in SAα2-3Gal sugar chain structure, on the adhesion of Candida albicans. First, the cells that have grown to the logarithmic phase are co-cultured with sialylated protein and Candida albicans for 4.5h. After a series of steps such as washing, fixing, and staining, the cells treated with different concentrations of D and bacterial cells were used as the experimental group, and the remaining groups were used as the control group. With the increase of the concentration of D, the number of Candida albicans decreased, and the aggregation phenomenon between the bacteria gradually weakened. Correspondingly, the number of bacteria attached to the cell surface also decreased, while the aggregation of the other control groups did not occur. Obvious and the number has not changed significantly. The adhesion rates of the experimental group and the control group were statistically analyzed with the blank control group. It was found that 25μg/mL saliva D and 200μg/mL E had no significant effect on the adhesion of Candida albicans, while 100μg/mL and 200μg D/mL had a significant inhibitory effect on the adhesion of Candida albicans compared with the control group, and the difference was statistically significant (p<0.001).

In summary, when glycoprotein D rich in SAα2-3Gal sugar chain structure is in the range of 50μg/mL～100μg/mL, it has a significant inhibitory effect on the adhesion of Candida albicans to CAL-27 cells, and with sialic acid The increase in protein concentration, the more obvious its inhibitory effect on the adhesion of Candida albicans, which is concentration-dose dependent. At the same time, the aggregation phenomenon of Candida albicans is also continuously weakened.

3. Conclusion

Candida albicans mainly infects host cells through processes such as adhesion, hyphae growth, and biofilm formation. When Candida albicans forms a biofilm on the outermost side, it can not only reduce its sensitivity to antibacterial drugs, but also avoid the host. Attacks by the immune system pose a serious threat to human health. The first step for Candida albicans to infect the host is adhesion. The adhesion is mainly through the combination of glycoproteins on the surface of the bacteria and glycoprotein receptors on the surface of the host cell. The adherent substances mainly include mannan, adhesin and chitin. Wait. At present, most of the clinical use is antibacterial drugs, which makes Candida albicans become resistant to antifungal drugs, which can also affect human health. Therefore, the development of new non-toxic and harmless drugs to inhibit the growth and adhesion of Candida albicans has become a hot research topic at this stage.

Experimental results show:

Sialylglycoprotein A, which is rich in SAα2-3Gal sugar chain structure, has a significant inhibitory effect on the growth and adhesion of Candida albicans. Within a certain concentration of sialoglycoprotein (400μg/mL), with the increase of glycoprotein concentration, it has a certain inhibitory effect on the growth of Candida albicans, and when the sialoglycoprotein concentration reaches 400μg/mL, white Candida hardly grows, which can also indicate that 400μg/mL is the bactericidal concentration of sialoglycoprotein. In addition, the proteins (BSA, asialoprotein, sialic acid monomer) in other control groups have no obvious inhibitory effect on the growth of Candida albicans, which further shows that the inhibitory effect of sialoglycoprotein on the growth of Candida albicans is mainly SAα2-3 is at work.

Sialylglycoprotein D, which is rich in SAα2-3Gal sugar chain structure, has a significant inhibitory effect on the growth and adhesion of Candida albicans. When the concentration of glycoprotein D rich in SAα2-3Gal sugar chain structure is in the range of 50μg/mL～100μg/mL, it has a good inhibitory effect on the growth and adhesion of Candida albicans. And when the concentration of sialoglycoprotein reached 200μg/mL, Candida albicans hardly grew, which can also indicate that 200μg/mL is the bactericidal concentration of glycoprotein D rich in SAα2-3Gal sugar chain structure.

Therefore, it can also be expected that milk products (such as pure milk) sprayed on the skin surface, oral cavity and other parts with potential for the growth of Candida albicans can also inhibit Candida albicans to a certain extent.

Claims

A preparation for inhibiting Candida albicans, which is characterized in that its active ingredient contains SAα2-3Gal sugar chain structure.
The preparation for inhibiting Candida albicans according to claim 1, wherein the preparation type is spray or film coating.
The preparation for inhibiting Candida albicans according to claim 1, wherein the SAα2-3Gal sugar chain structure is derived from milk.
The preparation for inhibiting Candida albicans according to claim 3, wherein the active ingredient to which the SAα2-3Gal sugar chain structure belongs is a glycoprotein enriched in SAα2-3Gal sugar chain structure separated and purified from milk.
The preparation for inhibiting Candida albicans according to claim 4, wherein the glycoprotein rich in SAα2-3Gal sugar chain structure is based on the lectin MAL-II-magnetic particle complex or serotonin-magnetic particle The complex is separated and purified from milk.
Use of the active ingredient with SAα2-3Gal sugar chain structure in the preparation of a preparation for inhibiting Candida albicans.
The use according to claim 6, characterized in that the active ingredient with SAα2-3Gal sugar chain structure is a glycoprotein enriched in SAα2-3Gal sugar chain structure separated and purified from milk.
The use according to claim 7, characterized in that the active ingredient with the sugar chain structure of SAα2-3Gal is obtained by separating and purifying the lectin MAL-II-magnetic particle complex or serotonin-magnetic particle complex from milk The glycoprotein rich in SAα2-3Gal sugar chain structure.
Use of a milk product in the preparation of a preparation for inhibiting Candida albicans, the milk product retains a glycoprotein containing a sugar chain structure of SAα2-3Gal.
The use of the milk extract in the preparation of a preparation for inhibiting Candida albicans, the milk extract is a glycoprotein rich in SAα2-3Gal sugar chain structure obtained by separation and purification.