CN111249444B - A preparation for inhibiting Candida albicans - Google Patents

Abstract

The application discloses a preparation for inhibiting candida albicans. The active component of the preparation comprises SA alpha 2-3Gal sugar chain structure (especially glycoprotein rich in SA alpha 2-3Gal sugar chain structure), and the inhibition effect on the growth of Candida albicans is obviously enhanced along with the increase of the concentration of sialoglycoprotein; when the concentration of the sialoprotein is in the range of 100 mu g/mL-200 mu g/mL, the inhibitor has a remarkable inhibiting effect on the adhesion of the Candida albicans to CAL-27 cells, and when the concentration of the sialoprotein reaches 200 mu g/mL, the Candida albicans hardly grows and can play a bactericidal effect. The preparation can be directly sprayed on skin surface, oral cavity, etc. with potential Candida albicans growth parts, and the active ingredient of the preparation is preferably glycoprotein rich in SA alpha 2-3Gal sugar chain structure separated and purified from milk.

Description

A preparation for inhibiting Candida albicans

technical field

The present application relates to a preparation for inhibiting Candida albicans.

Background technique

Candida albicans, also known as Candida albicans, is the most common opportunistic pathogenic fungus. It mainly exists in the respiratory tract, oral cavity and epidermis of the human body. Under normal conditions, it will not cause disease, but when the immune function of the human body is low, it will cause disease, and then pose a threat to the mucous membranes, tissues, skin and other organs of the human body, causing different diseases. The degree of Candida albicans disease can cause infection of organs such as the oral cavity in mild cases, and can cause systemic candida disease in the human body in severe cases.

Candida albicans mainly grows as single cells, and Gram staining is positive, but the coloring is not very uniform. Candida albicans causes diseases due to its good ability to adapt to the environment and numerous virulence factors that cause different degrees of infection to the host. Its ability to adapt to the environment includes the ability to absorb nutrients from the surrounding environment, flexible anti-stress response, and rapid adaptation to environmental changes. Its virulence factors mainly include extracellular hydrolytic enzymes, morphological transformation, and surface adhesins. After Candida albicans adheres to the host cell, it grows hyphae and secretes various enzymes, such as hydrolytic enzymes, proteases, and lipases. The hydrolytic enzymes secreted by it can help Candida albicans absorb 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 infection process, such as destroying the host's cell membrane to promote adhesion, digesting molecules for self-uptake Nutrition, destroying the host immune system and evading host attacks, etc.

Candida albicans is a single-celled fungus that grows in oval and oval shapes and can form germ tubes. Under normal circumstances, Candida albicans exists in three forms: spores, pseudohyphae and mycelia. Mycelia are parallel and straight hyphae, and pseudohyphae are long strings of yeast cells that are not separated from each other. , Pseudohyphae are easy to form branches, so it is easy to absorb nutrients from the outside world. The mycelial state of Candida albicans can cause tissue infection, and its phenotypic transformation means that the pathogenicity gradually increases. The speed of its phenotypic transformation is closely related to the pathogenicity. The formed mycelium can help It escapes the attack of immune cells by itself, so it is more pathogenic. Studies have shown that when Candida albicans transforms from white (White) to gray (Opaque), it can avoid the invasion of host cells and cause infection. Experiments have proved 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, thereby causing infection of host cells. It can be seen that Candida albicans can flexibly change among various 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 types of adhesins, they can adhere to different substrates, and produce different adhesins according to the substrate, which provides survival opportunities for fungi to adapt to the environment Base. The adhesion of Candida albicans to the host means the occurrence of infection. After adhering to the host cells, the bacteria will form hyphae, thereby exerting a pathogenic effect. If Candida albicans cannot form hyphae, its pathogenicity will be reduced. Significantly weakened.

According to the "IDF Global Diabetes Overview" survey, diabetes is one of the top ten causes of death in the world, and 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, and among all Among the patients, type 2 diabetes (T2DM) patients accounted for as high as 90%. In recent years, the number of fungal infections caused by Candida albicans has increased significantly, and the increase in salivary glucose levels in T2DM patients has significantly increased oral Candida albicans, 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 the host cells, the bacteria germinate and grow hyphae, which in turn cause pathogenicity. Currently, there is no effective treatment for oral Candida albicans infection in T2DM patients, 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 have been continuously recognized, such as glycoprotein sugar chains, proteoglycans, glycolipid sugar chains, and sugar-binding proteins, etc. participate in many important life activities, and are also associated with Many diseases, such as cancer, bacterial and viral infections, are closely related.

Contents 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, and as the concentration of sialic acid glycoprotein increases, it has an inhibitory effect on Candida albicans. The inhibition of Candida growth was significantly enhanced.

Specifically, the following solutions 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 glycoprotein rich in SAα2-3Gal sugar chain structure). The preparation can directly act on the skin surface, oral cavity and other sites with potential growth of Candida albicans.

Preferably, the formulation type 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 rich in SAα2-3Gal sugar chain structure isolated 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 having the SAα2-3Gal sugar chain structure is a glycoprotein rich in the SAα2-3Gal sugar chain structure isolated and purified from milk.

Further preferably, the active ingredient having the SAα2-3Gal sugar chain structure is rich in the SAα2-3Gal sugar chain structure obtained by separating and purifying the lectin MAL-II-magnetic particle complex or the serotonin-magnetic particle complex from milk of glycoproteins.

In the third aspect, the use of 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 through separation and purification.

Description of drawings

Figure 1 shows the results of Candida albicans culture. Wherein, (a) culture until milky white round colonies grow, (b) transfer to an agar plate, after the streaked bacterial solution dries up, continue to cultivate until milky white colonies grow.

Figure 2 is the result of CAL-27 cell culture (40×).

Fig. 3 is the influence 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: Control group and saliva with the same concentration (400 μg/mL) Acid monomer, BSA, asialoprotein B1 (NaIO ₄ oxidation), asialoprotein (sialidase) C1, sialoglycoprotein A.

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

Fig. 5 is the statistical result of the effect of sialylated glycoprotein on the adhesion of Candida albicans.

Detailed ways

The experimental analysis related to the present invention will be introduced in detail below. Of course, the applicant's research and development work on the present invention is not limited thereto.

1. Experimental part

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

Extraction method 1: Separation and purification of glycoproteins rich in sialylation from materials rich in sialic acid sugar chain structures based on serotonin-magnetic particle complexes. This sialylated glycoprotein contains SAα2-3Gal and SAα2-6Gal sugar chain structures. For details, please refer to the Chinese patent literature (Serotonin-Magnetic Microparticle Complex and Method for Enriching Sialylated Glycoprotein, Application No.: 201711206127.9).

Extraction method two: the lectin MAL-II and the SNA-magnetic particle complex were separated and purified to obtain glycoproteins rich in SAα2-3Gal sugar chain structure and glycoprotein rich in SAα2-6Gal sugar chain structure respectively. For details, please refer to the Chinese patent literature (buccal tablets for preventing influenza virus, application number: 201610654636.7).

Using the above-mentioned extraction method 1, one extract was obtained from milk, which was divided into 3 parts, which were recorded as A, B and C respectively. Extract B was oxidized with 1mmol/mL sodium periodate solution to remove the SA at the end of its sugar chain, and then recorded as B1; extract C was treated with α2-3Neuraminidase 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 2, two kinds of extracts, glycoprotein rich in SAα2-3Gal sugar chain structure and glycoprotein rich in SAα2-6Gal sugar chain structure, were separated and purified from milk, respectively marked as D and E.

(2) Tested strains

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

(3) cell line

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

(4) Preparation of culture medium and experimental solution

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

2) 10×PBS buffer solution: 0.1mol/L Na ₂ HPO ₄ , 1.37mol/L NaCl, 0.027mol/L KCl, 0.0176mol/L KH ₂ PO ₄ were dissolved in ultrapure water, and the pH was adjusted to 7.4, Store at room temperature and 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 later use.

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

(5) Preservation, activation and inoculation of strains

1) Preservation of strains

Take a sterile 50mL centrifuge tube, add 10mL liquid Sabouraud medium, use an inoculation loop to draw a small amount of Candida albicans standard strain and inoculate it into the centrifuge tube with liquid Sabouraud medium. Cultivate for 24h. Take 1mL of Candida albicans solution and add it to a 1.5mL sterile centrifuge tube, collect the bacterial cells after low-temperature centrifugation, add sterile liquid Sabouraud medium to resuspend, then add 80% glycerin solution, mix well and place in- Store in the 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 ultraviolet lamp and blow it to promote its solidification, then take out the preserved Candida albicans strains from the -80°C refrigerator, use a pipette gun to draw a small amount of bacterial liquid and transfer it Spread evenly on the culture medium plate with a coating stick. After the bacterial solution dries, cover the petri dish and place it upside down in a 37°C constant temperature incubator to cultivate until milky white round colonies grow (Figure 1a). Then use the inoculation loop to dip a little, then quickly transfer to the agar plate and streak. In this process, do not use too much force to prevent the medium from being scratched. After the streaked bacteria solution on the medium dries up, use the same conditions Continue to cultivate until milky white colonies grow (Fig. 1b).

Take a 50mL sterile centrifuge tube and add 10mL of liquid Sabouraud medium, use an inoculation loop to pick colonies on the activated medium plate and transfer them to liquid Sabouraud medium, place on a constant temperature shaker at 37°C, with a rotation speed of 160rpm Cultivate for 24h. After the cultivation, the concentration of Candida albicans suspension was adjusted to 1×10 ⁵ CFU/mL with liquid Sabouraud medium for 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, closely arranged, and paving stone-like (Figure 2). Cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution, and cultured in a constant temperature cell incubator with 5% CO ₂ and 37°C.

The frozen CAL-27 cells were taken out from the liquid nitrogen tank and transferred to a constant temperature water bath at 37°C to allow them to thaw quickly. Discard the supernatant after centrifuging at low speed in a centrifuge, add high-sugar DMEM medium and pipette evenly, then transfer the liquid to a culture bottle and culture under the above-mentioned culture conditions.

2) Cell replacement and passage

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

Medium replacement: first pour out the old culture medium in the culture bottle gently, rinse with sterile 1×PBS 2-3 times, wash off the excess culture medium, then add fresh culture medium, shake the culture bottle gently, put in Incubator, take logarithmic phase cells for subsequent experiments.

Subculture: 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, wash with sterile 1×PBS 2-3 times, then digest the cells with 0.25% trypsin, add trypsin, shake the culture bottle gently, and put it in the incubator to react for 5 minutes , then add the medium to stop the reaction, blow gently with the tip of the pipette, transfer all the mixed cell culture medium to a 15mL centrifuge tube, centrifuge to discard the supernatant, add fresh medium and blow evenly to resuspend the cells, and then Evenly distribute to each culture bottle, add a little fresh medium, and continue to culture in a 5% CO ₂ , 37°C constant temperature incubator until the logarithmic phase.

3) Cell starvation culture

Before carrying out the adhesion experiment, the cells should be starved for culture first. Serum-free culture can make the cells in a starvation state. After adding sialic acid protein, it can absorb better and help the protein to play a 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, digest the cells with the same method as above, add fresh medium and blow evenly to ensure The cells are in a suspended state, suck 200 μl into the cell culture dish, make up the volume to 990 μl with high-sugar DMEM medium, and culture under the same conditions above for 24 hours. After the cells adhere to the wall, slowly absorb the old medium and replace it with The serum-free DMEM medium was shaken gently, and the culture was continued at a constant temperature of 37°C in a 5% CO ₂ incubator, and the logarithmic phase cells were taken for subsequent experiments.

(7) 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 filtered through a 0.22 μm filter membrane to obtain a sterile glycoprotein solution, which was stored at -20°C for future use. Adjust the concentration of BSA and asialoprotein solution to 0.8 mg/mL, and store at -20°C for later use.

Place the 96-well plate in a clean bench, add 100 μl of the prepared bacterial suspension (10 ⁵ CFU/mL) to wells 1-8 in sequence, add 100 μl, 50 μl, and 25 μl to wells 1-4 , 12.5 μl of sialylated glycoprotein solution, so that the final protein concentration is 400, 200, 100, 50 μg/mL, respectively. Add 100 μl of BSA solution to well No. 5, add 100 μl of asialoprotein ( _NaIO oxidation) solution to well No. 6, and add 100 μl of sialoglycoprotein (0.8 mg/mL) treated with neuraminidase to well No. 7 ) solution, the final protein concentration in wells 5-7 was 400 μg/mL. Add 100 μl of sterile water to the No. 8 well, and use sterile water to make up the total volume of the solution to 200 μl in the remaining wells. The final concentration of the bacterial solution in each well is 10 ³ CFU/mL～10 ⁴ CFU/mL. The wells added with sialic acid protein were used as the experimental group, and the remaining wells were used as the control group. The whole experiment was repeated three times. Then the solutions in each well were mixed and blown evenly, and then cultured in a shaker at 37°C at a constant temperature with a rotation speed of 120rpm. The OD value of each well was measured at 595nm with a microplate reader every 12h and recorded.

Because the sealing of the 96-well plate is good, the CO ₂ produced by Candida albicans is discharged through the space around the plate during the cultivation process. At 37°C, the water vapor in the plate will also be taken out of the plate, so that This will result in a reduction in volume in the edge wells of the 96-well plate. Therefore, some points should be paid attention to in the above experiment process: First, add 200 μl 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 during the shaking of the 96-well plate, resulting in protein concentration. Or the high concentration of the medium will affect the measurement results. In this way, not only the loss of water in each well can be reduced, but also the cause of bacterial contamination can be analyzed; secondly, the joint between the 96-well plate and the cover is wrapped with a sealing film to prevent the temperature from being too high. The high concentration of high medium will affect the results; in addition, after adding each group of solutions to the 96-well plate, the shaking speed should not be too fast during the culture process on the shaker, generally between 100-130rpm, to avoid liquid spraying between the wells Affect the experimental results; Finally, before each determination of the OD value, use a pipette gun to blow and suck the solution in each well evenly, because with the increase of the culture time, the precipitation of the bacterial solution increases, and the solution can be mixed before measurement, which can improve Accuracy of measurement results.

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

For the growth curve of Candida albicans, the stage at the end of the logarithmic growth phase and the beginning of the stable phase is generally selected for research on various characteristics, 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 logarithmic phase, and subsequent adhesion experiments were carried out.

Under the ultra-clean workbench, take a 15mL sterile centrifuge tube, add 2mL sterile liquid Sabouraud medium and bacterial solution, and culture on a constant temperature shaker at 37°C with a shaking speed of 160rpm until the end of logarithmic growth. Centrifuge at low temperature for 20min at 4000rpm, discard the supernatant, add 1mL sterile 1×PBS buffer solution to the pellet, vortex and mix, and centrifuge again under the above conditions, repeating three times, the purpose is to wash away excess medium. After centrifugation, discard the supernatant, add 1 mL of sterile 1×PBS buffer to resuspend, adjust the concentration of Candida albicans suspension to 1×10 ⁶ CFU/mL, and set aside at 4°C.

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

1) Fix: use a pipette gun to slowly suck off the excess culture medium along the wall of the petri dish, add 1mL sterile PBS to rinse the excess culture medium and pour it off, then quickly add 1mL of 4% paraformaldehyde solution, and keep at room temperature React for 30 minutes. In this process, in order to prevent the culture dish from drying out, it should be cleaned quickly to prevent the cells from leaving the culture medium and deforming.

2) Cleaning: After the above reaction is finished, suck up the paraformaldehyde with a pipette gun, wash excess paraformaldehyde and unfixed cells with sterile 1×PBS buffer solution, and shake for 3 min×3 times.

3) DAPI staining: After washing, the excess 1×PBS buffer solution was sucked off, 200 μl of DAPI staining solution was added, and reaction was carried out at 4° C. in the dark for 20 minutes.

4) Washing: After the dyeing is finished, wash, and the washing steps are the same as (2). After washing, add 200 μl of sterile 1×PBS buffer solution to the cell culture dish to keep the bottom of the culture dish moist and avoid cell deformation.

After completing the above steps, take photos under an inverted fluorescence microscope in the dark, and take 3 times for each petri dish under different viewing angles, and finally calculate the average value.

(9) Data analysis

The average value and SD value of OD ₅₉₅ measured in the experiment were plotted using Graphpad Prism8 software. The brightness and grayscale of the pictures taken by the fluorescence microscope were adjusted with PhotoShop software, and then the cells and bacteria were counted with Image J software. 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 (with 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 one extracted sialylated glycoprotein

1) Effect on the growth curve of Candida albicans

In this experiment, Candida albicans was mixed with different concentrations of sialoglycoprotein A in a 96-well plate, and the absorption peak at 595 nm was measured with a microplate reader every 12 hours. The experiment was repeated three times, and Graphpad was 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 (Fig. 3a). It can be seen from the figure that within 12 hours, the OD ₅₉₅ value of Candida albicans treated with different concentrations of sialic acid protein has no significant change; when it grows to 16 hours, except for the OD ₅₉₅ value of the blank control group, the OD 595 value of each experimental group ₅₉₅ values did not change significantly; while in the range of 18h-24h, the OD ₅₉₅ value of the blank control group continued to rise, and gradually reached the logarithmic end of the growth of the bacteria, and the OD ₅₉₅ values of the 50μg/mL and 100μg/mL experimental groups also The OD ₅₉₅ values of the 200μg/mL and 400μg/mL experimental groups were almost unchanged; when the culture time was longer than 24h, only the 400μg/mL experimental group had no change in the number of bacteria, and the other groups All grew to the stationary phase. During the whole process, the OD ₅₉₅ value of Candida albicans after 400 μg/mL sialic acid protein treatment was almost unchanged, which may be due to the high protein concentration, which had a bactericidal effect on the bacteria.

The OD ₅₉₅ values of each group after the stable period were analyzed by variance, and the experimental group was compared with the control group. growth was significantly inhibited (p<0.001). From the above results, within the same time frame, with the increase of sialylated glycoprotein concentration, the OD ₅₉₅ value decreased significantly, and the growth of Candida albicans decreased, indicating that sialylated glycoprotein A had a significant effect on the growth of Candida albicans. inhibitory effect in a dose-dependent manner. According to the same determination method as 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 ( FIG. 3 b ). It can be seen from the figure that the growth curve of Candida albicans after adding 400 μg/mL of BSA and sialic acid monomer is almost the same as the normal growth curve of Candida albicans, while adding 400 μg/mL of asialoprotein B1 and sialidase treatment The growth curve of Candida albicans after the glycoprotein C1 was significantly different from the normal growth curve of Candida albicans (p<0.001), but at the same concentration of each protein (400μg/mL), there was a significant difference between the sialic acid protein and other control groups. Difference (p<0.001).

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

2) The 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 had grown to the logarithmic phase were co-cultured with sialylated protein and Candida albicans for 4.5 hours, and after a series of steps such as washing, fixing and staining, the Photographs were taken under a fluorescence microscope to obtain the experimental results shown in Figure 4. The cells treated with different concentrations of sialic acid protein and bacteria were used as the experimental group, and the other groups were used as the control group. The cells and bacteria were counted by Image J software, and the adhesion rate was calculated (adhesion rate = the number of cells with bacterial adhesion on the surface/total number of cells), and the histogram was drawn by using Graphpad Prism8 software (Figure 5). It can be seen from Figure 4 that with the increase of the concentration of sialic acid protein, the number of Candida albicans decreased, and the aggregation phenomenon between the cells gradually weakened. Correspondingly, the number of cells adhered to the cell surface also decreased, while the rest of the control The coagulation phenomenon of bacteria in the group was not obvious and the number did not change 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 of 50 μg/mL sialoglycoprotein A and 200 μg/mL asialoprotein (B1 and C1) to Candida albicans There was no significant effect, but 200 μg/mL and 400 μg/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 100 μg/mL-200 μg/mL, it has a significant inhibitory effect on the adhesion of CAL-27 cells to Candida albicans, and with the increase of the concentration of sialoprotein A, it has a significant inhibitory effect on the adhesion of CAL-27 cells. The inhibitory effect on the adhesion ability of Candida albicans is more obvious, which is concentration-dose dependent, and at the same time the aggregation phenomenon of Candida albicans is also weakened continuously.

(2) Glycoprotein D rich in SAα2-3Gal sugar chain structure and glycoprotein E rich in SAα2-6Gal sugar chain structure were separated and purified from milk by using the above extraction method 2.

1) Effect on the growth curve of Candida albicans

In the same way as above, Candida albicans was mixed with different concentrations of glycoprotein D rich in SAα2-3Gal sugar chain structure in a 96-well plate, and the absorption peak at 595nm was measured with a microplate reader every 12 hours, and the experiment was repeated. three times. Within 12 hours, there was no significant change in the OD ₅₉₅ value of Candida albicans treated with different concentrations of sialic acid protein; at 16 hours, there was no significant change in the OD ₅₉₅ value of each experimental group except for the increase in the OD ₅₉₅ value of the blank control group; In the range of 18h-24h, the OD ₅₉₅ value of the blank control group continued to rise, and gradually reached the logarithmic end of the growth of the bacteria, and the OD ₅₉₅ values of the 25μg/mL and 50μg/mL experimental groups also continued to increase, but they were all smaller than the control group group, while 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 200μg/mL experimental group had no change in the number of bacteria, and the rest of the groups all grew to the stationary phase. During the whole process, the OD ₅₉₅ value of Candida albicans after 200 μg/mL sialoprotein D treatment was almost unchanged, which may be due to the high protein concentration, which had a bactericidal effect on the bacteria.

The OD ₅₉₅ values of each group after the stable period were analyzed by variance, and the comparison between the experimental group and the control group showed that 25 μg/mL, 100 μg/mL, and 200 μg/mL sialoglycoprotein D had significant effects on the growth of Candida albicans. Significant inhibitory effect (p<0.001). From the above results, within the same time frame, as the concentration of sialylated glycoprotein increases, the OD ₅₉₅ value decreases significantly, and the growth of Candida albicans decreases, indicating that glycoprotein D rich in SAα2-3Gal sugar chain structure has an The growth of Candida albicans was significantly inhibited, and it was concentration-dose dependent. According to the above-mentioned same assay method, the influence of each control group on the growth of Candida albicans was detected, and the growth curve of Candida albicans after adding 200 μg/mL of BSA and sialic acid monomer was almost the same as the normal growth curve of Candida albicans, while adding Glycoprotein E rich in SAα2-6Gal sugar chain structure at 200μg/mL has an effect on the normal growth curve of Candida albicans, but there is no significant difference.

In summary, glycoprotein D rich in SAα2-3Gal sugar chain structure has a significant inhibitory effect on the growth of Candida albicans within the concentration range (200 μg/mL), and when the concentration reaches 200 μg/mL, the bacteria Body barely 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 can inhibit Candida albicans in is a main factor.

2) The effect on the adhesion of Candida albicans

In the same way as above, the effect of glycoprotein D rich in SAα2-3Gal sugar chain structure on the adhesion of Candida albicans was detected. First, the cells that had grown to the logarithmic phase were co-cultured with sialylated protein and Candida albicans for 4.5 hours. After a series of steps such as washing, fixing, and staining, the cells treated with different concentrations of D and bacteria were used as the experimental group, and the rest of the groups were used as the control group. With the increase of the concentration of D, the number of Candida albicans decreased, and the coagulation phenomenon between the cells also gradually weakened. Obvious and the number did not change significantly. Statistical analysis of the adhesion rates of the experimental group and the control group and the blank control group showed that 25 μg/mL saliva D and 200 μg/mL E had no significant effect on the adhesion of Candida albicans, while 100 μg/mL and 200 μg 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 the glycoprotein D rich in SAα2-3Gal sugar chain structure is in the range of 50 μg/mL-100 μg/mL, it has a significant inhibitory effect on the adhesion of Candida albicans to CAL-27 cells, and along with sialic acid As the protein concentration increases, its inhibitory effect on the adhesion of Candida albicans is more obvious, which is concentration- and dose-dependent. At the same time, the aggregation phenomenon of Candida albicans is also weakened.

3. Conclusion

Candida albicans mainly infects host cells through 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 hide from the host. The immune system's attack on it poses 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 the glycoprotein on the surface of the bacteria and the glycoprotein receptor on the surface of the host cell. The adherent substances mainly include mannan, adhesin and chitin. wait. At present, most of the antibacterial drugs are used clinically, which makes Candida albicans resistant to antifungal drugs, and will 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 research hotspot at this stage.

Experimental results show that:

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

The sialoglycoprotein D 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 50 μg/mL-100 μg/mL, it has a good inhibitory effect on the growth and adhesion of Candida albicans. And when the concentration of sialoglycoprotein reaches 200 μg/mL, Candida albicans hardly grows, which also shows that 200 μg/mL is the bactericidal concentration of glycoprotein D rich in SAα2-3Gal sugar chain structure.

Claims (3)

1. The use of an active ingredient with a SAα2-3Gal sugar chain structure in the preparation of a preparation for inhibiting Candida albicans; The active ingredient with SAα2-3Gal sugar chain structure is a glycoprotein with SAα2-3Gal sugar chain structure isolated and purified from milk. 2. The use according to claim 1, characterized in that: the active ingredient having the SAα2-3Gal sugar chain structure is separated from milk based on the lectin MAL-II-magnetic particle complex or serotonin-magnetic particle complex The purified glycoprotein with SAα2-3Gal sugar chain structure. 3. The use according to claim 2, characterized in that: the type of preparation is a spray or a coating.

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