CN111996129A - New strain of cicada fungus and its use in anti-tumor and bacteriostasis - Google Patents

Abstract

The invention relates to a new strain of cicada fungus and application thereof in the aspects of tumor resistance and bacteriostasis, wherein the new strain is separated from Fujian Wuyi mountain, an original strain is obtained by tissue separation and purification of a sporocarp, the new strain is identified as a new strain of cicada fungus Isaria cicada fungus by morphology and molecular biology, is named as cicada fungus Isaria cicada fungus HMGIM-N140534, is preserved in 7 and 8 days in 2020 to Guangdong province microorganism strain preservation center (Guangzhou, China), and has the preservation number as follows: GDMCC No. 61083. The strain of the cordyceps sobolifera is a new strain which is collected from the field and has stronger activity, the new strain is collected and separated from the field and is preserved in a patent strain preservation center, the strain is confirmed to be the cordyceps sobolifera through the traditional identification and the molecular identification, the mass preparation can be realized through artificial fermentation, and the strain has obvious inhibiting effect on antitumor and candida albicans and has development prospect.

Description

A new strain of cicada and its use in antitumor and antibacterial

technical field

The present invention relates to a new strain of rare medicinal bacteria and its application, in particular to a new strain of Cicada flower and its application in anti-tumor and antibacterial.

Background technique

Edible and medicinal fungi have become the fifth largest planting industry in my country after grain, vegetables, fruit and oil. According to the statistics of China Edible Fungi Association, in 2018, the total output of edible and medicinal fungi in 28 provinces, autonomous regions and municipalities directly under the Central Government continued to grow. It reached 37.8903 million tons (fresh products), and the output value was as high as 293.838 billion yuan. Edible and medicinal mushrooms have balanced nutritional components, rich secondary metabolites, and contain a variety of natural active ingredients, such as polysaccharides, flavonoids and terpenoids, alkaloids, etc. It has significant effects on viruses, anti-tumor, and enhancing immunity. Some edible and medicinal mushroom resources have been developed into cosmetics, health food, medicines and other products. my country's edible fungus industry accounts for more than 75% of the world's total, employing more than 20 million people. It is an industry with great development potential.

Today, with the booming development of the edible and medicinal mushroom industry, more and more rare edible and medicinal mushroom species have gradually entered people's field of vision, and many original rare species have been gradually domesticated, such as bamboo fungus, tea mushroom, lily umbrella, Morels etc. However, there are also a large number of wild edible and medicinal fungi that have not been studied because they have not been recognized by humans. According to research, there are currently more than 3 million species of fungi in the world, only 1% of which are known. Among them, there are about 14,000 known large fungi, 1,789 edible fungi and 798 medicinal fungi have been confirmed in China. Among them, less than 100 kinds of wild edible and medicinal fungi have been domesticated by humans, and there are only more than 30 varieties of large-scale cultivation. Humans still have a long way to go from the research and utilization of large fungi. With the gradual improvement of people's living standards, the requirements for the quality of life are higher, and large fungi have a very good effect on human health and are increasingly valued by people.

Cicada flower, also known as Cicada flower, belongs to Cordyceps family Cordyceps genus Cordyceps fungus. It is a dry complex formed by parasitic nymphs of cicadas by Isariacicadae Miquel (formerly known as Paecilomyces cicadae). Cicada flower is a traditional and precious Chinese medicinal material. "Compendium of Materia Medica" records that it is "sweet, cold, and non-toxic. It is mainly used for children's Tianzhu, epileptic axolotl, night cry and palpitations." It has various effects such as sedation and hypnosis, and improvement of renal function. Among them, there are many reports on the improvement of renal function with cicada flower. As a precious Cordyceps fungus, the wild resources of cicada flower are becoming increasingly scarce. At present, the research and demand for artificial cultivation of cicadas are increasing day by day.

SUMMARY OF THE INVENTION

In view of the above deficiencies, the present invention provides a new strain of wild and rare medicinal mushroom Isaria cicadae with in vitro antitumor activity and bacteriostatic effect, and the antitumor use of its fermented extract and its fermentation broth for inhibiting Candida albicans. use.

The present invention achieves the above object through the following schemes:

The invention provides a new strain of cicada, which is isolated from Wuyi Mountain in Fujian. The original strain is obtained by tissue separation and purification of fruiting bodies. It is identified as a new strain of Isaria cicadae by morphology and molecular biology, and the strain is named as cicada. The flower Isaria cicadae HMGIM-N140534 has been deposited in the Guangdong Provincial Microbial Culture Collection Center on July 8, 2020. The address is: 5th Floor, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou, China. The preservation number is: GDMCCNO.61083 .

The strain isolated by the inventor, and the strain was sequenced, the sequencing result was sequenced in GenBank, and it was found that the similarity to Isaria cicadae was as high as 100%. Combined with morphological identification, the macroscopic and microscopic characteristics of the fungal specimen were found. Consistent with the description of Isaria cicadae, the identification result is P Isaria cicadae Miq.

On this basis, the inventors also artificially cultivated the above-mentioned new strains of Cicada cicadas, and extracted the new strains of Cicada cicada HMGIM-N140534 for solid fermentation products and liquid fermentation broths. All showed significant effects, and further, its fermentation broth also showed significant effects on inhibiting Candida albicans.

The cicada flower strain in the present invention is a new strain with strong vitality collected from the wild. The new strain is isolated from the field and stored in the patent strain collection center. It is confirmed as cicada flower through traditional identification combined with molecular identification. Artificial fermentation can realize large-scale preparation, and has a significant inhibitory effect on anti-tumor and Candida albicans, and is a new strain with development prospects.

Description of drawings

Figure 1 is a picture of wild fruiting bodies of the cicada flower Isaria cicadae.

Figure 2 shows the results of ITS sequencing.

FIG. 3 is a technical roadmap of Test Example 2.

FIG. 4 is a schematic diagram of the extraction method of the crude polysaccharide extract of Test Example 2. FIG.

FIG. 5 is the comparison result of tumor mass in mice in the animal experiment of Test Example 2.

FIG. 6 is the comparison result of tumor volume in mice in the animal experiment of Test Example 2.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

In the first aspect, the present invention provides a new strain of cicada, which is isolated from Wuyi Mountain, Fujian. The original strain is obtained by tissue separation and purification of the fruiting body. It is identified as a new strain of Isaria cicadae by morphology and molecular biology. The strain is named Isaria cicadae HMGIM-N140534, and it has been deposited in the Guangdong Provincial Microbial Culture Collection Center on July 8, 2020. The address is: Guangzhou, China, and the deposit number is: GDMCC No.61083.

The inventors sequenced the strain, performed sequence Blast on the sequencing results in GenBank, and found that the similarity with the cicada flower Isaria cicadae was as high as 100%. Combined with the morphological identification, the macroscopic and microscopic characteristics of the fungal specimen were consistent with the description of Isaria cicadae. The identification result is P Isaria cicadae Miq.

The Isaria cicadae conidia of the present invention is composed of sporophore bundles growing from the head of the cicada pupa. The surface of the parasite is brownish yellow and covered with gray or white hyphae. The sporophore bundles are 1.6-6cm long, branched or unbranched. The upper fertile part is 5-8mm long, 2-3mm in diameter, generally oblong, oval or spindle-shaped or spike-shaped, with a large number of white powdery conidia. The sterile stalk is 1-5cm long, 1-2mm in diameter, yellow to yellowish brown. Conidiophores 5-8×2-3μm, bottle-shaped, inflated in the middle, tapering or suddenly narrowing at the ends, often clustered on fascicles. Conidia 5-14×1.8-3.5μm, oblong, fusiform or nearly semicircular, with 1-3 oil droplets. Scattered on cicada pupae in loose soil. medicinal. Domestic records are distributed in central China, and this strain was collected from Fujian in the southeast.

In a second aspect, the present invention provides an artificial culture method for the above-mentioned new strains of Cicada, including: strain isolation, strain purification, and solid fermentation, wherein the solid fermentation includes packing the solid fermentation medium into a polypropylene strain bag, and after sterilization Cooling, aseptic operation accesses the purified strain, 25 ℃ of constant temperature dark culture 44-46 days, humidity 50%-60%, keep carbon dioxide concentration below 4000ppm, obtain solid fermentation culture, wherein, solid fermentation medium comprises 1: 1 (g/ml) of rice and water.

Among them, the above-mentioned constant temperature and dark culture at 25°C was carried out for 45 days.

Wherein, the above strain isolation includes: wiping the collected wild cicada flower with 75% alcohol on the surface under aseptic conditions, tearing it open, and inserting 0.2-0.5mm×0.2-0.5 mm into the separation medium by aseptic operation. The internal mycelium tissue of mm was cultured in the dark at a constant temperature of 25°C, and the transfer can be carried out after the mycelium has grown to the slant.

Preferably, the time for the above-mentioned mycelia to become full is between 10 days and 15 days.

Wherein, the above-mentioned strain separation medium is a comprehensive PDA medium and 0.5% by weight of silkworm pupae powder.

Specifically: by weight percentage, the strain isolation medium is 20% potato, 2% glucose, 2% agar, 0.3% potassium dihydrogen phosphate, 0.15% magnesium sulfate, trace vitamin B1, 0.5% silkworm pupae powder.

Wherein, the above-mentioned purification includes: transferring the strains infected with the bacteria after separation, culturing in the dark at a constant temperature of 25°C, and picking and transferring the tip hyphae when the hyphae grow but the bacteria have not yet grown.

Wherein, the above-mentioned purified medium is Red Bengal medium.

Specifically: in weight percentage, each 1000ml of purified medium includes: peptone 0.5%, glucose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate (MgSO ₄ ·7H ₂ O) 0.05%, agar 2%, 1/3000 Red Bengal solution 10%, chloramphenicol 0.01%, and the rest is water.

Preferably, the water in the above-mentioned culturing method is distilled water.

In a third aspect, the present invention provides the use of the extract of the solid fermentation product of Isaria cicadae HMGIM-N140534 in anti-tumor.

In a fourth aspect, the present invention provides the use of the extract of the solid fermentation product of Isaria cicadae HMGIM-N140534 in preparing an antitumor drug.

Wherein, the extract of the solid fermented product of Isaria cicadae HMGIM-N140534 is the ethyl acetate extract of the solid fermented product of Isaria cicadae HMGIM-N140534.

Wherein, the tumor is breast cancer.

In a fifth aspect, the present invention provides the use of the extract of the liquid fermentation broth of Isaria cicadae HMGIM-N140534 in anti-tumor.

In a sixth aspect, the present invention provides the use of the extract of the liquid fermentation broth of Cicada flower Isaria cicadae HMGIM-N140534 in the preparation of antitumor drugs.

Wherein, the extract of the liquid fermentation broth of Isaria cicadae HMGIM-N140534 is the crude polysaccharide extract of the liquid fermentation product of Isaria cicadae HMGIM-N140534.

Wherein, the crude polysaccharide extract is the crude polysaccharide extract obtained by extraction with ethyl acetate.

Wherein, the tumor is breast cancer.

In a seventh aspect, the present invention provides the use of the liquid fermentation broth of Isaria cicadae HMGIM-N140534 in inhibiting Candida albicans.

In an eighth aspect, the present invention provides the use of the liquid fermentation broth of the cicada flower Isaria cicadae HMGIM-N140534 in preparing a drug for inhibiting Candida albicans.

In the present invention, it is confirmed through functional experiments that the ethyl acetate extract of the solid fermented product of Isaria cicadae HMGIM-N140534 has significant tumor inhibition effect in vitro, and the crude polysaccharide extract of the liquid fermented product of Isaria cicadae HMGIM-N140534 has significant inhibition in vivo The role of mouse breast cancer cells, while liquid fermentation broth can strongly inhibit Candida albicans.

Studies have proved that the artificially cultured cicada flower of the present invention also has the same or similar effect as the wild cicada flower, especially the cicada flower cultivated by solid fermentation is more advantageous.

Example 1

A new strain of cicada, isolated from Wuyi Mountain, Fujian, the original strain was obtained by tissue separation and purification of fruiting bodies. It was identified as a new strain of cicada Isaria cicadae by morphological and molecular biology, and the strain was named Isaria cicadae HMGIM-N140534, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on July 8, 2020, the address is: Guangzhou, China, and the deposit number is: GDMCC NO.61083.

The cicada flower Isaria cicadae conidia consists of sporophore bundles that grow from the head of the cicada pupa. The surface of the parasite is brownish yellow and covered with gray or white hyphae. The sporophore bundles are 1.6-6cm long, branched or unbranched. The upper fertile part is 5-8mm long, 2-3mm in diameter, generally oblong, oval or spindle-shaped or spike-shaped, with a large number of white powdery conidia. The sterile stalk is 1-5cm long, 1-2mm in diameter, yellow to yellowish brown. Conidiophores 5-8×2-3μm, bottle-shaped, inflated in the middle, tapering or suddenly narrowing at the ends, often clustered on fascicles. Conidia 5-14×1.8-3.5μm, oblong, fusiform or nearly semicircular, with 1-3 oil droplets. Scattered on cicada pupae in loose soil. medicinal. Domestic records are distributed in central China, and this strain was collected from Fujian in the southeast.

Example 2

Identification of Cicada (Cicada) Isaria cicadae Miq.

In July 2014, Cao Renrun and Chen Shengzhen collected a cicada flower (preliminary identification) specimen in Wuyi Mountain, Fujian. The PDA pure culture was obtained by the tissue separation method, the mycelia were collected by liquid culture, dried at low temperature (40° C.), ground with liquid nitrogen, and the DNA genome was extracted by using the Ezup column fungal genome DNA extraction kit to obtain Refrigerate the DNA solution at -20°C for later use. The ITS-PCR experiment was carried out using the universal primers ITS1/ITS4 (ITS1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGATATGC) of the fungal ribosomal intergenic region, and the amplification was carried out on a Biometra PCR machine. The composition of the PCR reaction solution (50 μl in total) was:

TaKaRaTaq(5units/μl)0.25μl

10×PCR Buffer 5μl

4μl of dNTP Mixture (2.5mM each)

DNA template 2μl

Primer 1 (10μmol·L ^-1 ) 5μl

Primer 2 (10μmol·L ^-1 ) 5μl

Sterilized distilled water 28.75μl

The reaction conditions were: 94°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, 30 cycles; 72°C for 10 min. PCR products were sent directly for bidirectional sequencing. Its ITS sequence is shown in Figure 2.

The sequencing results were sequenced in GenBank, and it was found that the similarity to Isaria cicadae was as high as 100%. Combined with morphological identification, the macroscopic and microscopic characteristics of the fungal specimen were consistent with the description of Isaria cicadae, and the identification result was P Isaria cicadae Miq. The strain N140534 was deposited in the Guangdong Provincial Microbial Culture Collection Center (Guangzhou, China) on July 8, 2020, and the deposit number is GCMCC NO: 61083.

Example 3

artificial cultivation

One: culture medium

1. Separation medium (by weight percentage)

Comprehensive PDA (potato 20% + glucose 2% + agar 2% + potassium dihydrogen phosphate 0.3% + magnesium sulfate 0.15% + vitamin B1 trace) + 0.5% silkworm chrysalis powder

2. Purified medium (by weight percentage)

Recipe: Bengal red medium (peptone 0.5% + glucose 1% + potassium dihydrogen phosphate 0.1% + magnesium sulfate (MgSO ₄ 7H ₂ O) 0.05% + agar 2% + 1/3000 Bengal red solution 10% + chloramphenicol Vegetarian 0.01% + distilled water)

3. Solid fermentation medium

Recipe: 50g rice, 50ml distilled water

2. Method:

1. Bacteria isolation

Divide the separation medium into test tubes, sterilize at 0.11MPa atmospheric pressure and 121°C high temperature, high pressure and moist heat for 30min, take it out and cool it and place it on an inclined plane. After the collected wild cicada flowers were wiped with 75% alcohol under sterile conditions, they were torn open, and 0.2-0.5 mm × 0.2-0.5 mm of internal fungal tissue was inserted into the separation medium by aseptic operation. Place in a 25°C incubator for constant temperature and dark cultivation. After the mycelium grows on the slope, it can be transferred. The growth time is about 10-15 days.

2. Strain purification

The purified medium was prepared according to the formula, divided into test tubes, sterilized at 0.11MPa atmospheric pressure and 121°C high temperature, high pressure and moist heat for 30 minutes, and the bacteria infected with bacteria after separation were transferred, and placed in a 25°C incubator for constant temperature dark cultivation, and the bacteria Picking and transfer of tip hyphae are carried out when the filaments are growing and the bacteria are not yet growing.

3. Solid fermentation

A 5-meter-thick polypropylene seed bag (15×30cm) was used to load the solid fermentation medium according to the formula, sterilized at 0.11MPa atmospheric pressure and 121°C high temperature, high pressure and moist heat for 30min, and then took out the successfully purified bacteria by cooling and aseptic operation. kind. Place in a 25°C incubator for constant temperature dark cultivation, humidity 50%-60%, pay attention to keep carbon dioxide concentration below 4000ppm, and cultivate for about 45 days.

Test Example 1

Inhibitory tumor cell function of extract of solid fermented material

1. Method:

1. Preparation of ethyl acetate extract

Adopt the method of embodiment 3 to carry out the solid fermentation of Cicada flower strain, the culture medium is rice and water, after culturing for 45 days, the rice culture medium covered with mycelium is soaked in ethyl acetate, 10 hours later, ultrasonic extraction, ultrasonic 100min, extraction Twice, the two organic phases were combined. After the collected organic phase was subjected to suction filtration, the liquid obtained by suction filtration was rotary evaporated to a state of no droplets with a rotary evaporator, and was stored at low temperature (4° C.) for later use.

At the same time, other wild strains were fermented in the same way and used as a control in advance. Paclitaxel was used as a positive control at a concentration of 0.5 mg/ml.

2. Tumor cell culture

Breast cancer cells MCF-7 were cultured in DMEM medium containing penicillin, streptomycin and 10% fetal bovine serum (FBS). The DMEM cell suspension containing 10% FBS was seeded on a 96-well tissue culture plate at a cell concentration of 2×10 ⁴ cells/ml. Cultures were placed in a tissue culture incubator at 37°C, 5% _CO2 for 4 hours until use.

3. Tumor inhibition test

The ethyl acetate extract was dissolved in DMSO to prepare a stock solution with a concentration of 20 mg/ml, and the stock solution was diluted to a sample solution of 100 μg/ml with DMEM medium containing 10% fetal bovine serum (FBS). Carefully aspirate the cell culture medium from the 96-well plate, and add sample solutions with final concentrations of 100 μg/ml and 200 μg/ml. Three replicate wells per sample. At the same time, set up zero adjustment wells (medium) and control wells (cells, culture medium). Place in a 37°C, 5% _CO2 tissue culture incubator for 48 hours. After the incubation, the cells were collected and mixed with trypan blue 1:1 for exclusion staining. According to the fact that dead cells are stained blue and live cells are not stained because they have intact cell membranes, the number of viable cells is determined with a cytometer, and the sample is repeated three times.

4. Data analysis

Using SPSS.21 special statistical software for statistics, using paired t test, P<0.05 has a significant difference.

2. Results

SPSS.21 special statistical software was used to carry out statistics. At the concentrations of 100 μg/ml and 200 μg/ml, the comparison results of ethyl acetate extract of edible and medicinal bacteria inhibiting MCF-7 cells in vitro are shown in Table 1.

Table 1

As can be seen from Table 1, at the concentrations of 100 μg/ml and 200 μg/ml, the inhibition rates of Cicada HMGIM-N140534 on breast cancer cell MCF-7 were 92.13% and 99.84%, respectively, showing that it has a strong in vitro inhibition of tumor cells effect. At the same time, in the control experiment of in vitro inhibition of tumor cells by ethyl acetate extracts of 14 other edible and medicinal fungi, it surpassed other edible and medicinal fungi tested at the same time. Among them, Inonotus obliquus is a relatively well-known medicinal bacterium, and experiments under the same conditions show that its effect of inhibiting breast cancer cell MCF-7 in vitro is far less than that of the new strain of the present invention.

Test Example 2

In vivo inhibition of tumor cell function of crude polysaccharide extract from liquid fermentation broth

1. Method

The overall method flow is shown in Figure 3, and the details are as follows:

1. Preparation of crude polysaccharide extract

Specifically: by weight percentage, the strain isolation medium is 20% potato, 2% glucose, 2% agar, 0.3% potassium dihydrogen phosphate, 0.15% magnesium sulfate, trace vitamin B1, 0.5% silkworm pupae powder.

The bacterial strain adopts liquid fermentation, and the culture medium is: comprehensive PDA (by weight percentage, potato 20%+glucose 2%+ potassium dihydrogen phosphate 0.3%+magnesium sulfate 0.15%+vitamin B1 trace), fermentation condition is 25 ℃, shake Bottle fermentation, 140rpm, dark culture. After culturing for 7 days, the mycelia were collected by suction filtration, freeze-dried, pulverized, and extracted with ethyl acetate. The crude polysaccharide extract, the specific extraction process is shown in Figure 4.

2. Dosing

The extracted polysaccharide extract was sterile filtered and administered by intraperitoneal injection, and the administration started from the next day after animal modeling, for 30 consecutive days. According to the experimental design in Table 2, low-dose and medium-dose were selected to carry out the experiment, and paclitaxel was selected as the positive control. The animal experimental drug groups are shown in Table 2.

Table 2 Cicada flower inhibits breast cancer animal experiment grouping

3. Tumor cell culture

(1) Resuscitate tumor cells (mouse-derived breast cancer cells 4T1) one week in advance, and observe that the cells must meet the characteristics of rapid growth and strong vitality.

(2) On the day of the mouse modeling, the cells need to be processed first, trypsinized first, counted with a counting plate, and injected subcutaneously at 1.5×105 cells/mice, and the injection volume of each mouse is preferably 0.2ml, so The required concentration is 7.5 x 105. Be careful to move quickly to prevent cells from re-attaching.

(3) Distribute the tumor cells evenly by pipetting into 1.5ml small centrifuge tubes, put them in an ice box (to prevent tumor cells from dying), and bring them to the animal room.

(4) It is necessary to re-mix the cells before injecting tumor cells. The specific operation is to invert the centrifuge tube a few times and gently pipette with a pipette.

4. Animal experiments

The model object was an active mouse with a body weight of 18-22 g. The mouse breast cancer cell 4T1 cells were subcutaneously injected into the anterior back cavity of the mouse at 1.5×10 ⁵ cells/mice. From the next day after modeling, each group was intraperitoneally injected with 0.2 ml of the corresponding liquid every day according to the samples prepared above, and the positive control group was injected twice a week. After 7 days and after tumor formation, the mice were observed and recorded. They were sacrificed 30 days later, weighed and recorded on the first day, sacrificed by cervical dislocation on the second day, and the tumor volume was measured, the tumor mass was weighed, and the drug effect was evaluated.

5. Statistics

Tumor volume V=a*b2/2 (a-the longest diameter of the tumor; b-the shortest diameter of the tumor)

Tumor inhibition rate=(1-Qtest/Qcontrol)*100%(Qtest-tumor mass of test sample; Qcontrol-tumor mass of control)

2. Experimental results

After weighing the tumor weight, measuring the tumor volume and calculating the tumor inhibition rate, the results are shown in Table 3 and Figure 5 and Figure 6.

Table 3 Tumor mass, tumor volume and tumor inhibition rate in animal experiments

The capital letters in the values in the same column in the table represent p<0.01, indicating that the difference is extremely significant. The lowercase letters in the same column value represent p<0.05, the difference is significant.

Figure 5 Comparison of tumor mass in mice in animal experiments

Figure 6 Comparison of mouse tumor volumes in animal experiments

The results from Table 3, Figure 5 and Figure 6 show that the crude polysaccharide extract of the liquid fermentation mycelium of Cicada HMGIM-N140534 showed extremely significant differences in the inhibition of mouse tumor cell growth experiments, and it was dose-dependent Compared with the positive control paclitaxel, the effect of inhibiting tumor growth was more prominent at the moderate dose (100 mg/kg), and the tumor inhibition rate was 84%.

At the same time, the inventor also observed in the experiment that the mice injected with the crude polysaccharide extract of Cicada flower showed a certain neural excitability, especially in the first week of injection, the injection of low and medium doses of polysaccharide made the mice very excited and Dry stools and greatly reduced water intake. However, after a week, the symptoms of neural excitation gradually disappeared, and the final animal experiment results showed a good effect on tumor inhibition, which may be related to the neural excitation of cicada flower, and it is worth further research.

Test Example 3 Test for inhibiting Candida albicans

1. Preparation of culture medium

SDB medium: glucose 40g, peptone 10g, water 1000ml, pH5.6±0.2

SDA medium: glucose 40g, peptone 10g, agar 20g, water 1000ml, pH5.6±0.2

2. Sample preparation

Preparation of positive control substance: Weigh amphotericin, dissolve in an aqueous solution containing 10% Tween, sonicate, centrifuge, filter and sterilize the supernatant, and prepare a 25 μg/mL solution for later use.

3. Preparation of bacterial suspension

The test bacteria were taken out from the -80°C refrigerator and inoculated on fresh slant medium for activation. The fungi were picked with a sterile inoculating loop and inoculated into SDB medium, and cultured with shaking at 28°C and 20r/min for 48h. The fungus was evenly spread on the SDA plate, incubated at 28°C for 48h, and each concentration was repeated 3 times; the concentration of the bacterial solution was determined by the plate counting method.

4. Determination of the zone of inhibition

Using the coating plate method, the counted bacterial solution was diluted to make the final concentration of Candida albicans 10 ⁵ cfu/mL, 200 μL of the prepared bacterial suspension was drawn on the plate with a pipette, and evenly spread with a spreader ; Use a sterilized hole puncher to punch holes evenly on the bacteria-containing plate, pick out the medium in the holes, draw 200 μL of the reference substance/test article into the holes under aseptic conditions, and cultivate the fungus at 28°C for 2 days , observe the size of the inhibition zone. The positive control was amphotericin at 25 μg/mL.

2. Experimental results

The test results show that the inhibition zone of the fermentation broth of the strain of the present invention to Candida albicans is greater than that of the positive control, which shows that the fermentation broth of the strain of the present invention has an obvious inhibitory effect on Candida albicans.

The inventor has further studied the fermentation broth of the strain of the present invention on Staphylococcus aureus (ATCC 6538P), and found that the inhibition zone presented by the fermentation broth to Staphylococcus aureus≤positive control, indicating that the fermentation broth of the strain of the present invention has no obvious effect. The inhibitory effect of Staphylococcus aureus.

The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. The equivalent replacement or change of the solution and its concept shall be included within the protection scope of the present invention.

Claims (10)

1. a new strain of Cicada flower, it is characterized in that, be separated from Wuyi Mountain, Fujian, by carrying out tissue separation to fruit body and purifying to obtain original bacterial strain, be identified as Cicada flower Isaria cicadae new bacterial strain through morphology and molecular biology, this bacterial strain Named the cicada flower Isaria cicadae HMGIM-N140534, it has been deposited in the Guangdong Provincial Microbial Culture Collection Center (Guangzhou, China) on July 8, 2020, and the deposit number is: GDMCC NO.61083. 2. the artificial cultivation method of cicada flower Isaria cicadae HMGIM-N140534, is characterized in that, comprises: bacterial strain separation, bacterial strain purification, and solid fermentation, wherein, solid fermentation comprises that solid fermentation medium is packed into polypropylene strain bag, sterilization. Post-cooling, aseptic operation access to purified strains, 25 ℃ constant temperature dark culture for 44-46 days, humidity 50%-60%, keeping the carbon dioxide concentration below 4000ppm, to obtain a solid fermentation culture, wherein the solid fermentation medium includes 1 : 1 (g/ml) of rice and water. 3. artificial culture method according to claim 2, is characterized in that, above-mentioned bacterial strain separation comprises: after the wild cicada flower collected is wiped surface with 75% alcohol under sterile conditions, tear open, with aseptic operation mode Insert 0.2-0.5mm × 0.2-0.5mm of internal fungus tissue into the separation medium, cultivate in the dark at a constant temperature of 25°C, and transfer after the hyphae are full of slopes. 4. artificial cultivation method according to claim 3 is characterized in that, above-mentioned bacterial strain separation medium is the silkworm chrysalis powder of comprehensive PDA medium and 0.5% by weight; Comprehensive PDA medium is specially: in weight percentage, bacterial strain is separated The medium is 20% potato, 2% glucose, 2% agar, 0.3% potassium dihydrogen phosphate, 0.15% magnesium sulfate, trace vitamin B1, 0.5% silkworm pupae powder. 5. artificial culture method according to claim 2, is characterized in that, above-mentioned purifying comprises: transfer the bacterial classification that is infected with bacteria after separation, 25 ℃ of constant temperature dark culture, when mycelium grows and bacteria have not grown, carry out tip Mycelium picking and transfer. 6. artificial culture method according to claim 2, is characterized in that, wherein, above-mentioned purifying medium is Bengal red medium; Bengal red medium is specifically: by weight percentage, every 1000ml purifying medium comprises: peptone 0.5 %, glucose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, agar 2%, 1/3000 red Bengal solution 10%, chloramphenicol 0.01%, and the rest is water. 7. Use of the extract of the solid fermentation product of Isaria cicadae HMGIM-N140534 in the preparation of antitumor drugs. 8. purposes according to claim 7, is characterized in that, the extract of the solid fermented matter of Cicadae Isaria cicadae HMGIM-N140534 is the ethyl acetate extract of the solid fermented matter of Cicadae Isaria cicadae HMGIM-N140534; and/or , the tumor is breast cancer. 9. Use of the extract of the liquid fermentation broth of Isaria cicadae HMGIM-N140534 in the preparation of antitumor drugs, preferably, the extract of the liquid fermentation broth of Isaria cicadae HMGIM-N140534 is Isaria cicadae HMGIM -The crude polysaccharide extract of the liquid fermentation product of N140534; further preferably, the crude polysaccharide extract is the crude polysaccharide extract obtained by extraction with ethyl acetate. 10. Use of the liquid fermentation broth of Isaria cicadae HMGIM-N140534 in preparing a drug for inhibiting Candida albicans.

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