CN101487022B - Preparation method of fermentation liquid for inhibiting growth of liver cancer cells - Google Patents

Abstract

A preparation method in the technical field of molecular biology, the preparation method comprising the following steps: first step, separation of endophytic fungi: separation of endophytic fungi and purification by mycelium tip cutting method; second step, inducing spore production: Inoculate Trichoderma longibrachiata CCTCC M 208102 on PDA medium for culture, cut off a small piece of PDA medium with hyphae, put it on CMX medium, and cultivate it at room temperature. After 1 to 3 days, spores grow out ; The third step, liquid fermentation culture: pick spores and transfer them to CM liquid medium, 26°C to 28°C, 180rpm shaker culture, stop fermentation after 7 to 8 days, that is, it has the effect of inhibiting the growth of liver cancer cells of fermentation broth. The invention is suitable for industrial scale fermentation to produce a fermentation liquid with the effect of inhibiting the growth of the human liver cancer cell line HepG2 and having high efficiency and low toxicity.

Description

Preparation method of fermentation liquid for inhibiting growth of liver cancer cells

technical field

The invention relates to a preparation method in the field of biotechnology, in particular to a preparation method of a fermentation liquid for inhibiting the growth of liver cancer cells.

Background technique

Liver cancer is a disease with a high mortality rate in the world. The general treatment method is surgical resection. However, in many cases, it occurs centrally, and early metastasis of cancer cells is very easy to occur. This condition limits the application of surgical resection. Therefore, adjuvant therapy with some drugs is needed, but the effect of existing drugs on systemic chemotherapy for liver cancer patients is poor, so finding new effective chemotherapy drugs is of great significance to improve the survival rate of patients with advanced and recurrent liver cancer.

Plant endophytic fungi are a class of fungi that live in the stems and leaves of host plants and complete their life cycle. During the evolution process, the two have formed a stable ecological relationship. The metabolites of endophytic fungi can stimulate plant growth and development, and improve the resistance of host plants to biotic and abiotic stress, especially endophytic fungi can often interact with plants to produce the same or similar metabolites. Considering that microorganisms have the characteristics of rapid reproduction, easy cultivation, and large biomass, it will be a potential drug source to study the isolation of endophytic fungi from medicinal plants and obtain endophytic fungi that produce medicinal substances.

Juglans mandshurica Maxim is a deciduous tree of the Juglandaceae Juglandaceae. It is mainly distributed in the Xiaoxing'an Mountains, Wanda Mountains and Zhangguangcai Mountains in the east of Heilongjiang Province in my country. It is one of the precious tree species in my country. Catalpa is rich in iridoids, which not only have diuretic, hypoglycemic, sluggish and laxative effects, but also have anti-inflammatory, anti-oxidant, and neuroprotective effects; Juglans catalpa has anti-tumor activity, and may be used as an effective Inhibitors of telomerase activity. Juglans extract has an anti-tumor effect by inducing apoptosis. The anti-tumor research of Juglans catalpa indicates that its endophytic fungi may also have the ability to produce anti-cancer substances. However, there is no report on the isolation of endophytic fungi with antitumor effects from Juglans catalpa.

Contents of the invention

The object of the present invention is to provide a method for preparing a fermentation broth that inhibits the growth of liver cancer cells, aiming at the deficiencies of the prior art. In the present invention, pure endophytic fungal strains are obtained by separating and purifying the bark of Juglans catalpa, and are identified by traditional morphological methods and fermentation methods. Low toxicity fermented liquid.

The present invention is achieved through the following technical solutions:

Obtaining of the strain of the present invention: the endophytic fungus strain of Catalpa used in the present invention is isolated from the bark of Juglans catalpa. The separation method is as follows: take the bark of Juglans catalpa and scrape off the floating skin, peel off the surface layer after sterilization, cut out small pieces of tissue, put them on the water agar solid plate medium, and put them in an incubator for cultivation; take the newly grown hyphae from the cut , transferred to the medium for culture, after the colony grows, according to the difference in the shape, color and growth time of the colony, pick the mycelium at the edge of the colony on each plate and connect it to a new medium plate for Isolation and cultivation; from which endophytic fungi with anti-tumor activity were screened out, identified as Deuteromycotina, Hyphospora, Polygonales, Trichoderma longibrachiatum (Trichoderima longibrachiatum), which is now preserved in the Chinese typical Culture Collection Center, deposit number: CCTCC M 208102.

The present invention comprises the following steps:

Step 1. Isolation of endophytic fungi: Isolate endophytic fungi and purify them by cutting mycelium tips;

The second step, inducing spore production: Trichoderma longibrachiata CCTCC M 208102 is inoculated on the potato-agar medium (PDA medium) and cultivated, and a small piece of the PDA medium with mycelia is cut off and placed in CMX for cultivation (Leach, Lang and Yoder, 1982.Journal of General Microbiology, 128: 1719-1729 disclosed), cultured at room temperature, spores grow out after 1 to 3 days;

The third step, liquid fermentation culture: pick spores and transfer to CM liquid medium (Leach, Lang and Yoder, 1982.Journal of General Microbiology, 128:1719-1729), 26°C to 28°C, 180rpm Vibrating culture on a shaking table, stop the fermentation after 7 to 8 days, and obtain the fermentation liquid with the effect of inhibiting the growth of liver cancer cells.

The method for isolating endophytic fungi in the first step is as follows: take the bark of Juglans catalpa and scrape off the floating skin, rinse it with tap water for 20 minutes, place it on a sterile ultra-clean workbench, rinse it 3 to 4 times with sterile water, and then use Soak in 75% alcohol by volume for 3 to 5 minutes, then sterilize it with 1% NaClO solution for 20 minutes by mass percent, wash it repeatedly with sterile water for 4 to 5 times, and blot the bark dry with sterile paper. Sterile water, cut off the black surface layer, cut out 0.5cm ³ small pieces, put them on the water agar solid plate medium, and put them in a 25°C incubator for cultivation.

The purification method in the first step is as follows: take the newly grown mycelium at the cut, transfer it to fresh PDA medium in time for cultivation, and after the colony grows, according to the difference in colony shape, color and growth time The mycelium at the edge of the colony on each plate is picked and connected to a new PDA plate for isolation and culture, and then the tip of the cultured mycelium is cut off, transferred to a new medium for culture, and cut 3 to 5 times. Purified strains were obtained.

In the second step, the PDA medium consists of: 200g fresh potato boiled juice, 20g glucose, solid medium plus 1.5% agar, and distilled water until the total volume is 1000ml.

The biological characteristics of a kind of endophytic fungus Trichoderma longibranchus CCTCC M208102 isolated from the bark of catalpa of the present invention are as follows:

1. Morphological characteristics of the colony: On the solid medium, the colony produces white fluffy mycelium, which is distributed radially, and the mycelium is developed. In the liquid culture medium, uniform round bacterial balls are formed. The surface of the bacterial ball is rough and filamentous, and the texture is Loose, fermented liquid is yellow.

2. Morphological characteristics of spores: the spores are green in naked eyes, and ellipsoid in microscopic observation, with smooth surface. The main shaft of the conidiophores is long and strong, the secondary branches are short, and the branches are altar-shaped, the stalks are often solitary, and the spores are born on the top of the secondary branches.

3. Culture characteristics: The bacterium grows rapidly on the PDA plate. Colonies with a diameter of about 2 cm can be seen within 2 days of inoculation. The hyphae in the front view are white velvet-like, growing radially outward, and the bacterium secretes yellow pigment substances, making The opposite side of the culture medium is evenly light yellow. When cultured on CMX medium, spores can grow quickly, the sporulation clusters are loose, and the spores are green. After being fermented in the liquid culture medium, the thalline is round, the surface is rough and silky, the texture is loose, the fermentation liquid is light yellow, and has an aromatic smell.

4. The strain has good stability: the strain has been subcultured for 5 times, the fermentation broth still has good activity, the fermentation broth has good thermal stability, and it still has good activity when treated below 80°C for 2 hours . It can be stored at -20°C for more than 3 months.

The invention obtains pure endophytic fungus strains by separating and purifying from the bark of Juglans catalpa, and its fermentation liquid has high efficiency and low toxicity, has the effect of inhibiting the growth of human liver cancer cell strain HepG2, and shows better cancer cell selectivity. At the same time, considering that the components of the fermented liquid have better water solubility, the disadvantage of poor water solubility of current anticancer drugs is avoided, and the invention has good development prospects.

The bacterial strain used in the present invention is:

Classification and naming of strains: Trichoderma longibrachiatum (Trichoderima longibrachiatum),

Name of depository unit: China Center for Type Culture Collection (CCTCC),

Deposit date: June 27, 2008,

Deposit number: CCTCC M 208102.

Description of drawings

The morphological figure of Fig. 1 Trichoderma longibrachiata CCTCC M 208102 on the PDA plate.

Fig. 2 Microscopic view of fungal spores of Trichoderma longibrachiata CCTCC M 208102.

Fig. 3 Trichoderma longibrachiata CCTCC M 208102 fungal fermentation broth diagram.

Fig. 4 Schematic diagram of the inhibition rate of HepG2 and HL-7702 proliferation by fermentation broth with different dilution multiples.

Detailed ways

The embodiments of the present invention are described in detail below: the present embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and processes are provided, but the protection scope of the present invention is not limited to the following embodiments. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed.

Example 1

Isolation of endophytic fungi

Take the bark of Juglans catalpa and scrape off the floating skin, rinse it under tap water for 20 minutes, place it on a sterile ultra-clean workbench, rinse it with sterile water 3 to 4 times, soak it with 75% alcohol by volume for 3 to 5 minutes, and then Sterilize with 1% NaClO solution for 20 minutes, then use sterile water to wash repeatedly 4 to 5 times, blot the sterile water on the bark with sterile paper, cut off the black surface, and cut out 0.5cm×0.5 The small pieces of cm are placed on the water agar solid plate medium and cultured in a 25°C incubator.

Take the newly grown hyphae at the incision, and transfer them to fresh PDA medium for culture in time. After the colonies grow, pick the colony edges on each plate according to the differences in colony shape, color and growth time. The hyphae at the place were inoculated on a new PDA plate for isolation and culture. Cut the tip of the cultured hyphae, transfer it to a new medium for culture, and cut it 3 to 5 times to obtain purified mycelium. After the strains were numbered, they were transferred to PDA slant medium and cultured in a 28°C incubator for 4-5 days. During this period, the growth morphology of the strains was observed and recorded, and then stored in a 4°C refrigerator for later use.

Example 2

Identification of Endophytic Fungi in Catalpa

Colony morphology: On the solid medium, the colony produces white fluffy mycelium, which is distributed radially, and the hyphae are well developed, as shown in Figure 1. Uniform round fungus balls are formed in the liquid culture solution, the surface of the fungus balls is rough and filamentous, the texture is loose, and the fermentation broth is yellow.

Spore Morphology: The spores are green in naked eyes, and ellipsoid in microscopic observation, with smooth surface. The main shaft of the conidiophores is long and strong, the secondary branches are short, and the branches are altar-shaped, the stalks are often solitary, and the spores are born on the top of the secondary branches. Under the microscope, the main axis of the conidiophores is long, the secondary branches inserted are short, and the secondary branches are few, and the branches are altar-shaped and the stalk is often solitary; in addition, the conidia are single-celled and oval . as shown in picture 2.

Culture characteristics: The bacterium grows rapidly on the PDA plate. Colonies with a diameter of about 2 cm can be seen within 2 days of inoculation. The hyphae in the front view are white and fluffy, and grow radially outward. The bacterium secretes yellow pigments, making the medium The opposite side is evenly light yellow. When cultured on CMX medium, spores can grow quickly, the sporulation clusters are loose, and the spores are green. After being fermented in the liquid culture medium, the thalline is round, the surface is rough and filamentous, the texture is loose, and the fermentation liquid is light yellow, as shown in Figure 3, and has an aromatic smell.

The strain has good stability, subcultured for 5 times, the fermentation broth still has good activity, the fermentation broth has good thermal stability, and it still has good activity when treated below 80°C for 2 hours. It can be stored at -20°C for more than 3 months.

After identification, the endophytic fungus belongs to the subphylum Deuteromycetes, the class Hyphospora, and the order Trichoderma, and the genus Trichoderma longibrachiatum (Trichoderima longibrachiatum).

Example 3

Preparation of Fermentation Broth Inhibiting the Growth of Liver Cancer Cells

Cultivate the isolated Trichoderma longibrachiata CCTCC M 208102 on a PDA plate, then cut a small piece of the PDA medium with mycelium, put it on the CMX medium, and cultivate it at room temperature. After two days, spores can be seen grow out. Then pick the spores and transfer them to a Erlenmeyer flask equipped with 250ml of CM liquid medium, culture them on a shaking table at 26°C to 28°C at 180rpm, stop the fermentation after 7-8 days, and obtain the spores that have the effect of inhibiting the growth of human liver cancer cell line HepG2 fermentation broth. The bacterial fluid was collected for preliminary analysis of antitumor activity.

Example 4

In Vitro Anticancer Test of Fungal Fermentation Broth

The human liver cancer HepG2 cell line (purchased from the Institute of Molecular and Genetics, School of Life Sciences, Zhejiang University of Traditional Chinese Medicine) cultivated with RPMI 1640 medium (purchased from Gibco) containing 10% by weight of fetal bovine serum (FBS) and the human Normal liver cells HL-7702 (purchased from the Institute of Biochemistry and Cells, Chinese Academy of Life Sciences) were inoculated in a 96-well plate at 1×10 ⁴ cells/well, cultured in an incubator at 37°C and 5% CO ₂ , and after 24 hours, a series of gradients ( The dilution times of the fermentation broth were 40, 20, 10, 5) the diluted fermentation broth treated the cells, and each concentration had 6 replicate wells. The unfermented CM culture fluid was used as a blank control, and curcumin was used as a positive control. Cultivate in an incubator with 5% _CO2 at ℃, after culturing for 24 hours, add 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) and incubate for 4 hours Afterwards, the supernatant was discarded by suction, 150 μL/well dimethyl sulfoxide (DMSO) was added to shake for 10 min, and the OD value at 570 nm was detected on an enzyme-linked immunosorbent monitor. repeat three times.

It can be seen from Figure 4 that the MTT method combined with the time-lapse microscope observation records show that the fungal fermentation broth has a strong inhibitory effect on the human liver cancer cell line HepG2. Although the fermentation broth also has a certain inhibitory or killing effect on normal cells, it is not as good as that on liver cancer Strong cell action.

Claims (2)

1. a preparation method for inhibiting the growth of liver cancer cell fermentation liquid, is characterized in that, comprises the steps: The first step, inducing spore production: Trichoderima longibrachiatum (Trichoderima longibrachiatum) CCTCC M208102 is inoculated on the PDA medium for cultivation, and a small piece of the PDA medium with mycelia is cut off, placed on the CMX medium, and kept at room temperature Under culture, spores grow out after 1 to 3 days; The second step, liquid fermentation culture: pick the spores and transfer them to the CM liquid medium, 26°C to 28°C, 180rpm shaker culture, stop the fermentation after 7 to 8 days, and obtain the liver cancer cell growth inhibitor fermentation broth. 2. the preparation method of the fermented liquid that suppresses liver cancer cell growth according to claim 1 is characterized in that, in the first step, PDA culture medium consists of: the juice that 200g fresh potatoes boil, 20g glucose, solid culture medium adds 1.5% Agar, add distilled water to a total volume of 1000ml.

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