CN1618951A

CN1618951A - Brachyplast bundle spore mold

Info

Publication number: CN1618951A
Application number: CN 200310116787
Authority: CN
Inventors: 杨丽源; 陈有为; 夏国兴; 余斌; 李绍兰; 周斌; 李治滢; 张琦
Original assignee: JINHUA XIAYANG BIOTECHNOLOGY CO Ltd
Current assignee: JINHUA XIAYANG BIOTECHNOLOGY CO Ltd
Priority date: 2003-11-21
Filing date: 2003-11-21
Publication date: 2005-05-25
Anticipated expiration: 2023-11-21
Also published as: CN1244683C

Abstract

A calarisporium sp.Ym35852 (CCTCC No.M203064) is disclosed, which can be used as initial bacterial strain to ferment the endofunguses in rauwolfia plant for obtaining ceramide compound used to prepare the medicines for treating cancer or dermatopathy.

Description

Brachyplast bundle spore mould

Technical field

The present invention relates to microbial technology field, particularly relate to a kind of its fermented product and have microorganism medicinal, nourishing function.

Background technology

Ceramide (Ceramide) compounds; the compound medicinal, nourishing function that is that a class has; wherein part of compounds can preserve moisture and suppress the melanochrome generation, prevent pachylosis, is used for cosmetic or dermatology aspect skin, hair, nail and eyelashes are handled or treated and protect.This compounds obtains not appear in the newspapers as yet with microbial fermentation mode.

Summary of the invention

The purpose of this invention is to provide a kind of its fermented product and have microorganism-brachyplast bundle spore mould medicinal, nourishing function.

Mould Ym 35852 Calcarisporiumsp.Ym 35852 of brachyplast bundle spore mould called after brachyplast bundle spore of the present invention now are deposited in specified depositary institution of State Intellectual Property Office, and deposit number is CCTCC NO:M203064.

The mould fungus strain of brachyplast bundle spore of the present invention separates acquisition from apocynaceae plant Rauvolfia yunnanensis Rauwolfiayunnanaensis Tsiang plant living body.Now be deposited in the preservation of depositary institution of Patent Office of State Intellectual Property Office, depositary institution's title: Chinese typical culture collection center, preservation date are on August 6th, 2003, and deposit number is M203064.

The microscopic morphology feature of YM35852 bacterial strain of the present invention:

1, hyphae colorless has barrier film asexual generation.Conidiophore is coarse, colourless or pigment arranged.Bigger stalk is taken turns dendritic branch, and nascent branch becomes elongated sporogenous cell usually; Conidium (sympodulospore) is colourless smooth, monospore, Long Circle, on single the warty tooth at living sporophore branch estranged top, form evacuate bunch.5.0～6.25 * 2.5～3.75 microns.

2, sexual generation is not found.

The solid culture feature of brachyplast bundle spore mould of the present invention:

1, the PDA substratum was cultivated 4 days for 28 ℃, and colony diameter 47mm spread in 7 days.The bacterium colony circle, beige, central authorities are with yellow slightly, and suede is cotton-shaped, and there is powdery in the later stage, and densification is thicker, and neat in edge has protuberance.Back side chocolate.

2, malt extract medium was cultivated 4 days for 28 ℃, colony diameter 38mm, and 7 days 49mm spread in 11 days.The bacterium colony circle, canescence is with a small amount of black particle, the suede powdery, the edge is irregular, and is flat tightr.Back side black.

3, examine the Bake substratum and cultivated 4 days for 28 ℃, colony diameter 38mm, 7 days 54mm spread in 11 days.The bacterium colony subcircular, the edge is irregular, beige, suede is cotton-shaped, closely.Back side brown.

Brachyplast bundle spore mould YM35852 strain liquid fermentation culture of the present invention:

1,5～7 days substratum PDA shake-flask culture time.28 ℃ ± 2 ℃ of culture temperature.

2, fermentation culture feature: cultivated the 1st day, bacterial classification has odd white hypha point; Cultivated the 2nd day, and covered with white hypha; Cultivated the 3rd day, the fermented liquid mycelium is intensive, and it is filbert that mycelium is; Cultivated the 4th day, and covered with pitch black brown mycelium, the fermented liquid thickness was cultivated the 5th～7 day, and the intensive one-tenth of fermented liquid mycelium is block, the chocolate mycelium.

3, the fermentation of YM35852 strain liquid was carried out chemical extraction, column chromatographic isolation and purification to fermented liquid after 5～7 days, obtained 1 anti-skin pathogenic bacterium and people's myeloplast activity of tumor cells compound.The present structure of gained compound is still indeterminate.

Brachyplast bundle spore mould YM35852 strain liquid fermenting extraction process of the present invention is: bacterial classification → test tube slant → seed culture → fermentation culture → fermented product extraction → column chromatography → mixing cpd → purifying → monomeric compound.

In the above-mentioned steps, fermentation raw material is potato glucose substratum (PDA).This technology is suitable for imperfect fungi Moniliaceae filamentous fungus quasi-microorganism.

In the above-mentioned YM35852 liquid-state fermentation technology, the fermentation condition parameter is as follows:

(1) fermentation mode: liquid state fermentation.

(2) strain inclined plane: the test tube slant spawn culture adopts potato glucose solid medium (solid PDA).

(3) seed culture: seed culture is potato glucose liquid nutrient medium (liquid PDA).

Potato glucose substratum (PDA) preparation: get fresh potato 200 grams, peeling is cut into small pieces, and adds 1 liter in water and boils 30 minutes, filters and abandons potato, and filter adds water and complement to 1 liter night, adds glucose 20 grams, the PH nature.

Add agar 15 grams in the above-mentioned potato glucose substratum, be solid PDA substratum.

(4) fermentation culture: the substratum of fermentation is potato glucose liquid nutrient medium (liquid PDA), the PH nature.

(5) incubation time: test tube slant strain activation and culture 5 days, seed shaking table incubation time 72～96 hours, fermented incubation time 5～7 days.

(6) culture temperature: 28 ℃ ± 1 ℃ of test tube slant spawn culture temperature, 28 ℃ ± 2 ℃ of seed shaking table culture temperature, fermentation culture temperature are 28 ℃ ± 2 ℃.

(7) fermented product extracts: fermentation finishes, and collects fermented liquid and thalline respectively.Chemical extraction method is routinely extracted 3 merging respectively with industrial hexyl hexanoate and is drained, and gets paste and slightly carries compound.

(8) separating compound: adopt silica gel (Silica G) column chromatography method separation and purification to obtain ceramide (Ceramide) compound.

The present invention is the endogenetic fungus in the employing Radix Rauvolfiae plant materials obtains the ceramide compound as fermentation a starting strain.It is to be raw material with potato glucose substratum (PDA), by the liquid state fermentation mode, adopts the combinatorial chemistry means, the strain excellent YM35852 brachyplast bundle mould genus of spore (Calcarisporium sp.) of separation and Extraction ceramide (Ceramide) effective constituent.This bacterial strain is an important microbe of seeking human antitumor and dermatosis medicinal ingredients Biological resources.With the ceramide of this bacterial strain fermentation liquor separation and Extraction is the replenishing water and preserving moisture composition of the high-efficient and lasting that comes out newly developed, different fully with the now general composition principle of preserving moisture, it can be close with the keratoderma structure, can very fast infiltration skin, with the moisture combination in the stratum corneum, form reticulated structure, form one deck water-retaining film at skin surface, supply water for cell paste part and nutrient in moisture-locking, be particularly suitable for dryness, maturation or by the skin of sun damage.The external application cream series products that can contain ceramide by use reaches purpose that prevents anaphylaxis dermatosis and the effect that suppresses the melanochrome foxiness.Can induce liver tumor Bel7402 apoptosis: adopt mtt assay, fluorescent dye, flow cytometry, agarose gel electrophoresis, Western blot. method, the result shows: the ceramide that is extracted is 14.28 μ mol-L to the cytostatic IC50 value of Bel7402 ^-1Nucleus concentrates, and fluorescence obviously strengthens; DNA presents typical case's " ladder type " to be changed; The p53 protein expression is on a declining curve; The Bcl-2 protein expression obviously reduces; The Bax protein expression does not have obvious change.

Description of drawings

Fig. 1 is YM35852 radicula byssoidea and conidiophore (200X).

Fig. 2 is a YM35852 fungus conidium form (400X).

Embodiment

The embodiment of the invention:

Get numbering YM35852 bacterial classification, under aseptic condition (sterilisable chamber or Bechtop),, transfer and (in 18 * 180mm), put interior 28 ℃ ± 1 ℃ activation culture of incubator 5 days in sterilized solid PDA substratum test tube with a little bacterial classification of inoculating needle picking.

Take out 5 days bacterial classification of activation, under aseptic condition, insert the good bacterial classification of activation in sterilized seed liquid PDA substratum, cultivated 72～96 hours, be the YM35852 seed at 28 ℃ ± 2 ℃ following shaking tables with the same manner.

500 milliliters of glass triangle bottles are adopted in fermentation, 100 milliliters of the liquid PDA substratum that prepare of packing into, and 15 pounds of sterilizations were taken out to be cooled to 30 ℃ after 30 minutes.Under aseptic condition, get the YM35852 seed, be inoculated in 500 milliliters of glass triangle bottles that 100 milliliters of liquid PDA substratum are housed, the seed inoculum size is 5～10% (v/w), puts 28 ℃ ± 2 ℃ aerated culture 5～7 days.During fermentation, note the proving room temperature variation, check to have or not the microbiological contamination phenomenon.

Fermentation finishes, and collects fermented liquid and thalline respectively.With industrial hexyl hexanoate extractive fermentation liquid 3 times, merging is drained; Thalline is with industrial hexyl hexanoate soaked overnight 3 times, and merging is drained.Get paste and slightly carry compound.

Separating compound: slightly carry compound and adopt silica gel (Silica G) column chromatography, use trichloromethane respectively: methyl alcohol and sherwood oil: hexyl hexanoate=95: 5 wash-outs, separation and purification obtains ceramide (Ceramide) compound.This compound is pressed 0.2mg/ml dosage, and people's bone marrow leukemia cells growth inhibition ratio is reached 73.33%.

The 18SRNA partial sequence of bacterial strain of the present invention is analyzed as follows (700bp):

TGTTACGAACACCGCAACAGCTTTCGCTGGCGGACGGACTATATCTTAGGCCTGCGTGCGCAAGCCCA

CCCCTATCTAGTCTCTGAACCTTCCCCCTATAACGAGGGGGCTTGGCTGCGAGTGGCCCAACCCTCCA

GCAGTGTTACCGTACCCGGAGCAGTTACGCCCGGCCGCCGCGCCTATTTCTAGGGCGGTTTGGTCGCT

GGAGGTTTAAGGGTTTTCCCGCACTTTAGGGATGTCGCCGCTCCGTCTAGGGGAGCGACTAGCGGTCT

TAGGTACACCACCTTCCTGTAGACACAGGCCGCTTTCGGGGGCAGAGACGCTGCGGAATCTTGTTTAC

CCCTGGTGGTGCCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCAGAACCCA

AAGACTTTGATTTCTCGTAAGGTGCCGAACGAGTCAAAAAATAACATCGTCCGATCCCTAGTCGGCAT

AGTTTATGGTTAAGACTACGACGGTATCTGCAGGTACCCCGCGGCTTTTCAGCGGCGGCTCGACTATA

TTTTAAGGGCGCTACAGCGGAGCGCTCCCACCGACATTTAGTCTGTGAACTGCATCCTGGCGCCCCGA

GGCGCCGTACGGACTTGGCTGCGGATTGTCCAATCCCTCACACTATTACGACTCGCCGCTATTTCTAG

CCGCGAGGTGTGGTGAGGGC。

Claims

1, a kind of brachyplast bundle spore mould is characterized in that the mould Ym 35852Calcarisporium of called after brachyplast bundle spore sp.Ym 35852, now is deposited in specified depositary institution of State Intellectual Property Office, and deposit number is CCTCC NO:M203064.

2, according to the described brachyplast bundle of claim 1 spore mould, it is characterized in that the 18SRNA partial sequence of described bacterial strain is analyzed as follows:

TGTTACGAACACCGCAACAGCTTTCGCTGGCGGACGGACTATATCTTAGGCCTGCGTGC

GCAAGCCCACCCCTATCTAGTCTCTGAACCTTCCCCCTATAACGAGGGGGCTTGGCTGC

GAGTGGCCCAACCCTCCAGCAGTGTTACCGTACCCGGAGCAGTTACGCCCGGCCGCCGC

GCCTATTTCTAGGGCGGTTTGGTCGCTGGAGGTTTAAGGGTTTTCCCGCACTTTAGGGA

TGTCGCCGCTCCGTCTAGGGGAGCGACTAGCGGTCTTAGGTACACCACCTTCCTGTAGA

CACAGGCCGCTTTCGGGGGCAGAGACGCTGCGGAATCTTGTTTACCCCTGGTGGTGCCC

TTCCGTCAATTTCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCAGAACCCAAAGAC

TTTGATTTCTCGTAAGGTGCCGAACGAGTCAAAAAATAACATCGTCCGATCCCTAGTCG

GCATAGTTTATGGTTAAGACTACGACGGTATCTGCAGGTACCCCGCGGCTTTTCAGCGG

CGGCTCGACTATATTTTAAGGGCGCTACAGCGGAGCGCTCCCACCGACATTTAGTCTGT

GAACTGCATCCTGGCGCCCCGAGGCGCCGTACGGACTTGGCTGCGGATTGTCCAATCCC

TCACACTATTACGACTCGCCGCTATTTCTAGCCGCGAGGTGTGGTGAGGGC。

3, according to the described brachyplast bundle of claim 1 spore mould, it is characterized in that the solid culture of described bacterial strain is characterized as:

One, the PDA substratum was cultivated 4 days for 28 ℃, and colony diameter 47mm spread in 7 days, the bacterium colony circle, and beige, central authorities are with yellow slightly, and suede is cotton-shaped, and there is powdery in the later stage, and densification is thicker, and neat in edge has protuberance, back side chocolate;

Two, malt extract medium was cultivated 4 days for 28 ℃, colony diameter 38mm, and 7 days 49mm spread in 11 days, the bacterium colony circle, canescence is with a small amount of black particle, the suede powdery, the edge is irregular, and is flat tightr, back side black;

Three, examine the Bake substratum and cultivated 4 days for 28 ℃, colony diameter 38mm, 7 days 54mm spread in 11 days, the bacterium colony subcircular, the edge is irregular, beige, suede is cotton-shaped, closely, back side brown.