CN101487022B - Preparation of fermentation liquor for inhibiting liver cancer cell growth - Google Patents
Preparation of fermentation liquor for inhibiting liver cancer cell growth Download PDFInfo
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- CN101487022B CN101487022B CN2008100412148A CN200810041214A CN101487022B CN 101487022 B CN101487022 B CN 101487022B CN 2008100412148 A CN2008100412148 A CN 2008100412148A CN 200810041214 A CN200810041214 A CN 200810041214A CN 101487022 B CN101487022 B CN 101487022B
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Abstract
The invention discloses a preparation method in the technical field of molecular biology, which comprises following steps: (1) separation of endophytic fungi: the endophytic fungi are separated and purified by adopting a hyphal tip cutting method; (2) induction of spore bearing: long-branch trichoderma with the preservation number of CCTCC M 208102 is inoculated onto a PDA culture medium for being cultivated, a block of the hyphe-bearing PDA culture medium is cut, placed on a CMX culture medium and cultivated at room temperature, and spores are generated after 1 to 3 days; and (3) fluid fermented cultivation: the spores are selected, transplanted into a CM-containing fluid culture medium and oscillating cultivated on a shaking table at the temperature of 26 to 28 DEG C and the speed of 180rpm, and fermentation is terminated after 7 to 8 days, thus obtaining a fermented fluid with the inhibitive action of liver cancer cell growth. The preparation method is suitable for being used in the industrially scaled fermented production of the fermented fluid, which has the inhibitive action of HepG2 growth of liver cancer cell strains as well as high efficiency and low toxicity.
Description
Technical field
The present invention relates to a kind of preparation method of biological technical field, particularly, relate to a kind of preparation of fermentation liquid method that suppresses liver cancer cell growth.
Background technology
Liver cancer is the very high disease of whole world mortality ratio, and general methods of treatment is to adopt excision, but mostly is the center generation in many cases, and the early stage transfer of cancer cells very easily takes place, and this state of an illness has limited the application of excision.Therefore the assisting therapy that needs some medicines, but existing medicine is relatively poor to the systemic chemotherapy effect that hepatocarcinoma patient carries out, and therefore seeks new effective chemotherapeutic agent the survival rate that improves late period and recurrent hepatic cancer patient is significant.
Plant endogenesis epiphyte be class existence in the host plant cauline leaf and finish the fungi of its life cycle, the two has formed stable ecological relationship in evolutionary process.The metabolite of endogenetic fungus can grow by stimulating plant, improve host plant to the resistivity, particularly endogenetic fungus of biological and abiotic stress often can with plant interaction, produce same or analogous meta-bolites.Consider that microorganism has breeding rapidly, cultivate easily, characteristics such as biomass is big, research separates endogenetic fungus from medicinal plant, obtain to produce the endogenetic fungus of medicinal substance, will be a kind of very potential medicament sources.
Juglans mandshurica (Juglans mandshurica Maxim) is the deciduous tree of Juglandaceae white walnut, mainly is distributed in mountain areas such as the Xiaoxinanlin Mountains, east, China Heilongjiang Province, Wanda Mountain and Zhang Guangcai mountain range, is one of China's rare tree.Contain abundant iridoid in the Chinese catalpa, diuresis, hypoglycemic and slow and discharge function are not only arranged, also have anti-inflammatory, anti-oxidant, neuroprotective; Juglans mandshurica has anti-tumor activity, might be as effective telomerase activation inhibitor.Thereby the juglans mandshurica extracting solution plays antitumor action by cell death inducing.The antitumor research of juglans mandshurica indicates that its endogenetic fungus also may have the ability of producing cancer-resisting substance.Yet, the present report of also from juglans mandshurica, not isolating endogenetic fungus with antitumous effect.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of preparation of fermentation liquid method that suppresses liver cancer cell growth is provided.Utilization of the present invention obtains pure endogenetic fungal bacterial strain from juglans mandshurica bark part from purifying; and by traditional morphological method; fermentation process is identified, is fit to the industrial scale fermentative production and has the effect of inhibition human hepatoma cell strain HepG2 growth and the fermented liquid of high-efficiency low-toxicity.
The present invention is achieved by the following technical solutions:
The acquisition of bacterial strain of the present invention: Chinese catalpa endogenetic fungal bacterial strain used in the present invention makes by separating in the juglans mandshurica bark.Separation method is: get the juglans mandshurica bark and scrape off outer skin, the top layer of pruning after the sterilization cuts and organizes fritter, places on the water agar solid plate substratum, puts into incubator and cultivates; Get the mycelia that incision newly grows, be forwarded on the substratum and cultivate, after treating that bacterium colony grows, according to the difference of colonial morphology, color and the difference that grows the time, each dull and stereotyped mycelia that goes up the colony edge place of picking is connected to and carries out separation and Culture on the new culture medium flat plate respectively; Therefrom filter out endogenetic fungus, through being accredited as Deuteromycotina, hyphomycetes with anti-tumor activity, Moniliales, trichoderma long shoot wood mould (Trichoderima longibrachiatum) now is kept at Chinese typical culture collection center, and preservation is numbered: CCTCC M 208102.
The present invention includes following steps:
The separation of the first step, endogenetic fungus: separate endogenetic fungus and adopt hyphal tip cutting method to carry out purifying;
Second goes on foot, induces spore to produce: the mould CCTCC M 208102 of long shoot wood is inoculated into potato-nutrient agar (PDA substratum) goes up cultivation, there is the PDA substratum of mycelia to downcut a fritter with long, be put in CMX substratum (Leach, Lang and Yoder, 1982.Journal of General Microbiology, open among the 128:1719-1729) on, cultivate under the room temperature, there is spore to grow after 1 to 3 day;
The 3rd step, liquid fermentation and culture: the picking spore is transferred to CM liquid nutrient medium (Leach is housed, Langand Yoder, 1982.Journal of General Microbiology, open among the 128:1719-1729) in, 26 ℃ to 28 ℃, 180rpm shaking table shaking culture, stuck fermentation after 7～8 days promptly gets the fermented liquid with the effect of inhibition liver cancer cell growth.
The method of separating endogenetic fungus in the described the first step is as follows: get the juglans mandshurica bark and scrape off outer skin, use the tap water flushing to be placed on aseptic Bechtop in 20 minutes, with aseptic water washing 3 to 4 times, it is 75% alcohol-pickled 3 to 5 minutes with volume percent again, be 1% NaClO solution sterilization 20 minutes with mass percent then, re-use the sterilized water repetitive scrubbing 4 to 5 times, blot corticole sterilized water with aseptic paper, the melanic epipedon of pruning cuts 0.5cm
3Fritter, place on the water agar solid plate substratum, put into 25 ℃ of incubators and cultivate.
Purification process in the described the first step is as follows: get the mycelia that incision newly grows, in time be forwarded on the fresh PDA substratum and cultivate, after treating that bacterium colony grows, according to the difference of colonial morphology, color and the difference that grows the time, each dull and stereotyped mycelia that goes up the colony edge place of picking is connected on the new PDA flat board and carries out separation and Culture respectively, cut the tip of cultivating mycelia then, move on the new substratum and cultivate, cut the bacterial strain that can obtain purifying for 3 to 5 times.
The PDA substratum consists of in described second step: the juice that the fresh potato of 200g boils, and 20g glucose, solid medium add 1.5% agar, and adding distil water to cumulative volume is 1000ml.
The biological characteristics of a kind of mould CCTCC M208102 of endogenetic fungus long shoot wood that separates from the Chinese catalpa bark of the present invention is as follows:
1, colony morphology characteristic: on the solid medium, bacterium colony produces white velvet-like mycelium, distributes radially, and the mycelia prosperity forms uniform circular bacterium ball in the liquid medium, and bacterium ball surface irregularity has thread, and quality is loose, and it is yellow that fermented liquid is.
2, spore shape feature: it is green that naked eyes are seen spore, and the visible spore of microscopic examination is a spheroid shape, smooth surface.The main shaft of conidiophore is long and sturdy, and secondary branch is short, and branch is urniform, and handle obstructs usually Dan Sheng, and spore and is born in secondary ramose top.
3, cultural characteristic: this bacterium grows on the PDA flat board rapidly, and inoculating 2 days is the about 2 centimetres bacterium colony of visible diameter, and the anterior view mycelia is that white is velvet-like, radial to outgrowth, and thalline secretion xanthein class material makes the substratum reverse side evenly be faint yellow.Cultivate on the CMX substratum and can grow spore very soon, it is bunch loose to produce spore, and spore be a green.Fermentation back thalline is rounded in liquid medium, and surface irregularity has thread, and quality is loose, and it is faint yellow that fermented liquid is, and a kind of aromatic odour is arranged.
4, bacterial strain has stability preferably: bacterial strain succeeding transfer culture 5 times, and fermented liquid still has good active, and fermented liquid has thermostability preferably, is handling 2 hours below 80 ℃, still has active preferably.Can preserve more than 3 months at-20 ℃.
The present invention obtains pure endogenetic fungal bacterial strain by utilizing from juglans mandshurica bark part from purifying, and its fermented liquid high-efficiency low-toxicity has the effect that suppresses human hepatoma cell strain HepG2 growth, demonstrates cancer cell selectivity preferably.Consider that simultaneously the fermented liquid composition has better water solubility, avoided the drawback of present cancer therapy drug poorly water-soluble, have the excellent development prospect.
Bacterial strain used in the present invention is:
The strain classification name: trichoderma long shoot wood mould (Trichoderima longibrachiatum),
Depositary institution's title: Chinese typical culture collection center (CCTCC),
Preservation date: on June 27th, 2008,
Preserve number: CCTCC M 208102.
Description of drawings
The morphology figure of Fig. 1 long shoot mould CCTCC M 208102 of wood on the PDA flat board.
The mould CCTCC M 208102 fungal spore microscope figure below of Fig. 2 long shoot wood.
The mould CCTCC M 208102 fungal fermented filtrate figure of Fig. 3 long shoot wood.
The fermented liquid of the different extension rates of Fig. 4 is to HepG2 and HL-7702 proliferation inhibition rate situation synoptic diagram.
Embodiment
Below embodiments of the invention are elaborated: present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
The separation of endogenetic fungus
Get the juglans mandshurica bark and scrape off outer skin, flushing was placed on aseptic Bechtop in 20 minutes under tap water, with aseptic water washing 3～4 times, be 75% alcohol-pickled 3～5 minutes with volume percent, be 1% NaClO solution sterilization 20 minutes with mass percent then, re-use the sterilized water repetitive scrubbing 4～5 times, blot corticole sterilized water with aseptic paper, the melanic epipedon of pruning cuts the fritter of 0.5cm * 0.5cm, place on the water agar solid plate substratum, put into 25 ℃ of incubators and cultivate.
Get the mycelia that incision newly grows, in time be forwarded on the fresh PDA substratum and cultivate, after treating that bacterium colony grows, according to the difference of colonial morphology, color and the difference that grows the time, each dull and stereotyped mycelia that goes up the colony edge place of picking is connected on the new PDA flat board and carries out separation and Culture respectively.Cut the tip of cultivating mycelia, move on the new substratum and cultivate, cut the mycelia that can obtain purifying for 3～5 times.Behind strain number, go on the PDA slant medium, in 28 ℃ of incubators, cultivated 4～5 days, during observe and record strain growth form, put into 4 ℃ of refrigerators then and preserve standby.
Embodiment 2
The evaluation of Chinese catalpa endogenetic fungus
Colonial morphology: on the solid medium, bacterium colony produces white velvet-like mycelium, distributes radially, and the mycelia prosperity, as shown in Figure 1.Form uniform circular bacterium ball in the liquid medium, bacterium ball surface irregularity has thread, and quality is loose, and it is yellow that fermented liquid is.
Spore shape: it is green that naked eyes are seen spore, and the visible spore of microscopic examination is a spheroid shape, smooth surface.The main shaft of conidiophore is long and sturdy, and secondary branch is short, and branch is urniform, and handle obstructs usually Dan Sheng, and spore and is born in secondary ramose top.It is long that microscopically is seen the main shaft of this conidiophore, and it is short the secondary branch of giving birth to, and branch is few once more, and branch is urniform and handle obstructs usually Dan Sheng; In addition, conidium is unicellular, ovalize.As shown in Figure 2.
Cultural characteristic: this bacterium grows on the PDA flat board rapidly, and inoculating 2 days is the about 2 centimetres bacterium colony of visible diameter, and the anterior view mycelia is that white is velvet-like, radial to outgrowth, and thalline secretion xanthein class material makes the substratum reverse side evenly be faint yellow.Cultivate on the CMX substratum and can grow spore very soon, it is bunch loose to produce spore, and spore be a green.Fermentation back thalline is rounded in liquid medium, and surface irregularity has thread, and quality is loose, and it is faint yellow that fermented liquid is, and as shown in Figure 3, and a kind of aromatic odour arranged.
Bacterial strain has stability preferably, succeeding transfer culture 5 times, and fermented liquid still has good active, and fermented liquid has thermostability preferably, is handling 2 hours below 80 ℃, still has active preferably.Can preserve more than 3 months at-20 ℃.
Through identifying that this endogenetic fungus is a Deuteromycotina, hyphomycetes, Moniliales, trichoderma long shoot wood mould (Trichoderima longibrachiatum).
Embodiment 3
The preparation of fermentation liquid that suppresses liver cancer cell growth
The mould CCTCC M 208102 of long shoot wood that separates is cultivated on the PDA flat board, have the PDA substratum of mycelia to downcut a fritter with long then, be put on the CMX substratum, under room temperature, cultivate, as seen have spore to grow two days later.The picking spore is transferred in the triangular flask that 250ml CM liquid nutrient medium is housed then, and 26 ℃ to 28 ℃, 180rpm shaking table shaking culture, stuck fermentation after 7～8 days promptly gets the fermented liquid with inhibition human hepatoma cell strain HepG2 growth effect.Collect bacterium liquid and be used for the anti-tumor activity initial analysis.
Embodiment 4
The external anticancer test of fungal fermented filtrate
With containing weight percent is that the human hepatoma HepG2 cell's strain (available from Zhejiang University of Traditional Chinese Medicine's life science institute molecule and genetic research chamber) of cultivating of the RPMI RPMI-1640 (available from Gibco company) of 10% foetal calf serum (FBS) and people's normal cell lines of human liver HL-7702 (the biochemical and cell institute available from Chinese school of life and health sciences) are with 1 * 10
4Individual/hole is inoculated in 96 orifice plates, and 37 ℃, 5%CO
2Incubator in cultivate, behind the 24h with serial gradient (the fermented liquid extension rate is respectively 40,20,10,5) the fermentation liquor treatment cell after the dilution, blank is made with unfermentable CM nutrient solution in 6 multiple holes of each concentration, with curcumine as positive control, in 37 ℃, 5%CO
2Incubator in cultivate, be cultured to 24h after, add 3-(4,5-dimethylthiazole-2)-2, after 5-phenylbenzene tetrazole bromine salt (MTT) is hatched 4h, inhale and abandon supernatant liquor, add 150 μ L/ hole dimethyl sulfoxide (DMSO) (DMSO) vibration 10min, on the enzyme linked immunological monitor, detect the OD value at 570nm place.Triplicate.
As seen from Figure 4, mtt assay shows that in conjunction with timing microscopic examination record fungal fermented filtrate has stronger restraining effect to human hepatoma cell strain HepG2, though fermented liquid also has certain inhibition or lethal effect to normal cell, not as strong to the liver cancer cell effect.
Claims (2)
1. a preparation of fermentation liquid method that suppresses liver cancer cell growth is characterized in that, comprises the steps:
The first step, induce spore to produce: CCTCC M208102 is inoculated on the PDA substratum and cultivates with long shoot wood mould (Trichoderima longibrachiatum), there is the PDA substratum of mycelia to downcut a fritter with long, be put on the CMX substratum, cultivate under the room temperature, have spore to grow after 1 to 3 day;
Second step, liquid fermentation and culture: the picking spore is transferred to and is equipped with in the CM liquid nutrient medium, and 26 ℃ to 28 ℃, 180rpm shaking table shaking culture, stuck fermentation after 7 to 8 days promptly gets the fermented liquid with the effect of inhibition liver cancer cell growth.
2. the preparation of fermentation liquid method of inhibition liver cancer cell growth according to claim 1, it is characterized in that the PDA substratum consists of in the first step: the juice that the fresh potato of 200g boils, 20g glucose, solid medium adds 1.5% agar, and adding distil water to cumulative volume is 1000ml.
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| CN104387425A (en) * | 2014-10-11 | 2015-03-04 | 上海交通大学 | Method for condensation and separation of catalpa bungei essence Qiulingsu in catalpa bungei endophytic fungus broth and use thereof |
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| CN102382791A (en) * | 2011-11-21 | 2012-03-21 | 滨州职业学院 | Fermentation process of trichoderma |
| CN102660493B (en) * | 2012-04-12 | 2013-11-20 | 上海交通大学 | Trichoderma sporulation medium and sporulation method |
| CN103756917B (en) * | 2014-01-14 | 2016-05-04 | 上海交通大学 | The preparation method of the protoplast of Chinese catalpa endogenetic fungus |
| CN104610383B (en) * | 2015-01-21 | 2017-01-04 | 上海交通大学 | By the method for Chinese catalpa spirit element in extraction back extraction isolated and purified catalpa bungei endophytic fungi fermentation liquid |
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| CN104387425A (en) * | 2014-10-11 | 2015-03-04 | 上海交通大学 | Method for condensation and separation of catalpa bungei essence Qiulingsu in catalpa bungei endophytic fungus broth and use thereof |
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