CN106929464B - A method of promote sterol effective constituents in the lobate layer bacterium of currant to accumulate using plant source cigarette water - Google Patents
A method of promote sterol effective constituents in the lobate layer bacterium of currant to accumulate using plant source cigarette water Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 54
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- 150000003432 sterols Chemical class 0.000 title claims abstract description 18
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
Promoting the method that sterol effective constituents accumulate in the lobate layer bacterium of currant using plant source cigarette water the invention discloses a kind of, the lobate layer bacterium hypha body of the preferable currant grown after inclined-plane culture is inoculated into fluid nutrient medium, it is inoculated with 78 hypha bodies per 1L fluid nutrient mediums, plant source cigarette water is added into Liquid Culture system within the 3rd day in Liquid Culture, 0.5 1.5mL plant source cigarette water is added in per 1L liquid systems, plant source cigarette water is added into Liquid Culture system again within the 7th day in Liquid Culture, 0.15 0.25mL plant source cigarette water is added in per 1L liquid systems, Liquid Culture totally 9 10 days.Plant source cigarette water is added in the present invention during currant lobate layer bacterium fermented and cultured, the plant source cigarette water of low concentration is " a micro- adverse circumstance " to the growth of the lobate layer bacterium of currant, the synthesis for promoting sterol substance, to improve the quality as the medicinal lobate layer bacterium of currant.
Description
Technical field
The present invention relates to the methods that active material in a kind of promotion lobate layer bacterium of currant accumulates, and in particular to a kind of utilization
The method that plant source cigarette water promotes active material in the lobate layer bacterium of currant-sterol substance accumulation.
Background technology
The lobate layer bacterium of currant（Phylloporia ribis（Schumach:Fr.）Ryvarden）For Hymenochaetaceae leaf
Shape layer Pseudomonas fungi, the bacterium main product is in Pingyi, shandong Province, and parasitizing honeysuckle plant, dry or exposed root, locals are referred to as always
" honeysuckle flower moth ", medication is with a long history, can be used for anti-inflammatory, removing toxic substances, analgesic, local resident with its treat pharyngitis, laryngitis, hepatitis and
Cancer.Contain the active ingredients such as triterpenes, sterols, polysaccharide and styryl pyranone, tool in the lobate layer bacterium of currant
There is the effects that clearing heat and detoxicating, detumescence relieving sore-throat, hypoglycemic and antitumor.Ergosterol is also known as ergosterol, is the feature of Mycophyta
Sterol is widely present in medicinal fungi fructification and fermentation mycelium, and the substance is also contained in the lobate layer bacterium of currant.With
Ergosterol is the sterols chemical composition of representative proves there is the pharmacological actions such as anti-inflammatory, promoting immunity, anticancer through pharmacological testing, is
A kind of important active constituents of medicine.
With the utilization of the lobate layer bacterium of currant, market demand is very big, and wild resource much can not meet work
Industry metaplasia is produced, and artificial fermentation's culture technique has made a breakthrough, and is adopted an effective measure in artificial fermentation's incubation and is improved tea Fischer
The content of ergosterol constituents, is beneficial to the utilization of such bacterium in cotyledon shape layer bacterium.
Invention content
The object of the present invention is to provide it is a kind of using plant source cigarette water promote the lobate layer bacterium of currant in sterols effectively at
Divide the method for accumulation, this method that suitable plant source cigarette water is added during currant lobate layer bacterium fermented and cultured, improves
The content of sterol substance in the lobate layer bacterium of currant, this method have important meaning to the quality for improving the lobate layer bacterium of currant
Justice.
Plant source cigarette water refers to the aqueous solution for the cigarette formation soluble in water that vegetable material smoldering generates, the life of plant source cigarette water
Reason Ecology Action has become the hot spot of international ecological educational circles research, it can effectively improve crop seed germination rate, improves seedling and lives
Power, and the accumulation of pharmaceutically active substance in medicinal plant body is improved, it is shown in terms of improving crop and Chinese medicine quality huge
Big potentiality, however its to the accumulation of active material in fungus body not yet studies have reported that.Inventor has found that in people
The specifically fermentation period of the lobate layer bacterium of work culture currant is added suitable plant source cigarette water and is cultivated, this kind of training method is big
The content for improving sterols active material in the lobate layer bacterium of currant of amplitude, it was found that the another new application of cigarette water, simultaneously
Culture for the lobate layer bacterium of high-quality currant provides new thinking.
Specific technical solution of the present invention is as follows：
A method of promote sterol effective constituents in the lobate layer bacterium of currant to accumulate using plant source cigarette water, this method
It is by the lobate layer bacterium of currantPhylloporia ribis(Schumach.:Fr.) Ryvarden carries out inclined-plane culture, inclined-plane
After culture, gained mycelium is inoculated into fluid nutrient medium and carries out Liquid Culture, Liquid Culture process is as follows：By inclined-plane culture
The lobate layer bacterium hypha body of preferable currant grown afterwards is inoculated into fluid nutrient medium, per 1L fluid nutrient mediums inoculation 7-8
Hypha body is added plant source cigarette water on the 3rd day in Liquid Culture into Liquid Culture system, 0.5- is added in every 1L liquid systems
Plant source cigarette water is added on the 7th day, in Liquid Culture per 1L liquid bulks in 1.5mL plant source cigarette water into Liquid Culture system again
0.15-0.25mL plant source cigarette water, total 9-10 days of Liquid Culture are added in system.
In the method for the present invention, the lobate layer bacterium of currant is lobate with the currant grown on wild state plant of lower honeysuckle
Layer bacterium Phylloporia ribis (Schumach.:Fr.) Ryvarden fructifications are strain, or are with preserving number
The lobate layer bacterium Phylloporia ribis (Schumach. of currant of CGMCC NO 1195:Fr.) Ryvarden is bacterium
Kind.
In the method for the present invention, the plant source cigarette water is that cigarette caused by plant source smoldering 50-60min by 6-7kg leads to
Enter the aqueous solution obtained in 500ml cold water, the plant source is that mass ratio is 1:3:3 cercis limb, a ball plane tree are fallen
Leaf, tree-of-heaven limb.
Further, the preparation method of plant source cigarette water further includes step in detail below：By cercis limb, a ball plane tree
Fallen leaves, tree-of-heaven limb are dried, and are deposited in black room, for 24 hours with ultraviolet light, then use 80-90 DEG C of hot gas stream process 7-8h；
After processing, by cercis limb, ball plane tree fallen leaves, tree-of-heaven limb according to 1:3:3 mass ratio mixing, takes the mixing of 6-7kg
Object is as plant source, and cigarette is passed through in 500mL cold water caused by the plant source smoldering 50-60min by 6-7kg, gained it is water-soluble
Liquid is plant source cigarette water.Wherein, thermal current is hot-air or hot oxygen-enriched air.In general, plant source cigarette water obtained is at 4 DEG C
It is kept in dark place spare.
In the method for the present invention, 2 plant source cigarette water, when being the 3rd day for the first time, second are added altogether in Liquid Culture
Secondary when be the 7th day, plant source cigarette water is added at one time.
In the method for the present invention, when Liquid Culture, fluid nutrient medium used was grouped as by the group of following weight percentage：
Maltose 1.5-2.5%, monosodium glutamate 0.8-1.2%, KH2PO40.03-0.06%, MgSO4•7H2O 0.02-0.04%, glucose 0.8-
1.2%, yeast extract 0.2-0.4%, corn steep liquor 0.05-0.15%, mannitol 1.8-2.5%, CaCO31.8-2.5%, water surplus；PH
For 6.5-7.0.
In the method for the present invention, when Liquid Culture, before plant source cigarette water is added at the 1st time（When i.e. the 3rd day）, in illumination
It is cultivated under conditions of intensity 300-350Lux, 27-29 DEG C, 140-160rpm, after plant source cigarette water is added at the 1st time,
It is dark, 24-25 DEG C, cultivate 12-14h under conditions of 140-160rpm, then go to intensity of illumination 300-350Lux, 27-28 DEG C,
It is cultivated under conditions of 140-160rpm, until the 2nd addition plant source cigarette water.After plant source cigarette water is added at the 2nd time, black
Secretly, 24-25 DEG C, cultivate 12-14h under conditions of 140-160rpm, then go to intensity of illumination 300-350Lux, 27-28 DEG C,
It is cultivated under conditions of 140-160rpm, until Liquid Culture terminates.
In the method for the present invention, during entire Liquid Culture, it is 6.5-7.0 to keep the pH of Liquid Culture system.Especially
After plant source cigarette water is added, because cigarette water pH value itself is low, the too low influence bacterium of PH of entire culture medium after being added in order to prevent
Growth, need to adjust pH simultaneously.
In the method for the present invention, in order to avoid bringing the ingredient for being unfavorable for growth in plant source cigarette water into, first to plant source
After cigarette water carries out following processing, add in fluid nutrient medium：Plant source cigarette water is first subjected to high pressure sterilization at 121 DEG C
Then plant source cigarette boiling is boiled 30min, is added in fluid nutrient medium after cooling by 20min.
In the method for the present invention, when inclined-plane culture, the lobate layer bacterium strain of currant is added in slant medium, is trained at 29 DEG C
It supports 7 days.Slant medium used is grouped as by the group of following weight percentage：Potato 20%, agar 1.5%, glucose 2%,
Potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, vitamin 1 × 10-3%, water surplus, PH 6.5.
The present invention is added plant source cigarette water during currant lobate layer bacterium fermented and cultured, plant source cigarette water of the present invention by
Cercis limb, ball plane tree fallen leaves, tree-of-heaven limb are made, and the phenols containing lot of trace and esters are lived in the plant source cigarette water
The ingredients such as property substance.When plant source cigarette water concentration is low, fractions therein form the growth of the lobate layer bacterium of currant " micro-
Adverse circumstance " becomes active material in the lobate layer bacterium of " startup " currant --- " elicitor " of sterol substance biosynthesis program.
Sterol substance is served as " degeneration-resistant molecule " in the lobate layer thalline of currant, and the presence of plant source cigarette water promotes the lobate layer of currant
Bacterium improves the synthesis of internal sterol substance to resist " micro- adverse circumstance ", therefore our drug target active material sterol substance
Content can increase, to improve the quality as the medicinal lobate layer bacterium of currant.Present invention firstly provides plant source cigarettes
The effect that water accumulates the lobate layer bacterium active material of currant, this method raw material sources are extensive, easy to operate, easy to implement, right
It is significant to improve the lobate layer mycoplasma measurer of currant.
Description of the drawings
The structural schematic diagram of Fig. 1 cigarette water device for making of the present invention.
In figure, 1, tubbiness container made of iron, 2, lid, 3, lateral partitions, 4, lighting-up tuyere, 5, hide cigarette lid, 6, surge flask, 7, connect
Receive bottle, 8, Vacuum filtration device.
Specific implementation mode
Below by specific embodiment, invention is further explained, and the following description is only intended to explain the invention,
Its content is not limited.
Embodiment 1
Plant source cigarette water of the present invention may be used the device described in patent ZL. 2,013 1 0631035.0 and carry out plant
The preparation of source cigarette water can also use device shown in Fig. 1 to carry out the preparation of plant source cigarette water.Cigarette water device for making in Fig. 1
Including tubbiness container made of iron 1, end opening and lid 2 is carried thereon, for holding vegetable material, handle, tubbiness are carried on lid
It is equipped with the lateral partitions 3 for placing plant source in the cavity lower part of container made of iron 1, the hole of ventilation, tubbiness iron are carried in lateral partitions
Also there is matter container 1 lighting-up tuyere 4, lighting-up tuyere 4 to be located at below lateral partitions 3, and lighting-up tuyere, which is equipped with, can automatically adjust oxygen-supplying amount
Lighting-up tuyere valve, with cigarette lid 5 is hidden on lighting-up tuyere, the top of tubbiness container made of iron 1 is equipped with outlet flue, outlet flue and surge flask
6 are connected, and the buffered bottle 6 of the cigarette being discharged from outlet flue enters multiple concatenated receiving bottles 7, and cold water, right end are housed in receiving bottle 7
Receiving bottle connect with Vacuum filtration device 8.
The preparation method of plant source cigarette water is as follows：Cercis limb, ball plane tree fallen leaves, tree-of-heaven limb are taken, dries, dries
Afterwards according to 1:3:Mixture, is then deposited in black room, for 24 hours with ultraviolet light, then uses 80-90 by 3 mass ratio mixing
DEG C hot air treatment 8h；After processing, takes the mixture of 6kg as plant source, the plant source of 6kg is put into cigarette water device for making
In, lid is covered, lights a fire from lighting-up tuyere, lights plant source, lighting-up tuyere is covered with screening cigarette lid after igniting, controls oxygen-supplying amount, is prevented
Only cigarette is overflowed from lighting-up tuyere.The plant source lighted cannot adequately burn because oxygen is insufficient, and fire can extinguish quickly, only
It stays Mars, plant source to be only capable of burning by Mars, will produce a large amount of cigarette at this time, under the action of Vacuum filtration device, combustion
The cigarette that burning process generates is passed through receiving bottle.Cigarette caused by smoldering 60min is passed through in 500mL cold water and is absorbed, gained
Aqueous solution is plant source cigarette water, it is kept in dark place at 4 DEG C, spare.
Embodiment 2
Plant source cigarette water is prepared according to the method for embodiment 1, unlike：Plant source is that the mass ratio of 6kg is 3:1:1
Cercis limb, yulan limb, weeping willow limb.
Embodiment 3
The lobate layer bacterium of fermented and cultured currant, method are as follows：
1, the preparation of culture medium
The preparation of slant medium：Buy potato on the market, prune crust, be cut into 1cm square fritters, by potato block plus
Enter in water, boils 30min after 121 DEG C of sterilizings（Pay attention to the control of firepower, it can appropriate moisturizing）, filtrate is taken with filtered through gauze.It is filtering
Agar, glucose, potassium dihydrogen phosphate, magnesium sulfate, vitamin are added in liquid, is stirred continuously with glass bar, dissolves by heating, supplies water
Point, adjustment pH value to 6.5 is packed into test tube, and sterilize postcooling, bevel culture medium；Inclined-plane culture based formulas is（wt%）：Soil
Beans 20%, agar 1.5%, glucose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, vitamin 1 × 10-3%, water surplus.
Liquid Culture based formulas is（wt%）：Maltose 2%, monosodium glutamate 1%, KH2PO40.05%, MgSO4•7H2O 0.03%, Portugal
Grape sugar 1%, yeast extract 0.3%, corn steep liquor 0.1%, mannitol 2%, CaCO32%, water surplus；PH is 6.5-7.0.
, the lobate layer bacterium of fermented and cultured currant
2.1：Inoculation, which is operated on clean work station, to be carried out.The currant leaf grown on wild state plant of lower honeysuckle
Shape layer bacteriumPhylloporia ribis(Schumach.:Fr. when) Ryvarden fructifications are strain, first with ultraviolet before inoculation
Lamp sterilizes 40min to fructification, is inoculated in the portion of tissue that fructification edge is growing is trained containing inclined-plane under aseptic conditions
In the test tube for supporting base, cultivated 7 days at 29 DEG C.When the lobate layer bacterium of currant with preserving number for CGMCC NO 1195Phylloporia ribis(Schumach.:Fr. when) Ryvarden is strain, directly the strain is inoculated in and is trained containing inclined-plane
In the test tube for supporting base, cultivated 7 days at 29 DEG C.
2.2：Then the plant source cigarette water of Example 1 boils 30min, cools standby in 121 DEG C of high pressure sterilization 20min
With；
2.3：The lobate layer bacterium hypha body of the preferable currant of solid culture growing state in above-mentioned slant tube is taken to be connect
Kind, it is inoculated into fluid nutrient medium, pays attention to rejecting the slant medium that agar is contained in bottom, the diameter of hypha body is about 1-
2mm is as possible consistent the size of taken hypha body, 8 hypha bodies is inoculated with per 1L fluid nutrient mediums, after inoculation, in illumination
Intensity 320Lux, 28 DEG C, first cultivate 2 days under conditions of rotating speed 150rpm, the 3rd day by the plant source cigarette water after above-mentioned sterilization treatment
Be added in fluid nutrient medium, per 1L fluid nutrient mediums in 1mL plant source cigarette water is added, above-mentioned sterilization treatment is added the 7th day the 2nd time
Often 0.2mL plant source cigarette water is added in 1L fluid nutrient mediums in plant source cigarette water afterwards, after plant source cigarette water is added at the 1st time,
It is dark, 24-25 DEG C, cultivate 12h under conditions of 150rpm, then go to intensity of illumination 320Lux, 28 DEG C, under conditions of 150rpm
Culture, until the 2nd addition plant source cigarette water.After plant source cigarette water is added at the 2nd time, in dark, 24-25 DEG C, 150rpm
Under the conditions of cultivate 12h, then go to intensity of illumination 320Lux, 28 DEG C, cultivate under conditions of 150rpm, until Liquid Culture knot
Beam, it is 6.5-7.0 that entire Liquid Culture process, which keeps pH, and the time of entire Liquid Culture is 10 days.
2.4：After Liquid Culture, the zymotic fluid of gained is filtered, gained filter cake is dried at 60 DEG C, until complete
It does to get the lobate layer bacterium of currant.
Embodiment 4
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：Plant source cigarette water used is to implement
Plant source cigarette water prepared by example 2.
Embodiment 5
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：Fluid nutrient medium is：Maltose
1.5%, monosodium glutamate 1.2%, KH2PO40.03%, MgSO4•7H2O 0.04%, glucose 0.8%, yeast extract 0.4%, corn steep liquor 0.05%,
Mannitol 2.5%, CaCO31.8%, water surplus；PH is 7.0.
Embodiment 6
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：Fluid nutrient medium is：Maltose
2.5%, monosodium glutamate 0.8%, KH2PO40.06%, MgSO4•7H2O 0.02%, glucose 1.2%, yeast extract 0.2%, corn steep liquor 0.15%,
Mannitol 1.8%, CaCO32.5%, water surplus；PH is 6.5.
Embodiment 7
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：The process of Liquid Culture is：It takes
The lobate layer bacterium hypha body of the preferable currant of solid culture growing state is inoculated in slant tube, is inoculated into fluid nutrient medium
In, pay attention to rejecting the slant medium that agar is contained in bottom, the diameter of hypha body is about 1-2mm, makes taken hypha body as possible
Size be consistent, 8 hypha bodies are inoculated with per 1L fluid nutrient mediums, after inoculation, in intensity of illumination 350Lux, 27 DEG C, rotating speed
It first cultivates 2 days, the plant source cigarette water after above-mentioned sterilization treatment was added in fluid nutrient medium in the 3rd day, often under conditions of 140rpm
Addition 0.5mL plant source cigarette water in 1L fluid nutrient mediums, the 7th day the 2nd time plant source cigarette water being added after above-mentioned sterilization treatment, often
0.25mL plant source cigarette water is added in 1L fluid nutrient mediums, after plant source cigarette water is added at the 1st time, it is dark, 24-25 DEG C,
14h is cultivated under conditions of 140rpm, then go to intensity of illumination 350Lux, 27 DEG C, cultivate under conditions of 140rpm, until the 2nd
Secondary addition plant source cigarette water.After plant source cigarette water is added at the 2nd time, cultivated under conditions of dark, 24-25 DEG C, 140rpm
14h, then go to intensity of illumination 350Lux, 27 DEG C, cultivate under conditions of 140rpm, until Liquid Culture terminates, entire liquid
It is 6.5-7.0 that incubation, which keeps pH, and the time of entire Liquid Culture is 10 days.
Embodiment 8
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：The process of Liquid Culture is：It takes
The lobate layer bacterium hypha body of the preferable currant of solid culture growing state is inoculated in slant tube, is inoculated into fluid nutrient medium
In, pay attention to rejecting the slant medium that agar is contained in bottom, the diameter of hypha body is about 1-2mm, makes taken hypha body as possible
Size be consistent, 8 hypha bodies are inoculated with per 1L fluid nutrient mediums, after inoculation, in intensity of illumination 300Lux, 29 DEG C, rotating speed
It first cultivates 2 days, the plant source cigarette water after above-mentioned sterilization treatment was added in fluid nutrient medium in the 3rd day, often under conditions of 160rpm
Addition 1.5mL plant source cigarette water in 1L fluid nutrient mediums, the 7th day the 2nd time plant source cigarette water being added after above-mentioned sterilization treatment, often
0.15mL plant source cigarette water is added in 1L fluid nutrient mediums, after plant source cigarette water is added at the 1st time, it is dark, 24-25 DEG C,
12h is cultivated under conditions of 160rpm, then go to intensity of illumination 300Lux, 29 DEG C, cultivate under conditions of 160rpm, until the 2nd
Secondary addition plant source cigarette water.After plant source cigarette water is added at the 2nd time, cultivated under conditions of dark, 24-25 DEG C, 160rpm
12h, then go to intensity of illumination 300Lux, 29 DEG C, cultivate under conditions of 160rpm, until Liquid Culture terminates, entire liquid
It is 6.5-7.0 that incubation, which keeps pH, and the time of entire Liquid Culture is 10 days.
Comparative example 1
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：Plant is not used when Liquid Culture
The process of source Yan Shui, Liquid Culture is：Take the lobate layer bacterium mycelia of the preferable currant of solid culture growing state in slant tube
Group is inoculated with, and is inoculated into fluid nutrient medium, pays attention to rejecting the slant medium that agar is contained in bottom, the diameter of hypha body is big
About 1-2mm is as possible consistent the size of taken hypha body, and 8 hypha bodies are inoculated with per 1L fluid nutrient mediums, after inoculation,
There is light（320Lux）, 28 DEG C, cultivate 10 days under conditions of rotating speed 150rpm.
Comparative example 2
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：The process of Liquid Culture is：It takes
The lobate layer bacterium hypha body of the preferable currant of solid culture growing state is inoculated in slant tube, is inoculated into fluid nutrient medium
In, pay attention to rejecting the slant medium that agar is contained in bottom, the diameter of hypha body is about 1-2mm, makes taken hypha body as possible
Size be consistent, 8 hypha bodies are inoculated with per 1L fluid nutrient mediums, after inoculation, in illumination（320Lux）, 28 DEG C, rotating speed
It first cultivates 2 days, plant source cigarette water embodiment 1 sterilization treatment after was added in fluid nutrient medium in the 3rd day under conditions of 150rpm,
1.5mL plant source cigarette water is added in per 1L fluid nutrient mediums, continues in illumination after addition（320Lux）, 28 DEG C, rotating speed 150rpm
Under conditions of cultivate, until Liquid Culture terminates, it is 6.5-7.0 that entire Liquid Culture process, which keeps pH, entire Liquid Culture
Time is 10 days.
Comparative example 3
According to the lobate layer bacterium of method fermented and cultured currant of embodiment 3, the difference is that：When Liquid Culture, plant source cigarette
Water consumption is：The amount of 1st addition plant source cigarette water be per 1L fluid nutrient mediums in be added 4.5mL plant source cigarette water, the 2nd time plus
The amount for entering plant source cigarette water is per addition 2.5mL plant source cigarette water in 1L fluid nutrient mediums.
In the following, the lobate layer fungisterol substance content of the currant that above-described embodiment and comparative example fermented and cultured are obtained into
Row detection, to verify the facilitation that the method for the present invention promotes sterols content of material.Wherein, sterol substance is with ergot steroid
Alcohol is representative.
The detection method of content of ergosterol is as follows in the lobate layer bacterium mycelia of currant：
The lobate layer bacterium mycelia of the drying currant that each embodiment and comparative example obtains is taken, accurately weighed 0.5g, uses stone respectively
Oily 6 h of ether Soxhlet extraction, extract obtained water bath method, residue is dissolved with absolute ethyl alcohol, and is settled to 5.0ml to get sample
Product solution, sample solution is used（0.22μm）Filtering with microporous membrane detects the lobate layer bacterium of currant using high performance liquid chromatography
The content of middle ergosterol, chromatographic condition are as follows：
Chromatographic column：Zorbax eclips XDB C8(150 mm×4.6 mm)；Mobile phase：Methanol；Flow velocity：1.0 ml·
min-1；Detection wavelength：282 nm；Column temperature：30℃；Sample size：20μl.Under these conditions, sample solution, theoretical tray are injected
Number is more than 10000 in terms of ergosterol.
The content of ergosterol is as shown in table 1 below in each sample solution that liquid chromatographic detection obtains.
Claims (10)
1. a kind of promoting the method that sterol effective constituents accumulate in the lobate layer bacterium of currant using plant source cigarette water, by currant
Lobate layer bacteriumPhylloporia ribis(Schumach.:Fr.) Ryvarden carries out inclined-plane culture, it is characterized in that：It trains on inclined-plane
After supporting, gained mycelium is inoculated into fluid nutrient medium and carries out Liquid Culture, Liquid Culture process is as follows：After inclined-plane culture
The lobate layer bacterium hypha body of preferable currant of growth is inoculated into fluid nutrient medium, and 7-8 bacterium is inoculated with per 1L fluid nutrient mediums
Clusters are added plant source cigarette water on the 3rd day in Liquid Culture into Liquid Culture system, 0.5- are added in every 1L liquid systems
Plant source cigarette water is added on the 7th day, in Liquid Culture per 1L liquid bulks in 1.5mL plant source cigarette water into Liquid Culture system again
0.15-0.25mL plant source cigarette water, total 9-10 days of Liquid Culture are added in system；
The plant source cigarette water is that cigarette caused by plant source smoldering 50-60min by 6-7kg is passed through in 500mL cold water and obtains
Aqueous solution, the plant source be mass ratio be 1:3:3 cercis limb, ball plane tree fallen leaves, tree-of-heaven limb.
2. according to the method described in claim 1, it is characterized in that：When Liquid Culture, before plant source cigarette water is added in first time,
It is cultivated under conditions of intensity of illumination 300-350Lux, 27-29 DEG C, 140-160rpm, plant source cigarette water is added at the 1st time
Afterwards, 12-14h is cultivated under conditions of dark, 24-25 DEG C, 140-160rpm, then goes to intensity of illumination 300-350Lux, 27-
29 DEG C, cultivate under conditions of 140-160rpm, until the 2nd addition plant source cigarette water；After plant source cigarette water is added at the 2nd time,
12-14h is cultivated under conditions of dark, 24-25 DEG C, 140-160rpm, then goes to intensity of illumination 300-350Lux, 27-29
DEG C, cultivate under conditions of 140-160rpm, until Liquid Culture terminates.
3. method according to claim 1 or 2, it is characterized in that：The fluid nutrient medium is by following weight percentage
Group is grouped as：Maltose 1.5-2.5%, monosodium glutamate 0.8-1.2%, KH2PO40.03-0.06%, MgSO4·7H2O 0.02-0.04%,
Glucose 0.8-1.2%, yeast extract 0.2-0.4%, corn steep liquor 0.05-0.15%, mannitol 1.8-2.5%, CaCO31.8-2.5%,
Water surplus；PH is 6.5-7.0.
4. method according to claim 1 or 2, it is characterized in that：During entire Liquid Culture, Liquid Culture system is kept
PH be 6.5-7.0.
5. method according to claim 1 or 2, it is characterized in that：Plant source cigarette water is first subjected to high pressure sterilization at 121 DEG C
Then 20min boils 30min, added in fluid nutrient medium after cooling.
6. according to the method described in claim 1, it is characterized in that：The preparation method of plant source cigarette water includes step in detail below：
Cercis limb, ball plane tree fallen leaves, tree-of-heaven limb are dried, is deposited in black room, for 24 hours with ultraviolet light, then uses 80-
90 DEG C of hot gas stream process 7-8h；After processing, by cercis limb, ball plane tree fallen leaves, tree-of-heaven limb leaf according to 1:3:3 matter
Amount takes the mixture of 6-7kg as plant source, cigarette is passed through caused by the plant source smoldering 50-60min by 6-7kg than mixing
In 500 ml cold water, the aqueous solution of gained is plant source cigarette water.
7. according to the method described in claim 6, it is characterized in that：The thermal current is hot-air or hot oxygen-enriched air；Gained is planted
Material resource cigarette water is kept in dark place spare at 4 DEG C.
8. method according to claim 1 or 2, it is characterized in that：When inclined-plane culture, used medium is by following weight percent
The group of content is grouped as：Potato 20%, agar 1.5%, glucose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, vitamin 1 ×
10-3%, water surplus；PH is 6.5.
9. according to the method described in claim 1, it is characterized in that：When inclined-plane culture, condition of culture is：It is cultivated 5-7 days at 29 DEG C.
10. method according to claim 1 or 2, it is characterized in that：The lobate layer bacterium of currant is with wild state lower honeysuckle
The lobate layer bacterium Phylloporia ribis (Schumach. of currant grown on plant:Fr.) Ryvarden fructifications are
Strain, or with preserving number be CGMCC NO 1195 the lobate layer bacterium Phylloporia ribis (Schumach. of currant:
Fr.) Ryvarden is strain.
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