CN106754414A - A kind of siberian Ginseng P.E induction fermentation black fungus bacterial and its mycelia preparation - Google Patents
A kind of siberian Ginseng P.E induction fermentation black fungus bacterial and its mycelia preparation Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of agaric mycelium, specifically a kind of addition siberian Ginseng P.E induction fermentation black fungus bacterial prepares mycelial method.Meanwhile, the invention further relates to using the agaric mycelium obtained after the method culture black fungus bacterial.The black fungus bacterial culture medium, on the basis of ordinary culture medium, adds a kind of Chinese herbal medicine, mainly siberian Ginseng P.E.The present invention is first with wild black fungus bacterial as starting strain, optimization is improved to black fungus bacterial submerged fermentation culture medium, submerged fermentation is carried out by adding siberian Ginseng P.E in liquid medium within, not only improve mycelium and its polysaccharide yield, improve mycelium polysaccharides composition simultaneously, strengthen the pharmacological activity of mycelium polysaccharides.So, agaric mycelium prepared by the present invention can have broad application prospects at aspects such as dietary additive, drug additive of replenishing the calcium, Organism immunoregulation health products.
Description
Technical field
The invention belongs to edible mushroom field, it is related to one kind to prepare agaric mycelium using Chinese medicine-siberian Ginseng P.E
Method, also relate to by the method prepare with healthcare function agaric mycelium.
Background technology
Black fungus（Auricularia auricula）It is subordinate to Eumycota, Basidiomycotina, Auriculariale, Auriculariaceae, agaric
Category fungi, is mainly made up of mycelium and fructification.Wherein mycelium is the nutrition organs of black fungus, is planted equivalent to high green
The root of thing, stem, leaf, are the main bodys of black fungus；The fructification of black fungus, is that we cook edible part also known as basidiocarps.It is black
Agaric contains abundant carbohydrate（Colloid）, protein, dietary fiber, calcium, iron, vitamin etc., wash intestines, anti-with clearing stomach
The generation of the disease such as blood coagulation, pre- preventing thrombosis, prevents and treats atherosclerosis and coronary heart disease.
Wilsonii（Eleutherococcus senticosus）A kind of traditional Chinese medicine, with regulation body it is disorderly,
Replenish qi to invigorate the spleen, blood-enriching tranquillizing, it is antifatigue, improve the function such as body-internal-circulation system；Currently with Caulis Et Caulis Acanthopanacis Senticosi, do as black fungus
Cultivation base stock, the cultivation technique for producing wilsonii black fungus puts it in Heilongjiang Province, and cultural method has obtained " a kind of
The production method of black fungus "（CN1762193A）Patent right, but there is no be related to Chinese medicine-siberian Ginseng P.E be induction
Thing, the method that agaric mycelium is prepared by solution fermentation.
The content of the invention
Prepared using siberian Ginseng P.E induction fermentation black fungus bacterial it is an object of the invention to provide one kind mycelial
Method, with siberian Ginseng P.E as inducer, agaric mycelium is prepared by solution fermentation.
Present invention firstly provides a kind of siberian Ginseng P.E induction fermentation black fungus bacterial and its mycelia preparation, with
Wild black fungus bacterial is starting strain, and siberian Ginseng P.E is with the addition of in the medium, by liquid deep layer fermenting, obtains black wood
Ear mycelium.
To achieve these goals, the present invention is carried out essentially according to following step：
Siberian Ginseng P.E induction black fungus bacterial submerged fermentation of the invention produces mycelial method and comprises the steps：
A kind of method for preparing siberian Ginseng P.E induction fermentation black fungus bacterial zymotic fluid, black fungus bacterial seed culture fluid is accessed
Fermentation medium after sterilizing, inoculum concentration be 10~20% (v/v), ventilation be tank volume per minute 50~
100%, fermentation pressure tank is 0.02~0.05Mpa, and fermented and cultured 7~9 days, obtain black fungus at 25~28 DEG C
Bacterium karusen；Wherein described fermentative medium formula is：5~9g/L of glucose, the g/L of corn flour 8~12, bean powder 1
~3 g/L, the g/L of wilsonii 0.5~2.5, the g/L of defoamer 0.1~0.5, regulation pH values 5.5~6.5.
Described defoamer can be the materials such as vegetable oil (soya-bean oil, rapeseed oil) class, material of organic ethers.
Wherein, described black fungus seed culture fluid is preferably prepared by the following method and obtains：
Liquid seeds culture：Activated shaking flask strain is accessed into the liquid seed culture medium after sterilizing, inoculum concentration is 0.1~
2.0 %, ventilation is the 50~100% of tank volume per minute, and tank pressure is 0.02~0.05Mpa, at 24~28 DEG C
Culture 4~6 days, obtains black fungus liquid seeds nutrient solution；
Described liquid seed culture medium formula is preferably：The g/L of glucose 15~25, the g/L of farina 3.5~5,
Regulation pH values 5.0~6.5.
Described fermented and cultured terminal is：Reduced sugar is minimized, and reduced sugar controls 0.3% (g/100g) below；
Microscopy mycelia is elongated, without living contaminants.
Described siberian Ginseng P.E, by expanding connecing for siberian Ginseng P.E main chemical compositions and agaric mycelium
Contacting surface is accumulated, and adds the amount of siberian Ginseng P.E to be directly proportional to the mycelial biomass for producing.But, it is added in fluid nutrient medium
The ratio of siberian Ginseng P.E has certain limitation, and the bulking value concentration that siberian Ginseng P.E accounts for culture medium is 0.5~5
G/L, more preferably 1~2.5 g/L.
The drying of described fermentation mycelium：After fermentation 7-9 days, karusen obtains zymotic fluid and black fungus bacterial through filtering
Filament.It is after mycelium homogenate then freeze-dried（Condition is：- 10 DEG C~-50 DEG C of temperature, the handkerchief of vacuum 1.3~13）Or spray
Mist is dried（Condition is：180 DEG C of inlet temperature, 100 DEG C of outlet temperature）Or heated-air drying（Condition is：80 DEG C of hot blast temperature）, obtain
To agaric mycelium.
Advantages of the present invention and effect：
Biological enzyme mechanism and the cumulative function of product of the present invention according to organism, add Chinese medicine in ordinary culture medium
Material-siberian Ginseng P.E, agaric mycelium of preparation and products thereof have the natural attractive in appearance, unique flavor of color and luster, nutritious
The characteristics of with certain health-care effect.
Brief description of the drawings
Influence of Fig. 1 various concentrations siberian Ginseng P.E to agaric mycelium biomass.
Fig. 2 Blackfungus polyhexoses are to mouse macrophage RAW264.7 ability of cell proliferation.
Specific embodiment
The percent concentration is mass/mass (W/W) percent concentration, mass/volume (W/V) unless otherwise instructed
Percent concentration or volume/volume (V/V) percent concentration.
The acquirement approach of the various biomaterials described in embodiment is only to provide a kind of approach for obtaining of testing to reach
To specifically disclosed purpose, the limitation to biological material source of the present invention should not be turned into.In fact, used biomaterial
Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to carrying in embodiment
Show and be replaced.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed implementation method and specific
Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.
Embodiment 1：Influence of the various concentrations siberian Ginseng P.E induction fermentation to black fungus mycelial growth
1st, the preparation of black fungus zymotic fluid：The black fungus bacterial that will have been activated is inoculated in 1L seed culture mediums by inoculum concentration 1%, its
Middle seed culture medium is, in BD PDATMCulture medium (a kind of standard medium, produced in USA), 4 are cultivated at 25 ± 0.1 DEG C
My god, obtain black fungus liquid seeds nutrient solution；Black fungus seed culture fluid is accessed into the fermentation medium after sterilizing, fermented and cultured
Based formulas are：The g/L of glucose 7, the g/L of corn flour 10, the g/L of bean powder 2, siberian Ginseng P.E concentration are respectively 0.0,0.5,
1.0th, 1.5,2.0,2.5,3.0 g/L, regulation pH value are 5.5, and inoculum concentration is 8% (v/v), fermented and cultured 7 days at 28 DEG C.
2nd, dry mycelial weight is determined:100 mL karusens are taken, 3000 r/min are centrifuged 10 min, collect mycelium,
After washing repeatedly, in 55 DEG C of drying to constant weight, assay balance is weighed.
3rd, result
Various concentrations siberian Ginseng P.E is shown in Fig. 1 to the influence result that agaric mycelium grows, and as seen from the figure, wilsonii is extracted
Thing can significantly improve agaric mycelium yield, and promotion of the siberian Ginseng P.E addition in 1 g/L to black fungus growth is made
With reaching maximum, mycelium dry weight be without 1.29 times.
Embodiment 2：Influence of the siberian Ginseng P.E induction fermentation to agaric mycelium form and its polysaccharide yield
1st, the preparation of black fungus zymotic fluid：The black fungus shaking flask bacterial strain that will have been activated is inoculated in seed culture medium by inoculum concentration 1%,
Wherein seed culture medium is, in BD PDATMCulture medium (a kind of standard medium, produced in USA), cultivates at 25 ± 0.1 DEG C
4 days, obtain black fungus liquid seeds nutrient solution；Black fungus seed culture fluid is accessed into the fermentation medium (4L) after sterilizing, hair
Ferment culture medium prescription is：The g/L of glucose 7, the g/L of corn flour 10, the g/L of bean powder 2, the g/L of siberian Ginseng P.E 2.5, defoamer
0.2 g/L, regulation pH values 5.5, inoculum concentration is 12% (v/v), and ventilation is the 50~100% of tank volume per minute, tank pressure
0.02~0.05Mpa, fermented and cultured 7 days at 28 DEG C, reduced sugar minimizes 0.2% and slightly fluctuates；Microscopy mycelia is elongated,
Without living contaminants.Obtain final product black fungus zymotic fluid 3.1Kg.
2nd, prepared by agaric mycelium polysaccharide：After agaric mycelium is dried, solid-liquid ratio 1:40（m/v）, load 5L enzymes
In solution reaction kettle, 50 DEG C are warming up to（40~50 DEG C）, adjust pH 6.0（5.0~6.0）, it is subsequently adding thalline
Dry weight 1~2%（Specially 2.0g）Complex enzyme（Cellulase：Pectase：Papain=2:1:1, mass ratio）, stir
Mix enzymolysis 3 hours（3~6 hours）；20min is kept to go out enzyme activity at 95 DEG C, centrifuging and taking supernatant, concentration adds 4V bodies
Long-pending absolute ethyl alcohol, overnight alcohol precipitation at 4 DEG C, then removes ethanol, it is lyophilized after obtain mycelium polysaccharides.
3rd, monosaccharide composition analysis method：1-phenyl-3-methyl-5-pyrazolones ketone column front derivation high performance liquid chromatography（PMP-
HPLC）Method determines monose composition.2 mg testing samples are taken, the 0.2 mol/L trifluoroacetic acids of 1 mL are added, hydrolyzed at 120 DEG C
120 min, cooling, N2Drying, adds the 0.3 mol/L NaOH of 50 μ L, adds the 0.5 mol/L PMP of 50 μ L,
React 60 min at 70 DEG C, after derivative terminates, plus in the 0.3 mol/L HCl of 50 μ L and remaining alkali, plus 1 mL 0.1
Mol/L PBSs and 1 mL chloroforms are extracted, and collect upper strata aqueous phase, repeat extraction 3~4 times, upper machine analysis.
Liquid phase analysis condition is:Chromatographic column: ZORBAX Eclipse XDB -C18（250 mm × 4.6 mm, 5 μm）；
Mobile phase:(both volume ratios are 83 to the mixed solvent of the phosphate buffer of 0.1 mol/L, pH 6.8 and acetonitrile:17)；Post
Temperature：30 DEG C, ultraviolet detection wavelength: 248 nm；Flow velocity: 1 mL/min；Sampling volume: 10μL.
4th, inoxidizability method of testing：Using ABTS radical scavenging activities and oxygen radical removing ability（Oxygen
Radical absorbance capacity, ORACFL）Test.Test result is with water miscible vitamin e derivative
Trolox is standard control, calculates amount (μm ol) of the every gram of polysaccharide sample equivalent to Trolox materials, and final inoxidizability is with μ
Mol Trolox/g is represented.
5th, result
Addition siberian Ginseng P.E the results are shown in Table 1 to the influence that agaric mycelium grows.
Table 1：The influence that siberian Ginseng P.E induction fermentation grows to black fungus bacterial ball
As shown in Table 1, with the extension of fermentation time, small-sized bacterium ball（D<1）Number is being gradually reduced, medium-sized（1≤D≤2）With
Large-scale bacterium ball（2 <D≤3）Number gradually increases.Compared with the control, after addition siberian Ginseng P.E, the large-scale bacterium ball of black fungus
In the fermentation middle and later periods（5~9d）Accumulating rate substantially speeds.During fermentation 9d, the ratio of large-scale bacterium ball is up to 94.47%.Explanation
Siberian Ginseng P.E induction fermentation promotes agaric mycelium fast-growth, metabolite is entered in advance and is flowed to.
Agaric mycelium biomass and polysaccharide are constituted siberian Ginseng P.E and the influence of inoxidizability the results are shown in Table 2.
Influence of the siberian Ginseng P.E of table 2 to black fungus Fungal biodiversity and polysaccharide composition and inoxidizability
Learnt from table 2, with contrast ratio, siberian Ginseng P.E is added in zymotic fluid can be improved agaric mycelium biomass
33.3%, galactose content increases by 35.3%, glucose content and declines 39.1%, inoxidizability enhancing 5.8 in intracellular polyse composition
Times.
Case study on implementation 3, the siberian Ginseng P.E induction fermentation external immunity test of agaric mycelium polysaccharide
1st, material
（1）Siberian Ginseng P.E induction fermentation agaric mycelium polysaccharide is prepared by the inventive method
The induction fermentation gained agaric mycelium polysaccharide self-control of addition siberian Ginseng P.E
（2）Mouse macrophage RAW264.7 derives from University Of Science and Technology Of Tianjin
2nd, method
（1）Cytotoxicity test：Using MTT colorimetric methods.RAW264.7 cells are counted, cell concentration reaches 5 × 104Cell/mL,
Take 100 μ L and be added to 96 orifice plates, at 37 DEG C, in containing 5% CO2Adhere-wall culture 24h in cell culture incubator, removes supernatant culture
Base, the testing sample for adding concentration to be 50~250 μ g/mL, puts containing 5% CO2After cell culture incubator continues to cultivate 24h, add
10μL MTT（5mg/mL）, 37 DEG C are incubated 4h, remove supernatant, add 150 μ L DMSO, the first that dissolving living cells is generated with MTT
A ceremonial jade-ladle, used in libation material, shakes 20min, and light absorption value is determined under 490nm, is control with blank cultures.
Cell viability measurement（%）=sample OD490Nm values/blank OD490Nm value × 100
（2）NO burst sizes and cytokine secretion measure examination：NO burst sizes are tested using Griess methods.Count RAW264.7 thin
Born of the same parents, concentration reaches 2 × 106During cell/mL, take 100 μ L and be added to 96 orifice plates, under the conditions of 37 DEG C, containing 5% CO2Cell culture
Adhere-wall culture 24h in case, removes supernatant culture medium, the testing sample that concentration is 50~200 μ g/mL is added, with 1 μ g/mL
LPS and blank cultures are control, after putting cell culture incubator culture 24h, draw the cell supernatant of 50 μ L with 50 μ L's
Griess reagents（1% sulfanilamide (SN) and 0.1% NDP are dissolved in 5% phosphoric acid）Room temperature reaction 5min is placed in, OD values are surveyed in 540nm, with
NaNO2（10~100 μM）For standard items draw standard curve, wherein abscissa x is NaNO2Concentration, ordinate y is absorbance
Value.Cytokine TNF-α, IL-6, IL-10 burst size are tested using ELISA enzyme linked immunological kits.
3rd, result
The result of siberian Ginseng P.E induction fermentation agaric mycelium Polysaccharides on Mice macrophage RAW264.7 toxicity tests
See Fig. 2.As shown in Figure 2, polysaccharide no cytotoxicity in the range of 50-250 μ g/mL, and with promotion RAW264.7 cell propagation
Effect.
Siberian Ginseng P.E induction fermentation agaric mycelium polysaccharide activation mouse macrophage RAW264.7 release NO and
Cell factor（TNF-α, IL-6 and IL-10）The results are shown in Table 3.
The Blackfungus polyhexose active cell RAW264.7 of table 3 discharges cell factor
As known from Table 3, the notable activating macrophage RAW264.7 releases cell factor of 50 μ g/mL wilsonii Blackfungus polyhexoses
TNF-α, IL-6 and IL-10, promote the burst size of NO under 200 μ g/mL concentration.Illustrate that siberian Ginseng P.E induction fermentation is black
Auricularia mycelium polysaccharide has the immunocompetent ability of regulation to macrophage RAW264.7.
Mycelium dry product nutritional ingredient detection prepared by case study on implementation 4, siberian Ginseng P.E induction fermentation black fungus bacterial
Protein uses the methods of GB5009.5-2,010 first；Total reducing sugar uses GB/T15672-2009；Dietary fiber is used
GB5009.88 - 2014；Microelements of calcium uses GB/T5009.92-2003；Iron uses GB/T5009.90-2003.
Testing result shows the agaric mycelium nutritional ingredient of present invention preparation（It is shown in Table 4）Including：
Protein 15%（Weight/mass percentage composition）Or more, protein refers to mycelium protein；
Total reducing sugar 56%（Weight/mass percentage composition）Or more, total reducing sugar refers to soluble polysaccharide and monose, specifically include macromolecular glycan,
Glucose etc.；
Dietary fiber 42% or more, dietary fiber refers to without oligosaccharide, resistant starch, resistant maltodextrin etc. in thalline
Dietary fiber；
Microelements of calcium 500mg/100g or more, calcium refers to total calcium constituent in thalline；
Trace elements iron 25mg/100g or more, iron refers to total ferro element in thalline；
The nutritional ingredient of the agaric mycelium of table 4
Claims (9)
1. a kind of siberian Ginseng P.E induction fermentation black fungus bacterial prepares mycelial method, it is characterised in that：With wild black wood
Ear bacterium is starting strain, and siberian Ginseng P.E is with the addition of in the medium, by liquid deep layer fermenting, obtains black fungus mycelia
Body, comprises the following steps：
（1）Black fungus strain is inoculated on slant medium according to 3~5% weight ratio, is cultivated at 22~26 DEG C
7~12d, obtains the black fungus strain of activation；The composition and its content of described slant medium be：Farina 35
~50g/L, 15~25g/L of glucose, 15~20g/L of agar, remaining is water；
（2）The black fungus strain of activation is accessed, is smashed to pieces, the black fungus strain that will be activated according to the ratio of 3~6g/L is inoculated with
In liquid seed culture medium, 4~6d is cultivated under the conditions of 22~26 DEG C, rotating speed are for 120~200rpm, obtain liquid
Seed；The composition and its percentage by weight of described seed culture medium be：35~50g of farina/L, glucose
15~25g/L, remaining is water；
（3）According to the volume ratio of 10~20% (v/v) by step（2）The liquid seeds of gained are inoculated in fermentation medium
In, it is 0.02~0.05MPa in 25~28 DEG C, pressure, trains under the conditions of 50~100% that ventilation is tank volume per minute
7~9d is supported, siberian Ginseng P.E induction fermentation black fungus bacterial zymotic fluid can be obtained；The composition of the fermentation medium and its
Content is：5~9g/L of glucose, 8~12g/L of corn flour, 1~3g/L of bean powder, siberian Ginseng P.E 0.5~2.5
G/L, remaining is water.
2. according to the fermentation culture method described in claim 1, it is characterised in that its step（1）Described in black fungus strain
Size be 3~5 × 3~5mm.
3. according to the fermentation culture method described in claim 1 or 2, it is characterised in that its step（2）Described in liquid strain
Sub- culture medium liquid amount is the 40% of tank volume.
4. according to the fermentation medium described in claim 3, it is characterised in that the mass body of siberian Ginseng P.E in culture medium
Product is 0.5~2.5 g/L.
5. according to the fermentation culture method described in claim 3, it is characterised in that its step（3）Or（4）Described in liquid hair
The canned liquid measure of ferment culture medium is the 60~80% of tank volume.
6. the one kind according to claim 1 prepares siberian Ginseng P.E induction fermentation black fungus bacterial zymotic fluid method, and it is special
Levy is that described fermented and cultured terminal is：Reduced sugar is minimized and slightly has fluctuation, reduced sugar control less than 0.3%；Microscopy
Mycelia is elongated, without living contaminants.
7. the agaric mycelium that according to claim 1 prepared by method, it is characterised in that：Specifically, it may include below
Step：
（1）Fermentation：Liquid submerged femrentation culturing method according to claim 1 obtains wilsonii black fungus zymotic fluid；
（2）Dry：After fermentation 7-9 days, karusen obtains zymotic fluid and agaric mycelium through filtering, and mycelium homogenate is passed through again
Freeze-drying（Condition is：- 10 DEG C~-50 DEG C of temperature, the handkerchief of vacuum 1.3~13）Or spray drying（Condition is：Inlet temperature
180 DEG C, 100 DEG C of outlet temperature）Or heated-air drying（Condition is：80 DEG C of hot blast temperature）, obtain wilsonii agaric mycelium and do
Product.
8. agaric mycelium dry product according to claim 7 contains dietary fiber 35-50 g, calcium 400- per 100g
600mg。
9. the agaric mycelium dry product for being prepared according to claims 1,7 has the natural attractive in appearance, unique flavor of color and luster, nutrition rich
The characteristics of rich and certain health-care effect.
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| CN113303169A (en) * | 2021-07-11 | 2021-08-27 | 贵州大学 | Black fungus culture material and preparation method thereof |
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