CN104323222A - Mushroom powder rich in vitamin K2 and preparation method thereof - Google Patents
Mushroom powder rich in vitamin K2 and preparation method thereof Download PDFInfo
- Publication number
- CN104323222A CN104323222A CN201410473168.4A CN201410473168A CN104323222A CN 104323222 A CN104323222 A CN 104323222A CN 201410473168 A CN201410473168 A CN 201410473168A CN 104323222 A CN104323222 A CN 104323222A
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- CN
- China
- Prior art keywords
- farnoquinone
- fermentation
- mushroom powder
- preparation
- bacterium
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- Granted
Links
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The present invention discloses a mushroom powder rich in vitamin K2. The mushroom powder contains a plurality of nutrients, wherein the content of vitamin K2 is over 200ug/g. The mushroom powder also contains a plurality of proteins, dietary fibers and trace elements. The present invention also discloses a preparation method of the mushroom powder. According to the present invention, autonomously screened bacillus natto (preservation number CCTCC M2014405) is used as a starting strain, mushroom dregs extracted out of active polysaccharides are used as main nitrogen source, a large number of bacterial cells are bred through fermentation controlled by appropriate means. The mushroom powder rich in vitamin K2 is prepared through cell collection, membrane filtration or centrifugation, and spray drying. The mushroom powder has efficacy of treating and preventing osteoporosis, promoting cardiovascular health, repairing damaged cells, resisting tumor, and the like. The present invention establishes the foundation for industrial production of vitamin K2, comprehensive utilization of mushroom dregs and development of new health care foods, and provides a new approach for producing vitamin K2.
Description
Technical field
The present invention relates to a kind of bacterium mushroom powder being rich in farnoquinone and preparation method thereof, belong to biological technical field.
Background technology
Farnoquinone is the fat-soluble blood coagulation biostearin of a class, is aphthoquinone series compound, in the prevention and therapy of osteoporosis, has significant effect, is called as " forth generation anti-osteoporosis product ".Farnoquinone is a compounds---methylnaphthoquinone compounds, 14 kinds of forms are had according to the length difference of C-3 position isoprene side chains on its molecular structure, represent (number that n refers to isoprene unit on side chain) with MK-n, MK-4 (MK-4) and menaquinone-7 (MK-7) the most common, MK-4 is similar to vitamin K1 molecular weight, all there is very short serum half-life, realize blood circulation and need very large dosage, higher than MK-7 RD 1000 times.And MK-7 fast by intestinal absorption, can be transported by large lipid granule, stop in the circulatory system for a long time, blood halflife is long, has the ability to play a role in the bone except liver and artery.Farnoquinone prepared by the present invention belongs to MK-7 configuration.
Farnoquinone can improve rapidly due to vitamin K deficiency cause hemorrhage, therefore farnoquinone is used as treating hemorrhage anticoagulant always in decades, is not concerned as important nutrients a kind of in human body.Except as except anticoagulant, the most significant function of farnoquinone is for improving bone and cardiovascular health.According to report, farnoquinone is all right: 1) anti-arteriosteogenesis and osteoarthritis; 2) antitumor; 3) delay senility; 4) repair damaging cells, potential treatment parkinsonism medicine, is widely used in the field such as medicine, food.Along with going deep into and strengthening the research of farnoquinone physiological function, the application of farnoquinone is more and more extensive, and obtains the accreditation of authoritative institution.Within 2005, Japan's approval farnoquinone goes on the market as osteosporosis resistant medicament.In farnoquinone is also listed in by State Food and Drug Administration of China " nutritious supplementary pharmaceutical declare with evaluate specify (trying) " " vitamin, mineral cpd list " (state eat medicine supervise note [2005] No. 202), can be used as the raw material of health food or nutrition fortifier.In January, 2008, farnoquinone is assert by U.S. food Drug Administration (FDA) safety.2009, European Parliament and EU Council determined in its all member state, and farnoquinone can be used as the raw material (2009/345/EC) of health food or nutrition fortifier.
Grifola frondosus, mushroom, Agricus blazei, Hericium erinaceus, coprinus comatus are common medicine-food two-purpose bacterium, and they contain abundant nutriment.As: in every hectogram dried thin mushroom, containing 13 grams, protein, 1.8 grams, fat, calcium 124 milligrams, 415 milligrams, phosphorus, iron 25.3 milligrams, and abundant vitamin B1, B2, C, D etc.Lentinus edodes-protein is made up of 18 amino acid, accounts for 35.9% of essential amino acid total amount, and nutritive value is very high.Grifola frondosus is described as " edible mushroom prince " and " North China ginseng ", its dry product contains 25.2 grams, protein (wherein containing amino acid needed by human body 18 kinds 18.68 grams, wherein must base acid account for 45.5%) fat 3.2 grams, dietary fiber 33.7 grams, 21.4 grams, carbohydrate, and being rich in the minerals and vitamins of multiple beneficial, various nutrients occupies first of various edible mushroom.The title that Hericium erinaceus has " mountain delicacy hedgehog hydnum, seafood delights shark's fin ", containing Hericium erinaceus (Bull. Ex Fr.) Pers. Ketone, alkali and glucan, ergosterol, monkey mushroom rhzomorph and polysaccharide etc., has the effects such as tonifying spleen and stomach, aid digestion, kidney-nourishing is smart.Above-mentioned bacterium mushroom, after flooding active polysaccharide, still contains a large amount of protein in bacterium mushroom slag, dietary fiber isoreactivity material, fermenting raw materials is done with it, can provide growth of microorganism as nitrogenous source on the one hand, other active materials still retain on the other hand, improve the nutritive value of subsequent product.
The domestic patent prepared about farnoquinone mainly concentrates at present: (1) induction mutation of bacterium screens; (2) purification of farnoquinone; Do not utilize cheap raw material to carry out the patent of farnoquinone production, more do not utilize these type of dietotherapeutics such as grifola frondosus slag, mushroom residue, there is the bacterium mushroom slag of fine health-care effect and medical value do the example that raw material carries out farnoquinone production.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of bacterium mushroom powder being rich in farnoquinone.
The technical problem that the present invention also will solve is to provide the preparation method of above-mentioned bacterium mushroom powder.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of preparation method being rich in the bacterium mushroom powder of farnoquinone, take bafillus natto as starting strain, to extract the bacterium mushroom slag of active polysaccharide for main nitrogen, fermentation thalli cell, collecting cell, through membrane filtration or centrifugal, the spray-dried obtained bacterium mushroom powder being rich in farnoquinone of collection solid portion.
Wherein, described farnoquinone is MK-7 type.
Concrete preparation method, comprises the steps:
(1) utilize boulder crusher tentatively to be pulverized by the bacterium mushroom slag extracting active polysaccharide, then carry out fine crushing with micronizer, enter sieving machine and cross 200 mesh sieves, obtain original bacteria mushroom powder;
(2) carry out actication of culture by the bafillus natto access solid medium being kept at glycerine pipe, cultivate in 37 DEG C of incubators, 20 ~ 28h, continuously two generations of activation;
(3) bacterium on inclined-plane is washed down by the physiological saline after utilizing sterilizing, and access is equipped with in the 250mL shaking flask of 50mL seed culture medium, in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 120 ~ 200rpm, cultivates 24 ~ 48h, obtains primary seed solution;
(4) be equipped with in the 500mL shaking flask of 100mL seed culture medium by first order seed access, inoculum concentration 2 ~ 10% (v/v), in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 120 ~ 200rpm, is cultivated 24 ~ 48h, is obtained secondary seed solution;
(5) secondary seed access is equipped with in the fermentation tank of fermentation medium, inoculum concentration 2 ~ 10% (v/v), add defoamer, maintain pH about 7.0, throughput 0.2 ~ 2vvm, speed of agitator 200 ~ 600rpm, tank temperature 30 ~ 37 DEG C, cultivate 64 ~ 96h, fermenting and producing farnoquinone; The original bacteria mushroom powder that described fermentation medium obtains with step (1) is for main nitrogen;
(6) divide water by fermentation liquor tubular type diffusion barrier equipment or centrifuge, concentrated material is to more than 200g/L, and filtrate retains farnoquinone through nanofiltration;
(7) by concentrated material with retain the bacterium mushroom powder that the drying of material spray-dried machine obtains being rich in farnoquinone.
In step (1), described extraction the bacterium mushroom slag of active polysaccharide be extracted the mushroom residue of lentinan, the grifola frondosus slag extracting grifolan, the Agricus blazei slag extracting Agaricus Blazei Murrill polysaccharide, the coprinus comatus slag extracting coprinus comatus polysaccharide and any one or a few the mixture extracted in the Hericium erinaceus slag of hericium erinaceum polysaccharide.
In step (2), described bafillus natto, its Classification And Nomenclature is bafillus natto R127 (Bacillus natto R127), be preserved in China typical culture collection center (being called for short CCTCC), address: Wuhan, China Wuhan University, postcode: 430072, its deposit number is CCTCC NO:M 2014405, and preservation date is on September 9th, 2014.This bafillus natto obtains from the natto product that market, Nanjing is bought, and by ultraviolet mutagenesis, screening obtains the higher bacterial classification of proteinase activity.The farnoquinone prepared by this strain fermentation is MK-7 type.
Bafillus natto CCTCC NO:M 2014405 strain characteristic is as follows:
(1) colonial morphology: by bacterial classification dilution spread on solid medium, 30-37 DEG C of cultivation, after 24h, bacterium colony rough surface is opaque, dirty white or micro-yellow;
(2) thalli morphology: form and the structure of observing CCTCC NO:M 2014405 under an optical microscope, thalline is shaft-like, the long 0.5-3 micron of individual cells, and gram-positive bacteria is aerobic, containing gemma, is positioned at thalline central authorities or slightly inclined;
(3) Liquid Culture: quiescent culture surface can produce the biomembrane of one deck white, concussion cultured cell fast growth, without white films;
(4) metabolism: main metabolites is the farnoquinone of seven alkene methylnaphthoquinone configurations, this bacterial strain all can grow at 25-40 DEG C, and wherein 30-37 DEG C is optimum growth temperature; Thalline is adapted at growing in neutral pH nutrient solution.
(5) ectoenzyme: can produce protease, amylase, cellulase etc., the outer nitrogen-containing material of decomposable asymmetric choice net born of the same parents, as soy meal, bacterium slag etc.
Judge the microorganism of this bacterium as gemma Pseudomonas by form and cultural character, for identifying obtained bacterial strain further, using molecular biology method and its 16sRNA sequence (as shown in SEQ ID No:1) is compared.Find that this bacterium exists the homology of 96% with the 16SRNA sequence of Bacillus subtilis, therefore judge that this bacterial strain belongs to bacillus, called after Bacillus natto R127.
In step (2), (3) or (4), described solid culture based formulas is: 30g/L glucose, 40g/L peptone, 5g/L NaCl, 5g/L beef extract, 5g/L yeast extract, 30g/L agar, and solvent is water.Seed culture based formulas is 10-50g/L glucose, 20-80g/L peptone, 2-10g/L NaCl, 2-10g/L beef extract, 2-10g/L yeast extract, and solvent is water.Seed culture medium optimization formula is: 30g/L glucose, 40g/L peptone, 5g/L NaCl, 5g/L beef extract, 5g/L yeast extract, and solvent is water.Fermentative medium formula is 30-80g/L glycerine, 20-100g/L soy peptone, 0.2-1.5g/L dusty yeast, 0.1-1g/LK
2hPO
4, 0.1-1g/LCaCl
2h
2o, 0.1-1g/LMgSO
47H
2o, solvent is water.Fermentation medium optimization formula is: 50g/L glycerine, 30g/L soy peptone, 0.6g/L dusty yeast, 0.3g/LK
2hPO
4, 0.1g/LCaCl
2h
2o, 0.3g/LMgSO
47H
2o, solvent is water.
In step (5), described defoamer is silicone SE-2, and use amount is 0.2-3g/L, preferred 0.3g/L.
In step (5), fermentation cylinder for fermentation is produced farnoquinone grease and is adopted Intermittent fermentation or feed supplement formula fermentation process.
In step (5), fermentation tank adopts the gas stone (micropore distributor) of breeding fish as gas distributor.
In step (5), original bacteria mushroom powder before adding fermentation medium as main nitrogen, first through the pretreatment of hydrolysis by novo.Hydrolysising condition: temperature 30 ~ 60 DEG C, rotating speed 150 ~ 300rpm, time 1 ~ 3h, pH7 ~ 10, after being decomposed by insoluble albumen, then ferment.
The process that the present invention takes is equally applicable to bafillus natto with other bacterial strains of all belonging to and carry out mutagenic obtained bacterial classification on this basis, as Bacillus subtilis CICC10262, Bacillus subtilis CGMCC NO.8400 etc., the suitableeest bacterial classification is CCTCC NO:M 2014405.
What above-mentioned preparation method prepared is rich in the bacterium mushroom powder of farnoquinone also within protection scope of the present invention.
This bacterium mushroom powder has following nutritive peculiarity:
(1) farnoquinone of 100ug/g-300ug/g is rich in;
(2) granularity can cross 200 mesh sieves;
(3) containing dried bacillus bacterium powder;
(4) containing a certain amount of protein, dietary fiber, trace element (as vitamin D, mineral matter), a small amount of lipid material (as: ergosterol);
The above-mentioned bacterium mushroom powder being rich in farnoquinone has prevention of osteoporosis, cardiovascular health in preparation, delays senility, the anticancer or application of repairing in the health food of damaging cells function or medicine is also within protection scope of the present invention.
Bacterium mushroom powder prepared by the present invention is except containing except farnoquinone, also containing vitamin D, in calcium uptake, there is positive effect, vitamin D can promote that calcium is in the absorption of small intestine, calcium is converted into blood calcium, and farnoquinone can promote that blood calcium is to the conversion of bone calcium, allow the place that calcium current goes to this, prevent the generation of the problems such as the vascular sclerosis formed because of the accumulation of calcium at positions such as blood vessels, it is the perfect combination of replenishing the calcium, add the farnoquinone reported in the articles such as Science in the recent period and repair the effect in damaging cells etc., therefore bacterium mushroom powder provided by the present invention is in prevention of osteoporosis, cardiovascular health, delay senility, anticancer or repair the aspects such as damaging cells function there is health care.
Current calcium supplementing product is calcium tablet vitaminize D mainly, and vitamin D promotes calcium uptake, becomes the calcium in blood, and the object of replenishing the calcium is mended on bone, farnoquinone mainly promotes that blood calcium transforms skeletonization calcium, this bacterium mushroom powder provided by the present invention, be rich in the farnoquinone of more than 100ug/g, in addition, mushroom inherently containing a small amount of vitamin D, is also withed a hook at the end in bacterium mushroom slag, so, both containing VD in this product, again containing VK2, be excellent calcium supplementing product.
Beneficial effect: the invention provides a kind of bacterium mushroom powder being rich in farnoquinone, it is of high nutritive value, taste-aromatic, be suitable as prepare prevention and therapy osteoporosis, prevention cardiovascular health, delay senility, anticancer, health food or the medicine of repairing the function such as damaging cells.Present invention also offers the preparation method of this bacterium mushroom powder simultaneously, with bafillus natto (CCTCC NO:M 2014405) for starting strain, to extract the bacterium mushroom slag of active polysaccharide for main nitrogen, fermentation accumulated vitamins K2, collecting cell, obtains through membrane filtration or centrifugal, spraying dry the bacterium mushroom powder being rich in farnoquinone.The method obtain farnoquinone output with utilize the concentration of the nitrogenous source such as soy meal, soy peptone close, there is industrialization potential, containing 100-300ug/g farnoquinone in the bacterium mushroom powder of preparation, it is 45-125 microgram (adult human dose) that current MK7 is in the world used for osteoporotic effective dose, and this bacterium mushroom powder therefore eaten every day less than 1g can reach meal supplement requirement.In addition, in bacterium mushroom, self contains medium trace element to some extent, as vitamin D, with farnoquinone conbined usage, is excellent calcium supplementing product.
accompanying drawing explanation
Fig. 1 is fermented sample farnoquinone (MK-7) mass spectrogram.
Detailed description of the invention
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1: bacterial strain screening
Get 1g natto product to be dissolved in 100ml physiological saline, vibration mixes, by the order obtained 10 of sample suspension according to every grade of dilution 10 times
-1-10
-7individual concentration, respectively by 10
-3-10
-7draw 100ul dilution in five concentration to be added in screening and culturing base and to be coated with, each concentration is coated with three blocks of plates, 37 DEG C, cultivate 48h, select and can produce hydrolysis circle and larger bacterium colony, continue to rule in screening and culturing base, obtain pure bacterium, through the multiple sieve that ferments, high-efficient liquid phase analysis and Mass Spectrometric Identification, acquisition can produce the bacterial classification Bacillus natto R of farnoquinone, and farnoquinone that this bacterial strain produces is MK-7 configuration (as Fig. 1).
For improving the protelytic ability of bacterial classification, mutagenesis screening is carried out to gained Bacillus natto R: by the bacterium liquid brine after activation, bacteria suspension is irradiated under uviol lamp, irradiation distance 30cm, sample every 20s, finally select 180s, fatal rate carries out mutagenesis screening in the condition of about 80%, gained mutagenic bacteria liquid dilution spread is in screening and culturing base, the bacterial strain that screening transparent circle is larger, called after Bacillus natto R127, is preserved in Chinese Typical Representative Culture Collection, and its deposit number is CCTCC NO:M 2014405.
Screening and culturing base is: 4g casein is dissolved in 100ml water, 115 DEG C of sterilizing 20min, beef extract 3g, and peptone 10g, NaCl 5g, agar 20g are dissolved in 900ml water, 121 DEG C, and sterilizing 20min, is cooled to about 50 DEG C, both is mixed, is down flat plate stand-by.
HPLC method: chromatographic column: C18 post, column temperature: 50 DEG C, mobile phase: methyl alcohol, flow velocity: 1ml/min, UV detect wavelength 270nm, sample size: 30ul, detection time: 35min.
Embodiment 2: the basic growth performance of bacterial strain is investigated.
Key property investigation is carried out to the bacterial strain CCTCC NO:M 2014405 that screening obtains, different liquids seed culture medium is adopted to cultivate, investigate cell growth rate, result is as table 1, and used medium is (% represents the quality g containing material in every 100ml culture medium):
Culture medium 1:100mL bean sprout juice, 5% sucrose
Culture medium 2:1% glucose, 1% peptone, 0.5%NaCl
Culture medium 3:3% glucose, 4% peptone, 0.5%NaCl, 0.5% beef extract, 0.5% yeast extract
Culture medium 4:0.5% glucose, 1% peptone, 0.5%NaCl, 0.5% beef extract, 0.5% yeast extract
Table 1 different seed culture medium thalli growth information slip
Drawn by above-mentioned result of the test, except No. 2 culture mediums, other three kinds all can make bacterial classification grow preferably, select No. 3 culture mediums.
Embodiment 3: strain for accumulating farnoquinone ability is investigated.
Four kinds of different fermentations culture mediums are selected to ferment, the production capacity of Primary Study bacterial classification farnoquinone.Cultivation temperature 30 DEG C, rotating speed 120rpm.(% represents the quality g containing material in every 100ml culture medium):
Culture medium 1:5% glycerine, 3% soy peptone, 0.6g/L dusty yeast, 0.3g/L K
2hPO
4, 0.1g/LCaCl
2h
2o, 0.3g/LMgSO
47H
2o
Culture medium 2:5% glycerine, 3% analysis for soybean powder, 0.6g/L dusty yeast, 0.3mol/LK
2hPO
4, 0.1g/LCaCl
2h2O, 0.3g/LMgSO
47H2O
Culture medium 3:15g/L peptone, 25g/L dusty yeast, 70g/L glycerine, 0.5g/L K
2hPO
4, pH7.3
Culture medium 4:100mL soya-bean milk, 2% glucose, 0.5%NaCl, 2%NaOH adjust pH7.0
Table 2 different fermentations culture medium produces farnoquinone comparison sheet
Drawn by above-mentioned result of the test, No. 3 culture medium base consumption speed are lower, and total fat content of No. 2 culture medium extractions is up to 6.99g/L, and this is that soy meal is relevant with main nitrogen in its culture medium and raw material, soy meal contains more grease, is proposed when fermentation ends simultaneously.And for the ability of synthetic vitamin K2, the synthesis of all favourable farnoquinone of 1-3 culture medium, wherein No. 1 culture medium reaches 10.5mg/L, is maximum output, and follow-up study is further experiment based on this culture medium.And No. 2 culture mediums and No. 1 maximum difference are that soy peptone has changed soy meal into, both are more or less the same at output, and visible cell protein capacity of decomposition is stronger.
Embodiment 4: improve the fermentation of 50L tank gas distributor and produce farnoquinone.
Consider in large-scale fermentation, rotating speed can not reach 600rpm, therefore the gas stone (micropore distributor) of breeding fish is adopted to improve fermentation tank gas distributor at 50L fermentation tank, rotating speed adjustable reduces energy consumption at below 300rpm, this micropore distributor can by dispersed for bubble one-tenth minute bubbles, improve gas transfer efficiency, the results are shown in Table 3, fermentation period foreshortens to 48h, farnoquinone output reaches 40.32mg/L, glycerine is reduced to about 5g/L, add glycerine, output reaches 58.64mg/L, 1.955mg/g is reached relative to soy peptone conversion ratio, for reporting highest level so far, production cost reduces greatly.
Table 3 gas distributor improves
Embodiment 5: farnoquinone production technology
By CCTCC NO:M 2014405 bacterial classification according to solid culture based formulas be: 30g/L glucose, 40g/L peptone, 5g/LNaCl, 5g/L beef extract, 5g/L yeast extract, 30g/L agar, activate, 37 DEG C, cultivate 20h, with physiological saline, inclined-plane thalline is washed down, Liquid Culture is carried out in culture medium 3 in access embodiment 2, 37 DEG C, 120rpm, after cultivating 24h, accessing liquid amount according to 5% inoculum concentration is carry out two cultures in the 500ml shaking flask of 100ml, then according in the inoculum concentration access 15L seeding tank of 5%, 1vvm, 200rpm, tank temperature 37 DEG C, cultivate 24h, according to 5% inoculum concentration, seed liquor is accessed with in the 50L fermentation tank of micropore distributor, liquid amount 35L, culture medium is with the culture medium 1 in embodiment 3, rotating speed 200rpm, ventilation 1vvm, temperature 37 DEG C, detect glycerine Expenditure Levels, glycerine is reduced to about 5g/L, add glycerine, final farnoquinone output reaches 60.54mg/L, 2.018mg/g is reached relative to soy peptone conversion ratio, for reporting highest level so far, production cost reduces greatly.
After fermentation ends, zymotic fluid is carried out membrane filtration, collect filtrate and cell respectively, sampling detects two-part farnoquinone content.Cell utilizes homogenizer broken, filtrate proceeds nanofiltration again, trapped fluid is mixed into that broken Cell relay is continuous to be extracted, and stirs and evenly mixs, extract three times according to the volume ratio of bacterium liquid, isopropyl alcohol, n-hexane 1:1:2, collect n-hexane layer, carry out rotary evaporation, obtain the grease being rich in farnoquinone, this grease is carried out HPLC analysis, detect the content of farnoquinone, recovery rate 95.11%.
Embodiment 6: various bacterium mushroom powder raw material basis is investigated.
For better understanding material characteristic, formulate fermentating formula, first detect the content of total nitrogen, soluble protein in various bacterium mushroom powder raw material respectively with Kjeldahl's method, Coomassie Brilliant Blue, concrete numerical value is as follows:
Table 4 different bacterium mushroom slag basis table
% represents the quality g containing material in every 100ml culture medium
From upper table result: in bacterium mushroom slag, nitrogen content is between 2-5%, total protein content, at 15-30% albumen, is a kind of microorganism cultivation nitrogenous source preferably; After this type of raw material dissolves, pH is neutral, and can not affect greatly medium pH, be comparatively ideal alternative materials.
Embodiment 7: utilize Lenlinus edodes mushroom slag fermentation to prepare farnoquinone.
Utilize Lenlinus edodes slag as nitrogenous source, method to specifications carries out actication of culture and fermentation, comparatively soy peptone is low to consider in bacterium mushroom slag nitrogen content, normal fermentation generally selects the soy peptone of 3%, therefore in replacement test, increases 1-1.5 consumption doubly, this fermentation medium is: 50g/L glycerine, 50g/L mushroom residue, 0.6g/L dusty yeast, 0.3g/LK
2hPO
4, 0.1g/LCaCl
2h
2o, 0.3g/LMgSO
47H
2o, be ferment in the 500ml shaking flask of 100ml at liquid amount, select 120rpm, 150rpm, 180rpm, 210rpm tetra-kinds of rotating speeds, 37 DEG C ferment, and result is as table 5.
Utilize mushroom residue to carry out fermentation results under table 5 different rotating speeds condition to compare
Embodiment 8: utilize Grifolas frondosa germ mushroom slag fermentation to prepare farnoquinone
For improving output further, comparatively peptone is low to consider in Grifolas frondosa germ slag nitrogen content, and addition is too much, is unfavorable for the control of sweat, therefore intend adopting Grifolas frondosa germ slag and the composite mode of soy peptone to ferment, add the Grifolas frondosa germ slag of 50g/L, add the soy peptone of 15g/L, other compositions are with embodiment 2 simultaneously, 150rpm, 37 DEG C, fermentation, result is as table 6.
Grifolas frondosa germ slag fermentation results is utilized to compare under the different compound proportion of table 6
Embodiment 9: utilize other bacterium mushroom slag fermentations to prepare farnoquinone
According to above cultural method, select respectively extracted polysaccharide Agricus blazei, Hericium erinaceus, coprinus comatus bacterium mushroom slag is as nitrogen source fermentation, wherein in Agaricus blazei mushroom slag, nitrogen content is higher, containing protein more than 30%, substitute soy peptone according to equal proportion (3%) to ferment, Hericium erinaceus, coprinus comatus content are about 50g/L, and fermentation condition is with embodiment 3, and result is as table 7.
Table 7 utilizes other bacterium mushroom slag fermentations to produce the fermentation results of VK2
Embodiment 10: utilize mushroom residue fermentation to produce the 5L tank experiment of farnoquinone
Adopt as fermentation medium in embodiment 2, according to 10% inoculum concentration by the bacillus access 5L fermentation tank after activation, divide 200rpm and 600rpm two conditions to ferment respectively, throughput is 1.5vvm, temperature 37 DEG C, pH is maintained about 6.5 in sweat, when glycerol concentration is reduced to about 5g/l, terminate fermentation, weigh biomass, detect farnoquinone content, result is as table 8.
Farnoquinone situation is produced under table 85L fermentation tank different rotating speeds condition
Gained fermentation liquor tubular membrane filtered, obtain the concentrate of solid content more than 20%, nanofiltration obtains filtrate trapped substance, adopts spray dryer to carry out spray concentrate and trapped substance and does, obtain bacterium mushroom powder.
Embodiment 11: increase the ferment effect after bacterium mushroom powder raw material pre-treatment step
In bacterium mushroom slag material, the utilization of albumen needs bacillus self to secrete a large amount of protease to be hydrolyzed, thalline energy will certainly be consumed, probably reduce the ability of thalline synthetic vitamin K2, therefore intend carrying out a step pretreatment of raw material before fermentation, alkali protease is utilized to be hydrolyzed, hydrolysising condition: temperature 50 C, rotating speed 200rpm, time 2h, pH10, after being decomposed by insoluble albumen, then ferment, result is as table 9.
Before and after table 9 bacterium mushroom pulp water solution, farnoquinone volume variance is produced in fermentation
Embodiment 12: utilize bacterium mushroom slag fermentation to produce the technique of farnoquinone
Utilize boulder crusher tentatively to be pulverized by the bacterium mushroom slag extracting active polysaccharide, then carry out fine crushing with micronizer, enter sieving machine and cross 200 mesh sieves, obtain original bacteria mushroom powder; By CCTCC NO:M 2014405 according to solid culture based formulas be: 30g/L glucose, 40g/L peptone, 5g/LNaCl, 5g/L beef extract, 5g/L yeast extract, 30g/L agar, activate, 37 DEG C, 24h, in continuous activation two generation, utilize the physiological saline after sterilizing to be washed down by the bacterium on inclined-plane, access is equipped with in the 250mL shaking flask of 50mL seed culture medium (in embodiment 2 culture medium 3), in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 120rpm, cultivate 24h, obtain primary seed solution; After according to 5% inoculum concentration access be equipped with in the 500mL shaking flask of 100mL seed culture medium (in embodiment 2 culture medium 3) expand cultivate; By secondary seed in 5% ratio access band micropore distributor fermentation tank in, fermentation medium, with the culture medium 1 in embodiment 3, adds 0.3g/L defoamer silicone SE-2, maintains pH about 7.0, throughput 1vvm, speed of agitator 250rpm, tank temperature 37 DEG C, cultivates 96h, after fermentation ends, tubular type diffusion barrier equipment divides water, concentrated material is to more than 200g/L, and filtrate retains farnoquinone through nanofiltration, and trapped substance and the spray-dried machine drying of enrichment materials obtain the bacterium mushroom powder being rich in farnoquinone.Detect each nutrition content in bacterium mushroom powder, as table 10.
Table 10 ferments each nutrition content in gained bacterium mushroom powder
Claims (10)
1. one kind is rich in the preparation method of the bacterium mushroom powder of farnoquinone, it is characterized in that, take bafillus natto as starting strain, to extract the bacterium mushroom slag of active polysaccharide for main nitrogen, fermentation thalli cell, collecting cell, through membrane filtration or centrifugal, the spray-dried obtained bacterium mushroom powder being rich in farnoquinone of collection solid portion.
2. preparation method according to claim 1, is characterized in that, described farnoquinone is MK-7 type.
3. preparation method according to claim 2, is characterized in that, it comprises the steps:
(1) utilize boulder crusher tentatively to be pulverized by the bacterium mushroom slag extracting active polysaccharide, then carry out fine crushing with micronizer, enter sieving machine and cross 200 mesh sieves, obtain original bacteria mushroom powder;
(2) carry out actication of culture by the bafillus natto access solid medium being kept at glycerine pipe, cultivate in 37 DEG C of incubators, 20 ~ 28h, continuously two generations of activation;
(3) bacterium on inclined-plane is washed down by the physiological saline after utilizing sterilizing, and access is equipped with in the 250mL shaking flask of 50mL seed culture medium, in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 120 ~ 200rpm, cultivates 24 ~ 48h, obtains primary seed solution;
(4) be equipped with in the 500mL shaking flask of 100mL seed culture medium by first order seed access, inoculum concentration 2 ~ 10% (v/v), in the shaking table of 30 ~ 37 DEG C, with the rotating speed of 120 ~ 200rpm, is cultivated 24 ~ 48h, is obtained secondary seed solution;
(5) secondary seed access is equipped with in the fermentation tank of fermentation medium, inoculum concentration 2 ~ 10% (v/v), add defoamer, maintain pH about 7.0, throughput 0.2 ~ 2vvm, speed of agitator 200 ~ 600rpm, tank temperature 30 ~ 37 DEG C, cultivate 64 ~ 96h, fermenting and producing farnoquinone; The original bacteria mushroom powder that described fermentation medium obtains with step (1) is for main nitrogen;
(6) divide water by fermentation liquor tubular type diffusion barrier equipment or centrifuge, concentrated material is to more than 200g/L, and filtrate retains farnoquinone through nanofiltration;
(7) by concentrated material with retain the bacterium mushroom powder that the drying of material spray-dried machine obtains being rich in farnoquinone.
4. preparation method according to claim 3, it is characterized in that, in step (1), described extraction the bacterium mushroom slag of active polysaccharide be extracted the mushroom residue of lentinan, the grifola frondosus slag extracting grifolan, the Agricus blazei slag extracting Agaricus Blazei Murrill polysaccharide, the coprinus comatus slag extracting coprinus comatus polysaccharide and any one or a few the mixture extracted in the Hericium erinaceus slag of hericium erinaceum polysaccharide.
5. preparation method according to claim 3, it is characterized in that, in step (2), described bafillus natto, its Classification And Nomenclature is bafillus natto (Bacillus natto), be preserved in China typical culture collection center, its deposit number is CCTCC M 2014405, and preservation date is on September 9th, 2014.
6. preparation method according to claim 3, is characterized in that, described solid culture based formulas is 30g/L glucose, 40g/L peptone, 5g/LNaCl, 5g/L beef extract, 5g/L yeast extract, 30g/L agar, and solvent is water; Seed culture based formulas is 10-50g/L glucose, 20-80g/L peptone, 2-10g/L NaCl, 2-10g/L beef extract, 2-10g/L yeast extract, and solvent is water; Fermentative medium formula is 30-80g/L glycerine, 20-100g/L soy peptone, 0.2-1.5g/L dusty yeast, 0.1-1g/LK
2hPO
4, 0.1-1g/LCaCl
2h2O, 0.1-1g/LMgSO
47H
2o, solvent is water.
7. preparation method according to claim 3, is characterized in that, in step (5), fermentation cylinder for fermentation is produced farnoquinone grease and adopted Intermittent fermentation or feed supplement formula fermentation process; Fermentation tank adopts micropore distributor as the gas distributor of fermentation tank.
8. preparation method according to claim 3, is characterized in that, in step (5), original bacteria mushroom powder before adding fermentation medium as main nitrogen, first through the pretreatment of hydrolysis by novo.
9. the bacterium mushroom powder being rich in farnoquinone that in claim 1 ~ 8, any one preparation method prepares.
10. bacterium mushroom powder according to claim 9 in preparation, there is prevention of osteoporosis, cardiovascular health, delay senility, the anticancer or application of repairing in the health food of damaging cells function or medicine.
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