CN102399840A - Culture medium formulation for fermenting rainbow conk high-yield EPS and application thereof in cigarettes - Google Patents
Culture medium formulation for fermenting rainbow conk high-yield EPS and application thereof in cigarettes Download PDFInfo
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Abstract
The invention discloses a culture medium formulation for fermenting rainbow conk high-yield extracellular polysaccharide (EPS) and a method for carrying out liquid fermentation on rainbow conk culture. The fermenting and culturing method comprises the following steps of: inoculating a seed culture solution into a basic culture medium at an inoculating quantity of 4%; culturing the seed culture solution in the basic culture medium, wherein the formulation of the culture medium is 4-7 g/L of multivalent peptone and 50-55 g/L of maltose, and the initial pH value is 5; and culturing the seed culture solution under the condition of 150 r/min at 28 DEG C for 7-9 days. By using the culture medium formulation and the culture method, disclosed by the invention, the polysaccharide yield is 10.0982 g/L. The application of the EPS in the cigarettes is searched; and preparation is well made for large-scale fermentation and production of rainbow conk EPS and further application in medicine and food industry.
Description
Technical field
The invention belongs to the microbial fermentation technology field, be specifically related to a kind of liquid fermenting rainbow conk and produce the technology of exocellular polysaccharide and the application of exocellular polysaccharide.
Background technology
Rainbow conk (Coriolus versicolor) has another name called Corilus versicolor Quel., variegated rainbow conk, color suede bacterium and watt bacterium etc., is subordinate to Aphyllophorales, polyporaceae, trametes.Sporophore is generally less, and stockless is imbricate and arranges.Rainbow conk is purposes one of fungi the most widely in Chinese medicine and the food, has invigorating spleen to remove dampness, function such as relieving cough and asthma, clearing heat and detoxicating, antitumor.Contain multiple physiologically active ingredient in the rainbow conk, wherein one of topmost composition is exactly a krestin.The krestin molecular weight is the VISOSE that is rich in β-(1,3) glycosidic link more than 100,000, contains seminose, wood sugar, semi-lactosi, rhamnosyl and pectinose simultaneously.A large amount of research datas shows that the krestin composition is one type and has multiple bioactive polysaccharide material, has multiple pharmacology and physiological function, and effect is remarkable, and pharmaceutical use is high, can do the raw material of each similar drug, healthcare products, functional foodstuff.Big quantity research shows that krestin has the function of antibacterium, mould and virus, stimulates scavenger cell to transform; Increase the effect of interleukin-I, IFN-and tumour necrosis factor in the blood; Can generate by anticancer, be a kind of novel antineoplastic immune regulator, the ability immune cell activated; Enhance immunity function and, do not have toxic side effect to normal cell.Krestin is internationally recognized efficient immunopotentiating agent; It is a kind of orally active polysaccharide; Clinically can be used for treating chronic hepatitis B, alcoholic liver, strengthen the carcinosis radiotherapy and chemotherapy effect at positions such as Digestive tract, lung and uterine neck, mammary gland, can obviously improve patient's cellular immune function and humoral immune function; Enhancing body reduces infection with hemorrhage to chemotherapeutical tolerance, improves patient's quality of life.Krestin has liver protection function simultaneously, and significantly transaminase lowering has tangible repair to liver tissue lesions and hepatic necrosis.
Krestin comprises three types; The one, the Crude polysaccharides that from the sporophore of rainbow conk, extracts (CVPS); The 2nd, from rainbow conk deep layer culture bacteria filament, be separated to a kind of conjugated protein polysaccharide (PSK) (being called rainbow conk peptide sugar again), the 3rd, the rainbow conk exocellular polysaccharide (EPS) that from the fermented liquid of liquid fermenting rainbow conk, extracts.It is active that the polysaccharide that from prepared from coriolus versicolor mycelium and fermented liquid, extracts all has utmost point intensive anticancer, and anti-oxidant activity and immunoloregulation function are good immunostimulants, the effects such as amount that can improve animal body IgM; And that the antitumor action of PSK, immuno-potentiation and liver-protective effect also generally are used for is clinical.Clinically; That krestin has is safe, toxic side effect is little, widely applicable and to advantages such as body damage are little; Be applied to chronic hepatitis B as a kind of immunotherapy medication, the treatment and the assisting therapy of position Cancerous diseases such as Digestive tract, respiratory system disease and uterine neck and mammary gland.Krestin is developed as the anti-system aspect that natural drug immunopotentiating agent and regulator be applied to other diseases has more wide prospect.People have carried out a large amount of research to the process for extracting of manyzoned polypore sporophore and fermentation mycelium polysaccharide, function and application etc. in recent years, but the research that the rainbow conk of just fermenting is produced exocellular polysaccharide (EPS) and EPS function and application facet also seldom.
Along with deepening continuously with human of anti-smoking campaign in the world wide to self healthy concern; " smoking with healthy " has become the great difficult problem that the world of medicine and tobacco circle face jointly, is the main challenge that faces of tobacco industry and the focus of current social extensive concern.In recent years, along with the startup of fulfiling " Framework Convention on Tobacco Control " work, society constantly strengthens the concern of smoking and health problem, reduces the smoking health hazard and has become the responsibilities and obligations that tobacco industry must be born.At present domesticly on herbal medicine selection, drug mechanism and clinical application effect, the distinctive new cigarette of China has been carried out deep research; Develop successfully some disease of human body is had the commercial cigarette of certain curative effect, like " gold is holy ", " WUYESHENG ", " rivers and mountains " etc.These cigarette are except that having certain optimum effect alleviating the human respiratory disease, also can certain prophylactic effect be arranged to cardiovascular system diseases etc.Also have research to show; The cigarette that contains class Chinese herbal medicine effective ingredientses such as rainbow conk is when burning and sucking; Its effective constituent is distilled, gasify, volatilize and distillation forms particulate mutually and gas phase; Can catch the radical in the flue gas, blocking-up or reduce the generation of objectionable impurities, the harm that alleviates the cardiovascular and microcirculqtory system that smoking causes.Therefore, cigarette has become one of focus of each tobacco enterprise and scientific research institutions research at present with the research of herbal medicine.But at present, do not see that also fermentation product rainbow conk exocellular polysaccharide is used for the relevant report of cigarette.
Summary of the invention
One of the object of the invention is to provide the culture medium prescription of a kind of rainbow conk high yield EPS that ferments.
Another object of the present invention is to provide a kind of method of liquid fermenting rainbow conk culture and the method for separation and purification exocellular polysaccharide from rainbow conk cultivation and fermentation liquid.
A purpose more of the present invention is to disclose the application of rainbow conk exocellular polysaccharide in cigarette.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:
The culture medium prescription of fermentation rainbow conk high yield EPS of the present invention is: multivalence peptone 4-7g/L, SANMALT-S 50-55g/L, initial pH are 5.
Be preferably: multivalence peptone 5.870g/L, SANMALT-S 53.120g/L, initial pH are 5.
The method of liquid fermenting rainbow conk culture of the present invention, the inoculum size with 4% is inoculated in seed culture fluid in the basic medium and cultivates, and culture medium prescription is multivalence peptone 4-7g/L; SANMALT-S 50-55g/L; Initial pH is 5, at 28 ℃, cultivates 7-9 days under the 150r/min condition.
Be preferably: the inoculum size with 4% is inoculated in seed culture fluid in the basic medium and cultivates, and culture medium prescription is multivalence peptone 5.870g/L, and SANMALT-S 53.120g/L, initial pH are 5, at 28 ℃, cultivates 8 days under the 150r/min condition.
Described seed culture fluid is the once dull and stereotyped cultivation of slant culture warp of bacterial classification cfcc 87458 and the product of a liquid culture,
The slant culture based formulas is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Inoculating back 28 ℃ cultivated 4-6 days;
The plate culture medium prescription is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Cultivated 6-8 days at 28 ℃ the inoculation back;
The liquid culture based formulas is: multivalence peptone 4-7g/L, and SANMALT-S 50-55g/L, initial pH are 5, cultivated 4-6 days under the 150r/min condition at 28 ℃ the inoculum size inoculation back with 4%.
The method of separation and purification exocellular polysaccharide is from rainbow conk cultivation and fermentation liquid:
Fermented liquid steams the 1/4-1/5 that appearance is concentrated into original volume through revolving, and adds the absolute ethyl alcohol of 4 times of volumes then, and 4 ℃ are spent the night; The centrifugal 10min of 10000r/min must be precipitated as Crude polysaccharides;
The Crude polysaccharides sample is used dissolved in distilled water, add the chloroform-butanol solution of 1/4 volume, the volume ratio of chloroform and propyl carbinol is 4: 1; Place tool plug container; Behind the shake well 30min,, then water is separated with chloroform mutually through the centrifugal 5min of whizzer 1000r/min; Water is added the chloroform-butanol solution that is equivalent to its volume 1/4 again, repeat said process, amount to the repetition secondary; The exocellular polysaccharide composition that is purified into concentrates with Rotary Evaporators, and NaCl is removed in dialysis, must make with extra care the rainbow conk exocellular polysaccharide, and vacuum lyophilization is preserved.
The application of rainbow conk exocellular polysaccharide in cigarette, with the spraying into pipe tobacco or inject cigarette of purified rainbow conk exocellular polysaccharide, exocellular polysaccharide content is the 0.01%-0.04% of tobacco quality, puts into climatic chamber then 22 ℃ of temperature, 60% time balance 48h of humidity.
Used bacterial classification cfcc 87458 purchases and receives wound in Bei Jingbei and join Bioteknologisk Institut among the present invention, is preserved in China Forest microbial preservation administrative center.
The invention discloses the method for culture medium prescription and the liquid fermenting rainbow conk culture of a kind of rainbow conk high yield EPS that ferments, adopt the culture medium prescription and the cultural method of this invention, polysaccharide yield is 10.0982g/L.And the EPS physiologically active carried out pre-test, for large scale fermentation production rainbow conk EPS and the further application in the medicine food industry thereof are got ready.
The present invention has found the application method of a kind of rainbow conk exocellular polysaccharide in cigarette, for the application of exocellular polysaccharide in cigarette of fungies such as rainbow conk provides a kind of feasible program, has broad development space, with producing far-reaching economy and social effect.Utilize herbal medicine to erect and have the technology barriers of the low harm cigarette of traditional smoking cultural connotation and Chinese style suction flavor characteristic, improve the Chinese cigarette competitiveness of product in market, can tackle pressure and challenge that WHO and WTO bring.High-quality temperature compensation herbal medicine fermented extracted exocellular polysaccharides such as rainbow conk are added in the cigarette; Fragrance style, taste characteristic and the processing mode of the uniqueness that forms all meets the Chinese style cigarette developing direction that State Bureau proposes; Can play the effect of harm reduction gain to cigarette smoke; Meet national tobacco development in science and technology direction, help improving the technology content and the competitive power of China's tobacco industry product, make the cigarette human consumer when enjoying smoking enjoyment, reduce harm of smoking property as much as possible.
Description of drawings
The refining rainbow conk exocellular polysaccharide of Fig. 1 is crossed Sepharose CL-6B chromatography column result.
Fig. 2 crosses the infrared spectrogram that post gets rainbow conk exocellular polysaccharide component.
Fig. 3 makes with extra care the rate of transform of rainbow conk exocellular polysaccharide in cigarette smoke.
Embodiment
(1) slant strains is cultivated: the preparation solid medium, and the packing test tube, the slant culture based formulas is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Preparation inclined-plane, sterilization back, after the streak inoculation rainbow conk, in 28 ℃ of cultivations 5 days, freezing was rainbow conk bacterial strain bacterial classification.
(2) from the test tube of preserving bacterial strain, use inoculation to be hooked in the fritter that the test tube slant takes out 0.5 square centimeter, be inoculated in P days A solid plates.The plate culture medium prescription is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Will make inoculation comprise few substratum of trying one's best on the piece during inoculation, two of board joints are put into constant incubator, and cultivated 7 days at 28 ℃ the inoculation back.
(3) punch tool of use diameter 0.5cm takes off two thalline that are positioned at colony edge from the solid plate substratum, is inoculated in (250ml triangular flask in the liquid seed culture medium; 50 milliliters of nutrient solutions); The liquid culture based formulas is: multivalence peptone 5.870g/L, and SANMALT-S 53.120g/L, initial pH are 5; At 28 ℃, cultivated 5 days under the 150r/min condition.
(4) with 4% inoculum size seed culture fluid is inoculated in the basic medium and cultivates, culture medium prescription is multivalence peptone 5.870g/L, and SANMALT-S 53.120g/L, initial pH are 5, at 28 ℃, cultivate 8 days under the 150r/min condition.
After the rainbow conk fermentation ends, the fermented product of 50mL is separated mycelia and fermented liquid through the vacuum filtration method, fermented liquid steams the 1/4-1/5 that appearance is concentrated into original volume through revolving, and adds the absolute ethyl alcohol of 4 times of volumes then, and 4 ℃ are spent the night.The centrifugal 10min of 10000r/min must be precipitated as Crude polysaccharides; Take by weighing the 0.1g Crude polysaccharides, be dissolved in 10mL zero(ppm) water, use the phenol sulfuric acid process to measure to calculate that polysaccharide content is 10.10g/L in the fermented liquid.
(1) slant strains is cultivated: the preparation solid medium, and the packing test tube, the slant culture based formulas is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Preparation inclined-plane, sterilization back, after the streak inoculation rainbow conk, in 28 ℃ of cultivations 4 days, freezing was rainbow conk bacterial strain bacterial classification.
(2) from the test tube of preserving bacterial strain, use inoculation to be hooked in the fritter that the test tube slant takes out 0.5 square centimeter, be inoculated in P days A solid plates.The plate culture medium prescription is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Cultivated 6-8 days at 28 ℃ the inoculation back; Will make inoculation comprise few substratum of trying one's best on the piece during inoculation, two of board joints are put into constant incubator, cultivate 8 days for 28 ℃.
(3) punch tool of use diameter 0.5cm takes off two thalline that are positioned at colony edge from the solid plate substratum, is inoculated in (250ml triangular flask in the liquid seed culture medium; 50 milliliters of nutrient solutions); The liquid culture based formulas is: multivalence peptone 4g/L, and SANMALT-S 55g/L, initial pH are 5; At 28 ℃, cultivated 6 days under the 150r/min condition.
(4) with 4% inoculum size seed culture fluid is inoculated in the basic medium and cultivates, culture medium prescription is multivalence peptone 4g/L, and SANMALT-S 55g/L, initial pH are 5, at 28 ℃, cultivate 7 days under the 150r/min condition.
After the rainbow conk fermentation ends, the fermented product of 50mL is separated mycelia and fermented liquid through the vacuum filtration method, fermented liquid steams the 1/4-1/5 that appearance is concentrated into original volume through revolving, and adds the absolute ethyl alcohol of 4 times of volumes then, and 4 ℃ are spent the night.The centrifugal 10min of 10000r/min must be precipitated as Crude polysaccharides; Take by weighing the 0.1g Crude polysaccharides, be dissolved in 10mL zero(ppm) water, use the phenol sulfuric acid process to measure to calculate that polysaccharide content is 10.12g/L in the fermented liquid.
(1) slant strains is cultivated: the preparation solid medium, and the packing test tube, the slant culture based formulas is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Preparation inclined-plane, sterilization back, after the streak inoculation rainbow conk, in 28 ℃ of cultivations 6 days, freezing was rainbow conk bacterial strain bacterial classification.
(2) from the test tube of preserving bacterial strain, use inoculation to be hooked in the fritter that the test tube slant takes out 0.5 square centimeter, be inoculated in P days A solid plates.The plate culture medium prescription is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Cultivated 6 days at 28 ℃ the inoculation back; Will make inoculation comprise few substratum of trying one's best on the piece during inoculation, two of board joints are put into constant incubator, cultivate 6 days for 28 ℃.
(3) punch tool of use diameter 0.5cm takes off two thalline that are positioned at colony edge from the solid plate substratum, is inoculated in (250ml triangular flask in the liquid seed culture medium; 50 milliliters of nutrient solutions); The liquid culture based formulas is: multivalence peptone 7g/L, and SANMALT-S 50g/L, initial pH are 5; At 28 ℃, cultivated 4 days under the 150r/min condition.
(4) with 4% inoculum size seed culture fluid is inoculated in the basic medium and cultivates, culture medium prescription is multivalence peptone 7g/L, and SANMALT-S 50g/L, initial pH are 5, at 28 ℃, cultivate 9 days under the 150r/min condition.
After the rainbow conk fermentation ends, the fermented product of 50mL is separated mycelia and fermented liquid through the vacuum filtration method, fermented liquid steams the 1/4-1/5 that appearance is concentrated into original volume through revolving, and adds the absolute ethyl alcohol of 4 times of volumes then, and 4 ℃ are spent the night.The centrifugal 10min of 10000r/min must be precipitated as Crude polysaccharides; Take by weighing the 0.1g Crude polysaccharides, be dissolved in 10mL zero(ppm) water, use the phenol sulfuric acid process to measure to calculate that polysaccharide content is 10.09g/L in the fermented liquid.
(1) slant strains is cultivated: the preparation solid medium, and the packing test tube, the slant culture based formulas is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Preparation inclined-plane, sterilization back, after the streak inoculation rainbow conk, in 28 ℃ of cultivations 6 days, freezing was rainbow conk bacterial strain bacterial classification.
(2) from the test tube of preserving bacterial strain, use inoculation to be hooked in the fritter that the test tube slant takes out 0.5 square centimeter, be inoculated in P days A solid plates.The plate culture medium prescription is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Cultivated 6 days at 28 ℃ the inoculation back; Will make inoculation comprise few substratum of trying one's best on the piece during inoculation, two of board joints are put into constant incubator, cultivate 6 days for 28 ℃.
(3) punch tool of use diameter 0.5cm takes off two thalline that are positioned at colony edge from the solid plate substratum, is inoculated in (250ml triangular flask in the liquid seed culture medium; 50 milliliters of nutrient solutions); The liquid culture based formulas is: multivalence peptone 6g/L, and SANMALT-S 53g/L, initial pH are 5; At 28 ℃, cultivated 4 days under the 150r/min condition.
(4) with 4% inoculum size seed culture fluid is inoculated in the basic medium and cultivates, culture medium prescription is multivalence peptone 5g/L, and SANMALT-S 52g/L, initial pH are 5, at 28 ℃, cultivate 9 days under the 150r/min condition.
After the rainbow conk fermentation ends, the fermented product of 50mL is separated mycelia and fermented liquid through the vacuum filtration method, fermented liquid steams the 1/4-1/5 that appearance is concentrated into original volume through revolving, and adds the absolute ethyl alcohol of 4 times of volumes then, and 4 ℃ are spent the night.The centrifugal 10min of 10000r/min must be precipitated as Crude polysaccharides; Take by weighing the 0.1g Crude polysaccharides, be dissolved in 10mL zero(ppm) water, use the phenol sulfuric acid process to measure to calculate that polysaccharide content is 10.09g/L in the fermented liquid.
Take by weighing 1g the Crude polysaccharides sample; Add the 50mL dissolved in distilled water under the room temperature; Add chloroform-propyl carbinol (being mixed with volume ratio in advance is 4: 1 mixed solutions) solution of 1/4 volume, place tool plug container, behind the shake well 30min; Through the centrifugal 5min of whizzer 1000r/min, then water is separated with chloroform mutually.Water is added the chloroform-butanol solution that is equivalent to its volume 1/4 again, repeat said process, amount to the repetition secondary.With sulfuric acid-phynol method exocellular polysaccharide is detected.The exocellular polysaccharide composition that is purified into is concentrated into 10mL with Rotary Evaporators, and NaCl is removed in dialysis (dialysis tubing molecular weight 8000-14000Da), must make with extra care the rainbow conk exocellular polysaccharide, and vacuum lyophilization is preserved.
The exquisite rainbow conk exocellular polysaccharide that obtains with embodiment 5 is an analytic sample, carries out following analysis research.
1, the proximate analysis of rainbow conk exocellular polysaccharide is produced in fermentation
Take by weighing 80mg rainbow conk and make with extra care the NaCl damping fluid dissolving that exocellular polysaccharide is dissolved in 4mL 0.2mol/L, centrifugal, supernatant is crossed the filter membrane of 0.45 μ m; Get 2mL and go up Sepharose CL-6B gel column, with 0.2mol/L NaCl buffer solution elution, flow velocity is 0.6mL/min, utilizes the substep scoop to collect, the 5mL/ pipe; With sulfuric acid-phynol method exocellular polysaccharide is detected.Isolated exocellular polysaccharide component is concentrated into 10mL with Rotary Evaporators, and NaCl is removed in dialysis (dialysis tubing molecular weight 8000-14000Da), and vacuum lyophilization is preserved.
To make with extra care rainbow conk exocellular polysaccharide sample and cross Sepharose CL-6B post, and measure polysaccharide content with sulfuric acid-phynol method, number be that X-coordinate is that ordinate zou is mapped with SigmaPlot software with the polysaccharide absorbancy with pipe, and the result is illustrated in fig. 1 shown below.Can find out that from Fig. 1 as a result refining rainbow conk exocellular polysaccharide has been isolated a kind of main polysaccharide fraction through Sepharose CL-6B post.Repeated post 5 times, and each all the solution of crossing behind the post was detected, detected result is identical, 5 resulting components are collected concentrate, dialysis, lyophilize obtain a polysaccharide fraction.
2, rainbow conk exocellular polysaccharide component IR spectroscopy
Took by weighing post and get rainbow conk exocellular polysaccharide component 1mg, with carrying out ir spectra (IR) analysis behind Potassium Bromide (KBr) compressing tablet, the result sees Fig. 2.The analysis of characteristic absorbance crest can be known from collection of illustrative plates, at 3421.4cm-1 one characteristic broad peak is arranged, and belongs to the stretching vibration of O-H, is illustrated in this sugar to have O-H; At the 2926.1cm-1 place absorption peak being arranged, should be the C-H stretching vibration, is the charateristic avsorption band of carbohydrate, at 2361cm-1,2335.8cm-1 place absorption peak is arranged, and should be C=N or C=C stretching vibration, and Fermi resonance takes place; At 1645.7cm-1,1071.9cm-1 place absorption peak is arranged, explaining has carboxylic acid to exist in this component, and knowing this exocellular polysaccharide component by inference is acidic polysaccharose.
3, the mensuration of rainbow conk EPS antioxidant property
Measure the clearance rate of rainbow conk EPS to the OH radical with the phenanthroline method, the result sees table 1.
Table 1 polysaccharide is to the elimination efficiency of OH radical
The result shows: rainbow conk EPS has the activity of stronger removing hydroxyl radical free radical; And along with the ability that increasing of EPS concentration removed hydroxyl radical free radical is also strengthened gradually; But the clearance rate of OH radical has reached 61.99 when the EPS addition is to 1mg/mL in the reaction system; And improving EPS concentration again, its effect of removing the OH radical increases and is not obvious.
Adopt the DPPH method to measure the antioxygenic activity of polysaccharide, the result sees the following form 2.
Table 2 polysaccharide is to the clearance rate of DPPH radical
Can be known that by table 2 rainbow conk EPS has the activity of removing the DPPH radical, along with the increase of concentration, EPS also strengthens DPPH free radical scavenging effect, demonstrates dose-effect relationship preferably.
4, the mensuration of refining rainbow conk exocellular polysaccharide rate of transform in flue gas
Get refining exocellular polysaccharide storing solution of different volumes and adding distil water and add to 0.5mL; Be injected in the flower tobacco product that looses, make cigarette prop up middle exocellular polysaccharide content and reach 0.02%, 0.04%, 0.06%, 0.08%, 0.1% of cigarette quality respectively, and be the blank of injection 0.5mL zero(ppm) water, 10 every group; Put into climatic chamber (22 ℃ of temperature, humidity 60%) balance 48h; With smoking machine cigarette Zhi Jinhang is aspirated then; Cambridge filter after the suction is put into the 150mL triangular flask, add the ultrasonic 1h of 50mL absolute ethyl alcohol, outwell ethanol; Add the ultrasonic 0.5h of 50mL absolute ethyl alcohol then, outwell ethanol, add 50mL zero(ppm) water more ultrasonic twice; Each 1h, merging filtrate is measured polysaccharide content with sulfuric acid-phynol method; Calculate its rate of transform in flue gas, the result sees Fig. 3.
The rate of transform=(on the experimental group cambridge filter on polysaccharide amount-control group cambridge filter polysaccharide amount)/polysaccharide addition.
Can know by Fig. 3; The refining rate of transform of rainbow conk exocellular polysaccharide in flue gas the first variation tendency that afterwards reduces that increases occurred with its increase of concentration in cigarette, is 0.02% o'clock at the polysaccharide addition, and the rate of transform is 2.76%; At the polysaccharide addition is 0.06% o'clock; The rate of transform is 4.78%, and the polysaccharide addition reaches at 0.1% o'clock above 0.06%, and the rate of transform of polysaccharide in flue gas is 3.82%.
5, the refining sensory evaluation of rainbow conk exocellular polysaccharide in cigarette
Take by weighing 4 parts in Xuanwei, Yunnan HC1F thin material pipe tobacco, 10g/ part; Get refining exocellular polysaccharide storing solution of different volumes and adding distil water afterwards and add to 0.5mL; Evenly be sprayed at micro-sprayer and respectively organize in the pipe tobacco; Make exocellular polysaccharide content reach 0.0%, 0.01%, 0.04%, 0.08% of tobacco quality respectively, according to the standard fabrication cigarette of every 0.83g; Put into climatic chamber (22 ℃ of temperature, humidity 60%) balance 48h then.Ask the cigarette sensory evaluating smoking group of Light Engineering Institutes Of Zhengzhou's food and biological institute to be smoked panel test after ready to balance is good; Filter out aesthetic quality's sample preferably from fragrance matter, perfume quantity, concentration, exquisiteness, the aspects such as gas, stimulation, pleasant impression of mixing of cigarette, confirm addition and the effect thereof of rainbow conk exocellular polysaccharide in cigarette.
Organize the cigarette sensory evaluating smoking group of Light Engineering Institutes Of Zhengzhou's food and biological institute to carry out the test of smokeing panel test, the perfuming test smoking result of refining rainbow conk exocellular polysaccharide in cigarette seen table 3.The result shows: adding concentration is 0.04% o'clock, and refining rainbow conk exocellular polysaccharide can play in cigarette effectively covers assorted gas, and removing stimulates; Improve the effect of pleasant impression; The fine and smooth degree of fragrance is promoted to some extent, and flue gas state, mellow and full sense are better, and the Harmony of fragrance is good; It is fragrant to have tangible herbal medicine characteristic, helps to improve the sucking quality of cigarette.
The refining rainbow conk exocellular polysaccharide of table 3 is in the sensory evaluation result of cigarette
The rainbow conk fermented liquid separates through alcohol extracting, Sevag method deproteinization and through Sepharose CL-6B gel column and obtains a polysaccharide fraction, and drawing this sugar through IR spectroscopy is acidic polysaccharose; The result that the rate of transform is measured shows that the rainbow conk exocellular polysaccharide has the rate of transform preferably in flue gas; For the rainbow conk exocellular polysaccharide can the interpolation in the middle of cigarette provide theoretical support; Perfuming experiment has simultaneously proved that fermentation product rainbow conk exocellular polysaccharide can play the effect that sets off the fragrance style by contrast; Improved the sucking quality of cigarette, for the interpolation of rainbow conk exocellular polysaccharide in cigarette provides the feasibility evidence.In a word, this result of study is that rainbow conk exocellular polysaccharide and other application of medicinal fungi in relevant food industries such as cigarette provide certain theory and technical support.
Claims (7)
1. fermentative medium formula of rainbow conk high yield EPS that ferments, it is characterized in that: culture medium prescription is multivalence peptone 4-7g/L, and SANMALT-S 50-55g/L, initial pH are 5.
2. the fermentative medium formula of fermentation rainbow conk high yield EPS according to claim 1 is characterized in that: culture medium prescription is multivalence peptone 5.870g/L, and SANMALT-S 53.120g/L, initial pH are 5.
3. the method for a liquid fermenting rainbow conk culture is characterized in that: the inoculum size with 4% is inoculated in seed culture fluid to optimize in the substratum and cultivates, and culture medium prescription is multivalence peptone 4-7g/L; SANMALT-S 50-55g/L; Initial pH is 5, at 28 ℃, cultivates 7-9 days under the 150r/min condition.
4. the method for liquid fermenting rainbow conk culture according to claim 2; It is characterized in that: the inoculum size with 4% is inoculated in seed culture fluid to optimize in the substratum and cultivates; Culture medium prescription is multivalence peptone 5.870g/L, and SANMALT-S 53.120g/L, initial pH are 5; At 28 ℃, cultivated 8 days under the 150r/min condition.
5. according to the method for claim 3 or 4 described liquid fermenting rainbow conk cultures, it is characterized in that: described seed culture fluid is the once dull and stereotyped cultivation of slant culture warp of bacterial classification cfcc 87458 and the product of a liquid culture,
The slant culture based formulas is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Inoculating back 28 ℃ cultivated 4-6 days;
The plate culture medium prescription is: 250g/L potato, SANMALT-S 10g/L, peptone 4g/L, 2% agar; Cultivated 6-8 days at 28 ℃ the inoculation back;
The liquid culture based formulas is: multivalence peptone 4-7g/L, and SANMALT-S 50-55g/L, initial pH are 5, cultivated 4-6 days under the 150r/min condition at 28 ℃ the inoculum size inoculation back with 4%.
6. the method for a separation and purification exocellular polysaccharide from rainbow conk cultivation and fermentation liquid is characterized in that:
Fermented liquid steams the 1/4-1/5 that appearance is concentrated into original volume through revolving, and adds the absolute ethyl alcohol of 4 times of volumes then, and 4 ℃ are spent the night; The centrifugal 10min of 10000r/min must be precipitated as Crude polysaccharides;
The Crude polysaccharides sample is used dissolved in distilled water, add the chloroform-butanol solution of 1/4 volume, the volume ratio of chloroform and propyl carbinol is 4: 1; Place tool plug container; Behind the shake well 30min,, then water is separated with chloroform mutually through the centrifugal 5min of whizzer 1000r/min; Water is added the chloroform-butanol solution that is equivalent to its volume 1/4 again, repeat said process, amount to the repetition secondary; The exocellular polysaccharide composition that is purified into concentrates with Rotary Evaporators, and NaCl is removed in dialysis, must make with extra care the rainbow conk exocellular polysaccharide, and vacuum lyophilization is preserved.
7. the application of rainbow conk exocellular polysaccharide in cigarette; It is characterized in that: spraying into pipe tobacco or injecting cigarette purified rainbow conk exocellular polysaccharide; Exocellular polysaccharide content is the 0.01%-0.04% of tobacco quality, puts into climatic chamber then 22 ℃ of temperature, 60% time balance 48h of humidity.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102863549A (en) * | 2012-09-27 | 2013-01-09 | 上海师范大学 | Method of using by-product fermentation broth generated in production of posaverptidum to produce extracellular posaverptidum |
| CN102942614A (en) * | 2012-11-23 | 2013-02-27 | 重庆优宝生物技术有限公司 | Preparation method and application of high-purity polysaccharopeptide |
| CN105219657A (en) * | 2015-10-22 | 2016-01-06 | 江苏安惠生物科技有限公司 | Rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof |
| CN107409743A (en) * | 2017-03-24 | 2017-12-01 | 泰安市农业科学研究院 | A kind of artificial generation material pseudo-wild cultivating method of rainbow conk |
| CN112980901A (en) * | 2021-03-18 | 2021-06-18 | 天津质谱生物科技有限公司 | Method for preparing formanilide compounds by fermenting and culturing trametes robiniophila, trametes robiniophila and corious versicolor and application |
| CN114591843A (en) * | 2022-03-01 | 2022-06-07 | 北京建筑大学 | Solid culture medium of coriolus versicolor, preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55114288A (en) * | 1979-02-26 | 1980-09-03 | Takara Shuzo Co Ltd | Production of cholesterol esterase |
| CN1651076A (en) * | 2004-12-29 | 2005-08-10 | 江苏华源药业有限公司 | Method of extracting rainbow conk saccharide peptide for injection in rainbow conk saccharided peptide crude product obtained from submerged fermentation |
| CN101831471A (en) * | 2010-05-12 | 2010-09-15 | 浙江省农业科学院 | Method for producing polysaccharides by fermenting yellow serofluid with edible and medicinal fungi |
-
2011
- 2011-11-16 CN CN2011103622934A patent/CN102399840A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55114288A (en) * | 1979-02-26 | 1980-09-03 | Takara Shuzo Co Ltd | Production of cholesterol esterase |
| CN1651076A (en) * | 2004-12-29 | 2005-08-10 | 江苏华源药业有限公司 | Method of extracting rainbow conk saccharide peptide for injection in rainbow conk saccharided peptide crude product obtained from submerged fermentation |
| CN101831471A (en) * | 2010-05-12 | 2010-09-15 | 浙江省农业科学院 | Method for producing polysaccharides by fermenting yellow serofluid with edible and medicinal fungi |
Non-Patent Citations (3)
| Title |
|---|
| 余晓斌等: "液体发酵法生产云芝胞内糖肽", 《食品与生物技术学报》, no. 01, 30 January 2006 (2006-01-30) * |
| 徐恩彪: "蛹虫草、云芝发酵条件优化及其复方多糖降血糖作用的研究", 《中国优秀硕士学位论文全文数据库-工程科技I辑》, no. 5, 15 May 2011 (2011-05-15) * |
| 胡卫珍等: "不同云芝菌株获得云芝胞内多糖比较", 《食品与生物技术学报》, no. 05, 30 September 2005 (2005-09-30) * |
Cited By (8)
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| CN102863549A (en) * | 2012-09-27 | 2013-01-09 | 上海师范大学 | Method of using by-product fermentation broth generated in production of posaverptidum to produce extracellular posaverptidum |
| CN102942614A (en) * | 2012-11-23 | 2013-02-27 | 重庆优宝生物技术有限公司 | Preparation method and application of high-purity polysaccharopeptide |
| CN102942614B (en) * | 2012-11-23 | 2014-11-05 | 重庆优宝生物技术有限公司 | Preparation method and application of high-purity polysaccharopeptide |
| CN105219657A (en) * | 2015-10-22 | 2016-01-06 | 江苏安惠生物科技有限公司 | Rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof |
| CN105219657B (en) * | 2015-10-22 | 2018-11-23 | 江苏安惠生物科技有限公司 | Rainbow conk liquid fermentation high polysaccharide bacterial strain and its selection |
| CN107409743A (en) * | 2017-03-24 | 2017-12-01 | 泰安市农业科学研究院 | A kind of artificial generation material pseudo-wild cultivating method of rainbow conk |
| CN112980901A (en) * | 2021-03-18 | 2021-06-18 | 天津质谱生物科技有限公司 | Method for preparing formanilide compounds by fermenting and culturing trametes robiniophila, trametes robiniophila and corious versicolor and application |
| CN114591843A (en) * | 2022-03-01 | 2022-06-07 | 北京建筑大学 | Solid culture medium of coriolus versicolor, preparation method and application |
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