CN103130909A - Preparation method of selenium-rich Morchella polysaccharide - Google Patents
Preparation method of selenium-rich Morchella polysaccharide Download PDFInfo
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- CN103130909A CN103130909A CN2013100867873A CN201310086787A CN103130909A CN 103130909 A CN103130909 A CN 103130909A CN 2013100867873 A CN2013100867873 A CN 2013100867873A CN 201310086787 A CN201310086787 A CN 201310086787A CN 103130909 A CN103130909 A CN 103130909A
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Abstract
The invention discloses a preparation method of selenium-rich Morchella polysaccharide, and aims to overcome the problems of low selenium content, low inorganic selenium conversion degree, low safety coefficient and low polysaccharide content of the selenium-rich edible fungus at present. The preparation method comprises the following steps: 1, preparation of selenium-rich Morchella: preparing a culture medium, activating a strain, and performing deep fermentation to obtain selenium-rich Morchella mycelium pellets; 2, extraction of selenium-rich Morchella intracellular crude polysaccharide: cleaning the selenium-rich Morchella mycelium pellets, drying, pulverizing, screening, weighing, adding distilled water, centrifuging to take the supernatant, centrifuging to collect precipitates, and drying in a vacuum drying oven for 2 hours to obtain the selenium-rich Morchella intracellular crude polysaccharide; and 3, purification of the selenium-rich Morchella intracellular crude polysaccharide: separating through a macroporous resin AB-8 to obtain a selenium-rich Morchella polysaccharide elution curve, collecting eluate at eluting peaks of the curve, and performing further purification; and performing gel column chromatography, drawing a selenium-rich Morchella polysaccharide elution curve, and collecting eluate at eluting peaks of the curve to obtain the selenium-rich Morchella polysaccharide.
Description
Technical field
The present invention relates to a kind of preparation method of functional food technical field, or rather, the present invention relates to a kind of preparation method of rich selenium Morchella esculenta (L.) Pers polysaccharide.
Background technology
Selenium is the trace element of needed by human, and it keeps the eubolism of free radical by Selenoperoxidase, ubiquinone and vitamin-E, to keep the human normal physiological function.The Main Function of selenium has the following aspects: anti-cancer and kill cancer action; Antioxygenation; Strengthening immunity; Prevent diabetes; Prevent cataract; Prevent cardiovascular and cerebrovascular diseases; Prevent Keshan disease, Kaschin-Beck disease, sacroiliitis; Detoxifcation, toxin expelling; Prevent and treat hepatopathy, protection liver.
But selenium can't synthesize in vivo, can only be by external picked-up.Contain abundant organoselenium in animal viscera and sea-food, but contain more cholesterol in animal viscera, be unfavorable for especially the elderly and cardiovascular patient; And in sea-food, the utilizability of selenium is also very low, because contain methyl mercury in sea-food, can be combined into protein-S-Se-mercury with selenium, becomes inactive state, the detoxification of mercury, selenium also has been cancelled.Therefore, organoselenium is the best benefit selenium sources of the mankind.
Recent domestic is all in the exploitation of being devoted to organoselenium and popularization, and the developed countries such as Japan, the U.S. have forbidden adding the inorganic seleniums such as Sodium Selenite in food.Estimate, organoselenium will replace Sodium Selenite use in food-processing as foodstuff additive.
Polysaccharide be the more important thing is to have participated in the cell comings and goings in vivo not only as energy substance.A lot of pharmacology and clinical trial certificate, polysaccharide has multiple physiologically active, is a kind of immunopotentiating agent of wide spectrum, has cancer-resisting, regulates immunologic function, delays senility, the physiological action such as anti-infective, radioprotective.
The research of selenium polysaccharide still is in the starting stage at home and abroad, but due to the physiologically active of selenium polysaccharide uniqueness, has become in recent years study hotspot.
Morchella esculenta (L.) Pers mycelium all contains abundant nutritive ingredient, its aminoacids content is first of edible mushrooms, contain altogether 19 seed amino acids, wherein 8 kinds of essential amino acids account for 47.47% of total amount, in morel, the useful linolic acid of human body being accounted for 56% of fatty acid total amount, is a kind of high nutrition high-grade nutrient excellent tonic product low in calories.But because morel has special biological characteristics, realize morel commercialization cultivation difficulty greatly, tank fermentation method is a kind of important channel of suitability for industrialized production Morciiella Esculeuta Mycelia.
The organic state micro element of edible mushrooms enrichment is easy to absorbing of human body; And the trace element of enrichment has good stability, and is convenient to store, transport, and can avoid some nutritive ingredient loss wherein; The trace element of picked-up also has no side effect to human body in limited field.
Utilize at present edible mushrooms to be carrier, contain by executing Se-rich lucid ganoderma, selenium-rich gold needle mushroom, mushroom with abundant selenium, Se-rich xianggu and the Hericium erinaceus (Bull. Ex Fr.) Pers. Rich in selenium etc. that selenium fertilizer is cultivated, low to the enrichment degree of selenium, low to the transforming degree of inorganic selenium, the edible fungi polysaccharide extraction yield is low low with degree of purification; And all unrealized morel commercialization cultivations both at home and abroad, the while does not utilize the method for submerged fermentation to obtain the report of rich selenium Morchella esculenta (L.) Pers polysaccharide yet.
Summary of the invention
Technical problem to be solved by this invention is to have overcome prior art to have that fruit body of edible fungi is low to the enrichment degree of selenium, low to the transforming degree of inorganic selenium, the low problem low with degree of purification of edible fungi polysaccharide extraction yield, and a kind of preparation method of rich selenium Morchella esculenta (L.) Pers polysaccharide is provided.
For solving the problems of the technologies described above, the present invention adopts following technical scheme to realize: the preparation method's of described a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide step is as follows:
1. the preparation of rich selenium morel
(1) preparation substratum:
A. prepare slant medium;
B. prepare submerged fermentation culture medium;
(2) actication of culture:
By after ultra-violet sterilization 30min, No. 926, the Morchella crassipes of use transfering loop picking preservation under gnotobasis is transferred on fresh slant medium, cultivates 24h in 25 ℃ of constant incubators with all utensils;
(3) submerged fermentation:
After actication of culture, get approximately 5mm
210 access submerged fermentation culture mediums of the bacterial classification of size, the Sodium Selenite that adds simultaneously, make that in submerged fermentation culture medium, selenium concentration reaches 80 μ g/ml, 250mL triangular flask charging 100mL, be placed in the shaking culture case, culture temperature is 26 ℃, the oscillation frequency 100rpm of shaking culture case, incubation time 5 days obtains rich selenium Morciiella Esculeuta Mycelia ball.
2. the extraction of Crude polysaccharides in rich selenium morel born of the same parents
(1) the rich selenium Morciiella Esculeuta Mycelia ball warp rear weighing of cleaning, dry, pulverize, sieve, the ratio that is 1:30 in mycelium pellet and distilled water adds distilled water, carry out the microwave cooperating ultrasonic-assisted extraction, then be placed in water-bath, at 90 ℃ of lower hot water lixiviate 140min, with revolution in whizzer the centrifugal 15min of vat liquor with 4000r/min, get supernatant liquor;
(2) supernatant liquor after cryoconcentration, under agitation adds 95% ethanol of 3 times of volumes, and alcohol is analysed liquid standing 12h in 4 ℃ of refrigerators, adopts whizzer separated and collected throw out;
(3) throw out of collecting is redissolved in distilled water, it is the abundant mixing of ratio of 5:1 in sample and Sevag reagent, be placed in separating funnel, discard the protein denaturation layer on lower floor's organic layer and two-phase interface after standing 20min, get upper strata liquid and repeat this step process 7～8 times, until between organic layer and water layer without white gluey deposits yields, this moment, supernatant liquid was Crude polysaccharides solution in the morel born of the same parents;
(4) get in the morel born of the same parents of upper strata that Crude polysaccharides solution is concentrated, collecting precipitation thing after alcohol precipitation, then wash 2 times with dehydrated alcohol, acetone, ether respectively, carefully scrape throw out in weighing disk at last, be placed in 40 ℃ of dry 2h of vacuum drying oven, namely obtain Crude polysaccharides in solid-state rich selenium morel born of the same parents.
3. the purifying of Crude polysaccharides in rich selenium morel born of the same parents
1) adopt the AB-8 macroporous resin to separate, obtain rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, carry out next step purifying;
2) Sephadex G-100 gel filtration chromatography is drawn rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collects the elutriant at curve elution peak position, gets rich selenium Morchella esculenta (L.) Pers polysaccharide.
Preparation slant medium described in technical scheme refers to: adopt by weight potato 20%, add distilled water, potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with by weight glucose 2%, agar 1.5%, KH
2PO
40.3%, MgSO
40.15%, adding distil water is settled to 100mL, mixes, and is sub-packed in test tube, 10 milliliters of every pipe liquid amounts, and 121 ℃ of sterilization 30min become test tube 10 ° of angles to put to clean work station with horizontal plane, cool to prepare the actication of culture use.
Preparation submerged fermentation culture medium described in technical scheme refers to: adopt by weight potato 10%, add distilled water, potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with by weight sucrose 3%, peptone 0.1%, yeast extract paste 0.5%, KH
2PO
40.05% and MgSO
40.05%, the adding distil water constant volume mixes, and makes that in submerged fermentation culture medium, selenium concentration reaches 80 μ g/mL, is that in the triangular flask of 250mL, liquid amount is 100mL at capacity, and 121 ℃ of sterilization 30min cool in clean work station and prepare submerged fermentation and use.
employing AB-8 macroporous resin described in technical scheme separates, obtain rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, carrying out next step purifying refers to: be to draw supernatant liquor after the Crude polysaccharides solution centrifugal in the rich selenium morel born of the same parents of 1mg/mL with the concentration that obtains, inject the AB-8 macroporous resin column, each injection rate is 5mL, carry out wash-out with distilled water, the volume of distilled water is 3 to 5 times of macroporous resin column volume, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, can obtain rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, carry out next step purifying.
Sephadex G-100 gel filtration chromatography described in technical scheme, draw rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, getting rich selenium Morchella esculenta (L.) Pers polysaccharide refers to: the rich selenium Morchella esculenta (L.) Pers polysaccharide solution that will obtain after separating through the AB-8 macroporous resin, get supernatant liquor after centrifugal, inject SephadexG-100 gel column chromatography, each injection rate is 5mL, the distilled water of 3 to 5 times that is Sephadex G-100 gel column volume with volume carries out wash-out, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, draw rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, through dialysis, alcohol precipitation, after lyophilize, namely get rich selenium Morchella esculenta (L.) Pers polysaccharide.
Compared with prior art the invention has the beneficial effects as follows:
1. the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide of the present invention as rich selenium carrier, utilizes the preparation of submerged fermentation technology to morel, extracts and the rich selenium Morchella esculenta (L.) Pers polysaccharide of purifying, and the Organic Selenium element of morel enrichment is easy to absorbing of human body;
2. little selenium of the rich selenium Morchella esculenta (L.) Pers polysaccharide enrichment of the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide of the present invention preparation has good stability, utilize morel as rich selenium carrier, be convenient to store, transport, avoid wherein nutritive ingredient loss, rich selenium Morchella esculenta (L.) Pers polysaccharide is a kind of organic selenium compounds, had both the activity of selenium and polysaccharide by the combination of polysaccharide and selenium, thereby inorganic selenium has been converted into organoselenium, organism has been had low toxicity.
3. the rich selenium Morchella esculenta (L.) Pers polysaccharide of the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide of the present invention preparation has better reducing blood-fat and anti-fatigue ability than common Morchella esculenta (L.) Pers polysaccharide, is suitable for developing the special efficacy protective foods of reducing blood-fat antifatigue.
Description of drawings
The present invention is further illustrated below in conjunction with accompanying drawing:
The typical curve that Fig. 1 makes for selenium content in the preparation method who adopts a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide of the present invention calculates rich selenium Morchella esculenta (L.) Pers polysaccharide;
Fig. 2 is that common Morchella esculenta (L.) Pers polysaccharide is at 4000～400cm
-1Carry out the RI spectrogram that Infrared spectroscopy obtains in wavelength region;
Fig. 3 for the rich selenium Morchella esculenta (L.) Pers polysaccharide of preparation method's preparation of adopting a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide of the present invention at 4000～400cm
-1Carry out the RI spectrogram that Infrared spectroscopy obtains in wavelength region;
Fig. 4 is the functional sequence block diagram that the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide of the present invention prepares rich selenium Morchella esculenta (L.) Pers polysaccharide.
Embodiment
Below in conjunction with accompanying drawing, the present invention is explained in detail:
Technical problem to be solved by this invention is to have overcome prior art to have that fruit body of edible fungi is low to the enrichment degree of selenium, low to the transforming degree of inorganic selenium, the low problem low with degree of purification of edible fungi polysaccharide extraction yield, and a kind of preparation method of rich selenium Morchella esculenta (L.) Pers polysaccharide is provided.
One. prepare the preparation of rich selenium Morchella esculenta (L.) Pers polysaccharide
1. prepare the rich selenium morel of preparation equipment and material used:
1) bacterial classification that adopts is No. 926, the Morchella crassipes that is purchased from Mianyang, Sichuan edible mushrooms institute;
2) medicine and the reagent that adopt are that potato, yeast extract paste, peptone, Sodium Selenite, glucose, sal epsom, dipotassium hydrogen phosphate, hydrochloric acid, toluene, sulfuric acid, agar powder, nitric acid, perchloric acid, EDTA, O-Phenylene Diamine, the concentration that is purchased from Tianjin Fengchuan Chemical Reagent Science ﹠ Technology Co., Ltd. is selenium standardized solution and the distilled water of 100 μ g/ml;
3) prepare the rich selenium morel instrument equipment of preparation:
The electronics universal furnace that Tianjin Tai Site Instr Ltd. produces;
The model that Switzerland METTLER TOLEDO company produces is the analytical balance of GB204;
The model that Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd. produces is the climatic chamber of SP-250C;
The model that Shanghai City Shen An medical apparatus and instruments factory produces is the portable Sterilizers of stainless steel of DSX-280A;
The model that Shanghai Precision Scientific Apparatus Co., Ltd produces is the pH meter of PHS-3D;
The model that Harbin City Dong Lian Electron Technology Co., Ltd produces is the shaking culture case of HZQ-F100;
It is the double two-sided clean work station of SW-CJ-2F that the Ji state purifies the model of producing.
2. prepare to extract Crude polysaccharides equipment and material used in rich selenium morel born of the same parents:
1) extract Crude polysaccharides instrument equipment in rich selenium morel born of the same parents:
The model that Beijing Medical Centrifugal Machine Factory produces is the low speed centrifuge of DT5-1;
The microwave oven of U.S. that the microwave oven electrical equipment Manufacturing Co., Ltd that De Shun district, Foshan City is beautiful produces;
The model that city of Kunshan ultrasonic instrument company produces is the numerical control ultrasonic cleaner of KQ-250DB;
The model that Switzerland METTLER TOLEDO company produces is the analytical balance of GB204;
The model that changzhou state China Instr Ltd. produces is 78 magnetic stirring apparatus;
The model of Shanghai City laboratory apparatus head factory production is the vacuum drying oven of ZK-82;
The model that Tianjin Tai Site produces is the electric-heated thermostatic water bath of DK-98-1.
2) extract Crude polysaccharides material therefor in rich selenium morel born of the same parents:
The anthrone that AR China Medicine (Group) Shanghai Chemical Reagent Co., produces;
95% ethanol that the AR Beijing Chemical Plant produces;
The 98%H of AR Beijing Chemical Plant
2SO
4
The chloroform that AR Changchun chemical reagent factory is produced;
The propyl carbinol that the AR Beijing Chemical Plant produces.
3. prepare the rich selenium Morchella esculenta (L.) Pers polysaccharide of preparation equipment and material used:
1) the rich selenium Morchella esculenta (L.) Pers polysaccharide equipment used of preparation:
The model that Shanghai Techcomp Instrument Ltd. produces is the spectrophotometer of UV2300;
2) the rich selenium Morchella esculenta (L.) Pers polysaccharide material therefor of preparation:
The model that Nanhua, Shanghai medical apparatus and instruments factory produces is the chromatography column macroporous resin of AB-8;
The model that Beijing ancient cooking vessel state biotech company produces is the chromatography column Sephadex of G-100.
Two. the preparation method's of rich selenium Morchella esculenta (L.) Pers polysaccharide step is as follows:
1. the preparation of rich selenium morel
1) preparation substratum
(1) preparation slant medium
Adopt by weight potato 20%, 100mL distilled water, potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with by weight glucose 2%, agar 1.5%, KH
2PO
40.3%, MgSO
40.15%, adding distil water is settled to 100mL, mixes, and is sub-packed in test tube, 10 milliliters of every pipe liquid amounts, and 121 ℃ of sterilization 30min become test tube 10 ° of angles to put to clean work station with horizontal plane, cool to prepare the actication of culture use;
(2) preparation submerged fermentation culture medium
Adopt by weight potato 10%, 500mL distilled water, potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with by weight sucrose 3%, peptone 0.1%, yeast extract paste 0.5%, KH
2PO
40.05% and MgSO
40.05%, adding distil water is settled to 500mL, mixes, and in the 250mL triangular flask of packing into, liquid amount is 100mL, and 121 ℃ of sterilization 30min cool in clean work station and prepare the submerged fermentation use.
2) actication of culture
All utensils are put to the clean work station by after ultra-violet sterilization 30min, with No. 926, the Morchella crassipes of transfering loop picking preservation, be transferred on fresh slant medium under gnotobasis, cultivate 24h in 25 ℃ of climatic chambers;
3) submerged fermentation
After actication of culture, get approximately 5mm
210 access submerged fermentation culture mediums of the bacterial classification of size, the Sodium Selenite that adds simultaneously, make that in submerged fermentation culture medium, selenium concentration reaches 80 μ g/ml, 250mL triangular flask charging 100mL, be placed in the shaking culture case, culture temperature is 26 ℃, shaking culture case oscillation frequency 100rpm, incubation time 5 days obtains rich selenium Morciiella Esculeuta Mycelia ball.
2. the extraction of Crude polysaccharides in rich selenium morel born of the same parents
1) rich selenium Morchella esculenta (L.) Pers mycelium after cleaning, dry, pulverize, sieving, is used the analytical balance weighing, and the ratio that is 1:30 in mycelium and distilled water adds distilled water, then puts into microwave oven, and microwave power is made as 700W, and the time is 40s; Then put into numerical control ultrasonic cleaner, ultrasonic power is set as 250W, and the time is 35min, processing is placed in thermostat water bath, at 90 ℃ of lower hot water lixiviate 140min, vat liquor is put into low speed centrifuge centrifugal 15min under the 4000r/min condition, get supernatant liquor;
2) supernatant liquor after cryoconcentration, under agitation adds 95% ethanol of 3 times of volumes, and alcohol is analysed liquid standing 12h in 4 ℃ of refrigerators, puts into low speed centrifuge centrifugation collecting precipitation thing;
3) throw out of collecting is redissolved in distilled water, be that the ratio of 5:1 is with the abundant mixing of magnetic stirring apparatus in sample and Sevag reagent, be placed in separating funnel, discard the protein denaturation layer on lower floor's organic layer and two-phase interface after standing 20min, get upper strata liquid and repeat this step process 7～8 times, until between organic layer and water layer without white gluey deposits yields, this moment, supernatant liquid was Crude polysaccharides solution in the morel born of the same parents;
4) get that upper strata morel intracellular polyse solution is concentrated, collecting precipitation thing after alcohol precipitation, then wash 2 times with dehydrated alcohol, acetone, ether respectively, at last throw out is carefully scraped in weighing disk, be placed in 40 ℃ of dry 2h of vacuum drying oven, namely obtain Crude polysaccharides in solid-state rich selenium morel born of the same parents.
3. the purifying of Crude polysaccharides in rich selenium morel born of the same parents
1) adopt the AB-8 macroporous resin to separate
with the centrifugal rear absorption supernatant liquor of Crude polysaccharides solution (concentration is 1mg/mL) in the rich selenium morel born of the same parents that obtain, injection AB-8 macroporous resin column (Φ 2cm * 30cm), each injection rate is 5mL, carry out wash-out (volume is approximately 3-5 times of column volume) with distilled water, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, can obtain rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve.Collect the elutriant at curve elution peak position, carry out next step purifying.
2) Sephadex G-100 gel filtration chromatography
the rich selenium Morchella esculenta (L.) Pers polysaccharide solution that will obtain after separating through the AB-8 macroporous resin, put into low speed centrifuge and get supernatant liquor after centrifugal, inject the Sephadex G-100 gel column (chromatography of Φ 2cm * 30cm), each injection rate is 5mL, carry out wash-out (volume is approximately 3-5 times of Sephadex G-100 gel column volume) with distilled water, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, draw rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve.Collect the elutriant at curve elution peak position, after dialysis, alcohol precipitation, lyophilize, namely get rich selenium Morchella esculenta (L.) Pers polysaccharide.
Embodiment:
The preparation of rich selenium Morchella esculenta (L.) Pers polysaccharide:
1. the preparation of rich selenium morel
1) preparation slant medium:
Adopt potato 20g, 100mL distilled water; Potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with glucose 2g, agar 1.5g, KH
2PO
40.3g, MgSO
40.15g adding distil water is settled to 100mL, mixes, and is sub-packed in test tube, 10 milliliters of every pipe liquid amounts, and 121 ℃ of sterilization 30min become test tube 10 ° of angles to put to clean work station with horizontal plane, cool to prepare the actication of culture use;
2) preparation submerged fermentation culture medium:
Adopt potato 50g, 500mL distilled water, potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with sucrose 15g, peptone 1g, yeast extract paste 5g, KH
2PO
40.5g with MgSO
40.5g adding distil water is settled to 500mL, mixes, in the 250mL triangular flask of packing into, liquid amount is 100mL, and 121 ℃ of sterilization 30min cool in clean work station and prepare the submerged fermentation use.
3) actication of culture
By after ultra-violet sterilization 30min, No. 926, the Morchella crassipes of use transfering loop picking preservation under gnotobasis is transferred on fresh slant medium, cultivates 48h in 25 ℃ of constant incubators with all utensils.
4) submerged fermentation
After actication of culture, get approximately 5mm
2Bacterial classification more than the 10 piece equivalent access submerged fermentation culture mediums of size, add simultaneously Sodium Selenite, make that in submerged fermentation culture medium, selenium concentration reaches 80 μ g/mL, 250mL triangular flask charging 100mL, be placed in the shaking culture case, 26 ℃ of culture temperature, shaking culture case oscillation frequency 100rpm, incubation time 5 days.Obtain rich selenium Morciiella Esculeuta Mycelia ball
5) mensuration of selenium content
Consult Fig. 1, the O-Phenylene Diamine ultraviolet spectrophotometry is adopted in the detection of selenium content, and O-Phenylene Diamine generates 3 with the selenite reaction in acidic medium, the 4-phendioxin, 2,5-diazonium kohlrabi selenium brain, with toluene extraction, utilize ultraviolet spectrophotometer in the absorbancy of 335nm place mensuration extraction liquid.
(1) sample preparation:
Accurately take the dried rich selenium Morchella esculenta (L.) Pers mycelium of 1g, put into the ground triangular flask after grinding, add vitriol oil 10.0mL, wetting sample, then add concentrated nitric acid-perchloric acid (2:1) mixing acid 20.0mL, placement is spent the night.Next day, heating in water bath was to the NO of brown color
2γ-ray emission after solution becomes brown color, continues heating, till eliminating to white cigarette, then adds the 10.0mL(concentrated hydrochloric acid: the hydrochloric acid of distilled water=1:9), continue to heat 5 minutes, and namely reach the digestion terminal point.Get in 10.0mL Digestive system and Erlenmeyer flask, add water 30.0mL, add respectively 5%EDTA2.0mL and 1% O-Phenylene Diamine 2.0mL, with HCI regulator solution pH value to 2.0, after mixing, the room temperature lucifuge is placed 60min, adds 10.0mL toluene, large forced oscillation 4min moves into standing 4min in separating funnel.Measure the absorbancy of test solution under the 335nm wavelength, do contrast with blank sample, measure the absorbancy of test solution, the contrast standard curve is obtained selenium concentration.
(2) making of selenium typical curve:
Configuration concentration is 0.00,3.00,6.00,9.00, and the selenium standardized solution of 12.00,15.00,18.00,21.00 μ g/ml is measured the absorbancy of each standardized solution.Take selenium content as X-coordinate, absorbancy is ordinate zou drawing standard curve, and typical curve is seen shown in figure.
The typical curve equation is Y=0.0343x-0.0215, R
2=0.9975 (2)
(3) mensuration of selenium content and calculating:
Liquid to be measured after treatments of the sample is diluted with suitable multiple, measure its absorbancy under similarity condition.Draw the selenium concentration of test fluid according to typical curve.Obtain selenium content in rich selenium Morchella esculenta (L.) Pers polysaccharide as 18 μ g/mL take this.
2. the extraction of Crude polysaccharides in rich selenium morel born of the same parents
1) rich selenium Morchella esculenta (L.) Pers mycelium after cleaning, dry, pulverize, sieving, is used the analytical balance weighing, and the ratio that is 1:30 in mycelium and distilled water adds distilled water, then puts into microwave oven, and microwave power is made as 700W, and the time is 40s; Then put into numerical control ultrasonic cleaner, ultrasonic power is set as 250W, and the time is 35min, processing is placed in thermostat water bath, at 90 ℃ of lower hot water lixiviate 140min, vat liquor is put into low speed centrifuge centrifugal 15min under the 4000r/min condition, get supernatant liquor;
2) supernatant liquor after cryoconcentration, under agitation adds 95% ethanol of 3 times of volumes, and alcohol is analysed liquid standing 12h in 4 ℃ of refrigerators, puts into low speed centrifuge centrifugation collecting precipitation thing.
3) throw out of collecting is redissolved in distilled water, be that the ratio of 5:1 is with the abundant mixing of magnetic stirring apparatus in sample and Sevag reagent, be placed in separating funnel, discard the protein denaturation layer on lower floor's organic layer and two-phase interface after standing 20min, get upper strata liquid and repeat this step process 7～8 times, until between organic layer and water layer without white gluey deposits yields, this moment, supernatant liquid was Crude polysaccharides solution in the morel born of the same parents.
4) get in the morel born of the same parents of upper strata that Crude polysaccharides solution is concentrated, collecting precipitation thing after alcohol precipitation, then wash 2 times with dehydrated alcohol, acetone, ether respectively, at last throw out is carefully scraped in weighing disk, be placed in 40 ℃ of dry 2h of vacuum drying oven, namely obtain Crude polysaccharides in solid-state rich selenium morel born of the same parents.
3. the purifying of Crude polysaccharides in rich selenium morel born of the same parents
1) adopt the AB-8 macroporous resin to separate
with the centrifugal rear absorption supernatant liquor of Crude polysaccharides solution (concentration is 1mg/mL) in the rich selenium morel born of the same parents that obtain, injection AB-8 macroporous resin column (Φ 2cm * 30cm), each injection rate is 5mL, carry out wash-out (volume is approximately 3-5 times of column volume) with distilled water, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, can obtain rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve.Collect the elutriant at curve elution peak position, carry out next step purifying.
2) Sephadex G-100 gel filtration chromatography
the rich selenium Morchella esculenta (L.) Pers polysaccharide solution that will obtain after separating through the AB-8 macroporous resin, put into low speed centrifuge and get supernatant liquor after centrifugal, inject the Sephadex G-100 gel column (chromatography of Φ 2cm * 30cm), each injection rate is 5mL, carry out wash-out (volume is approximately 3-5 times of column volume) with distilled water, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, draw rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve.Collect the elutriant at curve elution peak position, after dialysis, alcohol precipitation, lyophilize, namely get rich selenium Morchella esculenta (L.) Pers polysaccharide.
3) Purity
Utilize normal pressure Sephadex G-100 gel filtration chromatography method to carry out preliminary evaluation to the purity of rich selenium Morchella esculenta (L.) Pers polysaccharide.The rich selenium morel holosaccharide that to collect through Sephadex G-100 gel filtration chromatography, refill again Sephadex G-100 gel column (in Φ 2cm * 30cm), each injection rate is 5mL, carry out wash-out with distilled water, volume is 3-5 times of column volume, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, draw rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve.
Three. the structural analysis of rich selenium Morchella esculenta (L.) Pers polysaccharide
Consult Fig. 2 and Fig. 3, selenium as the existence form of the characteristic of selenium polysaccharide part in selenium polysaccharide may have Se-H and
Two kinds.With common morel and rich selenium Morchella esculenta (L.) Pers polysaccharide, respectively at 4000～400cm
-1Carry out Infrared spectroscopy in scope, as shown in FIG..
Rich selenium does not change the agent structure of water-soluble polysaccharide, shows both have same polysaccharide characteristic peak: at 3200～3800cm
-1There is the wide absorption peak of the last one at the place, be on polysaccharide-OH forms due to molecular linkage, interior hydrogen bond; 2921.9cm-1 be-CH3 and-CH2-stretching vibration absorb, strong peak in genus; Particularly at 1149.5～1026.1cm
-1In all have 3 charateristic avsorption bands of pyranoid ring in polysaccharide structures, this 3 peak is caused by two kinds of C-O stretching vibration: a kind of C of belonging to-O-H, another kind are the nonsymmetrical vibration absorption peaks of glycosidic link C-O-C; Selenium may be to be present on the side chain of water-soluble polysaccharide with Se-H form, because rich selenium makes 3 absorption peaks of pyranoid ring that red shift occur, namely by original 1149.5,1078.1,1026.1cm
-1Red shift to 1114.7,1078.1,1049.2cm
-1Fig. 2 is at 927cm
-1The place is in polysaccharide molecule-COOH in the out-of-plane deformation vibration absorption peak of O-H, and Fig. 3 does not have this characteristic.Fig. 3 is at 873cm
-1The absorption peak at place is that the flexural vibration by the C-H of pyranoid ring a-anomerism cause, illustrates and contains the pyranose that the a-glycosidic link connects in this polysaccharide chain.
Infrared spectra structural analysis by two kinds of form Morchella esculenta (L.) Pers polysaccharides before and after rich selenium is as can be known: rich selenium morel selenium polysaccharide has the general feature absorption peak of polysaccharide, and is the pyrans polysaccharide that α-glycosidic link connects.Selenium may be to be present on the side chain of water soluble selenium polysaccharide with Se-H form, because selenium has participated in the synthetic of polysaccharide, causes the structure of polysaccharide that change has occured after rich selenium.
Four. the function of rich selenium Morchella esculenta (L.) Pers polysaccharide
According to the requirement of in country's " protective foods check and evaluation technique standard ", test principle, test subject and result being judged, in order to study rich selenium Morchella esculenta (L.) Pers polysaccharide to the impact of mouse reducing blood lipid and antifatigue, be male mice with 48 Kunming, be divided at random 6 groups, measure mouse body weight, spleen weight, swimming with a load attached to the body time, blood lipids index level on an empty stomach after 20 days.
1. animal grouping and processing
48 of male Kunming mouses, body weight (20 ± 2) g, available from Changchun City Jilin University's experimentation on animals center, after the adaptability of 5 days are raised, be divided at random 6 groups: common control group, high fat control group, Morchella esculenta (L.) Pers polysaccharide group, the high fat group of Morchella esculenta (L.) Pers polysaccharide, rich selenium Morchella esculenta (L.) Pers polysaccharide group, the high fat group of rich selenium Morchella esculenta (L.) Pers polysaccharide.Common control group feed arm's length basis feed consists of fish meal, yolk, soybean cake powder etc.; High fat control group feed high lipid food, filling a prescription is cholesterol 1.2%, Sodium cholic acid 0.2%, lard 10%, basal feed 88.6%; Feed on the basis carrying out basis and high lipid food for all the other four groups, respectively gavage 100ml/ (kgd) Morchella esculenta (L.) Pers polysaccharide and rich selenium Morchella esculenta (L.) Pers polysaccharide.Wherein, rich selenium morel selenium content is 9 μ g/ml.
2. effect of relieving physical fatigue
The mouse of common control group, Morchella esculenta (L.) Pers polysaccharide group and rich selenium Morchella esculenta (L.) Pers polysaccharide group is carried out the quality of the internal organs such as body weight, spleen and measure, according to 5% of every Mouse Weight, appropriate galvanized wire is tied up in mouse tail root position.Be placed on swimming in the swimming trunk that water temperature (25 ± 1) ℃, the depth of water be no less than 30cm, record every mouse and begin to Post-dead duration from swimming, as the mice burden swimming time.
3. auxiliary antilipemic effect
Common control group, the high fat group of Morchella esculenta (L.) Pers polysaccharide and the high fat group of rich selenium Morchella esculenta (L.) Pers polysaccharide are recorded the body weight of mouse, divide cage to feed 20 days, finish respectively to organize mouse fasting 16h in experiment, pluck the eyeball blood sampling, collect blood sample in centrifuge tube, through the centrifugal 15min of 3000r/min, draw upper serum with liquid-transfering gun, utilize successively total cholesterol detection kit, triglyceride level detection kit, high density lipoprotein cholesterol detection kit that serum total cholesterol, triglyceride level and High-density Lipoprotein-cholesterol are measured.
This experiment utilizes UniCel DxC800Synchron full automatic biochemical apparatus that the serum urea level is measured.
4. result and discussion
1) observation of mouse growth developmental condition
The impact of table 1 different treatment on Mice Body weight
Experimental session, each organizes mouse Mao Zhibai and glossy, the behavior no abnormality seen, without rare just, semiliquid stool and the phenomena of mortality, grow normal.
The Mice Body qualitative change is as shown in table 1, and the Mice Body weight of different treatment group does not have significant difference, illustrates that DIFFERENT FEED has no adverse effects to Mice Body weight.
Table 2 effect of relieving physical fatigue experimental result
As can be seen from Table 2, compare with control group, the mice burden swimming time of Morchella esculenta (L.) Pers polysaccharide group, rich selenium Morchella esculenta (L.) Pers polysaccharide group obviously extends, and can judge that this experimental result is positive.Simultaneously, the serum urea of experimental group can judge namely that lower than control group this experimental result is positive.
Therefore, Morchella esculenta (L.) Pers polysaccharide, rich selenium Morchella esculenta (L.) Pers polysaccharide have significant alleviating physical fatigue effect.
2) variation of blood lipid level
The impact of table 3 different treatment on Mice Body weight
In whole experimentation on animals process, it is all normal that each organizes the mouse growth activity.The Mouse Weight amplification of high fat control group is maximum, is secondly the high fat group of Morchella esculenta (L.) Pers polysaccharide and the high fat group of rich selenium Morchella esculenta (L.) Pers polysaccharide.The Mouse Weight amplification of the high fat group of Morchella esculenta (L.) Pers polysaccharide and the high fat group of rich selenium Morchella esculenta (L.) Pers polysaccharide is significantly lower than high fat control group, illustrates that Morchella esculenta (L.) Pers polysaccharide, rich selenium Morchella esculenta (L.) Pers polysaccharide can alleviate high lipid food and feed the body weight of mouse and increase.
The impact of table 4 different treatment on lipid of mice
As can be seen from Table 4, high fat control group mice serum total cholesterol, triglyceride level and serum low-density LP content are significantly higher than control group, and the modeling success is described.All significantly lower than high fat control group, HDL-C content is significantly higher than high fat control group for the TC of Morchella esculenta (L.) Pers polysaccharide group and rich Morchella esculenta (L.) Pers polysaccharide group, TG content.Rich Morchella esculenta (L.) Pers polysaccharide group LDL-C illustrate that the ability of rich selenium Morchella esculenta (L.) Pers polysaccharide reduction LDL-C is better than Morchella esculenta (L.) Pers polysaccharide, and the comprehensive reducing blood-fat ability of rich selenium Morchella esculenta (L.) Pers polysaccharide group slightly is better than the Morchella esculenta (L.) Pers polysaccharide group lower than high fat control group and the high fat group of Morchella esculenta (L.) Pers polysaccharide.
Result shows: rich selenium Morchella esculenta (L.) Pers polysaccharide has the function of alleviating physical fatigue, and can significantly reduce TC, TG and the LDL-C of hyperlipemia in mice, rising HDL-C.
By setting up mouse anti-reflecting fatigue model and hyperlipidemia model and to the observation of the blood fat of 4 groups of mouse, finding that rich selenium Morchella esculenta (L.) Pers polysaccharide can effectively reduce the blood lipid level of hyperlipidemia mouse, improves blood lipid metabolism.Experiment results proved rich selenium Morchella esculenta (L.) Pers polysaccharide have better reducing blood-fat and anti-fatigue ability than Morchella esculenta (L.) Pers polysaccharide, be a kind of more satisfactory reducing blood-fat antifatigue special efficacy protective foods.
Claims (5)
1. the preparation method of a rich selenium Morchella esculenta (L.) Pers polysaccharide, is characterized in that, the preparation method's of described a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide step is as follows:
1) preparation of rich selenium morel
(1) preparation substratum:
A. prepare slant medium;
B. prepare submerged fermentation culture medium;
(2) actication of culture:
By after ultra-violet sterilization 30min, No. 926, the Morchella crassipes of use transfering loop picking preservation under gnotobasis is transferred on fresh slant medium, cultivates 24h in 25 ℃ of constant incubators with all utensils;
(3) submerged fermentation:
After actication of culture, get approximately 5mm
210 access submerged fermentation culture mediums of the bacterial classification of size, the Sodium Selenite that adds simultaneously, make that in submerged fermentation culture medium, selenium concentration reaches 80 μ g/ml, 250mL triangular flask charging 100mL, be placed in the shaking culture case, culture temperature is 26 ℃, the oscillation frequency 100rpm of shaking culture case, incubation time 5 days obtains rich selenium Morciiella Esculeuta Mycelia ball;
2) extraction of Crude polysaccharides in rich selenium morel born of the same parents
(1) the rich selenium Morciiella Esculeuta Mycelia ball warp rear weighing of cleaning, dry, pulverize, sieve, the ratio that is 1:30 in mycelium pellet and distilled water adds distilled water, carry out the microwave cooperating ultrasonic-assisted extraction, then be placed in water-bath, at 90 ℃ of lower hot water lixiviate 140min, with revolution in whizzer the centrifugal 15min of vat liquor with 4000r/min, get supernatant liquor;
(2) supernatant liquor after cryoconcentration, under agitation adds 95% ethanol of 3 times of volumes, and alcohol is analysed liquid standing 12h in 4 ℃ of refrigerators, adopts whizzer separated and collected throw out;
(3) throw out of collecting is redissolved in distilled water, it is the abundant mixing of ratio of 5:1 in sample and Sevag reagent, be placed in separating funnel, discard the protein denaturation layer on lower floor's organic layer and two-phase interface after standing 20min, get upper strata liquid and repeat this step process 7～8 times, until between organic layer and water layer without white gluey deposits yields, this moment, supernatant liquid was Crude polysaccharides solution in the morel born of the same parents;
(4) get in the morel born of the same parents of upper strata that Crude polysaccharides solution is concentrated, collecting precipitation thing after alcohol precipitation, then wash 2 times with dehydrated alcohol, acetone, ether respectively, carefully scrape throw out in weighing disk at last, be placed in 40 ℃ of dry 2h of vacuum drying oven, namely obtain Crude polysaccharides in solid-state rich selenium morel born of the same parents;
3) purifying of Crude polysaccharides in rich selenium morel born of the same parents
1) adopt the AB-8 macroporous resin to separate, obtain rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, carry out next step purifying;
2) Sephadex G-100 gel filtration chromatography is drawn rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collects the elutriant at curve elution peak position, gets rich selenium Morchella esculenta (L.) Pers polysaccharide.
2. according to the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide claimed in claim 1, it is characterized in that, described preparation slant medium refers to:
Adopt by weight potato 20%, add distilled water, potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with by weight glucose 2%, agar 1.5%, KH
2PO
40.3%, MgSO
40.15%, adding distil water is settled to 100mL, mixes, and is sub-packed in test tube, 10 milliliters of every pipe liquid amounts, and 121 ℃ of sterilization 30min become test tube 10 ° of angles to put to clean work station with horizontal plane, cool to prepare the actication of culture use.
3. according to the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide claimed in claim 1, it is characterized in that, described preparation submerged fermentation culture medium refers to:
Adopt by weight potato 10%, add distilled water, potato is cut into small pieces boils approximately 30min, boil till thoroughly well cooked but not mushy, four layers of filtered through gauze, then be equipped with by weight sucrose 3%, peptone 0.1%, yeast extract paste 0.5%, KH
2PO
40.05% and MgSO
40.05%, the adding distil water constant volume mixes, and makes that in submerged fermentation culture medium, selenium concentration reaches 80 μ g/mL, is that in the triangular flask of 250mL, liquid amount is 100mL at capacity, and 121 ℃ of sterilization 30min cool in clean work station and prepare submerged fermentation and use.
4. according to the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide claimed in claim 1, it is characterized in that, described employing AB-8 macroporous resin separates, and obtains rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, carry out next step purifying and refer to:
be to draw supernatant liquor after the Crude polysaccharides solution centrifugal in the rich selenium morel born of the same parents of 1mg/mL with the concentration that obtains, inject the AB-8 macroporous resin column, each injection rate is 5mL, carry out wash-out with distilled water, the volume of distilled water is 3 to 5 times of macroporous resin column volume, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, can obtain rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, carry out next step purifying.
5. according to the preparation method of a kind of rich selenium Morchella esculenta (L.) Pers polysaccharide claimed in claim 1, it is characterized in that described Sephadex G-100 gel filtration chromatography is drawn rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, get rich selenium Morchella esculenta (L.) Pers polysaccharide and refer to:
the rich selenium Morchella esculenta (L.) Pers polysaccharide solution that will obtain after separating through the AB-8 macroporous resin, get supernatant liquor after centrifugal, inject Sephadex G-100 gel column chromatography, each injection rate is 5mL, the distilled water of 3 to 5 times that is SephadexG-100 gel column volume with volume carries out wash-out, flow rate control is at 2mL/min, every 2min collects 1 pipe elutriant, utilize sulfuric acid-anthrone method to measure the polysaccharide content of respectively managing in elutriant, utilize the spectrophotometer of UV2300 to carry out the polysaccharide content detection to each pipe elutriant, with wash-out pipe number as X-coordinate, with absorbancy numerical value as ordinate zou, draw rich selenium Morchella esculenta (L.) Pers polysaccharide elution curve, collect the elutriant at curve elution peak position, through dialysis, alcohol precipitation, after lyophilize, namely get rich selenium Morchella esculenta (L.) Pers polysaccharide.
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Application publication date: 20130605 |