CN108503724A - Chinese caterpillar fungus culture medium polysaccharide and its isolation and purification method and application - Google Patents
Chinese caterpillar fungus culture medium polysaccharide and its isolation and purification method and application Download PDFInfo
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Abstract
The invention discloses a kind of Chinese caterpillar fungus culture medium polysaccharide and its isolation and purification method and purposes, which is made of the monosaccharide of following molar percentage：0.26% ribose, 0.11% rhamnose, 2.01% arabinose, 0.34% xylose, 29.62% mannose, 67.19% glucose, 0.47% galactolipin.Extracting method using the present invention does not influence the bioactivity of Chinese caterpillar fungus culture medium polysaccharide P2, and obtained polysaccharide sterling P2 purity is high, property is stablized, and has remarkable result at anti-oxidant, anti-trioxypurine, antibacterial aspect, is beneficial to body metabolism；Cost is relatively low, and polysaccharide P2 sterlings can be further used for the exploitation of health products, drug, cosmetics.
Description
Technical field
The present invention relates to a kind of Chinese caterpillar fungus culture medium polysaccharide and its isolation and purification method and applications.
Background technology
Polysaccharide (Polysaccharide) is also known as polysaccharide, the line being connected together by glycosidic bond by aldose or ketose
Property or branch chain polymer, be the polarity complexity macromolecular that the degree of polymerization is more than 10, molecular weight is generally more than tens thousand of, is to constitute
One of four big base substances of vital movement, the polysaccharide in organism, also can be with protein or fat other than existing with free state
Fat is combined into proteoglycan and lipopolysaccharides.
Cordyceps militaris (C.militaris) is also known as Cordceps militaris, and Ascomycotina, gang pyrenomycetes, Spheeriales are belonged on taxology,
Clavicipitaceae Cordyceps is belonged to northern Chinese caterpillar Fungus, is mainly distributed on the areas such as China northeast, North China, northwest.Cordyceps militaris can be parasitic
In on the larva of the insects such as Lepidoptera, coleoptera, Diptera or pupal cell, the carry out such as silkworm pupa, rice medium can be used artificial
Batch is cultivated.
Since natural Cordyceps militaris resource is limited, artificial cultivation is more at present, and there are mainly three types of cultural methods：(1) it adopts
Collect wild Cordyceps militaris, carries out separation, the purifying of strain, then Cordyceps militaris spawn is seeded in the solids such as the rice containing dried silkworm chrysalis meal
On culture medium, under certain temperature, humidity and illumination condition, culture 35-45d obtains fruiting bodies of cordyceps militaris；(2) by Cordyceps militaris
Strain is seeded in silkworm larva or pupal cell living, and under certain temperature, humidity and illumination condition, culture 35-45d obtains pupa worm
Grass seed entity；(3) using soy meal sucrose or corn steep liquor sucrose as culture medium, using liquid fermentation and culture Cordyceps militaris.
China has considerable scale using the production of manual method culture fruiting bodies of cordyceps militaris, but in the same of fructification harvest
When, a large amount of Chinese caterpillar fungus culture medium leftover bits and pieces is also produced, pollution and the very important resource wave of environment are not only caused
Take.
Studies have shown that containing the bioactive substances such as cordycepin, Cordyceps sinensis polysaccharide in Chinese caterpillar fungus culture medium leftover bits and pieces.Therefore it is
Cordyceps militaris resource is preferably developed and used, increases economic efficiency, improves Cordyceps militaris industrial chain, to Chinese caterpillar fungus culture medium leftover bits and pieces
Utilization and research and development have become necessity.
Chinese patent application (application number 201110086808.2) discloses one kind and extracting polysaccharide from Chinese caterpillar fungus culture medium
Method, using different enzyme solutions to remove the albumen in Chinese caterpillar fungus culture medium, starch, then through alcohol precipitation obtain Chinese caterpillar fungus culture medium
Thick many candies；Its operating process is complicated, and cost is higher, and cannot further isolate and purify polysaccharide, and it is higher to be unable to get purity
Polysaccharide component.
Invention content
The primary purpose of the present invention is that providing a kind of Chinese caterpillar fungus culture medium polysaccharide.
It is auxiliary using ultrasonic wave it is a further object of the present invention to provide the isolation and purification method of above-mentioned Chinese caterpillar fungus culture medium polysaccharide
It helps water extraction and alcohol precipitation method to discard culture medium to Cordyceps militaris and carries out Polyose extraction, and polysaccharide is detached by ion exchange chromatography
Purifying, thus extraction isolate that purity is higher, biologically active polysaccharide component.
It is still another object of the present invention to provide the purposes of above-mentioned Chinese caterpillar fungus culture medium polysaccharide.
The purpose of the invention is achieved by the following technical solution：
A kind of Chinese caterpillar fungus culture medium polysaccharide, is made of the monosaccharide of following molar percentage：0.26% ribose, 0.11% sandlwood
Sugar, 2.01% arabinose, 0.34% xylose, 29.62% mannose, 67.19% glucose, 0.47% galactolipin；
The average molecular weight of the Chinese caterpillar fungus culture medium polysaccharide is 16.6k Da；
The Chinese caterpillar fungus culture medium polysaccharide contains pyranose ring and β-type glycosidic bond.
The isolation and purification method of above-mentioned Chinese caterpillar fungus culture medium polysaccharide, includes the following steps：
(1) Chinese caterpillar fungus culture medium polysaccharide is extracted：By Cordyceps militaris rice medium leftover bits and pieces crushed after being dried, it is sieved to get a foothold
Expect dry powder；Weigh the distilled water that 15-16 times of quality is added in dry powder, ultrasonication 30min or more, the refluxing extraction at 70 DEG C
1.5-2.0h, extraction merge extracting solution, are concentrated after extracting solution filtering, obtain polysaccharide concentrate several times；In polysaccharide concentrate
95% (V/V) ethyl alcohol of 3-4 times of volume is added, is stood overnight in 4 DEG C after stirring；Precipitation drying is obtained into Cordyceps militaris after centrifugation
Culture medium polyoses extract；
Preferred 40 mesh of the mistake sieve of sieving described in step (1)；
Concentration described in step (1) preferably concentrates at 50-55 DEG C；
The preferred 5000r/min of centrifugation described in step (1) centrifuges 15min；
(2) decoloration of Chinese caterpillar fungus culture medium polysaccharide：Chinese caterpillar fungus culture medium polyoses extract is added into distillation water dissolution, adjusts pH
H is added dropwise to 8.0-8.5 in value2O2Solution is to colourless, 50-55 DEG C of heat preservation 2h or more；
H described in step (2)2O2Solution, concentration preferably 30% (V/V)；
(3) enzyme process combination Sevage methods take off albumen：
3-1：Papain solution is mixed with Chinese caterpillar fungus culture medium polysaccharide extraction liquid, the volume ratio of the two is 1.0:
1.5~1.0:1.7,60-70 DEG C of enzymolysis 2-3h；
The papain solution is formulated with the PBS buffer solution of pH value 6.0, wherein the concentration of papain
It is preferred that 250U/ml；
3-2：The Sevage reagents of 1/5 volume, shaken cultivation 30min or more, then repeatedly centrifugation are added in enzymolysis liquid
Until no albumen precipitation is precipitated, supernatant, drying is taken to obtain Chinese caterpillar fungus culture medium Thick many candies；
Sevage reagents described in step (3) are chloroform and n-butanols by volume 5:1 is formulated；
Shaken cultivation described in step (3), the preferred 150r/min of shaking speed；
The preferred 4000r/min of centrifugation described in step (3) centrifuges 20-30min；
(4) Chinese caterpillar fungus culture medium polysaccharide isolates and purifies：On after the distillation water dissolution of Chinese caterpillar fungus culture medium Thick many candies
DEAE Ago-Gels FF (DEAE Sephrose Fast Flow) ion exchange column, with 0.1mol/L NaCl solutions into
Row elution, obtains the Chinese caterpillar fungus culture medium polysaccharide P2.
Polysaccharide isolates and purifies the process for being not only a removal of impurities, while being also that mixing polysaccharide is separated into one-component
Process.Column chromatography can be divided into ion-exchange chromatography and gel filtration chromatography.Ion-exchange chromatography is according to ionic charge density
Different and carry out classification separation, the charge groups of anionite are positively charged, and counter ion is negatively charged, therefore this exchanger can
With in solution negative electrical charge compound or anion swap and react, and cation-exchanger is then on the contrary.
The Chinese caterpillar fungus culture medium polysaccharide of the present invention can be used as antioxidant or bacteriostatic agent application, can be used for preparing tool
There is the drug of anti-trioxypurine effect.
The present invention obtains Chinese caterpillar fungus culture medium Thick many candies by water extraction and alcohol precipitation method, is carried out using ion exchange column thick more
Isolating and purifying for sugar, has the following advantages and effect compared with the existing technology：
(1) present invention takes full advantage of the culture medium leftover bits and pieces cultivated Cordyceps militaris and generated, and meets the green to turn waste into wealth
Environmental protection concept establishes a complete feasible Chinese caterpillar fungus culture medium Polyose extraction, isolates and purifies, physicochemical property, structure and life
The technology path of object activity research, the perfect residue treatment technique of Tissue Culture of Cordyceps militaris industry, for carrying for edible fungi polysaccharide
It takes to isolate and purify and provides technological guidance.
(2) water extraction and alcohol precipitation method used in the present invention can complete the Polyose extraction operation of big flux, and cost is relatively low, repeatability
Good, yield is high, is suitable for industrialization large-scale production.
(3) the DEAE Sephrose Fast Flow physical and chemical stabilities used in the present invention and mechanical performance are more preferable, exchange and hold
Amount is big, can be varied less with the ionic strength of pH with incumbent firms, bed volume, since flow velocity and carrying capacity are high, is suitable for carrying out big
Measure the purification work of crude product.
(4) extracting method using the present invention does not influence the bioactivity of Chinese caterpillar fungus culture medium polysaccharide P2, obtained more
Sugared sterling P2 purity is high, property is stablized, and has remarkable result at anti-oxidant, anti-trioxypurine, antibacterial aspect, is beneficial to body metabolism；
Cost is relatively low, and polysaccharide P2 sterlings can be further used for the exploitation of health products, drug, cosmetics.
(5) present invention is creatively by the water extract-alcohol precipitation extracting method of polysaccharide and ion-exchange chromatography isolation and purification method knot
It is used for the research of Chinese caterpillar fungus culture medium leftover bits and pieces polysaccharide altogether, relatively and obtains preferably technological parameter, and determine DEAE
For Sephrose Fast Flow as chromatography column packing, the extraction separation and purification for Chinese caterpillar fungus culture medium leftover bits and pieces polysaccharide provides skill
Art instructs and new approaches.
Description of the drawings
Fig. 1 is the elution curve of Chinese caterpillar fungus culture medium polysaccharide P2.
Fig. 2 is the ultraviolet spectrogram of polysaccharide P2.
Fig. 3 is the infrared spectrogram of polysaccharide P2.
Fig. 4 is the ABTS free radical scavenging abilities of polysaccharide P2.
Fig. 5 is the bacteriostatic experiment result figure of polysaccharide P2；Wherein, Sa- is to the inhibition zone of staphylococcus aureus, and Pa- is to green
The inhibition zone of purulence bacillus, the inhibition zone that 2- polysaccharide P2 is generated, the inhibition zone of 0- control groups.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
In the present invention, the analysis of physical and chemical property of Chinese caterpillar fungus culture medium polysaccharide is carried out by China Guangzhou Analysis ＆. Test Center, report
It is respectively 2018001306-2b to accuse number.
Embodiment 1
A method of polysaccharide is extracted and isolated and purified from Chinese caterpillar fungus culture medium leftover bits and pieces, is included the following steps：
(1) Chinese caterpillar fungus culture medium polysaccharide is extracted：The abundant crushed after being dried of 55g Cordyceps militaris rice medium leftover bits and pieces is weighed,
40 mesh sieve is crossed, the distilled water that 800ml is added in 50g dry powder is weighed, ultrasonic 30min, the refluxing extraction 1.5h at 70 DEG C are extracted 3 times
Merging filtrate is concentrated into 100ml at 55 DEG C after vacuum filtration, obtains polysaccharide concentrate；400ml is added in polysaccharide concentrate
95% ethyl alcohol, being stirred continuously makes polysaccharide homogeneous precipitation, is stood overnight in 4 DEG C；5000r/min will precipitation after centrifuging 15min
Drying obtains Chinese caterpillar fungus culture medium polyoses extract；
(2) decoloration of Chinese caterpillar fungus culture medium polysaccharide：Distillation water dissolution is added to be made into Chinese caterpillar fungus culture medium polyoses extract dense
Degree is the polysaccharide extraction liquid of 0.05g/ml, and NaOH is added and adjusts pH value to 8.0,30% H is added dropwise2O2It is extremely colourless, it is kept the temperature at 50 DEG C
2h；
(3) enzyme process combination Sevage methods take off albumen：
Papain 0.1g accurately is weighed, the molten of final concentration of 250U/ml is dissolved into the PBS buffer solution of pH value 6.0
Liquid is mixed with Chinese caterpillar fungus culture medium polysaccharide extraction liquid, and the volume ratio of enzyme solution and Chinese caterpillar fungus culture medium polysaccharide extraction liquid is 1.0:
3h is digested at 1.5,64 DEG C；
Sevage reagent (the chloroforms of 1/5 volume are added in enzymolysis liquid:N-butanol=5:1), it is placed in shaking table 150r/min
4000r/min centrifuges 20min after vibrating 30min, and repeated centrifugation is repeatedly precipitated until no albumen precipitation, merging supernatant, 50 DEG C
Lower drying obtains Chinese caterpillar fungus culture medium Thick many candies；
(4) Chinese caterpillar fungus culture medium polysaccharide isolates and purifies：
It weighs 0.1g Chinese caterpillar fungus culture medium Thick many candies to be dissolved in 10ml distilled water, upper DEAE Sephrose Fast Flow
Ion exchange column is eluted with 0.1mol/L NaCl solutions, flow velocity 0.5ml/min, and 1 pipe is collected per 10min, with
Phend-sulphuric acid detects polyoses content by pipe, merges according to elution curve (Fig. 1), obtains Chinese caterpillar fungus culture medium polysaccharide P2.
Phend-sulphuric acid detects polyoses content concrete operations：Precision weighs 105 DEG C of dryings to the DEXTROSE ANHYDROUS mark of constant weight
Quasi- product 0.1g is placed in 100ml volumetric flasks plus distills water dissolution and constant volume, shakes up, the standard solution for being made into 1mg/ml is spare.
The solution is taken to be diluted to the standard solution of 10,20,40,60,80,100 μ g/ml various concentrations respectively.Above-mentioned solution is drawn respectively
Each 1ml is placed in test tube, and 6% phenol solution 0.5ml and mixing is added, adds 2.5ml concentrated sulfuric acid mixings, is stored at room temperature
20min measures absorbance using distilled water as blank control at 490nm, and using concentration of glucose as abscissa, OD values are vertical sit
Mark draws standard curve.Unknown sample measures polyoses content with calibration curve method.
It will be freeze-dried after the polysaccharide component afforded concentration, dialysis, obtain Chinese caterpillar fungus culture medium polysaccharide sterling, name
For P2.
Embodiment 2
Ultraviolet spectral analysis is carried out to the Chinese caterpillar fungus culture medium polysaccharide P2 that embodiment 1 obtains, respectively weighs 1mg polysaccharide samples,
Prepare 1mg/mL polysaccharide solutions, scanning uv-spectrogram within the scope of 200-800nm.
Fig. 2 be P2 ultraviolet spectrogram, the results show that P2 at 260nm, 280nm without apparent absorption peak, show P2
Without protein and nucleic acid substances.
Embodiment 3
Polysaccharide molecular weight analysis is carried out to the Chinese caterpillar fungus culture medium polysaccharide P2 that embodiment 1 obtains, specific experimental method is as follows：
Molecular weight is measured using gel permeation chromatography (GPC).The polysaccharide sample 2mg for weighing freeze-drying, adds 0.02M phosphorus
Acid buffer dissolves, and is configured to 2.0mg/mL solution, is filtered with 0.22 μm of sterilised membrane filter, took cleaner liquid for use.
Chromatographic condition：35 DEG C of column temperature；0.02mol/L phosphate buffers (pH value 7.0) are mobile phase, flow velocity 0.6ml/
Min, 20 μ L of sample size；Tsk gel guard column (40mm × 6.0mm), TSKG-4000K gel columns (300mm × 7.8mm) and
TSKG-2500K gel columns (300mm × 7.8mm)；2414 differential refraction detectors of Waters detect.Prepare a series of different points
The dextran solution (700,400,200,100,50,30,10,5kD) of son amount is used as standard specimen, does standard curve.The molecule of sample
Amount is calculated according to its corresponding elution volume reference standard curve.
The result shows that the average molecular weight of Chinese caterpillar fungus culture medium polysaccharide P2 is 16.6k Da.
Embodiment 4
Monosaccharide composition analysis is carried out to the Chinese caterpillar fungus culture medium polysaccharide P2 that embodiment 1 obtains, the specific method is as follows：
Polysaccharide sample 10mg is weighed, 5mL trifluoroacetic acids (4M), 110 DEG C of hydrolysis 2h are added.Hydrolyzate is in 50 DEG C of vacuum rotatings
It is evaporated to dryness, cleaning 3 times with Chromatographic Pure Methanol (is added Chromatographic Pure Methanol, then is spin-dried for, 3 times repeatedly, until being spin-dried for substance again
Without trifluoroacetic acid taste), obtain polysaccharide hydrolysis object.
10mg hydroxylamine hydrochlorides, 1mg internal standards inositol and 2mL pyridines are sequentially added in polysaccharide hydrolysis object, are sealed, 90 DEG C of water-baths
2m L 90 DEG C of water-bath 30min of acetic anhydride are added after 30min, 2m L water is added and terminates reaction.The extraction of 2m L dichloromethane, weight is added
2 times multiple, anhydrous sodium sulfate drying is added in combined dichloromethane phase, crosses 0.22 μm of organic miillpore filter, spare.
Using chromatographic, analytical column is HP-5MS quartz capillary columns (30m × 0.25mm × 0.25 μm).It rises
Warm program is as follows：Injector temperature is 250 DEG C, and 100 DEG C of initial column temperature keeps 0.5min；Then 140 are risen to 20 DEG C/min
DEG C, keep 5min；160 DEG C are risen to 3 DEG C/min speed；250 DEG C are raised to 10 DEG C/min speed again, keeps 5min.Sample introduction body
Product is 1 μ L；Split ratio is 10:1；Mobile phase is helium；Flow velocity is 1mL/min.
Various monosaccharide standards (rhamnose, arabinose, ribose, xylose, mannose, glucose and galactolipin) are according to phase
It is tested with step, according to identical detection level, by treated, standard items monosaccharide carries out gas chromatographic analysis.
Measure the monosaccharide composition result such as following table of Chinese caterpillar fungus culture medium polysaccharide：
The monosaccharide of 1 Chinese caterpillar fungus culture medium polysaccharide P2 of table forms
Embodiment 5
FTIR spectrum analysis is carried out to the Chinese caterpillar fungus culture medium polysaccharide P2 that embodiment 1 obtains：
2mg polysaccharide samples are weighed, the mixing in mortar is ground with the KBr (potassium bromide) after drying by it, through tablet press machine pressure
In flakes, using Fourier Transform Infrared Spectrometer, in 400-4000cm-1Wave-number range in scanning.
Fig. 3 is the infrared spectrogram of P2, in 3401cm-1The peak at place is the O-H stretching vibrations generation of P2, and 2929cm-1
The peak at place is to be vibrated to generate by C-H, 1642cm-1The peak at place is that the C=O stretching vibrations of P2 generate, these are all polysaccharide
Characteristic peak illustrates that P2 belongs to polysaccharose substance.
In the infrared spectrum of P2,1153cm-1The peak at place is the absorption peak of C-O on ring, 1080,1027cm-1Peak be by
Alcoholic extract hydroxyl group becomes what angular oscillation generated, these three peaks illustrate that there are pyranose ring, 862cm in P2-1The peak at place indicates exist in P2
β-type glycosidic bond.
Embodiment 6
Determination oxidative is carried out to the Chinese caterpillar fungus culture medium polysaccharide P2 that embodiment 1 obtains：
ABTS free radical scavenging abilities measure：
7mmol/L ABTS aqueous solutions and each 5mL mixing of 2.45mmol/L persulfate aqueous solutions are taken, dark place reaction is placed in
12h generates ABTS free radicals, and ABTS free-atom aqueous solutions are diluted and its light absorption value under 734nm wavelength conditions is made to be 0.70
±0.02.The Chinese caterpillar fungus culture medium polysaccharide P1 solution of 1mL different quality concentration is uniformly mixed with 2mL ABTS free-atom aqueous solutions,
Its light absorption value at 734nm is surveyed after 10min, is denoted as A1；2mL ABTS free-atom aqueous solutions measure after being mixed with 1mL distilled water
Light absorption value at 734nm, is denoted as A0；2mL distilled water and light absorption value of the 1mL polysaccharide solutions at 734nm are measured, A is denoted as2。
Clearance rate (%)：Y=[1- (A1-A2)/A0] × 100%
Fig. 4 is the ABTS free radical scavenging abilities of Chinese caterpillar fungus culture medium polysaccharide P2.As shown in Figure 4, concentration 1.0~
Within the scope of 5.0mg/ml, P2 has certain removing ABTS free radical abilities, and is proportionate with polysaccharide concentration.Work as polysaccharide concentration
For 5.0mg/ml when, the ABTS free radical scavenging activities of P2 are 38.0%.
Embodiment 7
Anti-trioxypurine research is carried out to the Chinese caterpillar fungus culture medium polysaccharide P2 that embodiment 1 obtains：
(1) foundation of hyperuricemia model
Male mice 60, raising after a week, are randomly divided into 6 groups, every group 10, are followed successively by blank group, model group, polysaccharide
(200mg/kg), low (100mg/kg) dosage groups of P2 and positive drug (50mg/kg) control in P2 high (400mg/kg), P2
Group.Every morning model group, positive controls, polysaccharide P2 senior middle schools low dose group gavage give xanthine 600mg/kg (ig)+abdomen
Chamber injects Oteracil Potassium 100mg/kg (ip).Modeling previous hour is deprived of food but not water, and blank group gives Isodose physiology salt
Water.Afternoon, the high, medium and low dosage groups of polysaccharide P2 used polysaccharide suspension to carry out gastric infusion, and positive controls gavage is given not fast
Then gavage gives the physiological saline of equal volume for alcohol suspension 50mg/kg, model group and blank group, and continuous seven days (2) are raw in vivo
Change the measurement of index
It weighs after administration in 7th day afternoon 1h, broken end takes blood, after being placed at room temperature for 40min, is centrifuged with 3000r/min
10min draws upper serum, using kit detection uric acid in serum (UA), serum creatinine (CREA) and serum urea nitrogen
(BUN) value investigates influence of the polysaccharide to hyperuricemia model mouse UA, CREA and BUN and renal function.
Measure the anti-trioxypurine exercising result such as following table of Chinese caterpillar fungus culture medium polysaccharide P2：
The anti-trioxypurine of 2 Chinese caterpillar fungus culture medium polysaccharide P2 of table acts on
As can be seen from Table 2, ratio, serum creatinine, serum uric acid and the serum urea nitrogen water of model group mouse are organized with normal
It is average significantly to increase, show modeling success.
Compared with model group, the low middle high dose group of polysaccharide P2 can make mice serum creatinine level decline 2.22% respectively,
10.06%, 19.01%, mice serum uric acid level can be made to decline 3.52%, 11.16%, 19.80% respectively, mouse blood can be made
Clear urea nitrogen levels decline 10.22%, 22.70%, 43.30% respectively.Illustrate under the experimental model, Chinese caterpillar fungus culture medium is more
Sugared P2 can reduce the serum creatinine, serum uric acid and serum urea nitrogen level of hyperuricemia.
Embodiment 8
Antibacterial research is carried out to the Chinese caterpillar fungus culture medium polysaccharide P2 that embodiment 1 obtains：
Bacteriostatic activity is measured using punch method to common pathogenic bacteria staphylococcus aureus (Sa), Pseudomonas aeruginosa (Pa).
Primary operational process is as follows：The culture dish for taking a diameter of 90mm, is down flat the bacteria suspension for taking 150 μ l after plate with liquid-transfering gun, and coating is equal
Each tablet makes a call to 4 holes with 8mm card punch after even, is separately added into the spare 1mg/ml Chinese caterpillar fungus culture medium polysaccharide P2 of 40 μ l, compares
40 μ l physiological saline are then added in group per hole.It is placed in culture in 37 DEG C of constant incubators and for 24 hours, distinguishes measurement experiment with crossing method
The diameter of each inhibition zone on tablet and control tablet, and calculate its average value.
The results are shown in Figure 5 for the bacteriostatic experiment of polysaccharide P2, is P2 respectively to the antibacterial result of staphylococcus aureus, golden yellow
Antibacterial result to Pseudomonas aeruginosa of color staphylococcus control group, P2, Pseudomonas aeruginosa control group, measure the diameter such as table 3 of inhibition zone.
3 antibacterial circle diameter of table (cm)
As shown in Table 3, Chinese caterpillar fungus culture medium polysaccharide P2 has staphylococcus aureus, Pseudomonas aeruginosa different degrees of suppression
It makes and uses, wherein the inhibiting effect for comparing Pseudomonas aeruginosa to the inhibiting effect of staphylococcus aureus is stronger.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (10)
1. a kind of Chinese caterpillar fungus culture medium polysaccharide, it is characterised in that be made of the monosaccharide of following molar percentage：0.26% ribose,
0.11% rhamnose, 2.01% arabinose, 0.34% xylose, 29.62% mannose, 67.19% glucose, 0.47% gala
Sugar.
2. Chinese caterpillar fungus culture medium polysaccharide according to claim 1, it is characterised in that：Average molecular weight is 16.6k Da.
3. Chinese caterpillar fungus culture medium polysaccharide according to claim 1, it is characterised in that：Contain pyranose ring and β-type glucosides
Key.
4. the isolation and purification method of claim 1-3 any one of them Chinese caterpillar fungus culture medium polysaccharide, it is characterised in that including with
Lower step：
(1) Chinese caterpillar fungus culture medium polysaccharide is extracted：By Cordyceps militaris rice medium leftover bits and pieces crushed after being dried, be sieved leftover bits and pieces is dry
Powder；Weigh the distilled water that 15-16 times of quality is added in dry powder, ultrasonication 30min or more, the refluxing extraction 1.5- at 70 DEG C
2.0h, extraction merge extracting solution, are concentrated after extracting solution filtering, obtain polysaccharide concentrate several times；It is added in polysaccharide concentrate
95% (V/V) ethyl alcohol of 3-4 times of volume, stands overnight after stirring in 4 DEG C；Precipitation drying is obtained into Cordyceps militaris culture after centrifugation
Quito sugar extract；
(2) decoloration of Chinese caterpillar fungus culture medium polysaccharide：Chinese caterpillar fungus culture medium polyoses extract is added into distillation water dissolution, adjust pH value to
8.0-8.5 H is added dropwise2O2Solution is to colourless, 50-55 DEG C of heat preservation 2h or more；
(3) enzyme process combination Sevage methods take off albumen：
3-1：Papain solution is mixed with Chinese caterpillar fungus culture medium polysaccharide extraction liquid, the volume ratio of the two is 1.0:1.5~
1.0:1.7 60-70 DEG C of enzymolysis 2-3h；
3-2：Be added the Sevage reagents of 1/5 volume in enzymolysis liquid, shaken cultivation 30min or more, then repeatedly centrifugation until
Until no albumen precipitation is precipitated, supernatant, drying is taken to obtain Chinese caterpillar fungus culture medium Thick many candies；
(4) Chinese caterpillar fungus culture medium polysaccharide isolates and purifies：By upper DEAE fine jades after the distillation water dissolution of Chinese caterpillar fungus culture medium Thick many candies
Sepharose FF ion exchange columns, are eluted with 0.1mol/L NaCl solutions, obtain polysaccharide.
5. isolation and purification method according to claim 4, it is characterised in that：Concentration described in step (1) is at 50-55 DEG C
Lower concentration.
6. isolation and purification method according to claim 4, it is characterised in that：H described in step (2)2O2Solution, it is a concentration of
30% (V/V).
7. isolation and purification method according to claim 4, it is characterised in that：Papain solution described in step (3)
It is formulated with the PBS buffer solution of pH value 6.0, wherein a concentration of 250U/ml of papain.
8. isolation and purification method according to claim 4, it is characterised in that：Sevage reagents described in step (3), are chlorine
Imitative and n-butanol by volume 5:1 is formulated.
9. application of the claim 1-3 any one of them Chinese caterpillar fungus culture medium polysaccharide as antioxidant or bacteriostatic agent.
10. claim 1-3 any one of them Chinese caterpillar fungus culture medium polysaccharide has effects that in preparation in the drug of anti-trioxypurine
Using.
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| CN116425901B (en) * | 2023-06-13 | 2023-08-18 | 西南民族大学 | Bitter bamboo shoot polysaccharide and preparation method and application thereof |
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