CN108159283A - The extracting method and purposes of the compound free amino acid of stem of noble dendrobium blade - Google Patents
The extracting method and purposes of the compound free amino acid of stem of noble dendrobium blade Download PDFInfo
- Publication number
- CN108159283A CN108159283A CN201810083227.5A CN201810083227A CN108159283A CN 108159283 A CN108159283 A CN 108159283A CN 201810083227 A CN201810083227 A CN 201810083227A CN 108159283 A CN108159283 A CN 108159283A
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- Prior art keywords
- amino acid
- extraction
- free amino
- acid
- resin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The present invention provides a kind of methods that compound free amino acid is extracted in blade from dendrobium candidum, include the following steps：Free amino acid in preliminary extraction dendrobium candidum blade obtains first extract, reextraction extracts residual ionization amino acid, it removes the impurity such as removing protein, polysaccharide and is cleaned, decolourized with activated carbon adsorption, purify free amino acid extracting solution crude product with acid cation exchange resin, molecular sieve is further purified, crystal refining, wherein obtaining crystal purity is up to more than 99.5%.
Description
Technical field
The present invention relates to a kind of methods that compound free amino acid is extracted in blade from the stem of noble dendrobium, relate more specifically to a kind of high
Compound free amino acid is extracted to yield high-purity from dendrobium candidum blade and its as chelates of composite amino acid or nutrition
The purposes of replenishers belongs to natural product extraction purification art.
Background technology
Amino acid is nutrition composition needed by human, is the base unit for building protein.Except from conventional food obtain with
Outside, it is also obtained frequently as nutritional supplement form for human body.As the nutritional supplement of amino acid composition, plant free amino acid
Compared with protein, free amino acid is more prone to be absorbed and used in vivo；And protein then needs to digest in alimentary canal
Catabolism is into free amino acid.Especially quality ist or meat intake deficiency person, obtain enough essential amino acids
It is very necessary.Compared with artificial synthesized nutriment or dietary supplements, people to be obtained from natural healthy replenishers and
Extraction produce are more keen to.Such as rich in free amino acid raise plant or plant parts such as leaf, flower, seed etc., be considered to
It can be used as the origin of amino acid of food-grade.
In general, protein is made of 20 kinds of standard amino acids, and only half of which or so oneself can be closed in human body
Into, other amino acid that cannot voluntarily synthesize are referred to as essential amino acid, i.e. lysine (Lysine), tryptophan
(Tryptophan), phenylalanine (Phenylalanine), methionine (Methionine), threonine (Threonine),
Isoleucine (Isoleucine), leucine (Leucine), valine (Valine) etc..These must be obtained from diet must
Amino acid is needed, otherwise may result in malnutritive even physical health issues.
The stem of noble dendrobium, a kind of medicinal plant, the Chinese medicine traditional as China usually take stem to give up leaf, and rich in free in leaf
Amino acid, therefore cause a large amount of unnecessary wastes.At present, medicinal dendrobium include the fresh stem of noble dendrobium, HERBA DENDROBII, dendrobium candidum,
Dendrobium loddigesii Rolfe, stem of Eyeshaped Dendrobium etc.；2010 editions pharmacopeia are the new of orchid family Dendrobium Sw dendrobium candidum by the independent list of dendrobium candidum
Fresh or dry stem.Research medicine shows that the stem of noble dendrobium has high medicinal and healthcare function, and effective component includes：Dendrobium polysaccharide,
Stem of noble dendrobium amino acid, alkaloid etc..
In the prior art, in terms of studies of medicinal plants Dendrobium Sw is concentrated mainly on pharmacological activity and the chemical constitution of polysaccharide.It is worth
It is noted that in addition to polysaccharide, another beneficial key component further includes amino acid in the stem of noble dendrobium.Amino acid in the stem of noble dendrobium is mainly with trip
Exist from form, be that content is only second to polysaccharide component, content will be far above other compositions such as alkaloid, dendrophnol, flavones
Class etc..Free amino acid is also one of principle active component in the stem of noble dendrobium.
Research shows that dendrobium candidum contains whole essential amino acids in addition to tryptophan, wherein primary amino acid
It is aspartic acid, glutamic acid, glycine, valine and leucine, this 5 kinds of amino acid account for 53% of total amino acid or so.Wherein,
The content of glutamic acid, aspartic acid and glycine accounts for 36% of total content or so.
For the distribution of amino acid, by taking dendrobium candidum as an example, total amino acid is put down in the blade of 11 strains of dendrobium candidum
Equal mass content is about 7.5%, stem 1.91%, and blade is significantly higher than stem, and the reason that amino acid composition is proposed close to FAO/WHO
Think the standard of protein, be one of the ideal source of current food grade plant free amino acid, there is very big potentiality to be exploited.
There are many extracting method about free amino acid, including ultrasonic extraction, zymohydrolysis extracting method, high-pressure water heating extraction
Method, solvent extraction method, microwave loss mechanisms etc..There are high-pressure water heating extraction, ultrasonic extraction, enzymolysis to carry using more at present
It follows the example of, but only few method extraction of green and economy and the free amine group acid system of purifying plant origin.It carries at present
Take the related art of amino acid for example：
CN105523875B is related to rose waste liquid technical field, is carried in a kind of rose waste liquid after essential oil is extracted
The method for taking rose amino acid；Preprocessed, enzymolysis, except obtaining rose amino acid after matter, purifying, decoloration and drying steps
Crude product.After adding in pectinase enzymatic hydrolysis in extraction process, viscosity is decreased obviously compared with rose waste liquid, production 1kg Rosa Damascanas
10.48kg be can extract in rose waste liquid to 12.78kg rose amino acid, substantially increase the absorption effect of rose amino acid
Rate and yield；Simple for process, safe and non-toxic and rose coarse amino acid purity is high, in obtained rose coarse amino acid
Rich in 16 kinds of natural amino acids and convenient for preserving, realizing turns waste into wealth the rose waste liquid after extraction essential oil, avoids rose
The waste of rare colored amino acid resource, rose coarse amino acid can be used for cosmetics and health products, improve the synthesis of rose
Utility value.
CN106692210A discloses a kind of method for extracting amino acid in cordyceps sinensis, includes the following steps：By cordyceps sinensis
100 mesh are ground into, cordyceps sinensis is put into conical flask, and conical flask is placed in concussion instrument to be extracted 3 times with 35-65 DEG C of water, and each water carried
The mass ratio of journey cordyceps sinensis and water is 13~5；Cordyceps sinensis drying after water carries is put into conical flask, and conical flask is placed in concussion instrument and uses
Solvent extraction 3~8 times, collects supernatant, and the mass ratio of solvent in supernatant, each alcohol extracting process cordyceps sinensis and alcohol is removed in rectifying
It is 11~4；Concentrate is drawn with suction pipe, suction pipe is slowly added into concentrate along chromatography column wall, dense with acetone after sample into after column
The ethyl acetate solution flushing for 1~6% is spent, each layer efflux at chromatographic column bottom is collected respectively, is extracted using this technology mode
Amino acid in cordyceps sinensis has many advantages, such as simple for process, easy realization and good separating effect.
CN104001006B discloses a kind of method that free amino acid is extracted from asparagus fern, and this method includes：By asparagus fern
, dries pulverizing clean with distilled water flushing after medicinal material peeling, filtering weigh Cochinchinese Asparagus Root and are put into micro-wave diminishing pot, by asparagus fern
The ratio 1 of powder and water：15~1：25 addition water are placed on heating extraction in microwave dissolver, filter constant volume after extracting solution, from
The heart takes its supernatant up to free amino acid extracting solution.By the free amino acid extracting solution in 4 DEG C of storages, with asparagine drug
For reference substance, the free amino acid is measured using ninhydrin colorimetry.The invention is using water as Extraction solvent, using microwave
Method extraction wherein free amino acid, the content for measuring amino acid of resolution extraction is about 5.75%, and extraction efficiency is high, simultaneously
With it is simple and easy to do, materials are few, extraction the used time it is short, extraction cost is low, advantages of environment protection, be conducive to industrialized production, have
There are preferable economic benefit, social benefit and ecological benefits.
CN106266032A discloses a kind of extracting method of Nakai leaves amino acid, which is characterized in that includes the following steps：
1) it crushes and impregnates：Nakai leaves are cleaned up, be polished into coarse powder and are impregnated 12-16 hours under clear water room temperature, it is thick to obtain Nakai leaves
Powder soak；2) filtering and concentrating：The pawpaw leaf coarse powder soak obtained in step 1) is subjected to secondary filter processing, takes filtered fluid
It is placed in enrichment facility and is concentrated under reduced pressure；3) alcohol leaching：It is 78- that concentrate in step 2), which adds in mass percent concentration,
80% ethyl alcohol is stirred, and then carries out a cold soaking processing, the cold soaking time is 40-60 minutes, then takes supernatant
It adds the ethyl alcohol that mass percent is 80-85% and carries out secondary cold soaking processing, the cold soaking time is 40-60 minutes；4) it extracts：It will
Alcohol immersion liquid in step 3), which is placed in reflux, first carries out alcohol reflux processing, then carries out boiling water refluxing extraction again；5) it hands over
It changes clothes de-：Reflux extracting liquid in step 4) is placed in storng-acid cation exchange resin and swaps processing and then uses alkaline
Solution is eluted, and the liquid being collected into is the amino acid extracted in Nakai leaves；6) finished product：Extracting solution is put in 60~70 DEG C of baking
It dries to constant weight in case.
Although the prior art provides the extracting method of many amino acid, current most methods are not appropriate for food
Grade purifying amino acid, this is mainly due to excessive or the chemical reagent such as organic solvent, bronsted lowry acids and bases bronsted lowry have been excessively used, high concentration chemistry
The use of reagent sometimes results in amino acid and resolves into amine or amino acid deamination, and then affects purity.
Even in addition, avoid the certain methods using chemical reagent, such as water seaoning, microwave method etc., recovery rate and
Extraction process is still to be improved, such as inevitably to introduce enzymolysis protein matter such as small molecule more for the protease wherein introduced
The impurity such as peptide, and most of amino acid prepared is feed grade.
At present, the amino acid content of each plant and type are different, and the extraction difficulty of different parts also differs, because
This is still without the plant free amino acid extracting method of a universality.Wherein, the food-grade amino acid especially food-grade stem of noble dendrobium
Amino acid extraction process report it is less, more do not develop a suitable industrialized production food-grade/pharmaceutical grade it is high-purity
Spend high yield route.
Therefore, the inexpensive extraction side of the green of the free amino acid based on the stem of noble dendrobium especially dendrobium candidum of high-purity high yield
Method still has demand.
Invention content
As described above, to overcome the free amino acid extracting method of dendrobium candidum in the prior art insufficient, the present invention is directed to
A kind of high-purity high yield dendrobium candidum free amino acid extraction route of food/pharmaceutical grade of suitable industrialized production is provided.
In addition, extracting method of the present invention is suitble to low cost production, product can both chelate micro- using conventional equipment and means
Secondary element prepares amino-acid chelate, can also be used as nutriment addO-on therapy, and purposes is very extensive.
Specifically, the invention mainly relates to following aspects.
The first aspect, the present invention provides a kind of method that compound free amino acid is extracted in blade from dendrobium candidum,
The extracting method includes the following steps：
A. it just carries：The free amino acid in dendrobium candidum blade is tentatively extracted using ultrasound-leaching method in deionized water,
Filtering obtains first extract；
B. second extraction：Filter residue is dissolved, reextraction is carried out using two phase aqueous extraction system combination ultrasonic method, extraction is remaining
Free amino acid obtains secondary first extract；
C. it cleans：Merge first extract and secondary raffinate, be concentrated in vacuo, further extracted in two phase aqueous extraction system pure
To change, split-phase, filtering, filtrate adds in purified reagent and stands overnight, and removes the impurity such as removing protein, polysaccharide, and cleaned with activated carbon adsorption,
Decoloration, so as to obtain dendrobium candidum free amino acid extracting solution crude product；
D. preliminary purification：Purify free amino acid extracting solution with acid cation exchange resin, further remove low molecule
The impurity such as oligosaccharide, the polysaccharide of amount collect each amino acid composition eluent, deamination concentration；
E. it is secondarily purified：The compound amino acid eluent of acquisition is further purified with absorption resin molecular sieve analog, removes stone
The low molecular weight impurities such as dry measure used in former times phenol, it is dry, obtain free amine group acid crude；
Wherein, the absorption resin molecular sieve analog is polystyrene based polymers resin, it is preferable that is handed over for styryl
Union II ethylene benzene copolymer resin；
F. crystal refining：Ethyl alcohol dissolves, and vacuum evaporating crystalization obtains compounded amino acid crystal crude product；
G. recrystallize 1-2 times, obtain compound free amine group acid treating crystal, wherein the crystal purity 99.5% with
On.
Wherein, in extracting method of the present invention, the dendrobium candidum physiological age is raw for 2-3, preferably 3 years
Raw, the stem of noble dendrobium age raw 1-3 is longer, and free aminoacid content is higher.
In extracting method of the present invention, step (a) is specific as follows：
The dendrobium candidum blade powder to finish and after drying crushing is crossed into 100-120 mesh sieve, 10-40 times of quality of addition
Distilled water is placed in flash extracter, under room temperature in the case where flash extracter rotating speed of flail is 15000-20000r/min just
Step extraction 30-90s, extracts 20-60min, after extraction, by leaching liquor while hot in water bath chader at 50-60 DEG C
Decompression filters, and collects filtrate and is cooled to room temperature, as first extract, filter residue is spare；
Wherein, the extraction time is preferably 30-60min；
Wherein, it is preferably 40-60s that the sudden strain of a muscle, which carries the time,.
In extracting method of the present invention, step (b) is specific as follows：
(1) aqueous two-phase extraction liquid is prepared：PEG4000-8000 polymer is added to the compound nothing of dipotassium hydrogen phosphate-sodium sulphate
In the aqueous solution of machine salt, it is configured to the extraction that polyethylene glycol mass fraction is 5-8%, complex inorganic salt catalyst mass fraction is 8-12%
Liquid；
(2) second extraction：Filter residue after just carrying adds in the extract liquor of 20-30 times of quality, and tune pH value is 6.8-7, is stirred
Uniformly, first it is placed in ultrasonic extraction 3-5 times at 40-50 DEG C in ultrasonic wave countercurrent extractor, each ultrasonic extraction time 5-10s,
Ultrasonic extraction interval 30-60s, ultrasonic power are set as 300-400W；Then uniform stirring 5-10min, after extraction, while hot
Decompression filters, and collects filtrate and is cooled to room temperature, and stands 1-3h split-phases, and free amino acid is extracted to upper strata aqueous phase, collects double
The upper strata extracting solution of aqueous phase system, as secondary raffinate, merges with first extract, is named as extracting solution 1；
Wherein, complex inorganic salt catalyst dipotassium hydrogen phosphate in the extract liquor：The mass ratio of sodium sulphate is 1:0.5-1；
Wherein it is preferred to the standing split-phase time is 2-3h.
In extracting method of the present invention, step (c) is specific as follows：
(1) secondary aqueous two-phase extraction liquid is prepared：PEG-6000 is added to the water-soluble of sodium chloride-sodium sulphate complex inorganic salt catalyst
In liquid, it is configured to the extraction fluid that polyethylene glycol mass fraction is 2-3%, complex inorganic salt catalyst mass fraction is 3-5%；
Extracting solution 1 after merging is concentrated into 1/3rd of original volume to a quarter, adds in the upper of 5-10 times of quality
State extraction fluid, it is 7 to adjust pH value, and it is simultaneously cold to collect filtrate for the uniform stirring 5-15min at 25-30 DEG C of temperature, micro-filtrate membrane filtration
But to room temperature, split-phase 1-3h is stood, gives up the PEG layers containing protein, collects the water layer extracting solution containing amino acid, be named as
Extracting solution 2；
(4) filtering and impurity removing：Extracting solution 2 is concentrated in vacuo to 1/10th volumes, carries out secondary filtration：First use 0.1-0.2
The micro-filtrate membrane filtration removal of impurities of micron, then with big molecular impurities such as ultrafiltration membrane Polysaccharide removing, albumen；
(5) it except big molecular impurity and decolourizes：Filtrate after filtering is added in into the purified reagent solution of 1-1.5 times of volume into one
Step removal residual protein and water-soluble polysaccharide, 4 DEG C stand overnight after 15-20min centrifuged with 8000-9000r/min, take supernatant
Liquid, by 10ml：The ratio of 1g adds in activated carbon stirring 15-20min adsorption-edulcorations and decolourizes, and micro-filtrate membrane filtration is to get free ammonia
Base acid extracting solution, concentrates filtrate to original volume 1/10th to ten/5th；
Wherein, complex inorganic salt catalyst sodium sulphate in the extraction system：The mass ratio of sodium chloride is 1:0.2-0.5；
Wherein, the purified reagent solution is 5-6% sulfosalisylics acid solution or absolute ethyl alcohol, it is preferable that is selected anhydrous
Ethyl alcohol.
In extracting method of the present invention, step (d) is specific as follows：
(1) the free amino acid concentrate of above-mentioned acquisition is adjusted into pH to 2.0-3.0, using flow velocity as 2-4mL/min loadings
In the styryl acid cation exchange resin crossed with HCl treatment, stopping 15-30min makes amino acid and resin-bonded, first
Exchange column is washed with the speed of 3-5ml/min with distilled water and removes impurity, up to no ultraviolet detection signal, wherein electronegative residual
Remaining polysaccharide component does not rinse by absorption；
(2) and then with the ammonium hydroxide of 1-2mol/L exchange column is eluted with the speed of 3-5ml/min, amino acid ultraviolet detection occurs
Signal starts to collect eluent, obtains compound amino acid eluent, and gained eluent is handled through Rotary Evaporators concentration deamination,
1st/10th to ten/5th of original volume is concentrated into, wherein compound amino acid purity detecting is more than 96%.
In extracting method of the present invention, step (e) is specific as follows：
Make the polyphenyl second that the amino acid concentrate that step obtains is 80-100 microns by the grain size that deionized water is impregnated
Then alkenyl polymer resin elutes resin column, the low molecular weight impurities example of doping with deionized water with the speed of 5-8ml/min
If stem of noble dendrobium phenols is adsorbed so as to remove, according to amino acid ultraviolet detection signal collection amino acid eluent, vacuum at 50-60 DEG C
It is dried to obtain compounded amino acid crude of the content more than 98%；
Wherein, polystyrene based polymers resin optimization styrene base crosslinking divinylbenzene copolymer resin.
Wherein, after the Contents of Amino Acids uses automatic amino acid analyzer with ion-exchange chromatography-ninhydrin column
Derivatization method, so as to the content of amino acid in determination sample.
In extracting method of the present invention, step (f)-(g) is specific as follows：
Crystallization：The 50-70% ethyl alcohol of 5-6 times of quality of the coarse amino acid that above-mentioned steps obtain is dissolved, is first heated dense
1/10th of original volume are reduced to, then using vacuum evaporating crystalization, obtains crystal of the purity more than 99%；
Recrystallization：Crystal crude product with 50-60% ethyl alcohol is dissolved again, one is recrystallized in vacuum crystallizer to twice, is obtained
To refined compounded amino acid crystal, purity is more than 99.5%.
Wherein, in terms of dendrobium candidum blade powder dry weight, the recovery rate of compound free amino acid is 50-60mg/g, average
Purity is more than 99.5%.
In the present invention, the vacuum concentration or the temperature of heating concentration are preferably 65 DEG C hereinafter, to ensure in concentrate
Amino acid physical property stablize.
The second aspect of the invention is provided through dendrobium candidum free amine group acid product made from said extracted method.
The third aspect of the invention provides the purposes of above-mentioned free amine group acid product, can be used as protein or ammonia
Base acid nutritional supplement can be used for chelated microelement so as to obtain amino acid derivativges, such as the compound ammonia of dendrobium candidum
The one kind therein such as base acid chelate, including but not limited to compound amino acid chelate calcium, compositing acid iron, compound amino acid zinc
Or several combination.
Beneficial effects of the present invention further include but are not limited to the following aspects：
1. it develops extraction purification in a kind of slave dendrobium candidum blade of new high yield high-purity (food/pharmaceutical grade) to swim
Cost effective method from natural biological amino acid, purity is up to more than 99.5%.
2. it for dendrobium candidum blade part bit optimization extraction process and parameter, and extracts and is set in route without costliness
Standby, sudden strain of a muscle, which is carried, to be combined with aqueous two-phase extraction so that extraction efficiency is high, and method is simple, and agents useful for same is at low cost.
3. the compound free amino acid of gained is practically free of biological phenol impurity similar in size property, also without rich in soil
The heavy metal classes element of collection.
4. most of solvent is recyclable, extraction can be industrialized on a large scale.
5. the feed grade compound amino acid of middle extraction compared with prior art, natural free amino acid that the present invention purifies and its
Derived product is the essential amino acid nutritional supplement that the mankind is suitble to eat.
In conclusion extracting method of the present invention is simple, and using a large amount of dendrobium candidum blades usually given up as raw material, raw material
It is at low cost, it is suitble to large-scale production, and food/pharmaceutical grade purity can be reached, has a good application prospect and market value.
Specific embodiment
Below by specific preparation example and embodiment, the present invention is described in detail, but these exemplary embodiments
Purposes and purpose be only used for enumerating the present invention, any type of any limit not is formed to the real protection scope of the present invention
It is fixed, it is more non-that protection scope of the present invention is confined to this.
Preparation example 1：Free amino acid in preliminary extraction dendrobium candidum blade
100 grams of the dendrobium candidum blade powder to finish and after drying crushing is sieved with 100 mesh sieve, adds in 3000ml distillations
Water is placed in flash extracter, tentatively extracts 50s in the case where flash extracter rotating speed of flail is 18000r/min under room temperature,
40min is extracted at 50 DEG C in water bath chader, after extraction, leaching liquor is depressurized to suction filtration while hot, collects filtrate simultaneously
It is cooled to room temperature, as first extract, filter residue is spare.
Preparation example 2：Second extraction
(1) aqueous two-phase extraction liquid is prepared：PEG4000 polymer is added to dipotassium hydrogen phosphate-sodium sulphate complex inorganic salt catalyst
Aqueous solution in, be configured to the extract liquor that polyethylene glycol mass fraction is 8%, complex inorganic salt catalyst mass fraction is 10%；Wherein,
Complex inorganic salt catalyst dipotassium hydrogen phosphate in the extract liquor：The mass ratio of sodium sulphate is 1:0.5；
(2) second extraction：Filter residue after just carrying adds in the extract liquor of 20-30 times of quality, and it is 6.8 to adjust pH value, and stirring is equal
It is even, ultrasonic extraction 3 times at 40 DEG C are first placed in ultrasonic wave countercurrent extractor, each ultrasonic extraction time 5s, between ultrasonic extraction
Every 30s, ultrasonic power is set as 300W；Then uniform stirring 5min, after extraction, decompression while hot filters, and collects filtrate simultaneously
It is cooled to room temperature, stands 3h split-phases, free amino acid is extracted to upper strata aqueous phase, collects the upper strata extraction of double-aqueous phase system
Liquid as secondary raffinate, merges with first extract, is named as extracting solution 1.
Preparation example 3：Removal of impurities
(1) PEG-6000 is added in the aqueous solution of sodium chloride-sodium sulphate complex inorganic salt catalyst, is configured to polyethylene glycol matter
Amount score is 3%, the extraction fluid that complex inorganic salt catalyst mass fraction is 5%；Wherein, sodium sulphate：The mass ratio of sodium chloride is
1:0.2；
Extracting solution 1 after merging is concentrated into a quarter of original volume, adds in the above-mentioned extract liquor of 5 times of quality, adjusts pH
It is 7 to be worth, the uniform stirring 5min at 30 DEG C of temperature, micro-filtrate membrane filtration, collects filtrate and is simultaneously cooled to room temperature, and stands split-phase 3h, house
The PEG layers containing protein are abandoned, the water layer extracting solution containing amino acid is collected, is named as extracting solution 2；
(2) extracting solution 2 is concentrated in vacuo to 1/10th volumes, carries out secondary filtration：First use 0.1-0.2 microns of micro-filtration
Membrane filtration cleans, then with big molecular impurities such as ultrafiltration membrane Polysaccharide removing, albumen；By filtrate 1.5 times of volumes of addition after filtering
Absolute ethyl alcohol, 4 DEG C stand overnight after with 8000r/min centrifuge 15min, supernatant is taken, by 10ml：The ratio of 1g adds in activated carbon
Stirring 15min adsorption-edulcorations simultaneously decolourize, and micro-filtrate membrane filtration concentrates filtrate to substance to get free amino acid extracting solution
Product 1/10th.
Preparation example 4：Cation exchange resin purifies
(1) the free amino acid concentrate of above-mentioned acquisition is adjusted into pH to 2.0, using flow velocity as 3mL/min loading hydrochloric acid
In processed styryl acid cation exchange resin, stop 20min make amino acid and resin-bonded, first with distilled water with
The speed washing exchange column of 3ml/min removes impurity, up to no ultraviolet detection signal, wherein electronegative remnants polysaccharide components are not
It rinses by absorption；
(2) and then with the ammonium hydroxide of 1mol/L exchange column is eluted with the speed of 5ml/min, amino acid ultraviolet detection signal occurs
Start to collect eluent, obtain compound amino acid eluent, gained eluent is handled through Rotary Evaporators concentration deamination, concentration
To 1st/10th to ten/5th of original volume, wherein compound amino acid purity detecting is 96.5%.
Preparation example 5：Molecular sieve is further purified
Make the styryl that the amino acid concentrate that preparation example 4 obtains is 80 microns by the grain size that deionized water is impregnated
Divinylbenzene copolymer resin is crosslinked, resin column is then eluted with the speed of 5ml/min with deionized water, it is ultraviolet according to amino acid
Signal collection amino acid eluent is detected, is dried in vacuo at 60 DEG C and obtains compounded amino acid crude of the purity 98.5%.
Preparation example 6：Crystallization and recrystallizing and refining
Crystallization：50% ethyl alcohol of 5 times of quality of the coarse amino acid that above-mentioned steps obtain is dissolved, first heating is concentrated to original
/ 10th of volume, then using vacuum evaporating crystalization, obtain crystal of the purity 99%；
Recrystallization：Crystal crude product with 60% ethyl alcohol is dissolved again, recrystallizes twice, obtains refined in vacuum crystallizer
5.8 grams of compounded amino acid crystal, purity is 99.8%.
Wherein, each key component and content such as following table are measured：
Table 1
As seen from the above table, containing most essential amino acid type in dendrobium candidum blade, wherein main ammonia
Base acid is aspartic acid, glutamic acid, glycine, valine and leucine, this 5 kinds of amino acid account for the 52.5% of total amino acid.
Preparation example 7：The chelated calcium preparation of the compound free amino acid of dendrobium candidum blade
It is raw material to choose amino acid crystals made from preparation example 6 and food-level calcium acetate, to above-mentioned raw materials by weight 300
Gram:100 grams of progress dispensings in the mixer of mixed raw material input food, will add in the stirring of 3.5L distilled water, must be suspended
Liquid adds the citric acid of weight ratio 1%, by homogenizer homogeneous, suspension is heated to 60 degrees Celsius and introduces colloid
Mill makes in mixed liquor solid particle degree below 50 microns.Gained mixed liquor is sprayed into high-pressure fluid nanometer mill chelate
To clear compound amino acid chelate calcium solution.Acquired solution is concentrated, is crystallized, dry and obtains finished product after being sieved.Yield
95.6%, purity 98.7%.
It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to limitation protection model of the invention
It encloses.In addition, it should also be understood that, after reading the technical contents of the present invention, those skilled in the art can make the present invention each
Kind change, modification and/or variation, all these equivalent forms equally fall within the guarantor that the application the appended claims are limited
Within the scope of shield.
Claims (10)
1. extracting the method for compound free amino acid in a kind of blade from dendrobium candidum, the extracting method includes the following steps：
A. it just carries：The free amino acid in dendrobium candidum blade, mistake are tentatively extracted using ultrasound-leaching method in deionized water
Filter obtains first extract；
B. second extraction：Filter residue is dissolved, reextraction is carried out using two phase aqueous extraction system combination ultrasonic method, extracts residual ionization
Amino acid obtains secondary first extract；
C. it cleans：Merge first extract and secondary raffinate, be concentrated in vacuo, the further abstraction purification in two phase aqueous extraction system,
Split-phase, filtering, filtrate add in purified reagent and stand overnight, and remove the impurity such as removing protein, polysaccharide, and cleaned, taken off with activated carbon adsorption
Color, so as to obtain dendrobium candidum free amino acid extracting solution crude product；
D. preliminary purification：Purify free amino acid extracting solution with acid cation exchange resin, further remove low molecular weight
The impurity such as oligosaccharide, polysaccharide collect each amino acid composition eluent, deamination concentration；
E. it is secondarily purified：The compound amino acid eluent of acquisition is further purified with absorption resin molecular sieve analog, removes dendrophnol
Etc. low molecular weight impurities, it is dry, obtain free amine group acid crude；Wherein, the absorption resin molecular sieve analog is polystyrene
Based polyalcohol resin, it is preferable that be crosslinked divinylbenzene copolymer resin for styryl；
F. crystal refining：Ethyl alcohol dissolves, and vacuum evaporating crystalization obtains compounded amino acid crystal crude product；
G. recrystallize 1-2 time, obtain described in compound free amine group acid treating crystal, wherein the crystal purity 99.5% with
On.
2. preparation method as described in claim 1, it is characterised in that：The step (a) is specific as follows：
The dendrobium candidum blade powder to finish and after drying crushing is crossed into 100-120 mesh sieve, adds in the distillation of 10-40 times of quality
Water is placed in flash extracter, is tentatively carried in the case where flash extracter rotating speed of flail is 15000-20000r/min under room temperature
30-90s is taken, 20-60min is extracted at 50-60 DEG C in water bath chader, after extraction, leaching liquor is depressurized while hot
It filters, collect filtrate and is cooled to room temperature, as first extract, filter residue is spare.
3. preparation method as described in claim 1, it is characterised in that：The step (b) is specific as follows：
(1) aqueous two-phase extraction liquid is prepared：PEG4000-8000 polymer is added to dipotassium hydrogen phosphate-sodium sulphate complex inorganic salt catalyst
Aqueous solution in, be configured to the extract liquor that polyethylene glycol mass fraction is 5-8%, complex inorganic salt catalyst mass fraction is 8-12%；
(2) second extraction：Filter residue after just carrying adds in the extract liquor of 20-30 times of quality, and tune pH value is 6.8-7, is stirred evenly,
Ultrasonic extraction 3-5 times at 40-50 DEG C, each ultrasonic extraction time 5-10s are first placed in ultrasonic wave countercurrent extractor, and ultrasound carries
Interval 30-60s is taken, ultrasonic power is set as 300-400W；Then uniform stirring 5-10min, after extraction, decompression while hot is taken out
Filter is collected filtrate and is cooled to room temperature, and stands 1-3h split-phases, and free amino acid is extracted to upper strata aqueous phase, collects aqueous two-phase body
The upper strata extracting solution of system, as secondary raffinate, merges with first extract, is named as extracting solution 1；Wherein, in the extract liquor
Complex inorganic salt catalyst dipotassium hydrogen phosphate：The mass ratio of sodium sulphate is 1:0.5-1；
Preferably, the standing split-phase time is 2-3h.
4. preparation method as described in claim 1, it is characterised in that：The step (c) is specific as follows：
(1) secondary aqueous two-phase extraction liquid is prepared：PEG-6000 is added to the aqueous solution of sodium chloride-sodium sulphate complex inorganic salt catalyst
In, it is configured to the extraction fluid that polyethylene glycol mass fraction is 2-3%, complex inorganic salt catalyst mass fraction is 3-5%；
Extracting solution 1 after merging is concentrated into 1/3rd of original volume to a quarter, adds in above-mentioned the two of 5-10 times of quality
Secondary extract liquor, it is 7 to adjust pH value, the uniform stirring 5-15min at 25-30 DEG C of temperature, micro-filtrate membrane filtration, collects filtrate and is simultaneously cooled to
Room temperature stands split-phase 1-3h, gives up the PEG layers containing protein, collect the water layer extracting solution containing amino acid, be named as extraction
Liquid 2；
(2) filtering and impurity removing：Extracting solution 2 is concentrated in vacuo to 1/10th volumes, carries out secondary filtration：First use 0.1-0.2 microns
Micro-filtrate membrane filtration removal of impurities, then with ultrafiltration membrane removal par-tial polysaccharide, the big molecular impurities such as albumen；
(3) it except big molecular impurity and decolourizes：The purified reagent solution that filtrate after filtering is added in 1-1.5 times of volume is further gone
Except residual protein and water-soluble polysaccharide, 4 DEG C stand overnight after with 8000-9000r/min centrifuge 15-20min, take supernatant, press
10ml：The ratio of 1g adds in activated carbon stirring 15-20min adsorption-edulcorations and decolourizes, and micro-filtrate membrane filtration carries to get free amino acid
Liquid is taken, concentrates filtrate to original volume 1/10th to ten/5th；
Wherein, complex inorganic salt catalyst sodium sulphate in the extraction system：The mass ratio of sodium chloride is 1:0.2-0.5；
Wherein, the purified reagent solution is 5-6% sulfosalisylics acid solution or absolute ethyl alcohol；Preferably, absolute ethyl alcohol is selected.
5. preparation method as described in claim 1, it is characterised in that：The step (d) is specific as follows：
(1) by the free amino acid concentrate of acquisition adjust pH to 2.0-3.0, using flow velocity as 2-4mL/min loading hydrochloric acid at
In the styryl acid cation exchange resin managed, stopping 15-30min makes amino acid and resin-bonded, first uses distilled water
Impurity is removed with the speed washing exchange column of 3-5ml/min, up to no ultraviolet detection signal, wherein electronegative remnants polysaccharide groups
Divide and do not rinse by absorption；
(2) and then with the ammonium hydroxide of 1-2mol/L exchange column is eluted with the speed of 3-5ml/min, amino acid ultraviolet detection signal occurs
Start to collect eluent, obtain compound amino acid eluent, gained eluent is handled through Rotary Evaporators concentration deamination, concentration
To 1st/10th to ten/5th of original volume.
6. preparation method as described in claim 1, it is characterised in that：The step (e) is specific as follows：
Make the grain size that the amino acid concentrate that step obtains is impregnated by deionized water polystyrene-based for 80-100 microns
Then fluoropolymer resin elutes resin column with deionized water with the speed of 5-8ml/min, the low molecular weight impurities of doping are adsorbed
So as to remove, according to amino acid ultraviolet detection signal collection amino acid eluent, it is dried in vacuo at 50-60 DEG C, obtains purity and exist
More than 98% compounded amino acid crude；Wherein, polystyrene based polymers resin optimization styrene base crosslinking diethyl
Alkene benzene copolymer resin.
7. preparation method as described in claim 1, it is characterised in that：Step (f)-(g) is specific as follows：
The 50-70% ethyl alcohol of 5-6 times of quality of the coarse amino acid that above-mentioned steps obtain is dissolved, heating is concentrated to original volume
1/10th, using vacuum evaporating crystalization, obtain crystal of the purity more than 99%；By crystal crude product with 50-60% ethyl alcohol again
It dissolves, recrystallization one is to refined compounded amino acid crystal twice, is obtained in vacuum crystallizer, and purity is more than 99.5%.
8. according to the compound free amine group acid product of dendrobium candidum that any one of claim 1-7 the methods obtain.
9. food, nutritional supplement or pharmaceutical composition containing free amine group acid product compound described in claim 8.
10. the chelates of composite amino acid that the compound free amino acid as described in claim 8 is prepared, which is characterized in that by
The compound free amino acid of the dendrobium candidum trace element chelated is formed with other.
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Cited By (3)
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| CN109403026A (en) * | 2018-10-31 | 2019-03-01 | 嘉兴珠韵服装有限公司 | A kind of preparation method of dyeing and finishing technology pretreating reagent |
| CN111876451A (en) * | 2020-08-10 | 2020-11-03 | 武汉轻工大学 | Method for extracting amino acid from chicken manure |
| CN115088847A (en) * | 2022-07-13 | 2022-09-23 | 呼伦贝尔市林海森林经营管理有限公司 | Method for extracting multiple amino acids from birch juice |
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| CN104761426A (en) * | 2015-03-31 | 2015-07-08 | 川渝中烟工业有限责任公司 | Method for extracting free amino acids from tobacco flower buds |
| CN106266032A (en) * | 2016-08-28 | 2017-01-04 | 安徽旺润生物科技有限公司 | A kind of amino acid whose extracting method of leaf of Fructus Chaenomelis |
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| CN109403026A (en) * | 2018-10-31 | 2019-03-01 | 嘉兴珠韵服装有限公司 | A kind of preparation method of dyeing and finishing technology pretreating reagent |
| CN111876451A (en) * | 2020-08-10 | 2020-11-03 | 武汉轻工大学 | Method for extracting amino acid from chicken manure |
| CN115088847A (en) * | 2022-07-13 | 2022-09-23 | 呼伦贝尔市林海森林经营管理有限公司 | Method for extracting multiple amino acids from birch juice |
| CN115088847B (en) * | 2022-07-13 | 2023-12-22 | 呼伦贝尔市林海森林经营管理有限公司 | Method for extracting multiple amino acids from birch juice |
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