CN108159283B - Extraction method and application of compound free amino acids of dendrobe leaves - Google Patents
Extraction method and application of compound free amino acids of dendrobe leaves Download PDFInfo
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- CN108159283B CN108159283B CN201810083227.5A CN201810083227A CN108159283B CN 108159283 B CN108159283 B CN 108159283B CN 201810083227 A CN201810083227 A CN 201810083227A CN 108159283 B CN108159283 B CN 108159283B
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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Abstract
The invention provides a method for extracting compound free amino acid from dendrobium officinale leaves, which comprises the following steps: primarily extracting free amino acid in the dendrobium officinale leaves to obtain primary extract, secondarily extracting and extracting residual free amino acid, removing impurities such as protein, polysaccharide and the like, removing impurities and decoloring by using activated carbon adsorption, purifying a crude product of the free amino acid extract by using acidic cation exchange resin, further purifying by using a molecular sieve, and refining by crystallization, wherein the purity of the obtained crystal is up to more than 99.5 percent.
Description
Technical Field
The invention relates to a method for extracting compound free amino acid from dendrobium officinale leaves, in particular to a method for extracting compound free amino acid from dendrobium officinale leaves with high yield and high purity and application of the compound free amino acid as a compound amino acid chelate or a nutritional supplement, belonging to the field of extraction and purification of natural products.
Background
Amino acids are essential nutrients for the human body and are the basic units for the construction of proteins. In addition to being obtained from daily food, it is also commonly obtained as a nutritional supplement for the human body. As a nutritional supplement of amino acid components, free amino acids of plants are more easily absorbed and utilized in the body than proteins; proteins are required to be enzymatically catabolized to free amino acids in the digestive tract. Especially those with poor quality or insufficient meat intake, it is necessary to obtain enough essential amino acids. Compared to synthetic nutritional or dietary supplements, people are keen on health supplements and extract products obtained from natural sources. For example, plants or plant parts such as leaves, flowers, seeds, etc., which are rich in free amino acids are considered useful as food grade sources of amino acids.
Generally, proteins are composed of 20 kinds of standard amino acids, and only about half of them are synthesized in the human body by itself, and other amino acids that cannot be synthesized by itself are called essential amino acids, i.e., Lysine (Lysine), Tryptophan (Tryptophan), Phenylalanine (Phenylalanine), Methionine (Methionine), Threonine (Threonine), Isoleucine (Isoleucine), Leucine (Leucine), Valine (Valine), and the like. These essential amino acids must be obtained from the diet otherwise malnutrition and even physical health problems may result.
Dendrobe, a medicinal plant, is a traditional Chinese medicinal material in China, and is usually prepared by cutting leaves from stems, while the leaves are rich in free amino acids, so that a great amount of unnecessary waste is caused. At present, the medicinal dendrobium comprises fresh dendrobium, dendrobium nobile, dendrobium candidum, dendrobium loddigesii, dendrobium candidum, dendrobium fimbriatum and the like; the 2010 version of pharmacopeia lists Dendrobium officinale as fresh or dry stem of Dendrobium officinale belonging to Dendrobium of Orchidaceae. Research medicines show that the dendrobium has extremely high medicinal and health-care functions, and the medicinal components of the dendrobium comprise: dendrobe polysaccharide, dendrobe amino acid, alkaloid and the like.
In the prior art, the research on medicinal dendrobium is mainly focused on the pharmacological activity and chemical structure of polysaccharide. It is noted that another beneficial major component in dendrobium nobile contains amino acids in addition to polysaccharides. The amino acids in herba Dendrobii mainly exist in free form, are second to polysaccharide component, and are much higher than other components such as alkaloid, dendrophenol, and flavonoids. Free amino acid is also one of the main effective components in dendrobium.
Researches show that the dendrobium officinale contains all essential amino acids of human bodies except tryptophan, wherein the main amino acids are aspartic acid, glutamic acid, glycine, valine and leucine, and the 5 amino acids account for about 53 percent of the total amino acids. Wherein, the content of glutamic acid, aspartic acid and glycine accounts for about 36 percent of the total content.
From the distribution of amino acids, taking the dendrobium officinale as an example, the average mass content of the total amino acids in the leaves of 11 lines of the dendrobium officinale is about 7.5%, the stem is 1.91%, the leaves are obviously higher than the stem, and the composition of the amino acids is close to the standard of ideal protein proposed by FAO/WHO, so that the dendrobium officinale leaf protein is one of the ideal sources of free amino acids of edible plants at present and has very large development potential.
There are many methods for extracting free amino acids, including ultrasonic extraction, enzymatic extraction, high-pressure hot water extraction, solvent extraction, microwave extraction, and the like. At present, high-pressure hot water extraction, ultrasonic extraction, enzymolysis extraction and the like are more applied, but few green and economic methods are available for extracting and purifying free amino acid from plants. The related prior art for extracting amino acids at present is for example:
CN105523875B relates to the technical field of rose waste liquid, in particular to a method for extracting rose amino acid from rose waste liquid after extracting essential oil; obtaining the rose amino acid crude product after the steps of pretreatment, enzymolysis, quality removal, purification, decoloration and drying. After pectinase is added in the extraction process for enzymolysis, the viscosity is obviously reduced compared with the rose waste liquid, 10.48kg to 12.78kg of rose amino acid can be extracted from the rose waste liquid for producing 1kg of rose essential oil, and the adsorption efficiency and yield of the rose amino acid are greatly improved; the process is simple, safe and nontoxic, the purity of the rose amino acid crude product is high, the obtained rose amino acid crude product is rich in 16 natural amino acids and is convenient to store, the waste liquid of rose after extracting essential oil is changed into valuable, the waste of rose amino acid resources is avoided, the rose amino acid crude product can be used for cosmetics and health care products, and the comprehensive utilization value of rose is improved.
CN106692210A discloses a method for extracting amino acids in cordyceps sinensis, which comprises the following steps: crushing the cordyceps sinensis into 100 meshes, putting the cordyceps sinensis into a conical flask, putting the conical flask into a shaking instrument, and extracting the cordyceps sinensis with water at 35-65 ℃ for 3 times, wherein the mass ratio of the cordyceps sinensis to the water in each water extraction process is 13-5; drying the cordyceps sinensis subjected to water extraction, putting the cordyceps sinensis into a conical flask, putting the conical flask into a shaking instrument, extracting the cordyceps sinensis with a solvent for 3-8 times, collecting supernatant, rectifying to remove the solvent in the supernatant, wherein the mass ratio of the cordyceps sinensis to the alcohol in each alcohol extraction process is 11-4; and (2) sucking the concentrated solution by using a suction pipe, slowly adding the concentrated solution into the suction pipe along the inner wall of the chromatographic column, flushing the sample after the sample enters the chromatographic column by using an ethyl acetate solution with the acetone concentration of 1-6%, and respectively collecting each layer of effluent liquid at the bottom of the chromatographic column.
CN104001006B discloses a method for extracting free amino acids from asparagus, which comprises: peeling radix asparagi, washing with distilled water, oven drying, pulverizing, filtering, weighing radix asparagi powder, placing into a microwave digestion tank, and mixing radix asparagi powder and water at a ratio of 1: 15-1: 25 adding water, heating and extracting in a microwave digestion instrument, filtering the extracting solution, fixing the volume, centrifuging and taking supernatant to obtain the free amino acid extracting solution. Storing the free amino acid extractive solution at 4 deg.C, and determining the free amino acid by ninhydrin colorimetry with asparagine as reference substance. The method takes water as an extraction solvent, adopts a microwave digestion extraction method to extract free amino acid, measures that the content of the amino acid is about 5.75 percent, has high extraction efficiency, has the advantages of simplicity, convenience, practicability, less material consumption, short extraction time, low extraction cost, environmental friendliness and the like, is beneficial to industrial production, and has better economic benefit, social benefit and ecological benefit.
CN106266032A discloses a papaya leaf amino acid extraction method, which is characterized by comprising the following steps: 1) crushing and soaking: cleaning papaya leaves, grinding into coarse powder, and soaking in clear water at normal temperature for 12-16 hours to obtain papaya leaf coarse powder soaking solution; 2) and (3) filtering and concentrating: carrying out secondary filtration treatment on the pawpaw leaf coarse powder soaking solution obtained in the step 1), and placing the filtrate in a concentration device for reduced pressure concentration; 3) alcohol leaching: adding 78-80% ethanol by mass into the concentrated solution in the step 2), stirring, performing primary cold soaking for 40-60 minutes, taking the supernatant, adding 80-85% ethanol by mass, and performing secondary cold soaking for 40-60 minutes; 4) extraction: placing the alcohol extract obtained in the step 3) into a reflux device, firstly carrying out ethanol reflux treatment, and then carrying out boiling water reflux extraction; 5) exchange elution: placing the reflux extracting solution in the step 4) in strong acid cation exchange resin for exchange treatment, and then eluting with alkaline solution, wherein the collected liquid is amino acid extracted from the pawpaw leaves; 6) and (3) finished product: and (5) drying the extracting solution in a drying oven at the temperature of 60-70 ℃ to constant weight.
Although the prior art provides a plurality of methods for extracting amino acids, most of the current methods are not suitable for food-grade purification of amino acids, mainly because chemical reagents such as organic solvents, acids and bases are excessively used or used in excess, and the use of high-concentration chemical reagents sometimes causes the decomposition of amino acids into amines or deamination of amino acids, thereby influencing the purity.
In addition, even in some methods that avoid the use of chemical reagents, such as water immersion, microwave method, etc., the extraction rate and extraction process thereof are still to be improved, for example, in which the introduced protease inevitably introduces impurities that decompose proteins such as small-molecule polypeptides, etc., and the produced amino acids are mostly feed-grade.
At present, the amino acid content and the variety of each plant are different, and the extraction difficulty of different parts is different, so that a universal plant free amino acid extraction method does not exist. Wherein, the extraction process of food-grade amino acid, in particular food-grade dendrobium amino acid, has less reports, and a food-grade/medicine-grade high-purity high-yield route suitable for industrial production is not developed.
Therefore, there is still a need for a green, low-cost extraction method of free amino acids based on dendrobium, especially dendrobium officinale, with high purity and high yield.
Disclosure of Invention
As mentioned above, in order to overcome the defects of the extraction method of free amino acid of Dendrobium officinale in the prior art, the invention aims to provide a high-purity high-yield extraction route of free amino acid of Dendrobium officinale which is suitable for industrial production and is in food/medicine grade.
In addition, the extraction method adopts conventional equipment and means, is suitable for low-cost production, can chelate trace elements to prepare amino acid chelate, can also be used as an additive component of nutriment, and has wide application.
Specifically, the present invention mainly relates to the following aspects.
In a first aspect, the invention provides a method for extracting compound free amino acid from dendrobium officinale leaves, which comprises the following steps:
a. carrying out initial extraction: primarily extracting free amino acids in the dendrobium officinale leaves by adopting an ultrasonic-leaching method in deionized water, and filtering to obtain primary extract;
b. secondary extraction: dissolving the filter residue, performing secondary extraction by adopting a double water phase extraction system and combining an ultrasonic method, and extracting the residual free amino acid to obtain secondary primary extract;
c. removing impurities: combining the primary extract and the secondary extract, concentrating in vacuum, further extracting and purifying in a two-aqueous-phase extraction system, splitting phases, filtering, adding a purifying reagent into the filtrate, standing overnight, removing impurities such as protein and polysaccharide, and adsorbing with activated carbon to remove impurities and decolor, thereby obtaining a crude product of the free amino acid extract of the dendrobium officinale;
d. primary purification: purifying the free amino acid extract by using acidic cation exchange resin, further removing low molecular weight oligosaccharide, polysaccharide and other impurities, collecting each amino acid component eluent, and performing deamination concentration treatment;
e. and (3) secondary purification: further purifying the obtained compound amino acid eluent by using an adsorption resin molecular sieve, removing low molecular weight impurities such as dendrophenol and the like, and drying to obtain a crude product of the free amino acid;
wherein, the adsorption resin molecular sieve is polystyrene-based polymer resin, preferably, styrene-based cross-linked divinylbenzene copolymer resin;
f. and (3) crystallization and refining: dissolving with ethanol, and vacuum evaporating for crystallization to obtain crude compound amino acid crystal;
g. recrystallizing for 1-2 times to obtain the compound free amino acid refined crystal, wherein the purity of the crystal is more than 99.5 percent.
In the extraction method, the physiological age of the dendrobium officinale is 2-3 years, preferably 3 years, and the longer the age of the dendrobium officinale growing in 1-3 years, the higher the content of free amino acid.
In the extraction method of the present invention, the step (a) is specifically as follows:
sieving the deactivated, dried and crushed dendrobium officinale leaf powder with a 120-mesh sieve with 100-fold sand, adding distilled water with the mass of 10-40 times, placing the powder into a flash extractor, preliminarily extracting the powder for 30-90s at the rotating speed of the flash extractor blade of 15000-fold sand 20000r/min at room temperature, then extracting the powder for 20-60min in a water bath oscillator at 50-60 ℃, after extraction, carrying out vacuum filtration on the extract while the extract is hot, collecting filtrate, cooling the filtrate to room temperature to serve as primary extract, and keeping filter residues for later use;
wherein, the leaching time is preferably 30-60 min;
wherein, the flash time is preferably 40 to 60 s.
In the extraction method of the present invention, the step (b) is specifically as follows:
(1) preparing aqueous two-phase extract: adding PEG4000-8000 polymer into aqueous solution of dipotassium hydrogen phosphate-sodium sulfate composite inorganic salt to prepare extract liquid with the mass fraction of polyethylene glycol of 5-8% and the mass fraction of composite inorganic salt of 8-12%;
(2) secondary extraction: adding 20-30 times of the extract liquor into the filter residue after primary extraction, adjusting the pH value to 6.8-7, uniformly stirring, placing the mixture in an ultrasonic countercurrent extractor, and performing ultrasonic extraction for 3-5 times at 40-50 ℃, wherein the ultrasonic extraction time is 5-10s each time, the ultrasonic extraction interval is 30-60s, and the ultrasonic power is set to be 300-400W; then uniformly stirring for 5-10min, after extraction, carrying out vacuum filtration while the solution is hot, collecting filtrate, cooling to room temperature, standing for 1-3h for phase separation, extracting free amino acid into an upper aqueous phase, collecting the upper extract of a double aqueous phase system, taking the upper extract as a secondary extract, combining the secondary extract with the primary extract, and naming the secondary extract as extract 1;
wherein, the extraction liquid contains compound inorganic salt dipotassium hydrogen phosphate: the mass ratio of the sodium sulfate is 1:0.5-1;
wherein, the standing phase separation time is preferably 2-3 h.
In the extraction method of the present invention, the step (c) is specifically as follows:
(1) preparing secondary aqueous phase extract: adding PEG-6000 into aqueous solution of sodium chloride-sodium sulfate composite inorganic salt to prepare secondary extraction liquid with the mass fraction of 2-3% of polyethylene glycol and the mass fraction of 3-5% of composite inorganic salt;
concentrating the combined extract 1 to one third to one fourth of the original volume, adding 5-10 times of the secondary extract, adjusting pH to 7, stirring at 25-30 deg.C for 5-15min, filtering with microfiltration membrane, collecting filtrate, cooling to room temperature, standing for 1-3 hr, discarding PEG layer containing protein, collecting water layer extract containing amino acids, and naming as extract 2;
(4) and (3) filtering and removing impurities: concentrating the extract 2 to one tenth volume in vacuum, and performing secondary filtration: filtering with 0.1-0.2 micrometer microfiltration membrane to remove impurities, and removing macromolecular impurities such as polysaccharide and protein with ultrafiltration membrane;
(5) removing macromolecular impurities and decoloring: adding 1-1.5 times volume of purification reagent solution into the filtered filtrate to further remove residual protein and water-soluble polysaccharide, standing at 4 deg.C overnight, centrifuging at 8000-: adding 1g of activated carbon, stirring for 15-20min, adsorbing, removing impurities, decolorizing, filtering with microfiltration membrane to obtain free amino acid extractive solution, and concentrating the filtrate under reduced pressure to one fifth to one tenth of the original volume;
wherein, the extraction system comprises the following components in percentage by weight: the mass ratio of the sodium chloride is 1: 0.2-0.5;
wherein, the purification reagent solution is 5-6% sulfosalicylic acid solution or absolute ethyl alcohol, and preferably, the absolute ethyl alcohol is selected.
In the extraction method of the present invention, the step (d) is specifically as follows:
(1) adjusting the pH value of the obtained free amino acid concentrated solution to 2.0-3.0, loading the free amino acid concentrated solution into styrene-based acidic cation exchange resin treated by hydrochloric acid at the flow rate of 2-4mL/min, staying for 15-30min to combine the amino acid with the resin, washing an exchange column with distilled water at the speed of 3-5mL/min to remove impurities until no ultraviolet detection signal exists, wherein residual polysaccharide components with negative electricity are not adsorbed and washed off;
(2) then eluting the exchange column with 1-2mol/L ammonia water at a speed of 3-5ml/min, collecting the eluent when an amino acid ultraviolet detection signal appears, obtaining a compound amino acid eluent, concentrating the eluent by a rotary evaporator to deaminate, and concentrating to one fifth to one tenth of the original volume, wherein the purity of the compound amino acid is detected to be more than 96%.
In the extraction method of the present invention, the step (e) is specifically as follows:
allowing the amino acid concentrated solution obtained in the previous step to pass through polystyrene-based polymer resin with the particle size of 80-100 microns soaked in deionized water, eluting a resin column with deionized water at the speed of 5-8ml/min, adsorbing and removing doped low-molecular-weight impurities such as dendrophenol, collecting amino acid eluent according to an amino acid ultraviolet detection signal, and performing vacuum drying at 50-60 ℃ to obtain a crude compound amino acid product with the content of more than 98%;
among them, the polystyrene-based polymer resin is preferably a styrene-based crosslinked divinylbenzene copolymer resin.
Wherein, the amino acid content is measured by an amino acid automatic analyzer through an ion exchange chromatography-ninhydrin post-column derivatization method, so that the content of the amino acid in the sample is measured.
In the extraction method of the present invention, the steps (f) to (g) are specifically as follows:
and (3) crystallization: dissolving the crude amino acid product obtained in the above steps with 5-6 times of 50-70% ethanol, heating and concentrating to one tenth of the original volume, and vacuum evaporating for crystallization to obtain crystal with purity of more than 99%;
and (3) recrystallization: dissolving the crude crystals with 50-60% ethanol again, and recrystallizing in a vacuum crystallizer for one to two times to obtain refined compound amino acid crystals with purity of over 99.5%.
Wherein, based on the dry weight of the dendrobium officinale leaf powder, the extraction rate of the compound free amino acid is 50-60mg/g, and the average purity is more than 99.5%.
In the present invention, the temperature of the vacuum concentration or the heating concentration is preferably 65 ℃ or lower, so as to ensure the stable property of the amino acid substance in the concentrated solution.
In a second aspect of the invention, a free amino acid product of dendrobium officinale obtained by the above extraction method is provided.
In a third aspect of the present invention, the use of the above-mentioned free amino acid product is provided, which can be used as a protein or amino acid nutritional supplement, and can also be used for chelating trace elements to obtain amino acid derivatives, such as dendrobium officinale complex amino acid chelate, including but not limited to complex amino acid chelate calcium, complex amino acid iron, complex amino acid zinc, and one or more combinations thereof.
The beneficial effects of the invention also include but are not limited to the following aspects:
1. develops a new low-cost method for extracting and purifying free natural biological amino acid from dendrobium officinale leaves with high yield and high purity (food/medicine grade), and the purity reaches more than 99.5 percent.
2. The extraction process and parameters are optimized for the leaf part of the dendrobium officinale, expensive equipment is not needed in the extraction route, the extraction efficiency is high due to the combination of flash extraction and aqueous two-phase extraction, the method is simple, and the cost of the used reagent is low.
3. The obtained compound free amino acid hardly contains biological phenol impurities with similar size and properties, and does not contain heavy metal elements enriched in soil.
4. Most of the solvent can be recycled and reused, and the industrial extraction can be carried out on a large scale.
5. Compared with feed-grade compound amino acid extracted in the prior art, the purified natural free amino acid and the derivative product thereof are the essential amino acid nutritional supplement for human body suitable for human consumption.
In conclusion, the extraction method is simple, takes a large amount of abandoned dendrobium officinale leaves as raw materials, is low in raw material cost, is suitable for large-scale production, can achieve food/medicine grade purity, and has good application prospect and market value.
Detailed Description
The present invention is described in detail below with reference to specific preparation examples and examples, but the use and purpose of these exemplary embodiments are merely to illustrate the present invention, and do not constitute any limitation to the actual scope of the present invention in any form, and the scope of the present invention is not limited thereto.
Preparation example 1: preliminarily extracting free amino acid in dendrobium officinale leaves
100 g of the deactivated, dried and crushed dendrobium officinale leaf powder is sieved by a 100-mesh sieve, 3000ml of distilled water is added, the mixture is placed in a flash extractor, primary extraction is carried out for 50s at the rotating speed of a flash extractor blade of 18000r/min under the condition of room temperature, then extraction is carried out for 40min at 50 ℃ in a water bath oscillator, after the extraction is finished, the extraction liquid is subjected to vacuum filtration while hot, the filtrate is collected and cooled to room temperature to serve as primary extraction liquid, and filter residue is reserved.
Preparation example 2: secondary extraction
(1) Preparing aqueous two-phase extract: adding PEG4000 polymer into aqueous solution of dipotassium hydrogen phosphate-sodium sulfate composite inorganic salt to prepare extract liquid with the mass fraction of polyethylene glycol being 8% and the mass fraction of composite inorganic salt being 10%; wherein, the extraction liquid contains compound inorganic salt dipotassium hydrogen phosphate: the mass ratio of the sodium sulfate is 1: 0.5;
(2) secondary extraction: adding 20-30 times of the extract liquor into the filter residue after primary extraction, adjusting the pH value to 6.8, uniformly stirring, placing in an ultrasonic countercurrent extractor, performing ultrasonic extraction for 3 times at 40 ℃, wherein the ultrasonic extraction time is 5s each time, the ultrasonic extraction interval is 30s, and the ultrasonic power is set to 300W; and uniformly stirring for 5min, after extraction, carrying out vacuum filtration while the extract is hot, collecting filtrate, cooling to room temperature, standing for 3h for phase separation, extracting free amino acid into an upper aqueous phase, collecting the upper extract of a double aqueous phase system as a secondary extract, combining the secondary extract with the primary extract, and naming the secondary extract as extract 1.
Preparation example 3: removing impurities
(1) Adding PEG-6000 into aqueous solution of sodium chloride-sodium sulfate composite inorganic salt to prepare secondary extraction liquid with the mass fraction of 3 percent of polyethylene glycol and the mass fraction of 5 percent of composite inorganic salt; wherein, sodium sulfate: the mass ratio of the sodium chloride is 1: 0.2;
concentrating the combined extract 1 to one fourth of the original volume, adding 5 times of the above extract, adjusting pH to 7, stirring at 30 deg.C for 5min, filtering with microfiltration membrane, collecting filtrate, cooling to room temperature, standing for 3 hr, discarding PEG layer containing protein, collecting water layer extract containing amino acids, and naming as extract 2;
(2) concentrating the extract 2 to one tenth volume in vacuum, and performing secondary filtration: filtering with 0.1-0.2 micrometer microfiltration membrane to remove impurities, and removing macromolecular impurities such as polysaccharide and protein with ultrafiltration membrane; adding 1.5 times volume of anhydrous ethanol into the filtered filtrate, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting supernatant, and mixing with 10 ml: adding activated carbon in a proportion of 1g, stirring for 15min, adsorbing, removing impurities, decolorizing, filtering with microfiltration membrane to obtain free amino acid extract, and concentrating the filtrate under reduced pressure to one tenth of the original volume.
Preparation example 4: purification by cation exchange resin
(1) Adjusting the pH value of the obtained free amino acid concentrated solution to 2.0, loading the free amino acid concentrated solution into styrene-based acidic cation exchange resin treated by hydrochloric acid at the flow rate of 3mL/min, standing for 20min to combine the amino acid with the resin, washing an exchange column by using distilled water at the speed of 3mL/min to remove impurities until no ultraviolet detection signal exists, wherein residual polysaccharide components with negative electricity are not adsorbed and washed down;
(2) then 1mol/L ammonia water is used for eluting the exchange column at the speed of 5ml/min, the eluent is collected when an amino acid ultraviolet detection signal appears, a compound amino acid eluent is obtained, the obtained eluent is concentrated and deaminated by a rotary evaporator to one fifth to one tenth of the original volume, wherein the purity of the compound amino acid is detected to be 96.5%.
Preparation example 5: further purification with molecular sieves
And (3) soaking the amino acid concentrated solution obtained in the preparation example 4 in deionized water to obtain styrene-based crosslinked divinylbenzene copolymer resin with the particle size of 80 microns, eluting the resin column with deionized water at the speed of 5ml/min, collecting amino acid eluent according to amino acid ultraviolet detection signals, and performing vacuum drying at 60 ℃ to obtain a crude compound amino acid product with the purity of 98.5%.
Preparation example 6: crystallization and recrystallization refining
And (3) crystallization: dissolving the crude amino acid product obtained in the step by using 50% ethanol with the mass 5 times that of the crude amino acid product, heating and concentrating the solution to one tenth of the original volume, and then performing vacuum evaporation crystallization to obtain crystals with the purity of 99%;
and (3) recrystallization: the crude crystals were redissolved with 60% ethanol and recrystallized twice in a vacuum crystallizer to obtain 5.8 g of refined compound amino acid crystals with a purity of 99.8%.
Wherein, the main components and the contents are measured as follows:
TABLE 1
As can be seen from the above table, the leaves of Dendrobium officinale Kimura et Migo contain almost all the essential amino acid species of human body, wherein the main amino acids are aspartic acid, glutamic acid, glycine, valine and leucine, and these 5 amino acids account for 52.5% of the total amino acids.
Preparation example 7: preparation of dendrobium officinale leaf composite free amino acid chelated calcium
Selecting the amino acid crystal prepared in preparation example 6 and food-grade calcium acetate as raw materials, mixing the raw materials according to the weight ratio of 300 g to 100 g, putting the mixed raw materials into a stirring mixer for food, adding 3.5L of distilled water, stirring to obtain a suspension, adding 1 weight percent of citric acid, homogenizing by a homogenizer, heating the suspension to 60 ℃, and introducing into a colloid mill to ensure that the solid particle size in the mixed solution is below 50 microns. Spraying the obtained mixed solution into a high-pressure fluid nano mill for chelation to obtain a clear compound amino acid chelated calcium solution. And concentrating, crystallizing, drying and sieving the obtained solution to obtain a finished product. The yield is 95.6 percent, and the purity is 98.7 percent.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.
Claims (5)
1. A method for extracting compound free amino acid from dendrobium officinale leaves is characterized by comprising the following steps:
a. carrying out initial extraction: primarily extracting free amino acids in the dendrobium officinale leaves by adopting an ultrasonic-leaching method in deionized water, and filtering to obtain primary extract;
b. secondary extraction: adding the filter residue after primary extraction into the extraction liquid, performing secondary extraction by adopting a double water phase extraction system and combining an ultrasonic method, and extracting the residual free amino acid to obtain secondary primary extraction liquid;
c. removing impurities: combining the primary extract and the secondary extract, concentrating in vacuum, further extracting and purifying in a two-aqueous-phase extraction system, splitting phases, filtering, adding a purifying reagent into the filtrate, standing overnight, removing protein and polysaccharide impurities, and adsorbing with activated carbon to remove impurities and decolor, thereby obtaining a crude product of the free amino acid extract of the dendrobium officinale;
d. primary purification: purifying the free amino acid extract by using acidic cation exchange resin, further removing oligosaccharide and polysaccharide impurities, collecting eluent of each amino acid component, and performing deamination concentration treatment;
e. and (3) secondary purification: further purifying the obtained compound amino acid eluent by using an adsorption resin molecular sieve, removing low-molecular-weight impurities of the dendrophenol, and drying to obtain a crude product of the free amino acid; wherein, the adsorption resin molecular sieve is polystyrene-based polymer resin;
f. and (3) crystallization and refining: dissolving with ethanol, and vacuum evaporating for crystallization to obtain crude compound amino acid crystal;
g. recrystallizing for 1-2 times to obtain the refined crystal of the compound free amino acid, wherein the purity of the crystal is more than 99.5 percent;
wherein the step (b) is specifically as follows:
(1) adding PEG4000-8000 polymer into aqueous solution of dipotassium hydrogen phosphate-sodium sulfate composite inorganic salt to prepare extract liquid with the mass fraction of polyethylene glycol of 5-8% and the mass fraction of composite inorganic salt of 8-12%; wherein, the extraction liquid contains compound inorganic salt dipotassium hydrogen phosphate: the mass ratio of the sodium sulfate is 1:0.5-1;
(2) adding 20-30 times of the extract liquor into the filter residue after primary extraction, adjusting the pH value to 6.8-7, uniformly stirring, placing the mixture in an ultrasonic countercurrent extractor, and performing ultrasonic extraction for 3-5 times at 40-50 ℃, wherein the ultrasonic extraction time is 5-10s each time, the ultrasonic extraction interval is 30-60s, and the ultrasonic power is set to be 300-400W; then uniformly stirring for 5-10min, after extraction, carrying out vacuum filtration while the solution is hot, collecting filtrate, cooling to room temperature, standing for 1-3h for phase separation, extracting free amino acid into an upper aqueous phase, collecting the upper extract of a double aqueous phase system, taking the upper extract as a secondary extract, combining the secondary extract with the primary extract, and naming the secondary extract as extract 1;
wherein the step (d) is specifically as follows:
(1) adjusting pH of the obtained free amino acid concentrated solution to 2.0-3.0, loading into styrene acidic cation exchange resin treated with hydrochloric acid at flow rate of 2-4mL/min, standing for 15-30min to combine amino acid with resin, washing the exchange column with distilled water at 3-5mL/min to remove impurities until no ultraviolet detection signal exists, wherein residual polysaccharide component with negative electricity is not adsorbed and washed off;
(2) eluting the exchange column with 1-2mol/L ammonia water at 3-5ml/min, collecting the eluate when the amino acid ultraviolet detection signal appears to obtain compound amino acid eluate, concentrating the eluate with rotary evaporator to remove ammonia, and concentrating to one fifth to one tenth of the original volume.
2. The method for extracting compound free amino acids from dendrobium officinale leaves as claimed in claim 1, wherein the method comprises the following steps: the step (a) is specifically as follows:
sieving the deactivated, dried and crushed dendrobium officinale leaf powder with a 120-mesh sieve of 100-.
3. The method for extracting compound free amino acids from dendrobium officinale leaves as claimed in claim 1, wherein the method comprises the following steps: the step (c) is specifically as follows:
(1) preparing secondary aqueous phase extract: adding PEG-6000 into aqueous solution of sodium chloride-sodium sulfate composite inorganic salt to prepare secondary extraction liquid with the mass fraction of 2-3% of polyethylene glycol and the mass fraction of 3-5% of composite inorganic salt; concentrating the combined extract 1 to one third to one fourth of the original volume, adding 5-10 times of the secondary extract, adjusting pH to 7, stirring at 25-30 deg.C for 5-15min, filtering with microfiltration membrane, collecting filtrate, cooling to room temperature, standing for 1-3 hr, discarding PEG layer containing protein, collecting water layer extract containing amino acids, and naming as extract 2;
(2) and (3) filtering and removing impurities: concentrating the extract 2 to one tenth volume in vacuum, and performing secondary filtration: firstly, filtering and removing impurities by using a microfiltration membrane of 0.1-0.2 micron, and then removing partial polysaccharide and protein macromolecular impurities by using an ultrafiltration membrane;
(3) removing macromolecular impurities and decoloring: adding 1-1.5 times volume of purification reagent solution into the filtered filtrate to further remove residual protein and water-soluble polysaccharide, standing at 4 deg.C overnight, centrifuging at 8000-: adding 1g of activated carbon, stirring for 15-20min, adsorbing, removing impurities, decolorizing, filtering with microfiltration membrane to obtain free amino acid extractive solution, and concentrating the filtrate under reduced pressure to one fifth to one tenth of the original volume;
wherein, the extraction system comprises the following components in percentage by weight: the mass ratio of the sodium chloride is 1: 0.2-0.5;
wherein the purification reagent solution is 5-6% sulfosalicylic acid solution or absolute ethyl alcohol.
4. The method for extracting compound free amino acids from dendrobium officinale leaves as claimed in claim 1, wherein the method comprises the following steps: the step (e) is specifically as follows:
and (2) enabling the amino acid concentrated solution obtained in the previous step to pass through polystyrene-based polymer resin with the particle size of 80-100 microns soaked in deionized water, then eluting a resin column with the deionized water at the speed of 5-8ml/min, adsorbing and removing doped low-molecular-weight impurities, collecting amino acid eluent according to an amino acid ultraviolet detection signal, and performing vacuum drying at 50-60 ℃ to obtain a crude compound amino acid product with the purity of more than 98%.
5. The method for extracting compound free amino acids from dendrobium officinale leaves as claimed in claim 1, wherein the method comprises the following steps: the steps (f) - (g) are as follows:
dissolving the crude amino acid product obtained in the above steps with 5-6 times of 50-70% ethanol, heating and concentrating to one tenth of the original volume, and vacuum evaporating for crystallization to obtain crystal with purity of more than 99%; dissolving the crude crystals with 50-60% ethanol again, and recrystallizing in a vacuum crystallizer for one to two times to obtain refined compound amino acid crystals with purity of over 99.5%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103553740A (en) * | 2013-10-14 | 2014-02-05 | 杨冬 | Preparation method of chelate containing high-concentration amino acid and microelement |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (2)
| Title |
|---|
| 丁亚平，吴庆生，杨道麒，于力文，徐云 .霍山石斛不同部位中必需微量元素与必需氨基酸的相关性研究.《安徽农业科学》.1994,第22卷(第03期),第265-267页. * |
| 霍山石斛不同部位中必需微量元素与必需氨基酸的相关性研究;丁亚平，吴庆生，杨道麒，于力文，徐云 ;《安徽农业科学》;19940930;第22卷(第03期);第265-267页 * |
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