CN103030569B - Preparation method of safflower pollen amino acids - Google Patents
Preparation method of safflower pollen amino acids Download PDFInfo
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Abstract
The invention relates to a preparation method of safflower pollen amino acids. The method includes: adding water to soaking dried safflower naps, and conducting centrifugation so as to obtain a soak solution; then adding water to the remaining safflower residue to soak it, and conducting centrifugation so as to obtain a soak solution, combining the two soak solutions, performing sieving, adding cellulase, carrying out uniform mixed suspension, laying the mixed solution aside, then performing quick stirring, conducting standing, subjecting the precipitate to pump filtration, washing, and pump filtration so as to obtain pure safflower pollen, carrying out wall-breaking treatment on the pollen, and then passing the obtained pollen solution to an activated carbon column and a cation exchange resin column in series, thus obtaining four pure amino acid products: leucine, histidine, lysine and arginine. The method completely adopts manual operation, and has a simple process and low cost. Compared with traditional safflower pollen collected and processed by bees, the safflower pollen amino acids obtained by the method are white crystals, have no special smell, and are completely pure varieties. Thus, the economic added value of safflower is enhanced, and the income of medicinal herb collectors is increased.
Description
Technical field
The present invention relates to the amino acid whose preparation method of a kind of safflower pollen, belong to healthcare products and cosmetic field.
Background technology
Safflower is Flos Carthami, belongs to composite family safflower and belongs to annual herb plant Carthamus tinctorius L..Its resource distribution is wider, in a lot of countries, all has cultivation.Approximately 1,100,000 hectares of whole world safflower Annual planting areas.Safflower major country of production is India, and approximately 760,000 hectares of Annual planting areas, account for the over half of world's total area and output, and China's safflower cultivated area is at 3～40,000 hectares, and main producing region concentrates on Xinjiang, is secondly Sichuan, Yunnan, Henan etc.China's safflower cultivation history is long, and Han dynasty just has about safflower cultivation and medicinal record, is the large Chinese medicinal materials of China's tradition.Its effect is promoting blood circulation and removing blood stasis, anti-inflammatory analgesic, and the effect such as have reducing blood-fat, hypotensive, the extravasated blood that disappears, detumescence pain, stimulate the circulation of the blood and cause the muscles and joints to relax is the key medicine of Chinese traditional treatment cardio-cerebrovascular disorder.Safflower flower suede is mainly medicinal, and Semen Flos Carthami is mainly edible oil processed, and its safflower pollen is used for healthcare products.
Pollen, English name Pollen, it is defined as the sporule of flowering plant, produces the microgametophyte containing three haploid nucleuses during sprouting.The main component of pollen is protein, amino acid, VITAMIN, trace element, organized enzyme, flavonoid, rapin, lipid, nucleic acid etc.Wherein amino acid whose content and ratio are to approach the aminoacid pattern that Food and Argriculture OrganizationFAO (FAO) is recommended most.It is pollen name that Latin Pollen (former meaning " powerful, brio ") is quoted in Sweden botanist woods in 1860 luxuriant growth, has ideally embodied the essence of pollen.
The pollen of selling on market is that beekeeper utilizes electric shock net to force the honeycomb doorway that honeybee is shaken off the pollen granule of adopting back to obtain mostly.Like this, the quality that impact is made honey and the procreation that affects bee colony.
In recent years, food chemistry additive application is improper, and health suffers damage.Therefore, people's back to nature, the theory of recovering one's original simplicity and hope are increasingly strong.
Summary of the invention
The object of the invention is, provides a kind of safflower pollen amino acid whose preparation method, and the method is by the dried floral suede of safflower, soaks, centrifugal, obtains soak solution; Again remaining safflower slag is soaked, centrifugal, obtain soak solution, merge secondary soak solution, sieve, add cellulase, suspendible is even, places then rapid stirring, standing, by throw out suction filtration, washing, suction filtration, obtains pure safflower pollen, then pollen is carried out to broken wall treatment, the use of connecting with cation exchange resin column by activated carbon column again, can obtain leucine, Histidine, Methionin and arginine four seed amino acid sterlings.The method adopts manual operation completely, and technique is simple, and cost is low, and the safflower pollen amino acid obtaining by the method for the invention is white crystal, and without special odor, product kind is completely pure, thereby has improved the economic value added of safflower, increases medicinal herb grower's income.Abandon the safflower pollen that honeybee is gathered and processed, its participation is made honey and meet the optimum procreation of bee colony, stablize honeybee industry, improved its economic value added and use value.
The amino acid whose preparation method of a kind of safflower pollen of the present invention, follows these steps to carry out:
A, by the dried floral suede of safflower, add by weight the deionized water that 5-20 doubly measures to soak, temperature room temperature-60 ℃, time 5-10 hour, the 800-1400 hurdle whizzer of walking around is centrifugal, obtains soak solution for the first time;
B, the deionized water immersion that will again add 4-8 doubly to measure by the weight ratio of step a dried floral suede in remaining safflower slag, temperature room temperature-60 ℃, time 5-10 hour, the 800-1400 hurdle whizzer of walking around is centrifugal, obtain soak solution for the second time, merge secondary soak solution, cross screen cloth 250-350 order, obtain filtering soak solution;
C, the soak solution of step b is placed in to diameter: high=1: the transparent or semitransparent cylindrical container of 1-3, add cellulase, suspendible is even, places and within 6-12 hour, obtain enzyme liquid under temperature 20-60 ℃ condition;
D, by the enzyme liquid of step c along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 10-60 minute, siphon goes out supernatant liquor, to in throw out, add by volume deionized water 10-50kg again, repeat stirring, standing and siphon operating process, to making pollen remain in container bottom;
E, steps d pollen throw out is leached to moisture with suction filtration, keep intact filter cake, more colourless to filtrate clarification with deionized water wash, then use ethanol and ether gradient suction filtration, pour out filter cake stand dry, obtain pure safflower pollen;
F, by the pollen of step e by ultrasonic, enzyme process or temperature differential method, or three kinds of integrated broken wall treatment of doing of method, amino acid in pollen is discharged, make the safflower pollen broken wall aqueous solution, at 10 ℃ of temperature, place, time 3-4 days, separates out a large amount of suspensions and throw out, filter, obtain safflower pollen hydrolyzed solution;
G, by step f safflower pollen hydrolyzed solution, adjust pH4.0 with ammoniacal liquor, then successively by conventional activated carbon column and 732 type cation exchange resin columns, coutroi velocity 12 ml/min, through paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction, detect again, obtain containing leucine, Histidine, Methionin and arginic elutriant;
H, contain leucine, Histidine, Methionin and the arginine elutriant of step g are detected by paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction according to a conventional method, refine successively:
Leucine is refining: pauly reacts the part concentrating under reduced pressure that is negative, add activated carbon decolorizing, place refrigerator hold over night, crystallization, filter, recrystallization is dissolved in 80 ℃ of hot water of temperature again, then carries out activated carbon decolorizing, places refrigerator hold over night, crystallization, filter, filtrate, by 95% washing with alcohol, dry, can obtain leucine sheet white crystals;
Histidine is refining: pauly reaction is positive partly concentrated except ammonia under temperature 70 C, 3 times repeatedly, dilute with water again, with hydrochloric acid, adjust pH3.0, add activated carbon decolorizing, concentrated, add concentration 70% ethanol of 2 times of amounts of volume ratio, standing, there is mass crystallization to separate out, filter, by concentration, be that 75% and 95% ethanol washs 2 times successively, crystallization is adjusted to pH2.5 by water dissolution, then add activated carbon decolorizing, have mass crystallization to separate out, filter, by concentration, be 75% and 95% washing with alcohol again, dry, can obtain L-Histidine hydrochloride sterling;
Methionin is refining: pauly reacts diminuendo, and the part concentrating under reduced pressure that ninhydrin reaction is positive, except ammonia, to small volume, adds water, adjust pH4.0 with HCl, add activated carbon decolorizing 30 minutes, filter crystallization, with concentration be 95% washing with alcohol, dry, obtain lysine hydrochloride sterling;
Arginine is refining: by positive Sakaguchi reaction elutriant part, merge decompression except ammonia, adjust pH4.0 with HCl, be evaporated to crystallization, crystallization, with concentration be 75% alcohol immersion, drain, by concentration, be 95% ethanol dehydration again, drain, obtain white crystals arginine monohydrochloride sterling.
The add-on of step c cellulase adds cellulase 0.005-0.01% by soak solution weight ratio.
Step g activated carbon column is connected with Zeo-karb use.
The amino acid whose preparation method of a kind of safflower pollen of the present invention, the method is in the post stamen of safflower flower suede, to adhere to a large amount of dried flower powder, adopt washing, aqueous suspension method elutes safflower pollen from post stamen, proportion according to safflower flower suede relic in water is less than the principle of safflower pollen, make safflower pollen first be deposited in container bottom, collecting precipitation, repeat aqueous suspension method, residual safflower flower suede relic is further separated, with the enzymolysis of cellulase, further enzymolysis is suspended in the tiny relic of safflower flower suede in liquid, make the cellulase hydrolysis of macromolecular safflower flower suede become micromolecular Mierocrystalline cellulose, make it all or part of soluble in water, the safflower flower suede of trying one's best out in most pollen, collect pollen, adopt gradient to reduce the method for solvent polarity, remove the moisture in safflower pollen, stand is dry, by ultrasonic, enzyme process, the integrated broken wall treatment of doing of temperature differential method or three kinds of methods, amino acid in pollen is discharged, the use of connecting with cation exchange resin column by activated carbon column again, can obtain leucine, Histidine, Methionin and arginic four seed amino acid sterlings.The safflower pollen amino acid obtaining by the method for the invention is white crystal, without special odor.
Accompanying drawing explanation
Fig. 1 is sample safflower pollen amino acid collection of illustrative plates of the present invention
Fig. 2 is standard substance safflower pollen amino acid collection of illustrative plates of the present invention
Embodiment
A, by the dried floral suede 10kg of safflower, add by weight the deionized water of 5 times of amounts to soak, room temperature, 5 hours time, the 800 hurdle whizzers of walking around are centrifugal, obtain soak solution 30kg for the first time;
B, will be in remaining safflower slag by step a dried floral suede weight ratio, again add the deionized water of 4 times of amounts to soak, room temperature, 5 hours time, the 800 hurdle whizzers of walking around are centrifugal, obtain soak solution for the second time, merge secondary soak solution 80kg, cross screen cloth 250 orders, obtain filtering soak solution;
C, by filtering soak solution 80kg, be placed in diameter: the high=transparent cylindrical container of 1: 1, by soak solution weight ratio, add cellulase 0.005%, suspendible is even, under 20 ℃ of conditions of temperature, places 6 hours;
D, by the enzyme liquid of step c along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 10 minutes, siphon goes out supernatant liquor (as producing carthamin yellow), to in throw out, add deionized water 10kg again, along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 10 minutes, siphon went out supernatant liquor, to making pollen remain in container bottom;
E, pollen throw out is leached to moisture with suction filtration, keep intact filter cake, colourless to filtrate clarification with deionized water wash, then use ethanol and ether gradient suction filtration, pour out filter cake stand dry, obtain pure safflower pollen;
F, the pollen of gained is done to broken wall treatment by conventional ultrasonic method, the amino acid in pollen is discharged, make the safflower pollen broken wall aqueous solution, at 10 ℃ of temperature, place 3-4 days, separate out a large amount of suspensions and throw out, filter, obtain safflower pollen hydrolyzed solution;
G, by well-established law, granulated active carbon is processed into column chromatography gac, dress post, 8 × 140 centimetres centimetres, then 732 type Zeo-karbs are processed into [H
+] type, dress post, 10 × 170 centimetres, activated carbon column and resin column series connection are used, by the safflower pollen hydrolyzed solution of broken wall, adjust pH4.0 with ammoniacal liquor, then successively by charcoal post and resin column, coutroi velocity 12 ml/min, when ninhydrin reaction is positive, with every part of 500 milliliters of collections, with paper chromatography detection (condition: propyl carbinol: Glacial acetic acid: water=4: 2: 1), merge same stream part, obtain I, II, III, IV component, this part is mainly containing leucine, again by hydrolyzed solution by activated carbon column and resin column, pauly reaction is negative, resin does not have saturated, Histidine does not occur, with 20 ml/min flow velocitys, wash resin column to effluent liquid pH6.8 with distilled water, use 0.1M ammoniacal liquor wash-out instead, flow velocity 12 ml/min, when reacting positive, pauly illustrates that Histidine has been eluted, continue fraction collection, to Methionin entirely by after wash-out, continue wash-out, can there is one section of clear area, use 2M ammoniacal liquor wash-out instead, flow velocity 20 ml/min, be washed till ninhydrin reaction feminine gender, arginine wash-out completes,
H, will contain leucine, Histidine, Methionin and arginic elutriant, and adopt paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction to detect, refine successively:
Leucine is refining: pauly is reacted to the part concentrating under reduced pressure that is negative, add activated carbon decolorizing, place refrigerator hold over night, crystallization, filter, recrystallization is dissolved in 80 ℃ of hot water of temperature again, then carries out activated carbon decolorizing, places refrigerator hold over night, crystallization, filter, filtrate, by 95% washing with alcohol, dry, can obtain leucine sheet white crystals;
Histidine is refining: pauly reaction is positive partly concentrated except ammonia under temperature 70 C, 3 times repeatedly, be diluted with water to again 5000mL, with hydrochloric acid, adjust pH3.0, add activated carbon decolorizing, be concentrated into 200mL, add 2 times of amount 75% ethanol of volume ratio, standing, there is mass crystallization to separate out, filter, by concentration, be that 75% and 95% ethanol washs 2 times successively, crystallization is adjusted to pH2.5 by water dissolution, then add activated carbon decolorizing, have mass crystallization to separate out, filter, by concentration, be 75% and 95% washing with alcohol again, dry, can obtain L-Histidine hydrochloride sterling;
Methionin is refining: pauly reacts diminuendo, and the part concentrating under reduced pressure that ninhydrin reaction is positive, except ammonia, to small volume, adds water 2000mL, adjust pH4.0 with HCl, add activated carbon decolorizing 30 minutes, filter crystallization, with concentration be 95% washing with alcohol, dry, obtain lysine hydrochloride sterling;
Arginine is refining: by positive Sakaguchi reaction elutriant part, merge decompression except ammonia, adjust pH4.0 with HCl, be evaporated to crystallization, crystallization, with concentration be 75% alcohol immersion, drain, by concentration, be 95% ethanol dehydration again, drain, obtain white crystals arginine monohydrochloride sterling.
The leucine, Histidine, Methionin and the arginine that by method of the present invention, obtain are white crystal, without special odor.
Embodiment 2
A, by the dried floral suede 10kg of safflower, add by weight the deionized water of 10 times of amounts to soak, 30 ℃ of temperature, 6 hours time, the 1000 hurdle whizzers of walking around are centrifugal, obtain soak solution 40kg for the first time;
B, will be in remaining safflower slag by step a dried floral suede weight ratio, again add the deionized water of 5 times of amounts to soak, 30 ℃ of temperature, 6 hours time, the 1000 hurdle whizzers of walking around are centrifugal, obtain soak solution for the second time, merge secondary soak solution 90kg, cross screen cloth 280 orders, obtain filtering soak solution;
C, by filtering soak solution 90kg, be placed in diameter: the high=translucent cylindrical container of 1: 2, by soak solution weight ratio, add cellulase 0.007%, suspendible is even, under 30 ℃ of conditions of temperature, places 9 hours;
D, by the enzyme liquid of step c along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 30 minutes, siphon goes out supernatant liquor (as producing carthamin yellow), to in throw out, add deionized water 30kg again, along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 30 minutes, siphon went out supernatant liquor, to making pollen remain in container bottom;
E, pollen throw out is leached to moisture with suction filtration, keep intact filter cake, colourless to filtrate clarification with deionized water wash, then use ethanol and ether gradient suction filtration, pour out filter cake stand dry, obtain pure safflower pollen;
F, the pollen of gained is done to broken wall treatment by conventional enzyme process, the amino acid in pollen is discharged, make the safflower pollen broken wall aqueous solution, at 10 ℃ of temperature, place 3-4 days, separate out a large amount of suspensions and throw out, filter, obtain safflower pollen hydrolyzed solution;
G, by well-established law, granulated active carbon is processed into column chromatography gac, dress post, 8 × 140 centimetres centimetres, then 732 type Zeo-karbs are processed into [H
+] type, dress post, 10 × 170 centimetres, activated carbon column and resin column series connection are used, by the safflower pollen hydrolyzed solution of broken wall, adjust pH4.0 with ammoniacal liquor, then successively by charcoal post and resin column, coutroi velocity 12 ml/min, when ninhydrin reaction is positive, with every part of 500 milliliters of collections, with paper chromatography detection (condition: propyl carbinol: Glacial acetic acid: water=4: 2: 1), merge same stream part, obtain I, II, III, IV component, this part is mainly containing leucine, again by hydrolyzed solution by activated carbon column and resin column, pauly reaction is negative, resin does not have saturated, Histidine does not occur, with 20 ml/min flow velocitys, wash resin column to effluent liquid pH6.8 with distilled water, use 0.1M ammoniacal liquor wash-out instead, flow velocity 12 ml/min, when reacting positive, pauly illustrates that Histidine has been eluted, continue fraction collection, to Methionin entirely by after wash-out, continue wash-out, can there is one section of clear area, use 2M ammoniacal liquor wash-out instead, flow velocity 20 ml/min, be washed till ninhydrin reaction feminine gender, arginine wash-out completes,
H, will contain leucine, Histidine, Methionin and arginic elutriant, and adopt paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction to detect, refine successively:
Leucine is refining: pauly is reacted to the part concentrating under reduced pressure that is negative, add activated carbon decolorizing, place refrigerator hold over night, crystallization, filter, recrystallization is dissolved in 80 ℃ of hot water of temperature again, then carries out activated carbon decolorizing, places refrigerator hold over night, crystallization, filter, filtrate, by 95% washing with alcohol, dry, can obtain leucine sheet white crystals;
Histidine is refining: pauly reaction is positive partly concentrated except ammonia under temperature 70 C, 3 times repeatedly, be diluted with water to again 5000mL, with hydrochloric acid, adjust pH3.0, add activated carbon decolorizing, be concentrated into 200mL, add 2 times of amount 75% ethanol of volume ratio, standing, there is mass crystallization to separate out, filter, by concentration, be that 75% and 95% ethanol washs 2 times successively, crystallization is adjusted to pH2.5 by water dissolution, then add activated carbon decolorizing, have mass crystallization to separate out, filter, by concentration, be 75% and 95% washing with alcohol again, dry, can obtain L-Histidine hydrochloride sterling;
Methionin is refining: pauly reacts diminuendo, and the part concentrating under reduced pressure that ninhydrin reaction is positive, except ammonia, to small volume, adds water 2000mL, adjust pH4.0 with HCl, add activated carbon decolorizing 30 minutes, filter crystallization, with concentration be 95% washing with alcohol, dry, obtain lysine hydrochloride sterling;
Arginine is refining: by positive Sakaguchi reaction elutriant part, merge decompression except ammonia, adjust pH4.0 with HCl, be evaporated to crystallization, crystallization, with concentration be 75% alcohol immersion, drain, by concentration, be 95% ethanol dehydration again, drain, obtain white crystals arginine monohydrochloride sterling.
The leucine, Histidine, Methionin and the arginine that by method of the present invention, obtain are white crystal, without special odor.
A, by the dried floral suede 10kg of safflower, add by weight the deionized water of 15 times of amounts to soak, temperature 50 C, 8 hours time, the 1200 hurdle whizzers of walking around are centrifugal, obtain soak solution 50kg for the first time;
B, will be in remaining safflower slag by step a dried floral suede weight ratio, again add the deionized water of 7 times of amounts to soak, temperature 50 C, 8 hours time, the 1200 hurdle whizzers of walking around are centrifugal, obtain soak solution for the second time, merge secondary soak solution 110kg, after 300 eye mesh screens, obtain filtering soak solution;
C, by filtering soak solution 110kg, be placed in diameter: the high=transparent cylindrical container of 1: 3, by soak solution weight ratio, add cellulase 0.008%, suspendible is even, places 10 hours under temperature 45 C condition;
D, by the enzyme liquid of step c along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 5 minutes, siphon goes out supernatant liquor (as producing carthamin yellow), to in throw out, add deionized water 40kg again, along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 5 minutes, siphon went out supernatant liquor, to making pollen remain in container bottom;
E, pollen throw out is leached to moisture with suction filtration, keep intact filter cake, colourless to filtrate clarification with deionized water wash, then use ethanol and ether gradient suction filtration, pour out filter cake stand dry, obtain pure safflower pollen.
F, the pollen of gained is done to broken wall treatment by conventional temperature differential method, the amino acid in pollen is discharged, make the safflower pollen broken wall aqueous solution, at 10 ℃ of temperature, place 3-4 days, separate out a large amount of suspensions and throw out, filter, obtain safflower pollen hydrolyzed solution;
G, by well-established law, granulated active carbon is processed into column chromatography gac, dress post, 8 × 140 centimetres centimetres, then 732 type Zeo-karbs are processed into [H
+] type, dress post, 10 × 170 centimetres, activated carbon column and resin column series connection are used, by the safflower pollen hydrolyzed solution of broken wall, adjust pH4.0 with ammoniacal liquor, then successively by charcoal post and resin column, coutroi velocity 12 ml/min, when ninhydrin reaction is positive, with every part of 500 milliliters of collections, with paper chromatography detection (condition: propyl carbinol: Glacial acetic acid: water=4: 2: 1), merge same stream part, obtain I, II, III, IV component, this part is mainly containing leucine, again by hydrolyzed solution by activated carbon column and resin column, pauly reaction is negative, resin does not have saturated, Histidine does not occur, with 20 ml/min flow velocitys, wash resin column to effluent liquid pH6.8 with distilled water, use 0.1M ammoniacal liquor wash-out instead, flow velocity 12 ml/min, when reacting positive, pauly illustrates that Histidine has been eluted, continue fraction collection, to Methionin entirely by after wash-out, continue wash-out, can there is one section of clear area, use 2M ammoniacal liquor wash-out instead, flow velocity 20 ml/min, be washed till ninhydrin reaction feminine gender, arginine wash-out completes,
H, will contain leucine, Histidine, Methionin and arginic elutriant, and adopt paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction to detect, refine successively:
Leucine is refining: pauly is reacted to the part concentrating under reduced pressure that is negative, add activated carbon decolorizing, place refrigerator hold over night, crystallization, filter, recrystallization is dissolved in 80 ℃ of hot water of temperature again, then carries out activated carbon decolorizing, places refrigerator hold over night, crystallization, filter, filtrate, by 95% washing with alcohol, dry, can obtain leucine sheet white crystals;
Histidine is refining: pauly reaction is positive partly concentrated except ammonia under temperature 70 C, 3 times repeatedly, be diluted with water to again 5000mL, with hydrochloric acid, adjust pH3.0, add activated carbon decolorizing, be concentrated into 200mL, add 2 times of amount 75% ethanol of volume ratio, standing, there is mass crystallization to separate out, filter, by concentration, be that 75% and 95% ethanol washs 2 times successively, crystallization is adjusted to pH2.5 by water dissolution, then add activated carbon decolorizing, have mass crystallization to separate out, filter, by concentration, be 75% and 95% washing with alcohol again, dry, can obtain L-Histidine hydrochloride sterling;
Methionin is refining: pauly reacts diminuendo, and the part concentrating under reduced pressure that ninhydrin reaction is positive, except ammonia, to small volume, adds water 2000mL, adjust pH4.0 with HCl, add activated carbon decolorizing 30 minutes, filter crystallization, with concentration be 95% washing with alcohol, dry, obtain lysine hydrochloride sterling;
Arginine is refining: by positive Sakaguchi reaction elutriant part, merge decompression except ammonia, adjust pH4.0 with HCl, be evaporated to crystallization, crystallization, with concentration be 75% alcohol immersion, drain, by concentration, be 95% ethanol dehydration again, drain, obtain white crystals arginine monohydrochloride sterling.
The leucine, Histidine, Methionin and the arginine that by method of the present invention, obtain are white crystal, without special odor.
Embodiment 4
A, by the dried floral suede 10kg of safflower, add by weight the deionized water of 20 times of amounts to soak, temperature 60 C, 10 hours time, the 1400 hurdle whizzers of walking around are centrifugal, obtain soak solution 70kg for the first time;
B, will be in remaining safflower slag by former dried floral suede weight ratio, again add the deionized water of 8 times of amounts to soak, temperature 60 C, 10 hours time, the 1400 hurdle whizzers of walking around are centrifugal, obtain soak solution for the second time, merge secondary soak solution 150kg, cross screen cloth 350 orders, obtain filtering soak solution;
C, by filtering soak solution 150kg, be placed in diameter: the high=translucent cylindrical container of 1: 3, by soak solution weight ratio, add cellulase 0.01%, suspendible is even, places 12 hours under temperature 60 C condition;
D, by the enzyme liquid of step c along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 60 minutes, siphon goes out supernatant liquor (as producing carthamin yellow), to in throw out, add deionized water 50kg again, along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 60 minutes, siphon went out supernatant liquor, to making pollen remain in container bottom;
E, pollen throw out is leached to moisture with suction filtration, keep intact filter cake, colourless to filtrate clarification with deionized water wash, then use ethanol and ether gradient suction filtration, pour out filter cake stand dry, obtain pure safflower pollen;
F, the pollen of gained is done to broken wall treatment by integrated temperature differential method, enzyme process and the ultrasonic method of routine, the amino acid in pollen is discharged, make the safflower pollen broken wall aqueous solution, at 10 ℃ of temperature, place 3-4 days, separate out a large amount of suspensions and throw out, filter, obtain safflower pollen hydrolyzed solution;
G, by well-established law, granulated active carbon is processed into column chromatography gac, dress post, 8 × 140 centimetres centimetres, then 732 type Zeo-karbs are processed into [H
+] type, dress post, 10 × 170 centimetres, activated carbon column and resin column series connection are used, by the safflower pollen hydrolyzed solution of broken wall, adjust pH4.0 with ammoniacal liquor, then successively by charcoal post and resin column, coutroi velocity 12 ml/min, when ninhydrin reaction is positive, with every part of 500 milliliters of collections, with paper chromatography detection (condition: propyl carbinol: Glacial acetic acid: water=4: 2: 1), merge same stream part, obtain I, II, III, IV component, this part is mainly containing leucine, again by hydrolyzed solution by activated carbon column and resin column, pauly reaction is negative, resin does not have saturated, Histidine does not occur, with 20 ml/min flow velocitys, wash resin column to effluent liquid pH6.8 with distilled water, use 0.1M ammoniacal liquor wash-out instead, flow velocity 12 ml/min, when reacting positive, pauly illustrates that Histidine has been eluted, continue fraction collection, to Methionin entirely by after wash-out, continue wash-out, can there is one section of clear area, use 2M ammoniacal liquor wash-out instead, flow velocity 20 ml/min, be washed till ninhydrin reaction feminine gender, arginine wash-out completes,
H, will contain leucine, Histidine, Methionin and arginic elutriant, and adopt paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction to detect, refine successively:
Leucine is refining: pauly is reacted to the part concentrating under reduced pressure that is negative, add activated carbon decolorizing, place refrigerator hold over night, crystallization, filter, recrystallization is dissolved in 80 ℃ of hot water of temperature again, then carries out activated carbon decolorizing, places refrigerator hold over night, crystallization, filter, filtrate, by 95% washing with alcohol, dry, can obtain leucine sheet white crystals;
Histidine is refining: pauly reaction is positive partly concentrated except ammonia under temperature 70 C, 3 times repeatedly, be diluted with water to again 5000mL, with hydrochloric acid, adjust pH3.0, add activated carbon decolorizing, be concentrated into 200mL, add 2 times of amount 75% ethanol of volume ratio, standing, there is mass crystallization to separate out, filter, by concentration, be that 75% and 95% ethanol washs 2 times successively, crystallization is adjusted to pH2.5 by water dissolution, then add activated carbon decolorizing, have mass crystallization to separate out, filter, by concentration, be 75% and 95% washing with alcohol again, dry, can obtain L-Histidine hydrochloride sterling;
Methionin is refining: pauly reacts diminuendo, and the part concentrating under reduced pressure that ninhydrin reaction is positive, except ammonia, to small volume, adds water 2000mL, adjust pH4.0 with HCl, add activated carbon decolorizing 30 minutes, filter crystallization, with concentration be 95% washing with alcohol, dry, obtain lysine hydrochloride sterling;
Arginine is refining: by positive Sakaguchi reaction elutriant part, merge decompression except ammonia, adjust pH4.0 with HCl, be evaporated to crystallization, crystallization, with concentration be 75% alcohol immersion, drain, by concentration, be 95% ethanol dehydration again, drain, obtain white crystals arginine monohydrochloride sterling.
The leucine, Histidine, Methionin and the arginine that by method of the present invention, obtain are white crystal, without special odor.
The amino acid of the safflower pollen obtaining by the method for the invention, detects as leucine, Histidine, Methionin and arginine through Xinjiang Uygur Autonomous Regions food and medicine inspection institute.
Concrete operation step:
Get 0.5 gram, the safflower pollen amino acid (leucine, Histidine, Methionin and arginine) that embodiment 1-4 produces arbitrarily, add 2 times to 10 milliliters of safflower pollen amino acid (W/W) deionized waters, fully dissolve;
Check:
Amino acid checks: extract reaction solution 2 milliliters, drip 10 of 3% triketohydrindene hydrate ethanol reagent, be heated to micro-boiling, liquid should manifest purple or blueness;
Safflower pollen amino acid analysis:
Test foundation: two the 6th > > of < < the Sanitation Ministry medicine standard (biochemical drug the first fascicle);
Test organization: Xinjiang Uygur Autonomous Regions food and medicine inspection institute
Instrument: German SYKAM-4300 amino acidanalyser, post-column derivation, the strong polarity ion exchange resin of sulfonic group, 50 ℃ of column temperatures, 130 ℃ of temperature of reactor, sodium citrate buffer solution gradient elution, flow velocity 0.45 ml/min;
Reagent: ninhydrin reagent, flow velocity 0.25 ml/min.
Result: detect 18 seed amino acids, amino acid total content is 0.29%, and wherein histidine content is up to 0.0574%, content has leucine, arginine and a phenylalanine more than 0.02%.In Table
Aminoacids content after table safflower pollen broken wall
Claims (3)
1. the amino acid whose preparation method of safflower pollen, is characterized in that following these steps to carrying out:
A, by the dried floral suede of safflower, add by weight the deionized water that 5-20 doubly measures to soak, temperature room temperature-60 ℃, time 5-10 hour, the 800-1400 hurdle whizzer of walking around is centrifugal, obtains soak solution for the first time;
B, the deionized water immersion that will again add 4-8 doubly to measure by the weight ratio of step a dried floral suede in remaining safflower slag, temperature room temperature-60 ℃, time 5-10 hour, the 800-1400 hurdle whizzer of walking around is centrifugal, obtain soak solution for the second time, merge secondary soak solution, cross screen cloth 250-350 order, obtain filtering soak solution;
C, the soak solution of step b is placed in to diameter: high=1: the transparent or semitransparent cylindrical container of 1-3, add cellulase, suspendible is even, places and within 6-12 hour, obtain enzyme liquid under temperature 20-60 ℃ condition;
D, by the enzyme liquid of step c along a direction rapid stirring, then stir in the other direction immediately, liquid is no longer rotated, standing 10-60 minute, siphon goes out supernatant liquor, to in throw out, add by volume deionized water 10-50kg again, repeat stirring, standing and siphon operating process, to making pollen remain in container bottom;
E, steps d pollen throw out is leached to moisture with suction filtration, keep intact filter cake, more colourless to filtrate clarification with deionized water wash, then use ethanol and ether gradient suction filtration, pour out filter cake stand dry, obtain pure safflower pollen;
F, by the pollen of step e by ultrasonic, enzyme process or temperature differential method, or three kinds of integrated broken wall treatment of doing of method, amino acid in pollen is discharged, make the safflower pollen broken wall aqueous solution, at 10 ℃ of temperature, place, time 3-4 days, separates out a large amount of suspensions and throw out, filter, obtain safflower pollen hydrolyzed solution;
G, by step f safflower pollen hydrolyzed solution, adjust pH4.0 with ammoniacal liquor, then successively by conventional activated carbon column and 732 type cation exchange resin columns, coutroi velocity 12 ml/min, detecting through paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction, obtain containing leucine, Histidine, Methionin and arginic elutriant;
H, step g contained to leucine, Histidine, Methionin and arginine elutriant adopt according to a conventional method paper chromatography, pauly reaction, ninhydrin reaction and Sakaguchi reaction to detect, refine successively:
Leucine is refining: pauly reacts the part concentrating under reduced pressure that is negative, add activated carbon decolorizing, place refrigerator hold over night, crystallization, filter, recrystallization is dissolved in 80 ℃ of hot water of temperature again, then carries out activated carbon decolorizing, places refrigerator hold over night, crystallization, filter, filtrate is 95% washing with alcohol, dry by volumetric concentration, can obtain leucine sheet white crystals;
Histidine is refining: pauly reaction is positive partly concentrated except ammonia under temperature 70 C,
, adjust pH3.0 with hydrochloric acid for 3 times repeatedly, then dilute with water, add activated carbon decolorizing, concentrated, adding 2 times of amount volumetric concentrations of volume ratio is 75% ethanol, standing, there is mass crystallization to separate out, filter, by volumetric concentration, be that 75% and 95% ethanol washs 2 times successively, crystallization adjusted to pH2.5 by water dissolution, then add activated carbon decolorizing, have mass crystallization to separate out, filter, then with volumetric concentration be 75% and 95% washing with alcohol, dry, can obtain L-Histidine hydrochloride sterling;
Methionin is refining: pauly reacts diminuendo, and the part concentrating under reduced pressure that ninhydrin reaction is positive, except ammonia, to small volume, adds water, adjust pH4.0 with HCl, add activated carbon decolorizing 30 minutes, filter crystallization, with volumetric concentration be 95% washing with alcohol, dry, obtain lysine hydrochloride sterling;
Arginine is refining: by positive Sakaguchi reaction elutriant part, merge decompression except ammonia, adjust pH4.0 with HCl, be evaporated to crystallization, crystallization, with volumetric concentration be 75% alcohol immersion, drain, by volumetric concentration, be 95% ethanol dehydration again, drain, obtain white crystals arginine monohydrochloride sterling.
2. method according to claim 1, is characterized in that the add-on of step c cellulase adds cellulase 0.005-0.01% by soak solution weight ratio.
3. method according to claim 1, is characterized in that step g activated carbon column is connected with Zeo-karb use.
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| CN105214335B (en) * | 2015-10-16 | 2017-05-03 | 新疆于田瑰觅生物科技股份有限公司 | Method for extracting pollen amino acid liquid from rose pollen |
| CN105254518B (en) * | 2015-11-27 | 2017-06-16 | 广州普星药业有限公司 | The decolouring purification process of DL lysines |
| CN108186429A (en) * | 2017-10-20 | 2018-06-22 | 新疆于田瑰觅生物科技股份有限公司 | A kind of method that amino acid is prepared from rose slag |
| CN108208686A (en) * | 2018-02-10 | 2018-06-29 | 中国农业科学院蜜蜂研究所 | A kind of wall-breaking method of Bee Pollen |
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