CN103641837B - A kind of method of chlorophyll copper sodium extracting from silkworm excrement - Google Patents
A kind of method of chlorophyll copper sodium extracting from silkworm excrement Download PDFInfo
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Abstract
The invention discloses a kind of method of chlorophyll copper sodium extracting from silkworm excrement; wherein; the method comprises carries out enzymolysis with enzyme to silkworm excrement; wherein, described enzyme selects one or more in the group that forms of free dimension element enzyme, hemicellulase, polygalacturonase, Lipase and proteolytic enzyme.Adopt method provided by the invention, Mierocrystalline cellulose, hemicellulose, pectin, protein, the fats composition that can effectively degrade in silkworm excrement raw material.And enzyme dosage is few, treatment process is simple, easy to operate, consuming time short, chlorophyll extraction yield is high, finished product chlorophyll copper sodium purity salt is high, environmentally safe, the yield that improves sodium copper chlorophyllin shorten chlorophyllous extraction time to a great extent, decrease the usage quantity of solvent, reduce the follow-up difficulty prepared in sodium copper chlorophyllin process, save production cost.
Description
Technical field
The present invention relates to a kind of method of chlorophyll copper sodium extracting from silkworm excrement.
Background technology
From silkworm excrement, extract chlorophyll then can prepare sodium copper chlorophyllin through a series of reaction, it is as the one of metalloporphyrin, and compared with chlorophyll, not only stability strengthens, and also has well water-soluble, easy to use.And sodium copper chlorophyllin with the nutritive element of institute's chelating in itself distinctive constructional feature and derivative to need painted, improve or keep the food color of natural green and improve in food use value own that there is advantageous advantage, be therefore widely used in the fields such as foodstuff additive, cosmetics additive, tinting material, medicine, photoelectric conversion material.Sodium copper chlorophyllin, as a kind of safe foodstuff additive and tinting material, can be used in the food such as carbonated drink, candy, fruity powder, fruit syrup, assembled alcoholic drinks, can, cake, also can be used for the painted etc. of toothpaste.Study discovery simultaneously, sodium copper chlorophyllin also has certain nourishing function, as: the functions such as anti-inflammatory, promotion liver function recovery, to treatment stomatocace, dispel halitosis and have obvious curative effects, therefore in medicine industry, sodium copper chlorophyllin becomes the important source material of production for treating hepatitis, stomach trouble and duodenal ulcer medicine.
Be at present that the method for raw material production sodium copper chlorophyllin mainly contains following several technical process with silkworm excrement:
1, impurity removing → pulverizing → extraction → concentrating and separating → copper generation → saponification salify → removal of impurities → drying → chlorophyll copper sodium product salt;
2, impurity removing → pulverizing → extraction → concentrated → saponification → acidifying copper generation → secondary saponification → refining → drying → chlorophyll copper sodium product salt;
3, impurity removing → pulverize → add copper compound to extract → concentrated → saponification salify → solvent wash → drying → chlorophyll copper sodium product salt.
The program of these three kinds of techniques is roughly the same, and the key distinction of first two technique is the sequencing in saponification and copper generation; The third technique is synchronously carried out with extraction process copper generation reaction, can effectively reduce chlorophyllous loss.
But owing to containing the macromolecular substance such as a large amount of Mierocrystalline cellulose, hemicellulose, pectin, protein in silkworm excrement, the extraction add chlorophyllous extraction difficulty, generally needing to use a large amount of solvent to come silkworm excrement raw material, this adds production cost and time loss on the one hand, simultaneously because solvent is acetone, a large amount of uses of acetone too increase the pollution to environment, are unfavorable for environmental protection.
Therefore be necessary to develop a kind of take silkworm excrement as simple, easy to operate, consuming time short, the cost less investment of the treatment process of raw material, chlorophyll copper sodium salt production process that environmental pollution degree is low.
Summary of the invention
The object of the invention is to overcome the above-mentioned defect existed in existing sodium copper chlorophyllin production method, a kind of macromolecular substance such as Mierocrystalline cellulose, hemicellulose, pectin, protein, fat can effectively removed before extraction in silkworm excrement is provided, treatment process is simple, easy to operate, short, cost less investment consuming time, acetone usage quantity are few, the method for environmental pollution degree is low from silkworm excrement chlorophyll copper sodium extracting.
In order to reach above object, the invention provides a kind of method of chlorophyll copper sodium extracting from silkworm excrement, wherein, the method comprises carries out enzymolysis with enzyme to silkworm excrement,
Wherein, described enzyme selects one or more in the group that forms of free dimension element enzyme, hemicellulase, polygalacturonase, Lipase and proteolytic enzyme.
Compared with prior art; the invention has the advantages that: utilize the silkworm excrement raw material of one or more enzymes in cellulase, hemicellulase, polygalacturonase, proteolytic enzyme, Lipase to drying to carry out pre-treatment, Mierocrystalline cellulose, hemicellulose, pectin, protein, the fats composition that can effectively degrade in raw material.And enzyme dosage is few, treatment process is simple, short, environmentally safe easy to operate, consuming time, shorten chlorophyllous extraction time to a great extent, decrease the usage quantity of solvent, reduce the follow-up difficulty prepared in sodium copper chlorophyllin process, saved production cost.In addition, according to method provided by the present invention, chlorophyll condensed cream Determination of Chlorophyll content is high, and finished product chlorophyll copper sodium purity salt is high.And the method easily realizes industrialization, economic benefit can be significantly improved.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
A method for chlorophyll copper sodium extracting from silkworm excrement, wherein, the method comprises carries out enzymolysis with enzyme to silkworm excrement,
Wherein, described enzyme is selected from one or more in the group be made up of cellulase, hemicellulase, polygalacturonase, Lipase and proteolytic enzyme.
Wherein, described silkworm excrement is the dry ight soil of Bombycidae animal bombyx mori (BombyxmoriL.) larva, in short cylindrical shape granule, and long 2-5 millimeter, diameter 1.5-3 millimeter.The present invention had no particular limits the silkworm age stage producing silkworm excrement, can be the silkworm excrement in the first to the 5th silkworm age stage.
Wherein, the enzyme that described cellulase can have a cellulose degradation ability by circumscribed beta-glucanase, Endo-β-glucanase, zytase and beta-glucosidase etc. forms, the cellulase used in the present invention can for the marketed cellulose enzyme finished product such as cellulose complex enzyme finished product bought, preferably, described cellulase is selected from one or more in the group be made up of circumscribed beta-glucanase, Endo-β-glucanase, zytase and beta-glucosidase.
In one embodiment of the invention, with the total amount of cellulase for benchmark, the content of circumscribed beta-glucanase is 1.0 × 10
-3-9 × 10
-2% by mole, the content of Endo-β-glucanase is 1.2 × 10
-3-7.6 × 10
-2% by mole, the content of zytase is 8 × 10
-4-1.2 × 10
-1% by mole, the content of beta-glucosidase is 1.0 × 10
-3-1.5 × 10
-1% by mole.
Wherein, the enzyme that described hemicellulase can have a hemicellulose degradation ability by xylosidase, arabinoxylanase, glucuroide, mannase and tilactase etc. forms, wherein arabinoxylanase comprises inscribe β-1, one or more in 4-D zytase (EC3.2.1.8), circumscribed β-Isosorbide-5-Nitrae-zytase (EC3.2.1.37) and xylobiase.The hemicellulase used in the present invention can for the commercially available hemicellulase finished product bought, such as hemicellulose prozyme finished product.Preferably, described hemicellulase is selected from one or more in the group be made up of xylosidase, arabinoxylanase, glucuroide, mannase and tilactase.
In one embodiment of the invention, with the total amount of hemicellulase for benchmark, the content of arabinoxylanase is 1.0 × 10
-3-8.0 × 10
-2% by mole, the content of glucuroide is 5.0 × 10
-3-1.1 × 10
-2% by mole, the content of mannase is 3.0 × 10
-3-1.5 × 10
-2% by mole, the content of tilactase is 8.0 × 10
-3-3.0 × 10
-2% by mole.
Wherein, the enzyme that described polygalacturonase can have a pectin degrading ability by protopectinase, pectinesterase lytic enzyme, polygalacturonase class and pectate lyase etc. forms, the polygalacturonase used in the present invention can for the commercially available polygalacturonase finished product bought, such as polygalacturonase prozyme finished product.
Preferably, described polygalacturonase is selected from one or more in the group be made up of protopectinase, pectinesterase lytic enzyme, polygalacturonase and pectin lyase.
In one embodiment of the invention, with the total amount of polygalacturonase for benchmark, the content of described protopectinase is 3.0 × 10
-3-1.5 × 10
-2% by mole, the content of pectinesterase lytic enzyme is 1.0 × 10
-3-1.2 × 10
-2% by mole, the content of polygalacturonase is 1.0 × 10
-3-8 × 10
-3% by mole, the content of pectin lyase is 8.0 × 10
-4-6.5 × 10
-3% by mole.
Wherein, described proteolytic enzyme can the enzyme of protein matter in enzymolysis silkworm excrement can be made up of thiol proteinase, aspartate protease, serine/threonine protein enzyme and metalloprotease etc., the proteolytic enzyme used in the present invention can for the commercially available proteolytic enzyme finished product bought, such as protease composite enzyme finished product.Preferably, described proteolytic enzyme is one or more in thiol proteinase, aspartate protease, serine/threonine protein enzyme and metalloprotease.
In one embodiment of the invention, with the total amount of proteolytic enzyme for benchmark, the content of thiol proteinase is 8.0 × 10
-4-8.0 × 10
-2% by mole, the content of aspartate protease is 1.0 × 10
-3-6.55 × 10
-2% by mole, the content of serine/threonine protein enzyme is 1.0 × 10
-3-5.0 × 10
-2% by mole, the content of metalloprotease is 1.0 × 10
-3-8.0 × 10
-2% by mole.
Wherein, described Lipase (No. CAS: 9001-62-1) can make fat splitting be lipid acid and glycerine, generates lipid acid, glycerine and monoglyceride or diester.
Wherein, in method provided by the present invention; relative to the silkworm excrement of 100g; the consumption that the consumption of water is 20-65g, the consumption of cellulase is 0.01-0.20g hemicellulase is 0.008-0.15g, the consumption of polygalacturonase is 0.005-0.15g, the consumption of Lipase is 0.005-0.1g, and the consumption of proteolytic enzyme is 0.003-0.18g.
Preferably; relative to the silkworm excrement of 100g; the consumption of water is 35-60g, the consumption of cellulase is 0.05-0.18g, the consumption of hemicellulase is 0.02-0.10g, the consumption of polygalacturonase is 0.01-0.10g, the consumption of Lipase is 0.01-0.06g, and the consumption of proteolytic enzyme is 0.005-0.15g.
In the present invention, in order to obtain best hydrolysis result, the contamination of enzyme can be allocated according to the chemical composition of different times, different silkworm age silkworm excrement.
When described silkworm excrement is the silkworm excrement in the first silkworm age stage, described enzyme can comprise cellulase, hemicellulase, proteolytic enzyme, polygalacturonase and Lipase;
Wherein, relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.05-0.1g, the consumption of hemicellulase is 0.03-0.075g, the consumption of polygalacturonase is 0.02-0.08g, the consumption of Lipase is 0.008-0.02g, the consumption of proteolytic enzyme is 0.03-0.07g.
Wherein, when described silkworm excrement is the silkworm excrement in the second silkworm age stage, described enzyme can comprise cellulase, proteolytic enzyme, polygalacturonase; Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.05-0.12g, the consumption of polygalacturonase is 0.07-0.15g, the consumption of proteolytic enzyme is 0.06-0.1g.
Wherein, when described silkworm excrement is the silkworm excrement in the 3rd silkworm age stage, described enzyme can comprise cellulase, hemicellulase, polygalacturonase; Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.05-0.11g, the consumption of hemicellulase is 0.05-0.08g, the consumption of polygalacturonase is 0.02-0.05g.
Wherein, when described silkworm excrement is the silkworm excrement in the 4th silkworm age stage, described enzyme can comprise cellulase, hemicellulase, polygalacturonase and Lipase; Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.01-0.03g, the consumption of hemicellulase is 0.003-0.008g, the consumption of polygalacturonase is 0.01-0.04g, the consumption of Lipase is 0.02-0.08g.
Wherein, when described silkworm excrement is the silkworm excrement in the 5th silkworm age stage, described enzyme can comprise cellulase, hemicellulase and polygalacturonase; Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.02-0.07g, the consumption of hemicellulase is 0.01-0.054g, the consumption of polygalacturonase is 0.05-0.13g.
Method provided by the present invention can comprise the following steps:
(1) silkworm excrement and enzyme and water are contacted at 25-40 DEG C also enzymolysis 2-8h and obtain enzymolysis product;
(2) by enzymolysis product granulation, the silkworm excrement particle that acquisition diameter is 0.2-0.8cm, length is 1-2cm;
(3) with the chlorophyll in acetone extraction silkworm excrement particle, extracting solution is obtained;
(4) saponification process is carried out to extracting solution and obtain saponification liquor;
(5) acidifying is carried out to saponification liquor and put Copper treatment acquisition sodium copper chlorophyllin.
Wherein, in order to improve the efficiency of enzymolysis, described silkworm excrement is the Powdered silkworm excrement of 0.005-0.02cm preferably past the diameter of pulverization process.
Wherein, step (1) can be carried out in the following manner, and contact also stirring and evenly mixing after enzyme and water being mixed in proportion with silkworm excrement and obtain silkworm excrement mud, the steady environment of perseverance silkworm excrement mud being placed in 25-40 DEG C is hydrolyzed 2-8h, control temperature between the reaction period also stirs, to make enzyme digestion reaction more abundant in good time.
In the present invention, the method for contact and stirring is had no particular limits, enzyme and water can be made into enzyme liquid in advance, then the mode adopting spray limit, limit to stir makes enzyme, water contacts fully with silkworm excrement.
Wherein, when described enzyme also comprises one or more in cellulase, hemicellulase, polygalacturonase, Lipase when comprising proteolytic enzyme; in order to avoid proteolytic enzyme is to the enzymolysis of the plain enzyme of dimension, hemicellulase, polygalacturonase, Lipase, described method comprises first silkworm excrement and cellulase, hemicellulase, polygalacturonase and one or more in Lipase being contacted and also adds proteolytic enzyme again after enzymolysis 2-5h and carry out enzymolysis 3-6h.
Wherein, step (2) can be carried out in the following manner; in order to effectively reduce the loss in leaching process; granulation again can be carried out to enzymolysis product; first enzymolysis product high for water content is dried to water content between 20%-45%, re-use high speed granulator and make that diameter is 0.2-0.8cm, length is the particulate material of 1-2cm.In whole process, enzymolysis product is in high speed rotating state, and temperature controls between 90-120 DEG C, and the time is 3-10min.This step will strictly get hold of Heating temperature and heat-up time, and temperature is too high, time overlength all can have a strong impact on chlorophyllous extraction yield.
Wherein, step (3) can be carried out in the following manner, the acetone of preferred employing 70%-90% is that solvent carries out refluxing extraction to completing the silkworm excrement particle after granulation: the temperature of refluxing extraction is 45-60 DEG C, the time is 1.5-2.5h, solid-liquid ratio is 1:2-3.5, add up extraction 3-5 time, finally determines the chlorophyll content in extracting solution.
Wherein, step (4) can be carried out in the following manner, and the extracting solution Rotary Evaporators vacuum concentration at 50-60 DEG C step (3) obtained, obtains chlorophyll condensed cream, then carry out saponification.Saponification Conditions is: the NaOH-95% ethanolic soln adding 5%-20%, and saponification temperature is 45-75 DEG C, and saponification time is 0.5-3.0h, and the mass ratio of reaction Determination of Chlorophyll condensed cream and NaOH-ethanolic soln is in the scope of 1:4-1:12.Whole saponification process constantly stirs, and stirring velocity is 100-150r/min.
Wherein, step (5) can be carried out in the following manner, first uses the ratio of 1:1(hydrogenchloride and water) HCl solution regulate the pH value of saponification liquor to be 2.5-4, then dose 10%-25%CuSO wherein
4solution, at 65-75 DEG C, constant temperature stirs 0.5-1.5h.Add the NaOH solution salify of 20% after reacting completely, stir 30min, filter, obtain sodium copper chlorophyllin crude product; Wherein relative to the saponification liquor of 100ml, CuSO
4the addition of solution is 4-15ml; Be 6-8ml relative to the addition of the NaOH of the saponification liquor 20% of 100ml.
The present invention can also comprise the purification step to sodium copper chlorophyllin crude product, described purification step can be carried out according to the method that this area is conventional, such as, described purification step can comprise: solvent wash method, recrystallization method, extraction process, membrane separation process, macroreticular resin absorbing method etc.
Below will be described the present invention by embodiment.In following examples,
Silkworm excrement derives from the dry ight soil of Qujing of Yunnan silkworm egg.
Cellulase compound enzymic preparation is purchased from taste bio tech ltd, Hebei hundred; hemicellulase compound enzymic preparation is purchased from taste bio tech ltd, Hebei hundred; polygalacturonase compound enzymic preparation is purchased from You Libao bio tech ltd, Beijing; protease composite enzyme preparation is purchased from You Libao bio tech ltd, Beijing, and Lipase is purchased from You Libao bio tech ltd, Beijing.In the present invention, the moiety of each prozyme is in table 1.
Table 1
In the present invention, chlorophyllous content records by the following method:
Raw material silkworm excrement Determination of Chlorophyll content assaying method: get silkworm excrement particle, crosses 80 mesh sieves after pulverizing with micromill.Get 0.3 powder in 100ml volumetric flask, after adding the ultrasonic 30min of 80 % by weight acetone soln, be settled to 100ml, pipette supernatant liquor ultraviolet visible spectrophotometry after placing 3h and detect, and record the absorbance at A663 and A645 place.
Silkworm excrement particle Determination of Chlorophyll content assaying method after enzymolysis granulation: adopt heating and refluxing extraction method to carry out, concrete, with 80 % by weight acetone solns, refluxing extraction is carried out to 500g silkworm excrement particle, totally three times, each 1,L80 % by weight acetone soln, merges each filtrate and constant volume.Precision pipettes after 1ml is settled to 100ml and adopts ultraviolet visible spectrophotometry to detect, and records the absorbance at A663 and A645 place, with the content of following formulae discovery raw material and each sample Determination of Chlorophyll.
Ca(chlorophyll-a concentration)=12.72A663-2.59A645 unit: μ g/ml
Formula 1
Cb(chlorophyll b concentration)=22.88A663-4.67A645 unit: μ g/ml
Formula 2
Chlorophyll content=(Ca+Cb) × V is total/1000m
Unit: mg/g
V is total=constant volume number × 100(extension rate)
M: the quality (g) extracting raw material
Formula 3
Yield=m1/m2 × 100% of condensed cream
M1: extracting solution concentrates the quality of rear chlorophyll condensed cream
M2: the quality of silkworm excrement raw material
Formula 4
Formula 5
The method for drafting of sodium copper chlorophyllin typical curve
Accurately take the sodium copper chlorophyllin standard substance 9.5mg of dry constant weight, the constant volume that adds water, in 100ml volumetric flask, obtains the sodium copper chlorophyllin standardized solution that concentration is 95mg/L.Precision pipette above-mentioned solution 0,2,4,6,8,10ml is in 10ml volumetric flask, respectively add water and be settled to 10mL, shake up rear detection, be blank with water, by ultraviolet visible spectrophotometry, measure absorbance (see table 1) in 630nm and 405nmnm wavelength place, wherein choose with the absorbancy at 630nm place as ordinate zou, concentration is X-coordinate, carries out regression treatment to measurement result, obtains typical curve y=0.0098x-0.005.
Table 1
| Concentration (mg/L) | 0 | 19 | 38 | 57 | 76 | 95 |
| 630nm absorbancy | 0 | 0.172 | 0.376 | 0.543 | 0.748 | 0.925 |
| 405nm absorbancy | 0 | 0.048 | 0.105 | 0.152 | 0.209 | 0.258 |
Embodiment 1
The present embodiment realizes with the silkworm excrement raw material in 5000g first silkworm age stage.
Precision takes cellulase compound enzymic preparation 3.10g, hemicellulase compound enzymic preparation 2.78g, polygalacturonase compound enzymic preparation 1.81g, Lipase 0.46g are dissolved in 3000g40 DEG C of water; be sprayed onto in the 5000g silkworm excrement powder that crushes after abundant dissolving; spray limit, limit stirring and evenly mixing; being dissolved in water by proteolytic enzyme 2.13g after 1 hour sprays in the feed again, makes it be hydrolyzed 5h after stirring.
Enzymolysis: the silkworm excrement mud after hydrolysis 5h is placed in the isoperibol of 30 DEG C and is hydrolyzed 6h, stir between the reaction period in good time, enzyme digestion reaction is fully carried out.
Granulation: reacted rear enforcement granulation again, by water content be first the pureed silkworm excrement of 65 % by weight at 105 DEG C, high speed rotating dries 5min, is 40 % by weight to silkworm excrement powder moisture, re-uses high speed granulator and makes that diameter is 0.5cm, length is the particle of 1.5cm.
Extraction: get the silkworm excrement particle 500g after granulation, the acetone extraction of employing 80 % by weight, temperature is 55 DEG C, the time is 2h, material liquid volume ratio is 1:3g/ml, adds up extraction 3 times.Merge No. three extracting solutions, finally measure chlorophyll extraction yield.
Saponification: said extracted liquid is adopted Rotary Evaporators vacuum concentration at 60 DEG C, obtains chlorophyll condensed cream 37.4g, calculates chlorophyll condensed cream yield, and detects cream Determination of Chlorophyll content, then carry out saponification.Add the NaOH-95% ethanolic soln 350ml of 15 % by weight, saponification 1h under 60 DEG C of conditions, employ magnetic stirring apparatus in whole saponification process and constantly stir with the rotating speed of 120r/min, until react completely.
Copper is put in acidifying: regulate the pH value of above-mentioned saponification liquor to be 3.5 with the HCl of 1:1, then dose the CuSO of 20 % by weight wherein
4solution 8.0mL, at 70 DEG C, constant temperature stirs 0.5h.Add the NaOH salify of 6.0mL20 % by weight after having reacted again wherein, stir 30min, filter, dry and obtain sodium copper chlorophyllin crude product 1.632g, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Embodiment 2
The present embodiment realizes with the silkworm excrement raw material in 5000g second silkworm age stage.
Precision takes polygalacturonase compound enzymic preparation 4.82g and cellulase compound enzymic preparation 3.37g and is dissolved in 3000g40 DEG C of water, is sprayed onto in the 5000g silkworm excrement powder crushed, spray limit, limit stirring and evenly mixing after fully dissolving; Again proteolytic enzyme 4.15g is dissolved in water, is sprayed in raw material and continues to stir 3h.
Enzymolysis: the silkworm excrement mud being added with enzyme is placed in the isoperibol of 30 DEG C and is hydrolyzed 6h, stir between the reaction period in good time, enzyme digestion reaction is fully carried out.
Granulation: reacted rear enforcement granulation again, by water content be first the pureed silkworm excrement of 65 % by weight at 105 DEG C, high speed rotating dries 5min, is 40 % by weight to silkworm excrement powder moisture, re-uses high speed granulator and makes that diameter is 0.5cm, length is the particle of 1.5cm.
Extract: get the silkworm excrement particle 500g after granulation, adopt the acetone extraction of 80 % by weight, temperature is 55 DEG C, the time is 2h, solid-liquid ratio is 1:3, add up extraction 3 times.Merge No. three extracting solutions, finally determine its chlorophyll extraction yield.
Saponification: said extracted liquid is adopted Rotary Evaporators vacuum concentration at 60 DEG C, obtains chlorophyll condensed cream 45.4g, calculates chlorophyll condensed cream yield, and detects cream Determination of Chlorophyll content, then carry out saponification.Add the NaOH-95% ethanolic soln 350ml of 15%, saponification 1h under 60 DEG C of conditions, employ magnetic stirring apparatus in whole saponification process and constantly stir with the rotating speed of 120r/min, until react completely.
Copper is put in acidifying: regulate the pH value of above-mentioned saponification liquor to be 3.5 with the HCl of 1:1, then dose the CuSO of 20% wherein
4solution 8.0mL, at 70 DEG C, constant temperature stirs 0.5h.Add the NaOH salify of 6.0mL20% after having reacted again wherein, stir 30min, filter, obtain sodium copper chlorophyllin crude product 2.066g, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Embodiment 3
The present embodiment realizes with the silkworm excrement raw material in 5000g the 3rd silkworm age stage.
Precision takes cellulase compound enzymic preparation 3.10g, hemicellulase compound enzymic preparation 2.78g, polygalacturonase compound enzymic preparation 1.81g is added in 3000g water, is sprayed onto in the 5000g silkworm excrement powder crushed, spray limit, limit stirring and evenly mixing after fully dissolving.
Enzymolysis: the silkworm excrement mud being added with enzyme is placed in the isoperibol of 30 DEG C and is hydrolyzed 6h, stir between the reaction period in good time, enzyme digestion reaction is fully carried out.
Granulation: reacted rear enforcement granulation again, by water content be first the pureed silkworm excrement of 65 % by weight at 105 DEG C, high speed rotating dries 5min, is 40 % by weight to silkworm excrement powder moisture, re-uses high speed granulator and makes that diameter is 0.5cm, length is the particle of 1.5cm.
Extract: get the silkworm excrement particle 500g after granulation, adopt the acetone extraction of 80 % by weight, temperature is 55 DEG C, the time is 2h, solid-liquid ratio is 1:3, add up extraction 3 times.Merge No. three extracting solutions, finally determine its chlorophyll extraction yield.
Saponification: said extracted liquid is adopted Rotary Evaporators vacuum concentration at 60 DEG C, obtains chlorophyll condensed cream 48.0g, calculates chlorophyll condensed cream yield, and detects cream Determination of Chlorophyll content, then carry out saponification.Add the NaOH-95% ethanolic soln 350ml of 15 % by weight, saponification 1h under 60 DEG C of conditions, employ magnetic stirring apparatus in whole saponification process and constantly stir with the rotating speed of 120r/min, until react completely.
Copper is put in acidifying: regulate the pH value of above-mentioned saponification liquor to be 3.5 with the HCl of 1:1, then dose the CuSO of 20 % by weight wherein
4solution 8.0mL, at 70 DEG C, constant temperature stirs 0.5h.Add the NaOH salify of 6.0mL20 % by weight after having reacted again wherein, stir 30min, filter, obtain sodium copper chlorophyllin crude product 2.825g, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Embodiment 4
The present embodiment realizes with the silkworm excrement raw material in 5000g the 4th silkworm age stage.
Precision takes cellulase compound enzymic preparation 0.78g, hemicellulase compound enzymic preparation 0.26g, polygalacturonase 0.95g, Lipase 3.34g are added in 3000g40 DEG C of water, be sprayed onto in the 5000g silkworm excrement powder that crushes after abundant dissolving, spray limit, limit stirring and evenly mixing, reaction 1h; Add 3.08g proteolytic enzyme again in water, be sprayed in silkworm excrement powder and stir evenly.
Enzymolysis: the silkworm excrement mud being added with enzyme is placed in the isoperibol of 30 DEG C and is hydrolyzed 6h, stir between the reaction period in good time, enzyme digestion reaction is fully carried out.
Granulation: reacted rear enforcement granulation again, by water content be first the pureed silkworm excrement of 65 % by weight at 105 DEG C, high speed rotating dries 5min, is 40 % by weight to silkworm excrement powder moisture, re-uses high speed granulator and makes that diameter is 0.5cm, length is the particle of 1.5cm.
Extract: get the silkworm excrement particle 500g after granulation, adopt the acetone extraction of 80 % by weight, temperature is 55 DEG C, the time is 2h, solid-liquid ratio is 1:3, add up extraction 3 times.Merge No. three extracting solutions, finally determine its chlorophyll extraction yield.
Saponification: said extracted liquid is adopted Rotary Evaporators vacuum concentration at 60 DEG C, obtains chlorophyll condensed cream 50.22g, calculates chlorophyll condensed cream yield, and detects cream Determination of Chlorophyll content, then carry out saponification.Add the NaOH-95% ethanolic soln 350ml of 15 % by weight, saponification 1h under 60 DEG C of conditions, employ magnetic stirring apparatus in whole saponification process and constantly stir with the rotating speed of 120r/min, until react completely.
Copper is put in acidifying: regulate the pH value of above-mentioned saponification liquor to be 3.5 with the HCl of 1:1, then dose the CuSO of 20% wherein
4solution 8.0mL, at 70 DEG C, constant temperature stirs 0.5h.Add the NaOH salify of 6.0mL20 % by weight after having reacted again wherein, stir 30min, filter, obtain sodium copper chlorophyllin crude product 3.143g, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Embodiment 5
The present embodiment realizes with the silkworm excrement raw material in 5000g the 5th silkworm age stage.
Precision takes polygalacturonase compound enzymic preparation 4.39g, cellulase compound enzymic preparation 2.45g, hemicellulase compound enzymic preparation 1.58g is added in 3000g40 DEG C of water, is sprayed onto in the 5000g silkworm excrement powder crushed, spray limit, limit stirring and evenly mixing after fully dissolving.
Enzymolysis: the silkworm excrement mud being added with enzyme is placed in the isoperibol of 30 DEG C and is hydrolyzed 6h, stir between the reaction period in good time, enzyme digestion reaction is fully carried out.
Granulation: reacted rear enforcement granulation again, by water content be first the pureed silkworm excrement of 65 % by weight at 105 DEG C, high speed rotating dries 5min, is 40 % by weight to silkworm excrement powder moisture, re-uses high speed granulator and makes that diameter is 0.5cm, length is the particle of 1.5cm.
Extract: get the silkworm excrement particle 500g after granulation, adopt the acetone extraction of 80 % by weight, temperature is 55 DEG C, the time is 2h, solid-liquid ratio is 1:3, add up extraction 3 times.Merge No. three extracting solutions, finally determine its chlorophyll extraction yield.
Saponification: said extracted liquid is adopted Rotary Evaporators vacuum concentration at 60 DEG C, obtains chlorophyll condensed cream 51.38g, calculates chlorophyll condensed cream yield, and detects cream Determination of Chlorophyll content, then carry out saponification.Add the NaOH-95% ethanolic soln 350ml of 15 % by weight, saponification 1h under 60 DEG C of conditions, employ magnetic stirring apparatus in whole saponification process and constantly stir with the rotating speed of 120r/min, until react completely.
Copper is put in acidifying: regulate the pH value of above-mentioned saponification liquor to be 3.5 with the HCl of 1:1, then dose the CuSO of 20 % by weight wherein
4solution 8.0mL, at 70 DEG C, constant temperature stirs 0.5h.Add the NaOH salify of 6.0mL20 % by weight after having reacted again wherein, stir 30min, filter, obtain sodium copper chlorophyllin crude product 3.090g, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Embodiment 6
Prepare chlorophyll copper sodium product salt according to the method identical with embodiment 1, the addition unlike enzyme is cellulase compound enzymic preparation 1.50g, hemicellulase compound enzymic preparation 1.50g, polygalacturonase compound enzymic preparation 1.50g, protease composite enzyme preparation 1.50g, Lipase 1.50g.
Calculate chlorophyll cream yield, cream Determination of Chlorophyll content, finished product chlorophyll copper sodium purity salt, chlorophyllous extraction yield.Obtain sodium copper chlorophyllin crude product 1.059g, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Embodiment 7
Preparing chlorophyll copper sodium product salt according to the method identical with embodiment 1, is the compound formulation of cellulase and proteolytic enzyme unlike enzyme used.
Calculate chlorophyll cream yield, cream Determination of Chlorophyll content, finished product chlorophyll copper sodium purity salt, chlorophyllous extraction yield.Obtain sodium copper chlorophyllin crude product 1.497g, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Embodiment 8
Preparing chlorophyll copper sodium product salt according to the method identical with embodiment 1, is cellulase, hemicellulase and polygalacturonase unlike enzyme used.
Calculate chlorophyll cream yield, cream Determination of Chlorophyll content, finished product chlorophyll copper sodium purity salt, chlorophyllous extraction yield.Obtain sodium copper chlorophyllin crude product 1.603g.Finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Comparative example 1
The present embodiment is for illustration of the method for existing chlorophyll copper sodium extracting from silkworm excrement
Extract: precision takes the raw material silkworm excrement in the first silkworm age stage of 5Kg, the removing grains of sand, pulverize after lime particle, get raw material silkworm excrement particle 500g, acetone with 80 % by weight is under optimum experimental condition, that is: temperature is 55 DEG C, the time is 2h, solid-liquid ratio is 1:3, accumulative extraction 3 times.Merge No. three extracting solutions, finally determine its chlorophyll extraction yield.
Copper is put in acidifying: adopt Rotary Evaporators vacuum concentration at 60 DEG C, obtain chlorophyll condensed cream 66.84g, calculate chlorophyll condensed cream yield, and detect cream Determination of Chlorophyll content, the HCl adding 1:1 after stirring with 70ml ethanol regulates its pH value to be 3.5, then doses the CuSO of 20% wherein
4solution 8.0mL, at 70 DEG C, constant temperature stirs 0.5h.
Saponification: above-mentioned copper is carried out saponification for liquid.Add the NaOH-95% ethanolic soln 350ml of 15%, saponification 1h under 60 DEG C of conditions, employ magnetic stirring apparatus in whole saponification process and constantly stir with the rotating speed of 120r/min, reacted rear hold over night.Filter, filter residue uses the organic solvent washing such as acetone, alcohol successively, and the oil-soluble impurities in removing sodium copper chlorophyllin, improves product purity, finally obtain chlorophyll copper sodium product salt 0.769g after drying, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Comparative example 2
Extract: precision takes the raw material silkworm excrement in 500g first silkworm age stage, the removing grains of sand, pulverize after lime particle, and the acetone with 80 % by weight is under optimum experimental condition, that is: temperature is 55 DEG C, the time is 2h, solid-liquid ratio is 1:3, add up extraction 3 times.Merge No. three extracting solutions, finally determine its chlorophyll extraction yield.
A saponification: said extracted liquid is adopted Rotary Evaporators vacuum concentration at 60 DEG C, obtains chlorophyll condensed cream 69.36g, calculates chlorophyll condensed cream yield, and detects cream Determination of Chlorophyll content.Add the NaOH-95 % by weight ethanolic soln 350ml of 15 % by weight wherein, saponification 1h under 60 DEG C of conditions, employs magnetic stirring apparatus and constantly stirs with the rotating speed of 120r/min, until react completely in whole saponification process.
Copper is put in acidifying: regulate the pH value of above-mentioned saponification liquor to be 3.5 with the HCl of 1:1, then dose the CuSO of 20 % by weight wherein
4solution 8.0mL, at 70 DEG C, constant temperature stirs 0.5h.
Secondary saponification: copper generation reacted after add wherein again 15 % by weight NaOH-95% ethanolic soln 350ml, stir 30min, filter, filter residue uses acetone, ethanol wash successively, oil-soluble impurities in removing sodium copper chlorophyllin, improve product purity, finally obtain chlorophyll copper sodium product salt 1.153g after drying, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Comparative example 3
Extract: precision takes 500g raw material silkworm excrement, the removing grains of sand, pulverize after lime particle, with 20 % by weight CuSO
4the acetone mixed solvent of-80 % by weight adopts optimum experimental condition under PH=3.5 condition, that is: temperature is 55 DEG C, the time is 2h, solid-liquid ratio is 1:3, accumulative extraction 3 times.Merge No. three extracting solutions, finally determine its chlorophyll extraction yield.
Saponification: said extracted liquid is adopted Rotary Evaporators vacuum concentration at 60 DEG C, obtains chlorophyll condensed cream 67.91g and calculates chlorophyll condensed cream yield, and detect cream Determination of Chlorophyll content.Add the NaOH-95% ethanolic soln 350ml of 15 % by weight wherein, saponification 1h under 60 DEG C of conditions, employ magnetic stirring apparatus in whole saponification process and constantly stir with the rotating speed of 120r/min, until react completely.Hold over night, filter, filter residue uses the organic solvent washing such as acetone, alcohol successively, oil-soluble impurities in removing sodium copper chlorophyllin, improve product purity, finally obtain chlorophyll copper sodium product salt 1.334g after drying, finished product chlorophyll copper sodium purity salt can be calculated by sodium copper chlorophyllin typical curve.The results are shown in Table 2.
Table 2
Can significantly be found out with comparing of comparative example 1-3 by embodiment 1, adopt existing three kinds of technique chlorophyll extraction yields also higher, cream yield is large; But cream Determination of Chlorophyll content is lower, interior impure many, cause the stickiness of cream to increase, mobility is bad, so that follow-up saponification, copper generation reaction and refining weak effect, finally causes the sodium copper chlorophyllin product production that obtains low, purity difference.
As can be seen from above-mentioned experimental result, compared with existing technique, in method of the present invention first with enzyme by carrying out again extracting after the macromolecular substance degraded in raw material, saponification, acidifying copper generation etc. step, effectively can overcome that the chlorophyll content existed in technique is in the past low, foreign matter content is high, the defect of sodium copper chlorophyllin poor product quality.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be undertaken combining the repetition in order to avoid unnecessary by any suitable mode, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (9)
1. the method for chlorophyll copper sodium extracting from silkworm excrement, it is characterized in that, the method comprises carries out enzymolysis with enzyme to silkworm excrement,
Wherein, described enzyme be selected from the group be made up of cellulase, hemicellulase, polygalacturonase, Lipase and proteolytic enzyme one or more,
Wherein, the method comprises the following steps:
(1) silkworm excrement and enzyme and water are contacted at 25-40 DEG C also enzymolysis 2-8h and obtain enzymolysis product;
(2) by enzymolysis product granulation, the silkworm excrement particle that acquisition diameter is 0.2-0.8cm, length is 1-2cm;
(3) with the chlorophyll in acetone extraction silkworm excrement particle, extracting solution is obtained;
(4) saponification process is carried out to extracting solution and obtain saponification liquor;
(5) acidifying is carried out to saponification liquor and put Copper treatment acquisition sodium copper chlorophyllin.
2. method according to claim 1, wherein, described proteolytic enzyme be selected from the group be made up of thiol proteinase, aspartate protease, serine/threonine protein enzyme and metalloprotease one or more; Described cellulase be selected from the group be made up of circumscribed beta-glucanase, Endo-β-glucanase, zytase and beta-glucosidase one or more; Described hemicellulase be selected from the group be made up of xylosidase, arabinoxylanase, glucuroide, mannase and tilactase one or more; Described polygalacturonase be selected from the group be made up of protopectinase, pectinesterase lytic enzyme, polygalacturonase class and pectate lyase one or more.
3. method according to claim 1; wherein; relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.01-0.2g, the consumption of hemicellulase is 0.008-0.15g, the consumption of polygalacturonase is 0.005-0.15g, the consumption of Lipase is 0.005-0.1g, the consumption of proteolytic enzyme is 0.003-0.18g.
4. according to the method in claim 1-3 described in any one; wherein, described method comprises first silkworm excrement and cellulase, hemicellulase, polygalacturonase and one or more in Lipase being contacted and adding proteolytic enzyme again after enzymolysis 2-5h and carries out enzymolysis 3-6h.
5. according to the method in claim 1-3 described in any one, wherein, described silkworm excrement is the silkworm excrement in the first silkworm age stage, and described enzyme comprises cellulase, hemicellulase, proteolytic enzyme, polygalacturonase and Lipase;
Wherein, relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.05-0.1g, the consumption of hemicellulase is 0.03-0.075g, the consumption of proteolytic enzyme is 0.03-0.07g, the consumption of polygalacturonase is 0.02-0.08g, the consumption of Lipase is 0.008-0.02g.
6. according to the method in claim 1-3 described in any one, wherein, described silkworm excrement is the silkworm excrement in the second silkworm age stage, and described enzyme comprises cellulase, polygalacturonase and proteolytic enzyme;
Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.05-0.12g, the consumption of polygalacturonase is 0.07-0.15g, the consumption of proteolytic enzyme is 0.06-0.1g.
7. according to the method in claim 1-3 described in any one, wherein, described silkworm excrement is the silkworm excrement in the 3rd silkworm age stage, and described enzyme comprises cellulase, hemicellulase and polygalacturonase;
Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.05-0.11g, the consumption of hemicellulase is 0.05-0.08g, the consumption of polygalacturonase is 0.02-0.05g.
8. according to the method in claim 1-3 described in any one, wherein, described silkworm excrement is the silkworm excrement in the 4th silkworm age stage, and described enzyme comprises cellulase, hemicellulase, polygalacturonase and Lipase;
Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.01-0.03g, the consumption of hemicellulase is 0.003-0.008g, the consumption of polygalacturonase is 0.01-0.04g, the consumption of Lipase is 0.02-0.08g.
9. according to the method in claim 1-3 described in any one, wherein, described silkworm excrement is the silkworm excrement in the 5th silkworm age stage, and described enzyme comprises cellulase, hemicellulase and polygalacturonase;
Relative to the silkworm excrement of 100g, the consumption of water is 20-65g, the consumption of cellulase is 0.02-0.07g, the consumption of hemicellulase is 0.01-0.054g, the consumption of polygalacturonase is 0.05-0.13g.
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