CN103013165A - Method for preparing monascus red pigment by utilizing immobilized enzymes - Google Patents
Method for preparing monascus red pigment by utilizing immobilized enzymes Download PDFInfo
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- CN103013165A CN103013165A CN2012105498014A CN201210549801A CN103013165A CN 103013165 A CN103013165 A CN 103013165A CN 2012105498014 A CN2012105498014 A CN 2012105498014A CN 201210549801 A CN201210549801 A CN 201210549801A CN 103013165 A CN103013165 A CN 103013165A
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- monascorubin
- immobilized enzyme
- enzymolysis
- monascus
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 47
- 241000228347 Monascus <ascomycete fungus> Species 0.000 title abstract description 16
- 239000001054 red pigment Substances 0.000 title abstract 9
- 239000007788 liquid Substances 0.000 claims abstract description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
- 108090000790 Enzymes Proteins 0.000 claims abstract description 25
- 238000002360 preparation method Methods 0.000 claims abstract description 23
- 238000005406 washing Methods 0.000 claims abstract description 19
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 239000002253 acid Substances 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 239000003513 alkali Substances 0.000 claims abstract description 6
- 238000001035 drying Methods 0.000 claims abstract description 6
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 79
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 35
- 229940088598 enzyme Drugs 0.000 claims description 24
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 19
- 238000003756 stirring Methods 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 239000006187 pill Substances 0.000 claims description 16
- 238000001556 precipitation Methods 0.000 claims description 16
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 15
- 235000010413 sodium alginate Nutrition 0.000 claims description 13
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 12
- 238000003916 acid precipitation Methods 0.000 claims description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 11
- 241000031003 Monascus ruber Species 0.000 claims description 11
- 239000000661 sodium alginate Substances 0.000 claims description 11
- 229940005550 sodium alginate Drugs 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 229920002521 macromolecule Polymers 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 235000017550 sodium carbonate Nutrition 0.000 claims description 9
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 238000000967 suction filtration Methods 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 230000001105 regulatory effect Effects 0.000 claims description 7
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 7
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 7
- 239000012670 alkaline solution Substances 0.000 claims description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000006386 neutralization reaction Methods 0.000 claims description 6
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 claims description 5
- 239000004382 Amylase Substances 0.000 claims description 5
- 108010065511 Amylases Proteins 0.000 claims description 5
- 102000013142 Amylases Human genes 0.000 claims description 5
- 229920002498 Beta-glucan Polymers 0.000 claims description 5
- 108010059892 Cellulase Proteins 0.000 claims description 5
- 229920000858 Cyclodextrin Polymers 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 5
- 239000004368 Modified starch Substances 0.000 claims description 5
- 229920000881 Modified starch Polymers 0.000 claims description 5
- 102000035195 Peptidases Human genes 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 108010059820 Polygalacturonase Proteins 0.000 claims description 5
- 235000019418 amylase Nutrition 0.000 claims description 5
- 229940106157 cellulase Drugs 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 235000019426 modified starch Nutrition 0.000 claims description 5
- 235000010987 pectin Nutrition 0.000 claims description 5
- 239000001814 pectin Substances 0.000 claims description 5
- 229920001277 pectin Polymers 0.000 claims description 5
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 5
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 3
- 239000004310 lactic acid Substances 0.000 claims description 3
- 235000014655 lactic acid Nutrition 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 2
- 239000001099 ammonium carbonate Substances 0.000 claims description 2
- 230000018044 dehydration Effects 0.000 claims description 2
- 238000006297 dehydration reaction Methods 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 235000011181 potassium carbonates Nutrition 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000000049 pigment Substances 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- 239000000084 colloidal system Substances 0.000 abstract description 2
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 238000000151 deposition Methods 0.000 abstract 2
- 230000008021 deposition Effects 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000003301 hydrolyzing effect Effects 0.000 abstract 1
- 239000002184 metal Substances 0.000 abstract 1
- 230000003472 neutralizing effect Effects 0.000 abstract 1
- 239000000203 mixture Substances 0.000 description 9
- 238000002156 mixing Methods 0.000 description 8
- 238000011084 recovery Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- 239000012295 chemical reaction liquid Substances 0.000 description 5
- 230000001143 conditioned effect Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000001376 precipitating effect Effects 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 239000006185 dispersion Substances 0.000 description 4
- 238000004945 emulsification Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- -1 carboxylate salt Chemical class 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 0 CCC*(C1C2C1*C1=CC(C(CC(C=O)OC)CC(*)=*)C3(C)O)C2OC#CC=C1C3=O Chemical compound CCC*(C1C2C1*C1=CC(C(CC(C=O)OC)CC(*)=*)C3(C)O)C2OC#CC=C1C3=O 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000113306 Monascus purpureus Species 0.000 description 1
- 235000002322 Monascus purpureus Nutrition 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
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- 235000012970 cakes Nutrition 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
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- 239000003638 chemical reducing agent Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000576 food coloring agent Substances 0.000 description 1
- 235000002864 food coloring agent Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for preparing a monascus pigment, in particular to a method for preparing a monascus red pigment by utilizing immobilized enzymes. The method comprises the steps as follows: taking a monascus liquid fermentation solution as a raw material, homogenizing, enzymatically hydrolyzing the immobilized enzymes, alkalifying and filtering to obtain a monascus red pigment solution; and carrying out acid deposition on the monascus red pigment solution, washing and neutralizing the deposits, dehydrating and drying to obtain the monascus red pigment. The method has the advantages as follows: on one hand, the fermentation solution is effectively dispersed, broken, emulsified and mixed by utilizing a colloid mill, so that the subsequent enzymatic hydrolysis efficiency is improved and the monascus red pigment in monascus is fully released; and on the other hand, an enzyme preparation can be recycled, the reaction conditions are moderate, the monascus red pigment is concentrated through the acid deposition method, the process is simple, the energy consumption is low, and the product is easy to separate. The production cost can be remarkably reduced through the monascus red pigment prepared with the method. The monascus red pigment has the maximum absorption wavelength of 499 nm, is good in water solubility and light, heat, acid, alkali and metal stabilities, and is favorable for the monascus pigment industry development.
Description
Technical field
The present invention relates to a kind of preparation method of monascorubin, particularly a kind of method of utilizing immobilized enzyme to prepare monascorubin.
Background technology
Along with the development of foodstuffs industry and the day by day raising of people's living standard, food color more and more is subject to people's attention as integral part important in the foodstuff additive, particularly in recent years, along with the attention of its people to food-safety problem, adopting natural pigment to replace synthetic colour has been trend of the times.Monascorubin is as the deep processed product of agricultural-food, and is safe, and good stability has good development prospect.
Chinese invention patent ZL200510044948.8 discloses a kind of method of utilizing enzymolysis process to extract water-soluble monascus pigment, a kind of method of utilizing enzymolysis process to extract water-soluble monascus pigment of the present invention comprises the steps: Red kojic rice → immersion alkaline hydrolysis → heating gelatinization → enzymolysis → filtering separation → Acid precipitation → washing → neutralization → drying → pulverizing → packing.This invention is take Red kojic rice as raw material, through soaking alkaline hydrolysis, heating gelatinization, the techniques such as enzymolysis, Acid precipitation, extraction water-soluble monascus pigment.This patent adopts free enzyme liquid to carry out enzymolysis because resolvase is dissolved in the reaction system, make enzyme can't be from reaction system Separation and Recovery, cause the production cost of monascorubin high, be unfavorable for continuous suitability for industrialized production.
Summary of the invention
The purpose of this invention is to provide a kind ofly take the monascus ruber liquid fermentation liquid as raw material, by homogenizing, immobilized enzyme enzymolysis, Acid precipitation enrichment, prepare the method for monascorubin.Dispersion, fragmentation, emulsification, mixed fermentation liquid improve follow-up enzymolysis efficiency effectively, allow simultaneously the monascorubin in the thalline fully discharge, and improve pigment lixiviate yield; And enzyme liquid is difficult for Separation and Recovery in the traditional technology of solve determining, and the purification enrichment process is complicated, and cost is high, the problem that product look valency is low, and can improve resource utilization.
The present invention is take the monascus ruber liquid fermentation liquid as raw material, by homogenizing, immobilized enzyme enzymolysis, alkalization, filter to get monascorubin solution; Monascorubin solution is by Acid precipitation, and washing of precipitate neutralization, dehydration, drying namely obtain monascorubin.
At first, utilize milling treatment of colloid Monascus anka Nakazawa et sato fermented liquid, utilize fluid foods to be subjected to the effect of centrifugal force, pressure by between the stator of high speed relative movement and rotor, shear, friction, high-frequency vibration, effectively pulverize, emulsification, homogeneous, dispersion, mixing etc., thereby obtain the material of retrofit, impel the colour component in the thalline to discharge fully, be conducive to simultaneously improve the efficient of later stage enzymolysis.
Utilize the immobilization prozyme that fermented liquid is carried out enzymolysis processing, one side proteolytic enzyme wherein can make the protein in the fermented liq system be hydrolyzed into rapidly the protein fragments of solubility, obviously improves the water-soluble of monascorubin, the quality of Effective Raise product; On the other hand, amylase and/or polygalacturonase and/or cellulase and/or zytase and/or beta-glucan enzyme can impel the degraded of somatic cells wall, increase the saturating property of thalline, are conducive to the release of monascorubin component, and then have improved the utilization ratio of resource.Process realizes the recycle of enzyme by reclaiming immobilized enzyme, reduces material consumption.
Enzymolysis solution opens the lactone bond in the monascorubin molecule wherein through alkalization, generates carboxylate salt, impels colour component to be dissolved in solution system, improves the yield of monascorubin.
Alkalization removes by filter insolubles wherein after finishing, and obtains monascorubin solution.Then utilize Acid precipitation that monascorubin is precipitated out from reaction solution, realize the purpose of enrichment; In alkali and the method for dissolving, make the monascorubin acid deposit generate carboxylate salt soluble in water again, improve the water-soluble and homogeneity of pigment, obtain monascorubin.
Its conversion process is as follows:
A kind of method of utilizing immobilized enzyme to prepare monascorubin of the present invention, concrete steps are as follows:
(1) preparation of immobilized enzyme
The macromolecular compound carrier adds the solution that water is mixed with 15 g/L～100g/L concentration, is under 20 ℃～60 ℃ conditions in temperature, stirs, and makes its dissolving; Then adding with macromolecular compound carrier mass ratio is the enzyme of 1:1～1:50, stirs, and places on 20 ℃～60 ℃ the constant-temperature table vibration 0.5h～6h; Above-mentioned mixed solution is dropwise splashed in the calcium chloride solution of 0.1mol/L～2.5 mol/L, under 3 ℃～5 ℃ conditions, leave standstill 30min～150min, its cohesion is solidified, collect gelled pill, and with the washing of the sodium chloride solution of 0.08 mol/L～0.3 mol/L, suction filtration is removed free water content, collects the gelled pill that obtains and is immobilized enzyme;
(2) preparation of monascorubin
1. raw material: take the monascus ruber liquid fermentation liquid as raw material;
2. homogenizing: fermented liquid is advanced colloidal mill grind, broken fineness is at 0.1 μ m～10 μ m, and colloidal mill action time is 5 min ~ 120min, gets liquid after the homogenizing;
3. enzymolysis: regulating step 2. the liquid after the homogenizing to pH 4.0～pH 6.0, then carry out enzymolysis to the immobilized enzyme that wherein adds step (1) preparation, the mass ratio of immobilized enzyme and homogenized liquid is 1:1～1:100, and enzymatic hydrolysis condition is 20 ℃～70 ℃, and enzymolysis time is 1h～48h;
4. immobilized enzyme reclaims: with the 3. reaction solution coarse filtration after the enzymolysis processing of step, reclaim immobilized enzyme, obtain simultaneously filtrate and be enzymolysis solution;
5. alkalization: add alkali lye in the enzymolysis solution that 4. obtains to step, regulate enzymolysis solution to pH 8.0～pH13.0, concentration of lye is 0.5mol/L～10.0 mol/L.
6. filter: with 5. reacting liquid filtering of step, remove insolubles, obtain monascorubin solution;
7. Acid precipitation: 6. add acid solution in the monascorubin solution of gained to step, regulator solution makes its precipitation to pH 1～pH3, leave standstill 0.5h～24h after, the collecting precipitation thing;
8. wash, neutralize: the throw out that 7. step is obtained is washed with water to pH5～pH6, with the alkaline solution dissolution precipitation thing of 0.01 mol/L～5.0 mol/L, and is adjusted to pH 7～pH12, obtains the monascorubin aqueous solution;
9. dry, pulverizing: the monascorubin aqueous solution that 8. step is obtained dewaters, drying treatment, and the dried powder that obtains is prepared monascorubin.
Wherein the described macromolecular compound carrier of step (1) is sodium alginate, and one or more the mixture in sodium alginate and gelatin, modified starch, pectin and the cyclodextrin;
Alkaline solution in the described alkaline hydrolysis of step (2) is one or more the mixture in sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution and the sodium hydrogen carbonate solution;
Acid solution in the described Acid precipitation of step (2) is one or more the mixture in hydrochloric acid, sulfuric acid, acetic acid, citric acid, phosphoric acid and the lactic acid;
The described washing of step (2), the used alkaline solution of neutralization are one or more the mixture in sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, solution of potassium carbonate, sodium hydrogen carbonate solution, potassium bicarbonate solution and the ammonium bicarbonate soln.
Above-mentioned enzyme for enzymolysis is immobilized enzyme, and the enzyme that is fixed is the prozyme that contains proteolytic enzyme and amylase and/or polygalacturonase and/or cellulase and/or zytase and/or beta-glucan enzyme.The immobilized enzyme enzymolysis process is the stirring enzymolysis that carries out in reactor, or immobilized enzyme is packed in the packed column, acidizing fluid is pumped into the circulation enzymolysis that packed column carries out again.
The monascorubin of the inventive method preparation, soluble in water; Possess good thermostability, under 0 ℃～120 ℃ environment on pigment without impact; Pigment is at pH〉have satisfactory stability in 3 scopes, Oxidizing and Reducing Agents does not have detrimentally affect to pigment, and dextrose plus saccharose solution is on not impact of pigment, and the use of various common foodstuff additive does not affect the quality of pigment substantially, and is stable to metal ion.
The monascorubin of the inventive method preparation can be used for the painted of textile industry, plastic cement industry and press; Can be used as food colorant and be used for the painted of the industry processing such as grain, meat product, fishery products, can be used for assembled alcoholic drinks, soda pop, fruit juice (flavor) type beverage, jam, candy, cake etc.; The processing that can be used for the industries such as pharmaceuticals and makeup is painted.
The present invention assists homogenizing technology and enzyme immobilization technology for the preparation of monascorubin colloidal mill, can reduce production costs, and improves pigment lixiviate yield, and reduces enzyme liquid to the contaminating impurity that pigment product causes, and is conducive to improve the quality of products.
The present invention compares with traditional method, has the following advantages:
1, utilizes colloidal mill that the monascus ruber fermented liquid is carried out homogenizing and process, effectively pulverize, emulsification, homogeneous, dispersion, mixing etc., improve the rate of releasing of the colour component in the thalline, be conducive to simultaneously improve the efficient of later stage enzymolysis.
2, utilize immobilized enzyme to carry out enzymolysis, Enzymic stability is increased, easy Separation and Recovery from reactive system, but saving resource reduces cost.
3, the preparation method of monascorubin of the present invention dissolves to realize purification enrichment to monascorubin by Acid precipitation and basic solution, and is capable of reducing energy consumption.
Adopt the monascorubin of the inventive method preparation, its maximum absorption wavelength is 499nm, and the look valency is higher than 200, and the monascorubin extraction rate reached is more than 95%.
The gordian technique explanation:
1, colloidal mill homogenizing technology
Colloidal mill is to utilize fluid or semi-fluid material to be subjected to the effect of centrifugal force, pressure by between the stator of high speed relative movement and rotor, shear, friction, high-frequency vibration, effectively pulverize, emulsification, homogeneous, dispersion, mixing etc., wherein broken fineness and colloidal mill action time, treatment effect impact on material is larger, a kind of method of utilizing immobilized enzyme to prepare monascorubin of the present invention, fermented liquid is advanced colloidal mill to be ground, broken fineness is at 0.1 μ m～10 μ m, colloidal mill action time is 5 min ~ 120min, gets liquid after the homogenizing.
2, immobilized enzyme zymolysis technique
The macromolecular compound carrier adds the solution that water is mixed with 15 g/L～100g/L concentration, is under 20 ℃～60 ℃ conditions in temperature, stirs, and makes its dissolving; Then adding with macromolecular compound carrier mass ratio is the enzyme of 1:1～1:50, stirs, and places on 20 ℃～60 ℃ the constant-temperature table vibration 0.5h～6h; Above-mentioned mixed solution is dropwise splashed in the calcium chloride solution of 0.1mol/L～2.5 mol/L, under 3 ℃～5 ℃ conditions, leave standstill 30min～150min, its cohesion is solidified, collect gelled pill, and with the washing of the sodium chloride solution of 0.08 mol/L～0.3 mol/L, suction filtration is removed free water content, collects the gelled pill that obtains and is immobilized enzyme; Used macromolecular compound carrier comprises sodium alginate, and one or more the mixture in sodium alginate and gelatin, modified starch, pectin and the cyclodextrin.Described enzyme is immobilized enzyme, and the enzyme that is fixed is the prozyme that contains proteolytic enzyme and amylase and/or polygalacturonase and/or cellulase and/or zytase and/or beta-glucan enzyme.
The immobilized enzyme enzymatic hydrolysis condition is 20 ℃～70 ℃, and pH 4.0～pH 6.0, enzymolysis time are 1h～48h; The mass ratio of immobilized enzyme and homogenized liquid is 1:1～1:100, and enzymolysis process is the stirring enzymolysis that carries out in reactor, or immobilized enzyme is packed in the packed column, acidizing fluid is pumped into the circulation enzymolysis that packed column carries out again.The enzymolysis yield that the immobilized enzyme recycling still keeps more than 90% for 20 times.
Embodiment
Below in conjunction with specific embodiment the present invention is further described, so that those skilled in the art more understands the present invention, but does not therefore limit the present invention.
Embodiment 1
(1) preparation of immobilized enzyme
The sodium alginate of 35 g is mixed with 25 g gelatin, be added in the 1L water, under 45 ℃ of conditions, stirring and dissolving, the prozyme of adding 35g, stirring and evenly mixing, on 45 ℃ of constant-temperature tables, then the 100r/min 0.5h that vibrates dropwise injects this mixed solution the calcium chloride solution of 1.5mol/L, under 4 ℃ of conditions, leave standstill 50min, its cohesion is solidified, collect gelled pill, with the sodium chloride solution washing of 0.2 mol/L, suction filtration is removed free water content, collects being fixed of gelled pill enzyme.
(2) preparation of monascorubin
The liquid fermentation liquid of monascus ruber is advanced colloidal mill carry out homogenizing and process, broken fineness is 5 μ m, and the treatment time is 30min; After homogenizing finished, regulator solution was to pH4.5, was that the amount of 1:50 adds the immobilization prozyme according to the mass ratio of weight in wet base and homogenized liquid, filtered the recovery immobilized enzyme after 8 hours in 50 ℃ of shaking table enzymolysis; Then be that the sodium hydroxide solution of 0.5mol/L is regulated enzymolysis solution to pH8.0 with concentration, filter, remove insolubles, get monascorubin solution,, left standstill 2 hours to pH 2.7 with the phosphoric acid solution conditioned reaction liquid of 5 mol/L, filter, collecting precipitation, water washing and precipitating with the sodium carbonate solution dissolution precipitation thing of 0.1 mol/L, and is adjusted to pH8.0 to pH5, spray-dried, namely get the monascorubin powder.
Embodiment 2
(1) preparation of immobilized enzyme
The sodium alginate soln of 4g is mixed with the modified starch of 2g, be added in the 100ml water, under 45 ℃ of conditions, stirring and dissolving, the prozyme of adding 2g, stirring and evenly mixing, on 37 ℃ of constant-temperature tables, then the 60r/min 1.5h that vibrates dropwise injects this mixed solution the calcium chloride solution of 1.5mol/L, under 4 ℃ of conditions, leave standstill 110min, its cohesion is solidified, collect gelled pill, with the sodium chloride solution washing of 0.2 mol/L, suction filtration is removed free water content, collects being fixed of gelled pill enzyme.
(2) preparation of monascorubin
The liquid fermentation liquid of monascus ruber is advanced colloidal mill carry out homogenizing and process, broken fineness is 1 μ m, and the treatment time is 20min; After homogenizing finished, regulator solution was to pH5.0, was that the amount of 1:30 adds the immobilization prozyme according to the mass ratio of weight in wet base and homogenized liquid, filtered the recovery immobilized enzyme after 6 hours in 55 ℃ of shaking table enzymolysis; Then be that the sodium hydroxide solution of 0.8mol/L is regulated enzymolysis solution to pH8.5 with concentration, filter, remove insolubles, get monascorubin solution,, placed 4 hours to pH 2.5 with the citric acid solution conditioned reaction liquid of 5.5 mol/L, centrifugal, collecting precipitation, water washing and precipitating with the sodium hydroxide solution dissolution precipitation thing of 0.1 mol/L, and is adjusted to pH8 to pH5, spray-dried, namely get the monascorubin powder.
Embodiment 3
(1) preparation of immobilized enzyme
The 100g sodium alginate is added in the 8L water, 40 ℃ of lower stirring and dissolving, adds the prozyme of 50g, stirring and evenly mixing, on 37 ℃ of constant-temperature tables, the 90r/min 0.6h that vibrates dropwise injects this mixed solution the calcium chloride solution of 2.0mol/L, under 3 ℃ of conditions, leave standstill 120min, its cohesion is solidified, collect gelled pill, with the sodium chloride solution washing of 0.15mol/L, suction filtration is removed free water content, collects being fixed of gelled pill enzyme.
(2) preparation of monascorubin
The liquid fermentation liquid of monascus ruber is advanced colloidal mill carry out homogenizing and process, broken fineness is 4 μ m, and the treatment time is 60min; After homogenizing finished, regulator solution was to pH4.5, was that the amount of 1:40 adds the immobilization prozyme according to the mass ratio of weight in wet base and homogenized liquid, filtered the recovery immobilized enzyme after 5 hours in 65 ℃ of shaking table enzymolysis; Then be that the potassium hydroxide solution of 2mol/L is regulated enzymolysis solution to pH10.0 with concentration, filter, remove insolubles, get monascorubin solution,, placed 3 hours to pH 2.0 with the hydrochloric acid soln conditioned reaction liquid of 5 mol/L, filter, collecting precipitation, water washing and precipitating with the sodium carbonate solution dissolution precipitation thing of 0.3 mol/L, and is adjusted to pH8.5 to pH5, spray-dried, namely get the monascorubin powder.
Embodiment 4
(1) preparation of immobilized enzyme
30 g sodium alginates and 20g cyclodextrin are added in the 1L water, 37 ℃ of lower stirring and dissolving, add the 30g prozyme, stirring and evenly mixing, on 37 ℃ of constant-temperature tables, the 120r/min 2.5h that vibrates, this mixed solution is dropwise injected the calcium chloride solution of 1.0mol/L, under 5 ℃ of conditions, leave standstill 30min, its cohesion is solidified, collect gelled pill, with the sodium chloride solution washing of 0.2 mol/L, suction filtration is removed free water content, collects being fixed of gelled pill enzyme, immobilized enzyme is packed in the glass column, make immobilized enzyme column;
(2) preparation of monascorubin
The liquid fermentation liquid of monascus ruber is advanced colloidal mill carry out homogenizing and process, broken fineness is 8 μ m, and the treatment time is 50min; After homogenizing finished, regulator solution pumped into immobilized enzyme column with homogenized liquid and carries out enzymolysis to pH4.5, and the enzymolysis effluent liquid is got back in the stainless steel reactor, circulating reaction like this, and 55 ℃ of hydrolysis temperatures, enzymolysis time 10 hours after enzymolysis finishes, reclaims immobilized enzyme; Then be that the sodium carbonate solution of 4mol/L is regulated enzymolysis solution to pH9.0 with concentration, filter, remove insolubles, get monascorubin solution,, placed 6 hours to pH 2.0 with the hydrochloric acid soln conditioned reaction liquid of 6mol/L, centrifugal, collecting precipitation, water washing and precipitating with the sodium hydrogen carbonate solution dissolution precipitation thing of 0.3 mol/L, and is adjusted to pH7.5 to pH5, spray-dried, namely get the monascorubin powder.
Embodiment 5
(1) preparation of immobilized enzyme
30 g sodium alginates and 20g pectin are added in the 1L water, 45 ℃ of lower stirring and dissolving, add the prozyme of 10g, stirring and evenly mixing, on 45 ℃ of constant-temperature tables, the 150r/min 1.5h that vibrates dropwise injects this mixed solution the calcium chloride solution of 2mol/L, under 4 ℃ of conditions, leave standstill 120min, its cohesion is solidified, collect gelled pill, with the sodium chloride solution washing of 0.1 mol/L, suction filtration is removed free water content, collects being fixed of gelled pill enzyme;
(2) preparation of monascorubin
The liquid fermentation liquid of monascus ruber is advanced colloidal mill carry out homogenizing and process, broken fineness is 0.5 μ m, and the treatment time is 15min; After homogenizing finished, regulator solution was to pH5.0, was that the amount of 1:80 adds the immobilization prozyme according to the mass ratio of weight in wet base and homogenized liquid, filtered the recovery immobilized enzyme after 2 hours in 60 ℃ of shaking table enzymolysis; Then be that the potassium hydroxide solution of 1 mol/L is regulated enzymolysis solution to pH9.0 with concentration, filter, remove insolubles, get monascorubin solution,, placed 6 hours to pH 2.5 with the sulphuric acid soln conditioned reaction liquid of 6mol/L, filter, collecting precipitation, water washing and precipitating with the sodium hydrogen carbonate solution dissolution precipitation thing of 0.1 mol/L, and is adjusted to pH8.5 to pH5, spray-dried, namely get the monascorubin powder.
In above-described embodiment 1～6, the macromolecular compound carrier comprises sodium alginate, and one or more the mixture in sodium alginate and gelatin, modified starch, pectin and the cyclodextrin; The enzyme that is fixed is the prozyme that contains proteolytic enzyme and amylase and/or polygalacturonase and/or cellulase and/or zytase and/or beta-glucan enzyme; The alkali that is used for alkaline hydrolysis comprises one or more mixture of sodium hydroxide, potassium hydroxide, yellow soda ash and sodium bicarbonate; Enzymolysis process is the stirring enzymolysis that carries out in reactor, or immobilized enzyme is packed in the packed column, acidizing fluid is pumped into the circulation enzymolysis that packed column carries out again; The acid that is used for Acid precipitation comprises one or more mixture of hydrochloric acid, sulfuric acid, acetic acid, citric acid, phosphoric acid and lactic acid; Be used for one or more the mixture that neutralization and the sedimentary alkali of dissolving acid comprise sodium hydroxide, potassium hydroxide, yellow soda ash, salt of wormwood, sodium bicarbonate, saleratus and bicarbonate of ammonia; The recycling number of times and should look particular case and do suitable adjustment of its kind and consumption and immobilized enzyme.
Claims (9)
1. method of utilizing immobilized enzyme to prepare monascorubin comprises following step: take the monascus ruber liquid fermentation liquid as raw material, by homogenizing, immobilized enzyme enzymolysis, alkalization, filter to get monascorubin solution; Monascorubin solution is by Acid precipitation, and washing of precipitate neutralization, dehydration, drying namely obtain monascorubin.
2. the method for utilizing immobilized enzyme to prepare monascorubin according to claim 1 specifically comprises following step:
(1) preparation of immobilized enzyme
The macromolecular compound carrier adds the solution that water is mixed with 15～100g/L concentration, is under 20 ℃～60 ℃ conditions in temperature, is stirred to its dissolving; Then adding with macromolecular compound carrier mass ratio is the enzyme of 1:1～1:50, stirs, and places on 20 ℃～60 ℃ the constant-temperature table vibration 0.5h～6h; Above-mentioned mixed solution is dropwise splashed in the calcium chloride solution of 0.1mol/L～2.5 mol/L, under 3 ℃～5 ℃ conditions, leave standstill 30～150min, its cohesion is solidified, collect gelled pill, and with the washing of the sodium chloride solution of 0.08 mol/L～0.3 mol/L, suction filtration is removed free water content, collects the gelled pill that obtains and is immobilized enzyme;
(2) preparation of monascorubin
1. raw material: take the monascus ruber liquid fermentation liquid as raw material;
2. homogenizing: fermented liquid is advanced colloidal mill grind, broken fineness is at 0.1～10 μ m, and colloidal mill action time is 5 min～120min, gets liquid after the homogenizing;
3. enzymolysis: regulating step 2. the liquid after the homogenizing to pH 4.0～6.0, then carry out enzymolysis to the immobilized enzyme that wherein adds step (1) preparation, the mass ratio of immobilized enzyme and homogenized liquid is 1:1～1:100, and enzymatic hydrolysis condition is 20 ℃～70 ℃, and enzymolysis time is 1～48h;
4. immobilized enzyme reclaims: with the 3. reaction solution coarse filtration after the enzymolysis processing of step, reclaim immobilized enzyme, obtain simultaneously filtrate and be enzymolysis solution;
5. alkalization: add alkali lye in the enzymolysis solution that 4. obtains to step, regulate enzymolysis solution to pH 8.0～13.0, concentration of lye is 0.5～10.0 mol/L;
6. filter: with 5. reacting liquid filtering of step, remove insolubles, obtain monascorubin solution;
7. Acid precipitation: 6. add acid solution in the monascorubin solution of gained to step, regulator solution makes its precipitation to pH 1～3, leave standstill 0.5h～24h after, the collecting precipitation thing;
8. wash, neutralize: the throw out that 7. step is obtained is washed with water to pH5～pH6, with the alkaline solution dissolution precipitation thing of 0.01～5.0 mol/L, and is adjusted to pH 7～12, obtains the monascorubin aqueous solution;
9. dry, pulverizing: the monascorubin aqueous solution that 8. step is obtained dewaters, drying treatment, and the dried powder that obtains is prepared monascorubin.
3. the method for utilizing immobilized enzyme to prepare monascorubin according to claim 2, it is characterized in that: the described macromolecular compound carrier of step (1) is sodium alginate, and at least a in sodium alginate and gelatin, modified starch, pectin and the cyclodextrin.
4. the method for utilizing immobilized enzyme to prepare monascorubin according to claim 2 is characterized in that: the alkaline solution in the described alkalization of step (2) is at least a in sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution and the sodium hydrogen carbonate solution.
5. the method for utilizing immobilized enzyme to prepare monascorubin according to claim 2 is characterized in that: the acid solution in the described Acid precipitation of step (2) is at least a in hydrochloric acid, sulfuric acid, acetic acid, citric acid, phosphoric acid and the lactic acid.
6. the method for utilizing immobilized enzyme to prepare monascorubin according to claim 2 is characterized in that: the described washing of step (2), the used alkaline solution of neutralization are at least a in sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, solution of potassium carbonate, sodium hydrogen carbonate solution, potassium bicarbonate solution and the ammonium bicarbonate soln.
7. a kind of method of utilizing immobilized enzyme to prepare monascorubin according to claim 2 is characterized in that: the described enzyme of step (1) is to contain at least a in proteolytic enzyme and amylase, polygalacturonase, cellulase, zytase, the beta-glucan enzyme.
8. a kind of method of utilizing immobilized enzyme to prepare monascorubin according to claim 2 is characterized in that: the stirring enzymolysis of the described enzymolysis process of step (2) for carrying out in reactor.
9. a kind of method of utilizing immobilized enzyme to prepare monascorubin according to claim 2, it is characterized in that: the described enzymolysis process of step (2) is: immobilized enzyme is packed in the packed column, acidizing fluid is pumped into the circulation enzymolysis that packed column carries out again.
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| CN104448919A (en) * | 2014-12-26 | 2015-03-25 | 天津商业大学 | Purifying method of hydroxy-containing pigment |
| CN110205342A (en) * | 2019-06-28 | 2019-09-06 | 福州大学 | A kind of method of pigment production increase during immobilization Fermentation Condition of Monascus spp |
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| US20230279233A1 (en) * | 2020-07-06 | 2023-09-07 | Chromologics Aps | Atrorosins as food colors |
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