CN113943765B - Modification method for stabilizing annatto in acidic aqueous phase - Google Patents

Modification method for stabilizing annatto in acidic aqueous phase Download PDF

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CN113943765B
CN113943765B CN202111267477.2A CN202111267477A CN113943765B CN 113943765 B CN113943765 B CN 113943765B CN 202111267477 A CN202111267477 A CN 202111267477A CN 113943765 B CN113943765 B CN 113943765B
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annatto
molecular sieve
aqueous phase
acidic aqueous
maltitol
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CN113943765A (en
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管永光
刘晓月
吕丹雨
王牧云
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Shanghai Jiaotong University
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    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/06—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical being a hydroxyalkyl group esterified by a fatty acid

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Abstract

The invention relates to a modification method for stabilizing annatto in an acidic aqueous phase, comprising the steps of: (1) Placing annatto and maltitol into a solvent for full dissolution, adding immobilized lipase and a molecular sieve A, and placing into an oscillation incubator for reaction to obtain a primary reaction mixture; (2) Adding a molecular sieve B into the preliminary reaction mixture, continuing to react, and separating and purifying the obtained reaction product to obtain the stable carmine maltitol ester with the acidic aqueous phase. The invention gives the annatto the property of stable existence in an acidic aqueous phase and has wider application potential in the field of food industry.

Description

Modification method for stabilizing annatto in acidic aqueous phase
Technical Field
The invention belongs to the technical field of pigment modification, and relates to a modification method for stabilizing annatto in an acidic aqueous phase.
Background
Annatto (bixin, C 25H30O4), also known as bixin, is a functional carotenoid extracted from the aril of annatto (Bixa orellana) seeds. As a natural yellow-orange pigment, it is widely used for coloring in the food industry. The annatto molecule consists of a polyunsaturated hydrocarbon chain containing 4 non-ortho methyl groups and 9 alternating double bonds between the chains, one end of the chain being a carboxylic acid group and the other end being a methyl ester group, so that the pigment has extremely high lipophilicity. Annatto consists mainly of 9' -cis-bixin, with minor amounts of all-trans-bixin and 9',13' -cis-bixin. In addition to its use as a natural colorant in foods, annatto has a variety of pharmaceutical activities including inhibition of DNA damage, inhibition of postprandial inflammation and liposome oxidation, antioxidant, anti-inflammatory and healing effects, and the like. Annatto has been approved and used by a number of countries and regions and is a safe food color. The united states food and drug administration has certified it as a "generally recognized safe" food, drug, and cosmetic colorant. The European Union food Law also recognizes the use of annatto as a food colorant, numbered E160 (b). The national department of health has been prescribed for the range of use and the maximum limit of annatto from 1997, and is mainly applicable to cheeses, sausages, smoked foods, dairy products and the like.
As mentioned above, annatto is very hydrophobic and insoluble in water, which limits its use in water-based foods. The annatto sold in the market at present is mainly annatto added with a large amount of maltodextrin, wherein the real pigment content is very low, the purity is not high, and the use in actual production is influenced. By introducing hydrophilic residues into the molecular structure of annatto, increasing the polarity of the molecules, and thus increasing the water solubility of annatto would be a good solution. Similar attempts have been made in the literature by researchers, humeau et al (Humeau C,Rovel B,Girardin M.Enzymatic esterification of bixin by L-ascorbic acid[J].Biotechnology Letters,2000,22(2):165-168.) using candida antarctica immobilized lipase to catalyze the reaction of annatto with ascorbic acid, which found that annatto can be transesterified to form a hydrophilic bixin derivative. In addition, the transesterification of annatto with sorbitol is also well catalyzed by the Norwegian 435 lipase, resulting in the formation of the sorbitol ester of annatto, which partially alters the chemical nature of annatto to render it a hydrophilic colorant. Therefore, the introduction of hydrophilic groups can improve the poor water solubility of annatto, so that the annatto is completely insoluble and can be stabilized in an aqueous phase, and the application of the annatto in the food industry is further expanded. However, the modified annatto is still difficult to stabilize in an acidic aqueous phase.
Disclosure of Invention
The invention aims to provide a modification method for stabilizing annatto in an acidic aqueous phase, which has the characteristic of being stable in the acidic aqueous phase by introducing maltitol residues into annatto molecules.
The aim of the invention can be achieved by the following technical scheme:
a modification method for stabilizing annatto in an acidic aqueous phase comprising the steps of:
(1) Placing annatto and maltitol into a solvent for full dissolution, adding immobilized lipase and a molecular sieve A, and placing into an oscillation incubator for reaction to obtain a primary reaction mixture;
(2) Adding a molecular sieve B into the preliminary reaction mixture, continuing to react, and separating and purifying the obtained reaction product to obtain the stable carmine maltitol ester with the acidic aqueous phase.
Further, in the step (1), the molar ratio of annatto to maltitol is 1:1-1:10.
Further, in the step (1), the solvent is a binary mixed solvent, specifically, a mixture of one of dimethyl sulfoxide or N, N-dimethylformamide and one of tert-amyl alcohol or tert-butyl alcohol.
Further, in the binary mixed solvent, the volume ratio of the dimethyl sulfoxide or the N, N-dimethylformamide to the tertiary amyl alcohol or the tertiary butanol is 1:4-1:9.
Further, the immobilized lipase is selected from one of NoveXin 435, candida rugosa lipase and burkholderia cepacia lipase.
Further, the addition amount of the immobilized lipase is 10-80% of the mass of annatto.
Further, the adding amount of the molecular sieve A is 20-100% of the mass of annatto.
Further, the reaction temperature in the step (1) and the step (2) is 40-60 ℃, and the reaction time in the step (1) is 0.5-5h; the reaction time in the step (2) is 24-72h.
Further, the adding amount of the molecular sieve B is 20-100% of the mass of annatto.
Further, the molecular sieve A isMolecular sieve B is/>Molecular sieves.
Further, the mobile phase for separation and purification is any one of acetonitrile and 1% acetic acid water, acetonitrile and pure acetic acid water, or methanol and 1% acetic acid water.
In this reaction, annatto is first combined with an immobilized lipase to form a complex, followed by a single molecule isomerization reaction to form an acylase intermediate, and the first product methanol is released. Then, the acylase combines with maltitol to form another binary complex, and the acyl enzyme is isomerized into esterase complex through single molecule, and finally the carmine maltitol ester and the immobilized lipase are released. After the maltitol is used as a hydroxyl donor and subjected to transesterification reaction with annatto, the annatto has more hydroxyl groups in the structure, so that the water phase stability of annatto can be improved. The use of a highly polar organic solvent alone can dissolve maltitol but can affect the catalytic activity of lipase or even deactivate lipase, while the use of a low polar organic solvent alone can have a low solubility of maltitol therein, resulting in a low reaction yield. Thus, a binary mixed solvent system was used to dissolve maltitol and annatto.The molecular sieve is used for removing water in a reaction system, particularly water in a solvent, and the water can cause the annatto to undergo hydrolysis reaction. /(I)The molecular sieve is used for removing the methanol which is a side reaction product, and the reaction is reversely carried out due to the existence of the methanol, so that the yield of the reaction is affected.
In the modification process, various process conditions are limited, wherein the molar ratio of the annatto to the maltitol is 1:1-1:10, and if the molar ratio is lower than 1:1, almost no product is generated, but the yield is higher than 1:10, so that the yield is not greatly improved, and the maltitol is wasted. The limitation of the addition amounts of the immobilized lipase, the molecular sieve A and the molecular sieve B is also because the yield is very low below the limit value, but is not greatly improved above the limit value, and the waste of the immobilized lipase, the molecular sieve A and the molecular sieve B is caused.
The second technical scheme of the invention provides the carmine-reducing maltitol ester with stable acidic aqueous phase, which is prepared by adopting the preparation method.
Compared with the prior art, the invention has the following advantages:
1) The stability of the annatto in an acidic aqueous phase is effectively improved by carrying out chemical modification on the annatto;
2) The method has the advantages that the specificity of enzymatic catalysis is utilized to accurately synthesize the carmine-reducing maltitol ester, no byproducts are generated, high pressure and high temperature are not used, the energy consumption is low, and the method belongs to green processing;
3) The purity of the synthesized reduced carmine maltitol ester reaches more than 98%, so that the problems of low purity and low content of pigments such as the commercial annatto are effectively solved;
4) The synthesized carmine-reducing maltitol ester can exist in an acidic aqueous phase stably, so that the application potential of the carmine-reducing maltitol ester in the field of food industry is effectively expanded;
5) The invention has simple process flow, easily controlled reaction conditions, no need of secondary treatment on reaction products, cost saving and realization of large-scale industrialized production.
Drawings
FIG. 1 is a chemical structural formula of the carmine maltitol ester of reduced annatto prepared in example 1;
FIG. 2 is an HPLC chart of the carmine maltitol ester of reduced annatto obtained in example 1;
FIG. 3 is an MS diagram of the carmine maltitol ester down-conversion prepared in example 1;
FIG. 4 is a 1 H NMR chart of the carmine maltitol ester of reduced annatto obtained in example 1;
FIG. 5 is a 1 H NMR chart of maltitol used in example 1;
FIG. 6 is a 1 H NMR chart of annatto used in example 1;
FIG. 7 is a degradation rate constant (K) and degradation half-life (t 1/2) of a sample solution prepared from the reduced carmine maltitol ester prepared in example 1 after heat treatment;
FIG. 8 is a graph showing the degradation rate constant (K) and degradation half-life (t 1/2) of a sample solution prepared from the carminemaltitol ester of reduced annatto prepared in example 1 after UV irradiation treatment;
FIG. 9 is a graph showing the peak area ratio of the products of the reduced carmine maltitol esters prepared using different solvents.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.
In the following examples, unless otherwise indicated, the starting materials or processing techniques are all conventional commercially available in the art.
Example 1:
0.2410g of maltitol was taken, mixed with 1mL of dimethyl sulfoxide and dissolved well. To this was added 4mL of t-amyl alcohol, and 0.0394g of annatto, sonicated for 5min to dissolve well. Next, 0.6g was added The molecular sieve and 0.4g of Norwestin 435 lipase are placed in a shaking incubator for reaction, the reaction temperature is set to 50 ℃ and the rotating speed is 195rpm. After 1h of reaction, 0.6g/>Molecular sieve, continuing to react in a shaking incubator, and separating and purifying the product after 48 hours.
The resulting reaction product was filtered through a 0.22 μm organic filter. Then, 600. Mu.L of the filtrate was sampled. The conditions for the medium pressure chromatography preparative liquid chromatography were set as follows: flow rate: 7mL/min, monitoring wavelength: 400nm, collection wavelength: 460nm, mobile phase: 1% (v/v) acetic acid in water (A) and acetonitrile (B), gradient conditions were 0min 28%B,30min 60%B,80min 68%B,230min 80%B,240min 100%B,280min 100%B, and the product was isolated and collected. 1.0mL of the above-prepared product was taken and its purity was measured by high performance liquid chromatography. The chromatographic conditions were set as follows: sample injection amount: 20 μl, flow rate: 1mL/min, column: WATERS ATLANTIS T3C 18 column (5.0 μm,4.6 mm. Times.250 mm), monitoring wavelength: 460nm, mobile phase: 1% acetic acid water (A) and acetonitrile (B), gradient conditions were 0min 33%B,10min 33%B,15min 67%B,40min 75%B, and the purity of the resulting product was examined. And (3) spin-evaporating and freeze-drying the obtained product to obtain the norbixin maltitol ester, wherein the specific chemical structure is shown in figure 1.
The resulting carmine maltitol ester was subjected to chemical structure characterization. As can be seen from the HPLC chart (i.e. FIG. 2), the retention time of the synthesized carminose maltitol ester is about 11min under the set liquid phase gradient condition, and the purity can reach extremely approximately 100% (the peak area ratio of the separated product in the liquid phase is 100%, and no other miscellaneous peaks exist). As can be seen from the MS plot (i.e. FIG. 3), a peak is shown at a mass to core ratio (M/z) of 705.3123, which corresponds to the excimer ion [ M-H ] – in electrospray negative ion mode, indicating that the molecular weight of the compound is 706.3123, which is the same as the theoretical molecular weight of norbixin maltitol ester. In addition, the fragment ion [ M-H-C 12H23O11]– ] at M/z= 362.9691, corresponding to the mass-to-core ratio after the maltitol radical has been removed, was also observed. The fragment ion [ M-H-C 12H23O11–(COO)2]– ] at M/z= 318.9795 is a carboxyl group which is removed.
As can be seen from the 1 H NMR spectrum (i.e., FIG. 4), the peaks shifted between 3.0 and 4.0ppm are signal peaks for maltitol (i.e., FIG. 5), and the peaks shifted between 5.0 and 8.0ppm are signal peaks for annatto (i.e., FIG. 6), indicating that the resulting synthetic product is norannatto maltitol ester.
The prepared carmine maltitol ester was fully dissolved in 2 PBS buffers (10 mM) with different pH values (5.5,7.0), the concentration was 0.25mg/mL, the prepared carmine maltitol ester solution was then separated into 5mL vials, the prepared two sample solutions with different pH values (5.5,7.0) were placed in an 80℃oven for heating, and samples were taken at 0, 15, 30, 45, 60, 75, 90min, respectively. After the sample cooled to room temperature, the dynamic isomerization and degradation degree of the sample was monitored by liquid phase, corresponding degradation kinetics curves were drawn, and the degradation rate constant (K) and degradation half-life (t 1/2) of the sample solution were calculated (see fig. 7).
The prepared sample solutions (pH 5.5) were irradiated with UV light of two different wavelengths, 365nm (UVA) and 254nm (UVC), respectively, the conditions of the UV light being set as follows: the power was 15W, the vertical distance between the tube and the sample was 10cm, and samples were taken at 0, 1,2,3, 4, 6, 8h, respectively. The dynamic isomerization and degradation degree of the sample were monitored by liquid phase, corresponding degradation kinetics curves were plotted, and the degradation rate constant (K) and degradation half-life (t 1/2) of the sample solution were calculated (see FIG. 8).
As shown in FIG. 7, after heat storage at 80℃for 90min, the reduced annatto maltitol ester was thermally degraded in both pH 7.0 and pH 5.5, and the kinetics of thermal degradation was in accordance with the first order kinetic equation. However, the degradation kinetic constant K (pH 5.5) is lower than the degradation kinetic constant K (pH 7.0), which indicates that the prepared carmine maltitol ester has better thermal stability under the acidic condition.
As shown in fig. 8, both 365nm (UVA) and 254nm (UVC) radiation can degrade norbivalve maltitol esters at pH 5.5, but the degradation kinetic constant K (UVC) is significantly lower than the degradation kinetic constant K (UVA) because much of the short wave UVC uv fails to cross the glass bottle wall, resulting in a lower dose of effective photodegradation.
In addition, under the condition of keeping the total amount of the solvent unchanged, the types of the solvents used in the modification process are changed into acetonitrile, acetone, pyridine, DMSO, tertiary amyl alcohol/THF (volume ratio is 8:2) and tertiary amyl alcohol/DMSO (volume ratio is 6:4, 7:3 and 9:1), and the peak area ratio (namely the content of the product obtained after the reaction) of the obtained product is shown in fig. 9.
Example 2:
An acidic aqueous phase stable carmine maltitol ester reducing the annatto, the preparation method comprising the steps of:
1) Dissolving annatto and maltitol in binary mixed solvent, and adding commercially immobilized lipase and Putting the molecular sieve into a shaking incubator for reaction;
2) The mixture was taken out after 0.5h of reaction, and added thereto Molecular sieve, continuing to react for 48 hours;
3) Separating and purifying the mixture after the reaction is finished by using a medium-pressure chromatographic column, wherein the mobile phase for separation and purification is acetonitrile and 1% of acetic acid water, and performing rotary evaporation and freeze drying on the obtained separation product to obtain the carmine maltitol ester with stable acidic aqueous phase.
In the step 1), the mol ratio of the annatto to the maltitol is 1:5, the binary mixed solvent is dimethyl sulfoxide and tertiary butanol, the volume ratio of the two is 1:4, the commercial immobilized lipase is NoveXin 435 lipase, the addition amount of the commercial immobilized lipase is 20% of the mass of the annatto,The molecular sieve addition amount is 60% of the mass of annatto. /(I)The molecular sieve addition amount is 40% of the mass of annatto.
Example 3:
An acidic aqueous phase stable carmine maltitol ester reducing the annatto, the preparation method comprising the steps of:
1) Dissolving annatto and maltitol in binary mixed solvent, and adding commercially immobilized lipase and Putting the molecular sieve into a shaking incubator for reaction;
2) The mixture was taken out after 5 hours of reaction, and added thereto Molecular sieve, continuing to react for 48 hours;
3) Separating and purifying the mixture after the reaction by using a medium-pressure chromatographic column, wherein the mobile phase for separation and purification is acetonitrile and pure water, and performing rotary evaporation and freeze drying on the obtained separated product to obtain the stable carmine maltitol ester with an acidic aqueous phase.
In the step 1), the mol ratio of the annatto to the maltitol is 1:7, the binary mixed solvent is N, N-dimethylformamide and tertiary butanol, the volume ratio of the two is 1:5, the commercial immobilized lipase is Burkholderia cepacia lipase, the addition amount of the commercial immobilized lipase is 30 percent of the mass of the annatto,The molecular sieve addition amount is 50% of the mass of annatto. /(I)The molecular sieve addition amount is 80% of the mass of annatto.
Example 4:
An acidic aqueous phase stable carmine maltitol ester reducing the annatto, the preparation method comprising the steps of:
1) Dissolving annatto and maltitol in binary mixed solvent, and adding commercially immobilized lipase and Putting the molecular sieve into a shaking incubator for reaction;
2) The mixture was taken out after 2 hours of reaction, and added thereto Molecular sieve, continuing reaction;
3) Separating and purifying the mixture after the reaction is finished by using a medium-pressure chromatographic column, wherein the mobile phase for separation and purification is acetonitrile and 1% of acetic acid water, and performing rotary evaporation and freeze drying on the obtained separation product to obtain the carmine maltitol ester with stable acidic aqueous phase.
In the step 1), the mol ratio of the annatto to the maltitol is 1:9, the binary mixed solvent is dimethyl sulfoxide and tertiary amyl alcohol, the volume ratio of the two is 1:6, the commercial immobilized lipase is candida rugosa lipase, the addition amount of the commercial immobilized lipase is 40 percent of the mass of the annatto,The molecular sieve addition amount is 80% of the mass of annatto. /(I)The molecular sieve addition amount is 60% of the mass of annatto.
Example 5:
compared to example 1, the vast majority are identical, except in this example: the molar ratio of annatto to maltitol was 1:1.
Example 6:
Compared to example 1, the vast majority are identical, except in this example: the molar ratio of annatto to maltitol was 1:10.
Example 7:
compared to example 1, the vast majority are identical, except in this example: the addition amount of the immobilized lipase is 10% of the mass of annatto.
Example 8:
Compared to example 1, the vast majority are identical, except in this example: the addition amount of the immobilized lipase is 80% of the mass of annatto.
Example 9:
compared to example 1, the vast majority are identical, except in this example: The addition amount of the molecular sieve is 20 percent of the mass of annatto, and the weight of the annatto is/are calculated The addition amount of the molecular sieve is 100% of the mass of annatto.
Example 10:
compared to example 1, the vast majority are identical, except in this example: The addition amount of the molecular sieve is 100 percent of the mass of annatto, and is/are The addition amount of the molecular sieve is 20% of the mass of annatto.
In addition, in the above embodiment, the reaction temperature may be arbitrarily adjusted in the range of 40-60 ℃ according to actual needs, and the reaction time may be correspondingly adjusted according to actual conditions.
In conclusion, the stability of the annatto in an acidic aqueous phase is effectively improved by carrying out chemical modification on the annatto; the method has the advantages that the specificity of enzymatic catalysis is utilized to accurately synthesize the carmine-reducing maltitol ester, no byproducts are generated, high pressure and high temperature are not used, the energy consumption is low, and the method belongs to green processing; the purity of the synthesized reduced annatto maltitol ester reaches more than 98%, so that the problems of low purity and low content of the commercially available annatto are effectively solved; the synthesized carmine-reducing maltitol ester can exist in an acidic aqueous phase stably, so that the application potential of the carmine-reducing maltitol ester in the field of food industry is effectively expanded; the invention has simple process flow, easily controlled reaction conditions, no need of secondary treatment on reaction products and cost saving, so the technology is suitable for large-scale industrialized production.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims (6)

1.A modification method for stabilizing annatto in an acidic aqueous phase, comprising the steps of:
(1) Placing annatto and maltitol into a solvent for full dissolution, adding immobilized lipase and a molecular sieve A, and placing into an oscillation incubator for reaction to obtain a primary reaction mixture;
(2) Adding a molecular sieve B into the preliminary reaction mixture, continuing to react, and separating and purifying the obtained reaction product to obtain the stable carmine maltitol ester with the acidic aqueous phase, namely finishing modification;
in the step (1), the molar ratio of the annatto to the maltitol is 1:1-1:10;
In the step (1), the solvent is a binary mixed solvent, specifically, the solvent is a mixture of dimethyl sulfoxide or N, N-dimethylformamide and tertiary amyl alcohol or tertiary butanol;
the immobilized lipase is selected from one of NoveXin 435, candida rugosa lipase or burkholderia cepacia lipase;
The molecular sieve A is a 4A molecular sieve, and the molecular sieve B is a 5A molecular sieve.
2. A modification method for stabilizing annatto in an acidic aqueous phase according to claim 1, characterized in that the volume ratio of dimethyl sulfoxide or N, N-dimethylformamide to t-amyl alcohol or t-butanol in the binary mixed solvent is 1:4-1:9.
3. A modification method for stabilizing annatto in an acidic aqueous phase according to claim 1, characterized in that the amount of immobilized lipase added is 10-80% of the mass of annatto.
4. A modification process for stabilizing annatto in an acidic aqueous phase according to claim 1, wherein the molecular sieve a is added in an amount of 20% to 100% of the mass of annatto.
5. A modification process for stabilizing annatto in an acidic aqueous phase according to claim 1, wherein the molecular sieve B is added in an amount of 20% to 100% of the mass of annatto.
6. The method for modifying annatto to stabilize in an acidic aqueous phase according to claim 1, wherein the reaction temperatures in step (1) and step (2) are both 40-60 ℃, and the reaction time in step (1) is 0.5-5h; the reaction time in the step (2) is 24-72h.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586364A (en) * 2012-03-07 2012-07-18 江南大学 Method for enzymatic reaction-based synthesis of maltitol esters in nonaqueous phase and purification preparation of the maltitol esters
CN112155146A (en) * 2020-09-16 2021-01-01 上海交通大学 Method for enabling bixin to stably exist in water phase

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3103352A1 (en) * 2015-06-12 2016-12-14 DSM IP Assets B.V. Acid stable beverages comprising bixin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586364A (en) * 2012-03-07 2012-07-18 江南大学 Method for enzymatic reaction-based synthesis of maltitol esters in nonaqueous phase and purification preparation of the maltitol esters
CN112155146A (en) * 2020-09-16 2021-01-01 上海交通大学 Method for enabling bixin to stably exist in water phase

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