A method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification

Abstract

The invention discloses a method for preparing a lutein crystalline product via aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification. Using dried marigold flower granules as raw material, antioxidant pretreatment is performed under light-shielded or yellow safe-light conditions. After compound-enzyme-assisted cell wall disruption, lutein esters are extracted under low-oxygen conditions using an aqueous ethanol and medium-chain triglyceride (MCT) oil system. The extract is stratified to separate a free oil phase, and entrained lutein esters are recovered by ethanol washing. After secondary clarification, the interfacial layer is merged into the deoiled extract. Finally, mild saponification, neutralization, soap removal, microfiltration, ultrafiltration, macroporous resin enrichment, low-temperature crystallization, and vacuum drying are conducted to obtain the lutein crystalline product. This method improves process stability and food-grade suitability.

Description

[0001] DESCRIPTION

[0002] A Method for Preparing a Lutein Crystalline Product by Aqueous Ethanol-MCT Oil Assisted Extraction and Pre-Deoiling Saponification

[0003] Technical Field

[0004] The present invention relates to the technical field of natural product extraction and carotenoid preparation, and specifically relates to a method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification.

[0005] Background Art

[0006] Marigold is one of the main natural sources of lutein, in which lutein mainly exists in the form of fatty acid esters. Owing to its significant coloring and nutritional functions, lutein is widely used in food, feed, nutritional supplements, and other fields. Preparing high-purity lutein crystals from dried marigold flower granules is an important technical route in the industry.

[0007] Existing marigold lutein preparation processes mostly adopt a route of direct saponification after extraction with an organic solvent. Some studies have attempted to add medium-chain triglyceride oil to the extraction system to promote the dissolution and migration of fat-soluble lutein esters. However, if such oily auxiliary agents are directly introduced into the saponification step, they react with alkali to generate by-products such as fatty acid salts, causing system emulsification, increasing alkali consumption, and contaminating and interfering with subsequent membrane separation and resin purification processes. Conventional approaches control the amount of oil used or strengthen post-purification, but it remains difficult to balance improved extraction efficiency with purification stability.

[0008] In view of the foregoing circumstances, a key problem to be solved in the art is how to use medium-chain triglyceride oil to assist in improving the extraction efficiency of lutein esters while avoiding adverse chain reactions caused by the entry of the oily auxiliary agent into the saponification system, such asDESCRIPTION

[0009] emulsification, increased alkali consumption, and contamination of subsequent purification media, so as to ensure continuous stability of the entire process without sacrificing extraction performance.

[0010] Therefore, it is necessary to develop a method capable of synergistically using aqueous ethanol and medium-chain triglyceride oil for assisted extraction and effectively pre-removing the oily auxiliary agent before saponification, so as to combine the mass-transfer enhancement effect in the extraction stage with the system stability in the saponification and purification stages and meet industrial preparation requirements for food- grade lutein crystalline products.

[0011] Summary of the Invention

[0012] An objective of the present invention is to compensate for deficiencies in the prior art by providing a method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification. By using aqueous ethanol and medium-chain triglyceride oil for synergistic extraction and carrying out pre-deoiling treatment and secondary clarification of the interfacial layer before saponification, the method can use MCT oil to improve the dissolution efficiency of lutein esters while effectively avoiding emulsification, increased alkali consumption, and membrane / resin contamination caused by the entry of the oily auxiliary agent into the saponification system, thereby balancing extraction efficiency with stability of the subsequent purification process and meeting the industrial preparation requirements of food-grade lutein crystalline products.

[0013] To solve the above technical problems, the present invention provides the following technical solution: a method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification, the method comprising the following steps:

[0014] SI, pulverizing dried marigold flower granules, and adding an antioxidant protective agent under light-shielded conditions or yellow safe-light conditions for pretreatment to obtain a pretreated raw material;DESCRIPTION

[0015] 52, adding a citric acid-sodium citrate buffer and a compound enzyme solution to the pretreated raw material for enzymolysis, using the buffer system to maintain the pH and aqueous-phase environment required during enzymolysis and improve cell wall disruption efficiency, thereby obtaining an enzymolyzed material;

[0016] 53, adding an assisted extraction system composed of an aqueous ethanol solution and medium-chain triglyceride oil to the enzymolyzed material, using the synergistic effect of plant tissue penetration by the aqueous ethanol solution and affinity migration of fat-soluble lutein esters by the medium-chain triglyceride oil to promote lutein ester release, and extracting under nitrogen protection and low-oxygen conditions to obtain an extract containing lutein esters;

[0017] 54, after extraction is completed, subjecting the extract containing lutein esters to stratification treatment, separating a free medium-chain triglyceride oil phase, washing the free medium-chain triglyceride oil phase with an aqueous ethanol solution, combining the hydroalcoholic phase and the washing liquid, reducing the amount of oil entering the saponification system through pre-deoiling treatment and lowering alkali consumption and emulsification risk in the subsequent saponification process, subjecting an interfacial layer formed during extraction to secondary clarification and incorporating only the clarified hydroalcoholic layer, and discarding residual oily matter and a turbid emulsified layer, to obtain a deoiled extract;

[0018] 55, adding a potassium hydroxide ethanol solution to the deoiled extract for saponification to obtain a saponified solution;

[0019] 56, after saponification is completed, adjusting the saponified solution to pH 6.5-7.0, diluting and stratifying to remove a fatty-acid-salt-enriched layer and residual oily matter, reducing interference of fatty acid salts and residual oily matter with subsequent membrane separation and resin enrichment by neutralization and soap removal, and collecting an intermediate hydroalcoholic phase;DESCRIPTION

[0020] 57, subjecting the intermediate hydroalcoholic phase sequentially to treatment with a microfiltration membrane and an ethanol-resistant ultrafiltration membrane to obtain a clarified lutein solution;

[0021] 58, enriching the clarified lutein solution through a weak-polarity macroporous adsorption resin, and eluting with an aqueous ethanol solution to obtain a lutein enriched solution;

[0022] 59, subjecting the lutein enriched solution to reduced-pressure concentration followed by low-temperature crystallization, filtration, washing, and vacuum drying to obtain the lutein crystalline product.

[0023] Further, in step SI, the wavelength of the yellow safe light is 560-590 nm, and the illuminance is not higher than 500 lx, so that a specific wavelength band of yellow safe light provides necessary operating illumination while reducing photooxidation and isomerization induction of lutein by short-wavelength light; the antioxidant protective agent is vitamin E and ascorbyl palmitate, and the total amount of vitamin E and ascorbyl palmitate added is 0.06%-0.10% of the mass of the dried marigold flower granules.

[0024] Further, in step S2, the compound enzyme solution is composed of cellulase, pectinase and hemicellulase, the mass ratio of cellulase, pectinase and hemicellulase is l:(0.8-1.2):(0.6-1.0), and cellulose, pectin and hemicellulose components in the marigold cell walls are directionally degraded by synergistic action of the three enzymes to promote exposure and release of intracellular lutein esters; the total amount of the compound enzymes added is 0.6%-1.0% of the mass of the dried marigold flower granules;

[0025] the liquid-to-solid ratio for enzymolysis is 2-4 mL / g, the temperature is 42-46°C, the pH is 4.8-5.2, and the time is 1.0-1.5 h.

[0026] Further, in step S3, the ethanol volume fraction in the aqueous ethanol solution is 70%-85%, and the volume ratio of the aqueous ethanol solution to the medium-chain triglyceride oil is 97:3-92:8; the amount of medium-chain triglyceride oil is limited to a relatively low proportion to control the total amountDESCRIPTION

[0027] of oil entering subsequent steps while exerting the auxiliary migration effect;

[0028] the liquid-to-solid ratio of the assisted extraction system to the dried marigold flower granules is 7-10 mL / g;

[0029] the low-oxygen condition means that the oxygen volume fraction in the headspace of the reaction vessel is not higher than 3%, and nitrogen protection is used to reduce the oxygen partial pressure in the extraction system and decrease oxidative degradation of lutein during extraction; the extraction temperature is 42-46°C, and the extraction time is 1.5-2.5 h.

[0030] Further, in step S4, the ethanol volume fraction in the aqueous ethanol solution for washing the free medium-chain triglyceride oil phase is 75%-85%, the number of washes is 1-2, and the amount used for each wash is 0.5-1.5 times the volume of the free medium-chain triglyceride oil phase being washed; the separated free medium-chain triglyceride oil phase is washed to recover lutein esters entrained therein and reduce loss of active components caused by the pre-deoiling treatment;

[0031] the secondary clarification is secondary standing clarification or secondary centrifugation.

[0032] Further, in step S5, the potassium hydroxide ethanol solution is added in an amount controlled at 1.2-2.0 times the saponification demand of lutein esters in the deoiled extract, so that the final concentration of potassium hydroxide in the saponification system is 0.08-0.16 mol / L; a potassium hydroxide system with a relatively low final concentration is adopted to reduce damage to the conjugated structure of lutein caused by a strongly alkaline environment; the saponification temperature is 42-46°C, and the saponification time is 0.8-1.2 h.

[0033] Further, in step S5, the saponification demand of the lutein esters is based on the total lutein ester content determined by high-performance liquid chromatography and is converted into the molar amount of potassium hydroxide required according to the lutein diester structure;

[0034] the actual addition amount of the potassium hydroxide ethanol solution isDESCRIPTION

[0035] corrected according to a bench- scale saponification curve. Correction of the actual alkali addition amount by the bench-scale saponification curve avoids over-saponification or incomplete saponification caused by batch-to-batch differences in raw materials.

[0036] Further, in steps S4 and S6, the stratification treatment is standing stratification or centrifugal stratification, wherein the conditions for centrifugal stratification are centrifugation at 3000-5000 rpm for 5-15 min;

[0037] in step S7, the pore size of the microfiltration membrane is 0.22-0.45 pm, and the molecular weight cut-off of the ethanol-resistant ultrafiltration membrane is 10-30 kDa. Microfiltration and ultrafiltration are combined in series to gradually remove suspended particles, colloids, and macromolecular impurities from the extract and reduce the contamination load on subsequent resin enrichment.

[0038] Further, in step S7, the ethanol-resistant ultrafiltration membrane is a ceramic membrane, a polyvinylidene fluoride membrane, a polyethersulfone membrane, or a polytetrafluoroethylene membrane.

[0039] Further, in step S8, the weak-polarity macroporous adsorption resin is a styrene-based weak-polarity macroporous adsorption resin, and the styrene-based weak-polarity macroporous adsorption resin is an AB-8 type or DI 01 type macroporous adsorption resin; effective separation from polar impurities is achieved by selective adsorption of lutein using the weak-polarity resin;

[0040] the ethanol volume fraction in the aqueous ethanol solution used for elution is 75%-85%;

[0041] in step S9, the temperature for reduced-pressure concentration is 30-40°C, the temperature for low-temperature crystallization is -5°C to 5°C, and the temperature for vacuum drying is 30-40°C.

[0042] Compared with the prior art, the method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification has the following beneficial effects:DESCRIPTION

[0043] First, in the present invention, an assisted extraction system composed of an aqueous ethanol solution and medium-chain triglyceride oil is used, and the extract is subjected to pre-deoiling treatment before saponification. The free oil phase is separated, and entrained lutein esters are recovered by washing with an aqueous ethanol solution. Thus, while medium-chain triglyceride oil promotes the migration and dissolution of lutein esters from marigold tissue, the amount of oil entering the saponification system is effectively controlled, thereby reducing alkali consumption during saponification and emulsification caused by fatty acid salt generation, avoiding contamination of subsequent microfiltration membranes, ultrafiltration membranes, and macroporous adsorption resin by oily by-products, ensuring continuous stability of the whole purification process, and improving the process reliability of the lutein crystalline product.

[0044] Second, in the present invention, a nitrogen-protected low-oxygen environment throughout the process and light-shielded or yellow safe-light operation are combined with synergistic cell wall disruption by cellulase, pectinase and hemicellulase in a citric acid-sodium citrate buffer system and mild saponification conditions. This reduces oxidative degradation and cis-trans isomerization of lutein during extraction and saponification and maintains a relatively high proportion of all-trans lutein in the product. At the same time, compound-enzyme cell wall disruption promotes release of intracellular lutein esters, and proteins, colloids and saponification by-products are removed by neutralization and soap removal as well as coupled membrane-resin purification, thereby obtaining a high-quality lutein crystalline product.

[0045] Other advantages, objectives and features of the present invention will be set forth to some extent in the following description and, to some extent, will be apparent to those skilled in the art based on study of the following text, or may be learned from practice of the present invention.

[0046] Brief Description of the DrawingsDESCRIPTION

[0047] To more clearly describe the technical solutions in the embodiments of the present invention or in the prior art, the drawings required for describing the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention, and those of ordinary skill in the art may obtain other drawings based on these drawings without creative labor.

[0048] FIG. 1 is a process flow block diagram of preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to the present invention;

[0049] FIG. 2 is a schematic flow diagram of post-extraction pre-deoiling and secondary clarification of the interfacial layer according to the present invention;

[0050] FIG. 3 is a schematic flow diagram of post-saponification neutralization and soap removal and coupled membrane-resin purification according to the present invention.

[0051] Detailed Description of the Embodiments

[0052] To further describe the technical means and effects adopted by the present invention to achieve the predetermined objectives of the invention, the following provides a detailed description of the specific embodiments, structures, features and effects of the present invention with reference to the drawings and preferred embodiments.

[0053] In the embodiments of the present invention, the lutein ester content in the dried marigold flower granules, calculated as lutein, is 12-18 g / kg. The medium-chain triglyceride oil is food-grade caprylic / capric triglyceride, in which the caprylic acid content in the fatty acid composition is not less than 55%, and the capric acid content is not less than 30%. In the compound enzyme preparation, the cellulase activity is not less than 10000 U / g, the pectinase activity is not less than 30000 U / g, and the hemicellulase activity is not less than 5000 U / g. In the antioxidant protective agent, vitamin E is a mixed tocopherol concentrate with aDESCRIPTION

[0054] purity of not less than 70%, and ascorbyl palmitate has a purity of not less than 95%.

[0055] To facilitate understanding of the basis for determining the amount of potassium hydroxide in the saponification step, the method for calculating the theoretical saponification demand of lutein esters is described herein. First, high-performance liquid chromatography is used to determine the total lutein ester content in the deoiled extract. The chromatographic conditions are as follows: a C30 chromatographic column, a methanol-methyl tert-butyl ether- water gradient elution system as the mobile phase, detection wavelength of 450 nm, and lutein dipalmitate as the standard for preparing a standard curve. The measured peak areas of the lutein esters are converted into moles of lutein diesters. In the saponification reaction of lutein diesters, complete hydrolysis of one mole of lutein diester consumes two moles of potassium hydroxide, and therefore the molar amount of potassium hydroxide required is converted according to the lutein diester structure, thereby obtaining the theoretical saponification demand. In actual production, because the fatty acid composition of different batches of marigold raw materials may fluctuate to some extent, and trace amounts of other saponifiable substances may remain in the deoiled extract, the theoretical saponification demand is used as a basis. A bench-scale saponification curve test is first carried out to determine the relationship between the actual alkali addition amount and the saponification conversion rate, and the addition amount of the potassium hydroxide ethanol solution is corrected accordingly to avoid incomplete saponification caused by insufficient alkali or lutein degradation caused by excessive alkali.

[0056] As shown in FIG. 1 , the overall process of the present invention sequentially comprises eleven unit operation steps: SI pulverization and antioxidant pretreatment of dried marigold flower granules, S2 compound-enzyme-assisted cell wall disruption, S3 aqueous ethanol-MCT oil assisted low-oxygen extraction, S4 pre-deoiling and oil-phase washing recovery, S5 mild saponification, S6DESCRIPTION

[0057] neutralization and soap removal, S7 membrane separation for impurity removal, S8 macroporous adsorption resin enrichment, and S9 low-temperature crystallization and vacuum drying.

[0058] As shown in FIG. 2, after extraction in step S3 is completed, the extract containing lutein esters first enters a stratification unit, where an upper free medium-chain triglyceride oil phase, an intermediate hydroalcoholic phase, and a small amount of bottom sediment are separated. In step S4, the free oil phase enters a washing unit, and after washing with an aqueous ethanol solution, a washing hydroalcoholic phase and an oil-depleted phase are separated; the washing hydroalcoholic phase is combined with the intermediate hydroalcoholic phase. An interfacial emulsion layer that may exist in the intermediate layer enters a secondary clarification unit; after standing or centrifugation again, only the clarified hydroalcoholic layer is incorporated, and residual oily matter and the turbid emulsified layer are discharged as waste.

[0059] As shown in FIG. 3, after the saponified solution in step S5 enters a stratification unit following neutralization and pH adjustment in step S6, the fatty-acid-salt-enriched layer and residual oily matter are removed. The intermediate hydroalcoholic phase enters a microfiltration membrane unit in step S7 to remove suspended particles, and the permeate enters an ethanol-resistant ultrafiltration membrane unit to remove colloids and macromolecular impurities. The ultrafiltration permeate, as a clarified lutein solution, enters a macroporous adsorption resin column in step S8 for enrichment. After elution with an aqueous ethanol solution, a lutein enriched solution is obtained, and finally low-temperature crystallization and vacuum drying are carried out in step S9.

[0060] Example 1

[0061] This example fully demonstrates the entire process of preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to the present invention, including nine steps: pulverization pretreatment, compound-enzyme-assisted cell wall disruption,DESCRIPTION

[0062] aqueous ethanol-MCT oil low-oxygen extraction, pre-deoiling and oil-phase washing, mild saponification, neutralization and soap removal, membrane separation, resin enrichment, and low-temperature crystallization and drying. It is used to illustrate the feasibility of the technical solution of the present invention and the connection among the steps.

[0063] Step SI, pulverization and antioxidant pretreatment: 10 kg of dried marigold flower granules were taken, pulverized with a pulverizer and passed through a 50-mesh standard sieve, and the undersize material was collected. The pulverization workshop was illuminated with a yellow safety lamp having a wavelength of 570 nm, with the illuminance controlled at 300 lx, and a yellow shading film was additionally installed outside the windows to block outdoor natural light. 5 g of vitamin E and 3 g of ascorbyl palmitate were added to the pulverized marigold flower granule powder, the total addition amount of vitamin E and ascorbyl palmitate being 0.08% of the mass of the dried marigold flower granules. The mixture was mixed in a horizontal ribbon mixer at 30 rpm for 15 min, and after uniform mixing, a pretreated raw material was obtained.

[0064] Step S2, compound-enzyme-assisted cell wall disruption: a citric acid-sodium citrate buffer and a compound enzyme solution were added to the pretreated raw material. The buffer was prepared by weighing 210 g of citric acid monohydrate and 294 g of trisodium citrate dihydrate, dissolving them in an appropriate amount of deionized water, and making up the volume to 100 L; the pH of the resulting buffer was 5.0. The compound enzyme solution was prepared by weighing 40 g of cellulase, 40 g of pectinase and 32 g of hemicellulase, with a mass ratio of 1:1:0.8, and a total compound enzyme addition amount of 80 g, accounting for 0.8% of the mass of the dried marigold flower granules; the three enzyme powders were dissolved in 10 L of the above buffer and stirred until completely dissolved. The buffer and the compound enzyme solution were added together to the pretreated raw material, and additional buffer was added so that the total liquid-to-solid ratio of the system was 3 mL / g, namely a total liquid amountDESCRIPTION

[0065] of 30 L was added. The material was transferred into a stainless steel enzymolysis tank with jacket insulation, stirring was started at 60 rpm, hot water was introduced into the jacket to raise the system temperature to 45°C, an online pH meter monitored the system pH at 5.0, and enzymolysis was carried out under these conditions for 1.2 h while stirring continuously, thereby obtaining an enzymolyzed material.

[0066] Step S3, aqueous ethanol-MCT oil assisted low-oxygen extraction: an assisted extraction system was added to the enzymolyzed material. The assisted extraction system was composed of an aqueous ethanol solution having an ethanol volume fraction of 80% and medium-chain triglyceride oil, at a volume ratio of 95:5. Specifically, 76 L of the 80% aqueous ethanol solution was first measured and added to the enzymolyzed material, and then 4 L of medium-chain triglyceride oil was measured and added. Based on the total added volume of the aqueous ethanol solution and the medium-chain triglyceride oil, the liquid-to- solid ratio of the assisted extraction system to the dried marigold flower granules was 8 mL / g. The enzymolysis tank was sealed, nitrogen with a purity of not less than 99.5% was introduced through a top gas inlet at a flow rate of 2 L / min to replace the air in the tank, and an online oxygen content analyzer was used to monitor the oxygen volume fraction in the tank headspace. When the oxygen volume fraction decreased below 3%, the exhaust valve was closed and a slight positive pressure was maintained. The extraction process was carried out under yellow safe-light conditions; the jacket maintained the system temperature at 45°C, the stirring speed was 40 rpm, and the extraction time was 2 h, thereby obtaining an extract containing lutein esters.

[0067] Step S4, pre-deoiling and oil-phase washing recovery: after extraction was completed, stirring was stopped, and the extract containing lutein esters was transferred to a tubular centrifuge for centrifugal stratification under conditions of centrifugation at 4000 rpm for 10 min. After centrifugation, the liquid was divided into three layers: the upper layer was a free medium-chain triglyceride oil phase,DESCRIPTION

[0068] the middle layer was a hydroalcoholic phase, and the bottom contained a small amount of sediment; a small amount of emulsified layer was present near the interface. The upper free medium-chain triglyceride oil phase was carefully separated, and its volume was measured to be about 3.8 L. The free medium-chain triglyceride oil phase was washed with an aqueous ethanol solution having an ethanol volume fraction of 80%, for 2 washes; the amount used for each wash was 1 time the volume of the free medium-chain triglyceride oil phase, i.e., 3.8 L of 80% aqueous ethanol solution was used for each wash. After stirring and washing for 5 min, centrifugation was again carried out at 4000 rpm for 10 min, and the washing hydroalcoholic phase was separated. The two washing hydroalcoholic phases were combined with the above-mentioned intermediate hydroalcoholic phase. The emulsified layer at the interface between the intermediate layer and the upper layer was collected and separately subjected to secondary centrifugation at 4000 rpm for 10 min; after centrifugation, only the clarified hydroalcoholic layer was incorporated into the combined liquid, and the residual oily matter and turbid emulsified layer were discarded, thereby obtaining a deoiled extract having a total volume of about 108 L. High-performance liquid chromatography was used to determine the total lutein ester content in the deoiled extract, and the theoretical saponification demand was calculated according to the lutein diester structure.

[0069] Step S5, mild saponification: according to the calculation result of the theoretical saponification demand, potassium hydroxide was weighed at 1.6 times the theoretical demand and dissolved in a small amount of 80% aqueous ethanol solution to prepare a potassium hydroxide ethanol solution. The deoiled extract was transferred to a saponification reaction tank, the potassium hydroxide ethanol solution was added, and 80% aqueous ethanol solution was supplemented so that the final concentration of potassium hydroxide in the saponification system was 0.12 mol / L. After the saponification tank was sealed, nitrogen replacement was performed, the oxygen volume fraction in the tank headspace was controlled below 3%, the jacket maintained the temperature at 45 °C, the stirring speed wasDESCRIPTION

[0070] 50 rpm, and saponification was carried out for 1 h to obtain a saponified solution.

[0071] Step S6, neutralization and soap removal: after saponification was completed, the pH of the saponified solution was adjusted to 6.8 with a citric acid ethanol-water solution. The citric acid ethanol-water solution was prepared by dissolving citric acid monohydrate in 80% aqueous ethanol solution to a mass fraction of 10%. After pH adjustment, 30 L of 80% aqueous ethanol solution was added for dilution, and the mixture was allowed to stand for 20 min. After standing, the saponified solution stratified; the upper layer was a fatty-acid-salt-enriched layer in the form of a milky-white paste, and the lower layer was an intermediate hydroalcoholic phase. The material was transferred to a tubular centrifuge and centrifuged at 4000 rpm for 10 min to remove the fatty-acid-salt-enriched layer and residual oily matter, and the intermediate hydroalcoholic phase was collected, with a volume of about 135 L.

[0072] Step S7, membrane separation for impurity removal: the intermediate hydroalcoholic phase was sequentially subjected to membrane separation treatment. First, dead-end filtration was carried out through a 0.22 pm polypropylene microfiltration membrane at an operating pressure of 0.15 MPa and a membrane area of 0.5 m2, and the permeate was collected in an intermediate storage tank. The microfiltration permeate was then subjected to cross-flow filtration through an ethanol-resistant polyvinylidene fluoride ultrafiltration membrane having a molecular weight cut-off of 10 kDa at an operating pressure of 0.3 MPa, a membrane area of 1.0 m2, and a cross-flow velocity of 2.5 m / s. The ultrafiltration permeate was collected to obtain a clarified lutein solution having a volume of about 130 L.

[0073] Step S8, macroporous adsorption resin enrichment: the clarified lutein solution was passed through a DI 01 type styrene-based weak-polarity macroporous adsorption resin column at a flow rate of 2 BV / h. The resin column had a diameter-to-height ratio of 1:8 and a resin loading volume of 5 L. After loading was completed, the resin column was first rinsed with 2 BV of deionizedDESCRIPTION

[0074] water to remove residual ethanol, and then eluted with 80% aqueous ethanol solution at a flow rate of 1.5 BV / h. The eluate was collected from the point at which the color changed from pale yellow to deep yellow and then became lighter, and the main peak fractions were combined to obtain a lutein enriched solution having a volume of about 25 L.

[0075] Step S9, low-temperature crystallization and vacuum drying: the lutein enriched solution was transferred to a rotary evaporator and concentrated under reduced pressure at a vacuum degree of -0.09 MPa and a water bath temperature of 35°C to one fifth of the original volume, and the concentrate was transferred to a crystallization kettle. Frozen water was introduced into the jacket of the crystallization kettle to lower the temperature of the concentrate to 0°C, and crystallization was maintained at 0°C for 8 h. After crystallization, the material in the kettle was discharged and vacuum-filtered through a Buchner funnel. The filter cake was washed twice with 200 mL of 80% aqueous ethanol solution precooled to 0°C. After washing, the filter cake was transferred to a vacuum drying oven and dried at a vacuum degree of -0.095 MPa and a temperature of 35 °C for 12 h to obtain a lutein crystalline product, which was weighed and hermetically packaged in an aluminum foil bag under nitrogen.

[0076] The lutein crystalline product obtained in this example had a total lutein content of 82.8%, an all-trans lutein proportion of 89.8%, a total lutein recovery of 80.6%, a saponification conversion rate of 86.9%, and a cis isomer proportion of 10.2%. The saponification and resin column operation remained stable throughout, indicating that the method of the present invention can stably prepare a high-quality lutein crystalline product under a preferred parameter combination.

[0077] Example 2

[0078] This example demonstrates implementation of the method of the present invention near the lower limits of the parameter ranges, using coarser pulverization, light-shielded operation, lower compound enzyme dosage and liquid-to-solid ratio, lower ethanol concentration and extraction temperature, aDESCRIPTION

[0079] smaller MCT oil proportion, and lower alkali concentration and saponification temperature, thereby verifying the applicability of the method under mild conditions.

[0080] Step SI, pulverization and antioxidant pretreatment: 10 kg of dried marigold flower granules were taken and pulverized to 40 mesh. All operations were carried out under light-shielded conditions; the workshop windows were completely covered with black shading curtains, and a red darkroom safety lamp provided the minimum operating illumination indoors. 4 g of vitamin E and 2 g of ascorbyl palmitate were added to the pulverized marigold flower granules, with a total addition amount of 0.06% of the mass of the dried marigold flower granules, and the mixture was uniformly mixed to obtain a pretreated raw material.

[0081] Step S2, compound-enzyme-assisted cell wall disruption: a citric acid-sodium citrate buffer and a compound enzyme solution were added to the pretreated raw material, and the buffer had a pH of 5.2. In the compound enzyme solution, the mass ratio of cellulase, pectinase and hemicellulase was 1:0.8:0.6; 32 g of cellulase, 25.6 g of pectinase and 19.2 g of hemicellulase were weighed respectively. The total amount of the compound enzymes added was 76.8 g, accounting for 0.6% of the mass of the dried marigold flower granules. Buffer was supplemented so that the liquid-to- solid ratio of the system was 2 mL / g, i.e., the total liquid amount was 20 L. The material was enzymolyzed at 42°C and pH 5.2 for 1.5 h with a stirring speed of 50 rpm to obtain an enzymolyzed material.

[0082] Step S3, aqueous ethanol-MCT oil assisted low-oxygen extraction: an assisted extraction system was added to the enzymolyzed material. The ethanol volume fraction in the aqueous ethanol solution was 75%, and the volume ratio of the medium-chain triglyceride oil to the aqueous ethanol solution was 3:97, namely 67.9 L of 75% aqueous ethanol solution and 2.1 L of medium-chain triglyceride oil were added. The total added volume of the assisted extraction system was 70 L, and the liquid-to- solid ratio was 7 mL / g. Nitrogen was introduced into the enzymolysis tank until the oxygen volume fraction in the tankDESCRIPTION

[0083] headspace was not higher than 3%, and extraction was carried out at 42°C for 2.5 h.

[0084] Step S4, pre-deoiling and oil-phase washing recovery: after extraction was completed, centrifugal stratification was carried out, the free medium-chain triglyceride oil phase was separated, the free oil phase was washed once with 75% aqueous ethanol solution, the washing amount being 1 time the volume of the oil phase, and after the interfacial layer was subjected to secondary centrifugation, only the clarified hydroalcoholic layer was incorporated to obtain a deoiled extract.

[0085] Step S5, mild saponification: a potassium hydroxide ethanol solution was added to the deoiled extract, the addition amount being controlled at 1.2 times the theoretical saponification demand of the lutein esters. The final concentration of potassium hydroxide in the saponification system was 0.08 mol / L, and saponification was carried out at 42°C for 1.2 h.

[0086] Step S6, neutralization and soap removal: after saponification was completed, the pH was adjusted to 6.7, and centrifugal stratification was performed to remove the fatty-acid-salt-enriched layer and residual oily matter.

[0087] Step S7, membrane separation for impurity removal: the intermediate hydroalcoholic phase was sequentially treated with a 0.22 pm microfiltration membrane and a 10 kDa ethanol-resistant ultrafiltration membrane, under the same operating conditions as in Example 1.

[0088] Step S8, macroporous adsorption resin enrichment: the clarified lutein solution was enriched through a DI 01 type macroporous adsorption resin and eluted with 75% aqueous ethanol solution, under the same operating conditions as in Example 1.

[0089] Step S9, low-temperature crystallization and vacuum drying: concentration, crystallization and drying steps were the same as in Example 1 , thereby obtaining a lutein crystalline product.

[0090] The product obtained in this example had a total lutein content of 82.2%, anDESCRIPTION

[0091] all-trans lutein proportion of 89.2%, a total lutein recovery of 80.1%, and a saponification conversion rate of 86.4%. All indicators were close to those of Example 1, and the system operated stably, indicating that the method of the present invention can still be effectively implemented under lower-limit parameter conditions and has a relatively wide operating window.

[0092] Example 3

[0093] This example demonstrates implementation of the method of the present invention near the upper limits of the parameter ranges, using finer pulverization, higher compound enzyme dosage and liquid-to- solid ratio, higher ethanol concentration and extraction temperature, a higher MCT oil proportion, and higher alkali concentration and saponification temperature, thereby verifying the applicability of the method under intensified conditions.

[0094] Step SI, pulverization and antioxidant pretreatment: 10 kg of dried marigold flower granules were taken and pulverized to 60 mesh under yellow safe-light conditions; the yellow safe light had a wavelength of 560 nm and an illuminance of 450 lx. 6 g of vitamin E and 4 g of ascorbyl palmitate were added, with a total addition amount of 0.10% of the mass of the dried marigold flower granules, and the mixture was uniformly mixed.

[0095] Step S2, compound-enzyme-assisted cell wall disruption: a citric acid-sodium citrate buffer and a compound enzyme solution were added to the pretreated raw material, and the buffer had a pH of 4.8. The mass ratio of cellulase, pectinase and hemicellulase was 1:1.2: 1.0; 50 g of cellulase, 60 g of pectinase and 50 g of hemicellulase were weighed respectively. The total amount of the compound enzymes added was 160 g, accounting for 1.0% of the mass of the dried marigold flower granules. Buffer was supplemented so that the liquid-to-solid ratio of the system was 4 mL / g, namely a total liquid amount of 40 L. Enzymolysis was carried out at 46°C and pH 4.8 for 1.0 h.

[0096] Step S3, aqueous ethanol-MCT oil assisted low-oxygen extraction: an assisted extraction system was added to the enzymolyzed material. The ethanolDESCRIPTION

[0097] volume fraction in the aqueous ethanol solution was 85%, and the volume ratio of the medium-chain triglyceride oil to the aqueous ethanol solution was 8:92, namely 92 L of 85% aqueous ethanol solution and 8 L of medium-chain triglyceride oil were added. The total added volume of the assisted extraction system was 100 L, and the liquid-to- solid ratio was 10 mL / g. Nitrogen was introduced until the oxygen volume fraction in the tank headspace was not higher than 3%, and extraction was carried out at 46°C for 1.5 h.

[0098] Step S4, pre-deoiling and oil-phase washing recovery: after extraction was completed, centrifugal stratification was carried out, the free oil phase was separated, washing was performed twice with 85% aqueous ethanol solution, and after secondary centrifugation of the interfacial layer, the clarified hydroalcoholic layer was incorporated to obtain a deoiled extract.

[0099] Step S5, mild saponification: a potassium hydroxide ethanol solution was added to the deoiled extract, the addition amount being controlled at 2.0 times the theoretical saponification demand. The final concentration of potassium hydroxide in the saponification system was 0.16 mol / L, and saponification was carried out at 46°C for 0.8 h.

[0100] Step S6, neutralization and soap removal: after saponification was completed, the pH was adjusted to 6.9, and centrifugal stratification was performed to remove the fatty-acid-salt-enriched layer and residual oily matter.

[0101] Step S7, membrane separation for impurity removal: the intermediate hydroalcoholic phase was sequentially treated with a 0.45 pm microfiltration membrane and a 30 kDa ethanol-resistant poly ethersulfone ultrafiltration membrane.

[0102] Step S8, macroporous adsorption resin enrichment: the clarified lutein solution was enriched through an AB-8 type styrene-based weak-polarity macroporous adsorption resin and eluted with 85% aqueous ethanol solution.

[0103] Step S9, low-temperature crystallization and vacuum drying: concentration, crystallization and drying steps were the same as in Example 1 , thereby obtainingDESCRIPTION

[0104] a lutein crystalline product.

[0105] The product obtained in this example had a total lutein content of 81.7%, an all-trans lutein proportion of 88.8%, a total lutein recovery of 79.5%, a saponification conversion rate of 86.0%, and a cis isomer proportion slightly increased to 11.2%. The system still operated stably, indicating that the method can also be normally implemented under upper-limit parameter conditions and that the product quality is within an acceptable range.

[0106] Comparative Example 1

[0107] In this comparative example, 80% aqueous ethanol solution was used as a single extractant without adding MCT oil, and the remaining steps were the same as in Example 1. Specifically, in step S3, only 80 L of 80% aqueous ethanol solution was added to the enzymolyzed material as the extraction solvent, the liquid-to-solid ratio was 8 mL / g, and extraction was carried out for 2 h under the same nitrogen protection and temperature conditions. Because no MCT oil phase was present, after the extract in step S4 was allowed to stand for stratification, no upper oil phase was present, and the extract directly entered step S5 for saponification, with the pre-deoiling and oil-phase washing steps omitted.

[0108] In this comparative example, the total lutein recovery decreased to 75.8%, about 4.8 percentage points lower than Example 1; the total lutein content was 78.2%, the all-trans lutein proportion was 85.8%, and the cis isomer proportion increased to 14.2%. This indicates that, when MCT oil assisted extraction is absent, the dissolution and migration efficiency of lutein esters from marigold tissue is significantly reduced, and the product quality decreases.

[0109] Comparative Example 2

[0110] In this comparative example, nitrogen protection and yellow safe-light or light-shielding control were not performed, and the remaining steps were the same as in Example 1. Specifically, the extraction process in step S3 and the saponification process in step S5 were carried out under ordinary indoor lighting. The enzymolysis tank and saponification tank were operated open to theDESCRIPTION

[0111] atmosphere without nitrogen replacement, and the tank headspace was a natural air atmosphere.

[0112] In this comparative example, the all-trans lutein proportion decreased to 84.0%, 5.8 percentage points lower than Example 1, and the cis isomer proportion increased to 16.0%. The degree of lutein oxidative degradation and cis-trans isomerization increased significantly, indicating that low-oxygen and light-shielding conditions play an important role in protecting the conjugated structure of lutein and maintaining the proportion of the all-trans configuration.

[0113] Comparative Example 3

[0114] In this comparative example, the compound enzyme solution was not added for enzyme-assisted cell wall disruption in step S2, and the remaining steps were the same as in Example 1. A citric acid-sodium citrate buffer was added to the pretreated raw material so that the liquid-to- solid ratio was 3 mL / g, and the mixture was stirred at 45°C and pH 5.0 for 1.2 h, without adding cellulase, pectinase or hemicellulase. The subsequent steps were the same as in Example 1.

[0115] In this comparative example, the total lutein recovery decreased to 74.9%, 5.7 percentage points lower than Example 1, and the total lutein content was 78.5%, indicating that when the cell walls of the dried marigold flower granules are not disrupted by enzymolysis, intracellular lutein esters are difficult to fully expose and release, and the compound-enzyme-assisted cell wall disruption step makes a substantial contribution to improving extraction efficiency.

[0116] Comparative Example 4

[0117] In this comparative example, after extraction in step S4 was completed, pre-deoiling treatment was not performed, and potassium hydroxide ethanol solution was directly added to the extract containing the free MCT oil phase for saponification. The remaining steps were the same as in Example 1. After simple filtration to remove large solid particles, the extract was directly transferred to the saponification tank of step S5, and the free MCT oil phase and the hydroalcoholic phase entered the saponification system together.DESCRIPTION

[0118] In this comparative example, the saponification conversion rate decreased to 83.6%, 3.3 percentage points lower than Example 1. Obvious emulsification occurred after saponification, and a slight pressure increase occurred during resin column operation. This indicates that, after the free MCT oil phase enters the saponification system, it reacts with alkali to form fatty acid salts and causes emulsification, which not only consumes alkali and lowers saponification efficiency but also increases the operating resistance of subsequent resin purification.

[0119] Comparative Example 5

[0120] In this comparative example, after saponification in step S6 was completed, neutralization and soap-removal stratification were not carried out, and the material directly entered the microfiltration of step S7, the ultrafiltration and resin enrichment of step S8. The remaining steps were the same as in Example 1. After saponification was completed, pH was not adjusted, standing stratification or centrifugation to remove the fatty-acid-salt-enriched layer was not carried out, and the saponified solution as a whole was pumped into the microfiltration membrane unit.

[0121] In this comparative example, a slight pressure increase occurred during resin column operation, the eluate had turbid entrainment, the total lutein content was 79.8%, and the all-trans lutein proportion was 87.8%. This indicates that, when residual fatty acid salts and residual oily matter in the saponified solution directly enter the membrane and resin units without neutralization and soap-removal treatment, membrane surface fouling and resin pore blockage occur, affecting purification process stability and product quality.

[0122] Example 4

[0123] This example demonstrates implementation of the method of the present invention with adjusted combinations near the middle values of the parameter ranges. Parameters such as enzyme mass ratio, liquid-to-solid ratio, volume ratio of ethanol to MCT oil, and alkali concentration were all selected as differentDESCRIPTION

[0124] values within the set ranges, so as to verify reproducibility of the method under parameter combination changes.

[0125] Step SI, 10 kg of dried marigold flower granules were taken, pulverized to 50 mesh, and vitamin E 5 g and ascorbyl palmitate 3 g were added under yellow safe-light conditions, with a total addition amount of 0.08%.

[0126] Step S2, a citric acid-sodium citrate buffer and a compound enzyme solution were added to the pretreated raw material. The mass ratio of cellulase, pectinase and hemicellulase was 1 : 1.1 :0.7, the total compound enzyme addition amount was 0.9%, the liquid-to-solid ratio was 3.5 mL / g, the pH was 5.1, and enzymolysis was carried out at 44°C for 1.3 h.

[0127] Step S3, an assisted extraction system composed of 80% aqueous ethanol solution and MCT oil was added to the enzymolyzed material. The volume ratio of the aqueous ethanol solution to MCT oil was 94:6, the liquid-to-solid ratio was 8.5 mL / g, nitrogen protection was used so that the oxygen volume fraction was not higher than 3%, and extraction was carried out at 44°C for 2.2 h.

[0128] Step S4, after extraction was completed, centrifugal stratification was carried out, the free oil phase was washed twice with 80% aqueous ethanol solution, and after secondary centrifugation of the interfacial layer, the clarified hydroalcoholic layer was incorporated to obtain a deoiled extract.

[0129] Step S5, the potassium hydroxide ethanol solution was added in an amount controlled at 1.5 times the theoretical saponification demand, and the final concentration of potassium hydroxide in the saponification system was 0.10 mol / L. Saponification was carried out at 44°C for 1.0 h.

[0130] Step S6, after saponification, the pH was adjusted to 6.8, and centrifugal stratification was performed to remove the fatty-acid-salt-enriched layer.

[0131] Step S7, the intermediate hydroalcoholic phase was sequentially treated with a 0.22 pm microfiltration membrane and a 10 kDa ethanol-resistant ultrafiltration membrane, under the same operating conditions as in Example 1.

[0132] Step S8, the clarified lutein solution was enriched through a D101 typeDESCRIPTION

[0133] macroporous adsorption resin and eluted with 80% aqueous ethanol solution, under the same operating conditions as in Example 1.

[0134] Step S9, concentration, crystallization and drying steps were the same as in Example 1, thereby obtaining a lutein crystalline product.

[0135] The product obtained in this example had a total lutein content of 82.5%, an all-trans lutein proportion of 89.5%, a total lutein recovery of 80.3%, and a saponification conversion rate of 86.7%. All indicators were comparable to those of Example 1 , and the system operated stably, indicating that the method of the present invention has certain adaptability to parameter combination changes and good process reproducibility.

[0136] Example 5

[0137] This example demonstrates implementation of the method of the present invention under light-shielded conditions and with fine adjustment of the parameter combination, using coarser pulverization, a medium compound enzyme dosage, a lower MCT oil proportion, and a medium alkali concentration, thereby further verifying the stability of the method under different light conditions and parameter combinations.

[0138] Step SI, 10 kg of dried marigold flower granules were taken, pulverized to 45 mesh, and vitamin E 4 g and ascorbyl palmitate 5 g were added under light-shielded conditions, with a total addition amount of 0.09%.

[0139] Step S2, a citric acid-sodium citrate buffer and a compound enzyme solution were added to the pretreated raw material. The mass ratio of cellulase, pectinase and hemicellulase was 1 : 0.9: 0.9, the total compound enzyme addition amount was 0.7%, the liquid-to-solid ratio was 3 mL / g, the pH was 5.0, and enzymolysis was carried out at 45°C for 1.2 h.

[0140] Step S3, an assisted extraction system composed of 80% aqueous ethanol solution and MCT oil was added to the enzymolyzed material. The volume ratio of the aqueous ethanol solution to MCT oil was 96:4, the liquid-to-solid ratio was 7.5 mL / g, nitrogen protection was used so that the oxygen volume fraction wasDESCRIPTION

[0141] not higher than 3%, and extraction was carried out at 45°C for 2 h.

[0142] Step S4, the pre-deoiling and oil-phase washing treatment was the same as in Example 1.

[0143] Step S5, the potassium hydroxide ethanol solution was added in an amount controlled at 1.4 times the theoretical saponification demand, and the final concentration of potassium hydroxide in the saponification system was 0.13 mol / L. Saponification was carried out at 45°C for 1.1 h.

[0144] Step S6, after saponification, the pH was adjusted to 6.7, and centrifugal stratification was performed to remove the fatty-acid-salt-enriched layer.

[0145] Step S7, membrane separation treatment was the same as in Example 1.

[0146] Step S8, resin enrichment was the same as in Example 1.

[0147] Step S9, concentration, crystallization and drying were the same as in Example 1, thereby obtaining a lutein crystalline product.

[0148] The product obtained in this example had a total lutein content of 82.4%, an all-trans lutein proportion of 89.3%, a total lutein recovery of 80.2%, and a saponification conversion rate of 86.5%. All indicators were close to those of Example 1 , and the system operated stably, further confirming that the method of the present invention can stably obtain qualified products under different operating conditions.

[0149] The product performance test method was as follows: the total lutein content was determined by high-performance liquid chromatography. The chromatographic conditions were as follows: a YMC Carotenoid C30 column, column temperature of 30°C, mobile phase A being a methanol-water-triethylamine system, mobile phase B being methyl tert-butyl ether, gradient elution program of 100% phase A at 0 min, 60% phase A and 40% phase B at 30 min, and 40% phase A and 60% phase B at 40 min, flow rate of 1.0 mL / min, detection wavelength of 450 nm, and injection volume of 20 pL. A lutein standard was used to prepare a standard curve, and total lutein content was calculated based on free lutein in the lutein crystalline product and residual luteinDESCRIPTION

[0150] esters convertible to lutein.

[0151] The all-trans lutein proportion and cis isomer proportion were qualitatively determined under the above chromatographic conditions using all-trans lutein standard and 13-cis lutein and 9-cis lutein reference standards, and their respective proportions were calculated by the peak area normalization method.

[0152] The total lutein recovery was expressed as a percentage calculated by dividing the mass of total lutein in the obtained lutein crystalline product by the mass of total lutein esters in the dried marigold flower granule raw material converted to lutein.

[0153] The saponification conversion rate was calculated as the ratio of the molar amount of free lutein after saponification to the total molar amount of free lutein and residual lutein esters converted to lutein.

[0154] Ethanol residue was determined by gas chromatography, using a DB-624 capillary column, headspace injection, an FID detector, and external standard quantification with an ethanol standard.

[0155] The state of the saponification system was determined by visually observing whether the saponified solution exhibited obvious emulsification or stratification. The operating state of the resin column was determined by monitoring the change in column inlet pressure during loading and elution and by observing whether the eluate appearance was clear.

[0156] Specifically, the main performance indicator test results of the examples and comparative examples are summarized in Table 1 and Table 2.

[0157] To facilitate comparison of the technical effects of the examples and comparative examples, key indicators such as total lutein content, all-trans lutein proportion, total lutein recovery, saponification conversion rate, cis isomer proportion, and system operating state are listed in the following tables.

[0158] Table 1 Performance test results of the examples are as follows:

[0159] ITEM EXAMPLE 1 EXAMPLE 2 EXAMPLE 3 EXAMPLE 4 EXAMPLE 5 Total lutein 82.8% 82.2% 81.7% 82.5% 82.4%

[0160]

[0161] DESCRIPTION

[0162] content

[0163] All-trans lutein

[0164] 89.8% 89.2% 88.8% 89.5% 89.3% proportion

[0165] Total lutein

[0166] 80.6% 80.1% 79.5% 80.3% 80.2% recovery

[0167] Saponification

[0168] 86.9% 86.4% 86.0% 86.7% 86.5% conversion rate

[0169] Cis isomer

[0170] 10.2% 10.8% 11.2% 10.5% 10.7% proportion

[0171] Ethanol

[0172] Within limit Within limit Within limit Within limit Within limit residue

[0173] System

[0174] Stable Stable Stable Stable Stable operating state

[0175]

[0176] Table 2 Performance test results of the comparative examples are as follows:

[0177] COMPARATIVE COMPARATIVE COMPARATIVE COMPARATIVE COMPARATIVE ITEM EXAMPLE 1 EXAMPLE 2 EXAMPLE 3 EXAMPLE 4 EXAMPLE 5 Total lutein

[0178] 78.2% 78.9% 78.5% 79.4% 79.8% content

[0179] All-trans lutein

[0180] 85.8% 84.0% 86.5% 87.5% 87.8% proportion

[0181] Total lutein

[0182] 75.8% 78.6% 74.9% 77.2% 77.8% recovery

[0183] Saponification

[0184] 86.2% 85.8% 86.1% 83.6% 86.3% conversion rate

[0185] Cis isomer

[0186] 14.2% 16.0% 13.5% 12.5% 12.2% proportion

[0187] Ethanol

[0188] Within limit Within limit Within limit Within limit Within limit residue

[0189] Emulsification

[0190] after Slight pressure System saponification; increase in resin Stable Stable Stable

[0191] operating state slight pressure column; turbid increase in resin eluate column

[0192]

[0193] It can be seen from Table 1 that the indicators of Examples 1 to 5 are relatively close, indicating that the method of the present invention can be stably implemented within the defined parameter ranges and has good processDESCRIPTION

[0194] reproducibility. The all-trans lutein proportion of Example 1 reached 89.8%, and the total lutein recovery reached 80.6%, both of which are at preferred levels.

[0195] It can be seen from the data of the comparative examples in Table 2 that, in Comparative Example 1 without adding MCT oil, the total lutein recovery was only 75.8%, significantly lower than 80.6% in Example 1, indicating that a small amount of MCT oil assisted the migration of lutein esters from marigold tissue. In Comparative Example 2 without nitrogen protection and light- shielding control, the all-trans lutein proportion decreased to 84.0% and the cis isomer proportion increased to 16.0%, indicating that low-oxygen and light- shielding conditions have a substantial effect on protecting the conjugated structure of lutein and reducing isomerization. In Comparative Example 3 without enzyme-assisted cell wall disruption, the total lutein recovery decreased to 74.9%, reflecting the promoting effect of compound-enzyme cell wall disruption on lutein ester release. In Comparative Example 4 without pre-deoiling after extraction, the saponification conversion rate decreased to 83.6%, obvious emulsification occurred after saponification, and a slight pressure increase occurred during subsequent resin column operation, indicating that the free MCT oil phase entering the saponification system produces a fatty acid salt emulsified layer, interferes with the saponification reaction, and affects resin column operation. In Comparative Example 5 without neutralization and soap removal after saponification, the resin column showed a slight pressure increase and the eluate had turbid entrainment, indicating that fatty acid salts and residual oily matter pose a contamination risk to the membrane and resin units.

[0196] Based on the above test results, the present invention combines a small amount of MCT oil assisted extraction, pre-deoiling before saponification, low-oxygen and light-shielded environmental control, compound-enzyme-assisted cell wall disruption, and neutralization and soap-removal stratification after saponification, thereby forming a connection among the steps, improving lutein recovery while maintaining a relatively high all-trans lutein proportion, reducingDESCRIPTION

[0197] cis isomer formation, and ensuring stable operation of subsequent membrane separation and resin enrichment processes. The obtained lutein crystalline product has a total lutein content of 81.7%-82.8%, a total lutein recovery of 79.5%-80.6%, and an all-trans lutein proportion of 88.8%-89.8%, and all indicators can meet the quality requirements of food-grade lutein products.

[0198] The above are merely preferred embodiments of the present invention and do not constitute any limitation to the present invention in any form. Although the present invention has been disclosed above by preferred embodiments, the present invention is not limited thereto. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, may make minor changes or modifications based on the technical contents disclosed above to form equivalent embodiments with equivalent changes. Any simple modification, equivalent change or modification made to the above embodiments based on the technical essence of the present invention without departing from the contents of the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. CLAIMS1. A method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification, characterized in that the method comprises the following steps:51, pulverizing dried marigold flower granules, and adding an antioxidant protective agent under light-shielded conditions or yellow safe-light conditions for pretreatment to obtain a pretreated raw material;52, adding a citric acid-sodium citrate buffer and a compound enzyme solution to the pretreated raw material for enzymolysis to obtain an enzymolyzed material;53, adding an assisted extraction system composed of an aqueous ethanol solution and medium-chain triglyceride oil to the enzymolyzed material, and extracting under nitrogen protection and low-oxygen conditions to obtain an extract containing lutein esters;54, after extraction is completed, subjecting the extract containing lutein esters to stratification treatment, separating a free medium-chain triglyceride oil phase, washing the free medium-chain triglyceride oil phase with an aqueous ethanol solution, combining the hydroalcoholic phase and the washing liquid, subjecting an interfacial layer formed during extraction to secondary clarification and incorporating only the clarified hydroalcoholic layer, and discarding residual oily matter and a turbid emulsified layer, to obtain a deoiled extract;55, adding a potassium hydroxide ethanol solution to the deoiled extract for saponification to obtain a saponified solution;56, after saponification is completed, adjusting the saponified solution to pH 6.5-7.0, diluting and stratifying to remove a fatty-acid-salt-enriched layer and residual oily matter, and collecting an intermediate hydroalcoholic phase;57, subjecting the intermediate hydroalcoholic phase sequentially to treatment with a microfiltration membrane and an ethanol-resistant ultrafiltration membrane to obtain a clarified lutein solution;58, enriching the clarified lutein solution through a weak-polarity macroporous adsorption resin, and eluting with an aqueous ethanol solution toCLAIMSobtain a lutein enriched solution;S9, subjecting the lutein enriched solution to reduced-pressure concentration followed by low-temperature crystallization, filtration, washing, and vacuum drying to obtain the lutein crystalline product.

2. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1 , characterized in that, in step S 1 , the wavelength of the yellow safe light is 560-590 nm, and the illuminance is not higher than 500 lx;the antioxidant protective agent is vitamin E and ascorbyl palmitate, and the total amount of vitamin E and ascorbyl palmitate added is 0.06%-0.10% of the mass of the dried marigold flower granules.

3. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in step S2, the compound enzyme solution is composed of cellulase, pectinase and hemicellulase, the mass ratio of cellulase, pectinase and hemicellulase is l:(0.8-1.2):(0.6-1.0), and the total amount of the compound enzymes added is 0.6%-1.0% of the mass of the dried marigold flower granules;the liquid-to-solid ratio for enzymolysis is 2-4 mL / g, the temperature is 42-46°C, the pH is 4.8-5.2, and the time is 1.0-1.5 h.

4. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in step S3, the ethanol volume fraction in the aqueous ethanol solution is 70%-85%, and the volume ratio of the aqueous ethanol solution to the medium-chain triglyceride oil is 97:3-92:8;the liquid-to-solid ratio of the assisted extraction system to the dried marigold flower granules is 7-10 mL / g;the low-oxygen condition means that the oxygen volume fraction in the headspace of the reaction vessel is not higher than 3%, the extraction temperature isCLAIMS42-46°C, and the extraction time is 1.5-2.5 h.

5. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in step S4, the ethanol volume fraction in the aqueous ethanol solution for washing the free medium-chain triglyceride oil phase is 75%-85%, the number of washes is 1-2, and the amount used for each wash is 0.5-1.5 times the volume of the free medium-chain triglyceride oil phase being washed;the secondary clarification is secondary standing clarification or secondary centrifugation.

6. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in step S5, the potassium hydroxide ethanol solution is added in an amount controlled at 1.2-2.0 times the saponification demand of lutein esters in the deoiled extract, so that the final concentration of potassium hydroxide in the saponification system is 0.08-0.16 mol / L, the saponification temperature is 42-46°C, and the saponification time is 0.8-1.2 h.

7. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in step S5, the saponification demand of the lutein esters is based on the total lutein ester content determined by high-performance liquid chromatography and is converted into the molar amount of potassium hydroxide required according to the lutein diester structure;the actual addition amount of the potassium hydroxide ethanol solution is corrected according to a bench-scale saponification curve.

8. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in steps S4 and S6, the stratification treatment is standing stratification or centrifugal stratification, wherein the conditions forCLAIMScentrifugal stratification are centrifugation at 3000-5000 rpm for 5-15 min;in step S7, the pore size of the microfiltration membrane is 0.22-0.45 pm, and the molecular weight cut-off of the ethanol-resistant ultrafiltration membrane is 10-30 kDa.

9. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in step S7, the ethanol-resistant ultrafiltration membrane is a ceramic membrane, a polyvinylidene fluoride membrane, a poly ethersulfone membrane, or a polytetrafluoroethylene membrane.

10. The method for preparing a lutein crystalline product by aqueous ethanol-MCT oil assisted extraction and pre-deoiling saponification according to claim 1, characterized in that, in step S8, the weak-polarity macroporous adsorption resin is a styrene-based weak-polarity macroporous adsorption resin, and the styrene-based weak-polarity macroporous adsorption resin is an AB-8 type or DI 01 type macroporous adsorption resin;the ethanol volume fraction in the aqueous ethanol solution used for elution is 75%-85%;in step S9, the temperature for reduced-pressure concentration is 30-40°C, the temperature for low-temperature crystallization is -5°C to 5°C, and the temperature for vacuum drying is 30-40°C.

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