阅读说明：本技术 一种隐黄质的制备方法 (Preparation method of cryptoxanthin ) 是由 刘贤军 刘霞 杨国强 严守彩 于 2021-06-29 设计创作，主要内容包括：本发明涉及类胡萝卜素制备技术领域,具体涉及一种隐黄质的制备方法；将万寿菊鲜花浆液稀释后进行PH值调整,通过加入水解酶、脂肪酶和碱性蛋白酶水解,经过超声粉碎过滤后,对滤渣进行萃取合并,将物料浓缩,再次PH值调整,并加热回流,获得产物脱水叶黄素,将产物脱水叶黄素异构化,通过PH值调整、通入气体、离心、静置、结晶分离以及真空干燥,获得隐黄质提取物；本发明采用生物酶解的方法提取万寿菊中的叶黄素,然后利用脂肪酶水解的方法对酯进行水解皂化,实验条件更温和、产品安全、减少环境污染、减少有机溶剂的使用,采用加压氢化的方法获得隐黄质,方法简单,易操作,工序少,减少了目标产物的损失,得率高。(The invention relates to the technical field of carotenoid preparation, in particular to a preparation method of cryptoxanthin; diluting fresh marigold flower slurry, adjusting the pH value, adding hydrolase, lipase and alkaline protease for hydrolysis, performing ultrasonic crushing and filtering, extracting and combining filter residues, concentrating materials, adjusting the pH value again, heating and refluxing to obtain a product dehydrated lutein, isomerizing the product dehydrated lutein, and performing pH value adjustment, gas introduction, centrifugation, standing, crystallization separation and vacuum drying to obtain a cryptoxanthin extract; the method adopts a biological enzymolysis method to extract the lutein in the marigold, then utilizes a lipase hydrolysis method to carry out hydrolysis and saponification on the ester, has mild experimental conditions, safe products, reduces environmental pollution and reduces the use of organic solvents, adopts a pressure hydrogenation method to obtain the cryptoxanthin, and has the advantages of simple method, easy operation, less working procedures, reduction of the loss of target products and high yield.)

1. A preparation method of cryptoxanthin is characterized by comprising the steps of diluting fresh marigold flower slurry, adjusting the pH value, adding hydrolase, lipase and alkaline protease for hydrolysis, carrying out ultrasonic crushing and filtering, extracting and combining filter residues, concentrating materials, adjusting the pH value again, heating and refluxing to obtain a product of dehydrated lutein, isomerizing the product of the dehydrated lutein, adjusting the pH value, introducing gas, centrifuging, standing, carrying out crystallization separation and carrying out vacuum drying to obtain the cryptoxanthin extract.

2. The method of claim 1, wherein the hydrolytic enzyme is selected from the group consisting of protease, cellulase and pectinase.

3. The method of claim 1, wherein the hydrolysate is ultrasonically pulverized by an ultrasonic pulverizer.

4. The process of claim 1, wherein the material is concentrated to 20% by a wiped film flash evaporator.

5. The method of claim 1, wherein the gas is hydrogen.

6. The method of claim 1, comprising the steps of:

s1, taking fresh marigold flower slurry, adding water, uniformly stirring, adjusting the pH value, heating in a water bath, adding protease, cellulase and pectinase, uniformly stirring, performing constant-temperature water bath dark oscillation for enzymolysis, adjusting the pH value with a sodium hydroxide solution, homogenizing for 3min, adding lipase and alkaline protease, uniformly stirring, performing dark reaction on a water bath constant-temperature oscillator for 2-4h, homogenizing for 2min, adding lipase twice, uniformly stirring, and reacting for 1-2h to obtain a feed liquid;

s2, treating the feed liquid by using an ultrasonic pulverizer, repeating for 2 times, oscillating for 0.5h in a water bath at constant temperature, and filtering;

s3, mixing the filter residue in the S2 with a solvent according to a material-liquid ratio of 1: (1-4), extracting for 2-4 times;

s4, combining the extraction liquid in the S3, and concentrating by a wiped film flash evaporator under the condition that the working temperature is 45-60 ℃;

s5, adding 50% sulfuric acid to adjust the pH value, and then heating and refluxing at 80-88 ℃ for 2-4h to obtain a product, namely dehydrated lutein;

s6, adding a certain amount of water for reflux reaction for 1-2h to isomerize;

s7, adjusting the pH value to 7.0 by using 2moL/L sodium hydroxide, putting the feed liquid into high-pressure equipment, introducing hydrogen, controlling the pressure to be 1.8-3.5MPa, keeping the pressure for 10-30min, and then performing centrifugal separation;

s8, dissolving the lower-layer centrifugate with ethanol, cooling, standing, recrystallizing, and centrifuging;

s9, vacuum drying at 50 deg.C for 3 hr to obtain cryptoxanthin extract.

7. The method of claim 6, wherein citric acid is added to adjust the pH to 4.0-6.0 in step S1.

8. The method of claim 6, wherein in step S8, the cryptoxanthin is dissolved in ethanol at 60-78 deg.C for 30-60min, cooled to 10-20 deg.C and left to stand for 2-4 h.

Technical Field

The invention relates to the technical field of carotenoid preparation, and particularly relates to a preparation method of cryptoxanthin.

Background

Cryptoxanthin is carotenoid, has good oxidation resistance, and has unique physiological functions in resisting cancer, enhancing immunity, reducing incidence of cardiovascular diseases, etc. In the aspect of anticancer, beta-cryptoxanthin is considered as a good anticancer factor, and particularly has a unique curative effect on lung cancer caused by smoking. In enhancing immunity, lymphocyte proliferation reaction can be stimulated by promoting antigen. It can also protect cells and DNA from free radical damage and can repair oxidized DNA. However, the human body cannot synthesize it by itself and is mainly obtained by food. Cryptoxanthin sources include natural sources and artificial synthesis. Cryptoxanthin, which is of natural origin, is mainly obtained by extraction of natural products, and synthesis is mainly obtained starting from isoprenoids or by conversion from carotenoids. The content of natural extraction is too low, a large amount of organic solvent is needed, the synthetic reaction is generally hydrogenation reaction through hydroboration, toxic solvents such as dichloromethane and borane are used in the reaction, the process is complex, the number of side reactions is large, and the safety of products is difficult to guarantee.

Chinese patent CN105073711A reports that beta-cryptoxanthin derived from plants and a preparation method thereof, and the process is complex and tedious, is not beneficial to industrial production and limits the development and application of the beta-cryptoxanthin.

Chinese patent CN109503447B reports a method for preparing cryptoxanthin from marigold extract, namely a method for obtaining cryptoxanthin from marigold extract through hydrolysis and acidification, wherein a large amount of alkali is needed in the hydrolysis and saponification process of marigold extract, most intermediate products including dehydrated lutein and cis cryptoxanthin are generated in the acid treatment process, and the yield of the product is greatly reduced.

Chinese patent CN106977440B reports a method for separating and purifying beta-cryptoxanthin, which mainly uses a silica gel chromatographic column to carry out primary separation on the beta-cryptoxanthin in capsanthin; saponifying the primarily separated beta-cryptoxanthin; extracting saponified beta-cryptoxanthin by a solvent for primary concentration; and further separating and purifying the beta-cryptoxanthin by using a silica gel chromatographic column. The method is used for separating, saponifying and separating cryptoxanthin from natural sources, the yield of the natural sources is low, and a large amount of organic solvents are used for saponification and column analysis.

In summary, the development of a cryptoxanthin preparation method remains a key problem to be solved urgently in the technical field of carotenoid preparation.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention provides a method for preparing cryptoxanthin from fresh marigold flowers, which has high safety, high yield and convenient production.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for preparing cryptoxanthin comprises diluting fresh flower slurry of marigold, adjusting pH, hydrolyzing with hydrolase, lipase and alkaline protease, ultrasonic pulverizing, filtering, extracting and mixing the residue, concentrating, adjusting pH, heating under reflux to obtain dehydrated lutein, isomerizing the dehydrated lutein, adjusting pH, introducing gas, centrifuging, standing, crystallizing, separating, and vacuum drying to obtain cryptoxanthin extract.

Preferably, the hydrolytic enzyme is a protease, a cellulase, and a pectinase.

Preferably, the hydrolysate is ultrasonically crushed using an ultrasonic crusher.

Preferably, the material is concentrated to 20% using a wiped film flash evaporator.

Preferably, the gas is hydrogen.

Preferably, the method comprises the following steps:

s1, taking fresh marigold flower slurry, adding water, uniformly stirring, adjusting the pH value, heating in a water bath, adding protease, cellulase and pectinase, uniformly stirring, performing constant-temperature water bath dark oscillation for enzymolysis, adjusting the pH value with a sodium hydroxide solution, homogenizing for 3min, adding lipase and alkaline protease, uniformly stirring, performing dark reaction on a water bath constant-temperature oscillator for 2-4h, homogenizing for 2min, adding lipase twice, uniformly stirring, and reacting for 1-2h to obtain a feed liquid;

and S2, treating the feed liquid by using an ultrasonic pulverizer, repeating for 2 times, oscillating for 0.5h in a water bath at constant temperature, and filtering.

S3, mixing the filter residue in the S2 with a solvent according to a material-liquid ratio of 1: (1-4), extracting for 2-4 times.

S4, combining the extracts in the S3, and concentrating the combined extracts by a wiped film flash evaporator under the condition that the working temperature is 45-60 ℃.

S5, adding 50% sulfuric acid to adjust the pH value, and then heating and refluxing at 80-88 ℃ for 2-4h to obtain the product of dehydrated lutein.

S6, adding a certain amount of water for reflux reaction for 1-2h for isomerization.

S7, adjusting the pH value to 7.0 by using 2moL/L sodium hydroxide, putting the feed liquid into a high-pressure device, introducing hydrogen, controlling the pressure to be 1.8-3.5MPa, keeping the pressure for 10-30min, and then carrying out centrifugal separation.

And S8, dissolving the lower-layer centrifugate with ethanol, cooling, standing, recrystallizing and centrifuging.

S9, vacuum drying at 50 deg.C for 3 hr to obtain cryptoxanthin extract.

Preferably, in the step S1, citric acid is added to adjust the PH to 4.0-6.0.

Preferably, in the step S8, ethanol is used for dissolving for 30-60min at the temperature of 60-78 ℃, and the solution is cooled to 10-20 ℃ and kept stand for 2-4 h.

Advantageous effects

Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:

1. the method adopts a biological enzymolysis method to extract the lutein in the marigold, and then utilizes a lipase hydrolysis method to carry out hydrolysis and saponification on the ester, so that the experimental conditions are mild, the product is safe, the environmental pollution is reduced, and the use of organic solvents is reduced.

2. The invention adopts a pressure hydrogenation method to obtain cryptoxanthin, has simple method, easy operation and few working procedures, reduces the loss of target products and has high yield.

Drawings

FIG. 1 is a flow chart of a process for the preparation of cryptoxanthin.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention will be further described with reference to the following examples.

Example 1

Referring to fig. 1, the preparation method of cryptoxanthin in the present embodiment includes the following steps:

taking 1kg of fresh marigold flower pulp (crushed and ground-milled), adding 1.5kg of water, stirring uniformly, adding citric acid to adjust the pH value to 4.5, heating in a water bath at 45 ℃, adding hydrolase (protease 600U/g, cellulase 700U/g and pectinase 500U/g), stirring uniformly, and then oscillating in a constant-temperature water bath in a dark place to perform enzymolysis reaction for 4 hours; adjusting pH to 7 with sodium hydroxide solution (2moL/L), homogenizing for 3min, adding lipase (addition amount is 800U/g) and alkaline protease (addition amount is 600U/g), stirring, reacting for 2h on a water bath constant temperature oscillator, and homogenizing for 4 min; adding lipase (with the addition of 1200U/g) for the second time, stirring uniformly, and reacting for 2 h; treating the feed liquid with an ultrasonic pulverizer, repeating for 2 times, with total working time of 6min, working time/interval time of 15s/30s and power of 600W, oscillating in 45 deg.C water bath at constant temperature for 0.5 hr, and filtering; and filtering residues by using a solvent ethanol and n-hexane (V: V1: 1) according to a material-liquid ratio of 1: 2, extracting for 3 times; mixing filtrates, concentrating with a film-scraping flash evaporator at 50 deg.C to 10%; adding 50% sulfuric acid to adjust pH to 1.5, heating and refluxing at 82 deg.C, and reacting for 3 hr to obtain dehydrated xanthophyll; adding a certain amount of water for reflux reaction for 2 hours to carry out isomerization; adjusting the pH value to 7 by using 2moL/L sodium hydroxide; putting the feed liquid into high-pressure equipment, introducing hydrogen, controlling the pressure at 3.5MPa, keeping for 25min, and carrying out hydrogenation reaction; carrying out centrifugal separation; dissolving the lower layer with ethanol at 60 deg.C for 30min, cooling to 15 deg.C, standing for 3 hr for crystallization, and centrifuging; vacuum drying at 50 deg.C for 3 hr to obtain extract 2.2 g.

HPLC detection is carried out on the obtained cryptoxanthin, and the content of the cryptoxanthin is 85 percent according to detection.

Example 2

Referring to fig. 1, the preparation method of cryptoxanthin in the present embodiment includes the following steps:

taking 1kg of fresh marigold flower slurry (crushed and ground), adding 2kg of water in a certain proportion, uniformly stirring, adding citric acid to adjust the pH value to 4.5, heating in a water bath to 45 ℃, adding hydrolase (protease 500U/g, cellulase 600U/g and pectinase 600U/g), uniformly stirring, carrying out enzymolysis reaction for 3 hours by constant-temperature water bath and dark oscillation, adjusting the pH to 7.5 and homogenizing for 4 minutes by using 2moL/L of sodium hydroxide solution, adding lipase (the addition amount is 700U/g) and alkaline protease (the addition amount is 700U/g), uniformly stirring, reacting for 3 hours on a water bath constant-temperature oscillator, homogenizing for 4 minutes, adding lipase (the addition amount is 1500U/g) twice, uniformly stirring, and reacting for 2 hours; treating the feed liquid with an ultrasonic pulverizer, repeating for 2 times, with total working time of 2-10min, working time/interval time of 15s/30s and power of 500W, oscillating in 45 deg.C water bath at constant temperature for 0.5h, filtering, and mixing the filter residue with ethanol or n-hexane as solvent at a ratio of 1: 3, extracting for 3 times; mixing filtrates, concentrating with a film-scraping flash evaporator at 45 deg.C to 20%; adding 50% sulfuric acid to adjust pH to 1.5, heating and refluxing at 85 deg.C, and reacting for 2 hr to obtain dehydrated xanthophyll; adding a certain amount of water for reflux reaction for 2 hours to carry out isomerization; adjusting the pH value to 7 by using 2moL/L sodium hydroxide; putting the feed liquid into high-pressure equipment, introducing hydrogen, controlling the pressure at 3.0MPa, keeping for 30min, and carrying out hydrogenation reaction; carrying out centrifugal separation; dissolving the lower layer with ethanol at 65 deg.C for 50min, cooling to 10 deg.C, standing for 4 hr, crystallizing, and centrifuging; vacuum drying at 50 deg.C for 3 hr to obtain cryptoxanthin extract 1.9 g.

HPLC detection is carried out on the obtained cryptoxanthin, and the content of the cryptoxanthin is 80 percent according to detection.

Example 3

Referring to fig. 1, the preparation method of cryptoxanthin in the present embodiment includes the following steps:

taking 1kg of fresh marigold flower slurry (crushed and ground), adding 1kg of water in a certain proportion, uniformly stirring, adding citric acid to adjust the pH value to 4.5, heating in a water bath to 45 ℃, adding hydrolase (protease 600U/g, cellulase 500U/g and pectinase 600U/g), uniformly stirring, carrying out enzymolysis reaction for 2 hours by constant-temperature water bath in a dark oscillation mode, adjusting the pH value to 7.5 and homogenizing for 3 minutes by using 2moL/L of sodium hydroxide solution, adding lipase (the addition is 900U/g) and alkaline protease (the addition is 700U/g), uniformly stirring, carrying out reaction on a water bath constant-temperature oscillator for 3 hours, homogenizing for 4 minutes, adding lipase (the addition is 1300U/g) twice, uniformly stirring, and reacting for 2 hours; treating the feed liquid with an ultrasonic pulverizer, repeating for 2 times, with total working time of 2-10min, working time/interval time of 15s/30s and power of 600W, oscillating in 45 deg.C water bath at constant temperature for 0.5h, filtering, and mixing the filter residue with ethanol or n-hexane as solvent at a ratio of 1: 4, extracting for 2 times; mixing filtrates, concentrating with a film-scraping flash evaporator at 50 deg.C to 20%; adding 50% sulfuric acid to adjust pH to 1.5, heating and refluxing at 85 deg.C, and reacting for 2 hr to obtain dehydrated xanthophyll; adding a certain amount of water for reflux reaction for 2 hours to carry out isomerization; adjusting the pH value to 7 by using 2moL/L sodium hydroxide; putting the feed liquid into high-pressure equipment, introducing hydrogen, controlling the pressure at 2.5MPa, keeping for 30min, and carrying out hydrogenation reaction; carrying out centrifugal separation; dissolving the lower layer with ethanol at 65 deg.C for 50min, cooling to 17 deg.C, standing for 3 hr, crystallizing, and centrifuging; vacuum drying at 50 deg.C for 3 hr to obtain cryptoxanthin extract 1.7 g.

HPLC detection is carried out on the obtained cryptoxanthin, and the content of the cryptoxanthin is 78 percent according to detection.

Example 4

Referring to fig. 1, the preparation method of cryptoxanthin in the present embodiment includes the following steps:

taking 0.5kg of fresh marigold flower slurry (crushed and ground), adding 0.5kg of water according to a certain proportion, uniformly stirring, adding citric acid to adjust the pH value to 6, heating in a water bath at 50 ℃, adding hydrolase (protease 800U/g, cellulase 600U/g and pectinase 700U/g), uniformly stirring, carrying out enzymolysis reaction for 6 hours by constant-temperature water bath in a dark oscillation mode, adjusting the pH to 8 and homogenizing for 3 minutes by using 2moL/L of sodium hydroxide solution, adding lipase (the addition is 1000U/g) and alkaline protease (the addition is 800U/g), uniformly stirring, carrying out reaction on a water bath constant-temperature oscillator for 4 hours, homogenizing for 2 minutes, adding lipase (the addition is 1500U/g) twice, uniformly stirring and carrying out reaction for 2 hours; treating the feed liquid with an ultrasonic pulverizer, repeating for 2 times, oscillating for 30min in a water bath at 45 ℃ at constant temperature, filtering, and mixing the filter residue with ethanol solvent according to a feed-liquid ratio of 1: 4, extracting for 3 times; mixing filtrates, concentrating with a film-scraping flash evaporator at 60 deg.C to 20%; adding 50% sulfuric acid to adjust pH to 1.5, heating and refluxing at 88 deg.C, and reacting for 4 hr to obtain dehydrated xanthophyll; adding a certain amount of water for reflux reaction for 2 hours to carry out isomerization; adjusting the pH value to 7 by using 2moL/L sodium hydroxide; putting the feed liquid into high-pressure equipment, introducing hydrogen, controlling the pressure at 1.8MPa, keeping for 10min, and carrying out hydrogenation reaction; carrying out centrifugal separation; dissolving the lower layer with ethanol at 78 deg.C for 60min, cooling to 10-20 deg.C, standing for 4 hr, crystallizing, and centrifuging; vacuum drying at 50 deg.C for 3 hr to obtain cryptoxanthin extract 2 g.

HPLC detection is carried out on the obtained cryptoxanthin, and the content of the cryptoxanthin is 75 percent according to detection.

From examples 1 to 4, it can be seen that the preparation method of cryptoxanthin of the present invention adopts a biological enzymolysis method to extract lutein from marigold, and then utilizes a lipase hydrolysis method to perform hydrolysis and saponification on esters, and the experimental conditions are mild, the product is safe, the environmental pollution is reduced, the use of organic solvents is reduced, and the cryptoxanthin is obtained by a pressure hydrogenation method, and the method is simple, easy to operate, less in process, less in loss of target products, and high in yield.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.