阅读说明：本技术 一种从海带中提取岩藻黄素的方法 (Method for extracting fucoxanthin from kelp ) 是由 李达谅 曾鹭 黄鹭强 邓威力 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种从海带中提取岩藻黄素的方法,属于岩藻黄素提取工艺领域,包括如下步骤：A1预处理；A2酶解；A3、萃取；A4固液分离；A5浓缩；A6分离提纯；A7再浓缩；A8、干燥和保存。本发明从海带中提取岩藻黄素的方法,操作工艺简单,反应条件温和,提取效果好。(The invention discloses a method for extracting fucoxanthin from kelp, which belongs to the field of fucoxanthin extraction processes and comprises the following steps: a1 pretreatment; a2 enzymolysis; a3, extracting; a4 solid-liquid separation; a5 concentration; a6 separating and purifying; a7 is re-concentrated; a8, drying and storing. The method for extracting fucoxanthin from kelp has the advantages of simple operation process, mild reaction condition and good extraction effect.)

1. A method for extracting fucoxanthin from kelp is characterized in that: the method comprises the following steps:

a1, pretreatment: washing fresh kelp with clear water, washing off surface salt and mucus, sucking surface water with filter paper, and shearing into 0.1-0.5 cm-sized pieces of kelp;

a2, enzymolysis: taking the crushed kelp, adding deionized water and a complex enzyme preparation, uniformly mixing, carrying out enzymolysis at 35-45 ℃, carrying out suction filtration, and drying the enzymolyzed kelp in the shade in a cool and ventilated place;

a3, extraction: drying the kelp in the shade according to the proportion of 1: adding absolute ethyl alcohol in a weight g/volume mL ratio of 5-30, mixing, adding an antioxidant, leaching for 30-60 min at normal temperature in a dark place, and then putting into an ultrasonic-microwave synergistic extraction instrument for ultrasonic-microwave synergistic extraction;

a4, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a5, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 30-50 ℃ to obtain a fucoxanthin crude extract;

a6, separation and purification: separating and purifying the obtained fucoxanthin crude extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 1-10: 1, collecting orange to orange eluent;

a7, re-concentration: putting the collected eluent into a rotary evaporator, and carrying out evaporation concentration at the temperature of 30-50 ℃;

a8, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

2. The method for extracting fucoxanthin from kelp according to claim 1, wherein: in the step A2, deionized water and kelp are mixed according to the ratio of 1: adding the mixture in a weight g/volume mL ratio of 20-50; adding the complex enzyme preparation according to the weight of the kelp in a volume ratio of 0.15-0.3%, uniformly mixing, and carrying out enzymolysis treatment at 37 ℃ for 5 hours; the compound enzyme preparation is prepared from the following components in a mass ratio of 1: 0.7-1.5 of cellulase and pectinase.

3. The method for extracting fucoxanthin from kelp according to claim 1, wherein: in the step A3, the kelp dried in the shade is dried according to the weight ratio of 1: adding absolute ethyl alcohol according to the weight g/volume mL ratio of 6, mixing, adding 0.05-0.5% volume of antioxidant according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, and leaching for 45min in a dark place at normal temperature.

4. The method for extracting fucoxanthin from kelp according to claim 1, wherein: the antioxidant is 2, 6-di-tert-butyl-p-cresol.

5. The method for extracting fucoxanthin from kelp according to claim 1, wherein: in the step A3, the ultrasonic power is set to be 50W, the microwave power is 200-500W, the extraction temperature is 30-50 ℃, and the extraction time is 3-10 min.

6. The method for extracting fucoxanthin from kelp according to claim 1, wherein: in the step A3, the microwave-ultrasonic synergistic extraction adopts an intermittent extraction mode, and the extraction is repeated for 1-3 times.

7. The method for extracting fucoxanthin from kelp according to claim 1, wherein: in both the steps A5 and A7, the evaporation temperature was set at 40 ℃.

8. The method of claim 1, wherein the fucoxanthin is extracted from the gulfweed by: the steps of enzymolysis, extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or darkness.

Technical Field

The invention belongs to the field of fucoxanthin extraction processes, and particularly relates to a method for extracting fucoxanthin from kelp.

Background

Fucoxanthin (also called Fucoxanthin) is a fat-soluble carotene, is golden brown, has extremely strong antioxidation because of the special allene carbon skeleton contained in the structure, and has pharmacological actions of resisting acne, tumor, malaria, inflammation, angiogenesis, obesity and the like, has protective effects on human cerebral vessels, eyes, skin, bones, livers and the like, and has wide development prospect in the fields of medicines, foods, cosmetics, nutrition and health care and the like. Currently, products containing 1% fucoxanthin are sold at a price of $ 175 per kilogram.

Fucoxanthin is mainly present in animals and plants such as various algae and marine phytoplankton which participate in photosynthesis, and is particularly abundant in phaeophyceae and diatoms in nature, and fucoxanthin which is present in sources other than aquatic sources has not been found. In the existing research, fucoxanthin is mostly extracted from brown algae plants such as kelp and undaria pinnatifida. However, the current fucoxanthin research faces the problems of little research result on fucoxanthin, immature application technology, high environmental sensitivity of the fucoxanthin, high extraction process requirement and the like. And the fucoxanthin product has low purity, which is concentrated at about 10%, the application range is limited greatly, and the commercial development is difficult.

Kelp is a common practical algae, is abundant in shallow sea near the coast of tropical zone and temperate zone, the output of the kelp in China is the first in the world, and tens of thousands of tons can be produced every year. The kelp is rich in fucoxanthin, and the content of fucoxanthin in the carotenoid of the kelp is more than 60%. However, in recent years, research on fucoxanthin in China is not many, and research on the separation and purification of fucoxanthin in algae such as kelp and hizikia fusiforme is partly conducted, and research on the property structure, biological effect, action mechanism, extraction process and the like of fucoxanthin is partly conducted, but the requirement for industrial production of fucoxanthin is not met. If the kelp can be processed to extract fucoxanthin, the additional economic value of the kelp can be improved, and the supply amount of the fucoxanthin can be increased.

The inventor researches and discovers that the fucoxanthin has the characteristics of photosensitivity and heat sensitivity and is easy to lose in the extraction process. The fucoxanthin in the brown algae is extracted by common solvent extraction method, supercritical CO2 extraction method, ultrasonic auxiliary extraction method and the like, and the extraction efficiency is low and the extraction effect is poor.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a method for extracting fucoxanthin from kelp, which has the advantages of simple operation process, mild reaction conditions and good extraction effect.

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

a method for extracting fucoxanthin from kelp comprises the following steps:

a1, pretreatment: washing fresh kelp with clear water, washing off surface salt and mucus, sucking surface water with filter paper, and shearing into 0.1-0.5 cm-sized pieces of kelp;

a2, enzymolysis: taking the crushed kelp, adding deionized water and a complex enzyme preparation, uniformly mixing, carrying out enzymolysis at 35-45 ℃, carrying out suction filtration, and drying the enzymolyzed kelp in the shade in a cool and ventilated place;

a3, extraction: drying the kelp in the shade according to the proportion of 1: adding absolute ethyl alcohol in a weight g/volume mL ratio of 5-30, mixing, adding an antioxidant, leaching for 30-60 min at normal temperature in a dark place, and then putting into an ultrasonic-microwave synergistic extraction instrument for ultrasonic-microwave synergistic extraction;

a4, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a5, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 30-50 ℃ to obtain a fucoxanthin crude extract;

a6, separation and purification: separating and purifying the obtained fucoxanthin crude extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 1-10: 1, collecting orange to orange eluent;

a7, re-concentration: and (3) putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 30-50 ℃.

A8, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

Wherein, in the step A2, deionized water and kelp are mixed according to the ratio of 1: adding the mixture in a weight g/volume mL ratio of 20-50; adding the complex enzyme preparation according to the weight of the kelp by 0.15-0.3% in mass proportion, uniformly mixing, and carrying out enzymolysis treatment at 37 ℃ for 5 hours; the compound enzyme preparation is prepared from the following components in a mass ratio of 1: 0.7-1.5 of cellulase and pectinase.

In the step A3, the kelp dried in the shade is dried according to the weight ratio of 1: adding absolute ethyl alcohol according to the weight g/volume mL ratio of 6, mixing, adding 0.05-0.5% volume of antioxidant according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, and leaching for 45min in a dark place at normal temperature.

Wherein the antioxidant is 2, 6-di-tert-butyl-p-cresol.

In the step A3, the ultrasonic power is set to be 50W, the microwave power is 200-500W, the extraction temperature is 30-50 ℃, and the extraction time is 3-10 min.

In the step A3, the microwave-ultrasonic synergistic extraction adopts an intermittent extraction mode, and the extraction is repeated for 1-3 times.

Wherein, in the steps A5 and A7, the evaporation temperature is set to be 40 ℃.

Wherein, the steps of enzymolysis, extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or dark.

In the invention, the kelp used is purchased from the same lot in Lianjiang county of Fujian province.

2, 6-di-tert-butyl-p-cresol, also called 2, 6-di-tert-butyl-4-methylphenol, BHT for short, has strong oxidation resistance, good heat resistance and stability, is an oil-soluble antioxidant widely used at home and abroad, and is often used as an antioxidant in the processing process of foods such as edible oil and fat, fried foods, biscuits, instant noodles and the like. Researches show that the antioxidant BHT has a good protection effect on fucoxanthin extracted from kelp, the protection effect is enhanced along with the increase of the concentration of the BHT, and the stability of the fucoxanthin is enhanced, so that the antioxidant BHT is added in the extraction process of the step A3, the extraction process of pigments is carried out under the protection of the BHT, the extraction rate of the fucoxanthin is enhanced, and the extraction effect is ensured.

The invention has the following beneficial effects:

1. in the invention, the salt and the mucus attached to the kelp can be removed through water washing in the pretreatment, the mucus contains a large amount of mannitol and polysaccharide, and the water washing can avoid the influence on the subsequent extraction process, thereby being beneficial to enhancing the extraction effect of fucoxanthin and improving the purity of fucoxanthin.

2. According to the invention, the fucoxanthin in the kelp is extracted by synergistic effects of enzymolysis of a complex enzyme preparation, ultrasonic extraction and microwave extraction in absolute ethyl alcohol, firstly, cell wall components in kelp cells are degraded by enzymolysis, and then, the kelp cells are acted by the synergistic extraction of the ultrasonic and the microwave, so that the rapid wall breaking of the kelp cells is accelerated, and the fucoxanthin in the cells is fully dissolved out in a short time.

3. In the invention, after the fucoxanthin is separated by silica gel column chromatography, firstly, a rotary evaporator is adopted for primary concentration, the volume of an extracting solution is reduced, then, a nitrogen blowing instrument is used, the sample is rapidly concentrated by using nitrogen in the nitrogen, the volatilization of an organic solvent is accelerated, the exposure time of the fucoxanthin in the sample under the air is reduced, the fucoxanthin is prevented from being degraded, the stability of the sample is ensured, and the accuracy of experimental data is further ensured; and can handle multiunit sample simultaneously, operating efficiency is high, has shortened experimenter's extraction time.

4. In the invention, antioxidant BHT is added in the extraction process, so that the fucoxanthin can be effectively protected after being dissolved out, the extraction rate of the fucoxanthin can be enhanced, and the extraction effect can be ensured.

Drawings

FIG. 1 is a process flow diagram of the present invention;

FIG. 2 is a HPLC peak profile of fucoxanthin standard;

FIG. 3 is a HPLC peak profile of a pure fucoxanthin prepared in example 3.

Detailed Description

In order to better understand the present invention, the following examples are provided for illustration purposes only and are not intended to limit the present invention.

Example 1

A method for extracting fucoxanthin from kelp comprises the following steps:

a1, pretreatment: washing fresh herba Zosterae Marinae with clear water, removing surface salt and mucus, sucking surface water with filter paper, and shearing into 0.1 cm-sized pieces of herba Zosterae Marinae;

a2, enzymolysis: taking the crushed kelp, adding deionized water and a complex enzyme preparation, and uniformly mixing, wherein the deionized water and the kelp are mixed according to the ratio of 1: 20 g/mL; adding the complex enzyme preparation according to the weight of the kelp by 0.15 percent in a mass ratio, uniformly mixing, and performing enzymolysis treatment at 35 percent for 5 hours; the compound enzyme preparation is prepared from the following components in a mass ratio of 1: 0.7 cellulase and pectinase;

a3, extraction: drying the kelp in the shade according to the proportion of 1: adding absolute ethyl alcohol according to the weight g/volume mL of 5, mixing, adding 0.05% of antioxidant 2, 6-di-tert-butyl-p-cresol according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, leaching for 30min at normal temperature in a dark place, and then putting the mixture into an ultrasonic-microwave synergistic extraction instrument for ultrasonic-microwave synergistic extraction; setting the ultrasonic power to be 50W, the microwave power to be 200W, the extraction temperature to be 30 ℃ and the extraction time to be 3 min;

a4, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a5, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 30 ℃ to obtain a fucoxanthin crude extract;

a6, separation and purification: separating and purifying the obtained crude fucoxanthin extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 1:1, collecting orange to orange eluent;

a7, re-concentration: putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 30 ℃;

a8, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

Wherein, the microwave-ultrasonic synergic extraction adopts an intermittent extraction mode, and the extraction is repeated for 1 time.

Wherein, the steps of enzymolysis, extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or dark.

Example 2

A method for extracting fucoxanthin from kelp comprises the following steps:

a1, pretreatment: washing fresh herba Zosterae Marinae with clear water, removing surface salt and mucus, sucking surface water with filter paper, and shearing into 0.5 cm-sized pieces of herba Zosterae Marinae;

a2, enzymolysis: taking the crushed kelp, adding deionized water and a complex enzyme preparation, and uniformly mixing, wherein the deionized water and the kelp are mixed according to the ratio of 1: 50 g/mL; adding the complex enzyme preparation according to the weight of the kelp by 0.3 percent in mass proportion, uniformly mixing, and carrying out enzymolysis treatment at 45 ℃ for 5 hours; the compound enzyme preparation is prepared from the following components in a mass ratio of 1: 1.5 cellulase and pectinase;

a3, extraction: drying the kelp in the shade according to the proportion of 1: adding absolute ethyl alcohol in a weight g/volume mL ratio of 30, mixing, adding 0.5% volume of antioxidant 2, 6-di-tert-butyl-p-cresol according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, leaching for 60min at normal temperature in a dark place, and then putting the mixture into an ultrasonic-microwave synergistic extraction instrument for ultrasonic-microwave synergistic extraction; setting the ultrasonic power to be 50W, the microwave power to be 500W, the extraction temperature to be 50 ℃ and the extraction time to be 10 min;

a4, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a5, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 50 ℃ to obtain a fucoxanthin crude extract;

a6, separation and purification: separating and purifying the obtained crude fucoxanthin extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 10: 1, collecting orange to orange eluent;

a7, re-concentration: putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 50 ℃;

a8, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

Wherein, the microwave-ultrasonic synergic extraction adopts an intermittent extraction mode, and the extraction is repeated for 3 times.

Wherein, the steps of enzymolysis, extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or dark.

Example 3

A method for extracting fucoxanthin from kelp comprises the following steps:

a1, pretreatment: washing fresh herba Zosterae Marinae with clear water, removing surface salt and mucus, sucking surface water with filter paper, and shearing into 0.2 cm-sized pieces of herba Zosterae Marinae;

a2, enzymolysis: taking the crushed kelp, adding deionized water and a complex enzyme preparation, and uniformly mixing, wherein the deionized water and the kelp are mixed according to the ratio of 1: 25 weight g/volume mL; adding the complex enzyme preparation according to the weight of the kelp by 0.2% in mass proportion, uniformly mixing, and carrying out enzymolysis treatment at 37 ℃ for 5 hours; the compound enzyme preparation is prepared from the following components in a mass ratio of 1:1 cellulase and pectinase;

a3, extraction: drying the kelp in the shade according to the proportion of 1: adding absolute ethyl alcohol according to the weight g/volume mL of 6, mixing, adding 0.25% volume of antioxidant 2, 6-di-tert-butyl-p-cresol according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, leaching for 45min at normal temperature in a dark place, and then putting the mixture into an ultrasonic-microwave synergistic extraction instrument for ultrasonic-microwave synergistic extraction; setting the ultrasonic power to be 50W, the microwave power to be 300W, the extraction temperature to be 40 ℃ and the extraction time to be 5 min;

a4, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a5, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 40 ℃ to obtain a fucoxanthin crude extract;

a6, separation and purification: separating and purifying the obtained crude fucoxanthin extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 510: 1, collecting orange to orange eluent;

a7, re-concentration: putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 40 ℃;

a8, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

Wherein, the microwave-ultrasonic synergic extraction adopts an intermittent extraction mode, and the extraction is repeated for 2 times.

Wherein, the steps of enzymolysis, extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or dark.

Comparative example 1

The difference between the embodiment and the embodiment 3 is that a complex enzyme preparation is not added, and the method specifically comprises the following steps:

a method for extracting fucoxanthin from kelp comprises the following steps:

a1, pretreatment: washing fresh herba Zosterae Marinae with clear water, removing surface salt and mucus, sucking surface water with filter paper, and shearing into 0.15 cm-sized pieces of herba Zosterae Marinae;

a2, extraction: the crushed kelp is processed according to the following steps of 1: adding absolute ethyl alcohol according to the weight g/volume mL of 6, mixing, adding 0.25% volume of antioxidant 2, 6-di-tert-butyl-p-cresol according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, leaching for 45min at normal temperature in a dark place, and then putting the mixture into an ultrasonic-microwave synergistic extraction instrument for ultrasonic-microwave synergistic extraction; setting the ultrasonic power to be 50W, the microwave power to be 300W, the extraction temperature to be 40 ℃ and the extraction time to be 5 min;

a3, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a4, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 40 ℃ to obtain a fucoxanthin crude extract;

a5, separation and purification: separating and purifying the obtained crude fucoxanthin extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 5: 1, collecting orange to orange eluent;

a6, re-concentration: putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 40 ℃;

a7, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

Wherein, the microwave-ultrasonic synergic extraction adopts an intermittent extraction mode, and the extraction is repeated for 2 times.

Wherein, the steps of extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or dark.

Comparative example 2

The difference between the embodiment and the embodiment 3 is that the microwave-ultrasonic extraction apparatus is not used for extraction, and the method specifically comprises the following steps:

a method for extracting fucoxanthin from kelp comprises the following steps:

a1, pretreatment: washing fresh herba Zosterae Marinae with clear water, removing surface salt and mucus, sucking surface water with filter paper, and shearing into 0.15 cm-sized pieces of herba Zosterae Marinae;

a2, enzymolysis: taking the crushed kelp, adding deionized water and a complex enzyme preparation, and uniformly mixing, wherein the deionized water and the kelp are mixed according to the ratio of 1: 25 weight g/volume mL; adding the complex enzyme preparation according to the weight of the kelp by 0.2% in mass proportion, uniformly mixing, and carrying out enzymolysis treatment at 37 ℃ for 5 hours; the compound enzyme preparation is prepared from the following components in a mass ratio of 1:1 cellulase and pectinase.

A3, extraction: drying the kelp in the shade according to the proportion of 1: adding absolute ethyl alcohol according to the weight g/volume mL of 6, mixing, adding 0.25% volume of antioxidant 2, 6-di-tert-butyl-p-cresol according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, and leaching for 45min at normal temperature in a dark place;

a4, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a5, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 40 ℃ to obtain a fucoxanthin crude extract;

a6, separation and purification: separating and purifying the obtained crude fucoxanthin extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 5: 1, collecting orange to orange eluent;

a7, re-concentration: and (3) putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 40 ℃.

A8, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

Wherein, the steps of enzymolysis, extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or dark.

Comparative example 3

The difference between the embodiment and the embodiment 3 lies in that the method does not undergo enzymolysis and ultrasonic-microwave extraction processes, and specifically comprises the following steps:

a method for extracting fucoxanthin from kelp comprises the following steps:

a1, pretreatment: washing fresh herba Zosterae Marinae with clear water, removing surface salt and mucus, sucking surface water with filter paper, and shearing into 0.15 cm-sized pieces of herba Zosterae Marinae;

a2, extraction: drying the kelp in the shade according to the proportion of 1: adding absolute ethyl alcohol according to the weight g/volume mL of 6, mixing, adding 0.25% volume of antioxidant 2, 6-di-tert-butyl-p-cresol according to the volume of the mixed solution of the kelp and the absolute ethyl alcohol, and leaching for 45min at normal temperature in a dark place;

a3, solid-liquid separation: centrifuging the extracted solution, and reserving supernatant;

a4, concentration: putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant at the temperature of 40 ℃ to obtain a fucoxanthin crude extract;

a5, separation and purification: separating and purifying the obtained crude fucoxanthin extract by silica gel column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 5: 1, collecting orange to orange eluent;

a6, re-concentration: and (3) putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 40 ℃.

A7, drying and storing: and putting the concentrated eluent into a nitrogen blowing instrument to evaporate the solvent to be dry, thus obtaining the pure fucoxanthin, and storing the pure fucoxanthin at the temperature of minus 20 ℃ in a dark place.

Wherein, the steps of extraction, solid-liquid separation, concentration, separation and purification and re-concentration are all carried out under the condition of weak light or dark.

Comparative example 4

This example is a method for extracting fucoxanthin by an enzymatic hydrolysis method in a conventional method, and specifically includes the following steps:

a1, weighing 10g of kelp pieces, adding ethanol solution to make the ratio of material to liquid 1:10(g: mL), adjusting pH with 0.1mol/L HCl and NaOH solution, and adding 0.1% cellulase and 0.1% pectinase (based on the mass of the raw material). Extracting at 40 deg.C in dark for 1.0h, vacuum filtering, extracting for 2 times, mixing filtrates, inactivating enzyme in boiling water bath for 5min, centrifuging at 4000r/min for 10min, collecting supernatant,

a2, putting the obtained supernatant into a rotary evaporator, and carrying out evaporation concentration on the supernatant to obtain a fucoxanthin crude extract;

a3, separation and purification: separating and purifying the obtained fucoxanthin crude extract by silica gel column chromatography, and collecting orange to orange eluent; and putting the obtained eluent into a rotary evaporator, evaporating and concentrating to dryness to obtain a pure fucoxanthin product, and storing at-20 ℃ in a dark place.

Specific test method

Samples treated in examples 1, 2 and 3 and comparative examples 1, 2, 3 and 4 were taken, dissolved in absolute ethanol and subjected to high performance liquid chromatography to determine the fucoxanthin content. The detection conditions of the high performance liquid chromatography are as follows: column type and barThe part is C18 column (2.1x 50mm), particle size 1.7um, with protective column; a detector: a diode array detector; detection wavelength: 447 nm; flow rate: 0.25 ml/min^-1(ii) a Column temperature: room temperature; mobile phase: a is acetonitrile (containing 0.05 percent of formic acid); b: ultrapure water (containing 0.05% formic acid); elution procedure: 0-4 min, 65-100% of A, 4-7 min 100% of A maintenance, 7-8 min, wherein 100% of A is eluted to 65% of A, 8-12 min, 65%.

FIG. 2 is an HPLC peak profile of fucoxanthin standard substance, FIG. 3 is an HPLC peak profile of pure fucoxanthin extracted from Laminaria japonica in example 3, and the specific results of fucoxanthin content in 7 groups of examples are shown in Table 1:

table 17 fucoxanthin content of group of examples

As can be seen from fig. 2 and 3, the HPLC peak profiles of the fucoxanthin pure product obtained by the treatment in example 3 and the fucoxanthin standard product showed consistent peak-out time and only a few miscellaneous peaks, indicating that the fucoxanthin extracted from the kelp by the method of the present invention has high purity. The peak profiles of example 1 and example 2 are substantially the same as example 3 and will not be described in detail.

As is clear from Table 1, in the above 7 groups of examples, the fucoxanthin content in example 3 was the highest, 25.33mg/100g, i.e., 25.33mg of fucoxanthin was extracted from 100g of kelp. Compared with the examples 1, 2 and 3, the fucoxanthin content of the examples 1, 2 and 3 is lower than that of the examples 1, 2 and 3, which further illustrates that the enzymolysis and microwave-ultrasonic synergistic extraction have positive effects in the pigment extraction process and contribute to the pigment extraction efficiency, and the combination of the enzymolysis and the microwave-ultrasonic synergistic extraction leads the extraction efficiency of the invention to be higher and the extraction effect to be better; comparative example 4 is a method for extracting fucoxanthin by a conventional enzymatic hydrolysis method, and the content of pigment is greatly different from that in examples 1 to 4, which shows that the method for extracting fucoxanthin in the present invention has high extraction quality and good extraction effect.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, so that any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the technical solution of the present invention without departing from the content of the technical solution of the present invention.