阅读说明：本技术 一种从马尾藻中提取岩藻黄素的方法 (Method for extracting fucoxanthin from gulfweed ) 是由 李达谅 邱淡静 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马尾藻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提取；A3固液分离；A4浓缩；A5分离提纯；A6再浓缩。本发明中从马尾藻中提取岩藻黄素的方法,操作工艺简单,成本低,适合大规模生产,并且能够保证提取效果。(The invention discloses a method for extracting fucoxanthin from gulfweed, which belongs to the field of fucoxanthin extraction processes and comprises the following steps: a1 pretreatment; a2 extraction; a3 solid-liquid separation; a4 concentration; a5 separating and purifying; a6 was re-concentrated. The method for extracting fucoxanthin from gulfweed has the advantages of simple operation process, low cost, suitability for large-scale production and capability of ensuring the extraction effect.)

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

a1, pretreatment: washing fresh gulfweed with clear water, washing away silt particles and mucus on the surface, draining off surface water, and shearing into crushed algae with the size of 0.1-0.5 cm;

a2, extraction: weighing the sheared and crushed sargassum, and mixing the materials according to a material-liquid ratio of 1 g: adding absolute ethyl alcohol in a proportion of 5-30 ml, uniformly mixing, and putting into an ultrasonic extraction instrument for ultrasonic-assisted extraction;

a3, solid-liquid separation: carrying out solid-liquid separation on the extract liquor through suction filtration;

a4, concentration: putting the separated extracting solution into a rotary evaporator, and carrying out evaporation concentration on the extracting solution at the temperature of 30-50 ℃ to obtain a fucoxanthin crude extract;

a5, separation and purification: separating and purifying the obtained fucoxanthin crude extract by column chromatography, wherein a chromatography eluent is petroleum ether, and a chromatography eluent is a mixture of the following components in a volume ratio of 2-10: 1, collecting orange to orange eluent;

a6, re-concentration: putting the collected eluent into a rotary evaporator, and carrying out evaporation concentration at the temperature of 30-50 ℃;

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.

2. The method of claim 1, wherein the fucoxanthin is extracted from the gulfweed by: and B, during the ultrasonic-assisted extraction in the step A2, setting the ultrasonic power to be 1300-1600W, setting the initial temperature to be 20-40 ℃, setting the extraction time to be 3-10 min, and repeatedly extracting for 1-3 times.

3. The method of claim 2, wherein the fucoxanthin is extracted from the gulfweed by: the ultrasonic power is set to 1500W, the initial temperature is set to 20 ℃, the extraction time is 5min, and the extraction is repeated for 2 times.

4. The method of claim 1, wherein the fucoxanthin is extracted from the gulfweed by: in the step A2, the feed-liquid ratio of the gulfweed to the absolute ethyl alcohol is 1 g: 10 ml.

5. The method of claim 1, wherein the fucoxanthin is extracted from the gulfweed by: in the concentration of the step A4 and the reconcentration of the step A6, the evaporation temperature is set to 40 ℃.

6. The method of claim 1, wherein the fucoxanthin is extracted from the gulfweed by: 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 darkness.

7. The method of claim 1, wherein the fucoxanthin is extracted from the gulfweed by: the Sargassum is one or more of Sargassum pallidum, Sargassum muticum, and Sargassum thunbergii.

Technical Field

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

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.

Sargassum is one of brown algae, belongs to Fucales of Phaeophyceae, and has the advantages of dual purposes of edible and medicinal purposes and great development potential. The previous sargassum is mainly applied to extraction of algin and mannitol, and fat-soluble components generated in the extraction process can be discarded as industrial waste, so that great waste of resources is caused.

Patent CN 103232552B discloses a method for preparing fucosan and fucoxanthin by an enzymatic method, which comprises the following steps: mixing the dry brown algae powder and the complex enzyme preparation uniformly, and adding deionized water for enzymolysis; acid treatment; obtaining a concentrated solution; obtaining fucosan; obtaining crude fucoxanthin and pure fucoxanthin. The brown algae fucosan and fucoxanthin obtained by the method have high yield. However, the enzymolysis method has high cost and is not suitable for large-scale production.

Therefore, a method for extracting fucoxanthin from gulfweed is urgently needed, has simple operation process and low cost, is suitable for large-scale production, and can ensure the extraction effect.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide a method for extracting fucoxanthin from gulfweed, which has the advantages of simple operation process, low cost, suitability for large-scale production and guaranteed extraction effect.

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

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

a1, pretreatment: washing fresh gulfweed with clear water, washing away silt particles and mucus on the surface, draining off surface water, and shearing into crushed algae with the size of 0.1-0.5 cm;

a2, extraction: weighing the sheared and crushed sargassum, and mixing the materials according to a material-liquid ratio of 1 g: (5-30) ml of absolute ethyl alcohol is added, and after uniform mixing, the mixture is placed into an ultrasonic extraction instrument for ultrasonic-assisted extraction;

a3, solid-liquid separation: carrying out solid-liquid separation on the extract liquor through suction filtration;

a4, concentration: putting the separated extracting solution into a rotary evaporator, and carrying out evaporation concentration on the extracting solution at the temperature of 30-50 ℃ to obtain a fucoxanthin crude extract;

a5, separation and purification: separating and purifying the obtained fucoxanthin crude extract by column chromatography, wherein a chromatography eluent is petroleum ether, and a chromatography eluent is a mixture of the following components in a volume ratio of 2-10: 1, collecting orange to orange eluent;

a6, re-concentration: putting the collected eluent into a rotary evaporator, and carrying out evaporation concentration at the temperature of 30-50 ℃;

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.

And B, during the ultrasonic-assisted extraction in the step A2, setting the ultrasonic power to be 1300-1600W, setting the initial temperature to be 20-40 ℃, setting the extraction time to be 3-10 min, and repeatedly extracting for 1-3 times.

In the step A2, the feed-liquid ratio of the gulfweed to the absolute ethyl alcohol is 1 g: 10 ml.

Wherein the ultrasonic power is 1500w, the initial temperature is set to 20 ℃, the extraction time is 5min, and the extraction is repeated for 2 times.

Wherein, the evaporation temperature is set to be 40 ℃ in the concentration of the step A4 and the re-concentration of the step A6.

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.

Wherein the Sargassum is one or more of Sargassum pallidum, Sargassum muticum, and Sargassum thunbergii.

The invention has the following beneficial effects:

1. in the invention, silt particles and mucus attached to the gulfweed 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. In the invention, organic solvent extraction and ultrasonic wave assistance are adopted in the extraction process to extract fucoxanthin in the gulfweed, and the ultrasonic wave is helpful for quickly breaking the cell walls of the gulfweed cells, so that the extraction time is shortened, the solvent extraction effect is enhanced, and the fucoxanthin in the cells is fully dissolved out; wherein, the organic solvent adopts absolute ethyl alcohol, has low toxicity, is convenient to recycle, and is more suitable for large-scale industrial production. Compared with the conventional enzymolysis method, the method avoids the influence of high temperature during enzyme inactivation on fucoxanthin, increases the extraction efficiency of the fucoxanthin, and is low in cost, low in toxicity, convenient to recycle and more suitable for large-scale industrial production because the absolute ethanol is adopted as the organic solvent compared with the enzyme with high price in the enzymolysis method.

3. In the invention, after the fucoxanthin is separated by 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 instrument, 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.

Drawings

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

FIG. 2 is a UV scan of the extract of example 1 of the present invention.

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 Sargassum comprises the following steps:

a1, pretreatment: washing fresh sargassum pallidum with clear water, washing off silt particles and mucus on the surface, draining off surface water, and shearing into 0.3 cm-sized crushed algae;

a2, extraction: weighing the sheared and crushed sargassum, and mixing the materials according to a material-liquid ratio of 1 g: adding absolute ethyl alcohol in a proportion of 10ml, uniformly mixing, and placing into an ultrasonic extraction instrument for ultrasonic-assisted extraction; setting the ultrasonic power to 1500W, setting the initial temperature to 20 ℃, extracting for 5min, and repeating the ultrasonic extraction for 2 times;

a3, solid-liquid separation: carrying out solid-liquid separation on the extract liquor through suction filtration;

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

a5, separation and purification: separating and purifying the obtained fucoxanthin crude extract by 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 steps of extraction, solid-liquid separation, concentration, separation and purification are all carried out under the condition of weak light or darkness.

Example 2

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

a1, pretreatment: washing fresh sargassum muticum with clear water, washing off silt particles and mucus on the surface, draining off water on the surface, and shearing into 0.5 cm-sized crushed algae;

a2, extraction: weighing the sheared and crushed sargassum, and mixing the materials according to a material-liquid ratio of 1 g: adding anhydrous ethanol in a proportion of 30ml, uniformly mixing, and placing into an ultrasonic extraction instrument for ultrasonic-assisted extraction; setting the ultrasonic power to 1600W, setting the initial temperature to be 40 ℃, extracting for 10min, and repeating the ultrasonic extraction for 3 times;

a3, solid-liquid separation: carrying out solid-liquid separation on the extract liquor through suction filtration;

a4, concentration: putting the separated extracting solution into a rotary evaporator, and carrying out evaporation concentration on the extracting solution at the temperature of 50 ℃ to obtain a fucoxanthin crude extract;

a5, separation and purification: separating and purifying the obtained fucoxanthin crude extract by 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;

a6, re-concentration: putting the collected eluent into a rotary evaporator, and evaporating and concentrating at the temperature of 50 ℃;

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 are all carried out under the condition of weak light or darkness.

Example 3

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

a1, pretreatment: washing fresh Sargassum thunbergii with clear water, washing off silt particles and mucus on the surface, draining off surface water, and shearing into 0.4 cm-sized crushed algae;

a2, extraction: weighing the sheared and crushed sargassum, and mixing the materials according to a material-liquid ratio of 1 g: adding 20ml of absolute ethyl alcohol, uniformly mixing, and placing into an ultrasonic extraction instrument for ultrasonic-assisted extraction; setting the ultrasonic power to 1400W, the initial temperature to 30 ℃, extracting for 7min, and repeating the ultrasonic extraction for 2 times;

a3, solid-liquid separation: carrying out solid-liquid separation on the extract liquor through suction filtration;

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

a5, separation and purification: separating and purifying the obtained fucoxanthin crude extract by column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 8: 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 45 ℃;

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 are all carried out under the condition of weak light or darkness.

Example 4

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

a1, pretreatment: washing fresh sargassum pallidum and sargassum muticum with clear water, washing away silt particles and mucus on the surface, draining off water on the surface, and shearing into 0.1 cm-sized crushed algae with scissors;

a2, extraction: weighing the cut chopped sargassum pallidum and sargassum muticum, and mixing the materials according to a material-liquid ratio of 1 g: adding absolute ethyl alcohol in a proportion of 5ml, uniformly mixing, and placing into an ultrasonic extraction instrument for ultrasonic-assisted extraction; setting the ultrasonic power to 1300W, the initial temperature to be 20 ℃, the extraction time to be 3min, and carrying out ultrasonic extraction for 1 time;

a3, solid-liquid separation: carrying out solid-liquid separation on the extract liquor through suction filtration;

a4, concentration: putting the separated extracting solution into a rotary evaporator, and carrying out evaporation concentration on the extracting solution at the temperature of 30 ℃ to obtain a fucoxanthin crude extract;

a5, separation and purification: separating and purifying the obtained fucoxanthin crude extract by column chromatography, wherein the chromatography eluent is petroleum ether, and the volume ratio of the chromatography eluent is 2: 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 30 ℃;

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 are all carried out under the condition of weak light or darkness.

Wherein the Sargassum is sargassum pallidum and sargassum muticum.

Comparative examples

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 fresh chopped Artemisia annua, adding ethanol solution to adjust the ratio of material to liquid to 1:10(g: mL), adjusting pH with 0.1mol/L HCl and NaOH solutions, 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

1. Taking the extract obtained by the solid-liquid separation in the step A3 in the example 1, and diluting the extract by the proportion of 5 percent, wherein the diluent is absolute ethyl alcohol; in an ultraviolet spectrophotometer, absolute ethyl alcohol is used as a blank control, spectral scanning is carried out within the wavelength range of 250-750 nm, and the absorption spectrogram is shown in figure 2:

as can be seen from fig. 2, the extract liquid in example 1 has a maximum absorption peak at 447nm, which is consistent with the result of the wavelength detected by the fucoxanthin standard substance chromatography in "determination of fucoxanthin content in brown algae by reverse phase high performance liquid chromatography" such as liu xiafang, and thus it is confirmed that the main component of the extract liquid of gulfweed in example 1 is fucoxanthin. In addition, a small absorption peak exists at 665nm in FIG. 2, which is the characteristic absorption peak of chlorophyll a. This indicates that the extract also contains a small amount of impurities such as a green leaf and the like, and may interfere with the detection result of fucoxanthin. The extracts of examples 2, 3 and 4 and the comparative example were identical to the results of example 1 and are not described in detail.

2. Samples treated by a nitrogen blowing instrument in examples 1, 2, 3 and 4 and comparative examples are respectively taken, dissolved by absolute ethyl alcohol and then subjected to high performance liquid chromatography detection to determine the fucoxanthin content.

The detection conditions of the high performance liquid chromatography are as follows: column type and conditions C18 column (2.1x 50mm), particle size 1.7um, with guard 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%.

The results of the fucoxanthin detection are shown in figure 1:

as can be seen from Table 1, the fucoxanthin contents of the gulfweed extracted in examples 1, 2, 3 and 4 and the comparative example are 0.6869mg/g, 0.5135mg/g, 0.4138mg/g, 0.4939mg/g and 0.4355mg/g respectively, wherein the fucoxanthin content in example 1 is high, that is, 0.6869mg of pure fucoxanthin can be extracted from each gram of fresh gulfweed, and the fucoxanthin contents in examples 1, 2 and 4 are higher than those in the comparative example, which shows that the fucoxanthin extraction method in the invention has high extraction quality and good extraction effect compared with the prior art.

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.