阅读说明：本技术 羊栖菜岩藻黄质的绿色制备方法 (Green preparation method of sargassum fusiforme fucoxanthin ) 是由 卢延斌 聂景贵 于 2020-11-30 设计创作，主要内容包括：本发明公开了一种羊栖菜岩藻黄质的绿色制备方法,依次进行以下步骤：选择新鲜的羊栖菜或者选择解冻后的冷冻羊栖菜；在容器中加入羊栖菜和固定提取溶剂混合均匀,于暗处浸泡2±0.5h；浸泡完成后,先预热,然后进行超声提取；提取完成后离心,离心所得的上清液中含有岩藻黄质。本发明的提取过程中不使用任何有毒溶剂,因此具有绿色环保的技术优势。(The invention discloses a green preparation method of sargassum fusiforme fucoxanthin, which sequentially comprises the following steps: selecting fresh Cyrtymenia Sparsa or frozen Cyrtymenia Sparsa after thawing; adding Cyrtymenia Sparsa and fixed extraction solvent into container, mixing, and soaking in dark place for 2 + -0.5 hr; after soaking, preheating, and then carrying out ultrasonic extraction; centrifuging after extraction, and centrifuging to obtain supernatant containing fucoxanthin. The extraction process of the invention does not use any toxic solvent, thus having the technical advantage of environmental protection.)

1. The green preparation method of sargassum fusiforme fucoxanthin is characterized by sequentially carrying out the following steps:

1) selecting materials:

the method comprises the following steps:

selecting fresh sargassum fusiforme, and directly carrying out the subsequent step 2);

selecting frozen sargassum fusiforme, unfreezing, sucking water on the surface of the sargassum fusiforme by using paper, and then performing the subsequent step 2);

2) and soaking: adding sargassum fusiforme and a fixed extraction solvent into a container according to the liquid-solid ratio of 40-60 mL/1g, uniformly mixing, and soaking in the dark for 2 +/-0.5 h;

3) preheating:

after soaking, preheating a container filled with the sargassum fusiforme and the fixed extraction solvent at a preheating temperature of 65-80 ℃ for 5 +/-1 min;

4) and (3) extracting:

after preheating, extracting at 65-80 ℃ for 20-30 min;

5) and centrifuging:

after the extraction is finished, the container filled with the extract is placed in a dark place to be cooled to room temperature, and then centrifugation is carried out;

the supernatant obtained by centrifugation contains fucoxanthin.

2. The green preparation method of fucoxanthin of sargassum fusiforme according to claim 1, which is characterized by comprising the following steps:

the fixed extraction solvent in the step 2) is ethyl lactate.

3. The green preparation method of sargassum fusiforme fucoxanthin as claimed in claim 2, wherein the extraction in step 4) is:

after preheating, an ultrasonic drill bit is stretched below the liquid level and is 1-2 cm away from the bottom of the container, and the ultrasonic conditions are as follows: the time is 20-30 min, the temperature is 65-80 ℃, the amplitude is 40-60%, the operation is 10s, and the interval is 10 s.

4. The green preparation method of sargassum fusiforme fucoxanthin as claimed in claim 3, wherein the centrifugation in step 5) is: the temperature is 4 +/-1 ℃, and the rotating speed is 8000 +/-800 rpm/min; the time is 10 +/-2 min.

5. The method for green production of fucoxanthin of sargassum fusiforme according to claim 4, wherein the method comprises the following steps:

the liquid-solid ratio in the step 2) is 40mL/1 g;

the preheating temperature of the step 3) is 75 ℃;

carrying out ultrasonic treatment in the step 4) for 25min at the temperature of 75 ℃; the amplitude was 50%.

6. The green preparation method of fucoxanthin of sargassum fusiforme according to claim 5, which is characterized by comprising the following steps:

in the step 2), the outer surface of the container is wrapped with tinfoil.

Technical Field

The invention relates to a green preparation method of sargassum fusiforme fucoxanthin, belonging to the technical field of extraction of functional ingredients of food.

Background

Sargassum fusiforme belongs to Sargassaceae of Phaeophyta, is mainly distributed in tropical and subtropical coastal areas, grows on rocks in low or middle tidal zone, and consists of a holdfast, a trunk, branches and algae leaves. Sargassum contains complex nutrients, such as polysaccharide, protein, lipid, pigment, trace elements, etc. Fucoxanthin is a major pigment of Hizikia fusiforme, and is a carotenoid having various beneficial biological activities, such as antioxidant, anticancer, anti-inflammatory, anti-obesity, anti-angiogenic and neuroprotective activities. Most of the existing methods for extracting fucoxanthin use organic solvents for extraction, however, the organic solvents have toxicity and are harmful to human health. Most of the existing methods for extracting fucoxanthin use slightly or highly toxic organic solvents such as acetone, methanol, ethyl acetate and the like, and the organic solvents have harm to the health of people. Therefore, how to separate fucoxanthin from sargassum fusiforme by a green method and apply the fucoxanthin to the fields of food, cosmetics, medicines and the like attracts wide attention.

Ethyl lactate is a non-corrosive, non-carcinogenic and non-teratogenic, biodegradable solvent. It is synthesized by esterification reaction of ethanol and lactic acid generated in biomass raw materials, can be continuously produced, and is recognized as a green solvent. Currently, ethyl lactate is commonly used to replace MEK, MIBK, and toluene, which are air pollutants, as paint strippers and graffiti removers, or to remove grease, adhesives, and solid fuels. As a new green extraction solvent, the research on extracting phytosterol, squalene and sclareol by using ethyl lactate has been carried out, but the extraction of carotenoids such as fucoxanthin in brown algae is not carried out at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing a green and efficient preparation method of sargassum fusiforme fucoxanthin.

In order to solve the technical problems, the invention provides a green preparation method of sargassum fusiforme fucoxanthin, which sequentially comprises the following steps:

1) selecting materials:

the method comprises the following steps:

selecting fresh sargassum fusiforme, and directly carrying out the subsequent step 2);

selecting frozen (about-20 ℃) sargassum fusiforme, unfreezing, then sucking water on the surface of the sargassum fusiforme by using paper (namely, the surface of the sargassum fusiforme has no obvious water stain), and then carrying out the subsequent step 2);

2) and soaking: adding sargassum fusiforme and a fixed extraction solvent into a container according to the liquid-solid ratio of 40-60 mL/1g, uniformly mixing, and soaking in the dark for 2 +/-0.5 h;

3) preheating:

after soaking, preheating a container filled with the sargassum fusiforme and the fixed extraction solvent in a preheating temperature (constant temperature water bath) of 65-80 ℃ for 5 +/-1 min;

4) and (3) extracting:

after preheating, extracting (ultrasonic extracting) at 65-80 ℃ for 20-30 min;

the step 3) and the step 4) are preferably carried out in a dark place;

5) and centrifuging:

after the extraction (ultrasonic extraction) is finished, the container with the extract is placed in a dark place to be cooled to room temperature, and then centrifugation is carried out;

the supernatant obtained by centrifugation contains fucoxanthin.

The invention relates to an improvement of a green preparation method of sargassum fusiforme fucoxanthin, which comprises the following steps:

the fixed extraction solvent in the step 2) is ethyl lactate.

The green preparation method of the sargassum fusiforme fucoxanthin provided by the invention is further improved as follows:

the extraction in the step 4) is as follows: after preheating, an ultrasonic drill bit is stretched below the liquid level and is 1-2 cm away from the bottom of the container, and the ultrasonic conditions are as follows: the time is 20-30 min, the temperature is 70-80 ℃, the amplitude is 40-60%, the operation is 10s, and the interval is 10 s.

The green preparation method of the sargassum fusiforme fucoxanthin provided by the invention is further improved as follows:

the centrifugation in the step 5) is as follows: the temperature is 4 +/-1 ℃, and the rotating speed is 8000 +/-800 rpm/min; the time is 10 +/-2 min.

The green preparation method of the sargassum fusiforme fucoxanthin provided by the invention is further improved as follows:

the liquid-solid ratio in the step 2) is 40mL/1 g;

the preheating temperature of the step 3) is 75 ℃;

carrying out ultrasonic treatment in the step 4) for 25min at the temperature of 75 ℃; the amplitude was 50%.

The green preparation method of the sargassum fusiforme fucoxanthin provided by the invention is further improved as follows:

in the step 2), the outer surface of the container is wrapped with tinfoil.

The invention aims at the supernatant fluid containing fucoxanthin obtained by centrifugation to carry out the following operations:

and (3) volume fixing: adding a fixed extraction solvent into the supernatant obtained by centrifugation to a constant volume until the volume of the fixed extraction solvent used in the synchronous step 2), and then uniformly mixing; as an extract;

and (3) detection: the extract was diluted 4-fold with absolute ethanol (i.e., the volume ratio of the extract: ethanol was 1: 3), 2mL was taken up with a syringe, passed through a 0.45 μm nylon membrane, and loaded into a sample bottle, and then the fucoxanthin content of the sample liquid was measured by HPLC.

Calculating the extraction rate: obtaining peak area of fucoxanthin according to HPLC chromatogram, and substituting into standard curve for detecting fucoxanthin, wherein y is 176603x-105594 (R)²0.9991, y (mu g/mL) is the concentration of fucoxanthin in the sample solution, and x is the peak area of fucoxanthin in the chromatogram. The extraction rate Y of fucoxanthin is 4Y liquid-solid ratio.

The preparation method of the sargassum fusiforme fucoxanthin disclosed by the invention has the following technical advantages:

1) in the extraction process of the invention, no toxic solvent is used, and only ethyl lactate single solvent is used for extracting the sargassum fusiforme fucoxanthin, but acetone, methanol, ethyl acetate and other slightly or highly toxic solvents are usually used in the traditional extraction method, so the invention can reduce the pollution to the environment. The fucoxanthin extracted by the invention is green and safe.

2) When ultrasound is used as an extraction method, the extraction effect of ethyl lactate is equivalent to that of ethanol/acetone (V: V, 3:1), but acetone has toxicity.

3) Ethyl lactate has a very high boiling point, is not easy to volatilize in the ultrasonic-assisted extraction process, and ethanol/acetone is easy to volatilize, can be continuously lost in the extraction process, and causes pollution to the environment.

4) The invention uses probe-type ultrasonic wave to assist extraction, the cavitation of the ultrasonic wave can strengthen the damage to cell walls, and compared with the traditional auxiliary extraction method (such as rotary constant-temperature oscillation), the invention not only can improve the extraction rate, but also can shorten the extraction time and save the cost.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a graph comparing fucoxanthin extraction rates of Hizikia fusiforme in a single factor test;

in fig. 1:

a is the extraction ratio comparison of different liquid-solid ratios;

b is the extraction ratio comparison of different extraction time;

c is the extraction ratio comparison of different extraction temperatures;

d is the comparison of extraction rates of different amplifications.

FIG. 2 is a chromatogram of sample solutions of example 5, comparative example 1, and comparative example 2.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

The source of the biological material is as follows:

fresh Cyrtymenia Sparsa is purchased from the downtown area of Wenzhou city, Zhejiang province, picked at about 3 months, and stored in a refrigerator at-20 deg.C.

The detection method comprises the following steps:

detecting the content of fucoxanthin in the extracting solution by using HPLC (high performance liquid chromatography), wherein the detection conditions are as follows:c18 column (250 mm. times.4.6 mm. times.5 μm), detection wavelength 449nm, mobile phase acetonitrile: methanol: ammonium acetate aqueous solution with mass concentration of 0.1% (V: V: V, 75:15:10), isocratic elution, flow rate of 1mL/min, sample amount of 20 μ L, column temperature of 30 deg.C, time of 15 min.

In the following cases, nothing is explicitly stated to be done at ordinary room temperature.

Example 1, a green preparation method of sargassum fusiforme fucoxanthin (study on the influence of different liquid-solid ratios on the extraction rate of sargassum fusiforme fucoxanthin), the following steps are sequentially carried out:

1) and unfreezing:

thawing frozen Cyrtymenia Sparsa in water, and sucking water on the surface with paper (i.e. no obvious water stain on the surface of Cyrtymenia Sparsa) for use;

2) and soaking:

weighing a certain amount of sargassum fusiforme, and respectively carrying out the following operations according to liquid-solid ratios of 10/1, 20/1, 30/1, 40/1, 50/1 and 60/1 (mL/g): adding into a beaker containing 100mL of ethyl lactate (as a fixed extraction solvent), uniformly mixing the feed liquid, wrapping the beaker with tinfoil to prevent fucoxanthin from being decomposed under light, and soaking in dark for 2h (i.e., soaking Cyrtymenia Sparsa in ethyl lactate for 2h in dark);

3) preheating:

after soaking, placing the beaker in a constant-temperature water bath kettle at the temperature of 65 ℃ and preheating for 5 min;

4) and (3) extracting:

after preheating, the beaker is arranged under an ultrasonic drill, the drill extends to the position below the liquid level and 1 cm away from the bottom of the cup, and the ultrasonic conditions are set as follows: the time is 20min, the temperature is 65 ℃ (same as the preheating temperature), the amplitude is 40%, the operation is 10s, and the interval is 10 s;

the step 3) and the step 4) are both carried out in a dark place.

5) And centrifuging:

after the ultrasonic treatment is finished, placing the beaker in a dark place, cooling to room temperature, pouring the beaker into a centrifuge tube, and setting the centrifugation conditions to be 10min, 4 ℃ and 8000 rpm/min;

6) and constant volume:

after centrifugation, pouring the supernatant into a 100mL measuring cylinder, metering the volume to 100mL by using ethyl lactate, pouring into a conical flask, and uniformly mixing to obtain an extracting solution;

7) and detecting:

the extract was diluted 4-fold with ethanol (i.e., the volume ratio of the extract: ethanol was 1: 3), 2mL was taken up with a syringe, passed through a 0.45 μm nylon membrane, and loaded into a sample bottle, and the fucoxanthin content in the sample liquid was measured by HPLC.

8) Calculating the extraction rate:

from HPLC chromatogramsObtaining the peak area of fucoxanthin, and substituting the peak area into a standard curve for detecting fucoxanthin, wherein y is 176603x-105594(R is²0.9991, y (mu g/mL) is the concentration of fucoxanthin in the sample solution, and x is the peak area of fucoxanthin in the chromatogram. The extraction rate Y of fucoxanthin is 4Y liquid-solid ratio.

Through detection, the extraction rate of fucoxanthin of the sargassum fusiforme is different under different liquid-solid ratios, the extraction rate continuously increases along with the increase of the liquid-solid ratio, and the rising trend is reduced when the extraction rate exceeds 40mL/g (figure 1A). For cost saving, 40mL/g is selected as the optimal liquid-solid ratio for extraction.

By comparing the HPLC chromatogram with the fucoxanthin standard product, the fucoxanthin is really obtained by the invention.

Example 2, study of the effect of different ultrasound times on fucoxanthin extraction rate of sargassum fusiforme:

in the step 2), setting a liquid-solid ratio of 30/1 (mL/g);

in the step 4), the ultrasonic time is respectively 10min, 15min, 20min, 25min, 30min and 35 min;

the rest is equivalent to embodiment 1.

Detection shows that the fucoxanthin extraction rate of the sargassum fusiforme is different at different time, the extraction rate continuously increases along with the increase of time, the maximum value is reached at 30min, but the extraction rate difference between 25min and 30min is not large (figure 1B). For the sake of extraction simplicity, 25min was chosen as the optimal sonication time for extraction.

Example 3, study of the effect of different extraction temperatures on fucoxanthin extraction rate of sargassum fusiforme:

in the step 2), setting a liquid-solid ratio of 30/1 (mL/g);

in the step 3), the preheating temperatures are respectively set to be 55, 60, 65, 70, 75 and 80 ℃;

in the step 4), the temperature of ultrasonic extraction is correspondingly set to be 55, 60, 65, 70, 75 and 80 ℃ respectively; the rest is equivalent to embodiment 1.

Through detection, the extraction rate of fucoxanthin of the sargassum fusiforme is different at different temperatures, wherein the extraction efficiency is highest under the condition that the temperature is 75 ℃ (figure 1C). Therefore, 75 ℃ was chosen as the optimum temperature for extraction.

Example 4, study of the effect of different ultrasound amplitudes on fucoxanthin extraction rate of hizikia fusiforme:

in the step 2), setting a liquid-solid ratio of 30/1 (mL/g);

in the step 4), the amplitudes of ultrasonic extraction are respectively 20, 30, 40, 50, 60 and 70 percent;

the rest is equivalent to embodiment 1.

Through detection, the extraction rate of fucoxanthin of sargassum fusiforme is different under different amplitudes, wherein the extraction efficiency is highest under the condition that the amplitude is 50% (figure 1D). Therefore, 50% was selected as the optimal ultrasound amplitude.

Example 5, the extraction rate of fucoxanthin from sargassum fusiforme under the optimal extraction conditions, the test steps are as follows:

setting a liquid-solid ratio of 40/1(mL/g) in the soaking in the step 2); namely, the amount of the sargassum fusiforme is 2.5g, and the volume of the ethyl lactate is 100 mL;

in the preheating of the step 3), the preheating temperature is 75 ℃, and the preheating is carried out for 5 min;

in the ultrasonic extraction of the step 4), the ultrasonic conditions are set as follows: the time is 25min, the temperature is 75 ℃, the amplitude is 50%, the operation is 10s, and the interval is 10 s;

the rest is equivalent to embodiment 1.

Through detection, under the optimal extraction condition, the extraction rate of the fucoxanthin obtained from the sargassum fusiforme is 696.73 mug/g.

Description of the drawings: the "sargassum fusiforme thawed and the surface water is sucked dry by paper" in the example 5 is changed into fresh sargassum fusiforme, the rest is identical to the example 5, and the obtained extraction rate has no obvious difference compared with the example 5.

Comparative examples 1,

The fixed extraction solvent in step 2) of example 5 is changed from ethyl lactate to ethanol/acetone (V: V, 3:1), and the volume and the amount are kept unchanged; correspondingly, the ethyl lactate used in the volume fixing in the step 6) is changed into ethanol, and the volume consumption is kept unchanged;

the rest is equivalent to example 5.

The detection shows that the extraction rate of fucoxanthin of the sargassum fusiforme is 691.04 mug/g. However, the acetone used in comparative example 1 is a highly toxic solvent and is extremely harmful to the environment and human health.

Comparative examples 2,

The steps 3) and 4) of example 5 were modified as follows:

after the soaking in the step 2) is finished, placing the beaker in a shaking table with the temperature reaching 75 ℃, setting the rotating speed at 120rpm/min, and extracting for 1 h; then carrying out subsequent centrifugation;

the rest is equivalent to example 5.

The detection shows that the extraction rate of fucoxanthin of the sargassum fusiforme is 617.31 mug/g.

Comparative examples 3,

The ethyl lactate in the step 2) and the step 6) in the example 5 is changed into limonene, and the volume consumption is kept unchanged;

the rest is equivalent to embodiment 1.

The detection shows that the extraction rate of fucoxanthin of the sargassum fusiforme is 485.67 mug/g.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.