阅读说明：本技术 一种角叉菜多糖提取物及其制备方法与应用 (Carrageenan extract and preparation method and application thereof ) 是由 孙云起 裴运林 刘涵 郭朝万 胡露 于 2021-08-26 设计创作，主要内容包括：本发明涉及一种角叉菜多糖提取物及其制备方法与应用。所述角叉菜多糖提取物的制备方法包括冷冻干燥、回流脱脂、浓缩、离心、加溶剂去褐藻胶、乙醇沉淀等步骤,制备方法操作简单,且制备得到的角叉菜多糖提取物的抗氧化效果显著,可应用于食品添加剂或药品添加剂领域。(The invention relates to a carrageenan extract and a preparation method and application thereof. The preparation method of the carrageenan extract comprises the steps of freeze drying, reflux degreasing, concentration, centrifugation, addition of a solvent to remove algin, ethanol precipitation and the like, the preparation method is simple to operate, and the prepared carrageenan extract has a remarkable antioxidant effect and can be applied to the field of food additives or medicine additives.)

1. A process for the preparation of a carrageenan extract, comprising the steps of:

(1) drying, pulverizing and sieving carrageen to obtain carrageen algae powder;

(2) carrying out reflux degreasing and drying on the carrageenin algae powder obtained in the step (1) to obtain degreased algae powder;

(3) carrying out water bath treatment on the degreased algae powder obtained in the step (2) to obtain a first mixed solution;

(4) centrifuging the first mixed solution obtained in the step (3), concentrating the supernatant, and adding a solvent to remove algin to obtain a second mixed solution;

(5) centrifuging the second mixed solution obtained in the step (4), concentrating the supernatant, adding a solvent, and precipitating to obtain a third mixed solution;

(6) centrifuging the third mixed solution obtained in the step (5), and collecting precipitates to obtain the carrageenan extract.

2. The method for preparing carrageenan extract according to claim 1, wherein said drying in step (1) is performed by freeze-drying for 20-30 h;

preferably, the mesh number of the screen in the screening of the step (1) is 40-80 meshes.

3. The process for preparing carrageenan extract according to claim 1 or 2, wherein the solvent used in the refluxing in step (2) comprises any one or a combination of at least two of ethanol, acetone, ethyl acetate, diethyl ether, methanol or petroleum ether, preferably a combination of ethanol, diethyl ether and ethyl acetate;

preferably, the refluxing time of the step (2) is 1-4 times, and the refluxing time is 3-12 h;

preferably, the drying of step (2) is carried out at 30-60 ℃.

4. The process for the preparation of carrageenan extract according to any of claims 1 to 3, wherein the water bath treatment of step (3) is carried out with the aid of ultrasound for a period of 40 to 80 min;

preferably, the liquid-material ratio in the water bath treatment in the step (3) is 30-70 mL/g;

preferably, the temperature of the water bath treatment in the step (3) is 70-100 ℃, and the time of the water bath treatment is 3-7 h.

5. The method for preparing carrageenan extract according to any of claims 1 to 4, wherein the centrifugation in step (4) is performed at 15-40 ℃, wherein the speed of the centrifugation is 3000-5000r/min and the time of the centrifugation is 10-30 min;

preferably, the final volume of the concentration of step (4) is 1/6-1/4 times the volume of the supernatant;

preferably, the solvent in step (4) is 20% -50% ethanol, preferably 25% -35% ethanol;

preferably, the solvent removal of the algin in the step (4) is carried out at 15-25 ℃ for 0.5-2 h.

6. The method for preparing carrageenan extract according to any of claims 1 to 5, wherein the centrifugation in step (5) is carried out at 15-40 ℃, wherein the speed of the centrifugation is 3000-5000r/min and the time of the centrifugation is 10-30 min;

preferably, the final volume of the concentration of step (5) is 1/6-1/4 times the volume of the supernatant;

preferably, the solvent in the step (5) is 80% -95% ethanol;

preferably, the volume of the solvent in the step (5) is 3-5 times of the volume of the supernatant after concentration, and the precipitation in the step (5) is carried out at 0-8 ℃ for 12-16 h.

7. The process for the preparation of carrageenan extract according to any of claims 1 to 6, wherein the centrifugation in step (6) is carried out at 15-40 ℃ and the speed of the centrifugation is 3000-; centrifuging for 10-30 min;

preferably, the step (6) further comprises washing the precipitate after collecting the precipitate, wherein the washing reagent is absolute ethyl alcohol;

preferably, the number of washes is 1-3;

preferably, the washing further comprises drying the precipitate, wherein the drying is performed in a freeze drying manner, and the drying time is 0.5-1 h.

8. A process for the preparation of a carrageenan extract according to any of claims 1 to 7, wherein said process for the preparation of a carrageenan extract comprises the steps of:

(1) freeze drying carrageen for 20-30 hr, pulverizing, and sieving with 40-80 mesh sieve to obtain carrageen algae powder;

(2) refluxing the carrageenin powder obtained in the step (1) for 1-4 times, wherein the refluxing time is 3-12h each time, and drying at 30-60 ℃ to obtain degreased algae powder;

the solvent used in the reflux is a combination of ethanol, diethyl ether and ethyl acetate;

(3) treating the defatted algae powder obtained in the step (2) for 3-7h in water bath at 70-100 ℃ to obtain a first mixed solution;

the water bath treatment is carried out under the assistance of ultrasonic waves, the ultrasonic time is 40-80min, and the liquid-material ratio of the water bath treatment is 30-70 mL/g;

(4) centrifuging the first mixed solution obtained in the step (3) at the rotating speed of 3000-;

(5) centrifuging the second mixed solution obtained in the step (4) at the rotating speed of 3000-;

(6) centrifuging the third mixed solution obtained in the step (5) at the rotating speed of 3000-.

9. A carrageenan extract, characterized in that it has been prepared by a process for the preparation of a carrageenan extract according to any of claims 1 to 8.

10. Use of a carrageenan extract according to claim 9 for the preparation of a food additive or a pharmaceutical additive.

Technical Field

The invention belongs to the technical field of polysaccharide, relates to an algae polysaccharide extract and a preparation method and application thereof, and particularly relates to a carrageenan extract and a preparation method and application thereof.

Background

With the development of medical technology, researchers have found that the causes and development of tumors, inflammations, aging, and the like are closely related to radical and lipid peroxidation, and thus, there is an increasing interest in finding substances capable of inhibiting radical and lipid peroxidation. In particular, the search for natural, non-toxic antioxidants has been an important research direction for pharmaceutical and food additives. With the concern of the biological activity of marine algae, research on algae extracts has been greatly advanced, and research shows that algae extracts have significant antioxidant activity, for example, red algae extracts not only have a certain inhibitory effect on lipid peroxidation induced by AAPH, but also have a significant inhibitory effect on DPPH.

Carrageenan is a low-fat, high-protein macroalgae belonging to the family Gigartinaceae, the phylum Rhodophyta, the genus Carrageenan, and is one of the important algae for carrageenan production. The carrageenin is rich in active substances, mainly comprises seaweed pigment, fatty acid, amino acid, sterol, polysaccharide, vitamin, etc., and has good bioactivity.

Polysaccharide substances are a class of biomacromolecules ubiquitous in biological organisms and are important components of cytoskeletons and various endogenous bioactive molecules. The polysaccharide can be used for regulating the physiological function of organisms in a free form or in a form of conjugate with protein, lipid and the like. Polysaccharide substances are widely present in plants, and researches show that nearly hundred kinds of polysaccharides are separated from plants at present, have no cytotoxicity and can be applied to organisms.

The carrageenan polysaccharide extract has the functions of resisting tumor, ulcer, virus, bacteria, blood sugar and blood fat, has antioxidant potential, and can be applied to the fields of food and medicine. The extraction method of carrageenan commonly used in the prior art comprises a dilute alkali solution extraction method, a dilute acid solution extraction method, a hot water extraction method and the like. However, due to the different conditions of reagents, temperature, etc., the chemical components of the carrageenan extracts obtained by different extraction methods are different, so that the efficacy of the carrageenan extracts is also different. For example, CN111743924A discloses a marine plant extract for treating diabetes, which is prepared from total polyphenol and/or polysaccharide of carrageenin and has the effect of inhibiting the activity of diabetes target alpha-glucosidase. CN100513427C discloses the manufacture of carrageenan and carrageenan products which produce carrageenan solutions that thicken, suspend, stabilize microparticles, colloidal dispersions and water/oil emulsions while also assisting in giving the food product a suitable mouthfeel.

At present, the prior art does not disclose a method for preparing a carrageenan extract which is simple to operate and has a remarkable antioxidant effect.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a carrageenan extract, a preparation method and an application thereof, wherein the preparation method is simple to operate, and the prepared carrageenan extract has remarkable antioxidant effect.

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

in a first aspect, the present invention provides a process for the preparation of a carrageenan extract, said process comprising the steps of:

(1) drying, pulverizing and sieving carrageen to obtain carrageen algae powder;

(2) carrying out reflux degreasing and drying on the carrageenin algae powder obtained in the step (1) to obtain degreased algae powder;

(3) carrying out water bath treatment on the degreased algae powder obtained in the step (2) to obtain a first mixed solution;

(4) centrifuging the first mixed solution obtained in the step (3), concentrating the supernatant, and adding a solvent to remove algin to obtain a second mixed solution;

(5) centrifuging the second mixed solution obtained in the step (4), concentrating the supernatant, adding a solvent, and precipitating to obtain a third mixed solution;

(6) centrifuging the third mixed solution obtained in the step (5), and collecting precipitates to obtain the carrageenan extract.

The preparation method of the carrageenan extract provided by the invention is simple to operate, and the prepared carrageenan extract has a remarkable antioxidant effect and can be applied to the field of food additives or pharmaceutical additives.

Preferably, the drying in step (1) is performed in a freeze drying manner, the drying time is 20-30h, for example, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, and the like, and other specific values in the numerical range can be selected at will, which is not described herein again.

Preferably, the mesh number of the screen in the screening in the step (1) is 40-80 meshes, for example, 40 meshes, 45 meshes, 50 meshes, 55 meshes, 60 meshes, 65 meshes, 70 meshes, 75 meshes, 80 meshes, and the like, and other specific point values in the numerical range can be selected at will, and are not described in detail herein.

Preferably, the solvent used in the refluxing in step (2) comprises any one or a combination of at least two of ethanol, acetone, ethyl acetate, diethyl ether, methanol or petroleum ether.

The combination of at least two of the above-mentioned compounds, such as the combination of ethanol and acetone, the combination of acetone and ethyl acetate, the combination of ethyl acetate and ethyl ether, the combination of methanol and petroleum ether, etc., can be selected in any combination manner, and will not be described herein any more, and the combination of ethanol, ethyl ether and ethyl acetate is preferred.

The use of a combination of ethanol, diethyl ether and ethyl acetate as solvent for the reflux defatting of carrageenan powder compared to other solvents has surprisingly the effect of further improving the antioxidant capacity of the carrageenan extract produced.

Preferably, the refluxing time in step (2) is 1-4 times, such as 1 time, 2 times, 3 times, 4 times, and each refluxing time is 3-12h, such as 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, etc., and other specific values in the value range can be selected, which is not repeated herein.

Preferably, the drying in step (2) is performed at 30 ℃ to 60 ℃, for example, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃, 42 ℃, 45 ℃, 47 ℃, 50 ℃, 52 ℃, 55 ℃, 57 ℃, 60 ℃ and the like, and other specific values in the numerical range can be selected, and are not described in detail herein.

Preferably, the water bath treatment in the step (3) is performed under the assistance of ultrasonic waves, the ultrasonic time is 40-80min, such as 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min, and the like, and other specific point values within the numerical range can be selected at will, and are not repeated herein.

Preferably, the liquid-to-material ratio in the water bath treatment in step (3) is 30-70mL/g, for example, 30mL/g, 35mL/g, 40mL/g, 45mL/g, 50mL/g, 55mL/g, 60mL/g, 65mL/g, 70mL/g, and the like, and other specific values in the numerical range can be selected, which is not described herein again.

Preferably, the temperature of the water bath treatment in the step (3) is 70-100 ℃, and the time of the water bath treatment is 3-7 h.

The specific value of 70-100 deg.C can be selected from 70 deg.C, 72 deg.C, 75 deg.C, 77 deg.C, 80 deg.C, 82 deg.C, 85 deg.C, 87 deg.C, 90 deg.C, 92 deg.C, 95 deg.C, 97 deg.C, 100 deg.C, etc.

The specific value of 3-7h can be selected from 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, etc.

Other specific point values within the above numerical ranges can be selected at will, and are not described in detail herein.

Preferably, the centrifugation in the step (4) is carried out at 15-40 ℃, the speed of the centrifugation is 3000-5000r/min, and the time of the centrifugation is 10-30 min.

The specific value of 15-40 deg.C can be selected from 15 deg.C, 17 deg.C, 20 deg.C, 22 deg.C, 25 deg.C, 27 deg.C, 30 deg.C, 32 deg.C, 35 deg.C, 37 deg.C, 40 deg.C, etc.

The specific value of 3000-5000r/min can be selected from 3000r/min, 3200r/min, 3500r/min, 3700r/min, 4000r/min, 4200r/min, 4500r/min, 4700r/min, 5000r/min, etc.

The specific value of 10-30min can be selected from 10min, 12min, 15min, 17min, 20min, 22min, 25min, 27min, 30min, etc.

Other specific point values within the above numerical ranges can be selected at will, and are not described in detail herein.

Preferably, the final volume of the concentration in step (4) is 1/6-1/4 times of the volume of the supernatant, such as 1/6 times, 1/5.8 times, 1/5.5 times, 1/5.2 times, 1/5 times, 1/4.8 times, 1/4.5 times, 1/4.2 times, 1/4 times, and the like, and other specific points in the value range can be selected, and are not repeated herein.

Preferably, the solvent in step (4) is 20% to 50% ethanol, and the 20% to 50% is a volume fraction of ethanol, such as 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, and the like, and other specific points in the numerical range can be selected, and are not described herein, and preferably 25% to 35% ethanol is preferred.

Compared with ethanol with other concentrations, the removal effect is the best when 25% -35% ethanol is used as a solvent to remove algin, and further the antioxidant capacity of the prepared carrageenan extract can be further improved.

Preferably, the solvent removal of the algin in the step (4) is carried out at 15-25 ℃ for 0.5-2 h.

The specific value of the temperature of 15-25 deg.C can be selected from 15 deg.C, 16 deg.C, 17 deg.C, 18 deg.C, 19 deg.C, 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, etc.

Specific values of 0.5-2h can be selected from 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h and the like.

Other specific point values within the above numerical ranges can be selected at will, and are not described in detail herein.

Preferably, the centrifugation in step (5) is performed at 15-40 ℃, the speed of the centrifugation is 3000-5000r/min, and the time of the centrifugation is 10-30 min.

The specific value of 15-40 deg.C can be selected from 15 deg.C, 17 deg.C, 20 deg.C, 22 deg.C, 25 deg.C, 27 deg.C, 30 deg.C, 32 deg.C, 35 deg.C, 37 deg.C, 40 deg.C, etc.

The specific value of 3000-5000r/min can be selected from 3000r/min, 3200r/min, 3500r/min, 3700r/min, 4000r/min, 4200r/min, 4500r/min, 4700r/min, 5000r/min, etc.

The specific value of 10-30min can be selected from 10min, 12min, 15min, 17min, 20min, 22min, 25min, 27min, 30min, etc.

Other specific point values within the above numerical ranges can be selected at will, and are not described in detail herein.

Preferably, the final volume of the concentration in step (5) is 1/6-1/4 times of the volume of the supernatant, such as 1/6 times, 1/5.8 times, 1/5.5 times, 1/5.2 times, 1/5 times, 1/4.8 times, 1/4.5 times, 1/4.2 times, 1/4 times, and the like, and other specific points in the value range can be selected, and are not repeated herein.

Preferably, the solvent in step (5) is 80% -95% ethanol, and the 80% -95% is a volume fraction of ethanol, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, and the like, and other specific points in the numerical range can be selected, which is not described herein again.

Preferably, the volume of the solvent in the step (5) is 3-5 times of the volume of the supernatant after concentration, and the precipitation in the step (5) is carried out at 0-8 ℃ for 12-16 h.

The specific numerical range of 3 to 5 times may be 3 times, 3.2 times, 3.4 times, 3.6 times, 3.8 times, 4 times, 4.2 times, 4.4 times, 4.6 times, 4.8 times, 5 times, or the like.

The specific value range of 0-8 deg.C can be selected from 0 deg.C, 1 deg.C, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, etc.

The specific numerical range of 12-16h can be selected from 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, 15.5h, 16h and the like.

Other specific point values within the above numerical ranges can be selected at will, and are not described in detail herein.

Preferably, the centrifugation in the step (6) is carried out at 15-40 ℃, and the speed of the centrifugation is 3000-; the centrifugation time is 10-30 min.

The specific value of 15-40 deg.C can be selected from 15 deg.C, 17 deg.C, 20 deg.C, 22 deg.C, 25 deg.C, 27 deg.C, 30 deg.C, 32 deg.C, 35 deg.C, 37 deg.C, 40 deg.C, etc.

The specific value of 3000-5000r/min can be selected from 3000r/min, 3200r/min, 3500r/min, 3700r/min, 4000r/min, 4200r/min, 4500r/min, 4700r/min, 5000r/min, etc.

The specific value of 10-30min can be selected from 10min, 12min, 15min, 17min, 20min, 22min, 25min, 27min, 30min, etc.

Other specific point values within the above numerical ranges can be selected at will, and are not described in detail herein.

Preferably, the step (6) of collecting the precipitate further comprises washing the precipitate, wherein the washing reagent is absolute ethyl alcohol.

Preferably, the number of washes is 1-3, such as 1, 2, 3.

Preferably, the washing further includes drying the precipitate, the drying is performed in a freeze-drying manner, the drying time is 0.5-1h, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, and the like, and other specific values in the value range can be selected, which is not described herein again.

In a second aspect, the present invention provides a carrageenan extract produced by the method of producing a carrageenan extract according to the first aspect.

In a third aspect, the present invention provides the use of a carrageenan extract as described in the second aspect for the preparation of a food additive or a pharmaceutical additive.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method of the carrageenan extract provided by the invention is simple to operate, and the prepared carrageenan extract has a remarkable antioxidant effect and can be applied to the field of food additives or pharmaceutical additives.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

The following examples, comparative examples refer to carrageenans produced in Qinghai plateau from Roswent agricultural specialties.

Example 1

This example provides a method for preparing a carrageenan extract, comprising the steps of:

(1) freeze drying carrageen for 24 hr, pulverizing, and sieving with 60 mesh sieve to obtain carrageen algae powder;

(2) refluxing the carrageenin powder obtained in the step (1) for 3 times, wherein the refluxing time is 4h each time, and drying at 40 ℃ to obtain degreased algae powder;

the solvent used in the reflux is 95% ethanol, the 95% ethanol is the volume fraction of the ethanol, and the liquid-material ratio of the solvent to the carrageenan powder is 15 mL/g;

(3) carrying out water bath treatment on the degreased algae powder obtained in the step (2) for 6 hours at 100 ℃ to obtain a first mixed solution;

the water bath treatment is carried out under the assistance of ultrasonic waves, the ultrasonic power is 300w, the ultrasonic time is 50min, and the liquid-material ratio of the water bath treatment is 50 mL/g;

(4) centrifuging the first mixed solution obtained in the step (3) at 25 ℃ at the rotating speed of 4000r/min for 20min, concentrating the supernatant to 1/5 volume, and treating the supernatant with 30% ethanol at 20 ℃ for 1h to obtain a second mixed solution;

(5) centrifuging the second mixed solution obtained in the step (4) at the rotating speed of 4000r/min for 20min at the temperature of 20 ℃, concentrating the supernatant to 1/5 volume, and treating the supernatant with 95% ethanol of 4 times of the volume of the concentrated supernatant for 12h at the temperature of 4 ℃ to obtain a third mixed solution;

(6) centrifuging the third mixed solution obtained in step (5) at 25 deg.C at 4000r/min for 20min, washing the precipitate with anhydrous ethanol for 3 times, and freeze drying for 0.5 hr to obtain carrageenan extract.

Example 2

This example provides a method for preparing a carrageenan extract, comprising the steps of:

(1) freeze drying carrageen for 28 hr, pulverizing, and sieving with 70 mesh sieve to obtain carrageen algae powder;

(2) refluxing the carrageenin powder obtained in the step (1) for 2 times, wherein the refluxing time is 6h each time, and drying at 55 ℃ to obtain degreased algae powder;

the solvent used in the reflux is absolute methanol, and the liquid-material ratio of the absolute methanol to the carrageenin powder is 15 mL/g;

(3) carrying out water bath treatment on the degreased algae powder obtained in the step (2) for 5 hours at 90 ℃ to obtain a first mixed solution;

the water bath treatment is carried out under the assistance of ultrasonic waves, the ultrasonic power is 300w, the ultrasonic time is 60min, and the liquid-material ratio of the water bath treatment is 60 mL/g;

(4) centrifuging the first mixed solution obtained in the step (3) at the rotating speed of 4500r/min for 15min at the temperature of 15 ℃, concentrating the supernatant to 1/6 volume, and treating with 20% ethanol at the temperature of 20 ℃ for 2h to obtain a second mixed solution;

(5) centrifuging the second mixed solution obtained in the step (4) at 35 ℃ at a rotating speed of 3000r/min for 30min, concentrating the supernatant to 1/6 volume, and treating the supernatant with 90% ethanol in volume which is 3 times of the concentrated supernatant at 0 ℃ for 14h to obtain a third mixed solution;

(6) centrifuging the third mixed solution obtained in step (5) at 15 deg.C at 5000r/min for 10min, washing the precipitate with anhydrous ethanol for 2 times, and freeze drying for 0.5 hr to obtain carrageenan extract.

Example 3

This example provides a method for preparing a carrageenan extract, comprising the steps of:

(1) freeze drying carrageen for 21 hr, pulverizing, and sieving with 50 mesh sieve to obtain carrageen algae powder;

(2) refluxing the carrageenin powder obtained in the step (1) for 1 time, wherein the refluxing time is 12h, and drying at 35 ℃ to obtain degreased algae powder;

the solvent used in the reflux is anhydrous acetone, and the liquid-material ratio of the anhydrous acetone to the carrageenan powder is 15 mL/g;

(3) carrying out water bath treatment on the degreased algae powder obtained in the step (2) for 7 hours at the temperature of 80 ℃ to obtain a first mixed solution;

the water bath treatment is carried out under the assistance of ultrasonic waves, the ultrasonic power is 300w, the ultrasonic time is 40min, and the liquid-material ratio of the water bath treatment is 70 mL/g;

(4) centrifuging the first mixed solution obtained in the step (3) at the temperature of 30 ℃ at the rotating speed of 3500r/min for 25min, concentrating the supernatant to 1/4 volume, and treating with 50% ethanol at the temperature of 20 ℃ for 0.5h to obtain a second mixed solution;

(5) centrifuging the second mixed solution obtained in the step (4) at the rotating speed of 4500r/min for 15min at 15 ℃, concentrating the supernatant to 1/4 volume, and treating the supernatant with 80% ethanol of 5 times of the volume of the concentrated supernatant for 16h at 8 ℃ to obtain a third mixed solution;

(6) centrifuging the third mixed solution obtained in step (5) at 35 deg.C at 3000r/min for 30min, washing the precipitate with anhydrous ethanol for 2 times, and freeze drying for 0.5 hr to obtain carrageenan extract.

Example 4

This example provides a process for the preparation of a carrageenan extract which differs from example 1 only in that the solvent "95% ethanol" used in the reflux in step (2) is replaced by the same amount of "dehydrated ether" added, otherwise the conditions are as in example 1.

Example 5

This example provides a process for the preparation of a carrageenan extract, which differs from example 1 only in that the solvent "95% ethanol" used in the reflux in step (2) is replaced by "anhydrous ethyl acetate" in the same amount, and otherwise the conditions are as in example 1.

Example 6

This example provides a process for the preparation of a carrageenan extract which differs from example 1 only in that the solvent "95% ethanol" used in the reflux in step (2) is replaced by the same amount of "a combination of 95% ethanol and anhydrous ethyl acetate" wherein the volume ratio of 95% ethanol to anhydrous ethyl acetate is 1:1, and otherwise the conditions refer to example 1.

Example 7

This example provides a process for the preparation of a carrageenan extract which differs from example 1 only in that the solvent "95% ethanol" used in the reflux in step (2) is replaced by the same amount of "a combination of 95% ethanol and anhydrous diethyl ether" wherein the volume ratio of 95% ethanol to anhydrous diethyl ether is 1:1, and otherwise the conditions are as in example 1.

Example 8

This example provides a process for the preparation of a carrageenan extract which differs from example 1 only in that the solvent "95% ethanol" used in the reflux in step (2) is replaced by the same amount of "a combination of anhydrous ethyl acetate and anhydrous diethyl ether" wherein the volume ratio of anhydrous ethyl acetate to anhydrous diethyl ether is 1:1, and otherwise the conditions are as in example 1.

Example 9

This example provides a process for the preparation of a carrageenan extract which differs from example 1 only in that the solvent "95% ethanol" used in the refluxing in step (2) is replaced by the same amount of "a combination of 95% ethanol, anhydrous ethyl acetate and anhydrous diethyl ether" wherein the volume ratio of 95% ethanol, anhydrous ethyl acetate and anhydrous diethyl ether is 1:1:1, and the other conditions refer to example 1.

Example 10

This example provides a method for preparing a carrageenan extract, which is different from example 1 only in that the solvent "30% ethanol" in step (4) is replaced by the same amount of "20% ethanol", and other conditions refer to example 1.

Example 11

This example provides a method for preparing a carrageenan extract, which is different from example 1 only in that the solvent "30% ethanol" in step (4) is replaced with the same amount of "50% ethanol", and the other conditions refer to example 1.

Comparative example 1

This comparative example provides a method for preparing a carrageenan extract, which is different from example 1 only in that the reflux defatting operation in step (2) is omitted, and the other conditions refer to example 1.

Comparative example 2

This comparative example provides a process for the preparation of carrageenan extract, which differs from example 1 only in that the procedure of removing algin by adding solvent in step (4) is omitted and the other conditions refer to example 1.

Test example 1

The extraction yield of the carrageenan extracts prepared in examples 1-11 and comparative examples 1 and 2 was evaluated.

The total sugar content is determined by phenol-sulfuric acid method, polysaccharide is hydrolyzed into monosaccharide under the action of sulfuric acid, and is rapidly dehydrated to generate uronic acid derivative, which reacts with phenol to generate orange yellow substance with characteristic absorption at 490nm, and the content is quantified compared with standard series. The carrageenan content was tested with glucose as standard.

The specific operation steps are as follows;

(1) dissolving 1mg of the prepared carrageenan extract in 1mL of distilled water to prepare a solution to be detected;

(2) transferring all the solution to be detected obtained in the step (1) to a 20mL glass test tube with a plug, supplementing the solution to 1mL by using distilled water, and adding 1mL of phenol (5%);

(3) adding 5mL concentrated sulfuric acid (vertical to the liquid surface, without contacting the wall of the test tube, to mix with the reaction solution), and standing for 10 min;

(4) the reaction solution was thoroughly mixed using a vortex shaker, and then the tube was placed in a water bath at 30 ℃ for 20min, and the absorbance was measured at 490 nm.

The polysaccharide content in the sample was calculated according to the following formula:

wherein: m is₁-the polysaccharide content corresponds to the sugar content (ug) on the standard curve;

V₁-sample volumetric volume (mL);

m₂-sample mass (g);

V₂- -volume of sample removed for colorimetric determination (mL);

0.9- - -conversion factor of glucose to dextran.

The results of comparing the extraction yields of the carrageenan extracts prepared in examples 1-11 and comparative examples 1 and 2 are shown in Table 1.

TABLE 1

Group of	Extraction rate
		Example 1	3.53％
Example 2	3.19％
		Example 3	3.24％
Example 4	2.89％
		Example 5	2.98％
Example 6	3.63％
		Example 7	3.66％
Example 8	3.65％
		Example 9	3.83％
Example 10	3.05％
		Example 11	3.01％
Comparative example1	1.03％
		Comparative example 2	1.15％

The results show that: the carrageenans extracts prepared in examples 1-11 were obtained with higher extraction yields, with the carrageenans extracts prepared in examples 6-8 being slightly higher than in example 1, the carrageenans extract prepared in example 9 being the highest extraction yield, the carrageenans extracts prepared in examples 10 and 11 being slightly lower than in example 1 and the carrageenans extracts prepared in comparative examples 1 and 2 being lower extraction yields.

The extraction yield of the carrageenan extract prepared in comparative examples 1 and 2 was low.

Test example 2

The antioxidant effect of the carrageenan extracts prepared in examples 1-11 and comparative examples 1 and 2 was evaluated using an in vitro test. The test method is as follows:

dissolving carrageenan extract in anhydrous ethanol to prepare a series of sample solutions to be tested, wherein the sample solutions are 1mg/mL, 2mg/mL, 4mg/mL, 8mg/mL, 16mg/mL and 24 mg/mL.

(1) DPPH radical scavenging ability test:

preparing 0.2mmol/L DPPH solution (the solvent is absolute ethyl alcohol);

sample measurement A₁: mixing 100 μ L sample solution to be detected and 100 μ L DPPH solution in 96-well plate, reacting in dark for 30min, measuring the light absorption value at 517nm, and recording as A₁；

Sample blank A₂: mixing 100 μ L sample solution to be tested with 100 μ L anhydrous ethanol in 96-well plate, reacting in dark for 30min, measuring light absorption value at wavelength of 517nm, and recording as A₂；

Reagent blank A₀: mixing 100 μ L DPPH solution and 100 μ L absolute ethanol in 96-well plate, and reacting in dark for 30%min, measuring the light absorption value at the wavelength of 517nm and recording as A₀；

(2) ABTS free radical scavenging ability test:

preparing 5mmol/L ABTS stock solution, diluting with anhydrous ethanol to make the absorbance value of the diluted solution at 734nm be 0.7 + -0.02 (at 30 deg.C), and obtaining ABTS working solution.

Sample measurement A₁: absorbing 190 μ L ABTS working solution, adding 10 μ L sample solution to be tested, oscillating for 10s, standing at 30 deg.C for 6min, measuring absorbance at 734nm, and recording as A₁；

Sample blank A₂: absorbing 190 μ L of anhydrous ethanol, adding 10 μ L of sample solution to be tested, oscillating for 10s, standing at 30 deg.C for 6min, measuring absorbance at 734nm, and recording as A₂；

Reagent blank A₀: absorbing 190 μ L ABTS working solution, adding 10 μ L anhydrous ethanol, oscillating for 10s, standing at 30 deg.C for 6min, measuring absorbance at 734nm, and recording as A₀；

(3) Hydroxyl radical scavenging ability test:

sample measurement A₁: add 60. mu.L of FeSO to 96-well plates₄Solution, 60 μ L salicylic acid solution, and finally 60 μ L H₂O₂Starting the reaction of the solution, uniformly mixing, and incubating for 15min at 37 ℃; adding 20 μ L of sample solution to be tested, incubating at 37 deg.C for 15min, measuring absorbance at 510nm, and recording as A₁；

Sample blank A₂: add 60. mu.L of FeSO to 96-well plates₄The solution is 60 mu L of salicylic acid solution, and finally 60 mu L of distilled water is added, and after uniform mixing, incubation is carried out for 15min at 37 ℃; adding 20 μ L of sample solution to be tested, incubating at 37 deg.C for 15min, measuring absorbance at 510nm, and recording as A₂；

Reagent blank A₀: add 60. mu.L of FeSO to 96-well plates₄Solution, 60 μ L salicylic acid solution, and finally 60 μ L H₂O₂Starting the reaction of the solution, uniformly mixing, and incubating for 15min at 37 ℃; adding distilled water 20 μ L, incubating at 37 deg.C for 15min, measuring absorbance at 510nm, and recording as A₀；

And (3) drawing a change curve by taking the concentration of the sample solution to be detected as an abscissa and the free radical scavenging rate as an ordinate so as to calculate the IC50 value of the carrageenan extract for scavenging free radicals.

The test results are shown in Table 2.

TABLE 2

The results show that: the carrageenan extracts prepared in examples 1-11 have a strong scavenging ability against DPPH free radicals, ABTS free radicals and hydroxyl free radicals, i.e. a significant antioxidant effect. Among them, the carrageenan extracts obtained in examples 6 to 8 had slightly better antioxidant effect than that of example 1, the carrageenan extract obtained in example 9 had the best antioxidant effect, the carrageenan extracts obtained in examples 10 and 11 had slightly lower antioxidant effect than that of example 1, and the carrageenan extracts obtained in comparative examples 1 and 2 had poor antioxidant effect.

Test example 3

The antioxidant effect of the carrageenan extracts prepared in examples 1 to 11 and comparative examples 1 and 2 was evaluated using an in vivo experiment based on a caenorhabditis elegans (hereinafter, referred to as nematode) model. The test method is as follows:

(1) preparing a nematode culture medium and a reagent:

(1M) Potassium phosphate buffer

KH₂PO₄ 108.39g

K₂HPO₄ 35.69g

Adding water to 1L, and adjusting pH to 6.0.

② M9 buffer solution

Adding water to 1L, and sterilizing at 121 deg.C for 15 min.

③ LB liquid culture medium

Tryptone 10g/L

Sodium chloride 10g/L

Yeast powder 5g/L

Preparing distilled water, adjusting pH to 7.0 with 1M sodium hydroxide solution, and sterilizing at 121 deg.C for 15 min.

(iv) Nematode Growth solid Medium (NGM) 1L

Shaking, sterilizing at 121 deg.C for 30min, keeping the temperature at 80 deg.C for 15min, and adding the following sterilized solutions (wherein cholesterol is sterilized by filtration, and the rest solutions are sterilized at high temperature).

Lysate

0.1g NaOH and 1.3mL NaClO were dissolved in 4mL distilled water and mixed well (ready for use).

(2) Basic nematode manipulation method

(ii) cultivation of Escherichia coli OP50

Putting strain OP50 on LB mediumPlate streaking, picking single colony in 10mL LB liquid medium, 37 ℃, 200rpm, shaking culture for 12h, to OD₆₀₀Equal to 0.4, for inoculation of NGM to feed normal group nematodes.

Coating of e.coli OP50

An appropriate amount of inoculum (typically 60mm diameter plate plus 100. mu.L) was added to each NGM plate and the inoculum was spread evenly on the NGM plate using a sterile spreader or the bottom of a glass test tube, taking care that the inoculum edge should be about 0.5cm from the plate edge. The bacterial-coated NGM plates were kept overnight at room temperature (21-25 deg.C) in a cold room or 4 deg.C freezer until use.

③ synchronization of nematodes

Washing young adults into an aseptic EP tube by using M9 buffer solution, adding a proper amount of lysate to crack nematodes, placing the nematode on a low-speed centrifuge at 3000rpm for 1min, discarding the supernatant, washing the nematodes for 2 times by using M9, centrifuging the nematode, discarding the supernatant, sucking the nematodes at the bottom of the EP tube by using a pipette gun to drip into an aseptic area of the NGM, basically developing fertilized eggs in the cracked nematodes into L4 larvae after about 48 hours, and completing synchronization for the experiment.

Cracking of nematode

The young adults on the NGM plate are repeatedly washed twice by 1mL of M9 buffer solution and then transferred to a sterile 2mL centrifuge tube, 1mL of prepared lysate is added, after full oscillation for 3-5min, the mixture is centrifuged for 1min at the rotating speed of 3000rpm, and the supernatant is discarded. The nematodes were washed with 1mL of M9 buffer, centrifuged 2 times under the same conditions, the supernatant discarded, and 0.3-0.4mL of egg-containing buffer remained. Then gently blow and beat the mixed eggs by using a pipette gun, and suck about 100 mu L of buffer solution containing the eggs to a sterile area close to OP50 on an NGM plate. After about 48 hours, the nematode oosperm basically develops into L4 larvae, and synchronization is completed. The larvae in stage L4 were picked and transferred to drug plates for each index measurement.

(3) Preparation of carrageenan-containing NGM:

dissolving 1mg of carrageenan in 1mL of sterile water to prepare a mother solution, and uniformly mixing 98 μ L of E.coli OP50 bacterial solution and 2 μ L of the mother solution. A pipette gun was used to aspirate 150. mu.L of mixed inoculum and gently pipette the mixed inoculum into the center of each NGM plate. The blank group was uniformly coated with 100-. And (4) drying the bacterial-coated NGM plate in the dark, sealing the membrane, and storing at 4 ℃ for later use.

(4) And (3) accumulation test of active oxygen in the nematode:

the placebo and the test group after 96h carrageenan treatment were transferred to NGM plates to remove e. After 3 transfers, the nematodes were transferred to 96-well fluorescent plates containing 50. mu. L M9 buffer, 80 per well, in triplicate. At the same time, 50. mu.L of 100. mu.M H in M9 buffer was added₂DCF-DA solution with 50. mu.L of H without nematodes₂DCF-DA solution and 50. mu. L M9 buffer served as blanks. Immediately, the 96-well plate is placed into an enzyme-labeling instrument to measure the fluorescence intensity, the reaction temperature is 25 ℃, the emission wavelength is 528nm, and the excitation wavelength is 485 nm. The assay was performed every 20min for 6h with shaking before each reading. Because the accumulation of ROS in the nematode is in direct proportion to the generation amount of the fluorescent substance DCF, the fluorescence intensity of each group can directly reflect the accumulation amount of ROS in the nematode.

(5) CAT activity test in the nematode body:

respectively selecting 200 nematodes from a blank group of nematodes and a test group of nematodes treated by carrageenan for 96h, placing the nematodes into sterile water for washing, centrifuging at a low speed to remove supernatant, repeatedly carrying out three times, fixing the volume to 0.5mL, and carrying out low-temperature grinding for 1min in a grinding instrument. Grinding, adding 1% chap solution to a constant volume of 1mL, centrifuging at low temperature (4 ℃, 12000r/min, 15min), collecting supernatant, and standing at 4 ℃ for testing. The determination of the enzyme activity is carried out according to the specification requirements of BCA and CAT test boxes of Nanjing institute of Biotechnology.

The test results are shown in Table 3.

TABLE 3

The results show that: the carrageenans extracts prepared in examples 1-11 all significantly reduced the accumulation of active oxygen in the nematode while significantly enhanced CAT activity in the nematode, i.e., had significant antioxidant effects. Among them, the carrageenan extracts obtained in examples 6 to 8 had slightly better antioxidant effect than that of example 1, the carrageenan extract obtained in example 9 had the best antioxidant effect, the carrageenan extracts obtained in examples 10 and 11 had slightly lower antioxidant effect than that of example 1, and the carrageenan extracts obtained in comparative examples 1 and 2 had poor antioxidant effect.

In conclusion, the preparation method of the carrageenan extract provided by the invention is simple to operate, and the prepared carrageenan extract has a remarkable antioxidant effect and can be applied to the field of food additives or pharmaceutical additives.

The applicant states that the present invention is illustrated by the above examples of the carrageenan extract and the method of preparation and use of the present invention, but the present invention is not limited to the above examples, i.e. it is not meant that the present invention must be practiced by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.