Kappaphycus alvarezii polysaccharide extract, preparation method thereof and application of Kappaphycus alvarezii polysaccharide extract in preparation of antioxidant products

Abstract

The invention belongs to the technical field of marine organism extraction and food processing, and discloses a kappaphycus alvarezii polysaccharide extract with an anti-oxidation effect as well as a preparation method and application of the kappaphycus alvarezii polysaccharide extract. The preparation method of the kappaphycus alvarezii polysaccharide extract comprises the following steps: drying, dehydrating and crushing kappaphycus alvarezii, soaking the kappaphycus alvarezii in ethanol, then adding cellulase for enzymolysis extraction, centrifuging to take supernate, and concentrating and performing alcohol precipitation to obtain the kappaphycus alvarezii polysaccharide extract. The invention also discloses an application of the kappaphycus alvarezii polysaccharide extract in preparation of an antioxidant. The kappaphycus alvarezii is used as a raw material, an extraction preparation method is optimized, and the kappaphycus alvarezii polysaccharide extract with remarkably improved oxidation resistance is obtained by utilizing a cellulase enzymolysis extraction process, so that algal polysaccharides are efficiently released; and the preparation method is simple in process flow and easy to popularize and apply, and has a wide application prospect in preparation of the kappaphycus alvarezii polysaccharide extract with antioxidant activity. The method can be applied to research and development in the fields of foods, functional nutritional products and health foods.

Description

Technical Field

[0001] The invention belongs to the technical field of marine biological extraction and food processing, and more specifically, relates to a Kappaphycus sphaerocephala polysaccharide extract, a preparation method thereof and application of the extract in preparing antioxidant products. Background Art

[0002] Oxidative free radicals are important factors that lead to the occurrence, development and prognosis of many diseases. Therefore, the study of oxidative free radical scavenging has far-reaching biological significance for human health. Antioxidant active substances can exert their effects through various mechanisms, including scavenging free radicals, activating antioxidant stress pathways, regulating antioxidant enzyme activity and reducing oxidative damage, etc., and have received widespread attention in the research field. Among the many antioxidant substances from natural sources, seaweed polysaccharides have gradually become one of the important directions of antioxidant activity research due to their good antioxidant activity, low price and low toxic side effects.

[0003] Kappaphycus alvarezii, as a red algae, is an important tropical marine biological resource with both medicinal and edible value. Kappaphycus alvarezii is rich in nutrients, including antioxidant and anti-free radical effects, anti-tumor and anti-viral effects, and hypoglycemic and hypolipidemic effects. In the field of cosmetics, Kappaphycus alvarezii extracts have been used to develop products with skin tightening effects. These products can not only quickly tighten and lift the skin from the surface of the skin, but also penetrate into the dermis of the skin to repair the skin, replenish skin moisture and collagen, and enhance skin elasticity; at the same time, they can also resist damage to the skin caused by pollutants in the environment.

[0004] However, the existing extraction methods and application research are not enough to meet the needs of industrial production. Traditional extraction methods often have low extraction efficiency and yield, and may destroy the active structure of polysaccharides. Therefore, developing new extraction technologies to improve the extraction efficiency and biological activity of polysaccharides is of great significance for fully utilizing the antioxidant potential of Kappaphycus longifolia. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a method for preparing an antioxidant polysaccharide extract of Kappaphycus longifolia, which can efficiently extract and maintain the antioxidant activity of polysaccharides, and provide a new type of natural antioxidant for the fields of food, functional nutritional products and health food.

[0006] The first object of the present invention is to provide a Kappaphycan extract having antioxidant effect.

[0007] The second object of the present invention is to provide a method for preparing the Kappaphycan extract.

[0008] The third object of the present invention is to provide the application of the Kappaphycan extract.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A Kappaphycan extract is prepared by the following method:

[0011] S1. Wash and dry the long heart Kappaphycus algae, crush it, soak it in ethanol, filter and dry it to obtain algae powder;

[0012] S2. Add water to the algae powder in step S1, adjust the pH value to 5-6, then add cellulase for enzymolysis, and centrifuge after the enzymolysis to obtain a Kappaphycan solution;

[0013] S3. The Kappaphycan solution of step S2 is subjected to alcohol precipitation, washed after the alcohol precipitation, and dried to obtain the Kappaphycan extract.

[0014] Preferably, the material-liquid ratio of algae powder to water is 1:60-120, and the amount of cellulase added in step S2 is 2000-5000 U / g.

[0015] Preferably, the enzymatic hydrolysis conditions in step S2 are: enzymatic hydrolysis for 2 to 5 hours in a water bath at a temperature of 45 to 55°C.

[0016] Preferably, after the enzymatic hydrolysis is completed, the enzyme is inactivated and then centrifuged at 25° C. and 4000 rpm for 10 min.

[0017] Preferably, in step S1, the algae are passed through a 400-mesh sieve, 3 times the volume of 80% ethanol is added and soaked for 24 hours, filtered with a filter cloth, and dried to obtain algae powder.

[0018] Preferably, in step S3, the Kappaphycan solution of step S2 is first concentrated under reduced pressure, and then 3 volumes of ethanol are added, and the mixture is precipitated at 4° C. for 24 to 72 hours.

[0019] Preferably, the drying temperature in step S1 is 80° C., the ethanol concentration is 80%, and the soaking time is 24 to 72 hours.

[0020] After the above method is optimized, the yield of the Kappaphycus longifolia polysaccharide extract with antioxidant effect can reach 22.81%, the polysaccharide content is 55.69%, the uronic acid content is 4.41%, the sulfate content is 6.74%, and the ABTS content is 1. + The free radical scavenging rate was 74.03% (1.25 mg / mL), and the superoxide anion scavenging rate was 43.80% (0.8 mg / mL).

[0021] Therefore, the present invention also protects the use of the Kappaphycan extract in the preparation of antioxidant products.

[0022] Furthermore, the Kappaphycan extract increases ABTS + Free radical and superoxide anion scavenging rate.

[0023] Furthermore, the product is in the field of food, functional nutritional products and / or health food.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Compared with other traditional extraction methods such as acid extraction and alkali extraction, the polysaccharide yield and content of the Kappaphycus serrata polysaccharide extract prepared by the cellulase enzymatic extraction method of the present invention are improved, and the uronic acid content and sulfate content in the Kappaphycus serrata polysaccharide extract are effectively increased, and the ABTS + The scavenging rate of free radicals and superoxide anions shows good antioxidant activity. Therefore, the Kappaphycus longifolia polysaccharide extract has good development value and application prospects in preparing functional nutritional foods or health products with antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The yield and polysaccharide content of the polysaccharide extract of Kappaphycus sphaerocephala (based on Example 1 and Comparative Examples 1-2);

[0027] Figure 2 Analysis of the sulfate content and uronic acid content of the Kappaphycan extract (based on Example 1 and Comparative Examples 1-2);

[0028] Figure 3 This is an analysis of the antioxidant activity of the Kappaphycan extract (based on Example 1 and Comparative Examples 1-2).

[0029] Note: Comparison between cellulase extraction method and acid extraction method, #p<0.05, ##p<0.01; comparison between cellulase extraction method and alkali extraction method, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] Unless otherwise specified, the test methods used in the following embodiments and experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0032] Example 1

[0033] The long heart Kappa algae was cleaned and decontaminated, placed in an oven at 80°C for drying and dehydration, crushed with a pulverizer, passed through a 400 mesh screen, soaked in 80% ethanol for 24 hours, filtered with a filter cloth, and dried to obtain algae powder. A certain amount of algae powder was weighed, 60 times the volume of distilled water (solid-liquid ratio w / v=1:60) was added, the pH was adjusted to 5.5, preheated to 50°C, and then 4000U / g of cellulase was added, enzymolysis was performed in a water bath for 3 hours, enzyme was inactivated at 100°C for 10 minutes, and the long heart Kappa algae polysaccharide solution was collected by centrifugation (4000rpm, 25°C, 10min), and after reduced pressure concentration, 3 times the volume of anhydrous ethanol was added, precipitated at 4°C for 24 hours, centrifuged (4000rpm, 4°C, 15min), washed repeatedly with anhydrous ethanol 3 times, and the precipitate was collected and dried to obtain the long heart Kappa algae polysaccharide extract.

[0034] Example 2

[0035] The long heart Kappa algae was cleaned and decontaminated, placed in an oven at 80℃ for drying and dehydration, crushed with a pulverizer, passed through a 400 mesh screen, soaked in 80% ethanol for 36 hours, filtered with a filter cloth, and dried to obtain algae powder. A certain amount of algae powder was weighed, 90 times the volume of distilled water (solid-liquid ratio w / v=1:75) was added, the pH was adjusted to 5.5, preheated to 50℃, and then 2000U / g of cellulase was added, enzymolysis was performed in a water bath for 2 hours, enzyme was inactivated at 100℃ for 10 minutes, and the long heart Kappa algae polysaccharide solution was collected by centrifugation (4000rpm, 25℃, 10min), and then 3 times the volume of anhydrous ethanol was added after concentration under reduced pressure, alcohol precipitation was performed at 4℃ for 24 hours, centrifuged (4000rpm, 4℃, 15min), and washed repeatedly with anhydrous ethanol for 3 times, and the precipitate was collected and dried to obtain the long heart Kappa algae polysaccharide extract.

[0036] Example 3

[0037] The long heart Kappa algae was cleaned and decontaminated, placed in an oven at 80℃ for drying and dehydration, crushed with a pulverizer, passed through a 400 mesh screen, soaked in 80% ethanol for 24 hours, filtered with a filter cloth, and dried to obtain algae powder. A certain amount of algae powder was weighed, 120 times the volume of distilled water (solid-liquid ratio w / v=1:100) was added, the pH was adjusted to 5.5, preheated to 50℃, and then 5000U / g of cellulase was added, enzymolysis was performed in a water bath for 5 hours, enzyme was inactivated at 100℃ for 10 minutes, and the long heart Kappa algae polysaccharide solution was collected by centrifugation (4000rpm, 25℃, 10min), and after reduced pressure concentration, 3 times the volume of anhydrous ethanol was added, 4℃ alcohol precipitation was performed for 48 hours, centrifuged (4000rpm, 4℃, 15min), and washed repeatedly with anhydrous ethanol for 3 times, and the precipitate was collected and dried to obtain the long heart Kappa algae polysaccharide extract.

[0038] Comparative Example 1

[0039] Acid extraction method: Wash and remove impurities from Kappaphycus longifolia, place in an oven at 80°C for drying and dehydration, crush with a grinder, pass through a 400-mesh sieve, soak in 80% ethanol for 24 hours, filter with a filter cloth, and dry to obtain algae powder. Weigh a certain amount of algae powder, add 60 times the volume of 8% KOH solution, extract at 80°C for 3 hours, centrifuge (4000rpm, 25°C, 10min) to collect the Kappaphycus longifolia polysaccharide solution, concentrate under reduced pressure, add 3 times the volume of anhydrous ethanol, stand at 4°C for 24 hours, centrifuge (4000rpm, 4°C, 15min), wash repeatedly with anhydrous ethanol 3 times, collect the precipitate and dry it, and obtain the Kappaphycus longifolia polysaccharide extract.

[0040] Comparative Example 2

[0041] Alkaline extraction method: Wash and remove impurities from Kappaphycus longifolia, place in an oven at 80°C for drying and dehydration, crush with a grinder, pass through a 400-mesh sieve, soak in 80% ethanol for 24 hours, filter with a filter cloth, and dry to obtain algae powder. Weigh a certain amount of algae powder, add 60 times the volume of 0.2MHCL solution, extract at 80°C for 2 hours, centrifuge (4000rpm, 25°C, 10min) to collect the Kappaphycus longifolia polysaccharide solution, concentrate under reduced pressure, add 3 times the volume of anhydrous ethanol, stand at 4°C for 24 hours, centrifuge (4000rpm, 4°C, 15min), wash repeatedly with anhydrous ethanol 3 times, collect the precipitate and dry it, and obtain the Kappaphycus longifolia polysaccharide extract.

[0042] Effect experiment example 1:

[0043] The yield and polysaccharide content of Example 1 and Comparative Examples 1-2 were measured and analyzed, and the results are shown in Table 1.

[0044] Polysaccharide yield: yield (%) = weight of polysaccharide extract / initial weight of Kappaphycus longifolia × 100%

[0045] Table 1

[0046] serial number Yield (%) Polysaccharide content (%) Example 1 22.81 55.69 Example 2 20.37 50.22 Example 3 23.28 48.31 Comparative Example 1 22.67 43.18 Comparative Example 2 15.28 19.46

[0047] As can be seen from the results in Table 1, by comparing the treatment results of Examples 1-3 with the results of Comparative Examples 1-2, it can be seen that the yield and polysaccharide content of the Kappaphycus longifolia polysaccharide extract obtained by cellulase enzymatic hydrolysis can be improved to varying degrees. Figure 1 As shown, the cellulase hydrolysis method has a higher yield and no significant difference (p>0.05) compared with comparative example 1 (acid extraction method), but the cellulase hydrolysis method can significantly increase the polysaccharide content (p<0.05). The cellulase hydrolysis method can significantly increase the polysaccharide yield (p<0.05) and polysaccharide content (p<0.05) compared with comparative example 2 (alkali extraction method). It can be seen that the cellulase hydrolysis method can maintain a higher yield and polysaccharide content in optimizing the polysaccharide extraction process.

[0048] Effect experiment example 2:

[0049] The sulfate content and uronic acid content of the Kappaphycan polysaccharide extracts of Example 1 and Comparative Examples 1-2 were analyzed:

[0050] Determination of polysaccharide content: Accurately weigh 10 mg of glucose standard dried to constant weight, add appropriate amount of distilled water to dissolve and transfer to a 50 mL volumetric flask, dilute to the scale, prepare a glucose standard solution with a concentration of 0.2 mg / mL, and take 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mL respectively and place them in centrifuge tubes, and add distilled water to make up to a final volume of 2 mL. Then take 200uL into 2 mL centrifuge tubes respectively, add 100uL of 6% phenol to each tube, shake well and continue to add 500uL of concentrated sulfuric acid, mix well and cool to room temperature. Take 200uL from each tube into a 96-well plate and detect the absorbance at 490nm with an enzyme marker. Draw a standard curve with the mass of the glucose standard as the horizontal axis and the absorbance value as the vertical axis. Prepare 1 mg / mL polysaccharide sample solution, take 0.1 mL, add distilled water to make up to 2 mL, set up three groups of parallel, determine the absorbance value according to the above operation, and calculate the total sugar content in the sample according to the standard curve.

[0051] Sulfate content determination: accurately weigh 90.6 mg of potassium sulfate, dissolve it with 1M HCl solution and dilute to 100 mL to prepare a 0.6 mg / mL potassium sulfate standard solution; weigh 3 g of trichloroacetic acid and dilute to 100 mL with distilled water; weigh 0.5 g of gelatin particles, add distilled water and dissolve completely in a 60-70°C water bath and dilute to 100 mL, place in a 4°C refrigerator overnight, take 1 g of barium chloride and dissolve it in 0.5% gelatin solution to form a barium chloride-gelatin solution. Take 0, 40, 100, 160, and 200 uL of potassium sulfate standard solution respectively and place them in a test tube, add 1M HCl solution to 200 uL, add 3.8 mL of 3% trichloroacetic acid each, and 1 mL of barium chloride-gelatin solution. Oscillate and mix well, measure the absorbance at 360 nm with 1M HCl as a reference. Draw a standard curve with the mass of sulfate as the horizontal axis and the absorbance as the vertical axis. Weigh 4 mg of polysaccharide sample, add 2 mL of 1M HCl to dissolve, make three replicates, hydrolyze at 105°C for 3 h, cool to room temperature, take 100 uL, make up to 200 uL with 1M HCl, determine the absorbance value according to the above operation, and calculate the sulfate content in the sample according to the standard curve.

[0052] Determination of uronic acid content: Prepare 100μg / mL glucuronic acid solution, take 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mL standard solution into 10mL stoppered test tubes, add water to 1.0mL, add 6mL concentrated sulfuric acid, heat in a boiling water bath for 30min, add 0.25mL 0.1% carbazole solution after cooling, then boil in a boiling water bath for 15min, take out, cool to room temperature, detect at 525nm wavelength, and draw a standard curve. Take 5mg of polysaccharide sample, dissolve in 10mL water, take 0.5mL in a test tube, add 0.5mL water, add 6mL sulfuric acid, measure the absorbance according to the above steps, and calculate the sulfate content in the sample according to the standard curve.

[0053] The results are as follows Figure 2 As shown, the uronic acid content and sulfate content of the long-heart Kappaphycan extract obtained by cellulase enzymatic hydrolysis were 4.41% and 6.74%, respectively, which were effectively increased (p<0.05) compared with the uronic acid content of the long-heart Kappaphycan extract obtained by Comparative Example 1 (acid extraction method) and Comparative Example 2 (alkaline extraction method). Compared with Comparative Example 2 (alkaline extraction method), the cellulase enzymatic hydrolysis method can significantly increase the sulfate content of the polysaccharide (p<0.05).

[0054] Effect experiment example 3:

[0055] The antioxidant activity of the Kappaphycus sphaerocephala polysaccharide extracts of Example 1, Comparative Examples 1 and 2 was evaluated:

[0056] ABTS total antioxidant capacity determination: 50 μL of polysaccharide solution (final concentration gradient: 0.0625, 0.125, 0.25, 0.5, 1.0, 1.25 mg / mL) was taken into a 96-well plate, and then 150 μL of ABTS+ solution was quickly transferred to the 96-well plate using a multi-channel pipette, and reacted at room temperature in the dark for 6 min. A 734 Value, denoted as A sample The same volume of PBS solution was used to replace the sample solution as the control group, and its corresponding absorbance value was recorded as A control Calculate the sample's affinity for ABTS according to the following formula + Clearance rate:

[0057] ABTS + The clearance rate (%) = (1-A sample / A control )×100%

[0058] Determination of superoxide anion scavenging ability: Mix 50 μL of sample (final concentration gradient: 0.05, 0.1, 0.2, 0.4, 0.8 mg / mL) with 100 μL of Tris-HCl-EDTA solution (50 mM, pH 8.2). Add 50 μL of pyrogallol solution (5 mM), shake rapidly, react at 25°C in the dark for 5 minutes, and then add 20 μL of 4M HCl solution to terminate the reaction. The absorbance of the resulting solution was measured at 420 nm, where absorbances A1 and A2 are the absorbances of the sample and its background (solutions containing pyrogallol and pyrogallol-free, respectively), and A0 is the absorbance of the background solution (without sample). Its ability to scavenge superoxide anions was calculated by the following formula:

[0059] Superoxide anion clearance rate (%) = (1-(A1-A2) / A0) × 100%

[0060] The antioxidant activity evaluation showed that Figure 3 As shown in ABTS + In terms of free radical scavenging, compared with Comparative Example 1 (acid extraction) and Comparative Example 2 (alkaline extraction), the Kappaphycus polysaccharide extract prepared by cellulose enzymatic hydrolysis (at a concentration of 1.25 mg / mL) showed a more significant scavenging effect (p<0.01) and was dose-dependent. In terms of superoxide anion scavenging, compared with Comparative Example 1 (acid extraction) and Comparative Example 2 (alkaline extraction), the Kappaphycus polysaccharide extract prepared by cellulose enzymatic hydrolysis (at a concentration of 0.8 mg / mL) significantly improved the scavenging effect (p<0.05).

[0061] In summary, the yield, polysaccharide content, uronic acid content and sulfate content of Kappaphycus longifolia polysaccharide extract prepared by cellulose enzymatic hydrolysis were effectively improved, and the ABTS + Free radical and superoxide anion scavenging efficiency.

[0062] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A Kappaphycan extract, characterized in that: Prepared by the following method: S1. Wash and dry the long heart Kappaphycus algae, crush it, soak it in ethanol, filter and dry it to obtain algae powder; S2. Add water to the algae powder in step S1, adjust the pH value to 5-6, then add cellulase for enzymolysis, and centrifuge after the enzymolysis to obtain a Kappaphycan solution; S3. The Kappaphycan solution of step S2 is subjected to alcohol precipitation, washed after the alcohol precipitation, and dried to obtain the Kappaphycan extract.

2. The Kappaphycan extract according to claim 1, characterized in that The material-liquid ratio of algae powder to water is 1:60-120, and the amount of cellulase added in step S2 is 2000-5000 U / g.

3. The Kappaphycan extract according to claim 2, characterized in that The enzymatic hydrolysis conditions in step S2 are: enzymatic hydrolysis for 2 to 5 hours in a water bath at a temperature of 45 to 55°C.

4. The Kappaphycan extract according to claim 3, characterized in that After the enzymatic hydrolysis was completed, the enzyme was inactivated and then the mixture was centrifuged at 4000 rpm for 10 min at 25°C.

5. The Kappaphycan extract according to claim 1, characterized in that In step S1, the algae are passed through a 400-mesh sieve, 3 times the volume of 80% ethanol is added, and soaked for 24 hours, filtered with a filter cloth, and dried to obtain algae powder.

6. The Kappaphycan extract according to claim 1, characterized in that In step S3, the Kappaphycan solution of step S2 is first concentrated under reduced pressure, and then 3 times the volume of ethanol is added, and the mixture is precipitated at 4° C. for 24 to 72 hours.

7. The Kappaphycan extract according to claim 1, characterized in that The drying temperature in step S1 is 80° C., the ethanol concentration is 80%, and the soaking time is 24 to 72 hours.

8. Use of the Kappaphycan extract of claim 1 in the preparation of antioxidant products.

9. The use according to claim 8, characterized in that: The Kappaphycan extract increases ABTS + Scavenging rate of free radicals and superoxide anions.

10. The use according to claim 8, characterized in that: The product is in the field of food, functional nutritional products and / or health food.

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