Preparation method of scenedesmus polysaccharide

Abstract

The invention relates to the technical field of polysaccharide extraction, in particular to a preparation method of scenedesmus polysaccharide. The invention provides a preparation method of scenedesmus polysaccharide, the scenedesmus polysaccharide and application of the scenedesmus polysaccharide. According to the method disclosed by the invention, a compound enzyme-assisted enzymolysis treatment process is adopted, scenedesmus powder is taken as a raw material, and high extraction yield and purity of scenedesmus polysaccharide are kept by controlling process parameters such as different enzyme addition amounts and enzymolysis pH; a degreasing step is omitted, and meanwhile, the enzymolysis time is prolonged, so that a relatively good decolorizing effect is also ensured. Besides, anion exchange column chromatography and gel column chromatography are adopted, and the scenedesmus crude polysaccharide (TBP) is separated to obtain four components with high purity, namely TBP-1, TBP-2, TBP-3 and TBP-4. The preparation method of scenedesmus polysaccharide has the advantages of simple operation steps, low requirements on production equipment, environmental friendliness, low extraction cost and the like, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide extraction, in particular to a method for preparing Scenedesmus polysaccharide. Background Art

[0002] Scenedesmus belongs to the Chlorophyta, Chlorococcales, Scenedesmus family. It is a small algae with a palisade or elliptical body. It is one of the common freshwater algae. It has become a common microalgae species in microalgae biotechnology research because of its easy cultivation characteristics. Polysaccharides are one of the important active ingredients of microalgae. Due to their complex and special structure, they not only have a variety of biological activities, including antibacterial, antioxidant, anti-tumor, anti-radiation, anti-viral, anti-inflammatory, etc., but also have special functional properties, such as excellent emulsification and high viscosity. They can be used as emulsifiers and thickeners in the food industry. Microalgae polysaccharides have rich development potential in functional foods, food additives and food flavorings.

[0003] The extraction technology of polysaccharides directly affects the yield of polysaccharides. Traditional extraction methods include using hot water, acid, and alkali as solvents. Among them, the current conventional polysaccharide extraction method mainly uses ultrasonic-assisted and microwave-assisted extraction. During the production process of this production process, the cavitation effect generated by ultrasonic and microwave with too low a frequency cannot break the cell wall and cannot achieve the purpose of enhanced polysaccharide extraction. Too high a frequency will cause the breakage of the nucleoside bonds of the polysaccharide and lead to the degradation of the polysaccharide. Summary of the invention

[0004] In view of this, the present invention provides a method for preparing Scenedesmus polysaccharide. The method for preparing Scenedesmus polysaccharide of the present invention has the advantages of simple operation steps, low production equipment requirements, environmental friendliness, low extraction cost, etc., and is suitable for large-scale industrial production.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing Scenedesmus polysaccharide, comprising the following steps:

[0007] Step 1, taking seaweed of the Chlorophyta, Chlorococcales, Scenedesmus family, and Scenedesmus genus, drying it, hydrolyzing it with a composite enzyme, centrifuging it to obtain a supernatant, and concentrating it to obtain a concentrated supernatant; the composite enzyme comprises cellulase and papain;

[0008] Step 2, taking the concentrated supernatant in step 1, ethanol precipitation, centrifuging to obtain the precipitate, and freeze-drying to obtain the Scenedesmus polysaccharide;

[0009] The preparation method does not include a defatting step.

[0010] In some embodiments of the invention, the algae comprises Scenedesmus DL12.

[0011] In some specific embodiments of the present invention, the enzyme activity ratio of the cellulase to the papain comprises 1:1.

[0012] In some specific embodiments of the present invention, the enzymatic hydrolysis further includes an enzyme inactivation step; the enzyme inactivation temperature includes 100° C.; and the enzyme inactivation time includes 5 min.

[0013] In some specific embodiments of the present invention, the centrifugal rotation speed includes 8000 r / min, and the centrifugal time includes 10 min.

[0014] In some specific embodiments of the present invention, step 2 further comprises a purification step using an anion exchange column DEAE-52 cellulose before obtaining the scenedesmus polysaccharide.

[0015] In some specific embodiments of the present invention, the purified eluent comprises a 0-0.5 mol / L NaCl solution.

[0016] In some specific embodiments of the present invention, the purified eluent comprises water, 0.1 mol / L, 0.3 mol / L or 0.5 mol / L NaCl solution.

[0017] In some specific embodiments of the present invention, the added amount of the complex enzyme in step 1 ranges from 1% to 6%.

[0018] In some specific embodiments of the present invention, the added amount of the complex enzyme in step 1 is 5.35%.

[0019] In some specific embodiments of the present invention, the pH value of the enzymatic hydrolysis in step 1 is 4-6.5; the temperature of the enzymatic hydrolysis is 40° C.-65° C.; and the time of the enzymatic hydrolysis is 30 min-180 min.

[0020] In some specific embodiments of the present invention, the pH value of the enzymatic hydrolysis in step 1 is 6.1; the temperature of the enzymatic hydrolysis is 53.1° C.; and the time of the enzymatic hydrolysis is 84.2 min.

[0021] In some specific embodiments of the present invention, the ethanol in step 2 includes anhydrous ethanol.

[0022] In some specific embodiments of the present invention, the volume ratio of the concentrated supernatant to the anhydrous ethanol is 1:4.

[0023] In some specific embodiments of the present invention, the temperature of the alcohol precipitation is 4° C.; the time of the alcohol precipitation is 24 h.

[0024] The present invention also provides the scenedesmus polysaccharide obtained by the preparation method.

[0025] The present invention also provides a scenedesmus polysaccharide, which is extracted and separated from seaweed of the Chlorophyta, Chlorococcales, Scenedesmus family, and Scenedesmus genus; the molecular weight of the scenedesmus polysaccharide includes 4.51 kDa to 55.8 kDa, and the monosaccharides constituting the scenedesmus polysaccharide include: fucose, aminogalactose, rhamnose, arabinose, glucosamine hydrochloride, galactose, glucose, xylose, mannose, and glucuronic acid.

[0026] In some specific embodiments of the present invention, the molecular weight of the scenedesmus polysaccharide comprises 55.8 kDa.

[0027] In some specific embodiments of the present invention, the molecular weight of the scenedesmus polysaccharide comprises 6.45 kDa.

[0028] In some specific embodiments of the present invention, the molecular weight of the scenedesmus polysaccharide comprises 5.44 kDa.

[0029] In some specific embodiments of the present invention, the molecular weight of the scenedesmus polysaccharide comprises 4.51 kDa.

[0030] In some specific embodiments of the present invention, the molecular weight of the scenedesmus polysaccharide comprises 9.03 kDa.

[0031] In some specific embodiments of the present invention, the monosaccharides constituting the scenedesmus polysaccharide include, by mole fraction, 0.13-0.56 parts of fucose, 0.07-0.15 parts of galactosamine, 0.62-2.36 parts of rhamnose, 0.28-1.2 parts of arabinose, 0.71-1.62 parts of glucosamine hydrochloride, 0.99-3.86 parts of galactose, 0.92-3.43 parts of glucose, 0.2-0.92 parts of xylose, 0.56-1.35 parts of mannose, and 0.03-0.66 parts of glucuronic acid.

[0032] In some specific embodiments of the present invention, the monosaccharides constituting the lycosylvinyl sulfate polysaccharide include, by mole fraction, 0.56 parts of fucose, 0.15 parts of galactosamine, 0.72 parts of rhamnose, 0.54 parts of arabinose, 0.71 parts of glucosamine hydrochloride, 2.57 parts of galactose, 3.43 parts of glucose, 0.2 parts of xylose, 1.08 parts of mannose, and 0.03 parts of glucuronic acid.

[0033] In some specific embodiments of the present invention, the monosaccharides constituting the lysine polysaccharide include, by mole fraction, 0.23 parts of fucose, 0.07 parts of galactosamine, 0.62 parts of rhamnose, 0.86 parts of arabinose, 0.79 parts of glucosamine hydrochloride, 3.51 parts of galactose, 1.61 parts of glucose, 0.92 parts of xylose, 1.35 parts of mannose, and 0.04 parts of glucuronic acid.

[0034] In some specific embodiments of the present invention, the monosaccharides constituting the lysine polysaccharide include, by mole fraction, 0.13 parts of fucose, 0.11 parts of galactosamine, 1.27 parts of rhamnose, 0.28 parts of arabinose, 1.62 parts of glucosamine hydrochloride, 3.86 parts of galactose, 0.92 parts of glucose, 0.27 parts of xylose, 1.36 parts of mannose, and 0.19 parts of glucuronic acid.

[0035] In some specific embodiments of the present invention, the monosaccharides constituting the lysine polysaccharide include, by mole fraction, 0.25 parts of fucose, 0.08 parts of galactosamine, 2.36 parts of rhamnose, 1.2 parts of arabinose, 1.16 parts of glucosamine hydrochloride, 0.99 parts of galactose, 1.99 parts of glucose, 0.76 parts of xylose, 0.56 parts of mannose, and 0.66 parts of glucuronic acid.

[0036] The present invention also provides the use of any of the following items in the preparation of functional foods, food additives or food flavorings:

[0037] (I) Scenedesmus polysaccharide obtained by the preparation method; and / or

[0038] (II), the Scenedesmus polysaccharide; and / or

[0039] (III) the Scenedesmus polysaccharide.

[0040] The present invention provides, including but not limited to, the following beneficial effects:

[0041] The present invention adopts a composite enzyme-assisted enzymatic hydrolysis process, uses the algae powder of the Scenedesmus as a raw material, and maintains a high extraction rate and purity of the Scenedesmus polysaccharide by controlling the process parameters such as the amount of different enzymes added and the enzymatic hydrolysis pH; while omitting the defatting step, by extending the enzymatic hydrolysis time, a better decolorization effect is also guaranteed. In addition, the present invention uses anion exchange column chromatography and gel column chromatography to separate the Scenedesmus crude polysaccharide (TBP) to obtain four components with high purity, namely TBP-1, TBP-2, TBP-3 and TBP-4. The preparation method of the Scenedesmus polysaccharide of the present invention has the advantages of simple operating steps, low production equipment requirements, environmental friendliness, low extraction cost, etc., and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0043] Figure 1 The composite enzyme extraction process of Scenedesmus polysaccharide is shown;

[0044] Figure 2The effect of pH value on the extraction rate of TBP is shown;

[0045] Figure 3 The effect of enzyme addition on TBP extraction rate is shown;

[0046] Figure 4 The effect of enzymatic hydrolysis temperature on TBP extraction rate is shown;

[0047] Figure 5 The effect of enzymatic hydrolysis time on TBP extraction rate is shown;

[0048] Figure 6 Contour plots and response surface plots under different single factor interactions are shown;

[0049] Figure 7 The elution curve of DEAE-52 of Scenedesmus crude polysaccharide is shown;

[0050] Figure 8 The elution curve of the first purified Scenedesmus polysaccharide on Sephadex G-200 gel column is shown;

[0051] Fig. 9 The UV full wavelength spectrum scan of Scenedesmus polysaccharide is shown;

[0052] Fig.10 The logarithmic standard curve of dextran molecular weight is shown;

[0053] Fig.11 The relative molecular weight distribution diagram of Scenedesmus polysaccharide is shown;

[0054] Fig.12 shows the ion chromatogram of 16 mixed monosaccharide standards;

[0055] Fig.13 shows the ion chromatograms of the purified polysaccharides of four types of Scenedesmus;

[0056] Fig.14 Shown is the inhibitory effect of Scenedesmus polysaccharide (12 mg / mL) on the growth of different strains. DETAILED DESCRIPTION

[0057] The present invention discloses a method for preparing a polysaccharide of Scenedesmus, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0059] The composite enzyme extraction utilizes the singleness of different enzymes. After the multiple enzymes are combined, they can efficiently destroy structures such as cell walls, improve the permeability of cell membranes and cell walls, and promote the dissolution and release of polysaccharides, proteins and other contents, thereby increasing the product extraction rate and improving the overall efficiency. The enzyme extraction conditions are mild, do not destroy the structure of active ingredients, and are environmentally friendly. The present invention adopts a composite enzyme-assisted enzymatic hydrolysis treatment process, uses the algae powder of the scenedesmus as raw material, and maintains a high extraction rate and purity of the scenedesmus polysaccharide by controlling the process parameters such as the amount of different enzymes added and the enzymatic hydrolysis pH; while omitting the defatting step, by extending the enzymatic hydrolysis time, a good decolorization effect is also guaranteed. The process has the advantages of simple operating steps, low production equipment requirements, environmental friendliness, and low extraction cost, and is suitable for large-scale industrial production.

[0060] The present invention provides a composite enzymatic extraction process for Scenedesmus polysaccharide, and the process flow chart is as follows: Figure 1 The algae liquid of Scenedesmus is centrifuged to obtain algae mud, which is freeze-dried to obtain Scenedesmus powder, which is dissolved after adding complex enzymes, and subjected to enzymatic hydrolysis-assisted treatment after adjusting the pH, and the enzymatic hydrolysis solution is heated, maintained at a slight boiling reflux extraction, filtered to obtain a supernatant, and the enzymatic hydrolysis-assisted extraction is repeated three times to obtain an enzymatic hydrolysis-assisted extraction supernatant, which is concentrated after deproteinization, and ethanol is added to the concentrate for precipitation, which is centrifuged after standing, and vacuum freeze-dried to obtain Scenedesmus polysaccharide.

[0061] The multiple enzymatic extraction process of Scenedesmus polysaccharide is specifically implemented in the following steps:

[0062] (1) Centrifuge the Scenedesmus algae liquid at 8000 (r / min) to obtain algae mud, and then freeze-dry to obtain Scenedesmus algae powder.

[0063] (2) Place the Scenedesmus algae powder in an oven and dry it at 60°C until constant weight.

[0064] (3) Add 5.35% of the complex enzyme (cellulase and papain mixed in a ratio of 1:1) to the algae powder, and add ultrapure water at a solid-liquid ratio of 1:25 (g / mL) to dissolve evenly.

[0065] (4) Adjust the solution pH to 6.1.

[0066] (5) Place the solution in a water bath at 53.1°C for 84.2 min.

[0067] (6) After the water bath, raise the temperature to 100 degrees Celsius and inactivate the enzyme for 5 minutes.

[0068] (7) Centrifuge the solution at 8000 (r / min) for 10 min, take the supernatant, and repeat the operation three times to collect the supernatant.

[0069] (8) Concentrate the supernatant using a rotary evaporator.

[0070] (9) The concentrated supernatant was mixed with anhydrous ethanol in a volume ratio of 1:4, and the mixture was precipitated in a refrigerator at 4.0°C for 24 h. The crude polysaccharide precipitate was obtained by centrifugation at 8000 (r / min) for 10 min.

[0071] (10) After freeze-drying, the polysaccharide powder of Scenedesmus salsa was obtained.

[0072] Beneficial effects:

[0073] (1) The present invention uses the algae powder of Scenedesmus DL12 preserved in this laboratory as the research object, and adopts the composite enzyme method to extract Scenedesmus polysaccharide (TBP). The enzymatic hydrolysis pH, the amount of composite enzyme (cellulase and papain 1:1 compound) added, the enzymatic hydrolysis temperature and the enzymatic hydrolysis time for the optimal level of extraction rate of Scenedesmus polysaccharide (TBP) are obtained.

[0074] (2) The present invention adopts response surface methodology to optimize the design and determines that the optimal conditions for the complex enzyme method for extracting crude polysaccharides (TBP) from Scenedesmus are 6.1 enzymatic hydrolysis pH, 5.35% complex enzyme dosage, 53.1°C enzymatic hydrolysis temperature and 84.2 min enzymatic hydrolysis time, and obtains an extraction rate of 8.94±0.13%.

[0075] (3) The present invention uses anion exchange column chromatography and gel column chromatography to separate the crude polysaccharide (TBP) of Scenedesmus to obtain four components with high purity, namely TBP-1, TBP-2, TBP-3 and TBP-4.

[0076] The cellulase of the present invention at 1000 U / g was purchased from Shanghai Boer Chemical Reagent Co., Ltd., and the papain at 3 U / mg was purchased from Shanghai Boer Chemical Reagent Co., Ltd.

[0077] Unless otherwise specified, the raw materials and reagents used in the method for preparing Scenedesmus polysaccharide provided by the present invention can be purchased from the market.

[0078] The present invention will be further described below in conjunction with embodiments:

[0079] Example Extraction of Scenedesmus polysaccharide by complex enzyme method

[0080] The specific implementation steps of the composite enzymatic extraction process of Scenedesmus polysaccharide are as follows:

[0081] (1) Centrifuge the Scenedesmus algae liquid at 8000 (r / min) to obtain algae mud, and then freeze-dry to obtain Scenedesmus algae powder.

[0082] (2) Place the Scenedesmus algae powder in an oven and dry it at 60°C until constant weight.

[0083] (3) Add 5.35% of the complex enzyme (cellulase and papain mixed in a ratio of 1:1) to the algae powder, and add ultrapure water at a solid-liquid ratio of 1:25 (g / mL) to dissolve evenly.

[0084] (4) Adjust the solution pH to 6.1.

[0085] (5) Place the solution in a 53.1°C water bath for 84.2 min.

[0086] (6) After the water bath, raise the temperature to 100 degrees Celsius and inactivate the enzyme for 5 minutes.

[0087] (7) Centrifuge the solution at 8000 (r / min) for 10 min, take the supernatant, and repeat the operation three times to collect the supernatant.

[0088] (8) Concentrate the supernatant using a rotary evaporator.

[0089] (9) The concentrated supernatant was mixed with anhydrous ethanol in a volume ratio of 1:4, and the mixture was precipitated in a refrigerator at 4.0°C for 24 h. The crude polysaccharide precipitate was obtained by centrifugation at 8000 (r / min) for 10 min.

[0090] (10) After freeze-drying, the polysaccharide powder of Scenedesmus salsa was obtained.

[0091] Effect Example 1 Effect of enzymatic hydrolysis pH on the extraction rate of Scenedesmus polysaccharide (TBP)

[0092] The acid and alkali in the environment affect the conformation of the enzyme protein, which affects the activity of the enzyme. The pH value in Example 1 (4) was changed to extract the polysaccharide of Scenedesmus. The effect of pH value on the extraction rate of TBP is shown in Figure 2 As shown, from Figure 1 It can be seen that when the pH value is between 4 and 6, the activity of the complex enzyme increases significantly with the increase of pH value. When the pH value is 6.0, which is close to the optimal pH of the enzyme, the TBP extraction rate reaches a maximum value of 8.06%. However, as the pH continues to increase, the activity of the complex enzyme gradually decreases, and the corresponding TBP extraction rate shows a downward trend.

[0093] Effect Example 2 Effect of the amount of compound enzyme added on the extraction rate of Scenedesmus crude polysaccharide (TBP)

[0094] The effect of the amount of compound enzyme added on the TBP extraction rate was shown in Figure 1 (3). Figure 3 As shown, from Figure 2It can be found that with the increase of enzyme concentration in the system, the substrate algae powder and enzyme can completely react and contact, and the speed of releasing the algae powder content also increases accordingly. When the proportion of compound enzyme added is 5%, the enzyme and substrate algae powder can be completely contacted and reacted, and the TBP extraction rate reaches a maximum of 8.43%. However, with the continuous increase of enzyme addition, the TBP extraction rate shows a downward trend.

[0095] Effect Example 3 Effect of enzymatic hydrolysis temperature on the extraction rate of Scenedesmus crude polysaccharide (TBP)

[0096] Temperature has a dual effect on enzymes. On the one hand, it affects the reaction rate between the enzyme and the substrate, and on the other hand, it affects the conformation of the enzyme protein. The enzymatic hydrolysis temperature in Example 1 (5) was changed to extract the polysaccharide from Scenedesmus. The effect of enzymatic hydrolysis temperature on the extraction rate of TBP is shown in Figure 4 As shown, from Figure 3 It can be seen that the enzymatic hydrolysis temperature is between 40℃ and 55℃. As the temperature rises, the activity of the composite enzyme also increases, the efficiency of enzymatic hydrolysis is greatly improved, and the TBP extraction rate is improved. However, when the temperature rises further, the TBP extraction rate shows a downward trend.

[0097] Effect Example 4 Effect of enzymatic hydrolysis time on the extraction rate of Scenedesmus crude polysaccharide (TBP)

[0098] If the enzymatic hydrolysis time is too short, the enzyme and substrate will not react completely, and less content will be precipitated. If the enzymatic hydrolysis time is too long, some polysaccharides will be degraded by the enzyme, affecting the extraction rate. The enzymatic hydrolysis time in Example 1 (5) was changed to extract the polysaccharide from Scenedesmus. The effect of enzymatic hydrolysis time on the extraction rate of TBP is shown in Figure 2. Figure 5 As shown, Figure 4 It can be seen that when the enzymatic hydrolysis time is between 30 and 80 min, the time for the enzyme and substrate to react becomes more and more sufficient, and the extraction rate of TBP increases. However, as the enzymatic hydrolysis time continues to increase, the extraction rate of TBP shows a downward trend.

[0099] Effect Example 5 Optimization of Scenedesmus polysaccharide extraction conditions by response surface method

[0100] Select four factors and three levels for response surface optimization. Figure 6 , and 29 combinations of conditions were obtained as shown in Table 1. The optimal process obtained according to the constructed response surface model is as follows: 6.1 enzymatic pH, 5.35% compound enzyme addition, 53.1℃ enzymatic temperature and 84.2 min enzymatic time, and the ideal extraction rate of TBP is 8.94±0.13%. This parameter was experimentally verified, and the TBP extraction rate was 8.86±0.13%, which was less deviated from the ideal predicted value and within the error range, indicating that the obtained model matches the results and has certain feasibility.

[0101] Table 1 Response surface experiment design and results

[0102]

[0103] Effect Example 6: Concentration and preliminary purification of polysaccharide solution using anion exchange column chromatography

[0104] Anion exchange column chromatography utilizes the ionic interaction between the stationary phase and the sample solution and mobile phase to achieve the adsorption and separation of the target substance. Figure 7 As shown, after elution with different concentrations of NaCl, four polysaccharide components were obtained, namely TBP-1, TBP-2, TBP-3 and TBP-4.

[0105] Gel column chromatography separation uses the gel molecular sieving effect (three-dimensional porous network structure) to separate polysaccharides, and separates different polysaccharides according to the size and shape of the polysaccharide molecules and the speed of movement in the chromatographic column. The TBP-1, TBP-2, TBP-3 and TBP-4 samples obtained by anion exchange column chromatography were further purified by Sephadex G-200 gel column. The elution curve is shown in Figure 8 As shown, at this time

[0106] The purity of the polysaccharide component of Scenedesmus is relatively high.

[0107] The specific steps of anion exchange column chromatography are as follows:

[0108] Pretreatment of DEAE-52 cellulose: Take 100 g of DEAE-52 cellulose, add ultrapure water and soak for 4 h, filter and dissolve, repeat three times and then perform swelling treatment (12 h).

[0109] Filling column: Add sufficient ultrapure water to the pretreated DEAE-52 cellulose, stir evenly, and transfer to a D3.6×40.0 cm chromatography column.

[0110] Column balance: Open the upper and lower water inlets and outlets of the chromatography column, keep the upper and lower flow rates consistent, add 5 times the volume of ultrapure water of the chromatography column and balance for 12 hours to fully compact the filler and improve the column efficiency.

[0111] Sample loading: Take 100 mg of the crude polysaccharide of Scenedesmus after protein removal, dissolve it in ultrapure water to a concentration of 8 mg / mL, and slowly add the crude polysaccharide solution of Scenedesmus to the surface of the chromatography column with a rubber-tipped dropper.

[0112] Elution: Open the upper and lower water inlets and outlets of the chromatography column, maintain the flow rate at 1.5 mL / min, and use ultrapure water, 0.1 mol / L, 0.3 mol / L and 0.5 mol / L NaCl solutions to elute in sequence to achieve component separation.

[0113] Collection and monitoring: 60 tubes were collected for each elution gradient, each tube was 7.5 mL, and the phenol-sulfuric acid method was used to detect each tube, and the elution curve of the scenedesmus polysaccharide was drawn. The eluates with concentrated polysaccharide content were combined.

[0114] Concentration and dialysis: The combined eluate was placed in a dialysis bag (3000Da) and dialyzed in ultrapure water for 48 h, with the ultrapure water replaced every 16 h. After concentration, freeze-drying was performed to obtain the first round of purified polysaccharides of Scenedesmus.

[0115] The specific steps of gel column chromatography are as follows:

[0116] Pretreatment of Sephadex G-200: Take 50 g of Sephadex G-200, add ultrapure water and soak for 4 h, filter and dissolve, and repeat ultrasonication for 30 min three times to complete the pretreatment.

[0117] Packing column: Add 4 times volume of ultrapure water to the pretreated Sephadex G-200, stir evenly with ultrasound, and transfer to a D4.5×50.0 cm chromatography column using a glass rod.

[0118] Column balance: Open the upper and lower water inlets and outlets of the chromatography column, keep the upper and lower flow rates consistent, add 5 times the volume of ultrapure water of the chromatography column and balance for 12 hours to fully compact the filler and improve the column efficiency.

[0119] Sample loading: Take 100 mg of the polysaccharide from Scenedesmus eluted with DEAE-52, dissolve it in ultrapure water to a concentration of 8 mg / mL, and slowly add the crude polysaccharide solution of Scenedesmus to the surface of the chromatography column using a rubber-tipped dropper.

[0120] Elution: Open the upper and lower water inlets and outlets of the chromatography column, maintain the flow rate at 1.5 mL / min, and use ultrapure water to sequentially elute the scenedesmus polysaccharide eluted by DEAE-52.

[0121] Collection and monitoring: 60 tubes were collected for each elution gradient, each tube was 7.5 mL, and the phenol-sulfuric acid method was used to detect each tube, and the two-round elution curve of the Scenedesmus polysaccharide was drawn, and the eluates with concentrated polysaccharide content were combined.

[0122] Concentration and dialysis: The combined eluate was placed in a dialysis bag (3000Da) and dialyzed in ultrapure water for 48 h, with the ultrapure water replaced every 16 h. After concentration and freeze-drying, four purified Scenedesmus polysaccharides were obtained, which were named TBP-1, TBP-2, TBP-3, and TBP-4.

[0123] Effect Example 7 Analysis of Chemical Properties of Polysaccharide Components of Scenedesmus

[0124] UV full-band scanning can analyze the chemical properties such as material structure, purity, concentration, etc. by detecting the absorption or emission characteristics of chemical substances in the ultraviolet spectrum.

[0125] UV scanning results are as follows Fig. 9 As shown in the figure, TBP, TBP-1, TBP-2, TBP-3 and TBP-4 all have strong absorption peaks near the wavelength of 200 nm, indicating that the main component of the extracted water-soluble polymer is polysaccharide. TBP has a strong absorption peak at a wavelength of 280 nm, indicating that the crude polysaccharide contains impurities such as proteins and polypeptides. TBP-1, TBP-2, TBP-3 and TBP-4 have particularly weak absorption at a wavelength of 280 nm, indicating that the purified polysaccharide component has a high purity.

[0126] Effect Example 8 Relative molecular weight and monosaccharide composition of each polysaccharide component of Scenedesmus

[0127] Firstly, high performance gel chromatography was used to determine the relative molecular weight of each polysaccharide component of Scenedesmus, and then ion chromatography was used to determine the monosaccharide composition of each polysaccharide component of Scenedesmus.

[0128] According to the logarithm of molecular weight (lgMr) and peak time of standard dextran, the standard curve equation is y=-3.1656x+37.809 (R²=0.9797), and the relative molecular weights of TBP, TBP-1, TBP-2, TBP-3 and TBP-4 are 55.8 kDa, 6.45 kDa, 5.44 kDa, 4.51 kDa and 9.03 kDa respectively. Fig.10 , Fig.11 The elution peaks of TBP-1, TBP-2, TBP-3 and TBP-4 shown have single symmetry, indicating that TBP-1, TBP-2, TBP-3 and TBP-4 are polysaccharides with high purity and uniformity.

[0129] Fig.12 This is the ion chromatogram of 16 kinds of monosaccharide mixed standard. The monosaccharide composition and content of the polysaccharide can be determined based on the monosaccharide peak time and peak area. The peak at 2.0 min is the peak of sodium hydroxide, and the peak at 40.5 min is the peak of sodium acetate, which are solvent peaks. Fig.13 As shown in Table 3, TBP-1, TBP-2, TBP-3 and TBP-4 are composed of 10 monosaccharides, namely fucose, glucosamine hydrochloride, rhamnose, mannose, arabinose, galactose, glucose, xylose, ribose and glucuronic acid. As shown in Table 4, the main component of TBP-1 is glucose, the main components of TBP-2 and TBP-3 are galactose, and the main component of TBP-4 is rhamnose.

[0130] Table 2 Monosaccharide peak timetable of four polysaccharides

[0131]

[0132] Table 3 Monosaccharide molar ratio of four polysaccharides

[0133]

[0134] Comparative Example

[0135] Comparative Example 1 (ultrasonic extraction method):

[0136] The dried algae powder was dissolved in an appropriate amount of ultrapure water to a concentration of 5%, extracted under ultrasound at 60°C for 1 h, centrifuged (8000 rpm, 5 min) to collect the supernatant, and repeated three times to pool the supernatant, which was the crude extract of microalgae polysaccharides. The crude extract was concentrated and 4 times the volume of anhydrous ethanol was added, precipitated at 4°C for 12 h, centrifuged (8000 rpm, 5 min) to collect the precipitate, and freeze-dried to obtain crude microalgae polysaccharides.

[0137] Comparative Example 2 (hot water method):

[0138] The dried algae powder was dissolved in an appropriate amount of ultrapure water to a concentration of 5%, extracted at 95°C hot water for 2 h, centrifuged (8000 rpm, 5 min) to collect the supernatant, and repeated three times to pool the supernatant, which was the crude extract of microalgae polysaccharides. The crude extract was concentrated and 4 times the volume of anhydrous ethanol was added, precipitated at 4°C for 12 h, centrifuged (8000 rpm, 5 min) to collect the precipitate, and freeze-dried to obtain crude microalgae polysaccharides.

[0139] Comparative Example 3 (freeze-thaw method):

[0140] Weigh an appropriate amount of algae powder, freeze-thaw and grind it three times, add ultrapure water to make its concentration 5%, centrifuge (8000 rpm, 5 min) to collect the supernatant, repeat three times to collect the supernatant, this is the crude extract of microalgae polysaccharides, concentrate the crude extract and add 4 times the volume of anhydrous ethanol, precipitate at 4°C for 12h, centrifuge (8000 rpm, 5 min) to collect the precipitate, and freeze-dry to obtain crude microalgae polysaccharides.

[0141] Comparative Example 4 (single cellulase method):

[0142] The dried algae powder was dissolved in an appropriate amount of ultrapure water to a concentration of 5%, and 3% concentration of cellulase was added. The enzyme was hydrolyzed in a constant temperature water bath at 55°C for 90 min, the temperature was raised to 100°C, the enzyme was inactivated for 5 min, and the supernatant was collected by centrifugation (8000 rpm, 5 min). The supernatant was collected three times and the crude extract was concentrated. After adding 4 times the volume of anhydrous ethanol, the crude extract was precipitated at 4°C for 12 h, and the precipitate was collected by centrifugation (8000 rpm, 5 min). The crude microalgae polysaccharide was obtained after freeze-drying.

[0143] Comparative Example 5 (single papain method):

[0144] The cellulase in Comparative Example 4 was replaced by papain, and the remaining operations were the same as those in Comparative Example 4.

[0145] The extraction rates of polysaccharides from S. salsa using different traditional methods are shown in Table 4.

[0146] Table 4 Extraction rate of traditional method

[0147]

[0148] The extraction technology of polysaccharides directly affects the yield of polysaccharides. Currently, the most common extraction methods are ultrasonic method, hydrothermal method, single enzyme method, etc. Ultrasonic extraction method, hot water method, freeze-thaw method, and single enzyme method (cellulase, papain) were used to extract polysaccharides from Scenedesmus. The results are shown in Table 4. The advantages of various methods are different, and the overall polysaccharide extraction rate is low. Alkaline extraction or hot water extraction has black or green pigments attached, and the enzyme extract is light yellow.

[0149] Effect Example 9 Antibacterial Activity of Scenedesmus Polysaccharide

[0150] Microalgae polysaccharides have an antibacterial effect by inhibiting viral interactions or the positive charge on the cell surface, thereby inhibiting the attachment of viruses to host cells. Fig.14 The results show the inhibitory effect of Scenedesmus polysaccharide on 4 strains of tested bacteria. From the size of the inhibition zone, it can be seen that Scenedesmus polysaccharide has a better antibacterial effect on two Gram-positive bacteria: Bacillus subtilis and Staphylococcus aureus, and a weaker antibacterial effect on two Gram-negative bacteria: Escherichia coli and Pseudomonas aeruginosa.

[0151] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Scenedesmus polysaccharide, characterized in that: The steps include: Step 1, taking seaweed of the Chlorophyta, Chlorococcales, Scenedesmus family, and Scenedesmus genus, drying it, hydrolyzing it with a composite enzyme, centrifuging it to obtain a supernatant, and concentrating it to obtain a concentrated supernatant; the composite enzyme comprises cellulase and papain; Step 2, taking the concentrated supernatant in step 1, ethanol precipitation, centrifuging to obtain the precipitate, and freeze-drying to obtain the Scenedesmus polysaccharide; The preparation method does not include a defatting step.

2. The preparation method according to claim 1, characterized in that The enzymatic activity ratio of the cellulase to the papain is 1:

1.

3. The preparation method according to claim 1 or 2, characterized in that: The enzymolysis step also includes an enzyme inactivation step after the enzymolysis; the enzyme inactivation temperature includes 100° C.; and the enzyme inactivation time includes 5 minutes.

4. The preparation method according to any one of claims 1 to 3, characterized in that: Step 2 also includes a step of purifying the polysaccharide using an anion exchange column DEAE-52 cellulose before obtaining the polysaccharide.

5. The preparation method according to claim 4, characterized in that: The purified eluent includes a 0-0.5 mol / L NaCl solution.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The addition amount of the complex enzyme in step 1 ranges from 1% to 6%; The pH value of the enzymatic hydrolysis in step 1 is 4 to 6.5; the temperature of the enzymatic hydrolysis is 40° C. to 65° C.; and the time of the enzymatic hydrolysis is 30 min to 180 min.

7. Scenedesmus polysaccharide obtained by the preparation method according to any one of claims 1 to 6.

8. Scenedesmus polysaccharide, characterized in that The scenedesmus polysaccharide is extracted and separated from seaweed of the Chlorophyta, Chlorococcales, Scenedesmus family, and Scenedesmus genus; the molecular weight of the scenedesmus polysaccharide includes 4.51 kDa to 55.8 kDa, and the monosaccharides constituting the scenedesmus polysaccharide include: fucose, aminogalactose, rhamnose, arabinose, glucosamine hydrochloride, galactose, glucose, xylose, mannose, and glucuronic acid.

9. The scenedesmus polysaccharide according to claim 8, characterized in that The monosaccharides constituting the scenedesmus polysaccharide include, by mole fraction, 0.13 to 0.56 parts of fucose, 0.07 to 0.15 parts of galactosamine, 0.62 to 2.36 parts of rhamnose, 0.28 to 1.2 parts of arabinose, 0.71 to 1.62 parts of glucosamine hydrochloride, 0.99 to 3.86 parts of galactose, 0.92 to 3.43 parts of glucose, 0.2 to 0.92 parts of xylose, 0.56 to 1.35 parts of mannose, and 0.03 to 0.66 parts of glucuronic acid.

10. Use of any of the following in the preparation of functional foods, food additives or food flavorings: (I) Scenedesmus polysaccharide obtained by the preparation method according to any one of claims 1 to 6; and / or (II), the Scenedesmus polysaccharide according to claim 7; and / or (III) The Scenedesmus polysaccharide according to claim 8 or 9.

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