Method for ultrasonic compound enzyme-assisted extraction of North American plum-leaf crab polysaccharide

Abstract

The invention belongs to the technical field of natural product extraction and preparation, and particularly relates to a method for ultrasonic compound enzyme assisted extraction of North America crabapple polysaccharide. According to the method, North America crabapple is taken as a raw material, and the North America crabapple polysaccharide is extracted and prepared by optimizing process conditions of an ultrasonic compound enzyme-assisted extraction method by utilizing a response surface method. Compared with a conventional hot water extraction method, the polysaccharide extraction rate is remarkably improved. An infrared spectrum and a scanning electron microscope are used for determining the structure of the malus prunifolia fruit polysaccharide, and the result shows that the malus prunifolia fruit polysaccharide contains a pyranoid ring, a beta-glucosidic bond and an alpha-glucosidic bond which are related to various biological metabolism. And finally, determining superoxide anion removal capacity and cholesterol adsorption capacity of the North American malus prunifolia fruit polysaccharide to find that the North American malus prunifolia fruit polysaccharide has good antioxidant activity and blood fat reducing activity. Therefore, the North America plum-leaf crab polysaccharide can be widely applied to the fields of functional foods, nutritional supplements, natural plant antioxidants and the like in the future, so that the comprehensive utilization value of the North America plum-leaf crab is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and preparation of natural products, and specifically relates to a method for ultrasonic combined enzyme-assisted extraction of polysaccharides from Malus micromalus fruits. Background Art

[0002] Malus micromalus is a prominent species among many kinds of Chinese flowering crabapples. It was first introduced from Chinese flowering crabapples to North America and then introduced back to China as a new variety after being improved by North American plant scientists. After more than 200 years of collection and breeding work, many new varieties of Malus micromalus with high ornamental value have been cultivated. Due to its excellent leaf color, flower color and fruit color performance, Malus micromalus has great development potential in China.

[0003] Malus micromalus fruits not only have ornamental value, but also have very rich nutritional value. However, so far, the application of Malus micromalus fruits in the food field is very few. Its main research directions are in landscape art, botany and other aspects, and almost no scholars have studied Malus micromalus fruits with rich nutritional value. At the same time, due to the wide planting range of Malus micromalus in China, the fruits produced are often wasted in large quantities. Therefore, the present invention explores the value of Malus micromalus fruits in functional foods, gives full play to their intensive processing value, and creates economic benefits.

[0004] Plant polysaccharides refer to macromolecular compounds synthesized by plant cells and composed of multiple sugar molecules linked by glycosidic bonds. They are widely present in plants and serve as energy storage substances, structural materials or participate in the plant's defense mechanism. Plant polysaccharides have good antioxidant, hypoglycemic and hypolipidemic, anti-inflammatory, radiation protection, anticoagulant, antiplatelet and other effects.

[0005] At present, traditional extraction methods such as hot water extraction method have low extraction efficiency and low polysaccharide extraction rate; acid-base extraction method has a destructive effect on the polysaccharide structure and low extraction rate; single enzyme extraction method, ultrasonic extraction method, microwave extraction method, etc. all have lower extraction efficiency compared with the combination of multiple methods. Summary of the Invention

[0006] To solve the above problems, the present invention provides a preparation method for efficient extraction of polysaccharides from Malus micromalus fruits, which has the characteristics of improving the extraction rate of polysaccharides from Malus micromalus fruits without destroying the polysaccharide molecular structure.

[0007] The technical solution adopted by the present invention is:

[0008] A method for ultrasonic combined enzyme-assisted extraction of polysaccharides from Malus micromalus fruits, comprising the following steps:

[0009] 1) Pretreatment of raw materials: After the fresh Malus micromalus fruits are washed and the branches and leaves are trimmed, they are freeze-dried by a freeze dryer for 30 h until they become powdery, and then stored in a -80°C refrigerator for later use;

[0010] 2) Extraction: The extraction is carried out according to the technical route of Malus micromalus Makino jelly dry powder → dissolution → enzymatic hydrolysis → ultrasonic treatment → centrifugation → protein removal → decolorization → concentration → ethanol precipitation → freeze-drying → Malus micromalus Makino fruit polysaccharide.

[0011] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the dissolution is carried out by dissolving Malus micromalus Makino jelly dry powder and water at a solid-liquid ratio of 1 g: 35 mL.

[0012] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the enzymatic hydrolysis step is as follows: adding a composite enzyme at a mass-volume concentration of 3-5% to the solid-liquid mixture, carrying out enzymatic hydrolysis at an enzymatic hydrolysis temperature of 45-55 °C for 1 h, and inactivating at 95 °C for 5 min after enzymatic hydrolysis; the composite enzyme is: papain: cellulase: pectinase = 2:1:1 by mass ratio.

[0013] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the ultrasonic conditions are: ultrasonic power is 240-320 W, ultrasonic temperature is 45-55 °C, and ultrasonic time is 50 min.

[0014] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the centrifugation conditions are: centrifuging at 8000 r / min for 15 min and taking the supernatant.

[0015] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the protein removal step is: adding trichloroacetic acid at a mass-volume concentration of 5%, stirring with a stirrer for 1 h, then centrifuging at 8000 r / min for 10 min, and collecting the supernatant.

[0016] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the decolorization step is: adding activated carbon powder at a mass-volume concentration of 1.5%, stirring for 30 min and then centrifuging at 8000 r / min for 10 min, and taking the supernatant.

[0017] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the concentration step is: concentrating the volume to 1 / 5 using a rotary evaporator.

[0018] Furthermore, in the above method for ultrasonic composite enzyme-assisted extraction of Malus micromalus Makino fruit polysaccharide, in step 2), the ethanol precipitation step is: adding absolute ethanol, with the volume ratio of the concentrated solution to absolute ethanol being 1:4, carrying out ethanol precipitation for 12 h, centrifuging at 8000 r / min for 10 min, and taking the precipitate.

[0019] Further, in the method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits, in step 2), the freeze-drying step is as follows: After adding water, place it in a petri dish and freeze at -18°C for 12 h, then put it into a freeze dryer for freeze-drying for 30 h to obtain polysaccharides from Malus micromalus fruits.

[0020] The beneficial effects of the present invention are as follows: Compared with the traditional hot water extraction method, the ultrasonic composite enzyme-assisted extraction method significantly improves the extraction rate of polysaccharides from Malus micromalus fruits. Moreover, through infrared spectroscopy and scanning electron microscopy determination, it is confirmed that this method does not damage the structure of polysaccharides from Malus micromalus fruits. Finally, by measuring the biological activity of polysaccharides from Malus micromalus fruits, it is found that they have good antioxidant activity and lipid-lowering activity. In the future, it can be widely applied in the fields of functional foods, nutritional supplements, natural plant antioxidants, etc., so as to effectively improve the comprehensive utilization value of Malus micromalus fruits. Description of the Drawings

[0021] Figure 1 It is an analysis diagram of the standard curve of anhydrous glucose.

[0022] Figure 2 It is an analysis diagram of the interaction between enzymatic hydrolysis temperature and the addition amount of composite enzyme.

[0023] Figure 3 It is an analysis diagram of the interaction between enzymatic hydrolysis temperature and ultrasonic power.

[0024] Figure 4 It is an analysis diagram of the interaction between enzymatic hydrolysis temperature and ultrasonic temperature.

[0025] Figure 5 It is an analysis diagram of the interaction between the addition amount of composite enzyme and ultrasonic power.

[0026] Figure 6 It is an analysis diagram of the interaction between the addition amount of composite enzyme and ultrasonic temperature.

[0027] Figure 7 It is an analysis diagram of the interaction between ultrasonic power and ultrasonic temperature.

[0028] Figure 8 It is an analysis diagram of the Fourier infrared spectrum of polysaccharides from Malus micromalus fruits.

[0029] Figure 9 It is an analysis diagram of the scanning electron microscopy of polysaccharides from Malus micromalus fruits at different magnification.

[0030] Figure 10 It is an analysis diagram of the scavenging effects of polysaccharides from Malus micromalus fruits and ascorbic acid on superoxide anions.

[0031] Figure 11 It is an analysis diagram of the standard curve of cholesterol solution.

[0032] Figure 12Analysis chart of the adsorption capacity of Malus micromalus Makino polysaccharide and orlistat to cholesterol. Detailed implementation manners

[0033] Multiple embodiments of the present invention will be described in detail below. Unless otherwise specified, the methods used in the implementation process are conventional methods if not otherwise stated, and the reagents used are common reagents on the market or reagents prepared according to conventional methods. It should be noted that this description should not be regarded as a limitation of the present invention, but a more detailed elaboration of certain aspects, characteristics and implementation schemes thereof.

[0034] The drawings of the present invention are intended to provide those skilled in the art with a deeper understanding of the present invention. These drawings show schematic embodiments of the present invention and related descriptions, aiming to help explain the core concepts and applications of the present invention. However, these embodiments and drawings do not constitute a strict limitation of the present invention, but provide references for explaining and elaborating the features and advantages of the present invention. Therefore, those skilled in the art should understand that the actual scope of the present invention may exceed the content shown in these examples.

[0035] Example 1 Drawing of glucose standard curve

[0036] First, prepare a standard glucose solution of 0.1 mg / mL. Use a 1 mL pipette to accurately transfer 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of the standard glucose solution into 25 mL colorimetric tubes in 6 gradient steps, and make up to 1.0 mL with water. Add 1.0 mL of 5% phenol solution (prepared freshly), shake well, then add 5.0 mL of concentrated sulfuric acid quickly and shake well, and let stand for 30 min. Finally, measure the absorbance at a wavelength of 490 nm. Take the glucose concentration as the abscissa and the absorbance as the ordinate, and draw a standard curve.

[0037] According to Figure 1 As shown, through the phenol-sulfuric acid method, the regression equation of the standard curve of the anhydrous glucose solution measured by an ultraviolet-visible spectrophotometer is y = 9.69762x + 0.07906, and the goodness of fit R 2 = 0.998, indicating that within a certain concentration range, the linear correlation between the absorbance and the concentration of the anhydrous glucose solution is good, and the total sugar content of the measured sample can be calculated according to this standard curve.

[0038] Example 2 Optimal process for ultrasonic combined enzyme-assisted extraction of polysaccharide

[0039] Pick fresh fruits of Malus × micromalus cv. 'Jinyan Huahong' (a variety), soak and wash them in clean water, dry the excess water on the surface, and then put them into a freeze dryer for freeze-drying at -80°C for 30 h. After taking them out, use a mortar to grind them into powder, which is the freeze-dried powder of Malus × micromalus jelly. Take 1 g of the freeze-dried powder of Malus × micromalus jelly, dissolve it in water according to the solid-liquid ratio of 1:35 (g / mL). The addition amount of the composite enzyme (mass ratio, papain: cellulase: pectinase = 2:1:1) is 3-5% (mass-volume concentration) of the freeze-dried powder liquid. The enzymatic hydrolysis temperature is 45-55°C, and the enzymatic hydrolysis is carried out for 1 h. After enzymatic hydrolysis, inactivate at 95°C for 5 min. Then, carry out ultrasonic extraction for 50 min at an ultrasonic power of 240-320 W and an ultrasonic temperature of 45-55°C to continue extracting polysaccharides. After the extraction is completed, centrifuge at 8000 r / min for 15 min, take the supernatant, and then add trichloroacetic acid to deproteinize according to the mass-volume concentration of 5%. After stirring with a stirrer for 1 h, centrifuge at 8000 r / min for 10 min, and collect the supernatant. Add activated carbon powder to the supernatant for decolorization according to the mass-volume concentration of 1.5%. After stirring for 30 min, centrifuge at 8000 r / min for 10 min, and the supernatant is the polysaccharide extraction solution. Take 1 mL of the polysaccharide extraction solution and dilute it to 100 mL with deionized water to obtain a sample solution. Take 0.5 mL of the sample solution, add 0.5 mL of deionized water, then add 1.0 mL of 5% phenol solution (prepared freshly), shake well, then add 5.0 mL of concentrated sulfuric acid quickly and shake well. After standing for 30 min, measure the absorbance at a wavelength of 490 nm. Set a control group using the hot water extraction method (solid-liquid ratio 1 g:35 mL, 80°C, 2 h). Calculate the polysaccharide extraction rate according to the following formula:

[0040]

[0041] Where ω is the polysaccharide extraction rate of Malus × micromalus fruits, c is the polysaccharide concentration (mg / mL), k is the dilution factor, v is the volume of the sample solution, m0 is the weight of the freeze-dried powder of Malus × micromalus jelly, and 0.9 is the conversion correction coefficient of the glucose standard curve.

[0042] In the response surface experiment of the present invention, four factors, namely enzymatic hydrolysis temperature, composite enzyme concentration, ultrasonic power, and ultrasonic temperature, will be selected. Three levels will be selected based on the single-factor results, and a Box-Behnken four-factor three-level response surface experimental design and analysis will be carried out.

[0043] Through the response surface method, an experimental design for the ultrasonic composite enzyme-assisted extraction method of Malus × micromalus fruit polysaccharides is carried out. Taking A enzymatic hydrolysis temperature, B composite enzyme addition amount, C ultrasonic power, and D ultrasonic temperature as four single factors for analysis, the response surface experimental design table and experimental results are shown in Table 1 below. Using Design-Expert 13 software for Box-Behnken experiments, the following quadratic polynomial regression equation is obtained:

[0044] Y = 41.96 + 1.86×A + 0.6542×B - 1.31×C + 0.78×D - 0.08×AB - 0.95×AC - 0.2925×AD + 0.025×BC - 0.2125×BD - 0.04×CD - 2.16×A 2 - 2.23×B 2 - 2.57×C 2 - 2.25×D 2

[0045] Among them, A is the enzymatic hydrolysis temperature, B is the addition amount of complex enzyme, C is the ultrasonic power, D is the ultrasonic temperature, and Y is the extraction rate. From the coefficients of the linear terms, it can be concluded that the order of influence on the extraction rate is: enzymatic hydrolysis temperature > ultrasonic power > ultrasonic temperature > addition amount of complex enzyme.

[0046] Table 1 Design and results of response surface experiments

[0047]

[0048] Table 2 Data regression analysis table

[0049]

[0050]

[0051] According to Table 2, for the regression equation, R 2 = 0.9815, and the adjusted R 2 = 0.9631, indicating that the fitting effect of the entire model is good and has high statistical significance. The P value of the model < 0.0001 indicates extremely significant, and the P value of the lack-of-fit term > 0.05 indicates not significant. The P values of the linear terms A, B, C, D and the quadratic terms AB, 2 B 2 C 2 D 2 are all less than 0.05. Therefore, this model can be used for analysis and prediction.

[0052] The 3D stereograms and contour plots of the interaction effects among A (enzymatic hydrolysis temperature), B (addition amount of complex enzyme), C (ultrasonic power), and D (ultrasonic temperature) are as Figures 2 - 7 . Through Figures 2 - 7It can be concluded that the interactions between enzymolysis temperature and complex enzyme addition amount, between enzymolysis temperature and ultrasonic power, between enzymolysis temperature and ultrasonic temperature, between complex enzyme addition amount and ultrasonic power, between complex enzyme addition amount and ultrasonic temperature, and between ultrasonic power and ultrasonic temperature have a significant effect on the extraction rate. The effect of enzymolysis temperature is more obvious than that of complex enzyme addition amount. The effect of enzymolysis temperature is more obvious than that of ultrasonic power. The effect of enzymolysis temperature is more obvious than that of ultrasonic temperature. The effect of ultrasonic power is more obvious than that of complex enzyme addition amount. The effect of ultrasonic temperature is more obvious than that of complex enzyme addition amount. The effect of ultrasonic power is more obvious than that of ultrasonic temperature. The overall order of influence degree is: enzymolysis temperature > ultrasonic power > ultrasonic temperature > complex enzyme addition amount.

[0053] Finally, through the analysis of the response surface regression equation, the optimal extraction process level of the ultrasonic complex enzyme-assisted extraction method for Malus micromalus fruit polysaccharide was determined as follows: enzymolysis temperature 52.12 °C, complex enzyme addition amount 4.13%, ultrasonic power 269.53 W, ultrasonic temperature 50.70 °C, and the predicted extraction rate was 42.62%. Further, corresponding verification experiments were carried out on the stability and reliability of the optimal determination conditions. Considering the convenience and feasibility of actual operation, the verification conditions were appropriately adjusted to: enzymolysis temperature 52 °C, complex enzyme addition amount 4.13%, ultrasonic power 270 W, ultrasonic temperature 51 °C. Under this condition, the extraction rate was 42.42 ± 0.38%. The error between the actual measured value and the predicted value was small, indicating that the optimized extraction process of Malus micromalus fruit polysaccharide was accurate and reliable.

[0054] The extraction rate by the hot water extraction method (material-liquid ratio 1:35, 80 °C, 2 h) was 23.75 ± 0.44%. The extraction rate of the enzyme-assisted extraction method (material-liquid ratio 1 g:35 mL, 4% addition amount, 45 °C, 1 h), without ultrasonic treatment, was 29.85 ± 0.54%. It can be seen that the ultrasonic complex enzyme-assisted extraction method significantly improved the extraction rate of Malus micromalus fruit polysaccharide, providing a reference value for the efficient extraction of Malus micromalus fruit polysaccharide in the future.

[0055] Example 3 Preparation of Polysaccharide

[0056] Take 10g of freeze-dried powder of "Jinyanhuahong" (a variety) North American crabapple fruit, add water to dissolve it at a material-liquid ratio of 1:35 (g / mL), add the compound enzyme (mass ratio, papain: cellulase: pectinase = 2:1:1) in an amount of 4.13% (mass volume concentration) of the freeze-dried powder liquid, the enzymatic hydrolysis temperature is 52°C, the enzymatic hydrolysis is 1h, and the enzymatic hydrolysis is inactivated at 95°C for 5min. Then, ultrasonic power is 270W and the ultrasonic temperature is 51°C for 50min, and the polysaccharide is further extracted. After the extraction is completed, centrifuge at 8000r / min for 15min, take the supernatant, and then add trichloroacetic acid at a mass volume concentration of 5% to deproteinize. After stirring with a stirrer for 1h, centrifuge at 8000r / min for 10min, and collect the supernatant. Add activated carbon powder to the supernatant at a mass volume concentration of 1.5% for decolorization, centrifuge at 8000r / min for 10min after stirring for 30min, and take the supernatant as the polysaccharide extract. The polysaccharide extract was concentrated to 1 / 5 using a rotary evaporator, and then anhydrous ethanol was added at a volume ratio of 1:4, and the precipitate was centrifuged at 8000r / min for 10min. After adding water, the precipitate was placed in a petri dish and frozen in a -18℃ refrigerator for 12h, and then placed in a freeze dryer at -80℃ for 30h to obtain the "Golden Flower Red" North American crabapple fruit polysaccharide. Finally, a grinding dish was used to grind it into fine particles of North American crabapple fruit polysaccharide and put it into a sealed bag for use.

[0057] Example 4 Infrared Spectroscopy Determination of Polysaccharides

[0058] Weigh the Malus polysaccharide and potassium bromide powder in a mass ratio of 1:100, mix and grind thoroughly, dry and store, and simmer at 4000-400cm -1 Wavelength scanning analysis.

[0059] Infrared spectroscopy can be used to study the functional groups and isomers contained in polysaccharides. Figure 8 It can be seen that the polysaccharide of North American crabapple fruit is at 3373cm -1 There is a strong OH bond stretching vibration at 2935cm, which is the characteristic absorption peak of polysaccharides. -1 and 2854cm -1 They are the absorption peaks of CH bond stretching vibration. -1 The absorption peak is the symmetrical stretching vibration of a C=O on the CH3CO- group. -1 At 1735cm, the OH bond undergoes bending vibration. -1 and 1623cm -1 At 1500cm -1 、1600cm -1 There is no relevant signal peak at 1236cm -1and 1097 cm -1 The absorption peaks of C-H bonds and the characteristic peaks of C-O bonds on the sugar ring appeared respectively. The absorption peak at 1024 cm -1 is the bending vibration of -OH, the absorption peak at 917 cm -1 is the β-glycosidic bond, and the characteristic absorption peak of α-glycosidic bond exists at 835 cm -1 . The connection length of α-glycosidic bond is shorter, which can promote the close combination between sugar molecules and improve the molecular stability. At the same time, α-glycosidic bond has very important molecular functions in the biological system and participates in the biosynthesis processes of various metabolisms. In summary, the polysaccharide from Malus micromalus fruits contains pyran ring, β-glycosidic bond and α-glycosidic bond.

[0060] SEM determination of the polysaccharide in Example 5

[0061] Weigh 5 mg of the dried polysaccharide sample from Malus micromalus fruits, scan it under an acceleration voltage of 5 kV, and observe the morphology of the polysaccharide by scanning electron microscope.

[0062] According to Figure 9 shown, the microscopic morphology of the polysaccharide from Malus micromalus fruits magnified 50 times is in the form of irregular fragmentary layers, with a relatively rough surface, having protrusions and depressions. When the sample is magnified to 500 times, the surface of the polysaccharide is in the form of flocculent, pore-free and irregular sheet-like structures. When magnified to 2000 times and 5000 times, there are fewer particles on the surface of the polysaccharide, with surface protrusions and pores, and its porous structure can endow the polysaccharide with certain adsorption capacity.

[0063] Determination of the superoxide anion scavenging rate of the polysaccharide in Example 6

[0064] Dilute the 1 mg / mL polysaccharide sample solution and ascorbic acid solution from Malus micromalus fruits to 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0 mg / mL, and store them refrigerated for later use. In 1 mL of sample solutions with different concentrations, add 3 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer solution, heat in a water bath at 37 °C, after 10 min, add 0.5 mL of pyrogallol solution, after reacting for 5 min, quickly add 0.5 mL of concentrated hydrochloric acid to end the reaction. Use ascorbic acid as the positive control and measure the absorbance at 325 nm. Calculate the superoxide anion scavenging activity according to the following formula:

[0065]

[0066] where π is the superoxide anion scavenging rate, A i is the absorbance of the polysaccharide at different concentrations, A j is the absorbance of the reference substance, and A0 is the absorbance of the mixed solution with distilled water replacing the sample.

[0067] According to Figure 10As shown, polysaccharides from Malus micromalus fruits at different concentrations all have the effect of scavenging superoxide anion radicals. Generally speaking, with the increase of polysaccharide concentration, the scavenging rate of superoxide anion shows an obvious upward trend. When the mass concentration is 1.0 mg / mL, the scavenging rate of polysaccharides from Malus micromalus fruits on superoxide anion reaches the highest, which is 66.67 ± 1.89%. That is, polysaccharides from Malus micromalus fruits have good scavenging ability on superoxide anion and strong antioxidant ability.

[0068] Example 7 Preparation of Cholesterol Standard Curve

[0069] Mix 1 mL of standard cholesterol solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0 mg / mL, 0.2 mL of phthalaldehyde solution (1 mg / mL), and 4 mL of mixed acid solution (V 浓硫酸 :V 冰乙酸 = 1:1) evenly, incubate in a water bath at 37 °C for 10 min, and measure the absorbance at 550 nm. Take the concentration of the standard cholesterol solution as the horizontal axis and the measured absorbance as the vertical axis to draw the standard curve.

[0070] As Figure 11 shown, the obtained standard curve equation is y = 0.1971x + 0.0077, and the correlation coefficient R 2 = 0.9991, with a good linear relationship, and the effective concentration range is 0 - 1 mg / mL.

[0071] Example 8 Determination of Cholesterol Adsorption Capacity of Polysaccharides

[0072] Take 3 mL of polysaccharide solutions from Malus micromalus fruits at different concentrations (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL) in test tubes respectively, add 6 mL of cholesterol solution and mix evenly, shake at a constant temperature of 37 °C for 2 h, centrifuge, and measure the supernatant. Calculate the cholesterol adsorption rate according to the following formula:

[0073]

[0074] where ρ is the cholesterol binding rate, C is the amount of cholesterol added, and C1 is the remaining amount of cholesterol.

[0075] According to Figure 12 shown, polysaccharides from Malus micromalus fruits show certain cholesterol adsorption ability. When the sample concentration is 5 mg / mL, the cholesterol adsorption rates of the positive control and polysaccharides from Malus micromalus fruits are 75.85 ± 0.52% and 71.84 ± 0.3% respectively, indicating that the polysaccharide component from Malus micromalus fruits has good cholesterol adsorption effect, that is, it has certain hypolipidemic activity.

[0076] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits, characterized in that, It includes the following steps: 1) Pretreatment of raw materials: The fresh fruits of Malus micromalus are washed and trimmed, then freeze-dried by a freeze dryer for 30 h until powdered, and stored in a -80 °C refrigerator for later use. 2) Extraction: Extraction is carried out according to the technical route of Malus micromalus jelly dry powder → dissolution → enzymolysis → ultrasonic treatment → centrifugation → deproteinization → decolorization → concentration → ethanol precipitation → freeze-drying → Malus micromalus fruit polysaccharide.

2. The method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, wherein, In step 2), the dissolution is carried out by dissolving the Malus micromalus jelly dry powder and water at a solid-liquid ratio of 1 g:35 mL.

3. A method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the enzymolysis step is as follows: Add a complex enzyme to the solid-liquid mixture at a mass-volume concentration of 3-5%, enzymolyze at an enzymolysis temperature of 45-55 °C for 1 h, and inactivate at 95 °C for 5 min after enzymolysis; the complex enzyme is: papain: cellulase: pectinase = 2:1:1 by mass ratio.

4. A method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the ultrasonic conditions are: ultrasonic power of 240-320 W, ultrasonic temperature of 45-55 °C, and ultrasonic time of 50 min.

5. A method for ultrasonic compound enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the centrifugation conditions are: centrifuge at 8000 r / min for 15 min and take the supernatant.

6. The method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the deproteinization step is: Add trichloroacetic acid at a mass-volume concentration of 5%, stir with a stirrer for 1 h, then centrifuge at 8000 r / min for 10 min, and collect the supernatant.

7. A method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the decolorization step is: Add activated carbon powder at a mass-volume concentration of 1.5%, stir for 30 min, then centrifuge at 8000 r / min for 10 min, and take the supernatant.

8. A method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the concentration step is: Concentrate the volume to 1 / 5 using a rotary evaporator.

9. A method for ultrasonic compound enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the ethanol precipitation step is: Add absolute ethanol, and the volume ratio of the concentrated solution to absolute ethanol is 1:4, precipitate for 12 h, centrifuge at 8000 r / min for 10 min, and take the precipitate.

10. A method for ultrasonic composite enzyme-assisted extraction of polysaccharides from Malus micromalus fruits according to claim 1, characterized in that, In step 2), the freeze-drying step is: Add water, place it in a petri dish and freeze at -18 °C for 12 h, then put it into a freeze dryer and freeze-dry for 30 h to obtain Malus micromalus fruit polysaccharide.

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