Ultrasonic extraction of Spirulina polysaccharides was optimized by orthogonal experiments using factors such as time, pH and temperature. Use orthogonal table L2535, factor-level design.
Spirulina polysaccharide extraction 25 parts of spirulina dry powder, each mass is 10.00g, placed in 25 beakers after degreasing by ethanol, add 200mL of distilled water according to the orthogonal experimental design to adjust the pH, temperature, respectively, at a frequency of 28HZ Ultrasonic sonication for different time, the extract is centrifuged to precipitate, repeated twice, the supernatant is deproteinized with trichloroacetic acid, ethanol is precipitated, and dried to obtain crude polysaccharide. The extracted polysaccharide is dried. After the determination of the total sugar content by the sulfuric acid-anthrone method, the calculation method of the total sugar content: the total sugar content = the concentration of the polysaccharide in the solution / the crude extract mass concentration @ 100%, the polysaccharide extraction rate = the crude extract quality / Raw material quality @100%, polysaccharide extraction amount = crude extract quality @ total sugar content, Coomassie method to determine the protein content of the crude product.
According to the results of the orthogonal experiment, the difference between the ultrasonic extraction method and the conventional water extraction method was compared. In vitro anti-tumor effect of extracted spirulina polysaccharide on the growth of ovarian cancer SKV3 cells in vitro: Take SKV3 cells in logarithmic growth phase, adjust the cell concentration to 5@103 cells/mL with DMEM containing 10% fetal bovine serum. Placed on a 96-well culture plate, 1@104 cells/well, the final volume is 200L. After 12 hours of in vitro culture, the medium is replaced with a spirulina polysaccharide culture solution with a concentration of 10 to 100 ug/mL. After 48 hours of in vitro culture, it is detected by MTT assay. Tumor cell inhibition rate.
Inhibition rate (%) = (average OD value of the control group - average OD value of the experimental group) / average OD value of the control group @100%. Results Comparison of the extraction amount of polysaccharides from spirulina Orthogonal experiment showed that the dry powder of spirulina at pH 11 and 60e The amount of spirulina polysaccharide obtained by ultrasonic extraction for 60 min under water bath conditions was the highest. According to the results of the range analysis, the three factors (pH, extraction time, temperature) affecting the rate of Spirulina polysaccharides were most affected by pH, followed by extraction time and temperature. Compare the sugar content in the proposed rate and the crude product by the ultrasonic extraction method and the water bath extraction method (the results are shown).
The conditional optimization of the ultrasonic method is significantly higher than that of the simple water method. (The anti-tumor activity of the spirulina polysaccharide extracted by the two methods is compared. The results of the two extraction methods of the spirulina polysaccharide on the SKV3 cancer cell line show that: The crude extract obtained by the extraction method can effectively inhibit the growth of ovarian cancer SKV3 cell line (the inhibition rate of MTT method is 43.0133.497% and 39.26531293, respectively), and shows a good dose-effect relationship; the spiral extracted by ultrasonic method The anti-tumor effect of algal polysaccharide was higher than that of Spirulina polysaccharide obtained by water bath extraction method, but there was no significant difference between the two (P>0.05). The influence of time, pH and temperature on the extraction amount of spirulina 4 The composition of spirulina polysaccharide is very complicated, mainly composed of rhamnose, D-mannose, D-glucose, D-galactose, glucuronic acid, xylose, fucose and the like.
The most commonly used methods for determining sugar content are the anthrone-sulfuric acid colorimetric method and the phenol-sulfuric acid colorimetric method. However, in actual work, especially when determining the sugar content in natural products, it is found that the measured results often do not match the actual conditions. When the Spirulina polysaccharide was extracted by ultrasonic method, the ultrasonic time should not be too long. When the ultrasonic treatment was carried out for 60 min, the extraction amount reached the maximum. With the prolongation of the treatment time, the extraction rate decreased.
The reason may be that the ultrasonic wave has a strong mechanical shearing action, and the long-term action may break the polysaccharide of the macromolecule, thereby increasing the loss in the post-treatment process and affecting the rate of the polysaccharide. During the extraction of polysaccharides from Spirulina, when the temperature is too high, the local structure of the polysaccharides of macromolecules will change, which will affect the extraction effect. The effect of pH on the extraction of polysaccharides from Spirulina is complex, and our experimental results show that the maximum extraction is obtained at pH 11.
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