Determination of crude polysaccharides by UV-1100 ultraviolet spectrophotometry - Master's thesis - Dissertation

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Determination of crude polysaccharides by ultraviolet spectrophotometry

Key words: UV spectrophotometry; Lycium barbarum polysaccharide; Ganoderma lucidum polysaccharide; Aesthetic analysis instrument

The content of crude polysaccharide in food was measured by sulfuric acid-phenol colorimetry and glucose was used as a standard and dextran as a standard. The results show that the former results are slightly higher, about 4.8%. The method is simple, rapid, accurate and reproducible, and can be applied to the determination of various crude polysaccharides. A carbohydrate formed by linking ten or more monosaccharides through a glycosidic bond is called a "polysaccharide". It is generally a natural polymer compound. Polysaccharides include active polysaccharides and dietary fiber. Active polysaccharides specifically refer to polysaccharide compounds having a specific biological activity, such as fungal polysaccharides, plant polysaccharides and chitosan. These polysaccharides have complex and multi-faceted physiological activities and functions, such as: immune regulation function; anti-tumor effect; delaying aging; lowering blood fat, anti-thrombosis and other functions [1-2], thus causing more and more people attention. There are no nationally prescribed methods for the determination of polysaccharides. The methods reported in the literature are basically phenol-sulfuric acid method and anthrone-sulfuric acid method. Most of the standard curves are made of glucose as standard, instead of using glucose. Sugar (MW 500,000), because dextran standards are difficult to obtain in the country and expensive. However, according to a large number of research data at home and abroad, it shows that the water-soluble substances contained in mushrooms, Flammulina, Yunzhi, Dongcao, Xia Cao, Ganoderma lucidum, Poria, and Polyporus have physiological activities such as enhanced immunity, tumor suppression, sedative, cardiotonic and anti-inflammatory. Polysaccharides are composed of β(1-3) or β(1-6) glycosidic linkages to form dextran to form the main chain and branches, and some even β-glucans, then glucose is used as a standard and dextran is used. Whether the difference is the difference between the standard and the polysaccharide content in the food, the above experiment is carried out with lycium polysaccharide and ganoderma lucidum polysaccharide. 1. Materials and methods 1.1 Test material 枸杞 polysaccharide (polysaccharide content is 40-60%, its content is detected by national testing); Ganoderma lucidum polysaccharide (polysaccharide content is 40-60%, its content is detected by national testing); Glycan (MW500,000) 1.2 Reagent (1) Copper stock solution: Weigh 0.3g CuSO45H2O, 3g sodium citrate, dissolve in water and dilute to 100ml, mix and set aside. (2) Copper reagent solution: 50 ml of copper stock solution was taken, 50 ml of water was added, and after mixing, 12.5 g of solid anhydrous sodium sulfate was added and dissolved. Use a new match. (3) Detergent: 50 ml of water, add 10 ml of copper reagent solution, 10 ml of sodium hydroxide solution, and mix. (4) Ethanol solution (80%): 80 ml of absolute ethanol was added to 20 ml of water and mixed. (5) Sodium hydroxide solution (10%): Weigh 10 g of sodium hydroxide, dissolve in water and dilute to 100 ml, add solid anhydrous sodium sulfate to saturation, and set aside. (6) Sulfuric acid solution: 10 ml of concentrated sulfuric acid is added to about 80 ml of water, mixed, cooled and diluted to 100 ml. (7) Phenol solution (5%): 100 g of phenol was taken, distilled in an oil bath, and a fraction of 180 ° C to 182 ° C was collected. Weigh 5.0g of refined phenol, add to dissolve and dilute to 100ml, mix, and store the solution in the refrigerator for one month. (8) Glucose/Glucan standard stock solution: accurately weigh 1.0080g of glucose/glucan standard in dry to constant weight at 1050C, dissolve in water, and dilute to 100ml, mix and store in refrigerator. This solution contained 10.080 mg of glucose/dextran per ml. (9) Glucose/dextran standard use solution: Pipette 2.00ml of glucose/dextran standard stock solution, place it in a 100ml volumetric flask, add water to the mark, mix and store in the refrigerator. 1.3 instrument beauty analysis UV spectrophotometer UV-1100; UV-1200 centrifuge rotary mixer 1.4 experimental method 1.4.1 glucose standard curve preparation precision extraction glucose standard use solution 0,0.2,0.4,0.6,0.8,1.0,( Equivalent to glucose 0, 0.04032, 0.08064, 0.12096, 0.16128, 0.2016mg) respectively placed in a 25ml colorimetric tube, properly add water to 2.0ml, add 2.0ml of phenol solution, mix on a rotary mixer, carefully add Into the concentrated sulfuric acid 10ml, carefully mixed in a rotary mixer, boiled in a boiling water bath for 15min, after cooling, using a spectrophotometer at 490nm wave advantage with the reagent vacancy solution as a reference, 1cm cuvette to determine the absorbance value. The specific results are shown in Table 1. Table 1 Relationship __________________________________________________________________ standard solution and the concentrations of glucose standard (ml) 0 0.2 0.4 0.6 0.8 1.0 concentration (mg / ml) 0 0.02 0.04 0.06 0.08 0.10 Absorbance (A) 0 0.166 0.343 0.514 0.690 0.882 ___________________________________________________________________ quality glucose On the abscissa, the absorbance value is plotted on the ordinate and the standard curve is drawn (Fig. 1). FIG FIG 1 standard curve equation fro fro which the equation Y = 4.4122X-0.0147, correlation coefficient r = 0.9996.1.4.2 precision drawing dextran standard curve prepared using standard solutions 0,0.2,0.4,0.6,0.8 dextran, 1.0, (equivalent to dextran 0, 0.02, 0.04, 0.06, 0.08, 0.10 mg) were placed in a 25 ml colorimetric tube, water was added to 2.0 ml correctly, and 2.0 ml of phenol solution was added to the rotary mixer. Mix well, carefully add 10ml of concentrated sulfuric acid, carefully mix in a rotary mixer, boil for 15min in a boiling water bath, cool down, use a spectrophotometer at 485nm wave advantage with reagent vacancy solution as a reference, 1cm cuvette to determine absorbance value. The specific results are shown in Table 2. Table 2 Relationship between the concentration of dextran standard ______________________________________________________________________ standard solution (ml) 0 0.2 0.4 0.6 0.8 1.0 concentration (mg / ml) 0 0.02 0.04 0.06 0.08 0.1 absorbance (A) 0 0.083 0.171 0.256 0.352 0.456 __________________________________________________________________ to glucosamine The mass of the glycan is the abscissa and the absorbance is the ordinate. A standard curve is drawn (see Figure 2). Fig. 2 Standard return curve equation graph Its return equation Y=4.635X-0.0145, correlation coefficient r=0.9983. 1.5 Determination of sample 1.5.1 Sample extraction Weigh 2.0g of crude polysaccharide sample (M) mixed and placed in 100ml (V1) In a volumetric flask, add about 80 ml of water, heat on a boiling water bath for 2 h, cool to room temperature, add water to the mark, mix, filter, and filtrate to precipitate the polysaccharide. 1.5.2 Precipitating crude polysaccharides Take 1.5ml (V2) of the filtrate under 1.5.1 (for four sets of parallel experiments) or 4.0ml of liquid sample (V2) (that is, the tested polysaccharide is liquid rather than solid sample) and placed in 50ml centrifugation. Into the tube, 16 ml of absolute ethanol was added, and after mixing, it was centrifuged at 4000 rpm for 15 minutes, and discarded into the supernatant. The precipitate was washed with several milliliters of ethanol solution, centrifuged, and discarded to the supernatant for 3-4 manipulations. Next, the precipitate was dissolved in water and made up to 5.0 ml (V3), and after mixing, it was used to precipitate dextran. 1.5.3 Precipitated dextran Precision Take 1.5.2 solution 2ml (V4) in a 20ml centrifuge tube, add 2.0ml of NaOH solution, 2.0ml of copper reagent solution, boil for 3min in boiling water bath, and then centrifuge at 4000rpm for 15min after cooling. Discard the supernatant. The precipitate was washed with several milliliters of detergent, centrifuged and discarded into the supernatant. After repeated manipulations, the precipitate was dissolved in 2.0 ml of sulfuric acid solution and transferred to a 50 ml (V5) volumetric flask. The water was added to the mark and mixed. This solution is a sample assay solution. 1.5.4 Determination of the precision sample 1.5.3 sample solution 2.0ml (V6) placed in a 25ml colorimetric tube, add 2.0ml of phenol solution, mixed on a rotary mixer, carefully added 10.0ml of concentrated sulfuric acid After mixing on a rotary mixer, it was boiled in a water bath for 10 min, cooled to room temperature, and the advantage of the 490 nm wave was measured by a spectrophotometer. The reagent vacancy was used as a reference, and the absorbance value was measured in a 1 cm cuvette, and a sample vacancy test was performed at the same time. The glucose/glucan mass was determined from the glucose/dextran standard curve and the crude polysaccharide content in the sample was calculated (see Table 3). Table 3. dextran glucose meter and meter to determine the content of crude polysaccharide _________________________________________________________________________________ absorbance of sample No. barbarum polysaccharide content (mg / g) Ganoderan sample (mg / g) ______________ ________________________ _____________________ barbarum polysaccharides Ganoderan dextran as glucose in a glucose meter to meter to 1 meter glucan 0.150 0.146 0.453 0.431 0.423 0.145 0.148 0.440 0.417 0.4032 0.429 0.4083 0.413 0.3944 0.139 0.142 0.423 0.402 0.150 0.151 0.453 0.431 0.437 0.416 2 ___________________________________________________________________________________. formula X = (W1- W2)×V1×W3×W5/M×V2×V4×V6 where: X-----the content of crude polysaccharide in the sample (in terms of glucose/glucan), mg/gW1----sample determination solution Medium glucose/glucan mass, mgW2--- glucose/glucan mass in sample vacancy, mgM----sample quality, gV1----total volume of sample extract, mlV2---precipitated crude polysaccharide Sample extract volume, mlV3---- Volume of polysaccharide solution, mlV4---the volume of crude polysaccharide solution used to precipitate dextran, mlV5---total volume of sample solution, mlV6---measurement solution volume for sample, ml3. Stability test sample after color development, The absorbance was measured at intervals, and the results are shown in Table 4. Table 4 Absorbance stability test ________________________________________________________________________________ time 10min 20min 30min 1h 1.5h 2hA 0.150 0.151 0.148 0.151 0.150 0.147 ____________________________________________________________________________________ results show that the stable color reaction. 4. Recovery rate determination (using sample recovery method) to absorb 0.5ml of polysaccharide sample solution, add standard glucose solution 0, 0.2, 0.4, 0.6, 0.8, 1.0ml, respectively, placed in 25ml test tube, add 1ml each water, add phenol 2ml of solution, slowly add 10ml of concentrated sulfuric acid to the rotary mixer for careful mixing, boil for 15min in boiling water bath, cool and use spectrophotometer in the 485nm wave advantage with reagent vacancy solution as reference, 1cm cuvette to determine absorbance value . The results showed that the recovery was 93.15-99.7%. 5. Summary (1) It can be seen from Table 3 that when glucose is used as a standard and dextran is used as a standard to measure the content of crude polysaccharide in food, the former results are slightly higher, about 4.8% higher, but Glucan Sugar standards are expensive and difficult to buy in China, so glucose can be used instead of dextran as a standard. (2) The high molecular weight substance with a molecular weight > 10000 in food is precipitated in an 80% ethanol solution, separated from the water-soluble monosaccharide and oligosaccharide, and selectively precipitated from other high molecular substances with a basic divalent copper reagent. The polysaccharide of the glycan structure, the crude polysaccharide is hydrolyzed into a monosaccharide under the action of sulfuric acid, and the natural aldaldehyde derivative is rapidly dehydrated, and the colored compound is condensed with phenol, and the crude polysaccharide content of the sample is determined by spectrophotometry. (3) This method can be well used for the determination of crude polysaccharide content in foods, since it is not necessary to purify and decolorize the polysaccharide. (4) When washing the crude polysaccharide precipitate, be sure to wash the other sugar and carbohydrates stained on the wall of the centrifuge tube with 80% ethanol, otherwise the result will be affected. Key words: ultraviolet spectrophotometry; alfalfa polysaccharide; ganoderma lucidum polysaccharide; aesthetic analyzer ; UV-1100; UV-1200

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