专业技能英语实训报告专业:食品科学与工程班级：131姓名：成玲学号：2013035102Study on antioxidant activity of polysaccharide from Chinese Rice Wine invitroAbstract：The antioxidant activity of the polysaccharide from Chinese Rice Wine by alcohol precipitation was contrasted in the present study. The total antioxidant activity，DPPH radical，hydroxyl radical and superoxide anion free radical were selected as the antioxidant indicators in vitro and ascorbic acid（VC）as the positive control. The results showed that crude polysaccharide and refined polysaccharide from Chinese Rice Wine had antioxidant activity，and the antioxidant activity was related to concentration. The antioxidant ability of refined polysaccharide in total antioxidant activity，removing the DPPH radical and hydroxyl radical was better than that of crude polysaccharide. However，the antioxidant ability of crude polysaccharide in removing superoxide anion free radical was stronger than that of refined polysaccharide.Key words：Chinese Rice Wine；polysaccharide；alcohol precipitation；antioxidant ； Chinese rice wine is the oldest traditional brewing wine, and it is one of the world's three largest ancient wine full bodied, Yellow Wine the mellow, soft wine. Chinese rice wine with its wonderful color and its name, with its unique taste, rich nutrition and its health care function and known to the world. Research shows that Chinese Wine has antioxidant activity and antioxidant activity of substances containing the antioxidant activity and the Yellow Wine, such as polyphenols and peptide etc. A large number of studies have indicated that polysaccharide has antioxidant activity. The yellow rice wine contains polysaccharides, whether or not it has antioxidant activity, the research on this aspect has not been reported in any report. Therefore, Shaoxing yellow rice wine was used as raw material, and the polysaccharide was extracted from the raw materials, and the DPPH free radical, hydroxyl free radical and superoxide dismutase were extracted by the investigation of the crude polysaccharide and the refined yellow rice wine .Anion and its total antioxidant capacity, preliminary study of the antioxidant activity in vitro, which provides scientific basis for the further research and utilization of yellow rice wine.1 Materials and methods1.1 Materials and Experimental equipmentYellow Wine (brewing in the winter of 2010, Zhejiang Guyue Longshan, Shaoxing wine Limited by Share Ltd), 1, 1 -2-3phenyl 4-dinitrophenylhydrazine (1, 1-diphenyl-2-picrylhydrazyl, DPPH) America Sigma, bovine serum albumin, glucose, ascorbic acid, Coomassie brilliant blue G-250, adjacent benzene three phenol and concentrated sulfuric acid, salicylic acid, three chloroacetic acid, potassium ferricyanide, chloroform, positive butanol and other reagents, 722s (spectrophotometer Shanghai Precision Scientific Instrument Co. Ltd), IKA Bei RV10 rotary evaporator (Germany IKA group) , freeze dryer ( America LABCONCO), high speed (centrifuge Mizue Analysis Instrument Co Ltd), UV-2450 UV spectrometer( Shimadzu Corporation).1.2 Experimental methods1.2.1 Extraction and separation of Polysaccharide from Chinese rice wineThe wine Yellow Wine decompression at 55 DEG C, concentrated to the original volume of 1/3, adding 95% ethanol concentrated solution in ethanol concentration reached 30%, 4 C standing overnight, centrifugal (6000r/min, 15min), the supernatant was collected; then add 95% ethanol to the supernatant ethanol concentration reached 75%, stirring, 4 DEG C standing overnight, centrifugal (6000r/min, 15min), collecting alcohol precipitation; to add 30% ethanol alcohol in the resolution 4 C standing overnight, centrifugal (6000r/min, 15min), the supernatant supernatant was collected; 55 degrees of reduced pressure concentration obtained Yellow Wine crude polysaccharide (starch by freeze drying experiments show that Yellow Wine crude polysaccharide was non starch polysaccharide).After the crude polysaccharide was added to water, the Sevage method was used to remove the impurities, such as inorganic ions, amino acids, oligosaccharides, and so on. The 72h was used to remove the impurities such as inorganic ions, amino acids, oligosaccharides and so on.1.2.2 Determination of polysaccharide in Chinese rice wine1.2.2.1 Determination of total sugarDetermination of total sugar content in yellow rice wine by using phenol-sulfuric acid methods, referred to the grape sugar.1.2.2.2 Determination of reducing sugar contentDetermination of reducing sugar content in yellow rice wine by using DNS method, referred to the grape sugar.1.2.2.3 Determination of polysaccharidePolysaccharide = total sugar reducing sugar.1.2.3 Determination of total antioxidant capacity1mL of different concentrations of polysaccharide solution, adding concentration of PBS solution 1mL and 0.2mol/L mass fraction of 1% pH6.6 potassium ferricyanide 1mL, 50 DEG C water bath 20min, fast cooling after adding 10% three chloroacetic acid 1mL, (4000r/min, 5min), centrifugal supernatant 2mL, adding distilled water and quality volume fraction of 0.1% ferric chloride 0.4mL, mixing, static 10min, the absorbance was determined at 700nm. According to the same method, the absorbance value of the refined rice wine with different concentrations and the positive control VC were determined, and the average value of the 3 time was measured.1.2.4 Determination of free radical scavenging ability of DPPHReference CHIO the CW and other methods for determination of the LI XL, etc. The basic steps are: different concentrations of polysaccharide solution 2mL, adding volume concentration of DPPH 0.1mmol/L solution (prepared with ethanol), after mixing light reaction of 30min at room temperature (6000r/min, 10min), centrifugal supernatant was determined by A i in 517nm; and with 4mL ethanol and distilled water (two 2mL respectively)as zero. The blank control group was 2mL sample solution with 2mL absolute ethanol, the absorbance at 517nm wasA j, the blank control group was set to remove the light absorption value of the polysaccharide. The model control group was DPPH 2mL solution plus 2mL absolute ethanol, its absorbance at A c was 517nm. Using the same method to measure the absorbance of the refined rice wine with different concentrations and the positive control VC, the average value of the 3 times was measured. According to the following formula to calculate the sample to DPPH free radical scavenging rate:The sample to DPPH free radical scavenging rate (%) = [1-(A i-A j)/A c]*1001.2.5 Determination of hydroxyl free radical scavenging capacityThe Fenton reaction system model was used to determine the hydroxyl radical scavenging activity by a fixed time reaction method. The reaction system contained H2O2 9mmol/L, FeSO4 9mmol/L, 8.8mmol/L salicylic acid (prepared with anhydrous ethanol) and crude polysaccharide solution with different concentration of yellow rice wine. The volume of the reaction system was 1mL. With distilled water as the reference, the final addition of H2O2start up reaction, 37 C reaction 30min, the absorbance of the reaction system was measured at 510nm Ax. The polysaccharide was replaced by water, and the absorbance of the model control group was A0. Taking into account the absorbance value of the polysaccharide itself, with equal volume (9mmol/L) of FeSO4 9mmol/L, l mL salicylic acid ethanol, different concentrations of polysaccharide solution and distilled water as a mixture of the background absorption value of Ax0. Using the same method to measure the absorbance of the refined rice wine with different concentrations and the positive control VC, the average value of the 3 times was measured. The free radical scavenging rate of the sample is calculated as follows:Sample to hydroxyl radical scavenging rate (%) = [1-(A x-A xo)/A o]*1001.2.6 Determination of superoxide anion capacityBy the development of three phenol autoxidation method, the basic steps are: 50mmol/L pH8.2 Tris-HCl 4.5mL 4.2mL buffer solution and distilled water mixing, 25 DEG C water bath temperature 20min, add 25 degrees preheated 3mmol/L adjacent benzene three phenol 1mL immediately after removing (prepared with 10mmol/L HCl zero 10mmol/L tube for HCl instead of the adjacent benzene three phenol solution quickly, HCl) shake and pour into the cuvette (light size 1cm), at 325nm every 30 seconds to determine a light absorption value (A), the linear range increased to A per minute value is adjacent benzene three phenol self-oxidation rate (A1). Tris-HCl pH8.2 buffer solution 4.5mL and different concentrations of crude polysaccharide solution 4.2mL mix, the other steps as above, the linear range of A per minute increase in value that is added to the sample after the three phenol oxidation rate (A2). In the same method to measure the absorbance of refined rice wine with different concentrations and the positive control VC, 3 times in parallel. The scavenging rate of superoxide anion radical was calculated according to the formula:Scavenging rate of superoxide anion in samples (%) = [1-(A1-A2)/A1]*1001.2.7 Statistical analysisSPSS 17 software was used to analyze the data of single factor analysis of variance (ANOVA), and the results were expressed as mean plus or minus standard deviation, and the significant level was p < 0.05.2 Results and analysis2.1 Determination of polysaccharide in Chinese rice wineAccording to the 1.2.2 method to measure the content of total sugar and reducing sugar content in wine Yellow Wine and polysaccharide content results in table 1. Table 1 Cotent of total sugar，reducing sugar and polysaccharide in Chinese Rice Wine（x±SD）2.2 Total antioxidant capacity of Chinese rice wineIn the reaction system, the greater the absorbance measured at the wavelength of 700nm, the stronger the total antioxidant capacity was. From Figure 1, we can see that the crude polysaccharide and refined polysaccharide has a certain total antioxidant capacity, and its total antioxidant capacity increased with the increase of the concentration of the sample, and showed a dose effect relationship. Moreover, the total antioxidant capacity of the refined rice wine was stronger than that of the crude polysaccharide, the reason may be that the complex composition of crude polysaccharide influenced the total antioxidant capacity, and the exact reason was still to be further studied. Under the same conditions, the absorbance of 0.1g/LVC at 700nm in the reaction system was 1.040 + 0.005, and 2g/L refined polysaccharides was 1.109 + 0.026, the absorbance of crude polysaccharide at this time is only 0.213 + 0.009, which indicates that VC total antioxidant capacity of the strongest, refined Yellow Wine polysaccharide of Yellow Wine polysaccharide.Fig.1 Total antioxidant activity of polysaccharide from Chinese Rice Wine2.3 DPPH free radical scavenging ability of yellow rice wine polysaccharideDPPH method is a rapid, simple, sensitive and feasible method to evaluate the antioxidant activity of natural antioxidants, which is widely used in the home and abroad. DPPH radical is a stable organic base, characterized in ethanol solution the purple group absorption peak, when there is a free radical scavenger, due to its single electron pairing and the absorption gradually disappear, the fading degree of theirelectron number into the quantitative relationship, and thus can be used by spectrophotometry quantitative analysis. From Figure 2, we can see that different concentrations of samples on the DPPH free radical scavenging ability is different, with the increase of sample concentration, the higher the rate of free radical scavenging DPPH. In the reaction system, the control group VC in 0.1g/L, to reach the maximum clearance rate, and the refined Yellow Wine polysaccharide and VC required to achieve the same maximum clearance rate to 1g/L, while Yellow Wine crude polysaccharide scavenging rate was 30.20% + 0.48%, far lower than the maximum VC removal rate was 95.61% + 0.47%. Therefore, the effect of DPPH free radical scavenging effect of refined rice wine is better than that of yellow rice wine, but the removal efficiency of the two is less than VC.Fig.2 Scavenge effect of polysaccharide from Chinese Rice Wine on DPPH2.4 Free radical scavenging ability of yellow rice wine polysaccharideCompared with other free radicals, hydroxyl free radical is the most active oxygen free radicals in vivo, oxidative metabolism process in life activity, the amino acids, proteins and fats and nucleic acids and other molecules to oxidative damage, inducing apoptosis or transformed cells from normal and cancerous cells to unlimited growth control.We can see from Figure 3, Yellow Wine refined and crude polysaccharide had strong hydroxyl radical scavenging ability, and with the increasing concentration of polysaccharide, hydroxyl radical scavenging ability also increases, showing a good dose effect relationship. VC increased from 0.1g/L to 1g/L, the clearance rate from 20.01% + 1.92% to 97.89% + 0.10% rapid growth; and the refined polysaccharide concentration increased to 5g/L, removal rate of 85.11% + 4.51%, scavenging capacity growth rate slower than VC, and the concentration reached 5g/L after the clearance rate no longer increased basically; scavenging capacity of polysaccharides on Yellow Wine hydroxyl radical and refined polysaccharide is consistent with the trend, but the scavenging ability is weaker.Fig.3 Scavenge effect of polysaccharide from Chinese Rice Wine on hydroxyl radical 2.5 The ability of yellow rice wine to remove superoxide anionSuperoxide anion free radicals as precursors of most of the oxygen free radicals in the organism, through a series of reaction of other free radicals. Not only the free radicals derived from them can be toxic to the cells, but also the superoxide anion free radicals also have toxic effects. From the figure 4, we can see that the crude polysaccharide and refined polysaccharide have strong scavenging effect on the superoxide anion produced by the three phenol self-oxidation system. When the concentration of 0.1g/L was VC, the removal rate of the crude polysaccharide and the refined polysaccharide of yellow rice wine were 86.30% + 2.15%, 74.09% + 2.82%, 67.56% + 1.69%, respectively. VC at the concentration of 3g/L, can clear the reaction system of all superoxide anion free radicals, and yellow rice wine crude polysaccharide in the concentration of 3g/L, its clearance rate with the increase in the concentration of the increase is not maintained at about 94%. With the increase of the concentration, the scavenging ability of the purified polysaccharide was higher than that of the former two. Under the same concentration, the scavenging ability was weaker than the former two.Fig.4 Scavenge effect of polysaccharide from Chinese Rice Wine on Superoxide anionNote: at the same concentration, the different letters on the three column chart indicated that the ANOVA test was performed at the p<0.05 and the difference is significant.3 Conclusion and discussionIn the process of normal metabolism, superoxide anion, hydroxyl radical and other reactive oxygen species are produced, which are destructive to the body. Under normal circumstances, the body produces too much free radical will quickly be removed by the body of the scavenger, and thus the body free radicals in a dynamic equilibrium. If the imbalance or clearance is too slow, too many free radicals will make the biological macromolecules under attack, accelerate the aging process of the body, and induce inflammation, malignant tumors, immune disorders and other diseases. Therefore, it is important to study the natural antioxidants and foods that can clear the free radicals in the body. Research shows that the polysaccharide has strong antioxidant activity both in vitro and in vivo, polysaccharide compounds of different kinds of antioxidant effects of different reaction systems have different effect, also has selectivity on free radical scavenging species.The results of this study showed that Yellow Wine crude polysaccharide and refined Yellow Wine polysaccharide has the total antioxidant capacity, has good scavenging effect on DPPH free radical, hydroxyl radical, superoxide anion, and the concentration and clearance rate in a certain range of concentration and dose effect relationship. The total antioxidant capacity and scavenging ability of DPPH free radical and hydroxyl free radical of refined rice wine was stronger than yellow rice wine crude polysaccharide. After refining, the free radical scavenging activity was significantly increased, which showed that polysaccharide was the main component of scavenging free radicals, which was consistent with the report of Tao Mingxuan et al. According to Yang Fang reported on the two kinds of Morchella intracellular polysaccharide, purification of polysaccharide free radical scavenging ability in crude polysaccharide is probably due to the crude polysaccharide containing complex composition affects its antioxidant activity, or complex group of polysaccharides which has antagonistic oxidation, the exact cause is to further study. The ability of crude polysaccharide to remove superoxide anion was stronger than that of refined rice wine, which was the result of the removal of other free radicals.On the contrary, according to XQ ZHA and YF HUA and other [17-18] reports, this may be related to the structure of polysaccharides, because the polysaccharide structure is the basis of its biological activity. Therefore, the further research on the separation, purification and structure identification of rice wine and its related structure activity relationship should be carried out.Although Chinese rice wine crude polysaccharide and refined polysaccharide free radical scavenging effect was lower than that of VC, but Chinese rice wine polysaccharide can achieve free radical scavenging, blocking free radical chainreaction, can inhibit the oxidative damage caused by free radicals in a certain concentration range, the further research and application of Chinese rice wine polysaccharide provides a theoretical basis for.（注：可编辑下载，若有不当之处，请指正，谢谢!）精选文档。