Effects of Temperature and pH on the Physiological Activity of Grape Juice

Danlei LI Chunling XIAO

Abstract This study was conducted to investigate the effects of temperature and pH on polyphenol and flavonoid contents in grape juice and its antioxidant activity. Two groups （group A and group B） were set. Group A was set with different pH values： 3.0， 3.5， 4.0， 4.5 and 5.0; and group B was set with different temperatures： 65， 72， 79， 86 and 93. The grape juice was treated according to set methods， and two groups of samples were obtained. The contents of polyphenols and flavonoids in each test solution were determined， and the DPPH·free radical system and trivalent Fe reduction system were established to measure and compare the antioxidant activity of groups A and B. The results showed that within the set ranges， the effects of pH and temperature on the content of flavonoids in grape juice were not significant; the test solution with a pH of 4.5 and the test solution with a temperature at 72 had the strongest capacities of scavenging free radicals， and exhibited scavenging rates of 6.82% and 9.43%， respectively; the test solution with a pH of 4.5 and the test solution with a temperature at 65 had the strongest capacities of reducing ferric ions. It could be seen from the above four aspects comprehensively， within the set test ranges， the suitable pH was 4.5， and the suitable temperature was 65.

Key words Grape juice; Flavonoid; Polyphenol; Antioxidant activity

There are more than 40 species of Vitis known in China[1]， which is the origin center with the most abundant grape genetic resources in the world. At present， 20% of Chinaюs annual grape production is used as fresh food， and the remaining 80% is used for processing of fruit juice and wine. Grape polyphenol compounds are high in content and complex in variety， function and structure[2]. The type and content of phenolic substances are closely related to the color， taste and aroma and stability of grape juice and wine， which are important factors in determining the quality of wine and grape juice[3]. Medical research has proved that polyphenols are the most important active substances in wine， with a variety of physiological functions and pharmacological effects. Because of their strong antioxidant capacity and capacity of scavenging free radicals， they have a good health care effect on human body and can prevent the occurrence of some diseases such as cardiovascular disease， atherosclerosis， inflammation and Alzheimerюs disease. As important components of fruit juice， polyphenols have attracted attention from more and more people， and have been listed as food quality testing indexes and process control indexes of fruit juice[4]. Red grape juice contains more proanthocyanidins， flavonoids and polyphenols， which are good for heart health， and can prevent arterial stenosis， scavenge free radicals in human body and improve microcirculation[5]， while due to changes of external environment （including light， temperature and pH） in the processing and storage of grape juice， various components in grape juice will change， which directly affects its quality. On this basis， the effects of external environment conditions on the physiological activity of grape juice， including the contents of polyphenols and flavonoids， DPPH· scavenging capacity and ferric ion reducing capacity were studied， with an attempt to promote the further development and utilization of grape juice.

Materials and Methods

Materials and reagents

Yufeng grape was purchased from Wuyi Road Fruit Shop in Yaodu District， Linyi City. FolinCiocalteu was purchased from Tianjin Kermel Chemical Reagent Co.， Ltd. 1，1diphenyl2pierylhydrazy （DPPH·）， gallic acid and rutin standard （with purity of 99%） were purchased from Sigma. Ethanol， sodium carbonate， sodium nitrite， aluminum nitrate， sodium hydroxide， phosphate buffer， ferric chloride， anhydrous methanol， ascorbic acid， ferric trichloride， hydrochloric acid， 80% methanol， potassium ferricyanide and trichloroacetic acid were all analytically pure.

Main instruments

FA2204B electronic balance， Shanghai Techcomp Precision Balance Co.， Ltd. ; HH4 digital  constant temperature water bath， Jintan Ronghua Instrument Manufacturing Co.， Ltd.; UV1100 ultravioletuisible spectrophotometer， Shanghai Mapada Instrument Co.， Ltd.; pHS25 type acidimeter， INESA Scientific Instrument Co.， Ltd.

Experimental methods

Preparation of grape juice： grape ∪ breaking ∪ filtration with four layers of gauze∪ supernatant ∪ clarified grape juice.

Drawing of rutin standard curve： A certain amount of rutin standard （0.062 5 g） was accurately weighed， dissolved with 80% methanol solution and diluted to 250 ml， obtaining rutin standard solution with a mass concentration of 0.25 mg/ml， which was preserved in the shade at room temperature. Then， 0.0， 2.0， 4.0， 6.0， 8.0 and 10.0 ml of the standard solution were accurately pipetted and added with 1.5 ml of 5% NaNO2 solution， respectively， followed by shaking and standing for 6 min. Into each of the solution， 1.5 ml of 10% Al（NO3）3 solution was added， followed by shaking and standing for 6 min， and the obtained solution was added with 20 ml of 5% NaOH solution， and diluted with 80% methanol solution to 50 ml， followed by shaking and standing for 15 min. The absorbance value was determined at 510 nm repeatedly for 3 times， and the average was calculated. Taking the absorbance value as the ordinate and the mass concentration of the rutin standard solution as the abscissa， a standard curve was drawn[6].

Drawing of gallic acid standard curve： A certain amount of gallic acid standard （0.025 g） was accurately weighed， dissolved with distilled water and diluted to 50 ml， obtaining a gallic acid standard solution with a concentration of 0.5 mg/ml. Then， 0.0， 0.2， 0.4， 0.6， 0.8 and 1.0 ml of the standard solution were accurately pipetted and added 1 ml of Folin phenol reagent， respectively， followed by shaking well. Into each of the solution， 2 ml of 15% Na2CO3 solution was added， and diluted with distilled water to 25 ml， followed by shaking well， and the obtained solution was heated in a water bath at 75 for 10 min. The absorbance value was measured at a wavelength of 760 nm repeatedly for 3 times， and the average was calculated. Taking the absorbance value as the ordinate and the mass concentration of the gallic acid standard solution as the abscissa， a standard curve was drawn[7].

Preparation of test solution： The clarified grape juice was taken and divided into two groups， A and B. The grape juice of group A was divided into 5 groups， A1， A2， A3， A4 and A5， the pH values of which were adjusted to 3.0， 3.5， 4.0， 4.5 and 5.0， respectively， and the obtained solutions were then heated in a 50 water bath for 1 h. The grape juice of group B was adjusted to pH 4， and then divided into five groups of test solutions， B1， B2， B3， B4 and B5， which were heated in 65， 72， 79， 86 and 93 water baths for 1 h， respectively.

Determination of flavonoids in the sample： A certain amount of each tobetested solution （1 ml） was accurately measured， and determined for absorbance at 510 nm according to the abovementioned procedure for preparing the rutin standard curve. The content of flavonoids in each solution was calculated according to the regression equation obtained above.

Determination of polyphenols in the sample： A certain amount of each tobetested solution （1 ml） was accurately measured， and diluted with 70% ethanol by 25 times. A certain amount of each diluted solution （1 ml） was detected for absorbance， which was substituted into the standard curve equation. Ethanol was used as a blank control.

Determination of capacities of different test solutions to scavenge  1，1diphenyl2picrylhydrazyl （DPPH·）： A certain amount of 2.5 mg DPPH· was accurately weighed， dissolved in anhydrous methanol and diluted in a 100 ml volumetric flask to constant volume， obtaining a DPPH· methanol solution with a mass concentration of 25 g/ml， which was preserved in the dark （0-4）[8]. Then， 0.2 ml of each test solution was pipetted， and added with 7.8 ml of the DPPH· methanol solution with a concentration of 25 g/ml， followed by mixing rapidly. The solutions were measured for absorbance （516 nm） at 0， 5， 10， 15， 25， 35， 45， 55， 65 and 75 min， respectively， until the absorbance was relatively stable. The kinetic curve of scavenging DPPH· was drawn[9].

Determination of the reducing capacity： At first， 2.5 ml of 0.2 mol/L phosphate buffer solution （pH 6.6）， 2.5 ml of 1% potassium ferricyanide and 1 ml of corresponding test solution were mixed well and heated in a 50 water bath for 20 min. Each solution was cooled quickly， and added with 1 ml of 10% trichloroacetic acid solution， 2.5 ml of distilled water and 1.0 ml of 0.1% ferric trichloride solution， followed by mixing well and standing for 10 min at room temperature. The absorbance was determined in parallel at 700 nm for 3 times. The reducing capacity was judged according to the value of absorbance， and the greater the absorbance value， the stronger the reducing capacity. Ascorbic acid was used as a standard antioxidant[10].

Results and Analysis

Drawing of standard curve and establishment of regression equation

Taking the absorbance value as the ordinate and the mass concentration of the rutin standard solution as the abscissa， a standard curve was drawn （Fig. 1）， obtaining the regression equation of the absorbance value Y （A510） and the rutin mass concentration X （mg/50 ml）. The regression equation was Y=0.168 2X+0.001 7 （R2=0.996 6）.

Taking the absorbance value as the ordinate and the mass concentration of the gallic acid standard solution as the abscissa， a standard curve was drawn （Fig. 2）， obtaining the regression equation of the absorbance value Y （A760） and the gallic acid mass concentration X （mg/25 ml）. The regression equation was Y=3.020 3X+0.066 4 （R2=0.992 9）.

Flavonoid and polyphenol contents in different test solutions of group A

The determination results of total flavonoid and total polyphenol contents in the test solutions with different pH values are shown in Fig. 3 and Fig. 4， respectively. It could be seen from Fig. 3 that in the measured pH range， the flavonoid contents in the test solutions ranked as pH 4>pH 3.5>pH 5>pH 3>pH 4.5. The content of flavonoids was the largest at pH 4， and overall， pH had a little effect on flavonoids in the sample. It could be seen from Fig. 4 that in the measured pH range， the polyphenol contents in the test solutions were in order of pH 4.5>pH 4>pH 5>pH 3.5>pH 3. The test solution having a pH of 4.5 had the largest polyphenol content， and overall， the content of polyphenols increased from pH 3 to pH 4.5， and began to decrease at pH 5. It could be seen that the change of pH had a great effect on polyphenols in the test solution.

Flavonoid and polyphenol contents in different test solutions of group B

The determination results of total flavonoid and total polyphenol contents in the test solutions at different temperatures are shown in Fig. 5 and Fig. 6， respectively. It could be seen from Fig. 5 that in the measured temperature range， the flavonoid contents in the test solutions ranked as 72>65>86>79>93. The test solution at 72 had the largest flavonoid content， and the differences between various treatments were not large. It could be seen from Fig. 6 that the polyphenol contents in the test solutions were in order of 65 > 72 > 79 > 93 > 86 in the measured temperature range. The test solution at 65 had the largest polyphenol content， and the polyphenol content in the test solution decreased with the temperature increasing.

Determination of capacity of different test solutions to scavenge DPPH·

Fig. 7 is a graph showing the kinetics of DPPH· scavenged by the test solutions with different pH values. Fig. 7 shows that after adding the test solutions with different pH values to the DPPH· system， the absorbance value of the system decreased with time. From the respective of the decrease， the test solution with a pH of 3 had the largest decrease; and it could be seen from the final absorbance value that the test solution with a pH of 4.5 had the lowest absorbance value. Taken together， when the pH of the test solution was 4.5， the free radical scavenging capacity was the strongest， and the scavenging rate reached 6.82%. Fig. 8 is a kinetic curve of DPPH· scavenged by test solutions at different temperatures. It could be seen from Fig. 8 that after adding the test solutions at different temperatures in the system， the absorbance value of the system decreased continuously over time. In terms of the decrease， the test solution with a temperature at 72 had the largest decrease; and from the respective of the final absorbance value， the absorbance of the test solution with a temperature at 72 was the smallest. On the whole， the test solution with a temperature at 72 had the highest capacity of scavenging free radicals， and the scavenging rate reached 9.43%.

Determination of reducing capacity

The measurement results of the reducing capacities of group A and group B are shown in Fig. 9 and Fig. 10， respectively. Fig. 9 shows that the test solution with a pH of 5 had the highest absorbance value in the pH range. The absorbance value of the test solution continuously increased with the increase of pH value， and the reducing capacity increased continuously， indicating that the larger the pH value of the test solution was， the stronger reducing action it had on ferric ion. Fig. 10 shows that in the measured temperature range， the test solution with a temperature at 65 had the largest absorbance value. With the temperature increasing， the absorbance of the test solution decreased continuously， and the reducing capacity decreased continuously， indicating that the higher the temperature of the test solution， the weaker reducing action it had on ferric ion.

Conclusions

Different temperatures or different pH had different effects on various physiological activity of grape juice， and the results were different. In the pH range， the test solution with a pH of 4 had the largest flavonoid content， and the largest polyphenol content was detected in the test solution with a pH of 4.5. In the measured temperature range， the largest flavonoid content was determined in the test solution at 72， and the test solution at 65 had the largest polyphenol content. Overall， pH and temperature had little effects on the content of flavonoids in grape juice， while the changes of pH and temperature had great effects on polyphenols in grape juice. The higher the pH or temperature of the grape juice was the lower polyphenol content it had. The DPPH· scavenging capacities of the test solutions in groups A and B were determined， and the results showed that the test solution with a pH of 4.5 and the test solution with a temperature at 72 had the strongest capacities of scavenging free radicals， and exhibited scavenging rates of 6.82% and 9.43%， respectively. The reducing capacities of the test solutions in groups A and B were determined， and the results showed that the test solution with a pH of 4.5 and the test solution with a temperature at 65 had the strongest capacities of reducing ferric ions. It could be seen from the above four aspects comprehensively， within the set test ranges， the suitable pH was 4.5， and the suitable temperature was 65.

References

[1]KONG QS. Grapes in China[M]. Beijing： China Agriculture Science and Technique Press， 2004： 28-53.

[2]SONG LJ， DI Y， SHI B. The significance and development trend in research of plant polyphenols[J]. Progress in Chemistry， 2000（2）： 161-170.

[3]GYO AQ. Research on new antioxidants in red wine[D]. Yangling： Northwest A&F University， 2004.

[4]SHI HM， DING Y， SUN YX， et al. Research on the determination of antioxidation activity of polyphenols in grape wine[J]. LiquorMaking Science & Technology， 2011（7）： 95-97.

[5]LI XX， QIN XY， LEI SX. Grape juice production and research status[J]. Guangxi Journal of Light Industry， 2010， 26（2）： 1-3.

[6]ZHANG SY. Influence of different processing methods on total flavonoids content in peanut[J]. Journal of The Chinese Cereals and Oils Association， 2010， 25（8）： 104-108.

[7]ZHOU XJ， GAO YX， ZHANG YD， et al. Study on extraction technology and antioxidant activity of polyphenol from Amaranthus mangostanus L.[J]. Science and Technology of Food Industry， 2012， 33（6）： 327-331.

[8]GUO WL， LI M， XIE Q， et al. Study on antioxidant activities of grape skin pigment[J]. China Food Additives， 2007， 19（2）： 116-119.

[9]ZHU YH， TAN J. Antioxidant effect of corn peptide[J]. Journal of The Chinese Cereals and Oils Association， 2008， 23（1）： 36-43.

[10]LYU YH， SU P， NA Y， et al. Study on antioxidant activities of mulberry pigment in vitro[J]. Journal of Zhejiang University， 2007， 33（1）： 102-107.