CN107621456A - A kind of determination method of kiwi fruit polyphenol content - Google Patents

Abstract

The invention discloses a method for determining polyphenol content in kiwi fruit, which comprises the steps of extracting, measuring absorbance value, repeating extracting, calculating polyphenol equivalent, calculating polyphenol content and the like. The volume ratio of kiwi fruit juice and alcohol solution during extraction is 1:3, the extraction temperature is 61-64°C, and the extraction time is 45min; After completion, take the supernatant and measure its absorbance value at the characteristic absorption wavelength of 715nm; repeat the steps of leaching, centrifugation and absorbance value measurement 4 times, and bring the absorbance value measured 5 times into the pre-fitted standard curve In the equation y=2.8758x+0.4960, the equivalent value of gallic acid is calculated, and then the polyphenol content is calculated by the formula W=C×V/M. By using the method, the polyphenols in the kiwi fruit juice can be extracted to the greatest extent, the extraction rate is above 35%, and the measured polyphenol content is more accurate.

Description

A kind of determination method of kiwi fruit polyphenol content

technical field

The invention relates to an assay method, in particular to an assay method for kiwi fruit polyphenols.

Background technique

Kiwi fruit is widely planted in my country. Kiwi fruit contains a variety of nutrients such as vitamin C and polyphenols. Long-term consumption of kiwi fruit is of great help to human health.

Polyphenols have antioxidant effects, and proper intake can prevent diseases and enhance the body's resistance. However, excessive intake can cause liver and intestinal discomfort, and even serious liver damage. Therefore, the intake should be strictly controlled, and the safe oral dose of each person should not exceed 0.25g. Find a way to accurately measure the polyphenol content in kiwi fruit, and you can know how much kiwi fruit you eat each time is more appropriate. There are many determination methods of kiwifruit polyphenols content in the prior art, but one-step extraction is used in the determination, and the polyphenols in kiwifruit juice cannot be extracted, and the measured content varies greatly and is not accurate enough.

Contents of the invention

Aiming at the above-mentioned existing prior art, the present invention solves the technical problem that the determination of kiwi fruit polyphenol content is not accurate enough.

In order to achieve the above object, the technical scheme adopted in the present invention is: provide a kind of kiwi fruit polyphenol content assay method, concrete steps are:

(1) Extraction: put a certain amount of squeezed kiwi fruit juice and alcohol solution with a volume fraction of 50% in a 20mL volumetric flask, and extract at 61-64°C for 45min;

(2) Determination of the absorbance value: after the extraction is completed, take 10mL of the extract and centrifuge at 5000r/min for 15min; after the centrifugation is completed, take the supernatant and measure the absorbance value at the characteristic absorption wavelength of 715nm;

(3) Repeat extraction: get the centrifuged supernatant, repeat steps (1), (2) 5 times;

(4) Calculation of polyphenol equivalent: put the absorbance value measured 5 times into the pre-fitted standard curve equation y=2.8758x+0.4960, and calculate the gallic acid equivalent value, that is, the polyphenol content; In the curve, y represents the mass concentration of polyphenols, and x represents the absorbance value;

(5) Calculation of polyphenol content: the calculation formula of polyphenol content is: W=C×V/M; Wherein, W is the content of polyphenol, and C is gallic acid equivalent, promptly calculates according to the polyphenol content of standard curve, V is the volume of the extract, and M is the amount of liquid taken; the total polyphenol content can be obtained by adding the polyphenol content measured five times.

The beneficial effect of the invention is that the alcohol solution is used for leaching, which is friendly to personnel and the environment, and does not cause personnel injury and environmental pollution. The extraction temperature and time are moderate, and the polyphenols will not be decomposed or oxidized, which ensures the accuracy of polyphenol content determination. After the extraction is completed, the extract is centrifuged for 15 minutes under the action of a centrifuge, and the polyphenols and impurities in the extract are fully separated and then enter the supernatant, so that the measurement results are more accurate. The characteristic absorption wavelength is measured in advance by the gallic acid standard solution. When determining the polyphenol content, put the supernatant into the ultraviolet spectrophotometer and measure the absorbance directly at 715nm, which is convenient and time-saving. After the absorbance value is measured, it is brought into the pre-fitted standard curve equation, and the mass concentration of polyphenols can be obtained after calculation, which is convenient and quick. Repeatedly extracting the polyphenols in the supernatant liquid can extract the polyphenols in the supernatant liquid to the greatest extent. After adding the polyphenol content obtained by each extraction, a more accurate kiwi polyphenol content.

On the basis of the above technical solutions, the present invention can also be improved as follows.

Further, the method for determining the characteristic absorption wavelength in step (2) is: take 1mL of gallic acid standard solution, 1mL of folin phenol reagent and 14mL of water into a 20mL volumetric flask, and add 3mL of 20wt% of Sodium carbonate solution, after mixing evenly, make to volume; place it at 25°C for 1 hour, use a UV spectrophotometer to scan the full wavelength between 200 and 800nm, and measure the wavelength at the time of maximum absorption; the measured characteristic absorption wavelength is 715nm.

The beneficial effect of adopting the above further technical scheme is that the characteristic absorption wavelength of gallic acid is determined in advance, and can be directly used when measuring the polyphenol content of kiwifruit, saving a lot of time. After preparing the solution, place it under the condition of 25-30°C. The room temperature during the ripening period of kiwi fruit is just in this range. The prepared solution can be placed indoors without additional heating or cooling treatment. The operation is simple and energy saving. When determining the characteristic absorption wavelength, the whole wavelength is scanned in the range of 200-800nm, and the scanning wavelength range covers the range of ultraviolet light and visible light, so that the characteristic absorption wavelength of gallic acid can be accurately determined.

Further, the specific steps of standard curve fitting in step (3) are:

(1) Preparation of gradient concentration solution: put 100mg of gallic acid into a 20mL volumetric flask, dissolve it with 2mL of ethanol and make it to 20mL; pipette the above solutions 0mL, 0.2mL, 0.4mL, 0.6mL, 1mL and 2mL to 20mL capacity In the bottle, add water to 20mL to obtain a series of gradient concentration solutions; each take 0.2mL of the above gradient concentration solution, 1mL of Folin phenol reagent and 14mL water into a 20mL volumetric flask, mix well and add 3mL within 1 to 5min 20wt% sodium carbonate solution, mixed evenly and then constant volume; placed at 25°C for 1h;

(2) Draw a standard curve: each get 2 mL of the above-mentioned configured gradient concentration solutions in the quartz dish of the spectrophotometer, and measure the absorbance of these gradient concentration solutions at 715 nm; in the coordinate system, the absorbance is the abscissa, and the mass concentration Fitting is performed for the ordinate to obtain a standard curve; the equation of the standard curve is: y=2.8758x+0.4960, and the regression coefficient of the standard curve is: R ² =0.9991.

The beneficial effect of adopting the above-mentioned further technical scheme is: fitting the standard curve in advance, when measuring the polyphenol content of kiwi fruit, after obtaining the absorbance of the supernatant, bringing it into the standard curve to obtain the corresponding polyphenol mass concentration, saving time and effort.

Further, the alcohol solution in step (1) is a methanol solution.

The beneficial effect of adopting the above further technical solution is that methanol is relatively common in the laboratory, easy to obtain, friendly to personnel and the environment, and will not cause personnel injury or environmental pollution. In addition, 50% methanol solution has a good extraction effect, and the extraction rate of polyphenols can reach more than 30%.

Further, the extraction temperature in step (1) is 63°C.

The beneficial effect of adopting the above-mentioned further technical solution is that polyphenols will not be decomposed and oxidized at this temperature, which ensures the accuracy of polyphenol content calculation later. Moreover, at this temperature, the solubility of polyphenols in Xixia is relatively large, which is convenient for the extraction of polyphenols, and the extraction rate of polyphenols is above 30%.

Further, the volume of kiwi fruit juice and alcohol solution is 1:3 during extraction.

The beneficial effect of adopting the above further technical scheme is that when the ratio of the kiwi fruit juice to the alcohol solution is 1:3, the extraction rate of polyphenols is higher than 30%.

detailed description

Specific embodiments of the present invention will be described in detail below.

The invention provides a method for determining polyphenol content in kiwifruit, which comprises the steps of extracting, measuring absorbance value, repeating extracting, calculating polyphenol equivalent, calculating polyphenol content and the like. The extraction process is to put a certain amount of squeezed kiwi fruit juice and 50% alcohol solution in a 20mL volumetric flask, and extract at 61-64°C for 45 minutes. Alcoholic solution is methanol or ethanol solution. When the volume fraction is 50%, the extraction rates of methanol and ethanol are 35% and 28% respectively, and methanol has a higher extraction rate, so the present invention preferably uses methanol solution for extraction. In the range of 61-64°C, polyphenols not only will not decompose and oxidize, but also have greater solubility. Polyphenols can be extracted quickly and completely in this temperature range, and the extraction rate of polyphenols at 63°C Highest. During leaching, in order to achieve the maximum extraction rate, the volume ratio of kiwi fruit juice and alcohol solution is set to 1:2～1:4, and the ratio of 1:3 is preferably adopted in the present invention. After the extraction is completed, take 10mL of the extract and centrifuge at 5000r/min for 15min; after the centrifugation, take the supernatant and measure its absorbance value at the characteristic absorption wavelength of 715nm. The characteristic absorption wavelength has been determined before the determination of the polyphenol content of kiwifruit. The specific method for determining the characteristic absorption wavelength is: take 1mL of gallic acid standard solution, 1mL of folin phenol reagent and 14mL of water into a 20mL volumetric flask. Add 3mL of 20wt% sodium carbonate solution within 1 to 5 minutes, mix well and then make to volume; place it at 25 to 30°C for 1 hour, use a spectrophotometer to scan the full wavelength between 200 and 800nm, and measure the wavelength at the time of maximum absorption . The measured maximum absorption wavelength is the characteristic absorption wavelength of gallic acid, and it is found that there is a maximum absorption value at 715nm during the scanning process, so the characteristic absorption wavelength is 715nm. After measuring the absorbance of the supernatant, mix the supernatant with 50% alcohol solution, extract at 61-64°C for 45 minutes, take 10 mL of the extract, and offline at 5000 r/min for 15 minutes, take the upper layer Measure the absorbance of the clear liquid; repeat the above steps 4 times to completely extract the polyphenols in the extract. Put the absorbance values measured for a total of 5 times into the pre-fitted standard curve equation y=2.8758x+0.4960 to calculate the gallic acid equivalent value, that is, the polyphenol content; y in the standard curve represents the polyphenol quality Concentration, x represents the absorbance value. The calculation formula of polyphenol content is: W=C×V/M; Wherein, W is the content of polyphenol, C is gallic acid equivalent, namely the polyphenol content calculated according to the standard curve, V is the volume of extract solution, M is the amount of liquid taken; the total polyphenol content is obtained by adding the polyphenol content measured five times. The fitting steps of the standard curve are: (1) Prepare the gradient concentration solution: put 100mg of gallic acid into a 20mL volumetric flask, dissolve it with 2mL of ethanol and set the volume to 20mL; Put mL, 1mL, and 2mL into a 20mL volumetric flask, add water to make up to 20mL, and obtain a series of gradient concentration solutions; each take 0.2mL of the above gradient concentration solution, 1mL of Folin’s phenol reagent, and 14mL water into a 20mL volumetric flask, and mix evenly Then add 3mL of 20wt% sodium carbonate solution within 1 to 5min, mix well and then make to volume; place it at 25°C for 1h; (2) Draw a standard curve: take 2mL of the above-mentioned gradient concentration solution in the spectrophotometer In a quartz dish, at 715nm, measure the absorbance of these gradient concentration solutions; In the coordinate system, take the absorbance as the abscissa, and the mass concentration as the ordinate to fit, get the calibration curve; the equation of the calibration curve is: y= 2.8758x+0.4960, the regression coefficient of the standard curve is: R ² =0.9991.

The following examples are only used to illustrate the present invention in detail and do not limit the scope of the invention in any way.

(1) Embodiment one:

Put 5mL kiwi fruit juice and 15mL methanol solution with a volume fraction of 50% into a 20mL volumetric flask, and extract at 63°C for 45min. After the extraction is completed, take 10mL of the extract and centrifuge at 5000r/min for 15min; after the centrifugation, take the supernatant and measure its absorbance value at the characteristic absorption wavelength of 715nm. Repeat the extraction and centrifugation steps 4 times, and take the supernatant after each centrifugation to measure the absorbance value. Bring the absorbance values measured for a total of 5 times into the pre-fitted standard curve equation to calculate the equivalent value of gallic acid, that is, the polyphenol content, and then calculate the concentration of each extraction by the formula W=C×V/M Finally, the polyphenol content is the total content of kiwifruit polyphenols obtained by adding the contents of five times.

(2) Embodiment two:

Put 5mL kiwi fruit juice and 15mL ethanol solution with a volume fraction of 50% into a 20mL volumetric flask, and extract at 63°C for 45min. After the extraction is completed, take 10mL of the extract and centrifuge at 5000r/min for 15min; after the centrifugation, take the supernatant and measure its absorbance value at the characteristic absorption wavelength of 715nm. Repeat the extraction and centrifugation steps 4 times, and take the supernatant after each centrifugation to measure the absorbance value. Bring the absorbance values measured for a total of 5 times into the pre-fitted standard curve equation to calculate the equivalent value of gallic acid, that is, the polyphenol content, and then calculate the concentration of each extraction by the formula W=C×V/M Finally, the polyphenol content is the total content of kiwifruit polyphenols obtained by adding the contents of five times.

(3) Embodiment three:

Put 5mL kiwi fruit juice and 20mL methanol solution with a volume fraction of 50% into a 20mL volumetric flask, and extract at 63°C for 45min. After the extraction is completed, take 10mL of the extract and centrifuge at 5000r/min for 15min; after the centrifugation, take the supernatant and measure its absorbance value at the characteristic absorption wavelength of 715nm. Repeat the extraction and centrifugation steps 4 times, and take the supernatant after each centrifugation to measure the absorbance value. Bring the absorbance values measured for a total of 5 times into the pre-fitted standard curve equation to calculate the equivalent value of gallic acid, that is, the polyphenol content, and then calculate the concentration of each extraction by the formula W=C×V/M Finally, the polyphenol content is the total content of kiwifruit polyphenols obtained by adding the contents of five times.

(4) Embodiment four:

Put 5mL kiwi fruit juice and 15mL methanol solution with a volume fraction of 50% into a 20mL volumetric flask, and extract at 61°C for 45min. After the extraction is completed, take 10mL of the extract and centrifuge at 5000r/min for 15min; after the centrifugation, take the supernatant and measure its absorbance value at the characteristic absorption wavelength of 715nm. Repeat the extraction and centrifugation steps 4 times, and take the supernatant after each centrifugation to measure the absorbance value. Bring the absorbance values measured for a total of 5 times into the pre-fitted standard curve equation to calculate the equivalent value of gallic acid, that is, the polyphenol content, and then calculate the concentration of each extraction by the formula W=C×V/M Finally, the polyphenol content is the total content of kiwifruit polyphenols obtained by adding the contents of five times.

Result analysis:

As can be seen from the table, the extraction rate is the largest in Example 1, and the polyphenols extracted are the most. Therefore, the best conditions for the determination of polyphenol content in kiwifruit were: the solid-liquid ratio of kiwifruit juice to alcoholic solution was 1:3, the alcoholic solution was methanol, and the extraction temperature was 63°C. And as can be seen from the table, the result difference between embodiment three and embodiment one is the smallest, and the result difference between embodiment two and embodiment one is the largest, and the difference between embodiment two, three, four and embodiment is respectively: alcoholic solution kind is different, The solid-liquid ratio is not used and the extraction temperature is different. Therefore, it can be judged that the alcohol solution type, the solid-liquid ratio and the extraction temperature affect the extraction results in the following order: alcohol solution type > extraction temperature > solid-liquid ratio.

Claims (6)

1. a kiwi fruit polyphenol content assay method is characterized in that: the concrete steps are: (1) Extraction: put a certain amount of squeezed kiwi fruit juice and alcohol solution with a volume fraction of 50% in a 20mL volumetric flask, and extract at 61-64°C for 45min; (2) Determination of the absorbance value: after the extraction is completed, take 10mL of the extract and centrifuge at 5000r/min for 15min; after the centrifugation is completed, take the supernatant and measure the absorbance value at the characteristic absorption wavelength of 715nm; (3) repeated extraction: get the centrifuged supernatant, repeat steps (1), (2) 4 times; (4) Calculation of polyphenol equivalent: put the absorbance value measured 5 times into the pre-fitted standard curve equation y=2.8758x+0.4960 to calculate the gallic acid equivalent value; in the standard curve, y represents polyphenol The mass concentration of phenol, x represents the absorbance value; (5) Calculation of polyphenol content: the calculation formula of polyphenol content is: W=C×V/M; Wherein, W is the content of polyphenol, and C is gallic acid equivalent, promptly calculates according to the polyphenol content of standard curve, V is the volume of the extract, and M is the amount of liquid taken; the total polyphenol content can be obtained by adding the polyphenol content measured five times. 2. kiwi fruit polyphenol content assay method according to claim 1, is characterized in that: the determining method of characteristic absorption wavelength described in step (2) is: get 1mL gallic acid standard solution, 1mL folin phenol reagent and 14mL water Put it into a 20mL volumetric flask, mix it evenly, add 3mL of 20wt% sodium carbonate solution within 1-5min, mix it evenly, and then make it to volume; place it at 25-30°C for 1h, and use a spectrophotometer at 200-800nm Carry out full-wavelength scanning to determine the wavelength at the time of maximum absorption; the measured characteristic absorption wavelength is 715nm. 3. kiwi fruit polyphenol content assay method according to claim 1, is characterized in that: the concrete steps of standard curve fitting described in step (4) are: (1) Preparation of gradient concentration solution: put 100mg of gallic acid into a 20mL volumetric flask, dissolve it with 2mL of ethanol and make it to 20mL; pipette the above solutions 0mL, 0.2mL, 0.4mL, 0.6mL, 1mL and 2mL to 20mL capacity In the bottle, add water to 20mL to obtain a series of gradient concentration solutions; each take 0.2mL of the above gradient concentration solution, 1mL of Folin phenol reagent and 14mL water into a 20mL volumetric flask, mix well and add 3mL within 1 to 5min 20wt% sodium carbonate solution, mixed evenly and then constant volume; placed at 25°C for 1h; (2) Draw a standard curve: each get 2 mL of the above-mentioned configured gradient concentration solutions in the quartz dish of the spectrophotometer, and measure the absorbance of these gradient concentration solutions at 715 nm; in the coordinate system, the absorbance is the abscissa, and the mass concentration Fitting is performed for the ordinate to obtain a standard curve; the equation of the standard curve is: y=2.8758x+0.4960, and the regression coefficient of the standard curve is: R ² =0.9991. 4. the kiwi fruit polyphenol extraction process according to claim 1 is characterized in that: the alcoholic solution described in the step (1) is a methanol solution. 5. The process for extracting kiwifruit polyphenols according to claim 1, characterized in that: the extraction temperature in step (1) is 63°C. 6. the process for extracting kiwifruit polyphenols according to claim 1 is characterized in that: the volume ratio of kiwifruit juice and alcohol solution is 1:3 during leaching.