Black potato anthocyanin as well as extraction and purification method and application thereof

Abstract

The invention belongs to the technical field of agricultural and sideline product processing, and particularly relates to black potato anthocyanin and an extraction and purification method and application thereof. The extraction and purification method comprises the following steps: (1) mixing black potatoes with ethanol, pulping to obtain homogenate, adjusting the pH value of the homogenate to 3-4, carrying out ultrasonic treatment, centrifuging, taking supernatant, concentrating the supernatant, and freeze-drying to obtain a black potato anthocyanin crude product; and (2) dissolving the black potato anthocyanin crude product to obtain a black potato anthocyanin crude extract, adsorbing the black potato anthocyanin crude extract with macroporous resin for 1-12 hours, eluting with ethanol, collecting eluent, concentrating and drying to obtain the black potato anthocyanin. According to the extraction and purification method disclosed by the invention, the black potato anthocyanin with the content of 485.12 mg / g can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural and sideline product processing, and in particular relates to black potato anthocyanin and an extraction and purification method and application thereof. Background Art

[0002] In recent years, natural active ingredients from plants have been favored by food and pharmaceutical developers for their good biological activities, such as anti-oxidation and hypoglycemic effects. Anthocyanins are polyphenol compounds that are widely found in nature. Due to their good antioxidant and antibacterial effects, they are widely used by researchers in the fields of medicine, food and cosmetics.

[0003] Black potato is a variant of ordinary potato, rich in anthocyanins, with physiological functions such as anti-oxidation and hypoglycemic, and can be widely used in food or functional food. Anthocyanins can be extracted by direct extraction or auxiliary extraction, but different plants have different properties, and the methods of extracting anthocyanins are also different.

[0004] At present, no suitable method for extracting anthocyanins from black potatoes has been found. Summary of the invention

[0005] In view of this, the object of the present invention is to provide anthocyanidins from black potatoes and a method for extracting and purifying the anthocyanidins and an application thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for extracting and purifying anthocyanins from black potatoes, comprising the following steps:

[0008] (1) mixing black potato with ethanol, beating to obtain a homogenate, adjusting the pH of the homogenate to 3-4, ultrasonicating, centrifuging, taking the supernatant, concentrating the supernatant, and freeze-drying to obtain a crude black potato anthocyanin product;

[0009] (2) dissolving the crude black potato anthocyanin to obtain a crude black potato anthocyanin extract, adsorbing the crude black potato anthocyanin extract through a macroporous resin for 1 to 12 hours, eluting with ethanol, collecting the eluate, concentrating, and drying to obtain black potato anthocyanin.

[0010] Preferably, the initial water content of the black potato is 77-80%;

[0011] The mass volume ratio of the black potato and ethanol is 1g:1-2mL;

[0012] The initial concentration of ethanol in step (1) is 45-55%;

[0013] The initial pH of the ethanol in step (1) is 3.2 to 3.7.

[0014] Preferably, the ultrasonic time in step (1) is 25 to 35 minutes, the ultrasonic temperature is 23 to 27° C., and the ultrasonic power is 230 to 250 W.

[0015] Preferably, the centrifugal temperature in step (1) is 2-6°C, the centrifugal speed is 7500-8500 rpm, and the centrifugal time is 2.5-3.5 min.

[0016] Preferably, the macroporous resin in step (2) is macroporous resin AB-8; the sample loading during the adsorption in step (2) is 10 to 40 mL; during the ethanol elution in step (2), the initial concentration of ethanol is 80 to 100%, and the pH of ethanol is 3.5 to 4.5.

[0017] The invention also provides black potato anthocyanin prepared according to the extraction and purification method.

[0018] The present invention also provides the use of the black potato anthocyanin prepared according to the extraction and purification method in preparing products with antioxidant capacity.

[0019] Preferably, the product is food, medicine or cosmetics.

[0020] The invention also provides the application of the black potato anthocyanin in preparing products with antioxidant capacity.

[0021] Preferably, the product is food, medicine or cosmetics.

[0022] The present invention provides anthocyanidin from black potato and an extraction and purification method and application thereof. The extraction and purification method of the present invention comprises the following steps: (1) mixing black potato with ethanol, beating to obtain a homogenate, adjusting the pH of the homogenate to 3-4, ultrasonicating, centrifuging, taking a supernatant, concentrating the supernatant, and freeze-drying to obtain a crude product of anthocyanidin from black potato; (2) dissolving the crude product of anthocyanidin from black potato to obtain a crude extract of anthocyanidin from black potato, adsorbing the crude extract of anthocyanidin from black potato with a macroporous resin for 1-12 hours, eluting with ethanol, collecting the eluate, concentrating, and drying to obtain anthocyanidin from black potato. The extraction and purification method of the present invention has a higher content of anthocyanidin from black potato than that of the traditional aqueous solution extraction method and ethanol solution extraction method, and the content in the crude product reaches 57.20 mg / g. The crude product of black potato anthocyanin was purified by macroporous resin, and the adsorption rate of the macroporous resin could reach 96.43%, the resolution rate could reach 97.93%, and the content of anthocyanin in black potato anthocyanin could reach 485.12 mg / g. Therefore, it can be seen that the method of the present invention can be used to extract and purify black potato anthocyanin with high anthocyanin content.

[0023] The antioxidant properties of black potato anthocyanins were studied, and the results showed that black potato anthocyanins had good DPPH free radical scavenging rate, hydroxyl free radical scavenging ability, ABTS cation free radical scavenging rate and iron ion reducing ability. The scavenging rate of 0.2 mg / mL black potato anthocyanin solution on DPPH free radicals was 93.75%, the scavenging rate of 1 mg / mL black potato anthocyanin solution on ABTS free radicals was 85.4%, the scavenging rate of 1 mg / mL black potato anthocyanin solution on OH free radicals was 69.03%, and the total reducing ability of 0.1 mg / mL black potato anthocyanin solution was 0.46, indicating that it has good antioxidant activity, and the scavenging rate and reducing ability increased significantly with the increase of anthocyanin concentration (P < 0.05), showing concentration dependence. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The content of anthocyanins in black potatoes extracted by different methods;

[0025] Figure 2 This is the result of full wavelength scanning of black potato extract sample by UV spectrophotometer;

[0026] Figure 3 The adsorption effect of different sample loading amounts on macroporous resin AB-8;

[0027] Figure 4 This is the macroporous resin gradient elution curve of crude black potato anthocyanin;

[0028] Figure 5 This is the purification elution curve of crude black potato anthocyanin;

[0029] Figure 6 This is the Fourier infrared spectrum of anthocyanin in black potato;

[0030] Figure 7 The effect of light on the stability of anthocyanins in black potatoes;

[0031] Figure 8 The effect of temperature on the stability of anthocyanins in black potatoes;

[0032] Fig. 9 The effect of pH on the stability of anthocyanins in black potatoes;

[0033] Fig.10 The effect of metal ions on the stability of anthocyanins in black potatoes (Figure A shows the residual amount of anthocyanins after the reaction of metal ions and anthocyanins; Figure B shows the intuitive picture after the reaction of metal ions and anthocyanins);

[0034] Fig.11 The scavenging ability of black potato anthocyanin on DPPH free radicals;

[0035] Fig.12 The effect of anthocyanins from black potatoes on ABTS + Effect of free radical scavenging (Figure A shows the effect of Vc concentration on ABTS· + The effect of anthocyanin concentration on free radical scavenging; Figure B shows the effect of anthocyanin concentration on ABTS· + free radical scavenging effects);

[0036] Fig.13 The scavenging ability of black potato anthocyanins on hydroxyl free radicals;

[0037] Fig.14 The effect of anthocyanins from black potatoes on the ability to reduce iron ions. DETAILED DESCRIPTION

[0038] The present invention provides a method for extracting and purifying anthocyanins from black potatoes, comprising the following steps:

[0039] (1) mixing black potato with ethanol, beating to obtain a homogenate, adjusting the pH of the homogenate to 3-4, ultrasonicating, centrifuging, taking the supernatant, concentrating the supernatant, and freeze-drying to obtain a crude black potato anthocyanin product;

[0040] (2) dissolving the crude black potato anthocyanin to obtain a crude black potato anthocyanin extract, adsorbing the crude black potato anthocyanin extract through a macroporous resin for 1 to 12 hours, eluting with ethanol, collecting the eluate, concentrating, and drying to obtain black potato anthocyanin.

[0041] In the present invention, the initial water content of the black potato is 77-80%, preferably 78%; the mass volume ratio of the black potato and ethanol is 1g:1-2mL, preferably 1g:1mL; the initial concentration of ethanol in step (1) is 45-55%, preferably 50%; the initial pH of ethanol in step (1) is 3.2-3.7, preferably 3.5.

[0042] In the present invention, the ultrasonic time in step (1) is 25 to 35 minutes, preferably 30 minutes, the ultrasonic temperature is 23 to 27°C, preferably 25°C, and the ultrasonic power is 230 to 250W, preferably 240W.

[0043] In the present invention, the centrifugal temperature in step (1) is 2-6°C, preferably 4°C, the centrifugal speed is 7500-8500rpm, preferably 8000rpm, and the centrifugal time is 2.5-3.5min, preferably 3min.

[0044] In the present invention, the macroporous resin in step (2) is macroporous resin AB-8; the sample loading during the adsorption in step (2) is 10 to 40 mL, preferably 20 mL; during the ethanol elution in step (2), the initial concentration of ethanol is 80 to 100%, preferably 80%, and the pH of ethanol is 3.5 to 4.5, preferably 4.0.

[0045] The invention also provides black potato anthocyanin prepared according to the extraction and purification method.

[0046] The present invention also provides the use of the black potato anthocyanin prepared according to the extraction and purification method in preparing products with antioxidant capacity.

[0047] In the present invention, the product is food, medicine or cosmetics.

[0048] The invention also provides the application of the black potato anthocyanin in preparing products with antioxidant capacity.

[0049] In the present invention, the product is food, medicine or cosmetics.

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] The black potatoes used in the embodiments of the present invention are commercially available fresh Liangshan black potatoes;

[0052] Sources of reagents used in the examples of the present invention:

[0053] Hydrochloric acid and sodium hydroxide were purchased from Chengdu Kelong Chemical Co., Ltd.; anhydrous ethanol was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; macroporous resin AB-8 was purchased from Shanghai Yuanye Biological Co., Ltd.;

[0054] The instruments and equipment used in the embodiments of the present invention are:

[0055] Juicer JS42D-300 (A03) 83271 was purchased from Zhejiang Shaoxing Supor Home Appliances Co., Ltd.; electronic balance ZG-TP101 was purchased from Wuyi Zhuheng Electronics Co., Ltd.; analytical balance ME204E / 02 was purchased from Mettler-Toledo Instrument (Shanghai) Co., Ltd.; digital display constant temperature water bath HH-6 was purchased from Guohua (Changzhou) Instrument Manufacturing Co., Ltd.; UV-visible spectrophotometer UV-1900 was purchased from Aoyi Instrument (Shanghai) Co., Ltd.; refrigerator BCD-211TMBA was purchased from Qingdao Haier Co., Ltd.; glass cuvette 10 mm, purchased from Aoyi Instrument (Shanghai) Co., Ltd.; quartz cuvette 10 mm, purchased from Aoyi Instrument (Shanghai) Co., Ltd.; electric constant temperature blast drying oven LDO-9240A purchased from Shanghai Longyue Instrument Equipment Co., Ltd.; rotary evaporator RE-52AA purchased from Zhengzhou Yarong Instrument Co., Ltd.; vacuum pump SHZ-D (Ⅲ) purchased from Zhengzhou Yarong Instrument Co., Ltd.; desktop refrigerated centrifuge Centrifuge5810R purchased from Germany's Ebende Co., Ltd.; pH meter PHS-3E purchased from Shanghai Yidian Scientific Instrument Co., Ltd.; vacuum freeze dryer ALPHR 1-4LSC purchased from Germany's Christ Group; constant flow pump YZ1515X-B purchased from Baoding Lange Constant Flow Pump Co., Ltd.; ultrasonic cleaning machine SB-5200DT purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0056] Example 1

[0057] The moisture content of fresh black potatoes was measured. Weigh the aluminum dishes and number them, wash them, place them in a 105°C drying oven, dry them for 2 hours, then seal the container lid, take them out and place them in a dryer to cool, weigh them, and repeat several times until the weight is constant. Dry them again under the same conditions. Then weigh about 5g (0.0001g) of the sample (black potato) in the aluminum dishes, the sample thickness is less than 5mm, cover it with a lid, weigh it, place it in a 105°C oven to dry for 3 hours, seal the container lid, cool and weigh it until the weight is constant. The results showed that the moisture content of the black potatoes was 78.22±0.91%, indicating that the moisture content of the black potatoes is relatively high and relatively fresh.

[0058] (1) Aqueous solution extraction method

[0059] Select fresh black potatoes that are not rotten, unsprouted, and have full grains. Wash the black potatoes, cut them into small pieces of uniform size and set aside.

[0060] Specific operation: Mix black potato with water at a mass volume ratio of 1g:1mL, place in a juicer to make pulp, adjust the pH of the obtained homogenate to 3.5 with a hydrochloric acid solution with a concentration of 1mol / L, and place the homogenate in a 30℃ constant temperature water bath for 1h. Centrifuge at 4℃ and 8000rpm for 3min to take the supernatant, then concentrate the supernatant at 40℃, freeze-dry into powder, and finally obtain the crude black potato anthocyanin, which is stored in a -80℃ refrigerator away from light.

[0061] (2) Ethanol solution extraction method

[0062] Select fresh black potatoes that are not rotten, unsprouted, and have full grains. Wash the black potatoes, cut them into small pieces of uniform size and set aside.

[0063] Mix black potato with 50% ethanol at a mass volume ratio of 1g:1mL, place in a juicer to make pulp, adjust the pH of the obtained homogenate to 3.5 with a hydrochloric acid solution with a concentration of 1mol / L, and place the homogenate in a 30℃ constant temperature water bath for 1h. Centrifuge at 4℃ and 8000rpm for 3min, take the supernatant, and then concentrate the supernatant at 40℃, freeze-dry into powder, and finally obtain the crude black potato anthocyanin, which is stored in a -80℃ refrigerator away from light.

[0064] (3) Ultrasonic-assisted acidic ethanol extraction

[0065] Select fresh black potatoes that are not rotten, unsprouted, and have full grains. Wash the black potatoes, cut them into small pieces of uniform size and set aside.

[0066] Black potato is mixed with 50% ethanol (the pH of the ethanol is adjusted to 3.5 with hydrochloric acid) at a mass volume ratio of 1 g: 1 mL, and the mixture is placed in a juicer for pulping. The pH of the homogenate is adjusted to 3.5, and the mixture is subjected to ultrasound at 25°C and 240 W for 30 min. The mixture is centrifuged at 4°C and 8000 rpm for 3 min, and the supernatant is taken. The supernatant is then concentrated at 40°C and freeze-dried into powder to obtain the crude black potato anthocyanin product, which is stored in a -80°C refrigerator away from light.

[0067] The content of anthocyanins in crude black potato anthocyanins obtained by different extraction methods was determined according to the following method: pure water was used as the reference zero sample on the spectrophotometer. A certain mass of crude black potato anthocyanins was weighed and dissolved to 10 mL with potassium chloride-hydrochloric acid solution of pH 1.0 and pH 4.5, respectively, in three groups, and the content of anthocyanins in crude black potato anthocyanins extracted by different methods was determined under the condition of maximum absorption wavelength (532 nm), and all samples were also required to measure the absorbance value at 700 nm.

[0068]

[0069] Where A=[(A 最大吸收-A 700 ) pH1.0 -(A 最大吸收 -A 700 ) pH4.5 ];

[0070] MW: anthocyanin mass fraction;

[0071] DF: dilution factor;

[0072] ε: molar absorption coefficient (26900L·cm -1 ·mol -1 );

[0073] L: optical path (1 cm);

[0074] V: volume of solution;

[0075] W t : Sample quality.

[0076] The results of the test are as follows Figure 1 shown.

[0077] Figure 1 It was shown that there were significant differences in the extraction rates of anthocyanins from black potatoes using the three methods (P<0.05), among which the ultrasonic-assisted acidic ethanol extraction method had the highest anthocyanin content of 57.20 mg / g, followed by the ethanol extraction method, and the aqueous solution extraction method had the lowest anthocyanin content. From the above results, it can be seen that ethanol solution is more conducive to the extraction of anthocyanins from black potatoes than aqueous solution. Since ultrasound helps the dissolution of anthocyanins in black potatoes, and the acidic environment can effectively protect anthocyanins from being destroyed, the ultrasonic-assisted acidic ethanol extraction method showed the highest extraction rate. Therefore, the subsequent experiments selected the ultrasonic-assisted acidic ethanol extraction method (the method of the present invention) to extract fresh black potatoes.

[0078] The crude anthocyanins from black potatoes extracted by the three methods were diluted with 5% hydrochloric acid ethanol solution and the absorbance at 350-700 nm was scanned by UV-visible spectrophotometer. Figure 2 As shown. The results show that the anthocyanin solution has a maximum absorption peak at 532nm, while the maximum absorption wavelength of anthocyanins disclosed in the prior art is generally 510-540nm. The black potato extracts extracted by different methods have a maximum absorption wavelength of 532nm. Explanation: The absorbance of the black potato extract at 532nm may be anthocyanins.

[0079] Example 2

[0080] (1) Pretreatment of macroporous resin AB-8

[0081] Soak the macroporous resin AB-8 in 4% sodium hydroxide solution for 24 hours, and repeatedly wash the macroporous resin AB-8 with pure water until it becomes neutral. Then soak the macroporous resin in 95% ethanol for 12 hours, and then wash it with pure water until the outflow liquid has no ethanol smell, and then it can be put into use.

[0082] (2) Resin enhanced regeneration method:

[0083] Soak the macroporous resin in 4% hydrochloric acid solution for 6 hours, and repeatedly wash the macroporous resin AB-8 with pure water until it becomes neutral. Then, wash it according to the pretreatment steps of the macroporous resin AB-8, and finally, it can be put into use.

[0084] (3) Adsorption and desorption experiments of macroporous resin

[0085] Accurately weigh 2.0 g of macroporous resin AB-8 into a blue-capped bottle, add 30 mL, 80 mg / mL crude extract of black potato anthocyanins obtained by ultrasonic-assisted acidic ethanol extraction in Example 1 (the crude black potato anthocyanins are dissolved in a potassium chloride-hydrochloric acid solution), place it on a constant temperature shaker at 25°C and a speed of 250 rpm for 12 h, filter it, elute it with 80% ethanol at pH = 4.0, and calculate the adsorption rate and resolution rate.

[0086]

[0087] Among them, c 初始 is the initial concentration of anthocyanins in the crude extract of black potato anthocyanins, (mg / mL);

[0088] c 吸附后 is the equilibrium concentration of anthocyanins in the crude extract of black potato anthocyanins (the concentration of anthocyanins remaining in the blue-capped bottle after adsorption), (mg / mL);

[0089] c 解析液 is the concentration of anthocyanins in the analytical solution after elution equilibrium (mg / mL);

[0090] V 解析液 is the volume of the eluent (mL);

[0091] V 吸附后 : The volume of the initial sample black potato anthocyanin crude extract (mL).

[0092] The results showed that the adsorption rate of black potato anthocyanins by macroporous resin AB-8 was 96.46%, and the desorption rate was 97.93%. This indicated that macroporous resin AB-8 had good adsorption / desorption capacity for black potato anthocyanins extracted by ultrasound-assisted acidic ethanol, and could be used for the subsequent separation and purification of black potato anthocyanins.

[0093] (4) Effects of sample loading and adsorption time on the adsorption effect of macroporous resin

[0094] Accurately weigh 2.0g of macroporous resin AB-8 in 4 150mL conical flasks, add 80mg / mL different volumes (10mL, 20mL, 30mL, 40mL) of crude extract of anthocyanins from black potatoes obtained by ultrasonic-assisted acidic ethanol extraction in Example 1 (the crude anthocyanins from black potatoes were dissolved in a potassium chloride-hydrochloric acid solution), shake at 25°C for 12h, the shaking rate was 250rpm, the anthocyanin content was measured, and the adsorption rate was calculated according to the above formula. The results are as follows Figure 3 shown.

[0095] Figure 3 The results show that there are differences in the adsorption rate of different sample amounts on the macroporous resin AB-8. With the increase of the sample amount, the adsorption rate decreases successively, but the adsorption rate increases sharply in 0-1h. The adsorption rate of 10mL sample amount is the largest (>90%) at 1h. The adsorption rate of 10mL sample amount increases slowly in 1-2h, and the adsorption rate of 30mL and 40mL sample amount increases rapidly in 1-6h. The adsorption rate of 30mL and 40mL sample amount gradually tends to equilibrium during 6-12h. When the sample amount is 30mL and the time is 8h, the adsorption rate of anthocyanin reaches 96.57%. However, the adsorption rate of 40mL sample amount is less than 90% at 12h. The adsorption rate of 10mL sample amount is the largest at 6h, reaching 97.86%, reaching adsorption equilibrium. Finally, it is appropriate to select a sample amount of 10mL and an adsorption time of 6h.

[0096] (5) Determination of the concentration of the dynamic gradient eluent of the macroporous resin

[0097] After loading the macroporous resin AB-8, prepare 100 mg / mL crude extract of anthocyanins from black potato obtained by ultrasonic-assisted acidic ethanol extraction in Example 1, 5 mL sample volume, and static adsorption for 8 hours in the dark. Elution was performed in sequence with 0%, 20%, 40%, 60%, 80%, and 100% ethanol (acidic ethanol) at pH=4, flow rate: 1.5 ml / min, collection frequency: 5 ml / time, and the optimal elution concentration was determined by absorbance at 530 nm.

[0098] Figure 4The results show that the absorbance of 0%, 20%, 40%, and 60% ethanol acidic eluents at 530nm are basically close to 0, while the 80% ethanol acidic (pH = 4) eluent has a higher absorbance, indicating that the 0-60% ethanol acidic (pH = 4) eluent cannot effectively elute the black potato anthocyanidin adsorbed on AB-8, while the 80% ethanol acidic (pH = 4) eluent has a larger absorption peak at 530nm, indicating that the 80% ethanol acidic (pH = 4) eluent can effectively elute the black potato anthocyanidin on the macroporous resin. In summary, the 80% ethanol acidic (pH = 4) solution is used as the eluent to purify the black potato anthocyanidin.

[0099] Example 3

[0100] Purification of crude anthocyanins from black potato

[0101] After loading the macroporous resin AB-8, prepare 100 mg / mL crude extract of anthocyanins from black potato obtained by ultrasonic-assisted acidic ethanol extraction in Example 1, 10 mL sample volume, and static adsorption for 6 hours in the dark. Then use 80% ethanol (acidic ethanol) with pH = 4 to elute in sequence, flow rate: 1.5 ml / min, collection frequency: 5 ml / time, collect anthocyanin samples by absorbance at 530 nm, concentrate, and freeze-dry for use. The results are as follows Figure 5 shown.

[0102] Figure 5 It shows that the absorbance of the eluent received at 140mL is less than 0.01 at 530nm. From 140mL to 155mL, the absorbance at 530nm rises rapidly to the highest point, and the absorbance at the highest point is 1.43. From 155mL to 175mL, the absorbance at 530nm begins to drop rapidly to an absorbance of 0.27, and then from 175mL to 225mL, the absorbance at 530nm slowly decreases to 0.05. It can be seen that the macroporous resin AB-8 elutes the anthocyanins from black potato. With the increase of the analytical solution, the anthocyanin content in the eluent of black potato anthocyanins is getting higher and higher. Considering the optimal volume of the analytical solution is 225mL, the analytical equilibrium is reached at this time. After sample purification, the anthocyanin content of black potato is 485.12mg / g.

[0103] Example 4

[0104] The purified black potato anthocyanin from Example 3 was placed in a mortar and ground uniformly, and infrared spectrum scanning was performed using a Fourier transform infrared spectrometer at 25°C with a scanning wavelength range of 400 cm -1 -4000cm -1 The results are as follows Figure 6 shown.

[0105] Figure 6Display, located at about 1612cm -1 Corresponding to the stretching vibration of (C=O), identifying the characteristic absorption of aldehydes and ketones in molecules; at 1568cm -1 and 1482cm -1 These bands may be related to (C=C), identifying the benzene ring; at 1218cm -1 The left and right dense bands are related to the vibration of (CO), judging the presence of phenols; 927cm -1 It may be related to the in-plane bending vibration of (OH), which is used to determine the cis-trans and substitution types, or it may be caused by the stretching vibration of (COC). These peaks in the freeze-dried anthocyanidin powder are the main peaks in cyanidin-3-glucoside and cyanidin-3-galactoside, and it can be determined that the extracted black potato extract is anthocyanidin.

[0106] Experimental Example 1

[0107] Effect of light on the stability of anthocyanins in black potatoes

[0108] The purified black potato anthocyanin from Example 3 was prepared with distilled water to a certain concentration, and the anthocyanin content was measured and calculated. One group was placed under sufficient light, and the other group was placed under light-proof conditions. The change rate of anthocyanin was measured every 2 hours until 8 hours. The results are as follows Figure 7 shown.

[0109] Figure 7 It shows that with the extension of the illumination and light-proof treatment time, the entire curve of anthocyanin decomposition shows a similar downward trend, that is, the anthocyanin content gradually decreases, but the anthocyanin content of the illumination treatment decreases faster than that of the light-proof treatment; and the decline trend of the illumination and light-proof treatments from 0 to 4 hours is relatively rapid, and the difference in anthocyanin content between the two treatments is not significant; the decline trend of the illumination and light-proof treatments from 4 to 8 hours is relatively slow, but the decline of the illumination treatment is more obvious than that of the light-proof treatment. The above results show that anthocyanins will decompose in both illumination and light-proof storage, but the decomposition of anthocyanins caused by light is more obvious than that of light-proof storage. It can be seen that light will accelerate the decomposition of anthocyanins and affect the stability of black potato anthocyanins, so during the storage of black potato anthocyanins, light-proof storage should be considered.

[0110] Experimental Example 2

[0111] Effect of temperature on the stability of anthocyanins in black potatoes

[0112] Phosphate buffer solutions with pH values ​​of 1.0, 3.0, 5.0, 7.0, and 9.0 were prepared, and equal masses of the purified black potato anthocyanins from Example 3 were weighed and placed in phosphate buffer solutions with different pH values. Anthocyanin solutions of a certain concentration were obtained, and a certain volume of anthocyanin solutions were taken and placed in water baths at 30°C, 55°C, and 80°C for 12 hours. The anthocyanin content was determined by the pH differential method every 2 hours to obtain the changes in anthocyanin content at different pH values ​​and temperatures. The results are shown in FIG. Figure 8 shown.

[0113] Figure 8 It shows that under constant temperature water baths at 30℃, 55℃ and 80℃, the anthocyanin content gradually decreases with the increase of treatment time; and at 0-2h, the anthocyanin content at the three treatment temperatures decreases at a faster rate; at 2-8h, it decreases more slowly. At the same time, it was found that after the same concentration of anthocyanin solution was treated at 30℃, 55℃ and 80℃ for 8h, the anthocyanin residue in the solution was the highest at 30℃ (4.732mg / mL), followed by 50℃, and the lowest was 80℃. The above results show that high temperature will accelerate the decomposition of anthocyanins, and the higher the temperature, the worse the stability of anthocyanins, and the longer the treatment time, the worse the stability of anthocyanins.

[0114] Experimental Example 3

[0115] Effect of pH on the stability of anthocyanins in black potatoes

[0116] The purified black potato anthocyanidin in Example 3 was prepared into a 4.95 mg / mL solution using phosphate buffers of pH=2, pH=5, pH=8 and pH=11, and the anthocyanidin residue in the buffer was measured every 2 hours. Fig. 9 shown.

[0117] Fig. 9 It shows that under the treatment of buffer solutions of pH=2, pH=5, pH=8 and pH=11, the residual anthocyanin in the black potato anthocyanin solution shows a downward trend with the increase of treatment time; and the anthocyanin content decreases rapidly within 0-2h, and the anthocyanin content tends to be stable at 2-8h. These results show that different pH values ​​will cause the decomposition of black potato anthocyanin, and the anthocyanin change rate is more obvious within 2h. Under the treatment of buffer solutions of pH=2, pH=5, pH=8 and pH=11, as the pH value increases, the anthocyanin content decreases more significantly, indicating that the higher the pH value, the easier it is for black potato anthocyanin to decompose. The above results show that black potato anthocyanin is easier to decompose in an alkaline environment than in an acidic environment, and the lower the pH value, the higher the stability of black potato anthocyanin.

[0118] Experimental Example 4

[0119] Effects of Metal Ions on the Stability of Anthocyanins from Potatoes

[0120] The purified black potato anthocyanin in Example 3 was prepared into an anthocyanin solution of 0.650 mg / mL using pure water. 15 portions of 1 mL were taken and divided into 5 groups. 1% CaCl2, FeCl3, KCl, NaCl and pure water were added in a ratio of 1:1. The reaction was carried out at room temperature for 5 minutes. The anthocyanin content of each group was measured and the color change was recorded by taking photos. The results are shown in Figure 2. Fig.10 shown.

[0121] Fig.10 Shown, through Fe 3+ The anthocyanin residue in the treated anthocyanin solution was the lowest, which was 0.306 mg / mL. 2+ and Na + The anthocyanin content of the treated samples was higher than that of the untreated samples, indicating that Ca 2+ and Na + Can prevent the degradation of anthocyanins in black potatoes; + There is no significant difference in the anthocyanin content between the anthocyanin solution treated with Fe and the anthocyanin content without adding metal ions (Figure A). 3+ The anthocyanin solution treated with Ca 2+ and Na + The anthocyanin solution treated with K was darker than that without adding metal ions. + The color of anthocyanin solution treated with Fe is similar to that of anthocyanin solution not treated with metal ions. 3+ It has a strong destructive effect on anthocyanins. 2+ and Na + It has a certain protective effect on anthocyanins. + The effect of Fe on anthocyanin was not obvious. 3+ It will reduce the stability of anthocyanins in black potatoes, Ca 2+ and Na + Will maintain the stability of black potato anthocyanins, K + It has no obvious effect on the stability of anthocyanins in black potatoes.

[0122] Experimental Example 5

[0123] Determination of DPPH free radical scavenging ability

[0124] DPPH free radical is widely used in the determination of antioxidant activity, with maximum absorption at 517nm. Preparation of 0.2mmol / L DPPH ethanol solution: Accurately weigh 0.005g of DPPH powder, fully dissolve it with anhydrous ethanol, and transfer it into a 100mL brown volumetric flask. After constant volume, a 0.2mmol / L DPPH standard ethanol solution is successfully prepared. Store it at 4℃ away from light, and it is best to prepare it before use.

[0125] The purified black potato anthocyanin in Example 3 was dissolved in a potassium chloride-hydrochloric acid buffer solution to prepare anthocyanin solutions of different concentrations for later use.

[0126] Experimental group: Pipette 2.0mL of anthocyanin solution with different concentrations (0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2mg / mL) into a 10mL centrifuge tube, then add 2.0mL of 0.2mmol / L DPPH ethanol solution respectively, react in the dark for 30min, and measure the absorbance value As at 517nm. Adjust to zero with distilled water. Control group: Replace 2.0mL of DPPH ethanol solution with 2.0mL of anhydrous ethanol, and measure the absorbance value Ac according to the above method. Blank group: Replace 2.0mL of anthocyanin solution with 2.0mL of distilled water, and measure the absorbance value A0 according to the above method. Take Vc as the positive control, and measure its DPPH free radical scavenging rate according to the above method. Each sample was measured in parallel 3 times.

[0127] The calculation formula is as follows:

[0128] DPPH free radical scavenging rate = [1-(As-Ac) / A0] × 100%.

[0129] The results of the test are as follows Fig.11 shown.

[0130] Fig.11 The results showed that in the concentration range of 0.02-0.20 mg / mL, the DPPH radical scavenging ability of black potato anthocyanin and Vc showed an upward trend, indicating that black potato anthocyanin and Vc have a certain scavenging ability against DPPH radicals in the concentration range of 0.02-0.20 mg / mL, and the DPPH radical scavenging rate increases with the increase of black potato anthocyanin concentration. In the concentration range of 0.02-0.10 mg / mL, the DPPH radical scavenging rate of Vc increased rapidly, and the DPPH radical scavenging rate reached 95.05% at 0.10 mg / L. After 0.10 mg / L, the DPPH radical scavenging rate of Vc showed a relatively gentle upward trend. In the concentration range of 0.02-0.20 mg / mL, the DPPH radical scavenging rate of black potato anthocyanin increased steadily, and the DPPH radical scavenging rate reached 93.75% at 0.20 mg / L. This indicates that the black potato anthocyanin sample has good DPPH free radical scavenging ability, and the DPPH free radical scavenging rate increases significantly with the increase of anthocyanin concentration (P<0.05), showing concentration dependence.

[0131] Experimental Example 6

[0132] ABTS cation free radical scavenger+ ) Determination of removal capacity

[0133] ABTS is a synthetic non-physiological free radical with a maximum absorption value at 734nm. After oxidation, it will become a stable blue cation, which can be reduced to colorless by anthocyanins. Preparation of 7mmol / LABTS cation free radical working solution: weigh 0.384g ABTS, dissolve it and dilute it to 100ml with deionized water to obtain 7mmol / LABTS solution; weigh 0.0662g potassium persulfate, dissolve it and dilute it to 100mL with deionized water to obtain 2.45mmol / L potassium persulfate solution. Mix the two solutions in equal volumes, stand at room temperature and avoid light for more than 12h, and use anhydrous ethanol to release the solution until the absorbance at 734nm is between 0.70±0.02, and the ABTS cation free radical working solution is obtained.

[0134] The purified black potato anthocyanin in Example 3 was dissolved in a potassium chloride-hydrochloric acid buffer solution to prepare anthocyanin solutions of different concentrations for later use.

[0135] Experimental group: Take 0.1mL of anthocyanin solution of different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mg / mL) in a 10mL centrifuge tube, add 3.9mL of ABTS cation radical working solution respectively, shake for 10s to fully mix, water bath at 27°C for 6min, and immediately measure the absorbance As at 734nm. Adjust to zero with distilled water. Blank group: Use 0.1mL of anhydrous ethanol instead of anthocyanin solution, and measure the absorbance A0 according to the above method. Take Vc as the positive control, and determine its ABTS cation radical scavenging rate according to the above method. Each sample was measured in parallel 3 times. The results are as follows Fig.12 shown.

[0136] The calculation formula is as follows:

[0137] ABTS cation radical scavenging rate = [(A0-As) / A0] × 100%.

[0138] Fig.12The results showed that within the concentration range of 0.01-0.10 mg / mL, the ABTS cation radical scavenging rate of Vc showed a steady upward trend. At 0.10 mg / mL, the ABTS cation radical scavenging rate was 72.15%, indicating that Vc has good ABTS cation radical scavenging ability, and the ABTS cation radical scavenging rate will increase with the increase of Vc concentration. Within the concentration range of 0.1-1.0 mg / mL, the ABTS cation radical scavenging rate of black potato anthocyanin also gradually increased. At 1.0 mg / mL, the ABTS cation radical scavenging rate reached 85.4%, indicating that black potato anthocyanin also has good ABTS cation radical scavenging ability. The results showed that both black potato anthocyanin and Vc have good ABTS cation radical scavenging rates, and the ABTS cation radical scavenging rates gradually increase with the increase of concentration (P < 0.05), showing concentration dependence.

[0139] Experimental Example 7

[0140] Scavenging ability of anthocyanins from black potato on hydroxyl radicals (·OH)

[0141] Hydroxyl radicals are free radicals with maximum absorption at 510nm in nature and very strong oxidizing ability. The concentration of ·OH in the system can be understood based on the absorbance value. The higher the ability to remove ·OH, the stronger the antioxidant effect. Preparation of 9mmol / L salicylic acid ethanol solution: accurately weigh 0.3355g of 3,5-dinitrosalicylic acid, dissolve it in anhydrous ethanol and dilute it to a 250mL volumetric flask. Preparation of 9mmol / L ferrous sulfate solution: accurately weigh 2.5000g of ferrous sulfate, dissolve it in deionized water and dilute it to a 250mL volumetric flask. Preparation of 8.8mmol / L H2O2 solution: measure 0.25mL of 30% H2O2 solution and dilute it to 250mL.

[0142] The purified black potato anthocyanin in Example 3 was dissolved in a potassium chloride-hydrochloric acid buffer solution to prepare anthocyanin solutions of different concentrations for later use.

[0143] Experimental group: Take 1.0mL of anthocyanin solution with different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1mg / mL) and dissolve it in a 10mL centrifuge tube, add 1.0mL of 9mmol / L ferrous sulfate solution, then add 1.0mL of 9mmol / L salicylic acid ethanol solution, mix well and let stand for 10min, then add 1mL of 8.8mmol / L H2O2 solution to start the reaction, mix well and keep in a constant temperature water bath at 37℃ for 30min, cool and measure the absorbance value As at a wavelength of 510nm. Adjust to zero with distilled water. Blank group: Use 1.0mL of distilled water instead of anthocyanin solution, and measure the absorbance value A0 according to the above method. Control group: Use 1.0mL of distilled water instead of 1.0mL of H2O2 aqueous solution, and measure the absorbance value Ac according to the above method. Using Vc as the positive control, the OH clearance rate was determined according to the above method. The determination was repeated three times. Fig.13 shown.

[0144] The calculation formula is as follows:

[0145] OH removal rate = [1-(As-Ac) / A0] × 100%.

[0146] Fig.13 The results showed that in the concentration range of 0.1-1.0 mg / mL, the ·OH scavenging ability of black potato anthocyanidins and Vc showed a steady upward trend, and at 0.1 mg / L, the ·OH scavenging rate of black potato anthocyanidins reached 69.03%, and the ·OH scavenging ability of Vc reached 91.35%. The results showed that black potato anthocyanidins and Vc had a certain scavenging ability against ·OH in the concentration range of 0.1-1.0 mg / mL, and the ·OH scavenging rate increased significantly with the increase of anthocyanidin concentration (P < 0.05), showing concentration dependence.

[0147] Experimental Example 8

[0148] Determination of total reducing capacity of anthocyanins in black potatoes

[0149] Potassium ferrocyanide and ferric chloride can react, and the reactant is Prussian blue, which has a maximum absorbance at 700nm. The ability of the sample to reduce iron ions can be determined by measuring the absorbance of the reaction system at 700nm. Preparation of 10% potassium ferrocyanide (K3[Fe(CN)6]) solution: accurately weigh 2.5g of K3[Fe(CN)6], dissolve it in deionized water and make up to 250mL. Preparation of 10% trichloroacetic acid solution: accurately weigh 2.5g of trichloroacetic acid, dissolve it in deionized water and make up to 250mL. Preparation of 1% ferric chloride (FeCl3) solution: accurately weigh 0.25g of trichloroacetic acid, dissolve it in deionized water and make up to 250mL. 0.2 mol / L (pH=6.6) phosphate buffer (PBS): weigh 3.1203 g of sodium dihydrogen phosphate and 7.1628 g of sodium dihydrogen phosphate respectively, dissolve them in deionized water to 250 mL respectively, and mix the two to adjust the pH to 6.6.

[0150] The purified black potato anthocyanin in Example 3 was dissolved in a potassium chloride-hydrochloric acid buffer solution to prepare anthocyanin solutions of different concentrations for later use.

[0151] Take 2.5mL of PBS solution in a 10mL centrifuge tube, add 1.0mL of anthocyanin solution of different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mg / mL), then add 2.5mL of potassium ferricyanide solution, mix thoroughly, place in a constant temperature water bath at 50°C for 30min, cool quickly, add 2.5mL of trichloroacetic acid to terminate the reaction, centrifuge at 4000rpm for 10min, take 2.0mL of supernatant in another 10mL centrifuge tube, add 2.0mL of ferric chloride solution, mix thoroughly, let stand for 10min, measure its absorbance at 700nm, record it as A. Adjust to zero with distilled water. Take Vc as the positive control, and measure its total reducing ability according to the above method. Perform three parallel measurements. The test results are as follows: Fig.14 shown.

[0152] Fig.14 It shows that within the concentration range of 0.01-0.10 mg / mL, the ability of black potato anthocyanidins and Vc to reduce iron ions showed a gradual upward trend, and the total reducing capacity of black potato anthocyanidins solution was 0.462 at 0.10 mg / mL. This shows that black potato anthocyanidins and Vc both have a certain reducing capacity for iron ions, and the reducing power increases with the increase of concentration, showing concentration dependence.

[0153] Example 5

[0154] Select fresh black potatoes that are not rotten, unsprouted, and have full grains. Wash the black potatoes, cut them into small pieces of uniform size and set aside.

[0155] Black potato was mixed with 50% ethanol (the pH of the ethanol was adjusted to 3.5 with hydrochloric acid) at a mass volume ratio of 1 g: 1.5 mL, and the mixture was placed in a juicer for pulping. The pH of the homogenate was adjusted to 3.5, and the mixture was subjected to ultrasound at 250 W for 25 min at 25°C, and centrifuged at 8000 rpm at 4°C for 3 min. The supernatant was taken, and the supernatant was concentrated at 40°C and freeze-dried into powder to obtain the crude black potato anthocyanin, which was then stored in a -80°C refrigerator away from light.

[0156] After loading the macroporous resin AB-8, the crude black potato anthocyanin was dissolved in potassium chloride-hydrochloric acid solution to prepare a 100 mg / mL black potato anthocyanin crude extract, 10 mL of sample volume, static adsorption for 6 h in the dark, and eluted with 80% ethanol (acidic ethanol) with a pH of 4, flow rate: 1.5 ml / min, collection frequency: 5 ml / time, and the optimal elution concentration was determined by the absorbance at 530 nm.

[0157] After loading the macroporous resin AB-8, prepare 100 mg / mL crude extract of black potato anthocyanin obtained by ultrasonic-assisted acidic ethanol extraction in Example 1, 5 mL sample volume, and static adsorption for 8 hours in the dark. And use 0%, 20%, 40%, 60%, 80%, 100% ethanol (acidic ethanol) with pH=4 to elute in sequence, flow rate: 1.5 ml / min, collection frequency: 5 ml / time, collect anthocyanin samples by absorbance at 530 nm, concentrate, freeze-dry, and obtain black potato anthocyanin for standby use.

[0158] The present invention provides anthocyanidins from black potato, and an extraction and purification method and application thereof. The extraction and purification method of the present invention has a higher content of anthocyanidins in black potato than that of the traditional aqueous solution extraction method and ethanol solution extraction method, and the content in the crude product reaches 57.20 mg / g. The crude black potato anthocyanidins is purified by macroporous resin, and the adsorption rate of the macroporous resin can reach 96.43%, and the resolution rate can reach 97.93%. The content of anthocyanidins in the black potato anthocyanidins finally obtained can reach 485.12 mg / g. Therefore, it can be known that according to the method of the present invention, black potato anthocyanidins with a high anthocyanidin content can be extracted and purified from black potato.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting and purifying anthocyanins from black potatoes, characterized in that: The steps include: (1) mixing black potato with ethanol, beating to obtain a homogenate, adjusting the pH of the homogenate to 3-4, ultrasonicating, centrifuging, taking the supernatant, concentrating the supernatant, and freeze-drying to obtain a crude black potato anthocyanin product; (2) dissolving the crude black potato anthocyanin to obtain a crude black potato anthocyanin extract, adsorbing the crude black potato anthocyanin extract through a macroporous resin for 1 to 12 hours, eluting with ethanol, collecting the eluate, concentrating, and drying to obtain black potato anthocyanin.

2. The extraction and purification method according to claim 1, characterized in that: The initial water content of the black potato is 77-80%; The mass volume ratio of the black potato and ethanol is 1g:1-2mL; The initial concentration of ethanol in step (1) is 45-55%; The initial pH of the ethanol in step (1) is 3.2 to 3.

7.

3. The extraction and purification method according to claim 2, characterized in that: The ultrasonic time of step (1) is 25 to 35 minutes, the ultrasonic temperature is 23 to 27° C., and the ultrasonic power is 230 to 250 W.

4. The extraction and purification method according to claim 3, characterized in that: The centrifugal temperature in step (1) is 2-6° C., the centrifugal speed is 7500-8500 rpm, and the centrifugal time is 2.5-3.5 min.

5. The extraction and purification method according to claim 4, characterized in that: The macroporous resin in step (2) is macroporous resin AB-8; the sample loading during the adsorption in step (2) is 10 to 40 mL; during the ethanol elution in step (2), the initial concentration of ethanol is 80 to 100%, and the pH of ethanol is 3.5 to 4.

5.

6. The black potato anthocyanin prepared by the extraction and purification method according to any one of claims 1 to 5.

7. Use of the black potato anthocyanins prepared by the extraction and purification method according to any one of claims 1 to 5 in the preparation of products with antioxidant capacity.

8. The use according to claim 7, characterized in that: The product is food, medicine or cosmetic.

9. Use of the black potato anthocyanin according to claim 6 in preparing products with antioxidant capacity.

10. The use according to claim 9, characterized in that: The product is food, medicine or cosmetic.

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