Method for detecting and analyzing anthocyanin substances in plum peel

Abstract

The invention discloses a method for detecting and analyzing anthocyanin substances in plum pericarp, and relates to the technical field of anthocyanin substances, and the method comprises the following steps: S1, taking the pericarp of mature plum fruit, adding liquid nitrogen, grinding by using a homogenizer, placing in a centrifuge tube, adding a solution, carrying out ultrasonic oscillation, storing at-20 DEG C, taking out, recovering to room temperature, extracting at 4 DEG C, centrifuging, and collecting supernatant; s2, collecting supernate, filling the supernate into completely activated macroporous resin of a glass chromatographic column for purifying and eluting, collecting eluent, evaporating to obtain extract, redissolving with a methanol-0. 1% formic acid aqueous solution, fixing the volume, and filtering. In the plum peel extraction process, a small amount of inositol is added, the extraction rate of anthocyanin substances can be increased, the inositol can inhibit the activity of glycosidase and polyphenol oxidase in the plum peel, so that the stability of the extracted substances is improved, and meanwhile, under the mixing action of multiple solvents, the effect of a surfactant can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of anthocyanins, and in particular to a method for detecting and analyzing anthocyanins in plum peels. Background Art

[0002] The main substances that determine the color of plum peel are anthocyanin, chlorophyll and carotenoids. When the chlorophyll content is high, the peel color is cyan, when the carotenoid content is high, the peel color is yellow, and when the anthocyanin content is high, the peel color is red. The different proportions of the three substances form fruits of various colors. The formation of anthocyanin requires anthocyanin, polysaccharides and photoreceptors. There are five main types of polysaccharides: glucose, rhamnose, galactose, xylose and arabinose. Among them, the main component of the photoreceptor is photosensitive pigment, which plays an important regulatory role in the formation of anthocyanin.

[0003] The existing technology has the following problems: Cyclohexanehexol is usually not added when anthocyanins are extracted from plum peel, resulting in a low extraction rate of anthocyanins; and when anthocyanin pigments are extracted, the extraction efficiency and the accuracy of the analysis results are low; at the same time, the selection of chromatographic columns and elution procedures is also inevitably related, and it is impossible to accurately characterize the anthocyanins in plum peel. Summary of the invention

[0004] The present invention provides a method for detecting and analyzing anthocyanins in plum peels to solve the problems raised in the above background technology.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: A method for detecting and analyzing anthocyanins in plum peels, the method comprising the following steps: S1 taking the peels of mature plum fruits, adding liquid nitrogen and grinding them with a homogenizer, placing them in a centrifuge tube, adding a solution, ultrasonically vibrating, storing them at -20°C, taking them out and restoring them to room temperature, then placing them at 4°C for extraction, and centrifuging them; S2 collects the supernatant, loads the supernatant into a fully activated macroporous resin in a glass chromatography column for purification and elution, collects the eluate, evaporates it to an extract, re-dissolves it with methanol-0.1% formic acid aqueous solution, makes up the volume, and filters it; S3 analyzes and detects the filtrate using ultra-high performance liquid chromatography-mass spectrometry.

[0006] The further improvement of the technical solution of the present invention is that the step S1 comprises the following steps: 5 g of the peel of the ripe plum fruit in the step S11 is added with liquid nitrogen and ground with a homogenizer, and then placed in a centrifuge tube, and 21 ml of methanol, 0.15% formic acid water, and cyclohexanehexol are added, wherein the volume ratio of methanol, 0.15% formic acid water, and cyclohexanehexol is 1:4:2.5, and ultrasonic vibration is performed for 10 minutes; The S12 is freeze-dried and stored at -20°C for 10 minutes, taken out and restored to room temperature, and then placed at 4°C for leaching for 24 hours. The centrifuge tube after leaching is rotated and centrifuged for 10-15 minutes at a speed of 3500-5000 r / min, the supernatant is collected, and methanol solution is added to the supernatant for repeated leaching until the precipitate is colorless.

[0007] The further improvement of the technical solution of the present invention is that the step S2 comprises the following steps: the step S21 uses a 15% sodium chloride solution to soak the HPD-450A macroporous resin for 5 minutes, then soaks it in an 8% sodium chloride solution for 2 minutes, rinses it with distilled water, and then soaks it in ethanol for 24 hours and washes it with distilled water until it is tasteless; The S22 was then soaked in 5% sodium hydroxide solution for 12 hours, washed with distilled water until neutral, and finally soaked in 5% hydrochloric acid solution for 12 hours, washed with distilled water until neutral, and then soaked in distilled water; The S23 is purified and eluted by the fully activated HPD-450A macroporous resin, and the eluate is collected and evaporated at 30° C. to an extract state, redissolved with methanol and 0.1% formic acid aqueous solution and fixed to 2 mL, filtered to obtain a filtrate, wherein the volume ratio of methanol to 0.1% formic acid aqueous solution is 1:4.

[0008] The further improvement of the technical solution of the present invention is that the step S3 comprises the following steps: the chromatographic column in the ultra-high performance liquid chromatography-mass spectrometry method of S31 is a Pheromon Kinetex series, with performance parameters of 100 mm×4.6 mm, 2.6 μm, a column temperature of 35°C, an injection volume of 5 μL, a flow rate of 1.0 mL / min, mobile phase A is a formic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted; The S32 gradient elution conditions are 1-3 min, 5%-50% B, 3-5.0 min, 50%-90, 5.0-6.0 min, 90% B; 6.0-6.2 min, 90%-5% B; 6.2-8 min, 5% B, and the injection volume is 5 ul; The conditions of the S33 mass spectrometry are: curtain gas CUR: 35, ion source voltage IS: 5500, ion source temperature TEM550, ion source power reaction gas GSI55.

[0009] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art: 1. The present invention provides a method for detecting and analyzing anthocyanins in plum peel. During the extraction process of plum peel, a small amount of cyclohexanehexol is added to improve the extraction rate of anthocyanins. Cyclohexanehexol can inhibit the activities of glycosidase and polyphenol oxidase in plum peel, thereby improving the stability of the extracted substances. At the same time, under the mixed action of multiple solvents, it can play the role of a surfactant and reduce its surface tension, thereby improving the extraction rate.

[0010] 2. The present invention provides a method for detecting and analyzing anthocyanins in plum peels. By freeze-drying the plum peels, the temperature of the extraction process no longer fluctuates greatly, thereby effectively curbing browning. The subsequent low-temperature rotary evaporation concentration also protects the pigment from oxidation to the greatest extent, greatly improving the extraction efficiency of anthocyanins and other pigment substances and the accuracy of the analysis results. The method of the present invention is simple and easy to implement, and is very suitable for promotion and use in the field of agricultural product and food detection.

[0011] 3. The present invention provides a method for detecting and analyzing anthocyanins in plum peel. By screening and optimizing conditions such as mobile phase, elution program, and chromatographic column, the selected method has good repeatability and stability, and can quickly and accurately perform qualitative and quantitative detection of anthocyanins in plum peel with a low detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0013] The present invention is further described in detail below in conjunction with embodiments: Example 1 like Figure 1 As shown, the present invention provides a method for detecting and analyzing anthocyanins in plum peel, comprising the following steps: S1 taking the peel of a mature plum fruit, adding liquid nitrogen and grinding it with a homogenizer, placing it in a centrifuge tube, adding a solution, ultrasonically oscillating, storing it at -20°C, taking it out and restoring it to room temperature, then placing it at 4°C for extraction, centrifuging, S2 collecting the supernatant, loading the supernatant into a glass chromatography column with a fully activated macroporous resin for purification and elution, collecting the eluate, evaporating it to an extract, re-dissolving it with a methanol-0.1% formic acid aqueous solution and fixing the volume, filtering, and S3 analyzing and detecting the filtrate by ultra-high performance liquid chromatography-mass spectrometry.

[0014] In this embodiment, (1) 5 g of the peel of a ripe plum fruit was taken, and after adding liquid nitrogen and grinding with a homogenizer, the peel was placed in a centrifuge tube, and 21 ml of methanol, 0.15% formic acid water, and cyclohexanehexol were added, with the volume ratio of methanol, 0.15% formic acid water, and cyclohexanehexol being 1:4:2.5. The peel was subjected to ultrasonic vibration for 10 min, and the peel was placed at -20°C for freeze drying and stored for 10 min. After being taken out and restored to room temperature, the peel was placed at 4°C for 24 h. The extracted centrifuge tube was centrifuged for 10-15 min at a speed of 3500-5000 r / min, the supernatant was collected, and the methanol solution was added to the supernatant for repeated leaching until the precipitate was colorless; (2) Collecting the supernatant and loading the supernatant into a fully activated macroporous resin in a glass chromatography column for purification and elution, collecting the eluate, evaporating it at 30°C to an extract, re-dissolving it with methanol and 0.1% formic acid aqueous solution and fixing the volume to 2 mL, filtering, and obtaining a filtrate, wherein the volume ratio of methanol to 0.1% formic acid aqueous solution is 1:4; (3) The filtrate was analyzed and detected by ultra-high performance liquid chromatography-mass spectrometry. The chromatographic column was of the Pheromones Kinetex series with performance parameters of 100 mm × 4.6 mm, 2.6 μm, column temperature of 35 °C, injection volume of 5 μL, flow rate of 1.0 mL / min, mobile phase A was formic acid aqueous solution, mobile phase B was acetonitrile, and gradient elution was adopted.

[0015] Example 2 like Figure 1 As shown, on the basis of Example 1, the present invention provides a technical solution: Preferably, step S1 includes the following steps: S11 5g of peel of mature plum fruit is added with liquid nitrogen and ground with a homogenizer, then placed in a centrifuge tube, 21ml of methanol, 0.15% formic acid water, and cyclohexanehexol are added, the volume ratio of methanol, 0.15% formic acid water and cyclohexanehexol is 1:4:2.5, ultrasonic vibration is performed for 10 minutes, S12 is placed at -20°C for freeze-drying and stored for 10 minutes, taken out and restored to room temperature, and then placed at 4°C for leaching for 24 hours, the centrifuge tube after leaching is rotated and centrifuged for 10-15 minutes, the speed is 3500-5000r / min, the supernatant is collected, and methanol solution is added to the supernatant and repeatedly leached until the precipitate is colorless.

[0016] In this embodiment, (1) 5 g of the peel of a ripe plum fruit was taken, and after grinding with a homogenizer after adding liquid nitrogen, the peel was placed in a centrifuge tube, and 21 ml of methanol and 0.15% formic acid water were added, with the volume ratio of methanol to 0.15% formic acid water being 1:4. The peel was subjected to ultrasonic vibration for 10 min, and the peel was placed at -10°C for freeze-drying and stored for 10 min. After being taken out and restored to room temperature, the peel was placed at 15°C for 24 h. The extracted peel was centrifuged for 10-15 min at a speed of 3500-5000 r / min, the supernatant was collected, and the methanol solution was added to the supernatant for repeated leaching until the precipitate was colorless; (2) Collecting the supernatant and loading the supernatant into a fully activated macroporous resin in a glass chromatography column for purification and elution, collecting the eluate, evaporating it at 30°C to an extract, re-dissolving it with methanol and 0.1% formic acid aqueous solution and fixing the volume to 2 mL, filtering, and obtaining a filtrate, wherein the volume ratio of methanol to 0.1% formic acid aqueous solution is 1:4; (3) The filtrate was analyzed and detected by ultra-high performance liquid chromatography-mass spectrometry. The chromatographic column was of the Pheromones Kinetex series with performance parameters of 100 mm × 4.6 mm, 2.6 μm, column temperature of 35 °C, injection volume of 5 μL, flow rate of 1.0 mL / min, mobile phase A was formic acid aqueous solution, mobile phase B was acetonitrile, and gradient elution was adopted.

[0017] Example 3 like Figure 1 As shown, on the basis of Example 1, the present invention provides a technical solution: Preferably, step S2 includes the following steps: S21 uses a 15% sodium chloride solution to soak the HPD-450A macroporous resin for 5 minutes, and then soaks it in an 8% sodium chloride solution for 2 minutes, rinses it with distilled water, and then soaks it in ethanol for 24 hours and washes it with distilled water until it is odorless, S22 soaks it in a 5% sodium hydroxide solution for 12 hours, washes it with distilled water until it is neutral, and finally soaks it in a 5% hydrochloric acid solution for 12 hours, washes it with distilled water until it is neutral, and then soaks it in distilled water, S23 activates the completely HPD-450A macroporous resin for purification and elution, collects the eluate, evaporates it at 30°C to an extract, re-dissolves it with methanol and 0.1% formic acid aqueous solution and fixes it to 2mL, filters it, and obtains a filtrate, wherein the volume ratio of methanol to 0.1% formic acid aqueous solution is 1:4.

[0018] In this embodiment, (1) 5 g of the peel of a ripe plum fruit was taken, ground with liquid nitrogen using a homogenizer, and then placed in a centrifuge tube. 21 ml of methanol, 0.15% formic acid water and cyclohexanehexol were added, and the volume ratio of methanol, 0.15% formic acid water and cyclohexanehexol was 1:4:2. Ultrasonic vibration was applied for 10 min. After the peel was taken out and restored to room temperature, it was placed at -5°C for 24 h. The centrifuge tube after leaching was centrifuged for 10-15 min at a speed of 3500-5000 r / min. The supernatant was collected, and methanol solution was added to the supernatant to repeat the leaching until the precipitate was colorless. (2) Collecting the supernatant and loading the supernatant into a fully activated macroporous resin in a glass chromatography column for purification and elution, collecting the eluate, evaporating it at 30°C to an extract, re-dissolving it with methanol and 0.1% formic acid aqueous solution and fixing the volume to 2 mL, filtering, and obtaining a filtrate, wherein the volume ratio of methanol to 0.1% formic acid aqueous solution is 1:4; (3) The filtrate was analyzed and detected by ultra-high performance liquid chromatography-mass spectrometry. The chromatographic column was of the Pheromones Kinetex series with performance parameters of 100 mm × 4.6 mm, 2.6 μm, column temperature of 35 °C, injection volume of 5 μL, flow rate of 1.0 mL / min, mobile phase A was formic acid aqueous solution, mobile phase B was acetonitrile, and gradient elution was adopted.

[0019] Example 4 like Figure 1 As shown, on the basis of Example 1, the present invention provides a technical solution: Preferably, step S3 includes the following steps: S31 the chromatographic column in the ultra-high performance liquid chromatography-mass spectrometry is a Pheromon Kinetex series, with performance parameters of 100mm×4.6mm, 2.6μm, column temperature of 35°C, injection volume of 5μL, flow rate of 1.0mL / min, mobile phase A is formic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted, S32 the gradient elution conditions are 1~3min, 5%~50%B, 3~5.0min, 50%~90, 5.0~6.0min, 90%B; 6.0~6.2min, 90%~5%B; 6.2~8min, 5%B, injection volume of 5ul, S33 the conditions of mass spectrometry are curtain gas CUR: 35, ion source voltage IS: 5500, ion source temperature TEM550, ion source power reaction gas GSI55.

[0020] In this embodiment, (1) 5 g of the peel of a ripe plum fruit was taken, and after adding liquid nitrogen and grinding with a homogenizer, the peel was placed in a centrifuge tube, and 21 ml of methanol, 0.15% formic acid water, and cyclohexanehexol were added, with the volume ratio of methanol, 0.15% formic acid water, and cyclohexanehexol being 1:4:2.5. The peel was subjected to ultrasonic vibration for 10 min, and the peel was placed at -20°C for freeze drying and stored for 10 min. After being taken out and restored to room temperature, the peel was placed at 4°C for 24 h. The extracted centrifuge tube was centrifuged for 10-15 min at a speed of 3500-5000 r / min, the supernatant was collected, and the methanol solution was added to the supernatant for repeated leaching until the precipitate was colorless; (2) Collecting the supernatant and loading the supernatant into a fully activated macroporous resin in a glass chromatography column for purification and elution, collecting the eluate, evaporating it at 30°C to an extract, re-dissolving it with methanol and 0.1% formic acid aqueous solution and fixing the volume to 2 mL, filtering, and obtaining a filtrate, wherein the volume ratio of methanol to 0.1% formic acid aqueous solution is 1:4; (3) The filtrate was analyzed and detected by ultra-high performance liquid chromatography-mass spectrometry. The chromatographic column was TSKgelODS-100Z produced by Tosoh Quartz Corporation. The performance parameters were 150 mm×4.6 mm, 5 μm, column temperature was 40°C, flow rate was 1 ml / min, mobile phase A was formic acid aqueous solution, mobile phase B was acetonitrile, and gradient elution was adopted.

[0021] In summary, if Figure 1 As shown, the extraction effects of Example 1 and Examples 2-4 were compared, and qualitative and quantitative tests were performed on mature fruit samples of plum varieties Dashi Zaosheng, Longyuan Qiuli, Black Baoshi, and Angelo. A control group was set up and the method in the comparative example was used for detection. The positive correlation coefficient between fructose and anthocyanins in plums was calculated. The specific results are shown in the table below. It can be seen from the table that the extraction method provided in Example 1 of the present invention significantly improves the extraction rate, and a variety of anthocyanins can be better separated and detected with high sensitivity.

[0022] The above generally describes the present invention in detail, but it is obvious to a person skilled in the art that some modifications or improvements can be made to the present invention. Therefore, modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing anthocyanins in plum peel, characterized in that: The detection and analysis method comprises the following steps: S1, taking the peel of a ripe plum fruit, adding liquid nitrogen and grinding it with a homogenizer, placing it in a centrifuge tube, adding a solution, ultrasonically vibrating, storing it at -20°C, taking it out and restoring it to room temperature, then placing it at 4°C for extraction, and centrifuging it; S2 collects the supernatant, loads the supernatant into a fully activated macroporous resin in a glass chromatography column for purification and elution, collects the eluate, evaporates it to an extract, re-dissolves it with methanol-0.1% formic acid aqueous solution, makes up the volume, and filters it; S3 analyzes and detects the filtrate using ultra-high performance liquid chromatography-mass spectrometry.

2. The method for detecting and analyzing anthocyanins in plum peel according to claim 1, characterized in that: The step S1 comprises the following steps: 5 g of the peel of the ripe plum fruit in the step S11 is ground with liquid nitrogen using a homogenizer, placed in a centrifuge tube, 21 ml of methanol, 0.15% formic acid water, and cyclohexanehexol are added, wherein the volume ratio of methanol, 0.15% formic acid water, and cyclohexanehexol is 1:4:2.5, and ultrasonically vibrated for 10 minutes; The S12 is freeze-dried and stored at -20°C for 10 minutes, taken out and restored to room temperature, and then placed at 4°C for leaching for 24 hours. The centrifuge tube after leaching is rotated and centrifuged for 10-15 minutes at a speed of 3500-5000 r / min, the supernatant is collected, and methanol solution is added to the supernatant for repeated leaching until the precipitate is colorless.

3. The method for detecting and analyzing anthocyanins in plum peel according to claim 1, characterized in that: The step S2 comprises the following steps: the step S21 uses a 15% sodium chloride solution to soak the HPD-450A macroporous resin for 5 minutes, then soaks it in an 8% sodium chloride solution for 2 minutes, rinses it with distilled water, and then soaks it in ethanol for 24 hours and washes it with distilled water until it is odorless; The S22 was then soaked in 5% sodium hydroxide solution for 12 hours, washed with distilled water until neutral, and finally soaked in 5% hydrochloric acid solution for 12 hours, washed with distilled water until neutral, and then soaked in distilled water; The S23 is purified and eluted by the fully activated HPD-450A macroporous resin, and the eluate is collected and evaporated at 30° C. to an extract state, redissolved with methanol and 0.1% formic acid aqueous solution and fixed to 2 mL, filtered to obtain a filtrate, wherein the volume ratio of methanol to 0.1% formic acid aqueous solution is 1:

4.

4. The method for detecting and analyzing anthocyanins in plum peel according to claim 3, characterized in that: The step S3 comprises the following steps: the chromatographic column in the S31 ultra-high performance liquid chromatography-mass spectrometry method is a Pheromon Kinetex series, with performance parameters of 100 mm×6 mm, 2.6 μm, a column temperature of 35°C, an injection volume of 5 μL, a flow rate of 1.0 mL / min, mobile phase A is a formic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted; The S32 gradient elution conditions are 1-3 min, 5%-50% B, 3-5.0 min, 50%-90, 5.0-6.0 min, 90% B; 6.0-6.2 min, 90%-5% B; 6.2-8 min, 5% B, and the injection volume is 5 ul; The conditions of the S33 mass spectrometry are: curtain gas CUR: 35, ion source voltage IS: 5500, ion source temperature TEM550, ion source power reaction gas GSI55.

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