Ultrasonic extraction method of betacyanin based on accumulation rule of nutritional ingredients of red beet

Abstract

The invention relates to a betacyanin extraction method, in particular to a betacyanin ultrasonic extraction method based on a red beet nutritional ingredient accumulation rule. The problems that impurities are too high and the extraction amount is small in a betacyanin extraction project formed by a common technology are solved. Comprising the following steps: adding an extracting agent into freeze-dried powder, carrying out ultrasonic extraction, filtering with gauze after extraction, carrying out suction filtration with filter paper, removing residual dietary fibers in beet juice, centrifuging the beet juice in a centrifugal machine for 10 minutes at the speed of 12,000 r / min after removal, taking 665 mu L of supernate, and fixing the volume to 10 mL. According to the method disclosed by the invention, relatively high extraction efficiency of the betacyanin is ensured, and certain data support is provided for stabilization and industrial research of the betacyanin.

Description

Technical Field

[0001] The invention relates to a method for extracting betalain, in particular to an ultrasonic extraction method for betalain based on the cumulative law of red beet nutritional components. Background Art

[0002] Red beet is a deep-rooted crop. Studies have shown that lower temperatures can promote the formation of red pigments in the roots. The taproot of red beet grows fast and develops well, and a certain amount of lateral roots can be emitted from the taproot. The taproots are wedge-shaped, conical, spindle-shaped and spherical. The root bark shows different degrees of red due to the amount of betalain. The root flesh is pink or red and white concentric circles, similar to the growth rings of a tree trunk. The petioles and veins are mostly purple or dark red. The leaves are thick, mostly tongue-shaped, shiny on the surface, and mostly emerald green.

[0003] Betalain is a water-soluble nitrogen-containing natural pigment synthesized in the cytoplasm of flowers, fruits and leaves, stored in vacuoles, and distributed in the epidermis or subepidermal tissues of plants. It is a derivative of betaine and can be divided into two categories: red betalain and yellow betalain. The ratio of the two determines the color of the pigment extract. The content of betalain accounts for more than 90% of betaine, while the content of betalain is less than 10%. Betaine is formed by the combination of betaine and dihydroxyphenylalanine, and betaine produces betalain when betaine combines with other amino acids. Betaine includes betaine glycosides, isobetaine, probetaine glycosides, isopretaine glycosides and degradation products of betaine, among which betaine glycosides are the main component, accounting for 75% to 95%. Betaine glycosides include betaine I and betaine II. These pigments are present in 10 Caryophyllaceae plants except the Sedum and Caryophyllaceae, and have also been found in some fungi. But because it was first discovered in beetroot, it was named beetroot red pigment.

[0004] Traditional extraction techniques include solvent extraction, continuous diffusion, decoction, etc. The most widely used is solvent extraction, which is based on the difference in solubility of each component in the raw material in different solvents to achieve the purpose of extraction and separation. The process is generally blanching, shredding, leaching, pressing and concentration. Usually, betalain can be extracted in water, but if necessary, citric acid and ascorbic acid can also be added to increase the extraction rate. Xiong Yong et al. used red beet as material to optimize the best extraction process of betalain: at room temperature, water was used as the extraction solvent, the material-liquid ratio was 1:5g / mL, the time was 5h, and the pH was 4.5. The solvent extraction method has the following characteristics: simple operation, low cost, safe and reliable, but the extraction efficiency is low and the extraction quality is poor. Continuous diffusion is also often used, but the pigment extracted by this method still contains high concentrations of sugar, salt and other impurities.

[0005] Therefore, it is urgent to propose an ultrasonic extraction method of betalain based on the cumulative law of red beet nutrients to solve the above technical problems. Summary of the invention

[0006] The purpose of the present invention is to solve the problem of excessive impurities and low extraction amount in the betalain extraction process formed by conventional technology. A brief overview of the present invention is given below to provide a basic understanding of certain aspects of the present invention. It should be understood that this overview is not an exhaustive overview of the present invention. It is not intended to determine the key or important parts of the present invention, nor is it intended to limit the scope of the present invention.

[0007] The technical solution of the present invention:

[0008] A method for ultrasonic extraction of betalain based on the cumulative law of red beet nutritional components comprises the following steps:

[0009] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0010] Step 2: Add 30 ml of extractant to 1.2 g of freeze-dried powder, perform ultrasonic extraction, filter with 200-mesh gauze after extraction, and then filter with filter paper to remove the residual dietary fiber in the beet juice. After removal, place the beet juice in a centrifuge at 12000 r / min, centrifuge for 10 minutes, take 665 μL of supernatant, and make up to 10 mL.

[0011] A flavonoid detection method based on the cumulative law of red beet nutritional components comprises the following steps:

[0012] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0013] Step 2: Add 0.1 g of freeze-dried powder to 1 ml of 60% ethanol, perform ultrasonic extraction for 30 min, centrifuge in a centrifuge, and take the supernatant to a fixed volume after centrifugation;

[0014] Step 3: According to the plant flavonoid content detection kit, add the sample to the reaction system and mix evenly. After mixing evenly, put it into a 37°C oven and dry it for 45 minutes. After drying, cool and centrifuge it to obtain the supernatant. Place the supernatant in an ELISA instrument to measure its absorbance at a wavelength of 470nm, and then convert the measured absorbance value into the flavonoid content in the sample according to the standard curve and the flavonoid content calculation formula.

[0015] A betaine detection method based on the cumulative law of red beet nutritional components comprises the following steps:

[0016] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0017] Step 2: According to the betaine kit, 0.2g-0.3g of freeze-dried powder was added to 1ml of 80% ethanol, and the mixture was placed in a 60°C water bath for extraction for 30min. 3mg of reagent 4 in the betaine kit was added to the extract, and the supernatant was obtained by centrifugation;

[0018] Step 3: Place the supernatant in a 70°C water bath to evaporate the methanol to 0.2 ml, then adjust the volume to 1 ml, add reagents and samples according to the requirements of the kit, and finally substitute the standard curve to calculate the concentration of betaine in the sample.

[0019] A total phenol detection method based on the cumulative law of red beet nutritional components comprises the following steps:

[0020] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0021] Step 2: Add 0.1 g of freeze-dried powder to 2.5 ml of 60% ethanol, perform ultrasonic extraction for 30 min, centrifuge in a centrifuge, and take the supernatant to a fixed volume after centrifugation;

[0022] Step 3: According to the plant total phenol content detection kit, add samples to the control tube, test tube, standard tube and blank tube respectively. Let it stand for 10 minutes after adding samples. After standing, place it in an enzyme reader at 760nm to measure the absorbance, and use the gallic acid standard curve to calculate the total phenol concentration.

[0023] A mineral detection method based on the cumulative law of red beet nutritional components comprises the following steps:

[0024] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0025] Step 2: Add 0.2g-0.3g of freeze-dried powder to a 50ml polytetrafluoroethylene digestion tube, then add 10mL nitric acid and 0.5mL perchloric acid and mix evenly. After mixing evenly, place it in a fume hood to react for 1h, then place it on an intelligent digestion instrument for acid digestion, react at 110℃ for 50min, then adjust to 180℃ for 2h, and transfer it to a volumetric flask after cooling;

[0026] Step 3: Add ionized water to the volumetric flask to make up to 25 ml, mix and set aside, and use 10 ml nitric acid-0.5 ml perchloric acid mixture as a blank reference. Inject the treated blank solution and sample solution into the inductively coupled plasma emission spectrometer respectively, measure the signal response value of the analysis line intensity of the element to be measured, and obtain the concentration of the element to be measured in the digestion solution according to the standard curve.

[0027] A soluble sugar detection method based on the cumulative law of red beet nutritional components comprises the following steps: grinding 0.5 g of red beet root into a homogenate in a mortar, adding 10 mL of phosphate buffer, placing the mixture in a centrifuge, and centrifuging the mixture at a speed of 12000 r / min for 20 min. After the centrifugation, taking 100 μL of supernatant, adding 300 μL of water, 100 μL of anthrone ethyl acetate reagent, and 1 mL of sulfuric acid, mixing the mixture, and placing the mixture in an enzyme reader at 630 nm to determine its absorbance, and calculating the soluble sugar content according to a sucrose standard curve.

[0028] A soluble protein detection method based on the cumulative rule of red beet nutritional components comprises the following steps: grinding 0.5 g of red beet root into a homogenate in a mortar, adding 10 mL of phosphate buffer, placing the mixture in a centrifuge, and centrifuging the mixture at a speed of 12000 r / min for 20 min. After the centrifugation, taking 20 μL of supernatant into a 96-well plate, adding 200 μL of a mixed solution of an alkaline solution and 4% copper sulfate, mixing the mixture, heating the mixture at 37°C for 30 min, cooling the mixture to room temperature after heating, placing the mixture in an enzyme marker at 562 nm to measure absorbance, and calculating the concentration of the soluble protein according to a BSA standard curve.

[0029] The present invention has the following beneficial effects:

[0030] The invention ensures a high extraction efficiency of betalain and provides certain data support for the stabilization and industrialization research of betalain. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the chemical structure diagram of betalain;

[0032] Figure 2 It is the full wavelength scan of the extract;

[0033] Figure 3 This is a schematic diagram of extracting betalain with different extractants;

[0034] Figure 4 This is a schematic diagram of the effect of extractants on the yield of betalain;

[0035] Figure 5 This is a schematic diagram showing the effect of the solid-liquid ratio on the betalain yield;

[0036] Figure 6 This is a schematic diagram showing the effect of extraction temperature on betalain yield;

[0037] Figure 7 This is a schematic diagram showing the effect of extraction time on betalain yield;

[0038] Figure 8 This is a schematic diagram of the effect of ultrasonic power on betalain yield;

[0039] Fig. 9 It is the relationship diagram between single factor and betalain yield;

[0040] Fig.10 It is a response surface and contour diagram of the effect of material-liquid ratio and time on yield;

[0041] Fig.11 It is the response surface and contour diagram of the interaction between material-liquid ratio and ultrasonic temperature;

[0042] Fig.12 It is the response surface and contour diagram of the interaction between solid-liquid ratio and ultrasonic power;

[0043] Fig.13 It is the response surface and contour diagram of the interaction between ultrasound time and ultrasound temperature;

[0044] Fig.14 is the response surface and contour diagram of the interaction between ultrasound time and ultrasound power;

[0045] Fig.15 is the response surface and contour diagram of the interaction between ultrasonic temperature and ultrasonic power;

[0046] Fig.16 This is a schematic diagram of the root growth of different germplasms of red beets;

[0047] Fig.17 This is a schematic diagram of the appearance of different germplasm red beet roots;

[0048] Fig.18 It is the accumulation graph of flavonoids in different accessions of red beet;

[0049] Fig.19 This is the accumulation graph of flavonoids in red beet at different growth periods of different germplasms;

[0050] Fig. 20 It is a graph of betalain accumulation in different germplasms of red beet;

[0051] Fig.21 It is a graph of betalain accumulation in red beets of different germplasms at different growth periods;

[0052] Fig. 22 It is the accumulation graph of betaine in different germplasm red beets;

[0053] Fig.23 This is the accumulation graph of betaine in red beets of different germplasms at different growth periods;

[0054] Fig.24 This is the total phenol accumulation graph of different accessions of red beet;

[0055] Fig.25 This is the total phenol accumulation graph of red beet in different germplasms and different growth periods;

[0056] Fig.26 It is a graph of mineral accumulation of different germplasms of red beet;

[0057] Fig. 27 It is a mineral element accumulation trend chart;

[0058] Fig.28 This is a graph of soluble sugar accumulation in different germplasms of red beet;

[0059] Fig.29 This is a graph of soluble sugar accumulation in red beets of different germplasms at different growth periods;

[0060] Fig.30 This is a graph of soluble protein accumulation in different germplasms of red beet;

[0061] Fig.31 This is a graph of soluble protein accumulation in red beets of different germplasms at different growth cycles;

[0062] Fig.32 It is a graph of ascorbic acid accumulation in different accessions of red beet;

[0063] Fig.33 This is a graph of ascorbic acid accumulation in red beets of different germplasms at different growth periods;

[0064] in Figure 3 a-0.5% citric acid, b-90% ethanol, c-1% citric acid, d-deionized water, E-10% acetone; f-30% ethanol, g-60% ethanol. DETAILED DESCRIPTION

[0065] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described below by the specific embodiments shown in the accompanying drawings. However, it should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0066] The invention selects 9 red beet germplasms, sows them in the field of Hulan Campus of Heilongjiang University, sows three ridges for each germplasm, the ridge length is 10m, the plant spacing is 20cm, and the ridge spacing is 65cm (5 seeds need to be sown for each plant to ensure the emergence rate). Weeds are treated before sowing, base fertilizer is applied with tillage during sowing, field emergence is observed in time after sowing, seedlings are seeded at appropriate times, irrigation is carried out at appropriate times, weeds are cleared, and pest and disease prevention work is done well. Samples are taken every 15d starting from July 14 of the same year until the last sampling on September 27, for a total of six times.

[0067]

[0068]

[0069] The accumulation law of the main nutrients of 9 red beets was studied and it was found that most of the nutrients showed a bimodal accumulation, and the accumulation amount was significantly different among germplasms and at different sampling periods. In the accumulation of mineral elements, K>Mg>Ca>Fe>Zn>Mn>B, and the accumulation amount was less affected by the sampling period. The contents of glutamic acid, aspartic acid, alanine and lysine in the 9 red beets were high, and only the accumulation amount of four free amino acids was significantly different among germplasms and at different sampling periods. The accumulation of betalain and ASA was greatly affected by genotype, and the accumulation trend was significantly different among germplasms, but was less affected by the period. The accumulation trends of soluble sugar, soluble protein and flavonoids were consistent, parabolic, and the peak period was around 120 days. The accumulation trends of betaine and total phenols were mostly "bimodal", and a few were "single peak" or "trimodal".

[0070] Specific implementation method 1: Combination Figure 1-Figure 2 The present embodiment is described. The present embodiment is a method for ultrasonic extraction of betalain based on the cumulative rule of red beet nutrients, comprising the following steps:

[0071] Step 1: Remove the leaves and wash the roots of ALTOF1 with a growth period of 150 days, wipe off the moisture, peel and slice, place it in a refrigerator at -20°C for pre-freezing for 24 hours, take it out after pre-freezing and place it in a vacuum freeze dryer for freeze-drying for 48 hours, grind it with a grinder after freeze-drying, sieve it, and obtain freeze-dried powder;

[0072] Step 2: Add the extractant to the freeze-dried powder, perform ultrasonic extraction, filter with 200-mesh gauze after extraction, and then filter with filter paper to remove the residual dietary fiber in the beet juice. After removal, place the beet juice in a centrifuge at 12000r / min, centrifuge for 10min, take 665μL of supernatant, and make up to 10mL.

[0073] The germplasm of the red beet is ALTOF1, and the betalain yield is optimal when the planting time is 75, 120, 105, and 105 days.

[0074] After filtering and centrifuging, the betalain extract is scanned at a wavelength of 300-800 nm using an ultraviolet spectrophotometer to determine the maximum absorption wavelength of the betalain.

[0075] The extracting agent is one of 10% ethanol, 30% ethanol, 50% ethanol, 0.5% citric acid, 60% acetic acid, 1% citric acid, 10% acetone, distilled water solution, and deionized water, preferably one of 0.5% citric acid, 60% acetic acid, and deionized water, and the soaking time is 1 hour.

[0076] The pigment concentration in ionized water is the highest, followed by 0.5% citric acid and 60% ethanol. When deionized water is used as the extractant, the absorbance of the pigment extract is the highest, proving that deionized water has the best extraction effect. In addition, the solubility of betalain is also high under weak acidic conditions. In alcohol solution, as the ethanol concentration increases, the solubility first increases and then decreases. Analyzing the test results, the use of deionized water is not only convenient for operation and improves test efficiency, but also saves resources and protects the environment. Therefore, this study selected deionized water as the extractant for subsequent experiments.

[0077] The ultrasonic temperature may also be 20°C, 30°C, 40°C, 45°C, 50°C, 60°C, preferably 40°C.

[0078] The extraction rate increases slowly when the extraction temperature is 20℃~40℃, and decreases rapidly after 40℃. A certain temperature can accelerate the dissolution of betalain and promote full interaction with the solvent, but high temperature may cause the molecular structure of betalain to degrade and accelerate oxidation. At this time, the dissolution rate of the pigment is less than the degradation rate. Therefore, the ultrasonic temperature of the response surface experiment selects 40℃ as the middle value of the horizontal factor.

[0079] The ultrasonic extraction time can also be 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, preferably 30 min.

[0080] When the ultrasonic time is within 30 minutes, the yield of betalain increases, but when it exceeds 30 minutes, the yield begins to decrease. Short-term ultrasonic treatment and the cavitation effect of ultrasound can promote the dissolution of betalain, but the heating effect and long ultrasonic treatment time will lead to the decomposition of the pigment structure and reduce the extraction rate. Therefore, the ultrasonic time of the response surface experiment is selected as 30 minutes as the middle value of the horizontal factor.

[0081] The ultrasonic power may also be 60W, 80W, 100W, 150W, 200W, 250W, preferably 150W.

[0082] The yield of betalain increases with the increase of ultrasonic power. The yield of betalain is the highest at 150W, and then begins to decline. When ultrasound passes through a liquid medium, it produces strong shock waves and high-speed jets, causing the swelling of the material and the expansion of the pores in the material, promoting more complete cell wall breakage and pigment dissolution. However, excessive ultrasonic power will increase the number of bubbles in the solvent during cavitation, blocking the transmission of ultrasonic energy, thereby weakening the extraction rate. Therefore, the ultrasonic power of the response surface experiment selects 150W as the middle value of the level factor.

[0083] The sieving can also adopt 40 mesh gauze.

[0084] According to the Lambert-Beer law A(λ)=εbC, the content of betacyanin is proportional to its absorbance, so the yield can be calculated using the formula:

[0085]

[0086] Wherein, Bp is betalain content (mg / g), A is absorbance (535nm), DF is dilution factor, Mw is relative molar mass of betaine (550g / mol), V is total volume of solution (L), ε is molar extinction coefficient of betaine (65000L / (mol·cm)), L is optical path length (1cm), and m is mass of sample (g).

[0087] After filtering and centrifuging, the betalain extract was scanned at a wavelength of 300-800nm ​​on an ultraviolet spectrophotometer. The experiment found that the absorption peak of betalain was the largest at a wavelength of 540nm. The absorbance at the maximum absorption wavelength of betalain was measured, and three groups of parallel experiments were performed to take the average value.

[0088] Analysis of the single factor test results showed that the four factors had a significant effect on the betalain extraction rate. Design-expert was used to design a four-factor three-level test group with betalain yield as the index. The test factor levels and coding are shown in Table 1. Table 1 Test factor level design coding table

[0089]

[0090] According to the factor levels in Table 3-1, 29 groups of experiments were designed by Design-expert. The experimental process refers to 2.3.4.1. The experimental conditions and results are shown in Table 3-2.

[0091] Table 2 Experimental design and results

[0092]

[0093]

[0094] The experimental data were analyzed using different models (linear, interactive, quadratic and cubic), and the results are shown in Table 3.

[0095] Table 3 Comparison of response surface fitting models

[0096]

[0097] The results showed that the quadratic regression model performed well and was significant (p<0.0001). Therefore, this study used the quadratic regression model to represent the effect of process variables on pigment extraction rate. By analyzing the experimental data in Table 3-2, the regression equation was obtained as follows: Yield = 2.42 + 0.39*A-0.14*B+0.38*C-0.045*D-0.16*AB-0.15*AC+0.020*AD+0.36*BC-0.13*BD+0.14*CD-0.48*A2-0.80*B2-0.50*C2-0.49*D2

[0098] Wherein: A refers to the material-liquid ratio, B refers to the extraction time, C refers to the extraction temperature, and D refers to the ultrasonic power.

[0099] The variance analysis of the model is shown in Table 4, and the credibility analysis is shown in Table 5. The experimental analysis of Table 4 shows that the model F is 62.33, p < 0.0001, the model is highly significant, the lack of fit term is not significant, and the residual probability distribution effect is good, so the fitting is effective, that is, this experimental method can be used to analyze the extraction effect of betalain. From the data analysis in the table, it can be seen that the linear term A of the solid-liquid ratio, the linear term B of the extraction time, the linear term C of the extraction temperature, the quadratic interaction term BC of the extraction time and the extraction temperature, and the quadratic term A of the solid-liquid ratio in the model are 2 , extract the time quadratic term B 2 , extract the temperature quadratic term C 2 , ultrasonic power quadratic term D 2 , p<0.01, all reached the extremely significant level. The quadratic interaction terms AB between solid-liquid ratio and extraction time, AC between solid-liquid ratio and extraction temperature, BD between extraction time and ultrasonic power, and BD between extraction temperature and ultrasonic power, p<0.05, all reached the significant level.

[0100] From the F value, we can see that the order of influence of the four factors on the yield of betalain is: solid-liquid ratio > ultrasonic temperature > ultrasonic time > ultrasonic power.

[0101] Table 4 Analysis of variance of regression model

[0102]

[0103] The credibility of the model is usually reflected by the coefficient of variation. The lower the coefficient of variation, the higher the credibility. As shown in Table 5, the coefficient of variation of the regression model in this experiment is 7.45%, which is a low value, indicating that the model is highly credible. 2 =0.9842, indicating that the correlation of the model is good. 2 =0.9684 shows that the model can explain 96.84% of the changes in the response values. Through this model, the effects of the four factors on the yield of betalain can be reasonably analyzed and predicted.

[0104] Table 5 Regression equation credibility analysis

[0105]

[0106] Through the model analysis of Design-Expert, the optimal process level is coded as: A solid-liquid ratio = 0.375g / mL, B extraction time = -0.059min, C extraction temperature = 0.307℃, D ultrasonic power = 0.014W, and the expected yield is 2.554mg / g. Three parallel tests were conducted with a solid-liquid ratio of 1:27g / mL, extraction time of 29min, extraction temperature of 43℃, and ultrasonic power of 150W. It was determined that the average yield of betalain extracted under these conditions was 2.548mg / g, which was only 0.235% error compared with the predicted value of the model.

[0107] The solid-liquid ratio was 1:27 g / mL, the ultrasonic extraction time was 29 min, the extraction temperature was 43 °C, and the ultrasonic power was 150 W. The final yield of betalain after optimization was 2.548 mg / g, which was higher than that of the single factor experiment. Therefore, the extraction of betalain from 9 red beet germplasms under this process condition can ensure a higher pigment extraction rate.

[0108] Specific implementation method 2: Combination Figure 1-Figure 2 The present embodiment is described. A flavonoid detection method based on the cumulative rule of red beet nutrients in the present embodiment comprises the following steps:

[0109] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0110] Step 2: Add 0.1 g of freeze-dried powder to 1 ml of 60% ethanol, perform ultrasonic extraction for 30 min, centrifuge in a centrifuge, and take the supernatant to a fixed volume after centrifugation;

[0111] Step 3: According to the plant flavonoid content detection kit, add the sample to the reaction system and mix evenly. After mixing evenly, put it into a 37°C oven and dry it for 45 minutes. After drying, cool and centrifuge it to obtain the supernatant. Place the supernatant in an ELISA instrument to measure its absorbance at a wavelength of 470nm, and then convert the measured absorbance value into the flavonoid content in the sample according to the standard curve and the flavonoid content calculation formula.

[0112] The red beet germplasm is Yeginas2, planted at 135d, Red Circle 30, planted at 135d, Y7792, planted at 135d, Meihe No. 5, planted at 150d, and Y7792, planted at 120d, at which time the flavonoid yield is optimal.

[0113] Specific implementation method three: Combination Figure 1-Figure 2 The present embodiment is described. A betaine detection method based on the cumulative rule of red beet nutrients in the present embodiment comprises the following steps:

[0114] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0115] Step 2: According to the betaine kit, 0.2g-0.3g of freeze-dried powder was added to 1ml of 80% ethanol, and the mixture was placed in a 60°C water bath for extraction for 30min. 3mg of reagent 4 in the betaine kit was added to the extract, and the supernatant was obtained by centrifugation;

[0116] Step 3: Place the supernatant in a 70°C water bath to evaporate the methanol to 0.2 ml, then adjust the volume to 1 ml, add reagents and samples according to the requirements of the kit, and finally substitute the standard curve to calculate the concentration of betaine in the sample.

[0117] The red beet germplasm is Yeginas2, the planting time is 135d, Hongyuan 30, the planting time is 135d, Y7792, the planting time is 135d, Meihe No. 5, the planting time is 150d, and Y7792, the planting time is 120d, at which time the betaine extraction yield is optimal.

[0118] Specific implementation method four: Combination Figure 1-Figure 2 The present embodiment is described. A total phenol detection method based on the cumulative law of red beet nutrients in the present embodiment comprises the following steps:

[0119] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0120] Step 2: Add 0.1 g of freeze-dried powder to 2.5 ml of 60% ethanol, perform ultrasonic extraction for 30 min, centrifuge in a centrifuge, and take the supernatant to a fixed volume after centrifugation;

[0121] Step 3: According to the plant total phenol content detection kit, add samples to the control tube, test tube, standard tube and blank tube respectively. Let it stand for 10 minutes after adding samples. After standing, place it in an enzyme reader at 760nm to measure the absorbance, and use the gallic acid standard curve to calculate the total phenol concentration.

[0122] The red beet germplasm is ALTOF1, and the total phenol extraction yield is optimal when the planting time is 75, 120, 105, and 105 days.

[0123] Specific implementation method five: Combination Figure 1-Figure 2 The present embodiment is described as follows: a mineral detection method based on the cumulative rule of red beet nutrients in the present embodiment comprises the following steps:

[0124] Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, grind with a grinder after freeze-drying, sieve to obtain freeze-dried powder;

[0125] Step 2: Add 0.2g-0.3g of freeze-dried powder to a 50ml polytetrafluoroethylene digestion tube, then add 10mL nitric acid and 0.5mL perchloric acid and mix evenly. After mixing evenly, place it in a fume hood to react for 1h, then place it on an intelligent digestion instrument for acid digestion, react at 110℃ for 50min, then adjust to 180℃ for 2h, and transfer it to a volumetric flask after cooling;

[0126] Step 3: Add ionized water to the volumetric flask to make up to 25 ml, mix and set aside, and use 10 ml nitric acid-0.5 ml perchloric acid mixture as a blank reference. Inject the treated blank solution and sample solution into the inductively coupled plasma emission spectrometer respectively, measure the signal response value of the analysis line intensity of the element to be measured, and obtain the concentration of the element to be measured in the digestion solution according to the standard curve.

[0127] The red beet germplasm is Yeginas2, the planting time is 135d, Red Circle 30, the planting time is 135d, Y7792, the planting time is 135d, Meihe No. 5, the planting time is 150d, and Y7792, the planting time is 120d, at which time the mineral extraction yield is optimal.

[0128] Specific implementation method six: Combination Figure 1-Figure 2 The present embodiment is described. The present embodiment is a method for detecting soluble sugar based on the cumulative law of red beet nutritional components. 0.5 g of red beet root is ground into a homogenate in a mortar, 10 mL of phosphate buffer is added, and the mixture is placed in a centrifuge. The mixture is centrifuged at a speed of 12000 r / min for 20 min. After the centrifugation, 100 μL of supernatant is taken, 300 μL of water, 100 μL of anthrone ethyl acetate reagent, and 1 mL of sulfuric acid are added. The mixture is mixed and placed on an enzyme reader to measure its absorbance at 630 nm. The soluble sugar content is calculated according to the sucrose standard curve.

[0129] The red beet germplasm is Yeginas2, the planting time is 135d, Hongyuan 30, the planting time is 135d, Y7792, the planting time is 135d, Meihe No. 5, the planting time is 150d, and Y7792, the planting time is 120d, at which time the soluble sugar yield is optimal.

[0130] Specific implementation method seven: Combination Figure 1-Figure 2 The present embodiment is described. The present embodiment is a method for detecting soluble protein based on the cumulative rule of red beet nutritional components. 0.5 g of red beet root is ground into a homogenate in a mortar, 10 mL of phosphate buffer is added, and the mixture is placed in a centrifuge. The mixture is centrifuged at a speed of 12000 r / min for 20 min. After the centrifugation, 20 μL of supernatant is transferred to a 96-well plate. 200 μL of a mixture of alkaline solution and 4% copper sulfate are added. The mixture is mixed and heated at 37°C for 30 min. After the heating, the mixture is cooled to room temperature and placed in an enzyme reader to measure the absorbance at 562 nm. The concentration of soluble protein is calculated according to the BSA standard curve.

[0131] The germplasm of the red beet is ALTOF1, and the yield of soluble protein extracted is optimal when the planting time is 75, 120, 105, and 105 days.

[0132] Specific implementation method eight: Combination Figure 1-Figure 2 The present embodiment is described. The present embodiment is an ascorbic acid detection method based on the cumulative law of red beet nutritional components. 0.5-0.7g of red beet root is ground in a mortar. The mortar is placed in an ice box during grinding to ensure a low-temperature environment. The slurry is ground into a homogenate and 1-1.2mL of extract is added. The slurry is placed in a centrifuge for centrifugation to extract the supernatant. When testing the sample, according to the ascorbic acid content detection kit, 10 reagents and the supernatant are added into 5 test tubes as required and mixed. After all the test tubes are mixed, they are placed in a 42°C water bath for heat preservation and heating for 40 minutes. After the heating is completed, they are cooled. After cooling, the absorbance is measured at 525nm on an enzyme reader, and the ascorbic acid content in the sample is calculated according to the formula.

[0133] The red beet germplasm is Yeginas2, the planting time is 135d, Hongyuan 30, the planting time is 135d, Y7792, the planting time is 135d, Meihe No. 5, the planting time is 150d, and Y7792, the planting time is 120d, at which time the soluble sugar yield is optimal.

[0134] The membership function was used to calculate the comprehensive nutritional scores of various germplasms, and the scores were ranked from high to low as follows: ALTOF1 120 days, Y7792 135 days, S214 135 days, Meihe 5 150 days, Hongyuan 30 135 days, American Hybrid 135 days, Jieluotai 105 days, 357357 (Red) 120 days, and Yeginas2 135 days. In terms of the accumulation of several nutrients, germplasms with higher comprehensive scores contain more antioxidant components.

[0135] It should be noted that in the above embodiments, as long as the technical solutions are not contradictory, they can be arranged and combined, and those skilled in the art can exhaust all possibilities based on the mathematical knowledge of arrangement and combination. Therefore, the present invention will no longer describe the technical solutions after arrangement and combination one by one, but it should be understood that the technical solutions after arrangement and combination have been disclosed by the present invention.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for ultrasonic extraction of betalain based on the cumulative law of red beet nutrients, characterized in that: The steps include: Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, and grind and sieve with a grinder to obtain freeze-dried powder; Step 2: Add an extractant to the freeze-dried powder, perform ultrasonic extraction, filter with gauze after extraction, and then filter with filter paper to remove the residual dietary fiber in the beet juice. After removal, place the beet juice in a centrifuge at 12000r / min, centrifuge for 10min, take 665μL of supernatant, and make up to 10mL.

2. The method for ultrasonic extraction of betalain based on the cumulative law of red beet nutrients according to claim 1, characterized in that: The liquid-to-solid ratio of the freeze-dried powder to the extractant is 1:27 g / mL, the ultrasonic extraction time is 29 min, the extraction temperature is 43° C., and the ultrasonic power is 150 W.

3. The method for ultrasonic extraction of betalain based on the cumulative rule of red beet nutrients according to claim 1, characterized in that: The extractant is one of 0.5% citric acid, 60% acetic acid, and deionized water.

4. A flavonoid detection method based on the cumulative law of red beet nutrients, characterized in that: The steps include: Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, and grind and sieve with a grinder to obtain freeze-dried powder; Step 2: Add 0.1 g of freeze-dried powder to 1 ml of 60% ethanol, perform ultrasonic extraction for 30 min, centrifuge in a centrifuge, and take the supernatant to a fixed volume after centrifugation; Step 3: According to the plant flavonoid content detection kit, add the sample to the reaction system and mix evenly. After mixing evenly, put it into a 37°C oven and dry it for 45 minutes. After drying, cool and centrifuge it to obtain the supernatant. Place the supernatant in an ELISA instrument to measure its absorbance at a wavelength of 470nm, and then convert the measured absorbance value into the flavonoid content in the sample according to the standard curve and the flavonoid content calculation formula.

5. A betaine detection method based on the cumulative law of red beet nutrients, characterized in that: The steps include: Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, and grind and sieve with a grinder to obtain freeze-dried powder; Step 2: According to the betaine kit, 0.2 g to 0.3 g of freeze-dried powder was added to 1 ml of 80% ethanol, and the mixture was placed in a 60°C water bath for extraction for 30 min. 3 mg of reagent 4 in the betaine kit was added to the extract, and the mixture was centrifuged to obtain a supernatant; Step 3: Place the supernatant in a 70°C water bath to evaporate the methanol to 0.2 ml, then adjust the volume to 1 ml, add reagents and samples according to the requirements of the kit, and finally substitute the standard curve to calculate the concentration of betaine in the sample.

6. A method for detecting total phenols based on the cumulative law of red beet nutrients, characterized in that: The steps include: Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, and grind and sieve with a grinder to obtain freeze-dried powder; Step 2: Add 0.1 g of freeze-dried powder to 2.5 ml of 60% ethanol, perform ultrasonic extraction for 30 min, centrifuge in a centrifuge, and take the supernatant to a fixed volume after centrifugation; Step 3: According to the plant total phenol content detection kit, add samples to the control tube, test tube, standard tube and blank tube respectively. Let it stand for 10 minutes after adding samples. After standing, place it in an enzyme reader at 760nm to measure the absorbance, and use the gallic acid standard curve to calculate the total phenol concentration.

7. A mineral detection method based on the cumulative law of red beet nutrients, characterized in that: The steps include: Step 1: Remove the leaves and wash the roots of red beets picked in the same growth period, wipe off the moisture, peel and slice, place in a refrigerator at -20°C for pre-freezing for 24 hours, take out the pre-freezing and place in a vacuum freeze dryer for freeze-drying for 48 hours, and grind and sieve with a grinder to obtain freeze-dried powder; Step 2: Add 0.2g-0.3g of freeze-dried powder to a 50ml polytetrafluoroethylene digestion tube, then add 10mL nitric acid and 0.5mL perchloric acid and mix well. After mixing well, place it in a fume hood to react for 1h, then place it on an intelligent digestion instrument for acid digestion. After reacting at 110℃ for 50min, adjust to 180℃ for reaction for 2h, and transfer it to a volumetric flask after cooling; Step 3: Add ionized water to the volumetric flask to make up to 25 ml, mix and set aside, and use 10 ml nitric acid-0.5 ml perchloric acid mixture as a blank reference. Inject the treated blank solution and sample solution into the inductively coupled plasma emission spectrometer respectively, measure the signal response value of the analysis line intensity of the element to be measured, and obtain the concentration of the element to be measured in the digestion solution according to the standard curve.

8. A method for detecting soluble sugar based on the cumulative law of red beet nutrients, characterized in that: Grind 0.5 g of red beetroot in a mortar into a homogenate, add 10 mL of phosphate buffer, place in a centrifuge, and centrifuge at a speed of 12000 r / min for 20 min. After centrifugation, take 100 μL of supernatant, add 300 μL of water, 100 μL of anthrone ethyl acetate reagent, and 1 mL of sulfuric acid, mix well, and place in an enzyme reader to measure its absorbance at 630 nm, and calculate the soluble sugar content according to the sucrose standard curve.

9. A method for detecting soluble protein based on the cumulative law of red beet nutrients, characterized in that: Grind 0.5 g of red beetroot in a mortar into a homogenate, add 10 mL of phosphate buffer, place in a centrifuge, and centrifuge at a speed of 12000 r / min for 20 min. After centrifugation, take 20 μL of supernatant to a 96-well plate, add 200 μL of a mixture of alkaline solution and 4% copper sulfate, mix well, and heat at 37°C for 30 min. After heating, cool to room temperature, place in an enzyme reader to measure the absorbance at 562 nm, and calculate the concentration of soluble protein according to the BSA standard curve.

10. A method for detecting ascorbic acid based on the cumulative law of red beet nutrients, characterized in that: Grind 0.5-0.7 g of red beetroot in a mortar and place the mortar in an ice box to ensure a low temperature environment. After grinding into a homogenate, add 1-1.2 mL of extract and place it in a centrifuge for centrifugation to extract the supernatant. When testing, add 10 reagents and the supernatant to 5 test tubes as required according to the ascorbic acid content detection kit. After all the test tubes are mixed, place them in a 42°C water bath for insulation and heating for 40 minutes. After heating, cool them down and measure the absorbance at 525 nm on an enzyme reader. Calculate the ascorbic acid content in the sample according to the formula.

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