CN-122016712-A - Far embryo end scraping method for measuring total phenol content of seeds and near infrared data processing method

CN122016712ACN 122016712 ACN122016712 ACN 122016712ACN-122016712-A

Abstract

The invention discloses a far embryo end scraping method and a near infrared data processing method for measuring total phenol content of seeds, which comprise the following steps of sample screening and sampling; removing oil interference components, extracting medium-high polar components, and finally measuring the total phenol content of the seeds. The infrared data processing method directly takes unground peanut seeds with skin as near infrared spectrum data acquisition background, adopts a special far embryo end scraping method to measure the total phenol content of the peanuts, constructs a data process to optimize, and has consistent compatibility for phenol components with the content of less than 1% in common peanuts, rare hybridization germplasm resources and early generation materials of hybridization breeding. The research result of the invention can provide technical support for rapid screening of peanut resources with high total phenol content and efficient cultivation of peanut varieties with high total phenol content.

Inventors

ZHAO XING
FAN WENXUAN
WANG JIN

Assignees

石家庄学院
河北省农林科学院粮油作物研究所

Dates

Publication Date: 20260512
Application Date: 20240408

Claims (10)

1. The far embryo end scraping method for measuring the total phenol content of seeds is characterized by comprising the following steps: ⑴ . Sample screening and sampling; ⑵ . Removing oil and fat interference components; ⑶ . Extracting medium-high polarity components; And finally, drawing a standard curve based on the extracted components, a blank control and a standard substance to determine the total phenol content of the seeds.
2. The method for scraping the distal embryo end for measuring the total phenol content of seeds according to claim 1, wherein the method comprises the following steps: ⑴ . Selecting mature full, centrally-sized, germination-free, breakage-free and spot-free seeds, taking the seeds, scraping the position of each seed, which is not more than 1/4 of the distal embryo end, with a blade, repeating the operation for multiple times, and mixing the obtained seed powder for later use; ⑵ . The method comprises the steps of removing oil and fat interference components, placing seed kernel powder into a centrifuge tube, precisely weighing, adding n-hexane, inserting the centrifuge tube into a foam floating plate, soaking at room temperature, performing ultrasonic assisted extraction, cooling to room temperature, centrifuging at room temperature, separating a n-hexane layer for fatty acid determination, adding n-hexane into a precipitate by a pipette gun twice, mixing with force until no particles or caking are generated at the bottom of the centrifuge tube, performing ultrasonic assisted extraction, performing secondary degreasing, cooling to room temperature, centrifuging, separating the n-hexane layer, and obtaining defatted seed kernel powder; ⑶ . Adding methanol/ethanol water solution into the precipitate of n-hexane removed in the previous step, shaking or ultrasonic-assisted mixing until no granule or agglomeration is present at the bottom of the centrifuge tube, soaking at room temperature, ultrasonic-assisted extracting, centrifuging at room temperature, and collecting supernatant to obtain extractive solution rich in medium-high polarity components to be detected; And finally, drawing a standard curve based on the extracted components, a blank control and a standard substance to determine the total phenol content of the seeds.
3. The method for scraping the distal embryo end for measuring the total phenol content of seeds according to claim 1, wherein the method comprises the following steps: ⑴ . Selecting mature full, centrally-sized, sprouting-free, breakage-free and spot-free kernels, taking 10-20 kernels, scraping not more than 1/4 of the distal embryo end of each seed by a blade, repeating the operation for multiple times, and mixing the obtained kernel powder for later use; ⑵ . Removing oil and fat interference components, placing 0.1 g of kernel powder into a specific centrifuge tube, allowing the range to be 0.1000-0.1050 g, precisely weighing, adding 1.6 ml of normal hexane, inserting the centrifuge tube into a foam floating plate, soaking 2h at room temperature, shaking uniformly every 30min, performing ultrasonic 250W and 40 kHz auxiliary extraction 20min, shaking uniformly every 5min, cooling to room temperature, centrifuging 5min at 5000 and rpm at room temperature, separating normal hexane layer, measuring fatty acid, adding 1.6 mL of normal hexane into precipitate again by a pipette, shaking uniformly at room temperature until no particles and no caking are generated at the bottom of the centrifuge tube, performing ultrasonic 250W and 40 kHz auxiliary extraction 20min, shaking uniformly every 5min, performing secondary degreasing, cooling to room temperature, centrifuging 5min at room temperature at 3000 rpm, separating normal hexane layer, and obtaining defatted kernel powder; ⑶ . Extracting medium and high polarity components, adding methanol/ethanol water solution into the precipitate obtained by removing n-hexane in the previous step, shaking uniformly with force or mixing uniformly with the aid of ultrasonic waves for 5min, shaking uniformly every 30: 30 s with force until no particles or caking are generated at the bottom of a centrifuge tube, soaking 2: 2h at room temperature, shaking uniformly every 30: 30min with force, extracting 20: 20 min with the aid of ultrasonic waves of 250W and 40 kHz, shaking uniformly every 5: 5min with force, cooling to room temperature, centrifuging for 15: 15 min at room temperature with 13000: 13000 rpm, and collecting supernatant to obtain an extract rich in medium and high polarity components to be detected; And finally, drawing a standard curve based on the extracted components, a blank control and a standard substance to determine the total phenol content of the seeds.
4. The method for remote embryo end scraping for total phenol content determination of seeds according to claim 3, wherein in step ⑵, the specific centrifuge tube is constructed as a centrifuge tube made of polypropylene and having a conical bottom apex angle of 120 DEG+ -10 DEG, a round bottom or a pointed conical bottom centrifuge tube is avoided, and the method is used for reducing sediment hardening and adapting to multiple extractions of sediment on the premise of ensuring the centrifugal efficiency, not losing sediment and fully separating supernatant.
5. The method according to claim 3, wherein in step ⑵, precipitation is not lost when separating the n-hexane layer, the n-hexane residue is minimized when separating the n-hexane layer for the second time, and the n-hexane is prevented from being volatilized before adding the aqueous methanol solution.
6. The method of claim 3, wherein in step ⑶, if the amount of protein in the extract is higher than a predetermined value, the extract is refrigerated overnight to precipitate the protein sufficiently and then the subsequent measurement is performed.
7. The method of claim 3, wherein in step ⑶, a 50% aqueous methanol solution is selected for the type and concentration of the aqueous methanol/ethanol solution.
8. The method of claim 3, wherein in step ⑶, according to the criterion of the feed-to-liquid ratio, the ratio of the feed-to-liquid ratio is 1:15, i.e. 1.5. 1.5 ml methanol/ethanol aqueous solution is added to each 0.1. 0.1 g seed according to the actual content of the component to be measured and the sensitivity achieved by the subsequent detection method.
9. The near infrared data processing method is characterized in that the method directly takes unground multi-sample shelled peanut seeds as a background to carry out multiple sample loading and near infrared spectrogram measurement to obtain average near infrared spectrum data, and simultaneously carries out total phenol content measurement of all peanut samples based on a far-embryo end scraping method to obtain multi-sample total phenol content data, and on the basis, model structure construction and optimization are carried out according to the following processes: the method comprises the steps of constructing a primary filtering algorithm for the mean near infrared spectrum data of the obtained peanut multi-sample modeling material according to the following processes, wherein a designated wavelength lambda is used as a basic continuous space variable of linear self-variation, the corresponding variable of the original near infrared spectrum data f (lambda) of the obtained peanut multi-sample modeling material is obtained according to the differential correspondence of the wavelength lambda, and then the corresponding variable of f (lambda) is distributed to the differential variable of the wavelength lambda, so that the chemical information in the spectrum data is enhanced, the influence of background noise is reduced, and the primary filtering algorithm is used for carrying out the external highlighting on the internal intrinsic slope of a spectrum curve; Further, the primary filtering algorithm is iterated as required to obtain a secondary filtering algorithm, and the secondary filtering is used for highlighting curvature changes in a spectrum curve; On the basis, the spectrum data of the peanut sample is further subtracted by the mean value and divided by the standard deviation to correct the scattering effect and the associated data variation in the spectrum data; The method has consistent compatibility for phenolic components with the content lower than 1% in common peanuts and rare hybridization breeding germplasm resources and hybridization breeding early generation materials.
10. The near infrared data processing method compatible with rare germplasm resources and micro peanut total phenol content analysis is characterized by comprising the steps of carrying out sample loading and near infrared spectrogram measurement for a plurality of times by taking unground multi-sample shelled peanut seeds as a background, naturally airing a peanut material for modeling until the water content is below 5%, selecting mature full and unbroken and rotten peanut seeds as measurement samples, placing the samples at a constant temperature of about 25 ℃ for more than 48 hours, carrying out spectral measurement on the samples by adopting a near infrared quality analyzer, scanning the samples with a wavelength range of 950-1650 nm and a resolution of 5 nm, after the instrument is preheated, flatly loading the samples to be measured into a sample cup, fully covering a bottom mirror surface, repeatedly loading the samples for 3 times, carrying out sample loading measurement for 3 times each time, and carrying out detection on each sample to obtain 9 pieces of near infrared spectrograms of the peanut seeds in total, and taking an average spectrum.

Description

Far embryo end scraping method for measuring total phenol content of seeds and near infrared data processing method The application relates to a separated application of patent application, which is named as a near infrared data model capable of being compatible with rare germplasm resources and analyzing the total phenol content of trace peanuts, and has the application number of 202410415055.2 and the application date of 2024-04-08. Technical Field The invention relates to a data model construction and analysis technology of biological materials, in particular to a far embryo end scraping method and a near infrared data processing method for measuring total phenol content of seeds. Background Peanuts are important oil plants and cash crops in China, are also important sources of various food-borne functional components, and have important roles in national economy and social development in China. Peanut germplasm improvement and cross breeding in China begin in the 50 s of the 20 th century, and 5 times of updating are realized at present. Along with the improvement of the diet concept of consumers, green high-quality foods are increasingly valued, peanut varieties in China are undergoing the 6 th update, and the update takes the high-oleic acid varieties as marks for replacing common oleic acid varieties, prolonging the shelf life and improving the health care effect, so as to continuously meet the increasing living standard and health care requirements of people. Research shows that compared with common peanuts, the high-oleic acid peanuts can play a positive role in preventing and controlling various diseases such as metabolic syndrome, obesity, cardiovascular diseases, diabetes and the like, so that the quality gap between peanut oil which is known as Chinese olive oil and the olive oil is reduced. However, olive oil is recognized as beneficial to human health, and the high oleic acid content is one of the reasons and the high phenolic content is another important reason. The phenolic components in the edible oil are beneficial to prolonging the shelf life of the edible oil, reducing the degradation of lipid nutrient substances in the edible oil in the cooking process, and has important significance for improving the nutrition and quality of the edible oil. Phenolic components are a large class of substances including a plurality of different aromatic secondary metabolites in plants, including flavone, phenolic acid, phenylpropane, quinine and other substances, and often are produced in early stages of plant development and/or when being stimulated by a plurality of microorganisms, and the content of the phenolic components is influenced by a plurality of factors such as genotype, geography, climate and the like. According to statistics, the numbers of scientific research type documents related to phenolic component research published in CNKI and PubMed databases in the last 20 years are 4987 and 4121 respectively, the amount of the documents is in an ascending trend, and 950 documents taking national grade foundation as fund are in the documents of CNKI databases. Therefore, the development and utilization of phenolic components have become a popular focus at home and abroad. References relevant to the invention include ① Wang Chuantang, zhang Jiancheng, tangyue, in the tree, wang Jiang, liu Feng, li Qiu, china high oleic peanut breeding status and hope, shandong agricultural science ,2018,50（6）：171-176.② Jurgoński A, Fotschki B, Juśkiewicz J. Disparate metabolic effects of blackcurrant seed oil in rats fed a basal and obesogenic diet. European Journal of Nutrition, 2015, 54(6): 991-999.③ Huth PJ, Fulgoni VL, Larson BT. A systematic review of high-oleic vegetable oil substitutions for other fats and oils on cardiovascular disease risk factors: implications for novel high-oleic soybean oils. Advances in Nutrition, 2015, 6(6): 674-693.④ Schwingshackl L, Lampousi A, Portillo M, Romaguera D, Hoffmann G, Boeing H. Olive oil in the prevention and management of type 2 diabetes mellitus: a systematic review and meta-analysis of cohort studies and intervention trials. Nutrition & Diabetes, 2017, 7(4): e262-e262.⑤ Wang Chuantang, zhang Jiancheng, tangyue, in the tree, wang Jiang, liu Feng, li Qiu, china high oleic peanut breeding status and hope, shandong agricultural science ,2018,50（6）：171-176.⑥ Veloso ACA, Rodrigues N, Ouarouer Y, Zaghdoudi K, Pereira JA, Peres AM. A Kinetic-Thermodynamic study of the effect of the cultivar/total phenols on the oxidative stability of olive oils. Journal of the American Oil Chemists' Society, 2020, 97(6): 625-636.⑦ Winkel-Shirley B. Flavonoid biosynthesis: a colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology, 2001, 126:485-493.⑧ Sobolev VS, Horn BW, Potter TL, Deyrup ST, Gloer JB. Production of stilbenoids and phenolic acids by the peanut plant at early stages of growth. Journal of Agriculture and Food Chemistry, 2006, 54(10): 3505-