CN102692387A - Method for determining total content of glucosinolates in plants - Google Patents
Method for determining total content of glucosinolates in plants Download PDFInfo
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Abstract
The invention relates to a method for determining total content of glucosinolates in plants, belonging to the field of food biotechnologies. The method is characterized in that the plants are taken as testing materials, raw materials are crushed and then homogenized by using an acetate buffer, a homogenate is subjected to high-temperature water bath enzyme deactivation, then mixed liquor of polyvinylpyrrolidone and lead acetate/barium acetate and a saturated sodium sulfate solution are sequentially added, and a glucosinolate extracting solution is produced through centrifuging after carrying out shaking to mix homogeneously; the glucosinolate extracting solution is subjected to alkaline degradation and neutralized by concentrated hydrochloric acid of 37%, a neutralized solution reacts with a potassium ferricyanide solution, and then, the decrement of a light absorption value of potassium ferricyanide in the position of 420 nm is determined by using a spectrophotometer; and a standard curve is made by taking sinigrin as a standard matter, and the total content of the glucosinolates in the plants is obtained through calculating. The method has the advantages of high accuracy, good reproducibility and short determination time; and compared with the high-performance liquid chromatography (HPLC), expensive instruments and equipment and special chromatographic separation conditions are not required, so that the method is applicable to the rapid detection of the total content of the glucosinolates in vegetable raw materials and products thereof of ordinary laboratories and food enterprises.
Description
One, technical field
The present invention relates to the assay method of glucosinolate total amount in the kind of plant, belong to technical field of food biotechnology.
Two, background technology
The sulphur glycosides is ubiquitous one type of secondary metabolite in the crucifer, edible be rich in glucosinolate (sulphur glycosides) but the generation of multiple cancers such as brassicaceous vegetable prevention of pancreatic cancer, lung cancer and the carcinoma of the rectum.This function is main relevant with the hydrolysate one isothiocyanic acid salt material of sulphur glycosides.After plant tissue was damaged, the sulphur glycosides contacted with myrosin, and hydrolysis produces materials such as isothiocyanate, thiocyanate, nitrile and oxazolidine.
At present; The mensuration of sulphur glycosides generally adopts X-ray fluorescence method (XRF) (ISO 9167-2:1994 (E) .Rapeseed-Determination of glucosinolates content.Part 2:Method using X-ray fluorescence spectrometry in the world; 5 pp.) and high performance liquid chromatography (HPLC) (ISO 10633-1:1995 (E) .Oilseed residues-Determination of glucosinolates content.Part 1:Method using high-performance liquid chromatography; 9 pp.); Though these two kinds of precision of method height; But its apparatus expensive, sample pre-treatments are complicated and consuming time, are difficult to widespread usage.In addition, sulphur glycosides kind has kind more than 120, and the sulphur glycosides standard items that can buy only two kinds of sinigrin and glucotropaeolin, so the HPLC method also will be carried out mass spectrum to every kind of sulphur glycosides and identified, and then has improved the experiment difficulty.Other methods of measuring the sulphur glycosides have sulfuric acid-thymol method (Tholen, J.T., Shen; S., TruscottS, R.J.W.The Thymol Method for Glucosinolate Determination [J] .Journal of the Science of Food and Agriculture; 1989,49:157～165.), the sulfate ion precipitation method (Mcghee; J.E., Kirk, L.D.; Mustakas, G.C.Methods for Determining Thioglucosides in Crambe abyssinica.J.AOCS.889 (42), 1965.); Glucose discharges an enzyme identification method (ISO/TC 34/SC2:Rapeseed-Determination of glucosinolates Content-Part 2:Spectrometric Method for Total Glucosinolates by Glucose Release.) and ELISA (Doorn, H.E., Holst; G.G.C.Kruk, v.N.C.M.E.Raaijmakers, Postma; E.Quantitative Determination of the Glucosinolates Sinigrin and Progoitrin by Specific Antibody ELISA Assays in Brussels Sprouts [J] .Journal of Agricultural and Food Chemistry.1998; 46:793～800.), these methods all have certain limitation, like the interference of sulfate ion in the sample and glucose; The sulfate ion precipitation method need high-temperature calcination and minute long, and the experimental implementation of ELISA is complicated, cost is high.In addition, said method all need utilize exogenous enzymes degraded sulphur glycosides, and enzyme is difficult to obtain and the sulphur glycosides is degraded not thorough.Therefore, set up a kind of simple, quick, accurate total sulfur glycosides assay method, significant to the brassicaceous vegetable quality control and the comprehensive exploitation of being rich in the sulphur glycosides.
The bibliographical information of at present domestic relevant sulphur glycosides mainly concentrates on the separation and Extraction aspect of single sulphur glycosides and hydrolysate thereof; Like patent (publication number CN101100476A; Open day on January 9th, 2008) " method of from Radish seed, separating 4-methylsulfinyl-3-butenyl group glucosinolate " disclosed; Patent (publication number CN101831479A, open day on September 15th, 2010) discloses " method of preparing 4-methylsulfinylbutyltbyoglucoside byoglucoside biotransformation " etc.; (Qian Hua such as Qian Hua; Zhao Baitao, Huang Xiaode. the mensuration [J] of β in the broccoli-glucosinolate total amount. Chinese wild plant resource, 2008; 27 (2): 52～57.) through 3; The glucose content that 5-dinitrosalicylic acid method is measured sulphur glycosides hydrolysis generation calculates total sulfur glycosides content in the broccoli, but this method needs exogenous enzymes hydrolysis sulphur glycosides, and it measures the result than actual higher.(Chu Qinghua such as Chu Qinghua; Ni Xinlu. the research [J] of glucosinolate total amount rapid assay methods in rapeseed and the grouts thereof. Chinese grain and oil journal, 2004,19 (1): 79～83.) adopt glucose release-enzyme identification method to measure total sulfur glycosides content in the rapeseed; This method process is loaded down with trivial details; Still need exogenous enzymes hydrolysis sulphur glycosides, and the sulphur glycosides needs ion exchange column when extracting, strengthened experimental cost.(Huang Jiying such as Huang Jiying; Wang Suizhang, Li Sumei. palladium bichloride-spectrophotometry rapeseed sulphur glycosides Study on content. Northwest Agricultural University's journal, 1995; 23 (6): 104～107.) utilize chlorination handle-spectrophotometry rapeseed sulphur glycosides content; But this method accuracy is low, and palladium bichloride costs an arm and a leg, and it is bigger that experimental result is influenced by the accuracy etc. of passivation, sulphur extraction time former times, weighing and the constant volume of instrument, endogenous enzymes.
Three, summary of the invention
The object of the invention is to overcome the shortcoming of prior art; A kind of degree of accuracy height, favorable reproducibility, minute weak point are provided; Need not the sulphur glycosides assay method of expensive instrument and equipment and special chromatographic separation condition, be suitable for common lab and food enterprise fast detecting total sulfur glycosides content in vegetable raw-material and the goods thereof.
The object of the invention is achieved through following technical scheme:
(1) extraction of sulphur glycosides:
Get an amount of plant sample, after the pulverizing, press solid-liquid ratio (g/ml) and added acetate buffer (0.2mol/L in 1: 100～1: 200; PH 4.2), fully grind homogenate, homogenate is in 70～100 ℃ of water-bath 15min enzyme that goes out; Add 6g polyvinylpyrrolidone and 60～100ml lead acetate/barium acetate mixed liquor by every 1g sample successively after the cooling; After room temperature leaves standstill 15min, add the saturated metabisulfite solution of 60～100ml, the vibration mixing by every 1g sample; In the centrifugal 15min of 10000g, collect supernatant and be sulphur glycosides extract.
(2) alkaline degradation of sulphur glycosides:
Get above-mentioned sulphur glycosides extract 2～4ml and mix with equal-volume 2mol/L NaOH or KOH solution, behind the room temperature reaction 30min with 37% concentrated hydrochloric acid neutralization reaction liquid.
(3) mensuration of sulphur glycosides:
Get the potassium ferricyanide solution that reactant liquor 2～4ml and isopyknic 2mmol/L after the above-mentioned neutralization be dissolved in phosphate buffer (0.2mol/L, pH 7.0) and mix, room temperature is measured 420nm place light absorption value after leaving standstill 15min in 15s.Do sulphur glycosides typical curve with allyl sulfide glycosides (Sinigrin), horizontal ordinate is an allyl sulfide glycosides concentration, and ordinate is the light absorption value reduction Δ A of 420nm place
420(A
420max-A
420), record total sulfur glycosides content in the sample through calculating.A
420maxPotassium ferricyanide solution light absorption value when adding the extract of sulfur-bearing glycosides not, the experiment blank is the mixed liquor of sulphur glycosides extract and phosphate buffer.
(4) calculating of sulphur glycosides:
According to the Δ A that records
420Substitution typical curve equation is calculated as follows total sulfur glycosides content in the sample:
Y: sulphur glycosides total amount (μ mol/g) in the sample;
A: calculate gained sulphur glycosides concentration (μ mol/ml) by typical curve;
V: whole sulphur glycosides extracting liquid volume (ml);
M: institute's sample thief quality (g).
Compared with present technology, the present invention has the following advantages:
(1) degree of accuracy of the present invention is high, and favorable reproducibility is through adding polyvinylpyrrolidone, the interference that other compositions during lead acetate/barium acetate mixed liquor and saturated metabisulfite solution have greatly been got rid of (like phenols, flavonoids, protein and carbohydrate etc.) are measured the sulphur glycosides.
(2) the present invention need not expensive instrument, equipment and special chromatographic separation condition, can measure batch samples at short notice, has greatly improved sulphur glycosides determination efficiency and has reduced cost of determination.
(3) reagent required for the present invention is cheap, and equal environment-protecting asepsis, is suitable for the detection to sulphur glycosides in the plant of common lab and food enterprise.
Four, embodiment
Below in conjunction with embodiment the present invention is done further description, but embodiment does not constitute the restriction that the present invention is required protection domain.
● embodiment 1
Get the 0.05g cabbage seed, add 5ml acetate buffer (0.2mol/L, pH 4.2) and fully grind homogenate; Mixed liquor is in 80 ℃ of water-bath 15min enzyme that goes out; The cooling back adds 0.3g polyvinylpyrrolidone and 1.5ml lead acetate/barium acetate damping fluid, and room temperature adds the saturated metabisulfite solution of 1.5ml after leaving standstill 15min, the vibration mixing; In the centrifugal 15min of 10000g, collect supernatant and be sulphur glycosides extract.Get sulphur glycosides extract 2ml and mix with 2ml NaOH solution, room temperature reaction 30min then uses 37% concentrated hydrochloric acid neutralization reaction liquid.Get afterwards among the 2ml with after reactant liquor mix with 2ml 2mmol/L potassium ferricyanide solution, leave standstill behind the 15min in the centrifugal 5min of 8000g, get supernatant and in 15s, measure 420nm place light absorption value, after calculating, obtain total sulfur glycosides content in the sample.
● embodiment 2
Get 0.1g wild cabbage bud seedling, add 10ml acetate buffer (0.2mol/L, pH 4.2) and fully grind homogenate; Mixed liquor is in 100 ℃ of water-bath 15min enzyme that goes out; The cooling back adds 0.6g polyvinylpyrrolidone and 5ml lead acetate/barium acetate damping fluid, and room temperature adds the saturated metabisulfite solution of 5ml after leaving standstill 15min, the vibration mixing; In the centrifugal 15min of 10000g, collect supernatant and be sulphur glycosides extract.Get sulphur glycosides extract 4ml and mix with 4ml NaOH solution, room temperature reaction 30min then uses 37% concentrated hydrochloric acid neutralization reaction liquid.Get afterwards among the 4ml with after reactant liquor mix with 4ml 2mmol/L potassium ferricyanide solution, leave standstill behind the 15min in the centrifugal 5min of 8000g, get supernatant and in 15s, measure 420nm place light absorption value, after calculating, obtain total sulfur glycosides content in the sample.
● embodiment 3
Get the 0.05g broccoli, add 5ml acetate buffer (0.2mol/L, pH 4.2) and fully grind homogenate; Mixed liquor is in 80 ℃ of water-bath 15min enzyme that goes out; The cooling back adds 0.3g polyvinylpyrrolidone and 1.5ml lead acetate/barium acetate damping fluid, and room temperature adds the saturated metabisulfite solution of 1.5ml after leaving standstill 15min, the vibration mixing; In the centrifugal 15min of 10000g, collect supernatant and be sulphur glycosides extract.Get sulphur glycosides extract 2ml and mix with 2ml NaOH solution, room temperature reaction 30min then uses 37% concentrated hydrochloric acid neutralization reaction liquid.Get afterwards among the 2ml with after reactant liquor mix with 2ml 2mmol/L potassium ferricyanide solution, leave standstill behind the 15min in the centrifugal 5min of 8000g, get supernatant and in 15s, measure 420nm place light absorption value, after calculating, obtain total sulfur glycosides content in the sample.
● embodiment 4
Get the 0.05g horseradish, add 5ml acetate buffer (0.2mol/L, pH 4.2) and fully grind homogenate; Mixed liquor is in 80 ℃ of water-bath 15min enzyme that goes out; The cooling back adds 0.3g polyvinylpyrrolidone and 1.5ml lead acetate/barium acetate damping fluid, and room temperature adds the saturated metabisulfite solution of 1.5ml after leaving standstill 15min, the vibration mixing; In the centrifugal 15min of 10000g, collect supernatant and be sulphur glycosides extract.Get sulphur glycosides extract 2ml and mix with 2ml NaOH solution, room temperature reaction 30min then uses 37% concentrated hydrochloric acid neutralization reaction liquid.Get afterwards among the 2ml with after reactant liquor mix with 2ml 2mmol/L potassium ferricyanide solution, leave standstill behind the 15min in the centrifugal 5min of 8000g, get supernatant and in 15s, measure 420nm place light absorption value, after calculating, obtain total sulfur glycosides content in the sample.
● embodiment 5
Get the 0.05g cauliflower, add 5ml acetate buffer (0.2mol/L, pH 4.2) and fully grind homogenate; Mixed liquor is in 90 ℃ of water-bath 15min enzyme that goes out; The cooling back adds 0.3g polyvinylpyrrolidone and 1.5ml lead acetate/barium acetate damping fluid, and room temperature adds the saturated metabisulfite solution of 1.5ml after leaving standstill 15min, the vibration mixing; In the centrifugal 15min of 10000g, collect supernatant and be sulphur glycosides extract.Get sulphur glycosides extract 2ml and mix with 2ml KOH solution, room temperature reaction 30min then uses 37% concentrated hydrochloric acid neutralization reaction liquid.Get afterwards among the 2ml with after reactant liquor mix with 2ml 2mmol/L potassium ferricyanide solution, leave standstill behind the 15min in the centrifugal 5min of 8000g, get supernatant and in 15s, measure 420nm place light absorption value, after calculating, obtain total sulfur glycosides content in the sample.
More than specified embodiment of the present invention, but this instance of just lifting for the ease of understanding should not be considered to be limitation of the scope of the invention.Equally; Any person of ordinary skill in the field all can be according to the description of technical scheme of the present invention and preferred embodiment thereof; Make various possible being equal to and change or replacement, but all these changes or replacement all should belong to the protection domain of claim of the present invention.
Claims (3)
1. the assay method of glucosinolate total amount in the kind of plant; It is characterized in that: be the examination material with the plant; Raw material is used acetate buffer homogenate after crushed, and the homogenate high temperature bath goes out behind the enzyme; Add polyvinylpyrrolidone, lead acetate/barium acetate mixed liquor and saturated metabisulfite solution successively, the centrifugal sulphur glycosides extract that makes behind the vibration mixing.Sulphur glycosides extract after neutralizer reacts with potassium ferricyanide solution again, is measured the reduction of potassium ferricyanide light absorption value through alkaline degradation, the neutralization of 37% concentrated hydrochloric acid at the 420nm place with spectrophotometer.With allyl sulfide glycosides (Sinigrin) is standard items production standard curve, calculates total sulfur glycosides content in the plant.
2. according to claim 1, the assay method of glucosinolate total amount may further comprise the steps in the kind of plant:
(1) extraction of sulphur glycosides:
Get an amount of plant sample, after the pulverizing, press solid-liquid ratio (g/ml) and added acetate buffer (0.2mol/L in 1: 100～1: 200; PH 4.2), fully grind homogenate, homogenate is in 70～100 ℃ of water-bath 15min enzyme that goes out; Add 6g polyvinylpyrrolidone and 60～100ml lead acetate/barium acetate mixed liquor by every 1g sample successively after the cooling; After room temperature leaves standstill 15min, add the saturated metabisulfite solution of 60～100ml, the vibration mixing by every 1g sample; In the centrifugal 15min of 10000g, collect supernatant and be sulphur glycosides extract.
(2) alkaline degradation of sulphur glycosides:
Get above-mentioned sulphur glycosides extract 2～4ml and mix with equal-volume 2mol/L NaOH or KOH solution, behind the room temperature reaction 30min with 37% concentrated hydrochloric acid neutralization reaction liquid.
(3) mensuration of sulphur glycosides:
Get the potassium ferricyanide solution that reactant liquor 2～4ml and isopyknic 2mmol/L after the above-mentioned neutralization be dissolved in phosphate buffer (0.2mol/L, pH 7.0) and mix, room temperature is measured 420nm place light absorption value after leaving standstill 15min in 15s.Do sulphur glycosides typical curve with allyl sulfide glycosides (Sinigrin), horizontal ordinate is an allyl sulfide glycosides concentration, and ordinate is the light absorption value reduction Δ A of 420nm place
420(A
420max-A
420), record total sulfur glycosides content in the sample through calculating.A
420maxPotassium ferricyanide solution light absorption value when adding the extract of sulfur-bearing glycosides not, the experiment blank is the mixed liquor of sulphur glycosides extract and phosphate buffer.
(4) sulphur glycosides total:
According to the Δ A that records
420Substitution typical curve equation is calculated as follows total sulfur glycosides content in the sample:
Y: sulphur glycosides total amount (μ mol/g) in the sample;
A: calculate gained sulphur glycosides concentration (μ mol/ml) by typical curve;
V: whole sulphur glycosides extracting liquid volume (ml);
M: institute's sample thief quality (g).
3. like claim 1; The assay method of glucosinolate total amount in the 2 said kind of plant; It is characterized in that: plant can be the seed of plants such as wild cabbage, broccoli, cabbage, collard, cauliflower, leaf mustard, kohlrabi, brussels sprout, radish, turnip and horseradish, also can be the trophosome such as bud seedling and plant of these plants.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104327132A (en) * | 2014-10-23 | 2015-02-04 | 桂林莱茵生物科技股份有限公司 | Method for extracting high-content sinigrin |
| CN106644993A (en) * | 2015-10-28 | 2017-05-10 | 广州中大南沙科技创新产业园有限公司 | Spectrophotometer quantitative method of maca glucosinolate |
| CN107957460A (en) * | 2017-12-29 | 2018-04-24 | 中央军委后勤保障部军需装备研究所 | A kind of maca quality evaluating method based on aromatic series glucosinolate |
| CN111272694A (en) * | 2020-02-26 | 2020-06-12 | 北京市农林科学院 | Method for nondestructive rapid detection of content of 4-methyl thiooxybutyl thioglycoside in broccoli vegetable powder |
| CN115969893A (en) * | 2022-12-28 | 2023-04-18 | 河南理工大学 | Extraction method and application of total glucosinolates of common head cabbages |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104327132A (en) * | 2014-10-23 | 2015-02-04 | 桂林莱茵生物科技股份有限公司 | Method for extracting high-content sinigrin |
| CN106644993A (en) * | 2015-10-28 | 2017-05-10 | 广州中大南沙科技创新产业园有限公司 | Spectrophotometer quantitative method of maca glucosinolate |
| CN107957460A (en) * | 2017-12-29 | 2018-04-24 | 中央军委后勤保障部军需装备研究所 | A kind of maca quality evaluating method based on aromatic series glucosinolate |
| CN111272694A (en) * | 2020-02-26 | 2020-06-12 | 北京市农林科学院 | Method for nondestructive rapid detection of content of 4-methyl thiooxybutyl thioglycoside in broccoli vegetable powder |
| CN115969893A (en) * | 2022-12-28 | 2023-04-18 | 河南理工大学 | Extraction method and application of total glucosinolates of common head cabbages |
| CN115969893B (en) * | 2022-12-28 | 2024-01-26 | 河南理工大学 | Extraction method and application of total glucoside of common head cabbage |
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Application publication date: 20120926 |