CN115447886B - Total starch detection kit and detection method thereof - Google Patents
Total starch detection kit and detection method thereof Download PDFInfo
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- CN115447886B CN115447886B CN202210916449.7A CN202210916449A CN115447886B CN 115447886 B CN115447886 B CN 115447886B CN 202210916449 A CN202210916449 A CN 202210916449A CN 115447886 B CN115447886 B CN 115447886B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
- B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
- B65D25/00—Details of other kinds or types of rigid or semi-rigid containers
- B65D25/02—Internal fittings
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
- B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
- B65D81/00—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents
- B65D81/18—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents providing specific environment for contents, e.g. temperature above or below ambient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract
The invention discloses a total starch detection kit and a detection method thereof, the kit comprises a temperature control kit body with a square structure, wherein a plurality of mutually parallel adjusting holes are distributed on the front side wall of the temperature control kit body, a reagent taking and storing port is formed in the top surface of the temperature control kit body, a closed rubber plug is arranged at the reagent taking and storing port, a vertical shaft is arranged in the temperature control kit body, a plurality of rotating discs are arranged on the vertical shaft, 7 reagent storage grooves and 1 through hole are formed in each rotating disc, and 7 reagents are stored for storing starch detection reagent components. The detection method comprises the steps of storing a sample, preprocessing the sample before detection, and adopting a traditional pre-calibration and post-detection mode during detection. The kit is used for detection, reduces the pollution to the components of the starch detection reagent, and has the characteristics of long storage time, low detection cost, convenient detection, convenient carrying, storage and transportation, safety and reliability.
Description
Technical Field
The invention relates to the technical field of starch detection, in particular to a starch detection kit and a detection method thereof.
Background
Currently, the starch detection methods include an acid hydrolysis method, an enzymatic hydrolysis method and an optical rotation method, wherein in the enzymatic hydrolysis method, an enzyme-colorimetric method is commonly used, and the basic principle of the method is as follows: thermostable amylase hydrolyzes starch to soluble branched maltodextrins and debranched maltodextrins, and amyloglucosidase (saccharifying enzyme) hydrolyzes maltodextrins to D-glucose. Glucose oxidase catalyzes the oxidation of D-glucose (in the form of an aqueous solution) under aerobic conditions to produce D-glucono-delta-lactone and hydrogen peroxide. Hydrogen peroxide, 4-aminoantipyrine and phenol produce a red quinone imine, catalyzed by peroxidase. The absorbance of the quinone imine was measured at a wavelength of 505nm, and the starch content was proportional to the absorbance, thereby calculating the starch content.
The method is suitable for measuring starch in various foods by adopting an enzyme-colorimetric method, has higher measuring precision, but the used reagent is not easy to preserve, so that the application of the reagent is limited.
In the existing enzyme-colorimetric method, after a specified amount of sample is directly taken, the step of hydrolyzing starch by using heat-resistant amylase is carried out, and sugar in the sample is not treated before hydrolysis, so that the original sugar in the sample influences the detection result of the starch, and the measurement result of the starch is higher.
Disclosure of Invention
In order to overcome the defects, the invention aims to provide the total starch detection kit convenient for preserving the reagent, which has the characteristics of less pollution, long storage time, convenient detection, convenient maintenance, storage and transportation, safety and reliability.
Another object of the present invention is to provide a method for detecting total starch using a total starch detection kit, which has the characteristic of high detection accuracy.
In order to solve the technical problems, the invention adopts the following technical scheme:
the utility model provides a total starch detection kit, includes the control by temperature change kit body of square structure, be distributed on the preceding lateral wall of control by temperature change kit body and be parallel to each other, open on the top surface of control by temperature change kit body has reagent to get access opening, and reagent gets access opening department and is equipped with confined plug, is equipped with the vertical axis in the control by temperature change kit body, install a plurality of rolling discs on the vertical axis, on the vertical axis and be located between the adjacent rolling disc and be equipped with the spacer, the spacer passes through the screw fixation on the vertical axis, a part of a plurality of rolling discs stretches out to the control by temperature change kit body outside from corresponding regulation hole respectively, every the rolling disc on be equipped with 7 reagent storage tanks and 1 through-hole, the notch of 7 reagent storage tanks and the through-hole be located on the rolling disc and along the circumferencial direction of rolling disc, and dial the portion of rolling disc that is located the control by temperature change kit body with the hand, the rolling disc rotates corresponding angle, 7 reagent storage tank's notch and the through-hole all can be corresponding with the access opening of reagent; the notch department of 7 reagent storage tanks is equipped with the tectorial membrane plug, 7 reagent are deposited and are used for depositing starch detection reagent component.
Further, the edge of the disk surface of the rotating disk and the circumferential surface of the rotating disk are provided with 8 marks corresponding to the 7 reagent storage tanks and 1 through hole, the marks are provided with marks, a reference point is arranged on the temperature control kit body and positioned on one side of the adjusting hole, and when the marks are aligned with the reference point, the reagent storage tank or the through hole corresponding to the marks corresponds to the reagent taking and storing port above the marks.
Further, the marks at the marks corresponding to the through holes are numbered 0, and the marks at the marks corresponding to the 7 reagent storage tanks are numbered 1, 2, 3, 4, 5, 6 and 7 in sequence; the starch detection reagent component comprises 7 reagents, wherein the reagent 1 is 2.2mL of thermostable amylase, the reagent 2 is 16mL of amyloglucosidase, the reagent 3 is 60mL of extracting solution, the reagent 4 is 85mL of resistant starch buffer solution, the reagent 5 is 500mg/g of standard starch solution, the reagent 6 and the reagent 7 are respectively R1 and R2 double reagents, wherein in 22mL of R1 (reagent 6), the phenol content is 12mmol/L;12mL of R2 (reagent 7) containing 26KU of glucose oxidase, 4KU of peroxidase, 4.8mmol of 4-aminoantipyrine, 0.8mmol of potassium phosphate and 1g of sodium azide per liter; the reagent 1, the reagent 2, the reagent 3, the reagent 4, the reagent 5, the reagent 6 and the reagent 7 are respectively and sequentially placed in the reagent storage tanks 1, 2, 3, 4, 5, 6 and 7.
Further, the temperature control kit body is divided into a reagent placing cavity, a temperature control chamber, an airflow cavity and a battery placing cavity, the rotating disc is positioned in the reagent placing cavity, the temperature control chamber and the battery placing cavity are arranged at the left side of the reagent placing cavity, the battery placing cavity is arranged below the temperature control chamber, the airflow cavity is arranged at the rear part of the reagent placing cavity, a heat insulation board is arranged in the temperature control chamber, the temperature control chamber is divided into a heat dissipation cavity and a circulating air supply cavity by the heat insulation board, the circulating air supply cavity is communicated with the airflow cavity, an air guide baffle is arranged between the airflow cavity and the reagent placing cavity, a plurality of air guide holes are distributed on the air guide baffle, the air guide holes are arranged in a row and are positioned above the uppermost rotating disc, an air return port is formed in the baffle between the reagent placing cavity and the circulating air supply cavity, and a circulating fan is arranged in the circulating air supply cavity; the heat insulating plate is provided with a mounting hole, a semiconductor refrigerating and heating plate is mounted in the mounting hole, one end of the semiconductor refrigerating and heating plate is mounted on the inner wall of the mounting hole on the heat insulating plate through a rotating shaft, the other end of the semiconductor refrigerating and heating plate is connected with one end of an adjusting shaft, and the other end of the adjusting shaft extends out of the heat insulating plate from the outside of the temperature control kit body to be fixedly connected with an adjusting hand wheel.
Further, one side plate surface of the semiconductor refrigerating and heating plate is a refrigerating plate surface, the other side plate surface is a heating plate surface, and a plurality of radiating fins are distributed on the refrigerating plate surface and the heating plate surface.
Further, the plurality of fins form a cylindrical structure. The design does not affect the overturning adjustment of the semiconductor refrigerating and heating plate, and can ensure the maximization of the area of the radiating fin so as to improve the radiating effect.
Further, still include controller, a plurality of temperature sensor and a plurality of pilot lamp, a plurality of temperature sensor is corresponding to a plurality of pilot lamp, a plurality of temperature sensor is used for detecting the temperature of a plurality of rolling discs respectively, and a plurality of pilot lamps are installed on the preceding lateral wall of control by temperature change kit body, and a plurality of temperature sensor gives the controller with temperature information transfer, and when the temperature of rolling disc satisfied the default, the controller regulates and control the pilot lamp that corresponds into green light, and when the temperature of rolling disc did not satisfy the default, the controller regulated and control the pilot lamp that corresponds into red light to the power break-make of control semiconductor refrigeration heating board, battery in the battery place the chamber is controller, temperature sensor, pilot lamp and circulating fan power supply.
Further, the side wall of the temperature control kit body is provided with an air inlet and an air outlet which are communicated with the heat dissipation cavity, the semiconductor refrigeration heating plate is positioned between the air inlet and the air outlet, and the air outlet is provided with an air outlet.
Further, 8 clamping grooves are uniformly distributed on the circumferential surface of the rotating disc, a plurality of elastic metal cards are welded in the temperature control kit body, the elastic metal cards are arranged in a row from top to bottom and correspond to the rotating discs, and when the elastic metal cards are positioned in the clamping grooves of the rotating disc, the corresponding marks are aligned with the reference points. The cooperation of draw-in groove and elasticity metal card not only guarantees that the rolling disc rotates when next reagent storage tank, realizes accurate positioning, but also prevents the natural rotation of rolling disc.
The method for detecting the total starch by using the total starch detection kit comprises the following steps:
step 1, storing components of a starch detection reagent: the starch detection reagent components are stored in the total starch detection kit, and the temperature is controlled to be 2-8 ℃; the starch detection reagent component comprises 7 reagents, wherein the reagent 1 is 2.2mL of thermostable amylase, the reagent 2 is 16mL of amyloglucosidase, the reagent 3 is 60mL of extracting solution, the reagent 4 is 85mL of resistant starch buffer, the reagent 5 is 500mg/g of standard starch solution, the reagent 6 and the reagent 7 are respectively R1 and R2, wherein in 22mL of R1 (reagent 6), the phenol content is 12mmol/L;12mL of R2 (reagent 7) containing 26KU of glucose oxidase, 4KU of peroxidase, 4.8mmol of 4-aminoantipyrine, 0.8mmol of potassium phosphate and 1g of sodium azide per liter; the reagent 1, the reagent 2, the reagent 3, the reagent 4, the reagent 5, the reagent 6 and the reagent 7 are respectively and sequentially arranged in the reagent storage tanks 1, 2, 3, 4, 5, 6 and 7;
Step 2, preparation of starch detection reagent components before use: before the components of the starch detection reagent are used, an adjusting hand wheel on the total starch detection reagent box is rotated, a semiconductor refrigerating and heating plate is turned over, a refrigerating mode is changed into a heating mode, and when the temperature transmitted by a temperature sensor of the uppermost rotating plate in the total starch detection reagent box reaches more than 25 ℃, the semiconductor refrigerating and heating plate is closed, and heating is stopped;
step 3, sample pretreatment: all samples were crushed and sieved through a 35 mesh sieve for later use, and the specific pretreatment method was as follows:
A. for samples without resistant starch, D-glucose and maltodextrin, the treatment was performed as follows:
A1. taking 0.1g of sample in a digestion tube, rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference mark 3, enabling a reagent storage tank No. 3 to correspond to the reagent taking and storing port above the reference point, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-covered rubber plug at the reagent storage tank No. 3, adding 270ml of pure water into the reagent storage tank No. 3, diluting the reagent 3, then taking 5ml of diluted reagent 3 from the reagent storage tank No. 3, and adding the reagent 3 into the digestion tube;
A2. Rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference mark 1, enabling a reagent storage groove No. 1 to correspond to the reagent taking and storing port above the reagent storage groove No. 1, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-covered rubber plug at the reagent storage groove No. 1, extracting 0.02ml of reagent 1, adding the reagent 1 into a digestion tube in the step A1, placing the digestion tube in a water bath for boiling water bath for 15min, and forcefully oscillating once every 5min in the period; after boiling water bath is finished, placing the digestion tube into warm water for at least 2min, taking out, then rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 2 with the reference point, extracting 150 mu l of reagent 2 from a reagent storage groove No. 2 on the rotating disc at the uppermost layer of the total starch detection kit by using a pipette, adding the reagent 2 into the digestion tube, placing the digestion tube into a water bath kettle, and carrying out water bath at 60 ℃ for 30min;
A3. after the water bath is finished, placing the digestion tube into a centrifuge, centrifuging at a rotation speed of 5000rpm for 5min to separate mutually insoluble liquids, taking out supernatant in the digestion tube, diluting the supernatant by 8 times with pure water, and then entering a starch detection procedure of double reagents;
B. For samples containing resistant starch but no D-glucose and/or maltodextrin, the following steps were performed:
B1. taking 0.1g of crushed sample in a digestion tube, adding precooled 2M sodium hydroxide solution under ice bath condition, magnetically stirring for 20min, then rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference number 4, enabling a reagent storage tank No. 4 to correspond to a reagent taking and storing port above the reference point, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-coated rubber plug at the reagent storage tank No. 4, extracting 0.8mL of reagent 4, and adding the reagent into the digestion tube,
the pH value is 5.2, diluted reagent 3 is extracted from a reagent storage tank No. 3 and added into a digestion tube, and the liquid in the digestion tube is complemented to 5ml;
B2. beginning to treat 5ml of liquid in the digestion tube according to the step A2 in the step A;
C. for samples containing no resistant starch but D-glucose and/or maltodextrin, the following procedure was followed:
C1. taking 0.1g of crushed sample in a centrifuge tube, adding 10ml of 80% ethanol solution (v/v), incubating for 5min at 80 ℃, then placing the centrifuge tube in a centrifuge, centrifuging at 5000rpm for 5min to separate mutually insoluble liquids, discarding supernatant in the centrifuge tube, adding 10ml of 80% ethanol solution (v/v) in the centrifuge tube, placing the centrifuge tube in a vortex meter, and vortex shaking and mixing for 2min; and taking out the centrifugal tube, putting the centrifugal tube into a centrifugal machine, centrifuging at a rotation speed of 5000rpm for 5min, separating the mutually insoluble liquid, and discarding the supernatant in the centrifugal tube.
Water solubility of starch: amylose is insoluble in cold water and soluble in hot water; amylopectin is insoluble in water at normal pressure and dissolves in water only when heated and pressurized. Since the solution is insoluble in an ethanol solution having a concentration of 30% or more, the use of an 80% ethanol solution is intended to dissolve and extract D-glucose and/or maltodextrin, and when a sample contains a soluble saccharide, the detection result of starch becomes high, which affects the detection accuracy.
C2. Diluting the reagent 3 in the reagent storage tank No. 3 in the mode of the step A1 in the step A, adding 5mL of diluted reagent 3 into a centrifuge tube, and then starting the treatment operation from the step A2 in the step A;
D. for samples containing resistant starch and containing D-glucose and/or maltodextrin, the following was done:
D1. taking 0.1g of crushed sample in a centrifuge tube, adding 10mL of 80% ethanol solution (v/v), incubating for 5min at 80 ℃, then placing the centrifuge tube in a centrifuge, centrifuging at 5000rpm for 5min to separate mutually insoluble liquids, discarding supernatant in the centrifuge tube, adding 10mL of 80% ethanol solution (v/v) in the centrifuge tube, placing the centrifuge tube in a vortex meter, and vortex shaking and mixing for 2min; taking out the centrifuge tube, putting the centrifuge tube into the centrifuge, centrifuging at 5000rpm for 5min to separate the mutually insoluble liquid, and discarding the supernatant in the centrifuge tube;
D2. And then processing according to the step B;
step 4, starch detection of double reagents: the starch detection of the sample pretreated in the step 3 comprises the following steps:
s1, scaling: rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 5 with the reference point, enabling a No. 5 reagent storage groove to correspond to the reagent taking and storing port above the reference mark, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-covered rubber plug at the No. 5 reagent storage groove, extracting 16 mu L of reagent 5, and adding the reagent 5 into a 1.5mL centrifuge tube; then 16. Mu.L of 0mg/g starch standard solution was added to a 1.5mL centrifuge tube; rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 6 with the reference point, enabling a No. 6 reagent storage groove to correspond to the reagent taking and storing port above the reagent storage groove, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-covered rubber plug at the No. 6 reagent storage groove, extracting R1 into a test tube, rotating the rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 7 with the reference point, enabling a No. 7 reagent storage groove to correspond to the reagent taking and storing port above the reagent storage groove, enabling the liquid shifter to pass through the rubber plug at the reagent taking and storing port and the film-covered rubber plug at the No. 7 reagent storage groove, extracting R2 into the test tube, mixing R1 and R2 reagents in a volume of 2:1, adding 500 mu L of R1 and R2 reagent mixed liquid into the 1.5mL of the test tube, incubating for 20min at room temperature or 37 ℃, detecting 505nm absorbance by using a spectrophotometer, and establishing a standard curve of starch concentration and absorbance by multiple experiments;
S2, sample detection: taking a 1.5mL centrifuge tube, adding 16 mu L of the sample pretreated in the step 3, then adding 500 mu L of the R1 and R2 mixed reagent, incubating for 20min at room temperature or 37 ℃, detecting the absorbance at 505nm by using a spectrophotometer, and obtaining the starch concentration of the sample according to the starch concentration and the standard curve of the absorbance;
and 5, when the starch detection reagent components on the uppermost rotating disc of the total starch detection reagent kit are used, aligning the reference point with the reference point, enabling the through hole on the uppermost rotating disc to correspond to the reagent taking and storing port above the uppermost rotating disc, enabling the liquid shifter to penetrate through the rubber plug at the reagent taking and storing port and the through hole, extracting the starch detection reagent components on the second rotating disc, and so on until the starch detection reagent components on all the rotating discs in the total starch detection reagent kit are used, taking out the rotating discs in the total starch detection reagent kit, and putting corresponding new starch detection reagent components into the reagent storage grooves in each rotating disc after cleaning and drying treatment, and putting into next experimental work.
The invention has the beneficial effects that:
1. the starch detection reagent component is packaged on the turntable in the temperature control kit body, so that the starch detection reagent component is in a closed environment of 2-8 ℃, and whether liquid is added into the temperature control kit body or the starch detection reagent is extracted outwards, the starch detection reagent component is carried out in a closed space, the temperature control kit does not need to be opened, and a film coated rubber plug at a reagent storage groove does not need to be opened.
2. The turnover semiconductor refrigerating and heating plate is adopted, so that the conversion of a cold mode and a hot mode can be conveniently carried out through the regulating hand wheel, the starch detection reagent component can be effectively stored in the refrigerating mode, and the starch detection reagent component can be quickly heated to a normal temperature state through the heating mode and can be timely put into use.
3. The cooling plate surface and the heating plate surface of the semiconductor cooling and heating plate are additionally provided with cooling fins, and a plurality of cooling fins form a cylindrical structure, so that the area of the cooling fins can be maximized on the premise that the overturning adjustment of the semiconductor cooling and heating plate is not affected, and the heat dissipation effect is improved.
4. The automatic temperature control kit comprises a rotating disc, wherein 8 clamping grooves are uniformly distributed on the circumferential surface of the rotating disc, a plurality of elastic metal cards are welded in the temperature control kit body, and the clamping grooves are matched with the elastic metal cards, so that accurate positioning is realized when the rotating disc rotates to the next reagent storage groove, and natural rotation of the rotating disc is prevented.
5. Because the side wall of the temperature control reagent box body is provided with the air inlet and the air outlet which are communicated with the heat dissipation cavity, cold air or hot air can be discharged out of the temperature control reagent box body in time, so that the refrigerating and heating effects of the semiconductor refrigerating and heating plate are improved.
6. Because the turntable in the temperature control kit body can be disassembled and assembled for replacement, the temperature control kit body can be repeatedly used, and has the characteristics of low use cost, convenient detection and convenient maintenance.
7. Because the temperature control kit body can store five groups of starch detection reagent components at one time, the transportation is safer and more reliable under the protection of the temperature control kit body, and the phenomenon that the starch detection reagent components are damaged and leaked in the transportation is avoided.
8. In the detection method, the pretreatment of the sample is carried out before the detection, especially before the amylase hydrolyzes the starch, the sugar and the dextrin in the sample are removed, and the refining operation is carried out in the pretreatment of the sample according to the classification, so that the influence of the original sugar in the sample on the starch detection is avoided, and the accuracy of the starch detection is greatly improved.
9. In the pretreatment of the sample, when starch is hydrolyzed, a boiling water bath mode is adopted, so that the pretreatment time can be greatly shortened, the detection efficiency is improved, the time of the boiling water bath is accurately controlled, the thorough and sufficient starch hydrolysis can be ensured, the influence on glucose due to overlong heating time can be avoided, and a foundation is laid for the accurate detection of starch.
10. When the kit is used for detecting the total starch, the kit is convenient to carry and transport, so that the kit can be stored in places without refrigeration environments, the detection is not limited by application environments, and the problem of limited application of an enzyme-colorimetric method is solved greatly.
Drawings
The invention will be further described with reference to the accompanying drawings, in which embodiments do not constitute any limitation of the invention, and other drawings can be obtained by one skilled in the art without inventive effort from the following figures:
FIG. 1 is a schematic diagram of the structure of the present invention;
FIG. 2 is a top view of the device shown in FIG. 1;
FIG. 3 is a side view of the view shown in FIG. 1;
FIG. 4 is a schematic view of the interior of FIG. 1;
FIG. 5 is a schematic view of the interior of FIG. 2;
FIG. 6 is a schematic view of the heat shield of FIG. 4;
fig. 7 is a schematic view of the structure of the rotating disc shown in fig. 5.
In the figure: 1. a temperature control kit body; 2. A front sidewall; 3. An adjustment aperture; 4. A reagent taking and storing port; 5. A rubber plug; 6. a vertical axis; 7. a rotating disc; 8. a spacer bush; 9. a through hole; 10. a reagent storage tank; 11. laminating a rubber plug; 12. a reagent placement chamber; 13. a temperature control chamber; 14. a battery placement cavity; 15. an airflow cavity; 16. a heat insulating plate; 17. a heat dissipation cavity; 18. a circulating air supply cavity; 19. an air guide baffle; 20. a partition plate; 21. a mounting hole; 22. a semiconductor refrigerating and heating plate; 23. a rotating shaft; 24. an adjusting shaft; 25. an adjusting hand wheel; 26. a refrigeration plate surface; 27. heating the plate surface; 28. a heat sink; 29. a controller; 30. a temperature sensor; 31. an indicator light; 32. a battery; 33. an air inlet; 34. an air outlet; 35. an exhaust fan; 36. a clamping groove; 37. an elastic metal card; 38. marking; 39. a reference point; 40. a circulating fan; 41. an air return port; 42. a groove; 43. a handle.
Detailed Description
In order to make the technical solution of the present application better understood by those skilled in the art, the present application will be described in further detail with reference to the accompanying drawings and the specific embodiments, and it should be noted that the embodiments of the present application and features in the embodiments may be combined with each other without conflict.
As shown in fig. 1, 2, 3, 4, 5, 6 and 7, a starch detection kit comprises a square-structure temperature control kit body 1, wherein a plurality of mutually parallel adjusting holes 3 are distributed on the front side wall 2 of the temperature control kit body 1, a reagent taking and storing port 4 is formed in the top surface of the temperature control kit body 1, a closed rubber plug 5 is arranged at the reagent taking and storing port 4, a vertical shaft 6 is arranged in the temperature control kit body 1, a plurality of rotating discs 7 are mounted on the vertical shaft 6, a spacer bush 8 is arranged between adjacent rotating discs 7 and on the vertical shaft 6, the spacer bush 8 is fixed on the vertical shaft 6 through screws, a part of each rotating disc 7 extends out of the corresponding adjusting hole 3, 7 reagent storing grooves and 1 through holes 9,7 reagent storing grooves 10 and the through holes are arranged on the surface of the rotating discs and along the rotating discs, and the rotating discs 7 are positioned in the corresponding rotating directions of the rotating discs, and the rotating discs 7 can rotate by the corresponding through holes 9, and the corresponding rotating grooves of the rotating discs 7 can be rotated by the rotating discs 4; the notch department of 7 reagent storage tanks 10 is equipped with tectorial membrane plug 11, be used for depositing starch detect reagent in the 7 reagent storage tanks.
The top surface of the temperature control kit body 1 is also provided with a groove 42, and a handle 43 is arranged in the groove 42.
The temperature control kit body 1 is internally divided into a reagent placing cavity 12, a temperature control chamber 13, an airflow cavity 15 and a battery placing cavity 14, the rotating disc 7 is positioned in the reagent placing cavity 12, the temperature control chamber 13 and the battery placing cavity 14 are arranged on the left side of the reagent placing cavity 12, the battery placing cavity 14 is arranged below the temperature control chamber 13, the airflow cavity 15 is arranged at the rear part of the reagent placing cavity 12, the temperature control chamber 13 is internally provided with a heat insulation plate 16, the heat insulation plate 16 divides the temperature control chamber into a heat dissipation cavity 17 and a circulating air supply cavity 18, the circulating air supply cavity 18 is communicated with the airflow cavity 15, an air guide baffle 19 is arranged between the airflow cavity 15 and the reagent placing cavity 12, a plurality of air guide holes are distributed on the air guide baffle 19, the air guide holes are arranged in a row and are positioned above the rotating disc at the uppermost layer, a return air port 41 is arranged on a baffle 20 between the reagent placing cavity 12 and the circulating air supply cavity 18, and the circulating air supply cavity 18 is internally provided with a circulating fan 40; the heat insulating plate 16 is provided with a mounting hole 21, a semiconductor refrigerating and heating plate 22 is mounted in the mounting hole 21, one end of the semiconductor refrigerating and heating plate 22 is mounted on the inner wall of the mounting hole 21 on the heat insulating plate 16 through a rotating shaft 23, the other end of the semiconductor refrigerating and heating plate 22 is connected with one end of an adjusting shaft 24, and the other end of the adjusting shaft 24 extends out of the heat insulating plate 16 to the outside of the temperature control kit body 1 and is fixedly connected with an adjusting hand wheel 25.
The semiconductor refrigeration and heating plate 22 has a refrigeration plate surface 26 on one side and a heating plate surface 27 on the other side, and a plurality of cooling fins 28 are distributed on both the refrigeration plate surface 26 and the heating plate surface 27, and the plurality of cooling fins 28 form a cylindrical structure. The design does not affect the overturning adjustment of the semiconductor refrigerating and heating plate, and can ensure the maximization of the area of the radiating fin so as to improve the radiating effect.
The temperature control device further comprises a controller 29, a plurality of temperature sensors 30 and a plurality of indicator lamps 31, the plurality of indicator lamps 31 correspond to the plurality of temperature sensors 30, the plurality of temperature sensors 30 are respectively used for detecting the temperatures of the plurality of rotating discs 7, the plurality of indicator lamps 31 are arranged on the front side wall 2 of the temperature control kit body 1, the plurality of temperature sensors 30 transmit temperature information to the controller 29, when the temperatures of the rotating discs meet preset values, the controller 29 regulates the corresponding indicator lamps 31 to green light, when the temperatures of the rotating discs 7 do not meet preset values, the controller regulates the corresponding indicator lamps 31 to red light, when all the indicator lamps 31 regulate the green light, the controller 29 interrupts the power supply of the semiconductor refrigerating and heating plates 22, and the batteries 32 in the battery placing cavity 14 supply power to the controller 29, the temperature sensors 30, the indicator lamps 31 and the circulating fan 20.
The side wall of the temperature control kit body 1 is provided with an air inlet 33 and an air outlet 34 which are communicated with the heat dissipation cavity, the semiconductor refrigeration heating plate 22 is positioned between the air inlet 33 and the air outlet 34, and the air outlet 34 is provided with an air outlet 35.
The circumference of the rotating disc 7 is uniformly provided with 8 clamping grooves 36, a plurality of elastic metal cards 37 are welded in the temperature control kit body 1, the plurality of elastic metal cards 37 are arranged in a row from top to bottom and correspond to the plurality of rotating discs 7, and when the elastic metal cards 37 are positioned in the clamping grooves 36 of the rotating discs, the corresponding marks 38 are aligned with the reference points 39. The cooperation of the clamping groove 36 and the elastic metal card 37 not only ensures that the accurate positioning is realized when the rotating disc 7 rotates to the next reagent storage groove, but also prevents the natural rotation of the rotating disc 7.
The edge of the disk surface of the rotating disk 7 and the circumferential surface of the rotating disk are provided with 8 marks 38 corresponding to the 7 reagent storage tanks 10 and 1 through hole 9, the marks 38 are provided with marks, the marks at the marks corresponding to the through holes are numbered 0, the marks at the marks corresponding to the 7 reagent storage tanks are numbered 1, 2, 3, 4, 5, 6 and 7 in sequence, the temperature control kit body is provided with a reference point 39 at one side of the regulating hole 3, and when the marks 38 are aligned with the reference point 39, the reagent storage tank 10 or the through hole 9 corresponding to the marks 38 corresponds to the reagent access port 4 above the marks.
The method for detecting the total starch by using the total starch detection kit comprises the following steps:
step 1, storing components of a starch detection reagent: the starch detection reagent components are stored in the total starch detection kit, and the temperature is controlled to be 2-8 ℃; the starch detection reagent component comprises 7 reagents, wherein the reagent 1 is 2.2mL of thermostable amylase, the reagent 2 is 16mL of amyloglucosidase, the reagent 3 is 60mL of extracting solution, the reagent 4 is 85mL of resistant starch buffer, the reagent 5 is 500mg/g of standard starch solution, the reagent 6 and the reagent 7 are respectively R1 and R2, wherein in 22mL of R1 (reagent 6), the phenol content is 12mmol/L;12mL of R2 (reagent 7) containing 26KU of glucose oxidase, 4KU of peroxidase, 4.8mmol of 4-aminoantipyrine, 0.8mmol of potassium phosphate and 1g of sodium azide per liter; the reagent 1, the reagent 2, the reagent 3, the reagent 4, the reagent 5, the reagent 6 and the reagent 7 are respectively and sequentially arranged in the reagent storage tanks 1, 2, 3, 4, 5, 6 and 7;
step 2, preparation of starch detection reagent components before use: before the components of the starch detection reagent are used, an adjusting hand wheel on the total starch detection reagent box is rotated, a semiconductor refrigerating and heating plate is turned over, a refrigerating mode is changed into a heating mode, and when the temperature transmitted by a temperature sensor of the uppermost rotating plate in the total starch detection reagent box reaches more than 25 ℃, the semiconductor refrigerating and heating plate is closed, and heating is stopped; when in use, the kit is convenient to carry and transport, and can be used for storing the reagent box in places without refrigeration environments, so that the detection is not limited by application environments, and the problem of limited application of an enzyme-colorimetric method is solved greatly.
Step 3, sample pretreatment: when starch is hydrolyzed, the mode of boiling water bath is adopted, so that the pretreatment time can be greatly shortened, the detection efficiency is improved, the time of boiling water bath is accurately controlled, the thorough and sufficient starch hydrolysis can be ensured, the influence on glucose due to overlong heating time can be avoided, and a foundation is laid for accurate detection of starch. All samples were crushed and sieved through a 35 mesh sieve for later use, and the specific pretreatment method was as follows:
A. for samples without resistant starch, D-glucose and maltodextrin, the treatment was performed as follows:
A1. taking 0.1g of sample in a digestion tube, rotating a rotating disc 7 at the uppermost layer of the total starch detection kit, aligning a reference point with a reference mark 3, enabling a reagent storage tank No. 3 to correspond to the reagent taking and storing port above the reference point, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-covered rubber plug at the reagent storage tank No. 3, adding 270ml of pure water into the reagent storage tank No. 3, diluting the reagent 3, then taking 5ml of diluted reagent 3 from the reagent storage tank No. 3, and adding the diluted reagent 3 into the digestion tube; the reagent 3 is specifically calcium chloride extract, and because calcium ions and hydroxyl groups on starch molecules form a complex, starch has higher affinity with water and is easy to dissolve in the water.
A2. Rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference mark 1, enabling a reagent storage groove No. 1 to correspond to the reagent taking and storing port above the reagent storage groove No. 1, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-covered rubber plug at the reagent storage groove No. 1, extracting 0.02ml of reagent 1, adding the reagent 1 into a digestion tube in the step A1, placing the digestion tube in a water bath for boiling water bath for 15min, and forcefully oscillating once every 5min in the period; after boiling water bath is finished, placing the digestion tube into warm water for at least 2min, taking out, then rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 2 with the reference point, extracting 150 mu l of reagent 2 from a reagent storage groove No. 2 on the rotating disc at the uppermost layer of the total starch detection kit by using a pipette, adding the reagent 2 into the digestion tube, placing the digestion tube into a water bath kettle, and carrying out water bath at 60 ℃ for 30min; because starch particles have lattice structures, amylase is difficult to act, and the heating gelatinization of starch can destroy the lattice structures of the starch, so that the starch is easy to act by the amylase, the time of the boiling water bath can ensure that the starch is thoroughly and fully hydrolyzed, and the influence on glucose due to overlong heating time can be avoided. In the starch enzymolysis process, the viscosity can be rapidly reduced, the fluidity is enhanced, and the hydrolysis sequence of amylase to starch is as follows: starch-blue dextrin-red dextrin-maltose-glucose.
A3. After the water bath is finished, placing the digestion tube into a centrifuge, centrifuging at a rotation speed of 5000rpm for 5min to separate mutually insoluble liquids, taking out supernatant in the digestion tube, diluting the supernatant by 8 times with pure water, and then entering a starch detection procedure of double reagents;
B. for samples containing resistant starch but no D-glucose and/or maltodextrin, the following steps were performed:
B1. taking 0.1g of crushed sample in a digestion tube, adding precooled 2M sodium hydroxide solution under ice bath condition, magnetically stirring for 20min, then rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference number 4, enabling a reagent storage tank No. 4 to correspond to a reagent taking and storing port above the reference point, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-coated rubber plug at the reagent storage tank No. 4, extracting 0.8mL of reagent 4, and adding the reagent into the digestion tube,
the pH value is 5.2, diluted reagent 3 is extracted from a reagent storage tank No. 3 and added into a digestion tube, and the liquid in the digestion tube is complemented to 5ml;
B2. beginning to treat 5ml of liquid in the digestion tube according to the step A2 in the step A;
C. For samples containing no resistant starch but D-glucose and/or maltodextrin, the following procedure was followed:
C1. taking 0.1g of crushed sample in a centrifuge tube, adding 10ml of 80% ethanol solution (v/v), incubating for 5min at 80 ℃, then placing the centrifuge tube in a centrifuge, centrifuging at 5000rpm for 5min to separate mutually insoluble liquids, discarding supernatant in the centrifuge tube, adding 10ml of 80% ethanol solution (v/v) in the centrifuge tube, placing the centrifuge tube in a vortex meter, and vortex shaking and mixing for 2min; and taking out the centrifugal tube, putting the centrifugal tube into a centrifugal machine, centrifuging at a rotation speed of 5000rpm for 5min, separating the mutually insoluble liquid, and discarding the supernatant in the centrifugal tube.
C2. Diluting the reagent 3 in the reagent storage tank No. 3 in the mode of the step A1 in the step A, adding 5mL of diluted reagent 3 into a centrifuge tube, and then starting the treatment operation from the step A2 in the step A;
E. for samples containing resistant starch and containing D-glucose and/or maltodextrin, the following was done:
D1. taking 0.1g of crushed sample in a centrifuge tube, adding 10mL of 80% ethanol solution (v/v), incubating for 5min at 80 ℃, then placing the centrifuge tube in a centrifuge, centrifuging at 5000rpm for 5min to separate mutually insoluble liquids, discarding supernatant in the centrifuge tube, adding 10mL of 80% ethanol solution (v/v) in the centrifuge tube, placing the centrifuge tube in a vortex meter, and vortex shaking and mixing for 2min; taking out the centrifuge tube, putting the centrifuge tube into the centrifuge, centrifuging at 5000rpm for 5min to separate the mutually insoluble liquid, and discarding the supernatant in the centrifuge tube;
D2. And then processing according to the step B;
according to the technical scheme, before the amylase hydrolyzes the starch, sugar and dextrin in the sample are removed, and refining operation is performed in the sample pretreatment according to categories, so that the influence of original sugar in the sample on starch detection is avoided, and the starch detection precision is greatly improved.
Step 4, starch detection of double reagents: the starch detection of the sample pretreated in the step 3 comprises the following steps:
s1, scaling: rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 5 with the reference point, enabling a No. 5 reagent storage groove to correspond to the reagent taking and storing port above the reference mark, enabling a liquid shifter to pass through a rubber plug at the reagent taking and storing port and a film-covered rubber plug at the No. 5 reagent storage groove, extracting 16 mu L of reagent 5, and adding the reagent 5 into a 1.5mL centrifuge tube; then 16. Mu.L of 0mg/g starch standard solution was added to a 1.5mL centrifuge tube; rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 6 with the reference point, enabling a No. 6 reagent storage groove to correspond to the reagent taking and storing port above the reagent storage groove, enabling a pipettor to pass through a rubber plug 5 at the reagent taking and storing port and a film-covered rubber plug at the No. 6 reagent storage groove, extracting R1 into a test tube, rotating the rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 7 with the reference point, enabling a No. 7 reagent storage groove to correspond to the reagent taking and storing port above the reagent storage groove, enabling the pipettor to pass through the rubber plug at the reagent taking and storing port and the film-covered rubber plug at the No. 7 reagent storage groove, extracting R2 into the test tube, mixing R1 and R2 reagents in a volume of 2:1, adding 500 mu L of R1 and R2 reagent mixed solution into the 1.5mL centrifuge tube, incubating for 20min at room temperature or 37 ℃, and then detecting 505nm standard absorbance by using a spectrophotometer, and establishing a curve of starch concentration and absorbance through multiple experiments;
S2, sample detection: taking a 1.5mL centrifuge tube, adding 16 mu L of the sample pretreated in the step 3, then adding 500 mu L of the R1 and R2 mixed reagent, incubating for 20min at room temperature or 37 ℃, detecting the absorbance at 505nm by using a spectrophotometer, and obtaining the starch concentration of the sample according to the starch concentration and the standard curve of the absorbance;
and 5, when the starch detection reagent components on the uppermost rotating disc of the total starch detection reagent kit are used, aligning the reference point with the reference point, enabling the through hole on the uppermost rotating disc to correspond to the reagent taking and storing port above the uppermost rotating disc, enabling the liquid shifter to penetrate through the rubber plug at the reagent taking and storing port and the through hole, extracting the starch detection reagent components on the second rotating disc, and so on until the starch detection reagent components on all the rotating discs in the total starch detection reagent kit are used, taking out the rotating discs in the total starch detection reagent kit, and putting corresponding new starch detection reagent components into the reagent storage grooves in each rotating disc after cleaning and drying treatment, and putting into next experimental work.
According to the technical scheme, as the starch detection reagent component is packaged on the turntable in the temperature control kit body, the starch detection reagent component is in a closed environment at 2-8 ℃, and no matter liquid is added into the temperature control kit body or the starch detection reagent is extracted outwards, the starch detection reagent component is carried out in a closed space, the temperature control kit does not need to be opened, and a film coated rubber plug at a reagent storage groove does not need to be opened.
The foregoing embodiments are merely illustrative of the present invention and are not intended to limit the spirit and scope of the present invention, and those of ordinary skill in the art should not depart from the spirit and scope of the present invention as set forth in the appended claims.
Claims (7)
1. A total starch detection kit, characterized in that: the temperature control kit comprises a square body structure, wherein a plurality of mutually parallel adjusting holes are distributed on the front side wall of the temperature control kit body, a reagent access port is formed in the top surface of the temperature control kit body, a closed rubber plug is arranged at the reagent access port, a vertical shaft is arranged in the temperature control kit body, a plurality of rotating discs are mounted on the vertical shaft, a spacer bush is arranged between every two adjacent rotating discs and fixed on the vertical shaft through screws, a part of each rotating disc extends out of the temperature control kit body from the corresponding adjusting hole, 7 reagent storage grooves and 1 through hole are formed in each rotating disc, the notch of each 7 reagent storage grooves and the through hole are uniformly distributed on the disc surface of the rotating disc along the circumferential direction of the rotating disc, the part of the rotating disc, which is positioned outside the temperature control kit body, is rotated by hands, and the notch of each reagent storage groove and the through hole can correspond to the reagent access port; the notch of the 7 reagent storage tanks are provided with film-covered rubber plugs, and the 7 reagent storage tanks are used for storing starch detection reagent components;
The edge of the disk surface of the rotating disk and the circumferential surface of the rotating disk are provided with 8 marks corresponding to the 7 reagent storage grooves and the 1 through holes, marks are arranged at the marks, a reference point is arranged on the temperature control kit body and positioned on one side of the adjusting hole, and when the marks are aligned with the reference point, the reagent storage groove or the through hole corresponding to the marks corresponds to the reagent access port above the marks;
the marks at the marks corresponding to the through holes are numbered 0, and the marks at the marks corresponding to the 7 reagent storage tanks are numbered 1, 2, 3, 4, 5, 6 and 7 in sequence; the starch detection reagent component comprises 7 reagents, wherein the reagent 1 is 2.2mL of heat-resistant amylase, the reagent 2 is 16mL of amyloglucosidase, the reagent 3 is 60mL of extracting solution, the reagent 4 is 85mL of resistant starch buffer solution, the reagent 5 is 500mg/g of standard starch solution, the reagent 6 and the reagent 7 are respectively R1 and R2 double reagents, wherein in 22mL of R1 of the reagent 6, the phenol content is 12mmol/L;12mL of reagent 7 at R2 containing 26KU of glucose oxidase, 4KU of peroxidase, 4.8mmol of 4-aminoantipyrine, 0.8mmol of potassium phosphate and 1g of sodium azide per liter; the reagent 1, the reagent 2, the reagent 3, the reagent 4, the reagent 5, the reagent 6 and the reagent 7 are respectively and sequentially arranged in the reagent storage tanks 1, 2, 3, 4, 5, 6 and 7;
The temperature control kit comprises a rotating disc, wherein 8 clamping grooves are uniformly distributed on the circumferential surface of the rotating disc, a plurality of elastic metal cards are welded in the temperature control kit body, the elastic metal cards are arranged in a row from top to bottom and correspond to the rotating discs, and when the elastic metal cards are positioned in the clamping grooves of the rotating disc, the marks are aligned with the reference points correspondingly.
2. The total starch detection kit of claim 1 wherein: the temperature control kit body is internally divided into a reagent placing cavity, a temperature control chamber, an airflow cavity and a battery placing cavity, the rotating disc is positioned in the reagent placing cavity, the temperature control chamber and the battery placing cavity are arranged at the left side of the reagent placing cavity, the battery placing cavity is arranged below the temperature control chamber, the airflow cavity is arranged at the rear part of the reagent placing cavity, a heat insulating plate is arranged in the temperature control chamber, the heat insulating plate divides the temperature control chamber into a heat radiating cavity and a circulating air supply cavity, the circulating air supply cavity is communicated with the airflow cavity, an air guide baffle is arranged between the airflow cavity and the reagent placing cavity, a plurality of air guide holes are distributed on the air guide baffle, the air guide holes are arranged in a row and are positioned above the rotating disc at the uppermost layer, a return air inlet is formed in the baffle between the reagent placing cavity and the circulating air supply cavity, and a circulating fan is arranged in the circulating air supply cavity; the heat insulating plate is provided with a mounting hole, a semiconductor refrigerating and heating plate is mounted in the mounting hole, one end of the semiconductor refrigerating and heating plate is mounted on the inner wall of the mounting hole on the heat insulating plate through a rotating shaft, the other end of the semiconductor refrigerating and heating plate is connected with one end of an adjusting shaft, and the other end of the adjusting shaft extends out of the heat insulating plate from the outside of the temperature control kit body to be fixedly connected with an adjusting hand wheel.
3. The total starch detection kit of claim 2 wherein: one side plate surface of the semiconductor refrigerating and heating plate is a refrigerating plate surface, the other side plate surface is a heating plate surface, and a plurality of radiating fins are distributed on the refrigerating plate surface and the heating plate surface.
4. A total starch detection kit according to claim 3 wherein: the plurality of radiating fins form a cylindrical structure.
5. The total starch detection kit of claim 4 wherein: the temperature control kit comprises a battery, a plurality of rotating discs, a plurality of temperature sensors, a plurality of indicator lamps, a controller, a battery, a circulating fan and a battery, wherein the battery is used for supplying power to the battery, the temperature sensors and the battery.
6. The total starch detection kit of claim 5 wherein: the side wall of the temperature control kit body is provided with an air inlet and an air outlet which are communicated with the heat dissipation cavity, the semiconductor refrigeration heating plate is positioned between the air inlet and the air outlet, and the air outlet is provided with an air outlet.
7. A method for detecting total starch using the kit for detecting total starch as claimed in claim 6, comprising the steps of:
step 1, storing components of a starch detection reagent: the starch detection reagent components are stored in the total starch detection kit, and the temperature is controlled to be 2-8 ℃; the starch detection reagent component comprises 7 reagents, wherein the reagent 1 is 2.2mL of thermostable amylase, the reagent 2 is 16mL of amyloglucosidase, the reagent 3 is 60mL of extracting solution, the reagent 4 is 85mL of resistant starch buffer solution, the reagent 5 is 500mg/g of standard starch solution, the reagent 6 and the reagent 7 are respectively R1 and R2, wherein in 22mL of reagent 6, the phenol content is 12mmol/L;12mL of reagent 7 at R2 containing 26KU of glucose oxidase, 4KU of peroxidase, 4.8mmol of 4-aminoantipyrine, 0.8mmol of potassium phosphate and 1g of sodium azide per liter; the reagent 1, the reagent 2, the reagent 3, the reagent 4, the reagent 5, the reagent 6 and the reagent 7 are respectively and sequentially arranged in the reagent storage tanks 1, 2, 3, 4, 5, 6 and 7;
Step 2, preparation of starch detection reagent components before use: before the components of the starch detection reagent are used, an adjusting hand wheel on the total starch detection reagent box is rotated, a semiconductor refrigerating and heating plate is turned over, a refrigerating mode is changed into a heating mode, and when the temperature transmitted by a temperature sensor of the uppermost rotating plate in the total starch detection reagent box reaches more than 25 ℃, the semiconductor refrigerating and heating plate is closed, and heating is stopped;
step 3, sample pretreatment: all samples were crushed and sieved through a 35 mesh sieve for later use, and the specific pretreatment method was as follows:
A. for samples without resistant starch, D-glucose and maltodextrin, the treatment was performed as follows:
A1. taking 0.1g of sample in a digestion tube, rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference mark 3, enabling a reagent storage tank No. 3 to correspond to the reagent access port above the reference point, enabling a pipettor to pass through a rubber plug at the reagent access port and a film-covered rubber plug at the reagent storage tank No. 3, adding 270ml of pure water into the reagent storage tank No. 3, diluting the reagent 3, and then taking 5ml of diluted reagent 3 from the reagent storage tank No. 3, and adding into the digestion tube;
A2. Rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference mark 1, enabling a reagent storage groove No. 1 to correspond to the reagent access port above the reference point, enabling a pipettor to pass through a rubber plug at the reagent access port and a film-covered rubber plug at the reagent storage groove No. 1, extracting 0.02ml of reagent 1, adding the reagent 1 into a digestion tube in the step A1, placing the digestion tube in a water bath for boiling water bath for 15min, and strongly oscillating once every 5min during the process; after boiling water bath is finished, placing the digestion tube into warm water for at least 2min, taking out, then rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 2 with the reference point, extracting 150 mu l of reagent 2 from a reagent storage groove No. 2 on the rotating disc at the uppermost layer of the total starch detection kit by using a pipette, adding the reagent 2 into the digestion tube, placing the digestion tube into a water bath kettle, and carrying out water bath at 60 ℃ for 30min;
A3. after the water bath is finished, placing the digestion tube into a centrifuge, centrifuging at a rotation speed of 5000rpm for 5min to separate mutually insoluble liquids, taking out supernatant in the digestion tube, diluting the supernatant by 8 times with pure water, and then entering a starch detection procedure of double reagents;
B. For samples containing resistant starch but no D-glucose and/or maltodextrin, the following steps were performed:
B1. taking 0.1g of crushed sample in a digestion tube, adding precooled 2M sodium hydroxide solution under ice bath condition, magnetically stirring for 20min, then rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference point with a reference number 4, enabling a reagent storage tank No. 4 to correspond to a reagent storage port above the reference point, enabling a liquid shifter to pass through a rubber plug at the reagent storage port and a film-coated rubber plug at the reagent storage tank No. 4, extracting 0.8mL of reagent 4, and adding the reagent into the digestion tube,
the pH value is 5.2, diluted reagent 3 is extracted from a reagent storage tank No. 3 and added into a digestion tube, and the liquid in the digestion tube is complemented to 5ml;
B2. beginning to treat 5ml of liquid in the digestion tube according to the step A2 in the step A;
C. for samples containing no resistant starch but D-glucose and/or maltodextrin, the following procedure was followed:
C1. taking 0.1g of crushed sample, adding 10ml of 80% ethanol solution by volume percentage into a centrifuge tube, incubating for 5min at 80 ℃, then placing the centrifuge tube into a centrifuge, centrifuging at 5000rpm for 5min to separate the mutually insoluble liquid, discarding the supernatant in the centrifuge tube, then adding 10ml of 80% ethanol solution by volume percentage into the centrifuge tube, placing the centrifuge tube into a vortex meter, and vortex-shaking and mixing for 2min; taking out the centrifuge tube, putting the centrifuge tube into the centrifuge, centrifuging at 5000rpm for 5min to separate the mutually insoluble liquid, and discarding the supernatant in the centrifuge tube;
C2. Diluting the reagent 3 in the reagent storage tank No. 3 in the mode of the step A1 in the step A, adding 5mL of diluted reagent 3 into a centrifuge tube, and then starting the treatment operation from the step A2 in the step A;
D. for samples containing resistant starch and containing D-glucose and/or maltodextrin, the following was done:
D1. taking 0.1g of crushed sample in a centrifuge tube, adding 10mL of 80% ethanol solution by volume percent, incubating for 5min at 80 ℃, then placing the centrifuge tube in a centrifuge, centrifuging at 5000rpm for 5min to separate the mutually insoluble liquid, discarding the supernatant in the centrifuge tube, then adding 10mL of 80% ethanol solution by volume percent into the centrifuge tube, placing the centrifuge tube in a vortex meter, and vortex shaking and mixing for 2min; taking out the centrifuge tube, putting the centrifuge tube into the centrifuge, centrifuging at 5000rpm for 5min to separate the mutually insoluble liquid, and discarding the supernatant in the centrifuge tube;
D2. and then processing according to the step B;
step 4, starch detection of double reagents: the starch detection of the sample pretreated in the step 3 comprises the following steps:
S1, scaling: rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 5 with the reference point, enabling a No. 5 reagent storage groove to correspond to the reagent access port above the reference mark, enabling a liquid shifter to pass through a rubber plug at the reagent access port and a film-covered rubber plug at the No. 5 reagent storage groove, extracting 16 mu L of reagent 5, and adding the reagent 5 into a 1.5mL centrifuge tube; then 16. Mu.L of 0mg/g starch standard solution was added to a 1.5mL centrifuge tube; rotating a rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 6 with the reference point, enabling a No. 6 reagent storage groove to correspond to the reagent access port above the reagent storage groove, enabling a pipettor to pass through a rubber plug at the reagent access port and a film-covered rubber plug at the No. 6 reagent storage groove, extracting R1 into a test tube, rotating the rotating disc at the uppermost layer of the total starch detection kit, aligning a reference mark 7 with the reference point, enabling a No. 7 reagent storage groove to correspond to the reagent access port above the reagent storage groove, enabling a pipettor to pass through the rubber plug at the reagent access port and the film-covered rubber plug at the No. 7 reagent storage groove, extracting R2 into the test tube, mixing R1 and R2 reagents in a volume of 2:1, adding 500 mu L of R1 and R2 reagent mixed solution into the 1.5mL, incubating for 20 minutes at room temperature or 37 ℃, detecting absorbance at 505nm by using a spectrophotometer, and establishing a standard curve of starch concentration and absorbance through multiple experiments;
S2, sample detection: taking a 1.5mL centrifuge tube, adding 16 mu L of the sample pretreated in the step 3, then adding 500 mu L of the R1 and R2 mixed reagent, incubating for 20min at room temperature or 37 ℃, detecting the absorbance at 505nm by using a spectrophotometer, and obtaining the starch concentration of the sample according to the starch concentration and the standard curve of the absorbance;
and 5, when the starch detection reagent components on the uppermost rotating disc of the total starch detection reagent kit are used, aligning the reference point with the reference point, enabling the through hole on the uppermost rotating disc to correspond to the reagent access port above the reference point, enabling the liquid shifter to pass through the rubber plug at the reagent access port and the through hole, extracting the starch detection reagent components on the second rotating disc, and so on until the starch detection reagent components on all rotating discs in the total starch detection reagent kit are used, taking out the rotating discs in the total starch detection reagent kit, putting corresponding new starch detection reagent components in the reagent storage grooves in each rotating disc after cleaning and drying treatment, and putting into the next experimental work.
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| EP0289789A1 (en) * | 1987-04-06 | 1988-11-09 | Japan Tectron Instruments Corporation | Automatic analysis apparatus |
| JPH08160050A (en) * | 1994-12-09 | 1996-06-21 | Toshiba Corp | Automatic analyzing device |
| CN214030009U (en) * | 2020-11-22 | 2021-08-24 | 毛永 | Environmental monitoring sample storage device for civil engineering |
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