JP2007527499A - Portable device for measurement of total gossypol concentration - Google Patents

Abstract

Measuring gossypol in cottonseed using conventional methods such as spectroscopic methods, NMR methods, chemiluminescence methods, gas liquid chromatography, polarography, thin layer chromatography and paper chromatography is cumbersome and time consuming. Need a calibration system at high cost. The present invention has developed a low cost portable system where the concentration of total gossypol is displayed directly on the LCD at mgl- ¹ or ppm levels from the test solution and a standard for creating a calibration curve to measure absorbance. No solution is required.

Description

  The present invention relates to an apparatus for measuring gossypol in cottonseed deoiled cake, cottonseed and gossypol-containing compounds after color development with a green complex with iron [Fe (III)].

  Cottonseed products in the form of oil and flour are widely used as human and animal food. However, due to the presence of gossypol, a polyphenol compound, the use of cottonseed products to humans and their nutrition are limited. Gossypol toxicity also limits its applicability to many areas.

  Gossypol is a bioactive terpenoid substance present in isolated glands in many parts of cotton plants, including seed shells. Many gossypols are naturally present in the gland or are chemically anchored to the protein portion of the seed. This dye is a toxic compound in its free form and accumulates in human tissues.

  Cottonseed is processed into oil and shell flour for animal and human consumption. Gossypol toxicity is due to free gossypol, the opposite of fixed gossypol. The Food and Drug Administration (FDA) and the United Nations Protein Advisory Group have established limits on gossypol content in human food.

  Common symptoms associated with gossypol toxicity include decreased nutrient intake, weight loss, severe hypoprothrombinemia, diarrhea, erythrocyte fragility, and associated decreased blood hemoglobin levels, heart that worsens the liver and spleen And the accumulation of edematous fluid in the lung cavity. Gossypol exhibits phenolic properties as well as aldehydes.

  Several methods for measuring gossypol in various samples have been used / reported. The method used for the analysis of gossypol is a spectrophotometric method using a spectrophotometer that can be used for the measurement of gossypol (American Oil Chemists Association "official and experimental methods", American Oil Chemists Association: Champaign,; test Method Ba 8-58; Crouch, Floyd W; Bryant Melton F. Anal. Chem. 1982, 54, 242-246; Ponds, WA; Guthrie, JDJ Am. Oil Chem. Soc. 1949, 26, 671-676, Pons, WA; Hofpauer, CL; O'Connor, RTJ Am. Oil Chem. Soc 1951, 28, 8-12.) (Non-Patent Documents 1 to 4). These spectroscopic methods in either the UV or visible region require test equipment and create a calibration curve by plotting known concentration versus absorbance / transmittance values, so that standard test samples are is necessary. After preparing the test sample, the absorbance / transmittance value is recorded for analysis of the unknown sample and placed on the already plotted curve to obtain its concentration. Atnafusgeddo Admas, BS Chandravansi; Analytical Chem. Vol. 56, 1 Jan 1984.30-32 also uses a 1 cm path length quartz tube and a Beckman 26 UV-visible spectrophotometer. This is a spectroscopic method for measuring the total gossypol in the medium, and the absorbance is measured (Non-patent Document 5). In the present invention, we have developed a device in which the concentration of gossypol is displayed directly on the LCD. No calibration curve or absorbance value is required for system calibration.

  Weiss, AC; Zhang, BG; Benson, M ,; Lukfa, MJJ assoc off.Anal Chem. 1978, 61, 146-149; Abellanov, AA; Meltlyak, MN Rubin, BA Biokhmiya (Moscow) 1978, 43 1594-1601. 1978, 89, 211079h; Raja P. K; Kater, CJ Am. Oil Chem. Soc. 1967, 44, 465-466; Markman, AL Ratchkin; VP "Gossypol and its derivatives", scientific translation Israel Program for: Jerusalem, 1968; Chapter 8 references are other instrumental techniques including nuclear magnetic resonance (NMR), chemiluminescence, gas liquid chromatography, and thin layer chromatography (6) 10). These methods involve complex systems and are expensive, the analysis and interpretation of the results is cumbersome and not useful for general use where the system is dedicated to gossypol measurements. Polarography includes a system for measuring polarographic data together with a dropping mercury electrode, a calomel electrode, a supporting electrolyte, and a standard solution. Interpretation of results and the like is extremely troublesome and analysis time is extremely long. Chromatographic techniques involve expensive equipment, detectors, columns, and require trained personnel.

  Elsimar Metzkar Coutinho Science Inc. Contraceptive Method Vol. 65 Publication 4 pp. 259-263 is a document on the effect of gossypol as a contraceptive (Non-Patent Document 11). ). Kyon-Jun Lee and Conrad Dubrovski Journal of Chromatography B Vol 779, Publication 2,5 November 2002, 313-319, includes analysis of gossypol from aquaculture by chromatographic techniques (12) .

"Official and Experimental Methods", American Oil Chemists Association Anal. Chem. 1982, 54, 242-246 J. Am. Oil Chem. Soc. 1949,26, 671-676 J. Am. Oil Chem. Soc 1951, 28, 8-12. Analytical Chem. Vol. 56, 1 Jan 1984.30-32 Anal Chem. 1978, 61, 146-149 Biokhmiya, 1978,43 1594-1601 Chem. Abstr. 1978, 89, 211079h J. Am. Oil Chem. Soc. 1967,44, 465-466 "Gossypol and its derivatives", Israel program for scientific translation: Jerusalem, 1968 Contraception Vol. 65 Publication 4 259-263, Elsimar Metzkar Coutinho Science Inc. Journal of Chromatography B Vol 779, Publication 2,5 November 2002, 313-319

Many system methods are cumbersome, time consuming, expensive and require calibrated system curves. The novelty of the present invention is that the concentration of total gossypol from the test solution is displayed directly on the LCD in mgl- ¹ or ppm, and no calibration curve is required to measure the absorbance of a solution containing a known amount of gossypol. It is in.

The background art clearly shows the disadvantages of the prior art, and the inventors have thoroughly studied and recognized the limitations involved in the above process. In an attempt to eliminate such shortcomings, the inventors have devised a device for displaying the test solution directly on the LCD in mgl ^-1 or ppm and eliminating the need for a calibration curve. Although the functions of the components included in the apparatus are known individually, combinations and functional interrelationships for achieving the required results have been obtained for the first time. Accordingly, the novelty and inventive step of the present invention has been characterized.

(Object of the present invention)
The main object of the present invention is to provide a portable system for the measurement of total gossypol in cottonseed, cottonseed deoiled cake, cottonseed oil and gossypol compound after green color development.
Another object of the present invention is to provide a portable device useful for measuring deoiled cake and gossypol in cottonseed in the range of 5 ppm to 75 ppm in terms of milligram liter ^-1 (mgl ^-1 ) or ppm. There is to do.
Still another object of the present invention is to provide a displaying directly in mgl ^-1 to 3 _^1/2-digit on the LCD the total concentration of all gossypol.
Another object of the present invention is that the system is operated using a 635 nm (50 mW) LED light source, thereby eliminating the use of tungsten lamps and filters to reduce power consumption.
Another object of the present invention is to eliminate the cumbersome optics of spectrophotometers.
Yet another object of the present invention is that no calibration curve is stored in memory to display the actual concentration of gossypol.
Yet another object of the present invention is that the 100% transmission level of distilled water is adjusted from the light intensity.
Yet another object of the present invention is that the sample holder is specially designed to hold a borosilicate circular glass cuvette with a capacity of 6 ml and a diameter of 17 mm.
Yet another object of the present invention is that the system operates at ± 6V (battery is operated).

(Summary of the Invention)
The apparatus of the present invention is useful as a typical instrument for measuring gossypol concentration in deoiled cake / cotton seed / cotton seed oil. The instrument of the present invention is used in Lambert-Beer law.

(Description of the details of the invention)
The apparatus of the present invention includes the following subsystems. In the accompanying FIG. 1, a block diagram of the apparatus of the present invention is shown.
(1) A 5 mm LED light source has an intensity of 125 mcd, a viewing angle of 24 °, a peak wavelength of 635 nm, and a power loss of 50 mW.
The intensity of light from the LED is adjusted by a potentiometer (multi-rotation type) in order to obtain 100% transmittance from the light transmitted through distilled water in the cuvette.
(2) is a sample holder.
(3) is a photodetector.
(4) Electronic circuit (5) Concentration display (6) Power supply (7) Computer According to the present invention, gossypol in cottonseed and cottonseed shell flour is converted to milligram / liter or parts per million (ppm). To provide a device that is useful for measuring.

In an embodiment of the present invention, the device has a 5 mm size with an adjustable potentiometer (multi-turn) to obtain 100% transmittance or 0% absorbance from light transmitted through distilled water in a glass cuvette with a path length of 17 mm. A 635 nm light emitting diode (LED) was used.
In another embodiment of the invention, the cuvette holder is composed of a black painted aluminum alloy. The cuvette holder has two sub-mechanisms that are inclined with respect to the opposite end, one holding the LED and the other holding the detector. The cuvette used is composed of a screw lid type borosilicate glass having a diameter of 17 mm, a height of 5 cm and a capacity of 6 ml.
In another embodiment of the invention, the photodetector BPW21 has a TO5 package, an effective detection area of 5.9 mm in diameter, a wavelength range of 460-750 nm, and 7 nA for detecting transmitted / absorbed light through the test solution. The detection sensitivity of / lux is used.
In another embodiment of the present invention, the optical signal detected by the photodetector is converted into a voltage using a current-voltage converter. The current is proportional to the detected optical signal. As shown in FIG. 2, the output voltage of the I / V converter is given by the potential difference between resistors R3 and R4.

In another embodiment of the invention, the logarithmic amplifier is a TL441 IC.
In another embodiment of the present invention, the voltage output after logarithmic amplification (digit) is calibrated in terms of the Gossypol concentration of the test solution. This is basically a 10K POT as shown in the figure. The POT is set when a known standard sample is held so that an accurate measurement value is given in ppm displayed on the digital panel meter (DPM) when the unknown sample is held in the cuvette holder.
In other embodiments, the result of the concentration is displayed in 3 _^1/2 digit LCD.
In other embodiments, the concentration results were displayed on a computer through CC ⁺ Large.

In another embodiment of the present invention, for data acquisition and subsequent calculation of an ADAM acquisition module in which the analog voltage extracted from the IV converter is an input and the output is in digital form and connected to a PC serial port VisiDAQ software.
VisiDAQ is a data acquisition and control application software and application builder for all kinds of automation. The script designer is a text editor with several features that are useful for editing script code. The script source is compiled into p-code after editing so that it does not need to be recompiled during execution. The basic script syntax is compatible between Microsoft VBA (Visual Basic for Excel, Word, Access, etc.) and Microsoft Visual Basic. If only common functions are used, the visual basic source code can be obtained and compiled and executed under a basic script without changing the language. The two development environments are very similar. In the source code design stage, it features cut, copy and paste functions.

  The task designer uses a dataflow programming model that frees the user from the linear architecture of a text-based language. In order to generate process monitoring and control the application, the user constructs a block diagram without bothering with a detailed description of the many syntaxes of normal programming. Simply select an object (icon) from the function menu and connect them with wires so that data is passed from one block to the next.

  The task designer can edit multiple tasks at the same time. Each task is included in a task window and has specific properties such as scan speed, start / stop method, and so on. One strategy file is used to store all scan tasks associated with the control strategy. Because of a single strategy with only one task, it is executed in the same way as before. However, because of the strategy with one or more scan tasks, a top-level main script is required to manage the execution of all scan tasks.

  Large and complex tasks are divided into several small and simple tasks. This not only simplifies the editing job but also improves runtime performance in that fewer blocks are required to be processed in each scan.

  Virtual tags are a powerful feature in that they give developers the ability to create customized tags in the task designer without using user-defined DLLs. Virtual tags are created by the task designer and stored in the data center like other built-in blocks. Since the virtual tag is general-purpose regardless of the task, the virtual tag can be used to share data between a plurality of tasks.

  The data collection function generates a database file for each defined TAG point at a user specified time. Data collection functions are designed for report generation. The shortest time interval for data collection from a given TAG point is 10 minutes. High speed data collection is accomplished through other trend data collection functions.

  The invention is described in further detail in the following examples, which are provided to illustrate the invention, but should not be construed to limit the scope of the invention.

  20 mg of pure gossypol compound was dissolved in 2 ml of glacial acetic acid and the volume was made up to 100 ml with a hexane / isopropyl alcohol mixture. This solution corresponds to 200 ppm gossypol. Using complexing agent of a mixture of 3-aminopropanol, dimethylformamide and ferric nitrate (Anal Chemistry 1984, 56, 30-32, Ref. 5) to 5, 10, 20, 30, 40, 60, 70 ppm It was diluted appropriately to 25 ml correspondingly and the color was stained from this gossypol solution. The results were 5 ± 0.2, 10 ± 0.3, 20 ± 0.5, 30 ± 0.5, 40 ± 1.0, 60 ± 0.8, and 70 ± 1.0.

  In other experiments, 0.5 g of deoiled cake was collected in a flask and 10 ml of complexing agent from Example 1 was added. The volume was brought to 50 ml with a hexane / isopropyl alcohol mixture. Color was stained from 2 ml, 5 ml and 10 ml of the specimen. The results were obtained as 4ppm, 9.8ppm and 19.8ppm in the developer system. The results were compared to a standard spectrophotometer by measuring absorbance and calculated to be 4.3,9.8 and 20.0 ppm.

  In other experiments, 0.5 gm ground cottonseed was digested into 10 ml complexing agent to extract gossypol. The color was stained and the level of gossypol was measured at 20 ppm, representing 1% total gossypol.

(advantage)
The main advantages of the present invention:
-A low-cost and portable system-Specially designed sample holder to hold a 17 mm diameter glass cuvette-Gossypol concentration directly displayed in ppm after coloring-For creating calibration curves No standard solution is required

Projection view of cuvette sample holder. The figure showing the block diagram of an apparatus. The figure showing the block diagram of a sample holder.

Claims (20)

A direct display portable device for the measurement of gossypol concentration after coloring, said device comprising:
a) a light source that emits light of a specific wavelength;
b) means for adjusting the intensity of light from the light source;
c) a glass cuvette that holds the test sample and transmits the light;
d) a cuvette sample holder for holding the glass cuvette;
e) a photodetector for detecting light transmitted by the test sample;
f) means for converting the output current signal from the photodetector into a voltage;
g) a logarithmic amplifier for amplifying the output signal of the converter;
h) a calibration circuit that allows display on the display unit;
i) a device comprising a power supply for supplying power to different components of the device.   The apparatus of claim 1 wherein the gossypol is selected from the group of deoiled cake, cottonseed, and cottonseed oil. The measurement of gossypol concentration apparatus according to claim 1 carried out in at least 3 _^1/2 digit display said display unit mg / l and 100 parts per million in the (ppm).   The apparatus of claim 1, wherein the light source is a 5 mm LED having an intensity of 125 mcd, a viewing angle of 24 °, a peak wavelength of 635 nm, and a power dissipation of 50 mW.   The means used to adjust the intensity of light from the LED is a multi-rotation potentiometer that obtains 100% transmittance (0% absorbance) from light transmitted through distilled water in the cuvette. apparatus.   2. The device according to claim 1, wherein the cuvette used is composed of borosilicate glass.   The apparatus according to claim 1, wherein a path length of the glass cuvette is 17 mm.   2. The device according to claim 1, wherein the cuvette used is a screw cap type having a diameter of 17 mm, a height of 5 cm and a capacity of 6 ml.   2. An apparatus according to claim 1, wherein the cuvette holder is composed of a black painted aluminum alloy.   The photodetector BPW21 has a TO5 package, an effective detection area of 5.9 mm in diameter, a wavelength range of 460-750 nm, and a detection sensitivity of 7 nA / lux is used to detect transmitted / absorbed light through the test solution. The apparatus according to claim 1.   The apparatus of claim 1, wherein the current-to-voltage converter generates a voltage that is directly proportional to the current flowing through the converter, which current is also proportional to the light intensity entering the detector.   The apparatus of claim 1 wherein the means for converting the current signal to a voltage signal is a current / voltage converter.   The apparatus of claim 1, wherein the logarithmic amplifier is a TL441 IC.   The apparatus of claim 1, wherein the calibration circuit is 10 K POT, and after logarithmic amplification, the voltage output is calibrated in terms of Gossypol concentration in the test solution.   The apparatus of claim 1, wherein the power source is a 6V battery for the logarithmic amplifier, photodetector and other integrated chips, and a 9V battery for the display unit.   The apparatus according to claim 1, wherein a result is displayed on the display unit. The apparatus of claim 1, wherein the result is displayed on a computer through CC ⁺ Large.   The apparatus of claim 1 wherein the VISIDAQ software is used for data acquisition, calculation and application control.   The apparatus of claim 1, wherein the apparatus is based on Lambert-Beer law. The apparatus according to claim 1, wherein the apparatus is low-cost, portable, robust and measures gossypol with an error range of ± 2%.

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