KR102588448B1 - Methods for Discriminating Contamination of Non-Steamed Mature Silkworm in Steamed Mature Silkworm Using UV-VIS Spectroscopy - Google Patents

Abstract

The present invention relates to a method for determining the presence or absence of non-red ginseng in red ginseng using a UV-VIS spectrophotometer. The method of the present invention collects and analyzes the absorption spectrum in a specific area of a suspected red ginseng sample, quickly and accurately Since it is possible to determine whether or not red ginseng is mixed and its level, it can be usefully used in the production of high purity ginseng. In addition, among foods using red persimmon, it is possible to identify red persimmon foods that are labeled with false content, which can greatly contribute to managing the distribution of safe foods so that consumers can trust them.

Description

{Methods for Discriminating Contamination of Non-Steamed Mature Silkworm in Steamed Mature Silkworm Using UV-VIS Spectroscopy}

The present invention relates to a method for determining the presence or absence of non-red ginseng in red ginseng using a UV-VIS spectrophotometer.

The sericulture industry that produces silkworm cocoons is a traditional industry in Korea dating back to the Gojoseon Dynasty. In the 1970s, more than 1 million boxes of silkworms were raised annually on farms, but it is currently in a state of significant decline due to economic development and the influence of high-quality chemical fibers. Accordingly, the current sericulture industry has been converted into an industry that produces health functional food ingredients such as dried silkworms, pupae, and Cordyceps sinensis. Among them, the main product produced by farms is freeze-dried silkworms that are 5 instars and 3 days old. The reason why freeze-dried silkworms are used on the 3rd day of the 5th instar is because after the 3rd day of the 5th instar, from the 4th day of the 5th instar, the strong silk protein rapidly enlarges in the silkworm's body, making it very difficult to use it for food. Silk protein is stored in the silkworm's body in the form of silk glands, and when the silkworm becomes a grown-up silkworm (mature silkworm), it spits out this silk protein and builds a cocoon, which is the raw material for silk.

In this way, silkworms that have passed the edible period have not been used for any purpose other than for the production of silk. However, the present inventors have made such use for the first time in Korean Patent No. 10-1388455 and Korean Patent No. 10-1546446. A cosmetic composition for skin improvement has been developed by cooking silkworms containing silk protein at 55 to 125°C under pressurized or normal pressure conditions. In addition, Cooked Sleep was named “Hongjam” by the Rural Development Administration through a public contest. Recent research has shown that red ginseng processed for human consumption has various functional properties.

In order for this type of red silkworm to have excellent functionality, it must be manufactured using silkworms on the 7th or 8th day of the 5th instar. However, raising silkworms until the 7th or 8th day of the 5th instar requires a lot of effort, and freeze-drying the silkworms on the 3rd day of the 5th instar Since there is always a possibility that the powders are mixed, there is a need to develop technology to confirm whether freeze-dried 5-instar, 3-day-old silkworms are included in the red silkworm.

Under these circumstances, the present inventors made extensive research efforts to develop a method that can determine whether freeze-dried powder of 5-instar, 3-day silkworms and red silkworm (cooked silkworm) powder prepared from 5-day instar 7-day sleep are mixed. As a result, a determination method using a UV/VIS spectrophotometer was developed, and when this is applied to collect and analyze the absorption spectrum in a specific region of a suspected red persimmon sample, the presence and level of contamination of non-red persimmon samples can be quickly and accurately determined. By identifying this possibility, the present invention was completed.

Therefore, the purpose of the present invention is to provide a determination method using UV-VIS spectroscopy.

[Prior art literature]

[Patent Document]

Republic of Korea Patent No. 10-1388455

The terms used herein are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention provides a method for determining the presence or absence of non-red ginseng in red ginseng using UV-VIS spectroscopy, comprising the following steps:

(a) Preparing a 100% red persimmon sample as the target sample and comparative control group to be determined;

(b) preparing an extract by extracting each sample with one or more selected from the group consisting of 80 to 100% (v/v) methanol, 80% (v/v) ethanol, and Triton X-100;

(c) measuring the absorbance of the supernatant of each extract using a UV-VIS spectrophotometer; and

(d) Among the measured absorbance results, the absorbance value of each sample is integrated at a wavelength of 200 to 425 nm, a wavelength of 200 to 485 nm, or a wavelength of 550 to 755 nm to calculate the sum of absorbance, and if there is a difference, the sum of absorbance is calculated. A step of determining that the target sample is mixed.

In the present invention, when the extraction in step (b) is performed using 80% (v/v) methanol or ethanol, the discrimination in step (d) is performed at a wavelength of 200 to 425 nm.

Additionally, when the extraction in step (b) is performed using 100% methanol, the determination in step (d) is performed at a wavelength of 200 to 485 nm.

Additionally, when the extraction in step (b) is performed using Triton

In other words, when the sample to be determined is extracted with a specific solvent and the absorbance of its supernatant is measured, the absorption wavelength changes or the absorbance increases or decreases due to differences in component content in the resulting extract supernatant. Since the content of non-red ginseng in red ginseng is proportional to the concentration of non-red ginseng in the sample to be measured, the presence and/or level of non-red ginseng in the sample can be determined by measuring and comparing the absorbance of the extract supernatant. It becomes possible to measure.

More specifically, among the measured absorbance results, 'calculating the absorbance sum by integrating the absorbance value of each sample' at a wavelength of 200 to 425 nm, 200 to 485 nm, or 550 to 755 nm means the area of the absorbance spectrum of the corresponding wavelength. This means obtaining, and by comparing this, if there is a difference between the absorbance sum value of the target sample to be determined and the absorbance sum value of the 100% red persimmon sample as a comparative control group, it is necessary to determine whether non-red persimmon is mixed in the target sample. Rather, it is characterized by being able to determine the level of mixing at a low content of less than 2.5% by weight.

In other words, it provides a method to qualitatively and quantitatively identify the mixing of non-red ginseng in red ginseng by measuring the sum of absorbances in the specific wavelength range described above.

According to one embodiment, the step of measuring the absorbance may be measuring absorbance in a range of 200 to 425 nm, 200 to 485 nm, or 550 to 755 nm depending on the solvent.

There are no limitations to the absorbance measurement device that enables the absorbance measurement, but considering the aspect of enabling immediate identification in the field, a portable UV-VIS spectrophotometer is preferable.

Additionally, the extraction in step (b) may include additionally reacting the extract extracted using Triton X-100 with Triton X-100 at -50°C to 90°C for 15 to 60 minutes.

The extraction in step (b) of the present invention can be performed at room temperature for 2 to 10 minutes, and is not limited thereto, as long as the purpose of the present invention can be achieved.

According to one embodiment of the present invention, preferably,

When a red persimmon sample was extracted using 80% methanol and ethanol and the absorbance of its supernatant was measured with a UV-VIS spectrophotometer, a difference appeared at 320-365 nm, and the difference was the sum of absorbance at a wavelength of 200-425 nm. appeared;

In addition, when a 0.5% Triton Differences were observed in the sum of absorbances at different wavelengths;

In addition, when extracting a red ginseng sample for 3 minutes at room temperature using a 0.5% Triton Differences were seen at 320-395nm;

In addition, when extracting a red ginseng sample for 3 minutes at room temperature using a 0.5% Triton , differences appeared at 320-410 nm;

In addition, when extracting a red ginseng sample for 3 minutes at room temperature using a 0.5% Triton , differences appeared at 320-365 nm;

In addition, red ginseng samples were extracted for 3 minutes at room temperature using 0.5% Triton When measured, differences appeared at 320-365 nm;

In addition, when a 0.5% Triton

Additionally, when treated with various surfactants, differences appeared at 320-380 nm only when treated with a specific surfactant.

That is, according to an embodiment of the present invention, a sample (red ginseng) extract was prepared using a specific extraction solvent, the absorbance of the supernatant was measured, and compared with the absorbance value of a 100% red ginseng sample, the absorbance was found to be in a specific wavelength range. It was confirmed that a difference appeared.

Therefore, the determination method using the UV-VIS spectrophotometer of the present invention means that it is possible to quickly and accurately determine the purity of red persimmon by collecting and analyzing the absorption spectrum in a specific region of the red persimmon sample, which is difficult to discriminate.

The red silkworm of the present invention is manufactured by boiling raw silkworms in water or by cooking them with the heat of steam, and is characterized by being able to be consumed for dietary purposes while containing all silk proteins. The word "cooked sleep" used in this specification while referring to red jam is used interchangeably with red jam within the specification.

Maintaining the excellent functionality of red ginseng by cooking silkworms with silk protein, which were previously inedible and had no other purpose other than breeding them to make silkworm cocoons for silk production, and processing them into an edible state containing silk protein. To achieve this, it must be manufactured using the 7th or 8th day of deep sleep of the 5th instar, but freeze-dried powder of 5th instar 3rd day silkworms can be mixed during this manufacturing process.

Meanwhile, it is known in the art that the content of ingredients in each stage of silkworms is different when they are raw and when they are cooked. Therefore, based on this, the present invention is significant in that it is possible to determine whether red jam contains non-hongjam by confirming the different physicochemical characteristics between red jam and non-hongjam.

In this specification, cooked silkworm (hongjam), which is a processed cooked silkworm containing the silk protein, means manufactured through a process of cooking all silkworms such as three-sided silkworm and four-sided silkworm, and preferably 5th instar 4 of silkworms. It is possible to select and use one or more of the four sleeping silkworms up to the deep sleep stage, and it is more preferable to use silkworms in the deep sleep stage. It can be used on silkworms that are 5 days old and 3 days old, but because silkworms have less silk protein than silkworms that are 5 days old or older, there is a risk that it may not fully demonstrate its effectiveness as a red silkworm. Therefore, according to the invention of the present invention, it is possible to determine whether non-red silkworms of 5 instars and 3 days of age are mixed in the sample to provide high purity red silkworms made only from silkworms from 5 instars and 4 days of four-faced sleep to deep sleep. do.

On the other hand, a silkworm in the long sleep period refers to a silkworm whose body is full of silk protein before building a cocoon. Since most of the silkworm excretion during the long sleep period is excreted, it can be used as a pharmaceutical composition or food composition ( It is most suitable for use as an active ingredient in health functional foods.

The red silkworm can be prepared by boiling raw silkworms selected from silkworms ranging from four-faced silkworms of 5 instars and 4 days to deep sleep in water at a temperature of 55 to 125 ℃ for 7 minutes to 30 hours, or by cooking them with the heat of steam. , Preferably, the silkworms are cooked in the heat of steam at 80 to 125°C for 100 to 150 minutes (eg, 130 minutes) to ensure that all silk proteins are contained. In other words, if it is manufactured for more than 150 minutes with steam heat below 80℃, the manufacturing time is too long and there is a risk that the active ingredients contained in the silkworm itself, including silk protein, may be destroyed, and if it is manufactured over 125℃ and less than 100 minutes This is because, when manufactured, the temperature is too high and there is a risk of adversely affecting the active ingredients contained in the silkworm itself, which contains silk protein.

After the above-mentioned process, it can be dried in a conventional way and manufactured into powder form for use. Preferably, it is preferably dried and powdered through any one selected from hot air drying or vacuum freeze drying to prevent loss of active ingredients. To minimize this, vacuum freeze-drying is the most appropriate method.

Therefore, the red jam of the present invention may be hot air dried or vacuum freeze dried.

The powderization can be done using any method such as a household mixer, hammer mill, roller mill, or air jet mill (freeze grinding), but in order to minimize sample loss, prevent component distortion, and make fine powder, the roller mill method is recommended. good night. Usually, when grinding with a household blender or hammer mill, it is very difficult to grind the powder particles to a size of about 0.1㎜ or less, but when grinding repeatedly with a roller mill several times, the powdered particles can be finely powdered to an average size of about 10㎛, which is advantageous. It is expected to work well.

The silkworm is a silkworm breed selected from the group consisting of white daisy, golden silk, and orange silkworm, but is not limited thereto, as long as the purpose of the present invention can be achieved.

In the present invention, non-redjam refers to silkworms that are not suitable for producing redjam, and is preferably a 5-year-old, 3-day-old silkworm. According to the method of the present invention, it is possible to determine the presence or absence of non-red silkworm mixed in less than 2.5% by weight in the sample, providing excellent quality red silkworm manufactured only from silkworms from the four-faced sleep silkworm of 5 instars and 4 days to deep sleep. can do.

In addition, according to another aspect of the present invention, the present invention provides a method for determining the presence or absence of non-red ginseng in red ginseng using UV-VIS spectroscopy, comprising the following steps:

(a) Preparing a 100% red persimmon sample as the target sample and comparative control group to be determined;

(b) treating each sample with one type of surfactant selected from the group consisting of PEG200, PEG600, Tween 20, Tween 80, LFP-50P, Koremul pH4, CocoPG, CAPB, and Bardap26;

(c) measuring the absorbance of each sample treated with the surfactant using a UV-VIS spectrophotometer; and

(d) Among the measured absorbance results, the absorbance value of each sample is integrated at a wavelength of 200 to 425 nm, a wavelength of 200 to 485 nm, or a wavelength of 550 to 755 nm to calculate the sum of absorbance, and if there is a difference, the sum of absorbance is calculated. A step of determining that the target sample is mixed.

The method may include the step of additionally performing a ninhydrin color development reaction after step (b).

In other words, it provides a method to qualitatively and quantitatively identify the presence or absence of non-red ginseng in red ginseng by reacting with ninhydrin to develop color and measuring the absorbance of the color using an absorbance measuring device.

As long as the purpose of the present invention can be achieved, the concentration of ninhydrin reacting with the sample according to the present invention is not particularly limited.

Overall, the present invention is the first to demonstrate that it is possible to determine the presence (incorporation, mixing) of non-red ginseng contained in red ginseng, a new type of raw material for food and medicine, that is, the purity of red ginseng, using this method. In this case, it is expected that it will have a significant effect in contributing to the development of the sericulture industry and agriculture by creating utilization and high added value of red silkworms contained in high purity.

The present invention's method for determining the presence or absence of non-red ginseng in red ginseng using a UV-VIS spectrophotometer collects and analyzes the absorption spectrum in a specific area of a suspected red ginseng sample to quickly and accurately determine the presence and level of non-red ginseng incorporation. Therefore, it has the effect of being useful in the production of high-purity red ginseng. In addition, among foods using red persimmon, it is possible to identify red persimmon foods that are labeled with false content, which can greatly contribute to managing the distribution of safe foods so that consumers can trust them.

Figure 1 shows the color change of 80% MeOH extraction solutions according to the mixing ratio of red jam prepared from three varieties of silkworms and freeze-dried powder of 5-instar, 3-day silkworms.
Figure 2 shows the absorbance measurement results at 200 to 800 nm for a mixture of three varieties of red persimmon and 5-instar, 3-day-old silkworm freeze-dried powder extracted in 80% MeOH.
Figure 3 shows the color change of the extract obtained by extracting a mixture containing 90 to 100% of red perilla and 2.5 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder with 80% MeOH for 3 minutes at room temperature.
Figure 4 shows the absorbance at 200 to 800 nm for the extract of a mixture containing 90 to 100% of three varieties of Red Jam and 0 to 10% of 5-instar 3-day silkworm freeze-dried powder extracted with 80% MeOH for 3 minutes at room temperature. shows changes in
Figure 5 shows the color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 80% EtOH for 3 minutes at room temperature.
Figure 6 shows the change in absorbance at 200 to 800 nm for the extract of 90 to 100% of three varieties of Red Jam, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 80% EtOH for 3 minutes at room temperature. It shows.
Figure 7 shows the color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of red silkworm extracted with 100% MeOH for 3 minutes at room temperature.
Figure 8 shows the change in absorbance of an extract containing 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 100% MeOH for 3 minutes at room temperature.
Figure 9 shows the color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 100% EtOH for 3 minutes at room temperature.
Figure 10 shows the change in absorbance of the extract containing 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 100% EtOH for 3 minutes at room temperature.
Figure 11 shows the color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 0.5% Triton X-100 solution for 3 minutes at room temperature.
Figure 12 shows the change in absorbance of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 0.5% Triton X-100 solution for 3 minutes at room temperature.
Figure 13 shows the extract obtained by extracting 90 to 100% of red persimmon containing 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder with 0.5% Triton It shows the color change afterward.
Figure 14 shows the extract of 90 to 100% of red persimmon, including 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder, extracted with 0.5% Triton It shows the change in absorbance afterward.
Figure 15 shows the extract of 90 to 100% of Golden Silk Red Jam, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 0.5% Triton It shows the color change after reaction.
Figure 16 shows the extract of 90 to 100% of Golden Silk Red Jam, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 0.5% Triton It shows the color change after reaction.
Figure 17 shows the extract of 90 to 100% golden silk (GSHJ), including 0 to 10% 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 0.5% Triton It shows the color change after reacting with water for 60 minutes.
Figure 18 shows the extract of 90 to 100% golden silk (GSHJ) containing 0 to 10% 5-instar 3-day silkworm freeze-dried powder extracted with 0.5% Triton X-100 solution for 3 minutes at room temperature for 15 minutes and 30 minutes at 80°C. It shows the color change after reacting with water for 60 minutes.
Figure 19 shows the extract of 90 to 100% golden silk (GSHJ) containing 0 to 10% 5 instar 3 day silkworm freeze-dried powder extracted with 0.5% Triton X-100 solution for 3 minutes at room temperature for 15 minutes at -50°C. It shows the color change after reaction for 30 and 60 minutes.
Figure 20 shows the extract of 90 to 100% golden silk (GSHJ), including 0 to 10% 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 0.5% Triton It shows the color change after reaction for 30 and 60 minutes.
Figure 21 shows the extract of 90 to 100% of three varieties of red silkworm, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 0.5% Triton Shows color change.
Figure 22 shows the color change of the extract when various detergents were used in the extract of 90 to 100% GSHJ mixture containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder.
Figure 23 shows the color change of the extract when various detergents were used in the extract of 90 to 100% GSHJ mixture containing 0 to 10% 5-instar 3-day-old silkworm freeze-dried powder.
Figure 24 shows the color change after Ninhydrin reaction in extracts using various detergents in 90 to 100% GSHJ, including 0 to 10% 5-instar, 3-day-old silkworm freeze-dried powder.
Figure 25 shows the absorbance when the extract extracted with 0.5% Triton shows change.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

Samples and experimental methods

5 instar 3 day freeze dried silkworm manufacturing method

White jade silkworms were reared on mulberry leaves until they were 5 instars and 3 days old, washed and quickly frozen. The frozen 5-instar, 3-day-old silkworms were dried using a freeze dryer and then pulverized to prepare 5-instar, 3-day-old silkworm powder (FDSP) samples.

How to make Hongjam (cooked sleep)

White jade, golden silk, and orange jade were reared on mulberry leaves until deep sleep (5th instar, 7th to 8th day), washed, steamed at 100°C for 130 minutes using a pressure-free steamer, and then quickly frozen. Quick-frozen cooked mature silkworms were dried using a freeze dryer to remove moisture and then powdered to prepare steamed mature silkworm powder. The following used in this example was prepared. Named together:

GSHJ = Golden-silk Hong Jam (Golden-silk Hong Jam powder)

RSHJ = Red silk Hong Jam (red silk Hong Jam powder)

WJHJ = White Jade Hong Ham (White Jade Hong Ham powder).

Preparation of a mixture extract of 3 varieties of red ginseng and 5-instar 3-day silkworm freeze-dried powder and measurement method using a spectrophotometer

How to check the freeze-dried powder of 5-instar, 3-day silkworms contained in red jam at a ratio of 10% to 90%

Mix the freeze-dried material of 5-instar, 3-day silkworms made from red and white hyacinths made from 3 different breeds of mature silkworms using a weight ratio ranging from 100%:0% to 0%:100% at 10% intervals. did. The mixture of red silkworm and 5-instar 3-day-old freeze-dried silkworm powder was extracted by adding 9 times its weight of 80% methanol (MeOH) solution and stirring for 1 hour in a constant temperature water bath at 37°C. The extracted solutions were precipitated at 3000 rpm for 10 minutes at room temperature, and then the supernatant was obtained. The obtained supernatants clearly showed a difference in color to the naked eye. The absorbance of the supernatant was measured from 200 to 800 nm with a difference of 15 nm. The absorbance of the extracted solutions without dilution was too high, so they were diluted 1:10 with 80% MeOH and then the absorbance was measured using Thermo Fischer Multiskan Go. The value obtained by subtracting the absorbance of 80% MeOH from the absorbance of each extraction solution was calculated as the absorbance of the solution and displayed on a graph.

UV/VIS spectrophotometer (ultraviolet-visible spectroscopy) analysis conditions are shown in Table 1 below.

96 well plate UV/VIS spectrophotometer analysis conditions (Multiskan Go, Thermo-Fisher Scientific) Analysis conditions 25℃ Measurement wavelength range 200 to 800 nm Wavelength Resolution 15nm Absorption area/resolution 0 to 4.0,/0.003 96 well plates for UV/VIS spectrometer 85.4 mm x 126.6 mm

How to check 5-instar 3-day silkworm freeze-dried powder mixed at a ratio of 2.5% to 10%

If more than 10% of 5-instar, 3-day-old silkworm freeze-dried powder is included, contamination can be confirmed just by the color of the 80% MeOH extract, so this is a method to check whether less than 10% of 5-instar, 3-day-old silkworm freeze-dried powder is included. was additionally developed. For field use, 80% MeOH was added in a volume equivalent to 9 times the weight of a mixture of 90% to 100% red persimmon and 0 to 10% 5-instar 3-day silkworm freeze-dried powder, and then stirred for 3 minutes at room temperature. After extraction was performed, the solution was allowed to stand for 5 minutes to precipitate the mixture, and the supernatant was used to observe the color change and measure the change in absorbance at 200 to 800 nm.

Development of optimal extraction solution

In order to develop the optimal extraction solution, the following various solutions were extracted at room temperature for 3 minutes, stabilized for 5 minutes, and the absorbance was measured using the supernatant:

100% MeOH, 80% Ethanol (EtOH), 100% EtOH, 0.5% Triton Extraction was performed using 0.5% LFP-50P solution, 0.5% Koremul-pH4 solution, 0.5% CocoPG solution, 0.5% CAPB solution, and 0.5% Bardap26 solution.

Measurement of absorbance change using ninhydrin amino acid reaction color reaction (ninhydrin reactioin)

0.5% PEG 200 solution, 0.5% PEG600 solution, 0.5% Tween 20 solution, 0.5% Tween 80 solution, 0.5% LFP-50P solution, 0.5% Koremul-pH4 solution, 0.5% CocoPG solution, 0.5% CAPB solution, 0.5% Bardap26 After ninhydrin reaction was performed on the extract extracted using a solution, etc., the change in absorbance was measured using the method mentioned above.

Measurement of absorbance change using the presumptive color test, a method for measuring plant-derived compounds

The extract of 0.5% Triton Accordingly, a presumptive color test used to measure plant-derived compounds was performed on 0.5% Trioton The presumptive color tests used in the study include Chen test, Cobalt test, Froehde test, Lieberman test, Mandelin test, Mecke test, Marquis test, Zwikker test, Simon test, Dille-Koppanyi test, Dragnodoff test, and Biuret test.

Example 1. Color change of 80% MeOH extract solutions according to the mixing ratio of red ginseng prepared from 3 types of silkworms and freeze-dried powder of 5-instar 3-day silkworms.

The present inventors measured the color change of 80% MeOH extraction solutions according to the mixing ratio of red persimmon prepared from three types of silkworms and freeze-dried powder of 5-instar, 3-day-old silkworms.

As a result, as shown in Figure 1, the ratio of red persimmon and 5-instar 3-day silkworm freeze-dried powder was changed from 100% to 0% in 10% increments, and then extracted for 1 hour at 37°C using 80% MeOH. As a result of checking the color change of the extract, a dark green extract was observed in the case of 100% 5-instar, 3-day-old silkworm freeze-dried powder, and in the case of 100% red silkworm, a light yellow extract was observed, although there were differences depending on the variety. A dark green color was observed in 70-90% of the extracts including freeze-dried extracts of 5-instar, 3-day silkworms, but as the proportion of red silkworms increased, light green was observed, and in 30%, a light green color was observed. Also, at 20%, it was light green, and at 10%, it was a color intermediate between light green and golden sand. The extracts of white hyacinth and red hyacinth had the lightest golden sand color.

In other words, it was possible to confirm whether 90 to 10% of 5-instar, 3-day-old silkworms were included just by looking at the color of the extract.

Example 2. Absorbance measurement at 200 to 800 nm for a mixture of three varieties of red persimmon and 5-instar, 3-day-old silkworm freeze-dried powder extracted in 80% MeOH

The present inventors measured the absorbance at 200 to 800 nm of a mixture of three varieties of red persimmon and 5-instar, 3-day-old silkworm freeze-dried powder extracted in 80% MeOH.

A mixture of red silkworm and 5-instar 3-day silkworm freeze-dried powder made from golden silk, orange silkworm, and white silkworm was prepared with different mixing ratios from 100% to 10%, and then soaked in 80% MeOH at 37°C for 1 hour. The extracted solution was diluted to 1/10 using 80% MeOH, and then the change in absorbance from 200 to 800 nm was compared. The absorbance of each extract was measured after correction by measuring the absorbance of 80% MeOH.

As a result, as shown in Figure 2, looking at the change in absorbance of the extract of Golden Silk Jam (GSHJ) and 5-instar 3-day silkworm freeze-dried powder mixture (FDSP), the higher the ratio of 5-instar 3-day silkworm freeze-dried powder, the 275 The largest difference in absorbance was observed at ~365 nm (Figure 2A). In addition, looking at the change in absorbance of the extract of the mixture of RSHJ and 5-instar, 3-day-old silkworm freeze-dried powder, the higher the ratio of 5-instar, 3-day-old silkworm freeze-dried powder, the largest difference in absorbance was between 275 and 350 nm. was observed (Figure 2B). In addition, looking at the change in absorbance of the extract of the mixture of White Jade Jam (WJHJ) and 5-instar, 3-day-old silkworm freeze-dried powder, the higher the ratio of 5-instar, 3-day-old silkworm freeze-dried powder, the largest difference in absorbance was between 275 and 380 nm. was observed (Figure 2C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Among the specifically measured absorbance results, the absorbance values of each sample were integrated at wavelengths from 200 to 425 nm to calculate the absorbance values, and comparisons were made between samples. In the case of the sample containing 100% of the three types of red ginseng, the absorbance was GSHJ. 2.3 + 0.05, RSHJ was 2.5 + 0.13, and WJHJ was 2.1 + 0.11, which was 37.81 ~ 42.10% lower than the 90% red ginseng sample containing 10% FDSP. In the case of FDSP, the absorbance was 3.48 to 4.43 times higher than that of the three types of red persimmon samples. This result shows that when determining the quality of red ginseng when the ratio of non-red ginseng is less than 10%, the absorbance should be in the range of 2.0 to 2.8.

Example 3. Color change of the extract obtained by extracting a mixture containing 90 to 100% of red ginseng and 2.5 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder with 80% MeOH for 3 minutes at room temperature.

The present inventors confirmed the color change of the extract obtained by extracting a mixture containing 90 to 100% of red perilla and 2.5 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder with 80% MeOH for 3 minutes at room temperature.

As a result, as shown in FIG. 3, when there was a difference in composition ratio of 5%, the color could be distinguished with the naked eye, but it was difficult to distinguish color when there was a difference in composition ratio of 2.5%.

Example 4. A mixture containing 90 to 100% of three varieties of red persimmon and 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder was extracted with 80% MeOH for 3 minutes at room temperature. change in absorbance

The present inventors analyzed the absorbance at 200 to 800 nm for the extract of a mixture containing 90 to 100% of three varieties of red persimmon and 0 to 10% of 5-instar 3-day silkworm freeze-dried powder, extracted with 80% MeOH for 3 minutes at room temperature. Changes were confirmed.

As shown in Figure 4, looking at the results of measuring the change in absorbance of the extract of the Golden Silk Jam (GSHJ) mixture containing 0 to 10% of 5-instar 3-day silkworm freeze-dried powder, the absorbance was 320 to 365 nm. showed the biggest difference. Using the change in absorbance, a difference in mixing ratio of 2.5% could be measured (Figure 4A). Looking at the results of measuring the change in absorbance of the extract of the RSHJ mixture containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, the absorbance showed the greatest difference at 320 to 365 nm. Using the change in absorbance, a difference in mixing ratio of 2.5% could be measured (Figure 4B). Looking at the results of measuring the change in absorbance of the extract of the White Jade Jam (WJHJ) mixture containing 0 to 10% of 5-instar 3-day silkworm freeze-dried powder, the greatest difference in absorbance was shown at the light range of 320 to 365 nm. . Using the change in absorbance, a difference in mixing ratio of 2.5% could be measured (Figure 4C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Among the specifically measured absorbance results, the absorbance values of each sample were integrated at wavelengths from 200 to 425 nm to calculate the absorbance values to compare between samples. In the case of the sample containing 100% of the three types of red ginseng, the absorbance was GSHJ was 6.8 + 0.09, RSHJ was 6.1 + 0.06, and WJHJ was 6.2 + 0.07, which was 8.11 to 12.90% lower than the 97.5% red jam sample including 2.5% FDSP. This result indicates that the ratio of non-red ginseng is less than 2.5%. When determining the quality of red ginseng, the absorbance ranges from 6.4 to 7.2 for GSHJ, 5.7 to 6.5 for RSHJ, and 5.8 to 6.6 for WJHJ. It shows that it is sufficient to exist in .

Example 5. Color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 80% EtOH for 3 minutes at room temperature.

The present inventors confirmed the color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and extracting 90 to 100% of Red Jam with 80% EtOH for 3 minutes at room temperature.

As a result, as shown in Figure 5, when there was a difference in the red-silver ratio of 5% when viewed with the naked eye, the color could be distinguished, but when there was a difference of 2.5%, it was difficult to distinguish the color.

Example 6. Change in absorbance at 200 to 800 nm for the extract of 90 to 100% of three varieties of red jam, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 80% EtOH for 3 minutes at room temperature.

The present inventors examined the change in absorbance at 200 to 800 nm for the extract of 90 to 100% of three varieties of red jam, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, extracted with 80% EtOH for 3 minutes at room temperature. Confirmed.

As a result, as shown in Figure 6, looking at the absorbance of the 80% EtOH extract of the GSHJ mixture containing freeze-dried powder of 5-instar, 3-day silkworms, the greatest difference in absorbance was seen at light of 320 to 365 nm. A mixing ratio difference of 2.5% was also measurable (Figure 6A). Looking at the absorbance of the 80% EtOH extract of RSHJ mixtures containing freeze-dried powder of 5-instar, 3-day silkworms, the greatest difference in absorbance was seen at light between 320 and 365 nm. A mixing ratio difference of 2.5% was also measurable (Figure 6B). Looking at the absorbance of the 80% EtOH extract of the WJHJ mixture containing 5-instar, 3-day-old silkworm freeze-dried powder, the greatest difference in absorbance was seen at light between 320 and 365 nm. A mixing ratio difference of 2.5% was also measurable (Figure 6C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Looking at the results of comparison between samples by combining the absorbance from 200 to 425 nm, for samples containing 100% of the three types of red persimmon, the absorbance was 6.3 + 0.04 for GSHJ, 6.9 + 0.08 for RSHJ, and 6.7 + 0.12 for WJHJ. It was found to be 5.65% to 11.6% lower than the 97.5% red jam sample containing 2.5% FDSP. This result shows that when determining the quality of red ginseng when the ratio of non-red ginseng is less than 2.5%, the absorbance ranges from 6.1 to 6.7 for GSHJ, 6.6 to 7.2 for RSHJ, and 6.4 to 7.0 for WJHJ. It shows that it is sufficient to exist in .

Example 7. Color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of red ginseng extracted with 100% MeOH for 3 minutes at room temperature.

The present inventors confirmed the color change of the extract obtained by extracting 90 to 100% of Red Jam containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder with 100% MeOH for 3 minutes at room temperature.

As a result, as shown in Figure 7, when there was a 5% difference in the red-silver ratio, the color could be distinguished with the naked eye, but it was difficult to distinguish when there was a 2.5% difference.

Example 8. Change in absorbance of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 100% MeOH for 3 minutes at room temperature

The present inventors confirmed the change in absorbance of an extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 100% MeOH for 3 minutes at room temperature.

As a result, as shown in Figure 8, looking at the absorbance of the 100% MeOH extract of the GSHJ mixture containing freeze-dried powder of 5-instar, 3-day silkworms, the greatest difference in absorbance was seen at light of 290 to 440 nm. A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 8A). Looking at the absorbance of the 100% MeOH extract of RSHJ mixtures containing freeze-dried powder of 5-instar, 3-day silkworms, the greatest difference in absorbance was seen at light between 290 and 440 nm. A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 8B). Looking at the absorbance of the 100% MeOH extract of the WJHJ mixture containing freeze-dried powder of 5-instar, 3-day silkworms, the greatest difference in absorbance was seen at light between 290 and 440 nm. A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 8C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Among the specifically measured absorbance results, the absorbance values of each sample were integrated at wavelengths from 200 to 485 nm to calculate the absorbance values, and comparisons were made between samples. In the case of the sample containing 100% of the three types of red ginseng, the absorbance was GSHJ was 4.9 + 0.10, RSHJ was 7.0 + 0.26, and WJHJ was 5.4 + 0.45, which was 5.65% ~ 11.6% lower than the 97.5% red jam sample including 2.5% FDSP. This result indicates that the ratio of non-red ginseng is less than 2.5%, and when determining the quality of red ginseng, the absorbance ranges from 4.1 to 5.6 for GSHJ, 6.5 to 7.5 for RSHJ, and 4.9 to 5.9 for WJHJ. It shows that it is sufficient to exist in .

Example 9. Color change of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of red ginseng extracted with 100% EtOH for 3 minutes at room temperature.

The present inventors confirmed the color change of the extract obtained by extracting 90 to 100% of Red Jam containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder with 100% EtOH for 3 minutes at room temperature.

As a result, as shown in Figure 9, when there was a 5% difference in the red-silver ratio, the color could be distinguished with the naked eye, but it was difficult to distinguish when there was a 2.5% difference.

Example 10. Change in absorbance of the extract containing 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 100% EtOH for 3 minutes at room temperature

The present inventors confirmed the change in absorbance of an extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 100% EtOH for 3 minutes at room temperature.

As a result, as shown in Figure 10, the absorbance of the 100% EtOH extract of the GSHJ mixture containing freeze-dried powder of 5-instar, 3-day silkworm showed the greatest difference in absorbance at light of 290 to 440 nm. A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 10A). Looking at the absorbance of the 100% EtOH extract of the RSHJ mixture containing 5-instar 3-day silkworm freeze-dried powder, there was no significant difference in absorbance (Figure 10B). Looking at the absorbance of the 100% EtOH extract of the WJHJ mixture containing 5-instar, 3-day silkworm freeze-dried powder, there was no significant difference in absorbance (Figure 10C).

Therefore, 100% EtOH is unsuitable for use in the determination method of the present invention.

Example 11. Color change of the extract extracted from 90 to 100% of red ginseng containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder with 0.5% Triton X-100 solution for 3 minutes at room temperature.

The present inventors confirmed the color change of the extract obtained by extracting 90 to 100% of Red Jam containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder with 0.5% Triton X-100 solution for 3 minutes at room temperature.

As a result, as shown in Figure 11, when there was a 5% difference in the red-silver ratio, the color could be distinguished with the naked eye, but it was difficult to distinguish when there was a 2.5% difference.

Example 12. Change in absorbance of the extract containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 0.5% Triton X-100 solution for 3 minutes at room temperature

The present inventors confirmed the change in absorbance of the extract containing 0 to 10% of 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder and 90 to 100% of Red Jam extracted with 0.5% Triton X-100 solution for 3 minutes at room temperature.

As a result, as shown in Figure 12, looking at the absorbance of 0.5% Triton showed. A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 12A). Looking at the absorbance of 10.5% Triton The difference in mixing ratio of 2.5% was not clearly distinguished (Figure 12B). When looking at the absorbance of 0.5% Triton A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 12C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Among the specifically measured absorbance results, the absorbance values of each sample were integrated at wavelengths from 200 to 485 nm to calculate the absorbance values, and comparisons were made between samples. In the case of the sample containing 100% of the three types of red ginseng, the absorbance was GSHJ was 10.2 + 0.13, RSHJ was 10.6 + 0.08, and WJHJ was 10.8 + 0.12, which was 8.47% to 14.55% lower than the 97.5% red jam sample containing 2.5% FDSP. This result indicates that the ratio of non-red ginseng is less than 2.5%. When determining the quality of red ginseng, the absorbance ranges from 9.2 to 11.2 for GSHJ, 9.6 to 11.6 for RSHJ, and 10.2 to 11.4 for WJHJ. It shows that it is sufficient to exist in .

Example 13. The extract of 90 to 100% of red persimmon containing 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder was extracted with 0.5% Triton Color change after

The present inventors incubate the extract of 90 to 100% of red persimmon containing 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder with 0.5% Triton Color change was confirmed.

As a result, as shown in Figure 13, when there was a 5% difference in the red ginseng ratio, the colors of the extracts could be discerned with the naked eye, but when there was a 2.5% difference, it was difficult to discern.

Example 14. The extract of 90 to 100% of red persimmon containing 0 to 10% of 5-instar, 3-day silkworm freeze-dried powder was extracted with 0.5% Triton Change in absorbance after

The present inventors incubated 90 to 100% of red persimmon, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with 0.5% Triton The subsequent change in absorbance was confirmed.

As a result, as shown in Figure 14, looking at the absorbance before reacting 0.5% Triton Absorbance showed the greatest difference. A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 14A). When looking at the absorbance after reacting 10.5% Triton gave. The difference in mixing ratio of 2.5% was not clearly distinguished (Figure 14B). After reacting 0.5% Triton showed it A difference in mixing ratio of 2.5% was also clearly distinguishable (Figure 14C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Among the specifically measured absorbance results, the absorbance values of each sample were integrated at wavelengths from 200 to 485 nm to calculate the absorbance values, and comparisons were made between samples. In the case of the sample containing 100% of the three types of red ginseng, the absorbance was GSHJ was 9.8 + 0.08, RSHJ was 9.0 + 0.10, and WJHJ was 9.1 + 0.16, which was 11.85% ~ 18.48% lower than the 97.5% red jam sample including 2.5% FDSP. This result indicates that the ratio of non-red ginseng is less than 2.5%. When determining the quality of red ginseng, the absorbance ranges from 8.8 to 10.8 for GSHJ, 8.4 to 9.6 for RSHJ, and 8.1 to 10.1 for WJHJ. It shows that it is sufficient to exist in .

Example 15. 90 to 100% of Golden Silk Red Jam containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder was extracted with 0.5% Triton Color change after reacting for a minute

The present inventors extracted the extract of 90 to 100% of Golden Silk Red Jam, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with 0.5% Triton The color change after reaction was confirmed.

As a result, as shown in Figure 16, when there was a 5% difference in the red ginseng ratio, the colors of the extracts could be discerned with the naked eye, but when there was a 2.5% difference, it was difficult to discern.

Example 16. 90 to 100% of Golden Silk Red Jam containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder was extracted with 0.5% Triton Color change after reacting for a minute

The present inventors extracted the extract of 90 to 100% of Golden Silk Red Jam, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with 0.5% Triton The color change after reaction was confirmed with a spectrophotometer.

As a result, as shown in Figure 16, after reaction at room temperature for 15 minutes, a difference in components of 2.5% could be distinguished in the wavelength range of 320 to 395 nm (Figure 16A). After reacting at room temperature for 30 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 395 nm (Figure 16B). After reacting at room temperature for 60 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 395 nm (Figure 16C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Specifically, among the measured absorbance results, the absorbance value of each sample was integrated at a wavelength from 200 to 485 nm to calculate the absorbance value, and a comparison was made between samples. In the case of the 100% GSHJ sample, the absorbance was 100% after incubation for 15 minutes at room temperature. It was 10.2 + 0.08, 10.3 + 0.08 after 30 minutes of incubation, and 10.7 + 0.17 after 60 minutes, which was 13.81% ~ 16.08% lower than the 97.5% red perilla sample including 2.5% FDSP. This result shows that when determining the quality of red ginseng when the ratio of non-red ginseng is less than 2.5%, the absorbance is 9.2 to 11.2 after 15 minutes of incubation at room temperature, 9.3 to 11.3 after 30 minutes of incubation, and 9.7 after 60 minutes of incubation. It shows that it suffices to exist in the range of ~ 11.7.

Example 17. 90 to 100% of Golden Silk (GSHJ), including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, was extracted with 0.5% Triton Color change after reaction for 30 and 60 minutes

The present inventors extracted 90 to 100% of golden silk (GSHJ), including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with 0.5% Triton The color change was confirmed after reacting with powder for 60 minutes.

As a result, as shown in Figure 17, when there was a 5% difference in the red ginseng ratio, the colors of the extracts could be discerned with the naked eye, but when there was a 2.5% difference, it was difficult to discern.

Example 18. 90 to 100% of Golden Silk (GSHJ) containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder was extracted with 0.5% Triton Color change after reaction for 30 and 60 minutes

The present inventors extracted 90 to 100% of golden silk (GSHJ), including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with 0.5% Triton After reacting for 60 minutes, the color change was confirmed using a spectrophotometer.

As a result, as shown in Figure 18, after reaction at 80°C for 15 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 365 nm (Figure 18A). After reacting at 80°C for 30 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 365 nm (Figure 18B). After reacting at 80°C for 60 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 365 nm (Figure 18C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Specifically, among the measured absorbance results, the absorbance values of each sample were integrated at wavelengths from 200 to 485 nm to calculate the absorbance values, and comparisons were made between samples. In the case of the 100% GSHJ sample, the absorbance was 100% after incubation at 80°C for 15 minutes. was 9.7 + 0.17, 9.7 + 0.31 after 30 minutes of incubation, and 10.0 + 0.29 after 60 minutes, which was 13.81% ~ 16.08% lower than the 97.5% red perilla sample containing 2.5% FDSP. This result shows that when determining the quality of red ginseng when the ratio of non-red ginseng is less than 2.5%, the absorbance is 9.0 to 10.4 after 15 minutes of incubation at room temperature, 8.8 to 10.7 after 30 minutes of incubation, and 9.0 after 60 minutes of incubation. It shows that it suffices to exist in the range of ~ 11.0.

Example 19. 90 to 100% of Golden Silk (GSHJ), including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, was extracted with 0.5% Triton Color change after reacting with powder for 30 and 60 minutes

The present inventors extracted 90 to 100% of golden silk (GSHJ), including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with 0.5% Triton The color change was confirmed after reacting for 30 and 60 minutes.

As a result, as shown in Figure 19, when there was a 5% difference in the ratio of red jam with the naked eye, the colors of the extracts could be discerned, but when there was a 2.5% difference, it was difficult to discern.

Example 20. 90 to 100% of Golden Silk (GSHJ), including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, was extracted with 0.5% Triton Color change after reacting with powder for 30 and 60 minutes

The present inventors extracted 90 to 100% of golden silk (GSHJ), including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with 0.5% Triton The color change after reaction for 30 and 60 minutes was confirmed with a spectrophotometer.

As a result, as shown in Figure 20, after reacting at -50°C for 15 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 365 nm (Figure 20A). After reacting at -50°C for 30 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 365 nm (Figure 20B). After reacting at -50°C for 60 minutes, a 2.5% difference in components could be distinguished in the wavelength range of 320 to 365 nm (Figure 20C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Specifically, among the measured absorbance results, the absorbance values of each sample were integrated at wavelengths from 200 to 485 nm to calculate the absorbance values, and comparisons were made between samples. In the case of the 100% GSHJ sample, the absorbance was incubated at -50°C for 15 minutes. It was 10.7 + 0.04 after 30 minutes of incubation, 10.9 + 0.10 after 60 minutes, and 11.0 + 0.03 after 60 minutes, which was 11.24% to 12.09% lower than the 97.5% red perilla sample containing 2.5% FDSP. This result shows that when determining the quality of red ginseng when the ratio of non-red ginseng is less than 2.5%, the absorbance is 9.9 to 11.5 after 15 minutes of incubation at room temperature, 10.0 to 11.8 after 30 minutes of incubation, and 10.3 after 60 minutes of incubation. It shows that it suffices to exist in the range of ~ 11.7.

Example 21. The extract of 90 to 100% of three varieties of red silkworms containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder was extracted with 0.5% Triton X-100 solution for 3 minutes at room temperature and mixed with Bradford solution. Color change after

The present inventors mixed and reacted the extract of 90 to 100% of three varieties of red silkworms, including 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder, with a 0.5% Triton The color change was confirmed with a spectrophotometer.

As a result, as shown in Figure 21, in the case of the GSHJ mixture containing 5-instar 3-day silkworm freeze-dried powder, mixtures with a difference in mixing ratio of 2.5% in the wavelength range of 560 ~ 650 nm could be distinguished (Figure 21A ). In the case of the RSHJ mixture containing 5-instar 3-day silkworm freeze-dried powder, it was possible to distinguish mixtures with differences in the mixing ratio of 2.5% in the wavelength range of 560 to 650 nm (Figure 21B). In the case of the WJHJ mixture containing 5-instar, 3-day-old silkworm freeze-dried powder, it was possible to distinguish mixtures with differences in the mixing ratio of 2.5% in the wavelength range of 560 to 650 nm (Figure 21C). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Specifically, among the measured absorbance results, the absorbance value of each sample was integrated at a wavelength from 550 to 755 nm to calculate the absorbance value, and a comparison was made between samples. In the case of the sample containing 100% of the three types of red ginseng, the absorbance was GSHJ. 1.1 + 0.06, RSHJ was 1.1 + 0.08, and WJHJ was 1.2 + 0.10, which was 27.34% to 29.22% lower than the 97.5% red jam sample including 2.5% FDSP. This result indicates that the ratio of non-red ginseng is less than 2.5%. When determining the quality of red ginseng, the absorbance ranges from 1.0 to 1.2 for GSHJ, 1.0 to 1.2 for RSHJ, and 1.1 to 1.3 for WJHJ. It shows that it is sufficient to exist in .

Example 22. Color change of extracts extracted from 90 to 100% GSHJ mixture containing 0 to 10% 5-instar, 3-day-old silkworm freeze-dried powder with various surfactants

The present inventors confirmed the color change of the extract when various other surfactants were used instead of 90 to 100% GSHJ mixture Triton

As a result, as shown in Figure 22, when the color of the 0.5% PEG200, 0.5% PEG600, 0.5% Tween 80, 0.5% LFP-50P, 0.5% Koremul-pH4, 0.5% CocoPG, 0.5% Bardap26 extract was confirmed with the naked eye. There was no clear difference depending on the mixing ratio. However, in the case of 0.5% Tween20 and 0.5% CAPB, similar to 0.5% Triton

Example 23. Color change of the extract when various detergents were used in the extract of 90 to 100% GSHJ mixture containing 0 to 10% of 5-instar, 3-day-old silkworm freeze-dried powder.

The present inventors used various surfactants instead of Triton did.

As a result, as shown in Figure 23, when 0.5% PEG200 was used, a 2.5% difference in the mixture could be distinguished at 320 to 380 nm (Figure 23A). For more accurate judgment, a prediction model was developed that uses absorbance values to calculate absorbance values by integrating the absorbance values of each sample at each wavelength, as previously confirmed, and compares between samples. Looking at the results of comparison between samples by combining the absorbance from 200 to 425 nm of the 0.5% PEG200 extract, the absorbance of the 100% GSHJ sample was 10.6 + 0.20, which was 10.39% lower than the 97.5% GSHJ sample, resulting in a ratio of non-red ginseng. If it is included in less than 2.5%, it should be in the range of 10.0 to 11.2 when determining the quality of red ginseng.

Even when 0.5% PEG600 was used, a 2.5% difference in the mixture could be distinguished between 320 and 380 nm (Figure 23B). Looking at the results of comparison between samples by combining the absorbance from 200 to 425 nm of the 0.5% PEG600 extract, the absorbance of the 100% GSHJ sample was 10.3 + 0.16, which was 7.89% lower than that of the 97.5% GSHJ sample, resulting in a ratio of non-red ginseng. If it is included in less than 2.5%, the absorbance should be in the range of 9.8 to 10.8 to determine the quality of red ginseng.

Even when 0.5% Tween 20 was used, a 2.5% difference in the mixture could be distinguished between 320 and 380 nm (Figure 23C). Looking at the results of comparison between samples by combining the absorbance from 200 to 425 nm of the 0.5% Tween 20 extract, the absorbance of the 100% GSHJ sample was 10.8 + 0.02, which was 11.59% lower than that of the 97.5% GSHJ sample, When determining the quality of red ginseng when the ratio is less than 2.5%, the absorbance should be in the range of 10.2 to 11.4.

However, when 0.5% LFP-50P was used, there was no clear difference at 320 ~ 380 nm at 2.5% (Figure 23D), but the absorbance of the 0.5% LFP-50P extract from 200 ~ 425 nm was combined to compare between samples. Looking at the results, the absorbance of the 100% GSHJ sample was 9.9 + 0.13, which is 8.51% lower than that of the 97.5% GSHJ sample. Therefore, when determining the quality of the red ginseng when the ratio of non-red ginseng is less than 2.5%, the absorbance is lower than that of the 97.5% GSHJ sample. It should exist in the range of 9.6 to 10.2.

When 0.5% Tween 80 was used, a 2.5% difference in the mixture could be distinguished between 320 and 380 nm (Figure 23E). Looking at the results of comparison between samples by combining the absorbance from 200 to 425 nm of the 0.5% Tween 80 extract, the absorbance of the 100% GSHJ sample was 10.3 + 0.16, which was 7.89% lower than that of the 97.5% GSHJ sample, In cases where the ratio is less than 2.5% and the quality of red ginseng is determined, the absorbance should be in the range of 9.9 to 10.7.

When 0.5% Koremul pH4 was used, a 2.5% difference in the mixture could be distinguished between 320 and 380 nm (Figure 23F). Looking at the results of comparison between samples by combining the absorbance from 200 to 425 nm of the 0.5% Koremul pH4 extract, the absorbance of the 100% GSHJ sample was 10.1 + 0.15, which was 16.02% lower than that of the 97.5% GSHJ sample, In cases where the ratio is less than 2.5%, when determining the quality of red ginseng, the absorbance should be in the range of 9.1 to 11.1.

When 0.5% CocoPG was used, a 10% difference could not be distinguished (Figure 23G). Looking at the results of comparison between samples by combining the absorbance from 200 to 425 nm of the 0.5% CocoPG extract, the absorbance of the 100% GSHJ sample was 8.1 + 0.10, which was 4.92% lower than that of the 97.5% GSHJ sample, resulting in a ratio of non-red ginseng. If it is included in less than 2.5%, the absorbance should be in the range of 7.9 to 8.3 to determine the quality of red ginseng.

When 0.5% Cocamidopropyl Betaine (CAPB) was used, a 2.5% difference in the mixture could be distinguished at 320 to 380 nm (Figure 23H). Although the 10% difference was completely indistinguishable when using 0.5% Bardap26 (Figure 23I). Looking at the results of comparing the samples by combining the absorbance from 200 to 425 nm of the 0.5% Cocamidopropyl Betaine extract, the absorbance of the 100% GSHJ sample was 10.0 + 0.02, which was 7.77% lower than that of the 97.5% GSHJ sample, In cases where the ratio is less than 2.5%, when determining the quality of red persimmon, the absorbance should be in the range of 9.5 to 10.5.

When 0.5% Bardap26 was used, a mixture with a difference of 2.5% could not be distinguished between 320 and 320 nm. However, when comparing the samples by adding the absorbance of the 0.5% Bardap26 extract from 200 to 425 nm, it was 100% GSHJ. In the case of the sample, the absorbance is 7.7 + 0.08, which is 2.59% lower than that of the 97.5% GSHJ sample, so when determining the quality of red ginseng in the case where the ratio of non-red ginseng is less than 2.5%, the absorbance is in the range of 7.6 to 7.8. do.

Example 24. Color change after Ninhydrin reaction in extracts using various types of surfactants in 90 to 100% GSHJ, including 0 to 10% 5-instar, 3-day-old silkworm freeze-dried powder

The present inventors used various detergents to extract 90 to 100% GSHJ, including 0 to 10% 5-instar, 3-day-old silkworm freeze-dried powder, and confirmed the color change after Ninhydrin reaction using a spectrophotometer.

As a result, as shown in Figure 24, in the case of the extract of 0.5% PEG200, the difference in the 2.5% ratio was indistinguishable from 320 to 380 nm (Figure 24A). In order to make more accurate judgments, we developed a prediction model that uses absorbance values. Looking at the results of comparing the samples by combining the absorbance from 200 to 425 nm of the 0.5% PEG200 extract that underwent ninhydrin reaction, the absorbance of the 100% GSHJ sample was 20.0 + 0.68, which was 2.61% lower than that of the 97.5% GSHJ sample. , it was found to be unsuitable for determining that the ratio of non-red sleep is less than 2.5%.

In the case of the extract of 0.5% PEG600, a difference of 2.5% ratio was discernible between 320 and 380 nm (Figure 24B). For more accurate judgment, the absorbance of the 0.5% PEG600 extract subjected to ninhydrin reaction was combined from 200 to 425 nm and compared between samples. In the case of the 100% GSHJ sample, the absorbance was 10.1 + 0.15, which was 97.5% GSHJ sample. It is 5.66% lower than that, and when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 9.9 to 10.3.

In the case of the 0.5% Tween 80 extract, a difference of 2.5% was discernible between 320 and 395 nm (Figure 24C). For more accurate judgment, the absorbance of 0.5% Tween80 extract subjected to ninhydrin reaction was combined from 200 to 425 nm and compared between samples. In the case of the 100% GSHJ sample, the absorbance was 10.2 + 0.31, which was 97.5% GSHJ sample. It is 4.94% lower than that, and when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 10.0 to 10.4.

In the case of 0.5% Koremul pH4 extract, a difference of 2.5% was discernible between 320 and 410 nm (Figure 24D). For more accurate judgment, the absorbance of 0.5% Koremul pH4 extract subjected to ninhydrin reaction was combined from 200 to 425 nm and compared between samples. In the case of the 100% GSHJ sample, the absorbance was 9.3 + 0.12, which is 97.5% GSHJ. It is 18.21% lower than the sample, so when determining the quality of red ginseng in the case where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 8.3 to 10.3.

In the case of 0.5% CocoPG extract, the difference between the 2.5% ratio could not be distinguished (Figure 24E). For more accurate judgment, the absorbance of 0.5% CocoPG extract subjected to ninhydrin reaction was combined from 200 to 425 nm and compared between samples. In the case of the 100% GSHJ sample, the absorbance was 8.5 + 0.17, compared to the 97.5% GSHJ sample. It is 3.89% lower than that, and when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 8.4 to 8.6.

In the case of 0.5% CAPB extract, a difference of 2.5% ratio was discernible between 320 and 410 nm (Figure 24F). For more accurate judgment, the absorbance of 0.5% CAPB extract subjected to ninhydrin reaction was combined from 200 to 425 nm and compared between samples. In the case of the 100% GSHJ sample, the absorbance was 9.1 + 0.10, which was 97.5% GSHJ sample. It is 8.99% lower than that, and when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 8.7 to 9.5.

In the case of 0.5% Bardap26 extract, there was no difference in ratio within 10% (Figure 24G). For more accurate judgment, the absorbance of 0.5% CAPB extract subjected to ninhydrin reaction was combined from 200 to 425 nm and compared between samples. In the case of the 100% GSHJ sample, the absorbance was 9.3 + 0.19, which was 97.5% GSHJ sample. Although it was 2.28% lower than that of , it increased to 3.82% in the 95.0% sample and was not appropriate for judging the quality of red ginseng.

Example 25. When the extract extracted with 0.5% Triton Absorbance change

The present inventors treated the extract extracted with 0.5% Triton Changes were analyzed spectrophotometrically.

As a result, as shown in Figure 25, when Chen's test was performed, a ratio difference of 2.5% could be distinguished between 320 and 425 nm (Figure 25A). For more accurate judgment, the absorbance from 200 to 485 nm was combined and compared between samples among the results of Chen's Test on 0.5% Triton This is 9.59% lower than the 97.5% GSHJ sample, so when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 5.6 to 6.2.

When treated with Cobalt's test, a ratio difference of 2.5% could be distinguished between 320 and 425 nm (Figure 25B). For more accurate judgment, the absorbance of 0.5% Triton This is 18.42% lower than the 97.5% GSHJ sample, so when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 4.0 to 4.8.

When Froehde's test was performed, a ratio difference of 2.5% could be distinguished between 320 and 515 nm (Figure 25C). For more accurate judgment, the results of Froehde's Test on 0.5% Triton This is 15.04% lower than the 97.5% GSHJ sample, so when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 7.8 to 9.6.

When treated with Lieberman's reagent, a 10% difference was not discernible (Figure 25D).

Even in the case of Mandelin's test treatment, a difference of 10% was not discernible (Figure 25E).

Mecke’s test showed a 2.5% difference between 305 and 530 nm (Figure 25F). For more accurate judgment, the absorbance from 200 to 485 nm was combined and compared between samples among the results of Mecke's Test on 0.5% Triton This is 17.50% lower than the 97.5% GSHJ sample, so when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 9.5 to 11.5.

In the case of Marguis's test, a 2.5% difference was identified between 355 and 440 nm (Figure 25G). For more accurate judgment, the absorbance from 200 to 485 nm was combined to compare the results of Marguis's Test on 0.5% Triton This is 20.26% lower than the 97.5% GSHJ sample, so when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 6.5 to 7.7.

Zwikker reagents were indistinguishable in 2.5% (Figure 25H).

Simon’s test could not distinguish a difference of 2.5% (Figure 25I).

In the case of Dille-Koppanyi reagent, a difference of 2.5% could not be distinguished (Figure 25J).

Dragondoff reagent was unable to distinguish a 5% difference (Figure 25K).

In the case of treatment with Biuret reagent, a 2.5% difference could be clearly distinguished at a wavelength of 320 to 425 nm (Figure 25L). For more accurate judgment, the absorbance of 0.5% Triton This is 10.81% lower than the 97.5% GSHJ sample, so when determining the quality of red ginseng in cases where the ratio of non-red ginseng is less than 2.5%, the absorbance should be in the range of 4.4 to 4.6.

Therefore, if non-red ginseng is included at less than 2.5% as the standard for determining the ratio of red ginseng, Triton After processing, graph analysis and absorbance analysis can be performed using a spectrophotometer.

In other words, it can be seen that the spectrum between freeze-dried powder (non-hongjam) from 5-instar 3-day silkworms and red-jam powder (red-jam) prepared from deep sleep after 5-instar 7 days shows a difference in absorbance in a specific wavelength range, and the difference in absorbance is 200 ~ It was confirmed that an accurate measurement can be made by combining the absorbance up to 485 nm.

In other words, the method for determining the level of mixing of red persimmon and non-red persimmon using a UV/VIS spectrophotometer of the present invention collects and analyzes the absorption spectrum of commercially available red persimmon, in which it is difficult to determine the level of mixing of red persimmon and non-red persimmon, in a specific sample area, quickly and accurately. It was confirmed that it was possible to determine the level of mixing with non-hongjam.

As above, specific parts of the present invention have been described in detail, and those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A method for determining the presence or absence of non-red ginseng in red ginseng using ultraviolet-visible spectroscopy, comprising the following steps:
(a) Preparing a 100% red persimmon sample as the target sample and comparative control group to be determined;
(b) preparing an extract by extracting each sample with one or more selected from the group consisting of 80 to 100% (v/v) methanol, 80% (v/v) ethanol, and Triton X-100;
(c) measuring the absorbance of the supernatant of each extract using a UV-VIS spectrophotometer; and
(d) Among the measured absorbance results, the absorbance value of each sample is integrated at a wavelength of 200 to 425 nm, a wavelength of 200 to 485 nm, or a wavelength of 550 to 755 nm to calculate the sum of absorbance, and if there is a difference, the sum of absorbance is calculated. A step of determining that the target sample is mixed.
According to paragraph 1,
When the extraction in step (b) is performed using 80% (v/v) methanol or ethanol, the determination in step (d) is performed at a wavelength of 200 to 425 nm.
According to paragraph 1,
When the extraction in step (b) is performed using 100% methanol, the determination in step (d) is performed at a wavelength of 200 to 485 nm.
According to paragraph 1,
When the extraction in step (b) is performed using Triton
According to paragraph 4,
The extraction in step (b) further includes reacting the extract extracted using Triton X-100 with Triton X-100 at -50°C to 90°C for 15 to 60 minutes.
According to paragraph 1,
The method wherein the extraction is performed at room temperature for 2 to 10 minutes.
According to paragraph 1,
The Hongjam is made by boiling raw silkworms with silk protein selected from silkworms ranging from four-faced silkworms of 5 instars and 4 days to deep sleep by placing them in water at a temperature of 55 to 125 ° C. for 7 minutes to 30 hours or cooking them with the heat of steam. Manufactured, method.
According to paragraph 1,
The method wherein the red jam is hot air dried or vacuum freeze dried.
According to paragraph 1,
The method wherein the silkworm is a silkworm breed selected from the group consisting of white hyacinth, golden silk, and orange silkworm.
According to paragraph 1,
The above non-hongjam is a 5-year-old 3-day silkworm, method.
According to paragraph 1,
The method is to determine the inclusion of less than 2.5% weight of non-red ginseng in the sample.
A method for determining the presence or absence of non-red ginseng in red ginseng using ultraviolet-visible spectroscopy, comprising the following steps:
(a) Preparing a 100% red persimmon sample as the target sample and comparative control group to be determined;
(b) treating each sample with one type of surfactant selected from the group consisting of PEG200, PEG600, Tween 20, Tween 80, LFP-50P, Koremul pH4, CocoPG, CAPB, and Bardap26;
(c) measuring the absorbance of each sample treated with the surfactant using a UV-VIS spectrophotometer; and
(d) Among the measured absorbance results, calculate the sum of absorbance by integrating the absorbance value of each sample at 200 ~ 425 nm wavelength, 200 ~ 485 nm wavelength, or 550 ~ 755 nm wavelength, compare them, and if there is a difference, A step of determining that the target sample is mixed.
According to clause 12,
A method comprising: additionally performing a ninhydrin color reaction after step (b).