WO2016204350A1

WO2016204350A1 - Method for measurement of preharvest sprouting damaged grain using any one or more of the total dissolved solids and electric conductivity in wheat

Info

Publication number: WO2016204350A1
Application number: PCT/KR2015/011139
Authority: WO
Inventors: Yu-Young Lee; Dae-Wook Kim; Sun Lim Kim; Mi-Jung Kim; Yul-Ho KIM; Young-Up Kwon; Hyang-Mi PARK; Choon Woo Lee; Byung Joo Kim; Wook-Han KIM
Original assignee: Republic Of Korea(Management : Rural Development Administration)
Priority date: 2015-06-16
Filing date: 2015-10-21
Publication date: 2016-12-22

Abstract

Provided is a method for determining preharvest sprouting in wheat by measuring at least one selected from total dissolved solids and electric conductivity, and the method of determining preharvest sprouting in wheat of the present invention can not only determine the early stage of preharvest sprouting of wheat, which is difficult to determine by the naked eye during the progress of the preharvest sprouting of wheat, by measuring total dissolved solids and electric conductivity of a wheat seed, but also can determine the presence of the preharvest sprouting seeds among the wheat seeds, and thus the method can be effectively used for quality control of wheat, harvested in areas with the risk of preharvest sprouting, and can also contribute to the growth of domestic industry.

Description

METHOD FOR MEASUREMENT OF PREHARVEST SPROUTING DAMAGED GRAIN USING ANY ONE OR MORE OF THE TOTAL DISSOLVED SOLIDS AND ELECTRIC CONDUCTIVITY IN WHEAT

The present invention disclosed herein relates to a method for determining preharvest sprouting in wheat using at least one selected from total dissolved solids and electric conductivity, and more particularly, to a method for determining non-germinated seed in appearance, which is an early stage of preharvest sprouting seed, and preharvest sprouting seed in wheat, which are difficult to determine by the naked eye during the progress of the preharvest sprouting of wheat, by measuring at least one selected from total dissolved solids and electric conductivity.

The maturity period of wheat cultivated in Korea overlaps the rainy season every two or three years and thus a preharvest sprouting, a phenomenon that a seed is germinated while it is hung on the wheat grain, occurs, which considerably deteriorates the quality and utilization of wheat. The recent abnormal weather phenomena have been causing a change in the rainfall pattern during the maturity period of wheat, and thus there has been an urgent need for the development of a technology to reduce the damages due to the preharvest sprouting reflecting the environmental change described above. However, it is difficult to determine the early stage of the preharvest sprouting by the naked eye and thus a mixture between normal wheat and the preharvest sprouting could result in quality deterioration and thus there is a need for the development of a technology to determine the preharvest sprouting.

To this end, studies have been performed to induce preharvest sprouting during the actual wheat cultivation to thereby verify the physiological changes in wheat seed reacting to the preharvest sprouting, and verify the difference in resistance to the preharvest sprouting among genetic sources based on the same (The Korean Society of Crop Science, Journal of Crop Science and Biotechnology, Oct. 2012, 113-113), and physicochemical characteristics of wheat according to the degree of preharvest sprouting occurrence have been analyzed in order to reduce the damages by the preharvest sprouting of wheat and promote its utilization (the Korean Society of Crop Science, Journal of Crop Science and Biotechnology, Oct. 2012, 182-182).

At present, since the seeds with preharvest sprouting have ?-amylase activity, an enzymatic method using the same has been studied, but this method has a limitation in that there is a large variation with a very small value. Additionally, a falling number method employing the difference in amylolysis by ?-amylase has been used, but this method has a limitation in that it requires time and effort for measurement and a relevant device, and thus there is a need for the development of a method for simple measurement of the degree of preharvest sprouting.

Meanwhile, electric conductivity can be served as an index to determine the concentration of salts dissolved to be conductive for electricity using the principle in which the presence of dissociated ions in water can generate electricity, and electric conductivity is based on the inverse number of electric resistance measured in water temperature of 25°C, generally expressed as an abbreviated form, EC, and uses dS/m as its unit. In the Notice 2003-1 by the Korea Seed & Variety Service, there is described a method for measuring electric conductivity for determining seed viability, and a research report on the measurement of electric conductivity for confirming the relationship between of seed absorptivity of collected variety of bean and seed viability was previously also reported (KIM, Jina, Master’s Thesis, Gyeongsang National University, 2006).

Additionally, total dissolved solids (TDS) is also called dissolved solids, and refers to the total amount of organic materials such as lipids and amino acids dissolved and inorganic materials such as bases, metals, and ions, dissolved in water.

According to the method of measuring electric conductivity for verification of seed viability, the seeds which release many electrolytes are considered as having a low viability because they exhibit high leachate conductivity, whereas the seeds which release a few electrolytes (low conductivity) are considered as having high viability, thus indicating the lower the electric conductivity the higher the seed viability. However, there has been no report regarding the measured result of electric conductivity for determining preharvest sprouting of wheat.

The total dissolved solids and electric conductivity are currently used as an approved method for measuring seed viability (Notice 2003-1, the Korea Seed & Variety Service). However, the method determines as, the lower the total dissolved solids and electric conductivity the higher the seed viability, and there has been no report regarding a method for a simultaneous measurement and analysis of total dissolved solids and electric conductivity for determining the level of preharvest sprouting of wheat.

Accordingly, the present inventors, while endeavoring to reduce damages caused by preharvest sprouting of wheat by determining the early stage of preharvest sprouting, which is difficult to determine by the naked eye, and improve the quality of normal wheat, discovered that the total dissolved solids and electric conductivity of the normal seeds, non-germinated seeds in appearance, and preharvest sprouting seeds were different, thereby completing the present invention.

In order to solve the problems described above, the present invention provides a method for determining preharvest sprouting of wheat by a simultaneous measurement of total dissolved solids and electric conductivity, a device measuring the preharvest sprouting of wheat using the above method, or a kit including the method for determining preharvest sprouting of wheat as a manual.

The present invention provides a method for determining preharvest sprouting in wheat by measuring at least one selected from total dissolved solids and electric conductivity.

According to an exemplary embodiment of the present invention, the method for determining preharvest sprouting in wheat may include (a) measuring at least one selected from total dissolved solids and electric conductivity of a wheat seed sample for determining preharvest sprouting; and (b) determining the preharvest sprouting by comparing with the value of at least one selected from total dissolved solids and electric conductivity of a normal wheat seed.

According to another exemplary embodiment of the present invention, in step (a), at least one selected from total dissolved solids and electric conductivity of the wheat seed sample can be measured for a wheat seed sample, which is to be determined regarding the preharvest sprouting, while immersing the wheat seed sample in water for 0 to 40 hours by adding water thereto.

According to still another exemplary embodiment of the present invention, in step (b), based on Equation 1 and Equation 2 below, when at least one result value of Equation 1 and Equation 2 is between 0.96 and 1.05, it can be determined that the wheat seed is a normal wheat seed; and when at least one result value of Equation 1 and Equation 2 is between 0.01 and 0.95 or between 1.06 and 5.0, it can be determined that the preharvest sprouting was proceeded.

[Equation 1]

Result Value = total dissolved solids of a wheat seed sample for determining preharvest sprouting/ total dissolved solids of a normal wheat seed sample

[Equation 2]

Result Value = electric conductivity of a wheat seed sample for determining preharvest sprouting /electric conductivity of a normal wheat seed sample.

According to still another exemplary embodiment of the present invention, when at least one of the result values of Equation 1 and Equation 2 is in the range between 0.01 and 0.95, the seed can be determined as a non-germinated seed in appearance (early stage of preharvest sprouting), whereas when at least one of the result values of Equation 1 and Equation 2 is in the range between 1.06 and 5.0, the seed can be determined as a preharvest sprouting seed.

According to still another exemplary embodiment of the present invention, the wheat seed may be a Keumkang wheat seed.

The present invention also provides a device for measuring the wheat preharvest sprouting by the above method, or a kit including the above method for determining the wheat preharvest sprouting as a manual.

Furthermore, the present invention also provides a method for determining the mixed presence of the wheat preharvest sprouting seeds by measuring at least one selected from total dissolved solids and electric conductivity.

According to an exemplary embodiment of the present invention, the method for determining the mixed presence of the wheat preharvest sprouting seeds may include (a) measuring at least one selected from total dissolved solids and electric conductivity of a wheat seed sample for determining the presence of a mixture of the preharvest sprouting; and (b) determining the presence of a mixture of the preharvest sprouting by comparing with the value of at least one selected from total dissolved solids and electric conductivity of a group with 100 wt% normal wheat seeds.

According to another exemplary embodiment of the present invention, in step (a), total dissolved solids and electric conductivity can be measured for the wheat seed sample, which is to be determined regarding the presence of a mixture of the preharvest sprouting, while immersing the wheat seed sample for 0 hour to 40 hours by adding water thereto.

According to another exemplary embodiment of the present invention, in step (b), based on Equation 3 and Equation 4 below, when at least one result value of Equation 3 and Equation 4 is between 0.96 and 1.05, it may be determined that the wheat seeds are of 100 wt% normal wheat seeds; and when at least one result value of Equation 3 and Equation 4 is between 0.01 and 0.95 or between 1.06 and 5.0, it may be determined that the preharvest sprouting seeds are included among the normal wheat seeds.

[Equation 3]

Result Value = total dissolved solids of a group of wheat seeds for determining the presence of a mixture of preharvest sprouting seeds/total dissolved solids of a group with 100 wt% normal wheat seeds,

[Equation 4]

Result Value = electric conductivity of a group with wheat seeds for determining the presence of a mixture of preharvest sprouting seeds /electric conductivity of a group with 100 wt% of normal wheat seeds.

According to another exemplary embodiment of the present invention, when at least one result value of Equation 3 and Equation 4 is between 0.01 and 0.95 the seeds may be determined as a mixture between 10 wt% to 90 wt% of non-germinated seeds in appearance and normal wheat seeds, whereas when at least one result value of Equation 3 is between 1.06 and 5.0 the seeds may be determined as a mixture between 10 wt% to 90 wt% of early preharvest sprouting seeds and normal wheat seeds.

According to another exemplary embodiment of the present invention, the wheat seed may be a Keumkang wheat seed.

The method for determining preharvest sprouting in wheat of the present invention can not only determine the early stage of preharvest sprouting of wheat, which is difficult to determine by the naked eye during the progress of the preharvest sprouting of wheat, by measuring at least one of total dissolved solids and electric conductivity of a wheat seed, but also can determine whether preharvest sprouting seeds are included among the wheat seeds, and thus the method can be effectively used for quality control of wheat harvested in areas with the risk of preharvest sprouting and can also contribute to the growth of domestic industry.

The accompanying drawings are included to provide a further understanding of the present invention, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present invention and, together with the description, serve to explain principles of the present invention. In the drawings:

FIG. 1 is a picture showing normal seeds, seeds with early stage of preharvest sprouting, and seed with preharvest sprouting;

FIG. 2 is a data graph showing the measured values of ?-amylase according to the degree of progress of the preharvest sprouting;

FIG. 3 shows data graphs illustrating the measured values of total dissolved solids (a) and electric conductivity (b) according to the degree of damages of the preharvest sprouting;

FIG. 4 shows water absorption rate(a) and hardness values(b) of wheat seeds according to the degree of damages of the preharvest sprouting;

FIG. 5 shows images of starch structures of wheat seeds according to the degree of damages by the normal(a), non-germinated seed in appearance(b) and preharvest sprouting(c) observed under scanning electron microscope (SEM); and

FIG. 6 is a data graph showing the measured values of total dissolved solids according to the mixed ratio of wheat preharvest sprouting seeds.

Preferred embodiments of the present invention will be described below in more detail with reference to the accompanying drawings. The present invention may, however, be embodied in different forms and should not be constructed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.

As described above, the presence of preharvest sprouting in wheat has been determined by a method for measuring ?-amylase activity or Falling number (FN), however, these methods have limitation due to the requirement of time, effort, and equipments for the measurement, and thus there is a need for the development of a method for a convenient measurement of the degree of preharvest sprouting.

The present inventors attempted to solve the limitations described above by providing a method for determining preharvest sprouting of wheat via measurements of at least one of total dissolved solids and electric conductivity of wheat seeds. The method not only enables to determine the early stage of preharvest sprouting of wheat, which is difficult to determine by the naked eye during the progress of the preharvest sprouting of wheat, by measuring at least one of total dissolved solids and electric conductivity of wheat seeds, but also enables to determine whether preharvest sprouting seeds are included among the wheat seeds, and thus the method can be effectively used for quality control of wheat harvested in areas with the risk of preharvest sprouting.

Accordingly, the present invention provides a method for determining preharvest sprouting in wheat by measurements of at least one of total dissolved solids and electric conductivity.

As used herein, the term “'preharvest sprouting” refers to a sprouting from ears, which are at a stage near maturity, when the ears are placed in a wet state for a long period of time due to lodging or rainfall, and the grains with preharvest sprouting are not suitable for breeding or foods.

The methods for measurement using “total dissolved solids” or “'electric conductivity” are used for verification of seed vigor. The seeds with low seed viability (the germination vigor of seeds and growth vigor of seedling) undergo deterioration during storage and their cell membranes become destroyed and decomposed due to mechanical injuries. Therefore, when seeds with a poor structure of cell membranes are immersed in water, the contents become leached out, and their total dissolved solids or electric conductivity is measured by these methods.

In the present invention, the wheat species may preferably be a Keumkang wheat but is not limited thereto, and various species may be used.

The method of the present invention for determining preharvest sprouting in wheat may be characterized by including:

(a) measuring at least one selected from total dissolved solids and electric conductivity of a wheat seed sample for determining preharvest sprouting; and

(b) determining the preharvest sprouting by comparing with the value of at least one selected from total dissolved solids and electric conductivity of a normal wheat seed.

Specifically, step (a) of the above method of the present invention may be characterized by measuring at least one of total dissolved solids and electric conductivity of a wheat seed sample, which is to be determined for its preharvest sprouting, while adding water thereto for from 0 hour to 40 hours to immerse the wheat seed sample, preferably from 10 minutes to 12 hours, and more preferably from 1 hour to 12 hours. When the wheat seed sample is immersed longer than the above time period, normal wheat seeds will germinate by water and thus it will be difficult to determine the presence of preharvest sprouting by the measurements of total dissolved solids and electric conductivity.

Additionally, when step (b) is based on Equation 1 and Equation 2 below, when both result values of Equation 1 and Equation 2 lie between 0.96 and 1.05, it is determined that the preharvest sprouting is already proceeded.

[Equation 1]

[Equation 2]

Result Value = electric conductivity of a wheat seed sample for determining preharvest sprouting /electric conductivity of a normal wheat seed sample

It is characterized in that when at least one of the result values of Equation 1 and Equation 2 is between 0.01 and 0.95, the seed is determined as a non-germinated seed (at the early stage of preharvest sprouting), whereas, when at least one of the result values of Equation 1 and Equation 2 is between1.06 and 5.0, the seed is determined as a preharvest sprouting seed.

The normal wheat seeds used in the above determining method may be general seeds, which were confirmed that no preharvest sprouting occurred therein, that have been confirmed as normal seeds by the preharvest sprouting determining method, or those seeds which have been stored in conditions when no preharvest sprouting occurred therein, and those wheat seeds which can be compared for the purpose of determining preharvest sprouting may be used.

In an exemplary embodiment of the present invention, for the analysis of wheat characteristics according to the progress level of preharvest sprouting, an artificial rainfall was applied during the physiological maturity period of wheat for 4 days using an assembled preharvest sprouting-inducing house. As shown in FIG. 1, the seeds were classified into normal seeds (control), type I (early stage of preharvest sprouting; non-germinated seeds in appearance), and type II (preharvest sprouting seeds).

When the length, width, thickness, thousand grain weight, and the color differences including brightness, redness, and yellowness were observed for the understanding of wheat characteristics, it was confirmed that there was no difference or only a negligible difference between normal seeds and the seeds with preharvest sprouting except the redness, as shown in Table 1.

The non-germinated seeds in appearance correspond to those at early stage of preharvest sprouting, and they may deteriorate the quality of wheat, due to the difficulty in distinguishing the seeds having the progress of preharvest sprouting from normal wheat seeds, and thus for differentiation between the normal wheat seeds and the seeds having the progress of preharvest sprouting, α-amylase activity measurement method or a falling number (FN) measurement method should be used.

As shown in FIG. 2, when the α-amylase activity was measured according to the progress of preharvest sprouting, the wheat seeds having the progress of preharvest sprouting showed an increase in their α-amylase activity compared to that of the normal wheat seeds. However, there was a significant range of error and it was thus difficult to distinguish the real normal wheat seeds from the non-germinated wheat seeds in appearance.

In another exemplary embodiment of the present invention, at least any one of total dissolved solids and electric conductivity was measured for normal wheat seeds, non-germinated wheat seeds in appearance, and preharvest sprouting seeds, which were classified according to the degree of damages of the preharvest sprouting, after immersing them in water for 0 hour to 12 hours. As a result, it was confirmed that the longer the dissolution time the higher the value of Total dissolved solids, as shown in FIG. 3A. As shown in Table 2, the value of Total dissolved solids, after 12 hours of immersion, was 72.0 ppm for the normal seeds, 143.5 ppm for the seeds with preharvest sprouting, and 41.5 ppm for the non-germinated wheat seeds in appearance.

Additionally, as shown in FIG. 3B, the longer the elution time the higher the value of electric conductivity. As shown in Table 3, the value of electric conductivity, at 12 hours of immersion, was 110±0.2 μs/cm for the normal seeds, 256.7±5.8 μs/cm for the seeds with preharvest sprouting, and 53.3±0.1 μs/cm for the non-germinated wheat seeds in appearance.

In the present invention, Equation 1 and Equation 2 were derived based on the results of FIGS. 3A and 3B. When the values of the total dissolved solids according to each immersion time of the wheat seeds, which are to be determined for the presence of preharvest sprouting, were applied to Equation 1, based on the values of the total dissolved solids for the normal wheat seeds as the control group, it may be determined that the seeds are normal wheat seeds if the result value is between 0.96 and 1.05, whereas preharvest sprouting has already occurred to the seeds if the result value is between 0.01 and 0.95 or between 1.06 and 5.0 (Table 4).

When the values of the electric conductivity according to each immersion time of the wheat seeds, which are to be determined for the presence of preharvest sprouting, were applied to Equation 2, based on the values of the total dissolved solids for the normal wheat seeds as the control group, it may be determined that the seeds are normal wheat seeds if the result value is between 0.96 and 1.05, whereas preharvest sprouting has already occurred to the seeds if the result value is between 0.01 and 0.95 or between 1.06 and 5.0 (Table 5).

That is, the seeds may be determined as normal wheat seeds when both result values by Equation 1 and Equation 2 lie between 0.96 and 1.05, whereas the seeds may be determined that they are not normal wheat seeds but may have undergone preharvest sprouting when none of the result values by Equation 1 and Equation 2 lie between the above range.

In particular, when at least one of the result values of Equation 1 and Equation 2 is between 0.01 and 0.95, the seeds may be determined as non-germinated wheat seeds in appearance (at early stage of preharvest sprouting), whereas when at least one of the result values of Equation 1 and Equation 2 is between 1.06 and 5.0, the seeds may be determined as preharvest sprouting seeds.

Comparing the above result values, it was confirmed that at least one of the values of total dissolved solids and electric conductivity for the non-germinated wheat seeds in appearance was measured in overall as low compared to that of the normal wheat seeds, and this was confirmed to be different from the determining criteria for the total dissolved solids and electric conductivity measurements of verification of seed viability. That is, when wheat seeds are selected based on the existing criteria for verification of seed viability, the non-germinated seed in appearance, i.e., at early stage of preharvest sprouting, were measured to be higher in viability than that of the normal wheat seeds, and may thus mislead the determination as that the viability of the normal wheat seeds may be lower than that of the non-germinated seed in appearance, and there may be a problem that the non-germinated seed in appearance may be mixed with the normal wheat seeds thereby deteriorating the quality of the wheat seeds.

However, under the conditions established in the present invention, the seeds that have undergone preharvest sprouting can be distinguished among the wheat seeds, with ease and accuracy.

Additionally, FIG. 4 shows the measured values of water absorption rate and hardness of wheat seeds according to the degree of preharvest sprouting, and it was confirmed that the water absorption rate at room temperature of the non-germinated seed in appearance was lower than that of the normal seeds, whereas the hardness of the non-germinated seed in appearance was significantly higher than that of the normal seeds.

FIG. 5 shows images of starch structures of wheat seeds according to the degree of damages by the preharvest sprouting observed under scanning electron microscope (SEM). Although the materials in the body were depleted by the respiration of the non-germinated seeds in the case of the non-germinated seeds in appearance, there were observed structures starch became agglutinated and cell walls were hardened as they were dried again, and it was speculated from the above that, at the time of measuring the electric conductivity of the non-germinated seeds in appearance, the internal materials are dissolved in an amount less than that of the normal seeds.

The present invention also provides a method for determining the mixed presence of the preharvest sprouting wheat seeds by at least one measurement of the total dissolved solids and electric conductivity.

The method for determining the mixed presence of the preharvest sprouting wheat seeds may be characterized by including:

(b) determining the mixed presence of preharvest sprouting seeds by comparing with the value of at least one selected from total dissolved solids and electric conductivity of a group with 100 wt% normal wheat seeds.

Specifically, step (a) of the above method of the present invention may be characterized by measuring at least one of total dissolved solids and electric conductivity of a wheat seed sample, which is to be determined for its preharvest sprouting, while adding water thereto for from 0 hour to 40 hours to immerse the wheat seed sample, preferably from 10 minutes to 12 hours, and more preferably from 1 hour to 4 hours. When the wheat seed sample is immersed longer than the above time period, normal wheat seeds will germinate by water and thus it will be difficult to determine the presence of preharvest sprouting by the measurements of total dissolved solids and electric conductivity.

Additionally, in step (b), based on Equation 3 and Equation 4 below, when at least one result value of Equation 3 and Equation 4 is between 0.96 and 1.05, it may be determined that the wheat seeds are of 100 wt% normal wheat seeds; and when at least one result value of Equation 3 and Equation 4 is between 0.01 and 0.95 or between 1.06 and 5.0, it may be determined that the preharvest sprouting seeds are included among the normal wheat seeds.

[Equation 3]

Result Value = total dissolved solids of a group of wheat seeds for determining the presence of a mixture of preharvest sprouting seeds/total dissolved solids of a group with 100 wt% normal wheat seeds.

[Equation 4]

That is, when both of the result values by Equation 3 and Equation 4 are in the range of between 0.96 and 1.05, the seeds may be determined as 100 wt% of normal wheat seeds, whereas when neither of the result values by Equation 3 and Equation 4 are in the above range the seeds may be determined as those where preharvest sprouting seeds are mixed with the normal wheat seeds.

In particular, when at least one of the result values by Equation 3 and Equation 4 is in the range of between 0.01 and 0.95, the seeds may be determined as those where the non-germinated wheat seeds in appearance are mixed with the normal wheat seeds, whereas when at least one of the result values by Equation 3 and Equation 4 is in the range of between 1.06 and 5.0, the seeds may be determined as those where 10% to 90% of the preharvest sprouting seeds are mixed with the normal wheat seeds.

The above method for determining the mixed presence of the wheat seeds with preharvest sprouting may be performed in the same manner as in determining the preharvest sprouting in wheat. In an exemplary embodiment of the present invention, the total dissolved solids and electric conductivity were measured by mixing 10% to 90% of the seeds according to the degree of damages by the preharvest sprouting with the normal wheat seeds, and for comparison with the existing method, falling number and α-amylase activity were measured.

As shown in Table 6, it was confirmed that the falling number was lowered and α-amylase activity was increased depending on the mixed presence of the preharvest sprouting, whereas there was no correlation observed according to the mixed ration when the type II preharvest sprouting seeds were mixed with the normal seeds.

In contrast, when measured using the total dissolved solids and electric conductivity according to the method of the present invention, as shown in Table 7 and FIG. 6, it was confirmed that the measured values of the total dissolved solids decreased or increased depending on the mixed presence of the preharvest sprouting. Additionally, it was confirmed that when type I non-germinated seeds were mixed with the normal seeds, the measured values of the total dissolved solids decreased as the mixed ratio increased compared to that of the normal seeds, whereas when type II preharvest sprouting seeds were mixed with the normal seeds the measured values of the total dissolved solids decreased as the mixed ratio increased compared to that of the normal seeds.

Additionally, as shown in Table 8, it was confirmed that the values of electric conductivity, as in the case of the values of total dissolved solids, decreased or increased depending on the presence of the preharvest sprouting seeds, and when type I were mixed with the normal seeds the electric conductivity became lower than that of the normal seeds as the mixed ratio increased, whereas when type II preharvest sprouting seeds were mixed with the normal seeds the electric conductivity increased compared to that of the normal seeds as the mixed ratio increased.

When the values in Table 7 and Table 8 were applied to the Equation 3 and Equation 4, it was confirmed that when both result values of Equation 3 and Equation 4 were in the range of between 0.96 and 1.05 the seeds could be determined as 100 wt% of normal wheat seeds, whereas when the result values lied between 0.01 and 0.95 or between 1.06 and 5.0 the seeds could be determined as a mixture containing the preharvest sprouting seeds. In particular, it was confirmed that when the result values lied between 1.06 and 5.0 the seeds could be determined as having a mixture of normal wheat seeds and the preharvest sprouting seeds.

That is, it was confirmed that the method for determining the preharvest sprouting in wheat according to the present invention can easily distinguish between the wheat seeds at early stage of preharvest sprouting (non-germinated seeds in appearance) and the normal wheat seeds which are difficult to determine by the naked eye during the progress of the preharvest sprouting of wheat, by measuring total dissolved solids and electric conductivity of a wheat seed, and also can simply determine whether preharvest sprouting seeds are included among the normal wheat seeds using the method.

Furthermore, the present invention provides a device for measuring preharvest sprouting of wheat using the above method or a device for determining the presence of preharvest sprouting seeds of wheat.

Additionally, the present invention provides a kit including the above determining method as a manual.

[Example 1] Induction of preharvest sprouting in wheat and physical appearance of wheat seeds according to the progress of preharvest sprouting

1-1: Induction of preharvest sprouting in wheat

In the present invention, an induction of preharvest sprouting in wheat was performed in order to characterize the wheat seeds according to the progress of preharvest sprouting in wheat.

First, the experiments were performed in experimental field for wheat in the National Institute of Crop Science of Rural Development Administration (located in Suwon, Gyeonggi Province of Korea), between October 2013 and June 2014, and the experimental plot of wheat was sown 14 kg per 10a, and the wheats were grown using the fertilizers with the entire amounts of phosphoric acid and potassium as basal fertilizers (fertilizers provided before sowing wheat seeds) based on the standard amount of fertiilizer12 kg of N, 9 kg of P2O5, and 7 kg of K2O per 10a, and the experimental plots were performed via three repeats of completely randomized design. The Keumkang wheat, grown in the experimental plot, was provided with 63 mm of water daily in an assembled preharvest sprouting-inducing house (area: 28.5 m2/section, and height: 2.5m) using a spring cooler installed inside the preharvest sprouting house, the front side of which was shielded, starting from the 35th day from the wheat flowering for 4 days. During the period of inducing preharvest sprouting, the temperature inside the house was 23°C on average, varying within the range of from the lowest of 19°C to the highest of 26°C, and the relative humidity from the lowest of 79% to the highest of 94%. These conditions were very similar to the environmental conditions in late June in the central region of Korea during the period, which is the period when the preharvest sprouting of wheat occurs, in terms of daily average temperature and humidity during rainfall, which were about 22°C and 80%, respectively (between 1982 and 2011, Korea Meteorological Administration).

Then, the wheat seeds were classified into normal seeds (control), type I (seeds at early stage of preharvest sprouting; non-germinated seeds in appearance), and type II (seeds with preharvest sprouting) according to the progress level of preharvest sprouting (FIG. 1).

1-2: Characteristics of physical appearance in wheat according to the degree of preharvest sprouting

In order to study the characteristics of physical appearance of wheat, classified as in Example 1-1, according to the degree of preharvest sprouting, wheat grains were observed regarding their length, width, thickness, thousand grain weight, and color differences including brightness (value L), redness (value a), and yellowness (value b).

The length-to-width ratio was measured using a caliper (CD-15CP, Mitutoyo Corp., Japan), and for the thousand grain weight, the average value was calculated after measuring the weight of 1,000 grains.

For the color difference, brightness, redness, and yellowness were measured using a spectrophotometer (CM-3500d, Minolta, Japan), and the experiments were performed by repeating the experiment three times and the results were expressed as mean ± standard error. Table 1 means characteristics of physical appearances of wheat grains according to the degree of preharvest sprouting.

As a result, it was confirmed that there was no difference or negligible difference between the normal seeds and the seeds with the progress of preharvest sprouting except for redness, as shown in Table 1.

[Example 2] Measurement of α-amylase activity of wheat seeds according to the progress level of preharvest sprouting

In the present invention, in order to study whether it is possible to distinguish between different types of preharvest sprouting in wheat induced in Example 1-1 by the method for determining the preharvest sprouting in wheat, the α-amylase activity according to the degree of preharvest sprouting in wheat was measured.

First, the wheat seeds having induced preharvest sprouting induced in Example 1-1 was pulverized by a grain mill (Quadrumat Junior, Brabender GmbH & Co., Germany), passed through a 0.5 mm sieve, and 3 g of the milled wheat was subjected to the measurement of α-amylase activity using an α-amylase kit (Ceralpha Method, Megazyme international, Ireland).

As a result, as shown in FIG. 2, it was confirmed that the α-amylase activity of the wheat seeds with the progress of preharvest sprouting increased compared to that of the normal wheat seeds, however, due to the significant large error range, it was difficult to distinguish between the real normal wheat seeds and the non-germinated wheat seeds in appearance.

[Example 3] Measurement of total dissolved solids and electric conductivity of wheat seeds according to the progress level of preharvest sprouting

3-1: Measurement of total dissolved solids

For determination according to the progress level of preharvest sprouting in wheat, the total dissolved solids of the wheat seeds according to the types of preharvest sprouting of the wheat seeds induced in Example 1-1 was measured.

First, 5 g each of normal seeds (control), type I (early stage of preharvest sprouting; non-germinated seeds in appearance), and type II (seeds with preharvest sprouting) was prepared, added with 50 mL of distilled water and immersed for 12 hours, and their total dissolved solids were measured using a device for measuring total dissolved solids (Trans Pen-type TDS Tester, Korea) according to time zone, respectively. Table 2 means total dissolved solids according to the immersion time based on the degree of preharvest sprouting.

As shown in FIG. 3A and Table 2, as the dissolution time increased the values of total dissolved solids continued to increase, and in particular, at 12 hours of immersion, the values of Total dissolved solids were 63.7±1.2 μs/cm for the normal seeds, 138.3±3.1 μs/cm for the seeds with preharvest sprouting, and 35.0±1.0 μs/cm for the non-germinated wheat seeds in appearance.

3-2: Measurement of electric conductivity

For determination according to the progress level of preharvest sprouting in wheat, the electric conductivity of the wheat seeds according to the types of preharvest sprouting of the wheat seeds induced in Example 1-1 was measured.

First, 5 g each of normal seeds (control), type I (early stage of preharvest sprouting; non-germinated seeds in appearance), and type II (seeds with preharvest sprouting) was prepared, added with 50 mL of distilled water and immersed for 12 hours, and their total dissolved solids were measured using a device for measuring electric conductivity (Trans Pen-type Conductivity Tester, Korea) according to time zone, respectively. Table 3 means electric conductivity according to the immersion time based on the degree of preharvest sprouting.

As shown in FIG. 3B and Table 3, as the dissolution time increased the values of electric conductivity continued to increase, and in particular, at 12 hours of immersion, the values of electric conductivity were 110±0.2 μs/cm for the normal seeds, 256.7±5.8 μs/cm for the seeds with preharvest sprouting, and 53.3±0.1 μs/cm for the non-germinated wheat seeds in appearance.

3-3 : Equation derivation

Equation 1 and Equation 2 were derived based on the results of Table 2 and FIG. 3 above.

[Equation 1]

Result Value = total dissolved solids of a wheat seed sample for determining preharvest sprouting/total dissolved solids of a normal wheat seed sample.

[Equation 2]

Result Value = electric conductivity of a wheat seed sample for determining preharvest sprouting/electric conductivity of a normal wheat seed sample. Table 4 means comparison of result values on Equation 1, and table 5 means comparison of result values on Equation 2.

It was confirmed that when both result values by Equation 1 and Equation 2 lie between in the range of 0.96 and 1.05, the seeds may be determined as normal wheat seeds, whereas when both result values do not belong to the above range the seeds may be determined that they are not normal wheat seeds and preharvest sprouting might have been already proceeded therein.

In particular, when at least any one of the result values of Equation 1 and Equation 2 is in the range between 0.01 and 0.95, the seeds may be determined as the non-germinated wheat seeds in appearance (early stage of preharvest sprouting), whereas when at least any one of the result values of Equation 1 and Equation 2 is in the range between 1.06 and 5.0 the seeds may be determined as the seeds with the progress of preharvest sprouting.

[Example 4] Measurement of water absorption rate and hardness according to the degree of preharvest sprouting in wheat seeds

In the present invention, in order to confirm the correlation between the values of total dissolved solids and electric conductivity according to the degree of preharvest sprouting, the water absorption rate of wheat seeds was measured. For the measurement of the water absorption rate of wheat seeds at room temperature, 10 mL of distilled water was added into a test tube containing 2 g of wheat seeds and then the test tube was immediately placed into a homeostat at 21°C. The sample was taken out and water was decanted at 1 hour intervals according to the immersion time until 12 hours, and the wheat seeds were poured onto a filter paper to remove surface water and their weights were measured, and then the water absorption rate at room temperature was expressed as a weight increase rate. The hardness of the wheat seeds was measured 30 grains per hour using TA-XT2 Texture analyzer (Stable Micro System, England), a device for measuring physical property, after adding 5 g of the wheat seeds with 50 mL of water and immersing the seeds therein for 12 hours, under the measurement texture profile analysis (TPA) conditions; probe: 2 mm, graph type: force vs. time, distance threshold: 0.5 mm, contact force: 5 g, pre-test speed: 5 mm/s, post-test speed: 5 mm/s, and distance: 80%.

As a result, as shown in FIG. 4, it was confirmed that the water absorption rate at room temperature of the non-germinated seeds in appearance was lower than that of the normal seeds, whereas their hardness was higher than that of the normal seeds.

[Example 5] Confirmation of starch structure of wheat seeds according to the degree of preharvest sprouting

In order to confirm the correlation between the values of total dissolved solids and starch structure of the wheat seeds according to the degree of preharvest sprouting, the starch structure of wheat seeds was observed under a scanning electron microscopy (SEM, LEO 440, ZEISS, England). First, the wheat seeds were added into a Karnovsky fixative (Karnovsky, 1965) and fixed at 4°C for 24 hours, washed with 0.2 M Milonig’s phosphate buffer (pH 7.4), and then treated with 1% osmium tetroxide (OsO4) at 4°C for 2 hours. After washing with distilled water, dehydrated with 50% to 95 % ethyl alcohol for 80 minutes, respectively, with 100% ethyl alcohol for 2 hours, and four times with 100% amyl acetate for 120 minutes. The samples, upon completion of dehydration, were dried with Pelco™ CPD-2 critical point dryer (TedPella Inc., Redding, CA, USA) and then coated using the Pelco SC-4sputter coater (Ted Pella, Inc.). Then the surface images were observed using the coated samples.

As a result, as shown in FIG. 5, although the materials in the body were depleted by the respiration of the non-germinated seeds in the case of the non-germinated seeds in appearance, there were observed structures starch became agglutinated and cell walls were hardened as they were dried again, and it was speculated from the above that, at the time of measuring the electric conductivity of the non-germinated seeds in appearance, the internal materials are dissolved in an amount less than that of the normal seeds.

[Example 6] Determination of a mixed presence of wheat seeds according to the degree of preharvest sprouting

6-1: Confirmation of wheat seeds according to the mixed ratio of preharvest sprouting seeds

In order to confirm whether the method for determining preharvest sprouting in wheat using electric conductivity can be used for determining the mixed presence of the wheat seeds with preharvest sprouting among the normal wheat seeds, the non-germinated seeds in appearance or the preharvest sprouting seeds artificially prepared in Example 1-1 were mixed in different ratios.

Prior to the measurement of electric conductivity values, the α-amylase activity was measured in the same manner as in Example 4 and Example 2, and falling number was measured using Falling Number 1500 (Perten Instruments, Stockholm, Sweden) according to AACC method.

The falling number measures the α-amylase activity of grains. Due to the absence of ?-amylase, the normal seeds have high falling number. However, in the case of preharvest sprouting seeds, their starch is decomposed by α-amylase thereby deteriorating falling number. Table 6 means characteristics of wheat seeds according to the mixed ratio of preharvest sprouting seeds.

As a result, it was confirmed that falling number reduced and α-amylase activity increased according to the mixed presence of the preharvest sprouting seeds.

In particular, when the type II preharvest sprouting seeds were mixed with the normal seeds, falling number rapidly decreased and α-amylase activity increased but no correlation was observed regarding the mixed ratio. In contrast, when the type I non-germinated seeds in appearance were mixed with the normal seeds, α-amylase activity increased and falling number became lowered according to the increase in the mixed ratio.

6-2: Measurement of total dissolved solids according to the mixed ratio of preharvest sprouting seeds

The total dissolved solids of the wheat seeds mixed with preharvest sprouting seeds was measured in the same manner as in Example 3, and the total dissolved solids was measured for 0 hour to 4 hours of immersion time of the wheat seeds. Table 7 means measurement of total dissolved solids according to the mixed ratio of preharvest sprouting seeds.

As a result, as shown in Table 7, it was confirmed that the values of total dissolved solids reduced or increased according to the mixed presence of the preharvest sprouting seeds. Additionally, it was confirmed that when type I non-germinated seeds in appearance were mixed with the normal seeds, the measured values of total dissolved solids became lower than that of the normal seeds as the mixed ratio increased, whereas when type II preharvest sprouting seeds were mixed with the normal seeds, the measured values of total dissolved solids became higher than that of the normal seeds as the mixed ratio increased.

6-3: Measurement of electric conductivity according to the mixed ratio of preharvest sprouting seeds

The electric conductivity of the wheat seeds mixed with preharvest sprouting seeds was measured in the same manner as in Example 3, and the electric conductivity was measured for 0 hour to 4 hours of immersion time of the wheat seeds. Table 8 means measurement of electric conductivity according to the mixed ratio of preharvest sprouting seeds.

As a result, as shown in Table 8, it was confirmed that the values of electric conductivity reduced or increased according to the mixed presence of the preharvest sprouting seeds. Additionally, it was confirmed that when type I non-germinated seeds in appearance were mixed with the normal seeds, he electric conductivity became lower than that of the normal seeds as the mixed ratio increased, whereas when type II preharvest sprouting seeds were mixed with the normal seeds, the electric conductivity became higher than that of the normal seeds as the mixed ratio increased.

6-4: Equation derivation

In order to confirm whether the above results can be equally applicable in Equation 1 and Equation 2 derived from Example 3, Equation 3 and Equation 4 shown below were derived and the result values were calculated according to each equation.

[Equation 3]

Result Value = total dissolved solids of a group of wheat seeds for determining the presence of a mixture of preharvest sprouting seeds/total dissolved solids of a group with 100 wt% normal wheat seeds

[Equation 4]

Result Value = electric conductivity of a group with wheat seeds for determining the presence of a mixture of preharvest sprouting seeds/electric conductivity of a group with 100 wt% of normal wheat seeds. Table 9 means total dissolved solids according to mixed ratio of preharvest sprouting seeds - result values by Equation 3, and table 10 means electric conductivity according to mixed ratio of preharvest sprouting seeds - result values by Equation 4.

As a result, as shown in Table 9 and Table 10, when the result values of Equation 3 and Equation 4 are in the range of between 0.96 and 1.05 the seeds may be determined as 100 wt% normal wheat seeds, whereas when the result values are in the range of between 0.01 and 0.95 or between 1.06 and 5.0, the seeds may be determined that the preharvest sprouting seeds are mixed therein (in the case of measurement for 1 hour to 4 hours).

In particular, it was confirmed that when the result values are in the range of between 0.96 and 1.05, the seeds may be determined as a mixture of the preharvest sprouting seeds and the normal wheat seeds.

The above-disclosed subject matter is to be considered illustrative, and not restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other embodiments, which fall within the true spirit and scope of the present invention. Thus, to the maximum extent allowed by law, the scope of the present invention is to be determined by the broadest permissible interpretation of the following claims and their equivalents, and shall not be restricted or limited by the foregoing detailed description.

Claims

A method for determining preharvest sprouting in wheat by measuring at least one selected from total dissolved solids and electric conductivity.
The method of claim 1, comprising:

(a) measuring at least one selected from total dissolved solids and electric conductivity of a wheat seed sample for determining preharvest sprouting; and

(b) determining the preharvest sprouting by comparing with the value of at least one selected from total dissolved solids and electric conductivity of a normal wheat seed.
The method of claim 2, wherein step (a) is characterized in that at least one selected from total dissolved solids and electric conductivity of the wheat seed sample is measured for determining the preharvest sprouting while immersing the wheat seed sample for 0 hour to 40 hours by adding water thereto.
The method of claim 2, wherein step (b) is characterized in that:

based on Equation 1 and Equation 2 below, when at least one result value of Equation 1 and Equation 2 is between 0.96 and 1.05, it is determined that the wheat seed is a normal wheat seed; and

when at least one result value of Equation 1 and Equation 2 is between 0.01 and 0.95 or between 1.06 and 5.0, it is determined that the preharvest sprouting was proceeded;

wherein

[Equation 1]

result value = total dissolved solids of a wheat seed sample for determining preharvest sprouting/ total dissolved solids of a normal wheat seed sample,

[Equation 2]

result value = electric conductivity of a wheat seed sample for determining preharvest sprouting /electric conductivity of a normal wheat seed sample.
The method of claim 4, wherein when at least one result value of Equation 1 and Equation 2 is between 0.01 and 0.95 the seed is determined as a non-germinated (an early stage of preharvest sprouting) seed in appearance, whereas when at least one result value of Equation 1 and Equation 2 is between 1.06 and 5.0 the seed is determined as a preharvest sprouting seed.
A method for determining the presence of a mixture of the wheat preharvest sprouting seed by measuring at least one selected from total dissolved solids and electric conductivity.
The method of claim 6, comprising:

(a) measuring at least one selected from total dissolved solids and electric conductivity of a wheat seed sample to determine whether preharvest sprouting seeds are mixed therein; and

(b) determining the mixed presence of preharvest sprouting seeds by comparing with the value of at least one selected from total dissolved solids and electric conductivity of a group with 100 wt% normal wheat seeds.
The method of claim 7, wherein step (a) is characterized in that at least one selected from total dissolved solids and electric conductivity of the wheat seed sample is measured for the wheat seed sample, which is to be determined regarding the presence of a mixture of the preharvest sprouting, while immersing the wheat seed sample for 0 hour to 40 hours by adding water thereto.
The method of claim 7, wherein step (b) is characterized in that:

based on Equation 3 and Equation 4 below, when at least one result value of Equation 3 and Equation 4 is between 0.96 and 1.05, it is determined that the wheat seeds are of 100 wt% normal wheat seeds; and

when at least one result value of Equation 3 and Equation 4 is between 0.01 and 0.95 or between 1.06 and 5.0, it is determined that the preharvest sprouting seeds are included among the normal wheat seeds;

wherein

[Equation 3]

result value = total dissolved solids of a group of wheat seeds for determining the presence of a mixture of preharvest sprouting seeds/total dissolved solids of a group with 100 wt% normal wheat seeds,

[Equation 4]

result value = electric conductivity of a group with wheat seeds for determining the presence of a mixture of preharvest sprouting seeds /electric conductivity of a group with 100 wt% of normal wheat seeds.
The method of claim 9, wherein when at least one result value of Equation 3 and Equation 4 is between 0.01 and 0.95 it is determined that there is a mixture between 10 wt% to 90 wt% of a non-germinated seed in appearance and a normal wheat seed, whereas when at least one result value of Equation 3 is between 1.06 and 5.0 it is determined that there is a mixture between 10 wt% to 90 wt% of an early preharvest sprouting and a normal wheat seed.