JP2013213810A - Method for evaluating freshness degree of seafood - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quickly and non-destructively evaluating a freshness degree of seafood without any preprocessing.SOLUTION: A freshness degree of fresh seafood, as a freshness degree of a viable cell of the seafood, is non-destructively evaluated without any preprocessing, by an intensity measurement of fluorescent light of nicotinamide adenine dinucleotide acid that is one kind of coenzymes contained in the fresh seafood. Self-fluorescence beams of the coenzyme and amino-acid contained in the fresh seafood are simultaneously measured to evaluate the freshness degree of the fresh seafood from an intensity ratio of the fluorescent light beams thereof. For minimizing an error, self-fluorescence of a tryptophan that is one kind of amino acids contained in the biological tissue is measured as an evaluation index of an optical system.

Description

  The present invention relates to a method for quickly evaluating freshness of fresh fish and shellfish without any pretreatment.

  Conventionally, the freshness of fresh seafood has been evaluated using the K value as an index. This conventional method evaluates freshness according to the magnitude of the total ratio of inosine and hypoxanthine contained in ATP-related substances generated from ATP after death as the K value. Component analysis is performed with a liquid chromatograph, and the sum of the amounts of each component of the ATP-related substance in the sample is used as the denominator, and the total amount of inosine and hypoxanthine is used as the numerator for quantification.

  This technique requires special knowledge and skill for preprocessing, and has a problem that it cannot be measured without a certain level of skill and experience. As for the time required for the evaluation, it takes several hours for the sample pretreatment and the component analysis after the pretreatment, so it is difficult to evaluate the freshness by measuring the K value in a field where the freshness is to be known quickly, for example, in a distribution site. It has the disadvantage of being.

On the other hand, attention has been focused on a method for evaluating freshness from optical characteristics. For example, for fresh foods and cereals, there is a freshness evaluation method (fluorescence spectroscopy) using the fluorescence intensity of phenolic compounds, lignin, and proteins as an index. The following nonpatent literatures 1 and 2 by the present inventors exist as an example which disclosed this specific technique.
In general, the fluorescence analysis method is an excellent method capable of non-destructive, rapid, and high-sensitivity analysis, and it is considered that freshness can be evaluated without pretreatment using autofluorescence measurement. However, it is difficult to use at distribution sites.

  The reason is that because of high sensitivity, slight differences that disturb the measurement optical system, such as the shape of the sample, affect the fluorescence. In order to reduce the error, it is necessary to keep the measurement system in the same state, but in order to realize it, it means that the irradiation angle of the excitation light, the fluorescence generation range, and the fluorescence detection region are always kept constant. It is difficult to accurately fix a bent object or an object with unevenness, and there are many practical problems.

  As a reference material, while measuring the fluorescence intensity of the autofluorescence of a phenolic compound or lignin contained in food (agricultural products), the intensity of the standard fluorescent substance coexisted in the food is also measured, and the ratio of both fluorescence intensities. A method for evaluating the state of a food by using as an index is disclosed (see Patent Document 1). However, since these are intended for agricultural products, the substances to be detected in the freshness evaluation are different and cannot be used for the evaluation of the freshness of fresh seafood.

  In addition, a certain amount of the supernatant obtained by leaving a piece of meat homogenized with an aqueous solution of deproteinizing agent at the origin of the electrophoresis filter paper set in the electrophoresis frame and moistened with the electrophoresis buffer is micro-quantized. A method to determine the freshness of meat by comparing the origin and movement relative to each other by spot-dropping with a pipette, performing electrophoresis immediately, comparing and observing the size and density of the spots of nucleic acid-related compounds that emerge when irradiated with ultraviolet rays. It is disclosed (see Patent Document 2).

In addition, the method of measuring the freshness of seafood from the concentration ratio of ATP-related compounds contained in fish meat, specifically, the K1 value can be quickly calculated by the FIA method (flow analysis method) to shorten the freshness determination of seafood. A method of performing with time is disclosed (see Patent Document 3).
Furthermore, in Patent Document 4, a small amount of sample is cut from a fish product, and an effective amount of a coloring reagent containing at least one of a cell-permeable pigment and a cell-impermeable pigment is added to the sample, and this sample is added. A method of discriminating the freshness based on the fluorescence emitted from the sample after incubating for a certain time is disclosed. However, these do not meet the purpose of the present invention.

JP 2001-208745 A Japanese Patent No. 42911381 Japanese Patent No. 2857607 Special table 2008-500810

Tomoaki Sugawara and 5 other authors, “Evaluation of Fresh Scallops by Fluorescence Spectroscopy” Proceedings of the 2011 IEEJ Fundamental / Materials / Common Section Conference (issued September 21, 2011), page 174 Tomoaki Hagiwara and five other authors, “Evaluation of Fresh Squid Mantle by Fluorescence Spectroscopy” Proceedings of the 2011 Hokkaido Electrical and Information Society Conference (October 22, 2011), page 139

  In order to solve the problems of the prior art, an object of the present invention is to provide a method for quickly and freshly evaluating the freshness and quality of fish and shellfish without pretreatment, without pretreatment.

As a result of investigations to solve the above problems, the following inventions are provided.
1) To evaluate the freshness of fresh fish and shellfish without pre-treatment without losing the freshness of fresh fish and shellfish by fluorescence measurement of nicotinamide adenine dinucleotide acid, a coenzyme contained in fresh fish and shellfish A method for evaluating the freshness of seafood characterized by
2) A method for evaluating the freshness of fish and shellfish, characterized by simultaneously measuring autofluorescence of coenzymes and amino acids contained in fresh fish and shellfish and evaluating the freshness of fresh fish and shellfish from the intensity ratio of the fluorescence.

3) The method for evaluating freshness of fish and shellfish according to claim 2, wherein the measurement is performed using nicotinamide adenine dinucleotide acid as a coenzyme.
4) The method for evaluating the freshness of fish and shellfish according to claim 2 or 3, wherein autofluorescence of tryptophan, which is a kind of amino acid contained in a biological tissue, is measured as an optical system evaluation index in order to reduce errors.

  5) Based on evaluation of excitation light source, sample holder, fluorescence detector, apparatus for simultaneously measuring fluorescence of coenzyme and autofluorescence of amino acids contained in fresh seafood, apparatus for measuring intensity ratio of these fluorescence, and optical system An apparatus for measuring freshness of fish and shellfish, characterized by comprising an apparatus for correcting errors.

  The freshness evaluation method of the present invention can increase the fluorescence measurement accuracy by the evaluation index of the optical system, can perform freshness assessment of fresh fish and shellfish quickly and non-destructively without pretreatment, and can be used even in the distribution site of fish and shellfish. It has an excellent effect of being possible.

It is a figure which shows the fluorescence image of the scallop shell of the raw scallop which shines in blue when the scallop shell is irradiated with UV-A ultraviolet rays having a wavelength of 330 to 390 nm. It is a figure which shows the fluorescence spectrum of nicotinamide adenine dinucleotide acid contained in a scallop shell. It is a figure which shows the relationship between the storage time when a raw scallop shell is refrigerated at 10 degreeC, and the fluorescence intensity of nicotinamide adenine dinucleotide acid. It is a figure which shows the fluorescence spectrum of tryptophan. It is a figure which shows the fluorescence intensity of the nicotinamide adenine dinucleotide acid normalized with the fluorescence intensity of tryptophan about three types of samples (sample A, sample B, sample C) of a raw scallop. It is a figure which shows the fluorescence image of the squid which shines blue when a fresh squid is irradiated with UV-A ultraviolet-ray with a wavelength of 330-390 nm. It is a figure which shows the relationship between the refrigerated storage time of fresh squid, and the fluorescence intensity of nicotinamide adenine dinucleotide acid. It is a figure which shows the fluorescence intensity of the nicotinamide adenine dinucleotide acid normalized with the fluorescence intensity of tryptophan about three types of samples (sample A, sample B, sample C) of fresh squid.

It is a figure which shows the fluorescence intensity of nicotinamide adenine dinucleotide acid and tryptophan of a raw scallop. It is a figure which shows the fluorescence intensity of nicotinamide adenine dinucleotide acid normalized with the fluorescence intensity of the tryptophan of a raw scallop shell. It is a figure which shows the fluorescence intensity of fresh squid nicotinamide adenine dinucleotide acid and tryptophan. It is a figure which shows the fluorescence intensity of the nicotinamide adenine dinucleotide acid normalized with the fluorescence intensity of the fresh squid tryptophan.

  The method for assessing freshness of seafood according to the present invention is characterized in that freshness assessment is performed non-destructively and accurately with the state assessment of nicotinamide adenine dinucleotide acid, which is a type of coenzyme contained in fresh seafood, as an index. Yes.

In general, nicotinamide adenine dinucleotide acid is known as a coenzyme related to energy metabolism. Regarding fresh fish and shellfish, according to previous studies, the fluorescence of nicotinamide adenine dinucleotide acid contained in the tissue strongly depends on the freshness, and the fluorescence from nicotinamide adenine dinucleotide acid gradually weakens as the freshness decreases.
Therefore, measuring the fluorescence intensity of nicotinamide adenine dinucleotide acid can be said to be an extremely effective means for evaluating the freshness of fresh fish and shellfish.

  Since fluorescence analysis is highly sensitive, it can detect a slight difference in freshness. On the other hand, there is an error in the optical system as a factor that increases the error in the fluorescence measurement result. An evaluation device used at a distribution site may require evaluation of an optical system in order to reduce an error, and freshness evaluation based on the index may be required.

The fluorescence of tryptophan, which is a kind of amino acid contained in seafood, has little change with respect to deterioration of biological tissues such as heating and a decrease in freshness.
Therefore, in the present invention, it is effective to simultaneously measure autofluorescence of tryptophan, which is a kind of amino acid contained in a biological tissue, as an optical system evaluation index in order to reduce errors.

In the freshness evaluation method of the present invention, first, fresh fish and shellfish are irradiated with UV-B ultraviolet rays having a wavelength of 280 to 320 nm, fluorescence of a wavelength of 330 to 390 nm is measured, and the intensity value is optically evaluated. It is an index of the system.
Next, UV-A ultraviolet light having a wavelength of 330 to 390 nm, which serves as a reference for freshness, is irradiated, and fluorescence having a wavelength of 430 to 490 nm is measured. By correcting the fluorescence intensity at a wavelength of 430 to 490 nm with the intensity value measured by the optical system evaluation, the freshness can be evaluated with high accuracy.

Further, the value of the fluorescence intensity can be used as it is as the freshness evaluation by adjusting the increase or decrease of the light irradiation intensity or the sensitivity of the fluorescence detector so that the measured intensity of the optical system is constant.
As a method for selecting light of a specific wavelength from an excitation light source, there is a method of irradiating a sample with UV-B or UV-A using a spectroscope or a bandpass filter. Moreover, an ultraviolet LED device can also be used without a spectroscope or a bandpass filter.

In fluorescence measurement, a spectroscope, a cutoff filter, or the like can be used to detect light of a specific wavelength. As the detector, a photodetector such as a photomultiplier tube, a CCD device, a CMOS, or a photodiode can be used.
Regarding the measurement time, since the freshness changes with time, it is desirable to perform the evaluation of the optical system and the measurement of the freshness reference value simultaneously. By using a light source having a high ultraviolet intensity such as a xenon lamp or an ultraviolet LED, both the optical system evaluation measurement and the freshness evaluation measurement can be completed in several seconds to several tens of seconds.

  As for the optical system, in addition to the illumination light source and the fluorescence measurement detector arranged at 90 degrees, it is also possible to use a linear or coaxial arrangement. Since the correction is made by evaluating the optical system, the arrangement of the optical system can be freely designed.

  The present invention can simultaneously measure the autofluorescence of coenzymes and amino acids contained in fresh fish and shellfish, and evaluate the freshness of fresh fish and shellfish from the intensity ratio of the fluorescence, but it is normalized by the fluorescence intensity of tryptophan. It is desirable to use the fluorescence intensity of nicotinamide adenine dinucleotide acid.

As a standardization method, a value obtained by dividing the fluorescence intensity of nicotinamide adenine dinucleotide acid by the fluorescence intensity of tryptophan can be used.
As a more accurate standardization method, the correction coefficient of the optical system (for example, the reciprocal of tryptophan fluorescence intensity) is obtained from the fluorescence intensity of tryptophan, and the fluorescence intensity of nicotinamide adenine dinucleotide acid is evaluated using the correction coefficient. A technique can also be taken.

  The features of the present invention will be specifically described below. The following description is intended to facilitate understanding of the present invention, and is not limited thereto. That is, all modifications, other embodiments, and other examples based on the technical idea of the present invention are included in the present invention.

Example 1
The example of the freshness evaluation of a scallop shell is shown. The scallops were taken out from the live scallops and irradiated with UV-A ultraviolet rays having a wavelength of 330 to 390 nm.
FIG. 1 shows a fluorescent image of a scallop that glows blue. Blue fluorescence is derived from nicotinamide adenine dinucleotide acid, which is a type of coenzyme.
FIG. 2 shows the fluorescence spectrum of nicotinamide adenine dinucleotide acid contained in scallop shells.
FIG. 3 shows the relationship between the storage time and the fluorescence intensity of nicotinamide adenine dinucleotide acid when raw scallops are refrigerated at 10 ° C.

The longer the refrigerated storage time, the weaker the fluorescence derived from nicotinamide adenine dinucleotide acid. However, the difference in fluorescence intensity between samples is large, and the accuracy of evaluation is low.
As an improvement measure, we evaluated the optical system and examined tryptophan, a kind of amino acid contained in fresh fish and shellfish, to correct the fluorescence intensity.

When the shell pillar was irradiated with UV-B ultraviolet light having a wavelength of 280 to 320 nm, fluorescence having a wavelength of 330 to 390 nm was observed.
FIG. 4 shows the relationship (dependence) between the fluorescence spectrum from tryptophan contained in the raw scallop and the storage time of the fluorescence intensity. Since tryptophan is a more stable substance than nicotinamide adenine dinucleotide acid, as shown in the upper right diagram of FIG. 4, the fluorescence derived from tryptophan does not change much as the refrigerated storage time increases.
That is, the concentration of tryptophan does not change, but if the optical system is different, there is a difference in the fluorescence of tryptophan.

  For example, consider that a light source and a photodetector are arranged in front of the sample. If it is a flat sample, the brightest fluorescence is observed when incident vertically. That is, the fluorescence intensity depends on the positional relationship between the sample, the light source, and the photodetector. Ideally, it would be necessary to find a flat part of the sample and measure that part with fluorescence. However, this method is not convenient and is considered infeasible in the field. Therefore, when the fluorescence intensity of tryptophan is different, it is necessary to correct the optical arrangement by assuming that the optical arrangement is deviated from the ideal state.

FIG. 5 shows three samples (sample A, sample B, and sample C) prepared as an example of a freshness index corrected for the difference in optical system, and nicotinamide adenine dinucleotide acid normalized by tryptophan fluorescence intensity. The fluorescence intensity is shown.
A value (150/200 = 0.75) obtained by dividing the fluorescence intensity (eg, 150) of nicotinamide adenine dinucleotide acid by the fluorescence intensity (eg, 200) of tryptophan can be obtained.

To obtain more accuracy, calculate the correction coefficient of the optical system (for example, the inverse of tryptophan fluorescence intensity) from the fluorescence intensity of tryptophan, and evaluate the fluorescence intensity of nicotinamide adenine dinucleotide acid using the correction coefficient. You can also.
In the conventional method, the variation is as large as ± 30%, but in this example, it is as small as ± 11%, and according to this example, the evaluation accuracy can be improved.

(Example 2)
Next, a specific example of the freshness evaluation of the squid mantle is shown. Fresh squid was irradiated with UV-A ultraviolet rays having a wavelength of 330 to 390 nm. FIG. 6 shows a fluorescent image of a squid that glows blue. Blue fluorescence is derived from nicotinamide adenine dinucleotide acid, which is a type of coenzyme.

  FIG. 7 shows the relationship between the refrigerated storage time of fresh squid and the fluorescence intensity of nicotinamide adenine dinucleotide acid. As with scallops, the fluorescence intensity of nicotinamide adenine dinucleotide acid decreases with refrigerated storage time. However, the difference in fluorescence intensity between samples is large, and the accuracy of evaluation is low.

In FIG. 8, three types of fresh squid samples (sample A, sample B, sample C) are prepared as an example of a freshness index corrected for the difference in the optical system, and the fluorescence intensity of tryptophan is standardized as in the first embodiment. The fluorescence intensity of converted nicotinamide adenine dinucleotide acid is shown.
In the conventional method, the variation is as large as 11%, but in this example, it is as small as 5%. According to this example, it was possible to improve the evaluation accuracy.

(Example 3)
Next, an example of raw scallop shell will be described. FIG. 9 shows the fluorescence intensity of nicotinamide adenine dinucleotide acid (NADH) and tryptophan in raw scallops. For samples with high freshness (0 days of storage), there is a proportional relationship between the fluorescence intensity of tryptophan and NADH. Moreover, although the sample of low freshness (after 3 days of storage) has a slightly large variation, the fluorescence intensity of tryptophan and the fluorescence intensity of NADH are proportional.
From this, the evaluation accuracy of the present method for comparing the fluorescence intensity of NADH normalized by the fluorescence intensity of tryptophan is higher than the conventional method of comparing only the fluorescence intensity of NADH.

  In FIG. 10, the measurement result of a scallop shell is shown. The bar graph represents the mean value ± standard deviation (bar). In the result of high freshness (0 day), the standard deviation (9.2%) of this method is slightly smaller than the standard deviation (9.7%) of measurement data obtained by the conventional method. Also, even at low freshness (after 3 days), the standard deviation (15.5%) of the present method is smaller than the standard deviation (16.4%) of the conventional method. Highly accurate evaluation is possible.

Example 4
Next, a case of raw squid will be described. FIG. 11 shows the fluorescence intensity of nicotinamide adenine dinucleotide acid (NADH) and tryptophan of raw squid. For samples with high freshness (0 days of storage), there is a proportional relationship between the fluorescence intensity of tryptophan and NADH. In addition, the fluorescence intensity of tryptophan is proportional to the fluorescence intensity of NADH even in samples with low freshness (after 3 days of storage). Therefore, this method of comparing the fluorescence intensity of NADH normalized with the fluorescence intensity of tryptophan has higher evaluation accuracy than the conventional method of comparing only the fluorescence intensity of NADH.

  FIG. 12 shows the measurement results of Japanese squid. The bar graph represents the mean value ± standard deviation (bar). At high freshness (0 day), the standard deviation (15.4%) of this method is smaller than the standard deviation (18.6%) of measurement data obtained by the conventional method. Even at low freshness (after 3 days), the standard deviation (16.8%) of this method is smaller than the standard deviation (18.6%) of the conventional method. Accuracy can be evaluated.

  As shown in the above examples, by measuring fluorescence of nicotinamide adenine dinucleotide acid, which is a type of coenzyme contained in fresh seafood, the freshness of live cells in seafood is non-destructive, and the fresh fish It is effective to evaluate the freshness of a kind as a freshness index.

Although the above examples have been described for molluscs, it goes without saying that it can be applied to all seafood containing nicotinamide adenine dinucleotide acid and tryptophan.
Furthermore, in the case of only the fluorescence measurement of nicotinamide adenine dinucleotide acid, the variation tends to increase depending on the sample. By normalizing with the fluorescence intensity of tryptophan, the variation can be reduced and the evaluation accuracy can be improved. it can. The present invention includes these.

  The freshness evaluation method of the present invention can increase the fluorescence measurement accuracy by the evaluation index of the optical system, can perform freshness assessment of fresh fish and shellfish quickly and non-destructively without pretreatment, and can be used even in the distribution site of fish and shellfish. Because it has an excellent effect that it is possible, it is useful as a freshness assessment method for fresh seafood

Claims (5)

  To evaluate the freshness of fresh fish and shellfish without pre-treatment by measuring the fluorescence intensity of nicotinamide adenine dinucleotide acid, a kind of coenzyme contained in fresh fish and shellfish, without destroying the freshness of live cells of fish and shellfish A method for evaluating the freshness of seafood characterized by   A method for assessing freshness of seafood, characterized by simultaneously measuring autofluorescence of coenzymes and amino acids contained in fresh seafood and evaluating the freshness of fresh seafood from the intensity ratio of the fluorescence.   The method for evaluating freshness of fish and shellfish according to claim 2, wherein measurement is performed using nicotinamide adenine dinucleotide acid as a coenzyme.   4. The method for evaluating freshness of fish and shellfish according to claim 2, wherein autofluorescence of tryptophan, which is a kind of amino acid contained in a biological tissue, is measured as an optical system evaluation index in order to reduce errors.   Error correction based on evaluation of excitation light source, sample holder, fluorescence detector, coenzyme fluorescence and amino acid autofluorescence contained in fresh fish and shellfish, an intensity ratio of these fluorescences, and optical system A device for measuring the freshness of fish and shellfish, characterized in that it comprises a device for performing the processing.