NMR — SPECTROSCOPY TECHNIQUE FOR SALMON FISH SPECIES QUALITY ASSESSMENT

NMR-spectroscopy was used for development of the criteria which characterize the chilled and frozen fish quality. It has been shown that 1H-NMR experiments can be used as quality factor to measure the concentration of inosine, hypoxanthine and inosine-5’-monophosphate generated during the fish storage. The quality factor is expressed by the K1 correlates well with the sensory quality of chilled Atlantic salmon (Salmo Salar), whereas, quality factor H is more sensitive for measuring the quality characteristics of frozen pink salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka).


Introduction
One of the fundamental tasks of the Fisheries Industries is to ensure the preservation of the high-quality fishery products, the manufacturing of products with the predetermined customer properties and the guaranteed shelf life.
In order to comply with these fundamental task prerequisites, it is obligatory to be familiar with the processes occurring during the fish storage and processing, to know how to manage them and to have an opportunity to control the basic indexes characterizing its quality.
During fishing auctions in the European Community countries, the sensory quality of fish (fish quality index) is used for the evaluation of the freshness grade of the fishes. This quality index can also be used at fish unloading points, for calculation of the shelf life and for the storage conditions which are used for sorting before further processing [1]. Therefore, the sensory properties such as: taste, sight, smell, and touch are evaluated for the appearance, flavor, mucus condition, eyes, gills and abdominal cavity of the whole or gutted fish. On the basis of these indexes, the fish is referred to E (Extra) grade -that is the highest freshness grade, А, В or С grades (runoff product).
The rapid test quality index method (QIM) is used for the chilled fish [2]. The present method is based on consideration of the specific changes in the fish, namely appearance, flavor and consistence changes. The penalty points from 0 to 3 are calculated for each index, then the points are summarized by all the indexes and the total sensory estimate is obtained, that is known as the "quality index", linearly increasing with the fish storage time. Under modern conditions the method mentioned above allows for quick and objective evaluation of the fish quality by means of the special program, adapted for different computer-based systems. Evaluation can be performed at any stage: during the fishery products delivery at production site, fish storage or sale. The disadvantage of QIM is that it is applied only to the proven testing groups and it can be used only for whole fish.
It is well known that autolysis and bacterial deterioration lead to changes in concentrations of adenosine-5'-triphosphate [АТP], adenosine-5'-diphosphate [АDP], adenosine-5'-monophosphate [АМP] and inosine-5'-monophosphate [ІМР], which convert quantitatively in inosine [Ino] and hypoxanthine [Нх]. It has been reported that there was a good correlation relating the decrease of fish freshness with the increase in nucleotides formation for a large number of fish species [3,4,5]. In this context the quality factor K was used for the quantitative evaluation of fish freshness. The quality factor K is defined by the following equation [6,7,8]: It has been suggested that the determination of adenosine-5'-tripho products content in the fish can be used as the basis of the quality factor evaluating post-mortem fish quality changes during fish storage [8].
As a result, it became considerably interesting not only to anal metabolites in the chilled fish during their storage but also to characterize t from which the frozen product was manufactured.
According to Karube [9] for some fish species ATP decomposes very the definition of quality factor K can be modified by excluding the adeno suggested a new calculation quality factor К 1 defined as: Luong [10] proved that, for some species, quality factor K and K 1 reflect the alterations and proposed the quality factor H based on Нх considered as a good indicator of fish freshness under physiological and sens = + + * 100, % In recent years, it has been shown that NMR spectroscopy is a good to quality of fish raw materials [7,8]. The fish quality factor can be calculat NMR spectra and determining the concentration of ATP decay products.
In the work, NMR spectroscopy was used to assess the quality of sal The research was conducted on chilled Atlantic salmon (Salmo Salar) as the sold through retail chains as chilled and frozen pinc salmon (Oncorhynchu salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) which a processing as frozen.

Samples preparation
The chilled Atlantic Salmon (Salmo Salar) with storage duration of 9 d Faroe Islands (starting from the preparation date) and on the 18 day o preparation date) at the temperature of approximately 0 о С, frozen pinc salm gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorh storage duration about three months at the temperature of approximately min as objects for research. For the analysis were used average muscular sample of several chilled and frozen fish. The protein content was determined on the autoanalyzer «Kjeltec» F according to the Kjeldahl method [11]. Moisture content was determined in 7636 [11]. Lipids content was defined on the automated extractor SER 148 VELP company according to the Soxhlet method. The content of Total Vol (TVB-N) was determined on the semi-automatic distillation unit K-355 It has been suggested that the determination of adenosine-5'triphosphate degradation products content in the fish can be used as the basis of the quality factor K calculation, when evaluating post-mortem fish quality changes during fish storage [8].
As a result, it became considerably interesting not only to analyze the content of metabolites in the chilled fish during their storage but also to characterize the raw fish quality, from which the frozen product was manufactured.
According to Karube [9] for some fish species ATP decomposes very quickly to IMP and the definition of quality factor K can be modified by excluding the adenosine phosphates and suggested a new calculation quality factor К 1 defined as: It has been suggested that the determination of adenosine products content in the fish can be used as the basis of the quality evaluating post-mortem fish quality changes during fish storage [8].
As a result, it became considerably interesting not only metabolites in the chilled fish during their storage but also to chara from which the frozen product was manufactured.
According to Karube [9] for some fish species ATP decompo the definition of quality factor K can be modified by excluding th suggested a new calculation quality factor К 1 defined as: Luong [10] proved that, for some species, quality factor K reflect the alterations and proposed the quality factor H based considered as a good indicator of fish freshness under physiological In recent years, it has been shown that NMR spectroscopy is quality of fish raw materials [7,8]. The fish quality factor can be NMR spectra and determining the concentration of ATP decay produ In the work, NMR spectroscopy was used to assess the qualit The research was conducted on chilled Atlantic salmon (Salmo Sala sold through retail chains as chilled and frozen pinc salmon (Onco salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka processing as frozen.

Samples preparation
The chilled Atlantic Salmon (Salmo Salar) with storage duratio Faroe Islands (starting from the preparation date) and on the 18 preparation date) at the temperature of approximately 0 о С, frozen gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon storage duration about three months at the temperature of approxim as objects for research. For the analysis were used average muscula of several chilled and frozen fish.
The protein content was determined on the autoanalyzer «K according to the Kjeldahl method [11]. Moisture content was determ 7636 [11]. Lipids content was defined on the automated extractor Luong [10] proved that, for some species, quality factor K and K 1 do not adequately reflect the alterations and proposed the quality factor H based on Нх concentration to be considered as a good indicator of fish freshness under physiological and sensory points of view: It has been suggested that the determination of adenosineproducts content in the fish can be used as the basis of the quality evaluating post-mortem fish quality changes during fish storage [8].
As a result, it became considerably interesting not only metabolites in the chilled fish during their storage but also to chara from which the frozen product was manufactured.
According to Karube [9] for some fish species ATP decompo the definition of quality factor K can be modified by excluding th suggested a new calculation quality factor К 1 defined as: Luong [10] proved that, for some species, quality factor K reflect the alterations and proposed the quality factor H based considered as a good indicator of fish freshness under physiological a = + + * 100, % In recent years, it has been shown that NMR spectroscopy is a quality of fish raw materials [7,8]. The fish quality factor can be NMR spectra and determining the concentration of ATP decay produ In the work, NMR spectroscopy was used to assess the qualit The research was conducted on chilled Atlantic salmon (Salmo Sala sold through retail chains as chilled and frozen pinc salmon (Onco salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) processing as frozen.

Samples preparation
The chilled Atlantic Salmon (Salmo Salar) with storage duratio Faroe Islands (starting from the preparation date) and on the 18 preparation date) at the temperature of approximately 0 о С, frozen p gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon storage duration about three months at the temperature of approxima as objects for research. For the analysis were used average muscula of several chilled and frozen fish.
The protein content was determined on the autoanalyzer «K according to the Kjeldahl method [11]. Moisture content was determ 7636 [11]. Lipids content was defined on the automated extractor S In recent years, it has been shown that NMR spectroscopy is a good tool for assessing the quality of fish raw materials [7,8]. The fish quality factor can be calculated by analyzing the NMR spectra and determining the concentration of ATP decay products.
In the work, NMR spectroscopy was used to assess the quality of salmon species of fish. The research was conducted on chilled Atlantic salmon (Salmo Salar) as the most massive fish, sold through retail chains as chilled and frozen pinc salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) which are used in trade and processing as frozen.

Sample preparation
The chilled Atlantic Salmon (Salmo Salar) with storage duration of 9 days produced on the Faroe Islands (starting from the preparation date) and on the 18 day of storage (from the prepara- tion date) at the temperature of approximately 0 °С, frozen pinc salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) with storage duration about three months at the temperature of approximately minus 18 о С were used as objects for research. For the analysis were used average muscular samples of different parts, of several chilled and frozen fish.
The protein content was determined on the autoanalyzer «Kjeltec» Foss-2300 (Sweden) according to the Kjeldahl method [11]. Moisture content was determined in according to GOST 7636 [11]. Lipids content was defined on the automated extractor SER148/6 manufactured by VELP company according to the Soxhlet method. The content of Total Volatile Basic Nitrogen (TVB-N) was determined on the semi-automatic distillation unit K-355 (Buchi company, Switzerland).

Sample preparation for NMR-spectroscopy
The water soluble polar metabolites of fish samples were extracted by 7.5% solution of trichloroacetic acid (TCA), as it was described in the paper [7]. For this purpose 25 g of fish muscle was added to 50 mL of 7.5% TCA and homogenized using a vertical homogenizer. The homogenate was filtered through the paper filter. The filtrate was neutralized by 9 М solution of КОН up to рН value of 7.8. The solution was filtered through the paper filter (№ 1) and it was stored at the temperature minus 40 о С until the measurements conduction.

Proximate analysis
Proximate analysis chemical composition of the fish muscle of Atlantic salmon (Salmo Salar), pink salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) are presented in Table 1. The quality estimation of the fish samples studied were obtained from different conditions of storage (duration) and the sensory quality of fish were estimated and TVB-N values determined (see the results in Table 2).
As it is seen from the presented data on 18 day of storage the chilled fish had the off-odor and changed the color.
The TVB-N index was 31 g/100 g. According to Commission Regulation EC No 1022/2008/ЕС of 17 Dec. 2008, which makes amendments in Regulation ЕС № 2074/2005 in respect of the critical concentrations of Total Volatile Basic Nitrogen ( TVB-N) [13] the critical content of TVB-N should be 35 mg/100 g for salmon fishes. When the present value exceeds 35 mg/100 g, the product is considered inedible and unsuitable for industrial processing. According to the TVB-N index chilled Atlantic Salmon with storage duration of 18 days at 0 °С met the requirements applicable to food fishery products suitable for industrial processing and direct consumption. But at the same time on the basis of the sensory quality of fish it could not be recommended for direct consumption. Consequently, the TVB-N index which is recommended to be determined in case of discrepancies during evaluation of the sensory quality of the chilled or frozen fishery products, sometimes characterizes the product quality incorrectly.

NMR-spectra
The 1 Н-NMR spectra of chilled Atlantic salmon (Salmon Salar) with 9 and 18 days storage duration at 0 °С muscle TCA extract is presented in Figure 1. The similar 1 Н-NMR spectra were recorded for all fish samples.

Determination of adenosine-5'-triphosphate degradation products' content in fishery products
The analysis of 1 Н-NMR weak field spectra has allowed us to establish that all the samples exhibited the absence of adenosine-5'-triphosphate, adenosine-5'-diphosphate and adenosine-5'-monophosphate signals, whereas they were characterized by the diagnostic signals of inosine, hypoxanthine and inosine-5'-monophosphate.
This obtained data was in good agreement with the results published in the literature [14,15]. Indeed, these authors established that after post-mortem changes, the total decomposition of АТP occurred and the analyzed fish contained metabolites such as inosine-5'-monophosphate, inosine and hypoxanthine. Therefore, in our manuscript the quality estimation of fish, relied on the identification of three metabolites: inosine-5'-monophosphate, inosine and hypoxanthine.
On the basis of 1 Н-NMR spectra analysis, it is concluded that the value of the quality factor K for stored chilled and frozen raw materials can be calculated by the formula 2. The results are presented in Table 4.
It follows from the presented data that the chilled Atlantic salmon which were kept in storage for a duration of 18 days at 0 о C gave unsatisfactory sensory quality (signs of oxidation and offodor of rank fish) that had quality factor К 1 equal to 90%. This quality factor K 1 , which defines the fish quality, was indicative for the biochemical changes occurring in fish due to their storing conditions. Consequently, the higher the biochemical changes, and consequently the higher value of quality factor K 1 , indicate about loss of fish quality. This correlates well with degradation of the sensory quality of fish and the test of spoiled fish. The quality factor K 1 of more than 80% is the threshold quality value, when the product is considered to be unfit for food.
It is important to point out that quality factor K 1 for frozen samples of fish estimated under given above formula was too high, which did not correlate with satisfactory sensory quality of fish and the TVB-N indexes for these samples. For these reasons and as proposed by others [10,12,14] the quality factor H was used for characteristics of frozen fish, estimated under the formula 3.
Obtained results in table 4 show that quality factor H more objectively reflects quality of frozen samples. It can be concluded that Pinc salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) were frozen immediately after catching, as they have high sensory quality of fish and consequently low of quality factor H.
It allows to conclude that the following methodical approach can be used for characteristics of not only frozen fish but also for raw fish from which frozen pinc salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) were produced. Thus the NMR-spectroscopy allows for objective evaluation of the fish chemical quality. Consequently, reliable information concerning the product can be obtained, the shelf life can be predicted and the tailormade products manufacturing processes management can be performed on the basis of the comprehensive research and correlation with the organoleptic analysis as well as the nutritional quality and the processing properties evaluation.

Conclusion
It has been established that the fish quality index can be expressed by the quality factor K 1 which is calculated by means of analysis of 1 Н-NMR spectra and concentration measurement of inosine, hypoxanthine and inosine-5'-monophosphate generated during the fish storage. The quality factor K 1 correlates well with the sensory quality of chilled fish. For quality estimation of frozen pinc salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka) quality factor H is more informative, which correlate with low indicators of TVB-N and high sensory quality of fish.