Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain

*Sonia Nieto-Ortega, Rebeca Lara, Giuseppe Foti, Ángela Melado-Herreros and Idoia Olabarrieta*

## **Abstract**

Near-infrared spectroscopy (NIRS) has undergone a significant evolution in the last years due to the numerous scientific studies that revealed its potential for industrial applications, attracting a growing interest in the food sector. Furthermore, new advances have allowed the reduction in size and cost of the NIR devices, making them appropriate for on-site determinations. The complex structure of the fish value chain, combined to its high market value, makes this sector particularly vulnerable to fraud and adulteration practices. Also, the perishable nature of fish and fish products, as well as the lack of traceability, arises the urgent need for a fast, reliable and portable tool capable of precisely characterizing the quality and authenticity of the product while also ensuring its safety. In this chapter, the capabilities of NIRS combined to several chemometric techniques for quality, authenticity and safety applications are presented through an extensive review of the most recent research works.

**Keywords:** NIR applications, food testing and analysis, spectral analysis, industrial applications, fish chain, chemometrics

## **1. Introduction**

### **1.1 Principles of NIR spectroscopy and chemometrics**

Near-infrared spectroscopy (NIRS) is a technique that measures the absorption of the electromagnetic region from 750 nm to 2500 nm (**Figure 1a**), between the visible and the mid-infrared (MIR) region. Like MIR spectroscopy and some part of the far-infrared (FIR) spectroscopy, this technique detects molecular vibrations, which gives information about the different chemical bonds within the molecule and its environment [1, 2].

However, there are some differences between these spectroscopic techniques, based on the nature of the vibrational transitions. While in the MIR/FIR region (2500 nm– 50,000 nm), almost all fundamental transitions (v0 ! v1) can be observed, resulting in sharp and intense spectra, in the near-infrared (NIR) region (750 nm–2500 nm) only the much less intense overtones (v0 ! vn) of those transitions appear. NIR absorptions

#### **Figure 1.**

*a) Electromagnetic spectrum of UV-vis-IR. b) Vibrational transitions involved.*

are based on overtones and combination bands and, due to their lower transition probabilities, intensities usually decrease by a factor of 10 or 100 from the fundamental overtone to the next one (**Figure 1b**) [1, 3].

It could be logical to think that it would be better to use MIR bands (fundamental transitions) instead of NIR. However, NIR instruments are simpler and more affordable. Furthermore, NIR has a deeper penetration in skin and organic tissues (optical window) than MIR, so that a larger area or volume can be measured [4].

While MIR chemical information can be directly obtained from peak intensities, shape and energy, NIR spectra need special treatment to extract useful information that involves multivariate analysis of data, usually called chemometrics [5]. Chemometrics is the science that applies mathematics and statistical methods to process chemical data, in order to obtain useful information about the sample [6, 7]. These two sciences are closely related: NIRS would not have evolved without chemometrics, and chemometric algorithms usually use NIRS examples to illustrate their power. This process needs computational resources that only since the last few decades have been available. In NIR applications, chemometrics covers a wide range of multivariate methods, which involve preprocessing techniques, as well as qualitative and quantitative analysis [8].

NIRS data usually need to be preprocessed before the model construction, because the spectra usually contain plenty of noise and background information that must be filtered out to find the desired spectral signatures. Some of the most used preprocessing techniques are divided in: i) scatter correction methods and ii) spectral derivatives. The scatter correction methods include techniques such as multiplicative scatter correction (MSC), detrending, normalization, or standard normal variate (SNV). Regarding the spectral derivatives, the Savitzky-Golay (SG) derivative after smoothing is one of the most used [9]. Chemometric methods can be divided into **unsupervised**, when groups or patterns inside data are not known, and **supervised** (classification and regression) when categories or analytes are already quantified, and the objective is to predict them for new samples [10].

The rapid development of chemometric methods during the last decades has allowed the application of NIRS to different areas of food science [11], such as food fraud and authenticity [12, 13] and food quality [14, 15].

## **1.2 Fish value chain**

The demand for fish consumption has increased during the last years as a consequence of the better dietary habits of the population. Fish has become one of the most valuable foods, due to its higher protein content and the healthier fat profile that it has compared with meat [16]. However, fish and fish products present complex problematics in terms of food quality, authenticity and safety. In addition, a greater awareness of food quality and sustainability has led consumers to demand increasingly reliable information about the products purchased in the market, which has stimulated the food value chain to strengthen efforts in the quest for a better and more trusty traceability [17].

Fisheries and aquaculture production are very heterogeneous in terms of species and fish products and, due to its high economic value, are one of the most vulnerable targets to adulteration and fraud. These practices are favoured by the complex structure of the supply chain (**Figure 2**), the lack of transparency and the high percentage of the production destined to the elaboration of processed fish products (around the 50%) such as fillets, portions or elaborated products, in which the morphological characteristics are absent, making these frauds particularly elusive [18]. Furthermore, fish is a perishable product that may suffer a fast enzymatic decomposition and microbial spoilage compared with other foods [19]. This means that small changes in the preservation conditions along the value chain, such as temperature or salt level variations, can have a high impact on the fish quality and safety, resulting in a fast degradation, higher food waste and the risk of intoxications.

On the market side, consumers are demanding more transparency in the labelling of seafood. To ensure the prevention and prompt detection of illegal activities and the access to all the information about the nature, origin and characteristics of the fish, monitoring of all the processes and quality analysis must be done thoroughly through the whole production chain and market, until it arrives to the consumers [20, 21]. The implementation of reliable methods to ensure a complete and correct traceability of the product and to avoid fraud is of primary interest for both industry and consumers.

#### **Figure 2.**

*Flow chart of seafood value chain from catch to consumer, indicating the different points where the quality of the fish should be monitored. ) Monitoring of chain conditions and quality attributes; ) inspection technologies and quality control.*

### **1.3 NIRS in fish analysis**

Nowadays, the quality, authenticity and safety control procedures in fish industry are carried out using traditional laboratory techniques, making virtually impossible any real-time application. However, some rapid techniques, such as NIRS, have shown the capacity of obtaining valuable information in a rapid way, which is important for the fish chain analysis [22].

NIRS is a tool already well developed in agriculture [23] and other products [24, 25]. The capacity of this technique to detect vibrations of molecules with polar bonds, such as those with hydrogen as C-H (aliphatic compounds, fat, oils, proteins), N-H (proteins) or O-H (water, alcohols, acids), is especially useful to determine the composition of organic matter. This makes this technique suitable for the quantification of the chemical composition of samples with a high percentage of fat, protein or water content and allows the determination of other parameters that depend on many chemical and physical properties such as the species [26], freshness, storage conditions (unfrozen or frozen-thawed fish) [27] or even non-polar analytes in low concentrations through indirect ways.

For all the previously mentioned reasons, NIRS, coupled with chemometrics, is starting to be considered as essential as other analytical methods for fish analysis. Indeed, the number of Web of Science indexed articles in which NIRS techniques have been applied to determine fish quality, safety or authenticity have increased rapidly during the last 20 years. While this technique is mostly used for fish quality control and authenticity, applications such as fish safety have begun to be explored in the last 10 years (**Figure 3**).

#### **Figure 3.**

*Number of publications since 2001 regarding NIR and fish. Data extracted from Web of Science in April 2022. The keywords used in topic, common for all the sections, were 'fish' AND 'NIR' OR 'near infrared spectroscopy'. For quality control, two independent searches were made: i) 'composition' OR 'fat protein' AND 'quality' ii) 'freshness' AND 'storage' NOT 'safety'. For authenticity, four independent searches were made: i) 'specie\*' AND 'identification' OR 'substitution' OR 'authentication' ii) 'origin' iii) 'farm\* wild' OR 'production method\*' iv) 'fresh thawed' OR 'unfrozen thawed' OR 'fresh frozen' OR 'unfrozen frozen'. For safety, two independent searches were made: i) 'spoilage' OR 'toxic' OR '\*amine\*' AND 'safety' ii) 'micro\*' OR 'bacteria' AND 'safety'.*

## **2. Applications of NIRS in fish chain**

While writing this chapter, the authors reviewed the use of NIRS in the fish chain regarding three different application fields: fish quality control, fish authenticity and fish safety (**Figure 4**). The supplementary material, which shows a summary of the works that will be reviewed in this chapter, is available in the **Appendix**.

## **2.1 Fish quality control**

Assuring the quality of products is one of the objectives that any industry must consider, and it is of major importance in the fish chain, due to the problems that are usually associated with its evaluation at an industrial level [28]. Unlike other foodstuff, quality of fish and fish products is more difficult to control, due to several factors, such as the variations in species, sex, age or habitats [29]. In this sense, fish quality control refers specially to the determination of the proximate composition of the fish samples and the evaluation of the freshness of the products. From one side, the measurement of the proximate composition of fishery species is very important, since it varies from one species and one individual to another. Moreover, it also gives an idea of the sexual stage of the fish [30], and it changes depending on many factors (diet, origin, rearing systems). The proximate composition determines the organoleptic quality of a specific fish product [31]. At the same time, the determination of the freshness of samples is another indicator of the quality of the product. Freshness is an ambiguous term, which can be interpreted in different ways, and it should not be used unless it is properly defined. Through this chapter, the concept of freshness is going to be a quality descriptor of fish samples, and it will refer to the time past after the catch of the fish. If that time is short, the sample will be considered fresh, as it will retain its original characteristics. As time passes, samples will lose quality, due to the action of biochemical, chemical, physical and microbial processes, and therefore losing freshness [32]. Nowadays, NIRS is becoming an alternative as a quality control method, due to its advantages over traditional analysis [33].

## *2.1.1 Proximate composition*

Fish is mainly composed of water, protein, fat and other minoritarian compounds [28]. As fish proximate composition is a quantitative analysis, regression techniques (usually partial least square regression or PLSR) are used to determine the composition of fish by NIRS, as an alternative to laboratory determinations.

Many authors have determined the proximate composition of different fish species using NIRS. In one of the first studies, NIRS was used to determine moisture, fat and

#### **Figure 4.**

*Classification of the problematics considered and reviewed under this chapter.*

protein content in Atlantic salmon (*Salmo salar*) fillets and in minced samples [34]. Principal component regressions (PCRs) were used to perform the calibrations, which were evaluated using a full cross-validation. This study illustrated the potentiality of NIRS for predicting the proximate composition of minced fish, obtaining with the best models a root mean square error of cross-validation (RMSECV) of 6.6, 3.8 and 3.0 g/ Kg in fat, moisture and protein analysis, respectively. However, they failed in the analysis of the whole fish, with RMSECV higher than 13.2 g/Kg in all the models. Years later, another study performed PLSR directly in fish fillets of Chinese export tilapia [31]. The regressions showed good results for the determination of moisture and lipid content, with r<sup>2</sup> = 0.96 and 0.97 and standard error of cross-validation or SECV = 0.87 and 0.71, respectively. Even though the regression made on the protein content had a lower coefficient of determination in the cross-validation (r<sup>2</sup> = 0.31), it showed good results regarding the error (SECV = 0.55). This difference was due to the small protein differences that tilapia fillets have between them, and it could be solved adding samples with more variability to the calibration set. It is important to highlight that they performed an external validation and not only a cross-validation, for moisture and protein determination, using new model-outside samples, which presented variations that authors considered acceptable between predicted and measured values (a maximum relative error of 1.67% for moisture and 6.48% for lipid). Similar studies regarding the analysis of the proximate composition in fish samples are shown in **Table 1**.

## *2.1.2 Freshness*

In this chapter, freshness has been considered as a quality characteristic, and it will be defined as the time passed after the catch. It is also influenced by the temperature during the storage [32]. Freshness is usually evaluated using sensory analyses, which consider several parameters such as flesh elasticity, pupil colour and skin odour, among others [43].

Although principal component analysis (PCA) is not a classificatory nor a discriminant method (since it is a non-supervised technique, which only takes into account the spectral information), and it should not be used with that purpose, some authors have applied it in order to see the differences between fresh fish and fish samples that have been stored in the fridge for days. In this sense, some authors used short-wavelength near infrared spectroscopy (SW-NIRS) to differentiate fresh from non-fresh rainbow trout (*Oncorhynchus mykiss*) samples, in fillets and minced [44]. Fish samples were stored at 4°C for 8 days, or at room temperature (21°C) for 24 hours. PCA results showed a clear separation between control samples (1 day stored) and fish kept 4 days or longer in refrigeration. Regarding fish at room temperature, a segregation was found for samples stored 10 hours or longer in comparison to control samples (stored 0 hours). Other studies performed PCA to discriminate between fresh Atlantic salmon (*Salmo salar*) and salmon stored at 4°C for 9 days, finding a clear separation between fresh and non-fresh fillets [45]. A similar experiment used PCA to discriminate golden pompano (*Trachinotus ovatus*) samples regarding the storage time using visible/near-infrared spectroscopy (VIS/NIRS) [46]. However, they concluded that samples stored for 0, 2 and 3 days could not be well discriminated using this analysis. They also created models to predict the storage time, but the model which used VIS/NIRS spectroscopy was not validated (only a model combining VIS/NIRS data and electronic nose information was validated, obtaining an accuracy of 93.3%). Another study analysed the spectra of rohu fish (*Labeo rohita*) sliced into pieces, using SW-NIRS and PCA to distinguish between


## *Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*

*\*PUFAs: polyunsaturated fatty acids; MUFAs: monounsaturated fatty acids, FAs: fatty acids; MLR multiple linear regression; RMSEP: root mean squared error of prediction.*

### **Table 1.**

*Use of NIRS to analyse proximate composition of fish.*

fresh fish (after being collected) and fish stored in the fridge at 4°C during 4, 7 and 12 days [47]. In the PCA analysis, samples were well grouped by their freshness, so that four clusters were created (one per each measurement day).

Different studies aim not only to separate fresh samples from non-fresh ones, but also to make models to correlate spectral data with the storage time. One of them explored the suitability of SW-NIRS to estimate the freshness of the fillets of two fish species: cod (*Gadus morhua*) and salmon (*Salmo salar*) [32]. PLSR models were created to predict the number of days that the fish has been stored and validated using a

full cross-validation. Cod gave the best correlation with the visible wavelength range (r2 = 0.97, RMSE = 1.04 days) and salmon with the NIR range (correlation of 0.98 and RMSE = 1.20 days). A similar study predicted the freshness in cod (*Gadus morhua*) fillets with a handheld interactance probe, using VIS/NIRS. Fillets were stored in a cold room (between 2 and 4°C) for 13 days, making measurements at days 0, 2, 4, 7, 10 and 13 [48]. PLSR models were validated using a leave-one-out cross-validation. The best results were obtained selecting some wavelengths of the visible range (448, 487, 606 and 646 nm) and using SNV, giving a correlation coefficient of 0.93 and an RMSECV = 1.66 days. Other researchers used VIS/NIRS to perform a similar study on Atlantic salmon (*Salmo salar* L.) fillets [49]. In those, PLSR models using the wavelength region between 605 and 965 nm and the pre-treatment with SNV gave the lowest error in the leave-one-out cross-validation (RMSECV = 1.91 days). In another work the freshness of cod (*Gadus morhua*) was predicted using VIS/NIRS [50]. Authors built PLSR models to predict the quality index method (QIM), a sensory analysis made to determine the freshness of cod fillets. A full cross-validation was used to test the performance of the models, giving the analysis made with the visible part of the spectra the best results (correlation coefficients = 0.96 and RMSE = 2.6 points).

#### **2.2 Fish authenticity**

The globalization and expansion of the fish and aquaculture sector, in addition to the increasing public concern about food quality, have caused a growing interest in several issues related to fish authenticity. According to the European Regulation (EU) n. 1379/2013 [51], fishery and aquaculture products must be labelled with the commercial designation, proper scientific name of the species, production method (e.g. caught, farmed), fishing gear (e.g. hook, trap, trawl), catch or production area and storage method (unfrozen or frozen-thawed). Other claims, such as environmental and production techniques, can also be reported on the label, and all this information must be verified by effective methods. Special mention is the differentiation of farmed from wild-produced seafood, which is becoming a concern among consumers, since the quality and the price may be affected by the production method. The presence of errors in the label information about fish origin and production process is increasing, so that nowadays fish is the second most vulnerable category of food to fraud [52]. In order to protect consumers and with the aim to avoid it, the assessment of seafood origin is increasing as a security measure. Regulatory interventions are trying to stop the mislabelling or the substitution of wild with farmed fish, which is mitigating risks for the consumer's confidence and health [53].

Both the geographical origin and the production method, among others, can strongly affect the characteristics of the two types of products, whose discriminating properties are usually difficult to determine. Several analytical techniques have been traditionally used to assess fish authenticity. However, even though they are well established, there is still a necessity for faster, easier and more affordable methods [52].

#### *2.2.1 Species identification*

The substitution of valuable species with cheaper ones at any point of the supply chain is one of the most common frauds, sometimes in an unintentional way due to the similarities between species or the use of different names for the same species. However, the differences in the economic value, the exploitation of endangered species, the replacement with poisonous fish and their difficult identification, make the

substitution of fish species an extended problem, especially severe after processing, at the retailers and supermarkets [54]. The mentioned frauds particularly affect fish fillets and ready-to-eat products, such as fish patties, which cannot be recognized through the traditional morphological analysis.

The use of NIRS with the objective of discriminating between species has been explored during the last years [52]. For instance, in a preliminary study under industrial conditions, some authors used NIRS coupled with partial least square discriminant analysis (PLS-DA) and linear discriminant analysis (LDA) on PCA scores to discriminate between fishmeal made with different fish species: salmon (*Salmo salar*), blue whiting (*Micromesistius poutassau*), mackerel (*Scomber scombrus*) or herring (*Clupea harengus*) [55]. They obtained a correct prediction percentage > 80%, tested using cross-validation.

In other studies, authors measured the skin and meat of two different species of cod (winter cod and cod), mullet (red mullet and Atlantic mullet) and trout (samlet and salmon trout) with a handheld NIR spectrometer [56]. The data were analysed using PCA and soft independent model of class analogies (SIMCA), obtaining promising results with the last method. Other authors used Fourier transform NIR (FT-NIR) to distinguish fillets of red mullet (*Mullus surmuletus)* and plaice (*Pleuronectes platessa*), fish with higher economic value, from Atlantic mullet (*Pseudupeneus prayensis*) and flounder (*Platichthys flesus flesus*), species which are cheaper [57]. LDA and SIMCA were used to classify them, obtaining a sensitivity higher than 70% and a specificity of 100% for FT-NIR and SIMCA, and a 100% correct classification with LDA for both techniques.

The differentiation between different carp species: silver (*Hypophthalmichthys molitrix)* black (*Mylopharyngodon piceus*), bighead (*Aristichthys nobilis*), grass (*Ctenopharyngodon idellus*) and common carp (*Cyprinus carpio*) and also crucian (*Carassius auratus*) and bream (*Parabramis pekinensis*) has also been studied [58]. Only nine selected wavelengths in the 1000–1799 nm region after the pre-treatment were used. In this case, two models using PCA-LDA and the fast Fourier transformation pre-processing (FFT) before LDA analysis were built. They showed a 100% accuracy, specificity, sensitivity and precision. Fish surimi made from white croaker (*Argyrosomus argentatus*), hairtail (*Trichiurus haumela*) and redcoat (*Nemipterus virgatus*) was successfully classified by using LDA and multiplicative scatter correction (MSC) as pre-treatment. The results of the prediction of the calibration and the validation data sets were 98.5% and 100%, respectively [59].

More recently, an attempt to distinguish Atlantic cod (*Gadus morhua*) from haddock (*Melanogrammus aeglefinus*) was made. Raw fillets and patties were measured with a handheld and a benchtop instrument, with the spectra pre-processed using SNV, MSC or SG, and analysed with LDA and SIMCA. LDA with MSC pre-treatment obtained a 100% of correct classifications for both fillets and patties for the external validation set, regardless of the instrument. The SIMCA method also obtained a 100% of successful classification of fillets using SNV and SG (first and second derivative) for the benchtop instrument and using MSC for the portable NIR device. With SIMCA, 100% correct predictions of patties were obtained for all instruments and preprocessing techniques [60].

### *2.2.2 Farmed vs. wild fish and types of farming*

In the last years, there has been an increasing interest from farming industries, retailers and even from consumers in investigating the differences between cultured and wild fish [61]. Most of the studies which differentiate farm from wild fish are centred in fish species such as European sea bass (*Dicentrarchus labrax* L.), which is one of the most economically important fish species in the whole Mediterranean area [62]. The intense competition among producing countries in the Mediterranean Sea (Turkey and Greece are the largest producing countries and Italy, Spain and France are the world's leading importers) and the lowering of the market prices are requiring ways to differentiate sea bass quality. The differences in the production methods (farm and wild specimens) and, among farmed individuals, in the rearing systems and the feeding regimens used, may affect the flesh quality. Usually, farmed fish presents lighter fillet colour, softer texture and milder flavour than wild fish [36, 61]. Previous studies aimed to differentiate between farmed and wild sea bass were based on discriminating the two classes depending on their protein and amino acids composition, metallic ions and fatty acids groups, being usually the lipid profile the differential factor. However, the extraction of lipids and the transformation of the triglycerides into their corresponding fatty acid methyl esters (FAMEs) plus the analysis by chromatography with a flame ionization detector (GC-FID) are a tedious and time-consuming methodology, which not only generates toxic waste but can also provoke the oxidation of the polyunsaturated groups [63].

NIRS has shown its potentiality in some studies as a rapid method to classify sea bass according to rearing systems [53]. Some authors used an NIR spectrometer to discriminate between four types of sea bass rearing systems: extensive, semi-intensive, intensive and sea-cage in fresh minced fish and freeze-dried minced samples [36]. They performed a four-cluster SIMCA analysis, which was evaluated using a full crossvalidation. The best results were obtained with the freeze-dried minced fillets (83%, 80%, 74% and 83% of samples well classified for extensive, semi-intensive, intensive and sea-cage methods, respectively). Similar results were obtained by [37] in the discrimination of organic or semi-intensive sea bass. Some years later, NIRS was also used to discriminate between farm and wild seabass fillets, which were minced before the analysis [53]. They developed three different approaches: an authentication using measured chemical variables (PLS-DA\_mc), an authentication using estimated chemical variables (PLS-DA\_ec), an authentication with direct use of spectral data (PLS-DA\_NIR) and the WPTER algorithm. The evaluation of the validation data set gave very similar results for all the techniques in the classification of declared wild samples. Regarding the declared farming samples, PLS-DA\_mc classified correctly all the samples, while PLS-DA\_ec misclassified 2, and PLS-DA\_NIR and WPTER misclassified 3 (in all the cases out of 34). All the methods indicated that the most relevant spectral regions in the models were the ones related to the absorbance of the CH, CH2, CH3 and H2O groups, which corresponded to fat, fatty acids, and water content, highlighting that the difference between farm and wild sea bass is very related with the fat/water percentage and the lipid profile. Other authors also studied the differences between sea bass, according to the production method (wild or farmed) and the rearing system used (extensive, semi-intensive or intensive) [62]. In this study, an NIR spectrometer was used to analyse minced and homogenized fillets. In this case, cross-validation and an external validation were made. PCA was used as exploratory analysis and two different orthogonal partial least square-discriminant analysis (OPLS-DA) models were performed. The evaluation of the external validation showed an overall classification rate of a 100% for the production method and 94.44% for the farming system. Looking at the categories, all the farm and wild samples were correctly classified, while regarding the farming system, wild samples presented a classification rate of 100%, extensive samples 66.67%, semi-intensive samples 80.00% and intensive samples

### *Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*

100%. Although most of the studies regarding discrimination between farm and wild fish are performed in sea bass, other species have also been analysed. Segregation between wild and farm sole (*Solea solea*) and turbot (*Psetta maxima*) has also been performed [64]. Here, several chemometric methods were tested, obtaining the best results with logistic regression in sole (90% of precision, recall and F1-score) and with support vector machine in turbot (99% of precision, recall and F1-score) in the evaluation of the test set.

#### *2.2.3 Geographical origin*

NIRS is one of the most used techniques to determine the origin of a lot of types of foods (oil, coffee, wine, etc). However, especially in fish and fish products, its use for such purpose is a challenge due to the difficulty in proving the geographical origin of a particular fish. A very large number of samples must be measured for each location due to a high heterogeneity among them, depending on the season or different years, the available food, if the fish has been captured before or after the spawning season, etc. With the aim of determining the viability of NIRS regarding the geographical origin, it often requires multi-disciplinary methods that could consider all the environmental and genetic factors that determine fish characteristics. Even so, there are some studies that successfully applied NIRS to determine the geographical origin of fish products.

Some authors used NIRS to classify tilapia fillets in four different Chinese regions (Guangdong, Hainan, Guanxi and Fujian provinces) [31]. The classification models were carried out with SIMCA, achieving more than 80% correct classification for the Guangdong, Hainan and Fujian and 75% of the Guangxi samples by the cross-validation method. None of them were assigned to more than one category, less than 20% to none of the classes and only less than 2% were incorrectly classified. The differentiation between minced fish fillets made using European seabass (*Dicentrarchus labrax* L.) caught in three areas of the Mediterranean Sea has been also explored [62]. In this case, NIRS was used in combination with PLS-DA method. The external validation with a test set resulted in 100% of the eastern, 88% central and 85% western Mediterranean samples were correctly classified. In another study, NIRS was used successfully to assess the geographical origin (Morocco, Spain, Tunisia or Croatia) of minced semi-finished and finished salted anchovies (*Engraulis encrasicholus*) [65]. The combination of models based on orthogonal PLS-DA gave the correct identification of the origin of both types of anchovies with >98% sensitivity, > 99% specificity and > 99% accuracy in an external validation using a test set with the 20% of the samples. Moroccan samples were distinguished for the proteins and degradation compounds absorption bands, Tunisian anchovies for the unsaturated lipids, while Croatian and Spanish samples were classified for their differences on both types of absorption bands.

#### *2.2.4 Unfrozen vs. frozen-thawed fish*

Freezing, due to its advantages, is intensively used for preserving fish. It maintains the product safety, the sensory qualities and also the nutritional characteristics. However, freezing the products, as well as the storage and the thawing process, changes the physical and biochemical properties of the fish muscle [66]. Recent technological advances in freezing and the posterior storage have allowed to minimize the damage caused by the thawing process, while maintaining the product quality. The impact the freezing process does on the product depends on several factors inherent to fish characteristics (genetics, muscle type, husbandry) and the post-mortem treatments

(freezing rate, storage time, storage pressure and temperature, thawing method) [67]. Slow freezing leads to the formation of large extracellular ice crystals which damage the cell membranes and muscle proteins [68–70]. Also, in general, freezing-thawing cycles are related to the degradation of textural properties due to physical damages and protein denaturation and oxidation [66].

As it has been reported before, most of the analytical techniques used for determining whether the fish has been frozen or not, despite their accuracy, are destructive, time-consuming and expensive, making them not suitable for commercial applications [71]. As an alternative, NIRS, in combination with qualitative chemometric methods, has been adopted as an efficient solution for this determination.

Several authors have used NIRS and chemometrics to discriminate between unfrozen and frozen-thawed fish. FT-NIRS has been used, coupled with two different classification algorithms (LDA and SIMCA), to discriminate between unfrozen and thawed Atlantic mullet (*Pseudupeneus prayensis*) fillets [57]. LDA provided the most accurate results in the evaluation of the external validation set, with correct classification rates of 97.2% for unfrozen and 100% for thawed samples. The same classification algorithms were used in [72] to distinguish whether red sea bream (*Pagrus major*) samples were unfrozen or frozen-thawed using an NIR spectrophotometer in interactance mode. They observed that the change in total reflectance, the major effect of the freeze-thawing process, arises from changes in the scattered light at the surface of the fish due to alterations in its structure. The classification accuracy when using absorbance spectra without any pre-processing (100%) was much higher than for spectra pre-treated with the multiplicative scatter correction (MSC) (81%), confirming that light scattering contains most of the information. VIS/NIRS techniques were as well used to distinguish unfrozen from thawed swordfish (*Xiphias gladius L*) cutlets stored at different temperatures (10 and 18°C) for several days [73]. VIS/NIR spectra were collected using a portable spectrophotometer and a NIR monochromator. The correct classification percentage for VIS/NIR spectra was ≥96.7%, while for the NIR spectra was ≥90.0%, with no effect on the classification due to the temperature or days that the frozen samples were stored. In another reference, the authors distinguished unfrozen from frozen tuna fillets (*Thunnus thynnus*) which were frozen and, after 5, 21 and 35 days, thawed and then re-scanned [74]. In this study, the PLS-DA algorithm with double cross-validation was used to classify unfrozen from frozen-thawed fillets with a 92% and 82% of correctly classified samples, respectively. Some researchers have tried to discriminate between tilapia fillets (*Oreochromis*) that have been subjected to different freezing-thawing cycles, from one to seven [75]. In this project, NIR spectra were collected at each cycle and the test data analysed with PCA and discrimination analysis, obtaining a correct classification of 86% for once frozen-thawed fillets and 93% for samples subjected to several freezing cycles. Finally, in other recent study, unfrozen and frozen-thawed bigeye tuna (*Thunnus obesus*) fillets were also measured with NIRS for their discrimination [76]. To include variability, samples were injected with different commercially used concentrated solutions (NaCl, polyphosphates and protein hydrolysate). Three different models, based on PLS-DA, were developed giving NIRS the best results with an accuracy of 0.91 and an error rate of 0.10 for the prediction of the validation set.

### **2.3 Fish safety**

Fish and fish products are very perishable, being subject to a fast deterioration process and becoming easily unsuitable for consumption and, therefore, a possible

#### *Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*

threat to public health. The biological composition and characteristics of fish, i.e. low percentage of connective tissue, autolytic enzymes, neutral pH, high water activity [27] and the growth of spoilage bacteria, contribute to the short shelf-life of these products [45]. It is estimated that 25% of the fish losses are initiated by microbial and chemical deterioration [77].

Even though most studies regarding NIRS in the fish industry have been conducted to determine their quality and authenticity, the use of this technique in food safety evaluation is still relatively new but constantly increasing [25]. During the last years, many studies have been conducted in the field of fish safety, in order to determine the presence of spoilage compounds and microorganism in fish products.

#### *2.3.1 Detection of spoilage compounds*

Some toxic compounds produced during fish spoilage can be measured by NIRS to evaluate fish safety. An important group is formed by biogenic amines, with histamine being one of the most critical, as it is related with the most common foodborne illness associated with fish consumption [78]. They are a group of nitrogenous compounds, usually measured to evaluate the safety of aquatic products. Among them, the most common are histamine, putrescine, cadaverine and tyramine. Biogenic amines are formed by the decarboxylation of amino acids or by the amination and transamination of aldehydes and ketones. The consumption of fish products with excessive levels of biogenic amines may lead to food intoxication, so they have become an important index to evaluate fish safety [79].

NIRS has been used as a tool to determine the cadaverine content in homogenized and ultrafiltered solutions of Chub Mackerel (*Scomber japonicus*) stored at different temperatures (5 and 25°C) [80]. The r2 obtained in the regression analysis was 0.98 and 0.99 for the different dilutions made. However, authors mentioned that NIRS measurements of cadaverine content made directly in the fish were not successful. Histamine was also determined by this spectroscopic technique. It is the only biogenic amine for which international regulatory levels have been established [78]. The presence of this amine is due to the bacterial decarboxylation of histidine, an amino acid present in high levels in fish muscle, specially from Scomberiscida and Scombridae families (which include tuna). Studies have tried to predict this compound in order to assure fish safety. Histamine content has been measured in different dry extracts of skipjack tuna (*Katsuwonus pelmis*) using NIRS [81]. The standard error of prediction or SEP in all the cases (using different solvents and different pre-processing techniques) was between 2.94 and 3.47 in the validation set, with r2 between 0.63 and 0.79. Later on, in a similar experiment, histamine in raw (*Thunnus albacares*) and in processed tuna (*Katsuwonus pelamis* and *Thunnus albacares*) was detected, which were previously minced and homogenized [78]. Both models showed r2 values higher than 0.97 and RMSEP lower than 10 mg kg<sup>1</sup> , with the obtained results being better for processed tuna. Considering the mentioned studies, promising results have been seen in all the works regarding the detection of biogenic amines in fish by NIRS. However, more efforts are still needed to determine these compounds directly in the fish fillet, without the necessity of destroying the sample.

Biogenic amines are not the only compounds associated with the spoilage of fish. Trimethylamine concentration can also be used as an indicator of fish spoilage, since it is related with the putrefaction process and the fishy odour. This compound has been estimated in fresh fish (*Hypophthalmichthys molitrix*) using FT-NIRS and different chemometric algorithms [77]. In this case, measurements were made directly in the

fish, which was sliced in small cubes. Regression analysis showed RMSEP between 5.10 and 5.75 mg N/100 g and correlation coefficients of prediction between 0.94 and 0.98. K-value is another indicator of fish deterioration that is related to the degradation of ATP in the fish flesh. Likewise, this index was also estimated in fresh silver carps (*Hypophthalmichthys molitrix*) using FT-NIRS to make the measurements directly in the fish. Several chemometric techniques were compared, obtaining as best result an RMSEP of 0.036 and a correlation coefficient of prediction of 93.74% [82]. Another way to measure the spoilage of saithe (*Pollachius virens*) and hoki (*Macruronus novaezelandiae*) fillets has been also explored [33]. Here the authors developed PLSR models to predict free fatty acids (FFA), fluorescent interaction compounds (OFR) and thiobarbituric acid reactive substances (TBARS), in order to measure the lipid oxidation, one of the major deteriorative reactions in fish muscle. Results showed acceptable results for the test set in both species (r2 between 0.63 and 0.98), although some high errors were found (RMSEP of 2.90 and 3.43 g FFA/100 g lipids, 0.97 and 0.55 μmol malonaldehyde diethylacetal/kg of samples for TBARS and 5.23 and 1.20 for OFR in hoki and saithe, respectively). Similar results were found regarding FFA [83] and on finding lipid oxidation products in vegetable oils [84]. Other similar analysis were performed using NIRS to predict the pH, TBARS, total volatile basic nitrogen (TVB-N) and K-value of minced bighead carp (*Aristichthys nobilis*) [85]. PLSR together with competitive adaptive reweighted sampling (CARS) algorithm and pre-processing methods were used to make regressions, obtaining satisfactory results for the prediction of the test set (coefficients of prediction of 0.945, 0.932, 0.954 and 0.807 and RMSEP of 0.081, 2.099, 0.107 and 6.509 for pH, TVB-N, TBA and K value, respectively).

#### *2.3.2 Microorganisms*

Fish is one of the most vulnerable aquatic products, as it serves as a growth medium for microorganisms, which can be either pathogenic or cause fish spoilage. In this sense, spectroscopic techniques have shown potential for spoilage monitoring to confirm if fish is safe for consumption or not, since conventional methods to determine microorganisms are really time-consuming and tedious [86]. However, although some works have been done regarding the detection of microorganisms by NIRS and the potentiality of the technique is big, this is an application that must be studied carefully, and it must be validated using techniques with limits of detection more sensitive than NIR.

Some studies tried to use SW-NIRS to quantify microbial loads (expressed as total viable count) in rainbow trout (*Oncorhynchus mykiss*) [44]. Regression models were built based on spectra taken in the flesh and skin of fish fillets, stored at 4°C for 8 days, and also in minced samples stored at room temperature 24 hours. All the models gave goods results, the model made in the flesh being slightly better (r<sup>2</sup> = 0.97 and SEP = 0.38 log CFU/g) compared with the models of the skin and the minced samples (r2 = 0.94 and 0.82 and SEP = 0.53 and 0.82 log CFU/g, respectively). Similar experiments have used FT-NIRS to detect and predict microbial spoilage on Atlantic salmon fillets (*Salmo salar*) [45]. The results showed that the regression analysis was able to predict the numbers of bacteria (expressed as total aerobic plate counts) after 9 days of storage at 4°C, with an r2 of 0.64 and a RMSEP of 0.32 log cfu/g in the validation set. A portable spectrometer was also used to determine the total bacteria content in flounder fillets (*Paralicthys olivaceus*), expressed as the aerobic plate count of total bacteria, after 8 days of storage at 4°C [87]. Different models based on different chemometrics techniques were developed, obtaining the best results using a

combination of genetic algorithm and artificial neural networks, with an r<sup>2</sup> of 0.966 and an RMSEP of 0.083 in the test set.

## **3. Conclusions**

NIRS has been proved as an effective analysis tool for the fish value chain. Although the use of this technology in fish and fish products routine inspection has recently increased, more and more studies are available each year, some applications are more developed than others. The monitoring of the quality and authenticity of the fish chain by NIRS is well known, with applications that are nowadays implemented in the food industry. However, more efforts are still needed regarding the use of this technology for safety issues, where the limit of detection still represents a big challenge. Even if some studies have been carried out successfully during the last decade, these applications should be studied carefully, and more investigations must be done before trying to implement NIRS as an alternative to the already established destructive methods.

## **Acknowledgements**

The authors greatly acknowledge the Basque Government—Department of Economic Development, Sustainability and Environment—Vice. Dept. of Agriculture, Fishing and Food Policy, Directorate of Quality and Food Industries for the funding of the project ELIKatea 4.0 and for the scholarship of Sonia Nieto-Ortega.

**Appendix**


## *Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*



## *Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*


*Infrared Spectroscopy - Perspectives and Applications*


### *Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*



## *Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*

## **Author details**

Sonia Nieto-Ortega\*, Rebeca Lara, Giuseppe Foti, Ángela Melado-Herreros and Idoia Olabarrieta AZTI, Food Research, Basque Research and Technology Alliance (BRTA), Parque Tecnológico de Bizkaia, Derio, Spain

\*Address all correspondence to: snieto@azti.es

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Applications of Near-Infrared Spectroscopy (NIRS) in Fish Value Chain DOI: http://dx.doi.org/10.5772/intechopen.105736*

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**Chapter 6**
