Monitoring Red Sea Bream Scale Fluorescence As a Freshness Indicator

Article Monitoring Red Sea Bream Scale Fluorescence as a Freshness Indicator

Qiuhong Liao 1,*, Tetsuhito Suzuki 1, Kohno Yasushi 2, Dimas Firmanda Al Riza 1,3 , Makoto Kuramoto 4 and Naoshi Kondo 1

1 Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan; [email protected] (T.S.); dimasﬁ[email protected] (D.F.A.R.); [email protected] (N.K.) 2 Department of Planning and Environment, Ehime Research Institute of Agriculture, Forestry and Fisheries, Matsuyama, Ehime 799-2405, Japan; [email protected] 3 Agricultural Engineering Department, Faculty of Agricultural Technology, University of Brawijaya, Malang 65145, Indonesia 4 Advanced Research Support Centre, Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-090-8218-2560

Academic Editor: Maria Angeles Esteban Received: 26 May 2017; Accepted: 3 July 2017; Published: 10 July 2017

Abstract: Red sea bream (Pagrus major) scale fluorescence characteristics were identified as a potential rapid and non-destructive means for assessing the fish’s freshness. To investigate this, live red sea breams were purchased, slaughtered, and prior to measurement, stored at 22 ± 2 ◦C for 27 h. During subsequent storage, the K value of the dorsal meat—as a standard freshness indicator—along with front-face fluorescence spectra of representative dorsal scales, were measured simultaneously at 3 h intervals. Two major fluorescent peaks, A and B, were identified with excitation and emission wavelength pairs of 280/310 nm and 340/420 nm, which were mainly contributed to by tyrosine and collagen, respectively. Subsequent analysis showed that the fluorescence intensity ratio of peak B to 2 A(IB/IA) increased linearly during storage (R = 0.95) and is proposed as a potential non-destructive index of fish freshness. Thus, our results suggest that the fluorescence characteristics of fish scales can be used to assess fish carcass freshness during storage.

Keywords: freshness; scales; front-face ﬂuorescence; intensity ratio

1. Introduction Red sea bream is an important commercial marine culture fish species in Japan due to its palatability and being highly valued by Japanese consumers. According to the Ministry of Agriculture, Forestry and Fisheries in Japan, over the last decade, around 90,000 tons/year have been cultured or captured. With ever increasing demands for the delivery of high quality red sea bream, mariculture industry, retailers, consumers, and regulatory officials desire an improved, and preferably non-destructive, method for rapidly determining the quality and freshness of this high-end product. Freshness is a major determinant in most fish or fishery products, since it is the most relevant attribute that consumers use to evaluate taste and safety [1]. Various traditional methods, such as sensory [2], microbiological [3], and biochemical [4] methods have been investigated to evaluate the freshness of various commercial fish species. Of these, the most used method on a wide variety of fish species is the K value, a freshness index based on the adenosine triphosphate (ATP) degradation product ratio, due to its direct relationship to biochemical changes. The ATP degradation process is usually described as follows: ATP → adenosine diphosphate (ADP) → adenosine monophosphate

Fishes 2017, 2, 10; doi:10.3390/ﬁshes2030010 www.mdpi.com/journal/ﬁshes Fishes 2017, 2, 10 2 of 11

(AMP) → inosine monophosphate (IMP) → inosine (HxR) → hypoxanthine (Hx). The K value is calculated as the percentage rate of (HxR + Hx) to the sum of ATP and degradation products (ATP + ADP + AMP +IMP + HxR + Hx), and is widely used as a standard fish freshness indicator [5]. Recently, a wide range of new instrumental approaches, such as spectrophotometers [6], image analyzers [7], colorimeters [8] or electronic tongue [9], have been proposed as an alternative to the traditional complex chemical analyses. However, many of these traditional and new techniques still have drawbacks in terms of being time consuming, costly, requiring laboratory facilities, or are difficult to interpret. Given the potential rapid losses in fish freshness after capture and death, a simple, rapid and convenient means of freshness assessment is sought. In the last several decades, fluorescence spectroscopy has received particular attention due to its nondestructive nature, short sampling time, high sensitivity, and selectivity [10]. Fluorescence spectroscopy provides important information on the presence of fluorophores which change in response to the physical–chemical environment and provide a window into the freshness state of the sample. These fluorophores can be directly monitored and changes in fluorescence intensity of a given fluorophore can be directly correlated with fluorophore concentration [11]. Previous studies carried out by our research group have shown that fluorescence properties of fish eye fluid are closely related to freshness changes. Therefore, fluorescence spectroscopy offers the potential for fast and convenient assessment of fish freshness. Among fluorescence spectroscopy techniques, front-face fluorescence spectroscopy reduces the optical path for illumination, ensuring only the surface of the material is sampled, providing a potentially rapid, non-destructive and quantitative investigation of fluorophores of turbid or solid intact samples [12,13]. Scales attached to the surface of the fish body contain significant fluorescent organic substances and their changes during storage could be tracked using their fluorescence signals [14], making the scales a potential indicator of freshness decline. In the near future, these fluorescence data from scales with specific excitation and emission wavelength bands will be expected to be assessed with a device such as a multiplex sensor [15] or fluorescence imaging [16], making a simpler, faster and convenient method for intact fish freshness assessment based on the fish surface fluorescence. Therefore, the aim of this study is to identify and demonstrate the relationship between major fluorescence signal changes in red sea bream scales and freshness during storage using front-face fluorescence spectroscopy.

2. Materials and Methods

2.1. Red Sea Bream Red sea breams (Figure1) were purchased from Taisei Suisan, Ltd. (Ehime prefecture, Japan), from 17 August 2016 to 11 January 2017 with an average fork length of 42.6 ± 1.6 cm, weight of 1072 ± 106 g and age of 19–24 months. At the commencement of the storage experiment, fish were killed by puncturing the brains and spinal cord, cleaned, wrapped in a plastic bag to maintain humidity, and then stored at 22 ± 2 ◦C for 27 h. During this storage, samples (meat and scales) were collected every 3 h from the beginning of storage. Meat from the dorsal part of the fish was used for K value measurement (a standard freshness evaluation parameter). So-called “key scale” (short diameter: 0.71 ± 0.064 cm, long diameter: 0.92 ± 0.057 cm, thickness: 0.17 ± 0.055 mm) between the dorsal fin and the lateral line, which are representative of the features of the fish scales [17], were collected for fluorescence spectra measurement. K value measurement of meat was performed on 3 replicates and fluorescence spectra measurement of scales was performed on 9 replicates at each sampling time. In addition, scales collected at the beginning of storage were used for a water loss experiment (3 replicates) and scanning electron micrograph (SEM) observations. FishesFishes2017 2017, 2,, 102, 10 3 of3 of 11 11

(a) (b) Figure 1. Body of red sea bream (a) and “key scale” from fish body white border (b). Figure 1. Body of red sea bream (a) and “key scale” from fish body white border (b). 2.2. K Value Measurement 2.2. K Value Measurement In this study, the K value was measured using a Freshness Checker (QS 3201, QS solution, Miyagi,In this Japan), study, which the K is valuebased wason a measuredpaper electrophoresis using a Freshness method. Checker Approximately (QS 3201, 200 QSmg solution,meat from Miyagi,the dorsal Japan), part which of the is fish based was on cut a into paper small electrophoresis pieces and placed method. in Approximately0.6 mL of extraction 200 mg reagent meat A from(supplied the dorsal by QS part solution), of the fish then was neutralized cut into small using pieces reagent and placed5.61% inpotassium 0.6 mL of hydroxide. extraction reagentNext, the Asupernatant (supplied by was QS solution),dropped in then the neutralized electrophoresis using buffer reagent solution 5.61% potassiumsprayed electrophoresis hydroxide. Next, paper the to supernatantconduct at 800 was V dropped for 7 min. in After the electrophoresis completion of electrophoresis, buffer solution the sprayed paper electrophoresis was dried at 100 paper °C using to ◦ conducta forced at air 800 convection V for 7 min. dryer After (Paper completion dryer QS of electrophoresis,4201, QS solution, the Miyagi, paper was Japan). dried The at developed 100 C using spots a forcedon the air dried convection electrophoresis dryer (Paper paper dryer were QS then 4201, illuminated QS solution, at 254 Miyagi, nm and Japan). recorded The by developed a digital camera spots on(Canon the dried EOS electrophoresis M3, Tokyo, Japan). paper wereFinally, then the illuminated recorded spots at 254 were nm anddigitized recorded and bythe a K digital value camera for each (Canonspot calculated EOS M3, Tokyo,by a Spot Japan). Analyzer Finally, (QS the solution). recorded spots were digitized and the K value for each spot calculated by a Spot Analyzer (QS solution). 2.3. Scales Spectra Acquisition 2.3. Scales Spectra Acquisition 2.3.1. Front‐Face Fluorescence Spectra Measurement 2.3.1. Front-Face Fluorescence Spectra Measurement Front‐face fluorescence spectra of scales were measured using an F‐4500 fluorescence Front-face fluorescence spectra of scales were measured using an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Scales without prior preparation were measured at room spectrophotometer (Hitachi, Tokyo, Japan). Scales without prior preparation were measured at temperature using a quartz‐windowed standard surface fluorescence cell with a single‐position cell room temperature using a quartz-windowed standard surface fluorescence cell with a single-position holder, which was set at a 30° incidence angle to minimize the reflected light, scattered radiation, and cell holder, which was set at a 30◦ incidence angle to minimize the reflected light, scattered radiation, depolarization phenomena from the sample [18]. The scales were stacked up into a cell for front‐face and depolarization phenomena from the sample [18]. The scales were stacked up into a cell fluorescence spectra measurement immediately after removal from the fish body. A photomultiplier for front-face fluorescence spectra measurement immediately after removal from the fish body. voltage of 700 V was used to reduce detector saturation. The instrumental settings were: slit width 5 A photomultiplier voltage of 700 V was used to reduce detector saturation. The instrumental nm (for both excitation and emission), excitation wavelengths 200–600 nm (every 10 nm) and settings were: slit width 5 nm (for both excitation and emission), excitation wavelengths 200–600 nm emission wavelengths 210–700 nm (every 10 nm), and a scan speed of 12,000 nm/min. (every 10 nm) and emission wavelengths 210–700 nm (every 10 nm), and a scan speed of 12,0002.3.2. nm/min. Infrared Spectra Measurement for Compound Identification 2.3.2. InfraredIkoma et Spectra al. [19] Measurement found that the for infrared Compound (IR) spectrum Identification has characteristic peaks corresponding to the organic and inorganic components of the fish scale, which is a nanocomposite consisting of type Ikoma et al. [19] found that the infrared (IR) spectrum has characteristic peaks corresponding to I collagen and calcium‐deficient apatite containing carbonate ions. Thus, infrared spectroscopy was the organic and inorganic components of the fish scale, which is a nanocomposite consisting of type used to identify the main compounds of the red sea bream scale in the current study. I collagen and calcium-deficient apatite containing carbonate ions. Thus, infrared spectroscopy was Infrared spectra of the internal side (closest to skin) and the external side (exposed to air) of the used to identify the main compounds of the red sea bream scale in the current study. raw scales were obtained with a Thermo Scientific Nicolet iS5 FTIR (Fourier Transform Infrared) Infrared spectra of the internal side (closest to skin) and the external side (exposed to air) of spectrometer (Thermo Fisher Scientific, Kanagawa, Japan) equipped with an iD5 attenuated total the raw scales were obtained with a Thermo Scientific Nicolet iS5 FTIR (Fourier Transform Infrared) reflectance (ATR) accessory. The scale sample was placed on a prism (ZnSe) and compressed lightly spectrometer (Thermo Fisher Scientific, Kanagawa, Japan) equipped with an iD5 attenuated total using a pressure clamp. Spectra were measured from a wave number of 4000–500 cm−1 with a reflectance (ATR) accessory. The scale sample was placed on a prism (ZnSe) and compressed lightly resolution of 4 cm−1 with 32 scans for each spectrum. Spectra were collected and peaks detected using using a pressure clamp. Spectra were measured from a wave number of 4000–500 cm−1 with a Omnic software (Thermo Fisher Scientific). resolution of 4 cm−1 with 32 scans for each spectrum. Spectra were collected and peaks detected using Omnic software (Thermo Fisher Scientific).

FishesFishes2017 2017, ,2 2,, 10 10 44 of of 11 11

2.4. Scanning Electron Micrograph Image Acquisition 2.4. Scanning Electron Micrograph Image Acquisition In order to observe the morphology of the scales, the scales were cleaned with distilled water, In order to observe the morphology of the scales, the scales were cleaned with distilled water, dried, and then were coated with platinum (JEOL JFC 1600 Auto Fine Coater, Tokyo, Japan) for 2 min dried, and then were coated with platinum (JEOL JFC 1600 Auto Fine Coater, Tokyo, Japan) for 2 min at 10 mA, before being examined with a scanning electron microscope (SM‐200, Topcon, Japan) at an at 10 mA, before being examined with a scanning electron microscope (SM-200, Topcon, Japan) at an accelerating voltage of 20 kV. accelerating voltage of 20 kV.

3.3. Results Results and and Discussion Discussion

3.1.3.1. Time-Dependent Time‐Dependent Changes Changes of of K K Value Value TheTheK Kvalue value of of red red sea sea bream bream increased increased exponentially exponentially after after 27 27 h h storage storage at at 22 22± ± 22 ◦°CC (Figure(Figure2 2),), whichwhich is is in in agreementagreement withwith the K value observed observed in in DasyatisDasyatis brevis brevis musclemuscle when when stored stored at at 0 0°C◦ Cfor for 18 18days days [4]. [4 ].The The K Kvaluevalue began began from from 12.2% 12.2% at at the the start start of of storage storage to 83.2% after 2727 hh storage.storage. AccordingAccording toto Kato Kato et et al. al. [20 ],[20], fish fish with with a K value a K ofvalue less thanof less 20% than are classed 20% are as foodclassed suitable as food for raw suitable consumption; for raw withconsumption; a K value with less a than K value 40% less are than suitable 40% to are be suitable boiled to or be stewed; boiled withor stewed; a K value with ofa K over value 40% of over are classed40% are as classed spoiled as fish. spoiled Thus, fish. the Thus,K value the results K value indicate results that indicate the red that sea the bream red sea could bream still could be used still for be “Sashimi”used for “Sashimi” grade (raw grade consumption) (raw consumption) up until 9 h storageup until (a 9K hvalue storage under (a K 20%), value and under for cooking 20%), and grade for upcooking until 18 grade h storage up until (a K 18value h storage under (a 40%). K value For under our research 40%). For purposes, our research we term purposes, fish as beingwe term in thefish “freshas being stage” in the when “fresh the Kstage”value when is below the 40%, K value and is those below in the40%, “spoiled and those stage” in the when “spoiled the K value stage” is when over 40%.the K Thus, value the is over red sea 40%. bream Thus, monitored the red sea during bream this monitored storage experimentduring this remainedstorage experiment in the “fresh remained stage” forin the 18 h, “fresh while stage” fish stored for 18 for h, while more thanfish stored 18 h at for room more temperature than 18 h at (22 room± 2 temperature◦C) were found (22 ± to 2 be °C) in were the “spoiledfound to stage”. be in the “spoiled stage”.

FigureFigure 2. Changes 2. Changes in K invalueK value of red of redsea seabream bream during during storage storage at 22 at 22± 2± °C.2 ◦ValuesC. Values shown shown are are the the means means of three of three replicates with standardreplicates deviation with bars. standard deviation bars.

3.2. Fluorescence Spectroscopic Properties of Red Sea Bream Scales 3.2. Fluorescence Spectroscopic Properties of Red Sea Bream Scales For the red sea bream scales, three visible peaks were identified from the corrected fluorescence For the red sea bream scales, three visible peaks were identified from the corrected fluorescence landscape (Figure 3); the highest fluorescence intensity was observed at peak A with an excitation landscape (Figure3); the highest fluorescence intensity was observed at peak A with an excitation (Ex) wavelength of 280 nm and emission (Em) wavelength of 310 nm, with a weaker signal at a same (Ex) wavelength of 280 nm and emission (Em) wavelength of 310 nm, with a weaker signal at a same excitation and emission wavelength of 620 nm. Moreover, a relatively strong fluorescence signal was excitation and emission wavelength of 620 nm. Moreover, a relatively strong fluorescence signal was detected at peak B with an excitation wavelength of 340 nm and emission of about 420 nm. detected at peak B with an excitation wavelength of 340 nm and emission of about 420 nm.

Fishes 2017, 2, 10 5 of 11 Fishes 2017, 2, 10 5 of 11

FigureFigure 3. 3.Typical Typical front-face front‐face ﬂuorescencefluorescence landscape landscape of of the the red red sea sea bream bream scale. scale.

3.2.1. Identification of the Major Compound Contributing to Peak A (Ex/Em: 280/310 nm) 3.2.1. Identification of the Major Compound Contributing to Peak A (Ex/Em: 280/310 nm) A number of reports have noted that the fluorescence signal at peak A is caused by free tyrosine orA proteins number containing of reports tyrosine have noted residuals that the[21]. fluorescence Analyses of signalfish scales at peak reveal A a is 41% caused to 81% by freeorganic tyrosine or proteinsprotein content, containing including tyrosine 24% residualsof ichthylepidin [21]. and Analyses 76% of collagen of fish scales [22]; scale reveal ichthylepidin a 41% to contains 81% organic proteinabout content, 5% tyrosine including residuals 24% of(rare ichthylepidin tryptophan and residuals) 76% of [23], collagen while [ 22scale]; scale collagen ichthylepidin contains 2–3 contains abouttyrosine 5% tyrosine residuals residuals (tryptophan (rare tryptophannot detected) residuals) in 1000 total [23], residuals while scale [24]. collagen These organic contains proteins 2–3 tyrosine residualscontaining (tryptophan tyrosine notresiduals detected) contribute, in 1000 to total some residuals extent, to [24 the]. Thesefluorescence organic signal proteins at peak containing A. tyrosineHowever, residuals for the contribute, untreated scales, to some we extent, also need to theto consider fluorescence the mucus signal (and at epidermis) peak A. However, that covers for the the surface of the scales, since it reaches a considerable thickness on the surface of the scales [25]. The untreated scales, we also need to consider the mucus (and epidermis) that covers the surface of the mucus of fish, which is a complex jelly‐like fluid predominantly formed by macromolecules scales,(proteins) since it [26], reaches plays a a considerablecritical role in thicknesstheir defense on by the acting surface as a ofbiological the scales barrier [25]. [27]. The The mucus mucus of fish, whichshows is a complexstrong absorption jelly-like in fluid the protein predominantly region due formed to the tyrosine by macromolecules residuals which (proteins) are confirmed [26], to plays a criticalexist role in inmucus their proteins defense [28], by actingand free as tyrosine—an a biological essential barrier [content27]. The in mucus fish mucus shows fluid strong [29]. absorptionThus, in thethe protein free tyrosine region dueand toits theresidual tyrosine proteins residuals in the which mucus are were confirmed also deemed to exist to in contribute mucus proteins to the [28], and freefluorescence tyrosine—an signal essentialat peak A. content in fish mucus fluid [29]. Thus, the free tyrosine and its residual proteins inAdditionally, the mucus a were weak also signal deemed was observed to contribute in the to scale the fluorescencefluorescence spectra signal atat peakan excitation A. wavelengthAdditionally, of 280 a weaknm and signal emission was wavelength observed of in 620 the nm; scale this is fluorescence an artificial peak spectra due to at a an second excitation‐ wavelengthorder transmission of 280 nm of the and diffraction emission grating wavelength monochromators of 620 nm;from peak this A. is However, an artificial this peakartificial due to peak could be used to show the similar fluorescence behavior and to maintain a fixed fraction of peak a second-order transmission of the diffraction grating monochromators from peak A. However, A intensity [30]. Here, we use the intensities of the peak at Ex/Em of 280/620 nm rather than that at this artificialpeak A to peak represent could the be change used toin showfluorescence the similar intensities fluorescence during storage behavior because and the to intensities maintain at a fixed fractionpeak of A peakwere partly A intensity saturated. [30 ]. Here, we use the intensities of the peak at Ex/Em of 280/620 nm rather than that at peak A to represent the change in fluorescence intensities during storage because the intensities3.2.2. Identification at peak of A the were Major partly Compound saturated. Contributing to Peak B (Ex/Em: 340/420 nm) Previous research has indicated that a fluorescence signal at peak B with an Ex/Em wavelength 3.2.2. Identification of the Major Compound Contributing to Peak B (Ex/Em: 340/420 nm) pair of 340/420 nm can be assigned to collagen (Type I), reduced form of nicotinamide adenine dinucleotidePrevious research (NADH) has or indicatedvitamins [31,32]. that a fluorescence signal at peak B with an Ex/Em wavelength pair of 340/420Generally, nm the can elasmoid be assigned scales of to teleost collagen fish (Typeconsist I), of reducedcollagen fiber, form which of nicotinamide forms a highly adenine dinucleotideordered (NADH)three‐dimensional or vitamins structure [31,32]. together with calcium‐deficient hydroxyapatite Ca PO OH [33]. The internal and external layers of the red sea bream scale were measured Generally, the elasmoid scales of teleost fish consist of collagen fiber, which forms a highly ordered with a Fourier Transform Infrared (FTIR) in ATR mode (Figure 4). The FTIR spectrum of the internal three-dimensional structure together with calcium-deficient hydroxyapatite (Ca (PO ) (OH) ) [33]. side of the scale showed a characteristic peak at 3292 cm−1, which corresponds to the10 amide4 A6 arising2 The internalfrom the andN‐H externalstretching, layers and the of theabsorption red sea peak bream at scale3081 cm were−1 is measuredassigned to with the amide a Fourier B band Transform of Infraredcollagen; (FTIR) peaks in ATR at 1628, mode 1544 (Figure and 41238). The cm FTIR−1 are spectrumcharacteristic of theof C=O internal stretching side of vibrations, the scale showedN‐H a − characteristicbending vibrations peak at 3292 and the cm C1‐,N which stretching corresponds vibrations, to as the well amide as C‐ AN arisingstretching from and the N‐H N-H in‐plane stretching, and thebending absorption corresponding peak at to 3081 amide cm I,− II1 isand assigned III bands to of the collagen, amide respectively B band of collagen; [34]. The amide peaks III at band 1628, 1544 and 1238demonstrates cm−1 are the characteristic existence of a ofhelical C=O structure stretching [35]. vibrations, The FTIR spectra N-H bending of the external vibrations layer showed and the C-N stretchingstrong absorption vibrations, bands as well around as C-N 600 and stretching 1000 cm and−1, corresponding N-H in-plane to phosphate bending correspondinggroups in the apatite to amide I, II and III bands of collagen, respectively [34]. The amide III band demonstrates the existence of a helical structure [35]. The FTIR spectra of the external layer showed strong absorption bands around 600 and 1000 cm−1, corresponding to phosphate groups in the apatite lattice, and a peak at 1420 cm−1 Fishes 2017, 2, 10 6 of 11 correspondingFishes 2017, 2 to, 10 carbonate anions substituted for phosphate ions in the apatite lattice [33]. The6 of 11 FTIR spectraFishes results 2017, 2, on10 the internal and external sides of the red sea bream scale are in close agreement6 of 11 lattice, and a peak at 1420 cm−1 corresponding to carbonate anions substituted for phosphate ions in with the results of Ikoma et al. [19] which suggest that the fish scale consists of mainly type I collagen the apatite lattice [33]. The FTIR−1 spectra results on the internal and external sides of the red sea bream fibrilslattice, with aand partially a peak at calcified 1420 cm internal corresponding layer and to carbonate a well-calcified anions substituted external layer. for phosphate Importantly, ions in the IR thescale apatite are in lattice close [33]. agreement The FTIR with spectra the results onof theIkoma internal et al. and [19] external which suggestsides of thethat red the sea fish bream scale spectra of this collagen layer (internal side) is in agreement with the IR spectra of collagen isolated scaleconsists are ofin mainlyclose agreement type I collagen with thefibrils results with of a partiallyIkoma et calcified al. [19] whichinternal suggest layer and that a the well fish‐calcified scale from the scales of other fish species [36], confirming that collagen actually largely exists on red sea consistsexternal of layer. mainly Importantly, type I collagen the IR fibrils spectra with of athis partially collagen calcified layer (internal internal side)layer isand in aagreement well‐calcified with breamexternalthe scales. IR spectra layer. The current Importantly,of collagen fluorescence isolated the IR spectrafrom spectroscopy the of scales this collagen of device other layer hasfish aspecies (internal sufficient [36], side) intensityconfirming is in agreement of that excitation collagen with light whichtheactually could IR spectra penetrate largely of collagenexists into on the isolated red scale. sea frombream Therefore, the scales. scales collagen The of other current undeniably fish fluorescence species [36], contributes spectroscopy confirming to thethat device fluorescencecollagen has a signalactuallysufficient of peak largely Bintensity in theexists redof on excitation sea red bream sea breamlight scale, whichscales. in agreement couldThe current penetrate with fluorescence into the resultsthe spectroscopyscale. from Therefore, Santana device collagen ethas al. a [ 37]. Moreover,sufficientundeniably for intensity the contributes untreated of excitation to scales, the fluorescence light traces which of signal NADH could of peakpenetrate or vitamins B in the into red may the sea scale. be bream present Therefore, scale, in in theagreement collagen mucus or with the results from Santana et al. [37]. Moreover, for the untreated scales, traces of NADH or epidermis,undeniably also contributing contributes to slightly the fluorescence to this peak signal [32 of]. peak B in the red sea bream scale, in agreement withvitamins the resultsmay be from present Santana in the etmucus al. [37]. or epidermis,Moreover, also for contributingthe untreated slightly scales, to traces this peak of NADH [32]. or vitamins may be present in the mucus or epidermis, also contributing slightly to this peak [32].

FigureFigure 4. Fourier 4. Fourier Transform Transform Infrared Infrared (FTIR) (FTIR) spectra spectra of of the the redred sea bream scale: scale: the the upper upper part part is the is the internal layer, and the lower part is the external layer of the scale. internalFigure layer, 4. Fourier and the Transform lower part Infrared is the external(FTIR) spectra layer of of the the red scale. sea bream scale: the upper part is the internal layer, and the lower part is the external layer of the scale. 3.3. Time‐Dependent Changes in Fluorescence Intensity of Scales 3.3. Time-Dependent Changes in Fluorescence Intensity of Scales 3.3. Time‐Dependent Changes in Fluorescence Intensity of Scales 3.3.1. Time‐Dependent Changes in Fluorescence Intensity of Peak A (Ex/Em: 280/310 nm) 3.3.1. Time-Dependent Changes in Fluorescence Intensity of Peak A (Ex/Em: 280/310 nm) 3.3.1. AlthoughTime‐Dependent there was Changes a relatively in Fluorescence consistent Intensity decrease of Peakin peak A (Ex/Em: A fluorescence 280/310 nm) intensity with Although there was a relatively consistent decrease in peak A fluorescence intensity with storage, storage,Although there therewas awas large a errorrelatively bar mainlyconsistent associated decrease with in peakindividual A fluorescence differences intensity between with fish therestorage, wassamples a largethere (Figure was error 5). aSince barlarge the mainly error peak bar A associated fluorescence mainly associated with intensity individual with consists individual differencesof internal differences fluorescence between between fishof organic samplesfish (Figuresamplescompounds5). Since (Figure (from the 5). peak Since ichthylepidin the A fluorescence peak Aand fluorescence collagen) intensity intensityand consistssurface consists fluorescence of of internal internal (from fluorescencefluorescence the mucus of oforganic layer), organic compoundscompoundschanges (from in these(from ichthylepidin compounds ichthylepidin and will collagen)and be reflected collagen) and in surface andthe peaksurface fluorescence A intensity fluorescence changes (from (from the during mucusthe mucusstorage. layer), layer), changes in thesechanges compounds in these willcompounds be reflected will be in reflected the peak in Athe intensity peak A intensity changes changes during during storage. storage.

Figure 5. Changes in peak A fluorescence intensity during the storage time. Values shown are the means of 9 replicates with standard deviation bars. FigureFigure 5. Changes 5. Changes in peak in peak A ﬂuorescence A fluorescence intensity intensity during the the storage storage time. time. Values Values shown shown are the are the means of 9 replicates with standard deviation bars. means of 9 replicates with standard deviation bars.

Fishes 2017, 2, 10 7 of 11 Fishes 2017, 2, 10 7 of 11 Ichthylepidin belongs to scleroprotein, which is insoluble in most solvents, is hardly digested by enzymesIchthylepidin [38], and belongs is expected to scleroprotein, to be quite stable which during is insoluble storage, in most resulting solvents, in minimal is hardly changes digested in bytyrosine enzymes residuals. [38], and Since is expected collagen tocontains be quite traces stable of duringtyrosine storage, residual, resulting the change in minimal in collagen changes tyrosine in tyrosineresidual residuals. is not expected Since collagento significantly contains affect traces peak of tyrosineA fluorescence residual, intensity the change levels. in collagen tyrosine residualOn isthe not other expected hand, to the significantly outer mucus affect plays peak a Acritical fluorescence role in intensitylive fish levels.defense by trapping and immobilizingOn the other most hand, bacteria the outerand pathogens mucus plays before a critical they can role contact in live epithelial fish defense surfaces; by trapping continuous and immobilizingsecretion and most replacement bacteria andof pathogensmucus prevents before the they stable can contact colonization epithelial of surfaces; potential continuous infectious secretionmicroorganisms and replacement [39]. However, of mucusonce the prevents fish body the dies, stable the replacement colonization metabolism of potential stops, infectious and the microorganismsmicroorganisms [invade39]. However, the mucus once on the the fish scales body with dies, decreasing the replacement defensive metabolism capabilities stops, (secretion and the of microorganismsnew mucus). Thus, invade these the mucusmicroorganisms on the scales are with expected decreasing to multiply defensive rapidly capabilities and consume (secretion the of newfree mucus).tyrosine Thus,and proteins these microorganisms in the mucus, are which expected will tosignificantly multiply rapidly impact and total consume tyrosine the (i.e., free tyrosinepeak A) andfluorescence proteins inintensity the mucus, levels. which That will is, significantlythe free tyrosine impact in total mucus tyrosine is readily (i.e., peakutilized A) fluorescenceby the fast‐ intensitymultiplying levels. microorganisms, That is, the decreasing free tyrosine peak in mucusA intensity is readily during utilized storage. by However, the fast-multiplying the complex microorganisms,degradation of proteins decreasing and peak generation A intensity of free during tyrosine storage. result However, in the peak the complex A intensity degradation fluctuating of proteinsduring storage and generation [40]. of free tyrosine result in the peak A intensity fluctuating during storage [40].

3.3.2.3.3.2. Time-DependentTime‐Dependent Changes Changes in in Fluorescence Fluorescence Intensity Intensity of of Peak Peak B B (Ex/Em: (Ex/Em: 340/420 nm) nm) TheThe changeschanges inin peakpeak BB ﬂuorescencefluorescence intensity intensity of of red red sea sea bream bream scales scales during during storage storage is is shown shown in in FigureFigure6 .6. Intensity Intensity tended tended to to increase increase during during the the initial initial stage stage of of storage storage (ﬁrst (first 12 12 h h storage), storage), while while a a higherhigher value value was was apparent apparent after after 12 12 h h storage, storage, but but with with a a slow slow downward downward decline decline thereafter. thereafter.

FigureFigure 6. 6.Changes Changes inin peakpeak B ﬂuorescencefluorescence intensity during during storage. storage. Values Values shown shown are are the the means means of of 9 9 replicates with standard deviationreplicates bars. with standard deviation bars.

Since collagen was the biggest contributor to peak B, changes in collagen would essentially lead Since collagen was the biggest contributor to peak B, changes in collagen would essentially lead to the changes in peak B during storage. As the water holding capacity of fish decreases, in company to the changes in peak B during storage. As the water holding capacity of fish decreases, in company with moisture evaporation on the fish surface during storage [41], the scales on the fish surface will with moisture evaporation on the fish surface during storage [41], the scales on the fish surface will also lose moisture, which may be the main contributor to the increase in peak B fluorescence intensity also lose moisture, which may be the main contributor to the increase in peak B fluorescence intensity during this initial stage of storage (the first 12 h storage). In vitro scales lose water very fast; most of during this initial stage of storage (the first 12 h storage). In vitro scales lose water very fast; most the scale water content (23% by weight) was lost within 10 min of drying at room temperature of 22 of the scale water content (23% by weight) was lost within 10 min of drying at room temperature of ± 2 °C and humidity of 45%. The peak B fluorescence intensity was observed to linearly increase with 22 ± 2 ◦C and humidity of 45%. The peak B fluorescence intensity was observed to linearly increase the scale water loss (Figure 7), that is, the dryer the scale, the higher the peak B fluorescence intensity with the scale water loss (Figure7), that is, the dryer the scale, the higher the peak B fluorescence observed. Thus, fish scales can confidently be assumed to be losing moisture during storage which intensity observed. Thus, fish scales can confidently be assumed to be losing moisture during storage leads to increased peak B fluorescence from the scales at this initial stage of storage. In addition, scale which leads to increased peak B fluorescence from the scales at this initial stage of storage. In addition, moisture loss could slightly increase the peak A fluorescence intensity but not significantly; thus, this scale moisture loss could slightly increase the peak A fluorescence intensity but not significantly; thus, may not be the key factor causing the variation in peak A fluorescence intensity during storage (Figure 5).

Fishes 2017, 2, 10 8 of 11

FishesthisFishes may2017 2017, ,2 not 2, ,10 10 be the key factor causing the variation in peak A fluorescence intensity during storage88 of of 11 11 (Figure5). TheTheThe observed observedobserved peak peakpeak B BB fluorescence fluorescencefluorescence decrease decreasedecrease later laterlater in inin storage storagestorage (after (after(after 12 1212 h hh storage) storage)storage) may maymay be bebe mainly mainlymainly dueduedue to toto aggravated aggravatedaggravated bacteria bacteriabacteria activity activityactivity on onon fish fishfish scales scales collagen. collagen. SEM SEMSEM images imagesimages of ofof the thethe scale scalescale collagen collagencollagen layer layerlayer (Figure(Figure(Figure 88) 8)) clearlyclearly clearly showshow show thatthat that atat at thethe the spoiledspoiled spoiled stagestage stage (24(24 (24 hh h storage),storage), storage), thethe the collagencollagen collagen layerlayer layer was was much much rougher rougher andandand damaged damageddamaged than thanthan at atat the thethe fresh freshfresh stage stagestage (0 (0(0 h hh or oror 12 1212 h hh storage), storage),storage), due duedue mainly mainlymainly to toto the thethe bacterial bacterialbacterial collagenase collagenasecollagenase that thatthat areareare produced producedproduced by byby the thethe accelerated acceleratedaccelerated colonialization colonializationcolonialization and andand multiplication multiplicationmultiplication of of of bacteria bacteriabacteria entrapped entrappedentrapped in in in the thethe mucus mucusmucus ofofof the thethe fish, fish,fish, while whilewhile it itit was was still still alive, alive, gradually gradually digesting digesting the thethe scale scalescale collagen collagencollagen [42]. [[42].42]. Therefore, Therefore,Therefore, the thethe peak peakpeak B BB intensityintensityintensity which whichwhich was waswas mainly mainlymainly due duedue to toto the thethe changes changeschanges in inin collagencollagen tends tends tends to to decrease decrease later later in in storage. storage.

FigureFigureFigure 7. 7. Change7. Change Change in in Peakin Peak Peak B andB B and and A ﬂuorescenceA A fluorescence fluorescence intensities intensities intensities with with with water water water loss. loss. loss. Values Values Values shown shown shown are theare are meansthe the of 3 replicates with standardmeansmeans deviation of of 3 3 replicates replicates bars. with with standard standard deviation deviation bars. bars.

FigureFigureFigure 8. 8. 8. ScanningScanning Scanning electron electron micrograph micrograph (SEM) (SEM) of ofof the thethe internal internal (collagen) (collagen) layer layer layer of of of scales scales scales at at at different different different storage times. storagestorage times. times.

3.3.3.3.3.3. Ratio Ratio of of I IBB/I/IAA Related Related with with Freshness Freshness 3.3.3. Ratio of IB/IA Related with Freshness TheTheThe above above observations observations point point point to to to the the the conclusion conclusion conclusion that that that red red red sea sea sea bream bream bream scale scale scale fluorescence fluorescence fluorescence changes changes changes in in peakinpeak peak AA and Aand and peakpeak peak BB which Bwhich which isis is mainlymainly mainly duedue due toto to changeschanges changes inin in tyrosinetyrosine tyrosine andand collagen collagen content content in inin opposite oppositeopposite directionsdirectionsdirections during duringduring fish fishfish storage, storage,storage, suggesting suggestingsuggesting that that the thethe mean meanmean ratios ratiosratios of ofof these thesethese two twotwo peaks peakspeaks may maymay provide provideprovide a aa sensitivesensitivesensitive correlation correlationcorrelation with withwith fish fishfish freshness freshnessfreshness decline declinedecline during duringduring storage. storage.storage. Moreover, Moreover,Moreover, baseline baselinebaseline fluctuations fluctuationsfluctuations due duedue tototo experimental experimentalexperimental conditions conditionsconditions affect affectaffect both bothboth peaks peakspeaks similarly, similarly,similarly, thereby therebythereby canceling cancelingcanceling each eacheach other otherother out; out;out; the thethe ratio ratioratio of ofof thethe two two peaks peaks is is calculated. calculated. When When I BIB/I/IAA was was plotted plotted against against storage storage time, time, there there was was a a linear linear increase increase the two peaks is calculated. When IB/IA was plotted against storage time, there was a linear increase 2 (black(black(black line) line)line) with withwith RRR2 2 === 0.95 0.95 (Figure (Figure 9 9),9),), suggesting suggestingsuggesting a a fluorescence fluorescencefluorescence technique techniquetechnique based based on onon this thisthis ratio ratioratio for forfor quantitativequantitativequantitative detection detectiondetection of ofof the thethe fish fishfish freshness freshnessfreshness status statusstatus that isthatthat more isis intuitivemoremore intuitiveintuitive and responsive andand responsiveresponsive to fish freshness toto fishfish freshnessthanfreshness the exponentially than than the the exponentially exponentially increasing increasing increasingK value (Figure K K value value2). (Figure (Figure 2). 2).

Fishes 2017, 2, 10 9 of 11 Fishes 2017, 2, 10 9 of 11

FigureFigure 9. 9.Changes Changes of ofIB I/B/IIAA during the storage time. Values Values shown are are the means of of 9 9 replicates replicates with with standard deviation bars. standard deviation bars.

4.4. ConclusionsConclusions TheThe resultsresults havehave demonstrateddemonstrated thatthat aa fishfish freshnessfreshness index,index, basedbased onon front-facefront‐face fluorescencefluorescence spectroscopyspectroscopy ofof fishfish scales,scales, isis a a potentially potentially rapid, rapid, sensitive sensitive and and non-destructive non‐destructive meansmeans forfor assessingassessing fishfish freshness.freshness. TwoTwo major major fluorescence fluorescence signals signals at at peak peak A A and and BB fromfrom thethe scalesscales ofof redred seasea breambream withwith anan Ex/EmEx/Em of 280/310280/310 nm and 340/420 nm, nm, respectively, were observed.observed. Peak A waswas assignedassigned toto tyrosinetyrosine containing containing tyrosine tyrosine residuals residuals in in proteins proteins and and free free tyrosine tyrosine from from the the skeleton skeleton and and mucus mucus of the of scale.the scale. Peak Peak B was B thoughtwas thought to be to caused be caused by collagen by collagen (mainly (mainly Type Type I collagen), I collagen), which which forms forms the basic the skeletonbasic skeleton of the scale.of the When scale. theWhen intensity the intensity ratio of theseratio of fluorescence these fluorescence peaks (IB peaks/IA) was (IB/ plottedIA) was againstplotted storageagainst time,storage a linear time, increasea linear increase (R2 = 0.95) (R2 was = 0.95) found. was Thus,found. this Thus, ratio this offers ratio a offers novel a analytical novel analytical means formeans quantitative for quantitative measurement measurement of fish freshness of fish status. freshness In addition, status. this In methodology addition, this potentially methodology offers valuablepotentially information offers valuable to build information a further preparation-free, to build a further direct, preparation and non-destructive‐free, direct, and technique non‐destructive such as atechnique fluorescence such multiplex as a fluorescence sensor or multiplex imaging for sensor assessing or imaging fish freshness, for assessing which fish is currently freshness, not which offered is bycurrently other assessment not offered methods. by other assessment methods.

Acknowledgments:Acknowledgments:Financial Financial support support was was provided provided by Supportingby Supporting Program Program for Interaction-based for Interaction‐based Initiative Initiative Team StudiesTeam Studies (SPIRITS) (SPIRITS) in Kyoto in University. Kyoto University. We are grateful We are to grateful Katsuhiro to Kanamori Katsuhiro from Kanamori Panasonic from Corporation Panasonic (Kyoto, Japan) and Professor Takahashi Ryoji (Department of Chemistry, Graduate School of Science and Engineering,Corporation Ehime(Kyoto, University) Japan) and for Professor supplying Takahashi equipment, Ryoji Xu Kebing,(Department Motomu of Chemistry, Sakamoto andGraduate Matsui School Tomoya of forScience help and to gather Engineering, experimental Ehime data. University) We are thankfulfor supplying to Garry equipment, John Piller Xu (GraduateKebing, Motomu School ofSakamoto Agriculture, and KyotoMatsui University, Tomoya for Japan) help forto gather proof reading experimental of the manuscript.data. We are thankful to Garry John Piller (Graduate School of AuthorAgriculture, Contributions: Kyoto University,N.K. was Japan) the project for proof supervisor. reading Q.L.of the designed manuscript. the experiments presented in this paper, andAuthor performed Contributions: the experiments N.K. was and the analysis project supervisor. with the assistance Q.L. designed of M.K. the Q.L. experiments wrote and presented edited the in manuscript this paper, with the assistance of M.K, T.S. and D.F.A.R. and performed the experiments and analysis with the assistance of M.K. Q.L. wrote and edited the manuscript Conﬂictswith the assistance of Interest: ofThe M.K, authors T.S. and declare D.F.A.R. no conﬂict of interest. Conflicts of Interest: The authors declare no conflict of interest. References

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