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Article

Effect of Allyl-Isothiocyanate Release from Black Mustard (Brassica nigra) Seeds During Refrigerated Storage to Preserve Fresh Tench (Tinca tinca) Fillets

by
María José Rodríguez Gómez
1,*,
María Alejo Martínez
1,
Raquel Manzano Durán
1,
Daniel Martín-Vertedor
2 and
Patricia Calvo Magro
1
1
Centro de Investigaciones Científicas y Tecnológicas de Extremadura (CICYTEX), Instituto Tecnológico Agroalimentario de Extremadura (INTAEX), Área de Postcosecha, Valorización Vegetal y Nuevas Tecnologías, Avenida Adolfo Suárez s/n, 06007 Badajoz, Spain
2
Aquaculture Center ‘Las Vegas del Guadiana’, Regional Government of Extremadura, N-5, km 391.7, Villafranco del Guadiana, 06195 Badajoz, Spain
*
Author to whom correspondence should be addressed.
Fishes 2025, 10(8), 381; https://doi.org/10.3390/fishes10080381
Submission received: 30 June 2025 / Revised: 23 July 2025 / Accepted: 28 July 2025 / Published: 5 August 2025
(This article belongs to the Section Processing and Comprehensive Utilization of Fishery Products)
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Abstract

The aim of this study was to prevent the development of microorganisms in the refrigerated storage of tench by releasing allyl isothiocyanate (AITC) produced by black mustard seeds. Tench reared in an aquaculture centre were sacrificed and the fillets were separated. Different amounts of defatted mustard seed (300, 400 and 500 mg) were added to hermetic polypropylene trays. Microbiological, sensory, and gas chromatography with MS detection analysis were done. AITC release increased progressively until the third day of storage, significantly delaying the development of microorganisms in samples with higher mustard seed content. The tasting panel detected positive aromas at the beginning of the study, but these decreased and negative aromas appeared. The mustard seed treatment resulted in a higher positive aroma at the end of the storage, reducing rotting and ammonia odours. A total of 31 volatile compounds were detected and grouped into hydrocarbon, alcohol, benzenoid, isothiocyanate, ketone, acetate, aldehyde, and others. Butylated hydroxytoluene, an indicator of bacterial contamination, was the major aromatic compound found during storage. The release of AITC resulted in fewer organic compounds with negative aromas appearing during storage. PCA analysis allowed us to classify the assays during storage according to their volatile profiles, confirming the differences observed between treatments. Thus, adding mustard seed to fish packaging could be a viable alternative to extending the product’s shelf life and ensuring food safety.
Keywords:
Tinca tinca; allyl isothiocyanate; volatile organic compounds; refrigerated storage; sensory analysis
Key Contribution: AITC delayed microorganism growth, with fewer negative aroma compounds appearing during storage. PCA analysis classified the assays during storage according to their volatile profile.

Graphical Abstract

1. Introduction

Fish is a very important food in the human diet [1], and due to growing consumer demand there is currently a depletion of marine fish populations, making it necessary for governments to promote the production of inland fish as an element of food security [2]. For these reasons, one of the currently fastest growing productive sectors is aquaculture, mainly due to the application of new knowledge and technologies to intensive production [3].
The tench (Tinca tinca) is a warm-temperature, freshwater benthic fish endemic in Europe [4], which often takes on unpleasant odours and flavours which result from its natural habitat [5]. It is now found in freshwater regions around the world, including temperate and tropical areas [6], due to its adaptability to environments with high temperatures and even a limited availability of oxygen [7]. The tench is a fish rich in protein (17.7%) and essential amino acids [8] and poor in carbohydrates (3.3%) and fat (4.3%) with linoleic, oleic, and palmitic acid, among others, minerals (potassium, phosphorus, sodium, calcium, magnesium, and iron) and vitamins (A, niacin, C, riboflavin, and thiamine) [9]. They live in lentic or slow-flowing waters and play an important role in the environment, as they prevent the proliferation of algae and recirculate minerals and nutrients that are deposited at the bottom of lakes and streams in search of food, which helps reduce eutrophication. In several European countries, tench have been bred in agricultural ponds, as ornamental fish, and for recreational purposes. However, although tench has great potential for aquaculture [10], its cultivation is mostly managed extensively, which makes it difficult to compete with other intensive crops. In the Iberian Peninsula, tench is raised in the channels of the Tajo and Guadiana. The southwest of Spain is the main extensive producer of tench due to the existence of pastures, where the animals drink in ponds. These ponds have repopulated with tench due to their high adaptability. The use of intensive or semi-intensive environmentally friendly technologies and tench products valorisation could greatly increase consumer demand for this species [11].
In general, fish and fish products are highly perishable foods owing to the easy growth of spoilage microorganisms and the resulting limited shelf life of fish [12]. For fresh marketing, traditional methods such as refrigeration at low temperatures have been used. Other more recent methods such as the use of antimicrobial chemical agents can be used to improve stability, quality, and shelf life, as well as to reduce the microbial load and associated economic losses [13]. However, adverse effects of these chemical preservatives, such as allergenicity and carcinogenicity, on human health have been confirmed [13]. Therefore, natural alternatives for fish conservation must replace the use of synthetic chemical additives [14]. For this purpose, the allyl isothiocyanate (AITC) was generated from the hydrolysis of sinigrin by the myrosinase enzyme in the presence of water (Figure 1). Sinigrin is a glucosinolate compound [15] present in plants belonging to the Cruciferae family, including black mustard (Brassica nigra) [16], and it shows strong antimicrobial and antifungal activity [17]. Regulation CE 872/2012 adopts the list of flavouring substances included in Regulation CE 1334/2008 and supports the release of AITC for use in food preservation as an authorised GRAS (Generally Recognised As Safe) product due to the harmlessness of this substance and its strong activity in the gas phase. Furthermore, Regulation CE 1333/2008 regulates the use of substances added as preservatives. Thus, other authors have already demonstrated the inhibitory effects of AITC and the increase of the shelf life of fish fillets [18,19].
Therefore, the aim of this paper was to prevent the development of indicator microorganisms in tench by releasing AITC produced by mustard seeds during the refrigerated storage time of this product. Microbiological, sensory and gas chromatography with MS detection analysis were used to validate the results and PCA analysis was used to discriminate between the aromatic profiles of fresh and spoiled tench. The novelty of this work lies in the use of mustard seeds, a natural product, to preserve fresh tench (Tinca tinca) fillets.

2. Materials and Methods

2.1. Raw Material

The raw material was taken at the Las Vegas del Guadiana Aquaculture Center located in Villafranco del Guadiana (Badajoz, Spain). Tench had been reared for two years in 1500 m2 earthen bottom ponds at the aquaculture centre. At the end of this period, 62 tench (Tinca tinca) were submitted to a depuration pond of clean water for 24 h.
Mustard seeds were purchased from Merck KGaA (Darmstadt, Germany). Prior to the assays, they were defatted by hexane extraction in a Soxhlet system and ground to powder using a ball mill at 350 rpm for 30 min. Subsequently, they were sieved to obtain a size fraction of less than 500 μm. The mustard seed powder obtained was stored in airtight containers in a dry environment until use.

2.2. Experimental Design

After the tench were harvested, the fish were first stunned by immersion in ice-water slurry. Next, the fish were killed by a blunt blow to the head [20]. The total average weight of the fish was 91.5 ± 15.5 g. After being sacrificed, the fish were gutted and the skins removed. Loins were separated from each skinned tench (average weight per loin = 18 g) and placed into polypropylene trays (4 units per tray).
Four different assays were carried out to find out the antimicrobial effect of AITC release: (i) C: tench without mustard seeds; (ii) T1: tench in the presence of 300 mg of mustard seeds; (iii) T2: tench in the presence of 400 mg of mustard seeds, and (vi) T3: tench in the presence of 500 mg of mustard seeds. These trays were closed with 40 mm anti-fog film using a heat-sealing machine (Profi-3, ORVED, Barcelona, Spain). The trays from the different assays were subsequently stored at 4 ± 0.5 °C in the pilot plant facilities. The microbiological, sensory analysis, and volatile compounds measurements per treatment were carried out at 0-day (Day 0), and after four (Day 4), twelve (Day 12), and eighteen (Day 18) days of refrigerated storage. The experiments were performed in triplicate.

2.3. Analyses

2.3.1. Microbiological Analysis

For microbiological analysis, 10 g fish fillets from each tray (n = 5 trays per treatment) were transferred aseptically in a stomacher bag (VMR Blender bag, lateral filter, AVANTOR, EE.UU), containing 90 mL of peptone water (OXOID, CM0009, ThermoFisher, Barcelona, Spain). Serial 10-fold dilutions were prepared with the same diluent and duplicate 0.1 mL portions of the appropriate dilutions were spread on the following media: Plate count agar (VWR Chemicals, Barcelona, Spain) for the enumeration of mesophilic aerobic microorganisms, incubated at 30 °C for 3d and Violet Red Bile agar (OXOID, CM 0107, ThermoFisher, Spain) for total coliforms. After the solidified agar was covered with 5 mL of medium, it was incubated at 37 °C for 2 days. In this agar, colonies can be round. Purple-pink colonies may be surrounded by purple haloes, indicating lactose-positive organisms. Pale colonies with greenish haloes are lactose-negative organisms. Colonies were enumerated on the surface of the plate. The total microbial count per ml of sample was calculated by multiplying the average number of colonies per plate by the inverse dilution factor.

2.3.2. Sensory Analysis

A sensory panel with ten trained tasters belonging to the CICYTEX research centre (Extremadura, Spain) carried out evaluations to analyse the aromatic profile of the tench fillets tested. The sensory evaluation was carried out in standard tasting booths located at the CICYTEX facilities. The cooling trays corresponding to each experiment were removed, opened, and presented to each taster at room temperature for sensory evaluation. All tasters evaluated the olfactory profile for each treatment and sampling date. The aromatic intensity of the attributes assessed (fresh fish, rotten fish, river, sulphur, ammonia, pungent odour) was filled out on a document in which each attribute studied was evaluated on a 10 cm scale where the value of 0 indicated that the attribute was not detected and 10 indicated the highest olfactory intensity [21]. The results of each attribute evaluated were expressed as the average data from all the tasters. When the coefficient of variation was less than 20%, the results were considered valid. To comply with the Ethics Committee’s requirements for research involving human subjects, the tasters were provided with a structured document outlining the analysis assay before participating in the tasting evaluation.

2.3.3. Volatile Organic Compound Identification

Volatile organic compounds in a 2 g sample were analysed using solid phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS). A fused silica SPME covered with polydimethylsiloxane/divinylbenzene (PDMS/DVB) StableFlex fibre (65 μm, Supelco, Bellefonte, PA, USA) was used. This fibre was placed into 5 mL capacity vials with the sample. Subsequently, the sample fibre was inserted into the injection port of a Bruker Scion 456-GC triple quadrupole gas chromatograph (Bremen, Germany). The GC was equipped with a DB WAXETR capillary column (60 m × 0.25 mm × 0.25 µm) [22]. The fibre was heated to 250 °C for 40 min to thermally desorb the compounds from the fibre into the GC system. The identification of chemical compounds was achieved by comparing their mass spectra and linear retention indices (LRI) against known standards, the NIST mass spectral library, and values reported in the literature. Specifically, the LRIs were compared with those reported by Linstrom and Mallard (2003) [23]. Finally, an internal standard (2-octanol) was used to quantify the detected compounds. The volatiles were expressed in μg g−1.

2.3.4. Allyl-Isothiocyanate Quantification

The released of AITC from 300, 400, and 500 mg of mustard seeds introduced into the trays with tench samples was studied. For the quantification, samples were taken by solid phase microextraction (SPME) [24] for 17 days and continuously analysed using an Agilent CP3800-GC ion trap (Saturn 2200, Varian, Santa Clara, CA, USA) gas chromatograph equipped with a capillary column (30 m × 0.25 mm × 0.25 μm). Prior analysis, different amounts of AITC (Merck, Germany) were used to make a calibration curve. All experiments were performed in triplicate.

2.4. Statistical Analysis

A one-way ANOVA was used to determine if there were statistically significant differences between the means of multiple treatments groups on each sampling date. The Shapiro–Wilk test was used to assess if the data were normally distributed, while the Levene test was applied to check for equal variances across different groups. After that, when the data presented normal distribution and homoscedasticity, Tukey’s test was applied for multiple comparisons. When the data did not follow a normal distribution, a Kruskal–Wallis test by ranks was used to compare medians across multiple groups. If the variances of the groups were unequal, a Welch ANOVA test, followed by a Games–Howell post-hoc test, was applied to determine significant differences (p < 0.05). The statistical program used was XLSTAT-Pro 201,610 (Addinsoft 2009, París, France).

3. Results and Discussion

3.1. Allyl-Isothiocyanate Released from Mustard Seeds

The concentration of AITC released on the different sampling dates is shown in Figure 2. The identification and quantification of this compound was done independently of the other tench aromatic organic compounds present in the polypropylene trays. As can be seen in the Figure 2, the release of AITC occurs at the beginning of the study, since the moisture in the fish activates the seed immediately. From this date on, AITC release progressively increased, reaching its maximum amount after three days of storage in all treatments studied. From this point on, AITC content progressively decreased until the end of the study. The treatments that showed the highest concentrations of AITC were T2 and T3, containing 400 and 500 mg of mustard seed, respectively. At the beginning of the study, AITC values were more widely separated. However, at the day 5 of storage, the contents were quite similar. From day 10 of storage onward, no substantial differences were seen between the different treatments studied. These results agree with those found by Barea-Ramos et al. [25] for AITC released from mustard seeds in airtight containers with tomato samples. They obtained a maximum value of AITC at three days of storage. In this sense, when comparing the maximum AITC concentrations obtained, we observed that this value coincides with the lowest microbial counts (Figure 1). Thus, the maximum amount of AITC release was able to reduce the microbial load (due to its fungicidal or bactericidal effect) on Day 4.

3.2. Evaluation of the Antimicrobial Activity of Mustard Seeds

Due to fish being a highly perishable food that is very prone to microbial deterioration [26], an evaluation of the antimicrobial efficacy of AITC against aerobic mesophilic microorganisms (AMMs), and total coliform microorganisms (TCMs) in refrigerated tench fillets was done (Figure 3A,B). The initial count of AMMs (Day 0) in the control tench samples was less than 4 log CFU/g (Figure 3A), which is satisfactory for acceptable quality tench meat [27]. On the next sampling date (Day 4), the tench subjected to the T2 and T3 treatments (containing 400 and 500 mg of defatted mustard seeds, respectively) showed AMMs counts of 2.66 ± 0.69 and 2.17 ± 1.88 log CFU/g, respectively, lower than the C (3.26 ± 0.69 log CFU/g) and T1 (3.38 ± 0.69 log CFU/g) treatments, although statistics showed no difference between the treatments (p > 0.05). However, on Day 12 there was an increase in AMMs counts, although the T3 treatment showed the lowest content with respect to C treatment (p < 0.05). This indicates that the higher mustard seed content added to the containers was able to reduce the development of AMMs during up to 12 days of storage. The highest microbial load was found at the end of storage (Day 18), although we note that the T2 and T3 treatments showed significantly lower contents than the C treatment (p < 0.05). During storage, the autolysis process is promoted by proteolytic and lipolytic enzymes, which hydrolyse fish proteins and lipids [28] and can produce free peptides and amino acids, promoting microbial growth and the production of biogenic amines. The rate of degradation depends on the species and storage conditions [29]. Although bacteria increased gradually in both the untreated and treated samples, the tench with AITC maintained microbial populations at significantly lower levels during storage than those of the C treatment. Thus, the combined use of low temperatures (2 °C) and AITC released from seed can be an effective preservation alternative that prolongs the quality and safety of stored fish. The antimicrobial effect of AITC has also been demonstrated in other foods such as chicken meat, where it resulted in some inhibition of aerobic mesophilic counts such as Listeria monocytogenes, Salmonella Typhimurium and Pseudomonas lundensis [30]. In fact, four times the minimum inhibitory concentration of AITC provoked a reduction of 2.3 log of L. monocytogenes compared to the inoculated C treatment [31]. Furthermore, 1000 ppm of AITC reduced the growth of Staphylococcus aureus and Bacillus cereus and resulted in a reduction of 3 log CFU/g in AMMs [32]. In the case of TCMs (Figure 3B), the initial content was 2.69 CFU/g (Day 0). The lowest counts of TCMs occurred on Day 4 in samples treated with different concentrations of AITC. Thus, the T1, T2, and T3 treatments showed significantly lower values (2.31 ± 0.12, 1.34 ± 1.27 and 1.13 ± 0.98 log CFU/g of TCMs, respectively), than the C treatment (3.19 ± 0.05 log CFU/g) (p < 0.05). At the end of storage (Day 18), all of the treatments with AITC showed a lower content of TCMs compared to C, although significant differences were only found in the T3 treatment compared to C (p < 0.05). Thus, the maximum reduction was 4.50 log CFU/g in T3 compared with C. This could be because T3 contained the highest amount of AITC released from mustard seed. The data indicated that AITC showed a higher inhibitory effect against TCMs and lower inhibitory effect against AMMs. The reduction of these microorganisms during storage guarantees microbiological shelf life extensions.
Regarding the literature found of microbial inhibition by AITC in other foods, it should be noted that the inhibitory effect of AITC has been evaluated against TCMs in sausage batters (17.6% beef, 60.7% pork, and 17.6% lard) stored at 13 °C for 25 days. Furthermore, AITC at 750 and 1000 ppm was able to reduce the number of Escherichia coli O157 by 6.5 log CFU/g after processing for 21 and 16 days [33]. In other study, using AITC with high-pressure processing in raw ground chicken meat, a reduction of 5 log was observed in Salmonella survivals submitted to conservation treatment at 350 MPa for 4 min combined with 0.05% AITC in the packaging [34]. In fish, fillets of Gilthead Sea Bream (Sparus aurata) treated with 3.35 and 6.70 mg/l of AITC had lower counts of sulphite non-producing bacteria (6.55 ± 0.12 and 6.04 ± 0.05 log CFU/g, respectively) after 14 days of storage than the C sample (8.00 ± 0.05 log CFU/g). In the case of sulphite-producing bacteria, with the addition of AITC, the bacterial load never exceeded 3.5 log CFU/g until the end of storage [18]. The major effectiveness of AITC was observed on the growth of sulphite-producing bacteria that represent the predominant microbial spoilage organisms in fish stored under refrigerated conditions [35]. Other researchers have studied the fungicidal or bactericidal efficacy of AITC [36] in regulating certain microorganisms, such as Aspergillus flavus in Brazil nuts [37] or A. parasiticus in sliced bread [38] at a concentration of AITC ranging between 0.8 to 5 ppm.

3.3. Sensory Aroma of Tench During Storage Time

Organoleptic evaluation is a very important tool in assessing the freshness of farmed fish [39]. Thus, a descriptive olfactory sensory analysis was carried out on the tench samples to establish differences between the treatments tested on the different sampling dates (Table 1). Aromas related to the fish’s freshness (fresh fish), rearing ponds (river), microbiological spoilage (rotten fish, sulphur, and ammonium), and AITC preservative (sulphur and pungent) included in treatments T1, T2, and T3 were evaluated. The maximum aromatic intensity for fresh fish packaging at the beginning of the storage (Day 0) was 7.4. Also, the river odour was identified by the tasters (6.2). However, the other odours resulting from degradation were not detected at Day 0. As is well known, odour is considered one of the most significant characteristics of volatile compounds, which can be used to assess the fish freshness [40]. From the following sampling dates (Days 4, 12, and 18), the different treatments were compared to evaluate the effect of the different concentrations of AITC release by mustard seed. Thus, on Day 4 the fresh fish and river aromas were equal between treatments (p < 0.05), and slightly lower than Day 0. After 12 days of storage (Day 12), the C treatment retained the fresh fish odour compared to Day 4 (p > 0.05), however the fresh odour was lower compared to Day 0. In the T1, T2, and T3 treatments there was a loss of fresh fish odour compared to Day 4 (p < 0.05). However, when comparing freshness between treatments, no differences were found by Day 12 (p > 0.05). Likewise, the river odour remained constant in all treatments compared to Day 4 (p > 0.05). However, for the same sampling date (Day 12), the treatments containing higher amounts of AITC (T2 and T3) showed a lower river odour than the C treatment (p < 0.05). Other aromas corresponding to microbiological spoilage of fish (rotten and ammonia) or derived from AITC (sulphur and pungent) were not detected on Day 4 and 12. At the end of storage (Day 18) a loss of fresh fish odour was observed in the control and T1 treatments (p < 0.05). However, the T2 and T3 treatments maintained the fresh odour compared to day 12 (p > 0.05). This is consistent with a lower total presence of coliform microorganism (Figure 3B) in the treatments containing more mustard seed. On the other hand, rotten fish odour was detected in all treatments at the end of storage, with no significant differences between the treatments (p > 0.05). River odour was slightly lower at Day 18 compared to other sampling dates for the control treatment (p < 0.05), probably due to the appearance of other negative odours such as rotten fish. However, no differences were found for the T1, T2, and T3 treatments compared to the other sampling dates (p > 0.05). An ammonia odour from microbial spoilage was detected only at the end of storage in the C treatment and was not detected in the treatments including mustard seed. However, the characteristic pungent odour of AITC was found at the end of storage, although without differences between treatments (p > 0.05). As just shown, initially the fish evaluated by the tasting panel was characterised by high freshness, which is gradually lost through autolytic reactions and bacterial spoilage [41], resulting in an unpleasant odour with the production of ammonia and sulphuric chemicals [42]. Muscolino et al. [18] evaluated the sensory attributes of sea bream fillet packaging with and without the addition of AITC, reporting results like those found in our work. These authors demonstrated that the addition of AITC did not affect the natural odour of the fish. A higher concentration of AITC produced a marked AITC odour in the fish, but a few seconds after opening the package, the odour disappeared.

3.4. Volatile Compound Aroma of Tench During Storage Time

The flavours of freshwater fish, such as earthy/muddy, fishy, and grassy, come from multiple sources [43]. Therefore, the analysis of volatile compounds is a useful tool to determine the freshness status or spoilage of fish. A total of 31 volatile compounds were detected in the samples at different storage dates (Table 2). The compounds were divided into eight types of functional groups including hydrocarbon, alcohol, benzenoid, isothiocyanate, ketone, acetate, aldehyde, and other compounds. Most of these volatile compounds have been previously detected in other fish species [44]. In the same line, different researchers have indicated that volatile compounds that contribute to fish odour can be generally divided into three groups: (1) that of fresh fish odour, mainly related to C6-C9 alcohols and carbonyl compounds, although the existence of fish odour could reduce acceptability; (2) volatile compounds related to microbial spoilage, mainly ammonia, trimethylamine, hydrogen sulfide, and methylmercaptan; (3) lipid oxidation odour, mainly related to hexaldehyde and 2, 4, 7-decatrienal [40].
In the fresh unpackaged samples (Day 0), hydrocarbons, alcohols, and benzenoids were the predominant group compounds (74.3, 72.7, and 28.6 μg g−1 of the total volatile compounds, respectively). Aldehydes were also found in smaller concentrations (20.7 μg/g), although they represent a significant fraction due to their low detection thresholds [45]. Benzenoid compounds, including benzene, toluene, and derived compounds, are generally produced by lipid oxidation or the catabolism of aromatic amino acids such as tryptophan, phenyl alanine, and tyrosine [46], or brought by the environment, which may cause an unpleasant odour [47]. However, benzenoid compounds may also be related to floral, paint, and mothball odours [43,48]. In our study, the relatively high amount of benzenoid compounds might be partly attributed to the local pond culture conditions of the tench [48]. Furthermore, ten aromatic compounds, some of which are present in other aquatic products such as silver carp [44] were found. Among all of the detected compounds, the toluene derivative butylated hydroxytoluene (BHT) was the major aromatic compound found during storage. Zhang et al. [49] reported an increase of BHT in farmed sturgeon (Acipenser baerii) fillets inoculated with Shewanella putrefaciens. The presence of this compound may be an indicator of bacterial contamination. We observed a high significant proportion of BHT in the C treatment at Day 12 and Day 18 of storage with respect to the treatments including mustard seed (T1, T2, and T3). On Day 12, the proportion of BHT decreases significantly with the increasing amount of mustard seeds, indicating that the AITC generated in the container has retarded the development of microorganisms. This agrees with the TCMs (Figure 3B) performed on the samples on Day 12, where coliform counts were lower for the T1, T2, and T3 treatments, with T3 being significantly lower. On Day 18, the T3 treatment showed the lowest BHT content, with clear differences between the T1 and T2 treatments. Other benzenoid compounds like ethylbenzene, p-xylene, and o-xylene, present in our study in small proportions, were also detected in other fish species (Coilia ectenes) [50]. 1,3-dimethyl benzene was detected after 12 days of storage, with no differences between treatments. Thus, the effect of microbial activity and endogenous enzyme decomposition of endogenous enzymes can generate volatile compounds related to nitrogen, amines, ammonia, alcohols, sulphur-containing compounds, and others [39].
On the other hand, hydrocarbon compounds were found in a high proportion at the beginning of the study. These compounds may be related to a positive aroma derived from the oxidation and degradation of fatty acids [51]. 3-Methyl-hexane was the principal hydrocarbon compound detected and presented the highest concentration in the C treatment (Day 0). The T1 treatment (Day 4) also showed a high amount of this compound; however, it was not found in the rest of the treatments (T2 and T3) during storage.
Volatile compounds derived from alcohols such as 3-methyl-1-butanol and 1-octen-3-ol have also been proposed as indicators for bacterial species. Thus, Zhang et al. [49] reported that the spoilage characteristics of Pseudomonas mandelii could be related to unsaturated alcohols, which they considered to be derived from the oxidation of unsaturated fatty acids. Similarly, other studies used 1-octen-3-ol and (z)-2-penten-1-ol as the potential markers for spoilage in other aquatic products [52]. In our study, these compounds were detected in trace amounts at the end of storage, except for 3-methyl-1-butanol, which gives the fish fermented or rancid characteristics [53] and was found in significant concentration (64.6 μg/g) in the C treatment at the end of storage. Other alcoholic compounds detected in higher concentration in the C treatment were 2-ethyl-1-hexanol and 2,6-bis(1,1-dimethylethyl)-4-(1-oxopropyl) phenol, which are responsible for the off-odours of the water and fish [54]. Therefore, these changes in the alcoholic compounds were generally associated with a gradual deterioration in the flavour of the fish, manifesting as increased staleness, spoilage, or off-flavours.
On the other hand, aldehydes are very important compounds in fish odour due to their low detection thresholds. Several authors identified nonanal, among others, as contributors to the fishy odours in fish products [45,55]. Likewise, Zhou et al. [43] found nonanal as well as benzaldehyde and benzaldehyde derivatives, among others, in the white meat of silver carp. In our study, 2,5-bis[(trimethylsilyl)oxy] benzaldehyde (bitter and almond odour description) [56] was the main aldehyde compound found, followed by nonanal (green odour description) [56]. The C treatments showed a lower total aldehyde content compared to the rest of the treatments during storage. Similarly, Zhang et al. [49] found a decrease of most of the aldehydes in all spoiled samples of farmed sturgeon fillets compared with the control.
Other compounds belonging to the ketone family appeared after 12 days of storage. These compounds do not contribute significantly to the fishy odour owing to their high odour thresholds [56]. The major ketone compound found was 2-dodecanone, which was present in a high proportion in the C treatments and provides a woody smell [57]. 2-heptanone, related to a mouldy aroma [58], appeared in small proportions in the T1, T2, and T3 treatments.
On the other hand, sulphur-containing compounds could induce a foul sulphurous odour characteristic of fish flesh [59]. In our study, dimethyl trisulphide was found at the end of storage (C1, Day 18) probably originating from sulphur-containing amino acids. However, the main sulphur compound present in this study was allyl isothiocyanate (T1, T2, and T3 treatment), originating from the hydrolysis of sinigrin glucosinolate present in mustard seed. The highest concentrations of this substance were observed on Day 4. From this point on, the concentration progressively decreased during storage. The higher the concentration of seed added to the container with the tench, the higher the AITC content. This demonstrates that the release of this substance occurs exponentially at the beginning of seed activation, after which point the release progressively decreases. Finally, the volatile limonene is responsible for a positive citrus aroma and it was present at the beginning of the study (Day 0). This molecule was no longer present, or its concentration decreased during the storage time, being detected only in T3 on Day 18.
All of these results validated the sensory evaluation described in Section 3.4, where a loss of fresh fish odour and the appearance of rotten fish odour at the end of storage were observed.

3.5. Principal Component Analysis (PCA)

To assess the differences in the volatile profile during storage, a principal component analysis (PCA) was carried out at different sampling dates (4, 12, and 18 days) to reduce the number of dimensions to two principal components, which were plotted graphically based on the treatments applied (Figure 4). The results showed that the volatile compounds analysed were able to differentiate the samples according to the specific treatment on each sample date during the storage time. The two components explained 82.5–90.6% of the total variance. The various classification groups of the samples are shown in the different quadrants from left to right. After four days of tench storage in refrigeration chambers, the C treatment was completely separated from treatments T1, T2, and T3. Furthermore, the treatments with the highest amount of AITC (T2 and T3) showed very close clusters and separated from the T1 treatment. This indicates that the T2 and T3 treatments presented similar volatile profiles after four days of storage, with differences with respect to T1. In turn, T1 was different from C. As expected, this analysis was able to establish differences between treatments. However, the sensory analysis showed no differences on the fourth day between treatments in the different attributes evaluated. On the second sampling date (day 12), the aromatic profile again clearly separated treatment C from the T1, T2, and T3 treatments, and in addition the T2 and T3 treatments were separated from each other with respect to day 4 of storage. Although the groups were separated from each other on the third sampling date (Day 18), the T1 and T2 treatments were closer to each other and separated from T3. This is consistent with a lower content of benzenoid compounds such as BHT produced by bacteria [49], alcoholic compounds indicative of fish odour [54], and ketone compounds associated with the development of spoiled fish odour [58] in the T3 treatment. In addition, only the volatile limonene was found to be responsible for positive odours in T3. All this suggests that the high concentration of AITC in the T3 treatment controlled the microorganisms’ development and the discrimination of this group.

4. Conclusions

Microbial growth during storage was significantly reduced in samples with higher mustard seed contents, which caused the release of high concentrations of AITC from the start of the study. The results of the sensory evaluation indicate that the AITC generated in the packages was able to control microbial growth by improving the perception of fresh odours and reducing rotten and ammonia odours. Volatile aromatic compounds are also indicators of microbial preservation or deterioration developed in the packaging after storage. Significant changes were observed in the volatile organic compounds present in the tench samples during storage for periods of up to 18 days. PCA analysis corroborated the differences observed in the volatile profile between treatments during storage and allowed their classification. Therefore, adding defatted mustard seed to fish packaging could be a viable alternative to extending the product’s shelf life and ensuring food safety due to the reduction of some microorganisms. In a future project, the use of an optimal amount of mustard seeds and its application in packages containing tench, simulating realistic storage temperatures until they arrive to the final consumer, could be well suited to ensuring food safety conditions by testing typical spoilage bacteria, including Pseudomonas, Enterobacteriaceae, as well as other pathogens such as Listeria monocytogenes and Salmonella.

Author Contributions

M.J.R.G.: Investigation, Methodology, Validation, Writing—Original Draft, Writing—Review and Editing; Project administration; M.A.M.: Investigation, Methodology, Validation; R.M.D.: Methodology, Validation; D.M.-V.: Methodology, Validation, Writing—Original Draft. P.C.M.: Conceptualization, Investigation, Methodology, Supervision, Validation, Writing—Original Draft, Writing—Review and Editing, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the European FEDER Fund and Junta de Extremadura (TENCAEX II (Expte Nº: 2522999FP001), and the GR24122 (AGA002) 85% co-financed by the European Union, European Regional Development Fund, and the Regional Government of Extremadura).

Institutional Review Board Statement

This study was carried out under the 148b/2020 authorisation of the project “Comparative study of the growth and other biometric aspects and susceptibility to parasites in mixed and monosex culture of tench”.

Informed Consent Statement

Informed consent was obtained from all subjects (tasters) involved in the study.

Data Availability Statement

The original contributions presented in the study are included in the article; futher inquiries can be directed to the corresponding author.

Acknowledgments

The authors are grateful for the technical support of the TENCAEX II project staff, specially to Cesar J. Fallola Sánchez-Herrera and Juan Carlos Ramírez López from Aquaculture Centre of Vegas del Guadiana.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AITCAllyl Isothiocyanate
AMMSAerobic Mesophilic Microorganisms
BHTButylated Hydroxytoluene
PCAPrincipal Component Analysis
TCMsTotal Coliform Microorganisms

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Fishes 10 00381 g001
Figure 1. Enzymatic conversion of sinigrin to AITC, glucose, and sulphate.
Figure 1. Enzymatic conversion of sinigrin to AITC, glucose, and sulphate.
Fishes 10 00381 g001
Fishes 10 00381 g002
Figure 2. Release of allyl-isothiocyanate from mustard seed.
Figure 2. Release of allyl-isothiocyanate from mustard seed.
Fishes 10 00381 g002
Fishes 10 00381 g003
Figure 3. Effect of various AITC concentrations on (A) aerobic mesophilic, and (B) total coliform microorganism counts of tench meat kept for up to 18 days at 4 °C. C: tench without mustard seeds; T1: 300 mg, T2: 400 mg, and T3: 500 mg of mustard seeds. Different letters indicate significant differences between the sampling date. Multiple comparisons were analysed following a Kruscal–Wallis test (p < 0.05).
Figure 3. Effect of various AITC concentrations on (A) aerobic mesophilic, and (B) total coliform microorganism counts of tench meat kept for up to 18 days at 4 °C. C: tench without mustard seeds; T1: 300 mg, T2: 400 mg, and T3: 500 mg of mustard seeds. Different letters indicate significant differences between the sampling date. Multiple comparisons were analysed following a Kruscal–Wallis test (p < 0.05).
Fishes 10 00381 g003
Fishes 10 00381 g004aFishes 10 00381 g004b
Figure 4. Score plot of the principal component analysis (PCA) analysis of tench during its shelf life.
Figure 4. Score plot of the principal component analysis (PCA) analysis of tench during its shelf life.
Fishes 10 00381 g004aFishes 10 00381 g004b
Table 1. Descriptive sensory olfactory evaluation of tench fillets during shelf life. Results are expressed as mean ± SD of three samples replicates.
Table 1. Descriptive sensory olfactory evaluation of tench fillets during shelf life. Results are expressed as mean ± SD of three samples replicates.
TreatmentsFresh FishRotten FishRiver
Day 0Day 4Day 12Day 18Day 0Day 4Day 12Day 18Day 0Day 4Day 12Day 18
C7.4 ± 1.1 A6.6 ± 0.9 aAB 6.1 ± 1.7 aB 1.6 ± 1.1 bCndndnd3.8 ± 1.7 a 6.2 ± 0.8 A5.0 ± 2.9 aAB 5.5 ± 1.7 aAB3.5 ± 2.1 aB
T1 6.9 ± 1.0 aA5.1 ± 1.5 abB1.2 ± 0.7 bC ndnd2.5 ± 1.4 a 4.8 ± 2.0 aA 4.4 ± 1.4 abA2.7 ± 1.9 aA
T2 6.7 ± 1.1 aA 3.6 ± 0.9 bB2.9 ± 1.2 bB ndnd2.5 ± 1.5 a 4.9 ± 1.9 aA3.1 ± 1.6 bA3.3 ± 1.1 aA
T3 6.6 ± 1.5 aA 3.8 ± 1.4 bB2.8 ± 1.4 aB ndnd2.1 ± 1.3 a 4.6 ± 1.2 aA3.1 ± 1.5 bA2.7 ± 1.3 aA
TreatmentsSulphurAmmoniaPungent
Day 0Day 4Day 12Day 18Day 0Day 4Day 12Day 18Day 0Day 4Day 12Day 18
Cndndndnd ndndnd1.3 ± 0.9 ndndnd1.4 ± 1.0 a
T1 nd ndnd ndndnd ndnd1.0 ± 0.5 a
T2 nd ndnd ndndnd ndnd1.1 ± 0.8 a
T3 nd ndnd ndndnd ndnd1.2 ± 1.0 a
Values lower than 1 are considered as non-detected (nd). Different lower-case letters in the same column indicate significant differences among treatments. Multiple comparisons were analysed following a Kruscal–Wallis test (p < 0.05) for fresh fish (day 18), river (day 4), and pungent (day 18), and a Tukey test (p < 0.05) for fresh fish (days 4 and 12), rotten fish (day 18), and river (days 12 and 18). Different capital letters in the same row indicate significant differences among sampling dates. Multiple comparisons were analysed following a Kruscal–Wallis test (p < 0.05) for Control (fresh fish), T1 (fresh fish), and Control (river), and a Tukey test (p < 0.05) for T2 and T3 (fresh fish), and T1, T2 and T3 (river).
Table 2. Quantification of volatile organic compounds (μg/g) obtained by GC-MS in tench samples during its shelf life.
Table 2. Quantification of volatile organic compounds (μg/g) obtained by GC-MS in tench samples during its shelf life.
LRIVOCsDay 0Day 4Day 12Day 18
CCT1T2T3CT1T2T3CT1T2T3
Hydrocarbons
731.0Hexane, 3-methyl-73.2238.2147.2ndndndndndndndndndnd
890.5Hexane, 2,4-dimethyl-1.11.0ndndndndndndndndndndnd
957.71,3-cyclohexadienendndndndndndndndnd2.3ndndnd
967.13,5,5-trimethyl-1-hexenendndndndndndndndnd1.4ndndnd
TOTAL74.3239.2147.2ndndndndndnd3.7ndndnd
Alcohols
748.13-methyl-1-butanolndndndndndndndndndndnd11.9nd
862.91-hexanolndndndndndndndndndndndndnd
972.81-octen-3-olndndndndndndndndnd0.9nd0.9nd
10222-ethyl-1-hexanol7.726.623.27.5nd38.030.322.413.933.026.023.4nd
16262,6-bis(1,1-dimethylethyl)-4-(1-oxopropyl) phenol65.021.59.4ndnd15.110.09.68.014.410.45.3nd
TOTAL72.748.132.67.5nd53.040.331.921.9115.737.447.1nd
Benzenoids
765Toluene6.07.65.0ndndndndndndndndndnd
848Ethylbenzene1.61.71.5ndndnd1.71.11.1ndndndnd
857Benzene, 1,3-dimethylndndndndnd1.47.85.16.25.66.04.8nd
863o-xylene6.3nd6.3ndndndndndndndndndnd
880p-xylene2.52.8ndndndndndndndndndndnd
950Benzene-1-ethyl-4-methyl2.73.53.0ndndnd2.51.9ndndndndnd
980Benzene, 1,2,3-trimethyl-3.54.46.5ndndnd6.52.33.14.1ndnd1.6
1279Butylated hydroxytoluene4.26.7ndndnd40.731.728.926.6152.2103.264.047.4
1361Benzene, hexamethylndndndndndndndndndnd7.74.0nd
1300Indolendndndndndndndndndndndnd10.7
TOTAL28.627.523.4ndnd42.150.339.437.0162.0116.872.847.4
873Allyl isothiocyanatendnd19.416.324.5nd6.09.913.9nd2.34.55.4
9221-Isothiocyanatobutanendnd5.83.44.3nd5.213.210.2ndndndnd
TOTALndnd25.219.728.9nd11.223.124.1nd2.34.5nd
Ketones
8822-heptanonendndndndndndndndnd18.810.45.55.9
10822-dodecanonendndndndnd18.25.24.45.744.935.110.38.6
TOTALndndndndnd18.25.24.45.763.745.515.714.6
Acetates
742Acetic acidndndndndndndndndndnd188.3193.3nd
10724-hexen-1-ol, acetatendndndndndndndndndnd12.4ndnd
126611-tetradecen-1-ol, acetatendndndndndndndndndnd77.2ndnd
TOTALndndndndndndndndndnd277.8193.3nd
Aldehydes
1094Nonanal10.93.06.3nd4.32.47.26.36.9nd8.18.09.1
11182,5-bis[(trimethylsilyl)oxy]benzaldehyde9.86.38.67.08.75.05.75.58.77.710.39.76.2
TOTAL20.79.415.07.013.07.412.911.815.67.718.417.7nd
Others
954Dimethyl trisulfidendndndndndndndndnd5.6ndndnd
871Butanoic acid, 3-methyl-ndndndndndndndndnd17.2ndndnd
1017D-limonene2.21.91.51.31.2ndnd2.21.6ndndnd2.6
TOTAL2.21.91.5ndndndnd2.21.622.9ndnd2.6
LRI: Lineal Retention Index; nd: non-detected.
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MDPI and ACS Style

Rodríguez Gómez, M.J.; Alejo Martínez, M.; Manzano Durán, R.; Martín-Vertedor, D.; Calvo Magro, P. Effect of Allyl-Isothiocyanate Release from Black Mustard (Brassica nigra) Seeds During Refrigerated Storage to Preserve Fresh Tench (Tinca tinca) Fillets. Fishes 2025, 10, 381. https://doi.org/10.3390/fishes10080381

AMA Style

Rodríguez Gómez MJ, Alejo Martínez M, Manzano Durán R, Martín-Vertedor D, Calvo Magro P. Effect of Allyl-Isothiocyanate Release from Black Mustard (Brassica nigra) Seeds During Refrigerated Storage to Preserve Fresh Tench (Tinca tinca) Fillets. Fishes. 2025; 10(8):381. https://doi.org/10.3390/fishes10080381

Chicago/Turabian Style

Rodríguez Gómez, María José, María Alejo Martínez, Raquel Manzano Durán, Daniel Martín-Vertedor, and Patricia Calvo Magro. 2025. "Effect of Allyl-Isothiocyanate Release from Black Mustard (Brassica nigra) Seeds During Refrigerated Storage to Preserve Fresh Tench (Tinca tinca) Fillets" Fishes 10, no. 8: 381. https://doi.org/10.3390/fishes10080381

APA Style

Rodríguez Gómez, M. J., Alejo Martínez, M., Manzano Durán, R., Martín-Vertedor, D., & Calvo Magro, P. (2025). Effect of Allyl-Isothiocyanate Release from Black Mustard (Brassica nigra) Seeds During Refrigerated Storage to Preserve Fresh Tench (Tinca tinca) Fillets. Fishes, 10(8), 381. https://doi.org/10.3390/fishes10080381

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