Illegal Nitrite Treatment of Red Tuna and Prolonged Storage: What About Other Food Safety Risks?

Abstract

The illegal treatment of tuna with nitrite is a significant food safety concern. The risk may be due to not only the high nitrite levels and the possible formation of N-Nitrosamines but also to the possible increase in biogenic amines and microbial load and also pathogens. This study optimized the treatment of red tuna (Thunnus thynnus) with nitrite solutions and then determined several chemical (histamine, total volatile basic nitrogen (TVBN), biogenic amines, nitrite/nitrate, ascorbic acid, and sulfites) and microbiological (total microbial count, Enterobacteriaceae, Vibrionaceae, coagulase-positive staphylococci, Salmonella, Escherichia coli) parameters, comparing the results obtained with fresh samples with those obtained with treated samples after 5 days of storage (4 °C). The effect of such treatment on samples voluntarily contaminated with some pathogens was also investigated. The results indicate that if the products are characterized by suitable hygienic quality, the total microbial load and the levels of histamine and TVBN after 5 days of storage are below the legal limits, and no health risk subsists. The growth of pathogens/histaminogens (Salmonella and Morganella morganii) was also substantially inhibited during storage. Thus, this work confirmed that the high nitrite amounts and the possible development of N-nitrosamines represent the only significant food safety concerns.

1. Introduction

Proposed in all diets because of their high nutritional properties, seafood products have seen a marked increase in demand and consumption in recent years. The EFSA [1] and WHO [2] recommend consumption of one to two fish-based meals per week as fish is essential for a complete diet due to its high content of proteins, free amino acids, and other health-enhancing components such as vitamins, minerals, and omega-3 fatty acids [3].

Among fish products, the tuna supply chain is particularly market-driven by the increasing demand for processed/ready-to-cook tuna products. World tuna consumption is estimated at 0.45 kg per capita per year, which represents 2.2% of global fish consumption [4]. At the same time, tuna fish meat is a highly perishable foodstuff and easily subjected to microbial growth and decomposition. The fresh meat has a bright red color which quickly turns to brown during storage due to the autoxidation of myoglobin to metmyoglobin. In order to extend or even enhance the attractive aspect of fresh red tuna meat, different strategies were proposed to stabilize the red color, such as gassing with carbon monoxide (CO) or the addition of nitrate and/or nitrite salts and also by adding vegetable extracts with high nitrate concentration and in combination with reducing agents (ascorbic acid) to form nitric oxide (NO), which binds as a ligand to myoglobin. CO complexes are reported to be cherry red, while NO-complexes are described as pink red [5,6].

These procedures are illegal and the European Commission regulates these treatments, forbidding the addition of such components [7] due to possible economic fraud and potential health problems associated with consumption of illegally processed tuna [8].

Among the different possible adulterant solutions, the use of nitrite is particularly concerning for food safety. Indeed, nitrite salts are directly related to the so-called “blue baby syndrome”, an acute syndrome that can occur especially in infants when ingested nitrite reacts with the ferrous group of hemoglobin, which is oxidized to Fe³⁺ to form methemoglobin. This compound cannot transport oxygen throughout the body, and this deficiency leads to cyanosis and hypoxia [9]. The possible reaction of ingested nitrite with secondary amines to form N-nitrosamines is another toxic effect since these compounds, recently reviewed by the European Food Safety Authority, have been classified as pro-carcinogenic by the International Agency for Research on Cancer due to their role in gastrointestinal tumors and stomach cancer development [10,11,12]. It is worth noting that in the final remarks of the Scientific Opinion published by EFSA in 2023, entitled “Risk assessment of N-nitrosamines in food”, the CONTAM Panel concluded that “the MOE for TCNAs at the P95 exposure is highly likely (98–100% certain) to be less than 10,000 for all age groups, which raises a health concern”. This significant conclusion opens the scenario to new investigation on the presence on N-nitrosamines in food, focused on establishing specific legal limits in Europe, as have already been adopted in other countries worldwide [13,14].

Nitrite salts are food additives commonly used with specific legal limits as preservatives in food products such as processed meat. In drinking water, the World Health Organization (WHO) has set a fatal nitrite dose of 3 mg/L (65.2 μM). In the human body, concentrations from 33 to 250 mg of nitrite per kg of body weight are lethal, and those from 0.4 to 200 mg/kg of body weight are enough to cause methemoglobinemia [15,16]. European Regulation No. 1333/2008 [7] does not permit the addition of nitrite salts in both fresh and processed seafood. Only sodium and potassium nitrate can be used in pickled herring and sprat, with maximum level of 500 mg/kg until 9 October 2025 and 270 mg/kg after this date.

The illegal treatments of tuna with high amounts of nitrite to stabilize color characteristics obtain a significant improvement in the appearance and could mask a significant food safety risk since high levels of amines, particularly biogenic amines (BAs), can be produced during storage. Among these, histamine (2-(4-imidazolyl) ethylamine, HIM) is frequently implicated in poisoning outbreaks characterized by symptoms encountered with IgE-mediated food allergies [17]. Scombroid fish, such as tuna, may accumulate high HIM levels within fish muscles because of the presence of high concentrations of histidine, which is converted to HIM by the bacterial enzyme histidine-decarboxylase (HDS). Ingestion of food containing low amounts of histamine can cause an allergy-like syndrome referred as “histamine intolerance” [18]. However, in large doses, the normal metabolic mechanisms are insufficient for the detoxification. An HIM intake of 70–1000 mg per single meal may cause the so-called “scombroid poisoning”, characterized by an incubation period ranging from a few minutes to hours and symptoms such as hypotension, headache, tachycardia, nasal secretions, and possible death in sensitive subjects [19]. The nitrite-treated product, therefore, although fresh in appearance, may hide a state of degradation which makes it no longer fit for consumption.

The microbial count, growth, and the possible presence of pathogens are other concerns to consider as a possible consequence of the prolonged storage of adulterated red tuna samples.

With this background, a study of nitrite-treated tuna samples was carried out in the present work to produce data, both chemical and microbiological, that are useful and in the interest of health.

2. Materials and Methods

2.1. Treatment of Red Tuna with Nitrite Solutions

All fresh red tuna (Thunnus thynnus) samples were collected from local retailers and first analyzed for the detection of two food additives that could influence the study: sulfites and ascorbic acid.

The first phase of the work involved several simulation tests of red tuna treatment with nitrite solutions in order to define an optimized experimental protocol and achieve a proper stabilization of the red color of tuna during a 5-day period under refrigeration. Different procedures were tested, varying the nitrite concentration, the contact time, and mode, evaluating both immersion in and injection of nitrite solution. Finally, the optimized protocol involves the use of a 10% (100 g/L) sodium chloride and 0.4% (4 g/L) nitrite solution, proceeding with multiple injections of this solution into the samples. This treatment ensures the preservation of the original coloration of the tuna after 5 days stored at 4 °C, as shown in Figure 1. In this regard, the suitability of such treatment was evaluated by taking into account the overall appearance, consistency, and odor as generally comparable to the fresh sample.

All chemical and microbiological parameters were then quantified in the original tuna samples (non-treated, used as control), as well as in the adulterated fractions, comparing the results obtained for fresh samples with those obtained for treated samples after 5 days of storage at 4 °C. Overall, 3 different samplings were carried out. These samplings were scheduled in 3 different periods, so different samples collected from different local markets were analyzed, obtaining 3 independent analytical sessions.

2.2. Chemicals and Analytical Methods

The experimental design provided the evaluation of several parameters obtained by simulating the treatment of red tuna (Thunnus thynnus) with nitrite solutions and then determining the following chemical attributes: histamine (HIM), total volatile basic nitrogen (TVBN), biogenic amines, nitrite/nitrate, ascorbic acid, and sulfites, both before and after 5 days of storage under refrigeration. Regarding microbiological aspects, the following determinations were carried out: total microbial count at 30 °C and enumeration of Enterobacteriaceae, beta-glucuronidase-positive Escherichia coli, and coagulase-positive staphylococci, together with detection of Vibrio spp. and Salmonella spp. Overall, three samplings and 186 analytical determinations (114 chemical and 72 microbiological) were carried out. Further studies focused on clarifying the effect of such treatment on samples voluntarily contaminated with two pathogens, namely with Escherichia coli and Salmonella, were also carried out.

2.2.1. Histamine Analysis: HPLC Reagents, Equipment and Procedure

Trichloroacetic acid (TCA) of ACS grade (99%) was purchased from Carlo Erba Reagents (Rodano, Italy). Histamine dihydrochloride standard (≥99%), potassium phosphate bibasic (≥98.0%), sodium 1-decanesulfonate (≥99.0%), and potassium phosphate monobasic (≥98.0%) were supplied by Sigma–Aldrich (Steinhem, Germany). Acetonitrile and water of HPLC grade were purchased from Baker (Deventer, The Netherlands). The derivatization reagents N,N-dimethyl-2-mercaptoethylamine (Thiofluor™) and o-phtalaldehyde and potassium borate buffer (o-phtalaldehyde diluent, OD104) were provided by Pickering Laboratories (Mountain View, CA, USA).

The analytical determination of histamine was carried out by using HPLC/FLD (Agilent Technologies, Santa Clara, CA, USA) after on-line post-column chemical derivatization of the sample. Details about the sample pre-treatment step and instrumental method are reported elsewhere [20]. In 2017, the HPLC/FLD method used in this study was compared to EN ISO 112 19343: 2017 [21], confirming comparable analytical performances (i.e., limit of quantification and precision) [20].

2.2.2. TVBN Determination

Perchloric acid (HClO₄) (≥70%) and boric acid (H₃BO₃) (≥99.8%) were supplied by Honeywell Fluka (Morristown, NJ, USA). Ammonium chloride (NH₄Cl) (≥99.5%) was obtained from Sigma-Aldrich (Stenheim, Germany), while hydrochloric acid (HCl), anhydrous sodium hydroxide (NaOH) (≥99.8%), and ethanol were purchased from Carlo Erba Reagents (Rodano, Italy). Indicators methyl red and methylene blue also were provided by Carlo Erba Reagents (Rodano, Italy).

The determination of TVBN, according to Regulation No. EU 2019/627 [22], is based on steam distillation and allows the detection of the content of TVBN in the concentration range 5–100 mg/100 g. The method is based on the determination of the fraction of nitrogen produced by enzymes and bacteria that break down muscle proteins during the spoilage processes of fish products, resulting in the increase in the content of volatile nitrogen components.

The nitrogenous bases were extracted from the sample with a 6% aqueous solution of perchloric acid (HClO₄). After alkalization, the extract was subjected to steam distillation, and the volatile basic components were absorbed by a 3% boric acid (H₃BO₃) solution. The concentration of TVBN was then determined by titration of the absorbed bases with hydrochloric acid (HCl) 0.01 N. As required by Regulation EU No. 2019/627, the pH of the titration endpoint must be 5.0 (±0.1). The Tashiro’s solution (mixture in 95% ethanol of the two indicators methyl red and methylene blue) was the indicator used in this titration. This titration takes on a violet color in the acid range, gray in the neutral range, and green in the basic range. At the endpoint of the titration, therefore, the turning of the indicator from green to gray was observed.

2.2.3. Nitrite/Nitrate, Sulfites, Biogenic Amines, and Ascorbic Acid Determinations

The analytical determinations of nitrite/nitrate, sulfites, and biogenic amines were carried out by using ion chromatography. In particular, the analytical method described by Iammarino et al. [23], based on anion-exchange chromatographic separation and suppressed conductivity detection, was followed, using nitrite and nitrate (1000 mg/L) standard solutions supplied by CPAchem Ltd. (Bogomilovo, Bulgary) and Na₂CO₃ (mobile phase) purchased from VWR International s.r.l. (Milan, Italy). A standardized protocol, based on anion-exchange chromatography and already adopted for developing several food safety monitoring methods, was followed for the analytical determination of sulfites in seafood [24,25]. In this case, a carbonate-free sodium hydroxide (50%, w/w) was purchased from Sigma-Aldrich (Stenheim, Germany), and sodium sulfite (98%) was obtained from J.T. Baker (Deventer, The Netherlands). A well-established procedure based on cation-exchange ion chromatography with conductivity detection was followed for determining 5 biogenic amines, namely trimethylamine, cadaverine, putrescine, spermine, and spermidine [26]. The Dr. Ehrenstorfer™ reference materials were purchased from LGC Limited (Teddington, Middlesex, UK), with methanesulfonic acid used for mobile phase obtained from Sigma-Aldrich (Stenheim, Germany). All solutions used for ion-exchange chromatography were prepared with ultrapure water (minimal resistance 18.2 MΩ-cm) supplied by Milli-Q RG unit from Millipore (Bedford, MA, USA). Finally, the determinations of ascorbic acid were carried out by using a method based on HPLC/UV-DAD [27], using an L-ascorbic acid (≥99.0%) reference material and sodium acetate anhydrous purchased form Sigma-Aldrich (Stenheim, Germany), with phosphoric acid (85.0%), glacial acetic acid, and acetonitrile of HPLC grade supplied by J.T. Baker (Deventer, The Netherlands), and potassium phosphate monobasic and bibasic (≥98.0%) obtained from Carlo Erba Reagenti (Milan, Italy).

2.3. Microbiological Determinations

The bacterial determinations were carried out according to standardized and reference methods as follow: ISO 4833-1:2013/Amd 1:2022 [28], ISO 21528-2:2017 [29], ISO 16649-2:2010 [30], ISO 6888-2:2021/Amd 1:2023 [31], ISO 21872-1:2017/Amd 1:2023 [32], ISO 6579-1:2017/Amd 1:2020 [33]. These protocols were followed for evaluating total microbial count, enumerating Enterobacteriaceae, beta-glucuronidase-positive Escherichia coli, and coagulase-positive staphylococci (Staphylococcus aureus and other species), and detecting Vibrio spp. and Salmonella spp., respectively.

Twenty-five grams of tuna sample was placed in a Stomacher bag and 225 mL (1:10, w/v) of Buffered Peptone Water (BPW) was added and the mixture was blended using a BagMixer (Interscience, Milan, Italy). Then, serial dilutions (1:10 v/v) were plated on proper media in Petri dishes. More in depth, the following conditions and media were adopted: Violet Red Bile Glucose Agar (VRBGA) (Oxoid) incubated at 37 °C for 48 h for Enterobacteriaceae, plate count agar (PCA) (Oxoid, Hampshire, UK) incubated at 30 °C for 72 h for total microbial count, and Rabbit Plasma Fibrinogen Agar medium (RPFA medium) incubated at 37 °C for 24–48 h for staphylococci. Tuna dilutions in BPW were incubated at 37 °C for 18 h. Then, 0.1 mL of the culture was inoculated in 10 mL of Rappaport Vassiliadis Soy (RVS) broth (Microbiol, Cagliari, Italy) and incubated at 41.5 °C for 24 h. One milliliter of the same culture was transferred in 10 mL of Muller–Kauffmann Tetrathionate-Novobiocin (MKTTn) broth (Microbiol, Cagliari, Italy) and incubated at 37 °C for 24 h. At the end of the incubation, 10 µL of the culture from each tube was seeded using a sterile calibrated loop on Xylose Lysine Desoxycholate (XLD) (Microbiol, Cagliari, Italy) and Salmonella Detection Agar (SDA) (Microbiol, Cagliari, Italy) for the detection of Salmonella spp.

Vibrio spp. detection was performed by preparing a pre-enrichment of each sample (25 g) in 225 mL of alkaline peptone water (APW), which was then incubated at 37–41.5 °C overnight. Then, an inoculating loop of the culture broth was streaked on CHROMagar Vibrio (CHROMagar™, Paris, France) and thiosulfate citrate bile salt agar (TCBS) (Microbiol, Cagliari, Italy) plates and incubated for 24 h at 37 °C.

All presumptive colonies isolated from selective media were identified at species level with the MALDI-TOF-MS (Bruker Daltonics, Bremen, Germany) method.

Each tuna sample was analyzed in duplicate, both with and without the nitrite treatment, within 24 h of collection (t₀) and after 5 days of storage (t_5gg) in a refrigerator (5 ± 3) °C.

Enumeration of Selected Pathogens Added to Tuna

Tuna samples, treated or not, were also contaminated with low and high levels of Salmonella enteritidis ATCC^TM 13076^TM (10² and 10⁴ CFU/mL) [34] to evaluate the survival and possible growth of this pathogen in raw tuna treated with nitrite during prolonged refrigerated storage.

Slices of tuna samples were placed in a sterile container and mixed with each culture suspension of the Salmonella strain. Subsequently, inoculated samples were subjected to nitrite treatment and analyzed immediately and after 5 days of storage in a refrigerator (5 ± 3) °C to check the effect of nitrite treatment. The surface-plating method on selective media specific for Salmonella spp. (XLD agar plates) (Microbiol, Cagliari, Italy) was used to determine the populations of target pathogen in inoculated tuna samples.

The difference in the enumeration of Enterobacteriaceae between the samples with and without the nitrite treatment and the blank sample was calculated and correlated with the presence of Morganella morganii, a typical example of histamine-producing bacteria (HPB).

Briefly, tuna samples, treated or not, were contaminated with high levels of M. morganii ATCC 25830 (10⁶ CFU/mL). Slices of tuna samples were placed in a sterile container and mixed with each culture suspension of M. morganii strain. All samples contaminated and the blank were analyzed for the enumeration of Enterobacteriaceae. Isolated colonies were enumerated by spreading 1 mL of diluted samples in 12–15 mL of VRBGA and the plates were incubated (37 °C, 48 h).

2.4. Statistical Analysis

The statistical analysis was carried out by taking into account the data obtained for the fresh samples (control) as compared to those of nitrite-treated samples after 5 days of storage at 4 °C. Indeed, this can be the actual scenario when “nitrite-sophisticated” red tuna samples are put on the market in the place of fresh ones. Taking into consideration 3 samplings with duplicated analysis of each parameter, the one-way ANOVA was applied using a significance level of 0.05 (p < 0.05). This comparison was made to identify what parameters statistically differ between fresh samples and samples treated with nitrite after prolonged storage.

3. Results and Discussion

Three samplings in total were carried out, comparing the results obtained for fresh samples with those obtained for treated samples after 5 days of storage under refrigeration. Specifically, regarding chemical analysis, the presence and concentration of HIM, TVBN, biogenic amines, and nitrites/nitrates was evaluated, while microbiological investigations were focused on assessing total microbial count at 30 °C (Enterobacteriaceae, Vibrionaceae, coagulase-positive staphylococci, Salmonella and Escherichia coli).

All samples were preliminarily checked for the detection of two food additives which can influence these parameters: sulfites and ascorbic acid. The absence of sulfites was determined in all samplings, while ascorbic acid was quantified at a mean concentration of 245.5 mg/kg, below the 300 mg/kg limit specified by EU Regulation No. 1333/2008. Obviously, all samples were also analyzed for quantifying the nitrite concentration both in treated samples and those not treated. In Figure 2, some chromatogram examples are shown.

Histamine analysis is needed to assess the spoilage of fish products, in particular for fresh fish where the eventual use of illegal additives, such as nitrites, can alter sensory evaluations. HIM is used as a useful parameter of food quality, relating to freshness degree. A content lower than 10 mg/kg ensures a good quality level, while concentrations up to 30 mg/kg and 50 mg/kg are indexes of significant deterioration and definite decomposition, respectively [20]. The European Commission fixed some safety requirements for HIM presence in seafood [35]. More in depth, for fish species associated with high levels of histidine (including Scombridae, like tuna), nine samples have to be taken from each batch for analysis; the mean level must not exceed 100 mg/kg, no sample may have a level exceeding 200 mg/kg, and two samples may have a concentration higher than 100 mg/kg but lower than 200 mg/kg. Finally, the fishery products which have undergone enzyme ripening or treatment in brine may have HIM levels not higher than twice the limits listed above.

Figure 3 shows an example of HIM analysis performed using HPLC/FLD after on-line post-column chemical derivatization of the samples.

Other biogenic amines analyzed during this part of the study are shown in Figure 4, together with an example of a chromatogram obtained by ion chromatography.

The levels of these compounds contribute to the assessment of food quality by calculating the Biogenic Amines Index (BAI):

BAI = (cadaverine + putrescine + histamine)/(1 + spermine + spermidine)

A product can be described as fresh for BAI values between 0 and 1 mg/kg; for BAI values between 1 and 10 mg/kg, the product is initially altered, and it is decomposing for BAI values higher than 10 mg/kg.

The overall results obtained from these three samplings are reported in

Table 1. Samplings 1, 2, 3: chemical determinations. t₀ and t_5gg rely on samples being fresh and stored at 4 °C, respectively.

Sampling 1
Residual Nitrites = 152.1 mg/kg; Ascorbic Acid = 290.8 mg/kg; Sulfites = Absent
	HIM (mg/kg)	Nitrites (mg/kg)	Nitrates (mg/kg)	Trimethylamine (mg/kg)	Cadaverine (mg/kg)	Putrescine (mg/kg)	Spermine (mg/kg)	Spermidine (mg/kg)	BAI Index	TVBN (mg/100 g)
t0 untreated	7.4	0	0	2.8	0	0	7.2	2.2	0.71	0
t0 treated	6.9	180.0	15.0	3.6	0	0	4.7	1.2	1.00	0
t5gg untreated	24.1	0	1.2	24.1	0	0	3.8	1.3	3.95	11.9
t5gg treated	10.4	152.1	76.0	2.4	0	0	5.1	1.8	1.32	10.9
Sampling 2
Residual Nitrites = 84.0 mg/kg. Ascorbic Acid = 200.3 mg/kg; Sulfites = Absent
	HIM (mg/kg)	Nitrites (mg/kg)	Nitrates (mg/kg)	Trimethylamine (mg/kg)	Cadaverine (mg/kg)	Putrescine (mg/kg)	Spermine (mg/kg)	Spermidine (mg/kg)	BAI Index	TVBN (mg/100 g)
t0 untreated	0	5.6	0	7.1	0	0	0.3	0.4	0	0
t0 treated	0	74.2	5.7	2.1	0	0	5.6	0.6	0	0
t5gg untreated	29.6	0	0	12.4	0	0	3.1	0	7.22	24.0
t5gg treated	15.9	84.0	10.9	36.5	0	0	0	0	15.9	20.7
Sampling 3
Residual Nitrites = 200.9 mg/kg; Ascorbic Acid = 131.6 mg/kg; Sulfites = Absent
	HIM (mg/kg)	Nitrites (mg/kg)	Nitrates (mg/kg)	Trimethylamine (mg/kg)	Cadaverine (mg/kg)	Putrescine (mg/kg)	Spermine (mg/kg)	Spermidine (mg/kg)	BAI Index	TVBN (mg/100 g)
t0 untreated	0	0	0	0	0	0	9.1	7.9	0	0
t0 treated	0	212.0	0	0	0	0	10.5	1.1	0	0
t5gg untreated	34.3	0	0	0	41.5	0	18.8	34.4	1.40	26.0
t5gg treated	24.0	200.9	37.0	0	0	5.4	21.3	4.2	1.11	19.7

and

Table 2. Samplings 1, 2, 3: microbiological determinations. t₀ and t_5gg rely on the samples being fresh and stored at 4 °C, respectively.

Sampling 1
	Total Microbial Count	Enterobacteriaceae	Staphylococci	Escherichia Coli	Salmonella	Vibrio
t0 untreated	>103 CFU/g	>103 CFU/g	<10 CFU/g	absent	absent	absent
t0 treated	>103 CFU/g	>102 CFU/g	<10 CFU/g	absent	absent	absent
t5gg untreated	>105 CFU/g	>104 CFU/g	<10 CFU/g	absent	absent	absent
t5gg treated	>105 CFU/g	>103 CFU/g	<10 CFU/g	absent	absent	absent
Sampling 2
	Total Microbial Count	Enterobacteriaceae	Staphylococci	Escherichia Coli	Salmonella	Vibrio
t0 untreated	>105 CFU/g	>105 CFU/g	<10 CFU/g	absent	absent	absent
t0 treated	>105 CFU/g	>105 CFU/g	<10 CFU/g	absent	absent	absent
t5gg untreated	>106 CFU/g	>106 CFU/g	<10 CFU/g	absent	absent	absent
t5gg treated	>106 CFU/g	>106 CFU/g	<10 CFU/g	absent	absent	absent
Sampling 3
	Total Microbial Count	Enterobacteriaceae	Staphylococci	Escherichia Coli	Salmonella	Vibrio
t0 untreated	7.0 × 106 CFU/g	1.5 × 102 CFU/g	<10 CFU/g	absent	absent	absent
t0 treated	6.2 × 106 CFU/g	2.6 × 102 CFU/g	<10 CFU/g	absent	absent	absent
t5gg untreated	1.0 × 108 CFU/g	4.2 × 106 CFU/g	<10 CFU/g	absent	absent	absent
t5gg treated	1.0 × 108 CFU/g	4.0 × 106 CFU/g	<10 CFU/g	absent	absent	absent

.

It was noted that the residual concentration of nitrite in the samples, given the same treatment, is highly variable. This can be due to the different dimensions, shape, and thickness of the red tuna slices injected with nitrite solution and related solution release during refrigerated storage. In particular, the concentration detected at the end of the first sampling was almost twice that quantified on the second sampling (equal to 152.1 mg/kg vs. 84.0 mg/kg), and even greater is that found in the third sampling, 200.9 mg/kg. If compared to the available literature [5,6], these levels are significantly higher; however, the official control activity reported concentrations well higher than those registered in this study. This result confirms the stabilizing effect of these additives against various microorganisms, including histaminogens, regardless of the residual level of nitrite (Table 1). Indeed, no significant differences in chemical and microbiological parameters were observed between treated and untreated samples in the first sampling, while the increases in histamine, trimethylamine, TVBN, and BAI, although small, are greater in the second sampling, where the residual nitrite concentration is lower. In this case, as shown in Table 1, there was an almost 16-fold increase in histamine and 21-fold increase in TVBN for the treated sample. This is also true for trimethylamine, and the BAI exceeds a value of 10 in this case.

Graphically, this assessment is even more clearly evident: as shown in Figure 5, the increase in parameters, although small, was greatest where the residual nitrite concentration was the lowest.

However, it must be said that the values of HIM, TVBN, and total microbial load found in the treated samples after 5 days of storage are still abundantly within the normal ranges and as such they do not constitute a health risk.

The same trend can be observed for microbiological analysis (Table 2). The greatest variations in the parameters under investigation were again observed in the second sampling, in which case the levels of total bacterial count and Enterobacteriaceae were as much as 10 times higher for the treated sample. The absence of all pathogens was confirmed and there was no development of pathogenic microorganisms such as coagulase-positive staphylococci, Salmonella spp., Escherichia coli, or Vibrio spp. in these samples. The results of the third sampling show high levels of total bacterial count and Enterobacteriaceae that were not reduced by adding the treatment to the samples.

As appreciable from

Table 3. Statistical analysis.

Parameter	T0 (Control) Mean Value *	Nitrite-Treated Sample t5gg Mean Value *
Histamine (mg/kg)	2.8 a	16.8 b
TVBN (mg/100 g)	0.0 a	17.1 b
BAIindex	0.0 a	6.1 a
Total microbial count (CFU/g)	2.4 × 106 a	3.5 × 107 a
Enterobacteriaceae (CFU/g)	1.0 × 105 a	1.7 × 106 a

* n = 6 replicates; Values with different superscript ^a,b statistically differ (p < 0.05).

, where the mean values calculated considering all tests performed on fresh samples were compared to those obtained on nitrite-treated samples after 5 days of storage at 4 °C, although the latter results were higher, a statistically significant difference (p < 0.05) was verified only for the histamine and TVBN concentrations. Anyway, although statistically significant, this increase does not represent a food safety concern, since the final values are still largely below the legal limits in force at the European level.

Further studies in order to clarify the effect of nitrite treatment on red tuna samples in the presence of pathogen contamination were also investigated (

Table 4. Contamination tests with Salmonella and Morganella morganii.

Sample	Salmonella t0	Salmonella t5	Sample	Morganella morganii t0	Morganella morganii t5
Untreated sample (contam. 102 CFU/g)	3 × 10 CFU/g	3 × 10 CFU/g	Untreated sample not contaminated	3.0 × 104 CFU/g	2.8 × 107 CFU/g
NO2− treated sample (contam. 102 CFU/g)	2 × 10 CFU/g	0	Untreated sample (contam. 106 CFU/g)	4.5 × 106 CFU/g	2.7 × 107 CFU/g
Untreated sample (contam. 104 CFU/g)	4 × 10 CFU/g	1.5 × 102 CFU/g	NO2− treated sample (contam. 106 CFU/g)	1.7 × 106 CFU/g	9.0 × 105 CFU/g
NO2− treated sample (contam. 104 CFU/g)	4 × 10 CFU/g	2 × 10 CFU/g

). During these trials, the survival and the possible growth of Salmonella spp. and M. morganii were evaluated in tuna samples during storage of samples treated with nitrite and those not treated. As observed in Table 4, while the increase of contamination from 4 × 10 CFU/g to 1.5 × 10² and from 4.5 × 10⁶ CFU/g to 2.7 × 10⁷ CFU/g was verified for Salmonella and Morganella morganii, respectively, the treatment with nitrite caused the decrease in both species’ levels. More in depth, Salmonella was inhibited, and it was no longer detected after 5 days of storage, where the initial contamination level was 10² CFU/g.

The decrease in both species’ contamination level from 4 × 10 CFU/g to 2 × 10 CFU/g, and from 1.7 × 10⁶ CFU/g to 9.0 × 10⁵ CFU/g was verified for Salmonella and Morganella morganii, respectively. This means that the “stabilizing” effect of nitrite against microbial growth ensures that a possible contamination of tuna samples and the consequent prolonged storage after nitrite treatment do not pose a health risk due to the growth of Salmonella and/or Morganella morganii.

This finding was confirmed after chemical analysis of such samples voluntarily contaminated with Morganella morganii. Indeed, no HIM and other biogenic amines were detected in the fresh samples. After 5 days of storage, the final HIM concentration quantified in the non-treated sample was equal to 32.0 mg/kg, while a value of 11.0 mg/kg was quantified in the treated sample. Accordingly, the BAI calculated in the treated sample (0.7) result was lower than that of the non-treated sample (1.6).

4. Conclusions

In conclusion, in this work an experimental approach has been developed to study the effect of adulteration of red tuna (Thunnus thinnus) samples with high concentrations of nitrite to obtain a significant improvement in appearance, prolonging the shelf-life. Red tuna samples were treated using a 10% sodium chloride and a 0.4% nitrite solution, proceeding with multiple injections of this solution into the samples, which were then stored at 4 °C for 5 days. Chemical and microbiological parameters were then carried out for the original tuna samples, as well as for the adulterated fractions.

The obtained results showed that starting from products characterized by good hygienic and sanitary quality, the values of histamine, TVBN, and biogenic amines found in the treated samples, after 5 days of storage, do not constitute a health risk. As it regards microbiological determinations, in the same samples no significant increase in total microbial count, Enterobacteriaceae, and staphylococci was verified. The same finding was confirmed for Salmonella and Morganella morganii in voluntarily contaminated samples, confirming the stabilizing effect of nitrite.

Therefore, this study confirmed that no other food safety concern was highlighted in red tuna samples treated with nitrite and stored for a prolonged time, apart from nitrite presence and related food safety risk, other than the possible development of N-nitrosamines. This last point is worthy of future research, since it is likely due to the high levels of free amines which characterize food products such as red tuna.