Effects of different ozonized slurry‐ice treatments and superchilling storage (−1°C) on microbial spoilage of two important pelagic fish species

Abstract Combining different preservative treatments for improving quality and safety of fishery products increasingly receives global research attention. Consistent with this pursuit, the current research was undertaken to determine the effects of different ozonized slurry‐ice treatments and superchilling (−1°C) storage on microbial spoilage of European anchovy (Eugraulis encrasicolus) and sardine (Sardina pilchardus), which are two commercially important pelagic fish species. After the catch (within <5 hr) and at defined scheduled storage times, ozone has been discharged once on sardine (herein referred to as “One‐T”) and repeatedly/sequentially on European anchovy (herein referred to as “Seq‐T”). Microbiological analyses enumerated total viable count (TVC), Bacillus spp., Enterobacteriaceae, Lactobacillus spp., Moraxella spp., Shewanella spp., Listeria monocytogenes and Pseudomonas spp. Independent of potential antimicrobial effects of ozone during superchilling storage, no Listeria spp., Shewanella spp., Moraxella spp., and Bacillus spp. were found in all processed samples. While Enterobacteriaceae and Lactobacillus spp. were detected at below 1 log cfu/g, both TVC and Pseudomonas spp. proliferated at different rates throughout superchilling storage. The repeated ozone‐treated (“Seq‐T”) showed lower TVC and Pseudomonas spp. values compared with one‐time treated (“One‐T”) slurry‐iced and control samples. Thus, combined slurry‐ice and superchilling storage at Seq‐T produced improved antimicrobial activity over One‐T application. Largely, ozonized slurry‐ice outcomes/results appear promising thanks to superchilling storage.

Slurry-ice that constitute ice-water suspension and sub-zero temperature of up to −1.5°C, is among refrigeration candidates with a strong preservative potential for the fishery industry, which on one hand, is owed to the physical feature of ice-water suspension that increasingly facilitates the chilling rate (through rapid heat exchange within fish muscle) and on the other, the spherical microparticle nature that helps to reduce the skin/surface damage (Kılınc, Caklı, Cadun, Dıncer, & Tolasa, 2007;Múgica, Barros-Velázquez, Miranda, & Aubourg, 2008). In parts of Northern Europe, awareness among fisheries companies about the benefits of applying slurry-ice primarily to improve shelf quality appears to be on the increase, for example, fresh Cod species. However, this is much less among fishing communities of Mediterranean basin especially small highly perishable pelagic fishery products like European anchovy (Eugraulis encrasicolus), sardine (Sardina pilchardus), and so on. In particular, postharvest preservation challenge of rapid microbial degradation of these fish species directly affects the product/shelf quality (Bensid et al., 2014;Campos et al., 2005).
Whilst slurry-ice chilled storage applied to fishery products has been increasingly studied (Kılınc et al., 2007;Lin, Deng, Huang, & Guo, 2016;Múgica et al., 2008;Rodriguez, Carriles, & Aubourg, 2010;Zhang, Deng, & Wang, 2015), relevant information specific to ozonized slurry-ice to reduce microbiological contents in small pelagic fish (Campos et al., 2005(Campos et al., , 2006 appears inadequate. For instance, Campos et al. (2005Campos et al. ( , 2006 applied one time (postharvest) ozone treatment (biphasic water ratio mix of 40% ice -60% water) and concentration discharge (of range between 0.17 to 0.2 mg/L), then slurry-ice (−1.5°C), and refrigerated storage of 2°C to perishable pelagic sardine (Sardina pilchardus) and farmed turbot (Psetta maxima). These authors' reported such treatments with promising preservative potentials in reducing microbiological contents such as aerobic mesophiles, psychrotrophic bacteria and lactose-fermenting Enterobacteriaceae. In this context, there is need to formulate research questions that would help pave a way to supplement these previous studies, which had previously applied one-time postharvest ozone treatment of up to 0.2 mg/L concentration. Notably, the choice and use of research questions in science, according to Okpala (2017b), has been considered as very useful in the definition, collection and reporting of relevant information. Consequently, the doing of each research endeavor ought to be clearly defined with adequate and robust visualization.
Specifically, this current study was goaled to utilize a higher ozone concentration discharge of 0.3 mg/L, with the aim to determine the effects of either one-time postharvest ozone treatment ("One-T") and or repeated ozone treatments ("Seq-T") during superchilling storage (−1°C) on the microbial spoilage of European anchovy (E. encrasicolus) and sardine (S. pilchardus).

| Overview of experimental schedule
The experimental schedule of ozone treatment, slurry-ice and superchilling storage of anchovy and sardine, followed by subsequent analyses is given in Table 1.

| Sample collection up to treatment protocol
Directly onboard a "purse seine" fishing boat that traditionally are not equipped with slurry-ice machine, European anchovy (E. encrasicolus) and sardine (S. pilchardus) caught in the Strait of Sicily (Central Mediterranean) were dipped in 1:1 mixture of seawater and traditional flake-ice previously prepared in order to smoothen (by the water thermic effect) the characteristic damaging capability of this kind of ice.
Upon arrival at the land, both fish species were dripped and delivered to the laboratory of IAMC CNR within <5 hr. The quantity of the fish species were as follows: ~10 kg of sardine and ~15 kg of European anchovy.
At the laboratory, the chilling procedure previously described by Múgica et al. (2008) with some modifications applied. It involved a prototype facility (La Felsinea Electroindustriali S.r.L, Italy) that has been used to produce the slurry ice mix, which contained 50% ice-50% marine water. Previously described by Bono and Badalucco (2012) and with slight modifications, the ozone treatment method has been applied as follows: The first ~ 10 kg of sardine has been divided in two equal lots. One lot of ~ 5 kg labeled "One-T" was dipped in ~ 10 L slurry-ice (w/v of 1:2 with marine water) to ensure a rapid cooling of the core part of muscle. Subsequently, it was treated with 0.3 mg L −1 of ozone using SGC11 O 3 generator (Lenntech B.V., Rotterdamseveg, The Netherlands), and gently agitated for 15 min. At the next, maintaining in an appropriate amount of slurry-ice, the sample was kept at superchilling storage (−1°C) until required for analyses. Then, a second lot of ~ 5 kg dipped as abovementioned that had no ozone treatment and subject to only slurry-ice and superchilling storage (−1°C) served as "Control".
On the other hand and given the very high perishability of European anchovy (as compared to sardine) likely owed to some specific spoilage organisms (SSO), which are largely known to be directly involved in chemical and sensory changes during chilling storage, a more structured antimicrobial processing -based on repeated ozone treatment "Seq-T" during storage time, has been performed for this specific fish species of this current study. Using ~15 kg of European anchovy samples divided into three equal lots, one lot of ~ 5 kg labeled "One-T" was dipped in ~ 10 L slurry-ice (w/v of 1:2 with marine water) to ensure a rapid cooling the core part of muscle. When all treatments had completed, the samples continued with storage under superchilling condition (−1°C). Concurrently, microbiological quantification scheduled up to day 8 and 11 were performed on European anchovy and sardine, respectively. When required, the marine water slurry-ice has been supplemented during storage periods.

| Microbiological analysis
All processed and control samples was enumerated for total viable count (TVC), Bacillus spp., Enterobacteriaceae, Lactobacillus spp., Moraxella spp., Shewanella spp., Listeria monocytogenes and Pseudomonas spp. using standard references. All media employed for all microbiological enumeration was procured from Oxoid Ltd, UK.
Beyond TVC, the colonies were isolated on selective media and in most cases required further confirmation and identification using the required biochemical tests. At the predetermined time intervals, aseptically obtained sample flesh (E. encrasicolus and S. pilchardus) (~30 g) for all treatments and independently has been homogenized (~120 s) (Stomacher Lab-Blender 400, PBI International, Milan, Italy) in 270 mL of (w/v) sterilized saline peptone water with subsequent tenfold serial dilutions.
T A B L E 1 Experimental schedule of ozone treatment, slurry-ice and superchilling storage of anchovy and sardine samples, and subsequent microbial analyses Day 0 "One-T" "Seq-T" "One-T" N.R.

| Statistical analysis
One-way analysis of variance (ANOVA) was performed to establish treatment effects with storage time. To compare between treatments, Sample-T test has been applied. Mean differences were resolved post hoc using Tukey's honest significant difference (HSD) test. In all cases, the probability level of p < .05 was considered as statistically significant. In Figures 1-2

| RESULTS AND DISCUSSION
This current study has been designed to establish if and to what degree the ozonized slurry-ice treatment combining slurry-ice and superchilling storage would produce antimicrobial effects on these two commercially important pelagic fish species (European anchovy and sardine). This has been performed through the application of two different ozone treatments, namely: one-time "One-T" and repeated/ sequential "Seq-T". For emphasis, this latter/sequential approach (applied to the anchovy alone) aimed for any additional inhibitory effect of ozone on specific spoilage organisms (SSO) for example, Pseudomonas spp., which in abovementioned delicate fish species according to Gram (1995) are more directly involved in the production of deteriorative (chemical and sensory) attributes. Further, Pseudomonas spp., widely seen as ubiquitous and linked to its metabolic diversity, thrives at low temperatures to cause deteriorative spoilage (Gennari & Dragotto, 1992).
The TVC and Pseudomonas spp. continuously proliferated (p < .05) with superchilling storage even as ozone slurry-ice treatments were applied to both European anchovy and sardine samples (Figure 1 and 2). A closer look at the Figure 1b as  T" and "One-T" had less values than control, with "Seq-T" noticeably less compared to "One-T" (p < .05) (Fig. 1a). The result suggests that "Seq-T" approach supplemented with superchilling storage can be a promising candidate to decrease further the microbial proliferation of such delicate fishery products. Particularly, this result, on one hand, appears to strengthen the antimicrobial capacity of ozone treatment, and on the other, opens new scenarios regarding other potential uses even when applied at low dosage/quantities with storage period/time.
Indeed, the microbiological effect of "Seq-T" approach of this study can be likened to another sequential ozone-treated ice-stored crustacean product that had less microbial numbers compared to control at later stages of storage time compared with the earlier (Okpala, 2014a).
With respect to sardine samples ( Figure 1b) and somewhat different from the (above-described) anchovy ones, the "One-T" showed significantly lower TVC values at days 1 (p < .0001, F = 1643.57, R.sq in Pseudomonas spp seem apparent and at a faster rate over TVC especially between days 1 and 4 (Figures 1 and 2b). Despite such proliferation, ozone treatment can be said to show some reducing effects on Pseudomonas spp. and at the subsequent days. At the end of superchilling (−1°C) storage in ozonized slurry-ice (for emphasis: 50% ice-50% marine water), both Pseudomonas spp and TVC values of anchovy and sardine of between ~ 3.96 and 5.67 and 4.62 and 7.19 log cfu/g were noticeably below those of non-ozonized slurry-ice (i.e., control) of between ~ 4.95 and 5.77 and 5.34 and 7.29 log cfu/g, respectively.
In addition, not only being gram-negative and halophilic/salt-tolerant, Pseudomonas spp. can thrive in harsh conditions given their hardy cell walls (Van Eldere, 2003), which might be contributing in facilitating some resistance to the ozone treatment of this current study.
In view that either 10 6 or 10 7 cfu/g indicated respective upper acceptable limits for psychrotrophic and mesophilic bacteria (Bensid et al., 2014;Özogul et al., 2004), the microbial data outcome at end of storage of current study should be considered within the acceptable limit(s). Moreover, this tested superchilling approach method might have contributed to make the slurry-ice effective with storage time.
Superchilling storage stands as potential candidate to facilitate the ozone decomposition mechanism to quickly reach inhibition phase a bit activity of bicarbonate ions to facilitate inactivation of microbial cells.
And probably at such biochemical milieu, hydroxyl radicals get consumed without further regeneration of superoxide radical ion. In this context, ozone treatment improves the degree of oxidative destruction of microbial cell wall to cause cell lysis, which can result in the cell contents to leak into its surrounding medium (O'Donnell et al., 2012).
For the reason that the collapse of immune system of fishery product remains irreversible at postharvest and progressing with microbiological proliferation up to spoilage even under cold/iced storage, any variations in the detected/observed microbiological proliferation would continually depend on both innate microbial entities and cultivation environment (Okpala et al., 2014;Özogul & Balikci, 2013;Venugopal, 2005). Therefore, how microbiological proliferation develops in untreated pelagic fishery product such as European anchovy / sardine should never be underestimated even under slurry-iced situation(s).
The degree of attachment of microbial entities on food surface significantly can influence the bactericidal effects of ozone. Thus, the latter can be attributed to the unstable allotrope of oxygen, which makes its oxidizing properties very reliable (O'Donnell et al., 2012).
In the ozone and non-ozone processed samples of this current study, superchilling storage on proliferation of the above bacterial species in these two important pelagic fish species. Besides, Campos et al. (2005) seems to be the only relevant work that applied slurry-ice (−1.5°C) and refrigerated storage of 2°C to perishable pelagic sardine. However, existent relevant literatures (Erkan & Özden, 2008;Pons-Sánchez-Cascado et al., 2006;Stamatis & Arkoudelos, 2007)

| CONCLUSIONS
From the data provided by this current study, the ozonized slurry ice combined with superchilling storage shows improved antimicrobial potential on both European anchovy and sardine samples.
Specifically, the "Seq-T" performed better to reduce the microbial load of ozonized-slurry iced approach compared with the "One-T". The results of ozonized slurry-iced approach appear promising thanks to F I G U R E 3 Schematic illustration of estimated proliferation increment(s)/trend(s) of some (relevant/specific) bacterial species detected in (some) small pelagic fishery products as affected by different storage temperatures and times (days). Specifically, "A" refers to bacterial species of Enterobacteriaceae and Lactobacillus spp. in European anchovy and sardine samples of current study under slurry-iced superchilling storage condition; "B" refers to bacterial species of Enterobacteriaceae spp. in refrigerated sardine samples as reported by Stamatis and Arkoudelos (2007); "C" refers to bacterial species of Enterobacteriaceae spp. and Pseudomonas spp. in refrigerated sardine kept under ice (shorter period) as reported by Erkan and Özden (2008); "D" refers to bacterial species of Enterobacteriaceae spp. and Pseudomonas spp. in ice-stored/ refrigerated anchovy kept under flake ice as reported by Pons-Sánchez-Cascado et al. (2006); "E" refers to bacterial species of Shewanella putrefaciens and Pseudomonas spp. in refrigerated sardine samples as reported by Stamatis and Arkoudelos (2007) superchilling storage. To the best of our knowledge, this is the first report that investigated the effects of (different) ozonized slurry-ice and superchilling (−1°C) storage treatment conditions on microbial spoilage of two important pelagic fishery species (specifically, European anchovy and sardine). Given that the antimicrobial effects of ozonized slurry ice samples appears promising under superchilling storage (−1°C), future studies should be directed to define the best/optimized ozone treatment (e.g., low ozone dosage continuously applied with storage time or medium dosage sequentially or yet high dosage one time), which, not only accords with corresponding (potential) oxidative effects (in particular on unsaturated lipid component{s}) of fishery flesh product quality, but also, the acceptable safe level for on-board staff handling. And this should be performed so as to generate additional yet robust data to supplement the current information. Other such characteristic attributes as physicochemical and sensory properties of similar/other related pelagic species submitted to (different) combinations of slurry ice/ozone composition/concentrations equally deserves attention in future studies.