*Review* **State-of-Art on the Recycling of By-Products from Fruits and Vegetables of Mediterranean Countries to Prolong Food Shelf Life**

**Sara Nardella, Amalia Conte \* and Matteo Alessandro Del Nobile**

Department of Agricultural Sciences, Food and Environment, University of Foggia, Via Napoli 25, 71121 Foggia, Italy; sara\_nardella.547821@unifg.it (S.N.); matteo.delnobile@unifg.it (M.A.D.N.) **\*** Correspondence: amalia.conte@unifg.it; Tel.: +39-0881-589242

**Abstract:** Annually, 1.3 billion tons of food are wasted and this plays a major role in increasing pollution. Food waste increases domestic greenhouse gas emissions mainly due to the gas emissions associated with its production. Fruit and vegetable industrial by-products occur in the form of leaves, peel, seeds, pulp, as well as a mixture of them and represent the most abundant food waste. The disposal of agricultural by-products costs a large amount of money under certain governmental regulations. However, fruit and vegetable by-products are rich in valuable bioactive compounds, thus justifying their use as food fortifier, active food packaging or as food ingredients to preserve food quality over time. The present review collects the most recent utilization carried out at lab-scale on Mediterranean fruit and vegetable by-products as valid components to prolong food shelf life, providing a detailed picture of the state-of-art of literature on the topic. Bibliographic research was conducted by applying many keywords and filters in the last 10 years. Several scientific findings demonstrate that by-products, and in particular their extracts, are effectively capable of prolonging the shelf life of dairy food, fresh-cut produce, meat and fish-based products, oil, wine, paste and bakery products. All of the studies provide clear advances in terms of food sustainability, highlight the potential of by-products as a source of bioactive compounds, and promote a culture in which foods are intended to receive a second useful life. The same final considerations were also included regarding the current situation, which still limits by-products diffusion. In addition, a conclusion on a future perspective for by-products recycling was provided. The most important efforts have to be conducted by research since only a multidisciplinary approach for an advantageous investigation could be an efficient method to promote the scale up of by-products and encourage their adoption at the industrial level.

**Keywords:** fruit and vegetable by-products; food shelf life; sustainable food; by-products recycling

#### **1. Introduction**

In later years, the FAO stated that "food loss" comprises a decrease in the quantity or quality of food in the production chain [1]. It typically occurs during the animal and plant production, storage, processing, and distribution stages. In addition, it may be due to an incorrect agricultural practice as well as some phenomena, such as bruising or wilting or inadequate storage [2]. Globally, researchers estimate that, in food supply chains the percentages of food loss in production, postharvest, and consumption stages are 24, 24, and 35%, respectively [3]. "Food waste" is a component of the wider problem of food loss [4] and refers to the decrease in the quantity or quality of food resulting from decisions and actions by retailers, food service providers, and consumers [1]. This is regarding edible food, which is wasted in the second portion of the food supply chain until final consumption. In the UK, for example, the value of wasted products was estimated between USD 1500 to nearly 3000 per ton, depending on whether the waste is generated during processing or final consumption [5]. Food waste causes a large depletion of available land

**Citation:** Nardella, S.; Conte, A.; Del Nobile, M.A. State-of-Art on the Recycling of By-Products from Fruits and Vegetables of Mediterranean Countries to Prolong Food Shelf Life. *Foods* **2022**, *11*, 665. https:// doi.org/10.3390/foods11050665

Academic Editor: Luís Manuel Lopes Rodrigues da Silva

Received: 13 January 2022 Accepted: 16 February 2022 Published: 24 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and other environmental concerns, including the unnecessary gas emissions, which are measured by carbon footprint and water wastage [6,7]. Venkat [8] observed that grains, vegetables, and fruits generate 56% of the US waste, but register relatively low emission footprints.

The current linear model of the economy is based on the concept of the constant supply of products with a short useful life, forcing an increased production to satisfy the consumer's constant needs. It increases the indiscriminate exploitation of limited natural resources that would yield a significant environmental and economic crisis. In 2009, the FAO organized a forum on "How to Feed the World in 2050". Based on the prospects of that time period, the world population will increase up to 9.1 billion in 2050, accompanied by an increase of urbanization. This will lead to a rise in annual food production. However, it will only be possible with the implementation of the right investment and agricultural policies [9]. This is only one of many concrete examples, which demonstrate the real entity of the current growth trends. Apart from reducing losses and wastes at all levels of the food chain, it is possible to recycle rather than throw them [10]. Therefore, both economic and environmental impacts may be limited.

#### *Recycling of Fruit and Vegetable By-Products*

Within the framework of food loss, food industries annually produce tons of byproducts during food processing. The most abundant part is represented by fruit and vegetable by-products, which can occur during the pre- and post-harvesting process, preparation, and processing of fruits and vegetables. These industrial by-products are very different from one another due to the difference in industrial processes. For example, grape and olive pomace are derived from wine and oil production, while other fruit by-products are derived from the juice, jelly, and jam industry, for example, from apple, kiwifruit, citrus, passion fruit, mango, etc., as well as from the processing of potato, tomato or carrot.

By-products have phytochemical compounds with recognized antioxidant and antimicrobial properties [11–15]. Generally, food by-products are used as animal feed or for the production of biomaterials, biofuels, biogas, platform chemicals, and bio-fertilizers [16,17]. However, over time, thanks to the great potential of their active compounds, they have been utilized in several industrial fields (cosmetic, pharmaceutical, and food) [18].

In regards to the food industry, the goal was to re-introduce the by-products to the production line as raw materials, to obtain new functional products with health benefits or to enhance food preservation or develop active packaging, as presented in Figure 1 [19].

**Figure 1.** New directions in managing fruit and vegetable by-products.

The above picture represents the potential applications reported in the scientific literature in the field. In fact, many research studies report the usage of these by-products (i) as ingredients in different types of foods to increase the nutritional value, (ii) as natural preservatives to maintain food quality, (iii) as a source of bioactive agents to develop films and coatings with antimicrobial and/or antioxidant properties.

Specifically, with regards to the by-products for food preservation, it is interesting to observe that literature data are very abundant. Some of these studies are related to the active packaging systems, which are aimed at prolonging food shelf life. Additionally, other studies are focused on by-products, which are directly applied to food in order to preserve its quality during storage. Here, we provide some examples. Aloui et al. [20] developed a film incorporated with an extract of tomato pomace, while Kanmani and Rhim [21] added grapefruit seed extract into an agar film. Torres-León et al. [22] and Kanatt and Chawla [23] tested the effect of mango by-product-based films on peaches and chicken meat, respectively. Other authors investigated the usage of winemaking by-products on fruits and vegetables [24,25] and on fishery products [26,27]. Madzuki et al. [28] and Gallego et al. [29] tested tomato by-products added with film and coating on the deterioration of calamansi and pork meat, respectively. Moreover, there are various studies on different types of olive milling by-products [30,31]. In regards to by-products that are added to food in order to prolong the shelf life, the numerous applications found highlight that their effects are strictly dependent on the type of by-products and on the food characteristics. Therefore, the present review aims at collecting all of the information available in the literature during the last 10 years, which deal with specific applications of fruit and vegetable by-products to prolong food shelf life. To better organize the work, the studies were divided by the type of food, thus including dairy food, fresh-cut produce, vegetable-based processed food, meat, fish, and cereal-based products. In each paragraph, the reader can find details regarding the typology, concentration, and technique to apply by-products to food. In addition, the main results regarding the effects of by-products on food quality were highlighted. This provides a real map of the most effective by-products and food sectors, where by-products could find the concrete possibility of recycling. The final considerations regarding the current situation and future trends are also reported in the conclusions.

#### **2. Materials and Methods**

In this review, electronic literature searches were conducted using the online library "opac.unifg.it", which collects files from several databases including PubMed, Google Scholar, and Science Direct databases. Numerous search terms have been used, including keywords as "fruit by-products and food shelf life", "vegetable by-products and food shelf life", "antioxidant activity of food by-products", and "antimicrobial activity of food by-products". More specific search terms were also used, as "pomegranate by-products in food products", "grape pomace and food shelf life" or "fruit by-products and meat shelf life". The selected time interval was 2010–2022, since the treated subject is quite recent and the aim of the current review is to collect the latest lab-scale trends. The research process uncovered numerous articles, and among them, 86 articles were used for the current overview. Literature data focused on the potential re-utilization of fruit and vegetable byproducts, and how these resources can be used as ingredients in food products to prolong food shelf life. The studies cited in this overview are grouped by the type of food, in which by-products are added (dairy food, horticultural food, vegetable food, meat, fish food, and cereal food).

#### **3. By-Products and Dairy Food Applications**

Dairy products represent a large and differentiated group of food products. The quality of raw material is the first aspect that must be ensured when referring to dairy products. Furthermore, each type of product is associated with specific defects. However, it is possible to identify some factors that are common to a range of goods. In fact, high fat products are prone to oxidation. In addition, dairy products can be subjected to enzymatic, chemical degradation or spoilage by bacteria, molds, and yeasts that grow at a refrigerated temperature [32]. At present, there are many innovative treatments and packaging technologies, which can limit the degradation of dairy products and extend their shelf life. In this regard, fruit and vegetable by-products are a valid option. Only two studies regarding the actual use of fruit and vegetable by-products to prolong the dairy product shelf life are reported in the literature, one study with olive by-products and the other with grape by-products. The main research results are presented in Table 1.

#### *Olive and Grape By-Products*

Olive oil extraction produces a variety of solid and liquid by-products, which can be grouped in olive pomace, olive mill wastewaters, olive leaves, and olive stones, and seeds. In addition to the economic burden for producers, olive oil by-products represent a severe environmental problem for their safe disposal. Simultaneously, these are rich in bioactive molecules as phenolic compounds with antioxidant and antibacterial activity [33], which can be used in the food industry as a source of natural preservatives. Rolia et al. [34] used olive phenolic extract, obtained by liquid–liquid extraction with ethyl acetate, to limit the growth of spoilage bacteria in "Fior di latte" cheese. Phenolic extract was added to the brine of packaged cheese, in two different concentrations, 250 and 500 μg/mL. The maximum microbial load tolerated for *P. fluorescens* was reached at nearly 13 and 15 days of storage for 250 and 500 μg/mL loaded samples, respectively, compared to about 11 days, which was found for the untreated cheese. For Enterobacteriaceae, the threshold limit was reached at nearly 14 and 16 days of storage for 250 and 500 μg/mL added samples, respectively, whereas it was reached at about 10 days for the traditionally packaged cheese. Therefore, it can be asserted that this olive by-product extract can be advantageously used to extend the shelf life of "Fior di latte" from 2 to 4 days, by limiting the main spoilage microorganisms.

Grape processing by-products are generated from the winemaking process. It typically occurs as pomace consisting of a mixture of residual seeds and skins, which are rich in polyphenols, dietary fibers, citric acid, ascorbic acid, tocopherols, limonoid, and other trace compounds. These mixtures also have a strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, antiseptic, germicidal, fungicidal, and anti-viral properties [35]. For this reason, wine grape pomace can represent an excellent alternative to synthetic compounds in prolonging food shelf life. Tseng and Yanyun [36] examined the feasibility of using wine grape pomace as a source of antioxidant dietary fibers and polyphenols in yogurt for the improvement of its nutritional value and enhancement of its storability. In this study, dried whole grape pomace, pomace liquid extract (LE), and freeze-dried liquid extract (FDE) were investigated. Peroxide value, as an indicator of oxidation, increased during storage and after 3 weeks. In addition, 3% grape pomace added with yogurt showed the lowest oxidation value. The results are interesting, even though it is still necessary to further examine the mechanisms and methods of retention of total phenolic content and radical scavenging activity in the product.


**Table 1.** Applications of by-products to dairy food.

#### **4. By-Products Applied to Fresh-Cut Produce**

Minimally processed foods represent a great source of vitamins, minerals, dietary fiber, and other constituents. Their peculiarity is that as living entities, their metabolic processes, such as respiration, transpiration, and biochemical transformations continue after the harvest. Therefore, fresh produce quality decreases throughout time, until it becomes unacceptable for consumption. However, these processes can be slowed down by manipulating the process and storage conditions. Due to the advent of increasingly innovative technologies, it is possible to extend their shelf life over and beyond the harvest season [37]. This section is regarding the actual use of fruit and vegetable by-products, which are used as a dipping solution or food ingredient, with the aim of extending the fresh-cut food shelf life. All of the case-studies are presented in Table 2 and reported in detail below.

#### *Tomoto Papaya, Grapefruit, and Pomegranate By-Products*

Tomato by-products are a good source of chlorophylls and phenolic compounds with antioxidant and antimicrobial activities [38]. Martínez-Hernández et al. [39] investigated the effects of lycopene microsphere based dipping solutions on the evolution of the physicochemical, microbial, and bioactive quality of fresh-cut apples during refrigerated storage. Lycopene was obtained by thermal extraction from tomato by-products. The Browning Index showed that the treated samples of lycopene microspheres maintained a lower value until the 9th day of storage. In particular, this is shown in the sample dipped in the solution with the highest concentration of lycopene microspheres when compared to the water dipped control sample and the ascorbic acid dipped sample. Microbial loads of the control sample showed mesophilic, yeast, and mold increments after 9 days, similar to fresh-cut apples dipped in low lycopene microspheres solutions. Whereas, the sample dipped in the highest concentration of lycopene microspheres, showed no microbial increment during the storage period.

Papaya peel is also used to inhibit the food browning process. Papaya is a tropical fruit, in which its processing by-products mainly consist of peels and seeds. They are rich in bioactive compounds [40]. Faiq and Theerakulkait [41] evaluated the effects of dipping in the papaya peel crude extract and distilled water-based solution on the browning inhibition in potato, banana, and apple slices. Papaya peel extract was obtained by stirring a mixture of papaya peel with distilled water and then centrifuging. The authors reported that during storage, the extract had an higher percentage of browning inhibition in potato slices when compared to banana and apple slices.

In regards to grapefruit seed extract, Kim et al. [42] conducted a study on its antimicrobial efficacy against *E. coli* O157:H7, *S. typhimurium*, and *L. monocytogenes* inoculated in fresh-cut lettuce, alone and in combination with malic acid. Fresh-cut lettuce was stored at 5 ◦C, and its microbial quality was monitored over a period of 14 days. Regarding *E. coli* O157:H7, all of the treated samples showed more than 4.4 log CFU/g reductions. Concerning *S. typhimurium*, all of the treatments had more than 4.1 log reductions. Moreover, *L. monocytogenes* was subjected to a significant reduction of its viable cell concentration in all of the treated samples. Furthermore, it was observed that the combined treatment was more effective for the reduction of all the tested foodborne pathogens.

Pomegranate by-products mainly consist of seeds and peels, that originate from the industrial production of juices and jams. They are rich in active compounds, in particular, anthocyanins and hydrolysable tannins, with high antioxidant and antimicrobial activities [43]. Belgacem et al. [44] tested pomegranate peel extract as a natural antimicrobial to reduce the growth of *L. monocytogenes* inoculated on fresh-cut melons, apples, and pears via dipping. Fresh fruits were peeled, cut, and inoculated with the pathogen. Then, the inoculated samples were dipped in solutions at several extract concentrations. Regarding the fresh-cut apples, the sample without the extract showed an increase of the population of *L. monocytogenes* during 7 days of cold storage. On the contrary, the extract efficiently reduced the population of the pathogen. Moreover, the viable cell concentration reduction was reported as higher for samples with the higher concentration of added extract. In regards to fresh-cut pear, except for the sample at the lowest extract concentration, all of the other treated samples showed a significant reduction of *L. monocytogenes* when compared to the control sample. As expected, the sample at the highest concentration was the most effective. Moreover, in fresh-cut melon, after 7 days of storage, the sample at the highest concentration showed the most significant reduction of the tested pathogen. Elsherbiny et al. [45] also investigated the efficacy of a methanol extract of pomegranate peel applied as curative and preventive treatments to control *Fusarium* dry rot on potato tubers, using a dipping in five concentrations of extract solution or distilled water (control). The authors reported that, in curative application, the extract caused a significant reduction of dry rot development of potato tubers when compared to the control. The best reduction was obtained for the sample with the highest extract concentration. Moreover, it significantly reduced the diameter of dry rot lesions in potato tubers in the preventive application when compared to the control. Very recently, other authors tested pomegranate peel as it is, without producing any extract. In this case, Lacivita et al. [46] evaluated the effects of pomegranate peel powder at two concentrations, on the quality decay of a mixture of fresh-cut fruit salad (nectarine and pineapple) in fructose syrup, stored at 4 ◦C for 4 weeks. Microbial analysis showed that for the first 8 days, all of the samples maintained approximately the initial mesophilic and psychrotrophic bacteria concentration, whereas at the end of the storage period, the control sample had a significantly higher bacterial proliferation. The control salad was microbiologically acceptable for about 24 days, whereas both of the treated samples were below the microbial threshold until the end of storage. Therefore, the lowest concentration of pomegranate by-product is enough to ensure a longer microbial stability of fresh-cut fruit salad when compared to the control. In particular, at the end of the storage period, the treated salad had a significantly lower yeast concentration when compared to the control, especially the sample with the highest concentration of peel. It was also observed that the peel was very effective on lactic acid bacteria, which were lower than the control at the end of the storage. The overall sensory quality of the control sample lasted for 3 weeks, whereas the active samples lasted longer. In conclusion, it can be stated that the pomegranate peel capacity can be considered as a broad-spectrum since it had the capability of exerting a good antimicrobial and antifungal activity. These by-products can represent a great alternative to synthetic compounds, which are generally recognized by the scientific community as effective sanitizers of fresh-cut fruits [44].


**Table 2.** Applications of by-products to fresh-cut produce.

#### **5. By-Products Applied to Vegetable-Based Processed Products**

Various types of vegetable-based processed foods, such as wine, olive-based pâté, hazelnut paste, and different refined oils, are presented in this section. Each of these foods has a peculiar mechanism of deterioration and specific shelf life since chemical, microbiological, and physical changes can occur during storage. In recent times, many studies have been conducted on the feasibility of preserving these different food products with natural compounds derived from by-products. Table 3 briefly presents the case-studies that are detailed in the subsequent two paragraphs.

#### *5.1. Olive By-Products*

Sulfur dioxide (SO2) is commonly added to commercial wines at the time of bottling, in order to confer microbiological and oxidative stabilities [47]. Ruiz-Moreno et al. [48]

investigated the antioxidant and antimicrobial activities of hydroxytyrosol-enriched extract obtained from olive mill waste, as a potential alternative to SO2 in winemaking. The authors concluded that the hydroxytyrosol-enriched extract itself is not sufficient for the replacement of SO2 in wines, but a combination of SO2 and hydroxytyrosol-enriched extract could be a valid solution. In fact, the analyses showed that *Saccharomyces cerevisiae* delayed its growth only with SO2. *Hanseniaspora uvarum* and *Pediococcus damnosus* were completely inhibited by SO2, whereas the extract only delayed their growth. The extract resulted in inefficiency against *Lactobacillus plantarum*.

Bouaziz et al. [49] conducted a study on different types of olive leaf extracts (enzymatic hydrolysate leaf extract (EHE), acetylated hydrolysate extract (AHE), and pure oleuropein), in refined olive oil (ROo) and refined olive-pomace oil (RPo) during storage, then compared them to extracts with α-tocopherol. Both of the oils that were added with EHE showed the lowest oxidation value. The rise in oxidation of the control was similar to the AHE added with oil. Finally, the oxidative resistance improved with the addition of olive leaf phenolic extracts, especially for the EHE loaded sample. Furthermore, after 6 months of storage, the oxidative resistance was lower for the two refined oils added with oleuropein, whereas it fell to zero in the control sample, as well the extracts enriched with α-tocopherol and AHE. Therefore, it can be stated that enrichment with olive leaf extract reduced oil rancidity.

Other authors [50] evaluated the effect of olive leaf extract on non-thermally stabilized olive-based pâté during storage, as compared to the butylated hydroxytoluene (BHT). In regards to the microbial analysis, the absence of pathogens (*Clostridium* and *Listeria* spp.) and contaminants (*Pseudomonas* spp. and Enterobacteriaceae) was observed. As the samples of olive-based pâté were not subjected to thermal stabilization, cultivable bacteria, yeasts, and molds were detected during sample production and storage. However, their growth was affected by the addition of the extract and refrigeration storage. The main microbial groups registered a significant reduction of the viable cell concentration in samples added with the extract, especially with 1.0 g/kg. Therefore, the authors reported that the samples were suitable for consumption, from a microbiological point of view, during refrigeration storage for 120 days under modified packaging conditions, compared to the 90 days of the untreated or BHT-added samples.

#### *5.2. Potato, Grape, and Pomegranate By-Products*

Potato by-products usually occur, among other residues, as peels, which are derived from many industrial potato processes. Potato peel is a rich source of bioactive compounds due to its high content in phenolic compounds with recognized health-promoting properties [51,52]. Samotyja [53] evaluated the antioxidant properties of two different cultivars (Jazzy and Gala) of potato peel extracts on rapeseed and sunflower oil. In regards to rapeseed oil, potato peel extracts of both cultivars inhibited the formation of hydroperoxides, showing higher efficiency when compared to BHT and butylated hydroxyanisole (BHA). Among the tested extracts, 80% ethanol extract was the most efficient. The extracts also delayed the formation of conjugated diene hydroperoxides in oil. In regards to sunflower oil, ethanolic extracts showed a moderate activity against hydroperoxides formation. Moreover, by increasing the extract concentrations, the activity against hydroperoxides formation increased. All of the investigated extracts were capable of delay conjugated diene hydroperoxide formation. Furthermore, all of the extracts significantly reduced hexanal formation when compared to the control. Therefore, potato peel extracts, are not only capable of delaying primary oxidative changes, but also have a positive effect on retarding oil rancidity.


**Table 3.** Applications of by-products to vegetable-based processed food.

In regards to grape by-products, Spigno et al. [54] investigated the feasibility of using a grape marc phenolic freeze-dried extract to protect commercial hazelnut paste against oxidation, crude, and encapsulation into different nano-emulsion-based delivery systems. The authors asserted that phenolic grape marc can inhibit hazelnut paste oxidation and consequently improve its shelf life, even though the antioxidant effect was limited since the paste split in two phases after 5 weeks and the extract was separated. The extract did not solubilize completely in the dark fluid hazelnut paste added with emulsifiers. The encapsulation process reduced the antioxidant activity of almost all of the extracts. All of the tested formulations were no longer active after 83 days. The oil in water nano-emulsion resulted in the best encapsulation solution due to its efficiency.

Pomegranate peel extract exerted a significant role in the preservation of seed oil. This is the result of the case study by Drini´c et al. [55]. The authors investigated the effect of pomegranate peel extract, alone and combined with BHT, on the antioxidant stability of pomegranate seed oil. At the end of storage, oil with the combination of antioxidants (0.05% pomegranate peel extract and 0.01% BHT) had a significant lower oxidation when compared to the pomegranate peel extract added with oil. Thiobarbituric acid reactive substances (TBARS) showed that at the end of storage, oil added with pomegranate peel extract showed the highest percentage of oxidation inhibition, followed by the combination of antioxidants and BHT.

#### **6. By-Products and Meat Applications**

The shelf life of meat products is influenced by many factors, which are complex and interconnected. Microbial growth is one of them, and the types of microorganisms that can be found in meat depend, among other things, on animal species, personnel sanitation, type of packaging, storage time, and temperature [56]. Lipid oxidation is the major cause of chemical alteration during storage. It causes irreversible changes in taste, flavor, color, and texture of the products, resulting in a shelf life reduction. Moreover, protein oxidation causes the loss of sensory properties, essential amino acids, and protein digestibility. Modified atmosphere packaging and synthetic antioxidant and antimicrobial substances are widely used in the food industry to improve the meat product shelf life. Nevertheless, synthetic compounds are known as potentially toxic. Therefore, natural extracts can represent a good alternative to protect these foods.

#### *6.1. Olive Mill Wastewater, Pomace, and Seed Extract*

As previously reported, tomato pomace has shown good bioactive properties. Olive pomace is an important natural source of phenolic compounds. Pomegranate seeds are known to have powerful antioxidant compounds. Finally, grape pomace is very rich in antioxidant and antibacterial agents. All of these by-products were adopted, mainly in the form of extracts, with slight effects on the quality of meat-based food. In general, all of them, and in particular grape by-products, are capable of preserving meat products against lipid oxidation, protein oxidation, and microbial growth (Table 4). Nevertheless, in some cases, it is better to combine them with other preserving methods to increase their effects.

In the study by Andrés et al. [57], the authors evaluated the shelf life of lamb meat patties added with aqueous extracts from tomato, olive, pomegranate, and grape by-products stored in retail sale conditions. The authors reported that free thiols consistently decreased after 7 days of storage, even though the extract-loaded samples showed the highest values at the end of the observation period. Counts of mesophilic and psychrotrophic bacteria in lamb patties with extracts were significantly lower than the control and sodium ascorbate added samples, even though the antimicrobial effect of the extracts was less evident for Enterobacteriaceae and lactic acid bacteria. In a second study, Andres et al. [58] evaluated the in vitro antioxidant potential of tomato pomace extracts and then the effect on the shelf life of lamb meat packaged under modified packaging conditions (MAP). After 7 days of storage, the TBARS values of meat treated with the extract obtained using ethyl acetate and with the extract obtained using ethanol significantly increased, with no significant differences from the control. Free thiols significantly decreased during storage. Moreover, microbiological analysis showed that the aerobic count in lamb meat remained under the microbiological limits in fresh meat, for both mesophilic and psychrophilic bacteria. The final values of lactic acid bacteria were also substantially lower than the imposed limit.

Better results were found by Selani et al. [59], who investigated the effects of Isabel and Niágara grape seed and peel extracts on lipid oxidation of raw and cooked chicken meat vacuum-packed and stored at −18 ◦C for 9 months. The authors reported that both natural extracts were similar to commercial antioxidants in preventing lipid oxidation in raw and cooked chicken meat and their effects were more evident in cooked samples. According to Selani et al. [59], other authors also assessed the effects of grape seed extracts. Kulkarni et al. [60] compared three levels of grape seed extract as a commonly used antioxidant, in pre-cooked, frozen, and stored beef and pork sausage. Based on sensory characteristics, instrumental color, and TBARS values, the authors concluded that the sample added with 100 and 300 ppm of extract was generally as good as propyl gallate in maintaining product quality during the 4 months of storage.

Lorenzo et al. [61] evaluated the effect of grape seed and chestnut extracts and BHT on physico-chemical and microbiological changes, as well as lipid oxidation during the ripening process of dry-cured sausages. To this aim, the extracts were mixed with meat during the initial phases of processing. The authors asserted that the grape seed extract was the most effective antioxidant of dry-fermented sausages. In fact, they reported that the TBARS values decreased when compared to the control. However, compared to the control and chestnut extract-treated sausages, grape seed extract improved lipid stability. Total viable count and lactic acid bacteria increased during the first 19 days of ripening and remained stable until the end of the curing process. The highest lactic acid bacterial counts were observed in sausages of the chestnut extract group and control batch. At the end of the curing process, mold and yeast counts were higher in control and grape seed extract samples than in BHT and chestnut extract-treated sausages.

Grape pomace extract was found less effective than seed and peel extracts by Garrido et al. [62]. These authors evaluated the effects of two types of grape pomace extracts on the physico-chemical characteristics of pork burgers and their preservative capacity. They reported that total viable count, psychrophilic bacteria, and total coliform count were not affected by the extract addition. In fact, the samples were microbiologically unacceptable after 6 days. Nevertheless, compared to the extract obtained using methanolic extraction, the extracts obtained using the high–low instantaneous pressure and high–low instantaneous pressure + methanolic extraction showed a stronger antioxidant effect when added to meat.

*Foods* **2022**, *11*, 665


Garcia-Lomillo et al. [63] evaluated the effect of milled red and white grape skin wine pomaces on beef patties, which are stored for 15 days in high oxygen modified atmosphere against protein oxidation, in comparison to the protective effect of sulfites. The analysis on beef proteins showed that patties added with sulfites had the lowest accumulation of protein radicals. The addition of white grape skin wine pomace to the beef patties resulted in higher radical intensity, whereas the addition of red pomace caused no effect compared to the sample added with water (control). Moreover, while the radical intensity of control and white pomace added with patties increased during storage, those added with sulfites or red pomace were constant. The authors concluded that red pomace effectively protected against protein oxidation.

In regards to olive mill wastewater, Chaves-López et al. [64] evaluated its effect against the undesired household fungi that may grow on the surface of Italian dry fermented sausages during ripening, considering its impact on the microbiological and physicochemical characteristics. The authors reported that all of the microbial groups grew during the observation time, except for Enterobacteriaceae, which decreased. The treated samples showed an extra reduction of fungal growth, which was proportional to the extract concentration. In the enriched samples, only *Penicillium nalgiovense* and *Penicillium chrysogenum* were isolated. Micrococcaceae, yeasts, and molds decreased in the treated batches. However, compared to the control, their proteolysis index was slightly higher. In the presence of olive mill wastewater polyphenols, the volatile compounds derived from microbial esterification and lipid oxidation decreased. Furthermore, TBARS of fortified samples showed reduced values when compared to the control batch. Therefore, the authors found that the surface treatment of fermented sausages with 2.5% by-products addition was effective against some undesired fungal species.

#### *6.2. Olive Leaf Extracts*

Olive leaf extract was also greatly used at lab-scale to prolong the shelf life of several meat-based foods, which are considered a valid alternative to synthetic additives. The extracts are capable of maintaining both chemical and microbiological safety. Therefore, they can enhance meat quality in the same way or even better than synthetic compounds. In many cases, it was reported that their positive effect was more evident by increasing their concentrations (Table 4).

Specifically, Baker [65] conducted a study to establish the optimum concentration of olive leaf extract in minimizing the oxidative and microbiological deterioration of Karadi lamb patties. The author reported that compared to the untreated sample, the treated samples with 1, 2, 3% of extract had significantly lower TBARS values. The bacterial counts increased during storage. However, for the treated patties, microbial proliferations were significantly lower when compared to the control sample, until the end of the tested period. The authors found that 1% of treated patties also showed the best overall acceptability. In the same context, Djenane et al. [66] used Algerian wild olive leaf extracts as an enrichment of Halal minced beef at two different levels (1 and 5%, *v*/*w*) and evaluated their effects on microbiological safety during 6 days of retail. The authors reported that the addition of 5% extract showed the strongest antimicrobial activity. In addition, at the end of the storage period, the microbial count found in the treated samples was still not near the critical microbiological threshold. The TBARS values of all the tested samples also decreased by increasing the extract concentration. Other authors recently [67] assessed the effects of olive leaf extracts on physico-chemical properties and microbial quality of chilled poultry meat. To this aim, meat was dipped for 15 min in the extract at concentrations of 0.25%, 0.5%, and 1%. Herein, it can be asserted that olive leaf extract has the capability of maintaining the chemical and microbiological quality of chilled poultry meat. In fact, compared to the control (meat without the extract), the total volatile basic nitrogen (TVBN mg/100 g) in poultry meat after the treatment was significantly reduced, especially in 1% of the treated sample. Moreover, this last sample reached the unfit limit for TVN after 15 days over 6 days for the control. The TBARS values of all the samples increased during storage. However, compared to the control samples, the treated product had noticeably lower TBARS values. The extract also decreased the total aerobic plate count. Compared to the lower concentrations, 1% was more effective. Moreover, the extract, especially 1%, positively delayed the growth of psychrophilic, Enterobacteriaceae, Staphylococcal, as well as the mold and yeast counts. Elama et al. [68] investigated the effects of oleuropein from olive leaf extract on lipid peroxidation in frozen bovine hamburger, compared with sodium erythorbate. Oleuropein was mixed at different concentrations with meat during the hamburger preparation. The authors reported that the amount of oxidation products increased for both control and treated samples (with 0.25, 0.5, and 0.75%) during 6 months of storage. However, at the end of storage, the amount of oxidation products was found as lower in the treated samples than in the control. The sample which showed the best results was the one with 0.5% oleuropein.

The effects of destoned olive cake on the physico-chemical, microbiological, and sensory quality of beef patties during cold storage were also assessed by other authors [69]. Samples were prepared by mixing minced beef with olive by-products at several concentrations. Compared to the untreated sample, the DPPH radical scavenging activity values after 14 days of storage of fortified patties at a concentration above 2% were found as higher. The TBARS values of all the samples increased during the storage period. However, compared to the untreated samples, the TBARS values observed for the treated beef patties were lower. Moreover, it was found that the by-products effect is concentration dependent. Furthermore, the authors observed that all of the fortified samples had a significantly lower total plate count than the control sample. Therefore, the authors reported that the incorporation of destoned olive cake in beef patties could prolong their shelf life up to 14 days. To conclude the state-of-art of olive by-products, the case study by Nieto et al. [70] can be also cited. These authors evaluated the effects of different hydroxytyrosol extracts on chemical and sensory properties of low-fat frankfurter chicken sausages during 21 days of storage at 4 ◦C in a modified atmosphere. The chicken sausage formulation includes olive oil as a fat substitute, walnut as a macronutrient, and hydroxytyrosol extract as an antioxidant. The extracts were added to the homogenized meat, then mixed. The authors reported that the TBARS values of all the samples increased during the storage period. However, compared to the control sample, the oxidation was significantly lower in extracts added with sausages. Compared to the control with pork fat, control with walnut, and control with walnut and olive oil, the extracts significantly reduced the thiol concentration since the start of the storage period. Therefore, to conclude, the addition of walnuts and extracts was useful for the prevention of lipid and protein oxidation of sausages, especially when olive oil was used rather than pork fat.

#### **7. By-Products Applied to Fish-Based Products**

Fish food represents a highly healthy food, as they are an abundant source of high biological value proteins, long chain polyunsaturated fatty acids, and other nutritional components. In fact, color and lipid oxidation are the main factors for quality deterioration in fishery products during storage. The pH values in fish, which do not decrease as in meat products, cause enzymatic systems to be highly active, thus increasing their vulnerability to bacteria [71]. The shelf life of fish products is usually extended with refrigeration, freezing or thermal inactivation. Synthetic additives are frequently used to enhance their shelf life, although nowadays, food industries are searching for natural alternatives and have found them in by-products (Table 5).


**Table 5.** Applications of by-products to fish food.

#### *7.1. Peel, Seed, and Leaf Extracts*

Viji et al. [72] investigated and compared the effects of citrus peel extract and mint extracts to prolong the shelf life of washed, beheaded, and eviscerated Indian mackerel during chilled storage. The authors reported that TVBN increased significantly in all of the samples during storage. The rate of lipid hydrolysis was substantially lower in treated fish, whereas the level of free fatty acids and peroxide values in the sample remained slightly lower than the control sample. The TBARS values of all the samples increased gradually during storage. However, at the end of the observation period, the TBARS value of the sample with mint extract was the lowest one. Moreover, the extracts were capable of slowing down bacterial growth. Therefore, the treatments extended the shelf life of refrigerated mackerel by 2 and 5 days with citrus and mint extracts, respectively.

Özen and Soyer [73] evaluated the efficiency of green tea extract, grape seed extract, and pomegranate rind extract in limiting lipid and protein oxidation in minced mackerel, during 6 months of frozen storage. Peroxide values increased for minced mackerel added with the extracts and the control until the 4th month of storage, then declined rapidly. Among the extracts, pomegranate was the most effective. TBARS of the BHT treated sample showed only a slight increase, followed by the sample with pomegranate extract. However, at the end of frozen storage, compared to the control, all of the samples loaded with antioxidants showed significantly lower TBARS values. Compared to the control, the carbonyl content of all the antioxidant-loaded samples was significantly lower at the end of the 6th month. Furthermore, the changes in total protein solubility showed a progressive decrease throughout the storage period and compared to the control, the antioxidant treated samples showed a significantly higher total protein solubility. The authors concluded that these natural antioxidants improved the oxidative stability of fish and pomegranate extract was the most effective in protecting its quality. Prior to the Özen and Soyer study [73], Özen et al. [74] investigated the effects of natural extracts from by-products on lipid oxidation of fish during 3 months of frozen storage. The authors used grape and pomegranate seed extracts added to minced chub mackerel muscle. In addition, they reported that peroxide values increased for all of the tested samples during the storage period, especially for the pomegranate loaded sample, whereas no significant increase was observed for the sample with grape seed extract. Moreover, compared to the antioxidant

treated samples, the TBARS values of the control fish were significantly higher at the end of the tested period.

Hasani and Alizadeh [75] evaluated the effects of red grape pomace extract, added at two different percentages (2 and 4%), on quality changes in silver carp fillets during refrigerated storage. The TBARS values of all the samples increased during storage. However, compared to the control, the TBARS values of grape pomace extract-loaded sample were significantly lower, more evidently with 4% of extract.

Yerlikaya et al. [76] produced an ice with different citrus peel extracts and evaluated their effects on the shelf life and quality of common pandora during refrigerated storage. The authors reported that the TVBN concentrations in the fish stored in ice increased during storage in all of the samples. At the end of the storage, the grapefruit flavedo treatment had the highest TVBN value, whereas the bitter orange flavedo treatment showed the lowest value. Moreover, the TBARS values of samples treated with citrus extracts remained low. The count of total psycrophilic bacteria of all the samples significantly increased during storage, but the bitter orange extract-loaded samples showed the lowest value at the end of the storage.

No significant results were found by Ali et al. [77], who evaluated the supplementation of extracts from cabbage leaves and banana peels in fish-based products to prevent the formation of potential oxidized free fatty acid and peroxide compounds. Natural extracts were loaded at different concentrations (0.5, 1, and 1.5%) to fish meat balls and then stored both at 4 and −18 ◦C for 9 days and 2 months, respectively. All of the treated samples showed an increase of peroxide value and free fatty acid values, more evidently when stored at 4 ◦C. Contrary to Ali et al. [77], Abdel-Wahab et al. [78] investigated with success the antioxidant and antimicrobial properties of clove flower buds, sage leaves, kiwifruit peels aqueous extracts, as well as their mixture, on tuna fish fingers. The extracts mixed at concentrations of 0.1, 0.25, and 0.5% were mixed during processing. The authors observed that the TBARS values significantly increased in all of the samples during 30 days of storage. However, compared to the control sample, the treated fish reached significantly lower TBARS values. Moreover, compared to the 0.25 and 0.5% mixture of the added samples, the carbonyl content increased significantly in the other samples. TVBN values increased in all of the samples during storage. However, compared to the control, the extract mixture of the treated samples showed lower final values. Furthermore, the total bacterial count increased significantly in all of the samples during storage, less than two orders of magnitude in the mixtures of the treated samples. The control reached the threshold limit at day 3, and the BHT-treated fish at day 6. On the contrary, the 0.5% extract mixture of the treated sample reached the threshold limit at day 24, thus demonstrating the great effects of the extracts on product shelf life.

Miraglia et al. [79] asserted that the extract derived from olive mill wastewater was capable of delaying lipid oxidation, microbial growth, and TVBN on pink shrimp stored at 2 ◦C. The most remarkable reduction of microbial growth rate in the treated sample was observed for psychrotrophic bacteria.

#### *7.2. Peel Powders*

To date, a small number of examples are reported in the literature on the use of peel without any preliminary extraction, before direct application to fish products (Table 5). In this context, two articles can be cited by Panza et al. [80,81]. In the first article, the authors [80] developed a ready-to-cook cod stick breaded with different concentrations of dry olive paste powder and monitored the quality parameters during 15 days of refrigerated storage. The authors reported that compared to the raw and cooked breaded cod sticks without olive paste, the raw and cooked active samples showed an higher antioxidant activity. The microbial cell concentration increased during the storage, in both active and control fish. However, in the control sample, it was significantly higher than in the active samples. The control samples remained slightly lower than the active sample in the overall quality score. When the authors compared both microbiological and sensory limits to

determine the product shelf life, they found that the shelf life of active samples was about 12 days and it was longer by 3 days than the control fish. At a later date, Panza et al. [81] evaluated the effects of pomegranate peel powder on the same breaded cod sticks stored at refrigerated conditions. During the 17 days of storage, total mesophilic bacteria of the investigated breaded cod sticks, similar to the psychrotrophic count, gradually increased and reached the unacceptable limit after about 6 days of storage in the sample without the addition of the active powder. At the end of the storage period, the sticks breaded with pomegranate peel powder showed the lowest total bacterial count. The analysis on *Pseudomonas* spp. growth showed that the treated samples never reached the microbial acceptability limit during the storage period, whereas the control sample was already unacceptable after 9 days. Compared to the control, *Shewanella putrefaciens, Photobacterium phosphoreum,* and Enterobacteriaceae growth were limited in all of the treated samples. Therefore, the use of pomegranate peel powder also promoted a significant improvement of microbial stability of cod sticks.

#### **8. By-Products and Cereal Food Applications**

Cereals are a huge group of food products, among which bakery one represents the main portion. Its distinguished feature is that the recipes contain a significant proportion of wheat flour. Causes of the deterioration of bakery products are microbial spoilage or physical changes, which of course, also cause changes in their sensory properties, thus limiting the shelf life. Among the physical changes, loss or absorption of moisture from the atmosphere, which also causes crumb firming through starch retrogradation, are the most likely to occur during their storage. For this reason, a key function of the packaging of baked products is to control moisture transfer to and from the product [82]. Synthetic additives and modified atmosphere packaging are also widely used to prolong their shelf life. Recent trends include the use of natural compounds obtained from industrial byproducts, which are safer and can add value to food products. The three recent case studies are presented in Table 6.

**Table 6.** Applications of by-products to cereal-based food.


#### *Peel, Pomace, and Leaf Extracts*

Ismail et al. [83] evaluated the potential of pomegranate extracts and its residues in cookies as a natural preservative and promising food fortifier. The authors reported that higher free radical scavenging properties were observed for extract-loaded cookies when compared to the ones added with residues. Some fortified cookies showed a significant reduction of the microbiological load during the tested period. In addition, samples with extract-supplemented cookies showed a better inhibition rate to lipid oxidation.

Mehta et al. [84] produced bread and muffins by adding tomato pomace to investigate its effect on their nutritional properties and storage stability. The authors reported that the antioxidant activity of tomato pomace incorporated bread and muffin was higher when compared to their controls. In fact, tomato pomace added with bread showed a shelf life of 5 and 4 days at 10 and 25 ◦C, respectively, which was longer when compared to the control.

Difonzo et al. [85] evaluated the effects of olive leaf extract mixed in baked snacks, during both accelerated oxidation conditions and storage. Two different quality levels of oil were used for baked snacks preparation, EVO1 and EVO2. Results of the oxidative stability evaluation showed that the induction period of both oil-added baked snacks was higher when compared to the control. Snacks with EVO1 had a higher activity of hydrophilic fraction than the samples containing EVO2. Moreover, the authors reported that EVO1

showed a higher quality level than EVO2, as the amount of volatile compounds derived from oxidation was strongly lower in the EVO1 added snack.

#### **9. Conclusions**

The awareness of human, economic, and environmental impacts caused by food loss and waste encouraged researchers to investigate the feasibility of using industrial by-products to prolong food shelf life. Many compounds present in fruit and vegetable by-products have proven effective in prolonging food shelf life. In particular, the studies collected in this review demonstrated that by-products can be used for inhibiting oxidation processes, microbial growth, as well as physic degradation of food, without compromising sensory properties. Some critical considerations can be highlighted regarding the current situation and future trends.

**Current situation.** At present, it is still difficult to find these kinds of innovative products in the market. This represents a huge lack in the modern world, which is continuously aiming at sustainability. Several causes may lead to this issue. Considering this issue, presumably one of the main reasons is the high-risk investment associated with the recycling of by-products. The industrial implementation for recovery processes is complex and requires a careful evaluation. Currently, it seems as a significantly expensive investment, especially for small- and medium-sized enterprises, as the consumer is not inclined to pay the additional cost. Generally, people tend to assume an incoherent behavior on the matter. In fact, they demand an increased production at the lowest possible cost, and consider it even better when disposable. At the same time, they are increasingly interested in environmental and health matters. This is the reason that nowadays people demand natural preserving additives as a replacement of synthetic ones. However, they commonly tend to identify food by-products with something that is not safe or suitable for human consumption, which is at the end of its life cycle and cannot be reintegrated in the food chain. To date, most of the consumers would be discouraged from buying food with by-products.

**Future trends.** In a novel bio-economy perspective, the promotion of pathways that encourage the recovery of compounds, which are still presenting an added value that otherwise would be lost, is a priority. It is fundamental to dispel the misconceptions, which lead to identifying industrial by-products as trash. As in many other fields, information is the basis of concrete progress. In this perspective, a very important role is played by a holistic research approach, which is capable of identifying the advantages of by-products and their real efficacy. Research needs to simultaneously focus the attention on main interdisciplinary factors that could make industrial food by-products an effective entry point to mitigate the greater food waste problem. The recycling of by-products needs to be approved by the current legislation, and the prospect as new ingredients will depend on new safety and regulatory assessments. In addition, social, environmental, cultural, and psychological influences on consumers' food choices need to be considered. In particular, consumers should be increasingly aware of the great potential of by-products as a valid support in extending the food shelf life. In this regard, consumers will most likely be willing to pay for the additional cost.

**Author Contributions:** Conceptualization, A.C. and M.A.D.N.; data curation, S.N.; writing—original draft preparation, S.N.; writing—review and editing, A.C. and M.A.D.N.; supervision, A.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data are available on request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Fresh Fish Degradation and Advances in Preservation Using Physical Emerging Technologies**

**Jéssica Tavares 1, Ana Martins 1, Liliana G. Fidalgo 1,2, Vasco Lima 1, Renata A. Amaral 1, Carlos A. Pinto 1, Ana M. Silva <sup>3</sup> and Jorge A. Saraiva 1,\***


**Abstract:** Fresh fish is a highly perishable food characterized by a short shelf-life, and for this reason, it must be properly handled and stored to slow down its deterioration and to ensure microbial safety and marketable shelf-life. Modern consumers seek fresh-like, minimally processed foods due to the raising concerns regarding the use of preservatives in foods, as is the case of fresh fish. Given this, emergent preservation techniques are being evaluated as a complement or even replacement of conventional preservation methodologies, to assure food safety and extend shelf-life without compromising food safety. This paper reviews the main mechanisms responsible for fish spoilage and the use of conventional physical methodologies to preserve fresh fish, encompassing the main effects of each methodology on microbiological and chemical quality aspects of this highly perishable food. In this sense, conventional storage procedures (refrigeration and freezing) are counterpointed with more recent cold-based storage methodologies, namely chilling and superchilling. In addition, the use of novel food packaging methodologies (edible films and coatings) is also presented and discussed, along with a new storage methodology, hyperbaric storage, that states storage pressure control to hurdle microbial development and slow down organoleptic decay at subzero, refrigeration, and room temperatures.

**Keywords:** fresh fish; spoilage; shelf-life; chilling/refrigeration; freezing; edible coatings; hyperbaric storage

#### **1. Introduction**

Fish is a highly demanded and nutritious food product, yet perishability remains the biggest challenge for its preservation [1]. This food must be stored refrigerated or frozen, and, even under those conditions, it has a very short shelf-life, particularly for refrigeration (5–7 days and 9–12 months under refrigeration and frozen conditions, respectively) [2]. The deterioration of fresh fish during storage is attributed to different damage mechanisms, like microbiological spoilage, autolytic degradation, and lipid oxidation [3].

Fish products contain important nutritional and digestive proteins, including essential amino acids, lipid soluble vitamins, micronutrients, and highly unsaturated fatty acids. The muscle is mostly composed of water (75–85%), and it has a high water activity (0.98–0.99) [4]. Protein represents 20–22% of the muscle [5], while many types of lipids with different chemical composition, such as neutral/non-polar (triglycerides, diglycerides, etc.) and polar (free fatty acids, phospholipids, etc.) lipids, are also present [6]. Fish can be divided in four basic groups regarding its fat content: lean (<2% fat), low-fat (2–4% fat), medium-fat (4–8% fat), and high-fat (>8% fat) [7].

**Citation:** Tavares, J.; Martins, A.; Fidalgo, L.G.; Lima, V.; Amaral, R.A.; Pinto, C.A.; Silva, A.M.; Saraiva, J.A. Fresh Fish Degradation and Advances in Preservation Using Physical Emerging Technologies. *Foods* **2021**, *10*, 780. https://doi.org/ 10.3390/foods10040780

Academic Editor: Luis Guerrero Asorey

Received: 1 March 2021 Accepted: 30 March 2021 Published: 5 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### *1.1. Fish Spoilage*

After fish are caught, spoilage starts rapidly, and rigor mortis is responsible for changes in the fish after its death. A breakdown of various components and the formation of new compounds are responsible for the alterations in odor, flavor, and texture that happen throughout the spoilage process, and deterioration occurs very quickly due to various mechanisms triggered by the metabolic activity of microorganisms, endogenous enzymatic activity (autolysis), and by the chemical oxidation of lipids [1,8].

#### 1.1.1. Autolytic Enzymatic Spoilage

Initially, the main autolytic changes happening are the enzymatic degradation of adenosine 5 -triphosphate (ATP) and its related products, followed by the action of proteolytic enzymes [9], as reported in Table 1. The concentrations of ATP and its breakdown products (adenosine 5 -diphosphate (ADP), adenosine 5 -monophosphate (AMP), inosine 5 -monophosphate (IMP), inosine (INO), and hypoxanthine (Hx)) are one of the most effective and reliable indicators of fish freshness (K-value), varying according to the fish species, muscle types, and storage conditions [1,9]. The K-value, which increases with spoilage, is calculated according to the following ratio (Equation (1)):

$$\text{K}-\text{value} \left( \% \right) = \frac{100 \times (\text{INO} + \text{Hx})}{\text{ATP} + \text{ADP} + \text{AMP} + \text{IMP} + \text{Hx}} \tag{1}$$


**Table 1.** Enzyme action in chilled fish. Adapted from [10].

ATP: Adenosine triphosphatase; ADP: adenosine diphosphate; AMP: adenosine monophosphate; IMP: inosine monophosphate; Hx: hypoxanthine; TMAO: trimethylamine oxide.

High autolytic activity of the major muscle endogenous proteases causes the hydrolysis of key myofibrillar proteins, contributing to the weakening of the myofibril structure during post-mortem storage. The main proteolytic systems in place are the cytoplasmic calpains (at neutral pH) and the lysosomal cathepsins (at acid pH), such as cathepsins B, L, H, and D [11].

Trimethylamine (TMA) and its N-oxide compounds are usually used as indices for freshness in fishery products. The pathway of the production of formaldehyde and ammonia from TMA and its N-oxide is shown in Figure 1, which are associated with the formation of undesirable odors, and occur in fish by the action of several enzymes, such as trimethylamine N-oxide reductase (TMAO reductase), trimethylamine dehydrogenase (TMA dehydrogenase), dimethylamine dehydrogenase (DMA dehydrogenase), and amine dehydrogenase [5]. Total volatile base nitrogen (TVB-N) and trimethylamine-nitrogen (TMA-N) are quality indicators traditionally used for fish products [12]. TVB-N includes the measurement of volatile basic nitrogenous compounds associated with seafood spoilage, like TMA produced by bacteria, DMA derived from autolytic enzymes action, ammonia produced by the deamination of amino acids, and others [13]. A value of 35 mg N/100 g is proposed as the upper limit for the spoilage initiation [13]. However, some studies present

lower limits, depending on the results obtained and the studied fish species [13–15]. TMA-N typically has a fishy odor, and it is produced by the decomposition of TMA N-oxide (major constituent of non-protein nitrogen fraction) caused by bacterial spoilage and enzymatic activity. The upper limit of acceptability is typically around 10–15 mg TMA-N/100 g, however, like TVB-N, lower limits are suggested by other authors [12–14].

**Figure 1.** Degradation of trimethylamine and its N-oxide compounds [5].

1.1.2. Lipid Hydrolysis and Oxidative Spoilage

Fish quality can also be affected during storage at different temperatures by lipid oxidation through odors and lipid peroxide formation or by taste, texture, consistency, and nutritional value losses. Transition metals are primary activators of molecular oxygen, leading to oxidation, which consists of oxygen reacting with the double bonds of fatty acids, mostly of polyunsaturated fatty acids (PUFAs), that are highly susceptible to oxidation [16]. Lipid oxidation can occur either enzymatically or non-enzymatically in fish. In the enzymatic hydrolysis (lipolysis) process, glycerides are split by lipases, forming free fatty acids that are responsible for the common off-flavor (rancidity) and from the denaturation of sarcoplasmic and myofibrillar proteins [17]. Main lipolytic enzymes include triacyl lipase, phospholipase A2, and phospholipase B [3], and they can either be endogenous or derived from psychrotrophic microorganisms [17]. Furthermore, the presence of pro-oxidant enzymes, like lipoxygenases and peroxidases, facilitates lipid oxidation [18].

Non-enzymatic oxidation is triggered by the catalysis of hematin compounds, such as hemoglobin, myoglobin, and cytochrome, generating hydroperoxides. The peroxides are unstable and susceptible to hydrolysis, forming volatile compounds (like aldehydes, ketones, and alcohols), which causes off-flavors [19].

Lipid oxidation is relevant for fish quality due to the development of off-odors, especially in fatty fishes. Normally, the degree of lipid oxidation is given by a thiobarbituric acid (TBA) value that measures the malondialdehyde (MDA) content that is formed by the reaction with hydroperoxides (initial products of lipid oxidation). TBA values of 2–4 mg MDA/kg are within quality limits [13]. Nevertheless, this value might not reflect the real rate of lipid oxidation, because MDA can interact with other components of the fish body and produce secondary metabolites that include reactions with carbohydrates, furfural, alkenals, alkadienals, and other aldehydes and ketones [13].

So, in fish, lipid oxidation consists of a complex chain of reactions, with three distinct phases: primary (formation of hydroperoxides), secondary (e.g., hexanal and malondialdehyde formation), and tertiary/interaction compounds (new compounds are formed by the

breakdown of secondary oxidation products or through the reaction with other molecules, mostly nucleophilic type) [20].

#### 1.1.3. Microbial Spoilage

Microbial growth is the first mechanism deteriorating fish, being the spoilage factor that most affects the quality of fresh or lightly preserved fish [9]. Initially, the fish muscles are sterile, but after death, they are contaminated by the microbial population present at the fish skin [21]. The high water activity, low acidity (pH > 6), and high amount of nonprotein nitrogenous compounds typical of fish results in the fast growth of microorganisms, leading to undesirable changes in appearance, texture, flavor, and odor, reducing its quality. Spoilage created by microorganisms generates volatile amines, biogenic amines, organic acids, sulfides, alcohols, aldehydes, and ketones, which have unpleasant and unacceptable off-flavors [22]. The main compounds formed during microbiological spoilage are listed in Table 2. Biogenic amines, such as histamine, cadaverine, tyramine, and putrescine, are produced by the decarboxylation of specific free amino acids by microorganisms during storage, and are used to monitor fish safety and quality [23,24].

**Table 2.** Spoilage compounds produced by microorganisms during the storage of fresh fish. Adapted from [25,26].


TMA: trimethylamine; H2S: hydrogen sulphide; CH3SH: methylmercarptan; (CH3)2S: dimethylsulphide; Hx: hypoxanthine; NH3: ammonia.

The ability to produce histamine is known in different species of bacteria that have histidine decarboxylase [27]. Being extremely stable, histamine cannot be easily removed or destroyed by cooking, retorting, or freezing [28], and, among amines, it is toxicologically relevant, causing scombroid fish poisoning and food intolerance [29].

It should also be noted that the existing physicochemical conditions and the interactions between the microorganisms will impose a selection of the organisms capable of growing under such conditions. The initial microflora of the fish is dependent on different factors, such as the environment where the fish lives, the fishing season, water temperature, the method of capture, the handling on the ship, or the technological and sale process [21], but, regardless of the variety of microflora present in the fresh fish and the diverse parameters used for preserving, the species growing are consistent in the different products. From the different microbial species that can develop on fish, only one or a few will produce the off-odors and off-flavors, named specific spoilage organisms [30].

#### **2. Chilling, Superchilling, and Freezing**

The preservation of food products without using preservatives or additives has been increasingly demanded among consumers, and has brought additional challenges, especially to highly perishable foods, such as meat or fish. Low temperatures during the capture, transportation, and storage of the fish are of major importance worldwide. Chilling, superchilling, and freezing techniques allow for the preservation of fish for longer

periods without major changes in quality, and assure economic benefits for the fish companies [31]. Therefore, chilling is one of the most used methods for fish preservation, along with freezing and, recently, superchilling.

#### *2.1. Chilling*

Chilling is the process of cooling fish or fish products to a temperature approaching that of melting ice, using, for example, ice. Chilling promotes an increase of shelf-life by slowing physical and chemical reactions and the action of deteriorative microorganisms and enzymes [31,32].

Chilled fish can keep a high organoleptic quality, being highly attractive for consumers, however, it is susceptible to microbial safety problems due to the temperature range at which it is kept, since psychrotrophic pathogens can grow and proliferate without an obvious sensorial impact [33].

Usually done with ice, chilling can maintain the fish at temperatures close to 0 ◦C and extend the shelf-life up to 30 days (in fatty fish, this can be up to 40 days), depending on several factors, such as the water temperature (temperate or tropical waters) and the type of species (marine or freshwater species) [31]. The shelf-life of different fish species stored in ice in shown in Table 3. However, temperatures close to 0 ◦C are not easily possible at retail and consumer houses, and, therefore, refrigeration (storage above 0 ◦C and up to 5 ◦C), the most usual storage process for fresh fish, results in a much shorter shelf-life [34].


**Table 3.** Shelf-life of different fish species stored in ice. Adapted from [31,35].

na—not applicable.

#### *2.2. Superchilling*

Superchilling, also known as partial freezing or deep chilling, is characterized by low temperatures (between conventional chilling and freezing), in which a decrease of 1–2 ◦C occurs below the initial freezing point of the food product [32,36,37]. Most foods have a freezing point that varies from −0.5 to −2.4 ◦C and, specifically for fishery products, this parameter is between −0.8 and −1.4 ◦C [38,39].

The superchilling process results in the conversion of a small fraction of water (≈5–30%) into ice, forming a thin layer of ice (≈1–3 mm) on the surface of the food and an internal ice reservoir [40,41]. Thus, the combined effect of low temperature and internal/external ice on food produce slows deteriorative processes (such as microbial activity) and, for short periods, ice may not be necessary during transport or storage [32,36,40,42].

Ideally, in superchilling, a small amount of water content is transformed into ice and therefore, there is less freeze protein and structural damage (detachments and breaks of myofibers) by ice crystals compared to frozen storage. The shelf-life of superchilled food can be one and a half to four times longer when compared to the chilling process due to the reduction of microbial and enzymatic activity [32,37,39,41]. For example, according to [43], the shelf-life of fresh cod loins (*Gadus morhua*) has been extended from nine days in icechilled storage to 16 or 17 days in superchilled storage, which means that the shelf-life has increased by about 1.9 times. At superchilling temperatures, biochemical/physicochemical reactions are affected (at a higher rate or even accelerated over time), and there are some negative effects on quality parameters, for instance, changes in muscle texture [32,36,39]. Therefore, superchilled technology can be combined with other preservation methods, such as modified atmosphere packaging (MAP) and vacuum packaging (VP) as combined methodologies, to minimize possible detrimental effects [36]. The synergetic effect of MAP and superchilled technologies was described by [44], in which the extension of the shelf-life of fresh Atlantic salmon fillets (*Salmo salar*) was investigated. It was concluded that the salmon samples packaged in a MAP atmosphere (90% CO2 and a gas-to-product volume ratio of 2.5) increased from 11 to 22 days in terms of shelf-life when compared to the control samples (wrapped and exposed to atmospheric oxygen). Additionally, the sensorial and physicochemical properties (drip loss, pH, total volatiles amines, etc.) were evaluated, and all were within the acceptable limits, therefore, the shelf-life was determined only by microbial growth [44].

Superchilling has raised interest in its application to some food products, namely fishery products, due to the shelf-life extension and quality improvement, in comparison to traditional preservation methods. Table 4 presents the conditions for different superchilled fish species, including data from other preservation technologies and from combination with diverse packaging methods. Therefore, in general, the shelf-life is longer when superchilling technology is combined either with VP or MAP methods and when compared to the shelf-life obtained in each of these individually.

**Table 4.** The effect of superchilling, chilling, and/or freezing technologies and/or the synergistic effect with packaging (vacuum packaging or MAP: modified atmosphere packaging) on the quality and shelf-life of fish muscle foods.



**Table 4.** *Cont.*


**Table 4.** *Cont.*

Considering the information presented, it is necessary to understand how the technology influences the degree of superchilling, the growth of ice crystals, and the rate of biochemical reactions (like protein denaturation, enzymatic activity, or lipid oxidation) that indirectly influences quality parameters (like color, texture, flavor, drip/liquid loss, among others) of fish and fishery products [32,36–38].

Superchilling is also a promising and eco-friendlier technology due to an 18% reduction in environmental impact when compared to the conventional cold chain. Additionally, it improves the overall quality of food and extends its shelf-life by reducing microbiological contamination/propagation and, on an industrial scale, promotes higher production yields and reduces labor and transportation costs [32,42].

#### *2.3. Freezing*

Of all of the low-temperature preservation methods used, freezing (frozen storage) is the one that can maintain fish and fish products conserved for longer periods, but some quality parameters can be affected. It is typically applied at temperatures between −18 to −40 ◦C depending on the type of fish stored, and, contrary to what happens with chilling, for frozen storage, most deteriorative and pathogenic microorganisms are unable to proliferate at temperatures below −10 ◦C [32,52]. At this temperature, approximately 80% of the water is converted to ice, decreasing the water activity, which inhibits microbial activity [53,54].

The shelf-life of the frozen fish depends on several factors, such as the initial quality, storage conditions, and fish species, while the quality depends mainly on the storage temperature and temperature fluctuations [52,55]. Table 5 presents the shelf-life of some fish species stored at different freezing temperatures, according to fat content and fish size and shape. Notwithstanding the advantage over chilling regarding the inhibition of microbial growth, the impact of freezing temperatures in quality parameters is quite important when choosing the preservation technique. Some textural changes take place due to the formation of ice crystals that damage the tissues (mainly related to protein denaturation), which promotes dryness and toughness, and occurs more frequently in lean fish than in fatty or semi-fatty fish species. This can be minimized by fast-freezing processes, leading to smaller ice crystals and lower cell wall rupture and drip loss during the thawing process [52,53].


**Table 5.** Shelf-life of fish species stored at different freezing temperatures. Adapted from [56].

Flavor and odor changes also occur in frozen fish due to fatty acid oxidation and development of rancidity [53,57]. Color changes, especially in fatty fish, are directly related to lipid-protein cross links promoting the decrease in protein solubility. These reactions can be minimized by glazing and packaging, and through the exclusion of oxygen and light. In fish like salmon, due to the presence of carotenoids, oxidation occurrence promotes color changes [53,55].

The negative impacts of freezing in the quality parameters of the fish can be attenuated by adjusting and controlling storage temperature, rate of freezing, and fluctuation of temperature during storage through several types of freezing processes. Three basic methods can be used for fish or fish products: air blast freezers, contact or plate freezers, and immersion or spray freezers [56,58].

#### **3. Emergent Preservation Techniques**

Several strategies have been evaluated to increase the shelf-life of fresh fish with minimal impact on quality, particularly texture, and to extend shelf-life compared to refrigeration to try to avoid freezing preservation. These strategies rely on the application of additional hurdles prior to conventional storage, such as edible films and coatings, or even on the application of nonthermal preservation methodologies, such as hyperbaric storage, to slow down microbial proliferation, as in refrigeration, and also to possibly reduce microbial loads to more desirable levels and lessen degradation reactions to increase the shelf-life of fresh fish. This section will cover some of the main emergent approaches to preserve fresh fish, encompassing the main effects of each methodology on microbiological and chemical aspects of fresh fish [59].

#### *3.1. Edible Films and Coatings*

Edible films and coatings are other innovative strategies applied to food preservation that have been shown to be effective in protecting the sensorial and nutritional properties of food, while improving its safety and prolonging shelf-life by reducing/inhibiting microbial growth during the supply chain [60,61]. In fact, these technologies are similar to active packaging, however, they do not act as a package itself, even though the film/coating is in close contact with the food [62].

Edible films and coatings are defined as a thin layer of edible material, whereby, in the first phase, the film is produced separately (like solid sheets) and placed on the surface or between the food products (as wraps or separation layers, respectively). Meanwhile, edible coatings are formed directly on the surface of the food products by dipping (most used in fish and fishery products), spraying, or a fluidized bed, which are selected according to the characteristics of the food product and the film/coating [41,60,63,64].

These films and coatings are bio-based materials, and they are therefore named biopolymers due to their sustainable and eco-friendly source, as residues from the food industry and undervalued components of proteins (such as corn zein, gelatin, and casein), lipids (like shellac resin, waxes, and triglycerides), polysaccharides (such as starch, chitosan, and carrageenan), or their combinations [41,60,61,64].

In addition to high biodegradability, these biopolymers are edible, or can be washed or disintegrated due to further processing [41,65]. The most common natural polymers applied in fishery products are chitosan, alginate, whey proteins, gelatin, or their combinations [41]. Chitosan belongs to the group of polysaccharides, being one of the most abundant, and it has been investigated to be applied as a film/coating material for fishery products due to its non-toxicity, biocompatibility, biodegradability, biofunctionality, antimicrobial and antifungal properties, film-forming properties, selective gas permeability, and low-fat diffusion. Moreover, other protein and polysaccharide biopolymers have been developed for fish and fish-based products [41,63,66].

Similarly to active packaging films, active compounds, such as antioxidants, antimicrobials, and/or flavorants (namely essential oils, natural extracts from herbs and spices, enzymes, and protein hydrolysates, among others) as edible films and coating materials can be added to improve the safety, quality, and stability of foodstuff due to the low/reduced biological activity of biopolymers against spoilage microorganisms. Additionally, other additives, like plasticizers, and crosslinking agents (to improve or modify the physicochemical properties of films/coating polymers) are also incorporated [41,60,61,64].

Edible films and coatings, both biopolymers and active compounds, must comply with European Union Regulations, namely Commission Regulation (EC) No 450/2009, which establishes which active, intelligent, and article materials can enter in contact with food, and Commission Regulation (EC) No 1333/2008, related to food additives [67–69].

Edible films and coatings composed of biopolymers and enriched with active compounds have raised the interest of the food industry and technologists for application in fish, meat, and derived products in order to prevent lipid/protein/pigment oxidation, off-odors, off-flavors, moisture and color loss, oxygen penetration into the food matrix, and solute transport out of the food, and, therefore, improve preservation, quality, and sensorial properties of the products [64,67,68]. Besides this, these films/coatings add value to food products, as they increase their shelf-life by reducing/inhibiting the growth of spoilage and pathogenic microorganisms [64,67].

However, edible film and coating technologies have some associated concerns for both consumers (food safety) and the food industry, such as the initial investment and production cost, equipment and production process complexity, scale-up process, and associated regulations [41,64]. Table 6 shows some examples of edible films and coatings enriched with active compounds applied to fishery products and their effects on physicochemical, quality, and sensorial properties and the shelf-life of these products. Overall, the application of edible films and coatings enriched with active compounds in fish and fishery products enhanced or maintained its quality and sensorial properties, due essentially to its inhibitory action on the growth of spoilage and pathogenic microorganisms, throughout the storage period and, therefore, led to an extension of shelf-life. Ojagh et al. reported an extension from 12 days to 16 days (refrigerated storage, 4 ◦C) in rainbow trout (*Oncorhynchus mykiss*) coated with a film of chitosan and cinnamon oil [69]. Meanwhile, the shelf-life of beluga sturgeon (*Huso huso*) fillets covered with whey protein concentrate coating with 1.5% cinnamon essential oil (stored at 4 ◦C) was extended by eight days [70].


**Table 6.** Edible film and coating enriched with active compounds applied for fishery products.


**Table 6.** *Cont.*

\* The authors did not mention the scientific name of the species.

#### *3.2. Hyperbaric Storage: A Novel Methodology for Fish Preservation*

High-pressure processing is used as a promising "nonthermal" technique for food preservation that efficiently inactivates the vegetative microorganisms most commonly related to foodborne diseases. High-pressure processing is carried out with intense pressure in the range of 100–1000 MPa, allowing preservation with minimal effect on food taste and nutritional characteristics [78]. One of the advantages of high-pressure processing is that food products are treated instantly, regardless of their shape and size (isostatic principle). The application of elevated pressures (100–600 MPa) can be used for a variety of food processing and preservation applications, including high-pressure freezing and thawing, blanching, pasteurization, and commercial sterilization [79]. Research about the application of high pressure on fish muscles has been mainly focused on three main areas, including the extension of refrigerated/frozen shelf-life [80,81], pressure-induced texturation (gel-forming) [82], and high-pressure freezing/thawing [83].

#### 3.2.1. Hyperbaric Storage

Recently, a novel food preservation storage methodology based on storage under moderated pressure (hyperbaric storage, HS, from 25 to 100 MPa) has attracted interest due to its high potential energy savings and shelf-life extension. HS opened the possibility to store food products and other biomaterials above atmospheric pressure (AP, 0.1 MPa) as a possible enhancement of conventional refrigeration storage, increasing shelf-life and food quality. This methodology allows the storage of food under pressure at subzero (ST), low (LT), and room (RT) temperatures, HS/ST being particularly important for solid foods, on which freezing/thawing can cause substantial damages to cellular/tissue structures, leading to textural modifications [84].

The possibility to use HS for food preservation or other biomaterials occurred by chance, after the observation of recovered items from the research submersible Alvin (owned by Woods Hole Oceanographic Institution), which sank about 1540 m during ten months, containing two bottles filled with bouillon and a plastic box containing sandwiches and apples. The environmental conditions at a depth of 1500 m are fairly constant, and are estimated to be 3–4 ◦C and ≈15 MPa. After being recovered, the sandwiches appeared fresh by taste and smell, and apples showed no sign of obvious deterioration. The pH value of the apples was the same as fresh ones, and the tyrosinase activity was about half that of a fresh apple [85].

#### 3.2.2. Hyperbaric Storage at Subzero and Low Temperatures

Two freshly dressed whole fishes (pollock and cod, from the species *Gadus chalcogrammus* and *G. morhua*, respectively) were stored at 24.1 MPa and 1 ◦C during 12 and 21 days, respectively. According to an expert panel, both types of fish were assessed to have better sensory attributes than fish samples stored at the same temperature and AP. The ratings by the expert panel corresponded to the rating these fish samples would have received if they had been stored for a shorter period. This means that the pollock/cod samples stored for 12/21 days with HS at 1 ◦C received ratings typical of pollock/cod samples stored at AP at 1 ◦C for 6.7/8.2 days [86].

Some authors used HS/ST without freezing to extend fish shelf-life, while avoiding the damages caused by freezing. This advantage led to the first studies in HS/ST applied in fresh fish (Table 7). High-pressure application decreases the freezing and melting point of water to a minimum of −22 ◦C at 209 MPa [87], as pressure acts in opposition to the volume increase that occurs with the formation of type I ice crystals, resulting in tissue damage [88]. Charm et al. [86] showed that HS/ST (−3 ◦C and 22.8 MPa) for 36 days reduced microbial growth on cod fillets, while AP samples presented higher microbial loads. These samples were evaluated by an expert panel, and the HS/ST samples showed comparable or better quality than those stored at AP and −3 or −20 ◦C. These results suggested that HS/ST is a non-freezing storage method that improves the preservation of cod fillets compared to conventional methods of freezing or refrigeration, because HS/ST inhibits microbial growth and enzymatic activity and prevents damage caused by freezing/thawing. Furthermore, enzymatic degradation of nucleic acid-related substances (ATP, ADP, AMP, and IMP) from carp and chicken muscles stored under HS/ST for 50 days (−8 ◦C and 110 MPa, or −15 ◦C and 170 MPa) was slightly slower than for storage at −8 ◦C (AP), while the enzymatic activity was significantly reduced only by freezing at −18 ◦C (AP) [89].


**Table 7.** Main results of the application of hyperbaric storage (HS) on fresh fish.


**Table 7.** *Cont.*

\* The authors did not mention the scientific name of the species.

Recently, several studies were published regarding the use of HS/LT to preserve fresh fish. Cape hake loins, *Merluccius* spp. [91], and Atlantic mackerel, *S. scombrus*, fillets [93] were both stored at 50 MPa/5 ◦C for 7and 12 days, respectively, and showed almost no changes in microbial load or TVB-N content compared to the initial samples. For HS samples, an increase was found during storage for water content, water holding capacity, shear resistance, and whiteness. After cooking, HS samples presented a weight lost less than half of the control samples, with no differences in whiteness and only moderate differences by sensorial analysis [91,93].

The quality of Atlantic salmon (*S. salar*) was evaluated by HS/LT (40–60 MPa, 5–15 ◦C) during 10 days, and a slowdown of spoilage microbial growth was observed, while an additional longer storage experiment (50 days) at 60 MPa/10 ºC revealed microbial inactivation (Fidalgo et al., 2019) [94]. Furthermore, the established limit of total volatile base nitrogen was surpassed at 60 MPa/10 ◦C after 30 days (contrarily to six days at AP/10 ◦C), but with stable TMA-N content in the former. Formaldehyde and dimethylamine-nitrogen content increased after six days of HS/LT, but only the former progressively increased until the tenth day, indicating a possible formation by the action of enzymatic activity, but also by other chemical reactions. Additionally, HS/LT slightly increased secondary product content from the lipid oxidation, although to a lower extent compared to AP (at the different storage temperatures). This condition of 60 MPa/10 ◦C also showed no variations in drip loss, water holding capacity, or myofibrillar fragmentation index in Atlantic salmon, with low changes in muscle fibers, visible by scanning electron micrographs [96]. Furthermore, a decrease of resilience (a texture property) and a retention of fresh-like alcohols and aldehydes (not detected in AP samples after 15 days) were observed in these salmon samples stored at 60 MPa/10 ◦C for 30 days [96].

Proteolytic enzymes and muscle proteins of Atlantic salmon (*S. salar*) were studied under HS [95]. Generally, activities of acid phosphatase, cathepsin B and D, and calpains decreased when compared to fresh salmon, with a more pronounced effect of storage temperature of 37 ◦C, regardless of the pressure condition. However, activity recovery was observed for some enzymes, as the case of cathepsins B and D, and calpains, which showed an increase of residual activity for samples stored at 60 MPa/10 ◦C and 75 MPa/25 ◦C after 50 and 25 days, respectively. A pronounced increase of the myofibrillar fragmentation index was observed at 75 MPa (25/37 ◦C) after 10 days. Otherwise, at 60 MPa/10 ◦C, a decrease of myofibrillar fragmentation index values was observed after 50 days of storage. For sarcoplasmic proteins, no effect was observed at 60 MPa/10 ◦C during 30 days of storage, with a slight increase after 50 days. At 75 MPa/25 ◦C, a decrease of sarcoplasmic protein content was obtained after 10 days, with no further changes during the 25 days of storage [95].

Spoilage and inoculated surrogate pathogenic (*Bacillus subtilis* endospores, *Escherichia coli*, and *Listeria innocua*) microorganisms were monitored during HS using Atlantic salmon. HS/LT inhibited and inactivated the spoilage microorganisms, and *B. subtilis* endospores

reached counts below the detection limit after 30 days, verifying a similar reduction for *E. coli* and *L. innocua* counts [98].

#### 3.2.3. Hyperbaric Storage at Room Temperatures

Similarly, the concept of HS/RT arose as an opportunity to preserve food products, namely fresh fish products, with the published works demonstrating a great opportunity to preserve fresh fish. Tilapia fillets (*O. niloticus*) stored under HS/RT (100 MPa/25 ◦C) over 12 h revealed almost no changes in the microbial load of mesophiles (4.7 log CFU/g) and psychrophiles (4.59 log CFU/g), and a decrease to about 2.0 log CFU/g when stored at 200 MPa/25 ◦C for 12 h. Tilapia fillets stored at 200 MPa/25 ◦C for 12 h showed a lower K-value (40%) than samples stored at AP (92%). This result is significant, because a K-value above 60% indicates putrefaction, and only the HS/RT tilapia fillets were below this limit [90].

Recently, HS at 75 MPa caused a reduction in the initial microbial counts of Atlantic salmon, leading to an increase of the microbial shelf-life of at least 25 days, compared to three days of refrigeration, while, at 60 MPa, a microbial growth slowdown was observed, increasing the microbial shelf-life to at least six days. Additionally, besides the maintenance of muscle color during the 25 days, an enhancement of primary and secondary lipid oxidation products was observed, but to a lower extent compared to AP samples [92]. Later, vacuum-packaged fresh Atlantic salmon loins were studied for 30 days under HS/RT conditions, verifying the retention of important physicochemical properties for at least 15 days, such as fatty acids (n-3 polyunsaturated fatty acids) and fresh-like volatile compounds, or lower lipid oxidation and myofibrillar fragmentation index, while refrigeration after five days showed already volatile spoilage-like compounds due to microbial activity [97].

In addition, spoilage and inoculated surrogate pathogenic (*B. subtilis* endospores, *E. coli*, and *L. innocua*) microorganisms were also monitored during HS/RT using Atlantic salmon. HS/RT inhibited and inactivated the spoilage, and inoculated surrogate pathogenic (*B. subtilis* endospores, *E. coli*, and *L. innocua*) microorganisms reached counts below the detection limit after 30 days, showing that, besides shelf-life extension (due to microbial growth inhibition), it also increased microbial safety (by microbial inactivation) of vacuum-packaged Atlantic salmon [98].

This concept of HS/RT arose as an opportunity to preserve food products, providing an opportunity to reduce energy consumption, carbon footprint, and its associated costs (Figure 2).

**Figure 2.** Schematic representation of the energetic costs and environmental impact of hyperbaric storage at uncontrolled room temperature compared to conventional refrigeration [99,100].

Consequently, this method has attracted the attention of many researchers during the last few years, and some studies have been made recently to assess the feasibility of this technology for food preservation compared to refrigeration [101]. HS/RT has been shown to inhibit microbial growth at 50–100 MPa, to inactivate microorganisms at higher pressures (100–220 MPa), and to attenuate some of the physicochemical changes that occur during storage at AP, thereby yielding similar or better products than those obtained with refrigerated storage. HS/RT requires energy only during the short compression and decompression phases, and no additional energy is required to maintain the product while stored under pressure for prolonged periods. Energy cost savings with HS/RT were 101investigated by [99], who concluded that energetic costs of HS/RT were lower than refrigerated storage (Figure 3). Additionally, the carbon footprint associated to HS/RT is also lower than refrigeration. With regard to refrigeration, the two main sources of CO2 production are from energy utilization and the leakage of cooling gas, while, for HS/RT, the CO2 produced by energy consumption is negligible, and the main source of CO2 emissions are attributable to the production of construction materials used for the hyperbaric chamber, thereby demonstrating that HS/RT generates considerably less CO2 than conventional refrigeration processes [99].


**Figure 3.** Schematic representation of the advantages of hyperbaric storage at room temperature compared to conventional refrigeration [102].

#### **4. Conclusions**

As stated, fish is a highly perishable food characterized by a short shelf-life. Refrigeration is probably one of the most used methods for fish preservation, along with freezing, and, more recently, superchilling. However, several deteriorative fish quality changes occur during refrigerated storage, particularly in texture, color, and flavor, limiting shelflife. Frozen storage can avoid these changes (except for texture), but freezing/thawing largely alters the fish fresh-like characteristics. Emerging food packaging techniques, such as the use of edible films and coatings, also meet consumer demands due to their biodegradability and sustainability, while improving the safety and extending the shelf-life of fish and fishery products. Other emergent technologies are arising, as in the case of hyperbaric storage. This methodology uses different pressure and temperature conditions applied at subzero, low, and room temperatures, and has shown the possibility to increase fish shelf-life by microbial inhibition/inactivation, maintaining textural, sensorial, and nutritional characteristics when compared to conventional methods of storage, with the additional advantage of potentially high energy savings, especially when performed at naturally variable/uncontrolled room temperatures. However, currently available high pressure equipment was designed to operate at very high pressure (up to 600 MPa for short minutes), and not to perform hyperbaric storage (up to a maximum of 200 MPa, but for weeks/months), and so specific pressure requirements for hyperbaric storage are of interest to be built.

**Author Contributions:** Writing, original draft, J.T., A.M., L.G.F., and V.L.; Writing, review and editing, J.T., L.G.F., V.L., R.A.A., and C.A.P.; Validation, A.M.S. and J.A.S.; Resources, A.M.S. and J.A.S.; Project administration, A.M.S. and J.A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by project VALORMAR: Valorização Integral dos Recursos Marinhos: Potencial, Inovação Tecnológica e Novas Aplicações (POCI-01-0247-FEDER-024517).

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author, J.A.S., upon reasonable request.

**Acknowledgments:** Thanks are due to the University of Aveiro and FCT/MCT for the financial support for the Associate Laboratory LAQV-REQUIMTE (FCT UIDB/50006/2020) through national founds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement, and for financing the PhD grant of R.A.A. (SFRH/BD/146009/2019), V.L. (SFRH/BD/146080/2019), A.M. (SFRH/BD/146369/2019), and C.A.P. (SFRH/BD/137036/2018). The authors gratefully acknowledge the financial support of Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Competitividade e Internacionalização (POCI) through the research project VALORMAR: Valorização Integral dos Recursos Marinhos: Potencial, Inovação Tecnológica e Novas Aplicações (POCI-01-0247-FEDER-024517).

**Conflicts of Interest:** The authors have no conflict of interest to declare. There are no relevant financial or non-financial competing interests to report.

#### **References**


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