Evaluating the inhibition effect of a novel bio-packaging on histamine content in mackerel (Scomber scombrus): a new strategy with environment respect

Abstract This is the first study in which the impact of a new bio-packaging on histamine formation in mackerel was investigated. To monitor the preservation of fresh fish samples a treatment with innovative polymeric film and a soaking procedure in liquid new biomaterial was adopted. Ultra HPLC diode array detection (UHPLC-DAD) analyses for histamine monitoring were performed on fresh, packaged, and soaked mackerel samples at different time. The histamine content threshold value was up to 7 days, and after this period the results showed that histamine levels were affected by application of biomaterial. Significant increase was detected in sample that was not treated with biofilm. The new biofilm extended the shelf-life and reveals a promising packaging system to prevent histamine biosynthesis. Graphical Abstract


Introduction
Fish is a primordial food, with excellent nutritional properties since it is a good source of easily digestible proteins, vitamins, minerals, and polyunsaturated fatty acids to which are attributed positive effect on human health (Visciano 2015).the primary objective of fresh fish marketing is the maintenance of the hygienic and organoleptic characteristics of the product.the need to make fish and fish products available after some time with respect to the place of production has encouraged the creation of technologically advanced conservation systems and methods.Generally, thanks to the packaging, the foods are protected from the external environment and the organoleptic properties are preserved, to ensure the characteristics of the fresh product for consumers (Graci et al. 2017;di matteo et al. 2021).However, fishes belong to a food group for which the treatments to prolong their conservation raises a series of problems relating to their conservation and constitute a significant safety concern.it was evidenced that the consumption of seafood, particularly those belonged to Scombridae families are commonly involved in Scombroid poisoning (arulkumar et al. 2023).this poisoning, also known as histamine intoxication, is the major public health concern resulting from the ingestion of certain species of marine fishes that contain large amounts of histamine, a biogenic amine produced physiologically by many living organisms and responsible for inflammatory and allergic processes (Hungerford 2010).the severity of the toxic effect related with histamine intake depends on many factors, such as the histamine content and the level of other biogenic amines ingested, the activity of monoaminoxidase and the individual physiology and sensitivity (durak-dados et al. 2020;Kočar et al. 2021).Histamine is not present in living fish, except in very limited concentrations, but is produced post-mortem, by bacteria commonly found on the fish skin or in the marine and terrestrial environment (ozogul et al. 2018; arulkumar et al. 2023).it is produced before the deterioration of the fish and cannot be detected through the sensory characteristics (appearance, smell, taste).Histamine content in freshly caught fish is commonly regarded as one of the factors determining the quality and, the freshness/spoilage of fish meat, especially scombroid fish.thus, elevated histamine levels in fish species containing significant amount of endogenous histidine indicates that a problem raised in applying the cold chain and correct handing of the product, controlling bacteria growth, hindering and limiting the production of histamine (Cicero et al. 2020).
at the present the European union fixed risk level of histamine exposure only for fishes and specific fishery products and established that the average histamine content in fresh fish must be less than 100 mg kg −1 (EC 2013).Recently, in u.S.a. the Food and drug administration, updating guidance levels for histamine in certain fish species and fishery products, issued that if fish 'samples have 35 mg kg −1 (or more) histamine (lowered from 50 mg kg −1 )' , they may be considered adulterated, associated with decomposition, produced under insanitary conditions, and injurious to human health (Fda 2021).
Fish is a food product that readily spoils, and it is well known that the storage temperature and the time between capture and application of the refrigeration temperatures are decisive for fish's histamine contamination.normally, when properly harvested and handed, the fish have little histamine content and often it is not detectable.to assure fish quality/safety and extend its shelf-life, the histamine formation in raw fish could be controlled using conventional preservation methodologies that inhibit microbial growth and/or the decarboxylase activity of microorganisms (arulkumar et al. 2023).nowadays, to preserve sensorial and nutritional properties, and prevent food spoilage that may cause histamine accumulation, the conventional storage procedures are counterpointed by food industries with the use of novel packaging methodology and proper packaging materials (tavares et al. 2021).Currently, to respond to new demands related with environmental, social, and economic sustainability, innovation of packaging underwent a rapid evolution and allowed the use of new materials that ensure greater safety and integrity of the product, a longer shelf-life and greater sustainability.in this contest, searching for innovative preservation techniques having functional features, to reduce the impact of packaging on the environment pollution, and contemporary maintaining its high storage capacity, our research team has produced a new packaging material based on chitosan (Vadalà et al. 2021). in this paper the first results of the bio-packaging functionality on sample preservation were examined monitoring the histamine profile of fresh and packaged mackerel samples.Since histamine levels in seafood are closely related with the progress of spoilage, the aim of the present work was to provide an exhaustive overview on histamine development in mackerel (Scomber scombrus) preserved following different storage procedures with our innovative coating material.

Results and discussion
an innovative product made with natural, biodegradable and plastic-free material, such as chitosan, was employed to development of an efficient strategy to prevent the accumulation of histamine in mackerel.the content of histamine was evaluated on fresh and packaged mackerel samples by uHpLC coupled to a diode array detector (dad) (Galluzzo et al. 2021).as reported in the experimental section (see 'supplementary material'), mackerels were filleted with skin, separated in five groups, and stored at 5 °C for 7 days.one group of samples was stored without any preservation treatment (namely nm); the 2 nd and 3 rd group were nebulized with 20 mL and 30 mL of protective liquid biomaterial, respectively (namely nm20 and nm30, correspondingly); finally, the last two groups of mackerel samples were soaked for 2 or 4 min in 20 mL of protective liquid biomaterial, respectively (namely Sm2 and Sm4, correspondingly).Based on precedent studies on preservation of food products (tavares et al. 2021), the histamine content threshold value was up to 7 days.the data, summarized in tables S1, exhibited different trends of histamine content for fresh and differently packaged mackerel samples during the 7-days storage. in all the samples histamine was determined at levels lower than 100 mg kg −1 , the allowable limit by European Regulation (EC 2013) for scombroid fish.However, wide variations were observed in histamine contents, and in some samples, it was higher than 35 mg kg −1 , the allowable limit by u.S.a.Food and drug administration (Fda 2021).Histamine was not detected in mackerel when freshly caught (lower than the limit of quantification, LoQ = 0.30 mg kg −1 ), at the beginning of the storage period.at the end of the storage period there were significant differences among the five studied groups, and histamine reached the highest levels in the mackerel fillets that were no treated (93.95 ± 1.59 mg kg −1 ), while remained not detected in the sample Sm4 (< LoQ).among the treated groups, substantial differences were also found among the mackerel fillets that was soaked for 2 min (Sm2 = 2.95 ± 0.18 mg kg −1 ) and those were nebulized, which gave highest histamine levels (nm20 = 68.90± 0.56 mg kg −1 , and nm30 = 46.45± 0.63 mg kg −1 , respectively).
the data evidenced that histamine was not detectable only in the fresh mackerel and in the sample Sm4. according to ozogul et al. (2018), histamine may not be present in fishes when freshly caught and its formation and accumulation in fish depends on the level of free histidine in the muscle, the presence of bacteria with histidine decarboxylating enzyme (tao et al. 2009), time and temperature conditions during catching and processing (Chang et al. 2008).mackerel is one of the fish species least susceptible for histamine formation and the low levels in the mackerels indicate that the quality of the fishes collected was fine.the low levels of histamine detected after the storage, for the sample that was soaked for 4 min in the protecting biomaterial, revealed that the fillets of mackerel remained during the storage period in adequate conditions, leading to a good control of histamine development.in the samples stored Sm2 histamine content was significantly reduced respect to the other types of packaged samples, and the data proved that these samples, processed under different exposure time to the biomaterial, were stored in non-completely sterile conditions able to totally prevent the histamine formation.moreover, the significant increase of histamine observed after storage in samples processed nebulizing the biomaterial, revealed that even if we used the same material, the processing procedure in biomaterial application is important for fish safety and stability.as observed by other researchers (mohan et al. 2009, ozogul et al. 2018), the formation of biogenic amines such as histamine depends on packaging conditions that can alter o 2 levels and therefore, the growth of specific microorganisms which through the decarboxylation of histidine produce histamine.Consequently, the histamine levels raised significantly in samples packaged nm20 and nm30 since with this procedure o 2 was not wholly scavenged by biomaterial and/or the low thickness of the biofilm remains permeable to o 2 of the air.

Conclusions
using innovative technology, we have developed a biocompatible, and biodegradable material that does not contain aluminium or plastics of fossil origin (Vadalà et al. 2021) and meets the needs of consumers.product development was carried forward up to the limited pilot scale, as it was a research and innovation project.it has been found that the innovative biomaterial is suitable in inhibiting the formation of histamine in mackerel.Specifically, these preliminary results indicate that nebulizing biofilm is less effective in preventing the histamine evolution compared to the soaking biofilm procedure; therefore, soaking method can be recommended to prevent histamine formation.our primary goals were to minimize the development of histamine through gentle handling of the product, easy mechanized processing and improving the shelf-life of fish samples; but further work is underway to explore the biofilm activity against the evolution of other biogenic amines and to industrialize the developed technology.