Sepia ink has been proved to be a multifunctional marine material containing melanin, lipid, protein, polysaccharide and microelements [14]. The sepia ink polysaccharides (SIP) from Sepia esculenta ink is categorized as glycosaminoglycan mainly consisted of arabinose and aminogalactose [15]. MFESI and SIP have been proved to have antioxidant activity by our previous work based on in vivo and in vitro investigations, such as scavenging hydroxyl free radical and DPPH radical, preventing DNA from damage induced by H2O2 and ultraviolet radiation [16,17,18,19]. DPPH is a synthetic, stable free-radical containing three benzene rings and a lone electron in a nitrogen atom [20]. Aubergine DPPH captures a hydrogen atom from antioxidant to form yellow unfree DPPH-H [21]. In MFESI solution, many constituents, including polysaccharide, protein, lipid and melanin, can provide hydrogen. Consequently, DPPH was deleted by MFESI. And, higher concentration of MFESI provided more hydrogen, so antioxidant capacity increased with rising concentration of MFESI.
Hydroxyl radical is the most active one of reactive oxide species and reacts with biological macromolecules, such as protein, lipid and DNA through hydrogen abstraction, addition and electron transfer mechanisms [22]. We previously found that DNA breakage induced by hydroxyl originated from H2O2 exposed to UV can be prevented by SIP via inhibiting the activation of H2O2 by UV [17]. In this study, with the addition of MFESI into the Fenton reaction system, the reduction of hydroxyl radical content might correlate with suppression of Fenton reaction. However the accurate mechanism should be explained in the following work.
It is well known that oxidants, such as radicals, can lead to destruction of protein and lipid, resulting in cytolysis, which is a critical cause for shortening shelf-life of preserved food, especially fishes with large amount of polyunsaturated fatty acids. Our report revealed fresh-keeping effect of MFESI on marine fish demonstrated by elongated shelf-life that resulted from inhibition of bacteria growth and protein degradation [8].
Total volatile basic nitrogen (TVBN) is a group of nitrogen-containing compounds, including NH3 and amines, originated from protein degradation by enzymes and bacteria [23]. This study showed significant reduction of TVBN by MFESI in tilapia fillet during cold storage, which could be explained by the following mechanisms. Firstly, inhibition of bacteria by MFESI blocked protein degradation [8]. Secondly, SIP activated Nrf2/ARE pathway, an important antioxidation-associated signaling pathway, to delete oxidants [24]. Thirdly, SIP can prevent effectively cells from oxidants induced autophagy, ameliorating formation of autophagosome [15, 25]. Therefore, in our current research, a possible mechanism was that the liberation of hydrolases from lysosomes was inhibited by SIP, so that the degradation of protein and the formation of TVBN were weakened.
Apart from TVBN, two indicators of protein disruption are loss of sulphydryl group and production of carbanyl group. Determination of carbonyl is considered as a routine procedure for evaluating protein oxidation, but it is not very accurate to estimate the status of protein oxidation due to the presence of various originated carbonyls, such as derivatization agent and lipid-derived carbonyls [26]. Reactive oxygen, such as hydroxyl radical, can break peptide bond to form carbonyl [27]. Hydroxyl radicals were scavenged by MFESI, resulting in inhibition of production of carbonyl from proteins. Another indicator as a complementary technique of protein oxidation is loss of sulphydryl group of protein due to formation of disulphide bond, which partly results from lipid oxidation. NO induces nitrosation of protein sulfydryl, reducing protein sulfydryl that can be also caused by other oxidants [28]. SIP can reduce NO via activating Nrf2/ARE signaling pathway [24, 29, 30]. Therefore, MFESI decreased NO and oxygen radical contents, protecting protein from oxidation and consequently repressing increase of carbonyl and decrease of sulfydryl.
Additionally, lipid peroxidation is both a promoter of protein oxidation and another important cause of reducing quality and shelf-life of meats. Sepia ink and SIP possess antioxidant activities [16,17,18,19, 24, 31], which prevents lipid from oxidants mediated peroxidation [22]. As a secondary product and an indicator of lipid peroxidation, MDA content in fillet expresses degree of lipid oxidation. This study revealed that MFESI definitely inhibited lipid peroxidation in fillets, and the inhibition increased with the extract concentration.
Lipid oxidative products lead protein to oxidation degradation. Also, lipid oxidation and protein oxidation occur independently or parallel [32, 33]. Combining the data of lipid peroxidation and protein oxidation in tilapia fillets during cold storage, two important topics can be deduced reasonably. One is that MFESI definitely inhibited oxidation of lipid and protein. Another topic is that lipid peroxidation and protein oxidation occurred independently in the beginning stage (before 48 h). In the fillets treated with vehicle or low-dosage MFESI, protein oxidation was visible at 24 h whilst lipid peroxidation products were found at 48 h. Apparently, protein oxidation occurred before lipid peroxidation.
Aside from carbonyl formation and sulfhydryl reduction, protein oxidation brings about another outcome, alternation of water holding capacity (WHC). WHC expresses the capacity of muscle resisting water loss. There are two types of water forms, free and bound, accounting for 90 and 10%, respectively, in fish tissues. Free water can be influenced by various factors, such as protein structure and pH, and so on [13, 34]. Protein determines distribution of water in meat, affecting directly WHC of meat. Increase of WHC indicated that protein degradation was suppressed by MFESI during the cold-storage of fillet.
To further understand relationship among lipid peroxidation, protein oxidation and water-holding capacity, Pearson correlation was analyzed among all of the measured indicators in high dosage of MFESI treated fillets. The results indicated strong relationships among lipid peroxidation, protein oxidation and water-holding capacity in MFESI-administered tilapia fillet during cold storage.
Summarily, based on our previous findings about fresh-keeping effects of MFESI on marine fish, this study further investigated the involved mechanisms on freshwater fish through assessment of oxidation of lipid and protein, as well as WHC of fillets. Results revealed that MFESI prevented effectively fillets from protein oxidation and lipid peroxidation through eliminating radicals, WHC was maintained. Consequently, quality of fish meat was kept and shelf-time was extended undoubtedly. Sepia ink has a long history of being used in various ways in food and drugs [14], suggesting that it is edible safety. It is can be seen that MFESI is a potential natural preservative for fish and other foods.