Journal of the Science of Food and Agriculture J Sci Food Agric 86:2609–2620 (2006) Cryoprotective additives and cryostabilisation effects on muscle ﬁllets of the freshwater teleost ﬁsh Rohu carp (Labeo rohita) during prolonged frozen storage Shashi Kiran Jasra, 1∗ Barbara Zielinski 1 and Pardeep Kumar Jasra 2 1 Department of Biological Sciences, University of Windsor, Windsor, ON N9B 3P4, Canada 2 Department of Zoology, Dyal Singh College, University of Delhi, Delhi, India Abstract: The effects of various cryoprotective additives separately and in combination were studied on the myoﬁbrillar protein integrity, biochemical enzyme activity levels and muscle ultrastructure in the freshwater teleost ﬁsh Rohu carp (Labeo rohita). Fish muscle samples were divided into eight groups and immersed in different mixtures of cryoprotective additives (S1 – S8), then frozen at −20 or −30 ◦ C for 24 months. Electrophoretic studies revealed early (within 6 months) alteration of the myoﬁbrillar proteins myosin light chain, α-actinin and tropomyosin. Reduction of the storage temperature from −20 to −30 ◦ C slowed down the degradative processes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ﬁsh muscle treated with cryoprotective mixture S8 (40 g L −1 sorbitol/3 g L −1 sodium tripolyphosphate/4 g L −1 sodium alginate) showed minimal post mortem changes in myoﬁbrillar proteins. Ultrastructural results also revealed post mortem damage to the muscle, seen earliest (within 6 months) in the sample frozen-stored without additives (S2), as compared with the normal, unfrozen muscle (S1). The inﬂuence of cryoprotectants alone and in combination on ﬁsh muscle structural proteins, myosin and actin ﬁlaments (A and I bands), during prolonged frozen storage was investigated. After 12 months, samples frozen-stored with various cryoprotective additives (S2-S7), except S8, showed signs of myoﬁbrillar disintegration. Beyond that time the degradative processes started showing up in all samples, with minimal muscle ultrastructural damage in sample S8. Again, reducing the storage temperature from −20 to −30 ◦ C slowed down the degradative processes. Ultrastructural results correlated well with levels of biochemical enzymes (Ca 2+ myoﬁbrillar ATPase and succinic dehydrogenase) during frozen storage. This is the ﬁrst report of the cryoprotective effects of these additives on this popular edible ﬁsh species. Of the various combinations of additives tested, cryoprotective mixture S8 was found to preserve the muscle structure longest under frozen storage conditions. However, even this mixture was only effective for 18 months at −30 ◦ C. Beyond that time the myoﬁbrillar degradative processes were apparent with correlative electrophoretic, biochemical and ultrastructural studies.  2006 Society of Chemical Industry Keywords: cryoprotective additives; prolonged frozen storage; muscle ultrastructure; cryostabilisation; myosin and actin ﬁlaments; Z line disintegration; Ca 2+ m-ATPase INTRODUCTION Frozen storage is one of the most important techniques for long-term preservation of ﬁsh muscle. It prevents microbial spoilage and minimises the rate of biochemical reactions in the muscle. However, several structural and physicochemical changes still take place in the muscle, leading to deterioration of the muscle texture. This slowly renders the muscle unacceptable for consumption. 1–9 The principal causes of textural deterioration of ﬁsh muscle during prolonged frozen storage have been reported to be formation of ice crystals, disruption of cellular integrity and/or intermolecular crosslinking between adjacent molecules. 4–7 Fish myoﬁbrillar proteins, especially myosin and actin, are known to be highly susceptible to freezing and frozen storage. These proteins form intermolecular aggregates, leading to intramolecular conformational rearrangements. 1,3 There may be degradation of the Z disc in myoﬁbrils due to the presence of endogenous calcium-activated proteases or crosslinking of muscle proteins. As muscle contraction is regulated by Ca 2+ ions and the enzyme ATPase, removal of these ions would probably also reduce the availability of ATP (adenosine triphosphate) molecules. This may result in interactions among the various contractile proteins of the muscle. It may also promote changes in the conformation of protein molecules due to the formation of ionic crosslinkages between polypeptide chains or crosslinking reactions of myosin heavy chains. 10 – 12 With extended frozen storage, all these factors can signiﬁcantly inﬂuence the quality attributes of ﬁsh muscle foods. 13 – 15 A number of investigations have been carried out on a variety of biological systems regarding the cry- oprotective effects of certain chemical agents. 12,16 – 18 ∗ Correspondence to: Shashi Kiran Jasra, Department of Biological Sciences, University of Windsor, Windsor, ON N9B 3P4, Canada E-mail: sjasra@uwindsor.ca Contract/grant sponsor: University Grants Commission, India (Received 22 September 2004; revised version received 21 February 2006; accepted 25 July 2006) Published online 2 October 2006; DOI: 10.1002/jsfa.2658  2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00