Effect Of Egg White Protein And Oligosaccharides Mixture On The Functional And Structural Properties Of Fish Myofibrillar Proteins During Frozen Storage

The overall production of fish has increased immensely in the past few years up to 179 million tons?2018?,with growing population needs.Fish is a good source of essential nutrients such as,omega-3-fatty acids,amino acids,calcium,vitamin D and iodine that can assist in the treatment of several health diseases.Generally,fish is used in the production of various products such as,fish balls,sausages,snacks,kamaboko gel and crackers.Quality and acceptability of fish are very important issues that directly affect the consumer acceptance.To fulfill the public needs,preservation of fish quality has remained a big issue for researchers and industries during frozen storage.Myofibrillar proteins?MP? are the major proteins of fish muscles comprised of about 65-75% of total proteins,and responsible for the quality related changes.MP contribute to the better functional,gelling and structural properties of fish products.The change in MP resulted in the decreased textural,gelling and water binding properties.Additionally,these changes also affect the commercialization of fish products.These MP are greatly affected by the fluctuation of temperature during frozen storage and long term storage.In order to control protein oxidation and denaturation during frozen storage,various cryoprotectants have been reported in many studies such as,sucrose,sorbitol,phosphates and trehalose.These cryoprotectants can provide the protective shield to proteins,which prevent the dehydration,aggregation and denaturation via interaction with functional groups.The concept of new cryoprotective mixture has come after analyzing the more induced sweetness,increased caloric values,weak hydrogen and ionic interaction in fish products added with traditional or typical cryoprotectants.In order to overcome such issues,researchers and industries are focusing on the cryoprotectants with low sweetness,caloric values or similar sweetness level but better in hydrogen and ionic interactions than traditional cryoprotectants,which can preserve the quality of fish proteins for a prolonged time with minimal deteriorative changes.Additionally,these efficiently reduced the crystallization that can lead to protein denaturation and aggregation during frozen storage as compared to typical cryoprotectants reported in previous researches.In addition,stable secondary,tertiary structural properties of proteins during frozen storage,in contrast with typical cryoprotectants.Change in gelling and textural properties has also a direct relationship with the final product quality,which can be decreased during frozen storage besides the incorporation of typical cryoprotectants.Addition of EWP/?CD,EWP/XO and EWP/KC developed the dense and three-dimensional structure of MP gel.To the best of our knowledge and review of literature,this is the first time EWP/?CD,EWP/XO and EWP/KC mixtures were added in the MP during frozen storage.In addition,the reduced oxidative changes were reported with the association of stable functional,rheological and textural properties.Herein,this dissertation covered the following research contents and associated results which are as follow:1.The effects of egg white protein?EWP?and?-cyclodextrin??CD?mixture?3:1,EWP/?CD?at 0,2,4 and 6% concentrations on fish?Culter alburnus?MP was analyzed during frozen storage?-18?C? by analyzing functional,rheological and structural changes in MP and MP gels.During frozen storage,circular dichroism?CD?and Fourier transform infrared?FTIR?spectroscopy revealed that the addition of EWP/?CD?6%? significantly reduced the decline in?-helix contents.The loss of?-helix content is indicative of the exposure of hydrophobic residues,resulting in changes in secondary structural properties.EWP/?CD addition improved the MP secondary structural properties by inhibiting protein aggregation induced by exposure of hydrophobic residues.Particularly,the 6% EWP/?CD treatment significantly decreased the pores and spaces of MP gels formed during frozen storage,resulting in a denser network.Moreover,EWP/?CD?6%? significantly prevented the decline in textural properties of MP gel such as hardness,springiness,gumminess and cohesiveness as well as whiteness?87.5 to 79.02%?and WHC?98.01 to 82.47%? by improving the hydrogen bonding interactions between the amino acid side chains.In contrast with control MP?0%?,which showed significant decline in WHC from 97.08 to 77.14% due to shrinkage of myofilaments,protein-water or protein-protein interactions.Dynamic rheological analysis showed that 6% EWP/?CD also effectively inhibited the decrease in MP storage modulus G'and loss modulus G”,which can be attributed to the?-helix stability,rearrangement and cross-linking of protein molecules.The research provides useful information for increasing the commercial life of fish products by frozen storage and contributes to the potential application of EWP/?CD as a low-sweetness cryoprotectant for Culter alburnus fish.2.The effect of a cryoprotectant mixture?egg white protein and?-cyclodextrin?on the MP of Culter alburnus during a frozen storage at -18?C.Different concentrations of EWP/?CD?0,2,4 and 6%? were added into MP to analyze their effect against oxidative changes.During the study,it was observed that the addition of EWP/?CD?6%? significantly prevented the increase in carbonyls?26.26 to 37.69 nmol/mg? and surface hydrophobicity?11.51 to 20.31?g? in comparison with control MP?0%?,which showed the increase in carbonyls from 26.36 to 49.28 nmol/mg and surface hydrophobicity from 11.47 to 32.06?g respectively,resulted due to protein denaturation and unfolding of hydrophobic amino acid interactions.Meanwhile,Incorporation of EWP/?CD?6%? improved the stability in Ca²⁺-ATPase activity from 0.29 to 0.19 m mol/g as compare to control MP?0%? which showed remarkable decline in Ca²⁺-ATPase activity from 0.29 to 0.13 m mol/g due to myosin denaturation.Moreover,protein denaturation and aggregation also reduced the sulphydryl contents from 45.63 to 23.31 mmol/kg in control MP.Besides,EWP/?CD?6%? added MP samples showed stability against oxidative changes and significantly reduced the drop in emulsifying properties such as emulsion activity index?EAI?from 31.39 to 24.60 m2/g and emulsion stability index?ESI?from79.61 to 65.38%.On the other hand,control MP also showed significant decline in both EAI from 31.26 to 17.91 m2/g and ESI 79.37 to 56.73%.It can be concluded that EWP/?CD is an efficient approach against oxidative changes in MP from Culter alburnus.EWP/?CD?6%? proved to be efficient against protein denaturation and aggregation due to crystallization.This study also confirms the association of functional and structural changes with protein oxidation which was discussed in one of our previous works.3.The cryoprotective role of EWP/XO in the prevention of oxidative and structural changes of MP was investigated.Different concentrations of EWP/XO mixture?0,2,4 and 6%? were added to the MP of Culter alburnus fish during frozen storage?-18?C?.During the study,it was observed that addition of EWP/XO?6%?significantly reduced the decline in Ca²⁺-ATPase activity to 0.29 m mol/g,which is greatly related with tertiary structural changes.In contrast,control MP revealed remarkable decline in Ca²⁺-ATPase activity to 0.23 m mol/g resulted due to myosin denaturation and increased ionic strength.Meanwhile,EWP/XO?6%?restricted the increase in carbonyls from 28.31 to 39.67nmol/mg as compare to the control MP,which increased from 28.36 to 51.26 nmol/mg in line with frozen storage,which resulting into the major alterations in MPs such as aggregation,loss of functionality and fragmentation of protein.The addition of EWP/XO?6%?also efficiently controlled the decline in the sulfhydryl contents from 49.26 to 34.03mmol/kg by preventing oxidative changes in myosin molecules.Furthermore,through circular dichroism analysis it was confirmed that the addition of EWP/XO increased the secondary structural stability by preventing the reduction in?-helix content.Microstructural analysis confirmed the preservation of a well-structured protein network that reduced the porosity and protein aggregation of MP gel.It was concluded that 6% EWP/XO was an effective cryoprotectant mixture,which preserved the functional and structural properties of Culter alburnus during frozen storage period.4.The impact of egg white protein?EWP?and?-cyclodextrin??CD?mixture?3:1? on the structural and functional properties of MP from silver carp was investigated during frozen storage.Different concentrations of EWP/?CD were prepared and incorporated with MP.It was analyzed that the addition of EWP/?CD?6%? significantly enhanced the stability in?-helix content with the minimum increase of ?-turn and random coils frozen storage,which indicate reduced myosin denaturation and better gelling and textural properties of MP gel.Meanwhile,control MP showed prominent decline in?-helix content at the end of frozen storage period.Furthermore,rheological studies revealed that MP with increased EWP/?CD concentrations?6%? improved the inter-molecular protein interactions and enhanced the stability in viscoelastic properties from 625 to 387 Pa during the gelation process.Moreover,addition of EWP/?CD delayed the decline in textural properties such as hardness,springiness,gumminess and cohesiveness.Which is also confirmed from microstructural analysis which showed more compact and well-established network.Furthermore,these results were also coincide with addition of EWP/?CD reduced the water loss from 94 to 78%.Besides,control MP?0%? showed more coarser and porous structure.All experimental analyses confirmed that EWP/?CD?6%? showed better cryoprotective properties in the preservation of the functional and structural properties of silver carp MP during frozen storage,and could be effective in the industrial storage of fish and associated products.5.This section was focused on the cryoprotective effect of EWP and?-carrageenan?KC?mixture on MP of grass carp.Different concentrations?0,2,4 and 6%?of EWP/KC were added in MP to analyze its effect on oxidative changes,while alteration in intermolecular interaction forces was also studied during frozen storage.Addition of EWP/KC inhibited the changes in functional and structural properties resulted due to oxidative changes.EWP/KC?6%? significantly reduced the decline in sulphydryl contents,which is generally associated with decline in and Ca²⁺-ATPase activity.On the other hand,control MP showed substantial decline in sulphydryl content and Ca²⁺-ATPase activity,which are directly linked with reduced structural properties.Moreover,EWP/KC effectively prevented the increase in carbonyls,surface hydrophobicity and dityrosine contents by providing protective shield to MP against oxidative changes and restricted the formation of new compounds,which indicate improved hydrogen and ionic interactions between the amino acid side chains.Furthermore,CD analysis proved that EWP/KC lowered the decrease of?-helix and improved the secondary structure stability.In addition,fluorescence intensity showed the stable tryptophan residues,which correspond to stable tertiary structural properties than the control MP.Meanwhile,MP added with EWP/KC?6%? delayed the changes in intermolecular interaction forces such as ionic bond,hydrogen bond,hydrophobic bond and disulfide bond,associated with water binding and gelling properties of proteins resulted due to frozen storage.Thereby,reduced quality related changes in MP.Therefore,it can be determined from the current results that EWP/KC is an effective low sweetness cryoprotective mixture at the industrial level for prolonged safety and quality of fish and related products.