Effects Of Different Combinations Of Pressure And Freezing Factors From High Pressure Freezing On Denaturation Of Prawn(metapenaeus Ensis) Protein

High pressure freezing(HPF)is a novel rapid freezing technology,which refers to increase pressure during atmosphere liquid immersion freezing process.HPF has previously and definitely shown the large advantageous effects on forming small and uniform intracellular ice crystals by change the freezing point of water and ice formation path(includes pressure assisted freezing(PAF),pressure shifted freezing(PSF)).However,its effect on the protein denaturation is not clear.Because HPF process involves Pressure-Factors(pressure,time)and Freezing-Factors(temperature,phase transition,recrystallization,ice crystal types),which result in the protein denaturation are complicated.In the current study,the effects of different combinations of pressure and freezing factors from high pressure freezing on denaturation of prawn(Metapenaeus ensis)protein were investigated.Natural actomyosin solution(NAM)and polyphenol oxidase(PPO),which is the major component of prawn muscle and is the major factor induced melanosis phenomenon respectively,were treated by different pressure and freezing types.The combinations include freezing under various pressure(HPF),high pressure plus immersion freezing process(HPP-IF),as compared to high pressure process(HPP)and normal immersion freezing process(IF).Moreover,the possible mechanisms of pressure-factors and freezing-factors on NAM and PPO denaturation were analyzed as well.The main conclusions are as follows:The effects of high pressure freezing on the changes of physical and biochemistry of prawn were studied.And results shown that,the significant reduction of ice crystals morphology and distribution,water holding capacity,free water(T21)and uneasy flow water(T22)from prawn muscle between different frozen types were:PSF(200MPaP-20?/50min)>>PAF(100,150,200,300,400,500MPaP-20?/50min)>>IF(0.10MPaP-20?/50min).During PAF processing,with pressure increased,the changes of above index were aggravated;texture(includes hardness,springiness),total enthalpy of muscle exhibited an increase and reduce trend,respectively.300MPa was a critical point for HPF impacts on prawn.HPF could effectively improve the quality of frozen prawn:the characteristics of ice crystals directly and significantly affected prawn quality,including tissue microstructure,water holding capacity,texture and system water,however,these characters would be depended on protein denaturation caused by HPF under similar ice crystals size and distribution.The impacts of HPF(100,150,200,300,400,500MPaP-20?/30min)on denaturation of actomyosin from prawn were evaluated.Compared with untreated NAM,protein relative solubility,Ca2+-ATPase activity,total sulfhydryl content(T-SH)and a-helix of NAM treated by IF and HPF were significantly decreased.Surface hydrophobicity and fluorescence intensity of treated NAM were remarkably enhanced.The decrease and increase degree could be ranked as:untreated ?200MPaP-20?/30min(PSF)<100,150MPaP-20?/30min(PAF)<0.1MPa P-2?/30min(IF)?300MPa P-20?/30min(PAF)<400,500MPa P-20?/30min(PAF).SDS-PAGE pattern showed that there was no new covalent bonds except disulfide bond.All results indicated that 300 MPa was the key point for NAM denaturation induced by HPF.We found,compared with the Pressure-Factor,Freeze-Factor displayed stronger or weaker effects on protein denaturation during low pressure HPF(100-200 MPa)or high pressure HPF(300-500 MPa),respectively.And Freeze-Factor during HPF process could weaken the influence of high pressure over 0 ? on protein denaturation.The changes of secondary and tertiary structure were majorly in the period of ice formation and growth,B? D(PSF)or B?C?D(PAF).The influence of HPF(100,150,200,300,400,500MPaP-20?/30min)on the thermodynamics and conformation of polyphenol oxidase were analyzed,as compared to IF(0.1MPaP-20?/30min).There were no change in the activity of PPO treated by IF and low pressure(100-200 MPa)HPF,and PPO were activated during the process of high pressure(300-500 MPa)HPF.However,the thermodynamic parameters and conformation of all of the treated PPO(0.1-500 MPa)were significantly changed.The results suggested that(?)300 MPa might be regarded as a critical point in the denaturation of frozen PPO during HPF process;(?)the thermo stability of PPO were enhanced by HPF;(?)PPO with same activity might has various thermodynamic characteristics and conformation;(??)Pressure-Factors were the main strength in the denaturation of PPO during HPF process;(??)PPO might transfer into in a partially fold intermediate state.The denaturation of NAM during high pressure and subsequent freezing process(HPP-IF,100,200,300,400,500MPa P4?/25min--20?30min)were researched,compared with HPP and IF treatments.Remarkable changes in relative solubility and Ca2+-ATPase activity of all of treated PPO were found.The impacts of these treatments could be ranked as:100-200MPaP4?/25min(HPP)<0.1MPaP-20?/30min(IF)<100-200MPa0.1MPa P4?/25min--20?/30min(HPP-IF);with pressure increased to 300-500 MPa,IF<HPP<HPP-IF;300 MPa might be thought as the key point for PPO denatured by HPF treatments.The denaturation of NAM were intensified with pressure increased during various HPP-IF process.The subsequent freezing process enhanced the significant difference of NAM denaturation caused by HPP.The decrease in T-SH and a-helix and the increase in SoANS and fluorescence intensity of NAM treated by HPP-IF,besides,the degree of decrease was weaker than increase degree.Surface hydrophobicity played a dominant role in the denaturation of NAM and caused remarkable changes in tertiary structure.The denaturation of PPO caused by HPP-IF(100,200,300,400,500MPa P4?/25min--20?/30min)were evaluated.PPO kept original activity during IF and low pressure(100-200 MPa)HPP-IF and HPP process.PPO were activated with pressure increased under high pressure(300-500 MPa)HPP-IF and HPP process,and the activated degree caused by HPP was than caused by HPP-IF.300 MPa was the activated pressure point for PPO during various HPP-IF processes.Interestingly,the subsequent freezing process might cause a reversible denaturation of PPO after HPP treatments.The fluorescence intensity of PPO treated by HPP-IF,HPP,IF shown a significant reduction,compared with untreated PPO.Both the fluorescence and AFM images indicated that PPO with same activity could exhibit different conformation and molecule shape,and this phenomenon might happen in period of HPP or IF.