High pressure effect on fish muscle

Research about the application of HP on fish muscles has been mainly focused on three main areas, including the extension of refrigerated/frozen shelf-life [49, 50], pressure-induced texturation (gel-forming) [51] and HP freezing/thawing [52]. In this context and subsequently, general aspects about HP effects on fish muscle are presented below.

1.3.2.1. High pressure effects on microbial quality of fish

Many studies suggest that HP reduces the initial microbial load in many fish muscles [53–56]. Generally, a pressure level of 300 MPa or higher is adequate to reduce the initial microbial load and slows microbial growth during chilled storage in various fish muscles, such as hake [54], sea bass [57] and salmon [58]. The microbial inactivation in fish muscles is increased with the increment in pressure and holding time [57].

Furthermore, the effectiveness of HP on microbial inactivation depends on the microbiota characteristics in fish muscle. Psychrotrophic bacteria, the dominant bacteria during chilled fish storage, are the most sensitive to HP [53]. So, psychrotrophic Gram-negative Pseudomonas and H2S-producing bacteria (presumptive S. putrefaciens) are the most sensitive spoilage bacteria to HP, being these bacteria effectively inactivated in fish muscles at 100 MPa [56]. In contrast, Enterobacteriaceae and luminescent bacteria (presumptive P. phosphoreum) are more resistant to pressure than Pseudomonas and S.

putrefaciens, being only reduced considerably at 300 MPa [56]. Gram-positive microorganisms present in fish muscle, such as lactic acid bacteria, spore-forming bacteria Bacillus and Clostridium, have a greater resistance to HP inactivation [53, 55].

1.3.2.2. High pressure effects on enzymatic activity of fish muscle

HP inactivation of autolytic enzymes in fish muscles differs greatly among fish species and perhaps even within the same species. For instance, in cod muscle it was observed that a pressure level of 200 MPa (20 min) decreased the proteolytic activity of neutral protease (pH optima 6.6), being the acid (pH optima 3.3) and alkaline (pH optima 9.0) proteases resistant to pressure up to 400 and 500 MPa, respectively [59]. In this case, since the neutral protease is likely the most active enzyme and has most effect on fish muscles texture, HP inactivation of this enzyme could be of interest to control texture deterioration during storage [59].

Pressures between 100 and 300 MPa (up to 30 min), applied on enzyme extracts from bluefish and sheephead, decreased the enzyme activity, especially of cathepsin C, collagenase, chymotrypsin, and trypsin-like enzymes [35]. Teixeira et al. (2013) [60]

obtained similar results using sea bass muscle, with higher activity reduction observed at about 400 MPa for acid phosphatase, cathepsin D, and calpain. However, pressures up to 500 MPa increased cathepsins B, H and L activities in another study [61] using sea bass muscle, but calpains activity decreased, although an increment of calpains activity was afterwards observed during refrigerated storage [62, 63]. It was suggested that the increase of enzyme activity can be attributed to proteolytic enzymes releasing, due to perturbation of protein structure and the rupture of cell membrane by HP, contributing to the variation in enzyme activity [60, 64]. Moreover, the difference in enzyme structure also affects the behaviour of enzymes under pressure, since some proteases can have maximum activity and stability at a particular pressure-temperature combination due to structural modifications [63], as well as fish muscles can induce a protective effect on specific enzymes against HP, as observed in myofibril-bound serine proteinases of silver carp [65].

1.3.2.3. High pressure effects on physicochemical parameters of fish muscle HP can remarkably retard the development of total volatile base-nitrogen (TVB-N) and TMA in fish muscles, as was observed in tuna (220 MPa/15-30 min) [66] and red mullet (220 and 330 MPa/5 min) [67]. Total TVB-N is the total of TMA, dimethylamine (DMA), ammonia and other basic nitrogen compounds that result from fish decomposition.

In this way, a good correlation between the microbial inactivation by HP and the amines

of fish muscles, as observed in tuna treated at 200 and 300 MPa (5 min) during chilled storage at 2 ºC [68]. However, a pressure between 135 and 200 MPa (0.5 min) did not reduce considerably the K-value of farmed salmon (Oncorhynchus kisutch) muscle [69]. In addition, during trout storage at 3.5 ºC, the formation of biogenic amines (putrescine, cadaverine and tyramine) showed a strong correlation with the pressure levels applied, being reduced with the increase of pressure to 500 MPa (10 min) [70].

For consumer perception, colour is one of the most important sensory characteristics of fish muscles in determining their acceptability.Generally, L*- (lightness), a*- (redness) and b*- (yellowness) values are affected by HP, with lightness increasing in white fish muscles (cod, turbot, red mullet and sea bass) and redness reduction in red fish muscles (tuna, salmon and trout) [71]. The change in a*- and b*-values of fish muscles differed considerably with processing conditions and fish species. In salmonid muscle, red colour is the most important organoleptic property for customer acceptance [72], being dictated mainly by carotenoids (astaxanthin) and somewhat by haem proteins [73]. Generally, the redness of salmonid muscle was reduced with increase of pressure and time, as observed in salmon muscle treated from 100 to 200 MPa (10-60 min) [56]. The redness reduction can be attributed to myofibril denaturation including the host proteins for astaxanthin binding or the structural change of the astaxanthin-actinin embedding matrix under pressure [53, 74]. In addition, the oxidation of astaxanthin and myoglobin is accelerated by HP and coupled with haem compounds denaturation are also responsible for fish redness changes [73].

Sarcoplasmic proteins have been considered more susceptible to HP processing than myofibrillar proteins [75, 76]. However, there are some studies that suggested that myofibrillar proteins are more susceptible to pressure than sarcoplasmic proteins [64, 77], being the myosin the most readily denatured protein by HP [78]. According to Ko et al.

(2006) [64], the difference in the pressure denaturation of sarcoplasmic proteins was mainly attributable to the small molecular weight with less spatial structure compared to the heavier molecular weight of myofibrillar and more spatial structure.

HP treatment induces an increase in the hardness of fish muscles [59]. According to Angsupanich and Ledward (1998) [59], the unfolding of actin and sarcoplasmic proteins and the formation of new hydrogen-bonded networks could contribute to the increase in hardness and springiness.

After HP treatments, a significant increase in free fatty acids level was observed in carp muscle treated at 140-200 MPa (15-30 min) [79]. The authors hypothesised that the interactions changes (electrostatic, van der Waals, hydrogen bonding and hydrophobic forces between myofibrillar proteins and free fatty acids) could result in the release of free fatty acids rather than lipid fraction. In contrast, no significant changes were found in Coho salmon treated at 135-200 MPa (30 s) [69].

Lipid oxidation was accelerated in many fish muscles after HP and subsequent chilled storage including cod [59], red mullet [67], rainbow trout and mahi mahi (Coryphaena hippurus) [80]. However, the effects of HP on lipid oxidation of fish muscles also varied vastly depending on the pressure level and holding time, fish species and the type of muscle (dark or white muscle). There are several reasons for the HP effect on fish lipid oxidation. However, Wada and Ogawa (1996) [81] found that protein denaturation could be important in catalysing lipid oxidation in fish muscles, due to the interaction between proteins and lipids in fish muscles, which was destroyed leading to the increase in the surface between lipid and oxygen, thus allowing oxidation to occur easily. Atlantic salmon exhibits a fatty and dark muscle containing a higher amount of unsaturated lipids and pro-oxidants (iron and haem proteins). However, the lipid oxidation rate was observed to be similar in salmon muscle treated at 150 MPa (15 min) compared to the untreated control after 6 days of storage at 4 ºC [58]. Interestingly, a pressure level of 300 MPa significantly lowered the lipid oxidation during refrigerated storage. Possibly HP treatment can result in a higher availability of astaxanthin in fish muscle and exert a protective effect on fish lipids. Atlantic salmon contains a high amount of astaxanthin that has high antioxidant capacity, 10 times greater than -carotene, and up to 500 times greater than α-tocopherol [82].