高壓處理對魚制品影響的研究進展

Lisa Amanda Yakhin 王遠亮 李宗軍

（1.湖南農(nóng)業(yè)大學食品科技學院，湖南 長沙 410128；2.食品科學與生物技術(shù)湖南省重點實驗室，湖南 長沙 410128）

1 Introduction

Foods with fresh－like attributes are in demand nowadays.In other hand，food safety is another aspect which is more concerned.The use of good manufacturing practices（GMPs）is essential to control food borne diseases.However，these procedures alone may be insufficient to ensure microbial safety，making food－preservation methods necessary.Therefore，to satisfy market requirements，emerging or“new”technology is required.

One emerging technology promising agreat deal of attention is high pressure processing.Pressure application，are considered as a new alternative food processing technique that can be compared to heat processing.High pressure （HP）treatment is promising as a high interest technology，for it can preserve the essential function，flavor，and nutrition of food products.The initial investment is high，but it consumes less energy than thermal processing does，which becomes one of the reasons why it is commercially competitiveness.Heat processing usually takes more time for heat to penetrate，thus stirring might be necessary.On the other hand，the application of pressure is homogenous and there is no significant damage to food appearance during processing.

HP has two major applications in the food industry：the food preservation and food processing（texturation or tenderization）.High pressure treatment causes three main kinds of change in meat：enzymatic，protein modifications（mainly on myofibrils）and structural modifications（Ohshima，Ushio，＆ Koizumi，1993）.

High pressure processing（HPP）can also preserve food from its autolysis as well as microbial growth and its metabo－lism.HPP has been used commercially to produce food products such as raw oysters，guacamole，and ham and fruit juices in the US；also jams，jellies，fish and meat products，salad dressing，ham，fruit juices and yogurt in Europe and Japan.Some studies done on meat and fish have shown that high pressure might be a useful treatment for some products（Ohshima，et al，1993）.On fish products，high pressure were used with an aim of preservation（Chevalier，LeBail，＆Ghoul，2001；Ko ＆ Hsu，2001；Zhu，Ramaswamy，＆Simpson，2004）or texturation（LeBail，Chevalier，Mussa，＆ Ghoul，2002；Lakshmanan，Parkinson， ＆ Piggott，2007；Romero，Smiddy，Hill，Kerry，＆Kelly，2004）.

However，in spite of the general conception that HPP results in minimal changes in food products，it is also a common knowledge that the technology induces important changes in the texture and appearance of fish products.This paper reviews the changes induced by pressurization in fish，concerned in high pressure effects on biochemical，physical，and microbiological aspects of fish.

2 Biochemical Changes due to HP Treatment

2.1 Lipid Stability

Rancidity，malonaldehyde concentration and free fatty acid（FFA）content have been related to flavor loss.It limits the product shelf life of aquatic products，including frozen fish and seafood.HP treatments have no effect on the FFA content but influence the lipid oxidation.It was concluded that HP has no effect to hydrolysis mechanism which lead to FFA Production.100 ＆ 140MPa pressure level for 15min and 100MPa pressure level for 30min gave only a slight change on Thiobarbituric Acid （TBA）value；but started from 200MPa，TBA value was increased significantly（Chevalier，et al，2001）.

Purified marine lipids were stable against auto－oxidation at high pressure（Ohshima，et al，1993）.The presence of meat significantly accelerated the oxidation even without pressurization.It is suggested that accelerated oxidation in pressurized fishes may be due to denaturation of haem protein by pressure which releases metal ions promoting auto－oxidation of lipid in the pressurized fish meat；or there is an interaction between lipid and the protein which accelerated oxidation（Angsupanich，Edde，＆ Ledward，1999）.A study on turbot fillets showed that a pressure level of 140MPa at 4℃gave a minimize effect on lipid stability（Chevalier，Sentissi，Havet，＆Bail，2000）.

The TBA number of pressurized turbot （140MPa，－14℃）stored 2dchanged significantly（Chevalier，et al，2000）.Similar results was also found in pressurized cod muscle after 75dof storage（200MPa，20min，20℃）（Angsupanich ＆Ledward，1998）.The FFA content of pressurized shift freezing（PSF）samples did not show any systematic changes during frozen storage.A significant increase of TBA values was also shown in pressurized raw carp fillets along with pressure and pressurization time for all the pressure levels tested（100～200MPa，10～20min），except for the samples pressurized at 100MPa for 15min（Munoz，Chevalier，LeBail，Ramaswamy，＆Simpson，2006）.Some fish may be found more susceptible to pressure treatment because of different types of fat and content of unsaturated fats in different fish which responsible to the non－uniform sensitivity of fats to pressure.

2.2 Protein Stability

Myofibril protein was induced significantly by high pressure.Above 200MPa，the dissociation of oligomeric structure of proteins are usually induced，partial unfolding as well as denaturation of monomeric structures，protein aggregation and gelation were also happened（Mozhaev， Heremans，F(xiàn)rank，＆ Masson，1996）.These effects depends on the pressure level，temperature and protein concentration.

In turbot fillet，protein stability was induced significantly from 180MPa.Myosin was denaturated after 100MPa pressure was applied for 30min.Denaturation in actin was observed at 180and 200MPa pressure applied due to changes in actomyosin（Chevalier，et al，2001）.While in cod，myosin was denaturated at 100MPa pressure for 20min，because of new network due to hydrogen bonds gels formed under pressure（Angsupanich，et al，1998）.

150MPa was also known as the threshold over which protein denaturation might occur or the pressure which protein denaturation starts to occur.A protein denaturation of whiting fillets was confirmed to start from 150MPa from electrophoretic pattern of sarcoplasmic proteins extracted from fish meat（Chevalier，et al，1999）.The full denaturation of cod（Gadus morhua）muscle myosin was observed at 200MPa（Angsupanich，et al，1998）.

Fish enzymes were found more susceptible to hydrostatic pressure inactivation than their mammalian counterparts.Cathepsin C，collagenase，chymotrypsin－and trypsin－like enzymes from bluefish and sheephead fish，were found susceptible to 100～300MPa hydrostatic pressure treatments（Ashie ＆Simpson，1996）.

3 Physical Changes due to HP Treatment

3.1 Water Holding Capacity（WHC）

WHC is the ability of muscle to resist water loss.About 90%of water in fish tissues is held by capillary action，mainly in intracellular locations，the inter－filament spaces within the myofibrils，a substantial part in the extracellular space and the spaces between myofibrils.Appropriate selection of HPP can result in increasing WHC in foods and fresh cod（Hedges ＆ Goodband，2003）.An increase in moisture contents in both fresh salmon and cold smoked salmon （CSS）was noticed after pressure treatment（100～200MPa，10～20min）（Lakshmanan，et al，2007）.The moisture content was increased when oysters were pressurized （100 ～800MPa，10min，20 ℃）because of water absorption by protein（Romero，et al，2004）.Furthermore，application of HPP to fresh salmon reduced the WHC by 5%at 200MPa for 10and 20min，whereas HPP did not cause significant change in relation to WHC of CSS.The loss of water was higher in fresh Atlantic salmon muscle than that of CSS muscle samples.An increase was found in CSS WHC due to the salt addition or pressure applied（Hedges，et al，2003）.

A significant reduction of drip－loss by high pressure was also observed on liquid nitrogen frozen salmon in which mechanical cracking occurred and much of the drip appeared after Water Immersion Thawing（WIT）.High freezing rate or high pressurization rate reduced thawing drip loss of whiting fillets（Gadus merlangus），but drip loss was reduced only by prolonging holding time of pressure as compared to atmospheric pressure thawing（Chevalier，LeBail，Chourot ＆Chantreatu，1999）.

Pressure Assisted Thawing （PAT）had less thawing time and drip loss compared to atmospheric thawing （thawing at 0.1MPa）in aiguillat fish （best treatment was 150MPa，45min）.A higher drip volume was observed at 200MPa，explained as the higher protein denaturation which is believed to occur above 150MPa（Rouille，LeBail，Ramaswamy，＆ Leclerc，2002）.No significant differences in drip loss were found in PAT Atlantic salmon fillets （100～200MPa，20 ℃）frozen by conventional air freezing and plate freezing method（Zhu，et al，2004）.Pressure Shift Freezing（PSF）（140MPa，－14 ℃）reduced thawing and cooking drip of turbot fish compared to air blast freezing due to smaller and more regular intracelluar ice crystal（Chevalier，et al，2001）.

3.2 Toughness

As pressure employed in high pressure process increased，the hardness of samples was increased，particularly apparent for the High Pressure Thawing （HPT）at 200MPa.Pressurization，both PSF （200MPa，－18 ℃）and pressurization without freezing（200MPa，4℃）signifi－cantly increased the toughness of the nail of Norway lobster compared to untreated and air blast freezing（－30 ℃）samples（Chevalier，et al，2000）.High pressure thawed Atlantic salmon fillets（100～200MPa，20 ℃），salmon，whiting，haddock，cod and rainbow trout（200MPa，room temperature，60min）also had a tougher texture compared to atmospheric thawed fillet（Ko，et al，2001；Schubring，Meyer，Schluter，Boguslawski，＆ Knorr，2003）.Toughness was also found in cod（Gadus morhua）（Angsupanich，et al，1998）；cold smoked salmon（Gudbjornsdottir，Jonsson，Hafsteinsson， ＆ Heinz， 2010）； turbot（Scophthalmus maximus）after pressurization（Chevalier，Munoz，LeBail，Simpson，＆Ghoul，2000）.

Cold smoked salmon had a harder texture than control samples at pressures ≥ 300MPa，and a soft and jelly－like texture at pressures≤ 200MPa（Lakshmanan，Miskin，＆Piggott，2005）.The same results were obtained also for both fresh and smoked salmon samples treated at 100and 150MPa.At 200MPa，HPP brought slight dryness，hardness，pale color and lighter appearance to the samples.It is assumed that HPP brings some changes related to texture and WHC of fish through denaturing protein components（Lakshmanan，et al，2007）.

In bluefish（Pomatomus saltatrix）an increased in elasticity were found after pressurization conducted at room temperature（100MPa，5～30min，and 200MPa，5min）.In contrast，a decreased in elasticity were found after 200MPa pressurization for 15～30min（Ashie，et al，1996）.

The changes of texture profile were attributed to protein denaturation induced by high pressure（Chevalier，et al，2000）.The high pressure application related to the coagulation，stiffening and aggregation of myofibrillar structure（Chevalier，et al，2000；Jung，Ghoul，＆ Anton，2000）.

Though many high－pressure treated seafood have been reported to have harder textures or higher shear strengths than untreated controls，but studies include a formal sensory evaluation or the effect of high pressure－induced textural changes to consumer acceptance have not been reported.It has also been reported that the undesirable hardening following high－pressure treatment may also be reversed by subsequent cooking（Ohlsson ＆ Berngtsson，2002）.Thus the changes in texture may be particular significance for seafood that is not cooked prior to consumption.The development in high－pressure technology which enables the high pressure to be reached in 10sand lowering working temperature can also be considered to minimize the texture changes of fish.

The muscle of fish is softening quickly during the post mortem storage in chilling temperature.Softening of fish muscle is caused by biochemical－induced by enzymatic degradation of myofibrils and collagen，as well as physical separation of myotomes called gaping（Cheret，Chapleau，Delbarre－Ladrat，Verrez－Bagnis，＆de Lamballerie，2005）.A significant decrease in toughness of Biceps femoris pressurized muscles was also observed during storage and the decrease appeared to be delayed by pressure treatment（Jung，et al，2000）.

A research done in untreated sea bass（Dicentrarchus labraxL.）fillets showed a significant decrease of hardness，chewiness，cohesiveness，springiness，and gumminess during storage；it demonstrated that fish muscle became softer and tenderer during storage. Pressure treatment above 300MPa increased hardness after storage than untreated sample；thus it was considered that the pressure treatment might allow keeping hardness of fillet in a good range，not to be rejected as soft fish.This result proved the ability of highpressure to improve texture quality of chilled stored fish fillet（Cheret，et al，2005）.

3.3 Color

Changes in fish color and appearance affect consumer acceptance significantly.Color may changes during frozen storage due to lipid oxidation and pigment degradation process.It is well known that high pressure treatments can induce color modifications on meat and fish products（Cheftel ＆ Culioli，1997）.

Color of PSF turbot（140MPa，－14 ℃）was changed gradually with both the increase of the pressure level and pressure time，an increase in L＊values known as whitening effect was obtained（Chevalier，et al，2000）.Pressurized turbot fillets（100～200MPa，15～30min，4 ℃）lost their transparency with an increase of L＊values，for both increase of the pressure and pressure holding time，appeared as cooked（Chevalier，et al，2001）.These changes were attributed to the denaturation of myofibrillar and sarcoplasmic proteins，as it were also noticed in cod，taking place at 100～200MPa pressure levels at which the different myosin components were denaturated（Angsupanich，et al，1998）.Similar color change results were found in high－pressure processed（no freezing／thawing）cod（Gadus morhua）（Ohshima，et al，1993；Angsupanich，et al，1998）and raw turbot（Scophthalmus maximus）（Chevalier，et al，2001）.

Opaque appearance is happened because the incident light is unevenly scattered.Gradual disintegration of myofibrils during spoilage gives wider and more random intracellular distribution， resulting in unevenly scattered light（Charalambous，1993）.The disintegration of myofibrils by pressurization，resulting in high lightness value，appeared as cooked.

L＊value of carp（Cyprinus carpio）remained essentially unchanged at 100MPa regardless of processing time.However，at 140MPa and above，L＊values increased with pressure（Munoz，et al，2006）.Increased L＊value was also observed with increase in high pressure－low temperature（140MPa，4℃，15～30min）turbot fillets（Scophthalmus maximus）（Chevalier，et al，2001）；pressure shift freezing treated turbot fillets（140MPa，－14 ℃）（Chevalier，et al，2000）；sea bass（Dicentrarchus labrax L.）fillets（Cheret，et al，2005）；and oyster（Crassostrea gigas）（Romero，et al，2004）.At the higher pressure，immediate effect on the lightness was noticed in cold smoked salmon even in a very short holding time（400～900MPa，room temperature，10～60s）（Gudbjornsdottir，et al，2010）.

The a＊value is normally used as an index of visual redness.It was reported for mackerel and cod fish that the a＊values decreased after pressurization（Ohshima，et al，1993）.Similarly，redness of raw cod was lost after HP treatment at≥200MPa（Angsupanich，et al，1999）.However，when pressurization time was prolonged for 20min，the opposite effect，i.e.，increasing a＊values，was observed in carp fillets.Pressure－induced coagulation of sarcoplasmic and myofibrillar proteins were possibly responsible for the observed changes in the a＊values（Jimenez，Carballo，F(xiàn)ernandez，Barreto，＆Solas，1997）.On the contrary，there was no effect of pressure level and holding time on the redness（a＊value）（Chevalier，et al，2001；Gudbjornsdottir，et al，2010）.

The b＊values increased with pressure and holding times in observations made on turbot fillets（Chevalier，et al，2001）；Atlantic salmon（Salmo salar）（Zhu，et al，2004）；oyster（Crassostrea gigas）（Romero，et al，2004）；carp（Munoz，et al，2006）；and PSF treated turbot fillets（Chevalier，et al，2000）.

Oxidation of carotenoid pigments and lipids in tissues are responsible of the yellowing on fish（Angsupanich，et al，1998；Charalambous，1993）.Lipid oxidation was observed on turbot fillets（Scophthalmus maximus）（100～200MPa，4 ℃，15～30min）after high pressure－low temperature treatment.Little effect on lipid oxidation was also observed in cod muscle after 400MPa treatment.Significant increase in oxidation was seen at higher pressure.

Changes in color may limit the use of high－pressure treatment since color is very important for consumer acceptability.Nevertheless，the appearance changes in high pressure treated products are similar to heat treated products，thus the application of the technology can still be considered.HP－treated fresh meat with better hygienic quality with discolored appearance can still be produced for specific customers（e.g.food services companies）who are sensitive with food safety（Jung，Ghoul，＆ Anton，2003）.After heat treatment，the effect of pressure in cooked fish product toward color was slightly significant，but the difference was significantly smaller compared to uncooked products.Uncooked pressurized fish product had bigger difference compared to uncooked untreated fish product.The color differenceΔE＊between cooked fish fillets （rainbow trout，cod，haddock，salmon，whiting，redfish）thawed with high pressure treatment or conventionally，varied from negligible to significant（ΔE＊11.71）（Schubring，et al，2003）.

Working temperature is very important to color changes besides pressure.Low temperature gives less change in color.Decreasing the working temperature during high pressure treatment will likely reduce the color changes，as well as microstructure and texture changes.

4 Microbiological changes due to HP treatment

In relation to microbiological aspect，high pressure is a non－thermal preservation technique that depends on pressure，time，temperature and product characteristics which allows microorganisms’inactivation with little changes in texture，color and flavor compared to conventional technologies. Pressure at 250MPa did not inactivate L.monocytogenes，lag phases of 17and 10days were observed at 5and 10 ℃，respectively（Lakshmanan ＆ Dalgaard，2004）.

The resistance of microorganisms to pressure varies considerably and mainly depends on pressure，time and temperature.By increasing the pressure and time of treatment the number of L.monocytogenes decreased proportionally，which showed its sensitivity to pressure changes（Chen，Guan，＆ Hoover，2006）.

L.innocua was reduced significantly（P＜0.05）after 60sat 500MPa.One log reduction was observed and the number decreased during storage.No reduction was observed after 10sand it remained unchanged until after more than 26d.By increasing the treatment time to 20and 30sthe total number of bacteria decreased during the first 12dof storage but after that the bacteria recovered and the number increased.No L.innocua was detected 5dafter HPP treatment at 900MPa but after storage at 5.5 ℃ for 26～41d low level（0.3～20CFU／g）was detected（Gudbjornsdottir，et al，2010）.

Cold smoked dolphin fish processed under severe salting，smoking and 300MPa pressurization（200℃，15min）had L.monocytogenes count under the detection limit throughout 100dof storage（Montero，Gomez－Estaca，＆Gomez－Guillen，2007）.HPP can extend the microbial shelf life of cold smoked salmon when exposed to 900MPa to 26d compared to suggested limited shelf life for a period （14～21d）where growth of Listeria spp.is unlikely to take place or to reach levels＞100CFU／g（Huss，Jrgensen，＆ Vogel，2000）.

It is found that the total aerobic plate count were above，around and below 7log CFU／g for 300，400and 500MPa treatment，respectively，after 7dof storage.High－pressure was able to reduce the microbial growth and improved the microbiological quality of fresh food.A significant decrease of total aerobic plate count was reached when pressure increased from 200～500MPa（Cheret，et al，2005）.

5 Conclusion

The structure，the lipid and protein constituents of fish muscle are very sensitive to HP.Some effects of HP，such as lipid oxidation，protein denaturation，whitening effect，toughness increase，are detrimental；while some of them are very beneficial and potentially useful：inactivation or control enzymatic activity；reduced thawing rate，thawing drips as well as cooking drips due to small and homogenous ice crystal structure；improved texture quality；and reduced the number of microorganisms which improve the safety and shelf－life.However，inappropriate pressure level，holding time and working temperature will give undesirable effect to fish products，such as WHC decrease，toughness increase，color changes，as well as un－inactivated enzymes and microorganisms.All of the effects are depended on pressure level，temperature，time of treatment，protein concentration，F(xiàn)FA type and concentration，etc.

Changes in color and toughness may limit the use of high－pressure treatment，but as the changes in appearance are similar to heat treated products，there are still a big chance of the HPP application.Further studies should be done：①the effect of HPP after heat treatment；②the consumer’s sensory acceptance of cooked HP－treated fish product；③ HPP combined with other processes such as washing，smoking，mild heating，pre－packaging under vacuum，fermentation，and subsequent refrigerated storage；and④the vitamin content，protein and PUFA nutritive value of pressurized fish.A new development in HP technology also gives us a higher treatment pressure with significant shorter treatment time.Higher pressure，shorter time and lower working temperature will reduce undesirable changes of HPP.

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