Vol. 65, No. 2, 2000—JOURNAL OF FOOD SCIENCE 329© 2000 Institute of Food Technologists

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Comparison Of Air-blast And Pressure ShiftFreezing On Norway Lobster QualityD. CHEVALIER, M. SENTISSI, M. HAVET, AND A. LE BAIL

ABSTRACT: Pressure shift freezing (PSF, 200 MPa, �����18 �����C) of whole Norway lobster was compared with air-blastfreezing and with pressurized samples (200 MPa, 5 �����C) without freezing for its effect on the quality in texture,structure, water, and salt soluble protein extractabilities. For the pressurized Norway lobster meat either with PSF orwithout freezing, toughness increased while salt soluble protein extractability decreased. Conversely, air-blast freez-ing did not affect the textural quality of the meat. Scanning electron micrographs showed that PSF yielded smallerice crystals than air-blast freezing.

Key Words: high pressure, pressure shift freezing, texture, Norway lobster, ice crystals

Introduction

FREEZING IS A CONVENIENT PRESERVATION METHOD PARTICULARLY

used for fish and seafood, but it usually results in undesir-able physical, chemical, and structural changes leading to quali-ty losses in color, texture, and flavor (Krivchenia and Fennema1988). The freezing rate is a major parameter of the freezing pro-cess. A slow freezing causes formation of large ice crystals that ir-reversibly damage tissues. On the other hand, a high freezingrate produces small ice crystals resulting in a much preservedstructural quality of the food (Reid 1993).

Ultra-rapid freezing, as cryogenic freezing, can reduce damag-es due to large ice crystals. But it may cause lethal intracellular icecrystallization in living cells and mechanical cracking (Kalishveskyand others 1995). For conventional freezing, the velocity of thephase change front, which is related to supercooling, changes be-tween surface and center depending on the geometry and on heattransfer conditions. Supercooling (the difference between the ac-tual temperature of the sample and the expected solid-liquidequilibrium temperature at a given pressure) is the driving forceof the nucleation and is an important parameter that controls thesize and number of ice crystals. The ice nucleation rate is indeedincreased by a 10 factor for each degree of supercooling (Burkeand others 1975). Pressure shift freezing (PSF) permits to achievea large and uniform supercooling in the whole volume of the sam-ple. A much-preserved microstructure is, therefore, expectedwhen using PSF. The freezing point of water is depressed underpressure down to �21 �C at 210 MPa (Bridgman 1911). PSF is car-ried out in a high pressure vessel whose temperature is regulatedat subzero temperature (that is, �18 �C). The 1st step of the pro-cess consists in refrigerating the sample under pressure withoutice formation (that is, at 200 MPa). The pressure is then released toatmospheric pressure. The depressurization involves 3 mainphases. During the 1st one, the sample is cooled down withoutphase change. The 2nd phase corresponds to a sudden tempera-ture rise up to the phase change temperature at the current pres-sure. Partial freezing is initiated during this phase due to the highsupercooling of the sample. For example, Knorr and others (1998)observed 8 �C supercooling during PSF of potatoes (200 MPa, �18�C). During the last, the temperature increases according to thetemperature-pressure equilibrium relationship of liquid waterand ice I. Sanz and others (1997) showed that the ice to water ratiocan reach 0.36 for a sample of pure water at the end of the pres-sure release step indicating that only partial freezing can be ob-

tained during PSF. Freezing must, thereafter, be completedat atmospheric pressure.

This technique was first developed to cryoimmobilizebiological specimens for microscopy observations (Taylor1960; Moor 1987). PSF of meat and several vegetables wasstudied in recent years. Fuchigami and others (1997a,1997b, 1997c, 1998) found improvements of textural quali-ty and histological structure of carrot, tofu, and Chinesecabbage after PSF treatment compared with air-blast fro-zen samples. Koch and others (1996), Otero and others(1998), and Martino and others (1998) have compared PSFwith air-blast freezing of potatoes, eggplants, and porkmuscle, respectively. The main finding of these authors wasa significant improvement of the quality of PSF samples interms of ice crystal size. Martino and others (1998) havestudied pork meat but only for size and location of ice crys-tals and did not taken into account eventual texturalchanges. Presently no study has been done on the applica-tion of PSF in fish. The available studies on high pressureprocessing of fish and fishery products were usually relatedto the effect of pressure on bacteria (Ohshima and others1993), on texturization process (Ko and others 1990) and onpressure-assisted thawing (Chevalier and others 1999).

The main objective of this study was to compare differ-ent treatments (air-blast freezing, pressure shift freezing,and pressurization without freezing) on Norway lobstermeat texture and microstructure.

Results and Discussion

Temperature evolution during freezingThe average cooling rate during air-blast freezing was

0.9 �C/min calculated between 0 �C and �18 �C. The freez-ing temperature of individual tail muscle was �1.8 �C (Fig.1). Before application of pressure, the sample was cooledrapidly due to initial low temperature of the pressuriza-tion medium (Fig. 2). Application of the pressure causedrapid increase in the temperature of the sample and thepressurization medium (Fig. 2). These increases had thesame range (from �18 �C to �13 �C for the pressurizationmedium and from �2.5 �C to �2.5 �C for the sample) andwere due to the heat dissipated during compression. Dur-ing depressurization (Fig. 3), the temperature of the ambi-

JFS: Food Engineering and Physical Properties

330 JOURNAL OF FOOD SCIENCE—Vol. 65, No. 2, 2000

Food Engineering and Physical Properties

Air Freezing/Pressure Shift Freezing . . .

ent fluid and the sample cooled down to �21 �C and �18.8 �C, re-spectively. When the pressure reached the point where thephase change occurred from liquid to ice, the sample tempera-ture increased to �3.1 �C. This freezing temperature was slightlylower than that at atmospheric pressure (Fig. 1) due to ice crys-tallization upon pressure release.

Effect of different treatments on texture and proteinextractability

The shear force (N/m²) obtained after the 3 different treat-ments is compared in Table 1. This shear force represents thetoughness of the meat of Norway lobsters. There was no signifi-cant difference among the air-blast frozen Norway lobsters andthe control. A significant difference (p � 0.05) in toughness wasnoticed among control and PSF and pressurized samples. Bothtoughness of the PSF and pressurized without freezing meatsamples was higher than the toughness of the control samples,by about 2 and 1.3 times, respectively.

Protein extractability is indicative of protein denaturation infrozen fish. Hsu and others (1993) reported that salt solubleproteins denaturation in frozen pacific whiting (Merluccius pro-ductus) was inversely related to an increase in toughness. Pro-tein extractability is divided on water soluble protein extract-ability and salt soluble protein extractability, which reflect thestate of the sarcoplasmic proteins and the myofibrillar proteins,respectively. No significant difference in water soluble proteinextractability was observed among all the samples (Table 1).Conversely, large significant difference (p � 0.05) appeared insalt soluble protein extractability. There was a significant de-cline in salt soluble protein extractability for the 3 treatments.This extractability decreased by 26.6% after air-blast freezing.Both pressurization and PSF caused much larger decreases, by82.5% and 93%, respectively. As a first result, the decrease ofsalt soluble protein extractability of air-blast frozen sampleswas not correlated with an increase of toughness. Srinivasanand others (1997) showed that 2 opposing effects were both ac-

tive during a freezing-thawing cycle of prawns: tissue softeningcaused by proteases and ice crystal formation and tissue tough-ening caused by protein denaturation and cross linking. Thecounter-action of these 2 effects minimized the textural changein cooked Norway lobsters even if protein denaturation andcross linking had modified the extractability of salt soluble pro-tein. The sharp decline in salt soluble extractabilities for PSFand pressurized samples is accompanied by abrupt increase intoughness. This result can be explained by the pressure-in-duced denaturation of protein (Cheftel and Culioli 1997). Pres-sure induces denaturation of the main myofibrillar proteins, ac-tin and myosin and the loss of extractability is caused by cross

Fig.1— Temperature changes in central area of a whole Norway lob-ster during air-blast freezing (medium temperature: –30 °C; air ve-locity: 3 m/s). -�- : Norway lobster meat. -�- : cooling medium.

Fig. 3— Temperatures changes in central area of a whole Norwaylobster and the pressurization medium during pressure release. Thedepressurization rate was 10 MPa/s. The pressure evolution showsthe set point evolution. -�- : Norway lobster meat. -�- : pressuriza-tion medium. -�- : pressure.

Fig. 2—Temperature changes in central area of a whole Norway lob-ster and the pressurization medium during pressure shift freezingfrom 200 MPa. The pressurization rate was 100 MPa/min. The initialmedium temperature was –18 °C. The pressure evolution shows theset point evolution. -�- : Norway lobster meat. -�- : pressurizationmedium. -�- : pressure.

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linking of proteins mainly by electrostatic and disulfide bonds(Ikeuchi and others 1992).

Effects of air-blast freezing and PSF on structureThe pores observed in the frozen tissue were associated to ice

crystals (size, location). Scanning electron micrographs of air-blast frozen (Fig. 4) and PSF samples (Fig. 5) show a large differ-ence in tissue conformation. Larger ice crystals cavities (I) can beobserved in the air-blast frozen specimen compared with the PSFsample. The PSF section appeared to be more compact and

dense. This difference reflected the formation of smaller ice crys-tals during PSF that should cause less tissue structure damage.Similarly, Martino and others (1998) observed that mean ice crys-tal diameter of PSF was smaller than those of air-blast and liquidN2 frozen ones. In this present study, a contradiction is observedsince smaller ice crystals are observed with PSF but tissues aretougher than the air blast frozen ones. It seems that the pressurehas a great influence on protein denaturation, even at 200 MPa,and these modifications of proteins due to pressure cancel thebenefit of the formation of smaller ice crystals during pressureshift freezing.

Conclusion

THE SEM VISUALIZATION OF THE MICROSTRUCTURE OF MEAT

showed that PSF resulted in a reduction in the size of icecrystal and in a much preserved microstructure in comparisonwith air-blast frozen samples. This result did not yield an im-proved texture of the meat. Indeed, the toughness of the PSF (200MPa, �18 �C) frozen Norway lobster was significantly increased incomparison with non-pressurized samples. This result was ex-plained by the effect of high pressure on myofibrillar proteins. Itshould be interesting to study the PSF at lower pressure levels tominimize pressure effects on proteins (that is, below 150 MPa),while keeping a significant reduction in the size of ice crystals.

Table 1—Effects of air-blast freezing, pressure shift freezing andpressurization without freezing on toughness1 and water and saltsoluble protein extractabilities of Norway lobster meat2

Untreated Air-blast Pressure shift Pressurization(control) freezing freezing

Toughness 5.6 � 1.4a 5.1 � 1.2a 10.3 � 1.7b 7.0 � 1.9c

(� 104 N/m2) (n3 � 60) (n � 60) (n � 60) (n � 60)

Water soluble 58.7 � 1.92a 52.9 � 5.1a 51.2 � 1.3a 52.4 � 1.2a

protein (n � 9) (n � 9) (n � 9) (n � 9)(mg/g meat)

Salt soluble 46.7 � 3a 34.3 � 2.0b 3.2 � 1.6c 8.2 � 0.5d

protein (n � 9) (n � 9) (n � 9) (n � 9)(mg/g meat)

1 For toughness measurement, thawed Norway lobster was cooked in boiling water for 3 minand chilled in air at 4 °C overnight.2 Air-blast and pressure shift frozen samples were stored at – 20 °C for 12 h and thawed at 4 °Cfor 6 h.3 n represents the number of samples evaluated.abcd Means in the same row with the same superscript letter are not significantly different (p 0.05).

Fig. 4—Scanning electron micrograph of air-blast frozen Norway lob-ster flesh. The bar represents 10 mm and the I locates ice crystal mark.

Fig. 5—Scanning electron micrograph of PSF Norway lobster flesh.The bar represents 10 mm and the I locates ice crystal mark.

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Materials and Methods

MaterialNorway lobsters (Nephrops norvegicus) were caught off the

Britain coast and stored in ice for 12 to 15 h until being sold.Three kilograms of whole Norway lobsters (mean weight 20.7 g� 7) kept on ice were bought alive at the local wholesale marketfor each replicate in July 1998. Randomly selected whole Nor-way lobsters were placed in moisture-impermeable polyethyl-ene double bags (BB4L-Grace-Cryovac, France) and were vacu-um-packaged. They were stored at 4 �C for 1 h until being treat-ed. Four batches of 500 g whole Norway lobster were evaluatedfor each replicate. One batch was analyzed immediately asfresh, unfrozen control. The 3 others were used for air-blastfreezing, PSF, and pressurization without freezing. All experi-ments were performed in triplicate.

Pressurized and pressure shift frozen samplesHigh pressure treatments were carried out in a 3 L high pres-

sure unit (Alstom, France) with a vessel of 12 cm inside dia � 30cm high. The pressurization fluid was a 50% (v/v) ethylene glycolsolution. The vessel temperature was controlled by circulating atemperature controlled refrigerated fluid. Stainless steel shield-ed thermocouples (1 mm dia, Omega, Stamford, Colo., U.S.A.)were installed at the core of whole samples and in the pressuriza-tion fluid to monitor the temperature changes. A data logger(Model SA 32, AOIP, France) collected data. Samples were eitherpressure shift frozen or only pressurized without freezing to de-tect any effect due to pressurization. The pressurized sampleswere pressurized at 200 MPa for 30 min at 5 �C. The rate of pres-surization was 100 MPa/min. The PSF samples were prepared bycooling under pressure at 200 MPa, �18 �C with the same pres-surization rate. When the sample temperature reached �18 �C(30 min), a depressurization to atmospheric pressure was carriedout at 10 MPa/s resulting in supercooling of the sample. As freez-ing was partial at the end of the release of pressure (Le Bail andothers 1996; Sanz and others 1997), it was completed in a �20 �Cstorage room.

Air-blast freezingAir-blast freezing was carried out in an air-blast freezer (Ser-

vathin, France) at �30 �C and 3 m/s air velocity. Samples werestored in a �20 �C cold storage room when the center tempera-ture reached �20 �C. Temperatures of the samples were moni-tored using a temperature recorder (Model SA32, AOIP, France)with K-type thermocouples (1 mm dia, Omega, Stamford, Colo.,U.S.A.). The freezing rate was calculated from the cooling ratebetween 0 �C and �18 �C. This temperature range was chosenfollowing the International Institute of Refrigeration recommen-dations (International Institute of Refrigeration 1986).

Texture analysisThe frozen Norway lobsters stored at �20 �C for 12 h were

thawed for 6 h at 4 �C in an air-controlled room. Whole Norwaylobsters were cooked in boiling water (at atmospheric pressure)for 3 min and were chilled in air at 4 �C overnight. Norway lob-ster’s shells were removed before analysis. Individual tail mus-cles (20 per treatment for each replicate) were subjected to tex-tural analysis. Each tail was cut in 2 cm � 1 cm � 1 cm piecesfrom anterior part. The shear force required to rupture wasmeasured using a texture testing instrument (Lloyd Instru-ments LR5K, U.K.) with a 30� Warner-bratzler shearing device ata crosshead speed of 20 mm/min. The height of the major peak

represents the maximal shear force required to cut the musclesample. Shear forces (N) were normalized based on area (m²) ofthe muscle sample, which was subjected to the cross section ofthe blade.

Protein extractabilityAll preparations were done at 4 �C. A 60 g sample of Norway

lobster flesh obtained on average from 12 uncooked tails washomogenized in a warring blender (Waring, New Hartford,Conn., U.S.A.) for 8 s at 7940 � g. For water soluble protein ex-tractability, 20 g homogenized flesh were blended with 200 mLdistilled water for 10 s at 2560 � g with an Ultra Turrax (UltraTurrax T25 basic, Ika labortechnick, Germany). This mixturewas centrifuged at 5000 � g for 15 min in a GR 2022 centrifuge(Jouan, France). The same procedure was repeated with the re-sidual material. The 2 combined supernatants were used formeasurement of the water soluble protein concentration.

The extraction procedure for myofibrillar protein solubilitywas the same as that of water soluble protein except for 0.05 Mphosphate buffer, pH 7.5 in place of distilled water. After the 2nd

centrifugation, the residue was homogenized with 10 volumes of0.5 KCl/phosphate buffer, pH 5 and the mixture was centrifugedat 5000 � g for 15 min. This extraction was repeated with the re-sidual material. The supernatants of these 2 last centrifugationswere combined, and its protein content was then measured forthe salt soluble protein (that is, myofibrillar protein).

Measurement of protein concentrationThe protein concentration was determined by bicinchoninic

acid (BCA) procedure (Sigma Procedure n�TPRO-562, BCA-1,Sigma Biochemicals Co., St. Louis, Mo., U.S.A.). Two mL of BCAreagent (one part of C-2284 solution � 50 parts of B-9643 solu-tion) were added to 0.1 mL of the unknown protein solution. Thismixture was incubated at 37 �C for 30 min and then cooled to 20�C. The absorbance of the mixture was read at 562 nm in tripli-cate using a spectrophotometer UV1 (Unicam, U.K.). A calibra-tion curve was made with a standard solution of bovine serum al-bumin (P-014, Sigma Biochemicals Co., St. Louis, Mo., U.S.A.).

Microscopic observationsSections of 5 mm3 were removed at the center of the tail of

Norway lobsters with a chilled razor blade inside the cold storageroom. Three samples were taken from 3 different Norway lobstertails for each freezing treatment. These samples were placed inflasks containing a fixative solution at �20 �C. The isothermalfreeze substitution technique is an indirect method based onthe observations of the holes left by the ice in the tissue (Lampil-la and others 1985). The fixative consisted of 34% dimethyl sul-foxide, 2.4% cacodylate and 3% glutaraldehyde. Samples werefixed 7 d, and they were brought to room temperature, washed 3times for 15 min in a 0.1 M cacodylate buffer. Specimens werethen dehydrated in several increasing concentration ethanolbaths at �20 �C (70%, 95%, and 100% for 30 min, 1 h and 2 h, re-spectively). The ethanol was replaced with acetone and sampleswere dried to critical point. They were finally pulse-sputter-coat-ed with gold before being observed with a scanning electron mi-croscope (JSM-6300, Jeol, Peabody, Mass., U.S.A.).

Statistical analysisThe Statgraphics Plus version 2.1 software (Statistical

Graphics Corp., Princeton, N.J., U.S.A.) was used for analysis ofvariance. Whenever the F-test was not significant (p 0.05),Student T-test between 2 treatments was used.

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MS 1999-0432 received 4/12/99; revised 10/4/99; accepted 12/28/99.

Authors are with E.N.I.T.I.A.A., L.G.P.A., Rue de la Géraudière B.P. 82 225,F-44322 NANTES Cedex 03 - FRANCE. Send correspondence to Le Bail (E-mail: lebail@enitiaa-nantes.fr).