Z Lebensm Unters Forsch A (1999) 208 :349–354 Q Springer-Verlag 1999

ORIGINAL PAPER

Pilar Montero 7 M. Carmen Gómez-Guillén

Frozen storage of minced prawn flesh: effect of sorbitol, egg whiteand starch as protective ingredients

Received: 9 June 1998 / Revised version: 13 August 1998

P. Montero (Y) 7 M.C. Gómez-GuillénDepartamento de Ciencia y Tecnología de Carne y Pescado,(CSIC), Ciudad Universitaria s/n, E-28040 Madrid, Spain

Abstract The functionality of minced prawn flesh dur-ing frozen storage, with and without added ingredientsas protectants, was examined in terms of water-holdingcapacity, protein solubility, viscosity, texture and gel-forming capacity. Different lots containing sorbitol, eggwhite and starch were studied. Minced prawn showedhigh protein solubility which remained constant after 3months of frozen storage, regardless of the ingredientsused. A slight increase in shear strength during thestorage period was suppressed by the addition of ingre-dients; however, these were not effective at substantial-ly improving the gel-forming capacity of the mincedprawn. Viscosity was the property most affected byfrozen storage, and was improved by the addition of in-gredients.

Key words Frozen storage 7 Prawn 7 Mince 7Functionality 7 Gels

Introduction

The prawn is a highly prized species with a high level ofconsumer acceptance. A certain number of individualprawns, improperly handled during peeling and pack-aging, become broken and hence lose their commercialvalue. This portion can serve as raw material for a re-structured product with desirable properties (flavour,colour and texture) which can be used as an ingredientin the manufacture of high value-added products orshellfish-based ready-made dishes. Such products areoften commercialized in the frozen state, and thus it isimportant they retain their properties during the stor-age period. The major problems in frozen stored fishare losses in functional properties of muscle proteins,such as water-holding capacity, protein solubility andgel-forming capacity [1]. Textural hardening of fish dur-ing frozen storage is attributed to a freeze-induced pro-

tein aggregation caused by freeze syneresis due to theformation of large ice crystals [2]. The use of cryopro-tectants to prevent fish protein denaturation duringfrozen storage is a commonly used practice, and hasbeen well documented [1, 3, 4]. Sorbitol is one of themost used cryprotectants in fish mince. The basis of itscryoprotective effect is that it dissolves completely inthe extracellular fluid, interacting with myofibrillar pro-tein molecules, and increasing the hydration of the pro-tein molecule necessary for protein stabilization [5].Non-fish proteins, such as egg white, have also beenproposed for protein protection during frozen storage.In this connection, Yoon et al. [5] suggested that non-fish proteins with good water binding and dispersibilityreduced the amount of free water available for ice crys-tallization, preventing the formation of large ice crys-tals. A similar action could be assigned to somestarches which have been modified to be stable at froz-en temperatures. In addition, egg white has been re-ported to inhibit proteolytic activity when incorporatedinto surimi-based products [6].

Many studies on fish mince and surimi have re-ported the convenience of removing sarcoplasmic pro-teins by washing in order to enhance gel-forming capac-ity [7, 8]. However, according to Yoon et al. [9], sarco-plasmic proteins in unwashed mince retard sarcomereshrinkage resulting from freeze-induced contraction/protein cross-linking. This eliminates the need for amince washing step for products that are not based ongel-forming capacity; water-soluble substances are alsoretained, which in the case of prawn are highly desir-able in terms of flavour.

Some physicochemical and functional properties ofproteins from different prawn species during frozenstorage have been studied in whole tails [10, 11], butnot in minced flesh. Moreover, the use of sodium ace-tate as a prefreezing dip treatment in prawn has beenfound to enhance foaming and emulsification proper-ties, although it is detrimental to other properties suchas protein extractability or water-binding capacity[12].

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The aim of this experiment was to monitor changesin protein functionality of unwashed minced prawnduring frozen storage and to determine the contribu-tion of added ingredients as protectants to stabilize themince, in order to improve its technological suitabilitywith a view to the preparation of high-valued shellfish-based products or ready-made dishes.

Materials and methods

The species used in this experiment was prawn (Penaeus sp.).Fresh prawn tails (within 1 day after capture) were peeled, themuscle was finely chopped, and four lots (approx. 1.5 kg of mus-cle per lot) were made up with added protective ingredients asfollows: L1, control (muscle only); L2, muscle c 6% sorbitol; L3,muscle c 6% sorbitol c 2% egg white; L4, muscle c 6% sorbi-tol c 2% egg white c 6% starch. A modified waxy corn starch(acetylated starch adipate), which remains largely unaltered atfreezing temperatures, was used.

Each lot is divided into four samples (400 g per sample),which were immediately vacuum-packed in Cryovac BB-1 bags,tunnel-frozen (–40 7C setting) until the center reached –20 7C, andstored at –12 7C. Such a high temperature was selected to acceler-ate freeze-induced protein aggregation. The thawing of samplesfrom the different lots was carried out periodically during frozenstorage at room temperature.

For measurement of pH, a 5 g sample was homogenized with50 ml distilled water at room temperature and the pH measuredwith a PHM93 pH meter (Radiometer. Copenhagen).

For autolytic activity determination, 30 g of chopped prawnwas homogenized in an Omni-Mixer for 1.5 min with 0.15 MNaCl, ratio 1 :5 (weight:volume). Six-millilitre aliquots were incu-bated at 37 7C for 1 h. Autolytic activity was stopped by the addi-tion of 12 ml of 0.1 M trichloroacetic acid. A blank test was donewithout any incubation period. The peptide concentration (mg/mL) was determined according to Lowry et al. [13]. All determi-nations were performed in quadruplicate.

For the determination of protein solubility, 2 g of sample washomogenized with 30 ml of each solution (0.05 M NaCl and 0.8 MNaCl) in an Omni-Mixer model 17106 homogenizer (Omni Inter-national, Waterbury, USA) for 2 min at setting 5. The resultinghomogenates were stirred at 4–5 7C for 30 min and then centri-fuged for 30 min at 3000 g. The protein concentration in the su-pernatants was determined by the method of Lowry et al. [13].Soluble protein was expressed as the percent protein solubilizedwith respect to total protein, determined by the Kjehldahl meth-od.

Water-holding capacity (WHC, in %) and apparent viscosity(cP) were determined according to Gómez-Guillén et al. [14] andBorderías et al. [15], respectively. For evaluation of the gel-form-ing ability, gels were prepared periodically from L1, L2, L3 andL4 following the procedure described by Gómez-Guillén et al.[14], with 2% NaCl, 75% moisture and a single heating at 90 7Cfor 50 min. A puncture test and a Kramer test were determined asdescribed by Gómez-Guillén et al. [16], and were expressed aspenetration force (N) and shear strength (N/g), respectively.

One-way analysis of variance (ANOVA) was carried out. Thecomputer program used was Statgraphics (STSC, Rockville,USA). The difference of means between pairs was resolved bymeans of confidence intervals using a least significant difference(LSD) range test. The level of significance was set for P^0.05.

Results and discussion

The pH was considerably higher in lots containing pro-tective ingredients than in the control (Fig. 1). Values

Fig. 1 pH values of homogenates of minced prawn flesh, with orwithout different additives, during frozen storage (–12 7C). L1control (muscle only); L2 muscle c 6% sorbitol; L3 muscle c6% sorbitol c 2% egg white; L4 muscle c 6% sorbitol c 2%egg white c 6% starch

Fig. 2 Concentration of peptides (mg/mL) released during incu-bation, representing autolytic activity of minced prawn flesh, withor without different additives, during frozen storage (–12 7C). L1control (muscle only); L2 muscle c 6% sorbitol; L3 muscle c6% sorbitol c 2% egg white; L4 muscle c 6% sorbitol c 2%egg white c 6% starch

were within the alkaline range in all cases. There wereno significant changes over 3 months in frozen storage(see Table 1 for significant differences).

Changes in autolytic activity immediately after freez-ing and during frozen storage are shown in Fig. 2. Atthe outset, proteolytic activity was considerably higherin the control than in the lots with ingredients. Thiscould be due in part to a certain degree of a dilutioneffect of the muscle protein by the addition of ingre-dients. Of these, proteolysis was lowest in the lots con-taining egg white (L3 and L4), indicating that egg whitecould inhibit some proteases. An inhibitory effect ofproteolytic activity by the addition of egg white to fishmince or surimi has been reported [6]. After the first

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Table 1 Analysis of variance indicating significant differences(P^0.05) among the various lots along the storage period

pH Days of storagea

0 30 60 90

L1 a/x a/x a/x a/xL2 a/y b/y a/y ab/yL3 a/y a/y a/y a/yL4 a/y a/y b/x b/y

Protelytic activity

L1 a/x b/x c/x cb/xL2 a/y a/y a/x a/xL3 a/z b/z a/z ab/zL4 a/z b/z a/z b/z

WHC

L1 a/x b/x c/x d/xL2 a/x b/x b/xy a/yL3 a/x b/x ab/y a/yL4 a/x b/x ab/y a/y

SP in 0.05 M NaCl

L1 a/x b/x b/x b/xL2 a/y a/x a/x a/xL3 a/y a/x a/xy a/xL4 a/y a/x b/y a/x

SP in 0.8 M NaCl

L1 a/x a/x b/x a/xL2 a/x b/y a/y ab/xL3 a/x a/y a/y a/xL4 a/x a/y a/y a/x

Viscosity

L1 a/x b/x b/x c/xL2 a/y b/y b/y c/yL3 a/z a/x a/x b/yL4 a/v b/x c/z b/z

Shear strength

L1 a/x b/x c/x c/xL2 a/x b/x c/y a/yL3 a/x b/x a/z a/yL4 a/x a/y a/z a/y

Shear strength (gels)

L1 a/x b/x c/x c/xL2 a/y b/y c/y c/yL3 a/y b/z c/x c/xL4 a/y b/v c/z c/z

Penetration force (gels)

L1 a/x a/x a/x a/xL2 a/y b/y a/y b/yL3 a/y b/z c/x c/xL4 a/x b/z b/z c/z

a Different letters a, b, c . . . indicate significant differences withrespect to the time of storage for each lot. Different letters x, y,z . . . indicate significant differences among lots

Fig. 3 Water holding capacity (% of water retained with respectto total water content) of minced prawn flesh, with or withoutdifferent additives, during frozen storage (–12 7C). L1 control(muscle only); L2 muscle c 6% sorbitol; L3 muscle c 6% sorbi-tol c 2% egg white; L4 muscle c 6% sorbitol c 2% egg whitec 6% starch

month of storage there was a sharp decrease of proteo-lytic activity in the control (L1), attributed to a possiblecold-induced inhibition or denaturation of proteases,which were more vulnerable in the lot without protec-tants. In all other lots, the level of proteolytic activitywas low and remained constant (P^0.05) over 3months of storage. A decrease of proteolytic activity inprawn (Metapenaeus dobsoni) tails during frozen stor-age has also been reported [17], showing noticeable lev-els up to 300 days of storage.

The WHC was initially very high, decreasing slightlyin all lots over the first month of storage (Fig. 3), whichcould be attributable to initial protein conformationalchanges after the freezing process [2, 18]. After 3months, however, the WHC remained constant in alllots with ingredients, only the control exhibiting a de-crease (P^0.05). As well as retaining water (especiallystarch), these ingredients can act by hindering muscleprotein aggregation, which is normally produced in thefrozen state through the formation of ice crystals andthe concentration of solutes [18], thus enhancing theWHC of the muscle. No significant differences havebeen observed between the lot containing only sorbitoland those containing also egg white or egg white plusstarch. Chang and Regenstein [1] also found a constantwater uptake ability in cod mince with added kidneytissue and sucrose/sorbitol. The mechanism of water re-tention in fish mince by added carbohydrates and po-lyols is different for non-fish proteins or starch. Sorbitoldissolves completely in the extracellular fluid and thusits many functional –OH groups can interact with myo-fibrillar protein, increasing the hydration of the proteinmolecule [1, 5], whereas egg white and starch have ahigh water-binding capacity per se.

Protein solubility in 0.05 M NaCl was very high in alllots and remained constant (P^0.05) over three

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Fig. 4 Protein solubility (%) of minced prawn flesh in A 0.05 MNaCl and B 0.8 M NaCl, with or without different additives, dur-ing frozen storage (–12 7C). L1 control (muscle only); L2 musclec 6% sorbitol; L3 muscle c 6% sorbitol c 2% egg white; L4muscle c 6% sorbitol c 2% egg white c 6% starch

Fig. 5 Apparent viscosity (cP) of minced prawn flesh, with orwithout different additives, during frozen storage (–12 7C). L1control (muscle only); L2 muscle c 6% sorbitol; L3 muscle c6% sorbitol c 2% egg white; L4 muscle c 6% sorbitol c 2%egg white c 6% starch

months of frozen storage (Fig. 4A). Such high valueswere attributed to a large amount of low molecularweight proteins (mainly sarcoplasmic proteins and en-zymes) and probably also to a considerable quantity ofmyofibrillar proteins soluble at low ionic strength, con-sidering that the protein soluble in 0.05 M NaCl ac-counted for just over half of the protein soluble in0.8 M NaCl (Fig. 4B). A number of studies have re-ported a considerable degree of solubilization of myofi-brillar proteins at very low ionic strength (25 mMNaCl) [19, 20]. As well as being very high, the amountof soluble protein in 0.8 M NaCl remained very stablein all lots throughout frozen storage. This confirmedthat there was very little freeze-induced protein aggre-gation, even in the control. Such high solubility wouldaccount for the high WHC values noted above in alllots and gives an idea of the low degree of aggregationof the myofibrillar protein in the restructured product.In this connection, Yoon et al. [9] postulated that wa-ter-soluble sarcoplasmic proteins remaining in un-washed fish mince could act by reducing the contrac-

tion of muscle fibre units, and blocking the cross-link-ing of muscle fibrils.

The control containing no ingredients (L1) initiallyexhibited higher viscosity (P^0.05) than the other sam-ples (Fig. 5), probably owing to a higher proportion ofmyofibrillar protein. On the other hand, viscosity in L4,containing sorbitol, starch and egg white and hence alower muscle protein concentration, was relatively high,probably owing to the formation of a supersaturatedsolution of very high viscosity [5] and to swelling of thestarch, which has been found to adopt the form of hy-drated granules at temperatures below 50 7C [21]. Dur-ing the first month in storage, however, there was apronounced decrease in control viscosity owing to theabsence of protectants, but also it could be connectedwith the high level of proteolytic activity in this sampleat the outset of the storage period. The first month offrozen storage has been found to be precisely the peri-od where muscle proteins of freshwater prawn tails aremore susceptible to freezing-thawing processes [10].These molecular changes, however, were not detectedin the study of protein solubility. The decrease of vis-cosity in lot L4 during the first steps of storage was at-tributed to redistribution of the water available to thestarch through recrystallization, producing a change inits degree of hydration. Sorbitol alone (L2) maintaineda steady level of viscosity in the system over 2 months’frozen storage; however, at the end of the period,where the viscosity of the control lot fell, the lot with allthe ingredients (L4) presented the highest viscosity(P^0.05).

The shear strength of minced prawn (Fig. 6) at theoutset of storage was not significantly (P^0.05) af-fected by addition of ingredients at the given concen-trations. Lot L4 was the most stable over the storageperiod; after 90 days, no variations in shear strength

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Fig. 6 Shear strength (N/g) of minced prawn flesh, with or with-out different additives, during frozen storage (–12 7C). L1 control(muscle only); L2 muscle c 6% sorbitol; L3 muscle c 6% sorbi-tol c 2% egg white; L4 muscle c 6% sorbitol c 2% egg whitec 6% starch

Fig. 7 Shear strength of gels made from minced prawn flesh, withor without different additives, during frozen storage (–12 7C). L1control (muscle only); L2 muscle c 6% sorbitol; L3 muscle c6% sorbitol c 2% egg white; L4 muscle c 6% sorbitol c 2%egg white c 6% starch

Fig. 8 Force (N) required to penetrate gels made from mincedprawn flesh, with or without different additives, during frozenstorage (–12 7C). L1 control (muscle only); L2 muscle c 6% sor-bitol; L3 muscle c 6% sorbitol c 2% egg white; L4 muscle c6% sorbitol c 2% egg white c 6% starch

were induced by conformational changes in the pro-teins. The control lot (L1) showed higher values alongthe storage period, indicating the ability of sorbitolalone or in combination with egg white and starch toprevent textural hardening of minced prawn duringfrozen storage. These results are in agreement withthose reported by Yoon et al. [5] in red hake mince,and by Chang and Regenstein [1] for cod mince.

The gel-forming capacity of minced prawn duringfrozen storage was evaluated by thawing the mince andmaking gels from each lot periodically along the stor-age period. The measurements were done in terms offolding test, shear strength and penetration force of thegels. In the folding test, the minced prawn showed avery low gel-forming capacity, scoring values with ad-ded ingredients not higher than “3” in any case. Imme-diately after being frozen, gels with added ingredientspresented lower (P^0.05) values of shear strength thanthe control L1 (Fig. 7). However, during the first monthof frozen storage, shear strength increased considerablyin the lots containing egg white and starch (L3 and L4),scoring “3” in the folding test. As well as acting as pro-tein aggregation protectants and/or water binders,these ingredients act as gelation enhancers, especiallywhen added together (L4). The gelation enhancementof fish mince by the addition of egg white or starch hasbeen well documented [14, 22–24]. As the figure shows,although sorbitol effectively inhibits protein aggrega-tion, it is clearly detrimental to gel-forming ability as ithinders the formation of points of interaction betweenprotein molecules and hence formation of the gel net-work. After 3 months in frozen storage, the gels pre-pared from L2 (muscle c sorbitol) had the lowestshear strength (folding test 1), whereas L4 (muscle csorbitol c egg white c starch) showed the highest(P^0.05) (folding test 3). In the puncture test, the lotwith sorbitol (L2) again had the poorest gel-forming

ability, with the lowest penetration force values alongthe frozen storage (Fig. 8). The penetration force heldsteady in gels made from the control (L1) throughoutthe 3 months, which is consistent with the low level ofprotein aggregation found by analysis of the solubilityof L1 in 0.8 M NaCl (Fig. 4); according to Montero etal. [25], the puncture test is closely related to the degreeof freeze-induced protein aggregation produced by theproliferation of ice crystals. Once again the gels madefrom lot L4 had the highest penetration force valuesafter 2 months (P^0.05), thanks to the enhancement ofgel formation by egg white and starch, but decreased atthe end of the period.

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Conclusion

Minced prawn could be employed as an adequate rawmaterial in the preparation of shellfish-based ready-made dishes, since it showed a low degree of freeze-induced protein aggregation, increasing only slightly infirmess during frozen storage. The addition of ingre-dients as protectants (especially when added together)is recommended to improve mainly the water-holdingcapacity and viscosity in the course of frozen storage,and also to help maintain the texture, although no greatchanges were observed in this property. However, theadded ingredients were not effective at substantiallyimproving the gel-forming capacity, which has beenfound to be very low in this species, at the end of stor-age period.

Acknowledgements This research was financed by the EuropeanUnion under project TS-CT-94-0343 and by the spanish ComisiónInterministerial de Ciencia y Tecnología (CICyT) under projectALI 95-1715-CE.

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