J Sci Food Agric 1992, 58, 227-238

Effect of Chloride Salts on Protein Extraction and Interfacial Protein Film Formation in Meat Batters Andre Gordona and Shai Barbutb a Food Science Department, and Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario NlG 2W1, Canada

(Received 1 June 1990; revised version received 2 April 1991 ; accepted 6 August 199 1)

Abstract: Six comminuted chicken breast meat mixes and six meat batters were made with isoionic NaCl (25 g kg-'), MgCI,, CaCI,, KCl, LiCl (IS = 0.43) and 15 g kg-' NaCl (IS = 0.26). The quantity and type of proteins extracted and used for interfacial protein film (IPF) formation was determined and related to batter stability. The monovalent salts produced IPF which differed in individual protein content between salts but which all contained significantly larger amounts of protein (P < 0.01) than those using divalent salts. MgCI, extracted more protein than CaCl, and produced a different protein profile in the IPF. However, MgCl, formed unstable raw batters whereas CaCI, did not. In addition, a simple, rapid method for extracting and quantifying proteins from meat batters was developed to assist in direct determination of the actual soluble protein uptake by the fat phase during comminution.

Key words : Meat batter, protein, chlorides, film extraction.

INTRODUCTION

The mechanism by which comminuted meat batters are stabilised has been debated for some time. Comminuted meat batters are essentially mixtures of finely chopped lean meat and fat having a very homogeneous texture (eg frankfurters, bologna). There are two alternative theories for meat batter stabilisation : the emulsion theory, which is based on the formation of an interfacial protein film (IPF) around fat globules, and the gelation theory (physical entrapment) which centres on the entrapment of the fat within the heat-induced gel of the protein matrix upon cooking. Regardless of the mechanism by which batter stabilisation is achieved it is clear that the amount and type of protein extracted into the aqueous phase affects batter stability (Galluzzo and Regenstein 1978a,b,c; Schut 1978; Asghar et a1 1985; Smith 1988). The most important proteins in this respect are the salt- soluble myofibrillar proteins (Gillett et a1 1977). Protein extraction is therefore typically achieved by the use of high ionic strength (IS), normally supplied by sodium chloride (NaCI).

Sodium chloride has been used extensively for cen-

turies in the preparation of comminuted meat products because, in addition to extracting the myofibrillar proteins, it imparts a desirable flavour to these products, has some antimicrobial activity and improves their water holding capacity (WHC). The chloride ion is instru- mental in causing protein swelling and disruption of the myofibrillar structure (Offer and Trinick 1983; Lewis et a1 1986; Belton et a1 1987), both of which facilitate greater WHC and increase protein solubility. However, concern about the possible role of sodium in hyper- tension (Kent 1981) has led to a search for alternatives which has largely centred on other chloride salts (Terrell 1983). These have included the monovalent chlorides of potassium (KCl) and lithium (LiCI) as well as the divalent magnesium and calcium chlorides (MgCI, and CaC1,). Although LiCl is not 'Generally Recognized as Safe' (GRAS), it has been investigated with the hope of gaining better insight into the mechanisms by which NaCl acts (Hand et a1 1982; Gordon and Barbut 1989).

It has consistently been reported that the monovalent chlorides are similar to NaCl in their effect on water binding and texture in meat batters whereas divalent chlorides reduce water binding and can be detrimental to

227 J Sci Food Agric 0022-5142/92/$05.00 0 1992 SCI. Printed in Great Britain

17 JFA 58

228

texture (Seman et a1 1980; Hand et al 1982; Weinberg et ul 1984; Barbut et al 1988). This might suggest that all monovalent chlorides behave similarly in terms of protein extraction but differ from divalent chloride salts. However, i t has recently been shown that, in addition to the differences between monovalent and divalent chlorides, there were also microstructural and textural differences between batters made with different mono- valent chloride salts (Gordon and Barbut 1989; Gordon and Barbut 1990a). It was suggested that these differences may have been due to differences in the type and amount of protein extracted into the aqueous phase and forming the IPF. Barbut and Mittal (1988) have also indicated that substituting KCI or MgCI, for NaCl in meat batters resulted in rheological and textural differences, and Cheung and Cooke ( 1 971) have shown that Li', Na' and K' ions have different effects on myosin conformation. Unfortunately, there is little information on the quantity and types of protein extracted from meat by different chloride salts.

Research over the years has shown that myofibrillar proteins, mainly actomyosin and myosin, can be ads- orbed on to the surface of fat globules to form an IPF in meat systems. Most of this work has been done with model systems and has relied on the measurement of the so-called 'emulsifying capacity ' using methods devel- oped by several researchers (Hansen 1960; Swift et ul 1961; Hegarty et a1 1963; Saffle and Galbreath 1964). These systems and measurement tools have been refined and improved (Galluzzo and Regenstein 1978a,b; Gaska and Regenstein 1982a,b), and have been used to show that other soluble and insoluble components of the myofibrillar system are also capable of emulsifying fat (Perchonok and Regenstein 1986a,b). Schut and Brouwer (1971) and Schut (1978) have used meat systems which more closely resemble a commercial-type meat batter; however, they also measured the functionality of the proteins extracted using a modified emulsifying capacity test. The work of Gillett et a1 (1977) has shown that emulsifying capacity tests have several limitations in- cluding the dependence of the values obtained on the test parameters used and the non-linear relationship between emulsifying capacity and soluble protein content. Conse- quently, caution must be exercised when extrapolating the results obtained from model systems to the much more complex environment which exists in meat batters.

There are few reports on the type and amounts of proteins in the aqueous phase or in the interfacial film of commercial-type meat batters. Most of these studies have concentrated on myosin and actomyosin and have ignored the potentially important roles of other minor proteins. Furthermore, many of these studies have employed sodium dodecyl sulphate (SDS) gel electro- phoresis which disrupts native protein structure. Hence, the actual natural distribution of native proteins in these meat protein extracts is unknown. This study therefore had three main objectives: ( I ) to develop a

A Gordon, S Burbut

rapid method for the extraction, quantification and visualisation of native myofibrillar proteins; (2) to investigate the effects of five chloride salts on the quantity and type of proteins extracted from commercial-type meat batters; and (3) to determine the type and amount of proteins involved in IPF formation and to examine relationships between IPF proteins, aqueous phase proteins and batter stability.

MATERIALS AND METHODS

Product manufacture

Six comminuted chicken breast meat mixes (no fat added) and six batters containing chicken breast meat and pork fat were prepared with equal ionic strengths (IS) of NaCl (25 g kg-l), MgCl,, CaCl,, KCI and LiCl (IS = 0.43) and a reduced level of NaCl (I5 g kg-', IS = 0.26). The 15 g kg-' NaCl was used to provide a batter with borderline stability (Whiting 1984). Briefly, the breast meat was obtained (fresh) within 12 h post- slaughter from a local processor, hand deboned, trimmed to remove excess fat and connective tissue, ground successively through 9-mm and 3-mm plates and frozen at - 18°C for up to 1 month prior to use. The pork back fat was ground while still semi-frozen through a 9-mm plate and refrozen. Proximate analysis (AOAC 1980) of the meat and fat was determined in duplicate.

The meat batters (1-kg batches) were prepared by chopping the lean meat (690 g kg-' final mix) and the appropriate amount of salt (Table I ) at the high speed setting in a bowl chopper (Schneidmeister SMK40, Berlin, Germany) for 1.5 min after which the water (60 g kg-I), and then the fat (250 g kg-') were added. Total chopping time was 5 min. The fat content of the batters was based on the total weight of the meat block. The comminuted breast meat mixes were prepared in a similar manner except that no fat was added. All 12 treatments (meat batters and lean meat mixes) were made from the same meat source which was a composite of the breast meat from at least 30 birds. The chloride salts (Fisher, Ottawa, Ontario) varied among treatments (Table I). Air bubbles trapped during comminution were removed by tumbling under vacuum (68 kPa abs (0.15 atm) for 30 s) in a table-top vacuum tumbler (Lyco, Columbus, WI, USA). This improved the quality of sections used for image analysis by minimising the number of artefactual air spaces in the batters. The pH of all treatments was determined in duplicate (Chemcadet 5598, Cole Palmer, Chicago, IL, USA).

Protein extraction and protein concentration determination

A simple, rapid method for extracting and quantifying proteins from commercially prepared meat products was

E f e c t of chlorides on meat batter proteins 229

TABLE 1 Proteins in extracts (mg ml-') from meat by six chloride treatments

Treatment* Total protein Protein content of components?

157 kDa$ 240 kDa 260 kDa 300 kDa 425 kDa 520 kDa

15 g kg-' NaCl 13.52d 0.55d 2.1 9d 1.67' 0.53' 7.05' 2.03c 25 g kg-' NaCl 26.79" 0.86' 531a 1.83' 3.29" 9.05b 6.45" 13.5 g kg-' MgC1, 10.35' 0.86" 1.94" 1.86' 1.17' 4.52d 0.00' 15.8 g kg-' CaCl, 455f 0.00' 1.74l 1 .42f 1.41" 0.00" 0.OOd 31.9 g kg-' KCl 19.3OC 1.76b 2.35' 1.57e 1.39d 6.94" 5.29' 18.1 g kg" LiCl 25.78' 2.64" 2.3 1' 2.16" 2.19b 9.92" 6.57"

Means in the same column with different superscripts are significantly different (P < 0.05). * Ionic strength (IS) of 15 g litre-' NaCl = 0.26; all others = 0.43. t 157 kDa protein-M-protein (myosmesin) ; 240 kDa protein-synemin?; 260 kDa protein-unidentified ; 300 kDa protein-Z- protein (Z-nin) ; 425 kDa protein-myosin ; 520 kDa protein-actomyosin. § kDa = kilodaltons.

Protein extractwe procedure RAW BATTER + APPROPRIATE SALT SOLN

I !o~o~enise in \torndcher

SLURRY Centrifuge (30oMxg: 20 rnin)

+ 4 + CREAM LAYER SALINE PHASE RESIDUE

(30000xg; 20 min)

Hornogenise in stomacher (30 sl

RESIDUE I

t 'FRACTION A' PELLET

I Dilute x 18

2 Centnfuge (30000xg, 1.5 min)

t 'FRACTION B' PELLET

SALINE PHASE Resuspend in 0 6 M NaCI. 10 mM pho*phate buffer, pH 7 2

+ SUPERNATE

Resuspend in

I I 'FRACTION A' SOLN I 'FRACTION B' SOLN

1 AS FOR RAW BATTER

DISCARD

Fig. 1

developed. This was based on the method of Schut (1978) and was designed to produce more refined protein fractions with higher concentrations of the major myofibrillar proteins. The protocol used is outlined in Fig 1. Batters or comminuted meat mixes were hom- ogenised (Stomacher Lab Blender 400 UL, London) for 30 s with isoionic strength solutions of the salt with

which they were made and the resulting slurry was centrifuged at 30000 x g for 20 min (SS30, Sorvall RC2B). The slurry separated into three layers: a cream phase containing fat and fat-bound proteins, an aqueous (saline) phase and a residue. The saline phase was removed, clarified by centrifugation at 600 x g for 5 min, diluted with 12 volumes of water to IS of 0.04 to precipitate the fraction soluble at high IS (Galluzzo and Regenstein 1978a) and centrifuged at 30000 x g for 10 min. The pellet (fraction A, Fig 1) was resuspended in 0.6 M NaCl, 10 mM phosphate buffer (pH 7.2). The supernate was diluted with 18 volumes of water (Gal- luzzo and Regenstein 1978b) and centrifuged (30000 x g , 15 min), and the protein pellet soluble at low IS (fraction B) was resuspended in the buffer. The buffered protein solutions were stored at room temperature (22°C) and used for protein content determination and gel electrophoresis within 4 h. The residue from the initial extraction step for each treatment was re-extracted as outlined above (Fig 1) and the protein contents of the fractions soluble at high and low IS were added to those of the meat mix or batter from which the residue was originally obtained to give the total extractable protein (TEP). Hence, for the meat batter,

TEP in fraction A from batter

= protein in fraction A from batter

+protein in fraction A from batter residue (1)

and :

TEP (batter) = TEP batter (fraction A)

+TEP batter (fraction B) (2)

The TEP for the comminuted meat mixes were de- termined in a similar manner. The amount of protein in the IPF was determined by:

Protein in IPF = TEP (meat)-TEP (batter) ( 3 )

17-2

230 A Gordon, S Barbut

The total protein content of the extracts was de- termined using the BioRad protein assay (BioRad Laboratories 1977) which is based on the Coomassie Blue dye binding method (Peterson 1983). A 0.1-ml aliquot of each protein solution was added to 5 ml of dye reagent and mixed by gentle inversions, and the absorbance after 5 min was determined at 595 nm using a Shimadzu Recording Spectrophotometer (Shimadzu Co, Tokyo, Japan). The protein content of each solution was calculated from a standard curve constructed using bovine serum albumin (BioRad Laboratories, Rich- mond, CA, USA).

Native gel electrophoresis and densitometry

Native gel electrophoresis was done using the Phast System (Pharmacia LKB, Uppsala, Sweden). Samples (% 5 pg) were loaded on to gradient gels (10-15 YO) and run through the following programmed series of steps: ( I ) 400V, 10.0mA, 10Vh; (2) 400V, 1.0mA, 2 Vh; (3) 400 V, 10.0 mA. 278 Vh. The temperature of the electrophoresis chamber was kept at 15°C throughout the run, which took approximately 60 min. The sample buffer was the same as that used in the protein extraction procedure (0.6 hi NaCI, 10 mM phosphate buffer, pH 7.2). The buffer system in the gels was 0.1 12 M acetate, 0.1 12 M Tris, pH 6.4. Gels were stained with Coomassie Blue R-250 in 300 ml litre-' methano1/100 ml litre-' acetic acid, destained with the same methanol acetic acid mixture and preserved with a solution of 50 ml litre-' glycero1/100 ml litre-' acetic acid. The molecular weights of the protein bands were determined from a regression plot of the relative mobilities (R,) of the standard proteins against their log,, molecular weights. Each gel was scanned three times at 612 nm using a Zeineh Soft Laser Scanning Densitometer (Biomed Instruments Inc, Richmond, CA, USA) and peak areas were integrated with the Zeineh Videophoresis/ERIS program. This gave the content of each individual protein in each of the extracts.

Light microscopy

Specimens for light microscopy (LM) were processed as described by Gordon and Barbut (1 990b) for TEM but instead of ultrathin sections, 1-pm sections were cut on a Richert OMU3 microtome (Richert, Sidney, Australia). These were transferred to a 10 g kg-' toluidine blue in borax solution and stained by heating for approximately 60 s. Sections were then rinsed with three changes of double distilled water (DDH,O), transferred to a slide, heat fixed and viewed by LM. A Zidas computerised image analysis system (Carl Zeiss, Jena, Germany) attached to a Zeiss microscope was used to measure and record the area of fat globules in each treatment at x 400 magnification as described by Barbut (1989). The perimeter, minimum and maximum diameter and shape

factor of the globules were also determined. The shape factor indicates the degree of roundness of particles (that is, a circle has a shape factor of 1.0, and 0.0 represents a straight line and 0.5 an ellipse) and was calculated by the system based on the formula:

4nA Shape factor = - U2 (4)

where A = area, and u = circumference (Koolmes et a1 1989).

Experimental design and statistical analysis

The experiment was designed as a 2 x 6 factorial split plot with the meat as the main plot, protein source (comminuted meat mixes or meat batters) as the subplot factor and chloride salt as the second factor. There were two replicate experiments. The general linear models (GLM) procedure (Statistical Analysis System 1985, SAS Institute, Cary, NC, USA) was used to compute the analysis of variance and determine the significance of the main effects and their interactions. Means were com- pared using Tukey's test. The contents of individual proteins within each salt extract were compared across all treatments using the GLM procedure. Pearson's correlation coefficients were determined for all variables.

RESULTS AND DISCUSSION

The rapid protein extraction/analysis procedure

Most of the protein extraction procedures currently in use are very time consuming and are orientated towards producing 'pure' protein fractions with well defined components. These protocols are therefore not conducive to routine use for direct analysis of protein functionality in complex systems such as meat batters. Schut and Brouwer (1971) described an extraction procedure which approximated commercial conditions. However, the salt was added in the form of a solution in the initial stages of chopping, and consequently the NaCl concentration in the aqueous phase of the meat (ie the brine concentration) would be less than if salt had been added in its powder form (as is done in normal commercial operations). Salt concentration can adversely affect the types and ratios of proteins extracted (see differences between 15 and 25 g kg-' NaCI, Table 1). The procedure developed and used in this study (Fig 1) was based on that of Schut and Brouwer (1971) but the preparation of the meat batter simulated commercial practice; granular salt was added at the initial stages of com- minution. The salt soluble proteins were recovered by homogenisation of the raw batter, resulting in extracts with soluble protein profiles representative of those in the aqueous phase of the batters. In addition, the inclusion of comminuted meat (chopped without fat

E f e c t of chlorides on meat batter proteins 23 1

added) in the study allowed direct determination of actual soluble protein uptake by the fat during IPF formation in these commercial type meat batters.

The protocol began with a fractionation of the initial saline extract into fractions soluble in high and low IS. This was based on the procedures of Galluzzo and Regenstein (1978a,c) and was intended to concentrate actomyosin and myosin into fractions soluble in high IS (fraction A) and low IS (fraction B), respectively (Fig 1). The concentrations of these two proteins in the respective fractions were relatively high, but each fraction also contained several other proteins and therefore could not be taken as representative of these two proteins only. Consequently, the protein content of each individual component was combined across fractions to give the TEP for each meat source (comminuted meat or meat batter). The Coomassie Blue dye binding method (Peterson 1983) was used for protein content deter- mination since it is very rapid, requires only one reagent and compares favourably in terms of accuracy with the more laborious Lowrey assay (BioRad Laboratories 1977). Speed was desirable since we found that the extracts showed spontaneous precipitation if left to stand for more than 5 h at 22°C (room temperature). This precipitation was increased by refrigeration. Other workers have found that aggregation of myosin occurs fairly rapidly at 4°C depending on environmental conditions (Ishioroshi et a1 198 1 ; Nakayama et al 1983 ; Hermansson et al 1986). In addition, the extracts also contained several other proteins (Table 1) which were predisposed to aggregation. As a result, the extracts were kept at 22°C and used for gel electrophoresis and protein concentration determination within 4 h.

SDS gel electrophoresis would not be overly affected by the protein aggregation at 4°C because SDS disrupts protein structure and separates subunits which are not covalently linked (Greaser e t a1 1983). SDS solubilises all of the myofibrillar proteins and allows them to be

separated by gel electrophoresis (Sender 1971). However. myofibrillar systems contain numerous proteins, many of which are composed of multiple subunits (Asghar et al 1985). As a result of this, SDS gel electrophoresis of myofibrillar proteins often produces unidentified protein bands which increase in number with the increasing complexity of the protein extract being evaluated. In addition, the weights of the subunits of many myofibrillar proteins are often quite close (Asghar et al1985) and this adds to the difficulty in interpreting SDS electro- phoretograms of complex systems, especially with respect to the minor myofibrillar proteins. Native gel elec- trophoresis, although more difficult to run on most systems, gives a better idea of the exact, unaltered components of myofibrillar systems. This is particularly true of more complex mixtures such as those in this study.

Influence of monovalent chloride salts on protein distribution

The monovalent chloride salts all extracted six major proteins with molecular weights of 520, 425, 300, 260, 240 and 157 kDa from meat (Table 1). Myosin (425 kDa) was the major protein extracted from meat by the monovalent salts and was also the major constituent of the aqueous phase of meat batters (Table 2). Identi- fication of this protein was based on several studies which have reported molecular weights quite close to 425 kDa for myosin (Mihalyi and Szent-Gyorgyi 1953; Lowey e t aZl969; Kuehl and Adelstein 1969; Laki 1971). The 520 kDa band was assigned to actomyosin because of its abundance, its presence in the IPF (Table 3) as reported by Schut and Brouwer (1971) and Schut (1978), and its relative presence in comparison with myosin. This protein was concentrated in fraction A where high levels of actomyosin would be expected. In addition, work currently under way using high pressure liquid chromato-

TABLE 2 Protein content of the aqueous phase (mg m1-l) of meat batters prepared with different chloride salts

Treatment Total Protein content of components* protein

140 kDat 157 kDa 240 kDa 260 kDa 300 kDa 425 kDa 520 kDa

15 g kg-' NaCl 6.46' 1.00'' 0.51' 0.53' 069' 0.33' 2.57"' 0.75' 25 g kg-1 NaCl 10.31" 0.89'' 0.85' 0.64' 1.82" 051' 3.44" 1.56" 13.5 g kg-' MgCI, 4.83' 0.81' 0.67' 0.67' 0.58' 0.45' 1.55' 0.00' 15.8 g kg-' CaCl, 3.77' 0.84' 0.00" ND 1.42' 0.92" 0.00" 0.00' 31.9 g kg-' KCl 11.12" 1.15' 1.74" 0.69' 1.25' 0.79' 3.68" 1.77" 18.1 g kg-l LiCl 11.08" 1.59" 1.90" 1.31" 1.31b 1.11" 3.07" 0.75'

n-d Means in the same column with different superscript are significantly different ( P < 0.05). ND Not determined. * 140 kDa protein-C-protein; 157 kDa protein-M-protein (myosmesin) ; 240 kDa protein-synemin?; 260 kDa protein- unidentified ; 300 kDa protein-Z-protein (Z-nin) ; 425 kDa protein-myosin ; 520 kDa protein-actomyosin.

232 A Gordon, S Barbut

TABLE 3 Protein content of the interfacial protein film (mg ml-l) in meat batter made with different chloride salts

Treatment Total protein Protein content of components*

157kDa 240 kDa 260 kDa 300 kDa 425 kDa 520 kDa

15 g kg-' NaCl 8.08bc 0.04' 1.66' 0.42'' 0.19" 4.4O' 1.28" 25 g kg-' NaCl 17.97" O.Olb 4.67" 0.0 1 2.78" 5.61"' 4.89" 13.5 g kg-' MgCl, 5.5' 0.19' 1.26' 1.27" 0.7Td 2.06' 0 O O d 15.8 g kg-' CaCI, 0*49d O.OOb ND 0.00" 0.49de O.OOd O.OOd 31.5 g kg-' KCI 9.39' 0.03' 1.66' 0.32de 0.60' 3.26" 3.52' 18.1 g kg-' LiCl 1634" 0.74" 1 .ow 0.85' 1.08' 6.85" 5.82"

a-e Means in the same column with different superscripts are significantly different (P < 0.05). * 157 kDa protein-M-protein (myosmesin) ; 240 kDa protein-synemin? ; 260 kDa protein-unidentified ; 300 kDa protein-Z- protein (Z-nin) ; 425 kDa protein-myosin ; 520 kDa protein-actomyosin.

graphy (HPLC) has confirmed that this protein is actomyosin and has a molecular weight of 520 kDa (unpublished data). The 300-kDa protein was identified as Z-protein (Z-nin) which plays a structural role in the Z-disc of the Z-line of the thin filaments and has been reported to have a molecular weight in this range (Asghar et a1 1985). The 260 kDa protein was not identified but the 240 kDa protein could possibly be synemin which has a molecular weight in this range (Asghar et a1 1985). Synemin is located on the periphery of the Z-line and is associated with desmin or vimentin. It has been classified as a scaffolding protein but may have a regulatory function. The 157 kDa band was attributed to M-protein (myomesin). Ebashi and Non- omura (1973) reported that myomesin had a molecular weight of 155 kDa. Myomesin is thought to function in binding the myosin filaments together and is a common constituent of myofibrillar protein extracts (Asghar er a1 1985).

An interesting observation was that C-protein (mol- ecular weight 140 kDa) was not extracted from meat but was present in the extracts from the meat batters (Tables 1 and 2). C-protein is an integral part of the thick filaments in myofibrils (Starr and Offer 1978) and affects myosin filament configuration as well as actin-myosin binding (Moos 1981 ; Yamamoto et a1 1987). It may be that C-protein was an integral part of the matrix formed when raw meat was comminuted and was therefore not extracted into the aqueous phase. However, when fat was comminuted with meat to make a meat batter, C-protein was extracted into the aqueous phase of the batters (Table2). This could be due to a disruption of its relationship to actin and myosin by the lipid-protein interactions which took place during fat binding and IPF formation.

The monovalent chloride treatments differed from each other in the amount of total protein extracted from the meat (Table 1). LiCl and 25 g kg-' NaCl extracted

significantly more total protein from comminuted lean meat than KCl ( P < 0.01). These three isoionic strength treatments extracted more soluble protein from meat batter than the reduced NaCl treatment (Table 2) but did not differ significantly among themselves in this respect ( P > 0.05). This supports the argument of Regenstein (1988) that a relatively constant amount of soluble protein is left in the aqueous phase of batters after IPF formation. There were three major soluble proteins involved in IPF formation (Table 3). In order of importance these were myosin, actomyosin and the 240 kDa protein. Most of the other proteins were only present in the IPF in relatively small amounts although their exact levels varied among treatments. Only in the 25 g kg-' NaCl and LiCl treatment was another protein, Z-nin, a major contributor. These results show that both myosin and actomyosin play a major role in IPF formation in commercial type meat batters. Several studies have indicated that myosin was the major component of the IPF (Hansen 1960; Swift et a1 1961; Galluzzo and Regenstein 1978b), and others have shown that actomyosin is capable of forming an IPF in model systems (Schut and Brouwer 1971 ; Schut 1978; Galluzzo and Regenstein 1978~). However, the role of minor proteins such as Z-nin has not been investigated.

Both 2.5 YO NaCl and LiCl extracted significantly more actomyosin and myosin from lean meat ( P < 001) than KC1, and 25g kg-l NaCl extracted much more of the 240 kDa protein than the other monovalent chloride salts (Table 1). The relative levels of these three proteins in the IPF of batters made with LiCl, 25 g kg-' NaCl and KCl followed a similar pattern (Table 3). The differences induced by the various monovalent salts in the protein profiles in both the aqueous phase of batters and in the IPF strongly support earlier suggestions that differences in stability, texture and microstructure may be due to differences in protein extraction during comminution (Gordon and Barbut 1989, 1990~). It

Effect of chlorides on meat batter proteins 233

should be noted that differences in proteins extracted into the aqueous phase of meat systems, arising from the treatment used in their preparation, reflect differences in the protein composition of the matrix, since all treatments initially contained the same protein pool. It is the proteins of the matrix which gel during cooking and are mainly responsible for the final texture and WHC of finely comminuted meat products (Acton and Dick 1984; Lee 1985; Foegeding 1988). However, interaction be- tween matrix proteins, the proteins in the aqueous phase and the proteins of the IPF prior to and during cooking also affect product microstructure and texture (Schut 1978; Gordon and Barbut 1990b,c).

The differences between 25 g kg-' NaCl and LiCl in proteins extracted into the aqueous phase of meat batters and the amount of the 240 kDa protein in the IPF may account for the microstructural and textural differences reported in earlier studies (Barbut and Mittal 1988; Gordon and Barbut 1989,1990a). The smaller amount of total protein extracted by KCl and the many differences between this salt and 25 g kg-' NaCl in protein profiles induced in the aqueous phase, as well as in the IPF, appear to be the source of differences in terms of the characteristics of the products which each of these salts produces (Gordon and Barbut 1989 ; Gordon and Barbut 1990a). This has implications for the use of KCl to replace NaCl in comminuted meat products. The texture of these products may better approach that of 'normal' commercial products containing 25 g kg-' NaCl if the formulation can be manipulated to produce protein profiles similar to that resulting from 25 g kg-' NaCl in the aqueous phase, the IPF and hence the protein matrix.

The reduction of the NaCl level by 10 g kg-' resulted in an approximately 50% reduction in the TEP from meat and in the proteins forming the IPF (Tables 1 and 3). This was mainly due to significant reductions (P < 0.01) in the amounts of actomyosin, Z-nin and the 240 kDa protein extracted from meat. Several studies have shown that the reduction of NaCl levels from 25 to 15 g kg-l results in a change from a stable meat batter with desirable texture to a marginally stable batter with unacceptable texture (Whiting 1984, 1987; Gordon and Barbut 1989). It therefore appears that these three proteins play important roles in the stability and texture of normal commercial-type products. An interesting observation was that two of these proteins (Z-nin and the 240 kDa protein) were present in much larger amounts (P < 0.01) in the 25 g kg-' NaCl meat extract than any other (Table 1). This strongly implies that they may play a key role in giving the 'normal' commercial batter its unique texture. Their role in this respect merits further investigation.

The 15 g kg-' NaCl and KCl treatments produced similar protein profiles in the IPF. Both treatments resulted in an IPF with the same amounts (P > 0.05) of total proteins, myomesin and myosin (Table 3). All or some of the minor proteins in the IPF may possibly

influence IPF-mediated fat binding by the matrix during comminution (Gordon and Barbut 1990a) or cooking and thereby affect fat stabilisation. If this is so, then the similar levels of minor proteins in the IPF of the 15 g kg-' NaCl and KCl meat batters may explain some of the microstructural similarities reported between these treatments in raw (Gordon and Barbut 1990a) and cooked meat batters (Gordon and Barbut 1989). C- protein has been shown to affect the diameter and length of synthetic myosin filaments (Koretz 1982; Yamamoto et aZl987) and may affect actin-myosin binding (Krakol 1975; Moos 1981). Consequently, it may be that C- protein in the aqueous phase of batters could influence myosin-myosin or myosin-actin interaction during cook- ing and therefore affect the final texture of the product. Since the levels of C-protein in the aqueous phases of KCl- and 15 g kg-' NaCl-treated meat batters were the same (Table 2), any effects which it might have on texture would be similar for these two treatments.

Analysis of raw batter microstructure

The shape, size and organisation of fat particles within the meat batters produced by each chloride salt were examined with the aid of a digitising system (Table 4). The particle size distribution was estimated from the ratio of the minimum diameter (min dia) to the maximum diameter (max dia) of fat particles; the larger the ratio, the more even the particle size distribution should be. Barbut (1989) reported that the mean fat particle area (FPA) of a meat batter could be related to its stability. Koolmes et a1 (1989) indicated that fat particles with shape factors of 0.45 or greater were stable globules or fat cells while shape factors below 0.45 represented coalescence or unstable fat. Using these criteria, the chloride salt treatments were found to display a pattern of raw batter stability similar to that found previously (Gordon and Barbut 1989).

The LiCl and 25 g kg-' NaCl treatments were the most stable and were similar in their FPA (Table 4). These treatments were also similar in all other mor- phological characteristics investigated. The 15 g kg-' NaCl and KCl batters showed similarities in fat particle shape and other microstructural characteristics. The CaCl, raw batter was stable as indicated by its relatively small FPA (compared with most monovalent salt treatments) and high shape factor. In contrast, MgC1, produced a batter with a large FPA and a shape factor (< 0.45) which was much lower than all other treatments indicating extreme instability (Table 4). The FPA of the MgC1, batter, although high, was not significantly different (P > 0.05) from that of the 15 g kg-' NaCl treatment which was marginally stable. However, both perimeter and shape factor indicated clear differences between the MgCl, batter and all other treatments. This is because the FPA does not discriminate between large particles and coalesced fat, while both the perimeter and

234

TABLE 4 Morphological characteristics of fat particles as affected by chloride salts

A Gordon, S Barbut

Treatment Morphological characteristics of fat*

Mean fat Perimeter Shape Minimum Maximum Min dial particle area factor diameter diameter Max dia

15 g kg-' NaCl 248.79ab 55.86" 071*' 11.6Yb 3629ab 0.45" 25 g kg-' NaCl 55.21' 25.10" 0.81" 4.94* 10.29' 0.56"* 13.5 g kg-' MgCI, 356.76" 1 13.6 1 * 0.39d 15.59" 40.39" 0.42c 15.8 g kg-' CaCI, 1 74.49b' 44.51" 0.71* 8.60"* 23.39"*" 0.26'' 3 1.9 g kg-' KCI 1 55.96*" 51.83" 0.63" 10.64ab 30.30ab 0.48"*' 18.1 g kg-' LiCl 97.5 3 cd 41.40" 0.80" 6.37* 1 6 3 P 0.56"

a-d Means in each column with the same letter are not significantly different. * Area is in pm2; perimeter and diameter are in pm.

shape of fat 'particles' are clearly affected by the stability of the fat within the matrix. Hence, it appears that measurement of the mean perimeter and shape factor of particles may be more meaningful in relation to batter stability than is the FPA. Koolmes et aZ(1989) have also found that the shape factor is closely related to the morphology of the fat within the matrix and hence batter stability.

These results indicate that the raw batters obtained with monovalent chlorides were stable whereas that with MgCl, was not. These findings are consistent with the data presented in Tables 1 and 3 and the results of a previous study (Gordon and Barbut 1989). For the CaCl, batter, the poor fat particle size distribution indicates that a large number of these particles were either whole fat cells or cell clumps (see min dia/max dia; Table 4). This was also found in an earlier study (Gordon and Barbut 1990a). However, the microstructural results for CaC1, indicate that it produces a stable raw batter. This appears to conflict with the findings based on protein extraction if protein solubility is a prerequisite for raw batter stability.

Influence of divalent salts on protein distribution and microstructure

Both divalent salts extracted significantly less total protein from meat (P < 0.01) than the monovalent salts. However, MgCl, extracted much more total protein than CaCI, (Table 1). The major protein extracted by MgCl, was myosin, which was not extracted by CaCl,. In the latter treatment, very little soluble protein went into IPF formation when compared with the monovalent salts and MgC1, (Table3). However, the total amount of soluble protein used for IPF formation in the MgC1, batter was much less than the amount of soluble protein in the IPF of monovalent chloride salt batters (P < 0.01). Both divalent salts produced different distributions of

proteins between the aqueous phase, the IPF and therefore the matrix in raw batters.

Gordon and Barbut (1 989) showed that differences exist between MgCI, and CaCl, in terms of raw batter stability and the microstructure and texture of cooked batters although both produced unstable batters after cooking. Subsequently it was found that several micro- structural differences existed between raw MgCl, batters, in which widespread fat instability was evident, and CaC1, raw batters, which were relatively stable (Gordon and Barbut 1990a). The results of the quantification of the microstructural features of these treatments (Table 4) concur with our earlier findings. The difference between these treatments in the quantity and type of protein extracted from meat and forming the IPF gives further credibility to the hypothesis that CaCI, and MgCl, act by different mechanisms in destabilising meat batters. The MgC1, produced an IPF containing 2.97 mg ml-' of myosin and extracted 4.5 mg ml-l of myosin from meat but still caused raw batter instability. CaCl, extracted no myosin and < 4.6 mg ml-l of total protein from lean meat but produced a stable raw batter (Tables 1, 3 and 4). Using a timed emulsification procedure, Huber and Regenstein (1988) argued that extraction of 2.75 mg ml-' of myosin was enough to stabilise raw batters. However, the results obtained in this study suggest that this figure may not be valid in cases where divalent cations are present.

The protein profiles of the extracts made from meat using divalent salts showed that a major difference between the MgC1, and CaCI, was the retention of the majority of the protein in the matrix of the CaCl, batter. This included all of the myosin and actomyosin; none of these were extracted (Table 1). This probably accounts for the very dense gel matrix which CaCl, was shown to produce in raw batters (Gordon and Barbut 1990a). The inability of CaCl, to extract substantial amounts of myofibrillar proteins from meat may be due to its charge density or specific ion effects (Von Hippel and Wong

Efect of chlorides on meat batter proteins 235

1962) which might, instead, favour orderly gelation of the insoluble proteins in the raw batter. These proteins are often highly hydrated in salt-treated meat (Schut 1978) and are capable of forming a stable IPF (Schut and Brouwer 1971 ; Perchonok and Regenstein 1986a,b). Consequently, since the great majority of the insoluble proteins in CaCl, meat batter were probably in a swollen, hydrated state, it is likely that some were adsorbed to form an IPF. The relatively small FPA of CaCl, compared with MgCl,, and the high shape factor of the fat particles in this batter (Table 4), tend to support this scenario. The adsorbed protein would be mainly, but not exclusively, actomyosin (Schut 1978). It has recently been shown that the addition of CaCl, produced a coherent IPF around many fat globules in raw meat batters (Gordon and Barbut 199 1). Magnesium chloride extracted some proteins from meat but not enough to form stable protein films around all of the fat globules. This is manifested by the large amount of unstable fat which resulted in a high FPA, large perimeter and very limited organisation within the matrix of MgCl, raw batters (Table 4). The fat instability appeared to be a result of insufficient IPF formation combined with extensive pre-cooking protein matrix aggregation which resulted in widespread fat channelling in the raw batter. Magnesium ions have been shown to affect protein conformation in a way which favours structure for- mation (Szilagyi et a1 1975) and can bind to several sites within the intact myofibril. The extensive protein-protein aggregation which existed in the MgCl, batters could therefore be a result of these properties. A possible mechanism by which this occurs has been discussed previously (Gordon and Barbut 1990a). However, it is apparent that, regardless of the route by which de- stabilisation occurs, MgCl, produces the conditions conducive to extensive fat and water loss prior to cooking.

Relationship between extracted proteins and microstructural features

The interrelationships between proteins extracted from meat and those remaining in the aqueous phase of the batter or used in IPF formation were investigated (Tables 5 and 6). High correlations (r 3 0.90) were found between the total extracted proteins (TEP) from meat and the TEP from batters, and between myomesin from meat and C-protein, myomesin, the 240 kDa protein and Z-nin from batters (P < 0.01). The actomyosin extracted from meat was also highly correlated with the TEP from the batters (Table 5). These results suggest that the common practice of using the TEP from meat as an indicator of the likely protein content of the aqueous phase of meat batters (Asghar et a1 1985) is valid. Furthermore, they suggest that the amount of myomesin extracted from meat is a reliable indicator of the presence of most of the minor proteins in the aqueous phase of the batters. Myosin extracted from meat was less strongly related to the TEP of meat batters but still showed moderate correlation (r = 0.84, P < 0.01). The proteins extracted from meat and the proteins in the IPF showed very strong relationships for certain key components (Table 6). The TEP from meat was highly correlated with total IPF proteins (r = 0.97) and was a very good indicator of the presence of actomyosin in the IPF (r = 0.96). The 240 kDa protein, Z-nin, myosin and actomyosin extracted from meat were also found to be very good indicators of their presence in the IPF (Table 6). It was very interesting to note that, in contrast to the aqueous phase proteins, the myosin extracted from meat was more highly correlated with the total IPF proteins than was actomyosin. This reflects the pre- dominance of myosin in the IPF (Table 3) and is possibly a function of its preferential adsorbance by fat for IPF formation (Schut 1978). Another interesting observation

TABLE 5 Correlation coefficients for proteins from the aqueous phase of meat batters and comminuted meat

Proteins from Total Batter aqueous phase proteins Comminuted meat protein

140 kDa" 157 kDa 240 kDa 260 kDa 300 kDa 425 kDa 520 kDa ~~

Total protein 0.918** 0.746** 0.744** 0.725** 0.869** 0.578* 0.849** 0.707* 157 kDa 0.792** 0.933** 0960** 0.927** NS 0.911** 0.645* NS 240 kDa NS NS NS NS 0.830** NS* NS 0582* 260 kDa NS NS NS 0.763** NS 0.687* NS NS 300 kDa NS NS NS NS 0.889** NS NS NS 425 kDa 0.840** 0.842** 0.735** 0.776** 0.655* NS 0885** 0630* 520 kDa 0.962** 0.729** 0.780** 0.662* 0.895** 0.658* 0.832** 0775**

* Significant ( P < 0.05); ** significant ( P < 0.01). NS = Not significant. a 157 kDa protein-M-protein (myosmesin) ; 240 kDa protein-synemin?; 260 kDa protein-unidentified ; 300 kDa protein-Z- protein (Z-nin); 425 kDa protein-myosin ; 520 kDa protein-actomyosin.

236 A Gordon, S Barbut

TABLE 6 Correlation coefficients for proteins extracted from meat and the proteins in the IPF

Proteins extracted Total Proteins of the interfacial protein film (IPF) from meat

157 kDa 240 kDa 260 kDa 300 kDa 425 kDa 520 kDa

Total proteins 0.974** NS NS NS NS 0881** 0.959** 157 kDa 0.626* 0.738** NS NS NS 0.696* 0.773** 240 kDa 0.742** NS 0,962** -0'703* 0.913** NS NS 260 kDa 0.617* 0690* NS NS NS NS 0.600* 300 kDa 0.757** NS 0.738** NS 0.954** NS 0.708** 425 kDa 0.9 17** NS NS NS NS 0.960** 0.848** 520 kDa 0.896** NS NS NS NS 0.776** 0.980**

* Significant ( P < 0.05); ** significant ( P < 001). NS = not significant. a 157 kDa protein-M-protein (myosmesin) ; 240 kDa protein-synemin?; 260 kDa protein-unidentified ; 300 kDa protein-Z- protein (Z-nin) ; 425 kDa protein-myosin ; 520 kDa protein-actomyosin.

was that the 240 kDa protein from meat extracts was highly correlated ( r = 0.95) to the Z-nin in these extracts (data not shown). This appears to indicate that these proteins are closely related within the myofibrillar structure. This would be the case if the 240 kDa protein was synemin, as already suggested in this paper, since both synemin and Z-nin are located on or around the Z- disc (Asghar et a1 1985).

Investigation of the influence of the proteins in the IPF, in the batter aqueous phase or extracted from meat on morphological features of the raw batters did not reveal any strong relationships (data not shown). However, particle size distribution (min dia/max dia) and the FPA showed a moderate correlation with the levels of actomyosin in meat extracts and the IPF ( r 2 0.81 and 0.89 respectively, P c 0.05). The lack of strong relationships may have been due to the anomaly created by the CaCI, batter which did not extract much protein from meat and therefore had very little soluble protein forming an IPF but was still stable (Tables 1, 3 and 4). Gillett et al(l977) showed that protein solubility was an excellent predictor of batter stability. Fur- thermore, it has been shown that cooked CaCl, batters are extremely unstable (Gordon and Barbut 1989). Consequently, our present results suggest caution when using image analysis data to predict the stability of cooked meat batters as these may be affected by insoluble protein components which function in batter stabil- isation prior to cooking but are of less importance during cooking.

the actual soluble protein uptake by fat during IPF formation in commercial-type meat batters. It was found that the monovalent chloride salts (NaCl, KCl, LiCl) consistently extracted six native proteins from lean chicken breast meat. These proteins included actomyosin (520 kDa), myosin (425 kDa) Z-nin (300 kDa), myo- mesin (157 kDa) and a 240 kDa protein. In addition, C- protein (140 kDa) was extracted into the aqueous phase of meat batters (fat added). The monovalent salt treatments differed in the amounts and ratios of proteins extracted from lean meat. The differences between KC1 and 25 g kg-' NaCl suggested that commercial formula- tions containing KCl should be manipulated to produce protein profiles in the different phases similar to those produced by 25g kg-l NaCl and so achieve similar textural properties.

A reduction of 10 g kg-' in the NaCl level resulted in a 50% reduction in the total extracted proteins and in the proteins forming the IPF, mainly as a result of the lower levels of some of the minor proteins. All six proteins extracted from lean meat were involved in IPF formation but in the monovalent salt batters actomyosin, myosin and the 240 kDa protein were the major constituents of the IPF. This provides further evidence that MgCl, and CaCl, act by different mechanisms in destabilising meat batters. The results also indicate that while the extraction of soluble proteins may be an important determinant of final cooked product texture and stability, insoluble proteins can play a role in IPF formation and batter stabilisation in the raw state. Their role in this respect should therefore be further investigated.

CONCLUSIONS REFERENCES

A simple, rapid method for extracting and quantifying proteins from commercially prepared meat products was developed. The method allowed direct determination of

Acton JC, Dick R L 1984 Protein-protein interactions in processed meats. Recip Meat Conf Proc 37 36-43.

EfSect of chlorides on meat batter proteins 231

AOAC 1980 Oficial Methods of Analysis (13th edn). As- sociation of Official Analytical Chemists, Washington, DC.

Asghar A, Samejima K, Yasui T 1985 Functionality of muscle proteins in gelation mechanisms of structured meat products. CRC Crit Rev Food Sci Nutr 22 27-106.

Barbut S 1989 Effect of three chloride salts and chopping time on the microstructure and texture of meat batters. Can Inst Food Sci Technol J 22 284-290.

Barbut S, Mittal G S 1988 Rheological and gelation properties of meat batters prepared with three chloride salts. J Food Sci 53 12961299, 1311.

Barbut S, Maurer A J, Lindsay RC 1988 Effects of partial sodium replacement with other chloride salts on the physical and sensory properties of turkey frankfurters. Can Inst Food Sci Technol J 21 90-96.

Belton P S , Packer K J, Southon T E 1987 35Cl nuclear magnetic resonance studies of the interaction of chloride ions with meat in the presence of tripolyphosphate. J Sci Food Agric 40 267-275.

BioRad Laboratories 1977 BioRad protein assay. Tech Bull 1051. BioRad Laboratories, Richmond, CA.

Cheung H C, Cooke R 1971 Effect of alkali ions on myosin conformation. Biopolymers 10 523-528.

Ebashi S, Nonomura Y 1973 Proteins of the myofibril. In : The Structure and Function of Muscle, Vol3, ed Bourne G H. Academic Press. New York, pp 285-362.

Foegeding A 1988 Gelation in meat batters. Recip Meat Conf Proc 41 44-47.

Galluzzo S J, Regenstein J M 1978a Emulsion capacity and timed emulsification of chicken breast muscle myosin. J Food Sci 43 1757.

Galluzzo S J, Regenstein J M 1978b Role of chicken breast muscle protein in meat emulsion formation : myosin, actin and synthetic actomyosin. J Food Sci 43 1761.

Galluzzo S J, Regenstein J M 1978c Role of chicken breast muscle protein in meat emulsion formation : natural acto- myosin, contracted and uncontracted myofibrils. J Food Sci 43 1766.

Gaska M T, Regenstein J M 1982a Timed emulsification studies with chicken breast muscle ; soluble and insoluble myofibrillar proteins. J Food Sci 47 1438-1443.

Gaska M T, Regenstein J M 1982b Timed emulsification studies with chicken breast muscle : whole muscle, low-salt washed muscle and low-salt soluble proteins. J Food Sci 47

Gillett T A , Meiberg D E, Brown C L, Simon S 1977 Parameters affecting meat protein extraction and interpret- ation of model system data for meat emulsion formation. J Food Sci 42 1606-1610.

Gordon A, Barbut S 1989 The effect of chloride salts on the texture, microstructure and stability of meat batters. Food Microstr 8 271-283.

Gordon A, Barbut S 1990a The microstructure of raw batters prepared with monovalent and divalent chloride salts. Food Structure 9 279-295.

Gordon A, Barbut S 1990b Use of cold stage scanning electron microscopy to study meat batters. J Food Sci 55 1196-1 199.

Gordon A, Barbut S 1990c The role of the interfacial protein film in meat batter stabilization. Food Structure 9 77-85.

Gordon A, Barbut S 1991 Raw meat batter stabilization: the role of the interfacial protein film. Can Inst Food Sci Technol J24 136142.

Greaser M L, Yates L D, Krzywicki K, Roelke D L 1983 Electrophoretic methods for the separation and identification of muscle proteins. Recip Meat Conf Proc 36 87-91.

Hand L W, Terrell R N, Smith G C 1982 Effects of chloride salts on the physical, chemical and sensory properties of frankfurters. J Food Sci 47 1800-1802, 1817.

1460-1464.

Hansen L J 1960 Emulsion formation in finely comminuted sausage. Food Technol 14 565-569.

Hegarty G R, Bratzler L J, Pearson A M 1963 Studies on the emulsifying properties of some intracellular beef muscle proteins. J Food Sci 28 663-668.

Hermansson A M, Harbitz 0, Langton M 1986 Formation of two types of gels from bovine myosin. J Sci Food Agric 37

Huber D G, Regenstein J M 1988 Emulsion stability studies of myosin and exhaustively washed muscle from adult chicken breast muscle. J Food Sci 53 1282-1286, 1293.

Ishioroshi M, Samejima K, Yasui T 1981 Further studies on the roles of the head and tail regions of the myosin molecule in heat-induced gelation. J Food Sci 47 114-120, 124.

Kent S 1981 How dietary salt contributes to hypertension. Geriatric 36 14-18.

Koolmes P A, Moerman P C, Zijderveld M H G 1989 Image analysis of the fat dispersion in a comminuted meat system. Food Microstr 8 81-90.

Koretz J F 1982 Hybridization and reconstitution of thick filament structure. In : Methods in Enzymology, Vol 85B, eds Frederiksen D W & Cunningham L W. Academic Press. New York, pp 20-55.

Krakol I 1974 Some differences in the properties of myosin preparations with and without C-protein. In: Proteins of Contractile Systems. FEBS Ninth Meeting, Budapest, ed Biro E N A. American Elsevier Publishing Co, New York,

Kuehl W M, Adelstein R S 1969 Identification of E-N- monomethyl-lysine and e-N-trimethyl-lysine in rabbit skel- etal muscle. Biochem Biophys Res Commun 37 59-64.

Laki K 1971 Size and shape of the myosin molecule. In: Contractile Proteins and Muscle, ed Laki K. Marcel Dekker, New York, pp 98-1 10.

Lee C M 1985 Microstructure of meat emulsions in relation to fat stabilization. Food Microstr 4 63-72.

Lewis D F, Groves K H M, Holgate J H 1986 Action of polyphosphates in meat products. Food Microstr 5 53.

Lowey S, Slayter H S, Weeds A G, Baker H 1969 Substructure of the myosin molecule. I. Subfragments of myosin by enzymatic degradation. J Mol Biol42 1-29.

Mihalyi E, Szent-Gyorgyi A G 1953 Trypsin digestion of muscle proteins : ultracentrifugal analysis of the process. J Biol Chem 201 189-196.

Moos C 1981 Fluorescence microscope study of the binding of added C-protein to skeletal muscle myofibrils. J Cell Biol90 25.

Nakayama T, Niwa E, Hamada I 1983 New type of myosin gel induced by salts. Agric Biol Chem 47 227-233.

Offer G W, Trinick J 1983 On the mechanism of water holding in meats; the swelling and shrinking of myofibrils. Meat Sci 8 245-281.

Perchonok M H, Regenstein J M 1986a Stability at com- minution chopping temperatures of model chicken breast muscle emulsions. Meat Sci 16 17-29.

Perchonok M H, Regenstein J M 1986b Stability at cooking temperatures of model chicken breast muscle emulsions. Meat Sci 16 31-42.

Peterson G L 1983 Determination of total protein. Methods in Enzymology 91 95.

Regenstein J M 1988 Meat batter: why it is not an emulsion. Recip Meat Conf Proc 41 4-3.

Saffle R L, Galbreath J W 1964 Quantitative determination of salt soluble proteins in various types of meats. Food Technol

Schut J 1978 Basic meat emulsion technology. Meat Industry

Schut J, Brouwer F 1971 Differential adsorption of meat

69-84.

pp 187-192.

18 1943-1944. .

Res Conf Proc 1-15.

238 A Gordon. S Barbut

proteins during emulsification. 17th Europ Meeting Meat Res Workers Proc.

Seman D L, Olson D G, Mandingo R W 1980 Effects of reduction and partial replacement of sodium on bologna characteristics and acceptability. J Food Sci 45 11 1&1121.

Sender P M 1971 Muscle fibrils: solubilization and gel electrophoresis. FEBS Lett 17 106-1 10.

Smith D M 1988 Meat proteins: functional properties in comminuted meat products. Food Technol 42 116-121.

Starr R, Offer G 1978 The interaction of C-protein with heavy meromyosin and subfragment-2. Biochem J 171 81 3-816.

Swift C E, Lockett C, Fryar A J 1961 Comminuted meat emulsions. The capacity of meat for emulsifying fat. Food Technol 15 468-473.

Szilagyi L, Kurrenoy I, B a h t M, Biro E N A 1974 Influence of ions and of ATP on the conformation of HMM-studied by proteolysis. In : Proteins of Contractile Systemp, FEBS Ninth Meeting, Budapest, ed Biro E N A. American Elsevier Publishing Co, New York, pp 47-59,

Terrell R N 1983 Reducing the sodium content of processed meats. Food Technol37 6671.

Von Hippel P H, Wong K Y 1962 Effect of ions on the kinetics of formation and stability of the collagen fold. Biochemistry 1664674.

Weinberg Z G, Regenstein J M, Baker R C 1984 Effects of salts on heat initiated binding and water retention properties of comminuted cod muscle. J Food Biochem 8 215-227.

Whiting R C 1984 Stability and gel strength of frankfurter batters made with reduced sodium chloride. J Food Sci 49

Whiting R C 1987 Influence of various salts and water soluble compounds on the water and fat exudation and gel strength of meat batters. J Food Sci 52 1130-1 158.

Yamamoto K, Samejima K, Yasui T 1987 The structure of myosin filaments and the properties of heat induced gels in the presence and absence of C-protein. Agric Biol Chem 51

1350-1354, 1362.

197-203.