Humectants Improve Myosin Extractability and Water Activity of Raw, Cured Intermediate Moisture Meats
Mich io M u g u r u m a , ~ T e r u h i r o N i s h i m u r a , b R a i z a b u r o Umet su , ¢ Ichi ro G o t o ~ & M a m o r u Y a m a g u c h i ~
Department of Animal Science, Faculty of" Agriculture, Kyushu University, Fukuoka 812, Japan
b Kuju Agricultural Research Center, Kyushu University, Ooita 878-02, Japan Department of Veterinary Anatomy, College of Veterinary Medicine,
The Ohio State University, Columbus, Ohio 43210, USA
(Received 22 January 1987; revised version received 7 April 1987; accepted 22 April 1987)
SUMMAR Y
Glycerol. propylene glycol and sorbitol were incorporated into salt-based intermediate moisture meats manufactured from porcine M. longissimus thoracis and bovine M. biceps femoris by dry curing and air drying at 4°C. Moisture content and water activity (aw) in cured pork were reduced by the addition of propylene glycol and sorbitol. Propylene glycol ,'as more effective than sorbitol in lowering aw. The extractability of myosin heavy chain, used as an index of alteration of myofibrillar protein, decreased in intermediate moisture porcine meats with the addition of salt and was unaffected by sorbitol. However, use of glycerol and propylene glycol in cured and ah'-dried pork increased the extractability of myosin heavy chain. Whereas intact myofibrils could not be extracted from salt-cured, air-dried beef, myofibrils could be made frorn air-dried beef cured in the presence of tO% glycerol, 5% propylene glycol and 4% sorbitol. Such myofibrils contracted immediately on addition of MgZ+-ATP. In addition, even after storage for 5 months, including 30 days at 25°C, myosin heavy chain could be extracted from meat cured with this combination of humectants. In comparison with salt curing alone, curing meat with the above three humectants together, plus salt, results in intermediate moisture meats more like fresh meat.
I N T R O D U C T I O N
In the past two decades much research has focused on the application of intermediate moisture technology to meats (Ledward, 1981). Intermediate
moisture meats (IMM) have relatively long shelf-lives at normal room temperature due to the reduction in a w brought about by the addition of various humectants. The humectants most frequently used in intermediate moisture food are sodium chloride, glycerol, propylene glycol, sucrose, corn syrup, sorbitol and dextrose (Kaplow, 1970; Heidelbaugh & Karel, 1975; Erickson, 1982).
Previous studies (Muguruma et aL, 1986; Muguruma & Nakamura, 1986) showed that curing with sodium chloride (6%) reduced subsequent extractability of muscle structural proteins, especially myosin. However, incorporation of glycerol during curing of 6% salt-based IMM increased the extractability &muscle structural proteins and improved the water-binding capacity. This study was undertaken to determine the effect of humectants, especially propylene glycol (Haas et aL, 1975) and sorbitol (Leung et aL, 1984; Favetto & Chrife, 1985), on properties of glycerol-salt-based IMM. Also, the storage stability of cured and air-dried samples with reduced aw was evaluated. A combination of humectants was found that provided raw, salt-cured IMM which more closely resemble fresh meat in terms of protein extractability and myofibril properties than do meats cured with salt alone. The improved IMM may have improved organoleptic properties when consumed without rehydration (the usual practice) because of their improved structural properties. Acceptable products could be obtained with less curing salt (3"5 versus 6%), an advantage in view of the current interest in restricting dietary sodium intake. Finally, we are interested in the development of IMM that can be rehydrated during manufacture of processed meats (e.g. sausage). In developing these products, we want to begin with IMM that are as similar as possible to fresh meat.
MATERIALS AND METHODS
Materials
Samples were obtained from Landrace, Large White and Duroc mixed breed (LWD) pork (about 100kg live weight) and Japanese Black steers (519-1 + 25-9kg live weight, 23 + 1 months old). All chemicals used were reagent grade.
Preparation of intermediate moisture meats
The longissimus thoracis muscle from pork and bicepsfemoris muscle from steer were cut into 2.5 cm thick chops and cured, starting from about 15 h after slaughter, with 3"5 or 6% NaCI, 1% sucrose, 0-1% sodium ascorbate,
:kD'osin extractability and water activity 181
0-05% potassium nitrate, 0-03% sodium nitrite, 0-25% sodium tripoly- phosphate and 0-05% sodium hexametaphosphate (weight basis). Dry curing was done for 14 days at 4°C. The humectants propylene glycol and sorbitot were tested at concentrations ranging from 2 to 10%. Potassium sorbate (0-2%) was added as an antimycotic when sorbitol was used, as sorbitol plus sorbate were effective in inhibiting microbial growth in semi- moist dog food (Acott et al., 1976). After curing, residual cure mixture was brushed from the surface of each chop. One half of each cured sample was put into a nylon net bag, and then the bags were hung verticaUy to minimize surface contact. Samples were air-dried by an oscillating electric fan for 18-30 days at 4°C.
Chemical analysis
The pH, salt and moisture content of samples were determined by an established method (Koniecko, 1979). For determination of a w, one g of sample was placed in a Conway-type diffusion unit (Shibata Chemical, Japan) containing five different standard saturated solutions (a,,, = 0.980-0-807). Weighed samples were allowed to equilibrate in the incubator at 25°C for 24h. The increase or decrease of sample weight was determined with a precision of 0"1 mg, and aw was calculated by insertion into a standard curve.
Extractability of protein from cured and air-dried meats
Each sample of 1 g wet weight was minced with scissors and homogenized twice for 30 s with a 10-s interval at 0~C in 15 ml of extracting solution. The extracting solutions were as follows: (a) SSS-solution (25 mM KCI, 20 m~vx K phosphate, 2 mM MgC12, 2 mM EGTA, 1 mM NaN> pH 6-9) (b) GS solution (0"3M KC1, 0-15M K phosphate, pH 6-4) (c) GS-ATP solution (0"3M KC1, 0-15M K phosphate, 1 mM ATP, pH 6-4) (d) HS-solution (0-6M KC1, 0"IM K phosphate, 10mM sodium pyrophosphate, l mN MgC12, pH 6-4) (e) KI solution (0-6M KI, 6mN sodium thiosulfate, 2mM fi-mercaptoethanol, 0-5mM ATP, 20ram Tris-acetate, pH 7"5). The homogenate was then centrifuged at 25 000 x g for 20 min, and the protein concentration of the supernatant was determined by the biuret method (Gornall et al., 1949).
SDS-polyacrylamide gel electrophoresis
To examine the molecular species of extracted protein constituents, SDS- polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on gradient slab gels (7.5-17-5% acrylamide) (Laemmli, 1970) at 30mA. The
182 M. Muguruma et al.
gels were scanned densitometrically with a Hitachi 557 Double Wavelength Double Beam Spectrophotometer at 540 nm.
Preparation of myofibrils from cured and air-dried bovine muscle and determination of contractility of myofibril preparations
The a w of samples was adjusted to about 0-89, and the samples were then stored for 2 months at 4°C. Pieces of samples (3 x 3 x 10mm) were put into 50% glycerol, 4 mM EGTA, 5 mM K phosphate, pH 7.0 and stored for 2 months at -20°C. To prepare myofibrils, glycerinated pieces were soaked for 2 h in a solution containing 50 mM KC1, 4 mM EGTA, 5 mM K phosphate, pH7-0. The pieces were then transferred to a centrifuge tube and homogenized with about 10 volumes of SSS-solution with a poIytron homogenizer (Kinematica, Switzerland). The homogenate was centrifuged at 5000 x g for 15 min. The supernatant was discarded, and the precipitates containing myofibrils were washed three more times by resuspension in 10 votumes of SSS-solution. The contraction of myofibrils was evaluated according to the previous procedure (Muguruma et al., 1980). Photographs of the myofibrils were taken with a Nikon camera through a Nikon VFD- TR phase-contrast microscope.
Electron microscopy
Double fixation in 3% glutaraldehyde and 1.3% osmium tetroxide was followed by dehydration through graded ethanols and embedding in an Epon mixture. Thin sections were stained with uranyl acetate and lead citrate. Specimens were examined with a Hitachi H-300 S electron microscope operated at 75 kV.
Determination of lactate dehydrogenase activity
Lactate dehydrogenase (LDH) was prepared and assayed according to the method previously described (Yamada & Muguruma, 1978).
RESULTS AND DISCUSSION
Effect of propylene glycol and sorbitol on the properties of cured porcine meats
Moisture content and a w of 6% salt-cured porcine meats decreased with increasing content ofpropylene glycol or sorbitol added to cured meats during processing. Propylene glycol was more effective than sorbitol in
Fig. 1. Effect of concentration of propylene glycol and sorbitol on water activity, moisture content and pH of pork cured at 4~C for 14 days. Each point shows an average of five determinations under each condition. Vertical lines represent standard deviation at each
Fig. 2. Comparisons of amounts ofpro tdn extracted with five solutions 13om pork stored with various concentrations ofpropylene glycol and sorbitol. Each point shows an average of five determinations under each condition. Vertical l in~ represent standard deviation at each point. ,:~---~,, SSS solution; ~j--~,~ ~, GS solution" 0 - - 0 , GS-ATP solution: _/',--/"., HS
solution; D - - D , KI solution.
lowering a,,.. The pH of cured meat was increased slightly by the addition of sorbitol (Fizz. i', ~"~ . ~ _,. xn,. contents of sodium chloride in curea meats were not affected by propyiene glycol or sorbitoL
SSS-so!ution extracts water-so!ubie protein from muscle, and HS- solution can extract a structural protein, myosin, too. Kt solution extracts most structural proteins such as myosin, actin, tropomyosin, troponin, etc. GS-sotution has been used for extracting pure myosin. GS-ATP-solution can extract more myosin than GS-solution, because of dissociation of the rigor complex of actin and myosin by addit ion of ATP. Figure 2 shows the amounts of protein extracted with these five solutions from porcine meats cured with propylene glycol and sorbitoi at various concentrat ions and then stored. The species of protein extracted from cured porcine meats were determined by S D S - P A G E (Fig. 3). Addit ion of propylene glycol to cured
.... a __c__ b ._i_. c - - r - d - T - e -n
o , . Q ® ® ® ®® H-MHc
- . . , , o o , , , , , e e e e e g t t _
s ~ ~ ~ i ~ ~ i , ~ ~ ~ ~:!i~ ~ ~ ~ ~_!i,~ ~ ~ i ~ ~
_2L 12 3 1 2 3 1 2 3 1 ~ 3 ........ 3 N Fig. 3. Gels from S D S - P A G E of proteins extracted from cured pork; (a) SSS solution (b) GS solution; (c) GS-ATP so..m_on: (d) HS solution: (e) Kt solution: ( i t 6-,-.:z~ NaCI: (2) t + t0% propy!ene =lyco,,~, - ~ (3) I + 10% sorbitot: (M), . . . . myofibril s tandard: {MHO, myosin
heavy chain.
~_.~;u~,.------ ¢_,~..-.-:~c~c,,--c---, ~., a..--~c ".--~'a~er ectiri.--'_.r i g5
meats increased the * ' " * - ~;" "~'" ' ' ' extrac,~aDmt3, v,. s~It-Soii201e ~ u s c [ e 5-r uctur~.~ .Prote in .
. . . . . . ; ° ~ " ' ~ " ' ' , . . . . . ~ " * ' h a d egec:--*_ o n . , ~ . e , . , _~ . . . . . . . . . . : . , . ~ m ~ o ~ h . , . vop,.-..,,,,, ~ m y o s i n , o ~ t boi tJltOt n o th~ - x-,-~nof~ N{-'.--;_.~,, , :g . - ~
Effects of propylene glycol and sorbitol on the extraction of myosin from cured and air-dried porcine meats
The changes in ex t rac tab i l i ty of myos in heavy chain seem to be a useful index o f a R e r a f i o n of myof ibr i i ia r p ro te in in I M M ( M u g u r u m a e,.--a!.. 1986:
M u g u r u m a & N a k a m u r a . 1986). T o examine m o r e precisely the effects o f .~ropvlene. ~-ivcol~ and sorb i to! on the. extractabiiitv~ o f myos in , , _ ~ h . . . . . y~ chain, the p ro te ins ex t rac ted with. G S - s o l u d o n and GS-ATP-so iuv , o n m_m" :~ cured and air-dr ied porc ine meats were ana lyzed by S D S - P A G E (Fig. 4) and quan t i t a t ed by dens i tomet ry . T h e a m o u n t s o f myos in heavy chain ex t rac ted with GS-so tu t ion were 0-50% in meat cured with 6 % - '" . -,o~ ~a,,. 1 0~ ~o in meat
cured with 6% salt plus 5% propyiene glycol, and !-56% in meat cured with 6% salt plus 10% propy[ene glycol. On the other hand, the amounts of myosin neavv c,.am extracted with GS-ATP solution were 0-72% m meat cured with 6% salt, 4-76% in meat cured with 6% sait plus 5% propytene giycol, and 9.57% in meat cured with 6% salt plus 10% propyiene glycol In • ~ • " + t t . . . . tne meats cured wlm 6% sa,t m the presence of "~'+;, or 10% sorbitoi, however, the amounts of myosin heavy chain extracted with GS-solution or GS-ATP solution scarcely were changed.
Thus, propylene giycol markedly affected the extractabi!ity of myosin heavy chain even in extraction with GS-soiution. More myosin heavy chain was extracted t¥om cured and air-dried meats with GS solution in the presence than in the absence of ATP because of the dissociation of rigor complexes ofact in and myosin. These results were similar to those obtained with porcine meats cured with 6% satt plus i0% g!ycerot (Muguruma et aL, !986). The presence of propylene glycol as well as glycerol may maintain
i a b i
MHC
8 5 K Protein
Fig. 5. Gels from S D S - P A G E of protein extracted i?om cured beef with SSS solution (a) and GS solution (b); (t) 3-5% NaCI; (2) 1 + t0% glycerol; (3) ! + 5% propylene glycol; (4) t + 5% sorbitol; (5) t + I0% glycerol + 5% propylene glycol; (6) 1 + !0% glycerol + 4% sorbitol; (7) I + i 0 % glycol + 5% propylene glycol J- 4% sorbitol; MHC, myosin heavy
chain.
Myo-in ext~ra~i-::_.O, a:--~d weser actb:#_y 187
the muscle fiber in a condition in which the rigor complex~ of actLu and myosin can be dissociated by the addition of ATP,
Effect of glycerol, propylene glycol and sorbi.:ol on properties of cured bovine meats
Because of the alteration of myosin by 6% salt curing ( M u g u r u m a et al., !986). m e a t was cu red w!tn- ' 3"5% saL.'t - i he ex t rac tab i i i t ies ~~" w~+~.-v-,1,,~.> protein and myosin heavy chain f r o m m e a t cured with 3-5% salt were not affected by addition of 10% glycerol, 5% propylene gtycol or 4% sorbitol (Fig. 5). In the presence of glycerol, however, the density of a band having a molecular weight of 85 000 increased. The 85K band can be seen when fresh bovine meats are extracted with SSS-solution or GS-soiution. Evidently, the glycerol added to cured bovine meats maintains a condition ctose to that of the original raw meats. The LDH activity was halved by salt curing (Table 1), but the inhibition of the activity was lessened to 70% of the initiai value by the addition of glycerol. On the other hand, the addition ofpropylene glycol
TABLE 1 Efi:ect ofGlycerol, Propylene Glycol and Sorbitol on Lactate Dehydrogenase (LDH~ Activity
LDH activity was assayed in a solution containing 66ram Tfis-HCL pH 7.5, 0-t5mM N A D H , 2'5ram pyruvate and 5m:~ EDTA. The assav was carried out at 30:C in a final volume of 3-0ml, following the change of optical density at 340nm with a Hitachi two wavelength double beam spectrophotometer, type 557. One unit o fenzyme was defined as the amount of the preparation which catalyzes the t r a n s f o ~ a t i o n of I -emoI substrate/min. Each value is the mean for five preparations with the associated standard deviation.
or ~orb~tol ..~m~,~.:.~'~ .... . . reduced the L D H _.-~rivltv_~._ ._~, with ~-~o'-'-:~-ep,. ~,~,.~ -:,~vco~ having the greater eNect. However. glycerol mitigated the inhibition of L D H in cured meats by propyiene giycoi and/or sorbitol. Glycerol has been reported previously to protect against myosin denaturat ion (Nakano & Yasui, 1976) and against ATPase inhibition (Yasui eta!. , 1966).
Effect of glycerol, propylene glycol and sorbitol on the properties of myofibrils prepared from cured and air-dried bovine meats
Individual myofibri!s could not be separated from raw ham manut:actured by curing and cool ~ ' - ~ ' ~~ ~ ' ~mo~.ln~ (Muguruma eta!., i ~ o , or from porcine meats cured m the presence of giycerol and air-dried: these samD!es _a,.,. bands of myofibriis with an "adhesive appearance' in muscle fiber (Muguruma & Nakamura , 1986}.. under conditions allowing . . . . ,,;,,,,,~,~,~a'~-;~ to be ~pa ra ted from i~esh muscle. We ~-,.~-:, ~.,, ~ , ~ , ~ . " *.-~;h,,~.4 ,~,~ 'adhesive aooearance" of c~d~.~. - and air- dried muscle fibers to membrane m~,.:,~ivn~." ~ .... . . . ~ - - : therefore, to disrupt membrane function the fioe~ pieces were piaced in ~0% ~lvcerol at 0°C and stored for .z months at - 2 0 : C . To prepare myonurus [\"om curee- ann air- dried muscle, glycerinated fiber pieces were homogenized lbr 5 rain at i0 000 rpm with a Po!ytron homogenizer. Homogenizat ion broke the muscle fibers along the transverse axis only, in the eosition indicated by the arrow in Fig. 6-i. T~,~. side-by-side arrangement of mvofibn!s of samples cured with a 3-5% salt could not be separated ~,;, La~Lne~ homogenization. W n e ,
f ' r o - r ~ . . ~ - t . propyiene gNcol, was u~,.~a in curina~, =, muscle hoers were , a~ . . . . . . . . ed into ~mahe.~ p~eces, and the mvonbnis began to dissociate ,.m41~, . . . . . . . . a:~'. (Fig. ~,- zL Lon~itudina! dissociation was . . . . . . . . 4 -, -~ ". ~ oo~,~ . . . . also wne~t ~ivceroi. was added to .... 4 ~ -o). me other muscle nbers oniv separated ,.~rm~ agents (Fig. -7 - On "' hand, ~ . ~rans,er~,.,), into small fiber segments, when sorbitoi was added to curing agents (Fig. 6-3). The combined effect of t 0% glycero!. 5% propy!ene glycol and ~a%. sorbitol caused the mvofibriis, to separate parallel to the ion~itudinal axis (Fig. - ":' _ /-J~, as fresh myofibrils do.
On addition of Mg2÷-ATP intact myofibrils contract, and distinct contraction bands are observed {Fig. 7-2). Myofibrils from muscle cured and air-dried in the presence of giyceroi, propyiene glycol and sorbito! formed bundIes which could contract in the presence of Mg 2 +-ATP (Fig. 7-6). in a previous paper (Muguruma et al., 1986), we suggested that the adhesive appearance of fibers of raw ham cured with 6% salt might be due to the
. q 1 ÷ " " solubilization of myosin. However. with 3-5% ~.a,.~ curm~, myosin may not be solubiiized. Consequently, other factors may contribute to the adhesive appearance.
The uitrastructure of processed meat and meat cured ~' wan various humectants was studied in parallel with a light microscopic study. The
~,6~,..--,,-..-'.-.--~ e.~--,'_racL~i-_..iY~]- a ~ d ~---'fffer ~cf-t~S:!-- ! ~ 9
Fig. Ii. Phase-contrast micrographs ofmusc!e fiber prepared from cured, air-driied beef: (1) 3-5% NaCI: (2) 3-5% NaCI ~- 5% prop~!~n~ a~,-o~, t '~" ~ ,o NaC! + 4% sorbitoL The arrow indicates the position at which the muscle fibers are broken by homogenization. The
caiibration bar represents 10i~m- (Z) Z-iine.
c o m b i n e d use o f glycerol, p ropy l ene glycol, and sorbi to i was best in t e rms o f p rese rva t ion o f fine s t ructure , a n d resulted in easy d issocia t ion o f myofibr i ls and less r e loca t ion o f ex t rac ted musc~;e m ~ o m o a n s o n With oth, ,r t r ea tmen t s M e a t cured with giyceroi (Fig. 8-3) differed m ark ed iy f r o m mea t cured wi thou t glycerol (Fig. 8-2), especial ly in tha t Z-lines D o m mea t t rea ted with gtyceroI m o r e closely resembled Z-lines in the nat ive state (see Fig. 8-1, control) .
i90 M. ML~g~rz~_~---~a et ai.
Fig. 7. Phase-contrast micrographs showing the contraction of myofibfils !Yore cured, air- dried beef upon addition of Mg~"-ATP: (1,2) intact myofibrils; [3,4) 3'5% NaC1 + 10% glycerol; (5, 6) 3,5% NaCf + !0% glycerol + 5% propy!ene glycol + 4% sorbitol; {2, 4 and 6) indicate the myofibfils after addition of Mg-' +-ATP. The calibration bar represents I0 #m. (Z)
Z-line; (C) contraction band.
It has been suggested that intermediate filaments connect adjacent Z-lines of neighboring myofibrils whereby all the myofibrils are kept in register (Granger & Lazarides, 1978; Lazarides, t980). Intermediate filaments have been detected in the intermyofibfiiiar spaces but not intramyofibriitar structures (Tokuyasu, 1983). In the cured and air-dried bovine muscles, the structures of intermediate filaments may be changed t~om the native form due to the release of free water in lowering a,~,. Furthermore, the proteins desmin and vimentin (Lazarides & Granger, 1982) may be denatured. Another possibility is that the properties of the coilagenous fibrils which enclose the muscle fiber change (ishikawa, 1983). It is considered that the sarcoptasmic and myofibrillar proteins except myosin do degrade to some extent and the fragments are held in the highly crosslinked matrix. However, the possibility of the solubilization of myosin cannot be ruled out.
Fig. 8, Electron micrographs of muscle fibers prepared from cured, air-dried beef: (1) control; (2) 3-5% NaCI; (3) 3-5% NaCI + 10% glycerol. The caIibration bar represents 1 ~m.
(Z) Z-line.
:92 ,:_..r ,:-f:-<,:~e~---r~:a et aL
Fig. 8.--conM.
Effect of storage on the extractability of myosin from cured and air-dried bo~ine meats
The storage stability of IMM is very important. Figure 9 shows SDS-PAGE ~omuon from cured and air-dried ~:s of proteins . . . . . . . . d w:tn GS-ATP ~ ' "
beef which was stored for 3 montn~' - at 4°C and then for i month~ at 25°C and at pH 6"!-6-2. Even after 5 months of sample preparation and storage, myosin hea,,) ~ chain could be extracted from samples cured in the presence of glycerol. Myosin hea~)" chain could be extracted from samptes cured in the presence of propylene glycol before storage but not aRer storage. This result suggests that propyiene glycol could not prevent alteration of myosin in beef during storage at 25°C. Some of the myosin heavy chain was extractable from sample cured in the presence of sorbitol after storage at 25°C. Myosin heavy chain could be extracted from air-dried sample cured in the presence of 10% g!ycerol, 5% propylene glycol and 4% sorbitoi even after long-term storage.
Thus, the combined addition of !0% glycerol, 5% propylene glycol and 4% sorbi:ol is very effective in maintaining the contractility, structure and storage stability of IMM. Further studies are required to d e t e ~ i n e the effect of various mixed humectant systems on IMM during long-term storage.
w
i
- - ~ - M H C % ;
- :z- cc ~-- l _
- i l l ~ ~+~_~. ~ _
. . . . . .
I 2 3 4 Fig. 9. Gels from SDS-PAGE of proteins extracted with GS-ATP solution from cured, air- dried beef stored at 25:C for 30 days: (1) 3"5% NaCI - t0% glycerol; (2I 3.5% NaCI + 5% propylene glycol; (3) 3"5% NaCI + 4 % sorbitoI; (4) 3-5% NaC1 + 10% glycerol + 5%
propylene Nycol + 4% sorbitol. (MHC) myosin heavy chain.
A C K N O W L E D G E M E N T S
We would like to tnan~ Professor T. Fukazawa t:or mani, . . . . i~. t~ discussions and suggestions. This study was supported in part by the research grant from the Ito Science Foundat ion, Tokyo.
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