L’L'LLLLK 3"!"- :L' {LII “ ‘ ‘ 5 'I L 'r'L' I '_ i!‘ ._"_ '3' 'I."'.":“:L L'I' '3'?!“ I.’ :J L ' 'LLI‘ L \‘jI '(LJ"' LII‘LWIIIFIJ ‘ . “'L'LL'L'L-L'L'fil LIL II"; ILL: N ”(LI ~L-.""'.' "II': I 'I'I ’ . J:‘3;.‘"LL"'§:LI";<'._ "" I'I I' _,I I' I I 'LJ'0"""a '- “ELI“ LI'L J‘:(' L" L 'J‘L‘IE'L‘ ..-‘I‘ I I LII» «I‘" .‘S'I" ."‘I‘III. “‘ I:E' ' I L‘L’ILLL ""I‘L" IELLLLJ L;L:LL$L 'JJL'L I ' N .I" .I'I.‘ ': I'- _I' -' 'JLIL' “IL "L,""" J "'1' I "L" ‘ LLIL ,' f.“ I “7' h LILLII LIL“' 'L‘L" LII L:'.' L «,I‘IH'l-Ij; “3"" L'L'J LLL'L'IL‘I I hi” 'LI‘“ L ‘.;',.»“I; 3’3"" LLLLLLTLLL I'Ll'h LLLL LLLL'IILIL "I:L:'.'I""'§j'f""" LII.LI1.7'II‘IFI;":‘II I ‘ P ILI‘J L; . .I' gain? £2,356" FILL}! H“- pr -I. 4 'n' ‘3" It ‘J 4": , ”113:: ‘:|' I; « . 'IIIJLI '9‘ “and t .' L I 35')“; QIo-HI JI‘I‘ILI l'L'If'ILLLL. III LL IrL‘L {LL-”51,1 LIILLL'L 33? tII'ITLH ALI 3;; I'"L LL' ,LL‘III ILLHLL iL'LLLJLL' L L“, '3'." LI II'I . n...“ - ....._..- - 4 _ ..I - - h.— ~ -mn . » _ __~ ' ‘ ’ ‘3: ..~_r_‘. ~— #b—I‘ ‘ 5 —~- - “—‘9 _ I." < i - ‘- _ f'fiwfi— m. _. - -— a ' ‘ . ___.._...._—4-“—— -3 -. 1... _ ‘ > - ‘3 '5 - . M. _ _ «PA-r". @ - ~ 3 '-. n... 'J--.- '— _ ‘1 _. —— ._ ‘. A “L‘ - w ~ % ?:—_= ' 3L. a? £~ 'M _ M-_. .. m “a, _ ..- .. ’1 Jim I 7-“, OM .5“.._w N.- .‘A “TL”:— .. -- 'qu “ ' I“: ' . ';>&‘:;:":: I L‘1 :E’QLE‘J '53.; E17 I,“ 'i'. u : 97$ ' . I,It I . ‘IJ‘I If. .. 1:: “‘ , LL I L vii" LLL L"""" J I'LLL' 'I' 'LlJ LL" ’II .. -'1I,I'I}I ..,...-I .I ":"[JL~"-LLL" L321; in” ' ' 'L'jgg'v '{L I; 5 IE V! L 3&9 L} b3.) "Lia ‘~“,93:yd‘"-3 ‘ I" I4n%‘%I . 4%"? fig“ W . ‘- -. W .“3 it? h __' ‘J %‘ . .l J .31» A." ... . “25.31!” ‘le " "- In I-fIEI'nI‘ IIIIIII III: . ' ‘ "' "*L'J'LIIL‘LL": '1 " ' ' ’L 'ILJz‘IIIIif! "II;- ’ I;.;. ,; I'.‘I'.~F:'«. «I ‘ " ‘53?“ "33333: JILL? " ' ' II.I«..__., I I . _-I--; I I: v Ii‘IIfI" “15'?“ II III" ' -' ‘ 3 ' , I . .5 ,I“ quét‘i ‘ W'LUL {LIL} U LLIQ‘JII “I7"! HEEL? . " . I I I.'I ' . i‘I'KEJ'J:':'I' 1.333;?“ ' I " "- .- . - . - I .. V ., I'LI;‘LL:IV§?E'§E:H2 :. 123' "1:fo L1}! ' 1 :1?- . Xx L‘LJL‘ 'l ‘35! ,1 I '~ I':I‘-,~ “' |'.LIL"‘ LIIITI ‘I. .'. I“ " - i. 1’13) ”‘3" '. r' . ~ 9L ~' . . '4‘... 3 "w L by?! ‘ ("2‘ '00 fig" 1%: ILL I'lm LEI ‘ _‘ : WWT'.W=VI='7:;¥‘:WW‘"— _ II J . I V.. -.- A .. . .1 ’r _ A I ' : '. - .. . r..- .I-‘ SJ “I {LI}, I'N' .L " ' '3’:in IST-I ' 3‘7"": ' ! 1‘1. up: : -.q~_ :‘ :JI gs. .L‘ LLungLflLL: I '5.” {I ‘_ u. . II. II :3. ‘11," ,. ‘1 a? :5 " 'IL. ’ '4' .40' -‘..-;;~ k 'I-I L I ,E"I£" I,“ L'C'b _ ;.'%.. .21 f 39..."; -‘II' ‘ I I ‘ y‘l-HELJr'I. '. ‘ ‘1‘ I. L'I' ' ""I Li'i'L I I"-:. "I": .‘L"IE'?‘IV 'I""71mfll|:,": " ‘Ii‘ ‘« '» ’ ‘ ~r:' , ~'.I‘."' .'§‘13":‘."" viscera > dark muscle > white muscle. While in the round, the same order of the lipid oxidation was observed, but was markedly less pronounced than that of the minced except the skin. The preferential lipid oxidation in the skin was attributed to the property of Skin lipids being liable to autoxidation. They Suggested that a tendency toward lipid oxidation in both lean and fatty fish could be predicted in advance of the storage by measuring TBA values of fresh skin after having incubated its homogenate for 2 hours. Lee and Toledo (1977) reported that the lateral tissue (red muscle) along the visceral cavity and bone marrow exudate appeared to be most susceptible to oxidative ran- cidity. Rate of TBA value Change was very rapid when these 26 muscles had contacted with iron surfaces. Silberstein and Lillard (1978) studied the perooxidative effect of hemoglobin, myoglobin, and total heme and nonheme pigments in mechani- cally deboned mullet. They indicated that myoglobin has greater catalytic effect than hemoglobin. The rate of lipid oxidation increased as the ratio of hemoglobin to myoglobin decreased, i.e. oxidative influence of hemepro- tein on the minced fish was accelerated as the concentration of myoglobin increased. Protein Denaturation and Texture Deterioration Interaction between moisture, lipids and enzyme onggrotein denaturation and texture deterioration Shenouda (1980) suggested a diagram that illustrated the factors which affect directly or indirectly fish protein denaturation during frozen storage as follows: (Moisture) lEnzymei TMAO Ice crystal Intact formation Lipid ' DMA Dehydration Hydrolysis of Formaldehyde free fatty acids Increase sa t + Oxidation concentration - 4. lg T + + + {L \L V} J) ‘L ’ iv LProtein Denaturation and Texture Deterioration 27 Shenouda stated that change of the state and concentration of moisture, lipid stability, and enzyme activity could influence the muscle protein denaturation directly or in- directly through their effect on each other; as it is indicated in the above diagram, the horizontal arrow repre- sents the indirect factors and the vertical pathways represent the direct factors. The occurrence and accumu- lation of one of these factors could trigger or retard the reaction rate of others. For instance, high salt concen- tration resulting from protein denaturation could stimulate the hydrolysis of lipids and accelerate the liberation of free fatty acids. Freezing brings about crystallization of water present in the fish muscles (Sikorski et a1., 1976). The distri- bution and size of ice crystals depend upon the condition of the muscle, the rate of freezing, the storage time, and temperature fluctuations (Love, 1968). In pre-rigor fish, the crystallization of ice takes place intracellularly regardless of the rate of freezing. On the other hand, in post-rigor fish, if frozen slowly, ice crystals form in the extracellular fluids and grow at an expanse of water which diffuses out of the cells; while at faster freezing rates, a large number of tiny ice crystals grow both within and outside of the cells. As a consequence,the decreased amount of liquid water available to the proteins causes the increase in concentration of tissue salts. The 28 mechanical damage to the muscle structures due to the growing of ice crystals (Love, 1968; Kent, 1975) resulted from fluctuations in freezing temperatures (Dyer and Dingle, 1961). According to Castell et a1. (1970) and Sikorski et al. (1976), inorganic salts affect protein denaturation in frozen fish most probably by depressing the freezing point of the tissue fluids, causing dehydration, influencing the interfacial tension, and by ionic interaction with Charged groups of the polypeptide side chains. Inorganic salts can interfere with the conformation of protein by participating in the formation of lipid-protein complexes, reacting with nitrogen and oxygen functional groups of proteins, and inducing lipid oxidation, which in turn, may bring about protein insolubilization. Pigott and Shenouda (1975) repor- ted that the presence of Ca++ as well as a solution with high ionic strength favors polymerization of actin and binding of both neutral and polar lipids. Calcium ions enhance the polymerization of G-actin to F-actin and by so doing favor protein-lipid interaction. Buttkus (1971) added Cu++ ions to the homogenate of trout myosin, causing a rapid loss of -SH groups and aggregation of the myosin molecules. Many salts exhibit a solubility effect at low ionic strength (0.5-lu) by associating with ionic linkages of the protein, rupturing the bonds, and enforcing hydration of 29 the newly built associations (Lewin, 1974). At higher salt concentration, the competition of inorganic salts for water may result in a salting-out effect, whereby the number of hydrophilic groups associated with water that buttress the solubility of the macromolecule can be reduced, and decrease the amount of water for protein. In addition, at higher salt concentrations (above 1M), most inorganic salts cause a proportional increase in the surface tension of the solu- tions, resulting in an enhancement of hydrophobic interac- tions. Effect of Cryoprotective Agents on Prevention of Protein Denaturation Noguchi et a1. (l975a,b,c; 1976) have done intensive studies on the control of denaturation of fish muscle proteins during frozen storage by using cryoprotectants. They determined the cryoprotective effect of different compounds such as amino acids, carboxylic acids, carbo- hydrates and their derivatives;andimeyfoundthattme follow- ing compounds exhibited better cryoprotective effects among those tested: (1) glutamate, aspartate, proline, cysteine, glutamylcysteinylglycine, acetylglutamate; (2) malonic, maleic, malic, lactic, tartaric, gluconic, glycolic, methylmalonic and glutaric acid; and (3) glucose, fructose, sucrose, lactose, stachyose, melezitose, glycerol, ethylene, glycol, sorbitol, B-glycerolphosphate and glucose-l-phosphate. 30 These researchers proposed that the effective cryoprotective agents for fish muscle Should have the following proper- ties: (l) the molecule must possess an essential group of either -CO0H, -OH, or -OPO3H2, and more than one supplemen- tary group of the type -COOH, -OH, -NH -SH, -SO H, or 2’ 3 -OPO3H2; (2) the functional group of the compound must be suitably spaced and properly oriented to the protein mole- cules of the fish muscle; and (3) the molecules must be comparatively small. Matsumoto (1980) suggested that cryoprotectants acted as water structure modifiers by binding with protein mole- cules and preventing the unfolding Of globular proteins. This effect was due to increased hydration of the protein molecules and an increased resistance against displacement of water even when the system was frozen. Complex Character of Protein Deterioration in the Presence of Hydroperoxidizing Lipid in Frozen Fish The result of lipid hydrolysis is detrimental to quality in lean fish, which at higher concentrations of neutral lipids is dispersed in the muscle tissue in the form of droplets; their competition for hydrophobic binding sites may reduce the participation of polypeptide side Chains in hydrophobic interaction with fatty acids hydro- carbon residues. Lipid autoxidation which results in the formation of free radicals and reactive scission products 31 enhance protein Changes regardless of the concentration of neutral lipids in the muscle cells. Fish containing phos- pholipids with a large proportion of polyunsaturated fatty acids, which are most susceptible to autoxidation, should be especially liable to protein deterioration due to these effects (Sikorski et a1., 1976). Dyer and Dingle (1961) reported that formation of free fatty acids preceded the loss of extractability of myofibril- lar proteins and that the latter was more rapid in lean species. Ohta and Nishimoto (1964) demonstrated that addi- tion of fatty acids to minced mackerel flesh decreased the extractability of proteins from samples stored at -10 and -20°C at pH 6.4 to 6.8. This is because the interaction of free fatty acids with myofibrillar proteins produces a net- work of cross-links which increases the resistance of the muscle fibers of fragmentation and reduces the protein solubility. Fatty acids liberated from phospholipids as a result of hydrolysis,may attach themselves to appropriate binding sites of either neutral lipid droplets or hydrophobic, polar, or ionized fragments of the peptide Chains (Sikorski et a1., 1976). When they interact with neutral lipids, they become inactive; when they bind to polypeptide side chains of polar groups, they may decrease the protein solubility due to formation of intermolecular hydrophobic- hydrophilic or hydrophobic-ionic linkages, especially at 32 appropriate concentrations of inorganic ions (Pigott and Shenouda, 1975). Oxidized lipids in the lipid-protein system are known to induce polymerization and aggregation of the proteins, resulting in decreased solubility and formation of color complexes (Karel, 1973). According to Castell(l97l), lean fish muscle contains 0.5 to 1.0% unsaturated lipids, but during frozen storage it rarely goes rancid, as indicated by thiobarbituric acid values or rancid odor. The lipids oxidize, but instead of forming carbonyls and other compounds associated with rancidity, they become bound in lipid-protein complexes which account for the toughened texture of poorly stored frozen fish. Funes and Karel (1981) stated that the dominant mecha- nish of protein polymerization after exposure to peroxidizing linoleic acid is the transfer of free radical from lipid to protein, and subsequent free radical polymerization. Roubal and Tappel (1966) proposed a free radical displacement reaction as follows: P- + P-—————+ P-P' P-P- + P———-» P-P-P- where P = protein The concentration of protein radicals is higher with an increase in lipid oxidation and a decrease of water activity (Aw)' Extensive protein-protein cross-linking occurs in a food system at Aw values between 0.75 and 0.40, and 33 Significantly less at lower water activity. Schaich and Karel (1975) postulated that high water activity may reduce the concentration of radicals by favoring their cross- linking. The availability of water may control the rate of cross-linking via terminating the radical formation by donation of hydrogen atoms and/or by colligating two protein radicals together as follows: P. + P'————-OP-P Gardner (1979) summarized the following statement in his review of lipid hydroperoxide reactivity with proteins and amino acid: (1) production of lipid hydroperoxides in an inadequately processed foods is catalyzed by lipoxygenase; (2) the hydroperoxides and their scission products are potentially reactive compounds that bring about the degra- dation of food proteins and amino acids; (3) formation of lipid-protein complexes is resulted from the exposure of protein to peroxide lipids; (4) the degradation of food quality resulted from the interaction between lipid hydro- peroxide and proteins are characterized by protein-protein links, protein scission, protein-lipid aducts and amino acid damage; (5) formation of covalent bonds between protein and the secondary products of the lipid oxidation is another detrimental factor that reduce the product stability. Karel et a1. (1975) proposed a mechanism to illustrate the formation of lipid-protein polymers as follows: 34 LH + R' ; L' + LOO' + LOOH + RH + S PH + L' + LOO°————————» P' + LH + LOOH + POO' P' + POO'-——————4 POOP P' + L' 4; PL P00. + L. -——-—o POOL P00. + LH ——————» POOL + H. + POOH + L. PH + S 4‘: RHS + PHSPH where: LH = lipid PH = protein R’ = free radical L' = lipid free radical P' = protein free radical LOO' POO“ lipid peroxy free radical protein free radical S = lipid scission product Various aldehydes are the scission products of lipid hydroperoxides; they act covalently with proteins that contribute both flavor and color to a Specific food system (Eskin et a1., 1977). Non-enzymatic browning results from interaction between peroxidizing lipids and proteins. For example, the highly unsaturated nature of fish lipids results in the browning of fish. The aldehydes resulting 35 from lipid oxidation are usually volatile and often emanate potent odors. In browning reactions, these aldehydes produce gluey odors and fishy aromas (Gardner, 1979). In food systems with low water activity, the protein scission is more likely to occur than protein-protein cross-linking when peroxidized lipid is mixed with protein. At the low water activity state, protein peroxides were formed through oxygen attack on a-carbon-centered radicals (Gardner, 1979). Subsequent cleavage at the peroxide- bearing a-carbon resulted in protein scission and an increased amide content. Amino acid residues in protein are damaged from exposure to lipid hydroperoxide regardless of whether the mixture of peroxidized lipid and protein is incubated in an aqueous system or dehydrated state (Zirlin and Karel, 1969). Histidine, cystine/cysteine, methionine, lysine and tyrosine are the amino acid residues that are most sensitive to damage by lipid hydroperoxides. The formation of thiol radical from cysteine is facile. The oxidation of methio- nine sulfoxide is probably due to the ease of delocalization of electrons of sulfur. Products from the nonsulfur amino acids can be explained on the basis of radical formation at the a-carbon and to some extent on the side Chains (Tannen- baum et a1., 1969). Young and Karel (1978) postulated that the degradation of histidine involves the following sequence: (1) formation 36 of an a—carbon radical by deamination; (2) hydroperoxidation of the a-carbon, and (3) hydroperoxide hydrolysis which led to imidazole lactic acid and imidazole acetic acid. Forma- tion of lysine products was due to the radical attack at the a-Carbon, and side chain carbons. Among the side-Chain carbon the c-carbon was the most labile. Schiff base formation is a particularly important degradation reaction for lysine (Gardner, 1979). Enzymatic Activity of Trimethylamine Oxidase As Related to Protein Denaturation During Frozen Storage Distribution of trimethylamine oxide (TMAO) and trimethyl- amine oxidase (TMAOase) TMAO plays an important role in maintaining nitrogen balance in the marine fish. The magnitude of the natural occurrence of TMAO in marine fish differs in various Species; on the other hand, TMAO is scarcely found in freshwater fish. Both elasmobranchs (cartilage fish) and mollusks (squid) contain a higher amount of TMAO than the teleosts (bony fish). Among the teleosts, the gadoid family, such as cod, pollack, haddock, whiting and hake contain the highest amounts of TMAO; whereas the flatfish have the lowest amount; crustaceans, such as shrimp and crab, contain moderate amounts of TMAO; the bivalves (e.g. 37 clams, oysters) and echinoderms (e.g. starfish) contain very low levels of TMAO (Konosu et a1., 1974; Suyama and Suzuki, 1975). Disruption of fish muscle fibers during the mechanical deboning process leads to breakdown of TMAO to DMA and formaldehyde (FA) by the action of TMAOase. The distri- bution of TMAOase was detected by measuring its end products DMA and formaldehyde (Shenouda, 1980). The mechanism of formaldehyde and DMA formation was postulated by Harada (1975) as follows: + CH CH H C ' 3_ I 2|\ H3C\ _ CH3-N-O __, CH3-N-O ::.CH3-N-O _—. H C/C-CHZO -—- .3 CH3 CH3 CH3 H C H C 3 ‘N-CHZOH :23 3 :HN + HCHO H c’ ‘ H C 3 3 DMA FA According to Harada (1975) and Hiltz et a1. (1976), TMAOase exists only in a limited number of marine animals, and its activity varies widely among species, types of tissue, and storage temperatures. Fish, squid, bivalves, and gastropodes show some capacity to form DMA and formalde- hyde; while scallops, lobsters and shrimp were found lacking in TMAOase (Castell et a1., 1970). Godoid family was found to have the highest TMAOase activity; in which the formation 38 of DMA and formaldehyde was greatest in species that had large amount of dark lateral muscle in fillets. Harada (1975) reported that TMAOase is relatively heat-stable; its activity was maintained at temperatures up to 60°C for 5 minutes. Lall et a1. (1974) noted that heating the silver hake fillets or minced flesh to an internal temperature up to 60°C were not effective in inactivating the enzyme during subsequent frozen storage at -10°C, but when the internal temperature reached 80°C, the preheating treatment was highly effective in arresting the enzyme action. Effect of the Formation and Accumulation of Formaldehyde in Fish Muscle Formaldehyde is a very reactive compound capable of interacting with amino, amido, thiol, guanido, phenolic, imidazole, indolyl residues (Sikorski et a1., 1976). Therefore, extensive accumulation of formaldehyde in frozen minced fish is often accompanied by loss of extractability of myofibrillar proteins. The rate of formaldehyde accumu- lation is higher in dark muscle, intestine, kidney, blood, liver, and unwashed minced flesh than that of white muscle, washed muscle, and intact fillets (Babbit et a1., 1974; Dingle and Hines, 1975). Ishikawa et al. (1978a,b) pointed out that TMAO could act as a peroxide decomposer and, at the same time, Show a 39 synergistic effect on the activity of y-tocopherols in inhibiting the lipid oxidation. Thus, the depletion of TMAO concentration and formation of formaldehyde via the cata- lyzing reaction of TMAOase, would result in accelerating the hydrolytic decomposition of the fish lipids, especially the triglycerides, phospholipids, and sterol esters (Ostyakova and Kosvina, 1975). According to ChildS (1973), sensory evaluation revealed that tissue containing formal- dehyde became tougher and increased in its water-holding ability, but lacked juiciness. The texture was evaluated as being rubbery and spongy. Processing of Fish by Mechanically Deboninngachine The principle of the mechanical deboning machine in- volves squeezing the cleaned, gutted fish through a honeycomb of narrow orifices. This action results in the extrusion of the softer texture flesh, leaving behind most of the tougher skin, bones and scales as a wasted residue (Wong et a1., 1978). The size of the orifices can affect the amounts of bone residues left along with the minced flesh. There are large number of fine, long bones recovered from minced herring flesh Obtained through the use of drums with 5- and 7- mm openings, while the 2- and 3- mm perforated drums yielded minced flesh containing little or no scale fragment, and extremely short bones in minced pollock, rockfish and 39 synergistic effect on the activity of y-tocopherols in inhibiting the lipid oxidation. Thus, the depletion of TMAO concentration and formation of formaldehyde via the cata- lyzing reaction of TMAOase, would result in accelerating the hydrolytic decomposition of the fish lipids, especially the triglycerides, phospholipids, and sterol esters (Ostyakova and Kosvina, 1975). According to Childs (1973), sensory evaluation revealed that tissue containing formal- dehyde became tougher and increased in its water-holding ability, but lacked juiciness. The texture was evaluated as being rubbery and spongy. Processing of Fish by Mechanically Deboning Machine The principle of the mechanical deboning machine in- volves squeezing the cleaned, gutted fish through a honeycomb of narrow orifices. This action results in the extrusion of the softer texture flesh, leaving behind most of the tougher skin, bones and scales as a wasted residue (Wong et a1., 1978). The size of the orifices can affect the amounts of bone residues left along with the minced flesh. There are large number of fine, long bones recovered from minced herring flesh obtained through the use of drums with 5- and 7- mm openings, while the 2- and 3- mm perforated drums yielded minced flesh containing little or no scale fragment, and extremely Short bones in minced pollock, rockfish and 4O herring (Wong et a1., 1978). The variability of the bone residue present in minced fish appeared to be a function of processing methods and raw material, but not of species (Patashnik et a1., 1974). The yield of flesh recoverable from mechanically deboned fish of different Species ranged from 37 to 60%, in comparison to the fillet yields on the same species which ranged from 25 to 30% (Miyauchi and Steinberg, 1970). Rippen (1981) stated that the yield of mechanically deboned fish flesh depends on fish species and machine operation. The average yield of minced white sucker flesh is approxi- mately 50% of the round fish, containing 0.13% bone residues (Zapata, 1978). Su et a1. (l981a,b) stated that the presence of trace amounts of fish skin and internal organs, particularly liver, kidney and visceral tissue is responsible for increasing the alkaline protease levels in minced fish flesh. They indicated that alkaline protease activity in croaker kidney and liver is several thousand fold higher than in skeletal muscle. Addition of kidney and liver tissue to mechanically minced fish flesh that had been thoroughly washed, resulted in increased protease activity and degradation of fish tissue upon comminuting and cooking at 60°C. Failure to thoroughly wash eviscerated fish prior to mincing appears to result in the retention of tissue from intestinal organ. 41 ‘Okada et a1. (1973) and Miyauchi et a1. (1975) also reported that presence of blood pigments, particles of Skin, membrane from peritoneal cavity, scales and bone can con- tribute to the deterioration of fish flesh during subsequent storage. Contact of fish flesh with iron parts of a mechanical deboner can also accelerate oxidative rancidity and discoloration (Lee and Toledo, 1976). The mechanical deboning process not only breaks down the myofibrils at Z and M bands but alters the composition of fish muscle. Significant amounts of lipid and heme components are released from the bone marrow and subse- quently accumulated in the minced flesh (Schnell et a1., 1973). A higher content of dark muscle was obtained in the minced muscle in the fillet. The former accounts for a higher hemoglobin content (Froning, 1981). Functional Properties of Fish Muscle Proteins Functional Classification of Muscle Proteins According to 6011 et a1. (1974), the functional role of muscle proteins can be classified into 3 groups on the basis of their solubility: (l) the myofibrillar proteins are soluble in a salt solution with ionic strength greater than 0.30, (2) sarcoplasmic proteins are soluble at ionic strength less than 0.05, and (3) the stroma proteins or connective tissue proteins which are insoluble in neutral 42 solutions. Briskey and Fukazawa (1971) and G011 et a1. (1974) stated that myofibrillar proteins are responsible for the textural qualities of the comminuted fish and meat products, namely for the water binding capacity, gel forming ability, and emulsifying ability. In contrast, the sarcoplasmic proteins of fish muscle were considered to be responsible for the formation of undesirable flavor, color and protein denaturation due to postmortem enzyme activities (Bai and Radola, 1977)° Hater-holding Capacity The ability of processed meat to hold intrinsic and added water is an important factor in determining quality and product acceptability (Dawood, 1979). The rheological behavior of comminuted muscle products could be affected by the time of postmortem, pH, the ratio of myofibrillar to sarcoplasmic proteins, added ingredients such as sodium chloride, polyphosphate, starch, protein substitutes, etc. (Hamm, 1975; Lee and Toledo, 1979). Miller et a1. (1968) reported that pH affected water retention and swelling of meat products. The loss of. moisture by meat increased as its pH approached the isoelec- tric point of its protein at pH 5.5. The water holding capacity of meat increased as the pH was either decreased or increased away from the isoelectric point (Hamm and 43 Deatherage, 1960). Freshly slaughtered meat gradually decreased in pH with the onset of rigor mortis. Hater holding capacity reached a minimum when rigor mortis was complete. Aging the meat restored water binding ability, but never to the original levels of the living tissue. Treating meat with polyphosphate in the presence of the proper types and levels of alkaline-metal ions and optimum ionic strength and pH, restored its original water holding capacity (Ellinger, 1972). According to Hamm (1975), the retention of high water holding capacity could be due to binding of Chloride ions from the added sodium chloride by the muscle proteins. This is true when ATP is Still present in the muscle tissue before on-set of rigor mortis. Hamm postulated that the combined action of the salt and ATP caused the peptide chain to unfold, leaving such large distances between the chains that the divalent cations released by breakdown of ATP were unable to cross-link the chains. Thus, water was able to reach the numerous hydrogen bonding sites necessary for complete hydration of the proteins. The ATP present in pre-rigor muscle as well as the inorganic polyphosphates used in the fish industry to treat fillets before freezing for the purpose of reducing drip after thawing are capable of complexing divalent cations which inhibit the hydration of myofibrillar proteins, thereby increasing water holding capacity and reducing 44 thawing-drip loss (Love and Abel, 1966). Ellinger (1972) found that sodium polyphosphates were highly effective in increasing the water holding capacity of the Japanese kamaboko, a steamed fish paste, by aiding in formation of protein gels which were important to its formation. Seafood, both fin fish and shell fish, can benefit from phosphate treatment. Seafood readily loses large amounts of its fluid content. Treatment with phosphates can retain its natural moisture content and remain more succulent (FMC Food Chemical Codex,l980). Bindigg ngperties and Gel Forming Ability The binding property of fish muscle is attributed to the myofibrillar proteins and degree of hydration (Sato and Nakayama, 1970). Native tropomyoshiand actin promoted the binding capacity of meats when pyrophosphates were incor- porated into the sausage system; the promotion of binding effect was due to the interaction between myosin and F-actin which resulted in increasing the viscosity of the meat pro- tein (Nakayama and Sato, 1971a). The binding property of heated actomyosin gel increased with increasing myosin con- centration (Nakayama and Sato, 1971b). It also increased when F-actin was present in an optimum ratio of myosin, and a greater binding effect was found when tropomyosin was present (Nakayama and Sato, 1971c). 45 Nebb et a1. (1976) reported that mechanically deboned tissue had higher amounts of sarcoplasmic protein content as compared with the hand-deboned tissue; stroma protein was somewhat higher in hand-deboned tissue than in mechani- cally deboned tissue. Nishimoto and Koreeda (1979) indi- cated that the washed minced fish muscle contained higher actomyosin to sarCOplasmic protein ratio which resulted in a greater gel-forming capacity. As a consequence, the insoluble sol formed by sodium chloride extraction, from mechanically deboned fish muscle may have resulted in a less firmer texture since the mechanically deboned muscle had a lower ratio of actomyosin/sarcoplasmic protein as compared with hand-deboned tissue. Itoh et al. (1980a) found that the solubility of acto- myosin decreased and the molecular weight of protein molecules increased during the gel formation. These changes are impaired to some extent by adding the SH rea- gents to actomyosin, indicating that SH groups are involved in the changes to higher molecular weight protein molecules during the gel formation. Itoh et al. (1980b) further suggested that the formation of the polymeric molecules of protein resulted from the formation of intermolecular -S-S bonds in the heated actomyosin gel. Rheological Property and Structure of Kamaboko Yutaka et a1. (1981) studied the species variations in the gel forming Characteristics of fish meat paste. They 46 concluded that two reactions occurred in the gel-forming processes: (1) Suwari, a structure-setting reaction pro- ceeding at temperatures below 50°C, promoted specially at 30-40°C, and (2) modori, a structure-disintegrating reaction occurring at temperatures between 50 and 70°C, Optimum at 60°C. Their results agree with Cheng et a1. (1979) findings Based on these two phenomena the gel-forming characteris- tics of fish meat paste were summarized into the following 4 categories: (1) difficult-setting and difficult-disinte- grating group, e.g. meat from shark, chicken and rabbit, etc., (2) difficult-setting and easy-disintegrating groups e.g° the red meat fish, (3) easy-setting and easy-disinte- grating group, e.g. sardines, croaker and cold water fish, and (4) easy-setting and difficult-disintegrating group. Takagi (1973a) Studied the viscoelastic network struc- ture of kamaboko. He indicated that the kamaboko forming ability of brayed fish meat decreases with higher degree of modori, since modori of the brayed meat lowers the intensity of the entanglement between chain segments in kamaboko. Kamaboko prepared from undenatured brayed meat formed a cross-linked gel, while kamaboko prepared from brayed meat that has undergone a higher degree of modori resulted in a non-crosslinked gel. The average molecular weight of the network chain molecules in kamaboko de- creased with the degree of progressing modori phenomenon. 47 Takagi (1973b), in his further study, indicated that kamaboko is an irreversible hydrogel formed by heat dena- turation that contains some proteins other than the myofibrillar proteins. Myofibrillar proteins are distinc- tive components of the stable cross-linked network in kamaboko. Hhen brayed fish muscle is heated, it changes into a rubber-like kamaboko in which cross-linking and entanglements are formed. However, dense entanglements between network segments which are loosened in the earlier time scale of tensile stress relaxation are obstructed when water soluble protein coexists with the network-forming proteins. Miyake (1965) employed electron microscopy to observe the muscle destruction during the processing of fish sausage. He concluded that mechanical grinding could not disperse myofilaments from myofibril; and addition of sodium Chloride caused rapid dispersion of myofilaments from swollen myofibril. These facts suggested that in fish paste, swelled and well dispersed filaments are held together loosely; while in the heat coagulated product, protein filaments build a kind of network structure holding water, being connected with newly formed free radicals. Miyake et a1. (1971) studied the fine structure of kamaboko by electron microscopy. They found that the dispersed phase of kamaboko of high elasticity differs from those of low elasticity. Insufficient grinding of fish 48 paste before heating resulted in a kamaboko of less fine structure; in which the z-lines still remained and between them were found the aggregates of myofilaments. Sufficient grinding of fish muscle resulted in a finer structure of kamaboko; segments of the z-lines, aggregates of myofila- ments and fine filaments were well dispersed. These facts suggested that the myofilaments of the network structure might be the aggregates of polymers of myosin, binding with actin filaments which extend on each side of the segments of the z-lines. The mechanism of setting (suwari) and deterioration (modori) phenomena that take place in fish paste products was postulated by Niwa and Miyake (1971a). They found that the actomyosin was related to the setting phenomenon, in which no changes in the conformation of polypeptides were observed in either raw meat or ground meat; a Slight disordered arrangement of protein molecules was detected when meat was ground with sodium chloride, though a d-helical structure was retained. A part of the d-helical structure of polypeptides was transformed into a random coiled one at the advanced stage of setting. A possible scheme for gel formation at the setting process is illus- trated as follows (Niwa and Miyake, 1971b): 49 grindingw grinding . with NaCl ((((€“\ 1. d-hel1x in 2. orientation of 3. orienta- raw meat d-helix is not tion of disturbed the coil is dis- turbed, but con- formation of the coil does E not occur. ’ . CH3- -¢-H WC N H N- ° R- - i- 7 g sci incubation l R-C-H N-H at 38°C ‘ i;° 4. Trans conformation 5. Aggregation of the of a-helix to ran- polypeptide dom coil begins to chains. occur. 6. Formation of protein network, the d-helix of the chain still remains. 50 Tanikawa (1971) suggested that the elasticity of cooked fish gels are dependent upon several factors including the quantity and quality of myofibrillar proteins, fat content, fishing season and location, pH of the preblended fish muscle, kind of fish muscle (white or dark), physiological state of fish muscle (prerigor, on-set of rigor, or post rigor), freezing of fish and addition of ingredients or additives in the preblended fish muscle (e.g. sodium chloride, phosphate, sugar, starch and oil etc.). Tanikawa (1971) pointed out that fish of white muscle having high myosin and low fat content produces better gel elasticity than those of red muscle and/or fish from spawning season due to their high fat content. Addition of oil to the minced flesh or the fat from tissue itself tends to dilute the myosin content in the pre-blended fish paste or mixture that causes the weakening of gel strength. Products made from prerigor fish had better gel elasticity than those from rigor and post-rigor muscle. However, the elasticity of croaker and shark, is not weakened even after loss of freshness. Aged shark produced stronger gel elasticity than those from the fresh shark. This is probably due to their longer postmortem period and higher stroma protein content that affected the myosin extractability. The strongest elasticity is obtained from the fish muscle having a pH value between 6.5 to 7.0. The Optimum pH of dark muscle fish for the formation of gel elasticity is 6.2 to 6.7, and 51 for white muscle fish is 7.0 to 7.5. The basic amino acids having pH values of 7.5 to 8.1 up to 0.2 to 0.5% of the total fish paste provides stronger gel elasticity. Freezing of fish caused denaturation of muscle proteins and loss of water holding capacity; as a consequence, it brought about reduced gel forming ability. Addition of 3% salt and 0.3% sodium polyphosphate increased the extractability of myo- fibrillar proteins, therefore, bringing about enhanced gel elasticity. Addition of starch and/or gluten enforced gel strength. Excessive disruption of myofibrils by the grinding action or mechanical mincing resulted in decreased water holding capacity which reduced gel forming ability. EXPERIMENTAL Food Materials and Ingredients Nhite suckers used in this study were harvested commer- cially by trap net from Saginaw Bay, Lake Huron. Two batches were purchased from Bay Port Fish Co., Bay Port, Michigan on November 18 and 22, 1980. Suckers were boxed in ice for O to 3 days at time of purchase, were re—iced and trans— ported without additional refrigeration to the Meat Labora- tory of the Michigan State University. Suckers were stored at 2°C and processed the next day. Most suckers weighed in the range of l to 2 pounds. The total suckers from each batch weighed approximately 250 pounds. Suckers used for fractionation of myofibrillar proteins in experiment I were boxed in ice while they were still living, and immediately shipped within 3 hours to the food laboratory of the Michigan State University, afterwhich they were stored at 2°C for 0 to 6 days. Most of the dry ingredients were obtained from the local stores. Potato starch was purchased from Randall Health Foods, East Lansing, Michigan. Sucrose, salt, mono- sodium glutamate and soy sauce were bought from Meijer Thrifty Acres, Okemos, Michigan. Instant onion and lamb 52 53 sauce were donated by Dr. L.J. Minor from L.J. Minor's Corp., Cleveland, Ohio. Sodium hexametaphosphate was donated by FMC. Co., Philadelphia, Pennsylvania. Chemicals and Laboratory Materials Solvents and Chemicals All chemicals used in this study were reagent grade. All solvents and acids were freshly redistilled before use. They were all purchased from the general store of Michigan State University. Reference Standards Standard mixture of fatty acid methyl esters, standard polar lipid mixtures, lyso polar lipid mixture, and B-choles- tane standards were Obtained from Supelco Inc., Bellefonte, PA. Both high and low molecular protein standard kits were obtained from Pharmacia Fine Chemicals. Standard protein solution for Biuret analyses was obtained from Sigma Chemical Co., Missouri. Hypoxanthine anhydrous and xanthine oxidase were obtained from Sigma Chemical Co. where hypoxanthine anhy- drous was used to prepare a series of hypoxanthine standard solutions. 54 Others Column packing materials for gas liquid Chromatography were obtained from Supelco Co. Precoated Silica gel G plates for thin layer chromatograph (TLC) were obtained from Applied Science Laboratories Inc. Methanoliobase and BF3- Methanol were obtained from Supelco Co. . Methods This study is divided into two parts. The first part of this experiment was conducted to determine the effect of the freshness of sucker flesh as well as the washing tech- nique on the extractability of salt soluble proteins, and their composition as well as the gel forming ability of the minced sucker flesh. The second part of this study involved the effect of washing technique on the lipid composition, Cholesterol level, as well as TBA values; and further deter- mination of the effect of additives and frozen storage on lipid stability of both raw fish pastes and cooked fish balls during frozen storage at -23°C for six months. Duplicate determinations were performed for all chemical tests. Experiment I The sucker flesh used in this part of the study was prepared from five suckers daily by a hand-deboning operation. The deboned sucker flesh was ground through a Kitchen Aid Meat Grinder, Model K-SA. The freshly ground sucker flesh 55 was immediately assigned for hypoxanthine test and extrac- tion of myofibrillar proteins. The sucker flesh used for gel forming ability was held in a bag made of 4 layers of cheese cloth and washed in a Kenmore household clothes washer, using a gentle cycle and spun at regular cycle. The purpose of washing and spinning was to remove the sarco- plasmic constituents, blood residues, unbound lipids and the excessive water since these materials are factors that would weaken the gel strength of the cooked fish paste. The washed sucker flesh was blended with salt, monosodium glutamate, sucrose, sodium hexametaphosphate and potato starch; afterwhich the blended fish paste was made into fish balls following the procedure and formulations shown in Figure l and Table 2 (Experiment II). Moisture Analysis Moisture content determination for unwashed sucker flesh was carried out in a Hotpack vacuum oven, model 633, at 100°C and a vacuum of 27 in Hg following the official AOAC method (1975). Samples were dried to a constant weight 56 for 10 hrs and the dried weight was converted to the per- centage of the wet weight. For the routine test, the moisture content of the washed minced sucker flesh was determined in a microwave oven in order to obtain a value that is Close to the unwashed sample (10.5%) at a rapid speed, so that the processing of fish paste could be con- tinued soon after washing. Quantitation of Total Sglt Extractable Muscle Protein The determination of total salt extractable muscle proteins from ice-stored suckers was conducted according to the method described by Iwata and Okada (1971) as follows: duplicate portions of 20 g coarsely minced sucker flesh were homogenized with 180 m1 of 0.45 M KCl-phosphate buffer solution (I = 0.5, pH 7.2) for 90 sec, respectively. The homogenates were allowed to stand at room temperature for one hour; after which, the homogenates were centrifuged at 15,000 xg for 30 min and filtered through 8 layers of cheese cloth. The total volume of the supernatant was measured and recorded. Total protein concentration in the supernatant was determined by using micro-Kjeldahl analysis. Fish Muscle Protein Fractionation The procedure for fish muscle protein fractionation was that reported by Cheng et al. (1979). Duplicate samples were extracted twice. Extractions were carried out at 20 to 3°C with cold extracting solutions. 57 Three solutions were prepared for muscle protein frac- tionation: solution A contained 0.1 M NaCl and 0.02 M sodium phoSphate buffered at pH 7.0; solution B contained 0.6 M NaCl and 0.1 M sodium phosphate buffered at pH 7.0; solution C contained 20% trichloroacetic acid (TCA). Sarcoplasmic proteins were prepared by homogenizing 20 g minced sucker with 200 ml solution A for three 15 second periods at 5 second intervals and centrifuging at 15,000 xg for 15 minutes. The homogenates were filtered through 8 layers of cheese cloth. The supernatant contained mainly sarcoplasmic proteins and minor non-protein-nitrogen materials. The residues which remained on the cheese Cloth were suspended in 150 m1 of solution A for 30 min at 4°C. The suspensions were centrifuged at 15,000 xg for 20 min and filtered through 8 layers of cheese cloth. The second supernatant was collected and combined with the first super- natant. The total volume of supernatant was measured and recorded. Myofibrillar proteins prepared from the residue of sarcoplasmic fractionation were suspended in 200 ml of solution B and extracted for 3 hours at refrigerated tem- perature by placing the container in a sodium chloride-ice slurry bath. The solutions containing muscle protein residues were stirred constantly with a magnetic stirrer. They were centrifuged at 15,000 xg for 15 min and filtered through 8 layers of cheese cloth. The supernatants were 58 collected, and the residues were resuspended in 100 ml solution B for another 30 min with constant stirring in the same manner as the former step. The second supernatants were collected and combined with the first one. The total volume of myofibrillar protein supernatant was measured and recorded. Non—protein-nitrogen (NPN) was prepared by mixing the sarcoplasmic supernatant with an equal volume of 20% TCA. The mixture was allowed to stand overnight in the refrigera— tor and filtered through 8 layers of cheese cloth. The supernatant contained NPN. The total volume of NPN was measured and recorded. The nitrogen content of the sarco- plasmic fraction, NPN, and myofibrillar proteins were deter- mined by the micro-Kjeldahl method (AOAC, 1975). Preparation of Salt (Sodium Chloride) Extractable Protein for Sodium Dodecylsulfate (SDS) Polyacrylamide Gel Electro- phoresis Two portions of 40 g minced suckers were extracted with 200 ml 0.17% NaCl solution and stirred constantly with an electric magnet stirrer at 4°C. The suspensions were filtered through 8 layers of cheese cloth and washed with 400 m1 of 0.17% NaCl solution while filtering. The super- natant was discarded. The residues were then resuspended with 200 ml of 3.9% salt solution and homogenized in a Haring blender for 30 seconds. The homogenates were 59 centrifuged at 14,500 xg for 20 min and filtered through 8 layers of cheese Cloth. The supernatants were collected. The residues were resuspended in 200 ml 3.9% salt solution with constant stirring by a magnetic stirrer for 5 min at 4°C. The solution was centrifuged and filtered and the supernatant was collected. Both supernatantswere combined. The total volume of the supernatant was measured and recorded. The protein concentration of the salt extractable pro- tein was determined by the Biuret method described by Layne (1957). The freshly extracted salt-soluble protein was mixed with an equal volume of glycerol and stored at -18°C for sodium dodecylsulfate polyacrylamide gel electrophoresis (SOS-PAGE). SOS—PAGE was conducted by the method Of Weber and Osborne (1969). The following solutions were prepared for SOS-PAGE: (1) Tris-glycine stock solution (0.5 M Tris and 1.5 M glycine), (2) 25% acrylamide, 0.25% N,N-methylene- bisacrylamide (Bis) solution, (3) 2.5% sodium dodecylsulfate (SDS) solution, (4) 1% ammonium persulfate solution.(5) chember buffer solution (0.1% SDS, 0.2 M Tris-glycine, pH 8.8), (6) tracking dye solution, (7) staining solution and (8) destaining solution. The detailed methods and formula- tions of these solutions are shown in Appendix A. Acrylamide (10%) gels were prepared by using 10 ml 25% acrylamide Bis, 5 ml tris-glycine buffer, 1.25 ml glycerol 60 and 6.75 ml of deionized water was pipetted into a 40 m1 glass-stOppered flask which was connected with a vacuum degassing apparatus. The mixture was well mixed by an electric magnetic stirrer during degassing. After degassing, the following solutions were added inthis order: 1 ml 2.5% 505, 0.01 ml TEMED and 1 ml 1% ammonium persulfate. The solutions were mixed gently by swirling briefly to avoid formation of air bubbles and foam. The solution was immediately transferred to the treated glass tubes by using a 10 ml syringe. The tubes were filled up to a height of 8 cm. Tubes with gels were carefully overlayed with water to about a height of 1 cm, and were allowed to polymerize without disturbing for 20 to 30 minutes. After the gels had solidified, the top water was decanted. The tube- stoppers were removed from the bottom of the tubes. Protein solution for SOS-PAGE was prepared by mixing the salt soluble proteins and glycerol and diluted with tracking dye buffered solution to contain 0.4 mg protein/ml. The tracking dye-protein mixture was heated in a boiling water bath for 5 min. Standard protein mixtures of both high and low molecular weight were prepared in the same manner as the salt soluble proteins. The chamber buffered solution was filled to two-thirds of the lower chamber. The tubes containing polymerized gels were loaded on the electrophoresis chamber. The same buffered solution was filled in the upper Chamber to 1 cm above the tubes. All 61 the air bubbles that formed on the upper end of the tubes were expelled carefully by using a glass-rod without breaking the gels. Each gel was then loaded with 30 ul sample. The entry of the sample into the gels was conducted at a current of 0.2 mA per gel. After the dye had completely entered, the current was increased to 0.5 mA per gel and the migration continued until the dye front reached to 1 cm above from the end of the tube. It took approximately 10 to 12 hours for the total run. The gels were removed from running tubes by squirting water from a syringe between the edge of the gel and the tube wall. They were immersed in staining solution for 10 hours, followed by destaining in a diffusion chamber Model 172A for 3 days. Gels were scanned using a Beckman DB-G Grating Spectro- photometer Model 2400 equipped with a Gilford Gel Scanner (Model 2520) and a Photometer 252. This system was connected with a Hewlett-Packard Integrator (Model 33805). The gels were scanned at a rate of 1.0 cm/min and a chart speed of 2.0 cm/min start delay and slope sensitivity settings were 0 and 3.0 mV/min, respectively. SOS-PAGE gels were scanned at a wavelength of 550 nm. The relative areas of individual protein peaks were recorded. The relative mobility of the bands was assessed from the total length of the gel, tracking dye migration distance and from the distances migrated by individual proteins. A standard molecular weight calibration curve was established by plotting the log 62 molecular weights of standard proteins versus their relative mobilities on semi-log paper. The unknown proteins were identified by comparing their relative mobilities to that of the standard proteins. Experiment 11 Sucker flesh used for experiment 11 was prepared by using a mechanical deboner as described in the following section. Treatments for this experiment were designed to assess the lipid stability among the washed samples pre- blended with salt, sucrose, monosodium glutamate (MSG). sodium hexametaphosphate (SHMP) and potato starch as Shown in Table 3. The shelf life of the raw fish paste and/or cooked fish balls were monitored by analyses of lipid oxi- dation using TBA test and chromatographic methods, in a system, where lipids and proteins play a concurrent action during frozen storage. In addition, preliminary experiments were conducted to quantitate the effect of washing on the lipid compounds and stability. Preparation of Minced Suckers and Mechanical Deboner Operation Suckers were manually deheaded, gutted and split dorso- ventrally parallel to the backbone. The split suckers were washed under cool running tap water, using a hand brush to facilitate removal of liver, intestine and kidney material. They were immediately layered with crushed ice until being deboned within half an hour. 63 The mechanical deboner was a Bibun Model 5013 (Bibun Co., Fukuyama Kiroshima, Japan) belt type machine equipped with a 3 mm perforation size drum. The dressed suckers were passed through the machine flesh side to the drum and the minced flesh was recovered in a plastic lug. The minced sucker was passed twice through the deboner in order to insure that a minimum of small bone residue and scales remained in the minced flesh. The minced flesh yield after mechanical deboning was approximately 49% of the round weight. Hashing of the Minced Sucker Flesh Samples of minced flesh were collected after the second pass through the mechanical deboner (unwashed samples) and analyzed for moisture, total fat, protein, nonprotein nitro- gen and TBA. The remaining minced sucker flesh was weighed into 2.5 lbs aliquots and placed in bags made Of 4 layers of cheese cloth. Four bags of minced sucker were washed per load in a Kenmore household clothes washer using a gentle cycle and spun at regular cycle. Washing and spinning not only removed the excessive amount of water but also removed the undesirable sarcoplasmic constituents, contaminants such as blood and internal organs, and the unbound lipids which cause degradation of both protein and lipid components in the sucker flesh. COnsequently, it twingsabout the weakening of the gel strength and undesirable flavor of the cooked 64 products. The washed samples were stored at 2°C in the refrigerator for further processing into fish paste and fish balls. These products were vacuum packed in one pound lots in cryovac (polyvinylidene Chloride) bags which were then stored at -23°C for O to 6 months. The average yields of washed samples were approximately 55% of the minced sample. Analyses of the minced unwashed sucker flesh Showed a moisture content of 80.75% and a protein content of 16.00%. Formulation of Fish Paste and/or Fish Balls In this study, the formulation of fish pastes (Table 3) was derived from the formulation of Kamaboko as shown in Table 2. Kamaboko is a steamed Japanese style fish paste product. lhmng and Jeng (1979) reported that fish balls con- taining less than 6% starch and less than 4% sugar and fat were highly accepted by sensory evaluation. Products with 3% salt were indicated too salty by pretesting panels. Therefore, in this study, the level of salt was adjusted to 2.5%. This level of salt allowed extraction of 95% total myosin in the fish paste system. According to Noguchi and Matumoto (1970), 0.3 M Na-glutamate is as effective as l M glucose in preventing the denaturation of myofibrillar pro- teins as well as in producing high gel-strength of kamaboko. The preventive effect of Na-glutamate on protein denaturation was found to approach the maximum beyond 0.025 M (0.42%). By computing the 0.025 M ratio Of Na-glutamate to sucrose, a 65 Table 2. Formulation of kamaboko. Ingredients %/100 9 fish Minced fish 100.00 Salt 3.01-3.15 Sodium glutamate 0.21-0.40 Sugar 4.53-14.72 Starch 3.15-14.72 Sweet sake 2.40 Egg white (or 8.53 Polyphosphate) 0.2-0.3% Water 0% if starch is 10% 5% if starch is 15 to 20% aTanikawa (1971). Table 3. Formulation of fish paste and/or fish balls. Ingredients %/100 g minced fish Washed, minced sucker Salt (sodium chloride) Monosodium glutamate (MSG) Sucrose Sodium hexametaphosphate Potato starch 100.00 2.50 0.40 2.00 0.30 10.00 66 0.08 M (2.74% sucrose has an equivalent effect as 0.025 M Na-glutamate. In this study, the formulation of Chinese style fish paste was adjusted for these requirements as previous researchers had suggested. Formulation of raw fish paste and/or fish ball is shown in Table 3. According to the Food Chemical Codex, 0.3% sodium hexametaphosphate is under the maximum allowance (0.5%) as a food additive by USDA regulations (Code of Federal Regulation, 1971). At this level, it gives the maximum swelling ability of myo- fibrillar proteins in a gel type product. Processing of Fish Pastes and Fish Balls There are 3 consecutive steps of processing fish pastes as shown in the following flow chart (Figure l). Duplicate samples of four portions of 2,400 g washed minced sucker flesh were blended with four series of mixed additives (Table 4) separately for preparing fish pastes. These 4 additive mixtures are: (1) 2.5% sodium chloride, 0.4% MSG, 0.3% sodiunlhemmetaphosphate, 10% potato starch, (2) 2.5% sodium chloride, 0.4% 115(3, 2% sucrose, 10% potato starch, (3) 0.4% M556, 2% sucrose, 0.3% sodium hexametaphosphate, 10% potato starch, (4) 2.5% sodium chloride, 0.4% MSSG, 2% sucrose, 0.3% sodium hexameta- phosphate and 10% potato starch. Duplicates of 7.000 g washed minced sucker flesh were also blended with mixed additives of 2.5% sodium Chloride, lst stage 2nd stage 3rd stage Figure 1. 67 minced sucker flesh wash and drain washed minced sucker blend t 3 min gel-like paste add sodium chloride (salt) I add hexametaphosphate, monosodium glutamate and sucrose blend for another 2 min firm gel-like paste 1 add potato starch blend to just well-mixed, about 1.5 min 1 firmer gel-like paste——1 small ball- in diameter) boiled at the internal reaches 85°C Sapproximately 15 min). 1 formed into shape (3 cm vacuum packaging in dough, then cryovac bags 100°C unt11 I temperature Cool to room temperature Flow chart for processing fish paste and fish balls. 68 pcmspmmsm pom; use mrpmm mpcdwemaecw _Fe cow: o.o_ m.o o.N e.o m.~ swan uuxooo mpcmwedtmcw PPM 50.: o.o_ m.o o.N e.o m.~ ea _umz “segue: o.o_ m.o o.~ «.0 - me macadam pzoepwz o.o_ m.o - e.o m.N Na azzm “soguwz o.oP - o.N 4.0 m.~ Pa mopmma shad 2mm Aazzmv manganese Awmzv APuazv ucmspmmch cugmum -mummem: mpmsmp:_w mvwgopgu ucmspmmgp mo cowgmcwaeou owopod Ezruom mmoguzm E=PGOmocoz Ezwuom Low muou cmxusm vmucw: a cop so; pcmwvmgmcH ucmogmm .mmpmma meoam umocwe nmgmmz co acmEpmmgu mo cmwmmo .e mpnme 69 0.4% NISG, 2% sucrose, 0.3% sodium hexametaphosphate and 10% potato starch for making fish balls. Minced fish, additives and starch were blended in a Kitchen Aid mixer (Model KS-A), at 168 rpm Speed for fish pastes. Ingredients for fish balls were blended in a Kitchen Aid Mixer (Model N-50, Hobart Mfg. Co., Troy, Ohio). At first stage, the minced flesh and sodium chloride were blended for 3 min or until a gel was developed which ensured all the myofibrillar proteins, especially myosin was extracted by salt. At the second stage, hexametaphosphate, sucrose and monosodium glutamate were added, blending continued for another 2 minutes to allow the swelling of muscle proteins and even distribution of the myofibrillar proteins throughout the entire network of the gel-like paste. At the third stage, potato starch was added to enhance the gel strength of the fish paste. Extraction of Total Lipids The procedure of extraction of lipids from minced sucker flesh was essentially based on the method of Bligh and Dyer (1959), modified by Peng and Dugan (1965) who adapted the washing system from Folch et a1. (1957). The samples of frozen sucker pastes and fish balls were defrosted in a microwave oven for 4 min or until the internal temperature reached 2°C right before lipid extraction. Each 100 g of freshly minced sucker (both washed and unwashed), unfrozen 70 fish pastes and fish balls, and defrosted samples were homo- genized in a Haring blender for 2 minutes with a solvent system containing 100 ml of chloroform, 200 m1 of methanol, and deionized water to make the total moisture content equivalent to 80% of the sample weight. Then 100 m1 of chloroform was added to the mixture. Blending continued for 30 seconds, 100 m1 of deionized water was added and blending continued for another 30 seconds. The homogenate was filtered through Nhatman No. 1 paper on a porcelain Buchner funnel which was fitted on a filter flask with slight vacuum suction to ensure maximum recovery of solvent. The homo- genate residue and filter paper were blended with 200 ml of chloroform for 1 minute and filtered through the same Buchner funnel. The blender was then rinsed with 50 m1 of chloroform and 40 ml of methanol, separately. The collected filtrates were transferred quantitatively to a 1000 ml separatory funnel. An0.2% potassium chloride solution (0.74% w/v) of the total filtrate (v/v) was added to the filtrate and mixed well to facilitate the separation of the water soluble protein component from the fat soluble phase. The mixture was left in a freezer at -23°C overnight for complete extraction and separation. The lower phase, comprised mainly of chloroform soluble lipids, consisted approximately of chloroform: methanolzwater = 86:14:l. The lower phase was collected and filtered through a glass funnel lined with No. 4 Nhatman filter paper containing approximately 30 g of anhydrous 71 sodium sulfate into a glass stoppered round bottom flask. The upper phase (mainly water soluble components and minor methanol soluble lipid components) was washed with 3 por- tions of 30 m1 chloroform, the lower chloroform phase was collected and filtered in the same manner as in the first chloroform soluble phase. These two extractions were combined. The total volume of the chloroform phase was measured and recorded. Duplicate 10 ml aliquots were taken from the chloroform-lipid extracts into weighed Erlenmeyer flasks, the solvent was evaporated by allowing the Erlen- meyer flasks to stand on the top of a steam bath until the volume was reduced to approximately 1 ml, and the flasks were placed in a vacuum oven at 70°C for 2 hours to complete the solvent evaporation. The flasks with fat residue were then put into a desiccator over calcium chloride for 24 hours and weighed. The percentage of lipid content in the extracts was computed from their weight differences. Concentration of the Lipid Extracts The chloroform phase was held in a 500 ml round bottom flask and evaporated by a Rinco rotating high vacuum type evaporator almost to dryness, and transferred to a weighed glass vial fitted with a teflon lined screw cap. The flask was rinsed with small amounts of chloroform with the aid of a disposable pipet, and combined with the concentrated lipid in the same vial. The lipid containing small amounts Of 73 Preparation of silicic acid columns. For each column (2.5x25 cm.) a 60 m1 of silicic acid slurry was prepared by mixing 30 g of activated silicic acid and 60 m1 of chloroform in a filtering flask with the aid of a magnetic stirrer until a homogeneous translucent mixture was obtained. The silicic acid Slurry was then degassed by a vacuum set-up which consisted of an additional trapping flask, to avoid water entering into the flask containing the silicic acid slurry. The column containing 10 ml of chloroform was packed with degassed slurry, by pouring the slurry Slowly into the column along the edge of a glass rod, in such a way that no air bubbles would be trapped. The silicic acid was allowed to settle and elute first with chloroform, followed with methanol and finally with chloroform, to check whether undesirable air bubbles and channeling existed in the column. A thin layer of glass-wool and 2 cm layer of anhy- drous sodium sulfate were laid on top of the silicic layer. The column was ready for lipid fractionation. Since the packed silicic column should never be allowed to dry out, the solvent layer was kept 3 cm above the sodium sulfate layer, so that the undesirable channeling could be avoided. Approximately 0.5 g of lipid extracts wereredissolved in 1-2 ml of chloroform and applied onto the column. The loading capacity of silicic acid for lipid is approximately 0.02 g lipid per gram silicic acid. The column flow rate was adjusted to approximately 3 ml per minute by applying a 74 constant pressure with a stream of nitrogen gas on top of the column. Neutral lipids were eluted first with chloroform until a negative Salkowski test was achieved, in which 1 ml of chloroform-lipid eluent was carefully added to an equal volume of concentrated sulfuric acid in a test tube. The development of a characteristic yellow to brown band in the test tube indicated the presence of lipid in the chloroform. Acetone, which acted as a scavenger for oxidized materials (Peng, 1965), was eluted next to remove pigmented materials. Phospholipids were eluted with methanol until a negative ninhydrin test was reached. Purity of neutral lipid was further tested by the ninhydrin test, and the phospholipids by micro thin layer chromatography with a developing system containing petroleum etherzethyl etherzacetic acid (90:10:l by volume). The Slides were sprayed with sulfuric acid followed by charring. Migration of the original spot, indi- cated presence of neutral lipids. The ninhydrin test was done by adding ninhydrin to the methanol eluant in a 15 m1 vial, covered with a teflon screw cap, placed on a block of heating module at 150°C, and Shaken vigorously periodically. The development of a purplish color indicated the presence of phosphatidyl ethanolamine and its lyso derivatives. The neutral and phospholipid fractions were used to prepare fatty acids for esterification, or dried for gravi- metric determination of total neutral and phospholipids. 75 Total lipid was calculated from the sum of these two values. Classification of Phospholipids Approximately 100 mg of phospholipids were dissolved in chloroform and separated into their components using preparative thin layer chromatography (TLC). Twenty micro- 1iter of phospholipid solution were Spotted on each plate using a stream of nitrogen gas using a Hamilton micro- syringe. A standard polar lipid mixture (cholesterol, phosphatidyl ethanolamine, phosphatidyl choline) and standard lysopolar lipid mixture (lysophosphatidyl ethanolamine, lysophosphatidyl serine, lysophosphatidyl choline) were spotted on the right hand side of the plate. The plates were developed in a standard TLC developing tank saturated with solvents containing chloroform:methanol:waterzacetic acid - 60:30:5:O.5. After the solvent had ascended 17 cm on the plates, the plates were removed from the TLC tank and allowed to dry at room temperature under the ventilation hood. The spots of lipid components were detected by spraying with different indicators: e.g., ninhydrin solu- tion for phosphatidyl ethanolamine and Dragendorf agent for choline (Skidmore and Entenman, 1962). The relative Rf values in this developing system were found to be 0.80 for phosphatidyl ethanolamine, 0.47 for phosphatidyl choline (lecithin), 0.15 for lysophosphatidyl ethanolamine, and 0.07 for lysophosphatidyl choline. 76 Preparation of Methyl Esters Methylation of total lipids, neutral lipids and phos- pholipids from all minced sucker products were conducted according to the method described by Morrison and Smith (1964). Lipids were converted to methyl esters by treatment of lipids with boron trifluoride-methanol reagent (14% BF3 in methanol), in which BF3-methanol acts as acid catalyst in the esterification of fatty acids. The methylated fatty acid esters was preserved under nitrogen and covered tightly and stored at -23°C before injecting into a gas Chromato- graph. Total Cholesterol Determination Total cholesterol level present in total lipid extracts was determined by using the base-catalyzed trans- esterification method according to Luddy et a1. (1960, 1968). Anhydrous methanolic-base (0.5 N sodium methoxide) obtained from Supelco Inc. was used for transesterification of the lipid sample, in which methanoliC-base acted as catalyst converting cholesterol into Cholesteryl esters, glycerides and phospholipids quantitatively to their methyl esters. The procedure is as follows: into a 15 ml teflon lined screw cap vial were placed 30 mg of total lipids, 1 ml of benzene and 1 ml of the methanolic-base reagent. The vials was sealed and heated in a temperature block heater at 80°C for 20 min and removed from the heating block. The 77 vials were allowed to cool to room temperature, and 3 m1 of deionized water and 3 ml of diethylether then were added and the resulting solution was mixed. The mixture was allowed to stand until two layers of separation were clear. The top benzene-ether layer was pipetted into another Clean vial and was once more washed with 3 ml of deionized water; afterwhich the top phase was dried over anhydrous Nazso . This extract 4 was ready for analysis by GLC. Gas Chromatography Analyses of Methyl Esters The qualitative determination of the components of fatty acid methyl esters and cholesterol ester were conducted using a Hewlett-Packard Model 5830A gas chromatograph equipped with a Hewlett-Packard k8850 Terminal. Nitrogen was used as the carrier gas at a flow rate of 30 ml per minute for analyzing fatty acid and 40 ml per minute for Cholesterol. Temperature was programmed from 150° to 210°C at a rate of 2°C per minute for fatty acid analysis, choles- terol was analyzed at 260°C isothermally. Temperatures for flame ionization detector and injection ports for fatty acid determination were maintained at 350°C and 210°C respectively; while the temperatures for cholesterol were 350° and 260°C, respectively. The components of the fatty acid methyl esters were reported as % area of the total lipid esters. A sample of 0.5 ul was injected for each analysis. Fatty acid methyl esters were identified by comparing their relative retention time with the mixture of standard 78 fatty acid methyl esters of marine source; or by comparing their relative retention times (relative to methyl palmi- tate), using a plot of logarithm of the relative retention time versus the number of carbon atoms. The percentage of each fatty acid ester was computed by dividing the individual peak area by the sum of total peak areas. 2-Thiobarbituric Acid (TBA) Test A distillation method for quantitative determination of malonaldehyde in rancid foods from Tarladgis et a1. (1960) was used to determine the oxidative rancidity of minced sucker products. Interfering yellow and Orange colors were found in the malonaldehyde-TBA distillates at the second month of frozen storage, indicating that interactions of carbonyl compounds with either carbohydrates or proteins had occurred. Therefore, separation of yellow and orange pigments from the pink malonaldehyde-TBA reactant was conducted according to the method described by Yu and Sinnhuber (1962), prior to measuring their optical density by a Beckman DB-G grating Spectrophotometer. Evaluation of Gel Strength Related Internal Structure Gel strength was determined by using the Allo-Kramer Shear Press, Model SP12, equipped with a Food Technology Inc. Texturecorder, Model TR3. Three shear press measure- ments of coOked fish balls were determined on cooked fish 79 balls made from suckers stored in ice from O to 6 days. Unfrozen fish balls from experiment I were prepared in the afternoon, boiled, cooled, packed in polyvinyl bags and stored at 4°C overnight (10 hours). Two fish balls weighing from 46 to 65 g were placed side by side into the cell per- pendicular to the slots. A 3000 lb maximum load transducer was used with a range setting of 10 and a downward stroke of 30 seconds. Pounds of force per gram of sample required to shear were calculated as follows: 3000 lb peak height Pounds of force = 10 x 100 g sample sample weight (g) The frozen fish balls from experiment 2 were measured the same way for 6 consecutive months of frozen storage. On each month, samples were defrosted in a microwave oven setting for 3 min before measuring the Shear press values. Sensory Evaluation Fifteen taste panelists (7 ethnics and 8 Americans) from faculty, staff and students at the Department of Food Science and Human Nutrition evaluated fish balls at the 0, 3rd, and 6th month of storage. Fish balls were sliced, stir-fried and seasoned with soy sauce, lamb sauce and instant onion right before serving. Each panelist was asked to rate the samples from 2 batches on the following parame- ters: off odor and flavor, textural resilience, and 80 acceptability on a 9-point scale (Appendix C). Statistical Analysis Analyses Of variance were computed by submitting raw coded data into STAT 4 packaged program interfaced with the CDC cyber 7500 system at Michigan State University. Bonferroni t statistics and Tukey's test statistics were used to compare the test statistics of mean values among different treat- ments. Possible correlations among proximate composition of fish muscle proteins and shear force data were determined by measuring the product-moment correlation described by Gill (1978). RESULTS AND DISCUSSION The Effects of Freshness of Minced Sucker Flesh and Hashing Technique on the Extractability of Myofibrillar Proteins and the Gel Forming Ability of Fish Pastes. Identification of salt extractable myofibrillar proteins in sucker flesh. Figure 2 shows the relative mobility of 5 high and 4 low molecular weight proteins used as standard, obtained by sodium dodecyl sulfate-polyacrylamide gel electrophore- sis (SDS-PAGE). The calibration curve was constructed by plotting relative electrophoretic mobility (Rf) vs. log subunit molecular weights of the myofibrillar proteins from sucker flesh. The molecular weights of the unknown proteins were determined by comparison of the electropho- retic mobility of each unknown protein with the mobility of standard proteins of known molecular weight (Appendix A2). According to this procedure eight major myofibri- llar proteins were identified in sucker flesh as shown in Table 5. Figure 3 illustrates the scanning of a SDS- PAGE gel with the major protein bands showing different peaks. The relative concentrations of the myofibrillar 81 82 mmmcomoeuzsmc mmmpumd n o mmmpmamu n m :mE=n~< n e cwsanmwom_r n m a mmmpxeosamosm n.m couwnwzcw :Pmogzpn w :wuwggmu n N mmmgcxgcm owconemun u cwpznopmocagp n _ .w.w In .me mumEmngumz—oa mop m :o mwg=u cowumcnw—mu oé m6 m.o ~.c m.o m.o. ¢.o m.o ~.o P.o cameo peeve came»: mcwmuoca ccmucmum mcwmuoea emppwgnwmozz H :mmozsoaoeala + C d d I d I d u d :Pmoxsomoeprm :wpu< :wuumuw m+ xmpasou :wmoxsonogp 14A+4 cwcwuomno :wmyogauu :wmpoealz cwmnu A>mmz :wmoxz N+ F + 4 .N mesmwm ooo.op ooo.om m. ooo.om mw m. ooo.oe mm 25.8 m. ooo.om mu. 25.2 M 25.8 m. ooo.om MW ooo.oopa+ ooo.oo~ ooo.oom ooo.oo¢ 83 Table 5. Relative electrophoretic mobility and approximate subunit molecular weight of the major myofibrillar protein in the washed and the unwashed minced sucker flesh that identified on SOS-PAGE . Components 0f salt Electro- Molecular Unwashe Hashed extractable proteins phoretic weight of sample samplez mobility subunits % % (Rf) (daltons) Myosin heavy chain 0.11 200,000 18.32 21.76 Unidentified protein 0.14 180,000 1.87 2.30 Unidentified protein 0.15 170,000 1.45 1.37 Unidentified protein 0.16 165,000 1.96 1.98 M-line protein 0.18 155,000 1.55 1.92 C-protein 0.21 140,000 0.13 0.82 Unidentified protein 0.23 125,000 2.08 5.88 Unidentified protein 0.25 115,000 1.36 - o-Actinin 0.27 100,000 0.70 0.97 Unidentified 0.31 94,000 2.39 0.51 Tropomysin complex 0.38 70,000 0.88 - Unidentified 0.41 68,000 1.70 0.43 Unidentified4 0.44 57,000 16.80 23.31 G-Actin 0.49 47,000 11.82 2.15 G-Actin 0.53 42,000 9.92 10.91 B-tropomyosin 0.57 35,000 4.62 6.01 o-tropomyosin 0.65 32,000 3.55 5.07 Myosin light Chain 0.72 20,000 5.15 6.59 Myosin light chain 0.82 18,500 6.74 7.88 Unidentified protein 0.87 14,000 7.01 0.13 110% polyacrylamide gel in 0.1% SDS. 2The unwashed sample was extracted with 3.9% NaCl solution with ionic strength equal to 0.66. 3The washed sample was first extracted with 0.17% NaCl solution to remove mainly water soluble components and followed by extraction with 3.9% NaCl solution. 4Possible contamination of the sarcoplasmic reticulum pro- teins and vimentin (a Z—disc protein). '(sasauauaued u; uaAIB st pueq uLaqoud patgtauapt uoea do; saufiLaM Jelnoalom anewtxoudde aul) alosnm aaxons paoutm pausemun pue pausem won; spueq utaaoud uoCem aua go uotaeatjtnuapt 011M slab ang-sgs Io Butuueos 'g eunfitg 84 Washed sample Unwashed sample -————:22: Start to scan (beg1nn1ng 0f the gel) iii: Myosin Heavy Chain g 5;... (200,000) M-line Protein (155, 000) C- -protein (140, 000) a—actinin (100,000) Tropomyosin Complex (70, 000) Unidentified (57,000) G-actin (47,000) Actin (42,000) B-tropomyosin (35,000) o—tropomyosin (32,000) Myosin Light Chain (20,000) Myosin Light Chain (18,500) Unidentified (14,000) Stop to scan (the end of the gel) 85 pm newest? 323C295 98 5:38 9:3,. 9: co 338233 #53:; 5.; 3.33 .mo.cvd . mmmmnunm covuumcm uwsmmpaoogmm Eoee cowuzpom ovum owpmumoLo—guwcu xom sow: umpomsuxme .om.onH .0.“ :a pm Cowman mumgamoga z_.o use Pumz 20.0 cue: umuumeuxmm .mo.ouH .o.~ In on eoeoeeae demeamoea seweom z~o.o eea Fonz z_.o gem: eoeoeeoxmm .o.m :8 .m.onH cowu=FOm commas mpmcamognrpux zmv.o sue: cmuumeuxmp mm.o heme.o_ mm.o Ammn.m mo.o Ampp.qm m~.o Hoom.mm m No.0 Home.op mo.o hmmp.m Fm.o Amun.mm Fm.o Aueo.mm m m_.o Hamm.op mp.o Hmm¢.w om.o Hamm.mm mp.o Ammm.wp e m~.o Ammm.op mm.o num¢.op mm.o Ammp.¢m mm.o Ame~.m_ m n_.o neum.o_ N_.o Hamm.pp o¢.o Ammo.¢~ Pp.o “www.mm N mp.o Hmmm.o_ _N.o humo.m_ mm.o Hmo_.em om.o Aauo.Fm P _F.o.nm<¢.o_ mm.o Amvm.op mm.o hmm~.m~ mm.o Ammo.¢e o x . ammoguw: x .mcmmoeuw: :_muoca N.Ncmmoepmc cwmpoeq N . :mmocpwc evapoea mow pm :wmpoeaucoz gmppwcnweoxz uwsmmpaooemm mpnmu wepxm ppmm Pouch mama emuumspxm mcwmuoga Emppwgawwoxs mo cowumm>mu ugmucmum use mmmucmugma mmmgm>< .m>mu m on 0 Com mom cw cmgoum meuzm smote Eoew .0 «Bo.— 86 proteins were calculated from the area under the band peaks. The effect of washing technique and ice-storage on the extractability of myofibrillar proteins are shown in Tables 5 and 6, respectively. The myosin molecule has been reported to be comprised of one myosin heavy chain and three myosin light chains with subunit molecular weights approximately 21,000, 18,500, and 17,000 daltonS. In the present study, only DTNB-light Chain (21,000 daltons) and light chain alkali A (18,500 daltons) were retained. Effect of washing and_sajt_concentration on myofibrillar protein extractability. Washing minced sucker flesh with 0.1M sodium Chloride solution (ionic strength = 0.03) followed by 0.6M salt extraction (ionic strength = 0.66), removed a substantial amount of water soluble constituents as compared with those samples extracted with 0.6M salt solution only. these water soluble constituents included sarcoolasmic proteins, blood materials and proteins with electrophore- tic mobilities of 0.25, 0.31, 0.38 (tropomyosin complex), 0.41,0.49, (G-actin) and 0.87 on a 10% polyacrylamide gel (Table 5). This result is in agreement with the finding of Wilkinson et a1. (1972). Washing of minced sucker flesh reduced the relative percentage of the extractable G-actin from 11.82% to 2.15%. Loss of G-actin can be explained by the extraction method used which called for 87 a salt solution on low ionic strength. Seraydarian et a1. (1967) reported the depolymerization of F-actin into G-actin and isolation of G-actin was accomplished by ex- traction at low ionic strength (< 0.001M) at 0°C and slightly alkaline pH. In this study, prewashing with 0.17% NaCl solution increased the relative concentration of myosin heavy chain, myosin light chains, M-line protein, C-protein, a-actinin, o-actin, B-tropomyosin, o-tropomyo- Sin and a few unidentified proteins with electrophoretic mobilities of 0.14, 0.23 and 0.44. From these results, it is postulated that prewashing of minced sucker flesh with Chilled water would remove more G-actin and other water soluble muscle components than washing with 0.17% salt solution. In the present study, a 0.6M salt solution with an ionic strength of 0.66 was used to extract the myofibri- llar proteins. The freshly minced sucker flesh had pH values ranging from 6.7 to 6.8. The addition of 0.6M sodium chloride to the extraction system caused a drop in pH to 6.2. By increasing the salt concentration, an increase in ionic strength and a decrease in pH of myo- fibrillar protein was caused. Thus, addition of sodium chloride to the extraction solution would favor the po- lymerization of F-actin and prevent removal of G-actin from the sucker flesh, causing an increase in the myo- fibrillar protein extractability. 88 The lower extractability of myofibrillar proteins at low ionic strength has been attributed to the strong asso- ciation between myofibrillar proteins (G011 et a1., 1970). Dawood (1979) reported that only 13.69% of the total pro- tein of sucker muscle was extracted with plain water. However, a rapid increase in myofibrillar protein extracta- bility occurred on increasing the concentration of sodium chloride from 0 t0 3%. This is in agreement with Dyer et a1. (1950) who noted that high ionic strength is required to solubilize myofibrillar proteins. Most of the myo- fibrillar are soluble only in solution with ionic strength ranging from 0.4 to 1.5. In addition to the ionic strength, the pH of the extraction solution and the muscle protein solubility are other factors affecting the muscle protein extractability. Protein solubility increase on both the acidic and basic side of its isoelectric zone which ranges from 5.5 to 6.0 for fish protein (Meinke et a1., 1972). The effect of ice-storage on salt-extractability of myofibrillar proteins. Data consisting of the total salt extractable proteins, myofibrillar proteins and non-protein nitrogen of sucker flesh stored in ice are presented in Table 6. Orthogonal tests showing significant differences among extractable of salt soluble proteins and myofibrillar proteins during 6 days of ice-storage are presented in Tables 7 and 8, 89 respectively. The initial total salt extractable muscle protein was 44.95%. The extractability of muscle protein fell to 15.24% by the third day and increased to 29.20% upon further storage in ice. It is known that total salt extractable proteins are comprised of partial sarcoplasmic proteins and total myo- fibrillar proteins (Cheng et a1., 1979). The myofibrillar protein extractability decreased linearly during a period of 0 to 6 days in ice-storage (Table 6). There were no Significant changes in the sarcoplasmic fraction and non- protein nitrogen content during this time (Table 6). This result suggests that a decrease in myofibrillar protein extractability caused a decrease in the ratio of myofibri- llar proteins to sarcoplasmic proteins; consequently, this changes would decrease the gel forming ability as shown by Nishimoto and mneemi (1979). The maximum extractability of myofibrillar proteins was on day 0. Possibly the suckers were still in pre-rigor or at the on-set of rigor since the first extraction was taken within 5 hours of ice-packaging. The lowest levels of total salt extractable proteins were obtained on the third day of storage in ice. This suggests that suckers were at the maximum stage of rigor on this day. As such, muscle fibers demonstrated increased resistance to fragmentation and reduced protein solubility. Anderson and Ravesi (1970) reported that protein ex- tractability of cod muscle aged in ice decreased at a slower rate than that during frozen storage. Their finding was in 90 Table 7. Orthogonal test of statistics for extractability of myofibrillar protein in ice-stored sucker flesh. ._r 3:123:32: ”22:22.11 231.2: .1322. 21 Treatmeht 6 279.87 46.65 2.761 2.08 Linear 1 262.15 262.15 15.492 14.60 Quadratic w 1 57.36 57.36 3.393 2.96 Error 21 355.25 16.92 1Significant1y different at a<0.02. 2Significant1y different at a<0.01. 3Significant1y different at a<0.02. Table 8. Orthogonal test of statistics for salt-extrac- table proteins in ice-stored sucker flesh. F0.001, Source of Degree of Sum of Mean F-ratio V' 71 1, . variation freedom square square Treatment 6 2233.59 372.27 4136.33 5.88 Linear 1 468.84 468.84 5209.33 14.60 Quadratic 1 1575.77 1575.77 17508.56 14.40 Error 21 1.88 0.09 1All means are Significantly different at o<0.001. 91 agreement with Dawood (1979) who reported that the amount of myofibrillar proteins extracted from pre-rigor sucker muscle was higher than that from refrigerated sucker, and the latter was higher than that from freezer storage. Effect of ice-storage on gel forming ability of sucker muscle. Table 9 Shows the changes in the relative concentration of myofibrillar proteins from ice-stored sucker flesh. Myosin heavy and light chains make up aoproximatly 60% 0f the total myofibrillar proteins . Actin is the second major myofibrillar protein, constituting 16% of the total. Total myosin concentration remained constant during the first four days of storage; it decreased slightly on the fifth day and dropped to 55% on the sixth day of storage. These results suggest that sucker muscle had passed post- mortem rigor, and possibly, gone through autolytic degra- dation. This could result in release of both C- and M- proteins from the thick filaments, perhaps increasing C- and M-protein extractability. 0011 et a1. (1977) explained that release of M-protein from M-zone situated in the cen- ter of the thick filaments causes the disruption of hexa- gonal lattice structure of the myofibrils. Consequently, a loss of the water holding ability and subsequent weaken- ing of the gel forming ability would be expected. According to Regenstein and Stamm (1979a,b), the water 92 .Aa apaxpa-802 < epax_a cameo DemeF camozzm .Aud-mzegv cameo eeme_-mzeo .mmsupmn acmemeewn ozp seem mcowpmcwsgmumu mumuwraau pcmmmgame mommcw>aoe game»: m m e m N _ o mc_mvoen emppwenwm mama .uo_ema mmmcopm .ll teas Co mpcmcoasou .Ammmapcce mwwuepme we» cw mmmcegu .m anmp 93 holding Capacity (WHC) of raintrout white muscle and lobs- ter tail muscle did not<flmnge from pre- to post-rigor. The trout muscle WHC values were similar to those of post-rigor chicken breast muscle and were not affected by the addition of pyrophosphate. In contrast, the WHC values of lobster muscle was like the WHC values of pre-rigor Chicken breast muscle. The pre-rigor lobster muscle showed a large in- crease in WHC values with addition of pyrophosphate (205% of control). Their findings suggest that WHC for pre- and post-rigor muscle were species specific (Regenstein, 1977). Results in Table 10 indicate that the gel strength of the cooked fish paste made from sucker flesh aged in ice from 0 to 4 days increased slightly, with a decrease beginning on the fiftfl1 day. However, the gel strength obtained during this period was not significantly different. Table 10. Means and standard deviations of shear press values as an index for measurement of gel strength of fish balls made from fresh suckers stored in ice from 0 to 6 days. fiv—v ‘w— Wfi 1 W . i h iv“ f“ Storage period, Pound shear force1 day per 9 sample 0 1.22:0.11 1 1.3510.03 2 1.5110.04 3 1.8210.06 4 1.8910.05 5 1.56:0.05 6 1.07:0.09 1 Average of 3 determinations from each batch. 94 Data on the correlations between individual components of myofibrillar proteins and the gel strength of the cooked fish pastes are presented in Table 11. Results Show that the gel strength of the heated fish pastes were directly related to the concentration of myosin, C-protein, actin, mule-tropomyosin in the salt soluble fraction of the sucker muscle. Myosin heavy or light chains alone were not strong- ly correlated with gel strength, whereas myosin heavy and light chains together had a Significant effect on the gel strength. As indicated in Table 11, the r values for total myosin, myosin heavy chain, myosin light chain-DTNB and myo- sin light chain alkali A~are 0.98, 0.55, 0.08, and 0.35 respectively. Mannherz and Goody (1976) reported that myosin heavy chain and myosin light chain alkali A are responsible for the binding of F-actin molecules while myosin light chain-DTNB is not responsible for myosin function. Myosin and actomyosin are considered to be the essential myofibri- llar proteins that are needed for the best performance in gel formation and a firm product structure (Nakayama and Sato, 1971 a,b,c; Cheng etal., 1979). Also, at pre-rigor stage, more myosin and actin are bound together than in ri- gor and post-rigor stages. The high correlation of B-tropo- myosin with gel strength (r=0.82) shown in this study is in agreement with the findings of Nakayama and Sato (1971c), and Cheng and Parrish (1979). 95 Table 11. The correlation between individual components of myofibrillar proteins and the gel strength of the cooked fish paste. Components of myofibrillar proteins Product-moment correlation, r Total myosin 0.98 Myosin heavy chain 0.55 Myosin light chain (DTNB-LC) 0.08 Myosin light chain (LC-Alkali A) 0.35 M-protein -0.66 C-protein 0.56 o-Actinin -0.53 Actin 0.14 B-tropomyosin 0.82 o-tropomyosin -0.64 1-lSrSl, where upper limit implies that the values of myo- fibrillar proteins and gel strength of heated fish paste are directly related linearly in perfect harmony, the lower limit implies a perfect linear inverse relation, and zero implies no linear interdependence of the two variables. 96 Myosin heavy chain alone makes up approximatly 40% of the total sucker myofibrillar proteins, and contains all of the -SH groups of the myosin molecules. Itoh et a1. (1980 a,b) proposed that the -SH groups are involved in the Changes of native actomyosin property during heat treat- ment and gel formation. They found that the solubility of actomyosin decreased and the molecular weight of actomyosin increased during gel formation. They further suggested that the formation of the polymeric molecules of actomyosin could result from the formation of intermolecular disulfide bonds (-S-S-) in the heated actomyosin gel. Similarly, Buttkus (1971) proposed that intermolecular disulfide bonds are involved in the mechanism of protein denaturation. The study on the effect of ice-storage on the myofi- brillar protein extractability and gel forming ability lead to the conclusion that the myofibrillar proteins of the pre- rigor sucker flesh are more extractable and provide for better ge1 forming ability upon cooking than those in the rigor and post-rigor stages. After washing, the ratio of myofibrillar protein to sarcoplasmic proteins increased, re- sulting in greater gel forming capacity than that before washing. In general, fish have a relatively shorter postmortem duration than the mammalian animals. The acceptable shelf life for ice-stored sucker usually does not exceed one week. This varies in different species and size of fish. After 97 harvesting, the fish should be processed as soon as possi- ble. The location of the processing plant should be either on—board ship or near the unloading dock, so that the best quality of minced sucker flesh could be obtained before reaching the on-set of rigor stage. Prior to freezing, blending of minced sucker flesh with certain additives such as SHMP and MSG could also be a way of extending the quality stability of sucker flesh as a raw material for making fish paste products in later study. 98 The Effect of Washing on the Lipid Composition, Cholesterol Level and TBA Values of Mechanically Deboned Sucker Flesh Data on the changes in lipid content, cholesterol level and TBA values in minced sucker flesh before and after washing are shown in Table 12. The washing technique removed primarily the unbound lipids and free cholesterol which had been released from the muscle tissues and bone marrow during the mechanical deboning process. As a result, a reduction in TBA values from 1.35 mg to 0.62 mg per kg of sucker flesh was realized. Total lipid content was decreased from 1.739 to 0.59 per 1009 of sucker flesh. The cholesterol level was reduced from 44.90mg to 15.47mg per 1009 of sucker flesh. Data indicating the relative percentage of fatty acid composition in minced sucker flesh of both washed and un- washed samples are presented in Table 13. The results indicate that washing changes the relative percentage of fatty acid composition in total lipid as well as in its neutral lipid and phospholipid fractions. Fatty acid content changes in total lipids after washing. Changes in the relative percentage of total saturation and unsaturation in total lipid were negligible. Never- theless, the relative concentration of mono- and dienoic £99 .mco.u~:weLouou ouou._a=v uo mo=_o> soc» cue—ouao new: ouogo>o use — mc.o«~o.o c—.c«mn.— mo.o«~n.c op.o»om.— «o.voo.o mo.c«~o.— oo.oAme.o —_.c«nm.c ~¢.pflmc.m_ oo.p«oo.~— om.~“on.m— o_.—«om.n_ Km.—«cm.vq oe.o»—m.om oo.~«ov.~e o~._wom.—e ~o.cacm.o mc.oAn~._ coacu>< ~o.oAme.o mo.erm._ _ma_ .m. __ca< ~o.oaee.o ~c.c«~o._ owe. .- .sez no.0Ame.o .o.oame.. omm_ .mp .562 mcvgmu: souu< mc—gmuz acowom m=_=mux Louu< m:_gma3 ogoeom ocvzmax Luuu< mcvgmuz «Looms suuom oPQEum ux\oe .mo:.o> .¢o —~.~A-.wm om.—A-.mm m_.e«—o.mm ~m.voe.me oo.—«om.u— .n._noo.w_ muvu< u.o:m»—oa a mp.on—o.m Nv.ommm.m om.paem.m m~.voa.— mm.oflmo.m mm.o“o~.m muvu< upocm_a u co.o«~m.mm ~o.~n~o.om me.—n~w.- um.~w¢m.m~ om.0no~.mm mm.meo~.mm mv_u< u.o:oocox a mo.n«oc.m~ mo.eumm.mu w_.~«-.ow Ne.mwm~.o~ ~o.¢nmo.mn n~.maoo.m~ mu_u< xuuou .ammca a m—..«_m.e~ mo.oA~o.e~ mo.~«mm.o_ me.o«-.m~ e¢.cA~m._~ wo.o«eo.o~ muru< xuuum .uam u mm.—«_~.op oc.on_o.m_ o~._wom.m~ ce.owm~.om cm.o«~s.m we.onmo.n onmw o—.c«mv.v mo.ouo~.o ce.oAmc.m No.0Amm.m w_.cfl_~.~ ~_.o«m_.~ mumw n—.oflo_.~ mp.oN-.o w~.cume.a sp.owmv.o mm.oflo~.o m~.c»-.o mup~ um.cAo—.pp o_.oum—.o— so.o«nv.m Kc.oum_.op sn.o«~m.~ mn.oama.w muom pp.9umm.o om.oeno.o .o.o-m.o so.owom.o mc.o«mm.o No.o«_~.~ «new v—.cnm—._ oo.cum¢.— m_.—no~.— mo.ero.o ~o.cnw—.o mo.ou~c.~ enm— vo.cno~.o —m.oAmm.o . A t - A . po.o«om.o .c.o«~e.o nnmp c—.o«~m.o mo.o«o~.o om.—«nn.— so.oAc¢.o um.on~c.— mc.o«mm.o mnom No.cnoe.~ mm.c«e~.n oo.o«~o.— o—.o«mm._ om.ouyo.c cm.c«_e.m Nump op.o~mm.— -.o«m_.— m—.o«~v.— c~.oAm~.— no.OAmm.o sc.ono~.o _”v~ ~o.o«-.o mm.o«mm.o —o.oem~.~ om.oAm¢.w mo.o«¢m.n nn._«—m.m _”_~ mc.o«o~.c ——.cA—m.o op.oA—c.c o_.o“mm.o o—.oflmm.— oc.o»mv.— —"c~ mo.o«mm.—p —v.ouss.~p F~.ouco.m oe.o~m~.m e—.oAm_.—~ m~.o«—~.c~ .uw— mo.oAmc._ vo.oA-.— so.ceoo.o m—.oumo.o o—.cemc.~ n~.ca~o.~ —u~— m~.oA~—.—p om.o-m.~— ~c.o«mm.n -.oAmo.m cm.o«-.c~ on.—Hmw.e~ .Hmp mo.c“a~.c ec.oum¢.o mc.OA—m.c ~m.oA~m.o oc.o»mc.o mo.oaoo.o —“mp op.erc.o ~c.oumo.o . a n 1 A . .o.c«~e.— oc.o«nm.— pav— m—.cn—o.c mo.voo.o oo.oAso.o s A . ~o.oApm.— mp.voe.— cupm mo.o«oo.¢ ~—.ouom.e mm._umm.o .o.c«m~.w oo.o“—o.m v~.o«op.n cum— ~o.cuom.o p_.cwoo.— o_.omm~.o —_.voc.— m—.o«~o.o wo.onuo.— ous— v~.ono~.n— -.o«mm.c— o~.ouo~.pg mo.c«on.m— m~.oamc.~— am.o»—c._— onc— vo.cnmm.o ec.cemv.o oo.o«m—.o mo.o~m~.c m—.o“~m.o oo.cA—o.o camp me.onm_.— oo.cwmn.— ~o.oA~—.c w—.0nmm.o m~.owss.~ o..onmo.m one. acvzmu: mcvzmo: mc_2mo3 oceans: mcpgmo: m:_:ma3 Louu< mgoumm Lmuu< ogoumm Luuu< ugouum mu_u< 3::— _38. 32:233.: 3::— Zzeoz 3»: .a:.gmo3 scams ace acumen gmo—w Luxuaw couc*s :— :c_uwmoaeou u.uo macaw no «co—uo_>ou eunucoum new menu: .n—_o_aah 102 Fatty acid content changes in phospholipid through washing. Washing of minced sucker flesh caused an increase in relative total unsaturation and a decrease in proportion of total saturation in the phospholipid fraction. The re- lative percentage of monoenoic acid declined as the amount of di-and polyenoic acids increased in phospholipids due to washing. The reduced relative concentration in mono- enoic acid was due to a decrease in fatty acids, C16:1, C18:1, C21:1, and C24:1. An increase in the relative con- centration of fatty acids, C18:2, C20:2, C18:4, C20:4 and C21:5 could be accounted for by an increase in the relative concentration of both di- and polyenoic acids. A decrease of docosahexaenoic acid (C22:6) from 30.75% to 28.26% after washing was probably due to the mechanical disruption of myofibrils resulting in the release of C22:6 acid from tissue phospholipids. Washing the minced sucker flesh before processing and subsequent frozen storage is a practical way to improve quality and storage life. The washing technique removed not only sarcoplasmic constituents and blood materials, but also greatly reduced the unbound lipids, free cholesterol content and susceptibility to oxidation as indicated by TBA values. The fatty acid ratio of total unsaturation to saturation in total lipid increased slightly through washing, while in the phospholipid fraction, a shift from more saturation to less saturation through washing was observed. 103 Effect of Frozen Storage on the Lipid of Sucker Paste Products Changes in quality of the frozen sucker paste products during storage were determined by analysis of fatty acid composition in total lipids, neutral lipids and phospho- lipids, analyses of phospholipid classes and TBA test as well as the sensory evaluation. Changes due to lipid hydrolysis. Data indicating the Changes in relative percentage of neutral lipid and phospholipids of the sucker paste pro- ducts are presented in Table 14. As the relative percen- tage of total phospholipids decreased, the total neutral lipid increased for all treatments. The magnitude of differences in the proportion of neutral lipids to phospho- lipids increased as the storage time increased. This evi- dence is further confirmed by the TLC analyses of phospho- lipid classes Shown in Table 15 which suggests that decom- position of phosphatidyl ehtanolamine (PE) to lysophospha- tidyl ethanolamine (LPE) began during the first month and continued upon further storage while phosphatidyl choline (PC) remained unchanged. This result is in agreement with Braddock (1970) who reported that the hydrolysis rate of PE to LPE in Coho salmon during 6 month frozen storage was greater than that of PC. In contrast, Bosund and Ganrot 104 .muvav—ogqmozq to» muapeumcmqam acmcouewc guy: pc.oVa an mucmsuuocu ocean muucogouu.u acuu.u.:m*m~xxz> .mu.n.— —oeu:oc so; muawcumcuaam acogomeeu :0.) pc.ovd an mucoEuoocu ocean mmucugomw.u u:uu.m+cm.mmuuaa .zouaa sumo soc» covuacvsgouuu or» we omego>

e—.em -.onuom.mv sv.onxom.mv sv.oea_o.vm mp.o«xom.me m—.onuee._m mo.oAx—o.me no.onaoo.¢m em.o«»w~._e «w.o«o-.wm o hm.on>o~.om mm.onom~.me -.o«xmo.me -.oAa~m.mm .m.oazmm.mv pn.onu~e.om ¢¢._erm.me oo.—«umc.pm _~.ou-—.~e —~.onumm.sm m o~.on>~o.~m o~.o«omn.~c o—.o«»oo.se o_.o«nma.~m om.oexoo.om om.oecoo.om mv.onxom.oe ov.o«oo~.om n—.cu-m._e m_.ouamm.mm v v_.o«>mo.mm ~—.onumn._e m—.caxms.me a_.oAnv~._m m~.—Azoc.cm v~.oun_m.me ~m.c«3me.om eo.c«asm.me oo.c«xmm.~e eo.o«avo.~m m o~.c«>vm.mm No.cuuoo.ov o~.o«3om.cm o~.o«aom.oe cm.—«3mm.—m om.—«nmo.mo oo.~n3o~.—m so.—«aom.m¢ n~.on3cm.oo m~.o«oo~.—m N mm.o«>om.om -.o«uco.oe ms.onzmm.mm m~.onu~p.oe oq.o«xmm.nm o¢.onum_.oe mo..n3¢~.~m mo.—«no~.~v mm.o«3_m.oo No.—nuae.cm _ m_.oe>mv.oo -.onamm.am ow.o«3o~.mm ow.oAme~.oo Nm.o«xco.¢m -.onevm.me no.~«zm_.om No.~nopm.mv mm.—nzma.nm an.—«umc.ov o e.g.. e.g.. own“. uwnpp e_a._ cwa__ away. u_a__ c.a._ u_q__ rosamoge paeuaoz -osqmomn Foeuaoz -osqmoge .ocuaoz tosnmosa pocuaoz -ocqmogq vacuum: azzm-oaoco=m-umz-_uez a::m-omocoem-omz-_uez a::m-omoco=m-umz axxm-um=-puaz unoccem-umz-_uez mmwuwu a__em eu.d eoxeou . eemaa em.a see oaecoem .uom~- as wooeoam cu~oum uo mgucos o mcwcac muuavosa manna Luxuam c. newn._ogamogn use «e.g.. .acuamc c, mmmcugo amoucuueoa ouocoeutonoen use we meoyuo_>oe unaccoam ecu menu: .c. «pace 105 Table 15. Qualitative changes of phospholipid classes by TLC analyses Treatment Phospholipid Storage period, months Classes 1 2 3 4 5 6 Raw Fish Pastes: NaCl-MSG-Sucrose PE X X X X X X PC X X X X X X LPE X X X X X X LPC NaCl-MSG-SHMP PE X X X X X X PC X X X X X X LPE X X X X X X LPC MSG-MSG-SHMP PE X X X X PC X X X X X X LPE X X X X X X LPC NaCl-MSG-Sucrose-SHMP PE X X X X X X PC X X X X X X LPE X X X X X X LPC Cooked Fish Balls PE X X X X X X (NaCl-MSG-Sucrose-SHMP) PC X X X X X X LPE X X X X X X LPC PE = Phosphatidyl ethanolamine. PC = Phosphatidyl choline. LPE Lyso-phosphatidyl ethanolamine. LCP = Lyso-phosphatidyl choline. X = Presence of a specific phospholipids. 106 (1969 a,b) showed that in frozen stored Baltic herring, the hydrolysis rate of PC was faster than that of PE in both white and dark muscle. This suggests that the rate of hydrolysis of PE and PC to their lyso-derivatives differs in various species. The decomposition of PE to its lyso-derivative in raw fish pastes could be attributed to enzymatic lipolysis while the decomposition of PE occurring in cooked fish balls was not totally enzymatic lipolysis. Between the time of processing and cooking, the change in PE was brou- ght about by emzymatic lipolysis. After cooking, the tissue enzyme syshmiwas inactivated; thus it is unlikely that further lipolytic decomposition of PE to LPE occurred during frozen storage. Nevertheless, the presence of LPE in the cooked fish balls was continually monitored by TLC analyses during 6 months of frozen storage. Presumably, this LPE originated from lipolysis of PE before cooking. The hypothesis is that hydrolysis of PE to LPE indicates a decrease in the relative phospholipid content of raw fish pastes due to enzymatic action; this Should result in free fatty acid (FFA) production and formation of lyso-deriva- tives. The FFA release from PE could be a factor contri- buting to further oxidation and protein denaturation ( Roubal, 1967; Awad et a1., 1969; Braddock and Dugan, 1969; Shenouda, 1980). TBA values in this study, however, remained low during the 6 months of storage. Buttkus 107 (1967) has postulated that a reaction between myosin and malonaldehyde may occur during storage and caused a de- cline in TBA value. The following explains why there was no apparent PC decomposition: (1) low phospholipase activity or lyso- lecithinase may be active, (2) PC, per se, functions as a natural emulsifier which prevents separation of PC from fish muscle protein, and (3) SHMP is known to have emulsi- fying ability in maintaining a lipid-protein-water emul- sion in which phosphates prevent the protein from dena- turing, thus maintaining the hydrated form of protein and preventing PC-protein separation in the sucker paste system. Comparison of the relative percentage of fatty acids of phospholipids in the fresh minced sucker flesh and frozen stored sucker pastes is shown in Table 16. The Changes in fatty acid contents in the phospholipid fraction mainly occurred between the initial time of processing and the first month of frozen storage. Upon further storage, the changes were small suggesting that changes during fro- zen storage were minor. The primary change occurring be- tween 0 time and one month of storage could be caused by the prolonged exposure of raw samples to the open atmos- phere at room temperature during processing that promoted lipid autoxidation, and /or lipolysis of raw fish pastes. 108 .mu>.u.uua o: mc_=_uucou o—anm vegan: uo muvav_o:amozn c. uv'uo anus» mo «mongoosoa —o_u—c~ n .omocaum ho gage: n z .ucusueoc» r » .omucoum Co gases sax—m we use we» u< a .omucoam mo gucos amt.» be use as» u< u w .omocoum Go gazes vg.:u mo new use u< n m — o me.m~ “m.o~ e~.m~ ~o.m~ am.m~ em.¢~ m_.o~ so.m~ om.n~n~.e~ mm.m~ ~v.v~ mo.m~ oo.m~ oo.m~ o~.m~ : cnww o~.m mm.m c~.m mo.m o_.m ma.e o_.m mo.m vc.e mo.m om.m m~.m a¢.¢ mn.m m_.m we.m : mU- oo.— ae.— om.c oe.o “c.~ o—.— mm.o -.m mm.o -.c m~.~ mm.~ we.c mn.m m~._ n¢.o x mup~ mm.P_ mo... mo.—— -.p_ cw... am... no... e~.pp m~.——no._— o~._— eo.~_ e—.._ mv.—_m~.~— ~¢.m muow me._ do.— mm._ -.— os._ Ne._ em.p mP.~ c~.~ ve.p mm._ -.~ o~.— ~m.~ mu.p se._ : .neu mm.m 59.x ~N.m «m.m ~o.m m~.m .o.m mm.m oo.m m~.m oe.m ~—.m me.w pv.m mm.m m~.~ .u—N ~o.o— m~.a ~c.m .~.m oo.m Ne.a om.m mm.o m~.op om.m o_.o om.m um.m oe.m mm.a «a.» x pum— m¢.c oe.o mm.o me.o .o.o o~.o m¢.o mm.o m~.o o~.c mm.o mm.o No.0 oo.o ~o.o no.9 z —"~_ ou.m _m.m mm.o mo.m ——.m oo.m e~.m om.o aw.“ .m.m o~.e mv.m oe.m -.m .o.m mm.m .uop mo.o cc.o ~c.o cp.c mo.c ~—.o _—.o co.o m—.o m_.o oo.o oo.o m—.o oo.c oo.o oc.o : pug. mm.e m—.m en.m op.m o~.m .m.m om.m m¢.m om.e mm.m .o.e om.v mm.e m~.o mp.m ma.m cum— -.o~ e..m_ oo.n_ mm.c~ —m.o_ m_.m_ em.o~ ~m.m— mm.m_om.mp mp.m_ em.w_ ~_.o~ m~.m_ -.m— os.p_ : ouc— on.o -.o m~.c ~v.o o~.o om.o cv.c —~.o mm.o oe.c o~.o o_.o ne.o m~.o e~.c n_.o : cum— o¢.c Fe.o no.9 mm.o e¢.o mm.o mm.o ~m.o cc.o _o.o mm.o .v.o mm.c ee.c me.c h_.o b one. o m p m m _ o m _ m m p em on «P to case: «coauuuch dxzwomocuamromn—ueza::m-omocu=m-umz-—uez mzzw-uwucuafldwx a::m-umz-puez omocusm1umx-puez _opu.:. an mo.ovd an mc_u< e‘ m‘ m. —. .mv.ue xuuaw moucucoum_v » a accused acne—evcmwm 9» a m—pom gm_m coxoou «gamed zm_u sea .uomm- an mongoum cones» we «canoe o mcvsav m__un zm_e umxcou use acumen gave yo mv.n._o=nmo;n «nu :. mufiuo zuaeo vogue—om as» ho omoucouewa ouocoeusoaogn c, momcagu .o_ u_nu» 109 Analysis of fatty acid changes immediately after processing were not conducted in this study. Therefore, it is not possible to state whether changes of fatty acid contents were caused by processing or by the initial slow rate of freezing. A further study is needed to confirm fatty acid changes in sucker paste products caused by processing per se. The relative percentage of fatty acids, C16:0, C16:1, and C20:5 in raw fish pastes generally increased from the initial value to one month of storage while the relative percentage of C1820, C21:5 and C22:6 acids decreased. In cooked fish balls , the relative percentage of palmitic acid(C16:0) increased greatly from an initial 11.74% to 17.60% during the first month and after that, the rate of increase in 016:0 acid was less pronounced. A slight in- crease in the relative concentration of fatty acids C16:1, C18:1 and C2025 was also observed in cooked fish balls be- tween the initial value and one month of frozen storage. The relative concentration of C21:5 and C22:6 acids in both raw and cooked fish pastes decreased significantly from initial to one month of storage. The Changes reflected losses of Specific fatty acids from phospholipid fraction, which could be caused by enzymatic lipolysis and intercon- version of longer chained to Shorter chained acids (Hardy et a1., 1979). A significant increase in the relative per- centage of palmitic acid (C16:0) at an early stage of 110 freezing and prior to freezing could possibly be resulted from hydrolysis of PE to LPE and release of C1620 acid as mentioned previously and from hydrolysis of C21:5 and C22:6 acids. According to Mai and Kinsella (1979b), the major com- ponents of FFA found in white sucker flesh were palmitic acid (C16:0), palmitoleic acid (C16:1), oleic acid (C1821), arachidonic acid (20:4), eicosapentaenoic acid (C2025), and docosahexaenoic acid (C22:6). This evidence is consistant with the finding of this investigation. Braddock (1970) noted a preferential hydrolysis of PE in Coho salmon containing C1620 and C22:6 acids. The re- lease of C1620 acid from phosphoglyceride molecules through phospholipase action preferentially occurred in the Sn-2 position. It is also known that polyunsaturated fatty acids, C1620 and short chain fatty acids of fish accumulate mainly at the Sn-2 position of a triglyceride and /or phos- phoglyceride molecules (Brockerhoff et a1., 1968; Braddock and Dugan, 1972; Dugan, 1976). Whether the C1620 acid found in phospholipids of sucker flesh is actually accumu- lated at the Sn-2 position needs further study. The rate of enzymatic hydrolysis of lipid is pH and temperature de- pendent, and may be retarded by the hydrolysis end product concentration. The freezing temperature fluctuation obser- ved in the present study could be one of the factors that caused a slight Change in quality of sucker paste products. 111 Oxidative changes. The changes in fatty acid composition in total lipids, neutral lipids and phospholipids of the frozen sucker paste products used in the present study are shown in Table 16 to 21 and Appendix B1 to 816. The primary oxidative changes in fatty acids probably occurred during processing and early stage of frozen storage as mentioned previously in this study. During the first month of storage, the changes in the relative percentage of total saturated fatty acids in the total lipids of the raw fish pastes were slight while the relative percentage of the total unsatura- ted fatty acids tended to decrease. This decrease was pri- marily due to a decrease in polyenoic acids. Cooked fish balls showed a slight increase in total unsaturated fatty acids and a decrease in the total saturated fatty acids during the first month of frozen storage. After 6 months, the relative percentage of total saturated fatty acids in total lipids increased slightly while the relative percen- tage of total unsaturated fatty acids tended to decrease. The lower dienoic acid content refleCted to the decrease in total unsaturated fatty acids for both raw and cooked samples after 6 months of storage. However, the raw fish paste that contained MSG-Sucrose-SHMP showed a reverse trend, a slight increase in total unsaturated fatty acids and a slight decrease in total saturated fatty acids. This finding suggests that treatmnet with MSG-Sucrose-SHMP 112 Table 17. Changes in proportionate percentage of fatty acid composition of total lipids in sucker paste products during 6 months of frozen storage. , 0 Fatty Acid Classes Month of storage at 23 C 01 1 3 6 : saturated fatty acids 24.3121.l3 Raw Fish Pastes NaCl-MSG-Sucrose 26.4lzl.03 25.13:2.8l 25.77:l.06 NaCl-MSG-SHMP 26.08:2.08 26.22:0.72 26.97:1.56 MSG-Sucrose-SHMP 26.02:1.30 24.52:0.91 23.91:0.4l NaCl-MSG-Sucrose-SHMP 26.56:3.89 25.72:1.35 25.93:1.07 Cooked Fish Balls 23.03:2.02 26.04:0.34 25.26:0.64 % unsaturated fatty acids 75.69t3.03 Raw Fish Paste NaCl-MSG-Sucrose 73.38:3.32 73.23:12.84 74.31:l.92 NaCl-MSG-SHMP 73.97:7.51 74.06:5.25 73.4422.ll MSG-Sucrose-SHMP 73.91:10.79 75.53:10.26 76.51:1.63 NaCl-MSG-Sucrose-SHMP 73.01:l3.24 74.3826.94 74.022l.70 Cooked Fish Balls 77.06:4.92 74.52:1.92 74.9922.84 % Monoenoic acids 33.92:0.64 Raw Fish Pastes NaCl-MSG-Sucrose 32.48:l.55 36.54:6.27 34.2520.80 NaCl-MSG-SHMP 33.93:3.93 33.67:3.40 32.31:0.59 MSG-Sucrose-SHMP 35.95:5.27 36.39:5.21 35.13:1.35 NaCl-MSG-Sucrose-SHMP 35.42:6.93 37.83:4.08 32.04:0.84 Cooked Fish Balls 34.07:2.70 32.16:l.59 32.51:1.67 % Dienoic acids 3.01:0.18 Raw Fish Pastes b c a NaCl-MSG-Sucrose 2.47 20.34 1.51 20.81 3.18 20.33 NaCl-MSG-SHMP 2.72 20.20 2.45 20.15 2.79 20.22 MSG-Sucrose-SHMP 3.06 20.32 3.20 20.40 3.17 20.28 NaCl-MSG-Sucrose-SHMP 2.82a20.82 2.86c20.29 2.77b20.43 Cooked Fish Balls 3.26 30.32 2.44 20.14 2.96 20.22 % Polyenoic acids 38.7722.21 Raw Fish Pastes NaCl-MSG-Sucrose 38.43:l.43 35.1825.76 36.88:0.79 NaCl-MSG-SHMP 37.32:3.38 37.94:l.70 37.34:l.30 MSG-Sucrose-SHHP 34.90:5.20 35.94:4.65 38.21:l.76 NaCl-MSG-Sucrose-SHMP 34.77:5.49 33.69:2.57 39.21:0.43 Cooked Fish Balls 39.73:1.90 39.9220.19 39.52:0.95 abc Figures at the horizontal line with different superscripts have significant difference at o<0.05 during storage time (Turkey's test of statistics) 0=Initial level of fatty acids without adding additives. Analyses of variance were not significant. Analyses of variance established a significant difference among time (o<0.05). 1 2 3 113 renders the paste more stable to lipid oxidation. In this case, the prevention of polyunsaturated fatty acid(PUFA) oxidation could be attributed to the metal chelating effect of SHMP. The polyenoic acid content of the total lipid decreased slightly but not significantly in raw fish pastes containing NaCl-MSG-Sucrose and NaCl-MSG-SHMP, while little change in the polyenoic acid content of cooked fish balls and raw fish pastes containing MSG-Sucrose-SHMP and NaCl- MSG-Sucrose-SHMP were found after six months of storage at -23°C. Changes in the relative percentage of some major fatty ackm of the total lipids (Table18) were not signifi- cant among treatments throughout six months of frozen sto- rage period except linoleic acid (C1822) which fluctuated in the raw fish pastes containing NaCl-MSG-Sucrose. The fluctuation may be related to the oxidative products re- sulting in the formation of TBA-malonaldehyde complex and oxidative products other than malonaldehyde which reacted slowly with the TBA reagent to yield a yellow compound (Yu and Sinnhuber, 1962). Linoleic acid is also known to interact with myosin in frozen muscle resulting in muscle protein insolubilization (King et a1., 1962; Braddock, 1970). The rate at which the relative percentage of total neutral lipids increased and phospholipid decreased was faster in raw fish pastes than that in the cooked fish balls; and among raw fish pastes the rate of change is 114 gazes: .oueeeum we genes sex—m we» we eeo 0:» uwuweee e: ecwcweuceu zmeww Lexuem eeecwe vogue: we newew— pesueec :w mewue auuew we emeeceuwen pewuw:_ n o -.m ewne awuw e_.e me.m em.m ee.w ma.e ee.e mm.m w..e me.e .e.e ee.m mm.m ww.m w GHNN ee.~ ~_.~ ~a.. se.. Ne.. ee.~ .w._ Nm._ me._ ee.~ ee.~ Na._ _m._ e~.~ mm._ e_.e z w enm— me.e me e Nm e wm.e ee.e .m.e en.e ee.e me.e me.e ee.e ~e.e ee.e .e.e me.e em.e : mum. ee.o .m.e ew.e ee.e em.e ~m.e .m.e me.e _e.e mm.e we.e me.e em.e ae.e _e.e Ne.p z NHeN em.e ee.e ~e.e me.e ee.e ao.e ee.e ee.e me.e em.e ww.e an.e ~m.e ~e.e we.m .e.e w Nue. em.e we.e meme mm.e em.e ee.e em.e se.. ea.e em.e me.e we.e Ne.e em.e o..e mm.e w _"e~ e~.m wo.m em e .m.m ~_.e .o.m ee.e ee.e ew.m an.” ~e.m aw.” .m.e ee.n we.m me.. w _n_~ e...~ _~.e~ .e.m_ m~.e~ mm._~ me..~ ee.a_ e~.m_ ew.e~ me..~ mo.e~ mm.e~ e_.e~ Na.e~ e~._~ m_.F~ w _"m_ me.e~ e~.- ee.e~ m..n~ wm.m~ em.e~ ee.- .a._~ .e.m~ ew.e~ .e.m~ ma.m~ am.- ~e.m~ ee.e~ -.e~ w _"e_ mm.e meme None ee.e ~mne em.e ee.e em.e .m.e em.e me.e mm.e ~e.e we.e mm.e ne.e z w _”m_ ee.p mm F em _ a... an _ me._ we._ ee.o e~.. ~e.. ee._ Ne._ _m.F _e._ em._ ~e.. z w ."e_ on." .m.m me.m mm.m ~m.n em.m mm.m we.“ ae.m ~n.m we.” aw.” Ne.” cu.” mm.m .e.m w one. ee.. e... o... m_._ .e._ we.e em.e me.e wm.e me.e we._ me.e we._ me.. ee._ we.e z cue. em.~_ e~.~_ we.~_ m_.~_ we... em.~_ Ne.m_ em.np ew.~p me.~P me.~p e~.~_ ee.~_ ea... me.~_ ee.~_ : w one. em.e em.e mm.e mm.e ~m.e em.e .e.e me.e em.e ee.e mm.e mm.e em.e mm.e mm.e we.e : cum. em.~ em.~ mm.~ ee.~ ne.~ .m.~ Ne.~ e~.~ .m.~ mw.~ ee.~ ~w.~ em.~ ew.~ ww.~ ww.~, w one. o gaze: acesueeew me a. m. «w .w m. aw nw N. .w m. e. m. N. .w Hawawe. . 111 aewo< ewue me ove we speed gazes sue gazes ecm geces um— xuuew meucewewwwe usages .eewgee emeceum acousee aceewwwce—m .uemN- we emegeum we usages e ucwwee uppea gmww vexeee eee meumee gexeem res we newew— peguee: on» cw mewue Agnew eeuuepem as» we emeuceugee ounce—ugeeewe as» we meaeegu .ou eweew 119 fatty acids and monoenoic acids increased rapidly from 0 to one month of storage but only increased slightly upon fur- ther storage (Table 211- This suggests that further lipolytic degra- dation of phospholipids may have been retarded by either low temperature or its hydrolysis end products after the first month of storage. A further minor change in phos- pholipid after the first month of storage could be attri- buted to autoxidation which might have been promoted by NaCl. A slow rate of increase in TBA values in this study indicates that slight autoxidation may have occurred du- ring frozen storage. Production of malonaldehyde in frozen fish paste pro- ducts in this study was relatively low (Table 22). During six months of storage at -2300. the TBA values of raw fish pastes containing NaCl-MSG-Sucrose, NaCl-MSG-SHMP, MSG- Sucrose-SHMP, and NaCl-MSG-Sucrose-SHMP ranged from 0.58 to 1.36, 0.33 to 0.67, 0.24 to 0.49, and 0.26 to 0.65 res- pectively; while the TBA values for the cooked fish balls ranged from 0.04 to 0.47. These TBA values could be corre- lated with the acceptable quality of fish Since they were below TBA values of 2 (Yu and Sinnhuber, 1957). Evidence showed that raw fish pastes except those with no NaCl added had higher TBA values than cooked fish balls. Among raw fish pastes, samples containing NaCl with or without SHMP had higher TBA values than those containing only SHMP. This result suggests that SHMP may have a preventive effect on 1120 gaze: n : .uceENeecw n w .me>waweee sew: eeeee e» Lewce cam—w Lexoem eezmex we newewp pewueo: cw emeueeucee «ewes muuew wewuwc_a ezzm eee.emece:w .cm: .—uez new: eeeee eemee :mww eexeeu r m—pee gmww ezzm eee .emeceem .umz .puez new: eeeee meemee gmww sea u e. .ezzm eee emeceem .omz sew: eeeee Noumea gmww 3e: . .nzzm ecu .omz .puoz zap) touts mmumoa :mww 3mg emegeem ecu .um: .—uez sew: eeeee meumee zmww sex III N ‘ Ne e.ee... Ne .NNG .. N_.NAee.me mm._NNm.ee em.eNMN.e. eN.NAm..m. a..NN a... eoxeee em.one.m. ee.NNNN.N. ac .NN. m. N_.Nae..m. NN._A_N... .m._NNN.N. .. N..e._e... mm..._m.e. .N.eNN_.m. .e._ANN.N. .e.eame.m. _e..amN._. N. ._._W.N.N. ee.e.ee.m. N. a... .. ee._N_N.ee a...aep.m. ._._ama.m. N. mN._.ae.N. __._aea.me em .NN. .. NN._A._... N....Nm.e. me.Namm.m. _. menace :mww 3e: . . m—.eA—m.mm z mewu< owecexwed a mo.o«o~.~ mo.oNv—.~ wm.cnwp.~ m_.owmm.~ m—.on..~ om.oao_.m m—pem zmww eoxeeu .N.eam_.N e_.oN.e.N ee.e..e.. eN.eNNe.N eN.eN...N .N.enoe.N .. mm.ewee.N .e.eapm._ ...eAmN.N _..eaee.N No.0. e.N me.eANN.N N. w..eune.~ .m.one_.~ e_.onem.~ eo.ewe_.~ e~.on.o.~ eo.eNeN.~ N. en.eNNN.N ...e..m.N NN.eNNN.N N_.onea._ e. ewe. N om.o.N_.N _. neumee gmww xem . . cm.pwvm.m w mewu< uwocowa a .e...me.eN Ne.eeme.eN ee._am_.eN Ne.eA.N.eN ae.enNN.mN me._nNe.eN m__.N e... eoxeae N..eNNe.eN me.eNeN.eN .N.eN_..eN m..eane.mN em._NNm.eN em.eame.eN e. .N.eae..mN me._amo.mN me._aee.eN N_._Ne_.mN .N._NNN.eN e_.NNeN.oN N. ee._NNe.NN .N.e..e.eN .N.e.Nm.eN NN._NNe.mN .N.N.NN.eN Ne.e.NN.eN N. em.eANe.NN Ne.eNN..eN N..eANe.eN .m.eNNN.mN .e eaoe NN ee.e.ee.eN _. nuance :mww rec . . .e._NNe.NN z “v.6. o.eeoeeo= N mm.pemm.mw we.~nee.nw .N.¢Nee.nw ee.~NeN.mw .N._Nm..ew .m.enwm.cw m__em gmww eexeeu .N._aeN.N. om.NNN_.NN .a...em.nN N_.NNeN.NN .e.mfl.e.mN ee.NAmm.m. .. mN.NN_e.NN Ne.N.N_..N m_.NNme..N Ne.NNN...N m.....e... e...NNa.NN N. .N.NAN..NN .e._NNN.NN Ne._N.N.nN m_.NNNN..N m....ee.e. NN..Nee.e. N. wo.n«e~.mw ~m.~Nm—.mw ~e.~wem.ew om.pnmo.mw we anm as mm.mamm.ew p. weaned gmww 3e: . . m_.NANN.eN z w me.o< we... .eamee N __.owm~.e~ n—.cnoe.o~ me.oNe~.m~ wm.cnme.e~ em.oeee.m~ eo.nnmm.m~ m—pem gmww eexeeu .N._Amo.NN Ne._a.e.NN .N.ene..eN Nm.eame.eN N..eNNN.eN NN._NOm.eN .. .e._aNe.NN mm._NNN.eN ee._N_e.eN .N.e._e.eN em.e.mm.eN _..Nn__.NN n. Ne.eaN_.NN Nm..N_N.NN ea.enme.eN a...N.N.mN P.._a_o.mN Nm.e.eN.mN N. .e._NNN.eN mN.e.Nm.eN m..eame.mN .N._Ane.mN N. e... eN me.eaoe.mN .. meeme; zmww rem No.NNme.e. x w me.o< as... .eam N e m cos.» c x w . . . _ .2. new...“ . e eue ueNN- em eeeceem we mgecez e. “ewe—cw eeeeww.eewm ewe< weeew Iii .uemw- we eoeceem we menses c ocwcee meeeeeee eumee Lexus. cw mewewpegemeze we :ewuwmeeseu ewue weeew we emceeeugee euezeweLeeege cw meeeegu ._~ eweew .w_m xweceee< e. eeumwp ewe :eng me_escew use Eeww eeee_=ewee meepe> eg muewwumweeem aeowewwwe guwz meeemwweee eoxoee sea oe.e..oeN.e Ne.eN.oeN.e e_.ea.oeN.e Ne.e waee.e mm.en.a_N._ e mN.ewoa...e Ne.eewa_e.e NN.eN.oNe.e N..eA.aNe.o .e._a.aeN._ m ._.ea.oNe.e .N.eaxeme.e eN.eNxoeN.e mN.eaxaee.e o~.eaxaee._ . ._.enonN.e .N.eaxaee.e ._.euxaen.o .N.en aNm.e _N.enxa_e.e N mn.enxeN..e eN.eN a.e.e eN.e.xeN..e .N.eN a...e .N.eN .NN.. N ee.eaxo.e.e .N.ea ae..e NN.eA amn.e Nm.en eme.e e...“ me... _ me.ee eee.e mN.eNeeN..e N_.eaoaeN.e eN.en emN.e eN.eN aem.e e azzm-omecosm-emz-.eaz axzm-eaeco=m-emz-_eaz axzm-omcco=m-emz .a::m-em:-_eaz oaecosm-emz-_eez “geese .eewsee ooeLeem Newsweecw .Aepe5em ox\eexgee—ece_ee may meueeege woxuem eeuews one we comes—e> ee eweeceum ecu meow: .- e—aew 11 illiiill.| 122 lipid oxidation while addition of NaCl may promote lipid oxidation in sucker paste system. Thus, the antioxidant effect through metal chelating action of SHMP is much greater than the prooxidant effect of NaCl, hence, the SHMP retarded the prooxidant effect of NaCl. Occurrence of yellow and /or orange pigments was found in the TBA reaction solution of the frozen sucker paste products during frozen storage. The formation of yellow pigment could be due to complexing of aldehyde with proteins, sucrose and other food constituents that were present in sucker paste products (Turner et a1., 1954; Yu and Sinnhuber, 1962). Marcuse and Johnasson (1973) reported that alkanals resulting from lipid oxidation formed a yellow pigment when reacting with TBA reagent, while only malonaldehyde, 2,4- alkadienals and, to a lesser extent, 2-alkenals produced the red 530 nm pigment. The low concentration of TBA values found in these fish paste products could be interpreted as the result of a relatively low rate of lipid oxidation, and could be attributed to the washing of the preliminary raw sucker flesh, the presence of SHMP as a metal-chelating agent. vacuum packaging which provides an oxygen free enviroment, relatively low storage temperature (-23°C), inactivation of enzymes by heat treatment for cooked fish balls, and a dark storage room. The fluctuation of TBA values from month to month could be influenced by complex factors such 123 as duration of the fish sawed hiice prior to processing (Deng, 1978), storage condition before and after processing (Deng, 1978), mechanically deboned process (Lee et a1., 1975; Lee and Toledo, 1977), kind and amount of fatty acids in the system (Shono and Toyomizu, 1971; Takama, 1974; Fisher and Deng, 1977; Toyomizu and Hanaoka, 1980a,b), pre- sence of natural antioxidant and additives (Biggar et a1., 1975; Iredale and York, 1977; Morris and Dawson, 1979), presence of matal iron and copper in muscle food system and washing water (Tappel, 1953; Castell and Bishop, 1969; Castell, 1971; Lee and Toledo, 1977; Fisher and Deng, 1977), ratio of sarcoplasmic proteins to myofibrillar proteins in the system (Shenouda, 1980; Froning, 1981), and possible incccuracies in analyses. Changes in cholesterol. Analyses of variance indicated a significant change in cholesterol (Table 24) in sucker paste with added MSG- Sucrose-SHMP, NaCl-MSG-Sucrose-SHMP and cooked fish balls during six months of storage at -23°C. However, there were no significant differences among the 5 treatments. The slight decrease in cholesterol level shown in Table 23 could possibly be attributed to a slight oxidative reaction of cholesterol; however, this is unlikely since no evidence has been reported in the current literature to support the finding that cholesterol oxidation takes place in a f00d 124 .meewueewseeuee ezu we eewe> emege>< e .emeweum we mguces e mcwwee Amo.ove new eueewewwwe aceewwwcmwm me: a. wees: e:_e> we» memes eeweemceeem acewewwwo e.e eexeeu sea 3.3.3.: 8.2... .4: 2.3.3.2 8.3.8.2 2.3%.... e mm._neeN.m_ eo._.e N... eN.eA em.m. ee._NaeN.m_ eN.eNeem.m. m e..enaee.e_ em.enaee.m_ e..eheoe.._ ee.eNGeN... ee.Nnae_.e. . Ne.eanmN.e_ e..ew:e..m. ea.eneem.N. eN..neee.m_ em.eneem.N_ N mN.eNaNm.N_ NN.e.aea.N_ ee._aeee.mp ee.eaaee.e. e...NeeN.N_ N .m.ena.N.e. ee.enaee.m. em.eaaem.a_ ee.enaem.N_ en._eae_.N_ . azzm-omeco=m-emz-_eez axzm-omecosm-emz-_eez azzm-omecosm-emz azzm-emz-peez ooecosm-emx-.eaz meeeee .eswuemeweem Awexeem e co—\peweume—eze may uemwr ee «museum we mguees e.m:wg=e mueeeege enema sexeem cw ewe>ew peweume—esu we memcegu .NN eweew 125 eegwm w_.o mm e_.m weeewmem zucez ow.o me.o ew.o om me.w x “seaweeew ww.o mo.o wo.o w wo.o euuem mooc.ov aw.ww mm.w m wm.e ence: ow.o ww._ m_.o e ww.o acespeeew mewumweeum d we xuw_weeeeed eeeeem Eeeeeew eeeeem eecewee> eeceewwwcmwm eweswxewee< mewumwuepm w new: we eeemeo we Eem we eeweem .mowemweaem a we Ne._weaaeca eeceewwwcmwm epeewxeeeee mew ece eewe> weeeemewege cow eeeewee> we memawec< .em eweew 126 system at the freezing temperature which was used in this study. Evidence that cholesterol oxidation takes place in a food system at a frying temperature (180°C) has been reported by Mai et al.(1978) using deep-fat fried fresh water fish and Ryan et al.(1981) using heated tallow for an extended period. Further study is needed to understand the possible mechanism involved in cholesterol change under different conditions. The decrease of cholesterol levels shown in Table 23 did not follow a linear relationship. The slight decline in the cholesterol concentration also may be due to random sampling and variation in the analytical procedures, since lipid oxidation in sucker paste products was not seriously reflected by low TBA values. Sensory evaluation of flavor and texture. The parameters of sensory scores used for this study are shown in Appendix C. Rancid and putrefactive charac- teristics were used to describe a specific off-flavor of the frozen fish balls. Sensory scores, shear values and TBA values are shown in Table 25 for comparison of subjec- tive, objective, and chemical tests. Mean sensory scores for flavor acceptability decreased as the intensity of off-flavors increased with length of storage. This trend is consistent with the TBA values which increased during storage (Figure 4). Although off-flavor development occurred during storage, it should be noted that the sensory 127 .mo.ovnepe pcecewwwe zwuceewwwcmwm ewe pewwemceezm pcewewwwe mcweeen csewee esem esp cw mweeE=z one .eweem wcweetm e :e Pe>ew eweepeepeo n m N .u xweceee< cw czezm me o e» w Eeww eweem neweeum e an eewemees ewe: meweem agemcem w mo.oNNeoN.o we.ouewm.m mN.o«ewe.N mm.o«ewm.o mN.o«umm.e wN.o«ecm.N m w_.oNNeNN.o eN.o«eeN.w ow.oweew.N mN.oneNe.e Nm.onemw.e e_.owemw.N m mo.o_wemo.o eo.oneee.w oo.o«eoo.w wm.o«eww.w wN.oneew.w PN.o«eom.m o .3. .3388. .3. .5388. 85.. :8. Aewesem ax wee we>eww Nwe>ewwuwwo wewszew Fewepxew Amwasem m gaze: eezceewecewes mev \ eewew .ewv meewe> e.m~ No.NH~o.mm .oem Page» mo.onom._m _o..no~.F~ NN.OH~P._N oo.onk_.mm mo.~amm.- m~.onmm.om oHNN o..on~N.¢ NN.OHNL.¢ mo.CHNc.e mo.ohmm.e om.oao_.¢ _o.onom.¢ mHNN eo.on~e.o wN.OHmo.o ¢_.on_m.o _o.onmm.o mm.onoo.o em.onm~.o mnpm wP.OHoo.__ mm.¢nmm.__ Np.onmm.op so.onoe.PF me.¢nmm.o_ mo.oa~o.__ muom oo.onom.o mo.onpe.o eo.onmm.o No.03mm.o _o.onmm.o mN.cH~N.o «new _o.onfio.F m_.on¢o._ Po.onmm.o No.onmm.o No.onN~._ m¢.onmm._ euwp om.onmo.o w~.onfis.o oo.onnp.o Fo.0hmo.o mo.cnmm.o op.onmm.o mum_ No.onoe.o mo.onmm.o No.0Hoe.o eo.oamm.o eo.oh.¢.o op.onmm.o Nuom cm.oaom.m mm.cnmm.~ Fe.oheo.m o..onmo.~ _N.onom.m op.ohmu.~ mum, mo.onmo.F Fo.oho_._ mo.onso.F so.ohom.o ¢~.onmm.o NF.OH__._ Fuem em.on~¢.e co.onmo.u ¢¢.ono_.m mm.ohmm.~ eu.onme.o oo.onme.~ _H_N mo.on_m.o mm.onme.o mm.onme.o N_.ohm¢.o m_.oamm.o m_.onmm.o _“oN o~.onoe.__ LN.onNm._F NM.OH58.__ o¢.onmN._F om._hmm.m_ mm.ohmm.__ Pump mo.oaom.o om.onmk.o No.0nmm.o o_.onmm.o mm.onfip._ op.onko._ _HNF o~.onmm.m w~.onmm.m om.oamm.m mm.oamw.m _m._nem.m_ e~._aao.op _uop mp.0hmm.o ~_.onpm.c oo.ono~.o ~_.onmw.o mo.ohom.o mo.onem.o _no_ mo.onmm.o mo.onmm.o No.0nmm.o mo.ohm_.o N_.onmo.o P_.oaom.o _n¢_ co.onm¢.o m_.onN~.o mo.onm~.o mo.OHNN.o No.onem.o No.onme.o oupm _F.onom.e mo.oneo.¢ wo.0HNm.¢ _F.onoe.¢ m_.onom.e mm.onpe.¢ onm_ No.onom.o mo.onm~.o mo.on¢w.o mo.ohao.o ep.onmm.o eo.oncm.o OHNP Am.oa_m.NF _m.OH¢m.mF we.ohm¢.mp o_.onem.mp mm.onmm.N_ ma._hN~.m_ one: _o.ohom.o mo.on¢¢.o oo.ono¢.o Po.oahm.o No.ONm¢.o Fo.ono¢.o oum_ mo.on_o._ ~_.onoo._ mo.onoo._ ao.onfim.o mm.on¢¢._ m:.onp_.F on§_ o m e m N _ 141/11:11:11: cocoa .Qomm- be catapm ms_p S. OJ .0 E 3 C C O .Q L. (U U .uomm- pm mmmcoum mo mgucoe m mcwcsu azzm-mmocu:muemzupomz spwz umucanmLa Appma gmwwv mumma cmxuam umxoou cw cwavp page» we cowuwmoaeou uwom xuumm eo mmmacmucmq mumcowucoqoca cw mmmcmgo m m x 'U c Q! Q q <- 169 __._Hm_.m_ om._Hmm.mF “K.FHmm.mp ¢~.onmo.m_ Fu.ohpm.m_ mm.owom.mp owocmxpoa em.onmo.m mm.onnp.m mm.onmm.e mm.OH_m.¢ No.onxm.e P¢.onmm.m uwocmwo N¢.Nhem.¢m mm.anm.em mm.onmm.em mo._nom.mm No._n~m.¢m mm._nmm.¢m owocmocoz N_.vnmo.mm mm.mnom.mm ow.mhmo.mn «P.Nwom.m~ mv.mno~.m~ mm.mnmm.m~ .uemca Page» mm.onmm.om mN.onmN.p~ em.onmo._m aw.owom.om o¢.onmm.om No.0nom.om .pmm —muop om.onmo.m mo.onwm.m um.ohmm.m mp.onmm.m mF.0hme.m Np.onmm.m mumm so.oh¢m.m mo.on_o.m mo.ono~.~ mp.on~¢.m mo.onom.m co.onma.m mumm eo.onmv.o m¢.onem.o mm.onop.F _o.onm~.o oo.on-.o oo.onm~.P mupm m_.ono~.n ma.ono¢.~ _m.on-.~ _m.oneo.~ mm.oumm.~ so.OHmo.~ muom em.onmw.o Fo.onom.o o_.0ho~.o mo.owe~.o No.onmm.o mo.oncm.o «now _o.on~m._ Np.onmm._ No.ohmw.P mo.0hoo.m mo.Ohmp.~ ¢_.0hmm._ eump No.OH¢¢.o mo.onme.o po.onma.o mo.ohm¢.o oo.onmm.o mo.onm¢.o muwF m_.onum.o mo.onpe.o Pp.onpm.o mo.onmm.o No.0hpm.o em.on_o.o mnom mm.OHwe.¢ m_.oncm.e ~_.OHFN.¢ om.onmm.e mo.onm~.e Np.onmo.m NumF Fo.onom.o om.on¢w.o mo.onmo.o mm.on~m.o Fo.on~¢.o mo.onm¢.o Fuem No.on~_.e oo.onnm.m mo.onmm.m oo.on¢m.m em.ohm_.¢ mN.onua.m _HFN ee.on¢m._ ~_.onmk.p a..onmm.P m_.oh¢~._ oo.on¢a.~ __.on¢m.~ _uom m_._hmm._m om.onmo.pm ao.onmm.om m~.0hwo.p~ mm.on~m.o~ N~.onem._~ Pum_ oo.onmm.P .P.onmm._ mo.onoo._ m_.Ohmm.F _o.onmm._ Fm.onme.. _nn_ «F.0hfim.mm m_.on_m.¢~ mm.onwu.em m~.oneu.¢~ em.onmv.m~ mo.ono¢.¢~ Fum— _o.onmm.o mo.onme.o oo.onom.o mo.on¢m.o mo.on~o.o co.onmm.o Pump mo.on~m.F mo.onpe._ mp.on_N.F wo.o«~¢._ mo.ouoa.~ Fo.onom.p _H¢F mo.onmp._ mP.OHm~._ No.OHPF.P _o.onmp._ m_.on~m.P m_.oH¢N._ ou—N ao.onmm.m mo.onmm.m mo.onmm.m P_.on~m.m oo.owm~.m wo.onmm.m oum_ po.onpo._ mo.onmm.o Po.onnm.o Nm.onmm.o mo.onno._ .o.onoo._ o"~_ m_.onmm.F— _~.onmn.mp No.0hmw.~— mo.onm¢.mp op.onmo.__ om.ohmo.m_ OHQF No.onmm.o m_.onm¢.o op.0h¢¢.o o~.onm¢.o mo.onmm.o No.oumm.o cum? co.onmm.~ mm.on_x.~ mo.onpm.m _o.onm~.~ _o.onmm.m mo.onn~.~ one, o m a m N F Longs: coacmu spcoe .oomm- pm mangopm mo mswh qumum Apumm .oomm- um mmmcopm mo mgucos m mcwcsu mmogoamiwmz-Pumz saw: nmucmpnmca mumma Luxuzm cw muwawp Pmcuzm: mo cowuvmoqsou uwom xuumm mo mumpcmucma mpmcompcoaoga cw mmmcmgu .om chcmqa< 170 pm.ohxo.FN mv.on~m.mp m~.ohmm.om m~.onmm.om mm.onmm.om NN.OHm~.mF urocmxroa Ne.onm~.m m~.ohmm.¢ o¢.onwm.m m~.onmm.m -.onom.m ¢~.onpm.m uwocmwo em.owmm.mm mp.pnmm.mm mm._hpm.mm mw.on¢m.mm mm.on¢m.mm mw.onv_.em owocmocoz mm.phmn.om mm.ano.m~ em.mhum.m~ mm.Pn~m.mm mw.an~.mn em.mn¢m.mm .pmmc: Peach .o.on_F._N wo.onwm.PN mo._nmo.pm om.onmm._m om.onmm.P~ em.onpo.Fm .amm Fmpo» _p.ono_.o m_.onem.m mo.on-.m mm.0hoo.o m~.o«mo.m co.onoo.m onmm op.onoo.~ mo.on¢n.~ om.onpm.m No.ono~.m o_.oh_n.~ mo.0hwm.~ mumm wo.onmc.o mo.on_¢.o up.onom.o op.onmm.o “F.0hwm.o mp.cnmo.o mnpm mp.ohmm.~ op.onem.u N0.0hom.m oo.onm~.n mo.onmm.m om.ono~.n muom mo.onmm.o _o.onom.o e_.onme.o mo.ono¢.o mo.ohoa.o oo.oamm.o enom eo.onmm.P Fo.onmo.w co.OH~m.p mo.onmm.F mo.ohmo.~ No.onom.~ ¢"m_ oo.onwm.o mo.onmm.o op.on_acmuwmwcmwm mcm maqwcumcmazm ucmcmmmwu new: «UNFQ mo consaz 177 5555 55.5H55.55 55.thw.me 55._n_m.mq NF.N555.55 mm._nPN.55 5m._hmm.me uwocms_oa 55.oan.N 55.5055.N 55.5055._ 5N.55N5.N 5N.5555.N NN.5355.N 5555555 N5.55N5.5N 55.555N.5N Fm.on_5.5N 5N.OHNN.5N om._nNm.5N 55.5055.5N uwoemocoz 55.535N.NN 55.NHN_.NN 55.Fhom.mN N_.mhoN.mN 55.5HN5.MN 55.Namm.MN .pmmCD _5pop 5N.:HN5.NN N5._HN5.NN 5N.5555.5N N5.5HN5.5N NN.oan.5N NN._hom.5N 55m Peace _m.onNo.mN NP._n5N.mN N5.535N.5N 55.5555.5N 55.5555.5N N5.5555.5N 5HNN 55.5555.m 5N.5355.m 55.5355.m 5_.5hop.m __.55N5.5 55.5555.5 muNN 55.5H55.5 Nm.5Hmo._ 55.505N.N 55.:HN5.N 55.onmm._ mm.5n5_.F m"_N 55.55NN._P No.53_m.PF 5N.505_.FP No.555N.PF 5_.5hNN.FP 55.5555._F mHON 55.5555.5 55.5555.5 55.5H55.5 No.55N5.5 m_.5355.5 m_.55_m.5 5H5N 5550155 55.. UN55 555... 55.5 555“ $5 N55... 555 2.3535 5;: N5.5055.5 mo.onmo.o 55.55w5.5 mo.onmo.5 55.5m5_.5 55.5HN_.5 mum_ 55.5555.5 55.on_5.5 N5.5555.5 55.5555.5 N:.5h55.5 5..onmN.o NHON mm.5n55.~ 55.5555._ No.5355._ 5_.5055.P 55.5H5N._ m_.5555._ NHNF 55.5HNN._ 5N.5355._ 5_.5n55.N 55.5h5N._ 55.5nNF.N 5_.5HN5.F _"5N 55.5HN5.5 55.5355.w NN.5355.5 55.5HN5.5 55.5555.5 5..5th.m F"_N 55.5555.5 55.5355.5 55.5nNm.5 55.5H55.5 N5.5h55.5 po.onmo.o PuoN 5N.53_N.5 55.5HNP.5 55.5555.5 55.5555.m 55.5HNN.5 N5.5HN5.5 _”w. N_.5hme.5 N5.5H55.5 5_.5ame.5 _5.55_5.5 55.5355.5 55.5n5N.5 FHN_ 5_.5355.m No.5h55.m 5_.55Nm.m 55.5555.m mm.5555.m 5N.5555.m _"5_ m_.onN5.5 Np.onmm.5 N5.5HNN.5 mp.ohom.5 NN.5HNN.5 Np.on_5.5 .um. 55.555_.5 _5.5hN5.5 - h - N5.5555.5 mo.5n55.5 .5.55N_.5 F"5_ N_.on5N.5 N5.ono_.5 _5.5555.5 N5.5HP..5 55.5HNN.5 5..5355.5 ouFN 55.5n5_.m mm.ohpm.5 55.on_m.5 _N.555N.m 5m.onmp.m 5N.onpm.m 5:55 55.5hNN.5 55.53_N.5 N5.5555.5 No.555N.5 55.5HNN.5 55.5apm.5 5”N_ 55.5055.5N N5._n.m._N P_.5H5N.5N _N.5H_m.5p 5_.55F5.5N mm.onm_.5_ 5H5. 55.55N5.5 55.5H5N.5 P5.505N.5 55.555N.5 _5.onNm.o _5.5055.5 5Um_ _5.5hmm.5 55.OHmm.o mo.onmq.o 55.5H55.5 N5.5H55.5 55.5Hmm.5 5"5_ 0 m e m N — Lassa: :ongmu cucoe .quN- “a mmmcoum mo wave muwum 55555 F .uomN- pm wmmcoum mo mzpcoe o mcwcau 521mlmmocoamuwmz-_omz saw; umucmpnmca mummq meusm :_ mqump -ogqmoca No covamoasou uwum Apumm No mmmpcmucma mumcowucoaocn cw mmmcmgu .¢_m xwucmaa< 178 .mo.ova um newcomevu xpgcmuwwwcmwm mum muawgumcmaam pcmcmmmwu saw: «HNFQ mo cmnssz macaw mm._hmm.mN N5.N355.MN _N.5h55.mN m5.Nn5N.mN _N._Hm_.5N .m.5th.5N .55m== _epo+ 55.5555.55 N5._HN5.55 m_.Nn55.me mm._nNm.55 55.555N.55 5N.Nnme.me u_o:aNFON 55.555N.N No.one_.N Nm.5HNP.N 5_.5nmm.N m_.onm_.N 55.5355.m acocwwo 55._555.5N N5.on55.5N 55._n55.5N 55.5h5N.5N 55.53NN.mN mo._hN5.5N avocaocoz __.5nmN.5N 55.5h55.5N 55.5H5N.5N Nm.5h55.5N 5m.5n55.mN 55.mnmm.mN .5mm .muoe mm.5nm¢.mN 55.55N5.5N 55.5nmm.mN 55.5hNN.5N 5,.5H5N.5N 55.555N.5N QNNN m5.555N.m 55.555_.m 5_.5h5_.m 55.5nmm.m N5.Ohmp.m 5N.5n5N.m muNN 55.5555._ No.555N._ 55.5HN5._ 55.5n55._ No.5n55.N 55.5n55.5 m”_N 55.5hwm.__ 55.55_N.F_ mm.onmm.FF 55.onmm.__ 5N.5h_5.N_ N5.5h55._P 5:5N _5.5n55.5 Po.onme.o 55.5h55.5 N5.onme.5 N5.onme.5 No.55_m.5 «HON 5.3 $5 85.. :5 :5H 55.5 553585 55:85 25555 2: .5.o&55.5 N5.55m5.5 55.5mw5.o No.5hmo.5 _5.5355.5 mm.onmm.5 Num_ mo.onmm.5 _5.5555.5 N5.55N5.5 55.5Hmm.5 mo.onwe.o No.5nN5.5 NH5N m5.55N5._ No.5355._ 55.5a55._ _P.505N._ 5_.555N._ N5.555N.N Nnmp 5_.oam5.p 55.535N._ NN.505N._ 5N.5H55.F 5_.5355.F 55.onmm._ .H5N 5_.onmm.m mo.onmm.5 5N.5nN5.m NN.5nN5.m N_.OHNN.N N_.5nNN.5 _”PN 55.5555.5 55.5nwm.5 P_.55Nm.5 55.5055.5 No.onpe.o N5.53N5.5 _"5N 5N.55N5.5. mm.5055.5 N_.53NN.5 55.555N.5 __.5555.5 NF.5nN5.5 Pump 55.5355.5 mo.Ohmm.5 N5.55N5.5 N5.5h55.5 mo.OHmm.5 55.5hmm.5 PHN_ 5N.555N.m m:.5n5m.m 55.5nN5.5 55.5H_N.m m..onmo.m Nm.5n55.5 Pump 5_.5me.5 _,.5355.5 55.5h5m.5 NN.5555.5 mo.5noo.5 _5.5th.5 _um_ No.5Hmo.o 55.55m5.5 55.5555.5 _5.5555.5 55.55_o.o 55.5nN5.5 _"5_ _5.53NF.5 55.5355.5 5N.5H_N.5 _5.5n5_.5 Fo.oa_,.5 55.5hPN.5 oupN _5.5nmm.5 No.5hem.5 _5.5n55.5 mm.5hmp.m 5_.5055.m 55.5nem.5 5nm_ N5.50NN.5 N5.555N.5 N_.5355.5 _5.5355.5 No.5noN.o _5.5355.5 ouNP 55.5nNN.5N _5.5n5_.5N 5_.555N.5F 5N.5355.w~ 55.53N5.5_ 55.5355.N_ 5:55 .5.5H55.5 55.55Nm.5 5..535N.5 55.55NN.5 55.505N.5 No.5nmN.5 ohm— m5.5555.5 N5.5hN5.5 N5.5HN5.5 mo.oa_5.5 55.5355.5 5_.5n55.5 5:55 5 m 5 m N _ :ucos .uomN- um mmmcoum mo mswp Lwnszc cont—mu _m5_um 5555: .oomN- “a mmmcoum mo msucos m newczu mzzmummocozm -wmz-pumz zuwz kucm_amca AmFan cmwmv mumma cmxusm umxooo :5 muwawp nogamoca No cowuwmoaeou cwom xpumm mo mmmgcmucma mumcowugoaoca cw mmmcmgu .m.m x_o=aaa< 179 .sucoz x acwsumogh u z» .guuam u m .cuco: a : .ucuEuooL» u 55 5_.5 55.5 55.5 5N.5 55.5 55.5 55.5 N5.5 55.5 .5.5 55.5 55.5 55555 .5_55 5 NN.5 55.5 55.5 55.5 55.5 N5.5 55.5 N5.5 5N.5 55.5 55.5 5_.5 55555 .55.5 5 5_.5 55.5 55.5 55.5 55.5 55.5 55.5 _5.5 55.5 55.5 55.5 55.5 55.55 .555: 5 55.5 5.5 85 85 55.5 55.5 55.5 .55 55.5 5.5 55.5 5.55585 5:55 55555 55.5 ...5 .5.5 .5.5 55.5 55.5 55.5 .5.5 5N.5 55.5 55.5 55.555555 55555 .555 5 55.5 5_.5 55.5 5N.5 5_.5 55.5 N5.5 .5.5 .5.5 _5.5 .5.5 5N.5 5UNN 5_.5 N5.5 55.5 .5.5 55.5 5_.5 55.5 5_.5 5N.5 5N.5 55.5 5N.5 5uNN 55.5 5N.5 .5.5 .5.5 55.5 55.5 55.5 55.5 N5.5 55.5 55.5 5N.5 5u_N 55.5 55.5 5N.5 N_.5 55.. N5.5 5N.5 .5.5 55.5 .5.5 55.5 5N.5 5N5N 55.5 5_.5 5_.5 5_.5 55.5 55.5 __.5 55.5 55.5 55.5 55.5 55.5 5N5N N_.5 N5.5 N5.5 55.5 55.5 55.5 .5.5 55.5 55.5 5N.5 55.5 55.5 5:5. N5.5 5N.5 55.5 55.5 55.5 55.5 55.5 5..5 55.5 55.5 55.5 55.5 555. 5_.5 55.5 5_.5 55.5 55.5 55.5 _5.5 55.5 55.5 55.5 55.5 55.5 NH5N N5.5 55.5 N5.5 .N.5 55.5 .5.5 55.5 .5.5 5N.5 55.5 55.5 55.5 N55— 55.5 55.5 .5.5 55.5 55.5 55.5 55.5 .5.5 5N.5 55.5 55.5 55.5 _“5N .5.5 .5.5 5_.5 55.5 N5.5 55.5 55.5 .5.5 55.5 .5.5 55.5 55.5 _"_N 55.5 5..5 5_.5 5N.5 55.5 55.5 55.5 .5.5 N5.5 .N.5 55.5 5..5 .u5N N5.5 NN.5 .5.5 55.5 .5.5 .5.5 55.5 .5.5 .5.5 55.5 55.5 N5.5 .55. 55.5 5_.5 55.5 N5.5 .N.5 55.5 55.5 55.5 55.5 N_.5 55.5 55.5 ."55 55.5 N5.5 5_.5 55.5 N5.5 55.5 N5.5 _5.5 55.5 N5.5 55.5 5N.5 .55. 55.5 55.5 55.5 55.5 55.5 55.5 .5.5 .5.5 N5.5 55.5 55.5 55.5 _”5_ 55.5 ...5 .5.5 55.5 55.5 5_.5 N5.5 .5.5 55.5 55.5 55.5 55.5 .55. 55.5 55.5 N_.5 5_.5 5_.5 5_.5 5_.5 5N.5 55.5 55.5 55.5 5_.5 5"_N 5N.5 5N.5 55.5 5N.5 55.5 55.5 .5.5 .5.5 55.5 .5.5 55.5 55.5 555. 55.5 N5.5 55.5 55.5 55.5 55.5 .5.5 55.5 55.5 55.5 55.5 55.5 5“5_ 55.5 55.5 _5.5 55.5 55.5 5N.5 _5.5 .5.5 55.5 N5.5 55.5 .5.5 5:5. 55.5 .N.5 .5.5 55.5 55.5 55.5 N5.5 .N.5 55.5 55.5 55.5 55.5 5H5. 5_.5 NN.5 55.5 55.5 NN.5 N5.5 N_.5 _5.5 55.5 _5.5 5N.5 55.5 5H5, 5555555555555 555555 55555521 555555 555551 555555555> 5° mucaom .uomN- 55 5555055 55 55555: 5 55.555 acoeuuocu 5=o5co> :55: 55555555255 5.55 55555 :5 5555535 no 5555555555 5 55 55555555055 oucuu5v—ca55 maus5xoL55< .o_m xpuco55< 180 Appendix 817. The formula for computing corrected TBA values of sucker products. x Transmittance Correct extinction at Code I - 532 nm (or corrected Ys32 nh Y460 hm R"'s32/"460 TBA '°‘"°) 1 0 x - 0.7773 k 83 79 0.7773 532 460 11/18 Fish Balls 0 9145 a o 2 K - x .0.06651 80 81 0.6651 532 460 11/18 :1 0 9268 0 3 K - x 11 18 2c 85 76 0.6144 532 0.6144 460 I ' 0 9324 11/18 73‘ 83° 742 0.6330 K532 ' 0.6330“460 11/18 34' 821 693 0.5419 K532 - 0.5419‘460 ""'079IUT""" 0 0 x x 11 22 Fish Balls 87 79 - 0.5330 532 - 0.5330 460 ’ . Ti9414 11/22 11° 66° 603 0.8341 K532 - 0.8341K460 . O 1 11/22 42‘ 62° 55 2 0.8056 K532 - 0.8056K460 “"'UT§TTI“"" 11/22 33‘ 823 733 ' 0.6218 K532 - 0.6218‘460 """0T93T6""" 11/22 14‘ 74‘ 68° 0.7719 K532 - 0.7719K460 019151 aCalculation for the corrected extinction formula: e.g. 11/18 Balls x - K532 - 0.7773 y --- (1) ‘ y - K460 - 0.11 x ----- (2) combine formula (1) 3 (2), then x , K532 - 0.7773‘460 0.914s bRaw fish paste added with NaCl-MSG-Sucrose. cRaw fish paste added with NaCl-MSG-SHMP. dRaw fish paste added with MSGoSucrose-SHMP. eRaw fish paste added with NaCl-MSG-Sucrose-SHMP. 181 Appendix C. Name: 1. SENSORY EVALUATION FORM Date: You are given 2 samples for this sensory evaluation. Please mark down the sample number on the horizontal line for the most appropriate strength of each quality charac- teristic. Rancid odor: Not detectable Detectable Strong 4 :1 J; 1 l 1 l Putrifactive odor: Not detectable detectable strong Putrifactive flavor: Not detectable detectable strong 1_ L L J 1 L I Rancid flavor: i I L Not detectable detectable strong Texture resiliance: Heak i #4 strong 4‘ 1_ _i J 4 i Acceptability: Texture: Not acceptable J— L L Flavor: Not acceptable acceptable L J L acceptable