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V '7-7 4 I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII lllllllllllfllllllllllllllljlllzlsllll WW“ ~ Michigan State University This is to certify that the thesis entitled STABILITY OF LIPIDS IN RESTRUCTURED BEEF STEAKS presented by Rhonda Lynn Crackel has been accepted towards fulfillment of the requirements for M.S. degfiein Food Science Major professor Dan: September 10, 1986 0-7639 MSUium Am...,...-... . - r1 RETURNING MATERIALS: MSU Place in booE drop to remove this checkout from w your record. FINES will be charged if book is returned after the date stamped below. £58 2 5 IVE] M”; 7% 9/ 3%2‘5“'i¥31 ’9 ’JAN 0 6'?ng STABILITY OF LIPIDS IN RESTRUCTURED BEEF STEAKS BY Rhonda Lynn Crackel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1986 4914.955??? ABSTRACT STABILITY OF LIPIDS IN RESTRUCTURED BEEF STEAKS BY Rhonda Lynn Crackel The effect of antioxidants on the stability of lipids in restructured beef steaks was investigated. Two natural formulations--containing mixed tocopherols, ascorbyl palmitate and citric acid-~and TBHQ were evaluated. Ilipid oxidation was monitored over 12 months of frozen storage by a modified TBA procedure, gas chromatographic analysis (If volatiles and sensory evaluation. TBHQ significantly (p <0.05) lowered TBA numbers iju raw and freshly cooked samples and in samples that were cocfl chicken dark meat. These trends were indicated by increased TBA numbers and decomposition of constituent lipids. Evidence suggests changes occurring in the total lipid content of raw meat during frozen storage are due mainly to losses in the triglyceride fraction whereas the levels of phospholipids remain relatively constant even over extended storage periods (Igene gt gt., 1979b). Contradictory data 12 from Keller and Kinsella (1973) indicated negligible decreases in total lipids but significant losses of phospho- lipids during frozen storage of hamburger. These trends may have been caused by the grinding of the meat which promoted oxidation of the membrane-bound phospholipids (Sato and Hegarty, 1971). Lipid Oxidation in Meats Mechanism of Lipid Oxidation The autoxidation of unsaturated fatty acids is generally accepted to proceed according to a free radical chain mechanism. The susceptibility and rate of oxidation of fatty acids varies according to their degree of un- saturation and the presence of activating agents such as heat, light, enzymes or metal catalysts or inhibiting substances. The autocatalytic theory was extensively reviewed by Dugan (1961) and Lundberg (1962) and may be summarized as follows: I. Initiation RH + O ------ > R° + OH° 2 RH represents an unsaturated fatty acid containing a labile hydrogen on a reactive methylene group adjacent to a double bond. R’ is the free radical formed by abstraction of the labile hydrogen. II. Propagation ------ > ROO' R + 02 13 R00' + RH ------ > ROOH + R’ Reaction of the lipid free radical with oxygen yields a peroxy radical which may abstract a labile hydrogen from another unsaturated fatty acid. A hydroperoxide, ROOH, and another free radical, R’, which is capable of perpetuating the chain reaction are formed. Free radicals may also combine to form stable non- reactive products which terminate the chain reaction. III. Termination R’+R' ------ >RR R' + ROO' ------ > ROOR ROO' + ROO' ------ > ROOR + O 2 Products of Lipid Oxidation Hydroperoxides, ROOH, are the primary initial products of lipid oxidation. Many secondary products are formed through subsequent reactions as shown in Figure 1 (Gray, 1978). The decomposition of hydroperoxides may produce alcohols, ketones, acids and lactones via either of two pathways. At low peroxide concentrations, hydroperoxide decomposition is mainly monomolecular (Dugan, 1961; Lundberg, 1962). ROOH ------ > R0' + OH' At higher concentrations, bimolecular decomposition occurs. 2ROOH ------ > ROO‘ + R0‘ + H20. The decomposition of hydroperoxides is of great impor- tance in the development of WOF. Hydroperoxides themselves are odorless; however, their breakdown to aldehydes, ketones 14 .mmcvxocmaoccxg pm» mo cowpwmoceocmc co mcpzoc meow .H mczmwd 15 mwo_mm0>._0 .. IO .0 mw0_mm0>..0 . IO w0_0< I mDZDOQEOU .. IO mw0.mm0>._ C.OD>INDJ< mmD>ImDJ<._—2mm wmo_x0n.w wmn_mm0>._0.0._.m¥ mmD>ImDJ< $300.05 .850 5 :o n :o .c 538.5 / \ cozmu mums—>JOQ I mmo.x0mma.o \ :8 20:82:50 co_mm_.... costs“. / \\ mmExOmchmoE .2“. cozmntoEzoa I mmmE>JOQ mum—10.1 .mmm—ZE 16 and acids may result in rancid off-odors and flavors. While many of these secondary products are very reactive and may not accumulate appreciably in an oxidized product, con- centrations as low as 1 ppb may exceed flavor thresholds (Sato gt gt., 1973). Hexanal has been cited as one of the major secondary products formed during the oxidation of linoleic acid (Gaddis gt gt., 1961; Frankel gt gt., 1981). El-Gharbawi and Dugan (1965) reported the formation of hexanal during the storage of freeze-dried beef, while Cross and Ziegler (1965) measured both hexanal and pentanal in cooked, uncured pork. Love and Pearson (1976) detected hexanal in headspace samples taken from oxidizing PE and cooked meat and reported higher concentrations with increasing oxidation. Work by Fritsch and Gale (1977) suggests that hexanal production is indicative of lipid oxidation in dry cereals and may be related to the development of rancid odors. Other aldehydes have also been linked to WOF. Ruenger gt gt. (1978) reported that heptanal and n nona-3-6-dienal are major flavor compounds related to WOF in turkey. Bailey gt gt. (1980) identified 2,4 decadienal along with hexanal in cooked beef. Alone and in combination, these two compounds have been highly correlated with flavor scores of oxidized oils (Dupuy gt gt., 1976; Rayner gt gt,, 1978). Another aldehyde of major importance in oxidizing foods is malonaldehyde, a three-carbon dialdehyde produced during autoxidation of polyunsaturated fatty acids. A mechanism 17 for the formation of malonaldehyde was proposed by Dahle gt gt. (1962) and modified by Pryor gt gt. (1976). The revised scheme suggests a prostaglandin-like endoperoxide mechanism in which malonaldehyde is formed via the cleavage of a cyclic endoperoxide. The formation of malonaldehyde during lipid oxidation is the basis for several chemical methods of monitoring oxidation. One such method, the thiobarbituric acid (TBA) test, involves the condensation of two molecules of thiobarbituric acid with malonaldehyde to form a pink pigment which can be quantitated by measuring absorbance at 532 nm. This method has been criticized, however, as being nonspecific for malonaldehyde and therefore an unreliable indicator of oxidation. Consequences of Lipid Oxidation As shown in Figure 1 (Gray, 1978) the hydroperoxides from lipid oxidation may undergo any of several secondary reactions. Although the formation of off—flavor compounds is a major determinant of product quality, several other reaction may affect a product's safety and acceptability. Secondary reactions may lead to the formation of potentially toxic compounds, result in a loss of nutrients or promote further oxidative reactions. Pearson gt gt. (1983) reviewed the safety implications of lipid oxidation in muscle foods. They reported that evidence of the direct toxicity and mutagenicity of malonaldehyde is conflicting and inconclusive although data suggest that hydrolysis products may be mutagenic. 18 Additionally, malonaldehyde has been implicated as a catalyst of the formation of carcinogenic N—nitrosamines. It has also been reported that malonaldehyde and other lipid oxidation products can react with amino acids to form Schiff bases or may crosslink with proteins thus reducing the nutritional quality of a muscle food (Chic and Tappel, 1969; Braddock and Dugan, 1973; Gardner, 1979). Another consequence of lipid oxidation is the loss of the desirable red color of raw meat due to the formation of metmyoglobin (Meth). Haurowitz gt gt. (1941) demonstrated in model systems that free radicals from oxidizing lipids could destroy heme pigments. A positive correlation between metmyoglobin accumulation and lipid oxidation in raw meat was reported by Hutchins gt gt. (1967). Many other studies confirmed the interdependence of the two reactions (Greene, 1969; Greene gt gt., 1971; Greene and Price, 1975; Benedict gt gt., 1975). Through the use of antioxidants these researchers were able to prevent lipid oxidation and the conversion of myoglobin to metmyoglobin. Work with enzymic lipid peroxidation of microsomal fractions produced similar results (Lin and Hultin, 1977); the oxidation of oxymyoglobin to metmyoglobin was prevented by BHA or glutathione peroxidase, which functions to decompose lipid hydroperoxides to non-reactive compounds. From his work with glutathione peroxidase, Hultin (1980) concluded that lipid oxidation precedes pigment oxidation 19 since no metmyoglobin was formed in a system treated with this enzyme. Catalysis of Lipid Oxidation Heme Compounds The catalytic effect of iron porphyrins--hemoglobin, myoglobin and cytochromes--on lipid oxidation was first cited by Robinson (1924). Numerous studies have confirmed that heme compounds function as prooxidants when in contact with purified lipids (Tappel, 1952; Banks gt gt., 1961; Tappel, 1962; Liu, 1970; Hirano and Olcott, 1971), and it has been suggested they might function as prooxidants in meats (Younathan and Watts, 1958; Liu and Watts, 1970; Greene and Price, 1975). The valency of iron in the proposed catalytic species has been widely disputed. Comparing cured and uncured cooked meats, Younathan and Watts (1958) reported that Fe3+ hemes were the active catalysts rather than the ferrous form of nitric oxide hemochromogen. However, Brown gt El“ (1963) reported that hemes containing either Fe2+ or Fe3+ exhibited catalytic activity. This was confirmed by Hirano and Olcott (1971) who also reported that rates of oxidation did not differ when Fe2+ or Fe3+ hemes were added to linoleate emulsions. Work by Greene and Price (1975) demonstrated that either ferrous or ferric compounds might function as 3+ catalysts, but that the Fe is more active. 20 Labuza (1971) suggested that the ionic state of the metal is not as important in catalysis as is the ring structure of the heme. He proposed that the iron in heme may be sterically hindered by the large protein portion of the molecule. The rapid rate of oxidation in cooked meat would result from the denaturation of myoglobin and subsequent exposure of the iron. He also suggested that heating may release either the pigments or the lipids from protected compartments, causing more intimate contact between the two compounds. Tappel (1962) proposed that the most probable mechanism of catalysis involves the formation of a coordinate complex between a hematin compound and lipid hydroperoxide. The close proximity and high electronegativity of the two oxygens would favor scission of the ROOH to form a free radical. The radical could abstract a labile hydrogen from an unsaturated fatty acid and propagate lipid oxidation. In this mechanism there would be no change in the valence of the heme iron. Kanner and Harel (1985) reported the initiation of membranal lipid oxidation by "activated" metmyoglobin and methemoglobin (MetHb)--heme pigments combined with hydrogen peroxide. Heme pigments or H202 alone did not promote oxidation. However, the interaction of H202 with Meth led very rapidly to the production of an activated species which initiated lipid peroxidation. They proposed that the autoxidation of oxyhemoglobin and oxymyoglobin leads to the 21 formation of methemeproteins and the superoxide radical, O —, which dismutes to H202. A reactive porphyrin cation 2 radical, P‘+--FeIV+=O, results from the reaction of Meth or MetHb with H202. This radical may then react to produce lipid free radicals which can initiate lipid oxidation. In addition to their catalytic activity, heme compounds may also exhibit antioxidative properties. This depends largely on the proportion of heme and lipid in the system (Lewis and Wills, 1963). Kendrick and Watts (1969) examined the critical linoleate:heme ratios necessary for maximum catalysis of lipid oxidation. As the concentration of heme compounds was increased to optimal levels, lipid oxidation increased to a maximum. At heme concentrations exceeding the critical levels, oxidation decreased to zero. At these inhibitory heme concentrations, a red pigment, believed to be a stable heme—hydroperoxide complex, was formed. They also discovered that breakdown products from the reaction of hemes with H202 exhibited antioxidant properties. Similar work by Hirano and Olcott (1976) demonstrated that high concentrations of heme compounds inhibit lipid oxidation, while heme compounds at lower concentrations function as prooxidants. They proposed that compounds formed at high heme concentrations may act as free radical sinks thus decreasing lipid oxidation. Much of the data regarding heme catalyzed lipid oxidation is confusing and contradictory. Love (1983) 22 explained that this is most likely due to the use of dif- ferent types of model systems and experimental techniques for monitoring lipid oxidation. Additionally, results obtained from a model system may not be representative of the reactions and mechanisms found in meat systems. She suggests carefully designed and controlled studies are needed to clarify the role of heme compounds in lipid oxidation in meat. Metals and Non Heme Iron Heavy transition metals such as cobalt, copper, iron, manganese and nickel possessing two or more valency states generally increase the rate of oxidation of food lipids (Ingold, 1962). They may alter the rates of initiation, propagation and termination reactions as well as the rate of hydroperoxide decomposition. Their basic function is to increase the rate of formation of radical species. Heaton and Uri (1961) reported that metals in their higher valency state may initiate lipid oxidation directly via the following reaction: M(n+1)+ (n+1)+ +RH ------ >M H+R° Metals in lower valency states may also initiate oxidation directly although they are thought to activate oxygen first with subsequent reactions producing free radicals. n+ (n+1)+... M + O < ------ > (M 02') ------ > free radicals 2 Ingold (1962) suggests that while these direct metal— lipid reactions may be significant in the early stages of oxidation, at later stages other reactions are more 23 important. The metal catalysed decomposition of hydro- peroxides occurs much more rapidly that thermal decompo- sition and is the source of many free radicals as shown below. Mn+ + ROOH ------ > M(n+l)+ + RO' + OH- M(n+l)+ + ROOH ------ > Mn+ + ROO' + H+ Iron is the heavy metal most prevalent in meats. While most of the iron in meats is found in heme compounds, a number of compounds contain nonheme iron. Feritin, hemosiderin and transferrin function in the storage and transport of nonheme iron (Pearson gt gt., 1977), and several enzymes of the electron transport system contain nonheme iron. Several researchers have proposed that non- heme iron may also be a catalyst of lipid oxidation (Wills, 1965; Liu, 1970; Liu and Watts, 1970; Sato and Hegarty, 1971; Love and Pearson, 1974; Igene gt gt., 1979a). Work by Wills (1966) showed that both heme and nonheme iron may catalyze lipid oxidation but that nonheme iron was a more active prooxidant at acid pH while heme compounds were less pH sensitive. This was confirmed by Liu (1970) and Liu and Watts (1970) who demonstrated that nonheme iron exhibited catalytic activity below pH 6.4 while Meth was active over the pH range 5.6 to 7.8. Sato and Hegarty (1971) presented evidence that nonheme iron is the major catalyst of lipid oxidation in cooked meat. Beef muscle was extracted with water prior to cooking to remove any potential prooxidants. Addition of myoglobin 24 or hemoglobin to the cooked, extracted fibers had little effect on lipid oxidation. However, FeCl and FeCl both 2 3 promoted lipid oxidation with the ferrous form being more active. Love and Pearson (1974) reported similar results. Addition of nonheme Fe2+ to extracted muscle residue, at levels as low as 2ppm, resulted in increased TBA numbers while Meth added at 1 to 10 mg/g did not promote oxidation. They also found, as did Sato and Hegarty (1971), that low levels (5 ppm) of ascorbic acid added with the Fe2+ enhanced its catalytic effect presumably by maintaining the nonheme iron in the ferrous state. Igene gt gt. (1979a) also concluded that myoglobin is not the principal prooxidant in cooked meat. They reported that addition of a pigment extract to cooked beef muscle residue enhanced lipid oxidation. However, treatment of the extract with EDTA lessened its prooxidant activity. Treat- ment of the extract with H202 to destroy hemes increased its catalytic activity, which could also be suppressed by EDTA. Igene gt gt. (1979a) reported that over 90% of the iron in the fresh meat pigment was present as bound heme iron. Cooking destroyed the heme molecule and increased the concentration of nonheme iron in the extract from 8.72% to 27.0%. Thus, they concluded that the increased rate of lipid oxidation in cooked meat is due to the release of nonheme iron during cooking which catalyzes oxidation rather than the meat pigments per se. 25 Microsomal Enzymes While it is generally accepted that lipid oxidation in muscle foods is essentially nonenzymatic in nature, there is evidence of enzymatic lipid peroxidation systems associated with muscle microsomes. Lin and Hultin (1976) demonstrated that microsomes from chicken leg and breast muscle produced malonaldehyde in the presence of ADP, NADPH, ferric chloride and oxygen. Further studies (Lin and Hultin, 1977) showed that peroxidizing microsomal fractions could oxidize myo- globin in vitro, and that inhibition of microsomal oxidation inhibited pigment oxidation. Hultin (1980) reported that addition of glutathione and glutathione peroxidase to an oxidizing microsomal system could decrease myoglobin oxidation. Since glutathione/glutathione peroxidase are known to decompose lipid hydroperoxides without the production of free radicals, Hultin concluded that lipid peroxidation may precede pigment oxidation. Earlier studies of the oxidation of myoglobin (Govindarajan gt gt., 1977) showed that addition of lipase to ground beef increased pigment oxidation while addition of phospholipase A inhibited both pigment and lipid oxidation. The latter observation was unexpected since free fatty acids oxidize faster than the esterified form. Govindarajan gt gt. (1977) suggested that phospholipase A might inactivate an enzymatic system which destabilizes myoglobin. Hultin (1980) reported similar data but suggested that phospholipase A inhibition may be caused by free fatty acid 26 inhibition of the oxidative enzyme system in the microsomal membrane. He also proposed that, under proper conditions, native phospholipases might be used to produce a natural antioxidant system. Sodium Chloride (Salt) It is widely recognized that sodium chloride may initiate color and flavor changes in meat although the mechanism remains unclear. Early work suggested that NaCl catalyzed oxidation by lipoxidase (Lea, 1937) or by myoglobin (Tappel, 1952; Banks, 1961). Chang and Watts (1950) reported that salt had no greater effect on rancidity in the presence of hemoglobin or muscle extract than in their absence. They also demonstrated that the catalytic effect of NaCl depended on its concentration and the amount of moisture in the system. Aqueous salt solutions were prooxidative only at concentrations of NaCl above 15% while dry NaCl readily promoted oxidation of lard. The mechanisms of NaCl-induced rancidity in pork were examined by Ellis gt gt. (1968). They reported that increasing levels of NaCl accelerated autoxidation but did not alter the decomposition of hydroperoxides to mono- carbonyls. However, in samples containing high proportions of lean, lower conversion of peroxides to monocarbonyls was observed. They postulated that NaCl may activate a component in the lean which results in a change in oxidation characteristics of the adipose. 27 Other researchers have proposed that salt catalyzed rancidity may be related to traces of metal impurities in the salt. Olson and Rust (1973) reported, though, that using a purified low—metal salt to cure hams did not improve taste panel scores over those for hams cured with conven- tional salt. They did find, however, that hams cured with salt containing antioxidants were preferred over the control samples and hams cured with low-metal salt. Sodium chloride has also been reported to inhibit lipid oxidation under certain conditions. Mabrouk and Dugan (1960) observed that autoxidation of aqueous emulsions of methyl linoleate was suppressed by increasing concentrations of dissolved NaCl in the system. They suggested that the inhibition might result from decreased solubility of oxygen in the emulsions. Measurement of Lipid Oxidation Numerous chemical and physical methods have been devel— oped for monitoring oxidation in oils and lipid-containing foods. These have been reviewed extensively by Gray (1978) and Melton (1983). In this review, several methods commonly used for measuring lipid oxidation in meat systems will be addressed. TBA Test One of the most commonly used methods for monitoring lipid oxidation in meat products is the 2-thiobarbituric acid test or TBA test (Gray, 1978; Melton, 1983). The 28 premise of this method is the condensation of two molecules of TBA with one molecule of malonaldehyde to produce a red complex which is quantitated spectrophotometrically in the region of 530 to 532 nm (Sinnhuber gt gt., 1958). The extent of lipid oxidation is expressed in terms of 3 TBA number having units of mg of malonaldehyde per kg of sample. The malonaldehyde found in oxidized meat is a secondary oxidation product formed mainly from polyunsaturated fatty acids containing three or more double bonds (Dahle gt gt., 1962; Pryor gt gt., 1976). There are several ways in which the TBA test can be performed on muscle foods: 1) directly on the product, followed by extraction of the red pigment (Sinnhuber gt gt., 1958); 2) on a portion of the steam distillate of the food (Tarladgis gt gt., 1960) or 3) on an extract of the food (Witte gt gt., 1970). The most frequently used method involves steam distillation of volatiles--namely malonaldehyde--from the food and reaction of a portion of the distillate with TBA reagent to produce the character- istic red complex. This method has been widely used to monitor lipid oxidation and the development of WOF in poultry, beef and pork products (Melton, 1983). The TBA test initially was reported to be specific for malonaldehyde (Sinnhuber gt gt., 1958). However, several researchers have reported that TBA can react with other oxidation products such as 2,4 alkadienals and 2 alkanals to produce compounds with the same absorption maximum as the 29 malonaldehyde-TBA complex (Jacobson gt gt., 1964; Marcuse and Johansson, 1973). Additionally, the amount of malonaldehyde in an oxidized sample depends on the fatty acid profile since oxidizing PUFAs produce more malonaldehyde than mono or diunsaturated fatty acids (Pryor gt gt., 1976; Pearson gt gt., 1983). Thus, the TBA method may be best used to assess the extent of lipid oxidation in general and to compare samples of the same composition at different stages of oxidation (Gray, 1978). The original TBA distillation method has been modified by several researchers. Tarladgis gt gt. (1964) demon- strated that the usual acid and heat treatment was not necessary for the condensation reaction of TBA with malonaldehyde, nor for maximum color development. The acid- heat treatment was found to alter the Em530 value without accelerating the condensation reaction any more that heat alone. Use of an acidic TBA reagent was also found to con— tribute to the appearance of an absorption peak at 450 nm. These researchers proposed that acid may be responsible for the degradation of TBA which, when reacted with extracts or distillates from samples, produces peaks at 390 and 452 nm. To minimize these interfering reactions, Tarladgis gt gt. (1964) recommended a modified distillation method in which distillates of samples are reacted with TBA without the use of any acid. Zipser and Watts (1962) modified the TBA method for analysis of nitrite-cured meats. Residual nitrite may react 30 with malonaldehyde during distillation to form oximes which result in lower TBA values. Sulfanilamide, added prior to distillation, reacts with the nitrite to form diazonium salts which do not interfere with the malonaldehyde-TBA reaction. Shahidi gt gt. (1985) have pointed out, however, that the use of sulfanilamide when residual nitrite is not present may also lead to erroneously low TBA numbers. In general, the TBA numbers obtained by the distil- lation method are higher than those determined utilizing the extraction method. According to Witte 23.21- (1970) the distillation method produces TBA numbers which may be twice as large as those produced by an extraction method. Data from Siu and Draper (1978) showed that TBA numbers from the distillation procedure were approximately 1.5 times higher than those obtained from muscle extracts. They suggested the discrepancies in TBA values may result from: a) incom— plete extraction of malonaldehyde using the filtration method, b) the release of bound malonaldehyde from proteins and amino acids during distillation or c) the formation of malonaldehyde during distillation. The possibility of lipid oxidation and malonaldehyde formation during sample distillation was further investigated by Rhee (1978). She reported that chilled blending and addition of propyl gallate (PG) and EDTA to distillation mixtures substantially reduced the TBA numbers of catfish samples but did not show any significant effect on beef, pork and chicken samples. Based on these findings, 31 Rhee (1978) recommended the addition of PG and EDTA to samples, particularly fish, to minimize further lipid oxidation during the TBA distillation procedure. Pikul gt gt. (1983) evaluated the influence of butylated hydroxytoluene (BHT) on the TBA assay of fat extracted from chicken breast and leg meat. Samples analyzed without added BHT reportedly yielded six times higher malonaldehyde concentrations than those treated with BHT during extraction and 75pg BHT/mg of fat during the TBA analysis. Thus, these workers recommended that BHT or other suitable antioxidant should be added during sample prep- aration or before the critical heating step of any TBA assay to prevent sample autoxidation and erroneously high TBA values. The TBA test has also been modified to make it specific for malonaldehyde. Kakuda gt gt. (1981) used high performance liquid chromatography (HPLC) to quantitate malonaldehyde in aqueous distillates from freeze dried chicken samples having TBA numbers ranging from 3.96 to 16.60. They reported a linear relationship, with an r2 of 0.946, between the TBA absorbance at 532 nm and the HPLC peak height of malonaldehyde. Kakuda gt gt. (1981) also reported that the HPLC method required less time than the standard TBA method, was more sensitive and was not affected by the presence of other TBA reactive substances or side reactions. 32 Another HPLC method was developed by Csallany gt gt. (1984) to quantitate malonaldehyde in rat liver and beef, pork and chicken muscle. They found that malonaldehyde levels as measured by the TBA method were four to five times higher than those obtained by the HPLC method. This was attributed to the artifactual formation of malonaldehyde during heating steps or to the presence of interfering color-reactive compounds. Gas Chromatogtaphic Analyses Gas chromatography (GC) has been extensively used to monitor the products of lipid oxidation in model systems in order to elucidate the mechanisms of oxidation (Gray, 1978). Methods have also been developed which utilize secondary oxidation products as indices of lipid oxidation in foods. Hexanal, a major secondary product of linoleate oxidation, and other aldehydes have been identified in ox- idizing food systems including freeze dried beef (El-Garbawi and Dugan, 1965), cooked pork (Cross and Ziegler, 1965), cooked beef (Love and Pearson, 1976; Bailey gt gt., 1980), dry cereal (Fritsch and Gale, 1977) and cooked turkey (Ruenger gt gt., 1978). These volatile compounds have been used successfully to follow lipid oxidation in vegetable oils (Dupuy gt gt., 1976: Waltking and Zmachinski, 1977) and to predict flavor scores of various oils (Dupuy gt gt., 1977; Jackson and Giacherio, 1977; Warner gt gt., 1978). Several GC methods have been developed for following lipid oxidation. Fritsh and Gale (1977) utilized a 33 headspace gas-sampling technique to measure hexanal in cereals. Volatiles were released from the cereal by the addition of boiling water, the system was sealed and a sample of headspace gas was withdrawn for CC analysis. A direct GC method for the analysis of vegetable oils was developed by Dupuy gt gt. (1976) and later modified for aqueous and nonaqueous systems by Legendre gt gt. (1979). With this technique, the food to be analyzed is placed in an external inlet assembly where the volatiles are stripped from the sample by heat and an inert carrier gas. The volatiles condense onto a column which is then temperature programmed in an appropriate manner for GC analysis. Legendre gt gt. (1979) reported that this procedure is rapid, efficient and sensitive enough that sample volatiles can be analyzed without the need for prior enrichment. Bailey gt gt. (1980) used this direct sampling method to analyze volatile flavor components from cooked Boston butts. Data obtained from freshly cooked samples compared to that from meat stored at 4°C for one day following cooking indicated that concentrations of hexanal and 2-pentyl furan increased significantly making them excellent indices of oxidative changes. Other aldehydes increased in concen- tration over the storage period, but not as substantially. A third GC method, summarized by Bailey gt gt. (1980), involves the solvent extraction of volatiles using a Likens— Nickerson extraction apparatus. Volatiles released by boiling an aqueous homogenate of the sample were extracted 34 into a suitable organic solvent in a closed continuous extraction system. The solvent containing the extracted volatiles was concentrated and analyzed using conventional GC methods. This method was utilized by Bailey gt gt. (1980) to monitor lipid oxidation in roast beef stored at 4°C. Again, they observed an increase in the concentration of hexanal and 2-pentyl furan over time. Pentanal also increased during the storage period. From this work, Bailey gt gt. (1980) concluded that gas chromatographic methods are more informative than the TBA test since selected individual compounds can be quantitated. Secondary lipid oxidation products other than aldehydes have been used to follow lipid oxidation. Saturated hydro- carbons are produced during the early stages of oxidation when aldehydes are either absent or not detectable (Horvat gt gt., 1964; Selke gt gt., 1970). Warner gt gt. (1974) reported significant correlations between the concentration of pentane in headspace samples and the sensory scores from trained panelists for both aged vegetable oils and potato chips. Pentane formation has also been used to follow lipid oxidation in muscle foods. Seo and Joel (1980) monitored lipid oxidation in freeze dried pork by measuring levels of hydrocarbons in the headspace over the samples. The samples were then evaluated by a sensory panel to assess the degree of rancid odor. The overall pattern of pentane production was found to be significantly correlated with rancidity 35 scores. Additionally, 5ul pentane/10 9 meat appeared to be the threshold for rancid odor. The authors suggested that while pentane was not directly responsible for rancid odors, pentane levels were indicative of the degree of lipid oxidation. Sensory Evaluation The most critical test of a food product's quality is its acceptance by the consumer. Consequently, many researchers have attempted to correlate data obtained from chemical measurements of lipid oxidation with the devel— opment of off—flavors and odors. Tarladgis gt gt. (1960) reported a high correlation between TBA numbers of ground pork and the sensory scores from panelists trained to detect rancid odors. -Additionally, they reported the fedge”of TBA"\\ numbers at which rancid odors were first perceived by panelists to be between 0.5 and 1.0. 7 Other researchers have shownthat TBA numbers are related to sensory scores of oxidized and warmed over flavors in meat (Watts, 1962; Igene and Pearson, 1979; Igene gt gt., 1979a). Greene and Cumuze (1981) examined the rela- tionship between TBA numbers of cooked beef and the sensory score for oxidized flavor obtained from untrained panelists. They reported significant but low correlation coefficients for sensory scores versus TBA numbers and attributed this, in part, to variability in panelist scoring. When the panel was reduced to those statistically shown to be consistent in their scoring, correlation coefficients increased. The TBA 5.? .- l l I \ 36 /‘ / "threshold range for the "discriminating" panelists was 0.6 to 2.0 which Greene and Cumuze considered in close agreement with the results of Tarladgis gt gt. (1960). Similar studies by Igene gt gt. (1985a) examined the relationship between TBA numbers and sensory scores from a trained panel for WOF in cooked chicken white and dark meat. The meat was treated with chelators and/or antioxidants to produce samples having a wide range of TBA numbers. These researchers reported that TBA values were closely related to panel scores and that changes in TBA numbers accounted for over 75% of the variation in WOF scores. Attempts have also been made to correlate data from GC analyses with flavor scores. The presence and concentration of certain volatiles have been correlated to flavor scores of vegetable oils (Evans, 1969; Dupuy gt gt., 1976; Warner gt gt., 1974), while Fritsch and Gale (1977) reported that hexanal in dry cereal was indicative of lipid oxidation and could be responsible for off flavors. Ruenger gt gt. (1978) reported that heptanal and n-nona-3,6-dienal are related to WOF in turkey. Seo and Joel (1980) correlated pentane con- centrations with rancid odor in freeze dried pork, although they reported that pentane was not responsible for the off odors. Bailey gt gt. (1980) found that hexanal and 2-pentyl furan were excellent indices of oxidation in meat products but did not address their contribution to warmed over flavor. Clearly, further research is needed to identify 37 those volatiles responsible for warmed-over flavor and to establish threshold values for their detection by sensory evaluation. Lipid Stability in Restructured Meats Manufacture of Restructured Meats The technology for manufacturing restructured meats was developed in the 1970‘s as a means for utilizing lower grades and cheaper cuts of meats that would normally be processed into roasts or ground meat (Booren gt gt., 1981). The goal was to manufacture a product which: (i) resembles an intact muscle in textural properties, (ii) is uniform, (iii) has desirable color and (iv) is completely edible. The methods for producing restructured meats have been well summarized and reported by several sources (Breidenstein, 1982; Seideman and Durland, 1983). The basic procedure of restructuring requires a reduction of particle size of the meat, blending of these particles and reforming them to the desired final shape. Flake-cutting, slicing and sectioning are three comminution methods often used in manufacturing restructured products. Textural properties and other sensory charac— teristics of the finished product depend largely on the comminution method used and final particle size. It has been reported that flaking yields restructured products with improved texture, cohesiveness, juiciness and tenderness over those produced by grinding. Additionally, products 38 made with small and medium size flakes receive higher sensory scores than those made with large flakes (Seideman and Durland, 1983). Slicing has also been used to produce desirable restructured meat products (Ockerman and Organisciak, 1979). Nobel gt gt. (1982) compared intact steaks and restructured steaks made from beef sliced 2.5, 5.0 or 7.5mm in thickness. Slice thickness had little effect on sensory attributes although restructured steaks were judged more tender and palatable than intact muscle steaks. A procedure utilizing sectioning involves the cutting of large muscles into chunks of uniform or varying size. This method yields steaks having textural properties more nearly resembling those of intact muscle steaks than does the flaking process. Products manufactured by this procedure must have a lower fat content than flake-cut products, however, because fat particles are much larger and more noticable. Additionally, mechanical tenderization of the larger muscles may be necessary prior to sectioning to assure the desired tenderness of the finished product (Seideman and Durland, 1983). Following particle reduction, the meat is mixed or blended. This serves to extract muscle proteins which promote binding between the meat pieces. Myofibrillar proteins, especially myosin, are the major proteins responsible for binding (Macfarlane gt gt., 1977). The binding of meat pieces depends upon the presence of a 39 protein-containing exudate at the particle surfaces (Booren gt gt., 1982). The mechanical action of tumbling, massaging or vacuum mixing disrupts the muscle cells thereby releasing the protein which acts as the binding agent. The extraction of myofibrillar protein is enhanced by the addition of NaCl alone or with phosphates (Pepper and Schmidt, 1975; Siegel gt gt., 1978;). According to Theno gt gt. (1978a,b) salt functions to solubilize proteins while phosphates cleave actomyosin thus increasing the surface area so that greater amounts of myofibrillar proteins may be extracted. Salt and phosphates have also been reported to act synergistically to increase the water holding capacity of restructured meat (Shults gt gt., 1972). It has been proposed that polyphosphates cleave the actomyosin complex formed at rigor into components with increased water holding capacity (Shults and Wierbicki, 1973; Theno gt gt., 1978b). In addition, phosphates alter the ionic strength of the sarcoplasm which increases the electrostatic repulsions between muscle filaments thus increasing the amount of space available for water binding. This increased water binding results in improved processing yields, reduced cooking losses, increased juiciness and enhanced texture and palatability scores (Schwartz and Mandigo, 1976; Neer and Mandigo, 1977; Huffman gt gt., 1981a). Salt has also been found to improve the flavor of restructured meats. Several studies on both beef and pork 40 products have reported consumer preference for samples containing between 0.5% and 2.0% added NaCl over samples without added salt (Cross and Stanfield, 1976; Schwartz and Mandigo, 1976; Huffman gt gt., 1981b). After mixing or blending, the meat particles and protein exudate are stuffed into casings or extruded into the preliminary shapes commonly referred to as logs, which are then frozen to increase the binding of particles. Meat logs are usually tempered to -3°C to -5°C then hydraulically pressed into the final shape. Logs can then be cleaved into individual servings with very close control of portion size possible. Catalytic Factors Restructured meat products are susceptible to lipid oxidation as evidenced by increased TBA numbers and pigment oxidation (Schwartz and Mandigo, 1976; Huffman gt gt., 1981b). Because of their manufacture, restructured meats may be less stable than intact muscle products. Two major factors influencing the development of WOF in restructured meat are reduction of particle size and the use of additives, especially NaCl. Comminution of raw meat both disrupts the membranal structures and incorporates oxygen into the tissue. Membrane-bound lipids consist largely of phospholipids which, because of their high degree of unsaturation are especially susceptible to lipid oxidation (Igene gt gt., 1980). Grinding, chopping or emulsifying exposes these 41 labile phospholipids not only to oxygen but to other tissue catalysts such as enzymes, heme pigments and metal ions. Sato and Hegarty (1971) demonstrated that chopping of meat and exposure to air caused the development of rancidity in fresh meat within one hour. Salt has also been shown to initiate undesirable reaction in restructured meat products. Schwartz and Mandigo (1976) reported that increased salt levels in flaked and formed pork produced increased TBA numbers and decreased raw color scores. Work by Huffman gt gt. (1981b) demon- strated that TBA values of restructured pork chops increased linearly in response to increasing salt levels. This trend was accompanied by decreasing raw color scores especially in samples held for 30 days at -15°C. Despite its detrimental effects, salt may actually inprove certain characteristics of restructured meats. Small amounts of added salt are necessary to assure proper binding of the meat particles in the finished product. Consumer evaluation of restructured beef steaks (Cross and Stanfield, 1976) showed that steaks containing 0.75% added salt were preferred over the controls containing 0% salt. Schwartz and Mandigo (1976) reported that added salt improved the aroma, flavor and eating texture of flaked and formed pork. Neer and Mandigo (1977) reported similar trends which were enhanced synergistically by the use of tripolyphosphate. Data from Huffman gt gt. (1981b) showed the addition of salt to restructured pork chops increased 42 cohesiveness and improved flavor, juciness and textural properties. Based on these observations, the addition of salt to restructured meat products at levels between 0.5 and 1.0% has been recommended (Schwartz and Mandigo, 1976; Huffman gt gt., 1981b). Prevention of Lipid Oxidation in Meat As previously noted, the lipids in muscle foods, especially restructured meat products, are susceptible to oxidation. Many studies have indicated that oxidation can be controlled by the use of compounds possessing antioxidant activity. These compounds may function as free radical scavengers, chelating agents, oxygen scavengers or by stabilizing otherwise catalytic species. Labuza (1971) reviewed these various types of antioxidants and the mech— anisms by which they function. Several other researchers have investigated the effects of antioxidants, phosphates, ascorbate and nitrites on lipid stability in meats. Antioxidants In food systems, the most effective antioxidants function by interrupting the free radical chain mechanism of lipid oxidation. An antioxidant, AH, apparently reacts with radicals according to the following scheme (Dugan, 1976): R’ + AH ------ > RH + A' R0' + AH ------ > ROH + A' ROO’ + AH ------ > ROOH + A’ R’+A° ------ > RA RO' + A' ------ > ROA 43 The free antioxidant radicals thus produced are thermo— dynamically unable to initiate other autoxidative chain reactions (Pokorny, 1971). Antioxidants may be classified as either synthetic or naturally occurring (Pearson and Gray, 1983). Butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and several other synthetic phenolic antioxidants have been widely studied in meat systems and, in general, have been shown to be effective in retarding lipid oxidation. Greene (1969) reported that BHA and propyl gallate (PG) offered substantial protection to fresh meat pigments and effectively inhibited lipid oxidation in raw ground beef. Greene gt gt. (1971) demonstrated that BHA or PG prevented lipid oxidation and reduced pigment oxidation in ground beef for up to 8 days of refrigerator storage. When the combi- nation of antioxidants and ascorbic acid was used both lipid and pigment oxidation were effectively retarded. Olson and Rust (1973) used an antioxidant salt mixture containing BHA, BHT, citric acid and propylene glycol in the dry curing of hams. Improved flavor scores and decreased rancidity were noted in hams cured with the antioxidant mix over those cured using regular flake salt or low metal salt. Chastain gt gt. (1982) utilized synthetic phenolic antioxidants in the manufacture of restructured beef/pork steaks formulated to contain 20% fat and 0.75% salt. The antioxidants BHA and tertiarybutyl hydroquinone (TBHQ) were added at the 0.02% level based on the fat content of the 44 steaks. The addition of antioxidants alone or in combi- nation decreased TBA numbers, increased sensory scores for flavor and acceptability and decreased discoloration of the raw meat relative to control (NaCl only) samples. BHA was more effective in inhibiting discoloration while TBHQ offered greater protection against rancidity. These researchers suggested that a combination of antioxidants offers the best possibility for protection against both types of oxidative changes. Chen gt gt. (1984) examined the effects of salt and antioxidants on the TBA numbers of beef. They reported that salt coated with Tenox 4—-BHA, citric acid, propylene glycol——and a mixture of BHA and BHT in salt completely inhibited lipid oxidaiton in cooked meat. However, d—tocopherol coated on salt demonstrated only marginal antioxidant properties. This ineffectiveness could be related to the concentration used astx-tocopherol has been reported to act as a prooxidant at high concentrations (Cillard gt gt., 1980; Labuza, 1971). Earlier work by Cort (1974) showed, however, that in pork and beef fats d—tocopherol was as effective as BHT in preventing lipid oxidation while Y -tocopherol was more effective than either BHT or BHA. Several researchers have investigated the antioxidant activity of other naturally occurring substances. Pratt and Watts (1964) demonstrated that a number of plant extracts prevented lipid oxidation in cooked meat. The antioxidant 45 activity was found to be related to the content of flavonoids, a major group of plant phenols. Flavonoids have also been cited as antioxidative constituents in soybean flour and protein isolates (Hayes gt gt., 1977). Pratt gt gt. (1981) identified three isoflavones and several phenolic acids which exhibited antioxidant activity from soy protein hydrolyzates. Proteins from other plant sources have also been shown to contain natural antioxidants. Rhee and Ziprin (1981) reported that glandless cottonseed, peanut or soy proteins and aqueous extracts of these proteins effectively retarded lipid oxidation in beef. They suggested that small amounts of oilseed protein ingredients could be added to sauces and gravies to improve the oxidative stability of precooked meat products. Similar research by Rhee and Smith (1983) demon— strated that defatted glandless cottonseed flour added at a level of 2 to 3% effectively inhibited salt-catalyzed lipid oxidation and discoloration in raw ground beef. These results suggest that glandless cottonseed flour could be added to meat products containing (0.5% NaCl to prevent oxidative deterioration. Phosphates and Other Chelating Agents Phosphates are usually added to processed meats because they increase the water holding capacity and yield of the finished product. The addition of phosphates to cooked meats has also been shown to delay or prevent lipid oxidation (Tims and Watts, 1958; Sato and Hegarty, 1971). 46 Pyrophosphate, tripolyphosphate and hexametaphosphate all offer protection while orthophosphates do not (Sato and Hegarty, 1971). Tims and Watts (1958) proposed that the mechanism by which phosphates prevent oxidation in meat products is related to their ability to sequester heavy metal ions which are known prooxidants (Uri, 1961). Other chelating agents have been shown to prevent lipid oxidation in meat presumably because of their ability to chelate nonheme iron and copper. Liu (1970) and Liu and Watts (1970) demonstrated that ethylenediaminetetraacetic acid (EDTA) eliminated Fe2+ catalyzed oxidation in a linoleate model system and in raw beef. Sato and Hegarty (1971) found that EDTA at a concentration of 2.5 mg/g suppressed lipid oxidation in cooked ground beef. Igene gt gt. (1979a) reported that the addition of 2% EDTA to meat pigments extracted from cooked beef significantly reduced their catalytic activity. Treatment of the pigment extract with H202 to release free irom from heme compounds increased its prooxidant activity which again could be lessened by EDTA. Igene gt gt., (1979a) concluded that EDTA effectively chelated nonheme iron and thereby significantly reduced lipid oxidation in cooked meat. Although EDTA has been proven effective in model systems, it has not been approved for commercial use in meat products. Citric acid has also been evaluated as an antioxidant in meat systems, but was found to be much less effective than phoSphates or EDTA. Sato and Hegarty (1971) reported 47 minimal inhibition of lipid oxidation in cooked ground beef when sodium citrate was added at the level of 5mg/g. Benedict gt gt. (1975) also reported only a slight decrease in lipid oxidation in meat treated with 0.0005% citric acid. MacDonald gt gt. (1980) demonstrated that 1000mg/kg levels of citric acid could reduce TBA numbers in refrigerated hams but that 50 mg/kg nitrite was more effective. I5 MATERIALS AND METHODS Materials Beef Choice Yield Grade 2 three piece chucks 126 (NAMP, 1981) were purchased from Ada Beef Inc. (Ada, MI). The subprimals were purchased vacuum packaged within 48 hours of slaughter. Replicates were established using three con— secutive slaughter weeks. Each replicate was processed within 24 hours of purchase. Vacuum Packaging Polyethylene-laminated nylon pouches (3 mil) were obtained from Koch (Kansas City, MO). These pouches have an oxygen transmission rate of 9 m1/m2/24 hours at 40C (320F). Antioxidants Tenox TBHQ was supplied by Eastman Chemical Products Inc. (Kingsport, TN). Antioxidant—coated salts were supplied by Diamond Crystal Salt Company (St. Clair, MI). The two formulations used in the manufacture of restructured steaks are shown in Table 2. 48 49 Table 2. Composition1 ofzantioxidant-coated salts used in the manufacture of restructured beef steaks. Antioxidant System Component APMT-l APMT—2 NaCl 93.8 93.60 AP3 3.0 1.70 Mixed Tocs4 0.6 1.75 Veg. Oil 0.3 0.75 Citric Acid 0.3 0.28 Silicon Dioxide5 2.0 1.92 l Expressed as percentage of total 2 Added to yield a final concentration of 0.06% (fat basis) AP + mixed tocopherols 3 Ascorbyl palmitate 4 Mixed tocopherols 5 Added as an anticaking agent 50 Fatty Acid Methyl Esters Individual esters and standard mixtures were purchased from Supelco (Bellefonte, PA) and Alltech Associates, Inc. (Deerfield, IL). Reaggnts and Solvents All reagents and solvents utilized in this study were reagent grade and/or HPLC grade with the exception of propylene glycol (Fisher Scientific) which met USP/FCC specifications. Experimental Manufacture of Restructured Steaks Restructured beef steaks were manufactured using the method of Booren gt gt. (1981). The cuts were trimmed of excess fat and connective tissue. The trim fat was used as the fat fraction for the restructured steaks. This was estimated visually to contain 90 to 95% fat. The fat content of the lean fraction (8 to 10%) was determined using a Hobart F101 Fat Tester. The fractions were wrapped in plastic film to exclude as much air as possible and crust frozen for 4 hours at -3OOC. The fat fraction was then sliced into 1.5mm slices using a Hobart Food Slicer. The lean fraction was ground through a 24x48mm kidney plate. The fractions were formulated into 35 lb meat blocks containing 18% fat. Antioxidants, either coated on salt or dissolved in propylene glycol, were dispersed in the fat fraction 51 immediately prior to vacuum blending. Salt was added to yield a final concentration of 0.75% in the meat block. Each meat block was placed in a Keebler mixer (Keebler Inc., Chicago, IL) and vacuum mixed for 12 minutes. The blended samples were stuffed into fibrous, moisture-proof casings (5 inch diameter) and frozen overnight at —30°C. The following morning, the logs were unwrapped and portioned with a meat saw into approximately 12 ounce steaks. Individual steaks were vacuum packaged and placed in frozen storage for further analysis. Freezer temperatures ranged from approximately -20 to -240C during the 12 month storage period. Measurement of Lipid Oxidation Lipid oxidation occurring in restructured beef steaks during frozen storage was quantitated by the TBA test, GC analysis of volatiles and sensory evaluation. A major objective of this study was to determine the correlation among the data obtained from each method of analysis. Raw and cooked samples from all treatments were analyzed by the TBA method (Tarladgis gt gt., 1960, 1964). For all analyses requiring cooked samples, steaks were prepared using a modified oven broiling technique described by Paul gt gt. (1956) to an internal temperature of 70°C (158°F). Tem- perature was monitored by copper constantan thermocouples placed in the center of individual steaks. Samples to be analyzed were ground twice through a 3/16 inch plate, mixed thoroughly and analyzed within 30 minutes. 52 Cooked samples containing NaCl only or NaCl plus TBHQ were utilized for CC analysis of volatiles. Volatiles were extracted with ethyl ether in a Likens-Nickerson apparatus, concentrated and analyzed with a Hewlett Packard 5840A gas chromatograph as described under "Methods of Analysis". Cooked samples were also evaluated by trained sensory panelists. Panelists were selected and trained using the following procedures. Volunteers were selected for training based on their performance in triangle tests. Initial training sessions utilized triangle tests, group dis— cussions, sample ranking and sample rating to familiarize the panelists with restructured steak and oxidized flavor (WOF). A reinforcing training session was conducted approximately mid-way through the storage study. Research samples were evaluated using a rating pro- cedure. Steaks were cooked approximately one hour before each morning tasting session. At each session panelists were presented with four coded samples representing the four experimental treatments and were asked to rate them on a 15 cm scale with left and right anchor points of "No WOF" and "Very Strong WOF". A sample form is shown in Appendix A. Panelists were instructed not to rank the samples since the various antioxidant treatments could exhibit the same degrees of oxidation. Portions of steaks from each treatment were held at 4°C for four hours, reheated and evaluated in afternoon sessions. A panelist's score for each sample was determined by measuring the 53 distance from the left end of the scale to the vertical mark made by the panelist. Evaluation of the TBA Test A study was designed to evaluate the applicability of the TBA test for assessing lipid oxidation in meat systems. This involved two phases in which: 1) the use of acetic acid TBA reagent (Tarladgis gt gt., 1960) was compared with aqueous TBA reagent (Tarladgis gt gt., 1964) and 2) the use of antioxidants during assays was evaluated. Several modifications of the traditional distillation TBA method were suggested by Tarladgis gt gt. (1964), one of which was the use of an aqueous TBA solution rather than one containing 90% glacial acetic acid. To compare these two methods the following procedures were used. Standard curves were established using solutions of tetramethoxypropane (TMP) ranging from 0 to 2x10-8 moles/5 ml. TMP was used because it undergoes hydrolysis to form malonaldehyde when treated with heat and/or acid. The malonaldehyde thus produced reacts with TBA to form the characteristic pink pigment. Both acidic and aqueous TBA reagents were reacted with aliquots of TMP solutions, and absorbance was measured at 532 nm. The recovery of malonaldehyde during distillation was also determined. Aliquots of TMP were distilled and col- lected using the traditional TBA method (Tarladgis gt gt., 1960). Aqueous TBA reagent was used to develop the pink color. Percent recovery was determined using the 54 calibration curve for aqueous TBA. Using this recovery value and the proper standard curves, the multiplicative factors for acid and aqueous TBA assays were determined. The second phase of this study was designed to examine the potential formation of TBA reactive substances (TBARS) during the TBA assay. Several researchers have suggested that further lipid oxidation may occur during heating steps of the TBA method (Siu and Draper, 1978; Rhee, 1978; Pikul gt gt., 1983). To prevent this, they advocated using anti- oxidants during any heating step and possibly during sample blending and preparation. The methods of Rhee (1978) and Pikul gt gt. (1983) were modified and applied to beef, fish, and chicken light and dark meat. Prior to sample homogenization 1.0 ml of various ethanolic TBHQ solutions was added to give a final anti— oxidant concentration of 0.01% based on the fat content of the meat. Samples were then distilled in the usual manner. Distillates were developed using both acidic and aqueous TBA reagents. Methods of Analysis Proximate Analysis Moisture content was determined using AOAC procedure 24.002 (1975). A variation of AOAC procedure 24.005 (1975) was used for crude fat determination. 55 Thiobarbituric Acid Test (TBA Test) The TBA distillation method of Tarladgis gt gt. (1960) was modified (Tarladgis gt gt., 1964) and used to measure the development of oxidative rancidity. The modified method utilized an aqueous solution of TBA in place of the acetic acid/H20 reagent. Lipid Extraction and Fractionation Total lipid was extracted from raw samples containing TBHQ using the procedure of Bligh and Dyer (1959). The dry column method of Marmer and Maxwell (1981) was used for simultaneous extraction and class separation, into neutral and phospholipid fractions, of lipids from raw and cooked steaks. Columns were prepared by pouring a bed of 5.09 of a CaHPO 'ZHZO/Celite 545 (9:1) mixture into 11x300 mm glass 4 columns. Sample size and solvent volume were decreased by one half due to the size of the chromatographic columns used. A 2.59 meat sample was added to 109 anhydrous NaZSO4 and ground with a mortar and pestle. This mixture was reground with 7.59 Celite 545 to a uniform free-flowing powder. This powder was quantitatively transferred to the column and tamped (moderately) into place. The mortar, pestle and other utensils were rinsed with methylene chloride to remove trace amounts of lipids. The rinsings were added to the column along with enough methylene chloride to wet the entire column bed. The column was then charged with 75 ml methylene chloride, and the neutral 56 lipids were eluted. When the last of the methylene chloride reached the top of the column, the column was charged with 75 ml methylene chloride/methanol (9:1) to elute the phos— pholipids. This fraction was collected until the column was stripped of solvent. The two fractions were concentrated to 50 ml, quantitated and used for fatty acid determinations. Fatty Acid Analysis Neutral and phospholipid fractions from raw and cooked beef steaks were methylated according to the boron trifluoride-methanol procedure of Morrison and Smith (1964). The methylated samples were analyzed using a Hewlett Packard 5840A gas chromatograph equipped with a flame ionization de— tector and a Hewlett Packard 5840A GC integrator. The fatty acid esters were separated on a glass column (3m x 2mm id) packed with 10% SP-233O on 100/120 Supelcoport (Supelco, Bellefonte, PA). The GC was operated using a temperature program with the initial temperature at 150°C, held for one minute then increased at 1.50C/min to a final temperature of 225°C which was maintained for 10 minutes. The injection port temperature was 200°C, the flame ionization detector temperature 300°C and the nitrogen carrier gas flow rate was 25 ml/min. The component fatty acids were identified by comparing retention times to those obtained from lipid standards assayed under identical conditions. 57 GC-MS Analysis of Fatty Acid Methyl Esters Methyl esters of fatty acids from the phospholipid fraction of restructured beef steaks were analyzed by GC-MS. The gas chromatographic conditions were as previously de- scribed. Effluent from the GC passed into a Hewlett Packard 5985A mass spectrophotometer operated under the following conditions: electron impact voltage, 70eV; electron multiplier voltage, 2400 eV; threshold 0.6; source tem- perature, 200°C; analog/digital measurements, 3/sec and ion detection in the positive mode. Extraction of Volatiles from Cooked Steaks Two hundred grams of ground, cooked meat were weighed into a 2000 ml boiling flask, 750 ml of water were added, and the mixture was attached to a Likens-Nickerson extraction apparatus. Ethyl ether (25 ml) was used as the extracting solvent. The system was allowed to reflux for 6 hours followed by concentration of the ether fraction which contained the extracted volatiles. The ether was first dried over anhydrous sodium sulfate then concentrated under a stream of nitrogen to a final volume of 0.5 ml. Extracts were stored in screw-top vials at -20°C until analysis. Chromatographic Analysis of Extracted Volatiles The extracts were analyzed for hexanal using a Hewlett Packard 5840A gas chromatograph fitted with a 3m x 2mm (i.d) glass column packed with 10% Carbowax 20M TPA on Chromosorb WHP, 80/100 mesh (Supelco, Bellefonte, PA). Injection 58 volume was 0.5 ul. The GC was operated isothermally at 50°C, with an injection port temperature of 200°C, a de- tector temperature of 3000C and N2 carrier gas at 25 ml/min. Hexanal was tentatively identified by comparing the reten- tion time of the suspect peak to that obtained from a hexanal standard assayed under identical conditions. The GC analyses were terminated after hexanal eluted (approximately 15 minutes), and the oven temperature was raised to 190°C and held for 15 minutes to drive off the remaining volatiles from the column. Sensory Evaluation Freshly cooked steaks and cooked meat held 4 hours at 40C were evaluated by trained sensory panelists. At each session, all panelists were presented with four coded samples representing the four experimental treatments. All samples were reheated in sealed containers and served warm. Samples were evaluated under red light to mask any differ- ences in appearance. Panelists were asked to rate the intensity of warmed-over flavor using a 15 cm scale with anchor points 1 cm from each end. The descriptors at the left and right anchor points were "No WOF" and "Very Strong WOF", respectively. Statistical Analysis Data from TBA tests were analyzed by the ANOVA and Scheffe procedures of the Statistical Package for the Social Sciences, SPSS (Nie gt gt., 1975). SPSS/PC--SPSS for the 59 IBM PC/XT--was used for all computations (Norusis, 1984). Application and interpretation of these procedures was in accordance with Gill (1978). Sensory scores were analyzed by the ANOVA procedures outlined by Larmond (1977). RESULTS AND DISCUSSION Evaluation of the TBA Test As stated in the review of literature, the original TBA distillation procedure has been modified and refined by several researchers. Tarladgis gt gt. (1964) found that the usual acid—heat treatment was not necessary for maximum color development but, rather, that it altered the spectral properties of the pigment and contributed to the appearance of an absorption peak at 450 nm. In the preliminary stage of the current study, a similar phenomenon-—the presence of a yellow pigment absorbing at 450 to 452 nm--was observed during TBA assays of muscle products. In light of these observations, a study was designed to compare the original acetic acid-heat TBA assay (Tarladgis gt gt., 1960) to the modified aqueous TBA system (Tarladgis gt gt., 1964). Malonaldehyde calibration curves were established using solutions of tetramethoxypropane (TMP) containing 0 to 2x10"8 moles/5 ml. Aliquots of these TMP solutions were reacted with both acidic and aqueous TBA solutions. The con— centrations used and their respective absorbance values are 60 61 shown in Table 3. Plots of these data show a linear re- sponse with r2 values of 0.999 for both acidic and aqueous TBA (Figure 2). The recovery of malonaldehyde during distillation was subsequently determined. Aliquots of TMP were distilled and collected using the traditional TBA method. Aqueous TBA reagent was used to develop the pink pigment. The TMP concentrations utilized and corresponding absorbance values are found in Table 4. From the appropriate calibration curve recovery was found to range from 68.6 to 77.2 percent with an average of 73.1 percent. This value is slightly higher than that established by Tarladgis gt gt. (1960). Using the equations of Tarladgis gt gt. (1960) shown in Appendix B, the multiplicative factors for acidic and aqueous TBA assays were calculated to be 7.1 and 6.2, respectively. To express TBA numbers, with units of mg malonaldehyde/1000 9 sample, absorbance values are multiplied by the above factors. The appearance of a yellow pigment was not observed in any reactions utilizing TMP as the source of malonaldehyde. However, distillates from meat samples often produced the yellow pigment when developed with acetic acid TBA reagent. This was in agreement with the findings of Tarladgis gt gt. (1962), Tarladgis gt gt, (1964) and Igene gt gt. (1985a). To further investigate this, distillates from beef, fish and chicken were divided into two lots, and identical samples were reacted with both acidic and aqueous TBA Table 3. Absorbance of TMP standards developed with acidic and aqueous TBA reagents. _9 TMP 10 moles/5 ml Absorbance1 Acidic TBA at 532 nm Aqueous TBA 0.00 0.000 0.000 0.92 0.010 0.016 1.85 0.025 0.029 4.62 0.060 0.073 9.24 0.127 0.147 18.48 0.264 0.295 1 Analyses were performed in triplicate. Table 4. Recovery of malonaldehyde during TBA 1'2 distillation. _9 TMP 3 10 moles/5 m1 Absorbance %Recovery 6.09 0.075 77.2 7.31 0.080 68.6 9.09 0.106 73.1 9.14 0.107 73.4 1 Color was developed using aqueous TBA reagent. 2 Analyses were performed in duplicate. 3 Measured at 532 nm 63 Figure 2. Calibration curves for malonaldehyde-TBA complexes developed under aqueous and acidic conditions. TMPCONCENTRATION (10‘9 moles / 5 ml) 20.00 18.00 16.00 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 64 O AQUEOUS TBA . ACIDIC TBA \O 1 i 0.10 0.20 ABSORBANCE AT 532 nm 0.30 65 reagents. The sample type, treatments and absorbance values are summarized in Tables 5 and 6. Absorbance at 532 nm was consistently higher for the aqueous samples while absorbance at 450 nm was generally greater in samples developed with acidic TBA reagent. These trends were apparent in both raw and cooked samples. Figure 3 shows the spectral scans of a distillate from beef developed with both types of TBA reagents. In the sample developed with aqueous reagent a very sharp ab- sorption peak was noted at 532 nm while only a small peak was present at ca. 450 nm. However, in the sample developed with acidic TBA reagent, a substantial peak appeared at 450 nm and the absorbance at 532 was suppressed. The appearance of this yellow pigment has been noted by several other researchers. Tarladgis and Watts (1960) reported the formation of a pigment absorbing at 450 nm during the TBA assay of oxidized oleic, linoleic, linolenic and arachidonic acids but found none from the reaction of acidic TBA reagemt with pure malonaldehyde solutions. They also noted that absorption at 450 nm increased over time when the TBA chromagen was held at room temperature but were unable to identify the compound responsible for the peak. In further studies, Tarladgis gt gt. (1962) inves— tigated some of the side reactions which occur during the acid-heat portion of the TBA test. They reported that the visible, UV and IR spectra of acetic acid TBA reagent were altered by heat and oxidizing agents. Absorbance at 450 nm 66 Table 5. Absorbance values1 of distillates2 from various raw muscle products developed with acidic and aqueous TBA reagents. Absorbance TBA Sample Reagent 451 nm 532 nm Chicken breast H20 0.107 0.071 Chicken breast a01d 0.222 0.024 Chicken thigh H20 0.064 0.061 Chicken thigh ac1d 0.120 0.021 Beef H20 0.082 0.108 Beef ac1d 0.401 0.052 Fish H20 0.048 0.112 Fish a01d 0.055 0.099 1 Analyses were performed in duplicate. 2 Distillates from each muscle type were subdivided and developed with each type of TBA reagent. 67 Table 6. Absorbance values1 of distillates2 from various cooked muscle products developed with acidic and aqueous TBA reagents. Absorbance TBA Sample Reagent 451 nm 532 nm Chicken breast H20 0.109 0.138 Chicken breast ac1d 0.227 0.073 Chicken thigh H20 0.133 0.386 Chicken thigh ac1d 0.051 0.196 Beef H20 0.075 0.082 Beef ac1d 0.228 0.050 Fish H20 N. A N. A Fish ac1d N.A N.A 1 Analyses were performed in duplicate. 2 Distillates from each muscle type were subdivided and developed with each type of TBA reagent. N.A = not available 68 Figure 3. Spectra of distillates from beef developed with aqueous and acidic TBA reagents. ABSORBANCE 69 AQUEOUS 350 ACIDIC 1 l J o o o In In in v to co WAVELENGTH (nm) 750 70 increased substantially upon heating the reagent 5 to 35 minutes. The addition of H202 to the TBA reagent magnified this increase of the 450 nm peak. Data from UV and IR spectra presented further evidence that the structure of TBA was altered by acid-heat treatment and H202. These re- searchers postulated that TBA may undergo partial hydrolysis or may be oxidized at the C-SH or CH2 groups although they were unable to identify the specific compounds produced in the acetic acid TBA system. These researchers did suggest, however, that TBA should not be heated in the presence of acid or oxidizing agents. They also suggested that hydro- peroxides present in oxidized samples could affect TBA in the same manner as does hydrogen peroxide. Jacobson gt gt. (1964) modified the TBA test to cor— relate absorbance at 452 nm with flavor scores of various fats and oils. They used a moderate temperature for color development which favored formation of the yellow pigment. They also found, though, that recrystallization of TBA and redistillation of solvents markedly decreased artifactual absorbance at 452 nm. However, they did not address the possible interaction of the 452 nm and 532 nm absorbance peaks. Marcuse and Johansson (1973) studied the production of a 450 nm absorbance peak from the reaction of TBA with alkanals, alkenals and 2,4 alkadienals. Maximum production of the yellow pigment was promoted by heating the reaction mixture at 50°C for 2 hours. Conditions similar to those of 71 the traditional TBA assay-—95°C for 35 minutes--produced very little absorbance at 450 nm. As with Jacobson gt gt. (1964), this research was designed to study the appli- cability of utilizing the absorbance at 450 nm as an index of rancidity rather than to study the interference of a 450 nm peak with the 532 nm absorbance peak. It does, however, support the findings of the current study that formation of the yellow pigment is minimal when aqueous TBA reagent is heated in the presence of TBA reactive substances (TBARS) under the conditions normally used during TBA assays. While preliminary data from the current study shown in Table 7 indicate that the yellow pigment interferes with TBA assays of fresh products, the data in Table 8 show that interference is not as substantial as TBA absorbance values increase. Distillates from fresh beef which produced the yellow pigment yielded lower absorption values at 532 nm than those possessing only minor absorption peaks at 450 nm (Table 7). In oxidized samples, absorbance at 532 nm changed little despite large differences in absorbance at 450 nm (Table 8). This demonstrates that at high con- centrations of TBARS absorbance at 532 is not suppressed by the 450 pigment. This also suggests that results of the acetic acid TBA assay are less reproducible for fresh products than for more highly oxidized samples. In light of this evidence, and since formation of the yellow pigment is decreased when aqueous TBA reagent is used regardless of the 72 Table 7. Absorbance values1 of distillates from fresh raw beef developed with acetic acid TBA reagent. Sample2 Visual Appearance Absorbance3 la Orange 0.010 b Pink 0.015 2a Orange 0.045 b Pink 0.060 3a Orange 0.073 b Pink 0.091 1 Values represent the mean of three measurements. 2 Samples from a single source were analyzed in quadruplicate on successive days. Values from two replications were chosen for illustrative purposes. Measured at 532 nm 73 Table 8. Absorbance values1 of distillates from various muscle products developed with acetic acid TBA reagent. Absorbance Sample2 451 nm 532 nm Chicken breast 0.032 0.075 Chicken breast 0.227 0.073 Chicken thigh 0.051 0.196 Chicken thigh 0.301 0.200 Fish 0.023 0.150 Fish 0.111 0.149 1 Values represent the mean of three measurements. Samples of each muscle type are from a single source and were analyzed in quadruplicate. Values from two replications were chosen for illustrative purposes. 74 degree of sample oxidation, all further TBA assays of the current study utilized aqueous TBA reagent. Effect of TBHQ on Formation of TBARS During the TBA Assay. The ability of TBHQ to prevent artifactual formation of TBARS during the TBA assay was evaluated. The effects of TBHQ added prior to sample blending on the 532 nm absorbance values of various raw and cooked muscle samples are pre- sented in Table 9. The addition of 0.01% TBHQ (fat basis) to the distillation mixtures did not have any significant effect (p <0.05) on the TBA results of beef or cooked chicken thigh. However, TBA absorbance values of fish and chicken breast were significantly lower when TBHQ was added prior to blending and distillation of both raw and cooked samples. TBHQ also significantly lowered the TBA absorbance value of raw chicken thigh meat. These data from the cur- rent study along with those of Rhee (1978) and Pikul gt gt. (1983) show that chicken and fish appear to be susceptible to oxidative changes during the TBA distillation procedure. Rhee (1978) reported that addition of propyl gallate (PG) and EDTA prior to sample distillation significantly reduced the TBA numbers of both raw and cooked catfish. No effect was noted for beef, pork or chicken breast. Further experimentation demonstrated that the addition of PG and EDTA during the blending process was the most effective one- step method for minimizing further oxidation of fish lipids 75 Table 9. Absorbance valuesl’2 of distillates from various muscle samples distilled with and without added TBHQ. Absorbance at 532 nm Sample -TBHQ +TBHQ Beef a a Raw 0.083 0.090 (0.009) (0.021) Cooked 0.082a 0.084a (0.003) (0.016) Chicken breast a b Raw 0.096 0.073 (0.007) (0.003) Cooked 0.184a 0.155b (0.004) (0.017) Chicken thigh a b Raw 0.068 0.046 (0.005) (0.003) Cooked 0.335a 0.300a (0.049) (0.017) FiSh a b Raw 0.138 0.089 (0.027) (0.012) Cooked 0.318a 0.270b (0.033) (0.009) 1 Analyses were performed in triplicate. 2 Standard deviations are shown in parentheses. a b ' Values in the same row bearing the same superscript are not significantly different from each other at p <0.05. 76 during the TBA test. Similar, but not as marked, results were observed for other meat samples. Pikul gt gt. (1983) evaluated the influence of BHT on the TBA assay of fat extracted from chicken breast and leg meat. Samples analyzed without added BHT yielded 6 times higher TBA numbers than samples treated with BHT during lipid extraction and TBA assay. These researchers advised using antioxidants to prevent artifactually high TBA results. This protective effect of antioxidants is significant for fish and chicken most likely because of the lability of their component lipids. Poultry and fish are known to contain proportionately more phospholipids than red meats (Igene gt gt., 1979b), and these phospholipids are highly unsaturated. Additionally, the depot fats of these species are unsaturated with poultry containing substantial amounts of C18 and C20 polyunsaturates while fish contain pentaenoic and hexaenoic acids (Pearson gt gt., 1977). In contrast, beef contains a smaller amount of phospholipids, and polyunsaturated fatty acids comprise less that 3 percent of its triglyceride fatty acids. Evaluation of Restructured Steaks Proximate Analysis Restructured beef steaks were manufactured as detailed previously. The moisture and fat contents of each treatment and replication are shown in Table 10. As evident from the 77 Table 10. Moisture and lipid content of restructured beef steaks. % Lipid Sample1 %Moisture2 Goldfisch3 Dry3 Column Control 1 65.6 12.3 2 63.0 16.4 3 63.1 15.0 APMT-l 1 65.7 14.6 2 63.3 14.9 3 62.3 15.5 APMT-2 1 64.8 14.6 2 61.3 17.7 3 60.6 16.6 TBHQ 1 61.9 16.4 18.1 2 62.4 19.2 20.3 3 60.8 18.0 18.1 Samples were taken from each of three replications within each treatment group. Analyses were performed in triplicate. Analyses were performed in duplicate. 78 table, the target lipid content, 18%, was not always achieved. This was due to the unavoidable loss of a small amount of fat on the sides and blades of the vacuum mixer. Additionally, lipids contents determined by the dry column procedure were slightly higher than those obtained by the AOAC method. This is in agreement with the results of Maxwell gt gt. (1980) who reported more complete extraction of phospholipid by the dry column method. The fatty acid composition of neutral and phospholipid fraction of samples taken from each replication are summarized in Tables 11 and 12. Because these analyses were not performed at the initial stage of the study, TBHQ— treated samples were later chosen for analysis since their composition would have changed the least over time. The values shown in the tables were calculated as area percentages of total fatty acids identified with lipid standards. Two peaks in the chromatograms of neutral lipid fatty acids could not be identified. One, eluting shortly after C may have been C the other, eluting between C 15:0; may have been C 14:1’ and C 17:0 18:0’ 17:1. Nevertheless, the fatty acid profiles obtained were very similar to those found in the literature (Hornstein and Crowe, 1967; Igene gt gt., 1981), and especially to those reported by Marmer gt gt. (1984) who also used the dry column method for lipid extraction and class separation. This clearly demonstrates the high reproducibility of dry column methodology. 79 Table 11. Fatty acid composition of the neutral lipid fraction from restructured beef steaks. Samplel Fatty Acid TBHQ-1 TBHQ-2 TBHQ-3 Area Percent 14:0 3.2 3.1 3.0 14:1 0.7 1.1 1.0 16:0 25.6 25.3 24.6 16:1 5.5 5.5 5.2 17:0 1.3 1.2 1.4 18:0 13.5 13.4 13.9 18:1 46.4 46.7 47.2 18:2 3.0 2.6 2.9 18:3 0.8 0.9 0.6 20:0 tr 0.2 0.2 % Sat. 43.6 43.2 43.1 % Monounsat. 52.6 53.3 53.4 % Di and polyunsat. 3.8 3.5 3.5 % Total Unsat. 56.4 56.8 57.0 1 Duplicate samples were taken from each of three replications containing TBHQ. tr = trace amounts 80 Table 12. Fatty acid composition of the phospholipid fraction from restructured beef steaks. Sample1 Fatty Acid TBHQ-1 TBHQ-2 TBHQ-3 Area Percent 14:0 0.2 tr tr 14:1 tr tr tr 16:0 16.7 15.5 15.7 16:1 1.8 2.1 2.0 17:0 0.2 tr 0.3 18:0 14.6 14.6 14.4 18:1 23.4 26.5 25.4 18:2 25.1 20.3 23.1 18:3 0.9 1.1 0.8 20:0 tr tr tr 20:2 0.7 0.8 0.6 20:4 12.8 13.1 11.8 22:4 3.6 6.0 5.9 22:6 tr tr tr % Sat. 31.7 30.1 30.4 % Monounsat. 25.2 28.6 27.4 % Diunsat. 25.8 21.1 23.7 % Polyunsat. 17.3 20.2 18.5 % Total unsat. 68.3 69.9 69.6 |—' Duplicate samples were taken from each of three replications containing TBHQ. tr =trace amounts 81 Notable discrepancies were found, however, between experimental data and the values Igene gt gt. (1981) reported for 014:0’ C14:1 and C16:2‘ Igene gt gt. found a higher proportion of C14:1 and no C14:0 while the current data indicate approximately 3 percent C14:0 and only 1 percent C14:1. Additionally, they reported approximately 1 percent Cl6:2 in beef triglycerides. Attempts were made to substantiate this claim, but no commercial source of C16z2 could be located. The fatty acid profiles of phospholipids shown in Table 12 are very representative of previously reported values (Hornstein and Crowe, 1967; Terrell and Bray, 1969; Marmer gt gt., 1984). Slight variability was found even among literature values depending on muscle type analyzed, animal feeding history, animal sex and age at time of slaughter. However, experimental data again agreed closely with those of Marmer gt gt. (1984) although the phospho- lipids extracted from restructured steaks were richer in C and C22,4. These findings were not unusual, though, 20:4 as Hornstein and Crowe (1967) and Terrell and Bray (1969) reported 6 to 16 percent arachadonic acid in beef phOSpholipids. The chromatograms obtained for the phospholipid frac- tion contained 3 peaks, comprising approximately 10% of the total peak area, which could not be identified. The major peak eluted between C14:0 and C16:0' while the two lesser peaks eluted near C18:0 and C20:4, respectively. Mass 82 spectroscopy of the major peak indicated that the compound most likely was not a fatty acid methyl ester. Other researchers have reported the presence of compounds iden— tified as dimethylacetals (DMAs) of long chain aldehydes in the fatty acid profiles of polar lipids esterified by acid- catalyzed procedures (Maxwell and Marmer, 1983; Wood, 1983). DMAs arise from the hydrolysis of plasmalogens which contain a vinyl ether linkage in addition to the usual ester linkages. Since bovine polar lipid fractions may contain up to 30% plasmalogen, DMAs would be major components in chro- matograms of phospholipid fatty acids (Maxwell and Marmer, 1983). Wood (1983) reported up to 8 percent C DMA and 16:0 4 percent C18:0 DMA in the total phospholipids of beef. To prevent the acid-catalyzed hydrolysis of plasmalogen lipids, modified esterification methods have been utilized. Christopherson and Glass (1969) used methanolic KOH or sodium methoxide to prepare milk fat methyl esters. Maxwell and Marmer (1983) modified this method for use with phospho— lipid concentrates and reported that room temperature alkaline transesterification converts phospholipids to methyl esters without generating DMAs from plasmalogens. Further examination of the data in Tables 11 and 12 reveals that, despite slight discrepancies between liter- ature and experimental values for individual fatty acids, the proportions of saturated and unsaturated component fatty acids in each lipid fraction are typical and in close 83 agreement with data from the literature (Igene gt gt., 1981; Marmer gt gt., 1984; Eichhorn gt gt., 1985). These researchers reported approximately 45% saturates and 55% unsaturated fatty acids in the neutral lipids and 33% saturates and 67% unsaturates in the phospholipid fraction. A more significant difference in composition between the two fractions is evident in the di- and polyunsaturated fatty acids. Beef neutral lipids contain only about 4% di- and polyunsaturated fatty acids while the phospholipids contain approximately 40%. This high degree of unsaturation accounts, in part, for the lability of beef phospholipids to oxidation and their role in the development of WOF (Igene and Pearson, 1979). Lipid Stability The influence of antioxidants on the stability of lipids in restructured beef steaks was evaluated using the TBA procedure, quantitation of hexanal and sensory eval- uation. The effectiveness of two natural antioxidants and TBHQ was monitored over 12 months of frozen storage. TBA Test The modified distillation procedure of Tarladgis gt gt. (1964) was used to quantitate TBARS in restructured steaks during the storage period. Analysis of variance (ANOVA) and Scheffe's test were used to determine if any significant differences existed among the TBA numbers from the various samples. Initially, ANOVAs were performed using data from 84 all sampling points to determine the significance of treatment, time and the time/treatment interaction. For raw and freshly cooked samples both treatment and time were significant factors. For cooked and stored samples, only treatment was significant. In all cases the time/treatment interaction was not significant. Additional ANOVAs were performed, and Scheffe's procedure was used to determine which treatments were significantly different at each sampling period. In this manner, changes in the effectiveness of each antioxidant could be monitored over the course of the storage period. As shown in Figure 4 and Table 13, the TBA numbers for all raw samples increased over time with the salt-only control oxidizing most rapidly and to the greatest extent. TBA values for all antioxidant—treated samples were lower than control values throughout the entire study, however, only TBHQ-treated samples had TBA numbers which were significantally lower (p (0.05) than control samples over the major portion of the storage period. Additionally, the natural antioxidants were effective, statistically, only at 0 and 4 months. At all other sampling periods, TBA numbers from natural antioxidant samples were not different from those of control samples. There was also no significant difference in the effectiveness of the two natural anti- oxidants at any sampling period. th By the 12 month of storage there were no statistical differences among the TBA numbers from any of the four Figure 4. 85 Changes in TBA numbers of raw restructuged beef steaks over 12 months of storage at —20 C. TBA NUMBER 1.00 0.80 0.00 _ 86 U CONTROL A APMT-1 A APMT-2 0 TBHQ l J J l l 2 4 6 8 10 MONTHS OF FROZEN STORAGE 12 WWII! Table 13. TBA numbers 87 1’2 of raw restructured begf steaks after 0, 2, 4, 8 and 12 months at -20 C. Treatment Months of Storage Control APMT-l APMT—2 TBHQ 3 TBA Number 0 0.32a 0.22b 0.22b 0.25ab (0.03) (0.02) (0.04) (0.02) 2 0.428 0.32ab 0.348 0.16b (0.08) (0.04) (0.08) (0.02) 4 0.39a 0.26bC 0.35ac 0.19b (0.03) * * (0.02) 8 0.57a 0.41a 0.438 0.21b (0.09) (0.01) (0.07) (0.07) 12 0.75a 0.46a 0.63a 0.28a (0.28) (0.10) (0.26) (0.06) 1 Values represent means of three replications which were analyzed in duplicate. 2 Standard deviations are shown in parentheses. 3 mg malonaldehyde/kg sample * Value is from one replication only. a,b,c Values in the same row bearing the same superscript do not differ significantly at p <0.05. 88 treatments. However, as shown in Figure 4, TBA numbers from raw antioxidant-treated samples were substantially lower than that of the control sample. This apparent contra— diction is readily explained by the data in Table 13. Because of the large variances associated with the data at this sampling period, statistical differences among the treatments were not evident. Data for freshly cooked samples were analyzed in a similar manner (Table 14 and Figure 5). At all sampling periods, except month 4, all antioxidant treatments yielded significantly lower TBA numbers than did the control sample. At month 4, only TBHQ-treated samples had statistically lower TBA numbers. In addition, there were no differences among the various antioxidants; hence the natural anti- oxidants were just as effective as TBHQ in retarding lipid oxidation in freshly cooked meat that had been previously frozen for up to 12 months. The protection offered by the various antioxidant treatments may be more evident in the cooked meat system than in raw samples due to the high degree of oxidation which occurs in the salt-only control during and immediately following cooking. TBA numbers from the control samples were increased approximately two to three fold by the cooking process while values from antioxidant—treated steaks increased only approximately 50 percent. This demonstrates that all three antioxidants have good, or at least adequate, carry-through and are capable of retarding lipid oxidation 89 Table 14. TBA numbersl’2 of restructured bees steaks cooked after 2, 4, 8 and 12 months at -20 C. Treatment Months of Storage Control APMT-l APMT-2 TBHQ TBA Numbers'4 2 1.04a 0.45b 0.42b 0.27b (0.23) (0.08) (0.03) (0.07) 4 0.738 0.53ab 0.48ab 0.30b (0.11) (0.15) (0.10) (0.02) 8 1.178 0.70b 0.58b 0.33b (0.28) (0.09) (0.05) (0.06) 12 1.168 0.62b 0.49b 0.32b (0.28) (0.07) (0.10) (0.05) 1 Values represent means of three replications which were analyzed in duplicate. 2 Standard deviations are shown in parentheses. 3 mg malonaldehyde/kg sample 4 Corrected for loss of fat during cooking a,b,c Values in the same row bearing the same superscript do not differ significantly at p <0.05. 90 Figure 5. TBA numbers of restructured beef steaks cooked after 2, 4, 8 and 12 months at —20 C. TBA NUMBER 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 91 U CONTROL A APMT-1 A APMT-2 O TBHQ *5 1 1 l l l l 2 4 6 8 10 12 MONTHS OF FROZEN STORAGE 92 catalyzed by cooking. This catalysis of lipid oxidation could be related to the release of non-heme iron during cooking as postulated by Igene gt gt. (1979a). Cooked samples were held for 4 hours at 4°C, reheated and analyzed. This procedure was used to simulate a pre- cook or cook-and-hold situation which could be found in the food service industry. As shown in Table 15 and Figure 6, TBA numbers of antioxidant-treated samples were lower than those of control samples at all sampling periods. The protective effect of TBHQ was statistically significant throughout the entire storage study. The TBA numbers for both APMT-treated samples were significantly lower than control values at 4 months, while values from APMT-2 samples were lower at 8 months. Again, the efficacies of the two natural antioxidants were not different (p <0.05) from that of TBHQ.' Comparison of the magnitude of the TBA numbers from the various treatments is one way of determining the effec- tiveness of the antioxidants. Examination of the rates of lipid oxidation during the 4 hours of refrigerator storage may also demonstrate the effectiveness of the antioxidants in relation to each other. Figure 7 graphically shows the lipid oxidation which occurred in the various samples after they were cooked and held 4 hours at 4°C. The greater the slope of the line connecting the points representing TBA numbers, the more rapidily lipid oxidation occurred. 93 Table 15. TBA numbersl'2 of restructurgd beef steaks cooked and held 4 h8urs at 4 C after 2, 4 and 8 months at -20 C. Treatment Months of Storage Control APMT-l APMT-Z TBHQ TBA Number3’4 2 1.84a 0.79ab 0.85ab 0.38b (0.68) (0.24) (0.27) (0.10) 4 1.498 0.67b 0.74b 0.35b (0.35) (0.10) (0.08) (0.02) 8 2.048 0.98ab 0.79b 0.44b (0.74) (0.14) (0.13) (0.05) 1 Values represent means of three replications which were analyzed in duplicate. Standard deviations are shown in parentheses. mg malonaldehyde/kg sample Corrected for loss of fat during cooking Values in the same row bearing the same superscript do not differ significantly at p <0.05. 94 Figure 6. TBA numbers 05 restructured beef steaks cookedO and held at 4 C after 2, 4 and 8 months at —20 C. TBA NUMBER 2.00 1.80 1.60 1.40 1.00 0.80 0.60 0.40 0.20 0.00 95 ll D CONTROL _ A APMT-1 A APMT-2 O TBHQ \ N )— 1 l 1 l J 2 4 6 8 10 MONTHS OF FROZEN STORAGE l' " 96 .ooom- ea meoeoe ace e .N tance Goa pa omoLOpm to meson a no>o mxcopm coon ownauosnumwt ooxooo to wrongs: