Lipid-protein interactions as agents of quality deterioration in intermediate moisture meats: An appraisal

MeatScience4 (1980)79-88

LIPID-PROTEIN INTERACTIONS AS AGENTS OF QUALITY DETERIORATION IN INTERMEDIATE MOISTURE MEATS: AN APPRAISAL

Z. A. OBANU

Food Science and Technology, Department of Food and Home Sciences, University of Nigeria, Nsukka, Nigeria & D. A. LEDWARD& R. A. LAWRIE

Food Science Laboratories, Department of Applied Biochemistry and Nutrition, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, Leics., LEI 2 5RD, Great Britain (Received: ! 8 December, 1978)

SUMMARY

The literature on lipid-protein interactions which lead to loss o f solubility, complex jbrmation, chain scission and loss of specific amino acids in intermediate moisture jbods is reviewed. This knowledge is used to explain reported observations on the quality and nutritive value o f proteins in intermediate moisture meats as well as the conflicting reports on the significance o f oxidative rancidity and non-enzymic browning in intermediate moisture Jbod systems.

LIPID OXIDATION

Both plant and animal proteins are closely associated with lipids which contain varying types and amounts of unsaturated fatty acids. Hence these are capable of being oxidised and thus may affect the quality (and especially the flavour) of the food. Even lean meat contains at least the minimum amount of lipids essential for the metabolic machinery of the cell and it is by no means necessary for the proportion of lipid in a food to be high before autoxidative troubles can become serious (Lea, 1962). The nature and content of lipids in meat from various muscles and species of animals of different sex, age and management have been reviewed (Lawrie, 1979). 79 Meat Science 0309-1740/80/0004-0079/$02-25 © Applied Science Publishers Ltd, England, 1980 Printed in Great Britain

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Based on the fat content and degree ofunsaturation of the component fatty acids, the rate of oxidation of intramuscular fat would tend to be higher in meat from nonruminant animals (e.g. pork and whale meat) than in meat from ruminants (beef and mutton), younger animals, the less improved breeds of meat animal (which predominate in the developing countries) and animals on a low plane of nutrition or receiving large amounts of unsaturated fat in their diets. The oxidative deterioration of food lipids involv.es, primarily, autoxidative mechanisms which are accompanied by various secondary reactions of an oxidative or non-oxidative character (Lundberg, 1961 ; Schultz et al., 1962). Through research initiated in the laboratories of the British Rubber Producers' Association (Farmer et al., 1942, 1946; Bolland, 1946, 1969; Bateman et al., 1948, 1953) it is known that autoxidation of lipids proceeds by a free radical mechanism (Lundberg, 1962). The initial products consist largely of hydroperoxides (Frankel, 1962). The autoxidation and reactions of the free radicals and hydroperoxides areaffected by the water content of the food. To date contradictory views on the relative significance of lipid oxidation and non-enzymic browning in intermediate moisture foods are to be found in the literature. Some workers (for example, Loncin et al., 1968; Brockmann, 1970, 1973a,b) believe that lipid oxidation is not important in intermediate moisture foods while other workers (for example, Labuza et al., 1969, 1971a,b, 1972a; Chou et al., 1973) believe that oxidative rancidity occurs very rapidly and may be the reaction limiting storage. Studies by Labuza and co-workers suggest that lipid oxidation may be faster in desorption-processed foods than in those equilibrated by absorption (Chou et al., 1973; Labuza & Chou, 1974). However, Brockmann (1973b) observed that all intermediate moisture (IM) foods prepared by desorption equilibration (Hollis et al., 1968, 1969; Pavey, 1972; Johnson et al., 1972) retained normal sensory properties (except for a slight sweetness due to glycerol), and acceptability, throughout six months' storage at 38 °C. Data on oxidation rates at intermediate moisture levels are, however, sparse and conflicting and further studies are needed to clarify the situation. Oxidative rancidity would be expected to be more serious in intermediate moisture foods, such as meat, which contain haematin pigments (Watts, 1954, 1961, 1962; Kendrick & W a t t s , 1969; Liu, 1970) since several haematin compounds catalyse the oxidation of unsaturated lipids and other olefins (Tappel, 1953, 1955a). The catalytic effect is very dependent on the molar ratio of. fatty acid:haematin pigment, the effect apparently being maximal at ratios of about 500:1 (Lee et al., 1975). At higher pigment levels (lower ratios) the catalytic effect is less and at ratios of about 150:1 the haematin pigments actually inhibit the oxidation, complete inhibition occurring at a ratio of 89:1 in mechanically deboned meat (Lee et al., 1975). However, most meat products contain fatty acid:haematin ratios that give rise to marked catalysis. For example, Tappel et al. (1961) showed that when pork

LIPID-PROTEIN INTERACTIONS AS AGENTS OF QUALITY DETERIORATION

81

adipose tissue and rendered lard were stored in the presence of oxygen (at 38 °C for one day or at 25 °C for two days) 7-7g of adipose tissue of surface area 13-4cm 2 absorbed 640/~1 Of oxygen per hour and increased its peroxide value by 7-1 meq/kg while a portion of rendered lard of identical size and shape absorbed no oxygen and exhibited only an increase of 0-6 meq/kg in peroxide value. This study implicated haemoglobin, myoglobin and cytochromes as catalysts of lipid oxidation since they are present in adipose tissue but not in lard. The pro-oxidant effect of haematin compounds in fat oxidation is reciprocal since unsaturated fatty acids also accelerate the oxidation--and hence the destruction--of haematin pigments (Niell & Hastings, 1925; Haurowitz et al., 1941). It is now known (Greene & Price, 1975) that haematin pigmentsmay be more active catalysts when the iron is in the ferric state than when it is in the ferrous state while the reverse is true for non-haematin iron. This means that I M meat products, which are normally pasteurised or cooked, may be more susceptible to oxidative rancidity and discoloration since haematin iron is oxidised to the ferric state during heating.

FORMATION OF LIPID--PROTEIN COMPLEXES

Peroxidising lipids can interact with proteins to form lipid-protein complexes, although the mechanisms are not clearly understood. Gamage et al. (1973) have shown that both radical and non-radical intermediate products of peroxidised lipids can polymerise with bovine pancreatic ribonuclease. They indicated (Gamage & Matsushita, 1973) that no general rule describes the reaction of oxidised lipids with different proteins. Possible mechanisms (Karel et al., 1975) are summarised in Fig. 1. Although covalent bonds between lipids and proteins are uncommon in natural systems, Tappel (1965) and Roubal & Tappel (1966) have observed that peroxidised lipids can form rather stable complexes with mammalian proteins ultimately to yield insoluble lipid-protein complexes. The proteins in intermediate moisture meats have been shown to form complexes of increasing molecular weight as storage advances and concomitantly the proteins become increasingly insoluble even in the presence of sodium dodecyl sulphate containing fl-mercaptoethanol indicating that covalent linkages form (Obanu et al., 1975b, 1976a,b; Obanu, 1976a,b, 1977c; Neale et al., 1978). Such covalent bonds have been reported to form in reactions between - - S H groups (including those from cysteine; Gardner et al., 1973) and linoleic acid hydroperoxide (Wills, 1961; Tsen & Hlynka, 1962; Bloksma, 1963; Karel, 1972). Andrews et al. (1965) and Shin et al. (1972) have described the formation of inter- and intra-molecular crosslinks between proteins and lipids through a Schiff's base reaction with malonaldehyde. Buttkus (1967) showed that malonaldehyde reacts readily with myosin in solutions at 20 °, 0 ° and - 20 °C and its

82

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interaction with collagen has been shown to yield a highly crosslinked protein (Svadlenka et al., 1975).

NON-ENZYMIC BROWNING (NEB) REACTIONS

There is much evidence to indicate that peroxide formation in oxidising lipids fosters reactions with proteins in lipid-protein systems (Lundberg, 1961, 1962; Schu!tz et al., 1962; St. Angelo & Ory, 1975). Reactions similar to those leading to Maillardtype pigments (Hodge, 1953; Hurst, 1972) are possible between proteins and peroxidising lipids. Oxidised lipids emulsified in aqueous dispersions of proteins have been observed to give brown copolymers (Tappel, 1955b; Venolia & Tappel, 1958) or complexes (Narayan & Kummerow, 1958, 1963). Pokorny et al. (1975) have shown that the course of non-enzymic brbwning is more rapid in mixtures of polyunsaturated lipids and casein than in such mixtures in which the free amino groups were blocked by treatment with formaldehyde. It is now well known that the decomposition of lipid hydroperoxides releases several carbonyl compounds which readily enter NEB pathways (Pokorny et al.,

LIPID--PROTEIN INTERACTIONS AS AGENTS OF QUALITY DETERIORATION

83

1973a,b). The browning mechanism seems to operate through the reaction of both peroxides and carbonyl fission products with proteins, forming colourless (or slightly coloured) intermediates or premelanoidins (Pokorny et al., 1974) which are subsequently transformed into the brown insoluble melanoidins, the transformation being influenced by temperature (Pokorny et al., 1973c, 1974). Thus Obanu et al. (1976a) observed visible browning of intermediate moisture pork after three weeks' storage at 38 °C but not before six weeks at 28 °C.

ANT1OXIDATION BY NON-ENZYMIC BROWNING INTERMEDIATES

The colourless NEB intermediates of premelanoidins are known to have an antioxidant effect. This has been attributed to their ability to bind metal catalysts (E1-Zeany et al., 1973) and to react with peroxides and free radicals produced during lipid oxidation (Eichner, 1975). These premelanoidins could be responsible for the non-:'ancidity of IM meats even after prolonged storage at 38°C (Brockmann, 1973b; Obanu et al., 1975a, 1976a; Obanu, 1977a). The evolution ofcarbonyls from lipid oxidation during cook-soak equilibration (Obanu, 1977a) and during storage (Obanu et al., 1975a; Obanu, 1977a), together with products of glycerol oxidation (Obanu et al., 1977) during processing and storage, would supply NEB-reactive carbonyls to form the antioxidant premelanoidins. This may be a primary route for the rapid decrease of carbonyls in most IM meats within three to six weeks' storage at 38°C (Obanu et al., 1975a, 1976a; Obanu, 1977a). If so, their relative rates of production and reactivity in various IM meats at 28 °C and 38 °C could account for the differences in thiobarbituric acid (TBA) number between samples (Obanu et al., 1975a, 1976a) and between storage at 28°C and 38°C (Obanu, 1977a).

COLOUR AND FLAVOUR CHANGES IN INTERMEDIATE MOISTURE MEATS

The extent to which fission products from lipid oxidation are incorporated in nonenzymic browning could explain, at least in part, the controversy as to whether nonenzymic browning (Loncin et al., 1968; Brockmann, 1969, 1973a,b,c) or rancidity (Labuza, 1972, 1973, 1974; Labuza et al., 1972b; Chou & Labuza, 1974b) is the most important deteriorative change in IM foods. It is well established that the Q10 for lipid oxidation is far less than for non-enzymic browning (Labuza, 1972). Thus, in unheated lipid-oxidation model systems (studied by Labuza and co-workers) peroxidising lipid intermediary products, in the absence of browning reactions, would lead to rancidity. However, with heated food systems (e.g. the desorptionprocessed IM foods studied by Brockmann and co-workers) premelanoidins may form. These, by antioxidant interaction with lipid oxidation intermediates, may prevent rancidity while leading to the browning of the food system. This

84

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phenomenon would inhibit rancidity but promote non-enzymic browning and protein insolubilisation. Evidence in support of this hypothesis has recently been obtained by May (1978) who found that the decrease in solubility in SDS/flmercaptoethanol of intermediate moisture meats stored at 38 °C was far more rapid in samples subjected to cook-soak equilibration (at 70 °C) than samples equilibrated without heat treatment (Fig. 2). Thus Obanu and his co-workers observed browning as the quality-limiting factor during the storage of various cook-soak equilibrated intermediate moisture meats at 38 °C (Obanu, 1976a,b, 1977b; Obanu & Ledward, 1975; Obanu et al., 1975a, 1976a) while rancidity was insignificant (Obanu, 1976a,b, 1977a; Obanu et al., ~1975b, 1976b).

CHANGES IN PROTEIN QUALITY OF INTERMEDIATE MOISTURE MEATS

It is evident that the probability of proteins crosslinking through and with lipid oxidation products is very high in intermediate moisture meats. The active role of TBA-reactive malonaldehyde in intermediate moisture meats via lipid-protein complex formation, non-enzymic browning and possibly other mechanisms, is indicated by the fluctuations in TBA number (Obanu et al., 1975a, 1976a; Obanu, 1977a). These reflect the intricate balance between malonaldehyde evolution and utilisation. A close relationship has been found between malonaldehyde--protein interaction and storage changes in proteins of fish leading to reduced water binding capacity, solubility and lysine availability (Kuusi et al., 1975). The

LIPID--PROTEIN INTERACTIONS AS AGENTS OF QUALITY DETERIORATION

85

malonaldehyde protein reacting sites, at room temperature, which have been detected (Buttkus, 1967; Chio & Tappel, 1969) involve lysine, histidine, arginine and methionine. The possible involvement of other amino acid residues has been indicated recently (Svadlenka et al., 1975). Results of amino acid analysis indicate that the bonding arising from reaction with malonaldehyde may be irreversible and strong enough to withstand acid hydrolysis yielding decreased amino acid values (Buttkus, 1967; Chio & Tappel, 1969; Taggert, 1971; Svadlenka et al., 1975). It is evident that this reaction may be partly responsible for the decrease in the amino acid contents of intermediate moisture meats (Obanu et al., 1975a, 1976b; Neale et a l , 1978) as determined after acid hydrolysis. In a typical experiment amino acid analysis of IM meat samples suggested that, after 12 weeks' storage at 38 °C, between 70 and 96 ~ of the original amino acids could be determined following acid hydrolysis (Obanu et al., 1975b) resulting in threonine and valine becoming the first and second limiting amino acids compared with the FAO/WHO chemical score (Neale et al., 1978). As proteolysis also takes place in intermediate moisture meats stored at 38 °C (Obanu et al., 1975a,b, 1976a) it is not unexpected that all amino acids, to a greater or lesser extent, become unavailable (Obanu et al., 1976b) as the liberated amino groups may take part in this type of reaction. The total destruction of amino acids, and an increase in covalent mal0naldehyde-amino acid compounds, which are not utilisable by any organism (Svadlenka et al., 1975), would contribute to the observed decrease in nutritive value of intermediate moisture meats as shown by the decrease in available lysine, protein efficiency ratio and net protein utilisation (Obanu et al., 1976a; Obanu, 1976b, 1977c; Neale et al., 1978). Since small amounts of crosslinking in a polymer can result in a large decrease in solubility, it is not surprising that protein insolubility in SDS-plus-fl-mercaptoethanol (Obanu et al., 1975b, 1976a; Obanu, 1976a,b) parallels the decrease in nutritive value resulting from the protein crosslinking (Obanu et al., 1975a, 1976b; Neale et al., 1978). For example, in a typical experiment a reduction in SDS/fl-mercaptoethanol solubility from 88 ~ to 16 ~ corresponded to a decrease in NPU from 83 ~ (typical of meat) to 32 ~ which is more characteristic of cereal protein. These decreases were obtained during 12 weeks' storage at 38 °C--a maximal tropical temperature---and one needs to ask whether these changes would be significant under more temperate storage conditions. Certainly the changes are very temperature dependent and at 28 °C there is only a slight decrease in SDS/fl-mercaptoethanol solubility during 12 weeks' storage (from about 90 ~ to 80 ~ in chicken and sow IM meats (Obanu et al., 1976b). Thus one would anticipate, at this temperature, only small changes in N PU of an order of magnitude similar to that found in freshly canned meats (Burger & Waiters, 1973). However, tremendous variations between IM meats are found and recent work has suggested that, even at 1 °C, marked decreases in SDS/flmercaptoethanol solubilities may occur in some IM beef samples. At 17°C a

86

z . A . OBANU, D. A. LEDWARD, R. A. LAWRIE

d e c r e a s e f r o m 90 ~ t o 40 ~ s o l u b i l i t y o v e r 18 w e e k s w a s f o u n d while at 1 °C a d e c r e a s e f r o m 90 ~ to 80 ~ s o l u b i l i t y was o b s e r v e d o v e r a s i m i l a r s t o r a g e p e r i o d ( W e b s t e r , 1979). A l t h o u g h the d e c r e a s e at 1 °C m a y be a c c e p t a b l e , t h a t at 17°C w o u l d b e e x p e c t e d to l e a d to a significant d e c r e a s e in N P U , p o s s i b l y d o w n to a value o f a b o u t 50 ~ ( N e a l e et al., 1978). T h u s the m a g n i t u d e o f the q u a l i t y d e t e r i o r a t i o n in I M m e a t s d u e to l i p i d - p r o t e i n i n t e r a c t i o n s m a y n o t be insignificant even at low temperatures.

CONCLUSION

It will be a p p a r e n t , f r o m the p r e c e d i n g d i s c u s s i o n t h a t the d e t e r i o r a t i v e c h a n g e s in I M m e a t s m a y result f r o m several t y p e s o f r e a c t i o n s ; b u t t h a t these have n o t been c l e a r l y identified. W h i l e this m i g h t have been d u e to u n a w a r e n e s s o f the p o s s i b l e roles o f these r e a c t i o n s in d e t e c t i n g I M f o o d q u a l i t y , t h e i r c o n s i d e r a t i o n is n o w n e c e s s a r y . T h e relative significance o f each r e a c t i o n will g u i d e f u r t h e r studies w h i c h s h o u l d u n f o l d their m e c h a n i s m s . T h i s k n o w l e d g e is essential in devising a d e q u a t e c o n t r o l o f d e t e r i o r a t i o n in these i n t e r m e d i a t e m o i s t u r e ( a n d o t h e r susceptible) foods.

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