5.3.1 In soybeans and soy foods
The undesirable `green' and `beany' flavors of soybeans and soy protein are major factors limiting their acceptability and use in foods. In raw soybeans, these flavors can be generated very rapidly once the cellular structure is broken, as illustrated by the rapid appearance of strong beany flavor when chewing raw soybeans. In industrial processing such as oil and protein separation the seed is first cracked, conditioned, and flaked, and then extracted with a solvent. During the process, lipid oxidation by LOX and free radical auto-oxidation is inevitable. The volatile secondary oxidation compounds, such as aldehydes and ketones, have low flavor thresholds and strong affinities for the soy protein. Thus, developing soybeans without or with reduced LOX contents has been a very desirable goal to increase soybean and soy ingredient consumption in the Western countries.
Mutant soybean lines lacking LOX isozymes have been developed (Davies and Nielsen, 1986; Narvel et al., 1998). LOX-null seeds with unaltered yields, seed weights, and protein contents were developed by Narvel and co-workers (1998). Low 18:3 (<5%) and triple-null soybeans have been developed to improve the oxidative stability of soybean oil and reduce off-flavors. The study of agronomic performance showed that such modifications did not cause any obvious detrimental effects on agronomic traits including yield (Reinprecht et al., 2006), and it might be possible to use this novel germplasm to develop competitive soybean cultivars with improved oxidative stability. A new soybean variety, Zhonghuang 31, was developed in the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences through years of biochemical marker assisted selection for null trypsin inhibitor and LOX-null (Han et al., 2006). This line has a high and stable yield and good quality in general. In an triple-null soybean, the degradation of antioxidative compounds, such as vitamin E, vitamin C, and lutein in aqueous homogenate was much less than in commodity soybean (Nishiba and Suda, 1998). This suggests that the LOX-null soybean could become a superior food ingredient which not only improves the flavor of soy products but also its nutritional quality.
The oxidative stability of the extracted and purified oil from LOX-null beans are not expected to be different than the traditional oil if the fatty acid Lipoxygenase and lipid oxidation in foods 109
composition is unchanged. Numerous research publications have validated this prediction (Engeseth et al., 1987; King et al., 1998; Shen et al., 1996). LOX-null soybean lines that lack LOX-2, or LOX-2 and 3, and contain normal (8.0±8.6%) or low (2.0±2.8%) 18:3 were evaluated for their oil qualities and storage stabilities (Shen et al., 1996). In general, the absence of LOX-2 or LOX-2 and 3, although having a small effect on lipid oxidation, was not as important to oil quality as was the 18:3 content. Frankel and co-workers (1988) found that in a direct comparison of oil products from the LOX-1 null and commodity soybeans, there was no significant differences in either flavor quality or in flavor stability based on total volatiles, and in the analysis for 2,4-decadienal. There-fore, factors other than LOX appear to control the food quality of soybean oils and meals.
Soy flour and purified soy proteins from the LOX-null varieties are expected to have improved flavor quality because of the less oxidation thus less binding of the flavor compounds to proteins during seed processing and protein preparation. As expected, soymilk and tofu made from LOX-null soybeans were less beany than products made from commodity beans as demonstrated by many researchers and reviewed by Wilson (1996). Yuan and Chang (2005) compared the beany flavor profiles of soymilk made from two LOX-null soybean types with three other varieties. Beany flavor compounds, including hexanal, hexanol, 2-nonenal, 1-octen-3-ol, and 2,4-decadienal were quantified.
The results showed that the LOX-null varieties produced significantly lower levels of the beany flavor than the two low-18:3 varieties and a commodity soybean. However, the flavor of bread, meat patties, and beverage products made with LOX-null soybeans was not improved (King et al., 2001).
Soybean oil is a 18:2 type of oil, and it naturally oxidizes rapidly by auto-oxidation. This may be the reason that the LOX-null soybeans are not showing great promise in effectively and significantly improving the flavor quality of protein products. Oil composition modifications such as high oleic and mid-oleic together with low or ultra-low 18:3 have been developed to replace hydrogenated soybean oil for many purposes. These may results in much more improved soybean products than LOX-null varieties. These new protein products need to be tested systematically and compared with the LOX-null type of proteins.
A most recent publication showed that there may be another LOX enzyme present in LOX-null soybean (triple-null) and this enzyme may be responsible for the off-flavors in the LOX-null soybeans (Iassonova et al., 2009). Volatiles production in triple LOX-null soybean could be terminated by heat treatment, which suggests an enzymatic cause to the off-flavors. The source is LOX-like in that the volatile compounds produced are similar to LOX-generated products of polyunsaturated fatty acids. Oxygen was consumed when a LOX-null protein solution was incubated with crude soybean oil suggesting that the enzyme-catalyzed oxygen consuming reactions. The generation of flavor compounds was inhibited by the typical LOX inhibitors. The enzyme was more active with phosphatidylcholine than with soybean oil or its free fatty acids as substrates.
110 Oxidation in foods and beverages and antioxidant applications
This is the first recent report on another LOX enzyme in soybeans which has a unique substrate specificity. Further research is needed to fully characterize this enzyme.
It is logical to suspect that soybeans with triple-null LOXs may be more vulnerable to insect attack on the seeds because oxylipins are important molecules in plant defense. Defense against insect attack was investigated for the lipid oxidation products produced by soybean seed LOXs, to study the physiological role of the enzymes in seeds. Repellent effects against bean bugs, a major soybean pest, were observed in products of LOX oxidation of 18:2 such as hydroperoxides and hexanal (Mohri et al., 1990). Bean bugs preferred LOX-null (triple-LOX-null) and LOX-2-LOX-null seeds, more than commodity soybean seeds.
However, no significant difference was seen in feeding preference at the ripening period of the seed. The lipid oxidation products of LOXs, 18:2-hydroperoxide and hexanal, also repelled two species of leaf beetles that do not usually feed on soybean seeds. These results suggested that the lipid oxidation products repelled the tested insects and that the soybean seed LOX could act defensively, although the effect of the products on the pest insects was not great.
LOX catalyzed lipid oxidation also affects the functional properties of soy proteins. Native soy flour's three main LOX isoenzymes can improve dough characteristics by oxidizing unsaturated fatty acids to hydroperoxides. The hydroperoxides can oxidize proteins, which can lead to improved rheological properties. The hydroperoxides also can bleach carotenoids leading to a whiter product (Roozen et al., 1993). However, the fatty acid hydroperoxides can also form volatile compounds that detract from the flavor of bland-flavored products.
Oxidation of lipids leads to protein structural change in soybeans, thus soy protein functionality can be altered. The primary lipid oxidation product by the LOX-catalyzed reaction in soy protein is the 13-hydroperoxide of 18:2 (Wu et al., 2009). Incubation of soy protein with increasing concentration of this hydroperoxide resulted in generation of protein carbonyl derivatives, loss of protein sulfhydryl groups and loss of -helix structure. Surface hydrophobicity of the protein also decreased, indicating that aggregation had occurred. The extent of protein aggregation increased with exposure to the hydroperoxide in a dose-dependent manner that was quantified by size exclusion chromatogram. -Conglycinin was more vulnerable to hydroperoxide than glycinin. Aggregation of soybean proteins induced by lipid oxidation was also investigated by Huang and co-workers (2006). Soybean proteins obtained from the model systems with various levels of linoleic acid and LOX showed increased turbidity, protein oxidation, surface hydrophobicity, and decreased sulfhydryl content.
Besides developing LOX-null soybeans, other approaches can be used to reduce the beany flavor resulting from LOX activity. These measures inactivate the LOX during processing and minimize or mask the flavors, and they include heating of the soybean seeds or grinding the seeds at an acidic pH, with hydrogen peroxide, with calcium ion, in hot water, with added antioxidants, using steam or vacuum distillation or supercritical extraction to remove the volatiles, and adding other flavor compounds to mask the undesirable flavor Lipoxygenase and lipid oxidation in foods 111
(Wilson, 1996). The enzymes can be readily deactivated by heating and this is commonly done in the industry to stop LOX initiated oxidation. LOX-2 and 3 are more susceptible to heat treatment than LOX-1. LOX-2 and 3 can be inactivated by heating to 70 ëC for 20 min, whereas LOX-1 required 120 min at 70 ëC to be inactivated (Hildebrand and Kito, 1984).
5.3.2 In rice bran
Rice bran is a by-product from the milling of rice caryopsis. It contains significant quantity of oil, and it has been an important source of vegetable oil in Asian countries. The presence of lipid oxidative and hydrolytic enzymes is a significant problem for a successful processing and utilization of this by-product and oil. LOX activity in rice seeds is localized in the bran fraction, and LOX, together with lipases, plays critical role in product quality.
Shastry and Rao (1975) showed that rice bran extract contained three distinct protein bands with LOX activity, and the major band which was 3 to 5 times more intense on SDS-PAGE gel was the smallest molecule and it still contained five proteins. The purification of this major band, enzyme stability, optimum pH for activity, enzyme activity and kinetics, activators and inhibitors of this band were further studied. Cupric ion at 1mM concentration was found to completely inhibit the enzyme activity. John and co-workers (1981) also isolated and purified rice caryopsis LOX. The molecular weight was about 100KDa, and its pI was 4.8. The optimum activity for lipid oxidation was at pH 6.8±7.0 and 30 ëC. The enzyme was relatively stable at neutral pH and room temperature but was markedly inactivated by incubation at temperature of greater than 50 ëC.
The heat inactivation of rice bran LOX, as for other enzymes, requires high moisture content. Rice LOX was not deacitivated at 21% moisture content, microwave oven heated (at 850 W) for 3 min with internal temperature reaching 107 ëC (Ramezanzadeh et al., 1999), and it was suggested that much higher moisture content is needed for full inactivation. The fact of rice bran LOX showing 90% activity inhibition by free radical quencher, such as BHA, demonstrates the involvement of free radicals in this enzyme catalyzed oxidation (Jhon and Lee, 1985).
LOX-3 is the major isoenzyme component in rice bran (Shirasawa et al., 2008) and the LOX-3 null rice has less stale flavor during storage than normal rice. Thus, introduction of the LOX-3 null phenotype would facilitate the retention of high quality during storage. A molecular genetic tool was developed by Shirasawa and co-workers for breeding and screening LOX-3 null rice lines.
Using the Thai rice variety Daw Dam which lacks LOX-3 in its seed, and the Japanese rice varieties Koshihikari and Koganemochi, which have normal LOX-3 activity, the oxidative stability of lipids in rice bran fractions was studied during storage at 4 and 37ëC (Suzuki et al., 1996). FFA content in lipids differed between storage temperature and peroxide value differed among varieties. The results suggest that lipid oxidation occurred at lower levels in the Daw Dam bran fraction than in the varieties with LOX-3 in their seeds.
112 Oxidation in foods and beverages and antioxidant applications
Many studies have suggested that the main reason for rice bran deterioration is the effect of lipase, but information about LOX activity has been relatively limited. The relationship between LOX activity (LOX1, 2, and 3) and rice bran deterioration was studied with an accelerated-aging experiment (Zhang et al., 2009) using white or red coat rice from near isogenic lines. The results seem to indicate that the activity of some LOX and lipase is negatively correlated. In this study, several rice bran materials with different activities of LOX isoenzyme within the seed coat color group were studied for their LOX activity and FFA content. Red coat with high LOX-1, 2 and low LOX-3 activity gave low FFA value, and white coat with low LOX-1, 2 and high LOX-3 activity gave low FFA value, and these samples had the least deterioration. Therefore, the results indicate that high LOX isoenzyme activity may be beneficial to delay rice bran deterioration, since rice LOX may mainly act on FFA and there is a shortage of the substrate in high-LOX activity rice.
5.3.3 In meats
Rao and co-workers (1994) suggested that myoglobin has LOX-like activity because it forms ferryl myoglobin when treated with hydrogen peroxide, and ferryl myoglobin is able to initiate lipid oxidation by abstracting a hydrogen atom from a methylene group of the 1,4-pentadiene system in a fatty acid chain to start the lipid oxidation process (Rao et al., 1994; Baron and Andersen 2002).
Spectroscopic studies showed that the binding kinetics of linoleic acid to myoglobin is similar to that of LOX-oxidation and that linoleic acid reduced the ferryl species to the ferric state. The stereochemical results ruled out any role of singlet oxygen in myoglobin-catalyzed and hydrogen peroxide-dependent oxidation of 18:2. The myoglobin protein radical formed with hydrogen peroxide also played no role in the reaction because the rate of formation of the 9-ROOH was not affected if the protein radical was allowed to decay before the substrate was added. The 18:2 was oxidized within the heme crevice by reacting with the ferryl oxygen in an enzymatic fashion (Rao et al., 1994).
To examine if myoglobin acts as a LOX-like catalyst, various meat homogenates preparations were analyzed to identify the factors in raw chicken breast and beef loin that result in lipid oxidation (Min and Ahn, 2009). Chicken breast showed greater oxidative stability than beef loin during 10-day storage.
All fractions (meat homogenate, precipitate, and supernatant) from chicken breast showed lower amounts of free ionic iron and myoglobin and higher total antioxidant capacity than those from beef loin. Therefore, myoglobin was responsible for the high LOX-like activity and lipid oxidation potential of beef loin. In a similar study, the susceptibility to oxidation of meats from various animal species was reported (Min et al., 2008). Oxidation of raw beef increased significantly compared to chichen and pork during 7-day storage as a result of beef's high heme iron content and high LOX-like activities.
The LOX-like activity of myoglobin on an 18:2 emulsion was 8.39 units per mg myoglobin protein (Min and Ahn, 2009). This was derived by measuring the Lipoxygenase and lipid oxidation in foods 113
double bond conjugation in an 18:2 emulsion treated with meat extract. To fully understand the enzymatic nature of myoglobin, a more in-depth characterization is needed that fully describes the effects of temperature, pH, response to antioxidants, and substrate specificity of the `enzyme'.
5.3.4 In fish
The 12- and 15-LOXs have been identified in fish gill tissue that have properties similar to the plant LOXs. LOXs of Atlantic mackerel muscle had two prominent molecular weights of 119 and 125 kDa (Saeed and Howell, 2001).
The presence of LOXs indicates the possibility that lipid oxidation is initiated enzymatically in chilled and frozen mackerel fillets, and that this oxidative deterioration could be inhibited by antioxidants (BHT, ascorbic acid and tocopherols) and heating which are used widely in the food industry.
Fu and co-workers (2009) used a model system of minced silver carp to examine the effect of LOX and hemoglobin on the kinetics of lipid oxidation and fishy-odor formation. The major LOX in silver carp muscle was a 12-isoenzyme. Compared to hemoglobin, LOX caused faster lipid oxidation in the initial phase but lower lipid hydroperoxides and less thiobarbituric acid test response (TBARS). The LOX was affiliated with a strong fishy odor, while hemoglobin resulted in a severe oxidized odor.
LOXs are also present in fish skin tissue, and skin tissue LOX initiated lipid oxidation in trout skin and affected the conversion of docosahexenoic acid (DHA) and AA into polar products (German and Kinsella, 1985). A novel LOX also was found in sardine skin (Mohri et al., 1992) that oxidized 18:2 more efficiently than AA or eicospentenoic acid (EPA). Esterified fatty acids such as the methyl ester of 18:2 and glyceryltrilinoleate also were oxidized. This enzyme is believed to participate in the initiation of lipid oxidation in fish.
The role of LOX in causing lipid oxidation (measured as TBARS) in lake herring was studied by Wang and co-workers (1991). LOX activity was correlated to phospholipids content, which is highest in light muscle, lowest in skin and intermediate in dark muscle. This may be because of the enrichment of EPA and DHA in phospholipids. Heat could be used to inactivate this LOX (80 ëC for 1.5±2.0 min); however, when heating for too long (80 ëC for more than 5.0 min), non-enzymatic oxidation was accelerated.
In fish muscle, both enzymatic and non-enzymatic oxidation can take place, with low temperature promoting an enzymatic mechanism and high temperature a non-enzymatic mechanism (Frankel, 1998). As mentioned previoussly, LOXs occur mainly in fish gill and skin tissues, and they can be inactivated at above 60 ëC. But such thermal treatment increases the non-enzymatic reaction rate since the denatured metallo-proteins are also active catalyst for lipid oxidation, and the breakdown of the resulting hydroperoxides can cause appreciable flavor generation. Such heat generated non-enzymatic oxidation may exceed the rate of oxidation catalyzed by LOX in unheated tissue. A post-mortem reduction in the natural reducing agents of fish, such as ascorbate, NADH and glutathione may 114 Oxidation in foods and beverages and antioxidant applications
affect lipid oxidation by changing the balance of concentration and activity between heme iron and free iron.
Although lipid oxidation is considered a deteriorative process responsible for generating off-flavors, specific oxidation products are desirable flavor com-pounds particularly when formed in more precise reactions, such as the genera-tion of fresh fish flavor (German et al., 1992). The specificity and stability of endogenous LOX and the oxidation volatiles generated by the enzymes in the gills of marine and freshwater fish were important in the generation of fresh fish flavors. Understanding these enzyme systems may facilitate the development of methods for the generation of desirable flavors in seafood products.
5.3.5 In dairy products
Many enzymes in milk are known to initiate lipid oxidation. Xanthine oxidase is a flavin enzyme found in milk fat globule membranes that generate superoxide radical (O2ÿ·), a reaction by an O2taking up one electron. Superoxide dismutase catalyzes the dismutation of this radical to produce hydrogen peroxide, thus superoxide dismutase serves as an antioxidant in the presence of catalase which can remove hydrogen peroxide from the system. In the presence of Fe2+, H2O2
produces very reactive HO·by the Fenton reaction. This active free radical can react non-selectively with all organic molecules. Milk neutral lipid has little polyunsaturated fatty acid and inherently stable to oxidation. Copper and iron exposure and photo-oxidation have been the major causes of oxidation. Ribo-flavin generated singlet oxygen oxidation may play important role in milk lipid oxidation. Cupric catalyzed oxidation of milk fat seems to give rise to 1-octene-3-one or possibly 1,5-octadiene-1-octene-3-one from n-3 fatty acids. These compounds play a minor role in the oxidation of vegetable oil but they are the dominant flavor in milk fat.
5.3.6 In fruits and vegetables
Lipid derived flavors are produced in tomatoes during ripening via the action of endogenous enzymes such as LOX. They may be further influenced by non-enzymatic oxidative decomposition reactions which occur during processing and storage (Karmas et al., 1994). The generation of lipid oxidation compounds in fresh tomatoes during ripening by LOX activity is considered a desirable reaction.
Undesirable LOX catalyzed oxidation is observed during potato processing.
The impact of processing on lipid oxidation in potato flakes was investigated by Gosset and co-workers (2008). LOX activity was mostly related to lipid oxida-tion and flavor generaoxida-tion. However, non-enzymatic lipid oxidaoxida-tion products were also found during processing, and these auto-oxidative processes cannot be inactivated by the main endogenous non-enzymatic antioxidants in potato tubers, such as ascorbic acid, phenolic compounds and carotenoids, because these antioxidants are degraded during processing. Therefore, efficient exogenous antioxidants as well as adequate storage conditions should be used.
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5.3.8 In tree nuts
Almond seeds age rapidly during storage at high relative humidity (80%) and moderate temperature (20 ëC). The content of unsaturated fatty acids (18:2 and 18:3) decreased during accelerated aging, and the aged seeds contain high levels of malondialdehyde (Zacheo et al., 1998). LOX was believed to cause this oxidation because increased activity of this enzyme was observed during the aging experiment. In hazelnut, significant genotypic variability was found in total fat, fatty acid and alpha-tocopherol contents, as well as LOX activity in various varieties of the nuts (Pershern et al., 1995). Relationships were found between shelf-lives for hazlenuts and their polyunsaturated fatty acids, -tocopherol and LOX activity levels.