Oxidative Changes and Their Control in Meat and Meat Products

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Karl-Otto Honikel

Introduction

Oxygen is a rather reactive molecule and is able to combine with many compounds within a living organism and food. But due to this reactivity it is also essential for animal life because reactions with oxygen provide the tissues with chemical energy. But the main constituents of muscular and fatty tissues are in a healthy live animal rather unsusceptible for unwanted oxidative changes. The reason is the presence of antioxidative substances in sufficient concentrations which despite the prevailing high oxygen concentrations in the tissue control the oxidation processes.

Antioxidants are reduced chemical compounds which react with oxygen or other already oxidized constituents of tissues. In these reactions the antioxidants are oxidized. They can be either reduced again by other reduced substances or, if not possible, will loose their antioxidative character. The antioxidative compounds are either directly received via feed/food or formed in metabolism with the help of the reduced matter of the feed/food. If oxidative changes occur irreversibly in a live animal then the oxidized compounds are either degraded within the cells or removed via the bloodstream.

After death the antioxidants present in muscles like NADH (nicotine-adenine-dinucleotide), vitamins C and E and antioxidative enzymes (oxidor-eductases) like catalase or glutathione peroxidase are preventing uncontrolled oxidative changes for some time postmortem as long as the necessary substrates are not oxidized themselves. The oxidized antioxidants remain in the meat. In a carcass or a piece of intact meat, however, the concentration of oxygen in the interior is low as the oxygen present at death has been used up by metabolic processes and the myoglobin in the surface layers of the meat binds the oxygen in the surrounding air and forms oxymyoglobin. Hence in contrast to the high

Federal Research Centre for Nutrition and Food, Max Rubner Institute, EC Baumannstrasse 20, D-95326 Kulmbach, Germany e-mail: [email protected]

F. Toldra (ed.), Safety of Meat and Processed Meat,

Food Microbiology and Food Safety, DOI 10.1007/978-0-387-89026-5_12,

© Springer Science+Business Media, LLC 2009

oxygen concentration in a muscle of a living animal, its concentration in meat is low and should remain low during storage of meat.

The presence of oxygen cannot be excluded during the slaughter process, the meat handling and processing. During slaughter and the immediately following chilling the carcass is surrounded by air. Also unavoidable is air during wholesale or retail cutting of carcasses. Only during storage for ageing or after retail cutting, a vacuum or a modified atmosphere containing either only nitrogen or a mixture of nitrogen and carbon dioxide can be applied.

Often, however, meat in retail cuts is packaged under high oxygen concentrations as the bright red color of the formed oxymyoglobin on the surface is preferred on display of meat. Additionally, the display under light accelerates oxidative processes as oxygen is activated into its singlet state by light (see below). Due to this oxidation the antioxidative substances in the meat are used up and its antioxidative power is reduced.

During processing, on mincing, or even comminuting of meat in a bowl chopper the surface of the meat is greatly enhanced and oxygen is present throughout the mince or batter. Furthermore during the comminution process the cellular membranes are disrupted and oxygen penetrates more easily to the oxidizable compounds. This disintegration of membranes also occurs on heating of intact meat cuts. The action of oxygen during heating processes, however, can be retarded if a formation of mostly brown-looking Maillard reaction products occurs which have an antioxidative power.

A possibility for the exclusion of oxygen during the processing steps is to work under vacuum, the use of nitrogen or carbon dioxide gas as protecting gases which can be done with industrial manufacturing equipments. The addition of antioxidants is another possibility which can be applied for protection from oxidation. Especially vitamin E in feed protects the meat and meat products during storage and processing. Among others, vitamins C and E, isoascorbates, nitrites/nitrates, chelating agents, synthetic antioxidants, and natural antioxidants like spices/herbs or their extracts are used in meat product manufacturing.

Oxidative changes are not per se negative events. Oxidative changes may contribute to sensory flavor characteristics of meat and meat products. Unsatu-rated fatty acids can be oxidized creating a number of desirable products like ketones, aldehydes, alcohols, and acids which in small concentrations add to the flavor of the product (Farmer, 1992). This happens already during chilled storage of raw meat but mainly on heating. On storing the cooked meat in a chilled or frozen state, however, the oxidative processes continue, the concentrations increase, further reactions occur and on reheating an unpleasant flavor and even odor is noticed which is called warmed-over flavor (WOF, see Pearson, Gray, Wolzok, & Horenstein, 1983; Gray & Pearson, 1987).

Oxidation may also affect the color of meat forming the brown metmyo-globin where the Fe ion in the porphyrin ring of hem is oxidized to Fe3+ from Fe2+ which is present in the bright red oxymyoglobin and the red myoglobin without oxygen. Finally, the oxidation of meat compounds to other reactive

Table 12.1 Factors influencing the oxidation in meat and meat products

Status

Process steps

Animals

Feed stuffs

Health status

Slaughter process

Meat

Storage time

Storage temperature

Size of cut

Packaging of cuts (MAP, vacuum)

Display under light

Processing

Heating of meat

Cold storage of heated meat (WOF)

Mincing, comminution

Addition of antioxidants/prooxidants

Packaging of products (MAP, vacuum)

Display under light

Storage time

Storage temperature

MAP, modified atmosphere packaging; WOF, warmed-over flavour.

MAP, modified atmosphere packaging; WOF, warmed-over flavour.

compounds in higher concentrations like peroxides, ketones, aldehydes, and other reactive products are regarded as cancer inducing or promoting substances (WCRF, 2007).

Thus, the use of antioxidative substances and/or oxygen exclusion measures in the processing of meat is common and necessary. They are applied for reasons of safety (shelf life), flavor, odor and color of meat, and meat products beginning with the feed, during slaughter, storage, preparation, and processing (Table 12.1).

In the live animal, the presence of antioxidative substances like vitamins C and E and compounds of herbs and spices in feed will enhance the antioxidative status. Also a healthy animal is keeping the antioxidative conditions high. In the postmortem meat, temperature, time, size of cuts and packaging, and display are factors which influence the presence and reactivity of oxygen around or in the meat. During processing by any means, from heating of meat cuts to manufacturing of meat products, the oxygen may deploy its action or may be retarded or inhibited by the various procedures during processing and storage.

Chemical Reactions of Oxygen Induction by Light

Oxygen exists under normal conditions in the triplet electronic state (3O2). This is the low-energy state in which the two electrons with the highest energy have sensitizer + light sensitizereIcited (reaction 1)

sensitizereIcited + 3O2 sensitizer + JO2 (reaction 2)

R-CH2-CH=CH-CH2-R + R-CH=CH-CH-CH2 -R (reaction 3) Fig. 12.1 Sensitizing of oxygen parallel spins. There exists the principle of conservation of spin angular moments in reactions. Unsaturated bonds in fatty acids and the peroxides formed thereof are both in the singlet electronic state with nonparallel electron spins. Hence the reaction of the low-energy triplet oxygen with an unsaturated bond in a lipid is rather unlikely. The triplet electronic state can, however, be changed, e.g., by light in the presence of a sensitizer-like riboflavin or others in a muscle (reactions 1 and 2 in Fig. 12.1). Singlet oxygen (1O2) reacts much faster (about 1500-fold) with double bonds (Gordon, 2001).

Not only oxygen can be activated by UV or visible light but also the chemical double bonds like those in unsaturated fatty acids may form radicals (R*) or radical ions with hydrogen release or electron transfer which then react with oxygen to form peroxides (ROOH, Fig. 12.2). The autoxidation starts mainly by the transfer of a hydrogen atom of the unsaturated bond to an existing radical. The unsaturated compound itself becomes now a radical (R*). The addition of oxygen to an existing radical is resulting in a hydroperoxide radical (Fig. 12.2).

A second initiation step for autoxidation is the splitting of an existing peroxide which is usually present in small concentrations in fatty tissues (Fig. 12.2). Two radicals are formed which lead to the prolongation step multiplying and creating in this way an avalanche of radicals and oxidized products if the sequence of events is not inhibited or stopped by the reaction of two radicals. As shown in Fig. 12.2, the radical from molecule R* can be transferred via a peroxide radical to another molecule R1*.

Induction by Metal Ions

Meat, like all other living matter, contains ions of transition metals like iron, copper, cobalt, manganese, chromium, etc. They occur in widely varying concentrations of 0.5 mM for iron to, e.g., <1 mM for chromium. All of these ions exist in several states of oxidation in which they can change more or less easily. Their red/ox potential affects the velocity of autoxidation in food and the initiation X* + RH 2nd initiation ROOH 2 ROOH

propagation

ROO*

termination

ROOR

X* is the first radical formed by light, heat, sensitizer RH, R and Rj are synonymous for unsaturated fatty acids Fig. 12.2 Autoxidation sequence of reactions further breakdown of the formed peroxides to volatile compounds important for flavor and odor. The first step of radical formation catalyzed by metal ions is induced by light, energy (heat), or enzymes (lipoxygenases). The metal ions in the lower state of oxidation (Mn) react very quickly with hydroperoxides (ROOH) as an electron donor as shown in Fig. 12.3. The metal ion turns into Mn+X and a radical RO* is formed. The following reaction with a second peroxide forms a second radical (ROO*) and the metal ion is reduced to Mn. The reduced metal ion can react again as it is typical for a catalyst. In both reactions shown in Fig. 12.3 two radicals are coming into existence by one metal

Fig. 12.3 Metal ion-induced autoxidation ion and a water molecule from H + and OH~ = H2O is formed. The reactions in Fig. 12.3 show that the metal ion must be in the reduced state for starting the autoxidation. The red/ox state depends on many conditions like solvent, pH, and presence of electron donors (reduced compounds) such as ascorbate or cystein. pH 5-5.5 is optimal; a pH which exists in a piece of meat. Fe ions are usually more effective than Cu ions.

Meat has a further metal-induced oxidative power. Fe ions are bound in meat to myoglobin in its prosthetic group, the hem, which is built by the porphyrin ring with a central Fe ion. The iron can change its oxidative form from Fe2+ to Fe3+ even to Fe4+. In a muscle of a live animal the Fe ion is in the Fe2+ status (red color). Only with the low oxygen concentration postmortem the Fe ion is oxidized to Fe3+ (brown color) and further. The myoglobin-bound or more correct the prosthetic group (hem)-bound Fe ion which accelerates the metal-catalyzed lipid oxidation to hydroperoxides faster in comparison to free Fe ions if the meat is heated and the protein moiety is denatured (Tichivangana & Morrissey, 1985). This leads then to the above-mentioned warmed-over flavor.

The sequences of reactions are shown in Fig. 12.4. The formed H2O2 can react also with a free Fe2+ ion forming an OH* radical which can initiate lipid oxidation as shown in Fig. 12.3. The H2O2 reacts also with hem(Fe3+) and removes via a hem + (Fe4+) = O (ferryl radical) at the end of the reaction sequence a hydrogen ion (proton) from an double bond in the unsaturated fatty acid (RH). The unsaturated fatty acid forms a radical R* which can react with oxygen to form a hydroperoxide (Fig. 12.2).

(bright red) (brown)

Fig. 12.4 Hem-catalyzed lipid oxidation (adapted from Monahan, 2000)

Induction by Enzymes

The mode of action of, e.g., the enzyme lipoxygenase differs from that of the nonenzymatic initiation of lipid oxidation. The rather complicated mechanism starts with an oxidation step of the unsaturated fatty acid, releasing a methylene group-

bound hydrogen (—CH2--> —C*H—+ H*). A conjugated diene is formed

(Yanishlieva-Maslarova, 2001). Then the oxygen is taking up by the enzyme, a peroxide radical is formed and after the addition of an hydrogen and creating the hydroperoxide the compound is released from the enzyme (Grossmann, Bergmann, & Sklan, 1988). Lipoxygenase initiation of lipid peroxidation requires for the enzyme an activation by a preformed hydroperoxide. Also the fatty acid must exist in free form and not bound to glycerol in a glyceride (Kanner, Haral, & Hazan, 1986).

The discussed possibilities emphasize that the primary initiation of a radical-driven fat oxidation is most likely light or temperature (heat). All the reactions described in this chapter are just a part of the many theories and proven events which exist about fat oxidation in meat. For further details and information see Monahan (2000).

Lipid Oxidation in Meat

As said above fresh raw meat in the first days postmortem exhibits an acceptable stability against oxidative processes due to concentration of antioxidants which deteriorate with time of storage leading to discoloration (metmyoglobin), rancidity, and health hazardous oxidation products. As an example, the reduction of NADH from day 1 to day 5 in meat is shown in Fig. 12.5. Freezing

Fig. 12.5 Fluorescence of NADH in meat at 1 and 5 days postmortem (excitation wavelength 330 nm [adapted from Schneider et al., 2008])

and thawing, addition of salt, display in light and mincing are causing increased oxidative stress.

Rancidity

The most known oxidation of lipids is the sensory impression of rancidity and warmed-over flavor (WOF). Factors influencing the development of rancidity and WOF in meat and meat products, including restructured meats, have been extensively reviewed (Gray & Pearson, 1987). These include

(i) the composition and freshness of raw meat components;

(ii) cooking and/or heating of the product;

(iii) processing techniques which result in tissue membrane disintegration and subsequent exposure of the constituents to air;

(iv) storage; and

(v) various additives which may have prooxidant or antioxidant properties.

Pearson and Tauber (1984) have indicated that the freshness and bacterial quality of raw meat components play a major role in preventing and/or retarding oxidation in meat and meat products.

It is widely recognized that sodium chloride (salt) may initiate color and flavor changes in meat, but its action is still not fully understood. Early work suggested that salt catalyzed oxidation by lipoxidase or by myoglobin (Tappel, 1952). Chang and Watts (1950) reported, however, 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 salt depended on its concentration and the amount of moisture in the system. Aqueous salt solutions were antioxidative at concentrations of sodium chloride above 15%, but dry salt readily promoted oxidation in lard. The mechanisms of salt-induced rancidity in pork were examined by Ellis, Currie, Thornton, Bollinger, and Gaddis (1968) who reported that increasing levels of salt accelerated autoxidation. They postulated that salt may activate a component in the myofibrillar tissue which results in a change in the oxidation characteristics of adipose tissue.

The prooxidative activity of salt in processed meats can be minimized by the application of various ingredients. As a pronounced example, the antioxidative nature of nitrite in cured meats is well documented (Gray & Pearson, 1984). Also phosphates have been shown to moderate the oxidative effects of salt in pork patties (Keeton, 1983) and restructured pork (Schwartz & Mandigo, 1976).

Warmed-Over Flavor

Cooking of meat causes further oxidative stress since by heating besides membrane disintegration also antioxidative enzymes in the muscle, like catalase and superoxide dismutase, may denature and loose their activity, while iron-containing proteins at the same time become a source of catalytic iron or, like myoglobin and hemoglobin, may be transformed into partly denaturated forms with "pseudo peroxidase'' activity. Oxidative changes in heat-treated processed meat are influenced by a higher number of factors than in fresh meat, and minimization of lipid oxidation in precooked or heat-processed meat requires many factors to be considered at the same time. Such a multifactorial approach was described, defining the critical control points to be considered for processed meat (Skibsted, Mikkelsen, & Bertelsen, 1998).

The oxidative changes in cooked pieces of meat and mince occurring during chilled storage are called warmed-over flavor (WOF) as defined by Tims and Watts (1958): "WOF is the rapid development of off-flavors is in contrast to the more slowly developing rancidity encountered in raw meats or fatty tissues during refrigerated and frozen storage. Although WOF can occur in fresh meat, it most commonly occurs in meats that are cooked or in which the membranes are broken down by processes such as restructuring or grinding. Thus, any process that disrupts the integrity of the membranes encourages development of WOF.'' Consumers recognize WOF as an unpleasant flavor, calling it as "old, somewhat rancid or fishy.'' The highly unsaturated phospholipids, which are integral parts of the cellular membranes, have been identified as oxidation substrate and responsible for the development of WOF. Hence lean meats are equally exposed to development of WOF as more fatty meats (Mielche & Bertelsen, 1994).

Preprepared foods processed by heating experience a steadily increasing demand. Thus they require new production and packaging concepts. For such meals, the sensory quality of the meat is often central for the overall impression and control of the WOF is accordingly critical (Mielche & Bertelsen, 1994). Vitamin E (a-tocopherol) incorporated in the cellular structures during animal growth is found to be superior to this antioxidant-added postmortem during processing of the meat in preventing lipid oxidation processes (Mitsumoto, Arnold, Schaefer, & Cassens, 1993) and also in limiting the formation of cholesterol oxidation products in processed meats as discussed later (Monahan et al., 1992). Cholesterol oxidation products in the diet may constitute a health risk in promoting atherosclerosis and other lifestyle-related diseases (Maerker, 1980).

Cured meat products are surprisingly stable against lipid oxidation and development of WOF as long as the cured meat pigment is not oxidized by the combined action of oxygen and light (Skibsted, 1992). Protection of cured meat against oxidation seems to be possible if the meat used for curing is selected from animals raised on feed with an increased level of vitamin E or if the presence of oxygen is minimized during storage.

The catalytic effects of iron in meat on WOF are well documented. In the 1960s myoglobin was viewed as the major catalyst of lipid oxidation (Tappel, 1962). However, studies by Sato and Hegarty (1971), Love (1972) and Igene (1978) revealed that nonhem iron, rather than hem iron, was the active catalyst responsible for the rapid appearance of WOF in cooked meat. By the heating process, the iron in the hem moiety of myoglobin becomes ''free'' due to the denaturation of the protein moiety. Free iron is known to decompose lipid hydroperoxides, forming very reactive alkoxy radicals for the propagation reactions (Ingold, 1962). Also, the mechanism proposed by Tappel (1962) depends on the presence of lipid hydroperoxides which react with hem compounds and undergo homolytic decomposition. The ability of hem pigments and nonhem iron to accelerate the propagation step of the free radical chain mechanism (see Figs. 12.3 and 12.4) can explain the rapid rate of lipid oxidation in cooked meats (Tichivangana & Morrissey, 1985). Kanner and Harel (1985) have demonstrated that metmyoglobin, when activated by hydrogen peroxide, will initiate membrane lipid oxidation. They proposed that autoxidation of oxygenated hem pigments (oxymyoglobin and oxyhemoglobin) leads to formation of met-hem proteins and the superoxide radical (O2—), which dismutates to form hydrogen peroxide (see Fig. 12.4). A reactive porphyrin ferryl radical (P — Fe4+ = 0) results from the reaction of metmyoglobin and methemoglobin with hydrogen peroxide.

Rhee, Ziprin, and Ordonez (1987) studied the mechanism of lipid oxidation in meat systems and concluded that the hem pigment system (Fe III-Mb / H2O2, see Fig. 12.4), regardless of how it exerts its prooxidative effect, plays a major role in the catalysis of lipid oxidation in raw and cooked meat. They suggested that hydrogen peroxide-activated metmyoglobin was the primary initiator of lipid oxidation in raw meat, and that nonhem iron, released from metmyoglo-bin by the action of hydrogen peroxide, was the major catalyst of lipid oxidation in cooked meat.

Cholesterol Oxides

A very thorough study of the oxidation of lipids and cholesterol has been done in the thesis of Munch (2003) wherein he investigated various meat species. In Table 12.2 the oxidation at fatty acids measured as malondialdehyde (TBARS values) by heating of pork chops and roast beef is shown. Pan frying and water cooking enhanced the TBARS values by a factor of 3-4.

If the pork chops are reheated by cooking in water after 1-7 days of chilled storage the values of TBARS in pork chops of cooked meat rise immediately

Table 12.2 Concentration of malondialdehyde (TBARS value) (mg/kg muscle) in raw meat and after different treatments and cuts of different species (adapted from Munch, 2003) to well done (~80°C); the raw meat was used about 5 days post mortem

Treatment

Pork chop

Roastbeef

Raw

0.09

0.18

Pan fried

0.42

0.55

Water cooked

0.44

0.33

Table 12.3 Concentrations of malondialdehyde (TBARS values) after chilled storage lean pork chops and reheating by cooking in water bath or in microwave and stored at 8°C

Sample

TBARS (mg/kg product)

Reheating by cooking after storage for

1 day

0.59

2 days

0.64

7 days

1.45

Reheating by microwave after storage for

1 day

0.82

2 days

1.41

7 days

3.09

after cooking with 0.44 mg TBARS/kg meat (Table 12.2) and to 0.59/0.64/ 1.45 mg TBARS/kg meat after 1/2/7 days of chilled storage. If the reheating is done by microwave, then the values are even higher of 0.82/1.41/3.09 mg TBARS/kg meat after 1/2/7 days of storage (Table 12.3).

The results exhibit two things. Chilled storage of heated meat enhances the oxidation of fatty acids in muscle more than a storage of raw meat for some days as shown in Table 12.2 with raw meat at 5 days postmortem. But besides the storage time, the heating treatment is also important. Microwave heating nearly doubles the TBARS values of those of cooking in water.

Not only the fatty acids, but also the cholesterol is oxidized to a number of oxides which are shown in Table 12.4. In this table, different pork cuts have been analyzed for cholesterol oxides in raw, pan fried (80°C), and cooked in water (ca. 85°C). In the raw meat of all cuts, all oxides are lower than that in the heated samples. The minced meat has higher concentrations of 7 a-diol/7 b-diol and triol already in the raw meat which are further enhanced during the heating processes. The minced meat has a larger surface and thus the oxygen has an easier access to the phospholipids. The two different heating regimes (pan fried or cooked in water) enhanced in general the concentrations of the oxides; the cooking in water (no Maillard reaction products possible) exhibits higher oxide concentrations in most cases. If the pan-fried sample are stored up to 7 days and reheated, either by frying or by microwave heating (Table 12.5), then the observed results show a very similar behavior to those of Tables 12.2 and 12.3. Heating enhances cholesterol oxides and TBARS values, both during chilled storage, and reheating by frying, cooking, or microwave heating.

Figure 12.6 shows the results in a graph. It is apparent that some cholesterol oxides are increasing by factors of 50-60 in 7 days, others by factors of about 10; some of the possible oxides are not changed very much and remain altogether low in concentration.

The changes of cholesterol oxides during frozen storage at —20°C are shown in Table 12.6. Storage for 30 weeks leads only to slight increases of most cholesterol oxides. The concentrations at 30 weeks are much smaller than after chilled storage for 7 days without reheating. Reheating let the cholesterol

Table 12.4 Concentration of cholesteroloxides (mg/kg muscle) in raw and heated pork cuts (adapted from MUnch, 2003); raw cuts were analysed at 5 days post mortem, heated samples at the day of experiment

Cholesterol

Eye of

Leg

Shoulder

oxides

hind leg

bottom

Rump

Mince

blade

Chop

7a-diol

Raw

26

23

33

102

30

41

7b-diol

24

27

35

111

35

43

b-epoxide

n.d.

n.d.

n.d.

n.d.

n.d.

38

a-epoxide

5

35

6

n.d.

26

32

Triol

16

10

42

83

18

41

25-diol

11

18

22

23

19

17

7-keto

12

52

10

12

110

88

7a-diol

Pan fried

41

36

33

147

24

42

7b-diol

48

47

40

192

28

44

b-epoxide

n.d.

n.d.

n.d.

n.d.

n.d.

32

a-epoxide

40

77

30

n.d.

27

32

Triol

14

24

29

80

16

34

25-diol

20

16

27

34

20

21

7-keto

58

67

22

45

124

98

7a-diol

Cooked in

97

21

93

139

33

46

7b-diol

water

122

32

134

228

44

56

b-epoxide

n.d.

n.d.

n.d.

n.d.

34

56

a-epoxide

44

34

71

n.d.

88

32

Triol

8

10

37

53

20

34

25-diol

25

18

30

37

21

24

7-keto

106

56

74

22

205

104

n.d., not detectable.

n.d., not detectable.

Table 12.5 Concentrations of cholesterol oxides in lean pork chops, reheated by pan frying or microwave after storage for 1, 2 and 7 days at 8°C (adapted from MUnch, 2003)

Cholesteroloxides (mg/kg muscle)

Table 12.5 Concentrations of cholesterol oxides in lean pork chops, reheated by pan frying or microwave after storage for 1, 2 and 7 days at 8°C (adapted from MUnch, 2003)

Cholesteroloxides (mg/kg muscle)

7a-diol

7b-diol

b-epoxide

a-epoxide

20a-diol

triol

25-diol

7-keto

Reheating by

frying after

storage for

1 day

156

271

134

71

n.d.

41

35

353

2 days

459

760

351

133

n.d.

65

52

1004

7 days

974

1476

393

300

n.d.

59

59

1424

Reheating by

microwave after

storage for

1 day

187

314

116

78

n.d.

59

54

463

2 days

441

721

220

124

n.d.

60

51

990

7 days

1009

1476

354

246

4

87

63

1506

Gentamicin Normogram
Fig. 12.6 Changes of cholesterol oxides during chilled storage of pan-fried pork chops (for details see Table 12.5)

concentrations rise to those mentioned in Table 12.5. Due to the toxicological behavior of some cholesterol oxides, a reheating of chilled meat should be avoided if possible, if there is no protection against oxidation provided by other means as discussed below.

Prevention of Lipid Oxidation in Meat Vitamin E Supplementation

If vitamin E is added to animal feed, it becomes an integral part of cellular membranes, in contrast to the added vitamin E to a product during processing (Bertelsen, Jensen, & Skibsted, 2000). The location of the a-tocopherol, the main vitamin E compound, is in close proximity to the phospholipids as primary oxidation substrate and to the membrane cholesterol. It is the assumed basis for the pronounced antioxidative effects of vitamin E which is generally achieved.

In the review by Gray, Goman, and Buckley (1996), it is surprisingly concluded that feeding supranutritional levels of vitamin E to cattle does not provide any distinctive benefits for precooked beef. As said above, heating of meat liberates catalytically active iron from the hem group of myoglobin and

Table 12.6 Changes of cholesterol oxide concentrations (ng/kgmeat) during frozen storage (-20°C) in raw, pan fried and cooked pork chops (vacuum packaged), adapted from Munch (2003)

Treatment

Raw

Pan fried

Cooked in water

Storage weeks

0

4

11

30

0

4

11

20

30

0

4

11

20

30

7a-diol

47

49

69

75

52

64

80

86

86

57

76

73

92

187

7/?-diol

50

58

73

71

49

83

99

94

94

74

100

93

135

213

/i-epoxide

31

28

n.n.

33

13

13

13

70

8

60

69

32

86

70

a-epoxide

27

23

11

47

26

39

42

90

35

25

49

58

57

77

Triol

32

26

25

37

18

21

26

49

39

20

24

30

33

75

25-diol

17

18

19

20

14

18

20

14

21

15

23

22

23

23

7-keto

91

95

93

120

106

110

148

146

154

118

132

136

195

from other iron-containing proteins and transforms myoglobin and hemoglobin into prooxidative species, resulting in accelerated oxidation of lipids in the membranes (Geileskey, King, Coste, Pinzo, & Ledward, 1998; Mielche & Bertelsen, 1994). The higher content of hem and hem iron in beef and pork (Schricker & Miller, 1983) results in a higher load of prooxidative species in precooked products from these species compared to, for example, poultry and provides part of the explanation for the relatively lower vitamin E effect in beef and pork compared to poultry. The importance of prooxidative species originating from meat myoglobin and other iron-containing proteins is also evident from the studies performed with chicken, where a lower protection by vitamin E in general has been found for dark thigh meat compared to white breast meat (Jensen, Skibsted, Jakobsen, & Bertelsen, 1995; Galvin, Morrisey, & Buckley, 1998), despite the fact that the accumulation of vitamin E is higher in dark meat compared to white meat.

Protection in Meat

Supplementation of feed with vitamin E can be done in different ways. It can be done continuously in smaller amounts over the whole fattening period or within the last few weeks in higher doses. Table 12.7 shows such an experiment with pigs. Pigs were fed from about 27 kg live weight until slaughter (ca. 110 kg live weight) for about 100-110 days with no additional supplementation of vitamin (natural content of feed was 32 mg vitamin E/kg feed) and 100 or 200 mg vitamin E/kg feed, equivalent to about 22-44 g added vitamin E in total over the whole feeding period. A fourth group received additionally in the last 3 weeks before slaughter 1.2 g vitamin E/day equal to 21 x 1.2 g = 25.2 g total which is in between the feeding of groups 2 and 3 (Rosenbauer & Honikel, 2002, personal communication).

Figures 12.7 and 12.8 show that the concentrations of vitamin E increase in loin muscle and back fat in all supplementation regimes and are about twofold enhanced by 100 mg vitamin/kg feed respective 1.2 g/day in loin (2.5 vs. 5 mg vitamin E/kg and 10 vs. 20 mg vitamin E/kg back fat).

It is evident that the fatty tissue contains about five times higher vitamin E concentrations than the lean loin muscle (ca. 2% fat). As shown in Figs. 12.7 and 12.8 the additions of 200 mg vitamin E/kg to the feed enhanced the vitamin

Table 12.7 Supplementation of feedstuff with vitamin E

Group

Control II III IV

Number of samples

Vitamin E-additiona (mg/kg feed dry matter) Vitamin E-additiona in the last 3 weeks (g/day) prior to

slaughter a Additional to the basic content of 32mg vitamin E/kg feed (covering the requirement).

Fig. 12.7 Vitamin E level in fresh (2 days postmortem) and for 14 days at 6°C stored pork loin muscle (the line in the column indicates the lower half of the standard deviation, N = 10)
Fig. 12.8 Vitamin E concentration in fresh (2 days postmortem) and for 14 days at 6°C stored pork back fat (the line in the column indicates the lower half of the standard deviation, N = 10)

E concentration in loin and back fat by further 15-25%. During chilled storage for 14 days the vitamin E concentration fell by 10-25% (Rosenbauer & Honikel, 2002, personal communication). Figure 12.9 shows another experiment where 1 g vitamin E was fed per day for 1, 2, and 3 weeks prior to slaughter. Without supplementation the concentration in the muscle and liver was rather low. In liver and back fat it increased strongly with the time of supplementation.

The higher values of vitamin E in the tissues reduced the oxidation products measured as TBARS values from about 0.10 mg malondialdehyde/kg loin tissue with 2 mg vitamin E/g tissue to 0.06 mg malondialdehyde with 7 mg vitamin E/g loin tissue. With the time of storage, the TBARS increase and the values are the highest when the vitamin E concentration of tissue is the lowest (Fig. 12.10).

MS vitamin E / g tissue

Fig. 12.9 Supplementation of pig feed with 1 g vitamin E per day for 1-3 weeks prior to slaughter and its influence on the concentrations in liver, loin muscle, and back fat of pig (N = 9 animals)

liver muscle backfat

Fig. 12.9 Supplementation of pig feed with 1 g vitamin E per day for 1-3 weeks prior to slaughter and its influence on the concentrations in liver, loin muscle, and back fat of pig (N = 9 animals)

Fig. 12.10 Vitamin E level and TBARS values in pork loin muscles at storage at 4°C for 14 days (N = 10)

vitamin E [mg/kg tissue]

Fig. 12.10 Vitamin E level and TBARS values in pork loin muscles at storage at 4°C for 14 days (N = 10)

0 mg/kg 100 mg/kg 300 mg/kg 1.2 »'day control vitamin E-supplementation to feedstuff

Fig. 12.11 Course of TBARS values development in pork back fat during frozen storage for 26 weeks (at —20°C, vacuum packaged; N = 10; the line in the column indicates the lower half of the standard deviation)

0 mg/kg 100 mg/kg 300 mg/kg 1.2 »'day control vitamin E-supplementation to feedstuff

Fig. 12.11 Course of TBARS values development in pork back fat during frozen storage for 26 weeks (at —20°C, vacuum packaged; N = 10; the line in the column indicates the lower half of the standard deviation)

The results of Fig. 12.10 show that during a normal shelf life of fresh pork (<6 days) does not lead irrespective of vitamin E concentration to a sensory appearance of rancidity as the TBARS values are <0.15 mg malondialdehyde/ kg fresh unsalted tissue.

In frozen back fat, the TBARS values also increase despite higher vitamin E concentrations in the tissue (Fig. 12.11). Whereas after 10 weeks the increase is rather small (0.06-0.08 mg malondialdehyde/kg tissue in control samples) the further storage for additional 16 weeks resulted in a strong increase of TBARS values. The feed supplementation of 100 respective 200 mg vitamin E/kg has no protective effect any more at 26 weeks of frozen storage.

Protection in Meat Products

The production of raw ham and salami-type sausages takes months. Figure 12.12 shows that the concentration of vitamin E is high in raw hams even after 6 months; the vitamin E supplementation effect is still clearly visible.

The TBARS values (Fig. 12.13) are in the control samples at the edge of sensory detection at 6 months of ripening. Supplementation leads to lower values at 3 months. At 6 and 9 months there existed no effect of supplementation any longer. But the TBARS values are in all cases below sensory detectable values (<0.3 mg malondialdehyde/kg product) in salted products where the limit of sensory detection is higher than in unsalted fresh meat. Interestingly, in salami-type sausages the vitamin E concentration increased during storage up to 29 weeks due to the considerable weight loss of >30% (Fig. 12.14). Despite the considerable vitamin E concentration at 29 weeks, the sausages were

Fig. 12.12 Vitamin E concentrations in the fat of raw hams after 3 months fermentation and further 3 months (at 6 months in total) of storage (the line in the column indicates the lower half of the standard deviation, N = 6)

3 Hi lllli SB lllS

iojo~l I 11 11 11

0.10

0.05

0.00

0 mg'kg control

100 mg/kg 200 mg/kg 1.2 g/day vitamin E-supplementatlon to fi-tdstuff

Fig. 12.13 TBARS values in the lean tissue of raw hams after 3 months of fermentation and further 6 months (in total 9 months) of storage at 12°C in vacuum (N=6, the line in the column indicates the lower half of the standard deviation)

evaluated sensorial as rancid despite low TBARS values (Honikel & Rosenbauer, 1998). In consequence, it means that vitamin E protects against rancidity but not under all especially long-time storage conditions.

The short shelf life of cooked ham causes no problems in this respect. The supplementation led to higher and stable vitamin E concentrations (Fig. 12.15). The constant concentration at 3-4 and 13 days is most likely due to the

Fig. 12.14 Vitamin E concentrations in salami-type sausages immediately after start of production and after 13 and 29 weeks of ripening and storage at 12°C in vacuum package (N=2; the line in the column indicates the lower half of the variation)
Fig. 12.15 Vitamin E concentration in cooked ham after manufacturing (3-4 days) and storage for altogether 14 days at 5°C (N=10; the line in the column indicates the lower half of the standard deviation)

antioxidative action of the nitrite present in the product which was about 100 mg nitrite/kg addition and about 20-30 mg nitrite/kg left after heating. There was no rancidity development neither in the control samples nor in the supplemented ones detected (Rosenbauer, 2002).

In conclusion: In raw meat, cooked ham and raw hams (the latter two contained nitrite) enhanced vitamin E concentrations are not needed for

- backfat fresh

vitamin E intake [g/animal]

Fig. 12.16 Vitamin E intake of pigs by feed and tissue concentrations of fresh pig meat and back fat and in processed products

vitamin E intake [g/animal]

- backfat fresh

Fig. 12.16 Vitamin E intake of pigs by feed and tissue concentrations of fresh pig meat and back fat and in processed products sensory protection against rancidity. Salami-type sausages, if stored for long periods, are not protected against rancidity. WOF, however, can be prevented by vitamin E.

The vitamin E concentration from a nutritional point of view is enhanced in fresh meat and fat (Fig. 12.16). The addition of 1.2 g/day for 3 weeks or 100 mg respective 200 mg/kg feed with 20-45 g total addition during the feeding period led to an increase from 10 to 20/25 mg vitamin E/g fatty tissue and from 2 to 6 mg vitamin E/g muscular tissue. Processing did not change the increase to a large extent.

Other Measures Against Oxidation

Many studies have indicated that WOF development in meat products can be effectively controlled or retarded by the use of antioxidants or a proper packaging. These compounds can be used singly or in combination and can range from synthetic antioxidants to compounds in natural foods like herbs and spices whose structures are not always fully elucidated. In fresh meat the use of the compounds in the following chapters are prohibited or limited.

Packaging

Harte (1987) reviewed packaging techniques and indicated that the selection of a packaging system can significantly influence the oxidative stability of meat and meat products. Headspace control techniques (vacuum packaging, gas flushing and shrink- and skin-packaging), when used in conjunction with good oxygen and light barriers, can effectively control oxidative rancidity. Kingston, Monahan, Buckley, and Lynch (1998) reviewed the effectiveness of several packaging systems in controlling oxidation in precooked meat products. Packaging meat products in good oxygen-barrier materials can significantly retard autoxidation. The most protecting packaging materials are air tight metal and glass containers, the latter must be stored in the dark as light may induce radicals which also may lead to sensory changes. Antioxidant-impreg-nated films have also been used at the research level with some success in minimizing lipid oxidation in selected meat items.

Maillard Reaction Products

The antioxidant activity of Maillard reaction products is well established. For a review see Pokorny and Schmidt (2001). Rhee (1987) interestingly reported that the use of extracts from over-cooked, retorted, or pressure-cooked meat which contained brown Maillard products may not be economically feasible unless meat animal parts of little economic value are used to prepare the extracts.

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 also delays or prevents lipid oxidation (Sato & Hegarty, 1971). Ortho-, Pyrophosphates, tripolyphosphates, and hexameta-phosphates all offer protection but to a different extent. Phosphates appear to prevent autoxidation by chelating the heavy metal ions (Tims & Watts, 1958).

Other chelating agents have been shown to be effective as inhibitors of oxidation, presumably because of their ability to sequester transition metal ions like those of iron and copper. Liu and Watts (1970) demonstrated many years ago that ethylenediamine tetraacetic acid (EDTA) prevented Fe2 + -catalyzed oxidation in raw beef, while Sato and Hegarty (1971) showed that EDTA, at a concentration of 2.5 mg/g, suppressed lipid oxidation in cooked ground beef. These investigators concluded that EDTA effectively chelated free iron and thereby significantly reduced lipid oxidation in cooked meat. Although EDTA (E-385) has provided a good tool for studying the role of heavy metal ions in lipid oxidation, it has not been approved for commercial use in meat products.

Citric acid and citrates have also been evaluated as antioxidants in meat systems. Sato and Hegarty (1971) reported minimal inhibition oflipid oxidation in cooked ground beef when sodium citrate was added to the level of 5 mg/g. Macdonald, Gray, and Gibbins (1980) demonstrated that citric acid reduced

TBA numbers in refrigerated hams when used at the 1000 mg/kg level. However, this compound was not as effective as 50 mg/kg of nitrite.

At low levels (<100 mg/kg) ascorbic acid and ascorbate have been shown to catalyze WOF development in meat products (Sato & Hegarty, 1971). However, at levels in excess of 1000 mg/kg, ascorbate or isoascorbate is an effective inhibitor of oxidation. Sato, Hegarty, and Herring (1973) suggested that high levels of ascorbic acid shifted the balance between ferrous and ferric iron and acted as an oxygen scavenger. Kanner et al. (1986) demonstrated that iron in the presence of ascorbic acid stimulates membrane lipid peroxidation in muscles, presumably through the involvement of hydroxyl radicals. A synergistic relationship between ascorbic acid and phosphates in inhibiting lipid oxidation in meats was demonstrated a long time ago by Tims and Watts (1958) and Sato and Hegarty (1971). The latter investigators theorized that ascorbic acid functions by keeping a part of the iron in the reduced state. The combined actions of phosphates, ascorbate (or isoascor-bate), and nitrite assist in explaining the virtual absence of WOF in cured meats.

Nitrite/Nitrate

Nitrite is limited to the use in meat products. Sato and Hegarty (1971) reported that nitrite completely eliminates WOF at a rather high level of 2000 mg/kg and delays the development of WOF at the low level of 50 mg/kg. Fooladi, Pearson, Coleman, and Merkel (1979) demonstrated that a nitrite concentration of 150 mg/kg added to meat inhibited WOF development in cooked meat, with a twofold reduction of TBARS numbers for beef and chicken, and a fivefold reduction for pork. Nitrite is easily oxidized to nitrate and acts this way as an oxygen scavenger (Honikel, 2008). Some other possible mechanisms include

(i) The formation of a stable complex between the hem pigments and the nitrite, thereby preventing the release of nonhem iron and its subsequent catalysis of lipid oxidation (Igene, Yamauchi, Pearson, & Gray, 1985; Morrissey & Tichivangana, 1985); (ii) The formation of inactive "chelates" between nitrite and metal ions such as ferrous ions, thus rendering them unavailable for catalysis of oxidation reactions (Igene et al., 1985; Morrissey & Tichivangana, 1985); and (iii) The formation of nitric oxide myoglobin which has antioxidant properties per se (Kanner, Ben-Gara, & Berman, 1980; Morrissey & Tichivangana, 1985). Regardless of the mechanism of nitrite in preventing oxidation and WOF development in meat, there is little doubt about its effectiveness in decreasing lipid oxidation (Sato & Hegarty, 1971; Gray & Pearson, 1984, 1987).

Smoking

The antioxidant activity of smoke is provided by a number of compounds, including phenols, phenol aldehydes, and organic acids (Toth, 1984). Phenols with high boiling points, such as 2,6-dimethyoxyphenol and 2,6-dimethyoxy-4-ethylphenol, are particularly effective (Pearson & Tauber, 1984).

Synthetic Antioxidants

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 against oxidation of fresh meat pigments and effectively inhibited lipid oxidation in raw ground beef. Greene, Hsin, and Zipser (1971) further demonstrated that BHA or PG prevented lipid oxidation and reduced pigment oxidation in ground beef for up to 8 days of refrigerator storage. When a combination of antioxidants and ascorbic acid was used, both lipid and pigment oxidation were effectively retarded.

Natural Antioxidants

Many studies on lipid oxidation in meats have focused on the antioxidant activity of naturally occurring substances. These substances include various edible products from spices and herbs. Houlihan and Ho (1985) and Rhee (1987) have reviewed the antioxidative nature of these substances in some detail.

Many spices and herbs have been shown to function as antioxidants in fats and oils and in model food systems (Yanishlieva-Maslarowa & Heinonen, 2001). Rosemary, for example, contains a number of compounds possessing antioxidant activity, including carnosol, rosmanol, rosmariquinone, and ros-maridiphenol. Already in 1985, Barbut, Josephson, and Maurer demonstrated that a rosemary oleoresin, when added to turkey breakfast sausage at the 20 mg/ kg level, produced an antioxidative effect and did not adversely affect overall palatability of the product. The authors concluded that incorporation of rosemary oleoresin in meat products can substantially suppress lipid oxidation and increase shelf life at refrigerated temperatures.

Concluding Remarks

The chemically reactive oxygen, necessary for the energy turnover in living organisms, can show detrimental effects in meat and meat products during processing and storage. Rancidity, WOF development, cholesterol oxidation, and the loss of bright red color of fresh meat are the unwanted changes. These changes may appear to the consumers primarily as unpleasant sensorial deteriorations, but they can also be health hazardous. Radicals may initiate cancer; cholesterol oxides are held responsible for the development of artheriosclerosis. Hence the prevention or retardation of oxidative changes is required. The century-old methods of using herbs, spices, and nitrate/nitrite and the creation of the brown-reducing Maillard reaction products are accompanied today by oxygen exclusion in vacuum or MAP packaging, the use of natural antioxidants in feed of animals or during processing, ascorbic acid (vitamin C) vitamin E or phosphates and citrates, or the addition of synthetic antioxidants.

These measures are necessary as the shelf life of meat, meat preparations, and meat products has been extended largely in comparison to half a century ago. Usually a combination of several measures is necessary in order to safeguard a product during its shelf life. But there is always a limit of shelf life as antiox-idants due to the nearly ubiquitous presence of oxygen even in small concentrations leads to their oxidation. Only air tight metal or glass containers, the latter in the dark, prevent the presence of oxygen.

References

Barbut, S., Josephson, D. B., & Maurer, A. J. (1985). Antioxidant properties of rosemary clear esin in turkey sausages. Journal of Food Science, 50, 1356-1361.

Bertelsen, G., Jensen, C., & Skibsted, L. H. (2000). Alteration of cooked and processed meat properties via dietary supplementation of vitamin E. In E. Decker, C. Faustman, & C. J. Lopez-Bote (Eds.), Antioxidants in muscle foods (pp. 367-394). New York: Wiley Inter Science.

Chang, I., & Watts, B. M. (1950). Some effects of salt and moisture on rancidity of

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