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Effect of animal (lamb) diet and meat storage on myofibrillar protein oxidation and in vitro digestibility
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MEAT SCIENCE Meat Science 79 (2008) 777–783 www.elsevier.com/locate/meatsci
Effect of animal (lamb) diet and meat storage on myofibrillar protein oxidation and in vitro digestibility Ve´ronique Sante´-Lhoutellier, Erwan Engel, Laurent Aubry, Philippe Gatellier * INRA, UR370 QuaPA, 63122 Saint Gene`s Champanelle, France Received 4 May 2007; received in revised form 14 November 2007; accepted 17 November 2007
Abstract Effect of pasture- or concentrate-diet on myofibrillar protein oxidation and in vitro digestibility was measured in lamb meat (M. longissimus dorsi) during a refrigerated storage of 7 days under gas permeable film. Protein oxidation was measured by the carbonyl content determined chemically using 2,4-dinitrophenylhydrazine (DNPH) and specific targets of oxidation were identified by immunoblotting. Carbonyl content significantly increased during storage and diet affected protein oxidation where animals fed concentrate showed higher carbonyl group levels than animals fed pasture. To evaluate effect of diet and storage time on protein digestibility, myofibrillar proteins were exposed to proteases of the digestive tract (pepsin, and a mixture of trypsin and a-chymotrypsin) in conditions of pH and temperature which mimic digestive process. The myofibrillar protein digestibility was not influenced by the diet. Storage time had no significant effect on myofibrillar protein susceptibility to pepsin while an important increase in digestibility by trypsin and a-chymotrypsin was detected during storage. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Lamb meat; Myofibrillar proteins; Storage; Diet; Protein oxidation; Immunoblotting; Digestibility
1. Introduction In post-mortem muscle tissue, release of transition metals from carrier proteins (Kanner & Doll, 1991; Kanner, Hazan, & Doll, 1988) and decrease in antioxidant defense systems (Renerre, Dumont, & Gatellier, 1996; Renerre, Poncet, Mercier, Gatellier, & Metro, 1999) lead to formation of reactive oxygen species. These oxidative species which include hydroxyl, superoxide, peroxyl, and nitric oxide radicals can interact with lipids and proteins. Lipid oxidation in meat has been extensively described and its impact on meat quality through the formation of rancid odours and deterioration of flavour is well known (Asghar, Gray, Buckley, Pearson, & Boren, 1988). Less attention has been given to protein oxidation in meat. Nevertheless protein oxidation is responsible for many biological modifications such as pro-
*
Corresponding author. Tel.: +33 473 62 41 98; fax: +33 473 62 42 68. E-mail address: [email protected] (P. Gatellier).
0309-1740/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.11.011
tein fragmentation or aggregation, changes in hydrophobicity, and protein solubility, affecting technological properties such as gelation (Srinivasan & Xiong, 1996) and emulsification (Srinivasan & Hultin, 1997). Protein oxidation might also play a role in meat tenderness (Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004a) by controlling protease activity (Kristensen, Moller, & Andersen, 1997; Morzel, Gatellier, Sayd, Renerre, & Laville, 2006; Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004b). Meat proteins serve as an important source of energy and essential amino acids for humans. Nutritional quality of meat is largely dependent on protein digestibility because, to pass through the small intestine wall and to enter the bloodstream, proteins must be broken down into amino acids or small peptides. Little is known about the effect of oxidation on meat protein digestibility. Limited studies have been performed in model systems to link chemical oxidation of meat proteins with digestibility but results have been inconsistent and contradictory. Kamin-Belsky, Brillon, Arav, and Shaklai (1996) and Liu and Xiong
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(2000a) have demonstrated that chemical oxidation of myosin can affect its proteolytic susceptibility to enzymes of the digestive tract with increased or decreased digestibility depending of level of oxidation and presence or not of reducing agents. Moreover myosin is not representative of the whole meat proteins. The myofibrillar structure is a more complex system than myosin with many protein–protein interactions (Morzel et al., 2006) and even protein–lipid interactions (Chelh, Gatellier, & Sante´-Lhoutellier, 2007) which can affect its susceptibility to proteolysis (Morzel et al., 2006). So, in studies relating to digestibility, model systems using myofibrils are more suitable than those with purified myosin. Recently, we have demonstrated that chemical oxidation of myofibrillar proteins by hydroxyl radicals decreased their digestibility by the enzymes of the digestive tract (Sante´-Lhoutellier, Aubry, & Gatellier, 2007) and that loss of digestibility was correlated with oxidative parameters of proteins as hydrophobicity change, aggregation and carbonylation. Nevertheless, levels of oxidation induced by these chemical oxidations are considerably higher than those observed in aged meat (Martinaud et al., 1997) and so effects on protein digestibility may be overestimated. Thus, it would be of great interest to evaluate protein digestibility under lower oxidative conditions such as those generally observed during meat storage. Currently, no study has been performed about the effect of animal diet and meat ageing on myofibrillar protein digestibility. Therefore, our objective was to examine the impact of pasture- and concentrate-diet, which has been described to generate different levels of meat oxidation (Gatellier, Mercier, Juin, & Renerre, 2004; O’Sullivan et al., 2003), and a refrigerated storage of 7 days on both oxidation and in vitro digestibility of myofibrillar proteins of lamb meat. With this aim, myofibrils were exposed to proteases of the digestive tract (pepsin, and a mixture of trypsin and a-chymotrypsin) in conditions of pH and temperature which mimic gastric and intestinal fluids. 2. Materials and methods 2.1. Animals and diet We used 16 lambs (castrated males). After birth, animals were reared in a sheepfold with their dams for 51 days. After weaning, 8 animals remained in the sheepfold and were fed with high energy concentrate and 8 animals were reared on pasture. Concentrate was purchased from Guyomarc’h Nutrition Animale (France) and its composition provided by the retailer is given in Table 1.The pasture diet consisted essentially (more than 90%) of the graminae dactylis glomerata. Animals of each group were slaughtered at 220 days. Mean weights at slaughter were 28.1 kg (24.7– 32.2) for animals fed pasture and 33.6 kg (29.3–37.2) for animals fed concentrates. They were processed and eviscerated according to standard commercial procedures at the INRA experimental abattoir. Muscle longissimus dorsi of each animal was immediately taken and placed on a fibre
Table 1 Composition of concentrate used for finishing lamb Ingredients
Content
Beet pulp Barley Corn Maize Soya cattle cake Colza cattle cake Beet treacle Calcium carbonate Sodium bicarbonate Calcium phosphate Ammonium chloride Sodium chloride Magnesia Choline chloride Pam oil
Non-specified NS NS NS NS NS NS NS NS NS NS NS NS NS NS
Analytic constituents Proteins Fats Cellulose Ashes (inorganic matter)
15.00% 2.50% 9.50% 9.00%
Vitamins Vitamin A Vitamin D3 Vitamin E (alphatocopherol) Vitamin B1 (thiamin)
6.00 IU/kg 1.80 IU/kg 20 IU/kg 10 mg/kg
board tray, wrapped in air-permeable film and stored during 7 days in darkness at 4 °C to mimic commercial conditions of meat storage. 2.2. Determination of muscle antioxidant status Vitamin E content was determined according to the method of Buttriss and Diplock (1984). Activity of antioxidant enzymes was measured on a meat extract prepared on day 0 as previously described (Renerre et al., 1996). Total superoxide dismutase activity (Cu–Zn SOD and Mn SOD) was measured according to the procedures of Marklund and Marklund (1974) using inhibition of pyrogallol autoxidation. Catalase activity was measured by the rate of disappearance of H2O2 following the method of Aebi (1974). Glutathione peroxidase (GPx) activity was assayed with GSH reduction coupled to NADPH oxidation by glutathione reductase (Agergaard & Thode Jensen, 1982). 2.3. Isolation of myofibrils Myofibrils were prepared according to the method of Ouali and Talmant (1990) with some modifications as outlined by Martinaud et al. (1997). Myofibrils were prepared after 0, 2, 4 and 7 days of refrigerated storage. 2.4. Determination of carbonyl content Carbonyl groups were estimated using the method of Oliver, Alin, Moerman, Goldstein, and Stadtman (1987)
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with slight modifications (Martinaud et al., 1997). Carbonyl groups were detected by reactivity with 2,4-dinitrophenylhydrazine (DNPH) to form protein hydrazones. The results were expressed as nanomoles of DNPH fixed per milligram of protein. 2.5. SDS–PAGE Myofibrils were incubated for 5 min at 100 °C in a buffer containing 7.5% (v:v) glycerol, 1% (v/v) mercaptoethanol, 6% (w/v) SDS, 50 mM Tris–HCl (pH 6.8). SDS–PAGE was performed according to the method of Laemmli (1970) using 12.5% (6 8 cm, 0.75mm thick) polyacrylamide gels. The protein load was adjusted to 10 lg per lane. Two gels were run in parallel: one was Coomassie blue stained while the other was used for immunoblotting. Gels were scanned with a GS-800 calibrated densitometer (UMAX) controlled by a PDQuest application program and protein band intensity was evaluated with the gel scanning and measurement application: Sigma Gel software (Jandel Scientific, 1995). Proteins were identified by MALDI-TOF using a Voyager DE-Pro model of MALDI-TOF mass spectrometer (Perseptive BioSystems, Farmingham, MA, USA) and masses were assigned from NCBI database searches with the ‘‘Mascot” and ‘‘Profound” softwares according to the method of Morzel et al., (2004). 2.6. Immunoblotting Specific protein oxidation was evaluated by labeling protein carbonyls with DNPH followed by immunoblotting of proteins, separated by 12.5% SDS–PAGE, with the Oxyblot kit from Chemicon International. The kit uses a rabbit polyclonal antibody against DNPH. Gels were analyzed as described in Section 2.5. In order to standardize oxidation measurements, carbonylation levels were calculated by dividing the immunoblot band intensities by the corresponding Coomassie blue band intensities. Due to financial and time constraints, electrophoresis gels and immunoblottings were not performed on all 16 animals. Three animals of each diet group were examined, in duplicate, to test the reproducibility of the effects. For practical reasons, only one gel, corresponding to one animal representative of each group, is reproduced in this paper. 2.7. In vitro digestibility Myofibrillar protein digestibility was determined in vitro by a sequential exposure to digestive proteases. Myofibrillar proteins were washed in 33 mM glycine buffer at pH 1.8 (gastric pH) and the final concentration was adjusted at 0.8 mg/ml in the same buffer. Proteins were digested first by gastric pepsin (20 U/mg myofibrillar proteins) 1 h at 37 °C. Pepsin, from porcine gastric mucosa, was purchased from Sigma. Digestion was terminated by addition at various times (0, 10, 20, 30, 40, 60 min.) of
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15% (final concentration) trichloroacetic acid (TCA). After centrifugation for 10 min at 4000g, the content of hydrolyzed peptides in the soluble fraction was estimated by absorbance at 280 nm and the rate of proteolysis was expressed in optical density units by hour (DOD/hour). The non-soluble fractions of the 60 min pepsin hydrolyzate were then washed twice in 33 mM glycine buffer at pH 8 (duodenal pH) and final concentration was adjusted at 0.8 mg/ml in the same buffer. Proteins were then hydrolyzed 30 min at 37 °C by a mixture of trypsin and a-chymotrypsin (respectively, 6.6 U and 0.33 U/mg myofibrillar proteins). Trypsin and a-chymotrypsin from porcine pancreas were purchased from Sigma. Digestion was terminated by addition at various times (0, 5, 10, 20, 30 min.) of 15% (final concentration) trichloroacetic acid (TCA) and the rate of proteolysis was determined as previously described. 2.8. Statistical analysis All values are reported as the mean ± SEM of 8 independent determinations. Data were analysed under the SAS system. The unpaired Student t-test was used, to determine the levels of statistical significance between groups; with p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, ***. The relationships between the different parameters were assessed by calculation of Pearson correlation coefficients. To assess the effect of diet and storage time and their interactions, data were also analysed by a two-way analysis of variance (ANOVA). The mixed procedure with time repetition was used.
3. Results and discussion 3.1. Carbonyl group formation during storage In proteins, carbonyl groups are formed by oxidation of amino acids (Stadtman, 1990, 1993) and their determination is the most popular chemical test to evaluate protein oxidation. Fig. 1 shows the effect of animal feeding (concentrate/pasture) and storage time on carbonyl group content. Initial value of carbonyl content was around 2 nmoles/mg protein, a value close to those reported by Martinaud et al. (1997) on bovine myofibrils, Morzel et al. (2006) on pig myofibrils and Liu and Xiong (2000b) on chicken myosin. Carbonyl content slightly increased during the 7 days storage, by 13.0% in pasture fed group and 31.4% in concentrate fed group. At each day of measurement carbonyl levels were higher in the concentrate group and the difference was significant (p < 0.01) after 7 days of refrigerated storage. This increase in carbonyl content was similar to the 44% oxidation increase, described by Martinaud et al. (1997), in myofibrillar proteins of bovine M. longissimus lumborum stored 10 days under the same conditions. ANOVA (Table 2) reveals a significant effect of storage time and especially of diet on
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780
Carbonyls (nmol/mg protein)
4.0
concentrate
**
pasture NS
3.0
NS
NS
0
2
2.0
1.0
0.0 4
7
Days Fig. 2. Effect of animal diet (concentrate/pasture) and meat storage time on gel (12.5%) electrophoresis patterns of myofibrillar proteins (HC: heavy chain; LC: light chain).
Fig. 1. Effect of animal diet (concentrate/pasture) and meat storage time on myofibrillar protein oxidation measured by the carbonyl group content. Values are means ± SEM of 8 independent determinations and significance is noticed as: p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, *** .
3.2. Electrophoretic study of myofibrillar proteins Electrophoresis (Fig. 2) was performed in order to control the purity of the myofibril preparations and to observe modifications in protein patterns induced by animal diet and meat storage. Electrophoretic profiles show that myofibril preparations were free of sarcoplasmic protein contamination. Especially there was no trace of albumin (68 kDa), creatine kinase (42 kDa) and myoglobin (18 kDa), proteins mostly present in the sarcoplasmic fraction. Profiles were poorly affected by animal diet and storage time. No myofibrillar fragmentation, due to endogenous proteases, and no aggregation due to oxidation, was observed even after 7 days of refrigerated storage. Meat storage affected essentially the troponin T band and troponin I band which disappeared after 7 days of storage. Degradation of troponin T, during meat storage, has already been described in bovine muscle (Martinaud et al., 1997; Ouali & Talmant, 1990) with concomitant appearance of a ‘‘30 kDa component” which is used as a proteolysis index. No such component was identified in this study. We cannot reject the hypothesis that staining by Coomassie blue is not sensitive enough to detect myofibril fragments formed in very low amounts during meat storage.
carbonyl content without interaction between the two parameters. This result confirmed the protective effect of pasture diet against oxidation previously described on bovine meat by our laboratory (Gatellier et al., 2004) and others (Descalzo et al., 2000; O’Sullivan et al., 2003). In order to explain the possible protection on protein oxidation via the diet, the muscle antioxidant status was estimated. No diet effect was observed on the activity of antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) (results not shown), while an important difference was measured in vitamin E level (1.61 ± 0.76 ppm in concentrate group versus 6.42 ± 1.32 in pasture group; p < 0.001) which could, in part, account for differences in protein oxidation. Moreover a negative and significant correlation was observed between vitamin E level and carbonyl group amount after 7 days of storage (r = 0.65, p < 0.01) providing confirmation of the protective effect of vitamin E on protein oxidation. In pasture feeding, other antioxidants, which were not estimated in this study, such as vitamins from group A and C, carotenoids and flavonoids can also protect meat against oxidation (Wood & Enser, 1997).
Table 2 Effects of animal diet and storage time on carbonyl content and proteolytic activity on myofibrillar proteins Carbonyls
Diet Time Interaction
Pepsin
Trypsin + a-chymotrypsin
df
F
p
S
df
F
p
S
df
F
p
S
1 3 3
13.71 3.81 1.37
0.0024 0.0168 0.2665
**
1 3 3
1.47 0.18 0.92
0.2303 0.9074 0.4374
NS NS NS
1 3 3
1.77 37.15 3.32
0.1885 <.0001 0.0263
NS
*
NS
*** *
Values are degree of freedom (df), F value (F), and probability (p) from the two-way variance analysis. Significance (S) is noticed as: p > 0.05, NS; p < 0.05, * ; p < 0.01, **; p < 0.001, ***.
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3.4. In vitro digestibility of oxidized myofibrillar proteins Figs. 4 and 5 show the effect of animal diet and storage time on myofibrillar protein degradation by the digestive tract proteases. With pepsin, a stomach protease, myofibrillar
Pepsin digestibility (Δ OD/hour)
With chemical measurement of protein carbonyls using DNPH (Section 3.1) only global protein oxidation is determined. For this reason, immunoblot analysis was performed in parallel in order to determine specific protein target oxidation (Fig. 3). Among all myofibrillar proteins, only carbonylated myosin and actin were clearly identified. The coefficients of variation (VC = [standard deviation/ mean] 100) corresponding to protein band intensities were calculated from 3 animals. The reproducibility of the technique was evaluated by averaging out all the variation coefficients corresponding to each protein. We have observed that the reproducibility was lower for myosin (average VC = 35.4%) than for the actin band (average VC = 27.1%) probably because, as shown in Fig. 3, the myosin band intensity was less pronounced and its surface was less delimited. The low reproducibility observed turns this approach into a semi-quantitative technique. Due to the restricted number of animals tested by this technique no statistical analysis was performed and the effects of storage time and diet, measured on 3 animals, are mentioned for information only. At initial time (Day 0) the level of carbonylated actin was considerably higher than carbonylated myosin (approximately three times higher in concentrate fed group and eight times higher in pasture group). Currently we have no explanation for this difference in carbonylation between actin and myosin. We can hypothesize that structural difference (myosin is a fibrous protein while actin is globular) or difference in accessibility of oxidation sites (interaction of actin with myosin chains may mask oxidation sites of myosin) lead to different reactivity of the two proteins towards free radicals. This result was in good accordance with that of Dalle-Donne et al. (2001) who demonstrated that purified actin is particularly prone to chemical oxidation. Protein carbonylation was affected both by diet and storage time. In concentrate fed group, carbonylation of actin increased by 20% during the 7 days storage and myosin carbonylation remained stable, while in the pasture fed
group, during the same period, a 220% increase was measured in myosin carbonylation with no increase in actin carbonylation. Other myofibrillar proteins (a-actinin, troponin, tropomyosin and myosin-LC) were not observed on immunoblot. Smears appeared between myosin and actin which were not identified.
concentrate
0.25
pasture
** 0.20
NS
NS
NS
2
4
7
0.15 0.10 0.05 0.00 0
Days Fig. 4. Effect of animal diet (concentrate/pasture) and meat storage time on myofibrillar protein digestibility by pepsin. Values are means ± SEM of 8 independent determinations and significance is noticed as: p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, ***.
0.30
Trypsin + α -chymotrypsin digestibility ( Δ OD/hour)
3.3. Immunoblot analysis
781
0.25
NS
concentrate pasture
0.20
**
NS
0.15 0.10
NS
0.05 0.00 0
2
4
7
Days
Fig. 3. Effect of animal diet (concentrate/pasture) and meat storage time on immunoblotting of myofibrillar proteins.
Fig. 5. Effect of animal diet (concentrate/pasture) and meat storage time on myofibrillar protein digestibility by trypsin + a-chymotrypsin. Values are means ± SEM of 8 independent determinations and significance is noticed as: p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, ***.
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protein digestibility was minimally affected by diet and storage time. A significant effect of diet was measured only at the beginning of storage with a higher digestibility of proteins of the pasture fed group. ANOVA (Table 2) shows no significant effect of diet and storage time. With pancreatic proteases (trypsin + a-chymotrypsin) an important increase in myofibrillar protein digestibility was observed during storage in the two animal groups. A storage of 7 days increased more than four times myofibrillar protein digestibility. ANOVA (Table 2) confirms this time effect on digestibility with a significant interaction between time and diet effects. A significant increase of digestibility (p < 0.01) was already measured, by ANOVA, between day 0 and day 2. ANOVA shows no significant effect of animal diet on digestibility by pancreatic proteases. To establish links between protein carbonylation and digestibility, the relationships between carbonyl content and proteolysis rate were assessed by a correlation study. Pepsin and trypsin + a-chymotrypsin activities were not significantly correlated with carbonyl content (r = 0.232; p > 0.05 for pepsin and r = 0.167; p > 0.05 for trypsin + a-chymotrypsin). This result is quite surprising because in a preceding study, where myofibrillar proteins from pork muscle were exposed to a chemical oxidative system prior to hydrolysis by digestive proteases, negative and highly significant correlations were obtained between carbonyl level and protease activity (Sante´-Lhoutellier et al., 2007). However oxidation levels reached in this model system were considerably higher than those observed here. Moreover, it is worth stating at this point that carbonyl production is limited to only one group of amino acids and may not be representative of the whole oxidation phenomenon. Among other amino acids sensitive to oxidation, aromatic amino acids are largely present in recognition sites of proteases and evaluation of their oxidation state would be of great interest to understand the effect of oxidation on protein digestibility. The technique of front face fluorescence, which has been used with success in our laboratory to evaluate Schiff bases in non solubilized myofibrils (Chelh et al., 2007), will soon be tested in order to evaluate tyrosine and tryptophan oxidation products. Some authors have recently reported a biphasic curve when proteolysis was measured in relation to protein oxidation (Davies, 2001; Grune, Jung, Merker, & Davies, 2004). For these authors, low levels of oxidation induced subtle changes in protein structure which favoured their recognition by proteases and proteolytic susceptibility initially increased with oxidation. The formation of protein aggregates, observed at higher level of oxidation, can change both chemical and physical recognition sites and so can decrease proteolytic susceptibility. In our study, levels of oxidation reached during meat storage can also modulate protease activity. The activity of pancreatic proteases on myofibrillar proteins was probably situated in the initial increasing stage of the curve of Davis and Grune while pepsin activity had probably already reached the decreasing stage. This difference in proteolytic activity is probably
due to differences in enzymatic mechanisms and oxidative susceptibility of amino acids implicated in recognition sites of these enzymes. Trypsin and a-chymotrypsin are serine endopeptidases and their mixture can hydrolyse proteins at the carboxylic side of Tyr, Phe, Trp, Leu, Met, Ala, Asp, Glu, Arg and Lys while pepsin is an aspartic endopeptidase which cleaves preferentially at the carboxylic side of Phe, Met, Leu and Trp when they are bound to hydrophobic residues. 4. Conclusions In lamb meat, refrigerated storage significantly increases carbonyl group formation in myofibrillar proteins and is favourable to digestibility by pancreatic proteases. On the other hand diet has a significant effect on myofibrillar protein carbonylation, probably due to difference in vitamin E content of the meat, but not on digestibility. Protein carbonylation cannot explain differences in protein digestibility. The mechanisms by which oxidation modulates digestibility is complex and may implicate other groups of amino acids, especially aromatic amino acids which are particularly represented in recognition sites of proteases. Protein oxidation, during refrigerated storage of meat, is probably lower than that obtained in conditions generating high levels of free radicals such as cooking, irradiation, freezing/unfreezing cycles, and high oxygen packaging. This would be an obvious area to continue studying to get a better understanding of protein digestibility in relation to oxidative modifications in meat. Acknowledgements This study was a part of a bigger study (AUTMAT project) funded by unity Quality of Animal Products (INRA), whose objectives are focused on meat authentication with different physicochemical tools and on correlation between instrumental measurements of meat characterisation during storage. References Aebi, H. (1974). In H. U. Bergmeyer (Ed.), Methods enzymology analysis (pp. 673–682). New York: Academic Press. Agergaard, N., & Thode Jensen, P. (1982). Procedure for blood glutathione peroxidase determination in cattle and swine. Acta Veterinaria Scandinavia, 23, 515–527. Asghar, A., Gray, J. L., Buckley, D. J., Pearson, A. M., & Boren, A. M. (1988). Perspectives of warmed-over flavor. Food Technology, 42, 102–108. Buttriss, J. L., & Diplock, A. T. (1984). HPLC methods for vitamin E in tissues. In L. Parcker, A. N. Glazer (Eds.), Methods in enzymology, (Vol. 105, pp. 131–138). Chelh, I., Gatellier, P., & Sante´-Lhoutellier, V. (2007). Characterisation of fluorescent Schiff bases formed during oxidation of pig myofibrils. Meat Science, 76, 210–215. Dalle-Donne, I., Rossi, R., Giustarini, D., Gagliano, N., Lusini, L., & Milzani, A. (2001). Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment. Free Radical Biology and Medicine, 31, 1075–1083.
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Morzel, M., Chambon, C., Hamelin, M., Sante´-Lhoutellier, V., Sayd, T., & Monin, G. (2004). Proteome changes during pork meat ageing following use of two different pre-slaughter handling procedures. Meat Science, 67, 689–696. Morzel, M., Gatellier, Ph., Sayd, T., Renerre, M., & Laville, E. (2006). Chemical oxidation decreases proteolytic susceptibility of skeletal muscle myofibrillar proteins. Meat Science, 73, 536–543. O’Sullivan, A., Galvin, K., Moloney, A. P., Troy, D. J., O’Sullivan, K., & Kerry, J. P. (2003). Effect of pre-slaughter rations of forage and/or concentrates on the composition and quality of retail packaged beef. Meat Science, 63, 279–286. Oliver, C. N., Alin, B. W., Moerman, E. J., Goldstein, S., & Stadtman, E. R. (1987). Age-related changes in oxidized proteins. Journal of Biological Chemistry, 262, 5488–5491. Ouali, A., & Talmant, A. (1990). Calpain and Calpastatin distribution in bovine, porcine and ovine skeletal muscles. Meat Science, 28, 331–348. Renerre, M., Dumont, F., & Gatellier, P. (1996). Antioxidant enzyme activities in beef in relation to oxidation of lipid and myoglobin. Meat Science, 43, 111–121. Renerre, M., Poncet, K., Mercier, Y., Gatellier, P., & Metro, B. (1999). Influence of dietary fat and vitamin E on antioxidant status of muscles of turkey. Journal of Agricultural and Food Chemistry, 47, 237–244. Rowe, L. J., Maddock, K. R., Lonergan, S. M., & Huff-Lonergan, E. (2004a). Influence of early postmortem protein oxidation on beef quality. Journal of Animal Science, 82, 785–793. Rowe, L. J., Maddock, K. R., Lonergan, S. M., & Huff-Lonergan, E. (2004b). Oxidative environments decrease tenderization of beef steaks through inactivation of l-calpain. Journal of Animal Science, 82, 3254–3266. Sante´-Lhoutellier, V., Aubry, L., & Gatellier, P. (2007). Effect of oxidation on in-vitro digestibility of skeletal muscle myofibrillar proteins. Journal of Agricultural and Food Chemistry, 55, 5343–5348. Srinivasan, S., & Hultin, H. O. (1997). Chemical, physical, and functional properties of cod proteins modified by a non enzymatic free-radicalgenerating system. Journal of Agricultural and Food Chemistry, 45, 310–320. Srinivasan, S., & Xiong, Y. L. (1996). Gelation of beef heart surimi as affected by antioxidants. Journal of Food Science, 61, 707–711. Stadtman, E. R. (1990). Metal ion-catalysed oxidation of proteins: Biochemical mechanism and biological consequences. Free Radical Biology and Medicine, 8, 315–325. Stadtman, E. R. (1993). Oxidation of free amino acids and amino acids residues in proteins by radiolysis and by metal-catalyzed reactions. Annual Review of Biochemistry, 62, 797–821. Wood, J. D., & Enser, M. (1997). Factors influencing fatty acids in meat and the role of antioxidants in improving meat quality. British Journal of Nutrition, 78, S49–S60.
MEAT SCIENCE Meat Science 79 (2008) 777–783 www.elsevier.com/locate/meatsci
Effect of animal (lamb) diet and meat storage on myofibrillar protein oxidation and in vitro digestibility Ve´ronique Sante´-Lhoutellier, Erwan Engel, Laurent Aubry, Philippe Gatellier * INRA, UR370 QuaPA, 63122 Saint Gene`s Champanelle, France Received 4 May 2007; received in revised form 14 November 2007; accepted 17 November 2007
Abstract Effect of pasture- or concentrate-diet on myofibrillar protein oxidation and in vitro digestibility was measured in lamb meat (M. longissimus dorsi) during a refrigerated storage of 7 days under gas permeable film. Protein oxidation was measured by the carbonyl content determined chemically using 2,4-dinitrophenylhydrazine (DNPH) and specific targets of oxidation were identified by immunoblotting. Carbonyl content significantly increased during storage and diet affected protein oxidation where animals fed concentrate showed higher carbonyl group levels than animals fed pasture. To evaluate effect of diet and storage time on protein digestibility, myofibrillar proteins were exposed to proteases of the digestive tract (pepsin, and a mixture of trypsin and a-chymotrypsin) in conditions of pH and temperature which mimic digestive process. The myofibrillar protein digestibility was not influenced by the diet. Storage time had no significant effect on myofibrillar protein susceptibility to pepsin while an important increase in digestibility by trypsin and a-chymotrypsin was detected during storage. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Lamb meat; Myofibrillar proteins; Storage; Diet; Protein oxidation; Immunoblotting; Digestibility
1. Introduction In post-mortem muscle tissue, release of transition metals from carrier proteins (Kanner & Doll, 1991; Kanner, Hazan, & Doll, 1988) and decrease in antioxidant defense systems (Renerre, Dumont, & Gatellier, 1996; Renerre, Poncet, Mercier, Gatellier, & Metro, 1999) lead to formation of reactive oxygen species. These oxidative species which include hydroxyl, superoxide, peroxyl, and nitric oxide radicals can interact with lipids and proteins. Lipid oxidation in meat has been extensively described and its impact on meat quality through the formation of rancid odours and deterioration of flavour is well known (Asghar, Gray, Buckley, Pearson, & Boren, 1988). Less attention has been given to protein oxidation in meat. Nevertheless protein oxidation is responsible for many biological modifications such as pro-
*
Corresponding author. Tel.: +33 473 62 41 98; fax: +33 473 62 42 68. E-mail address: [email protected] (P. Gatellier).
0309-1740/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.11.011
tein fragmentation or aggregation, changes in hydrophobicity, and protein solubility, affecting technological properties such as gelation (Srinivasan & Xiong, 1996) and emulsification (Srinivasan & Hultin, 1997). Protein oxidation might also play a role in meat tenderness (Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004a) by controlling protease activity (Kristensen, Moller, & Andersen, 1997; Morzel, Gatellier, Sayd, Renerre, & Laville, 2006; Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004b). Meat proteins serve as an important source of energy and essential amino acids for humans. Nutritional quality of meat is largely dependent on protein digestibility because, to pass through the small intestine wall and to enter the bloodstream, proteins must be broken down into amino acids or small peptides. Little is known about the effect of oxidation on meat protein digestibility. Limited studies have been performed in model systems to link chemical oxidation of meat proteins with digestibility but results have been inconsistent and contradictory. Kamin-Belsky, Brillon, Arav, and Shaklai (1996) and Liu and Xiong
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(2000a) have demonstrated that chemical oxidation of myosin can affect its proteolytic susceptibility to enzymes of the digestive tract with increased or decreased digestibility depending of level of oxidation and presence or not of reducing agents. Moreover myosin is not representative of the whole meat proteins. The myofibrillar structure is a more complex system than myosin with many protein–protein interactions (Morzel et al., 2006) and even protein–lipid interactions (Chelh, Gatellier, & Sante´-Lhoutellier, 2007) which can affect its susceptibility to proteolysis (Morzel et al., 2006). So, in studies relating to digestibility, model systems using myofibrils are more suitable than those with purified myosin. Recently, we have demonstrated that chemical oxidation of myofibrillar proteins by hydroxyl radicals decreased their digestibility by the enzymes of the digestive tract (Sante´-Lhoutellier, Aubry, & Gatellier, 2007) and that loss of digestibility was correlated with oxidative parameters of proteins as hydrophobicity change, aggregation and carbonylation. Nevertheless, levels of oxidation induced by these chemical oxidations are considerably higher than those observed in aged meat (Martinaud et al., 1997) and so effects on protein digestibility may be overestimated. Thus, it would be of great interest to evaluate protein digestibility under lower oxidative conditions such as those generally observed during meat storage. Currently, no study has been performed about the effect of animal diet and meat ageing on myofibrillar protein digestibility. Therefore, our objective was to examine the impact of pasture- and concentrate-diet, which has been described to generate different levels of meat oxidation (Gatellier, Mercier, Juin, & Renerre, 2004; O’Sullivan et al., 2003), and a refrigerated storage of 7 days on both oxidation and in vitro digestibility of myofibrillar proteins of lamb meat. With this aim, myofibrils were exposed to proteases of the digestive tract (pepsin, and a mixture of trypsin and a-chymotrypsin) in conditions of pH and temperature which mimic gastric and intestinal fluids. 2. Materials and methods 2.1. Animals and diet We used 16 lambs (castrated males). After birth, animals were reared in a sheepfold with their dams for 51 days. After weaning, 8 animals remained in the sheepfold and were fed with high energy concentrate and 8 animals were reared on pasture. Concentrate was purchased from Guyomarc’h Nutrition Animale (France) and its composition provided by the retailer is given in Table 1.The pasture diet consisted essentially (more than 90%) of the graminae dactylis glomerata. Animals of each group were slaughtered at 220 days. Mean weights at slaughter were 28.1 kg (24.7– 32.2) for animals fed pasture and 33.6 kg (29.3–37.2) for animals fed concentrates. They were processed and eviscerated according to standard commercial procedures at the INRA experimental abattoir. Muscle longissimus dorsi of each animal was immediately taken and placed on a fibre
Table 1 Composition of concentrate used for finishing lamb Ingredients
Content
Beet pulp Barley Corn Maize Soya cattle cake Colza cattle cake Beet treacle Calcium carbonate Sodium bicarbonate Calcium phosphate Ammonium chloride Sodium chloride Magnesia Choline chloride Pam oil
Non-specified NS NS NS NS NS NS NS NS NS NS NS NS NS NS
Analytic constituents Proteins Fats Cellulose Ashes (inorganic matter)
15.00% 2.50% 9.50% 9.00%
Vitamins Vitamin A Vitamin D3 Vitamin E (alphatocopherol) Vitamin B1 (thiamin)
6.00 IU/kg 1.80 IU/kg 20 IU/kg 10 mg/kg
board tray, wrapped in air-permeable film and stored during 7 days in darkness at 4 °C to mimic commercial conditions of meat storage. 2.2. Determination of muscle antioxidant status Vitamin E content was determined according to the method of Buttriss and Diplock (1984). Activity of antioxidant enzymes was measured on a meat extract prepared on day 0 as previously described (Renerre et al., 1996). Total superoxide dismutase activity (Cu–Zn SOD and Mn SOD) was measured according to the procedures of Marklund and Marklund (1974) using inhibition of pyrogallol autoxidation. Catalase activity was measured by the rate of disappearance of H2O2 following the method of Aebi (1974). Glutathione peroxidase (GPx) activity was assayed with GSH reduction coupled to NADPH oxidation by glutathione reductase (Agergaard & Thode Jensen, 1982). 2.3. Isolation of myofibrils Myofibrils were prepared according to the method of Ouali and Talmant (1990) with some modifications as outlined by Martinaud et al. (1997). Myofibrils were prepared after 0, 2, 4 and 7 days of refrigerated storage. 2.4. Determination of carbonyl content Carbonyl groups were estimated using the method of Oliver, Alin, Moerman, Goldstein, and Stadtman (1987)
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with slight modifications (Martinaud et al., 1997). Carbonyl groups were detected by reactivity with 2,4-dinitrophenylhydrazine (DNPH) to form protein hydrazones. The results were expressed as nanomoles of DNPH fixed per milligram of protein. 2.5. SDS–PAGE Myofibrils were incubated for 5 min at 100 °C in a buffer containing 7.5% (v:v) glycerol, 1% (v/v) mercaptoethanol, 6% (w/v) SDS, 50 mM Tris–HCl (pH 6.8). SDS–PAGE was performed according to the method of Laemmli (1970) using 12.5% (6 8 cm, 0.75mm thick) polyacrylamide gels. The protein load was adjusted to 10 lg per lane. Two gels were run in parallel: one was Coomassie blue stained while the other was used for immunoblotting. Gels were scanned with a GS-800 calibrated densitometer (UMAX) controlled by a PDQuest application program and protein band intensity was evaluated with the gel scanning and measurement application: Sigma Gel software (Jandel Scientific, 1995). Proteins were identified by MALDI-TOF using a Voyager DE-Pro model of MALDI-TOF mass spectrometer (Perseptive BioSystems, Farmingham, MA, USA) and masses were assigned from NCBI database searches with the ‘‘Mascot” and ‘‘Profound” softwares according to the method of Morzel et al., (2004). 2.6. Immunoblotting Specific protein oxidation was evaluated by labeling protein carbonyls with DNPH followed by immunoblotting of proteins, separated by 12.5% SDS–PAGE, with the Oxyblot kit from Chemicon International. The kit uses a rabbit polyclonal antibody against DNPH. Gels were analyzed as described in Section 2.5. In order to standardize oxidation measurements, carbonylation levels were calculated by dividing the immunoblot band intensities by the corresponding Coomassie blue band intensities. Due to financial and time constraints, electrophoresis gels and immunoblottings were not performed on all 16 animals. Three animals of each diet group were examined, in duplicate, to test the reproducibility of the effects. For practical reasons, only one gel, corresponding to one animal representative of each group, is reproduced in this paper. 2.7. In vitro digestibility Myofibrillar protein digestibility was determined in vitro by a sequential exposure to digestive proteases. Myofibrillar proteins were washed in 33 mM glycine buffer at pH 1.8 (gastric pH) and the final concentration was adjusted at 0.8 mg/ml in the same buffer. Proteins were digested first by gastric pepsin (20 U/mg myofibrillar proteins) 1 h at 37 °C. Pepsin, from porcine gastric mucosa, was purchased from Sigma. Digestion was terminated by addition at various times (0, 10, 20, 30, 40, 60 min.) of
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15% (final concentration) trichloroacetic acid (TCA). After centrifugation for 10 min at 4000g, the content of hydrolyzed peptides in the soluble fraction was estimated by absorbance at 280 nm and the rate of proteolysis was expressed in optical density units by hour (DOD/hour). The non-soluble fractions of the 60 min pepsin hydrolyzate were then washed twice in 33 mM glycine buffer at pH 8 (duodenal pH) and final concentration was adjusted at 0.8 mg/ml in the same buffer. Proteins were then hydrolyzed 30 min at 37 °C by a mixture of trypsin and a-chymotrypsin (respectively, 6.6 U and 0.33 U/mg myofibrillar proteins). Trypsin and a-chymotrypsin from porcine pancreas were purchased from Sigma. Digestion was terminated by addition at various times (0, 5, 10, 20, 30 min.) of 15% (final concentration) trichloroacetic acid (TCA) and the rate of proteolysis was determined as previously described. 2.8. Statistical analysis All values are reported as the mean ± SEM of 8 independent determinations. Data were analysed under the SAS system. The unpaired Student t-test was used, to determine the levels of statistical significance between groups; with p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, ***. The relationships between the different parameters were assessed by calculation of Pearson correlation coefficients. To assess the effect of diet and storage time and their interactions, data were also analysed by a two-way analysis of variance (ANOVA). The mixed procedure with time repetition was used.
3. Results and discussion 3.1. Carbonyl group formation during storage In proteins, carbonyl groups are formed by oxidation of amino acids (Stadtman, 1990, 1993) and their determination is the most popular chemical test to evaluate protein oxidation. Fig. 1 shows the effect of animal feeding (concentrate/pasture) and storage time on carbonyl group content. Initial value of carbonyl content was around 2 nmoles/mg protein, a value close to those reported by Martinaud et al. (1997) on bovine myofibrils, Morzel et al. (2006) on pig myofibrils and Liu and Xiong (2000b) on chicken myosin. Carbonyl content slightly increased during the 7 days storage, by 13.0% in pasture fed group and 31.4% in concentrate fed group. At each day of measurement carbonyl levels were higher in the concentrate group and the difference was significant (p < 0.01) after 7 days of refrigerated storage. This increase in carbonyl content was similar to the 44% oxidation increase, described by Martinaud et al. (1997), in myofibrillar proteins of bovine M. longissimus lumborum stored 10 days under the same conditions. ANOVA (Table 2) reveals a significant effect of storage time and especially of diet on
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780
Carbonyls (nmol/mg protein)
4.0
concentrate
**
pasture NS
3.0
NS
NS
0
2
2.0
1.0
0.0 4
7
Days Fig. 2. Effect of animal diet (concentrate/pasture) and meat storage time on gel (12.5%) electrophoresis patterns of myofibrillar proteins (HC: heavy chain; LC: light chain).
Fig. 1. Effect of animal diet (concentrate/pasture) and meat storage time on myofibrillar protein oxidation measured by the carbonyl group content. Values are means ± SEM of 8 independent determinations and significance is noticed as: p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, *** .
3.2. Electrophoretic study of myofibrillar proteins Electrophoresis (Fig. 2) was performed in order to control the purity of the myofibril preparations and to observe modifications in protein patterns induced by animal diet and meat storage. Electrophoretic profiles show that myofibril preparations were free of sarcoplasmic protein contamination. Especially there was no trace of albumin (68 kDa), creatine kinase (42 kDa) and myoglobin (18 kDa), proteins mostly present in the sarcoplasmic fraction. Profiles were poorly affected by animal diet and storage time. No myofibrillar fragmentation, due to endogenous proteases, and no aggregation due to oxidation, was observed even after 7 days of refrigerated storage. Meat storage affected essentially the troponin T band and troponin I band which disappeared after 7 days of storage. Degradation of troponin T, during meat storage, has already been described in bovine muscle (Martinaud et al., 1997; Ouali & Talmant, 1990) with concomitant appearance of a ‘‘30 kDa component” which is used as a proteolysis index. No such component was identified in this study. We cannot reject the hypothesis that staining by Coomassie blue is not sensitive enough to detect myofibril fragments formed in very low amounts during meat storage.
carbonyl content without interaction between the two parameters. This result confirmed the protective effect of pasture diet against oxidation previously described on bovine meat by our laboratory (Gatellier et al., 2004) and others (Descalzo et al., 2000; O’Sullivan et al., 2003). In order to explain the possible protection on protein oxidation via the diet, the muscle antioxidant status was estimated. No diet effect was observed on the activity of antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) (results not shown), while an important difference was measured in vitamin E level (1.61 ± 0.76 ppm in concentrate group versus 6.42 ± 1.32 in pasture group; p < 0.001) which could, in part, account for differences in protein oxidation. Moreover a negative and significant correlation was observed between vitamin E level and carbonyl group amount after 7 days of storage (r = 0.65, p < 0.01) providing confirmation of the protective effect of vitamin E on protein oxidation. In pasture feeding, other antioxidants, which were not estimated in this study, such as vitamins from group A and C, carotenoids and flavonoids can also protect meat against oxidation (Wood & Enser, 1997).
Table 2 Effects of animal diet and storage time on carbonyl content and proteolytic activity on myofibrillar proteins Carbonyls
Diet Time Interaction
Pepsin
Trypsin + a-chymotrypsin
df
F
p
S
df
F
p
S
df
F
p
S
1 3 3
13.71 3.81 1.37
0.0024 0.0168 0.2665
**
1 3 3
1.47 0.18 0.92
0.2303 0.9074 0.4374
NS NS NS
1 3 3
1.77 37.15 3.32
0.1885 <.0001 0.0263
NS
*
NS
*** *
Values are degree of freedom (df), F value (F), and probability (p) from the two-way variance analysis. Significance (S) is noticed as: p > 0.05, NS; p < 0.05, * ; p < 0.01, **; p < 0.001, ***.
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3.4. In vitro digestibility of oxidized myofibrillar proteins Figs. 4 and 5 show the effect of animal diet and storage time on myofibrillar protein degradation by the digestive tract proteases. With pepsin, a stomach protease, myofibrillar
Pepsin digestibility (Δ OD/hour)
With chemical measurement of protein carbonyls using DNPH (Section 3.1) only global protein oxidation is determined. For this reason, immunoblot analysis was performed in parallel in order to determine specific protein target oxidation (Fig. 3). Among all myofibrillar proteins, only carbonylated myosin and actin were clearly identified. The coefficients of variation (VC = [standard deviation/ mean] 100) corresponding to protein band intensities were calculated from 3 animals. The reproducibility of the technique was evaluated by averaging out all the variation coefficients corresponding to each protein. We have observed that the reproducibility was lower for myosin (average VC = 35.4%) than for the actin band (average VC = 27.1%) probably because, as shown in Fig. 3, the myosin band intensity was less pronounced and its surface was less delimited. The low reproducibility observed turns this approach into a semi-quantitative technique. Due to the restricted number of animals tested by this technique no statistical analysis was performed and the effects of storage time and diet, measured on 3 animals, are mentioned for information only. At initial time (Day 0) the level of carbonylated actin was considerably higher than carbonylated myosin (approximately three times higher in concentrate fed group and eight times higher in pasture group). Currently we have no explanation for this difference in carbonylation between actin and myosin. We can hypothesize that structural difference (myosin is a fibrous protein while actin is globular) or difference in accessibility of oxidation sites (interaction of actin with myosin chains may mask oxidation sites of myosin) lead to different reactivity of the two proteins towards free radicals. This result was in good accordance with that of Dalle-Donne et al. (2001) who demonstrated that purified actin is particularly prone to chemical oxidation. Protein carbonylation was affected both by diet and storage time. In concentrate fed group, carbonylation of actin increased by 20% during the 7 days storage and myosin carbonylation remained stable, while in the pasture fed
group, during the same period, a 220% increase was measured in myosin carbonylation with no increase in actin carbonylation. Other myofibrillar proteins (a-actinin, troponin, tropomyosin and myosin-LC) were not observed on immunoblot. Smears appeared between myosin and actin which were not identified.
concentrate
0.25
pasture
** 0.20
NS
NS
NS
2
4
7
0.15 0.10 0.05 0.00 0
Days Fig. 4. Effect of animal diet (concentrate/pasture) and meat storage time on myofibrillar protein digestibility by pepsin. Values are means ± SEM of 8 independent determinations and significance is noticed as: p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, ***.
0.30
Trypsin + α -chymotrypsin digestibility ( Δ OD/hour)
3.3. Immunoblot analysis
781
0.25
NS
concentrate pasture
0.20
**
NS
0.15 0.10
NS
0.05 0.00 0
2
4
7
Days
Fig. 3. Effect of animal diet (concentrate/pasture) and meat storage time on immunoblotting of myofibrillar proteins.
Fig. 5. Effect of animal diet (concentrate/pasture) and meat storage time on myofibrillar protein digestibility by trypsin + a-chymotrypsin. Values are means ± SEM of 8 independent determinations and significance is noticed as: p > 0.05, NS; p < 0.05, *; p < 0.01, **; p < 0.001, ***.
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protein digestibility was minimally affected by diet and storage time. A significant effect of diet was measured only at the beginning of storage with a higher digestibility of proteins of the pasture fed group. ANOVA (Table 2) shows no significant effect of diet and storage time. With pancreatic proteases (trypsin + a-chymotrypsin) an important increase in myofibrillar protein digestibility was observed during storage in the two animal groups. A storage of 7 days increased more than four times myofibrillar protein digestibility. ANOVA (Table 2) confirms this time effect on digestibility with a significant interaction between time and diet effects. A significant increase of digestibility (p < 0.01) was already measured, by ANOVA, between day 0 and day 2. ANOVA shows no significant effect of animal diet on digestibility by pancreatic proteases. To establish links between protein carbonylation and digestibility, the relationships between carbonyl content and proteolysis rate were assessed by a correlation study. Pepsin and trypsin + a-chymotrypsin activities were not significantly correlated with carbonyl content (r = 0.232; p > 0.05 for pepsin and r = 0.167; p > 0.05 for trypsin + a-chymotrypsin). This result is quite surprising because in a preceding study, where myofibrillar proteins from pork muscle were exposed to a chemical oxidative system prior to hydrolysis by digestive proteases, negative and highly significant correlations were obtained between carbonyl level and protease activity (Sante´-Lhoutellier et al., 2007). However oxidation levels reached in this model system were considerably higher than those observed here. Moreover, it is worth stating at this point that carbonyl production is limited to only one group of amino acids and may not be representative of the whole oxidation phenomenon. Among other amino acids sensitive to oxidation, aromatic amino acids are largely present in recognition sites of proteases and evaluation of their oxidation state would be of great interest to understand the effect of oxidation on protein digestibility. The technique of front face fluorescence, which has been used with success in our laboratory to evaluate Schiff bases in non solubilized myofibrils (Chelh et al., 2007), will soon be tested in order to evaluate tyrosine and tryptophan oxidation products. Some authors have recently reported a biphasic curve when proteolysis was measured in relation to protein oxidation (Davies, 2001; Grune, Jung, Merker, & Davies, 2004). For these authors, low levels of oxidation induced subtle changes in protein structure which favoured their recognition by proteases and proteolytic susceptibility initially increased with oxidation. The formation of protein aggregates, observed at higher level of oxidation, can change both chemical and physical recognition sites and so can decrease proteolytic susceptibility. In our study, levels of oxidation reached during meat storage can also modulate protease activity. The activity of pancreatic proteases on myofibrillar proteins was probably situated in the initial increasing stage of the curve of Davis and Grune while pepsin activity had probably already reached the decreasing stage. This difference in proteolytic activity is probably
due to differences in enzymatic mechanisms and oxidative susceptibility of amino acids implicated in recognition sites of these enzymes. Trypsin and a-chymotrypsin are serine endopeptidases and their mixture can hydrolyse proteins at the carboxylic side of Tyr, Phe, Trp, Leu, Met, Ala, Asp, Glu, Arg and Lys while pepsin is an aspartic endopeptidase which cleaves preferentially at the carboxylic side of Phe, Met, Leu and Trp when they are bound to hydrophobic residues. 4. Conclusions In lamb meat, refrigerated storage significantly increases carbonyl group formation in myofibrillar proteins and is favourable to digestibility by pancreatic proteases. On the other hand diet has a significant effect on myofibrillar protein carbonylation, probably due to difference in vitamin E content of the meat, but not on digestibility. Protein carbonylation cannot explain differences in protein digestibility. The mechanisms by which oxidation modulates digestibility is complex and may implicate other groups of amino acids, especially aromatic amino acids which are particularly represented in recognition sites of proteases. Protein oxidation, during refrigerated storage of meat, is probably lower than that obtained in conditions generating high levels of free radicals such as cooking, irradiation, freezing/unfreezing cycles, and high oxygen packaging. This would be an obvious area to continue studying to get a better understanding of protein digestibility in relation to oxidative modifications in meat. Acknowledgements This study was a part of a bigger study (AUTMAT project) funded by unity Quality of Animal Products (INRA), whose objectives are focused on meat authentication with different physicochemical tools and on correlation between instrumental measurements of meat characterisation during storage. References Aebi, H. (1974). In H. U. Bergmeyer (Ed.), Methods enzymology analysis (pp. 673–682). New York: Academic Press. Agergaard, N., & Thode Jensen, P. (1982). Procedure for blood glutathione peroxidase determination in cattle and swine. Acta Veterinaria Scandinavia, 23, 515–527. Asghar, A., Gray, J. L., Buckley, D. J., Pearson, A. M., & Boren, A. M. (1988). Perspectives of warmed-over flavor. Food Technology, 42, 102–108. Buttriss, J. L., & Diplock, A. T. (1984). HPLC methods for vitamin E in tissues. In L. Parcker, A. N. Glazer (Eds.), Methods in enzymology, (Vol. 105, pp. 131–138). Chelh, I., Gatellier, P., & Sante´-Lhoutellier, V. (2007). Characterisation of fluorescent Schiff bases formed during oxidation of pig myofibrils. Meat Science, 76, 210–215. Dalle-Donne, I., Rossi, R., Giustarini, D., Gagliano, N., Lusini, L., & Milzani, A. (2001). Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment. Free Radical Biology and Medicine, 31, 1075–1083.
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