Robyn Warner, Primary Industries Research Victoria, Australia

Transcription

1 Robyn Warner, Primary Industries Research Victoria, Australia

2 Effects of nitric oxide and oxidation in vivo and postmortem on meat tenderness R. D. Warner, F.R. Dunshea, E. N. Ponnampalam and J.J. Cottrell International Congress of Meat Science and Technology, August 7 12, 2005

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4 Questions to answer Does nitric oxide influence meat tenderness? If nitric oxide does influence tenderness, what is the mechanism? Does in vivo and post mortem oxidation of proteins influence meat tenderness?

5 Background 50% of variation in meat tenderness is unexplained Large day of slaughter and abattoir effects in any statistical analysis even in a model slaughter system From farm to slaughter, animals are being constantly challenged physiologically (and psychologically) and are struggling to maintain homeostasis Post slaughter, meat is sometimes really tough or really tender and we don t know why Industry reports that meat fails to become more tender during ageing, often in the modified atmosphere packs How does nitric oxide and oxidation in vivo and post mortem influence meat tenderness?

6 Nitric Oxide Nitric oxide (N=O) is an inorganic, gaseous free radical whose predominant functions are that of a messenger and effector molecule. Science molecule of the year in 1992 Google 2,470,000 hits Pubmed 69,418 hits and 3,000 publications per year Pearce Wright (2004), A gas controlling a seemingly limitless range of functions including the brain, lungs, liver and process of learning. It controls the action of almost every orifice from swallowing to defecation

7 Production of Nitric Oxide L Arginine NADPH, O 2, BH 4, FAD, FMN, Heme, CaM Nitric Oxide Synthase (peroxynitrite) (nitroxyl) Citrulline + NO Cofactors/coenzymes required; Nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterine (BH 4 ), flavin adenine dinucleotide (FAD), flavine mononucleotide (FMN), oxygen (O 2 ), Heme binding of calmodulin (CaM) in a Ca 2+ dependent manner. Figure 1.10: Structure and binding domains of co factors on NOS dimer (Anon, 1999)

8 Forms of Nitric Oxide Synthase In mammals, NO is synthesised by a family of enzymes referred to as the nitric oxide synthases (NOS) Neuronal, Inducible and Endothelial NOS Neuronal NOS, nnos, NOS I, nnosµ (striated muscle isoform), in vitro half life of 50 min nnosα (brain isoform), in vitro half life of 12 min constitutive membrane bound, around dystrophin and possibly cytosolic? Mitochondria? Regulation is by binding of calcium to calmodulin

9 Forms of Nitric Oxide Synthase (cont.) Inducible NOS, inos, NOS II inducible, expressed during inflammation Calcium insensitive cytosolic Endothelial NOS, enos, NOS III, constitutive Regulation is by binding of calcium to calmodulin endothelium Mitochondria in skeletal muscle?

10 Nitric Oxide NO exerts several distinct effects on skeletal muscle function; excitation contraction coupling mitochondrial energy production glucose metabolism auto regulation of blood flow.

11 Main target sites of NO in skeletal muscle (Grozdanovic, 2001) Ryanodine receptor (RyR) Sarcoplasmic reticulum Ca 2+ ATPase (SERCA) Glyceraldehyde 3 phosphate dehydrogenase Phosphofructokinase Calpain Actomyosin ATPase Mitochondrial respiratory chain complexes M, mitochondrion; OR, oxidoreductase; ROS, reactive oxygen species; XO, xanthine oxidase

12 Mechanisms of NO dependent modifications of biological molecules, metal complexes (M) and O 2 species (Patel et al, 2000)

13 NO inhibitors and donors Inhibitors all arginine analogues L NAME= N(G) nitro L arginine methyl ester, highly selective for nnos and enos, not inos Donors arginine, etc, quite difficult with variable results

14 NO donors increased tenderness and NO inhibitors reduced tenderness when injected into hot boned beef loin Cook et al. (1998)

15 NO donor and NOS inhibitor had no effect on instrumental toughness in hot boned beef loins NO donor NOS inhibitor Cottrell et al. (2002c)

16 NOS activity post mortem Post mortem NOS activity in pork, chicken and turkey (dark and light) and trout is present when enzyme is isolated and measured with ample substrate and co factors (ideal conditions) In situ, unlikely to have NOS activity for any substantial period post slaughter due to unavailability of co factors. Since NOS mediated nitric oxide in muscle is a postmortem event of very short duration, future research on strategies to control peroxynitrite induced oxidation in muscle should focus on ante mortem muscle biochemistry, handling during slaughter, and pre rigor events (Brannan and Decker, 2001)

17 NO activity in vivo and meat tenderness

18 Inhibition of nnos pre slaughter decreases WBSF tenderness of LTL, variable effect on SM in lamb * P< P<0.01 Warner Bratzler shear (kg) * * * control L NAME * + 0 LTL_e1 LTL_e2 LTL_e3 SM_e1 SM_e2 SM_e3 expt1 Cottrell et al., (2002); expt2 Ponnampalam et al. (2005); expt3 Suster et al. (2005)

19 Two hypotheses 1. NO reduces tenderness by inhibiting calpain directly 2. NO reduces tenderness by reducing Ca2+ levels which reduces activity of calpain Proteoglycans Sarcolemma Costameres (ankyrin, desmin, dystrophin, talin, vinculin) Z disk Desmin Degradation of cytoskeletal proteins Arg O 2 Citrulline Ca 2+ Calpain RYR L NAME NOs 1 NO Ca2+ in cytoplasm SERCA Ca 2+ Sarcoplasmic reticulum LEGEND: NO, nitric oxide; NOS 1, nitric oxide synthase 1 (neuronal NOS); L_NAME, N G nitro L arginine methyl ester hydrochloride, an inhibitor of NOS 1; RYR, ryanodine receptor calcium channel; SERCA, sarco/endoplasmic reticulum calcium ATPAS.E

20 Evidence for effects of NO on calpain Inhibition of NOS (with L NAME) inhibits walking speed in rats and reduces muscle mass and muscles cross sectional area (Wang et al., 2001) Inhibition of NOS (with L NAME) increases accumulation of talin and vinculin by inhibiting calpain induced proteolysis in isolated myotubes (Zhang et al., 2004) NO inhibition of m calpain mediated proteolysis of talin in muscle cells occurs via S nitrosylation of the active cysteine site in calpain (Koh and Tidball, 2000) Inactivation of µ calpain by NO in human erythrocytes only occurs when intracellular ph drops (Michetti et al., 1995) such as during exercise

21 Evidence for effects of NO on Calcium NO increases RYR activity by S nitrosylation of cysteine 3635 in each subunit and this is modulated by O 2 tension using a C3635A mutant expressed in cultured muscle cells (Sun et al., 2001) Peroxynitrite inactivates SR Ca 2+ ATPase in vesicles from rabbit skeletal muscle (Viner et al., 1996). Numerous papers.

22 Other possibilities for effects of NO on tenderness Blood pressure and blood flow changes AMP activated protein kinase (AMPK) the activation of AMPK exerts long term effects at the level of both gene expression and protein synthesis. SR bound creatine kinase is inhibited by NO (nitrosylation of sulfhydryls) which reduces Ca 2+ uptake into SR (Wolosker et al., 1996) Other proteins

23 Summary of NO effects NOS activity pre slaughter appears to have an effect on meat tenderness and the effect varies between muscles We hypothesise that the effect of NO on meat tenderness is either by inhibiting calpain directly or by reducing Ca2+ levels which reduces activity of calpain Possibility of other mechanisms but ample evidence exists for effects of NO on calpain activity and on Ca 2+ levels in the muscle cell

24 Oxidation environments post slaughter and meat quality

25 Oxidation Negative effects of post mortem oxidation on rancidity, off flavours and odours and shelf life of meat are well known. Oxidation post mortem is promoted by high temperatures, metal ions, O 2, low ph, etc. Vitamin E is a potent anti oxidant as shown in numerous studies in beef, pork, lamb and other species Oxidative stress reversibly inhibits calpain activity in situ in human neuroblastoma calls (Guttman et al., 1998)

26 Some studies showed decrease in WBSF (increase tenderness) with Vitamin E feeding Change in WBSF with tream en * * Species and treatment *7d *14d Species Units fed Reference 1 Pig 110 Waylan Beef 500 Robbins Beef 300 Arnold Beef 1000 Rowe Beef pasture Realini Beef pasture Realini 2004

27 Effect of feeding Vitamin E on consumer panel tenderness varied from positive to negative 1 Beef 300 Arnold 1992 Change in Consumer assessment wit treament * * * * 2 Beef 500 Robbins Beef 500 Robbins Pig 110 Waylan Pig 110 Waylan Pig 900 Kerth Pig 900 Kerth Rabbit 200 Dal Bosco 2004

28 Effects of MAP on meat tenderness Author Sample VP vs Effect on tenderness Fu et al., 1992 Beef loin over 4 weeks storage Schluter et al., 1994 Sørheim et al., 2004 Pork loin and boston butt over 10 days storage Beef loin over 15 days storage Griffin et al., 1982 Beef loin over 30 days storage MAP; 02/CO2 varied from10/20 to 20/40 MAP 66%O2/ 26%CO2/ 8%N2 MAP 70%O2/ 30%CO2 Medium O2 barrier Reduced sensory tenderness No difference in sensory Less ageing as measured by WBSF and sensory No difference in sensory

29 MAP inhibits tenderisation during ageing over 15 days for beef loin Sensory tenderness x a MAP VP y a y a WBSF (N/cm2) a x MAP VP a y Time post slaughter (days) Time post slaughter (days) xy, if superscripts differ, means are different for VP at P<0.05 a, no difference in means for MAP, P>0.05 (SØrheim et al., 2004)

30 Irradiation causes oxidation of proteins and increased WBSF Rowe et al. (2004)

31 Irradiation causes oxidation of proteins and increased WBSF 4 Vitamin E tended to increase WBSF in non irradiated beef at 7 days WBSF (kg) Control Vitamin E Rowe et al. (2004)

32 Effects of NO on post mortem metabolism see paper

33 Answers to questions Does nitric oxide influence meat tenderness? If nitric oxide does influence tenderness, what is the mechanism? Does in vivo and post mortem oxidation of proteins influence meat tenderness and if so what is the mechanism?

34 Conclusions and future research NOS activity pre slaughter appears to have an effect on meat tenderness and the effect varies between muscles NO effects in vivo on meat tenderness may also vary with breed, temperament, stress and exercise. Release of NO in skeletal muscle between farm and slaughter warrants investigation Postmortem oxidative conditions can influence meat tenderness

35 Thank you

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