Acta agriculturae Slovenica, 90(november 2007)1, 5–16. Agris category codes: Q04 COBISS Code 1.01 RELATION OF MYOFIBRIL FRAGMENTATION TO TEXTURAL AND CHEMICAL PARAMETERS OF AGED PORK Longissimus dorsi * Dejan DOŠLER a) , Tomaž POLAK b) , Božidar ŽLENDER c) and Lea GAŠPERLIN d) a) Univ. of Ljubljana, Biotechnical Fac., Dept. of Food Science and Technology, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia, e-mail: dejan.dosler@bf.uni-lj.si. b) Same address as a) , Ph.D., e-mail: tomaz.polak@bf.uni-lj.si. c) Same address as a) , Prof., Ph.D., e-mail: bozidar.zlender@bf.uni-lj.si. d) Same address as a) , Assoc.Prof., Ph.D., e-mail: lea.gasperlin@bf.uni-lj.si. Received February 16, 2006, accepted March 30, 2007. Delo je prispelo 16. februarja 2007, sprejeto 30. marca 2007. ABSTRACT The relation of myofibrilar fragmentation (length of myofibrilar fragments, myofibrilar fragmentation index) to textural (Warner-Bratzler share force) and chemical parameters (non- protein nitrogen changes) of pork Longisimus dorsi muscles (acquired 24 h post mortem, normal meat quality) were investigated over a 16-day ageing period at 2 °C (± 1 °C). Ageing time significantly affected all parameters at the 1% level or less. The pH value for 16-day aged samples was slightly higher; the average values being 5.61 for nonaged and 5.67 for aged samples. Length of myofibrilar fragments being in average the highest (73 µm) for nonaged samples, and the lowest (15.7 µm and 12.4 µm) for 11 and 16 days aged ones. Myofibrilar fragmentation index increases significantly with storage: for nonaged samples below 30, after a 2-day ageing about 50, and after 5-day ageing 63.7 (determined as described by Olson et al., 1976), or after 11-day ageing 56.9 (determined as described by Hopkins et al., 2004) Indexes for Hopkins procedure were about 7% lower (P ≤ 0.001) compared to those for Olson procedure. Non-protein nitrogen after 11 and 16 days of storage was higher (10.78% and 10.93% of total nitrogen) compared to the nonaged pork (9.39% of total nitrogen). Warner-Bratzler share force was markedly affected by 16-day ageing (nonaged 51.3 N, 16 days 29.2 N). On the basis of instrumentally measured texture differences in thermally treated aged pork we concluded that myofibrilar fragmentation index was a suitable proteolysis rate pointer already from the second day on. The increase in non-protein nitrogen content indicates a release of free amino acids; so, it is a suitable measure of proteolysis after 5 days of ageing. Key words: pigs / meat / ageing / myofibrilar fragments / length / myofibrilar fragmentation index / non-protein nitrogen / Warner-Bratzler shear force POVEZAVA MED MIFIBRILARNO FRAGMENTACIJO, TEKSTRURNIMI IN KEMIJSKIMI PARAMETRI ZORENE PRAŠI ČJE MIŠICE Longissimus dorsi † IZVLE ČEK Namen raziskave je bil ugotoviti vpliv miofibrilarne fragmentacije (dolžina miofibrilarnih fragmentov, indeks miofibrilarne fragmentacije) na teksturne (Warner-Bratzler strižna trdnost) in * This article is part of a dissertation thesis 'Effects of quality, proteolysis and degree of doneness on heterocyclic aromatic amines formation in thermal aged pork Longissimus dorsi', issued by Dejan Došler, supervisor Assoc. Prof. Lea Gašperlin, Ph.D. † Prispevek je del doktorske disertacije Dejana Došlerja z naslovom ‘Vpliv kakovosti, proteolize in stopnje pe čenosti na nastanek heterocikli čnih aromatskih aminov v dolgi hrbtni mišici praši ča’, mentor doc. dr. Lea Gašperlin. http://aas.bf.uni-lj.si Acta agriculturae Slovenica, 90(november 2007)1. 6 kemijske parametre (neproteinski dušik) mišice Longissimus dorsi (LD) praši ča. V poskus so bile 24 ur post mortem vklju čene leve in desne LD normalne kakovosti šestih praši čev. Mišice smo razdelili na 3 dele, jih vakuumsko embalirali in zoreli 1-, 2-, 3-, 5-, 11- in 16 dni pri temperaturi 2 °C (± 1 °C). Čas zorenja je zna čilno (P ≤ 0,001) vplival na vse parametre. Vrednost pH se je v 16-ih dneh nekoliko pove čala, in sicer je bila povpre čno: pri nezorenih mišicah 5,61 in pri zorenih 5,67. Dolžina miofibrilarnih fragmentov je bila v povpre čju najve čja (73 µm) pri nezorenih vzorcih, medtem ko je bila pri 11- in 16 dni zorenih vzorcih zna čilno manjša (15,7 µm in 12,4 µm). Indeks miofibrilarne fragmentacije se z zorenjem zna čilno pove ča, in sicer je v povpre čju: pri nezorenih vzorcih pod 30, pri 2 dni zorenih pod 50 in pri 5 dni zorenih 63,7 (dolo čen po Olsonu in sod., 1976) oziroma pri 11 dni zorenih 56,9 (dolo čen po Hopkinsu s sod.). Indeks dolo čen po Hopkinsu (2004) je okrog 7 % (P ≤ 0,001) nižji glede na indeks dolo čen po Olsonu. Neproteinski dušik je pri 11- in 16 dni zorenih vzorcih ve čji (10,78 % in 10,93 %) od nezorenih vzorcev (9,39 % od celokupnega dušika). Warner-Bratzler strižna trdnost se v 16-ih dnevih zorenja zna čilno (P ≤ 0,001) spremeni. Povpre čna vrednost nezorenih vzorcev je 51,3 N in pri 16 dni zorenih vzorcih 29,2 N. Na osnovi instrumentalno izmerjene teksture termi čno obdelanih vzorcev lahko zaklju čimo, da je indeks miofibrilarne fragmentacije že po drugem dnevu zorenja ustrezni pokazatelj mikrostrukturnih proteoliti čnih sprememb, in da je vsebnost neproteinskega dušika ustrezni pokazatelj biokemi čnih proteoliti čnih sprememb šele po petem dnevu zorenja, ker so pove čane vrednosti neproteinskega dušika posledica sproš čanja prostih aminokislin. Klju čne besede: praši či / meso / zorenje / miofibrilarni filamenti / dolžina / miofibrilarna fragmentacija / indeks / neproteinski dušik / Warner-Bratzler strižna trdnost INTRODUCTION Important changes in chemical composition and structure of muscle tissues take place during ageing. It is known that the process of meat ageing differs for different muscles of the same animal, for different animal species and even for various meat qualities (Devine, 2004; Čandek- Potokar et al., 1999). The influence of protein proteolysis stages during ageing of beef meat on the increase in tenderness (Davey and Gilbert, 1969; Dransfield, 1994; Olson and Parrish, 1977; Olson et al., 1976), and the improvement of its taste and aroma (Nishimura et al., 1988; Smith et al., 1978) have been extensively studied, and recently, interest in different animal species such as pork, is increasing (Okumura et al., 2003). The ageing (conditioning) indicators for tenderness evaluation of aged beef have been reported. The sarcomere length in miofibrils (Strydom et al., 2005), the shear force value, the myofibrillar fragmentation index (MFI) (Olson et al., 1976), the 30 kDa component (Koohmaraie, 1994), phosphorylase b, creatine kinase and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (Okumura et al., 2003) were correlated with the tenderness of bovine muscles. In the case of pork, stored without as well as with vacuum packaging at low temperature, Okumura et al. (2003) have found some useful ageing indicators, such as MFI, the 32-kDa components, peptides P1 and P2 as well as GAPDH. In the present work, the myofibril fragmentation was examined in the pork Longisimus dorsi muscle (LD) stored under vacuum packaging at 2 °C for 16 days post mortem, and was compared with the results of texture evaluation. However, we anticipated that the myofibrilar fragment length (MFL) and Warner-Bratzler share force (WBSF) would decrease rapidly, while the MFI, the content of non-protein nitrogen fraction (NPN) would increase with ageing. On the other hand, as a contribution to the methodology of proteolysis evaluation we also wanted to compare two methods for MFI determination. Došler, D. et al. Relation of myofibril fragmentation to textural and chemical parameters of aged pork longissimus dorsi. 7 MATERIAL AND METHODS Animals and tissue sampling A total of six crossbred (Swedish Landrace × Large White × Duroc × Hampshire) barrows were included in the study. They were commercially slaughtered; their weight being between 80 and 85 kg (warm carcass weight), they contained 55–60% of lean meat, and had an ultimate pH (24 h post mortem) between 5.4 and 5.8. The carcasses were stored for 24 h at 4 °C (± 1 °C). Left and right LD muscles between 4 th thoracic and the last lumbar vertebrae were removed from carcasses and used in further study. Left and right LD muscles were cut into six samples and than vacuum packaged in polyethylene bags. Random sampling provided the part of muscle for a defined time of ageing; thus, the effect of sample location in the muscle was eliminated. After 1, 2, 3, 5, 11 and 16 days of ageing in the refrigerator at 2 °C (± 1 °C) each of 36 samples was divided into three sub- samples. On the first sub-sample (25 mm thick) thermal treatment was performed (grilling at 165 °C to the internal temperature of 70 °C (± 1 °C)). Grilled steaks were prepared for instrumental measuring of the texture (Warner-Bratzler Shear Force − WBSF) after 24 hours of cooling at 4 °C (± 1 °C). The second sub-sample was cut and immediately used for MFI determination. The third sub-sample was homogenized in a blender, repacked into polyethylene bags, frozen at −21 °C (± 1 °C), and was used for myofibrilar length (MFL) and non-protein nitrogen measurements, as well as for determination of water, protein, fat and ash content (carried out as well on nonaged samples − 24 h post mortem). Most of analyses were carried out in duplicate; WBSF was measured seven times and MFL 100 times on each sample. Determination of water, total protein, intramuscular fat and ash content The water content was determined on samples of 5 g of minced meat. Samples were dried in the oven at 105 °C according to AOAC 950.46 (Official Methods of Analysis, 1997). Total protein content (crude protein, N × 6.25) was assessed by the Kjeldahl method according to AOAC 928.08 (Official Methods of Analysis, 1997). The ash content was determined by mineralization of samples at 550 °C according to AOAC 920.153 (Official Methods of Analysis, 1997). Intramuscular fat content was determined by the method described in AOAC Official Method 991.36. Fat (Crude) in Meat and Meat Products (Official Methods of Analysis, 1997). The total lipids were extracted by hot treatment with petroleum ether as solvent. Determination of pH value pH value was measured directly using a spear combined glass-gel electrode type 03 (Testo pH electrode) with thermometer (type T, Testo penetration temperature probe) connected to pH meter (Testo 230, Testo). The pH meter was calibrated using pH = 4 and pH = 7 buffers and recalibrated after every 20 readings. Accuracy of reading was ± 0.01 pH unit. pH was measured six times per muscle; at day 1 (nonaged sample) and at 2, 3, 5, 11 and 16 days post mortem (aged samples). Determination of MFL Myofibrilar fragment suspension was prepared as outlined by Hopkins et al. (2000). A minced 0.5 g sample was placed in ice-cold vessels for homogenisation in 30 mL of ice-cold buffer. Homogenisation (Ultra-turrax T 25 with dispersing element S 25 N – 18 G at 15,000 rpm) was performed by two bursts of 30 s with a 30-s break on ice between them. The buffer Acta agriculturae Slovenica, 90(november 2007)1. 8 was 0.1 M KCl (Kemika, 112097), 1 mM EDTA (Merck, 1.08418), 1 mM NaN 3 (Merck, 1.06688), 7 mM KH 2 PO 4 (Merck, 1.05108) and 18 mM K 2 HPO 4 (Kemika, 1116108). Myofibril suspensions were filtered (1.0 mm mesh strainers) to remove connective tissue. An aliquot (a drop) of the myofibril suspension was placed on a microscopic slide and was examined microscopically (Nikon Microphot-FXA, 20×10 magnify) using a camera (Sony DXC- 930P) and a picture analysing programme (LUCIA_MTM). For each sample 6 different areas were chosen and 100 fragments were measured. MFL was expressed in µm. Determination of MFI MFI was determined according to the methods described by Olson et al. (MFI-Olson) (Olson et al., 1976) and Hopkins et al. (MFI-Hopkins) (2000) using a UV/VIS spectrophotometer (Ultrospec 2000, Pharmacia Biotech). It was expressed as absorbance of a myofibril protein solution (concentration 0.5 mg mL –1 ) at 540 nm multiplied by 100. Determination of total nitrogen and non-protein nitrogen All nitrogen contents were measured using Kjeldahl's method according to AOAC 928.08 (Official Methods of Analysis, 1997). Total nitrogen (TN) was measured on minced 1 g samples. Method for determination the non-protein nitrogen (NPN) was as fallows: a minced 5 g sample was homogenized with 40 ml of 3% (w/w) trichloroacetic acid (Ultra-turax T 25 with dispersing element S 25 N – 18 G, 120 s at 20,000 rpm). Then, the mixture was passed through a Sartorius no. 388 filter paper. Filtrate was analysed by BÜCHI Kjeldahl Line acording to AOAC 928.08 Kjeldahl method (Official Methods of Analysis, 1997). NPN was expressed as percent of TN. Determination of WBSF Steak (25 mm thick) was grilled at 165 ºC to 70 °C (± 1 °C) internal temperature and cooled for 24 h at 4 °C (± 1 °C). Seven cylinders (diameter 12.7 mm) were removed parallel to the longitudinal orientation of the muscle axis. Each cylinder was shared at the centre with a Warner-Bratzler shear ‘V’ slot blade (thickness of 3.0 mm and a triangular aperture of 60°) using a TA.XT plus texture analyser (Stable Micro Systems). The crosshead speed was 3.3 10 –3 m s –1 . Newtons (N) being the units of the measurement. Data analysis For statistical evaluation of experimental data, the computer program SAS/STAT (SAS Software, 1999) was used. Basic statistical parameters were calculated by the MEANS procedure. Data were tested for normal distribution and analysed by the GLM (General Linear Model) and TTEST paired procedures. For data analyses two statistical models were used. For analysing the data for pH value, WBSF, MFL, MFI and NPN the statistical model [1] was used. The statistical model [2] for MFI included the effect of analytical method. The models were described by the following equations: y ijk = µ + AT i + A j + e ijk [1] where y = the observation parameter, µ = general mean, AT i = effect of i th ageing time (i = 1, 2, 3, 5, 11 and 16 days post mortem), A j = effect of j th animal (j = l to 6), and e = residual random term with variance σ 2 e . y ijk = µ + AT i + M j + e ijk [2] Došler, D. et al. Relation of myofibril fragmentation to textural and chemical parameters of aged pork longissimus dorsi. 9 where y = the observation parameter, µ = general mean, AT i = effect of i th ageing time (i = 1, 2, 3, 5, 11 and 16 days post mortem), M j = effect of j th analytical method (j = Olson et al. (1976), Hopkins et al. (2000)) and e = residual random term with variance σ 2 e . Least square means for experimental groups were obtained using the LSM procedure and were compared at the 5% probability level. Relations between instrumental and chemical parameters were assessed by Pearson correlation coefficients using the CORR procedure. RESULTS AND DISCUSSION Proving the homogeneity of the samples Basic statistical parameters for chemical composition of raw pork LD muscle are shown in Table 1, the data clearly show the homogeneity of the samples. Table 1. Basic statistical parameters for chemical composition of pork LD day 1 post mortem (N = 6) Preglednica 1. Osnovni statisti čni parametri za kemijsko sestavo nezorene (prvi dan post mortem) prašičje LD mišice (N = 6) Parameter/(g/100 g) x Min. Max. SD CV/% Water Voda 74.2 73.2 75.6 0.83 1.11 IMF IMM 1.47 0.80 1.80 0.32 21.75 Protein Beljakovine 23.1 21.5 24.1 0.69 2.30 Ash Minerali 1.12 1.05 1.22 0.04 3.95 N – number of observations / število vzorcev, x – mean / povpre čje, Min. – minimal value / minimalna vrednost, Max. – maximal value / maksimalna vrednost, SD – standard deviation / standardni odklon, CV (%) – coefficient of variation / koeficient variabilnosti, IMF – intramuscular fat / IMM – intramuskularna maš čoba. The average pH value of all our measurements 24 h post mortem was 5.61 ± 0.19; colour of muscles was appropriate for normal muscle quality. pH value as physicochemical criteria showed pork meat quality to be normal. pH 24 values were comparable to pH 24 from pigs of different age at slaughter and different feed restriction investigated by Čandek-Potokar et al. (1998) or those from pigs being slaughtered without or under minimal stress investigated by Henckel et al. (2000). It is also known that pH increases for some tenths of a pH-unit due to ageing. In this study, an approximately 0.06-unit increase of pH value was determined after 16-days of ageing (Table 2). Myofibril fragmentation Meat tenderness is related also to structural (and biochemical) properties of skeletal muscle fibres, especially those of myofibrils and intermediate filaments. Histological studies dealt with Acta agriculturae Slovenica, 90(november 2007)1. 10 myofibrils breaking into shorter segments during post mortem storage of muscle, this phenomenon is called myofibril fragmentation. It is considered a useful ageing indicator of aged meat (Veiseth et al., 2001). Myofibril fragmentation can be estimated by different methods: by homogenization of muscle, determination of protein content and measurement of the turbidity of samples adjusted to a standard protein concentration, by examination of myofibrils under a light microscope (Takahashi et al., 1967; Moller et al., 1973), by passing homogenized muscle through a filter system and recording the weight of the sample product removed (Reagan et al.,1975; Purchas et al.,1997) or by measuring of myofibrilar fragment length (Fernandez and Tornberg, 1994). Positive correlation between the rate of myofibrilar fragmentation and the tenderness of the meat is well known. Table 2. Effect of ageing (1, 2, 3, 5, 11 and 16 days at 2 °C (± 1 °C)) on pH value, myofibrilar length, myofibril fragmentation index determined according to the methods described by Olson et al. (1976) and by Hopkins et al. (2000), non-protein nitrogen content, and Warner-Bratzler Shear Force of pork LD muscle (Model [1], N = 36) Preglednica 2. Vpliv časa zorenja na vrednost pH, dolžino miofibrilarnih fragmentov, indeks miofibrilarne fragmentacije (dolo čen po Olsonu s sod., 1976 in Hopkinsu s sod., 2000), neproteinski dušik in Warner-Bratzler strižno trdnost praši čje LD mišice (Model [1], N = 36) Ageing/days Zorenje/dnevi Effect of: Vpliv: 1 2 3 5 11 16 SE P value pH value Vrednost pH 5.62 b 5.61 b 5.52 c 5.62 b 5.64 ab 5.68 a 0.02 <0.001 MFL/µm DMF/µm 73.0 a 53.1 b 31.8 c 24.7 d 15.7 e 12.4 e 1.62 <0.001 MFI-Olson IMF-Olson 29.4 c 52.8 b 56.7 b 63.7 a 62.5 a 63.2 a 1.92 <0.001 MFI-Hopkins IMF-Hopkins 28.5 d 44.8 c 45.3 c 50.5 b 56.9 a 60.0 a 1.60 <0.001 NPN/(% of TN) 9.39 d 9.57 cd 9.73 c 10.12 b 10.78 a 10.93 a 0.10 <0.001 WBSF/N WBSS/N 51.3 a 37.9 b 35.8 b 32.7 c 31.5 cd 29.2 d 0.98 <0.001 N – Number of observations. SE – standard error. Least squares means with a different superscript within rows differ significantly (P ≤ 0.05). Levels of significance: statistically significant: P ≤ 0.05; highly statistically significant: P ≤ 0.001. MFL – myofibrilar fragment length. MFI-Olson – myofibril fragmentation index (Olson et al., 1976). MFI-Hopkins – myofibril fragmentation index (Hopkins et al.,2000). NPN – non-protein nitrogen. TN – total nitrogen. WBSF – Warner-Bratzler Shear Force. N – število obravnavanj. SE – standardna napaka ocene. Pri čakovane srednje vrednosti z razli čnimi nadpisanimi črkami a,b,c,d,e se statisti čno zna čilno (P ≤ 0.05) razlikujejo. Stopnja zna čilnosti: statisti čno zna čilna: P ≤ 0.05, statisti čno visoko zna čilna: P ≤ 0.001. DMF – dolžina miofibrilarnih fragmentov. IMF-Olson – indeks miofibrilarne fragmentacije (1976). IMF-Hopkins – indeks miofibrilarne fragmentacije (2000). NPN – neproteinski dušik. TN – celokupni dušik. WBSS – Warner-Bratzler strižna trdnost Generally, ageing can and does affect the MFL of pork meat (Table 2). The average MFL was 73 µm for day 1 post mortem and decreased significantly with the time of ageing. After 11 days Došler, D. et al. Relation of myofibril fragmentation to textural and chemical parameters of aged pork longissimus dorsi. 11 (15.7 µm) and 16 days (12.4 µm) of ageing the lowest MFL was measured, however the difference (between 15.7 µm and 12.4 µm) was not statistically significant. Therefore we assume that a great part of fragmentation measured occurred within day 3 and day 5 of ageing. The coefficient of variation for MFL was above 130%. This high variation in myofibril length was probably due to different activity of enzymes as well as due to nonenzyme process of decomposition (e.g. by calcium ions). 0 10 20 30 40 50 60 70 80 90 100 frequency/% 0-20 20-40 40-60 60-80 80-100 100-120 120-140 140-160 160-180 180-200 200-220 220-240 240-260 >260 1 2 3 5 11 16 myofibrilar lenght/µm ageing/days Figure 1. Relative frequency distribution of myofibrilar length of pork LD muscles as a function of ageing time. Slika 1. Relativna frekvenca porazdelitve dolžine miofibrilarnih fragmentov praši čje mišice LD v odvisnosti od zorenja. Myofibril breaking into shorter fragments during post mortem ageing of muscle is presented in Figure 1. In nonaged samples the size of fragments being between 0 and 20 µm (12%), 20 and 40 µm (33%), 40 and 60 µm (19%), 60 and 80 µm (10%), some of them being longer (28%). Generally, fragments isolated from samples 2, 3 and 5 days post mortem have the size of 0 to 20 µm, 20 to 40 µm and 40 to 60 µm. In samples at 11 and 16 days of ageing the average myofibrilar fragment length is in the range between 0 and 20 µm (73% and 88% of all measured fragments, respectively). The relative frequency in the first class (between 0 and 20 µm) progressively increases with post mortem time (Figure 1). Direct comparison of these data with those from literature is difficult, since the myofibril fragment length depends on ageing condition, species and process of homogenising. Early as well as recent studies dealt with the changes in sarcomere length (Herring et al., 1965; Rees et al., 2002) or myofibril fragment length for beef, lamb or chicken muscles. Myofibril fragmentation of beef and pork LD, as Strydom et al. (2005) and Čandek-Potokar et al. (1998) emphasize, was significantly influenced by ageing (beef: 2 days 34.2 µm, 14 days 24.7 µm; pork: 1 day 19.4 µm, 4 days 9.77 µm). Our results agree with their data in spite of the fact that measured lengths acquired on pork LD after 2 days of ageing are noticeably longer (53.1 µm). These differences are probably due to the use of different extraction procedure and different homogenisers (Olson et al., 1976; Hopkins et al., 2000; Culler et al., 1978). The fragmentation of myofibrils has been observed during post-mortem ageing for 16 days and its index increased (P < 0.001) until day 16 (Table 2). The present study shows MFI values Acta agriculturae Slovenica, 90(november 2007)1. 12 ranging from less than 30 to more than 60. The MFI values of nonaged samples were below 30, values about 50 have been reached at day 2 of ageing. Increase after day 5 of ageing in MFI- Olson was hardly found. MFI-Hopkins after day 11 of ageing remained unchanged. Absolute MFI values for 3-day aged pork LD acquired in this study are somewhat lower compared to those of Veiseth et al. (2001) for pork as well as those of Bruas-Reignier and Brun- Bellut (1996) for bulls reported for at the same post mortem time, but on the other hand are comparable to MFI for 2 to 20-day aged pork loins as investigated by Okumura et al. (2003). These differences are probably due to the use of different myofibril preparation, such as speed/time of homogenization and type of homogenizer particularly the blade type, used scaling factor 200, 150 or 100 and the state of the sample (fresh or frozen and thawed) (Hopkins et al., 2000; Hopkins et al., 2004), anatomical parts of the pigs taken as samples, days post mortem, etc. It should be emphasized that the difference between MFI determined on fresh and frozen muscles was not significant (Veiseth et al., 2001; Hopkins et al., 2000). Comparisons of the data in Table 2 with other published reports indicate that at day 1 post mortem ovine muscle exhibits 2 times higher MFI values (Hopkins et al., 2000) compared to pork LD muscle in this study. 10 20 30 40 50 60 70 80 024681 01 21 41 61 8 ageing/days MFI Hopkins Olson Ns Ns ** *** ** *** Figure 2. Comparison of MFI (myofibril fragmentation index) examined as described by Olson et al. (1976) and Hopkins et al. (2000), from aged pork LD muscles (mean ± standard deviation) (Model [2], Levels of significance: not significant: Ns – P > 0.05, statistically significant: ** P ≤ 0.01, statistically highly significant: *** P ≤ 0.001). Slika 2. Primerjava IMF (indeks miofibrilarne fragmentacije) praši čje mišice LD, dolo čenega po Olsonu s sod. (1976) in Hopkinsu s sod. (2000) (srednja vrednost ± standardni odklon) (Model [2], stopnja zna čilnosti: nezna čilna: Ns – P > 0,05, statisti čno zna čilna: ** P ≤ 0,01, statisti čno visoko zna čilna: *** P ≤ 0,001). Figure 2 shows the changes in MFI examined as described by Olson et al. (1976) and Hopkins et al. (2000), and prepared from pork LD muscles stored at 2 °C for 1–16 days after slaughter. Main differences between these two methods are in the amount of meat sample, volume of isolating medium (buffer) per mass unit of meat sample, the sequence of centrifugations and filtrations, time of homogenization and number of washings. Increases in MFI-Olson and MFI-Hopkins were continuously observed during storage for 16 days; on the average, the values (P < 0.001) for MFI-Hopkins are about 7% lower compared to those for Došler, D. et al. Relation of myofibril fragmentation to textural and chemical parameters of aged pork longissimus dorsi. 13 MFI-Olson, with the exceptions for the nonaged and 16-day aged samples, similar values were obtained. Repeatability of both MFI methods was established by analysing the same sample in six replicates, the coefficient of variation for MFI-Olson being 4.6% and that for MFI-Hopkins 9.0%. Textural and chemical parameters Table 2 shows also the effect of ageing time on textural properties of roasted pork meat. The WBSF values were significantly different for different ageing times. Significantly the highest values (51.3 N) across the fibres were determined 1 day post mortem. After 2 or 3 days samples were significantly tenderer, the lowest values (31.5 N and 29.2 N) were observed after 11 and 16 days of ageing. According to the statement of Van Oeckel et al. (1999), pork meat (stored for 48 h at 4 °C, frozen stored during several months at –18 °C and grilled until an internal temperature of 74 °C, followed by cooling in tap water for 40 min) had a WBSF value of 35.5 N. Results of our study (37.9 N) are in slight discrepancies with their data, since lower values were determined, very probably due to different genotypes, different cooling and different parts of LD (thoracis vs. lumborum) muscles used. On the other hand, Fortin et al. (2005) determined higher values (62.9 N) on grilled loins (2 days post mortem, internal temperature of 72 °C, cooled in an ice/water bath, chilled for 24 h). Significantly lower absolute values in our study could be explained mainly due to smaller diameter (12.7 mm vs. 19 mm) of cylinders. The non-protein nitrogen fraction (NPN) corresponds to the muscle’s soluble non-protein compounds containing nitrogen (creatine, creatine phosphate, nucleotides, urea etc., amino acids and small peptides derived from the protein metabolism post mortem) (Bruas-Reignier and Brun- Bellut, 1996; Mikami et al., 1991). Significant increase in NPN content during days 3–11 of ageing (Table 2, Figure 3) is in agreement with conclusions of Bruas-Reignier and Brun-Bellut (1996), who claimed that the increase in NPN can be considered an indicator of beef meat proteolysis. NPN content reached the highest value at about day 11 post mortem; by this time the maximum release of amino acids and small peptides has probably occurred. 8 9 10 11 12 024681 01 21 41 61 8 ageing/days NPN/% of TN Figure 3. Effect of ageing time on non-protein nitrogen content (NPN) (% of total nitrogen − % of TN) in aged pork LD muscles (mean ± standard deviation). Slika 3. Vpliv časa zorenja na vsebnost neproteinskega dušika (NPN) (% od celokupnega dušika − % od TN) v praši čjih mišicah LD (srednja vrednost ± standardni odklon). Acta agriculturae Slovenica, 90(november 2007)1. 14 Correlations Correlation coefficients between instrumental values and physicochemical proteolysis parameters of pork LD muscles are shown in Table 3. WBSF is associated with the MFL, strong positive correlation between the parameters was observed (R = 0.82 *** ). Furthermore, the WBSF was negatively correlated with variations in MFI values, MFI-Olson (R = –0.82 *** ) and MFI–Hopkins (R = –0.77 *** ), which is in agreement with the results of Culler et al. (1978). These results further on substantiate the fact that the usual term for the state of tenderness, namely, the expression myofibril fragmentation tenderness, is an appropriate one to describe tenderness of a conventionally aged pork LD muscle. Table 3 further on shows that the increase in the NPN content is not related to the extent of the myofibril fragmentation. The increase in MFI or decrease in MFL and WBSF, which occurred mainly during the first 5 days post mortem, are not directly related to the increase in amino acid content and small peptide content, which increased from day 5 to day 11 of pork ageing. Table 3. Pearson correlation coefficients between textural, chemical and myofibril fragmentation parameters of pork LD muscles (N = 36) Preglednica 3. Pearsonovi korelacijski koeficienti med teksturnimi, kemijskimi in fragmentacijskimi parametri praši čje mišice LD (N = 36) Parameters Parametri WBSF WBSS MFI-Olson IMF-Olson MFI-Hopkins IMF-Hopkins NPN MFL DMF 0.82 *** –0.84 *** –0.87 *** –0.65 *** WBSF WBSS 1.00 –0.82 *** –0.77 *** –0.58 *** MFI-Olson IMF-Olson 1.00 0.86 *** 0.58 *** MFI-Hopkins IMF-Hopkins 1.00 0.67 *** Levels of significance: statistically significant: * P ≤ 0.05 and ** P ≤ 0.01; highly statistically significant: *** P ≤ 0.001. WBSF – Warner-Bratzler Shear Force. MFL – Myofibrilar length. MFI-Olson – myofibril fragmentation index (1976). MFI-Hopkins – myofibril fragmentation index (2000). NPN – non-protein nitrogen. Stopnja zna čilnosti: statisti čno zna čilna: * P ≤ 0.05 in ** P ≤ 0.01, statisti čno visoko zna čilna: *** P ≤ 0.001. WBSS – Warner-Bratzler strižna trdnost. DMF – dolžina miofibrilarnih fragmentov. IMF-Olson – indeks miofibrilarne fragmentacije (1976). IMF-Hopkins – indeks miofibrilarne fragmentacije (2000). NPN – neproteinski dušik. CONCLUSIONS Generally, 16-day ageing of pork meat does affect the microstructure, texture and non-protein nitrogen content. Myofibrilar fragmentation increases significantly from the first day post mortem during the entire ageing period. Up to 60% decrease of MFL occurs within the first 3 days and up to 50% increase of MFI within the first 2 days of ageing. We assume that a great part of fragmentation occurred between day 2 and day 5 of ageing. It can be concluded that under the conditions of this experiment, 60% of tenderisation of pork LD occurs within 2 days post mortem and 84% within 5 days. WBSF is associated with the MFL (R = 0.82 *** ), MFI- Olson (R = –0.82 *** ) and MFI-Hopkins (R = –0.77 *** ). The increase in the NPN content was not related to the extent of myofibril fragmentation. Myofibrilar fragmentation, which occurred Došler, D. et al. Relation of myofibril fragmentation to textural and chemical parameters of aged pork longissimus dorsi. 15 mainly during the first 5 days post mortem, was not directly related to the increase in amino acid content and small peptide content, which both increased also after 5 days of ageing. As far as methods for MFI determination are concerned, it can be concluded that on the average the values for MFI-Hopkins are about 7% lower compared to those for MFI-Olson. ACKNOWLEDGEMENTS This research was financed by the Slovene Ministry of Education, Science and Sport (J4- 6475-0481-04/4.02). We want to express our gratitude to Milica Ka č, Ph. D. for her valuable comments on an earlier draft of this paper. REFERENCES Bruas-Reignier, F./ Brun-Bellut, J. Changes affecting the Longissimus dorsi, Triceps brachii caput longum and Rectus femoris muscles of young friesian bulls during meat ageing, Meat Sci., 43(1996), 335–344. Čandek-Potokar, M./ Lefaucheur, L./ Žlender, B./ Bonneau, M. Effect of slaughter weight and/or age on histological characteristics of pig Longissimus dorsi muscle as related to meat quality. Meat Sci., 52(1999), 195–203. Čandek-Potokar, M./ Žlender, B./ Lefaucheur, L./ Bonneau, M. Effects of age and/or weight at slaughter on Longisimus dorsi muscle: Biochemical traits and sensory quality in pigs. Meat Sci., 48(1998), 287–300. Culler, R.D./ Parrish Jr., F.C./ Smith, G.C./ Cross, H.R. Relationship of myofibril fragmentation index to certain chemical, physical and sensory characteristics of bovine Longisimus muscle. J. Food Sci., 43(1978), 1177–1177. Davey, C.L./ Gilbert, K.V. Studies on meat tenderness. 7. Changes in the fine structure of meat during aging. J. Food Sci., 34(1969), 69–74. Devine, C.E. Conversion of muscle to meat/Ageing. In: Encyclopedia of meat sciences (Eds.: Jensen, W.K./ Devine, C./ Dikeman, M.). Oxford, Elsevier, 2004, 336 p. Dransfield, E. Optimisation of tenderisation, ageing and tenderness. Meat Sci., 36(1994), 105–121. Fernandez, X. / Tornberg, E. The influence of high post-mortem temperature and differing ultimate pH on the course of rigor and ageing in pig Longisimus dorsi muscle, Meat Sci., 36(1994), 345–363. Fortin, A./ Robertson, W.M./ Tong, A.K.W. The eating quality of Canadian pork and its relationship with intramuscular fat. Meat Sci., 69(2005), 297–305. Henckel, P./ Karlsson, A./ Oksbjerg, N./ Søholm Petersen, J. Control of post mortem pH decrease in pig muscles: experimental design and testing of animal models. Meat Sci., 55(2000), 131–138. Herring, H.K./ Cassens, R.G./ Briskey, E.J. Sarcomere length of free and restrained bovine muscle at low tempereature as related to tenderness. J. Sci. Food Agric., 16(1965), 379–385. Hopkins, D.L./ Littlefield, P.J./ Thompson, J.M. A research note on factors affecting the determination of myofibrillar fragmentation. Meat Sci., 56(2000), 19–22. Hopkins, D.L./ Martin, L./ Gilmour, A.R. The impact of homogenizer type and speed on the determination of myofibrillar fragmentation. Meat Sci., 67(2004), 705–710. Koohmaraie, M. Muscle proteinases and meat aging. Meat Sci., 36(1994), 93–104. Mikami, M./ Yamada, Y./ Wakahara, Y./ Miura, H. Effects of electrical stimulation on the sarcoplasmic proteins, peptide and amino acid contents of beef. Anim. Sci. Technol., 62(1991), 519–528. Moller, A.J.T./ Vestergaard, T./ Wismer-Pederson, J. Myofibril fragmentation in bovine Longisimus dorsi as an index of tenderness. J. Food Sci., 38(1973), 824–825. Nishimura, T./ Rhue, M.R./ Okitani, A./ Kato, H. Components contributing to the improvement of meat taste during storage. Agric. Biol. Chemistry, 52(1988), 2323–2330. Official Methods of Analysis (16th ed.). Washington, AOAC, 1997. Okumura, T./ Yamada, R./ Nishimura, T. Survey of conditioning indicators for pork loins: changes in myofibrils, proteins and peptides during post mortem conditioning of vacuum-packed pork loins for 30 days. Meat Sci., 64(2003), 467–473. Olson, D.G./ Parrish, J.R./ Stromer, M.H. Myofibrilar fragmentation and shear resistance of three bovine muscles during post mortem storage. J. Food Sci., 41(1976), 1036–1041. Olson, D.G./ Parrish Jr., F.C. Relationship of myofibril fragmentation index to measures of beefsteak tenderness. J. Food Sci., 42(1977), 506–509. Acta agriculturae Slovenica, 90(november 2007)1. 16 Purchas, R.W./ Hartley, D.G./ Xun, Y./ Grant, D.A. An evaluation of the growth performance, carcass characteristics, and meat quality of Sahiwal-Friesian cross bulls. New Zealand J. Agric. Res., 40(1997), 497–506. Reagan, J.O./ Dutson, T.R./ Carpenter, Z.L./ Smith, G.C. Muscle fragmentation indices for predicting cooked beef tenderness. J. Food Sci., 40(1975), 1093–1094. Rees, M.P./ Trout, G.R./ Warner, R.D. Tenderness, ageing rate and meat quality of pork M. Longisimus thoracis et lumborum after accelerated boning. Meat Sci., 60(2002), 113–124. SAS Software. Version 8.01. Cary, SAS Institute, Inc, 1999. Smith, G.C./ Culp, G.R./ Carpenter, Z.L. Post mortem aging of beef carcasses. J. Food Sci., 43(1978), 823–826. Strydom, P.E./ Frylinck, L./ Smith, M.F. Should electrical stimulation be applied when cold shortening is not a risk? Meat Sci., 70(2005), 733–742. Takahashi, K./ Fukazawa, T./ Yasui, T. Formation of myofibrillar fragments and reversibile contraction of sarcomeres in chicken pectoral muscle. J. Food Sci., 32(1967), 409–413. Van Oeckel, M.J./ Warnants, N./ Boucqué, Ch.V. Pork tenderness estimation by taste panel, Warner–Bratzler shear force and on-line methods. Meat Sci., 53(1999), 259–267. Veiseth, E./ Shackelford, S.D./ Wheeler, T.L./ Koohmaraie, M. Technical note: Comparison of myofibril fragmentation index from fresh and frozen pork and lamb Longisimus. J. Anim. Sci., 79(2001), 904–906.