Effects of dietary α-tocopheryl acetate supplementation on α-tocopherol deposition in porcine m. psoas major and m. longissimus dorsi and on drip loss, colour stability and oxidative stability of pork meat

PII:

SO309-1740(96)00130-l

Meat Science, Vol. 45, No. 4, 491-500, 1997 0 1997 Elwier Science Ltd All rights reserved. Printed in Great Britain 0309-1740/97 $17.00+ 0.00

ELSEVIER

Effects of Dietary wTocophery1 Acetate Supplementation on wTocophero1 Deposition in Porcine m. psoas major and m. Zongissimus dorsi and on Drip Loss, Colour Stability and Oxidative Stability of Pork Meat C. Jensen,a J. Guidera,b I. M. Skovgaard,” H. Staun,d L. H. Skibsted,” S. K. Jensen,e A. J. Marller,” J. Buckleyb & G. BertelserP* aDepartment

of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark bDepartment of Food Technology, University College Cork, Cork, Republic of Ireland =Department of Mathematics and Physics, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK- 187 1 Frederiksberg C, Denmark dDepartment of Animal Science and Health, The Royal Veterinary and Agricultural University, Bulowsvej 13, DK- 187 1 Frederiksberg C, Denmark “Danish Institute of Animal Science, Research Centre Foulum, P.O. Box 39, DK-8830 Tjele, Denmark (Received 6 July 1996; revised version received 12 November 1996; accepted 14 November 1996)

ABSTRACT The effect of feeding supra-nutritional levels of a-tocopheryl acetate on its deposition in two porcine muscles of d$erent metabolic rates (m. longissimus dorsi and m. psoas major) and the effect on meat quality (lipid oxidation, colour stability and drip loss) was studied. Pigs were fed a standard diet supplemented with three levels: 100, 200 and 7OOmglkg of a-tocopheryl acetate from the time of weaning to slaughter at 90 kg live weight. Muscle a-tocopherol levels were linearly related to the logarithm of dietary a-tocopheryl acetate supplementation and the linear relationship was estimatedfor the two muscles. The levels of a-tocopherol in the two muscles differed by a parallel displacement with consistently higher a-tocopherol levels in m. psoas major compared to m. longissimus dorsi. Dietary a-tocopheryl acetate supplementation significantly reduced lipid oxidation as measured by thiobarbituric acid reactive substances (TBARS) in both raw and cooked meat during storage at 4°C for 6 days. Drip loss and colour stability of raw muscles were not affected by dietary a-tocopheryl acetate levels, IOOmg ar-tocopheryl acetate/ kg feed resulted in suficient cx-tocopherol levels in muscles to ensure minimum drip loss and optimum colour stability. 0 1997 Elsevier Science Ltd. All rights reserved

INTRODUCTION An

increasing

wtocopherol

body of experimental evidence indicates a protective role for muscle against lipid oxidation in fresh pork and pork products (Astrup, 1973;

*To whom correspondence

should be addressed. 491

492

C. Jensen

et al.

Monahan et al., 1990; Buckley et al., 1995). In addition a beneficial effect of muscle a-tocopherol on colour stability and drip loss has been shown for raw pre-frozen pork chops (m. longissimus dorsi) during subsequent chill storage (Asghar et al., 1991b). The rate of discolouration in fresh meat has been related both to the rate of pigment oxidation, determined by the activity of oxidative processes and their effect on the oxygen concentration at the meat surface, and to the effectiveness of the metmyoglobin enzymatic reducing systems (Ledward, 1991; Faustman and Cassens, 1989). Both systems are important in fresh meat; however, the activity of the oxidative systems decreases exponentially with time post-slaughter, whereas the reducing system decreases much more gradually and thus the latter system becomes dominant with time (Ledward, 1991). Muscles exhibit different colour stabilities and O’Keefe and Hood (1982) reported that the colour stability of beef muscles decreased in the order; m. longissimus dorsi > m. semimembranosus > m. gluteus medius > m. psoas major. The difference in colour stability among muscles has been explained by the differences in respiratory activity; muscles having higher oxidative activity are more colour labile (O’Keefe and Hood, 1982; Renerre and Labas, 1987). The mechanism of colour stabilisation performed by endogenous a-tocopherol is not clear, but discolouration and lipid oxidation in fresh meat are known to be closely related (Greene, 1971), and a decreased rate of lipid oxidation due to high levels of endogenous a-tocopherol in fresh meats has been shown to result in decreased myoglobin oxidation (Monahan et al., 1994; Faustman et al., 1989). Furthermore the activity of the metmyoglobin reducing systems is believed to be sustained for longer periods in meats with lower rates of lipid oxidation as the metmyoglobin reducing system is subjected to less free radical attack originating from the lipid oxidation processes (Faustman et al., 1989). Porcine tissue a-tocopherol levels have been found to respond to dietary o-tocopheryl acetate (Brekke et al., 1975; Jensen et al., 1988; Buckley et al., 1989). Various tissues (skeletal muscles, adipose tissue, liver, heart, kidneys and lungs) have been found to respond differently to the cr-tocopheryl acetate content of the diet depending on their metabolic activities. Thus ar-tocopherol concentration increased in the order: muscle < kidney < lung < heart < liver (Monahan et al., 1990; Asghar et al., 1991~). Recommendations for cr-tocopheryl acetate supplementation is 30-60 mg/kg for piglets and 1540mg/kg in fattening pigs (Albers et al., 1984), but several investigations have established a positive relationship between dietary a-tocopheryl acetate and meat quality (lipid oxidation, colour stability and drip loss), when raising levels to 100 mg and 200 mg/ kg compared to lOmg/kg (Buckley et al., 1989; Asghar et al., 1989; Monahan et al., 1990, 1992, 1994). In this study the effect of exceeding a dietary level of 200mg a-tocopheryl acetate/kg feed on meat quality (lipid oxidation, colour stability and drip loss) has been investigated in m. longissimus dorsi and m. psoas major, two muscles having different metabolic activities and colour stabilities.

MATERIALS

AND METHODS

Animals and diets

Sixty Danish Landrace pigs (30 males and 30 females) were fed a standard feed mix (Table 1) until an average weight of 50 kg was achieved, then pigs were randomly allocated to the six feeding groups described below, with five males and five females per group. The pigs were fed water and feed ad libitum. Two commercial vitamin E premixes with different carrier material were used, Rovimix supplied by Roche DK (Ballerup, Denmark) and E-vicin Staerk 25000 supplied by Ksge Stormalle (Koge, Denmark),

493

Dietary u-tocopheryl acetate supplementation

TABLE 1 Composition of Standard Feed Mix Ingredients

Starter Experimental (2&50 kg live weight) (50-90 live weight) (Percentage of diet)

Barley Soy meal roasted Rapeseed oil Minerals and vitamins

12.0 24.0 1.5 2.5

78.0 180 1.5 2.5

resulting in the following six feeding groups: (1) 100mg E-vicin/kg diet, (2) 100mg Rovimix/kg diet, (3) 200 mg E-vicin/kg diet, (4) 200 mg Rovimix/kg diet, (5) 700 mg E-vicin/kg diet, (6) 700mg Rovimix/kg diet (Table 2). Both premixes used all-rat-a-tocopheryl acetate as the tocopherol source to ensure optimum stability of the vitamin during storage. The tocopherol ester was in both cases manufactured by Hoffman-La Roche (Basel, Switzerland) and premixes differed only with respect to carrier material. Pigs were housed in controlled facilities at the Pig Experimental Station (Sjaelland II) at Roskilde, Denmark. The pigs were slaughtered at 90 kg at the abattoir of the Danish Meat Trade School at Roskilde. The carcasses were chilled overnight at 4°C and at 24 hr post mortem samples from m. longissimus dorsi (LD) and m. psoas major (PM) were removed.

Chill storage of raw meat Ten chops from each pig (1.5 cm thick) were sliced from LD and PM muscles and individually wrapped in polyethylene film (Oxygen Transmission Rate (OTR) > 4000 cm3/m2/ 24 h/atm) and placed in a chill cabinet under fluorescent light (Philips TLD 18W/36) at 4°C for six days. At the surface of the products an illuminance of 450 lux and a radiant TABLE 2 Mean a-Tocopherol Content of Feed, m. longissimus dorsi (N= 10) and m. psoas major (N= 10) of Pigs Fed Three Supplemental a-Tocopheryl Acetate Levels, Using Two Different Premixes (E-vicin and Rovimix) Premix’

Analysed u-tocopherol content Feed’ (mglkgj5

E-vicin

Rovimix

132 201 722 130 223 742

PM4 f l&C/&

54” 7.8b 11.4’ 5.1” 8.2b 11.1’

10.0” 11.46 16.3’ 9.8” 12.ob 15.1’

I Effect of premixes was not significant. 2 Effect of dietary or-tocopheryl acetate level was significant (P < 0.01). 3 m. longissimus dorsi. 4 m. psoas major. 5 Determined on a dry matter basis.

6 Determined on a wet wt basis. a,b~cMean values bearing different superscripts within a column are significantly different (P < O-01).

494

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et al.

flux density of 25mW/m* were measured. TBARS and colour were measured on two chops from each muscle at day 0, 1, 2,4 and 6 of chill storage. Pigment content, ar-tocopherol and drip loss were measured on the raw LD and PM muscle from individual pigs. Chill storage of cooked meat PM and LD muscles from eight pigs from the six experimental groups were trimmed of visible fat and connective tissue, then ground twice through a 4 mm plate and 20 g samples were vacuum-packed in laminate bags (OTR < 10 cm3/m2/24 hr/atm). The samples were pressed into uniform patties of 4mm height. The vacuum-packed patties were heated in a water bath at 80°C for 5min and cooled instantly in ice water. Patties were then packed in polyethylene bags (OTR = 3000 cm3/m2/24 hr/atm) and placed in a chill cabinet for six days at 4°C under the same fluorescent light conditions as the raw meat. TBARS were measured on two patties from each muscle at day 0, 1, 2, 4 and 6 of chill storage. Lipid oxidation The extent of lipid oxidation was measured as Thiobarbituric Acid Reactive Substances (TBARS) by the extraction method of Vyncke (1975). TBARS were measured in duplicate on each sample and expressed as pmoles malonaldehyde/kg dry matter. Colour The surface colour was measured during chill storage by a tristimulus calorimeter (Hunterlab D-25, Reston, Virginia, US), and the coordinates L*, a* and b* recorded. The haem pigment content was determined by the method of Homsey (1956) and expressed as ppm haem. Drip loss Drip loss was measured in duplicate on two chops from both muscles of the 60 pigs by the method of Barton-Gade et al. (1994) and was expressed as a percentage of the original weight. The drip loss was measured on non-illuminated chops after 48 hr at 4°C. Analysis of a-tocopherol Feed samples were saponified prior to extraction and HPLC analysis of a-tocopherol as described by Jakobsen et al. (1995); a-tocopherol content is the sum of cr-tocopherol originating from feed ingredients and from the supplementation with a-tocopheryl acetate. Muscle cr-tocopherol was extracted using the procedure of Buttriss and Diplock (1984) and quantified by reverse phase HPLC and fluorescence detection using an external standard. The mobile phase consisted of methanol:water (97:3) and the HPLC system included a pump (Shimadzu, LC-10, Kyoto, Japan) set at a flow rate of 1>7mL/min, a 250mm x 4~6mm, 5 p Cl8 stainless column (Chrohmpack, Melburg, an autosampler (Shimadzu, SIL-10, Kyoto, Japan) and a The Netherlands), fluorescence detector (Waters 470, Milford, Massachusetts). Excitation was at 292nm, emission at 330nm and integration was performed by the LClO software (Shimadzu, Kyoto, Japan).

Dietary a-tocopheryl acetate supplementation

495

Statistical analysis The measurements of a-tocopherol, pigment contents and drip loss were subjected to analysis of variance including the main effects of the factors: sex, brand names of premix, and amount of ar-tocopherol, as well as interaction between the latter two factors. The two muscles were compared using similar analyses of variance on the differences within individual pigs. Lipid oxidation and colour, quantified by the amount of TBARS and the Hunter a-value, respectively, were measured repeatedly on each pig on day 0, 1, 2, 4 and 6 during chill storage. The same model as above for analysis of variance formed the basis of analysis of these measurements, but with day included as a ‘repeated measurement factor’ using the Huyhn-Feldt correction for correlations between measurements from the same animal (Crowder and Hand, 1990). This analysis was performed by the procedure GLM in SAS@ (SAS/STAT User’s Guide, 1990). TBARS were logarithmically transformed beforehand to stabilise variances.

RESULTS Feed and muscle cr-tocopherol The a-tocopherol contents in the diets and muscles are shown in Table 2. Muscle cz-tocopherol levels were not significantly affected by sex and results were accordingly pooled. The o-tocopherol concentrations in muscles were significantly (P < 0.01) influenced by the concentration of a-tocopheryl acetate of the diets and cz-tocopherol levels in PM were significantly higher than LD. Muscle uptake of a-tocopherol was not significantly different for the two premixes and results were accordingly pooled for further statistical analysis. Effect of dietary treatment on lipid oxidation The oxidative stability of lipids in LD and PM muscles during chill storage was positively correlated to the levels of dietary ol-tocopheryl acetate (Fig. 1) and to the concentration of endogenous o-tocopherol. Feeding pigs levels of 200 and 700 mg o-tocopheryl acetate/kg feed significantly decreased TBARS development during chill storage. A similar positive relationship between dietary levels of a-tocopheryl acetate and lipid stability was seen in the cooked meat (Fig. 2) where TBARS in LD and PM muscles significantly decreased from pigs fed 700 mg a-tocopheryl acetate/kg feed compared to levels of 100 and 200 mg/ kg feed. Muscles from pigs fed 200 mg cr-tocopheryl acetate/kg feed showed lower TBARS values in the chill stored cooked patties compared to muscles from pigs fed 100 mg a-tocopheryl acetate. However, the difference between the two dietary levels was minor and statistically non-significant. Similarly, the TBARS development in the two muscles during chill storage was not significantly different, whereas TBARS increased to significantly higher values for cooked PM muscle compared to cooked LD muscles. Effects of dietary treatment on colour and drip loss PM was found to have higher pigment content than LD (Table 3), irrespective of supplementation levels. The drip loss in LD was approximately four times that in PM and no relationship to dietary a-tocopheryl acetate was found (Table 3). Surface colour was likewise not significantly influenced by dietary supplementation (results not shown).

C. Jensen et al.

496

DISCUSSION The present experiment showed a positive correlation between dietary intake of a-tocopheryl acetate and the levels of a-tocopherol found in porcine LD and PM muscle. In agreement with the results of Machlin and Gabriel (1982), the levels of endogenous

c

,.‘.‘..

0

1

2

3

4

5

6

7

5

6

7

Time (Days)

0

1

2

3

4

Time (Days) Fig. 1. Effect of dietary cr-tocopheryl acetate supplementation

on lipid oxidation measured by determination of TBARS of chill stored raw pork chops from (top) m. longissimus dorsi from pigs fed dietary levels of cu-tocopheryl acetate: 0 (100 mg/kg), A (200mg/kg) and 0 (7OOmg/kg) and (bottom) m. psoas major from pigs fed dietary levels of cu-tocopheryl acetate: l (lOOmg/kg), A (200 mg/kg) and n (700 mg/kg). Standard deviation is indicated by bars.

Dietary a-tocopheryl acetate supplementation

491

a-tocopherol found in the muscles could he approximated by a linear function of the logarithm of the concentration of cr-tocopheryl acetate administrated: y = a + b log(x) where x is the measured a-tocopherol content in the diets (mg/kg dry matter), y is the muscle a-tocopherol @g/g wet muscle) and a, b are constants depending on the muscle. Based on the present data the linear relationships for the two muscles were found to he: 1

0

1

2

3

4

5

6

7

8

Time (Days)

f

Q

B $ r” ; 9 E ,o iz

40 35 30 25 20

0

1

2

3

4

5

6

7

8

Time (Days)

Fig. 2. Effect of dietary a-tocopheryl acetate supplementation on lipid oxidation measured by determination of TBARS of chill stored cooked patties from (top) m. Zongissimusdorsi from pigs fed dietary levels of or-tocopheryl acetate: 0 (100 mg/kg), A (2OOmg/kg) and 0 (700mg/kg) and (bottom) m. psous major from pigs fed dietary levels of wtocopheryl acetate: l (lOOmg/kg), A (2OOmg/kg) and n (7OOmg/kg). Standard deviation is indicated by bars.

498

C. Jensen et al. PM cr-tocopherol content = (-6.4)

+ 7*7(log(dietary a-tocopherol))

with R2 =0.68 7 and LD ar-tocopherol content = (-10.3)

+ 7*6(log(dietary cy-tocopherol))

with R2 =0.64. The linear relationship is further illustrated in Fig. 3 and, as the factor b is approximately the same for the two muscles, the total uptake of cll-tocopherol in the two muscles can be considered as equal except PM has a consistently higher background level of (IItocopherol than LD. Yamauchi et al. (1984) found a positive relationship between the degree of respiratory activity of the muscle and the content of a-tocopherol in the membranal tissues since mitochondria isolated from porcine biceps femoris muscle was found to have a higher concentration of o-tocopherol than mitochondria from the less oxidative longissimus thoracis. Similar results have been found for broiler muscles, where membranal tissue isolated from the red leg musculature had a higher a-tocopherol content than that from the white breast muscle (Asghar et al., 1989; Yamauchi et al., 1984), and from beef muscles, where a higher level of cz-tocopherol was found in the microsomes of m. psoas major compared to m. gluteus medius and m. longissimus lumborum (Chan et al., 1996). Thus the higher content of a-tocopherol in PM muscle compared to LD (Table 2 and Fig. 3) can be explained both by a greater content of membranal tissue and the positive relationship between respiratory activity and membranal o-tocopherol content. The higher content of a-tocopherol in PM did not result in higher oxidative stability of fresh or cooked PM meat compared to LD meat. Chan et al. (1996) found beef psoas major muscle lipid to be less protected against oxidative attack, in spite of a higher muscle cr-tocopherol level, compared to gluteus medius and longissimus lumborum. For cooked meat the oxidative deterioration during chill storage was even more pronounced

0

2

3

Log(mg Dietary Alpha-Tocopheryl Acetate/kg feed) Fig. 3. Relationship between a-tocopherol muscle levels and log (dietary mg a-tocopheryl acetate/ kg feed) in m. longissimus dorsi from pigs fed: 0 (100 mg/kg), A 200 mg/kg and 0 (700 mg/kg) and in m. psous major from pigs fed: l (100 mg/kg), A 200 mg/kg and n (700 mg/kg).

499

Dietary a-tocopheryl acetate supplementation

TABLE 3 Effects of Dietary rr-tocopherol Supplementation on Initial Pigment Content (N= 20) and drip loss (N= 20) in Raw Meat ~Tocopheryl acetate supplementation (mglkg)

100 200 700

Drip loss (%)

Haem (ppm) LD’

PM’

LD’

PM2

31.7 32.9 34.7

52.9 54.0 53.6

3.7 4.0 4.4

1.1 1.1 1.1

’ m. longissimus dorsi. 2 m. psoas major.

The effect of dietary a-tocopheryl

acetate supplementation

was not significant.

for PM than for LD (Fig. 2). An important factor determining the rapid onset and development of oxidation in heated meats is the generation of pro-oxidative active species originating from haem-bound iron (Mielche and Bertelsen, 1994). In the present study, the content of haem was found to be significantly higher in the more oxidatively labile PM muscle compared to the LD muscle, supporting the role of haem-derived pro-oxidants (Table 3). Supplementation with o-tocopheryl acetate did not affect drip loss or colour stability of raw muscles during chill storage. In this study levels of 9.9 mg cz-tocopherol/kg muscle and 5.3 mg a-tocopherol/kg muscle were achieved for PM and LD, respectively, by feeding pigs 1OOmg cr-tocopheryl acetate/kg feed. These levels are higher than levels reported by Asghar et al. (1989) required to improve colour stability and decrease drip loss in porcine m. longissimus dorsi. In the study of Asghar et al. (1989) 10, 100 and 200mg cr-tocopherol/ kg feed resulted in LD levels of 0.54, 260 and 4.72mg a-tocopherol/kg muscle, respectively, and the latter two levels significantly improved colour stability and decreased drip loss of pre-frozen pork chops during chill storage under fluorescent light. Lanai-i et al. (1995) found muscle levels of 6.9 and 7.9 mg a-tocopherol/kg muscle when pigs were fed 198 and 207 mg ar-tocopherol/kg feed, respectively, and these levels enhanced colour stability in porcine LD during chill storage compared to unsupplemented samples. Thus, it may be concluded that each of the three supplemental levels used in the present study resulted in pig meat containing sufficient o-tocopherol for optimum drip and colour stability. Lanari et al. (1995) noted that the improvement in pork muscle colour stability produced by dietary a-tocopherol supplementation was not as profound as has been reported for beef muscle. In beef it has been shown that the advantageous effect of (Ytocopherol supplementation on the colour stability of m. psoas major, m. gluteus medius and m. longissimus lumborum are detected by both objective and subjective evaluation and that a-tocopherol supplemented meat was preferred by panellists (Chan et al., 1996). Further research is needed to evaluate whether the benefits of ar-tocopherol supplementation on pork meat colour and lipid stability are detectable by consumers. In conclusion, raising a-tocopheryl acetate supplementation levels from 100 to 200 or 700mg/kg enhanced oxidative stability of lipids in fresh chill stored LD and PM muscles and chill stored cooked PM patties.

ACKNOWLEDGEMENTS This work was partly sponsored by the AIR2-CT94 1577 contract (DIET-OX) and the Fstek program through LMC - Center for Advanced Food Studies. The authors are grateful to Annette Clausen and Astrid Beth Hansen for technical assistance.

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