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Reduced nitrite levels and dietary α-tocopheryl acetate supplementation: effects on the colour and oxidative stability of cooked hams
Meat Science 55 (2000) 475±482
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Reduced nitrite levels and dietary a-tocopheryl acetate supplementation: eects on the colour and oxidative stability of cooked hams N.M. Dineen a, J.P. Kerry a,*, P.B. Lynch b, D.J. Buckley a, P.A. Morrissey a, E.K. Arendt a a
Department of Food Science and Technology, University College Cork-National University of Ireland, Cork, Ireland b Teagasc, Moorepark, Fermoy, Co. Cork, Ireland Received 14 July 1999; received in revised form 5 November 1999; accepted 5 January 2000
Abstract The objective of the present study was to determine the eects of dietary vitamin E supplementation and reduced nitrite levels on the colour stability of cooked hams. Large white Landrace pigs (male n=6, female n=6) were each subdivided into two groups (n=3) and fed an a-tocopheryl acetate supplemented diet (1000 mg/kg feed) and a basal diet (10 mg/kg feed) for a period of 10 weeks. M. semitendinosus were removed from each pig, divided into light and dark pigmented fractions, vacuum packed and stored at 4 C for 24 h. Muscles were cured with input nitrite levels of 25 and 100 mg/kg meat and were tumbled and massaged for 17 h. Samples were cooked, sliced and overwrapped in a high oxygen permeable ®lm for a storage period of 10 days. Surface colour of hams was measured and expressed as Hunter `a' values. Concentrations of a-tocopherol were signi®cantly (P<0.001) greater in supplemented muscles compared to basal muscles for both male and female pigs. Hams manufactured from male and female supplemented pigs resulted in signi®cantly (P<0.001) higher Hunter `a' values than hams manufactured from male and female pigs receiving the basal diet. Muscles cured with 100 mg nitrite/kg meat formed products with signi®cantly (P<0.001) higher `a' values than those cured with the lower (25 mg/kg meat) nitrite level. Hams manufactured from supplemented muscles, treated with 25 mg nitrite/kg meat showed signi®cantly (P<0.05) higher Hunter `a' values than hams manufactured from basal muscles, treated with 100 mg nitrite/kg meat. Hams manufactured from female porcine muscles had signi®cantly (P<0.001) higher `a' values than hams from male muscles during the 10 days of simulated retail display. No such gender dierences were observed for TBARS values. # 2000 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Colour is an extremely critical component in the appearance of meat and has a substantial in¯uence on consumer purchase decisions (Sherbeck, Wulf, Morgan Tatum, Smith & Williams, 1995). The use of sodium nitrite in cured meats is responsible for the characteristic pink meat colour and ¯avour stability (Tichivangana, Morrissey & Buckley, 1984). Nitrite acts as a potent catalyst in lipid oxidation (Claus, Colby & Flick, 1994) and provides a preservative eect, especially against the outgrowth of spores of Clostridium botulinum (Cassens, 1995). However, the use of sodium nitrite in muscle * Corresponding author. Tel.: +353-21-902256; fax: 353-21276318. E-mail address: [email protected] (J.P. Kerry).
foods may lead to the formation of carcinogenic nitrosamines. Sarasua and Savitz (1994) observed a positive association between brain tumours and childhood consumption of ham, bacon and hot dogs. Bunin, Kuijten, Boesel, Buckley and Meadows (1994) reported an increased incidence of tumours in children whose mothers consumed cured meats. Cassens (1997) showed an approximate 80% reduction in the residual nitrite content of cured meats since the 1970s. However, the search for suitable alternatives to nitrite is still ongoing. a-Tocopherol is a highly eective lipid soluble, chain breaking antioxidant which is acceptable to the consumer (Faustman, Cassens, Schaefer, Buege, Williams & Scheller, 1989). a-Tocopherol, when incorporated in cell membranes by dietary means improves the antioxidant defense system and is an eective means of stabilising raw muscle during frozen storage and muscle
0309-1740/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00008-5
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food during processing, thus increasing the shelf-life of muscle foods during subsequent processing and distribution (Buckley, Morrissey & Gray, 1995). Localisation of a-tocopherol in membranes allows it to function eciently compared to other antioxidants (Lauridsen, Buckley & Morrissey 1997). a-Tocopherol protects post-slaughter animal tissues and muscle foods against lipid oxidation and indirectly delays oxymyoglobin oxidation and meat discolouration (Morrissey, Buckley, Sheehy & Monahan, 1994). Discolouration in retail meats during display conditions may be a combined function of muscle pigment oxidation (oxymyoglobin to methmyoglobin) and lipid oxidation occurring in membrane phospholipids (Sherbeck et al., 1995). Nitrosylmyoglobin, the cured meat pigment, is unstable in air and discolouration can be rapid. Light can catalyse the dissociation of nitric oxide from cured meat pigments and cause colour fading especially when oxygen is present (Varnam & Sutherland, 1995). The use of dietary vitamin E and nitrite in combination, to stabilise colour and inhibit lipid oxidation has received little attention with the exception of recent studies by Walsh, Kerry, Buckley and Arendt (1998), Walsh, Kerry, Buckley, Morrissey, Lynch and Arendt (1998) and Houben and Gerris (1998). Walsh, Kerry, Buckley and Arendt (1998) and Walsh, Kerry, Buckley, Morrissey et al. (1998) found that vitamin E supplementation had signi®cant bene®cial eects on the colour and oxidative stability of low nitrite cooked hams and turkey patties products. Houben and Gerris (1998) on examining the eect of dietary vitamin E on the colour stability of pasteurised ham reported that supplementation with vitamin E conferred only slightly bene®cial eects on ham quality. The objective of the present study was to examine the eects of reduced nitrite levels and dietary vitamin E supplementation on the colour and oxidative stability of cooked hams processed from both male and female porcine M. semitendinosus. Vitamin E supplementation levels used in this study were higher than those used previously by either Houben and Gerris (1998) or Walsh, Kerry, Buckley and Arendt (1998) and Walsh, Kerry, Buckley, Morrissey et al. (1998) and the low nitrite level was lower than that used by Walsh, Kerry, Buckley, Morrissey et al. (1998). 2. Materials and methods 2.1. Reagents All chemicals were `Analar' grade obtained from British Drug Houses, Poole, Dorset, UK or Sigma Chemical Co. Ltd, Poole, Dorset, UK. a-Tocopheryl acetate was obtained from Roche Products Ltd, Welwyn Garden City, Hertfordshire, UK. Salt (pure dry vacuum salt) was obtained from Runcorn, Cheshire,
UK. Nitrite (food grade) was obtained from British Drug Houses, Poole, Dorset, UK 2.2. Animals and diets Pigs (n=12) were selected at 32±36 kg live weight and were comprised of male (n=6) and female (n=6) animals. Half of each gender group were fed a basal diet of 10 mg a-tocopheryl acetate/kg feed and the remainder fed a supplemented diet of 1000 mg a-tocopheryl acetate/ kg feed for a period of 10 weeks. Pigs were penned in a slatted area at the Pig Husbandry Unit, Agriculture and Food Development Authority (Teagasc), Moorepark Research Centre, Fermoy, Co. Cork, Ireland. Temperature control was maintained using a mechanical ventilation system. Composition of the diets are shown in Table 1. Pigs were given water and fed ad libitum. Pigs were slaughtered at Cappoquin Pork and Bacon Factory, Cappoquin, Co. Waterford. 2.3. Sampling procedure Carcasses were split centrally and chilled at 4 C for 24 h. M. semitendinosus (n=12), were identi®ed and removed from the left hind limb of each pig. Dark fractions of the muscle were removed and excluded from analysis. Light fractions of the muscles were vacuum packed and stored at ÿ20 C until required. 2.4. Ham manufacture Male and female M. semitendinosus from both supplemented (1000 mg a-tocopheryl acetate/kg feed and basal (10 mg a-tocopheryl acetate/kg feed) dietary groups were cured with 25 and 100 mg nitrite/kg meat and an input salt level of 2% giving a sum of four treatments for each of the male and female pigs. The four cooked ham samples will from this point be referred to as Table 1 Composition of experimental diet kg/tonnea Component
Finisher
Barley Wheat Soya 50 Tallow Lysine synthetic Methionine synthetic Threonine synthetic Di calcium phosphate Limestone ¯our Salt Vitamins-minerals Mould curb Totals
350 399.75 215 10 2.5 0.75 1 5 11 3 1.5 0.5 1000
a
Kemin Industries Inc., Iowa, USA.
N.M. Dineen et al. / Meat Science 55 (2000) 475±482
(a) supplemented a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (b) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (c) basal atocopheryl acetate muscles plus 100 mg nitrite/kg meat and (d) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat. Muscles were pumped by hand to 115% of initial weight using a multi-needle injector pump (Dick, F., Postfach 209, 73703 Esslingen, Germany). Samples were tumbled and massaged for 10 min in every 30 min, at a temperature of 4 C for a total of 17 h at a speed of 10 rpm (Kerry, 1997). The meat was vacuum packed using a Webomatic vacuum packer (Webomatic vacuum packaging Systems, Werner Bonk, Mausegatt 59, D 463 Bochum 6, Germany) and held at 4 C for 36 h to allow for colour development. Samples were cooked at 80 C in a Memmert Oven (Memmert Gmbh, Aussere Rittersbacher Strasse 38, 91126 Schwabach, Germany) to an internal meat temperature of 72 C, measured using a Testo 700 digital thermometer probe (Testotherm Gmbh, Postfach 1140, D-7825 Lenzkirch/Schwartzwald, Germany). Cooked hams were cooled at 4 C for 6 hr, sliced and overwrapped in oxygen permeable (6000±8000 cm3/m2/24 h at standard temperature and pressure) cling®lm (Wrap Film Systems Ltd., Hales®eld 14, Telford TF7 4QR, Shropshire, England). All samples were stored in a display cabinet [Criobanc (ISS20250)] under ¯ourescent light (Osram Natura De Luxe [L36W/76-1]) at 4 C for 10 days.
477
5C18 (2500.4 mm) reverse phase column and a Waters model 486 UV-visible wavelength detector (Milipore Corporation, Milford, MA 01757, USA) set at 292 nm. The mobile phase used was methanol water (97:3) run at a ¯ow rate of 2 ml/min. Data were recorded on a Milipore Millenium 2010 chromatography management system (Milipore Corporation, Milford, MA 01757, USA). 2.8. Nitrite determination Nitrite levels were determined in triplicate on ham samples using the method of Follett and Ratcli (1963). 2.9. Statistical analysis a-Tocopherol concentrations, Hunter `a' values and TBARS values were analysed by repeated measures analysis of variance (ANOVA) using the SPSS statistical package [SPSS, Inc. (1997) SPSS Advanced Statistics 7.5, Chicago, Illinois]. 3. Results and discussion 3.1. Concentration of -tocopherol in muscle tissue
Surface colour measurements (Hunter L, a, b values) on ham slices were recorded in triplicate using a Minolta Chromameter (Model CR-300; Minolta Camera Co., Chuo-Ku, Osaka 541, Japan).
Mean a-tocopherol concentrations for male and female supplemented muscles were 5.470.29 and 5.490.25 mg/g muscle, respectively and for male and female basal muscles were 0.560.08 and 0.540.06 mg/g muscle, respectively. No signi®cant dierences in a-tocopherol levels were observed between supplemented male and female muscles or between basal male and female muscles.
2.6. Lipid oxidation determinations
3.2. Residual nitrite analysis
Lipid oxidation in all muscle samples was assessed by the 2-thiobarbituric acid distillation method of Tarladgis, Watts, Younathan and Dugan (1960) as modi®ed by Ke, Ackman, Linke and Nash (1977). Absorbance was read at 538 nm using a Beckman DU 640 spectrophotometer (Beckman Instruments, 2500 Harbour Boulevard, Fullerton, CA 92634-3100, USA). Results were expressed as mg malonaldehyde/kg tissue. Each sample was analysed in triplicate.
Residual nitrite levels, measured 4 days after initial injection, were in the range of 16±21 mg/kg meat for muscles cured with 25 mg nitrite/kg meat and 76±92 mg/ kg meat for muscles cured with 100 mg nitrite/kg meat. These results were in agreement with levels reported by Froehlich, Gullet and Usborne (1983) who estimated that nitrite losses ranged from 22±34% but disagreed with those of Cassens (1995) who reported that only about 10±20% of the originally added nitrite is analytically detectable and declines further during storage and distribution.
2.5. Colour measurement
2.7. -Tocopherol determination Meat samples (10 g) were taken from the centre of each supplemented and basal muscle. The method reported by Sheehy, Morrissey and Flynn (1994) was used for a-tocopherol extraction and HPLC analysis. HPLC was carried out using a Waters model S10 pump, Waters 717 autosampler, a Machery Nagel Nucleosil
3.3. Colour stability Hunter `a' values of overwrapped cooked ham slices from supplemented and basal male and female muscles, treated with high and low levels of nitrite are shown in Fig. 1a and b, respectively. Hunter `a' values decreased
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Fig. 1. (a and b). Eect of dietary a-tocopheryl acetate and reduced nitrite levels on the Hunter `a' values of overwrapped cooked ham slices manufactured from (a) male and (b) female M. semitendinosus and stored under retail display conditions at 4 C for 10 days. (*) Supplemented atocopheryl acetate muscles plus 100 mg nitrite/kg meat; (*) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (&) basal atocopheryl acetate muscles plus 100 mg nitrite/kg meat; (&) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat.
signi®cantly (P<0.001) during the 10 days storage period for each of the four treatments from both male and female muscles. Hams manufactured from supplemented a-tocopheryl acetate muscles cured with 100 mg nitrite/kg meat exhibited the greatest (P<0.001) degree of surface redness during retail storage, while hams manufactured from basal a-tocopheryl acetate muscles cured with 25 mg nitrite/kg meat showed poorest colour
stability. These ®ndings were in agreement with those reported by Walsh, Kerry, Buckley and Arendt (1998) and Walsh, Kerry, Buckley Morrissey et al. (1998). However, a recent study by Houben and Gerris (1998) reported that dietary supplementation with vitamin E did not appear to oer signi®cant advantages over currently used feeding regimes with regard to the quality of pasteurised ham.
N.M. Dineen et al. / Meat Science 55 (2000) 475±482
Repeated measures of analysis of variance was performed on the colour results obtained (Table 2). There were no signi®cant dierences at day 0 between any of the factors of interest in hams from either male or female pigs. From days 2±10, a clear diet eect emerged. Hams manufactured from supplemented muscles resulted in signi®cantly (P<0.001) higher Hunter `a' values than hams manufactured from basal muscles. This eect was not in¯uenced by time or nitrite levels and the trend was the same for hams produced from both male and female muscles. Hams from muscles cured with 100 mg nitrite/kg meat had signi®cantly (P<0.001) higher
Table 2 ANOVA analysis on Hunter `a' values of ham slices from M. semitendinosus Source of variation
F-value
P-value
Diet Gender Nitrite Day Daygender
135.42 31.22 90.73 77.10 4.14
<0.001 <0.001 <0.001 <0.001 0.007
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Hunter `a' values than muscles cured with 25 mg nitrite/ kg meat. This eect was not dependant on a-tocopherol level or time and the trend was common to hams from both male and female muscles. Hams manufactured from supplemented muscles cured with 100 mg nitrite/ kg meat showed the greatest (P<0.001) colour stability during storage while hams manufactured from basal muscles cured with 25 mg nitrite/kg meat performed least (P<0.001) favourably. However, hams manufactured from supplemented muscles cured with 25 mg nitrite/kg meat showed signi®cantly (P<0.05) greater colour stability during retail storage than hams manufactured from basal muscles cured with 100 mg nitrite/ kg meat. Walsh, Kerry, Buckley, Morrissey et al. (1998) found that ham slices produced from supplemented (500 mg a-tocopheryl acetate/kg feed) M.semimembranosus cured with 100 mg nitrite/kg meat had signi®cantly higher `a' values than hams processed from basal (10 mg a-tocopheryl acetate/kg feed) muscles cured with 50 mg nitrite/kg meat. Hams from supplemented muscles cured with 50 mg nitrite/kg meat had similar colour stability to hams from basal muscles cured with 100 mg nitrite/kg meat. O'Sullivan, Kerry, Buckley, Lynch and Morrissey (1998) found that Hunter `a' values of both
Fig. 2. Comparison of the Hunter `a' values of overwrapped cooked ham slices manufactured from male (&) and female (&) M. semitendinosus across each treatment level. (a) Supplemented a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (b) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (c) basal a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (d) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat.
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M. gluteobiceps and M. longissimus dorsi from the supplemented group (160 mg a-tocopheryl acetate/kg feed) were signi®cantly (P<0.05) more stable to colour change than those from the basal group (20 mg a-tocopheryl acetate/kg feed) during storage for 7 days at 4 C under ¯ourescent light. Walsh, Buckley and Arendt
(1998) found that colour stability of cooked cured turkey patties from supplemented vitamin E muscles (600 mg a-tocopheryl acetate/kg feed) was signi®cantly greater than those from basal muscles (20 mg a-tocopheryl acetate/kg feed). The signi®cant (P<0.001) eect of nitrite level on colour stability was in agreement with
Fig. 3. (a and b). Eect of dietary a-tocopheryl acetate and reduced nitrite levels on the TBARS values of overwrapped cooked ham slices manufactured from (a) male and (b) female M. semitendinosus and stored under retail display conditions at 4 C for 10 days. (*) Supplemented a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (*) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (&) basal atocopheryl acetate muscles plus 100 mg nitrite/kg meat; (&) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat.
N.M. Dineen et al. / Meat Science 55 (2000) 475±482
®ndings by Walsh, Kerry, Buckley and Arendt (1998), Walsh, Kerry, Buckley, Morrissey et al. (1998) and Deumier, Zakhia and Collignan (1996) who reported that cured venison tenderloins showed greater `a' values with increasing nitrite concentration. The in¯uence of porcine gender on Hunter `a' values was investigated separately among the four treatments. Hunter `a' values of cooked ham slices from male and female porcine M. semitendinosus are shown in Fig. 2. Hams produced from female porcine muscles had signi®cantly (P<0.01) higher `a' values than hams produced from male porcine muscles. The dierence in colour stability between male and female porcine M. semitendinosus can not be accounted for by dierences in muscle a-tocopherol concentrations. Muscles have dierent colour stabilities and the dierence in colour stability among muscles has been explained by the dierences in respiratory activity; muscles having higher oxidative activity are more colour labile (Renerre & Labas, 1987). However, this is not relevant to cooked cured muscles. It is not clear as to why hams from pig breeds that have been re®ned in their breeding should show such signi®cant colour dierences between gender. It is possible that biochemical dierences or metabolites present may be responsible for colour dierences observable in this study. 3.4. Oxidative stability TBARS values of overwrapped cooked ham slices from supplemented and basal male and female muscles, treated with high and low levels of nitrite are shown in Fig. 3a and b, respectively. TBARS values increased signi®cantly (P<0.001) during the 10 day storage period for each of the four treatments from both male and female muscles. As well as showing the highest colour stability, hams manufactured from supplemented atocopheryl acetate muscles cured with 100 mg nitrite/kg meat exhibited the greatest (P<0.001) degree of oxidative stability during retail storage while hams manufactured from basal a-tocopheryl acetate treated with 25 mg nitrite/kg meat showed the highest TBARS values. Repeated measures of analysis of variance were performed on the TBARS values obtained (Table 3). There
Table 3 ANOVA analysis on TBARS values of ham slices from M. semitendinosus Source of variation
F-value
P-value
Diet Gender Nitrite Day Daygender
129.82 0.09 87.69 69.22 0.17
<0.001 0.51 <0.001 <0.001 0.87
481
were no signi®cant dierences between any of the factors of interest in either male or female derived hams at days 0. Hams manufactured from supplemented atocopheryl acetate muscles showed signi®cantly (P<0.001) lower TBARS values than hams manufactured from basal muscles. The eect was not in¯uenced by time or nitrite level and the trend was the same for hams produced from both male and female muscles. Hams from muscles cured with 100 mg nitrite/kg meat had signi®cantly (P<0.001) lower TBARS values than hams from muscles cured with 25 mg nitrite/kg meat. This eect was not dependant on a-tocopherol level or time and the trend was also common to both male and female muscles. The eects of nitrite and a-tocopherol level on oxidative stability were signi®cant between days 2 and 10. Pfalzgraf, Frigg and Steinhart (1995) who observed that M. longissimus dorsi from pigs fed 200 mg a-tocopheryl acetate/kg feed were signi®cantly more stable (P<0.05) after 7 days of storage at 4 C compared to muscles from pigs fed a basal level (40 mg a-tocopheryl acetate/kg feed). Houben, Eikelenboom and Hoving-Bolink (1998) found that supplemented (200 IU a-tocopheryl acetate kgÿ1 feed) minced pork was more resistant to lipid oxidation than control meat when stored for 10 days at 7 C in an illuminated retail display cabinet. Freybler, Gray, Asghar, Booren, Pearson and Buckley (1993) found that TBARS values for microsomes and mitochondria from cured (156 mg/kg sodium nitrite) pork were signi®cantly (P<0.05) lower than those from uncured pork. Hams manufactured from supplemented muscles cured with 25 mg nitrite/kg meat showed signi®cantly (P<0.01) lower TBARS values during retail storage than hams manufactured from basal muscles treated with 100 mg nitrite/kg meat. This result was also found in the case of colour stability. Such a ®nding would suggest vitamin E as a partial substitute for nitrite in terms of maintaining the desirable colour and oxidative stability of cooked hams. The in¯uence of sex of the animal on TBARS values was found not to be signi®cant when investigated among each of the four treatments and results are not shown here. The oxidative stability of hams manufactured from male porcine muscles did not dier signi®cantly from hams manufactured from female muscles. A recent study by Brocks, Hulsegge and Merkus (1998) investigated the histochemical characteristics of Large White pigs and found that muscle properties were not the same in boars and gilts but sex dierences were only observable in meat colour. Acknowledgements This study was supported by funding from HomannLa Roche, Basel, Switzerland.
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Reduced nitrite levels and dietary a-tocopheryl acetate supplementation: eects on the colour and oxidative stability of cooked hams N.M. Dineen a, J.P. Kerry a,*, P.B. Lynch b, D.J. Buckley a, P.A. Morrissey a, E.K. Arendt a a
Department of Food Science and Technology, University College Cork-National University of Ireland, Cork, Ireland b Teagasc, Moorepark, Fermoy, Co. Cork, Ireland Received 14 July 1999; received in revised form 5 November 1999; accepted 5 January 2000
Abstract The objective of the present study was to determine the eects of dietary vitamin E supplementation and reduced nitrite levels on the colour stability of cooked hams. Large white Landrace pigs (male n=6, female n=6) were each subdivided into two groups (n=3) and fed an a-tocopheryl acetate supplemented diet (1000 mg/kg feed) and a basal diet (10 mg/kg feed) for a period of 10 weeks. M. semitendinosus were removed from each pig, divided into light and dark pigmented fractions, vacuum packed and stored at 4 C for 24 h. Muscles were cured with input nitrite levels of 25 and 100 mg/kg meat and were tumbled and massaged for 17 h. Samples were cooked, sliced and overwrapped in a high oxygen permeable ®lm for a storage period of 10 days. Surface colour of hams was measured and expressed as Hunter `a' values. Concentrations of a-tocopherol were signi®cantly (P<0.001) greater in supplemented muscles compared to basal muscles for both male and female pigs. Hams manufactured from male and female supplemented pigs resulted in signi®cantly (P<0.001) higher Hunter `a' values than hams manufactured from male and female pigs receiving the basal diet. Muscles cured with 100 mg nitrite/kg meat formed products with signi®cantly (P<0.001) higher `a' values than those cured with the lower (25 mg/kg meat) nitrite level. Hams manufactured from supplemented muscles, treated with 25 mg nitrite/kg meat showed signi®cantly (P<0.05) higher Hunter `a' values than hams manufactured from basal muscles, treated with 100 mg nitrite/kg meat. Hams manufactured from female porcine muscles had signi®cantly (P<0.001) higher `a' values than hams from male muscles during the 10 days of simulated retail display. No such gender dierences were observed for TBARS values. # 2000 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Colour is an extremely critical component in the appearance of meat and has a substantial in¯uence on consumer purchase decisions (Sherbeck, Wulf, Morgan Tatum, Smith & Williams, 1995). The use of sodium nitrite in cured meats is responsible for the characteristic pink meat colour and ¯avour stability (Tichivangana, Morrissey & Buckley, 1984). Nitrite acts as a potent catalyst in lipid oxidation (Claus, Colby & Flick, 1994) and provides a preservative eect, especially against the outgrowth of spores of Clostridium botulinum (Cassens, 1995). However, the use of sodium nitrite in muscle * Corresponding author. Tel.: +353-21-902256; fax: 353-21276318. E-mail address: [email protected] (J.P. Kerry).
foods may lead to the formation of carcinogenic nitrosamines. Sarasua and Savitz (1994) observed a positive association between brain tumours and childhood consumption of ham, bacon and hot dogs. Bunin, Kuijten, Boesel, Buckley and Meadows (1994) reported an increased incidence of tumours in children whose mothers consumed cured meats. Cassens (1997) showed an approximate 80% reduction in the residual nitrite content of cured meats since the 1970s. However, the search for suitable alternatives to nitrite is still ongoing. a-Tocopherol is a highly eective lipid soluble, chain breaking antioxidant which is acceptable to the consumer (Faustman, Cassens, Schaefer, Buege, Williams & Scheller, 1989). a-Tocopherol, when incorporated in cell membranes by dietary means improves the antioxidant defense system and is an eective means of stabilising raw muscle during frozen storage and muscle
0309-1740/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00008-5
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food during processing, thus increasing the shelf-life of muscle foods during subsequent processing and distribution (Buckley, Morrissey & Gray, 1995). Localisation of a-tocopherol in membranes allows it to function eciently compared to other antioxidants (Lauridsen, Buckley & Morrissey 1997). a-Tocopherol protects post-slaughter animal tissues and muscle foods against lipid oxidation and indirectly delays oxymyoglobin oxidation and meat discolouration (Morrissey, Buckley, Sheehy & Monahan, 1994). Discolouration in retail meats during display conditions may be a combined function of muscle pigment oxidation (oxymyoglobin to methmyoglobin) and lipid oxidation occurring in membrane phospholipids (Sherbeck et al., 1995). Nitrosylmyoglobin, the cured meat pigment, is unstable in air and discolouration can be rapid. Light can catalyse the dissociation of nitric oxide from cured meat pigments and cause colour fading especially when oxygen is present (Varnam & Sutherland, 1995). The use of dietary vitamin E and nitrite in combination, to stabilise colour and inhibit lipid oxidation has received little attention with the exception of recent studies by Walsh, Kerry, Buckley and Arendt (1998), Walsh, Kerry, Buckley, Morrissey, Lynch and Arendt (1998) and Houben and Gerris (1998). Walsh, Kerry, Buckley and Arendt (1998) and Walsh, Kerry, Buckley, Morrissey et al. (1998) found that vitamin E supplementation had signi®cant bene®cial eects on the colour and oxidative stability of low nitrite cooked hams and turkey patties products. Houben and Gerris (1998) on examining the eect of dietary vitamin E on the colour stability of pasteurised ham reported that supplementation with vitamin E conferred only slightly bene®cial eects on ham quality. The objective of the present study was to examine the eects of reduced nitrite levels and dietary vitamin E supplementation on the colour and oxidative stability of cooked hams processed from both male and female porcine M. semitendinosus. Vitamin E supplementation levels used in this study were higher than those used previously by either Houben and Gerris (1998) or Walsh, Kerry, Buckley and Arendt (1998) and Walsh, Kerry, Buckley, Morrissey et al. (1998) and the low nitrite level was lower than that used by Walsh, Kerry, Buckley, Morrissey et al. (1998). 2. Materials and methods 2.1. Reagents All chemicals were `Analar' grade obtained from British Drug Houses, Poole, Dorset, UK or Sigma Chemical Co. Ltd, Poole, Dorset, UK. a-Tocopheryl acetate was obtained from Roche Products Ltd, Welwyn Garden City, Hertfordshire, UK. Salt (pure dry vacuum salt) was obtained from Runcorn, Cheshire,
UK. Nitrite (food grade) was obtained from British Drug Houses, Poole, Dorset, UK 2.2. Animals and diets Pigs (n=12) were selected at 32±36 kg live weight and were comprised of male (n=6) and female (n=6) animals. Half of each gender group were fed a basal diet of 10 mg a-tocopheryl acetate/kg feed and the remainder fed a supplemented diet of 1000 mg a-tocopheryl acetate/ kg feed for a period of 10 weeks. Pigs were penned in a slatted area at the Pig Husbandry Unit, Agriculture and Food Development Authority (Teagasc), Moorepark Research Centre, Fermoy, Co. Cork, Ireland. Temperature control was maintained using a mechanical ventilation system. Composition of the diets are shown in Table 1. Pigs were given water and fed ad libitum. Pigs were slaughtered at Cappoquin Pork and Bacon Factory, Cappoquin, Co. Waterford. 2.3. Sampling procedure Carcasses were split centrally and chilled at 4 C for 24 h. M. semitendinosus (n=12), were identi®ed and removed from the left hind limb of each pig. Dark fractions of the muscle were removed and excluded from analysis. Light fractions of the muscles were vacuum packed and stored at ÿ20 C until required. 2.4. Ham manufacture Male and female M. semitendinosus from both supplemented (1000 mg a-tocopheryl acetate/kg feed and basal (10 mg a-tocopheryl acetate/kg feed) dietary groups were cured with 25 and 100 mg nitrite/kg meat and an input salt level of 2% giving a sum of four treatments for each of the male and female pigs. The four cooked ham samples will from this point be referred to as Table 1 Composition of experimental diet kg/tonnea Component
Finisher
Barley Wheat Soya 50 Tallow Lysine synthetic Methionine synthetic Threonine synthetic Di calcium phosphate Limestone ¯our Salt Vitamins-minerals Mould curb Totals
350 399.75 215 10 2.5 0.75 1 5 11 3 1.5 0.5 1000
a
Kemin Industries Inc., Iowa, USA.
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(a) supplemented a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (b) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (c) basal atocopheryl acetate muscles plus 100 mg nitrite/kg meat and (d) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat. Muscles were pumped by hand to 115% of initial weight using a multi-needle injector pump (Dick, F., Postfach 209, 73703 Esslingen, Germany). Samples were tumbled and massaged for 10 min in every 30 min, at a temperature of 4 C for a total of 17 h at a speed of 10 rpm (Kerry, 1997). The meat was vacuum packed using a Webomatic vacuum packer (Webomatic vacuum packaging Systems, Werner Bonk, Mausegatt 59, D 463 Bochum 6, Germany) and held at 4 C for 36 h to allow for colour development. Samples were cooked at 80 C in a Memmert Oven (Memmert Gmbh, Aussere Rittersbacher Strasse 38, 91126 Schwabach, Germany) to an internal meat temperature of 72 C, measured using a Testo 700 digital thermometer probe (Testotherm Gmbh, Postfach 1140, D-7825 Lenzkirch/Schwartzwald, Germany). Cooked hams were cooled at 4 C for 6 hr, sliced and overwrapped in oxygen permeable (6000±8000 cm3/m2/24 h at standard temperature and pressure) cling®lm (Wrap Film Systems Ltd., Hales®eld 14, Telford TF7 4QR, Shropshire, England). All samples were stored in a display cabinet [Criobanc (ISS20250)] under ¯ourescent light (Osram Natura De Luxe [L36W/76-1]) at 4 C for 10 days.
477
5C18 (2500.4 mm) reverse phase column and a Waters model 486 UV-visible wavelength detector (Milipore Corporation, Milford, MA 01757, USA) set at 292 nm. The mobile phase used was methanol water (97:3) run at a ¯ow rate of 2 ml/min. Data were recorded on a Milipore Millenium 2010 chromatography management system (Milipore Corporation, Milford, MA 01757, USA). 2.8. Nitrite determination Nitrite levels were determined in triplicate on ham samples using the method of Follett and Ratcli (1963). 2.9. Statistical analysis a-Tocopherol concentrations, Hunter `a' values and TBARS values were analysed by repeated measures analysis of variance (ANOVA) using the SPSS statistical package [SPSS, Inc. (1997) SPSS Advanced Statistics 7.5, Chicago, Illinois]. 3. Results and discussion 3.1. Concentration of -tocopherol in muscle tissue
Surface colour measurements (Hunter L, a, b values) on ham slices were recorded in triplicate using a Minolta Chromameter (Model CR-300; Minolta Camera Co., Chuo-Ku, Osaka 541, Japan).
Mean a-tocopherol concentrations for male and female supplemented muscles were 5.470.29 and 5.490.25 mg/g muscle, respectively and for male and female basal muscles were 0.560.08 and 0.540.06 mg/g muscle, respectively. No signi®cant dierences in a-tocopherol levels were observed between supplemented male and female muscles or between basal male and female muscles.
2.6. Lipid oxidation determinations
3.2. Residual nitrite analysis
Lipid oxidation in all muscle samples was assessed by the 2-thiobarbituric acid distillation method of Tarladgis, Watts, Younathan and Dugan (1960) as modi®ed by Ke, Ackman, Linke and Nash (1977). Absorbance was read at 538 nm using a Beckman DU 640 spectrophotometer (Beckman Instruments, 2500 Harbour Boulevard, Fullerton, CA 92634-3100, USA). Results were expressed as mg malonaldehyde/kg tissue. Each sample was analysed in triplicate.
Residual nitrite levels, measured 4 days after initial injection, were in the range of 16±21 mg/kg meat for muscles cured with 25 mg nitrite/kg meat and 76±92 mg/ kg meat for muscles cured with 100 mg nitrite/kg meat. These results were in agreement with levels reported by Froehlich, Gullet and Usborne (1983) who estimated that nitrite losses ranged from 22±34% but disagreed with those of Cassens (1995) who reported that only about 10±20% of the originally added nitrite is analytically detectable and declines further during storage and distribution.
2.5. Colour measurement
2.7. -Tocopherol determination Meat samples (10 g) were taken from the centre of each supplemented and basal muscle. The method reported by Sheehy, Morrissey and Flynn (1994) was used for a-tocopherol extraction and HPLC analysis. HPLC was carried out using a Waters model S10 pump, Waters 717 autosampler, a Machery Nagel Nucleosil
3.3. Colour stability Hunter `a' values of overwrapped cooked ham slices from supplemented and basal male and female muscles, treated with high and low levels of nitrite are shown in Fig. 1a and b, respectively. Hunter `a' values decreased
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Fig. 1. (a and b). Eect of dietary a-tocopheryl acetate and reduced nitrite levels on the Hunter `a' values of overwrapped cooked ham slices manufactured from (a) male and (b) female M. semitendinosus and stored under retail display conditions at 4 C for 10 days. (*) Supplemented atocopheryl acetate muscles plus 100 mg nitrite/kg meat; (*) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (&) basal atocopheryl acetate muscles plus 100 mg nitrite/kg meat; (&) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat.
signi®cantly (P<0.001) during the 10 days storage period for each of the four treatments from both male and female muscles. Hams manufactured from supplemented a-tocopheryl acetate muscles cured with 100 mg nitrite/kg meat exhibited the greatest (P<0.001) degree of surface redness during retail storage, while hams manufactured from basal a-tocopheryl acetate muscles cured with 25 mg nitrite/kg meat showed poorest colour
stability. These ®ndings were in agreement with those reported by Walsh, Kerry, Buckley and Arendt (1998) and Walsh, Kerry, Buckley Morrissey et al. (1998). However, a recent study by Houben and Gerris (1998) reported that dietary supplementation with vitamin E did not appear to oer signi®cant advantages over currently used feeding regimes with regard to the quality of pasteurised ham.
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Repeated measures of analysis of variance was performed on the colour results obtained (Table 2). There were no signi®cant dierences at day 0 between any of the factors of interest in hams from either male or female pigs. From days 2±10, a clear diet eect emerged. Hams manufactured from supplemented muscles resulted in signi®cantly (P<0.001) higher Hunter `a' values than hams manufactured from basal muscles. This eect was not in¯uenced by time or nitrite levels and the trend was the same for hams produced from both male and female muscles. Hams from muscles cured with 100 mg nitrite/kg meat had signi®cantly (P<0.001) higher
Table 2 ANOVA analysis on Hunter `a' values of ham slices from M. semitendinosus Source of variation
F-value
P-value
Diet Gender Nitrite Day Daygender
135.42 31.22 90.73 77.10 4.14
<0.001 <0.001 <0.001 <0.001 0.007
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Hunter `a' values than muscles cured with 25 mg nitrite/ kg meat. This eect was not dependant on a-tocopherol level or time and the trend was common to hams from both male and female muscles. Hams manufactured from supplemented muscles cured with 100 mg nitrite/ kg meat showed the greatest (P<0.001) colour stability during storage while hams manufactured from basal muscles cured with 25 mg nitrite/kg meat performed least (P<0.001) favourably. However, hams manufactured from supplemented muscles cured with 25 mg nitrite/kg meat showed signi®cantly (P<0.05) greater colour stability during retail storage than hams manufactured from basal muscles cured with 100 mg nitrite/ kg meat. Walsh, Kerry, Buckley, Morrissey et al. (1998) found that ham slices produced from supplemented (500 mg a-tocopheryl acetate/kg feed) M.semimembranosus cured with 100 mg nitrite/kg meat had signi®cantly higher `a' values than hams processed from basal (10 mg a-tocopheryl acetate/kg feed) muscles cured with 50 mg nitrite/kg meat. Hams from supplemented muscles cured with 50 mg nitrite/kg meat had similar colour stability to hams from basal muscles cured with 100 mg nitrite/kg meat. O'Sullivan, Kerry, Buckley, Lynch and Morrissey (1998) found that Hunter `a' values of both
Fig. 2. Comparison of the Hunter `a' values of overwrapped cooked ham slices manufactured from male (&) and female (&) M. semitendinosus across each treatment level. (a) Supplemented a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (b) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (c) basal a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (d) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat.
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M. gluteobiceps and M. longissimus dorsi from the supplemented group (160 mg a-tocopheryl acetate/kg feed) were signi®cantly (P<0.05) more stable to colour change than those from the basal group (20 mg a-tocopheryl acetate/kg feed) during storage for 7 days at 4 C under ¯ourescent light. Walsh, Buckley and Arendt
(1998) found that colour stability of cooked cured turkey patties from supplemented vitamin E muscles (600 mg a-tocopheryl acetate/kg feed) was signi®cantly greater than those from basal muscles (20 mg a-tocopheryl acetate/kg feed). The signi®cant (P<0.001) eect of nitrite level on colour stability was in agreement with
Fig. 3. (a and b). Eect of dietary a-tocopheryl acetate and reduced nitrite levels on the TBARS values of overwrapped cooked ham slices manufactured from (a) male and (b) female M. semitendinosus and stored under retail display conditions at 4 C for 10 days. (*) Supplemented a-tocopheryl acetate muscles plus 100 mg nitrite/kg meat; (*) supplemented a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat; (&) basal atocopheryl acetate muscles plus 100 mg nitrite/kg meat; (&) basal a-tocopheryl acetate muscles plus 25 mg nitrite/kg meat.
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®ndings by Walsh, Kerry, Buckley and Arendt (1998), Walsh, Kerry, Buckley, Morrissey et al. (1998) and Deumier, Zakhia and Collignan (1996) who reported that cured venison tenderloins showed greater `a' values with increasing nitrite concentration. The in¯uence of porcine gender on Hunter `a' values was investigated separately among the four treatments. Hunter `a' values of cooked ham slices from male and female porcine M. semitendinosus are shown in Fig. 2. Hams produced from female porcine muscles had signi®cantly (P<0.01) higher `a' values than hams produced from male porcine muscles. The dierence in colour stability between male and female porcine M. semitendinosus can not be accounted for by dierences in muscle a-tocopherol concentrations. Muscles have dierent colour stabilities and the dierence in colour stability among muscles has been explained by the dierences in respiratory activity; muscles having higher oxidative activity are more colour labile (Renerre & Labas, 1987). However, this is not relevant to cooked cured muscles. It is not clear as to why hams from pig breeds that have been re®ned in their breeding should show such signi®cant colour dierences between gender. It is possible that biochemical dierences or metabolites present may be responsible for colour dierences observable in this study. 3.4. Oxidative stability TBARS values of overwrapped cooked ham slices from supplemented and basal male and female muscles, treated with high and low levels of nitrite are shown in Fig. 3a and b, respectively. TBARS values increased signi®cantly (P<0.001) during the 10 day storage period for each of the four treatments from both male and female muscles. As well as showing the highest colour stability, hams manufactured from supplemented atocopheryl acetate muscles cured with 100 mg nitrite/kg meat exhibited the greatest (P<0.001) degree of oxidative stability during retail storage while hams manufactured from basal a-tocopheryl acetate treated with 25 mg nitrite/kg meat showed the highest TBARS values. Repeated measures of analysis of variance were performed on the TBARS values obtained (Table 3). There
Table 3 ANOVA analysis on TBARS values of ham slices from M. semitendinosus Source of variation
F-value
P-value
Diet Gender Nitrite Day Daygender
129.82 0.09 87.69 69.22 0.17
<0.001 0.51 <0.001 <0.001 0.87
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were no signi®cant dierences between any of the factors of interest in either male or female derived hams at days 0. Hams manufactured from supplemented atocopheryl acetate muscles showed signi®cantly (P<0.001) lower TBARS values than hams manufactured from basal muscles. The eect was not in¯uenced by time or nitrite level and the trend was the same for hams produced from both male and female muscles. Hams from muscles cured with 100 mg nitrite/kg meat had signi®cantly (P<0.001) lower TBARS values than hams from muscles cured with 25 mg nitrite/kg meat. This eect was not dependant on a-tocopherol level or time and the trend was also common to both male and female muscles. The eects of nitrite and a-tocopherol level on oxidative stability were signi®cant between days 2 and 10. Pfalzgraf, Frigg and Steinhart (1995) who observed that M. longissimus dorsi from pigs fed 200 mg a-tocopheryl acetate/kg feed were signi®cantly more stable (P<0.05) after 7 days of storage at 4 C compared to muscles from pigs fed a basal level (40 mg a-tocopheryl acetate/kg feed). Houben, Eikelenboom and Hoving-Bolink (1998) found that supplemented (200 IU a-tocopheryl acetate kgÿ1 feed) minced pork was more resistant to lipid oxidation than control meat when stored for 10 days at 7 C in an illuminated retail display cabinet. Freybler, Gray, Asghar, Booren, Pearson and Buckley (1993) found that TBARS values for microsomes and mitochondria from cured (156 mg/kg sodium nitrite) pork were signi®cantly (P<0.05) lower than those from uncured pork. Hams manufactured from supplemented muscles cured with 25 mg nitrite/kg meat showed signi®cantly (P<0.01) lower TBARS values during retail storage than hams manufactured from basal muscles treated with 100 mg nitrite/kg meat. This result was also found in the case of colour stability. Such a ®nding would suggest vitamin E as a partial substitute for nitrite in terms of maintaining the desirable colour and oxidative stability of cooked hams. The in¯uence of sex of the animal on TBARS values was found not to be signi®cant when investigated among each of the four treatments and results are not shown here. The oxidative stability of hams manufactured from male porcine muscles did not dier signi®cantly from hams manufactured from female muscles. A recent study by Brocks, Hulsegge and Merkus (1998) investigated the histochemical characteristics of Large White pigs and found that muscle properties were not the same in boars and gilts but sex dierences were only observable in meat colour. Acknowledgements This study was supported by funding from HomannLa Roche, Basel, Switzerland.
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