- Email: [email protected]
Effect of dietary vitamin E supplementation on the shelf life of cured pork sausage
Meat Science 63 (2003) 101–105 www.elsevier.com/locate/meatsci
Effect of dietary vitamin E supplementation on the shelf life of cured pork sausage C. Harmsa, H. Fuhrmannb, B. Nowakc, S. Wenzelc, H.-P. Sallmanna,* a
Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bu¨nteweg 17, D-30559 Hannover, Germany Veterinary-Physiological Chemical Department, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 43, D-04103 Leipzig, Germany c Department of Food Science, Meat Hygiene and Technology, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany b
Received 1 October 2001; received in revised form 4 March 2002; accepted 4 March 2002
Abstract This study examined the shelf life of cured sausage under different packaging conditions from vitamin E supplemented pigs. One group (n=6) of crossbred pigs received a normal fattening diet containing 20 ppm a-tocopherol for 39 days before slaughter. Another group was fed a diet containing 410 ppm a-tocopherol during the same period. After slaughter, cured sausages were produced, packaged under three different atmospheric conditions, ripened for 4 weeks and then stored for 8 weeks (9 C; 200 lux). The a-tocopherol content was recorded in the fat, liver, muscles and the sausage. TBARS, L*, a*, b* values and antioxidative capacity were evaluated in the sausage during storage. The results showed a transfer of vitamin E into tissues and sausage but no detectable effect on TBARS and colour stability. However, antioxidative effects of vitamin E were seen by provocation. Probably the effect of vitamin E in the sausage was masked by nitrite in the curing salt. The oxygen content of the packs had an influence on TBARS and colour stability. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cured pork sausage; Shelf life; Lipid peroxidation; Packaging; Vitamin E feed supplements
1. Introduction Meat colour and odour are two components which affect consumer acceptability of meat products. Several American and European studies on the use of vitamin E to prolong the shelf life of meat have dealt with its effects in fresh, frozen and processed pork, beef and poultry meat. All such studies showed the beneficial effects of vitamin E on lipid peroxidation in meat, usually evaluated by TBARS (thiobarbituric reactive substances) concentration (Faustman, Chan, Schaefer, & Havens 1998; Hoving-Bolink, Eikelenboom, van Diepen, Jongbloed, Houben, & van Diepen 1998; Liu, Scheller, Arp, Schaefer, & Williams 1996). However, studies focussing on colour stability were less clear.
* Corresponding author. Fax: +49-511-953-8585. E-mail address: [email protected] (H.-P. Sallmann).
Positive antioxidative effects of vitamin E were seen (Faustman et al., 1998; Monahan, Asghar, Gray, Buckley, & Morrissey 1994), but there are also reports of no influence of the vitamin on colour stability (Cannon et al., 1996; Houben & Gerris, 1998). In all studies vitamin E was added as a supplement to the feed of the animals, at different dosages for different times and there was a clear transfer of fed vitamin E into the tissue of the animals. The highest concentrations were found in liver and fat due to the metabolism of vitamin E and its fat solubility, respectively. Packaging conditions, in which the oxygen content was varied, also influenced colour stability (Andersen, Bertelsen, Ohlen, & Skibsted 1990; Miles, McKeith, Bechtel, & Novakofski, 1986). As little is known about the effects of vitamin E on meat products packed under different conditions, the present study looked at the oxidative and colour stability of the stored and packaged cured sausage made from pigs fed different vitamin E supplements.
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(02)00061-X
102
C. Harms et al. / Meat Science 63 (2003) 101–105
2. Materials and methods 2.1. Animals and diets Twelve hybrid crossbred pigs (castrates) were used in this study. The pigs were randomly divided into two groups. They were housed in an experimental animal house of the Department of Animal Nutrition, at the School of Veterinary Medicine Hannover (Germany). Average body weight at the beginning of the experiment was 87 kg. The two treatment groups received the experimental feed for 39 days prior to slaughter (at approximately 115 kg). The animals were fed twice a day with a total of 3 kg of liquid mealy feed with free access to water. The feed was identical for both treatment groups with a net energy content of 12.3 MJ/kg and a basic content of 10 mg vitamin E per kg feed. The rations of the control group and the experimental group were supplemented with 10 mg and 400 mg of vitamin E (DL-a-tocopherol acetate; Cuxavit E50, Lohmann, Cuxhaven, Germany) per kg diet, respectively. 2.2. Collection of tissue specimens Immediately after slaughter samples of m. longissimus dorsi, m. gluteobiceps, liver and fat tissue (5 g each) were taken from the carcasses and stored at 20 C for analysis. 2.3. Production and treatment of the sausage In a commercial food plant batches of 540 g of cured pork sausage were produced from the meat of each treatment group. The recipe is given in Table 1. The levels of nitrite in the sausage was 135ppm. The absence of ascorbic acid as an antioxidant was the only modification of the recipe normally used commercially. Ripening was carried out for 12 days according to a commercial ripening procedure (Table 2). Prior to packaging the sausages were sliced, alloted to units of 115 g each, and arranged steplike on trays. The Table 1 Recipe for the basic components of the sausagea
2.4. Measurements The samples from the carcasses and the sausages were analysed for vitamin E content weekly by the method of Rammell, Cuncliffe, and Kieboom (1983) using reversed phase high-pressure liquid chromatography (rpHPLC) and fluorescence detection. This procedure made it possible to separate isomers. The colour of the sausage slices was measured as L*, a*, b* values with a Minolta chromometer CR-100 according to the Commission Internationale de l’Eclerage 1976 (CIE). Lipid oxidation in the sausage was assessed by the TBARS values (thiobarbituric acid reactive substances) as adapted from the work of Uchiyama and Mihara (1978). The measurement of the antioxidative capacity of the samples during storage was performed by inducing oxidative stress in the material while observing luminescence suppression by vitamin E. 0.1 ml of the oxidative agent ABAP (azo-bis-amidinopropane-dihydrochloride; 1.94g /10 ml aqua bidest) was added to 0.9 ml of sausage tissue homogenate (2.5%) in potassium-phosphate buffer (30 mM, pH7.4). The measurement of luminescence and the injection of ABAP to each sample was carried out at 37 C in an Autolumat Lb 953 (EG&G Berthold, Wildbad, Germany). The resulting luminescence was observed for 120 min and the curve characteristics evaluated. 2.5. Statistical analysis Statistical evaluations were performed by the SAS program Ver. 6.12 (SAS Institute Inc., Cary NC, USA). Table 2 Ripening procedure
Components
kg
Meat of shoulder, air dried Pork meat Ventral meat Cooked pork fat Subcutaneous adipose tissue Enzymes Curing nitrite, iodinated Mustard seed Lactose/dextrose Spice mixture*
48.00 87.00 75.00 10.00 10.00 0.44 5.94 0.33 4.70 1.45
a
packaging material was a transparent PET/PE plastic wrap with an oxygen permeability of 10.6 cm3/m3/ bar in 24 h at 50% atmospheric humidity. A third of each sausage portion was vacuum packed, another third in N2/CO2 (80/20%) and a further third in air. The sausage was then stored for 8 weeks, at 9 C with continuous illumination of 200 lux (neon light) on the surface of the packages. Three packages of each of the six subgroups were analysed weekly.
70% pepper, 15% coriander, 5% ginger, 5% garlic, 5% pimento.
Step
Time (h)
Temperature ( C)
Humidity (%)
1 2 3 4 5 6 7 8 9 10
6 18 24 24 24 12 12 24 24 120
25 25 22 22 21 20 20 18 18 18
25.0 25.0 30.8 37.4 42.0 52.0 60.0 59.4 61.2 72.0–78.0
C. Harms et al. / Meat Science 63 (2003) 101–105
A three-way ANOVA was chosen to verify the influence of vitamin E supplementation, packaging conditions and storage on the data. Differences between data were tested using the Tukey-test or by comparing the least square means (significance P < 0.05).
3. Results 3.1. Vitamin E content Added vitamin E in the feed of the pigs increased the vitamin E contents of the tissue samples and the sausage. All tissue specimens from the supplemented group showed significantly higher vitamin E concentrations (up to seven-fold in liver) than the samples from the control animals (Fig. 1). There was up to a two-fold increase in vitamin E in the sausages made from the high-supplement group compared with the controls independent of the packaging. The lowest vitamin E values were found in the air-packed samples (Fig. 2). There was no change in vitamin E concentration in the sausages, from either treatment group in the three different packaging conditions, over the entire storage period (data not shown).
Fig. 1. a-Tocopherol content of different tissue samples of pigs fed a-tocopherol supplemented diets (meanS.E.M., n=6), § indicates significant differences between the supplemented groups (Tukey-test; P<0.05).
Fig. 2. a-Tocopherol content of sausages under different packaging conditions (meanS.E.M., n=6) at the end of storage; different upper or lower-case letters indicate significant differences between packaging conditions and tocopherol supplementation (Tukey-test; P<0.05).
103
3.2. Colour stability The colour stability of the sausages was not influenced by vitamin E content. However, packaging conditions did significantly affect the colour during storage. There was no colour change (E) in the vacuum-packed sausages (E < 1). But the colour changes in sausages packed under N2/CO2 (E=3.05) and air (E=6.41) were clearly visible during storage of eight weeks (Fig. 3). (Sensorially visible colour changes have E > 1; Yudd and Wyszecki, 1975). 3.3. Lipid peroxidation Lipid peroxidation in the sausages, as assessed by TBARS was not influenced by vitamin E content. The assumed oxygen contents of the different packages significantly affected TBARS, higher values being seen in the oxygen packed samples (Fig. 4). Storage time did not lead to the expected increase in TBARS. 3.4. Antioxidative capacity Storage time had no influence on the development of the antioxidative capacity of the sausage. Contrary to colour development and lipid peroxidation vitamin E content influenced the measured luminescence (see Fig. 5
Fig. 3. Change of colour (L*, a*, b*-values) of sausages under different packaging conditions (meanS.E.M., n=6) at the end of the storage period; different letters indicate significant differences between packaging conditions (Tukey-test; P <0.05).
Fig. 4. TBARS of sausages under different packaging conditions (meanS.D., n=6); filled symbols indicate a significant decrease between first and last sample of the specified packaging (P <0.05).
104
C. Harms et al. / Meat Science 63 (2003) 101–105
and Table 3). Sausages from the high supplemented group showed significantly lower light emission as shown by the areas under the curve (AUC) (Table 3) and lag phases and peak heights of the curves (Fig. 5). The lowest antioxidative capacity was measured in the sausage packed in air. Lag phase (tlag) before the beginning of light emission after catalysis with ABAP was shortest in the air packed sausage.
4. Discussion The vitamin E contents in the tissues confirmed results from other investigators (Morrissey, Buckley, Sisk, Lynch, & Sheehy 1996; O’Sullivan, Kerry, Buckley, Lynch, & Morrissey, 1997). The transfer of the vitamin differs between tissues. Liver is the main temporary store for vitamin E and thus shows the highest concentrations of this micronutrient; the vitamin E content in fat from the controls and supplemented animals is only 1:2, whereas in liver it is 1:7. The feeding time of 5 weeks was probably insufficent to concentrate vitamin E to the amounts in fat as found by Morrissey et al. (1996) in their long term (18 weeks) study. Vitamin E contents in the sausages and the fat are similar from both the control and experimental group. This is because nearly 50% of fat and fat tissue is incorporated into the sausage. During ripening and storage the vitamin E concentration in the sausages did not decrease. This agrees with results from Liu et al. (1996). The transfer of vitamin E had no detectable effect on colour stability and lipid peroxidation in the sausages. The curves of the TBARS results (Fig. 4) indicate differences between the packaging systems (LS mean: P < 0.001; n=30). The L*, a*, b* colour values showed similar variation due to packaging. The clear advantage of using packaging with low oxygen content confirms the results of Miles et al. (1986) for pork and of Ahn, Sell, Jefferey, Jo, Chen, Wu, and Lee (1997) for turkey. This study does not confirm the beneficial influence of vitamin E on colour and lipid peroxidation found for
fresh or frozen beef or pork (Faustman, Cassens, Schaefer, Buege, Williams, & Scheller, 1989; HovingBolink et al., 1998; Liu et al., 1996). We assume that nitrite, with its powerful antioxidative effect probably masked the effect of vitamin E. The antioxidative power of nitrite is based on its binding to the muscle pigment myoglobin and its conversion to nitrosyl myoglobin. In this structure myoglobin cannot act as a catalyst of lipid peroxidation (Gray & Pearson 1984; Kanner, Harel, & Granit 1991; O’Boyle, Rubin, Diosady, Aladin-Kassam, Comer, & Brightwell 1990). The results reported thus suggest that the well-known ‘‘scavenger’’ functions of vitamin E during peroxidation in fresh and frozen meat are not realized in un-pretreated sausage. Using catalytic test systems (chemiluminescence test with ABAP), the scavenging activity of the supplemented vitamin E could be detected in the stored sausage despite the presence of nitrite. This is obviously due to inhibition of peroxidation, which can be initiated by the free muscle pigments (ca. 35%, see Carballo, Cavestany, & Jime´nez-Colmenero, 1991). Reinforced radical production resulted in high chemiluminescence with the largest AUCs and short lag phases. In other words our data indicate the beneficial effects of packaging conditions on colour and lipid peroxidation in sausages when oxygen concentrations are low. This agrees with earlier studies (Andersen et al., 1990). The more oxygen in the packages, the more undesirable changes are seen for both parameters. Therefore beneficial effects of packaging is ranked as follows: vacuum is preferable to N2/CO2 atmosphere which is preferable to air. This confirms the results of other authors (Andersen et al. 1990; Houben & Gerris 1998). There have been no previous reports in the literature on luminescence measurements in meat and meat products stabilised with vitamin E. Studies conducted to check luminescence suppression used synthetic food antioxidants (Kahl, Weimann, Weinke, & Hildebrandt 1987; Noll, de Groot, & Sies 1987). The results reported showed reductions of luminescence by propylgallate, butylated hydroxytoluene and butylated hydroxyanisole (Kahl et al., 1987) and a clear dependence of radical production rate on oxygen content in the matrix (Noll et al., 1987). Table 3 Areas under the curve (AUC) of luminescence measurements (meanSD, n=3)a
Fig. 5. Development of luminescence after catalysis by ABAP in sausages made from the supplemented group and under different package conditions at the end of storage period; n=8.
a-Tocopherol in the diet
20 ppm
Package conditions
Mean
SD
mean
SD
Vacuum N2/CO2 Air
1.44a 1.43a 1.83b
0.11 0.14 0.29
1.33c 1.32c 1.52d
0.10 0.14 0.20
a
410 ppm
Values with different letters indicate significant differences (Tukey-test; P <0.05).
C. Harms et al. / Meat Science 63 (2003) 101–105
5. Conclusion and perspectives The safety consideration of nitrite contents in meat products, particularly in respect to cancer risk, make the reduction of curing salt advisible. For this reason and on the basis of the results reported in this work, further experiments with a gradual reduction of nitrite in cured sausages should be carried out. Antioxidants (natural or synthetic) may be added to the sausage material, either in vitro or as feed supplements for the fattening animals. Along these experimental lines the limits of colour transition of cured sausages which are still acceptable to consumers and antioxidative protection can be defined and determined. Such studies are currently underway in our laboratory.
Acknowledgements This work was conducted with the support of the Ahrberg Foundation. We gratefully acknowledge both the skilful technical help and assistance with graphics and text by Andrea Widdel.
References Ahn, D. U., Sell, J. L., Jefferey, M., Jo, C., Chen, X., Wu, C., & Lee, J. I. (1997). Dietary vitamin E affects lipid oxidation and total volatiles of irradiated raw turkey meat. Journal of Food Science, 62, 954–958. Andersen, H. J., Bertelsen, G., Ohlen, A., & Skibsted, L. H. (1990). Modified packaging as protection against photodegradation of colour of pasteurized, sliced ham. Meat Science, 28, 77–83. Cannon, J. E., Morgan, J. B., Schmidt, G. R., Tatum, J. D., Sofos, J. N., Smith, G. C., Delmore, R. J., & Williams, S. N. (1996). Growth and fresh meat quality characteristics of pigs supplemented with vitamin E. Journal of Animal Science, 74, 98–105. Carballo, J., Cavestany, M., & Jime´nez-Colmenero, F. (1991). Effect of light on colour and reaction of nitrite in sliced pork bologna under different chilled storage temperatures. Meat Science, 30, 235– 244. Faustman, C., Cassens, R. G., Schaefer, D. M., Buege, D. R., Williams, S. N., & Scheller, K. K. (1989). Improvement of pigment and lipid stability in Holstein steer beef by dietary supplementation with vitamin E. Journal of Food Science, 54, 858–862.
105
Faustman, C., Chan, W. K., Schaefer, D. M., & Havens, A. (1998) Beef color update: the role for vitamin E (1998). Journal of Animal Science, 76, 1019–1026. Gray, J. I., & Pearson, A. M. (1984). Cured meat flavour. Advances in Food Research, 29, 1–72. Houben, J. H., & Gerris, C. V. M. (1998). Effect of dietary supplementation with vitamin E on colour stability of packaged, sliced pasteurized ham. Meat Science, 50, 421–428. Hoving-Bolink, A. H., Eikelenboom, G., van Diepen, J., Jongbloed, A. W., Houben, J. H., & van Diepen, J. M. T. (1998). Effect of dietary vitamin E supplementation on pork quality. Meat Science, 49, 205–212. Kahl, R., Weimann, A., Weinke, S., & Hildebrandt, A. G. (1987). Detection of oxygen activation and determination of the activity of antioxidants towards reactive oxygen species by use of the chemiluminigenic probes luminol and lucigenin. Archives of Toxicology, 60, 158–162. Kanner, J., Harel, S., & Granit, R. (1991). Nitric oxide as an antioxidant. Archives of Biochemistry and Biophysics, 289, 130–136. Liu, Q., Scheller, K. K., Arp, S. C., Schaefer, D. M., & Williams, S. N. (1996). Titration of fresh meat color stability and malondialdehyde development with Holstein steers fed vitamin E-supplemented diets. Journal of Animal Science, 74, 117–126. Miles, R. S., McKeith, F. K., Bechtel, P. J., & Novakofski, J. (1986). Effect of processing, packaging and various antioxidants on lipid oxidation of restructured pork. Journal of Food Protection, 49(3), 222–225. Monahan, F. J., Asghar, A., Gray, J. I., Buckley, D. J., & Morrissey, P. A. (1994). Effect of oxidized dietary lipid and vitamin E on the colour stability of pork chops. Meat Science, 37, 205–215. Morrissey, P. A., Buckley, D. J., Sisk, H., Lynch, P. B., & Sheehy, P. J. A. (1996). Uptake of a-tocopherol in porcine plasma and tissues. Meat Science, 44, 275–283. Noll, T., de Groot, H., & Sies, H. (1987). Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation. Archives of Biochemistry and Biophysics, 252, 284–291. O‘Boyle, A. R., Rubin, L. J., Diosady, L. L., Aladin-Kassam, N., Comer, F., & Brightwell, D. (1990). A nitrite-free curing system and its application to the production of wieners. Food Technology, 48, 88–104. O‘Sullivan, M. G., Kerry, J. P., Buckley, D. J., Lynch, P. B., & Morrissey, P. A. (1997). The distribution of dietary vitamin E in the muscles of the porcine carcass. Meat Science, 45, 297–305. Rammell, C. G., Cunliffe, B., & Kieboom, A. J. (1983). Determination of alpha-tocopherol in biological speciemens by high performance liquid chromatography. Journal of Liquid Chromatography, 6, 1123–1130. Uchiyama, M., & Mihara, M. (1978). Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry, 86, 271–278. Yudd, D. B., & Wyszecki, G. (1975). Colour in business, science and industry. (3rd ed). New York: Wiley and Sons.
Effect of dietary vitamin E supplementation on the shelf life of cured pork sausage C. Harmsa, H. Fuhrmannb, B. Nowakc, S. Wenzelc, H.-P. Sallmanna,* a
Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bu¨nteweg 17, D-30559 Hannover, Germany Veterinary-Physiological Chemical Department, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 43, D-04103 Leipzig, Germany c Department of Food Science, Meat Hygiene and Technology, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany b
Received 1 October 2001; received in revised form 4 March 2002; accepted 4 March 2002
Abstract This study examined the shelf life of cured sausage under different packaging conditions from vitamin E supplemented pigs. One group (n=6) of crossbred pigs received a normal fattening diet containing 20 ppm a-tocopherol for 39 days before slaughter. Another group was fed a diet containing 410 ppm a-tocopherol during the same period. After slaughter, cured sausages were produced, packaged under three different atmospheric conditions, ripened for 4 weeks and then stored for 8 weeks (9 C; 200 lux). The a-tocopherol content was recorded in the fat, liver, muscles and the sausage. TBARS, L*, a*, b* values and antioxidative capacity were evaluated in the sausage during storage. The results showed a transfer of vitamin E into tissues and sausage but no detectable effect on TBARS and colour stability. However, antioxidative effects of vitamin E were seen by provocation. Probably the effect of vitamin E in the sausage was masked by nitrite in the curing salt. The oxygen content of the packs had an influence on TBARS and colour stability. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cured pork sausage; Shelf life; Lipid peroxidation; Packaging; Vitamin E feed supplements
1. Introduction Meat colour and odour are two components which affect consumer acceptability of meat products. Several American and European studies on the use of vitamin E to prolong the shelf life of meat have dealt with its effects in fresh, frozen and processed pork, beef and poultry meat. All such studies showed the beneficial effects of vitamin E on lipid peroxidation in meat, usually evaluated by TBARS (thiobarbituric reactive substances) concentration (Faustman, Chan, Schaefer, & Havens 1998; Hoving-Bolink, Eikelenboom, van Diepen, Jongbloed, Houben, & van Diepen 1998; Liu, Scheller, Arp, Schaefer, & Williams 1996). However, studies focussing on colour stability were less clear.
* Corresponding author. Fax: +49-511-953-8585. E-mail address: [email protected] (H.-P. Sallmann).
Positive antioxidative effects of vitamin E were seen (Faustman et al., 1998; Monahan, Asghar, Gray, Buckley, & Morrissey 1994), but there are also reports of no influence of the vitamin on colour stability (Cannon et al., 1996; Houben & Gerris, 1998). In all studies vitamin E was added as a supplement to the feed of the animals, at different dosages for different times and there was a clear transfer of fed vitamin E into the tissue of the animals. The highest concentrations were found in liver and fat due to the metabolism of vitamin E and its fat solubility, respectively. Packaging conditions, in which the oxygen content was varied, also influenced colour stability (Andersen, Bertelsen, Ohlen, & Skibsted 1990; Miles, McKeith, Bechtel, & Novakofski, 1986). As little is known about the effects of vitamin E on meat products packed under different conditions, the present study looked at the oxidative and colour stability of the stored and packaged cured sausage made from pigs fed different vitamin E supplements.
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(02)00061-X
102
C. Harms et al. / Meat Science 63 (2003) 101–105
2. Materials and methods 2.1. Animals and diets Twelve hybrid crossbred pigs (castrates) were used in this study. The pigs were randomly divided into two groups. They were housed in an experimental animal house of the Department of Animal Nutrition, at the School of Veterinary Medicine Hannover (Germany). Average body weight at the beginning of the experiment was 87 kg. The two treatment groups received the experimental feed for 39 days prior to slaughter (at approximately 115 kg). The animals were fed twice a day with a total of 3 kg of liquid mealy feed with free access to water. The feed was identical for both treatment groups with a net energy content of 12.3 MJ/kg and a basic content of 10 mg vitamin E per kg feed. The rations of the control group and the experimental group were supplemented with 10 mg and 400 mg of vitamin E (DL-a-tocopherol acetate; Cuxavit E50, Lohmann, Cuxhaven, Germany) per kg diet, respectively. 2.2. Collection of tissue specimens Immediately after slaughter samples of m. longissimus dorsi, m. gluteobiceps, liver and fat tissue (5 g each) were taken from the carcasses and stored at 20 C for analysis. 2.3. Production and treatment of the sausage In a commercial food plant batches of 540 g of cured pork sausage were produced from the meat of each treatment group. The recipe is given in Table 1. The levels of nitrite in the sausage was 135ppm. The absence of ascorbic acid as an antioxidant was the only modification of the recipe normally used commercially. Ripening was carried out for 12 days according to a commercial ripening procedure (Table 2). Prior to packaging the sausages were sliced, alloted to units of 115 g each, and arranged steplike on trays. The Table 1 Recipe for the basic components of the sausagea
2.4. Measurements The samples from the carcasses and the sausages were analysed for vitamin E content weekly by the method of Rammell, Cuncliffe, and Kieboom (1983) using reversed phase high-pressure liquid chromatography (rpHPLC) and fluorescence detection. This procedure made it possible to separate isomers. The colour of the sausage slices was measured as L*, a*, b* values with a Minolta chromometer CR-100 according to the Commission Internationale de l’Eclerage 1976 (CIE). Lipid oxidation in the sausage was assessed by the TBARS values (thiobarbituric acid reactive substances) as adapted from the work of Uchiyama and Mihara (1978). The measurement of the antioxidative capacity of the samples during storage was performed by inducing oxidative stress in the material while observing luminescence suppression by vitamin E. 0.1 ml of the oxidative agent ABAP (azo-bis-amidinopropane-dihydrochloride; 1.94g /10 ml aqua bidest) was added to 0.9 ml of sausage tissue homogenate (2.5%) in potassium-phosphate buffer (30 mM, pH7.4). The measurement of luminescence and the injection of ABAP to each sample was carried out at 37 C in an Autolumat Lb 953 (EG&G Berthold, Wildbad, Germany). The resulting luminescence was observed for 120 min and the curve characteristics evaluated. 2.5. Statistical analysis Statistical evaluations were performed by the SAS program Ver. 6.12 (SAS Institute Inc., Cary NC, USA). Table 2 Ripening procedure
Components
kg
Meat of shoulder, air dried Pork meat Ventral meat Cooked pork fat Subcutaneous adipose tissue Enzymes Curing nitrite, iodinated Mustard seed Lactose/dextrose Spice mixture*
48.00 87.00 75.00 10.00 10.00 0.44 5.94 0.33 4.70 1.45
a
packaging material was a transparent PET/PE plastic wrap with an oxygen permeability of 10.6 cm3/m3/ bar in 24 h at 50% atmospheric humidity. A third of each sausage portion was vacuum packed, another third in N2/CO2 (80/20%) and a further third in air. The sausage was then stored for 8 weeks, at 9 C with continuous illumination of 200 lux (neon light) on the surface of the packages. Three packages of each of the six subgroups were analysed weekly.
70% pepper, 15% coriander, 5% ginger, 5% garlic, 5% pimento.
Step
Time (h)
Temperature ( C)
Humidity (%)
1 2 3 4 5 6 7 8 9 10
6 18 24 24 24 12 12 24 24 120
25 25 22 22 21 20 20 18 18 18
25.0 25.0 30.8 37.4 42.0 52.0 60.0 59.4 61.2 72.0–78.0
C. Harms et al. / Meat Science 63 (2003) 101–105
A three-way ANOVA was chosen to verify the influence of vitamin E supplementation, packaging conditions and storage on the data. Differences between data were tested using the Tukey-test or by comparing the least square means (significance P < 0.05).
3. Results 3.1. Vitamin E content Added vitamin E in the feed of the pigs increased the vitamin E contents of the tissue samples and the sausage. All tissue specimens from the supplemented group showed significantly higher vitamin E concentrations (up to seven-fold in liver) than the samples from the control animals (Fig. 1). There was up to a two-fold increase in vitamin E in the sausages made from the high-supplement group compared with the controls independent of the packaging. The lowest vitamin E values were found in the air-packed samples (Fig. 2). There was no change in vitamin E concentration in the sausages, from either treatment group in the three different packaging conditions, over the entire storage period (data not shown).
Fig. 1. a-Tocopherol content of different tissue samples of pigs fed a-tocopherol supplemented diets (meanS.E.M., n=6), § indicates significant differences between the supplemented groups (Tukey-test; P<0.05).
Fig. 2. a-Tocopherol content of sausages under different packaging conditions (meanS.E.M., n=6) at the end of storage; different upper or lower-case letters indicate significant differences between packaging conditions and tocopherol supplementation (Tukey-test; P<0.05).
103
3.2. Colour stability The colour stability of the sausages was not influenced by vitamin E content. However, packaging conditions did significantly affect the colour during storage. There was no colour change (E) in the vacuum-packed sausages (E < 1). But the colour changes in sausages packed under N2/CO2 (E=3.05) and air (E=6.41) were clearly visible during storage of eight weeks (Fig. 3). (Sensorially visible colour changes have E > 1; Yudd and Wyszecki, 1975). 3.3. Lipid peroxidation Lipid peroxidation in the sausages, as assessed by TBARS was not influenced by vitamin E content. The assumed oxygen contents of the different packages significantly affected TBARS, higher values being seen in the oxygen packed samples (Fig. 4). Storage time did not lead to the expected increase in TBARS. 3.4. Antioxidative capacity Storage time had no influence on the development of the antioxidative capacity of the sausage. Contrary to colour development and lipid peroxidation vitamin E content influenced the measured luminescence (see Fig. 5
Fig. 3. Change of colour (L*, a*, b*-values) of sausages under different packaging conditions (meanS.E.M., n=6) at the end of the storage period; different letters indicate significant differences between packaging conditions (Tukey-test; P <0.05).
Fig. 4. TBARS of sausages under different packaging conditions (meanS.D., n=6); filled symbols indicate a significant decrease between first and last sample of the specified packaging (P <0.05).
104
C. Harms et al. / Meat Science 63 (2003) 101–105
and Table 3). Sausages from the high supplemented group showed significantly lower light emission as shown by the areas under the curve (AUC) (Table 3) and lag phases and peak heights of the curves (Fig. 5). The lowest antioxidative capacity was measured in the sausage packed in air. Lag phase (tlag) before the beginning of light emission after catalysis with ABAP was shortest in the air packed sausage.
4. Discussion The vitamin E contents in the tissues confirmed results from other investigators (Morrissey, Buckley, Sisk, Lynch, & Sheehy 1996; O’Sullivan, Kerry, Buckley, Lynch, & Morrissey, 1997). The transfer of the vitamin differs between tissues. Liver is the main temporary store for vitamin E and thus shows the highest concentrations of this micronutrient; the vitamin E content in fat from the controls and supplemented animals is only 1:2, whereas in liver it is 1:7. The feeding time of 5 weeks was probably insufficent to concentrate vitamin E to the amounts in fat as found by Morrissey et al. (1996) in their long term (18 weeks) study. Vitamin E contents in the sausages and the fat are similar from both the control and experimental group. This is because nearly 50% of fat and fat tissue is incorporated into the sausage. During ripening and storage the vitamin E concentration in the sausages did not decrease. This agrees with results from Liu et al. (1996). The transfer of vitamin E had no detectable effect on colour stability and lipid peroxidation in the sausages. The curves of the TBARS results (Fig. 4) indicate differences between the packaging systems (LS mean: P < 0.001; n=30). The L*, a*, b* colour values showed similar variation due to packaging. The clear advantage of using packaging with low oxygen content confirms the results of Miles et al. (1986) for pork and of Ahn, Sell, Jefferey, Jo, Chen, Wu, and Lee (1997) for turkey. This study does not confirm the beneficial influence of vitamin E on colour and lipid peroxidation found for
fresh or frozen beef or pork (Faustman, Cassens, Schaefer, Buege, Williams, & Scheller, 1989; HovingBolink et al., 1998; Liu et al., 1996). We assume that nitrite, with its powerful antioxidative effect probably masked the effect of vitamin E. The antioxidative power of nitrite is based on its binding to the muscle pigment myoglobin and its conversion to nitrosyl myoglobin. In this structure myoglobin cannot act as a catalyst of lipid peroxidation (Gray & Pearson 1984; Kanner, Harel, & Granit 1991; O’Boyle, Rubin, Diosady, Aladin-Kassam, Comer, & Brightwell 1990). The results reported thus suggest that the well-known ‘‘scavenger’’ functions of vitamin E during peroxidation in fresh and frozen meat are not realized in un-pretreated sausage. Using catalytic test systems (chemiluminescence test with ABAP), the scavenging activity of the supplemented vitamin E could be detected in the stored sausage despite the presence of nitrite. This is obviously due to inhibition of peroxidation, which can be initiated by the free muscle pigments (ca. 35%, see Carballo, Cavestany, & Jime´nez-Colmenero, 1991). Reinforced radical production resulted in high chemiluminescence with the largest AUCs and short lag phases. In other words our data indicate the beneficial effects of packaging conditions on colour and lipid peroxidation in sausages when oxygen concentrations are low. This agrees with earlier studies (Andersen et al., 1990). The more oxygen in the packages, the more undesirable changes are seen for both parameters. Therefore beneficial effects of packaging is ranked as follows: vacuum is preferable to N2/CO2 atmosphere which is preferable to air. This confirms the results of other authors (Andersen et al. 1990; Houben & Gerris 1998). There have been no previous reports in the literature on luminescence measurements in meat and meat products stabilised with vitamin E. Studies conducted to check luminescence suppression used synthetic food antioxidants (Kahl, Weimann, Weinke, & Hildebrandt 1987; Noll, de Groot, & Sies 1987). The results reported showed reductions of luminescence by propylgallate, butylated hydroxytoluene and butylated hydroxyanisole (Kahl et al., 1987) and a clear dependence of radical production rate on oxygen content in the matrix (Noll et al., 1987). Table 3 Areas under the curve (AUC) of luminescence measurements (meanSD, n=3)a
Fig. 5. Development of luminescence after catalysis by ABAP in sausages made from the supplemented group and under different package conditions at the end of storage period; n=8.
a-Tocopherol in the diet
20 ppm
Package conditions
Mean
SD
mean
SD
Vacuum N2/CO2 Air
1.44a 1.43a 1.83b
0.11 0.14 0.29
1.33c 1.32c 1.52d
0.10 0.14 0.20
a
410 ppm
Values with different letters indicate significant differences (Tukey-test; P <0.05).
C. Harms et al. / Meat Science 63 (2003) 101–105
5. Conclusion and perspectives The safety consideration of nitrite contents in meat products, particularly in respect to cancer risk, make the reduction of curing salt advisible. For this reason and on the basis of the results reported in this work, further experiments with a gradual reduction of nitrite in cured sausages should be carried out. Antioxidants (natural or synthetic) may be added to the sausage material, either in vitro or as feed supplements for the fattening animals. Along these experimental lines the limits of colour transition of cured sausages which are still acceptable to consumers and antioxidative protection can be defined and determined. Such studies are currently underway in our laboratory.
Acknowledgements This work was conducted with the support of the Ahrberg Foundation. We gratefully acknowledge both the skilful technical help and assistance with graphics and text by Andrea Widdel.
References Ahn, D. U., Sell, J. L., Jefferey, M., Jo, C., Chen, X., Wu, C., & Lee, J. I. (1997). Dietary vitamin E affects lipid oxidation and total volatiles of irradiated raw turkey meat. Journal of Food Science, 62, 954–958. Andersen, H. J., Bertelsen, G., Ohlen, A., & Skibsted, L. H. (1990). Modified packaging as protection against photodegradation of colour of pasteurized, sliced ham. Meat Science, 28, 77–83. Cannon, J. E., Morgan, J. B., Schmidt, G. R., Tatum, J. D., Sofos, J. N., Smith, G. C., Delmore, R. J., & Williams, S. N. (1996). Growth and fresh meat quality characteristics of pigs supplemented with vitamin E. Journal of Animal Science, 74, 98–105. Carballo, J., Cavestany, M., & Jime´nez-Colmenero, F. (1991). Effect of light on colour and reaction of nitrite in sliced pork bologna under different chilled storage temperatures. Meat Science, 30, 235– 244. Faustman, C., Cassens, R. G., Schaefer, D. M., Buege, D. R., Williams, S. N., & Scheller, K. K. (1989). Improvement of pigment and lipid stability in Holstein steer beef by dietary supplementation with vitamin E. Journal of Food Science, 54, 858–862.
105
Faustman, C., Chan, W. K., Schaefer, D. M., & Havens, A. (1998) Beef color update: the role for vitamin E (1998). Journal of Animal Science, 76, 1019–1026. Gray, J. I., & Pearson, A. M. (1984). Cured meat flavour. Advances in Food Research, 29, 1–72. Houben, J. H., & Gerris, C. V. M. (1998). Effect of dietary supplementation with vitamin E on colour stability of packaged, sliced pasteurized ham. Meat Science, 50, 421–428. Hoving-Bolink, A. H., Eikelenboom, G., van Diepen, J., Jongbloed, A. W., Houben, J. H., & van Diepen, J. M. T. (1998). Effect of dietary vitamin E supplementation on pork quality. Meat Science, 49, 205–212. Kahl, R., Weimann, A., Weinke, S., & Hildebrandt, A. G. (1987). Detection of oxygen activation and determination of the activity of antioxidants towards reactive oxygen species by use of the chemiluminigenic probes luminol and lucigenin. Archives of Toxicology, 60, 158–162. Kanner, J., Harel, S., & Granit, R. (1991). Nitric oxide as an antioxidant. Archives of Biochemistry and Biophysics, 289, 130–136. Liu, Q., Scheller, K. K., Arp, S. C., Schaefer, D. M., & Williams, S. N. (1996). Titration of fresh meat color stability and malondialdehyde development with Holstein steers fed vitamin E-supplemented diets. Journal of Animal Science, 74, 117–126. Miles, R. S., McKeith, F. K., Bechtel, P. J., & Novakofski, J. (1986). Effect of processing, packaging and various antioxidants on lipid oxidation of restructured pork. Journal of Food Protection, 49(3), 222–225. Monahan, F. J., Asghar, A., Gray, J. I., Buckley, D. J., & Morrissey, P. A. (1994). Effect of oxidized dietary lipid and vitamin E on the colour stability of pork chops. Meat Science, 37, 205–215. Morrissey, P. A., Buckley, D. J., Sisk, H., Lynch, P. B., & Sheehy, P. J. A. (1996). Uptake of a-tocopherol in porcine plasma and tissues. Meat Science, 44, 275–283. Noll, T., de Groot, H., & Sies, H. (1987). Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation. Archives of Biochemistry and Biophysics, 252, 284–291. O‘Boyle, A. R., Rubin, L. J., Diosady, L. L., Aladin-Kassam, N., Comer, F., & Brightwell, D. (1990). A nitrite-free curing system and its application to the production of wieners. Food Technology, 48, 88–104. O‘Sullivan, M. G., Kerry, J. P., Buckley, D. J., Lynch, P. B., & Morrissey, P. A. (1997). The distribution of dietary vitamin E in the muscles of the porcine carcass. Meat Science, 45, 297–305. Rammell, C. G., Cunliffe, B., & Kieboom, A. J. (1983). Determination of alpha-tocopherol in biological speciemens by high performance liquid chromatography. Journal of Liquid Chromatography, 6, 1123–1130. Uchiyama, M., & Mihara, M. (1978). Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry, 86, 271–278. Yudd, D. B., & Wyszecki, G. (1975). Colour in business, science and industry. (3rd ed). New York: Wiley and Sons.