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Effect of ascorbate and ascorbyl palmitate on lipid oxidation in semi-dry saugages manufactured from pork materials differing in stability towards oxidation
Meat Science 17 (1986) 199-211
Effect of Ascorbate and Ascorbyl Palmitate on Lipid Oxidation in Semi-dry Sausages Manufactured from Pork Materials Differing in Stability Towards Oxidation J. H. Houben Department of the Science of Food of Animal Origin, Meat Technology Section, Faculty of Veterinary Medicine, University of Utrecht, PO Box 80 175, 3508 TD Utrecht, The Netherlands
&
B. Krol Division for Nutrition and Food Research TNO, PO Box 360, 3700 AJ Zeist, The Netherlands (Received: 15 October, 1985)
S UMMA R Y Semi-dry fermented sausages were manufactured from three batches of pork slightly differing in their polyenoic fatty acid levels and ~-tocopherol contents. The sausage doughs were treated as follows: (1) control, (2) ascorbate, (3) ascorbyl palmitate and (4) a combination of ascorbate and ascorbyl palmitate. After brine fermentation, products ripened at 15°C were evaluated for rancidity by peroxide values and TBA numbers. During ripening important differences in stability towards oxidation were generally not observed. Both peroxide and TBA values gradually increased linearly with time. For the TBA values, gradients for the products derived from the three diet groups differed considerably. Only two of six batches containing increased levels of ~-tocopherol and product antioxidants exhibited slightly improved stability towards oxidation, as judged by peroxide and TBA values. Sensory evaluations, performed only on the product series containing ascorbate, failed to reveal any positive effect in favour of the increased :t-tocopherol content. 199
Meat Science 0309-1740/86/$03.50 © Else~ier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain
200
J. H. Houben, B. Krol
Thus, for semi-dry sausages manufactured from pork materials with a slightly increased sensitivity towards oxidation, results indicated that there was little advantage to increasing the levels of ~t-toeopherol in the meat by dietary means or to changing the current commercial practice of adding aseorbate to the sausage doughs.
INTRODUCTION Fermented sausages traditionally made in Europe usually are biologically stable but ultimately deteriorate due to proteolytic processes and by (oxidative) breakdown of components of lipids. Recently, acid type semi-dry fermented sausages have been developed (Krol, 1985); souring and lipid oxidation constitute the major causes of quality impairment in these sausages. Detailed information on lipid oxidation occurring during ripening of semi-dry sausages is not available in the literature. The present study was partly set up to collect relevant data in that area. Products manufactured from pork were investigated, because of its greater susceptibility towards oxidation in comparison with beef. Carcasses having depot fats with slightly increased levels of linoleic acid were used. Raw materials were obtained from pigs fattened on diets containing increasing amounts of corn cob mix (CCM; an ensiled corn and cob mixture). For some of the pigs receiving the highest amount of CCM, the diet was supplemented with 100 ppm dl-e-tocopheryl acetate. Houben & Krol (1985) reported on several analytical results for diets and meats and for the backfats on their stability with regard to oxidation. The present paper presents data on lipid oxidation in semi-dry sausages manufactured from these raw materials. Generally, ascorbic acid (sodium ascorbate) is added to meat products for its effect upon cotour formation and antioxidant activity (Bauernfeind & Pinkert, 1970; Kl~iui, 1972; Cort, 1974; Kanner & Mendel, 1977; Reinton & Rogstad, 1981). However, the solubility of ascorbic acid in lipids is poor. To improve the effectiveness of this compound, the use of ascorbic acid esters-such as the palmitate and the stearate--is sometimes recommended (Pongracz, 1973; Sedlacek, 1975; Kl~iui, 1976). No detailed studies comparing the effects of ascorbate and ascorbic acid esters with regard to lipid oxidation in cured meat products were found in the literature. According to Cort (1974) and Pongracz (1982) ascorbyl
Lipid oxidation in semi-dry pork sausages
201
palmitate acts as a (powerful) synergist, together with e-tocopherol. The concomitant availability of raw materials with an increased level of ~tocopherol offered the opportunity to evaluate the effect of the cited synergism under experimental conditions. Several batches of a Dutch-style semi-dry sausage were produced. After the brine fermentation step, the sausages were ripened in air at 15°C. During ripening, lipid oxidation was followed analytically by measuring peroxide and TBA values. The products containing ascorbate were also subjected to sensory evaluations.
MATERIALS AND METHODS
Raw materials The pigs were fattened on an experimental farm of the State Consultancy Service for Pig Production (Utrecht). The principal aim was to study the overall nutritive value of corn cob mix (CCM) under Dutch conditions (Arkes & Houben, 1983; A r k e s , 1984). Thirty castrated male pigs were randomly allotted in three equal groups. Feeds for the groups contained 0, 37-5 and 75-0% C C M on an energy basis. In addition to the highest C C M level, two male castrates were fed the same ration supplemented with 100ppm dl-~-tocopheryl acetate. Codes assigned to the feeding trial were as follows: C (control), M (medium CCM), H (high CCM) and HT (high C C M + dl-e-tocopheryl acetate). Raw materials derived from three pigs (average animals) from blocks C and H and from both HT animals were used for manufacturing the semi-dry sausages. Materials from the M block were used for the preparation of semi-dry sausages with the standard formulation only (data not presented here). For further details on diets and raw materials reference is made to H o u b e n & Krol (1985).
Manufacturing procedures After slaughtering, the carcasses were chilled overnight at 4 +_ I°C. Next day t h e carcasses were transported under refrigeration to our Institute and held at 4 ___I*C. Approximately 48 h post mortem, the carcasses were deboned. Coarsely cut mixed samples of lean meat and backfat were prepared for each block. Portions with standardized
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weights were packaged in polyethylene freezer bags and frozen at - 2 0 ° C . The freezing step was included since frozen raw materials were required for the preparation of the semi-dry sausages. After tempering the meats at about - 5 ° C , all sausage batches were prepared on the fourth day after slaughtering. Sausage doughs were manufactured with an Alexanderwerk chopper model SM 720/82. The composition of the mixes was: lean pork, 63-8%; backfat, 31-9%; sodium chloride, 1.0%; nitrite-containing salt, 1-0%; dextrose, 0-7%; sodium-L-glutamate, 0-2%; paprika, 0.1%; pepper, 0.3%; mace, 0.025%; cloves, 0-015%; and starter sausage, 1-0%. The nitrite-containing salt consists of sodium chloride, 99-4%, and sodium nitrite, 0-6%. The starter sausage was obtained from CIVO-TNO at Zeist. For each diet, four batches of about 8 kg were prepared. The batches contained, respectively: no ascorbic acid derivative (coded O); sodium-L(+)-ascorbate, 0-05% (coded A); L(+)-ascorbyl palmitate, 0-017% (coded P); sodium-L(+)-ascorbate, 0-042%, in combination with L(+)-ascorbyl palmitate, 0-017% (coded AP). Analytical grade antioxidants were purchased from Merck. The ascorbate was added at a level of 0.05%, the maximum level allowed by Dutch Food Regulations for cured meats (Anon, 1981). The palmitate was added at a level of approximately 500ppm on fat basis--a content generally used for stabilization. The quantities chosen for the combination of the salt and the ester delivered a total amount of ascorbic acid as calculated for the products series containing ascorbate only. The antioxidants were dispersed by spraying ethanolic solutions (20 ml/8 kg meat mix) on the doughs in the slowly revolving chopper bowl. In the doughs without ascorbic acid derivative(s) an equal volume of ethanol was added in the same way. The doughs were stuffed in collagen casings (diameter, 60 mm; approximate weight, 700 g). The sausages were fermented in a brine for 2 days, the brine temperature being maintained at 27 _+ I°C. The composition of the brine was: sodium chloride, 7-0%; nitrite-containing salt, 2.0%; potassium dihydrogen orthophosphate, 1-0%; and water, 90.0%. Next, the products were treated with natamycin to prevent mould growth and, finally, hung in a ripening r o o m at 15 +_ I°C. This product can be consumed after 2-3 weeks of ripening and has a pH of about 4.6. The sausages were not smoked, since smoke contains antioxidants (Eriksson, 1982); furthermore, compounds in woodsmoke may disturb the TBA test (Gray, 1978).
Lipid oxidation in semi-dry pork sausages
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At regular intervals during ripening, one sausage from each batch was removed for chemical and/or sensory analyses. Analyses were performed on two sections from each sausage. One section was obtained from the middle and the other near the upper end. From both sections approximately 5 m m was taken from the outside. Both samples were analyzed in duplicate for fat content, peroxide value and TBA number.
Methods of analysis Fat contents were determined according to ISO R 1443 (acid digestion and extraction according to Weibull-Stoldt). For the determination of peroxide values IUPAC II.D. 13 (fat extraction with chloroform) was applied (Paquot, 1979). TBA values were assayed by the distillation method according to Tarladgis et al. (1960), as modified by Tarladgis et al. (1964). The optical density (OD) was converted to milligrams of malonaldehyde per kilogram of sample by multiplying by a factor of 7.8. The results were expressed as milligrams of malonaldehyde per kilogram of fat.
Sensory evaluations For practical reasons only the A series was subjected to sensory evaluation. The middle half of each sausage was always evaluated. Judgements were performed by a CIVO-TNO expert panel consisting of from six to ten members. The panel members were carefully selected and trained to judge cured meat products. Evaluations were carried out by comparing the three products (after slicing) in one session, using blind codes. The products were served at room temperature. Scores on a scale from t (extremely poor) to I0 (excellent) were given for the following characteristics: external colour, internal colour, aroma/taste and consistency. Ranked data were tested for significance according to the method of Kahan et al. (1973). RESULTS A N D DISCUSSION
Backfat analyses The linoleic acid levels of the C, H and HT diets--as weight percentages of the fatty acid methyl esters--were 10-9, 14-0 and 13-7, respectively.
6 1 I 2 3 12 2 2 18 3 3 6 36 83
Diet Product antioxidant Diet/ripening time Diet/ripening time (t.in.) Diet/ripening time (Quad.)
Product antioxidant/ripening time Product antioxidant/ripening time (Lin.) Product antioxidant/ripening time (Quad.)
Diet/product antioxidant Residual Total
DF
Ripening lime Ripening time (Lin.) Ripening time (Quad.)
Souree
4.79990 0.723 54 2.32595
0.75743 1-61868 0.12509
1.521 30 9.64384 0.33848 I).629 92 0.441 92
[4.489 [9 57.901 51 12.721 05
MS
<0.001
0.438 0.101 0.914
0.137 <0.001 0-920 0.428 0.549
<0.00! <0.001 <0.001
P value
Peroxide values
6 36 83
18 3 3
2 3 12 2 2
6 1 1
DF
8.393E 5-662E 3.148E
1.155E 3.028E 1-146E
1.018E 5.371E 3.880E 1.748E 1.314E
1.446E 8.343E 9.605E
MS
0 - I 0
0 0 0
1 0 0 1 0
1 1 - 1
TBA values
<0.001
0.034 0.004 0.128
<0.001 <0.001 <0.001 < 0-00 I 0.113
P value
TABLE 1 Summary of the Results of the Analyses of Variance for Peroxide and TBA Values Measured During Ripening of Semi-dry Pork Sausages
Lipid oxidation in semi-dry pork sausages
205
Corresponding linolenic acid levels were 0-8, 1-2 and 1-3. The etocopherol levels (mg/kg backfat) were 2, 2 and 16, respectively. An oxidation stability test (Rancimat test) was performed at 100°C and showed induction times of 245, 238 and 485 min, respectively. Data for fatty acids, e-tocopherol and Rancimat tests are derived from Houben & Krol (1985).
Lipid oxidation measurements A summary of the analyses of variance performed on the peroxide and TBA values measured during ripening is given in Table 1. Peroxide values were not significantly affected by diet, but were so affected by the duration of ripening. A linear increase of peroxide values with time (P<0.001) was observed (PV=0-127 x number of weeks + 1-367; in meq/kg fat). Of the main effect s, the interaction of diets and product antioxidants for peroxide values was significant (P < 0-001), as shown in Fig. 1. Peroxide
value
Cmeq/kg
fat)
4.00
oc
3.00
2.00
1.00 L
0
A
AP
P
Fig. 1. Mean peroxide values measured during ripening of semi-dry pork sausages, as related to dietary and product antioxidant effects. Product antioxidant treatments are coded: O (no ascorbate or ascorbyl palmitate added); A (ascorbate); AP (ascorbate plus ascorbyt palmitate); and P (ascorbyl palmitate), lsd, least significant difference. Products are coded by the diet composition: (control), H (high in corn cob mix, CCM) and HT (high CCM supplemented with 100ppm dl-e-tocopheryl acetate).
206
J. H. Houben, B. Krol TBA v a l u e
i
Cmg malonaldehydelkg fat~
2 00
~
HT
1,00 -
i
J
i
4
8
12 >
'
1
16
Weeks rip e n in g at 15=C
Fig. 2. Course of the mean TBA values (all product treatments) measured during ripening of semi-dry,pork sausages, as related to diets. (For meaning of s~-mbols, see legend to Fig. 1.) TBA values were significantly (P < 0-001) affected by diets, ripening time and product antioxidants, with significant interactions between the three effects. Mean TBA values per diet treatment significantly increased linearly with time (P < 0.001), as shown in Fig. 2. The effect of the HT diet is particularly interesting, with a strong positive influence being found with the e-tocopherol levels in the tissues. Mean TBA values per product antioxidant treatment significantly increased linearly with time (P<0"01), as shown in Fig. 3. The ascorbic acid derivatives clearly retarded oxidation during the first 11 weeks of ripening, but, by 16 weeks, the protective action disappeared. A significant ( P < 0-001) interaction among diets and product antioxidants for TBA values occurred, as shown in Fig. 4. For diets C and H, the ascorbic acid derivatives generally did not inhibit lipid oxidation. For the H T products inhibitory effects were perceived for all product antioxidant treatments. Compared with the H-O, the HT-O products were remarkably unstable. According to
TBA value Crag m a l o n a l d e h y d e / k g fat] l
3.00
2.00
1.00
4
8
12 )
16
Weeks ripening at 15~C
Fig. 3. Course of the mean TBA values (all diet treatments) measured during ripening of semi-dry pork sausages, as related to product antioxidants. (For meaning of s)Tnbols, see legend to Fig. 1.)
TBA value Cmg matonaldehyde/kg T
fat]
4.00 I l.s.d.
oC o H • HT
3.00 -
2.00
-
i .000
A
AP
P
Fig. 4. Mean TBA values measured during ripening of semi-dry pork sausages, as related to dietary and product antioxidant effects. (For meaning of symbols, see legend to Fig. 1.)
208
J. H. Houben, B. Krol
Pongracz (1973) an optimal effect in stabilizing edible fats and oils with e-tocopherol will be obtained at a level of about 200 ppm. In model systems a shift from antioxidative to pro-oxidative action for this c o m p o u n d was found to occur at higher levels (Citlard et al., 1980a,b). In HT backfats, we only reached a content of 16rag a-tocopherol per kilogram of tissue. With regard to lipid oxidation, meat products are very complex systems. Sodium chloride behaves as a pro-oxidant, whereas sodium nitrite usually acts as an antioxidant (Hadden et al., 1975). Metal ions, especially copper and iron and even the iron present in metmyoglobin and some other haemoproteins, catalyze lipid o.~dation (Liu, 1970; Love & Pearson, 1974). Certain components in spices can inhibit these processes (Herrmann et al., 1981). However, it is difficult to explain the strong pro-oxidant action of e-tocopherol in the semidry sausage present in the HT-O products, which certainly was not expected.
Central versus end samples Overall means for peroxide values of the central and terminal sampling areas were 2.369 and 2.687 meq/kg fat, respectively; the least significant difference (lsd) for these means was 0-086 meq/kg fat. Data for the average TBA values of the same sample locations were, respectively, 1.950 and 2"321 mg of malonaldehyde per kilogram of fat (lsd = 0-092 mg of malonaldehyde per kilogram of fat). Means of peroxide values from weeks 7 and 11 were significantly (P < 0.05) higher for the end section as compared with the central section. The same was true for the TBA values from week 4 on. The observed difference in the progress of the oxidation processes probably can be attributed to a greater accessibility of oxygen at the looser upper end (wrinkled casing due to clipping, hanging and drying effects) in comparison with the evenly drying, tighter closed middle part.
Sensory evaluations Scores for external colour, internal colour and consistency did not reveal anything new. Mean scores for aroma taste are presented in Table 2. An analysis of variance for these data did not show any significant effects. Mean aroma/taste scores did not decrease significantly during the first 9 weeks of ripening. However, from weeks 4 (HT products) to 6 (C products) over 50% of the judges denoted rancid off-flavours.
Lipid oxidation in semi-dry pork sausages
209
TABLE 2 Mean Aroma/Taste Scores Obtained During Ripening of Semi-dry Pork Sausages (0-05% Ascorbate Added) as Affected by the Diets of the Pigs
Product 3 Cc H HT Average
6.8 7-1 6-2 6.70 b
4 6.7 7-2 6-2 6-70
Period of ripening (weeks at 15°C) 5 6 7 9 6-9 6-4 7.0 6-77
6-2 a 7-2 6-5 6.63
6-4 6-6 6-6 6-53
6.2 6-9 6-7 6.60
11
13
5.3 6.6 5-3 5-73
6-6 5.7 5-6 5-97
a Underlining a score means that over 50% of the judges denoted rancid off-flavours. b Least significant difference for the averages is 0-78. c Products are coded by the diet composition: C (control), H (high in corn cob mix, CCM) and HT (high CCM supplemented with 100 ppm dl-~-tocopheryl acetate).
Generally, the scores for aroma/taste passed the 6.0 mark after approximately 11 weeks, mean peroxide and TBA values determined for the A products being then 2.31 meq/kg fat and 1-82 mg of malonaldehyde per kilogram of fat. Peroxide and TBA values gradually increase with time, although not always consistently (Fig. 2; HT line). Although the lipid oxidation parameters give information about the progress of oxidation processes, the sensory evaluation data are more important in assessing the acceptability of meat products.
CONCLUSIONS Differences in stability towards oxidation for the sausages manufactured from the meats derived from the three groups of pigs were rather small. Nevertheless, TBA values for the sausages derived from the three diet groups varied considerably. Both peroxide and TBA values gradually increased linearly with time. In all cases, the ascorbic acid derivatives retarded lipid o~dation during a period of 11 weeks of ripening. However, the protective action disappeared during longer periods of storage. Important differences between the product antioxidant treatments were not observed. HT products without ascorbic acid derivatives compared with corresponding H products appeared remarkably unstable. However, HT products containing the ascorbic acid ester in some cases appeared more stable towards oxidation than comparable H products.
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J. H. Houben, B. Krot
Samples obtained from the top section of the sausages, from about 11 weeks on, consistently showed higher peroxide and TBA values than samples cut from the middle part. Considering the rather variable results obtained by determining peroxide and TBA values for differently formulated products, there appears to be no reason for establishing cut-off values for these parameters with regard to quality.
A C K N O W L E D G E M ENTS The authors are grateful to the State Consultancy Service for Pig Production, Utrecht, for the pig materials, to the Dutch C o m m o d i t y Board for Livestock and Meat and T N O for financial support, to Hoffmann-La Roche BV for fatty acid and tocopherol determinations and Rancimat tests and last, but not least, to Hanne Tjeerdsma-van Bokhoven and Gert Keizer for their skilled technical assistance.
REFERENCES Anon (1981). Vlees- en Vleeswarenbesluit (Warenwet). Arkes, J. G. (1984). Varkensproefbedrijf Noord en Oost Nederland, Proefverslag, No. 36. Arkes, J. G. & Houben, J. H. (1983). Boerderij, 68, 20-VA. Bauernfeind, J. C. & Pinkert, D. M. (1970). Adv. Fd Res., 18, 219. Cillard, J., Cillard, P., Cormier, M. & Girre, L. (1980a). J. Amer. Oil Chem. Soc., 57, 252. Cillard, J., Cillard, P. & Cormier, M. (1980b). J. Amer. Oil Chem. Soe., 57, 255. Cort, W. M. (1974). J. Amer. Oil Chem. Soc., 51, 321. Eriksson, C. E. (1982). Food Chem., 9, 3. Gray, J. I. (1978). J. Amer. Oil Chem. Soc., 55, 539. Hadden, J. P., Ockerman, H. W., Cahill, V. R., Parrett, N. A. & Borton, R. J. (1975). J. Fd Sci., 40, 626. Herrmann, K., Schiitte, M., Mfiller, H. & Bismer, R. (1981). Deutsche Lebensm.Rundschau, 77, 134. Houben, J. H. & Krol, B. (1985). Meat Sci., 13, 193. Kahan, G., Cooper, D., Papavasiliou, A. & Kramer, A. (1973). Food Technol., 27, 63. Kanner, J. & Mendel, H. (1977). J. Fd Sei., 42, 60. Ki/iui, H. (1972). Proc. Int. Symp. C.I.LA., 14, 62.
Lipid oxidation in semi-dry pork sausages
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K1/iui, H. (1976). Int. Flay. Fd Addit., 7, 165. Krol, B. (1985). Proc. Int. Syrup. Trends in Modern Meat Technology, Pudoc. Wageningen, 58. Liu, H. P. (1970). J. Fd Sci., 35, 590. Love, J. D, & Pearson, A. M. (1974). J. Agric. Fd Chem., 22, 1032. Paquot, C. (1979). Standard methods for the analysis of oils, fats and derivatives. (6th edn), Part 1, Pergamon Press, Oxford. Pongracz, G. (1973). Int. J. Vit. Nutr. Res., 43, 517. Pongracz, G. (1982), Fette Seifen Anstrichm., 84, 269. Reinton, R. & Rogstad, A. (1981). J. Fd Sci., 46, 970. Sedlacek, B. A. J. (1975). Nahrung, 19, 219. Tarladgis, B. G., Watts, B. M. & Younathan, M. T. (1960). J. Amer. Oil Chem. Soc., 37, 44. Tarladgis, B. G., Pearson, A. M. & Dugan, L. R. (1964). J. Sci. Fd Agric., 15, 602.
Effect of Ascorbate and Ascorbyl Palmitate on Lipid Oxidation in Semi-dry Sausages Manufactured from Pork Materials Differing in Stability Towards Oxidation J. H. Houben Department of the Science of Food of Animal Origin, Meat Technology Section, Faculty of Veterinary Medicine, University of Utrecht, PO Box 80 175, 3508 TD Utrecht, The Netherlands
&
B. Krol Division for Nutrition and Food Research TNO, PO Box 360, 3700 AJ Zeist, The Netherlands (Received: 15 October, 1985)
S UMMA R Y Semi-dry fermented sausages were manufactured from three batches of pork slightly differing in their polyenoic fatty acid levels and ~-tocopherol contents. The sausage doughs were treated as follows: (1) control, (2) ascorbate, (3) ascorbyl palmitate and (4) a combination of ascorbate and ascorbyl palmitate. After brine fermentation, products ripened at 15°C were evaluated for rancidity by peroxide values and TBA numbers. During ripening important differences in stability towards oxidation were generally not observed. Both peroxide and TBA values gradually increased linearly with time. For the TBA values, gradients for the products derived from the three diet groups differed considerably. Only two of six batches containing increased levels of ~-tocopherol and product antioxidants exhibited slightly improved stability towards oxidation, as judged by peroxide and TBA values. Sensory evaluations, performed only on the product series containing ascorbate, failed to reveal any positive effect in favour of the increased :t-tocopherol content. 199
Meat Science 0309-1740/86/$03.50 © Else~ier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain
200
J. H. Houben, B. Krol
Thus, for semi-dry sausages manufactured from pork materials with a slightly increased sensitivity towards oxidation, results indicated that there was little advantage to increasing the levels of ~t-toeopherol in the meat by dietary means or to changing the current commercial practice of adding aseorbate to the sausage doughs.
INTRODUCTION Fermented sausages traditionally made in Europe usually are biologically stable but ultimately deteriorate due to proteolytic processes and by (oxidative) breakdown of components of lipids. Recently, acid type semi-dry fermented sausages have been developed (Krol, 1985); souring and lipid oxidation constitute the major causes of quality impairment in these sausages. Detailed information on lipid oxidation occurring during ripening of semi-dry sausages is not available in the literature. The present study was partly set up to collect relevant data in that area. Products manufactured from pork were investigated, because of its greater susceptibility towards oxidation in comparison with beef. Carcasses having depot fats with slightly increased levels of linoleic acid were used. Raw materials were obtained from pigs fattened on diets containing increasing amounts of corn cob mix (CCM; an ensiled corn and cob mixture). For some of the pigs receiving the highest amount of CCM, the diet was supplemented with 100 ppm dl-e-tocopheryl acetate. Houben & Krol (1985) reported on several analytical results for diets and meats and for the backfats on their stability with regard to oxidation. The present paper presents data on lipid oxidation in semi-dry sausages manufactured from these raw materials. Generally, ascorbic acid (sodium ascorbate) is added to meat products for its effect upon cotour formation and antioxidant activity (Bauernfeind & Pinkert, 1970; Kl~iui, 1972; Cort, 1974; Kanner & Mendel, 1977; Reinton & Rogstad, 1981). However, the solubility of ascorbic acid in lipids is poor. To improve the effectiveness of this compound, the use of ascorbic acid esters-such as the palmitate and the stearate--is sometimes recommended (Pongracz, 1973; Sedlacek, 1975; Kl~iui, 1976). No detailed studies comparing the effects of ascorbate and ascorbic acid esters with regard to lipid oxidation in cured meat products were found in the literature. According to Cort (1974) and Pongracz (1982) ascorbyl
Lipid oxidation in semi-dry pork sausages
201
palmitate acts as a (powerful) synergist, together with e-tocopherol. The concomitant availability of raw materials with an increased level of ~tocopherol offered the opportunity to evaluate the effect of the cited synergism under experimental conditions. Several batches of a Dutch-style semi-dry sausage were produced. After the brine fermentation step, the sausages were ripened in air at 15°C. During ripening, lipid oxidation was followed analytically by measuring peroxide and TBA values. The products containing ascorbate were also subjected to sensory evaluations.
MATERIALS AND METHODS
Raw materials The pigs were fattened on an experimental farm of the State Consultancy Service for Pig Production (Utrecht). The principal aim was to study the overall nutritive value of corn cob mix (CCM) under Dutch conditions (Arkes & Houben, 1983; A r k e s , 1984). Thirty castrated male pigs were randomly allotted in three equal groups. Feeds for the groups contained 0, 37-5 and 75-0% C C M on an energy basis. In addition to the highest C C M level, two male castrates were fed the same ration supplemented with 100ppm dl-~-tocopheryl acetate. Codes assigned to the feeding trial were as follows: C (control), M (medium CCM), H (high CCM) and HT (high C C M + dl-e-tocopheryl acetate). Raw materials derived from three pigs (average animals) from blocks C and H and from both HT animals were used for manufacturing the semi-dry sausages. Materials from the M block were used for the preparation of semi-dry sausages with the standard formulation only (data not presented here). For further details on diets and raw materials reference is made to H o u b e n & Krol (1985).
Manufacturing procedures After slaughtering, the carcasses were chilled overnight at 4 +_ I°C. Next day t h e carcasses were transported under refrigeration to our Institute and held at 4 ___I*C. Approximately 48 h post mortem, the carcasses were deboned. Coarsely cut mixed samples of lean meat and backfat were prepared for each block. Portions with standardized
202
J. H. Houben, B. Krol
weights were packaged in polyethylene freezer bags and frozen at - 2 0 ° C . The freezing step was included since frozen raw materials were required for the preparation of the semi-dry sausages. After tempering the meats at about - 5 ° C , all sausage batches were prepared on the fourth day after slaughtering. Sausage doughs were manufactured with an Alexanderwerk chopper model SM 720/82. The composition of the mixes was: lean pork, 63-8%; backfat, 31-9%; sodium chloride, 1.0%; nitrite-containing salt, 1-0%; dextrose, 0-7%; sodium-L-glutamate, 0-2%; paprika, 0.1%; pepper, 0.3%; mace, 0.025%; cloves, 0-015%; and starter sausage, 1-0%. The nitrite-containing salt consists of sodium chloride, 99-4%, and sodium nitrite, 0-6%. The starter sausage was obtained from CIVO-TNO at Zeist. For each diet, four batches of about 8 kg were prepared. The batches contained, respectively: no ascorbic acid derivative (coded O); sodium-L(+)-ascorbate, 0-05% (coded A); L(+)-ascorbyl palmitate, 0-017% (coded P); sodium-L(+)-ascorbate, 0-042%, in combination with L(+)-ascorbyl palmitate, 0-017% (coded AP). Analytical grade antioxidants were purchased from Merck. The ascorbate was added at a level of 0.05%, the maximum level allowed by Dutch Food Regulations for cured meats (Anon, 1981). The palmitate was added at a level of approximately 500ppm on fat basis--a content generally used for stabilization. The quantities chosen for the combination of the salt and the ester delivered a total amount of ascorbic acid as calculated for the products series containing ascorbate only. The antioxidants were dispersed by spraying ethanolic solutions (20 ml/8 kg meat mix) on the doughs in the slowly revolving chopper bowl. In the doughs without ascorbic acid derivative(s) an equal volume of ethanol was added in the same way. The doughs were stuffed in collagen casings (diameter, 60 mm; approximate weight, 700 g). The sausages were fermented in a brine for 2 days, the brine temperature being maintained at 27 _+ I°C. The composition of the brine was: sodium chloride, 7-0%; nitrite-containing salt, 2.0%; potassium dihydrogen orthophosphate, 1-0%; and water, 90.0%. Next, the products were treated with natamycin to prevent mould growth and, finally, hung in a ripening r o o m at 15 +_ I°C. This product can be consumed after 2-3 weeks of ripening and has a pH of about 4.6. The sausages were not smoked, since smoke contains antioxidants (Eriksson, 1982); furthermore, compounds in woodsmoke may disturb the TBA test (Gray, 1978).
Lipid oxidation in semi-dry pork sausages
203
At regular intervals during ripening, one sausage from each batch was removed for chemical and/or sensory analyses. Analyses were performed on two sections from each sausage. One section was obtained from the middle and the other near the upper end. From both sections approximately 5 m m was taken from the outside. Both samples were analyzed in duplicate for fat content, peroxide value and TBA number.
Methods of analysis Fat contents were determined according to ISO R 1443 (acid digestion and extraction according to Weibull-Stoldt). For the determination of peroxide values IUPAC II.D. 13 (fat extraction with chloroform) was applied (Paquot, 1979). TBA values were assayed by the distillation method according to Tarladgis et al. (1960), as modified by Tarladgis et al. (1964). The optical density (OD) was converted to milligrams of malonaldehyde per kilogram of sample by multiplying by a factor of 7.8. The results were expressed as milligrams of malonaldehyde per kilogram of fat.
Sensory evaluations For practical reasons only the A series was subjected to sensory evaluation. The middle half of each sausage was always evaluated. Judgements were performed by a CIVO-TNO expert panel consisting of from six to ten members. The panel members were carefully selected and trained to judge cured meat products. Evaluations were carried out by comparing the three products (after slicing) in one session, using blind codes. The products were served at room temperature. Scores on a scale from t (extremely poor) to I0 (excellent) were given for the following characteristics: external colour, internal colour, aroma/taste and consistency. Ranked data were tested for significance according to the method of Kahan et al. (1973). RESULTS A N D DISCUSSION
Backfat analyses The linoleic acid levels of the C, H and HT diets--as weight percentages of the fatty acid methyl esters--were 10-9, 14-0 and 13-7, respectively.
6 1 I 2 3 12 2 2 18 3 3 6 36 83
Diet Product antioxidant Diet/ripening time Diet/ripening time (t.in.) Diet/ripening time (Quad.)
Product antioxidant/ripening time Product antioxidant/ripening time (Lin.) Product antioxidant/ripening time (Quad.)
Diet/product antioxidant Residual Total
DF
Ripening lime Ripening time (Lin.) Ripening time (Quad.)
Souree
4.79990 0.723 54 2.32595
0.75743 1-61868 0.12509
1.521 30 9.64384 0.33848 I).629 92 0.441 92
[4.489 [9 57.901 51 12.721 05
MS
<0.001
0.438 0.101 0.914
0.137 <0.001 0-920 0.428 0.549
<0.00! <0.001 <0.001
P value
Peroxide values
6 36 83
18 3 3
2 3 12 2 2
6 1 1
DF
8.393E 5-662E 3.148E
1.155E 3.028E 1-146E
1.018E 5.371E 3.880E 1.748E 1.314E
1.446E 8.343E 9.605E
MS
0 - I 0
0 0 0
1 0 0 1 0
1 1 - 1
TBA values
<0.001
0.034 0.004 0.128
<0.001 <0.001 <0.001 < 0-00 I 0.113
P value
TABLE 1 Summary of the Results of the Analyses of Variance for Peroxide and TBA Values Measured During Ripening of Semi-dry Pork Sausages
Lipid oxidation in semi-dry pork sausages
205
Corresponding linolenic acid levels were 0-8, 1-2 and 1-3. The etocopherol levels (mg/kg backfat) were 2, 2 and 16, respectively. An oxidation stability test (Rancimat test) was performed at 100°C and showed induction times of 245, 238 and 485 min, respectively. Data for fatty acids, e-tocopherol and Rancimat tests are derived from Houben & Krol (1985).
Lipid oxidation measurements A summary of the analyses of variance performed on the peroxide and TBA values measured during ripening is given in Table 1. Peroxide values were not significantly affected by diet, but were so affected by the duration of ripening. A linear increase of peroxide values with time (P<0.001) was observed (PV=0-127 x number of weeks + 1-367; in meq/kg fat). Of the main effect s, the interaction of diets and product antioxidants for peroxide values was significant (P < 0-001), as shown in Fig. 1. Peroxide
value
Cmeq/kg
fat)
4.00
oc
3.00
2.00
1.00 L
0
A
AP
P
Fig. 1. Mean peroxide values measured during ripening of semi-dry pork sausages, as related to dietary and product antioxidant effects. Product antioxidant treatments are coded: O (no ascorbate or ascorbyl palmitate added); A (ascorbate); AP (ascorbate plus ascorbyt palmitate); and P (ascorbyl palmitate), lsd, least significant difference. Products are coded by the diet composition: (control), H (high in corn cob mix, CCM) and HT (high CCM supplemented with 100ppm dl-e-tocopheryl acetate).
206
J. H. Houben, B. Krol TBA v a l u e
i
Cmg malonaldehydelkg fat~
2 00
~
HT
1,00 -
i
J
i
4
8
12 >
'
1
16
Weeks rip e n in g at 15=C
Fig. 2. Course of the mean TBA values (all product treatments) measured during ripening of semi-dry,pork sausages, as related to diets. (For meaning of s~-mbols, see legend to Fig. 1.) TBA values were significantly (P < 0-001) affected by diets, ripening time and product antioxidants, with significant interactions between the three effects. Mean TBA values per diet treatment significantly increased linearly with time (P < 0.001), as shown in Fig. 2. The effect of the HT diet is particularly interesting, with a strong positive influence being found with the e-tocopherol levels in the tissues. Mean TBA values per product antioxidant treatment significantly increased linearly with time (P<0"01), as shown in Fig. 3. The ascorbic acid derivatives clearly retarded oxidation during the first 11 weeks of ripening, but, by 16 weeks, the protective action disappeared. A significant ( P < 0-001) interaction among diets and product antioxidants for TBA values occurred, as shown in Fig. 4. For diets C and H, the ascorbic acid derivatives generally did not inhibit lipid oxidation. For the H T products inhibitory effects were perceived for all product antioxidant treatments. Compared with the H-O, the HT-O products were remarkably unstable. According to
TBA value Crag m a l o n a l d e h y d e / k g fat] l
3.00
2.00
1.00
4
8
12 )
16
Weeks ripening at 15~C
Fig. 3. Course of the mean TBA values (all diet treatments) measured during ripening of semi-dry pork sausages, as related to product antioxidants. (For meaning of s)Tnbols, see legend to Fig. 1.)
TBA value Cmg matonaldehyde/kg T
fat]
4.00 I l.s.d.
oC o H • HT
3.00 -
2.00
-
i .000
A
AP
P
Fig. 4. Mean TBA values measured during ripening of semi-dry pork sausages, as related to dietary and product antioxidant effects. (For meaning of symbols, see legend to Fig. 1.)
208
J. H. Houben, B. Krol
Pongracz (1973) an optimal effect in stabilizing edible fats and oils with e-tocopherol will be obtained at a level of about 200 ppm. In model systems a shift from antioxidative to pro-oxidative action for this c o m p o u n d was found to occur at higher levels (Citlard et al., 1980a,b). In HT backfats, we only reached a content of 16rag a-tocopherol per kilogram of tissue. With regard to lipid oxidation, meat products are very complex systems. Sodium chloride behaves as a pro-oxidant, whereas sodium nitrite usually acts as an antioxidant (Hadden et al., 1975). Metal ions, especially copper and iron and even the iron present in metmyoglobin and some other haemoproteins, catalyze lipid o.~dation (Liu, 1970; Love & Pearson, 1974). Certain components in spices can inhibit these processes (Herrmann et al., 1981). However, it is difficult to explain the strong pro-oxidant action of e-tocopherol in the semidry sausage present in the HT-O products, which certainly was not expected.
Central versus end samples Overall means for peroxide values of the central and terminal sampling areas were 2.369 and 2.687 meq/kg fat, respectively; the least significant difference (lsd) for these means was 0-086 meq/kg fat. Data for the average TBA values of the same sample locations were, respectively, 1.950 and 2"321 mg of malonaldehyde per kilogram of fat (lsd = 0-092 mg of malonaldehyde per kilogram of fat). Means of peroxide values from weeks 7 and 11 were significantly (P < 0.05) higher for the end section as compared with the central section. The same was true for the TBA values from week 4 on. The observed difference in the progress of the oxidation processes probably can be attributed to a greater accessibility of oxygen at the looser upper end (wrinkled casing due to clipping, hanging and drying effects) in comparison with the evenly drying, tighter closed middle part.
Sensory evaluations Scores for external colour, internal colour and consistency did not reveal anything new. Mean scores for aroma taste are presented in Table 2. An analysis of variance for these data did not show any significant effects. Mean aroma/taste scores did not decrease significantly during the first 9 weeks of ripening. However, from weeks 4 (HT products) to 6 (C products) over 50% of the judges denoted rancid off-flavours.
Lipid oxidation in semi-dry pork sausages
209
TABLE 2 Mean Aroma/Taste Scores Obtained During Ripening of Semi-dry Pork Sausages (0-05% Ascorbate Added) as Affected by the Diets of the Pigs
Product 3 Cc H HT Average
6.8 7-1 6-2 6.70 b
4 6.7 7-2 6-2 6-70
Period of ripening (weeks at 15°C) 5 6 7 9 6-9 6-4 7.0 6-77
6-2 a 7-2 6-5 6.63
6-4 6-6 6-6 6-53
6.2 6-9 6-7 6.60
11
13
5.3 6.6 5-3 5-73
6-6 5.7 5-6 5-97
a Underlining a score means that over 50% of the judges denoted rancid off-flavours. b Least significant difference for the averages is 0-78. c Products are coded by the diet composition: C (control), H (high in corn cob mix, CCM) and HT (high CCM supplemented with 100 ppm dl-~-tocopheryl acetate).
Generally, the scores for aroma/taste passed the 6.0 mark after approximately 11 weeks, mean peroxide and TBA values determined for the A products being then 2.31 meq/kg fat and 1-82 mg of malonaldehyde per kilogram of fat. Peroxide and TBA values gradually increase with time, although not always consistently (Fig. 2; HT line). Although the lipid oxidation parameters give information about the progress of oxidation processes, the sensory evaluation data are more important in assessing the acceptability of meat products.
CONCLUSIONS Differences in stability towards oxidation for the sausages manufactured from the meats derived from the three groups of pigs were rather small. Nevertheless, TBA values for the sausages derived from the three diet groups varied considerably. Both peroxide and TBA values gradually increased linearly with time. In all cases, the ascorbic acid derivatives retarded lipid o~dation during a period of 11 weeks of ripening. However, the protective action disappeared during longer periods of storage. Important differences between the product antioxidant treatments were not observed. HT products without ascorbic acid derivatives compared with corresponding H products appeared remarkably unstable. However, HT products containing the ascorbic acid ester in some cases appeared more stable towards oxidation than comparable H products.
210
J. H. Houben, B. Krot
Samples obtained from the top section of the sausages, from about 11 weeks on, consistently showed higher peroxide and TBA values than samples cut from the middle part. Considering the rather variable results obtained by determining peroxide and TBA values for differently formulated products, there appears to be no reason for establishing cut-off values for these parameters with regard to quality.
A C K N O W L E D G E M ENTS The authors are grateful to the State Consultancy Service for Pig Production, Utrecht, for the pig materials, to the Dutch C o m m o d i t y Board for Livestock and Meat and T N O for financial support, to Hoffmann-La Roche BV for fatty acid and tocopherol determinations and Rancimat tests and last, but not least, to Hanne Tjeerdsma-van Bokhoven and Gert Keizer for their skilled technical assistance.
REFERENCES Anon (1981). Vlees- en Vleeswarenbesluit (Warenwet). Arkes, J. G. (1984). Varkensproefbedrijf Noord en Oost Nederland, Proefverslag, No. 36. Arkes, J. G. & Houben, J. H. (1983). Boerderij, 68, 20-VA. Bauernfeind, J. C. & Pinkert, D. M. (1970). Adv. Fd Res., 18, 219. Cillard, J., Cillard, P., Cormier, M. & Girre, L. (1980a). J. Amer. Oil Chem. Soc., 57, 252. Cillard, J., Cillard, P. & Cormier, M. (1980b). J. Amer. Oil Chem. Soe., 57, 255. Cort, W. M. (1974). J. Amer. Oil Chem. Soc., 51, 321. Eriksson, C. E. (1982). Food Chem., 9, 3. Gray, J. I. (1978). J. Amer. Oil Chem. Soc., 55, 539. Hadden, J. P., Ockerman, H. W., Cahill, V. R., Parrett, N. A. & Borton, R. J. (1975). J. Fd Sci., 40, 626. Herrmann, K., Schiitte, M., Mfiller, H. & Bismer, R. (1981). Deutsche Lebensm.Rundschau, 77, 134. Houben, J. H. & Krol, B. (1985). Meat Sci., 13, 193. Kahan, G., Cooper, D., Papavasiliou, A. & Kramer, A. (1973). Food Technol., 27, 63. Kanner, J. & Mendel, H. (1977). J. Fd Sei., 42, 60. Ki/iui, H. (1972). Proc. Int. Symp. C.I.LA., 14, 62.
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K1/iui, H. (1976). Int. Flay. Fd Addit., 7, 165. Krol, B. (1985). Proc. Int. Syrup. Trends in Modern Meat Technology, Pudoc. Wageningen, 58. Liu, H. P. (1970). J. Fd Sci., 35, 590. Love, J. D, & Pearson, A. M. (1974). J. Agric. Fd Chem., 22, 1032. Paquot, C. (1979). Standard methods for the analysis of oils, fats and derivatives. (6th edn), Part 1, Pergamon Press, Oxford. Pongracz, G. (1973). Int. J. Vit. Nutr. Res., 43, 517. Pongracz, G. (1982), Fette Seifen Anstrichm., 84, 269. Reinton, R. & Rogstad, A. (1981). J. Fd Sci., 46, 970. Sedlacek, B. A. J. (1975). Nahrung, 19, 219. Tarladgis, B. G., Watts, B. M. & Younathan, M. T. (1960). J. Amer. Oil Chem. Soc., 37, 44. Tarladgis, B. G., Pearson, A. M. & Dugan, L. R. (1964). J. Sci. Fd Agric., 15, 602.