Release of copper from embedded solid copper

Meat Science 108 (2015) 21–27

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Release of copper from embedded solid copper bullets into muscle and fat tissues of fallow deer (Dama dama), roe deer (Capreolus capreolus), and wild boar (Sus scrofa) and effect of copper content on oxidative stability of heat-processed meat I. Schuhmann-Irschik a, M. Sager b, P. Paulsen a,⁎, A. Tichy c, F. Bauer a a b c

Institute of Meat Hygiene, Meat Technology and Food Science, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, 1210 Vienna, Austria Special Investigations in Element Analysis, Austrian Agency for Health and Food Safety, 1220 Vienna, Austria Bioinformatics and Biostatistics Platform, University of Veterinary Medicine Vienna, 1210 Vienna, Austria

a r t i c l e

i n f o

Article history: Received 16 September 2013 Received in revised form 20 March 2015 Accepted 13 May 2015 Available online 14 May 2015 Keywords: Copper fragments Meat Brine-curing Boiling pH TBARS Exposure assessment

a b s t r a c t When venison with embedded copper bullets was subjected to different culinary processing procedures, the amount of copper released from the embedded bullet was affected more by the retention period of the bullet in the meat during cool storage, than by the different heating protocols. The presence of copper fragments had no significant effect on levels of thiobarbituric acid reactive substances (TBARS). Conversely, TBARS in lean meat (fallow deer, wild boar, roe deer) were significantly affected by culinary treatment (higher TBARS in boiled and boiled-stored meat than in meat barbecued or boiled in brine). In pork–beef patties doped with up to 28 mg/kg Cu, TBARS increased after dry-heating and subsequently storing the meat patties. The amount of copper doping had no effect on TBARS for 0 and 7 days of storage, but a significant effect at day 14 (fat oxidation retarded at higher Cu doses). Evidence is presented that wild boar meat may be more sensitive to fat oxidation than pork–beef. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The use of lead-containing rifle bullets for hunting large game is under debate because of detrimental environmental effects (Cao, Ma, Chen, Hardison, & Harris, 2003; Evangelou, Hockmann, Pokharel, Jakob, & Schulin, 2012) and food safety issues (Hunt et al., 2009; Knott, Gilbert, Hoccom, & Green, 2010). Consequently, there is increased interest in “lead-free” rifle bullets, either of core-jacket type (i.e. tin core instead of lead) or “monoliths” composed of copper only or a copper-alloy (Irschik, Wanek, Bauer, Sager, & Paulsen, 2014). In a previous work, copper contents in large wild game killed with monolithic copper-bullets were studied. Using a non-fragmenting bullet construction, copper contents around the shot wound were not significantly different from those expected for venison. Copper fragments embedded into meat would release copper to the surrounding meat tissue, but only over a very limited distance. It was estimated that, per portion, copper contents would not exceed the recommended daily

⁎ Corresponding author. E-mail address: [email protected] (P. Paulsen).

http://dx.doi.org/10.1016/j.meatsci.2015.05.008 0309-1740/© 2015 Elsevier Ltd. All rights reserved.

intake for this metal (Irschik, Bauer, Sager, & Paulsen, 2013). The effect of culinary processing, was, however, considered only as regards marinating in red wine. A study of Mateo, Rodriguez-de la Cruz, Vidal, Reglero, and Camarero (2007) on the release of lead from fragments embedded in muscle from quails reported substantial increase in lead contents during culinary processing according to a traditional Spanish recipe. Currently, no data exist on the copper contents of meat with embedded copper fragments after culinary treatment. Recently, it has been shown that increased heavy metal contents in inner organs of wild boar are associated with higher levels of thiobarbituric acid reactive substances (TBARS) (Šuran, Prišc, Rašic, Srebočan, & Crnic, 2013). The aim of this study was, therefore, to determine if culinary processing of meat from different game species with embedded copper particles would be more prone to fat oxidation as compared with meat without embedded particles. In addition, oxidative stability of copper-doped meat patties was examined. Based on observations in fermented meats from wild boar which indicate that wild boar meat is more susceptible to rancidity than is pork (Paulsen, Vali, & Bauer, 2011), the oxidative stability of wild boar meat patties was compared to that of patties from a pork– beef mix.

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I. Schuhmann-Irschik et al. / Meat Science 108 (2015) 21–27

2. Materials and methods 2.1. Sample preparation for the determination of TBARS and copper content in meat from wild game, considering copper release from embedded fragments during meat storage and processing (experiment 1) Lean meat originated from fallow deer (Dama dama), roe deer (Capreolus capreolus) and wild boar (Sus scrofa), which had been shot at regular hunting events, and subsequently had been eviscerated and placed in a chiller within 3 h post-mortem. For each of the three species, the following experiment was conducted separately. Shoulder and hind leg muscles were excised from one carcass 24–48 h post-mortem. Subsequently, muscles were cut into 72 cubes of 30 g weight, and randomly assigned to 36 different groups (see below and Fig. 1). The 36 bags (each containing 2 meat cubes, thus forming the experimental unit, or “sample”) were randomly assigned to 12 groups in a 2-factorial design. An overview on the sequence of treatments is depicted in Fig. 1. Factor 1 was the presence of an embedded copper fragment being able to release copper to the surrounding meat. In particular, three variants were considered: (1a) no contamination (“blank”), (1b) contamination by inserting a .223 inch dia. copper bullet (3.5 g weight; Barnes TSX; Barnes Bullets, Mona, USA), in a 30 g meat cube, with the bullet remaining in the meat cube during storage plus subsequent culinary preparation, and (1c) contamination as under (b), but with the bullet being removed after storage i.e. prior to culinary preparation.

Factor 2 in the experiment was four different procedures of culinary treatment: (2a) storage in an open plastic bag under aerobic conditions for 7 days at 5 °C, followed by quick roasting (“barbecuing”; 5 min at 170 °C); (2b) storage with 20 ml of a brine simulant (Table 1, all reagents from Merck, Germany) in an open plastic bag under aerobic conditions for 7 days at 5 °C, followed by boiling in the plastic bag without addition of water (75 °C, 1 h);

Table 1 Brine, composed of wine simulant and vinegar (diluted acetic acid), quantities given for a total of 200 g; pH adjusted to 3.55. Wine simulant (Souci, Fachmann, & Kraut, 2000) Ethanol Lactic acid (1 mol/l) Glacial acetic acid Malic acid Tartaric acid Water

10.0 g 2.5 ml 0.07 ml 23 mg 150 mg Ad 100.0

Vinegar Glacial acetic acid Water

5.0 g Ad 100.0

(2c) storage in an open plastic bag under aerobic conditions for 7 days at 5 °C, followed by boiling in the plastic bag without addition of water (75 °C, 1 h); (2d) conditions as under (2c), but with subsequent storage of cooked meat cubes at 5 °C for 7 days.

For the determination of thiobarbituric acid reactive substances (TBARS), the two meat cubes per bag were combined, minced and an aliquot was analyzed at the same day, while the remaining sample material was preserved at − 20 °C for copper content determination. In addition, copper content in brine simulant and meat juice released during boiling was determined. All analyses were done in duplicate. Per combination of factors 1 and 2, three samples were studied. 2.2. Sample preparation for assessment of the effect of copper content on TBARS content of meat patties from a pork–beef mix (experiment 2) For this experiment, 2 kg minced beef and pork (10% fat) were obtained from a local supermarket. Four 500 g-batches were prepared, with 20 g/kg NaCl. To batches 1, 2 and 3, copper powder (particle size b 63 μm; Roth CP21.1, Roth, Karlsruhe, Germany) suspended in 10 ml water, was added to give final contents of 28, 14 and 7 mg/kg, respectively. Meat batter was mixed thoroughly to ensure uniform distribution of copper particles. The fourth batch received 10 ml water, but no copper doping (“blank”). From each batch, 15 meat patties

Fig. 1. Storage and processing of muscle cubes with and without embedded copper body (.223 inch dia. rifle bullet).


of ca. 30 g weight were formed. All patties were dry-heated at 175 °C for 30 min (core temperature ≥ 75 °C), then left to cool to room temperature. Per batch, five patties each were tested for TBARS immediately after cooling. The remaining 10 patties were wrapped in cling foil, and placed in a household refrigerator (5 °C) with a glass door to ensure exposure of patties to ambient light. After 7 days and 14 days of storage, five patties were removed for examination. This design included the amount of copper powder added and storage time after heating as factors, and TBARS as dependent variable.

2.3. Sample preparation for determination of TBARS content in patties from wild game meat as compared to such made from pork–beef mix (experiment 3) For the preparation of meat patties, 0.5 kg meat from wild boar (10% fat) was minced and 10 g NaCl added. From the batter, 15 patties (ca. 30 g weight) were formed. Likewise, patties were prepared from 0.5 kg of minced meat pork–beef mix (obtained from a local supermarket) with 10 g NaCl. The batches reflected a 2-factorial design, with meat species as one factor, and storage time (0, 7, 14 days with 5 patties per day and species examined) as the second factor. Storage was as described in Section 2.2. This experiment was not repeated.

2.4. Determination of copper and TBARS The determination of copper was done exactly as described by Irschik et al. (2013). In brief, samples were minced, homogenized and a 0.5 g aliquot was microwave digested, and copper content determined by AAS (atomic absorption spectrometry). Limit of determination was 0.08 mg/kg, recovery was 92%. Content of TBARS was determined according to Witte, Krause, and Bailey (1970), with some modifications. In brief, TBARS were extracted by homogenizing 10 g of the sample with 90 ml of ice-cold trichloroacetic acid (100 g/l) using an Ultra Turrax homogenizer (IKA t25; Jahnke & Kunkel, Staufen, Germany) for 1 min at maximum speed. The homogenate was made up to 100 ml with distilled water, and filtrated though a folded filter paper (MN 625 1/4; Macherey Nagel, Düren, Germany). From the filtrate, 5 ml was combined with 5 ml of 0.04 M thiobarbituric acid (TBA), mixed and held for 5 min at 100 °C. The liquid was rapidly cooled to ambient temperature, and extinction was measured at 530 nm (Hitachi U1100; Hitachi, Tokyo, Japan). A mix of 5 ml distilled water and 5 ml TBA served as control. Analytical reagents were obtained from Merck, Germany and Sigma-Aldrich, Germany. All results refer to fresh weight. TBARS were expressed as malondialdehyde (MDA) in mg/kg and were calculated according to: TBARS = (extinction530 nm ∗ molar mass of MDA ∗ 2) / (1.35 ∗ 105 ∗ sample weight in g); where 1.35 ∗ 105 mol/L−1 ∗ cm−1 was a constant. Extraction was carried out once per sample, whereas reaction with TBA and measurements were done in duplicate.

2.5. Statistical processing of data Data were analyzed by two-factorial univariate analysis of variance, with the Scheffé test (Scheffé, 1953) to discriminate among means (SPSS 21, IBM, USA). Significance was established at P b 0.05. Dependent factors were MDA and copper levels in experiment 1 (Section 2.1) and MDA levels in experiments 2 and 3 (Sections 2.2 and 2.3, respectively). Independent factors were the presence/absence of a copper bullet and culinary processing regime (experiment 1), copper doping and storage time (experiment 2) or meat species and storage time (experiment 3).

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3. Results and discussion 3.1. Release of copper from embedded fragments during storage and culinary processing of meat from different game species (experiment 1) After 7 days storage at 5 °C and subsequent culinary processing, average copper contents in meat without embedded bullets (“blanks”) ranged from 1.2–2.8 mg/kg fresh weight (Fig. 2). These results are in the range expected for meat from wild game (Chan, Brown, Lee, & Buss, 1996; Sager, 2005). In general, copper contents in meat stored with embedded bullets were significantly higher than those in blanks. As regards the influence of culinary treatment on copper contents, in most cases the removal of the bullet fragment prior to heating had no significant effect on copper contents, with exceptions of quick roasting of meat from fallow deer (Fig. 2a) and boiling and storage of meat from roe deer (Fig. 2b). In essence, the average copper content did not exceed the threefold copper content of blanks or 6 mg/kg fresh weight, irrespective of which treatment was studied. Considering all individual results, two-third of copper contents were below 3.8 mg/kg, and, thus, in the range expected for roast venison (3.6 mg/kg; Chan et al., 1996). Such contents in a 250 g food portion would still not exceed the recommended daily intake for copper (Irschik et al., 2013). The highest measured copper content was 6.8 mg/kg, and this was taken as a reference value (rounded to 7.0) for doping meat patties with copper (Section 3.3). 3.2. TBARS in meat from different game species, as affected by embedded copper fragments, storage and culinary processing (experiment 1) The presence of a copper fragment in the meat cube during storage or culinary processing did not affect TBARS contents significantly, thus, no attempt was made to relate the actual copper contents in the meat cubes with MDA levels. As regards the mode of culinary treatment, TBARS in meat quickroasted or boiled in brine were significantly lower than those of meat boiled without the addition of water and stored for 7 days at 5 °C (all 3 meat species). The treatment “boiled without addition of water” took an intermediate position according to meat species (Fig. 3). In line with previous studies (Andreo, Doval, Romero, & Judis, 2003; Ferioli, Caboni, & Dutta, 2008; Gray, Gomaa, & Buckley, 1996) highest MDA contents were found in meat that had been heat processed and then stored (Fig. 3). A statistically significant difference between heat treatments “boiling in brine” and “quick roast” vs. “boiling without addition of water” was observed in roe deer and wild boar samples. Since specieseffects may have contributed to these results, further studies are required. The bottom line of this trial was, however, that the presence of copper bullets did obviously not affect the fat oxidation parameter TBARS. 3.3. TBARS in minced meat patties, as affected by different levels of copper doping, heating and storage (experiment 2) The amount of copper powder added to the meat patties was based on the highest individual copper content found in meat cubes with an embedded bullet (Section 3.1), i.e. 7 mg/kg. To estimate worse conditions, also multiples of this amount of copper were considered. Pork–beef mix patties were used in this experiment as in pre-trials with meat from wild boar high initial TBARS were measured (data not shown). In detail, immediately after heat treatment, average TBARS contents were in the range of 0.029-0.039 mg malondialdehyde (MDA)/kg, without significant differences between different levels of copper doping. During storage, MDA contents increased significantly, with a strong positive correlation between storage time and MDA contents as observed in the previous experiment (r2 = 0.96). At day 7, there was no significant effect of the increase of copper doping on MDA contents. At day 14, MDA

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a)

8

blank bullet removed bullet remained

Cu, mg/kg fresh weight

7

i

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i

g

c

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Cu, mg/kg fresh weight

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boiling

boiling + storage (7 d)

fallow deer

7


6

b

b

d

i

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roe deer c) 8 Cu, mg/kg fresh weight

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5 4

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3 2

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g c

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1 0 quick roast

boiling in brine

boiling


wild boar Fig. 2. Copper contents in muscle and fat tissue of fallow deer, roe deer and wild boar as affected by embedded copper particle and culinary processing regime. Per mode of culinary treatment, different letters indicate statistically significant differences between samples with or without embedded copper particle (P b 0.05).

contents in groups with 14 and 28 mg/kg Cu were significantly different from groups with 7 or 0 mg/kg Cu added (Fig. 4). Similar to nickel and iron, copper is a strong pro-oxidant in fats and oils (Franzke, Grunert, Fitzner, & Rossow, 1972; Janíček, Zwain, Pokorný, & Davídek, 1963; Pokorný, Kondratenko, & Janíček, 1967; Pokorný, Zwain, & Janíček, 1963; Sedláček, 1971). Copper ions may oxidize fatty acids directly, and thus contribute to the initiation of fat oxidation. This reaction is considered comparably slow compared to the role of copper in degrading hydroperoxides, which, in turn promotes the formation of fatty acid radicals (Belitz, Grosch, & Schieberle, 2001). Comparably little information is available on the role of copper in meat, fish or products thereof (Lauritzsen, Martinsen, & Olsen, 1999). The pH optimum for pro-oxidative copper activity is in the 5.5–6.0 range (Belitz et al., 2001). While such pH conditions are generally

present in meats, the availability of copper in ionized form as well as the diffusion of oxygen in deeper layers of meat or meat products may become limiting factors for fat oxidation. The formation and fate of copper ions in meats are quite complex. Copper that occurs naturally in live tissues or fresh meat is protein or enzyme bound (Elias & Decker, 2010; Johnson, Fischer, & Kays, 1992), and thus, not pro-oxidative. Copper fragments embedded in tissues corrode and release copper ions, but apparently in a very limited circumference around the fragment (Irschik et al., 2013). During heat processing, copper is liberated from proteins. When copper was administered more evenly distributed as a powder, a copper-doping-related significant effect on fat oxidation was observed only after 14 days of storage. Interestingly, the fat oxidation parameters were lower in the copper-doped samples


25

a) 4,5 4


____c____

3,5

MDA mg/kg

3 2,5 2 1,5 1

____b____

____a,b___ ____a____

_

quick roast

boiling in brine

0,5 0 boiling


fallow deer b)

4,5 4


____c____

MDA mg/kg

3,5 3 2,5

____b____

2 1,5 1

____a____ ____a____

0,5 0 quick roast

boiling in brine

boiling


roe deer c)

4,5 4

____c____ blank bullet removed bullet remained

3,5

MDA mg/kg

3

____b____

2,5 2 1,5

____a____

____a____

1 0,5 0 quick roast

boiling in brine

boiling


wild boar Fig. 3. TBARS in lean muscle from fallow deer, roe deer and wild boar as affected by embedded copper particle and culinary processing regime. Statistically significant differences (P b 0.05) between the different culinary treatments are indicated by different letters.

(samples with 14 and 28 mg/kg significantly differing from those with 7 mg Cu /kg and from those with 0 mg Cu /kg added). In fact, several studies indicate that only low-dose copper (0.01–0.2 μg/kg) will promote fat oxidation in oils and fats. In contrast, higher doses slow down fat oxidation (Pokorný et al., 1963, 1967), which has been explained as being the result of a combination of copper-mediated degradation of peroxide radicals (retarding auto-oxidation) and a relative deficiency of oxygen (retarding the initiation phase). In sum,

levels of hydroperoxides remain low, which hinders progression of auto-oxidation of fats. 3.4. TBARS in minced meat patties from wild boar compared to those from a pork–beef mix, as affected by culinary processing (experiment 3) In previous studies in fermented meat products, evidence has been presented that fermented meat products from wild boar would be

26


c d e

e b

b

b

a

a

b

a

a

a

Fig. 4. TBARS in minced meat patties, as affected by different levels of copper doping (Cu powder) after dry-heating (15 min, 175 °C) and storage. Different letters indicate statistically significant differences between copper levels as well as between duration of storage (P b 0.05).

more prone to oxidative spoilage, compared to pork (Paulsen et al., 2011). This might be due to higher contents of unsaturated fatty acids (Sales & Kotrba, 2013; Valencak, 2010) and higher content of heme iron in meat from wild game (Hofbauer & Smulders, 2011), as these factors influence the shelf-life of fat (Gray et al., 1996; Love & Pearson, 1971; Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998; Sanders, 1989). As a similar mechanism might influence the results presented above, we examined the development of TBARS in dry-heated meat patties from wild boar and pork– (lean) beef mix during a 14-day storage period at 5 °C. It was assumed that the fat component in the pork–beef mix was nearly entirely from pork. In essence, significantly higher MDA contents were found in wild boar meat patties compared to those from pork–beef. Reduced oxidative stability of fat from wild boar has been reported previously, but for fermented meats only (Paulsen et al., 2011). As expected, there was a significant increase in MDA contents (Fig. 5) at days 7 and 14, which corroborates the observations described under Section 3.2. In addition to meat species and days of storage, also the interaction of the two effects had a significant effect on MDA levels. Further studies are necessary to explore the usefulness of wild boar tissues for the manufacture of meat products with extended shelf life.

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4. Conclusion In line with previous findings, copper contents in game meat increased when copper fragments remain embedded in raw meat for 7 days at 5 °C. In general, the nature of heat treatment and the removal of the fragment prior to heat treatment had no significant effect on copper contents. Average copper contents remained b 7 mg/kg fresh weight, which can be considered low as our experiments reflected a “worst case” scenario with one 3.5 g copper fragment per 30 g meat cube. Contents of TBARS were not affected by the presence/absence of embedded fragments. The mode of heat treatment, and, more consistently, storage after heating, had an effect on TBARS. The latter observation was confirmed when TBARS of meat patties (pork–beef) doped with up to 28 mg/kg copper powder were examined after a 14 days storage postheating. On the other hand, in the same experiment it was shown that higher copper doses could in fact retard fat oxidation. However, patties from wild boar meat may be more susceptible to fat oxidation than those manufactured from meat from domestic species. The latter hypothesis is currently under study.

day 0 day 7 day 14

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MDA mg/kg

14

c

12

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10 8 6

b

4 2

a

a

0 pork-beef mix patties (15 min, 175°C)

wild boar meat patties (15 min, 175°C)

Fig. 5. TBARS in minced meat patties, as affected by meat species (wild boar vs. pork–beef mix) and storage. Different letters indicate statistically significant differences between meat species as well as between duration of storage (P b 0.05).


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