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Extraction of meat juices for isotopic analysis
MEAT SCIENCE Meat Science 71 (2005) 334–341 www.elsevier.com/locate/meatsci
Extraction of meat juices for isotopic analysis Ines Thiem *, Matthias Lu¨pke, Hermann Seifert Unit General Radiology and Medical Physics, University of Veterinary Medicine, Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany Received 11 November 2004; received in revised form 5 February 2005; accepted 8 April 2005
Abstract So far no standard procedure exists to obtain water of meat for isotopic 18O/16O-water analysis. Fast extraction via heating the tissues is possible when considering certain boundary conditions. A specially designed vessel was tested with water and was then used for meat juice extraction. The reproducibility (r) of d18O-values was 0.12&. Meat samples of six different species were analysed. Water of pork samples was extracted after open storage. Here, decreases in meat weight correspond to decreases in extract yield and to an increase in the 18O/16O-ratio. The mean water contents in extracts was almost constant [93.2 ± 0.05 wt% (p > 0.05)]. The technique offers an opportunity to develop an automatic, mobile extraction device and to obtain extracts with no further influences on their quality. This method could also be useful for the determination of meat quality attributes as cooking loss or drip without evaporative losses. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Meat juice; Extraction; Oxygen isotope
1. Introduction Consumers demand food from defined sources. The need for reliable identification of its origin increases in times of global meat transfer and pandemic problems. Measurement of 18O/16O-ratios is already being used to determine the origin or adulteration of fruit juices (Roßmann, 1989). Oxygen in water had been considered as a useful parameter for origin analysis due to the known differences in the global and local distribution of 18O (and 16O) in precipitation and thus in ground water (Fo¨rstel & Hu¨tzen, 1982; IAEA/WMO, 2001). Some recent publications discussed the possibility of determining the 18O/16O-ratio in the water in
*
Corresponding author. Tel.: +49 511 856 7445; fax: +49 511 856 827445 E-mail address: [email protected] (I. Thiem). 0309-1740/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.04.023
meat for origin analysis (Boner & Fo¨rstel, 2004; Fo¨rstel & Lickfett, 2002; Hegerding, Seidler, Danneel, Gessler, & Nowak, 2002). The preparation of the water from meat varied between authors and the precision of the methods was not published. Additionally, the origin and the amount of water from within the meat samples varied. This paper describes a rapid extraction procedure for meat juices. We analysed the precision of the extraction using distilled water. After that, meat samples from different animal species were studied. Simulations of the meatÕs storage conditions were used to look for possible changes in the meat juice yield and in the water contents of these extracts. The 18O/16O-ratio in these extracts was determined to examine variations in the 18O/16O-ratio additional to geographical and seasonal differences (in beef; Boner & Fo¨rstel, 2004; Hegerding et al., 2002). The possibility of applying the 18O/16O-ratio for origin analysis of meat is discussed.
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2. Materials and methods
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18.5 °C. Each sample was extracted and the extracts were divided as described above.
2.1. Origin of samples 2.3. Extraction Samples of water, beef, pork and poultry were analysed. Distilled water for determination of the reproducibility was stored in a glass vessel (5 l) with a rubber seal (reservoir). British pork and beef (250 g of 10 animals each species) originated from local butchers. German beef (10 animals) originated from two abattoirs. The meat was packed immediately after slaughter. Pork of German landrace (14 animals) was cut from M. gluteus and M. biceps femoris from at least 1-cm depth, 2 h after the animalsÕ death. Avian muscle tissue originated from 31 broiler pullets, 6 Muscovy and 15 Peking ducks, 16 laying hens, 1 cock and 11 turkeys. The animals were slaughtered for health control of the stables population or deceased in the stable. Samples were prepared from the inner pectoral muscles. None of the tissue had been in contact with air or superficial contaminations. From ducks, only muscles of the hind legs were prepared as the pectoral muscles were too small for good comparison (longer preparation time, less weight). 2.2. Management of samples and extracts
Materials contacting meat or meat juices were cleaned, rinsed with distilled water and dried for 60 min at 90 °C. Before use, all materials were cooled to ambient temperature. New materials, especially rubber seals were degreased with ethanol, rinsed with distilled water and finally degassed for at least a week at 90 °C (Midwood, Haggarty, Milne, & McGaw, 1992). Rubber seals were used not more than four times. All meat samples were extracted using the principle of water release from meat during heating (modified, based on Hegerding et al., 2002; Honikel & Hamm, 1994). To prevent alterations in the 18O/16O-ratio, an air and vapour tight vessel was used for the extraction (Fig. 1). We chose a brass pipe (diameter 30 mm, length 120 mm, wall thickness 1.5 mm) supplied it with a lid at each end and each lid was equipped with a 3-mm deep, 35-mm diameter gap for a rubber seal. The seals were resistant to heat, water and water vapour. Each lid was fastened by three wing-bolts and nuts (soldered to the brass pipe). Before filling, the reactor was closed at one side. A meat sample (50 g) was filled into the
The aim of origin analysis via oxygen isotopes in water is to determine the natural differences between samples. To assure a good reproducibility, the water must not get lost during the extraction process. Therefore, the reliability of the experimental setup and the storage containers was tested with distilled water before meat extraction and analysis to ensure an unchanged 18 O/16O-composition. Five or 10 ml of distilled water was filled in glass vessels (1st hydrol. class; ScherfChroma, 16 ml volume; PE-lid with teflon/butyl-membrane) and stored for 6 months at 20 °C. These specimens were compared to 11 water samples, which were freshly taken from the reservoir. Five water samples were heated according to the procedure performed with meat and then they were stored for 6 months at 20 °C before isotopic measurement. Meat with no air contact was cut into cubes (1–2-cm length). Each meat sample was divided into batches of 50 g. These were extracted individually. Each extract was divided into two or three duplicates (5 g meat juice) for determination of the 18O/16O-ratio. After isotopic measurement, the water contents of the extracts were determined on three duplicates (1 g each). Ninety one pork samples (two individuals) of 50 g each were used to examine changes in the meatÕs properties under different storage conditions. Meat cubes (1–2-cm length) were arranged on 64 cm2, in a single circular layer. The samples were extracted after 11 different periods of storage at 0 and 21.5 °C, respectively, and after 37 periods at
Fig. 1. Dimensions of a brass vessel for heating up tissue for the extraction of 50 g meat.
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brass vessel. Immediately after that the second lid was fastened tightly. The vessel stayed in an oven (90 °C) for 60 min. Time and temperature were chosen according to Honikel and Hamm (1994) to assure breakdown of water binding proteins throughout the sample. After cooling to ambient temperature (60 min), the vessel was shaken to mix all the liquids separated from the meat pieces and condensed water on the vesselÕs walls. The extract was poured into glass vessels within 30 s after opening one lid. To determine the maximum meat juice yield, the remaining meat was compressed for 90 s using a stamp of 29.5-mm diameter (Fig. 2). Extracts were stored at 0 °C and warmed to ambient temperature before subsequent analysis. 2.4. Isotopic measurements Oxygen isotopes in the water molecules of the extracted meat juices were equilibrated over night with those in CO2 (Epstein & Mayeda, 1953). Each sample was measured three times (5 automated cycles per turn). All measurements were carried out on an Isotope Ratio
Mass Spectrometer (IRMS; MAT 251 Thermo Finnigan, Bremen). The internal standard was calibrated by measuring international standards (VSMOW, VSLAP and GIPS), which were obtained from the IAEA in Vienna (Austria). 3. Statistics The 18O/16O-ratio is defined as the difference to the international standard VSMOW (Vienna Standard Mean Ocean Water) the so called d18O-value in & (per mill; Craig, 1957; Schmidt, 1986). RSA and RST are the absolute isotope ratios of sample and standard; cj is an isotopeÕs concentration in the isotopic mixture of an element d ½‰ ¼
RSA RST 1000 ½‰; RST
cj with Rj ¼ Pn
i¼1 ci
. ð1Þ
Normal distribution was tested via v2-test. Deviations of normal distribution were recorded via the Kolmogoroff–Smirnoff-test (Lilliefors modification). Significance was tested by the bilateral StudentÕs t-test (p < 0.05) (Bronstein & Semendjajew, 1991). The 95% confidence interval c0.95 for determination of the expectation value l was calculated according to: c0.95 ¼ x
ta; n1 s pffiffiffi ; n
ð2Þ
where x is the mean value of samples as estimation of expectation value l; ta, n-1 is the factor of bilateral studentÕs t-distribution of normally distributed samples; s is the empirical standard deviation and n is the number of samples. For slope equations the regression r was determined. 4. Results
Fig. 2. Principle of increasing the yield of extracts by meat compression after heating, allowing the determination of the maximum extract yield.
Table 1 shows the comparison of water samples (after heating and/or storage in small containers) with water taken directly from the reservoir. There is no significant difference between the mean d18O-values of heated, not heated or freshly sampled water. The results for the meat juice yield at different heating temperatures are presented in Table 2. The amount of liquids poured from the reactor and the total amount are compared. These samples had only minimum air contact during preparation. It can be seen that higher heating temperatures result in significantly higher meat juice yields. At 60 °C the fast liquid yield (pouring meat juice from the brass vessel) was impossible. In the case of minced meat, no fast extraction was possible. The total amount of extract from minced meat was always less than the extract yield from diced samples.
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Table 1 d18O values of water samples which were heated and stored, stored or fresh Sample preparation
Volume of storage vessel
Number of samples, n
x of d18 O vs. VSMOW (&)
r (&)
Standard Heated and stored Stored without heating
5l 10 ml 5 ml 10 ml –
11 5 5 1 22
8.49 8.55 8.41 8.39 8.46
0.07 0.15 0.15 – 0.12
All samples
Mean value x and reproducibility r in &. No significant difference was found between the mean d18O-values.
Table 2 Meat juice yield (fast = poured within 30 s; total amount) by heating meat samples which had no previous air contact Temperature:
90 °C
Species:
Beef
Origin:
German
Structure:
Diced
Fast x (%) r (%)
60 °C Pork British
German
Diced
Minced
Diced
24.6c 8.0
23.9 4.8
25.4 –
Total yield x (%) r (%)
35.3d 6.3
35.8* 3.8
28.5* –
Number, n
24
25
2
Beef
Pork
British
British
Diced
Minced
Diced
Minced
Diced
Minced
33.7a,c 6.1
20.2a 3.6
19.6 –
– –
– –
– –
– –
42.7b,d 2.2
32.5*,b 4.3
28.0* –
22.4* 3.9
20.8* 5.1
21.7* 7.2
18.7* 9.1
20
15
3
32
6
27
5
Two final heating temperatures, different geographical origin, species and sample structure (minced or diced) were examined. Mean yield x and reproducibility r are given in wt% of the original meat weight. n represents the number of extracted meat samples. – means: no liquid via pouring was extractable. Asterisks and identical letters indicate significant differences between related results (p < 0.05). * A higher heating temperature results in a significantly higher yield (p < 0.05). a,b Significantly higher yields (afast extraction; btotal) from diced meat were found in extracts from German samples. c German pork (diced) showed significantly higher yields (cfast extraction; dtotal) than German beef (diced).
The following results were found for extracts from diced meat (at 90 °C): German pork samples had significantly higher meat juice yields than British ones (pork and beef, respectively). The mean liquid yield (poured and total, respectively) of German pork was significantly higher than it was for German beef.
For German pork samples stored for less than 10 h (Fig. 3), the corresponding mean total extract yield seemed to be similar to the average yield from nonstored meat (42.7 wt%). Applying a linear regression (y = mx + b) to the slopes, the decrease of the total extract yield during the first 10 h of storage was higher
Fig. 3. Meat juice yield from pork samples after open storage at 0, 18.5 or 21.5 °C in weight percent. The time axis is divided in two periods (0–10 and 15–150 h). Each symbol represents 1 meat sample (50 g at t = 0 min, cut in pieces). Extract yield decreases with increasing time of storage. This decrease is higher at ambient temperatures. For data to the slopes (m) see text.
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Fig. 4. Comparison of the weight loss and the total extract yield for pork samples (n = 11) during open storage at 21.5 °C. Each symbol represents 1 sample (50 g at t = 0 min, cut in pieces), the lines are linear ‘‘trends’’.
Fig. 5. 18O/16O-ratio of pork (2 animals) after up to 120 h of open storage at 0, 18.5 or 21.5 °C. 18O/16O-ratio shown as Dd 18O in &, meaning the difference between the 18O/16O-ratio after open storage to the mean d18O-value of the identical individual without storage. Number of samples per symbol: 2 at 0 °C and 18.5 °C; 4 or 6 at 21.5 °C. 0 °C intentionally left without line.
at ambient temperatures (21.5 °C: m = 0.48% per hour; 18.5 °C: m = 0.19% per hour) than it was at 0 °C (m = 0.10% per hour). After more than 3 days
of open storage, the slope of the change in the total extract yield at 21.5 °C was less steep (m = 0.39% per hour), while the slope at 0 °C stayed almost constant (m = 0.12% per hour). Fig. 4 shows a comparison of the weight loss of meat during open storage at 21.5 °C with the corresponding total extract yield. Comparing the ‘‘trend’’-equations of the weight loss (assuming a linear regression: y = mx + b) the slopes differ, depending on the storage temperature and the time period chosen. During the first 10 h of open storage, the weight loss was highest at 18.5 °C (1.32% per hour). At 0 °C, the slope (m) of the weight loss was 1.24% per hour, while it was only 0.88% per hour at 21.5 °C. After a longer storage time (3 and 4 days, respectively), the slopes (m) of the weight loss decreased to 0.6% per hour at 21.5 °C and 0.22% per hour at 0 °C, respectively. After open storage and extraction, the 18O/16O-ratios were determined (Fig. 5). The linear regressions of the first 10 h of open storage at different temperatures were compared (formulae 3, 4). Dd18 21.5 C :
m ¼ 0.402‰=h;
r ¼ 0.958
ð3Þ
Dd18 18.5 C :
m ¼ 0.277‰=h;
r ¼ 0.957
ð4Þ
To describe the slope of the 18O/16O-ratio in water of meat after storage the best mathematical match (up to 96 h) at 21.5 °C was a fourth grade polynomic formula (r = 0.995). Increasing storage times result in decreasing meat juice yield, increasing weight loss and increasing 18 O/16O-ratio values in the extracts and the slopes are steeper at higher temperatures. For poultry, the total meat juice yield is shown in Fig. 6. The amount of extract yield differs between the avian species. From hind leg muscles of ducks and hens, the highest amount was extracted. There was no significant difference in the mean extract yield between the pectoral muscles of slaughtered and deceased broiler chickens.
Fig. 6. Total meat juice yield by heating up poultry in weight percent. i represents the number of individuals, n the number of extracts. There are differences between the avian species. In chickens (cock, hens; broiler deceased/slaughtered) the extract yield is similar.
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Fig. 7. Water contents of extracts from different species in weight percent. Error bars represent reproducibility r; n, number of extract samples.
Normal distribution was found via the v2-test and Kolmogoroff–Smirnoff-test for all non-stored samples (poured and total meat juices yield). Fig. 7 shows the water contents of the extracts from the non-stored meat samples, extracted at 90 °C. There is no significant difference in the mean water contents between extracts from pork and beef. The mean water content of the extracts from pork, which were extracted after open storage of the meat, was 93.4%. The mean water content of all meat juice samples from poultry was 93.5%. Normal distribution was found for the water contents. After measuring 2184 meat juice samples, the 95% confidence interval c0.95 of the expectation value l of the water contents is 93.2 ± 0.05 wt%.
5. Discussion Samples of meat and water were used to establish a method for fast extraction of meat juice for 18O/16O-isotopic analysis. Some manipulations were performed to identify factors influencing the 18O/16O-ratio of water in meat extending former findings (Boner & Fo¨rstel, 2004; Fo¨rstel & Lickfett, 2002; Hegerding et al., 2002). Measuring the 18O/16O-ratio in meat water requires several preparation steps. All handling must prevent loss or addition of water molecules since undefined water exchange could alter the original isotopic ratio. Vessels for meat juice extraction and for storage of extracts were tested for applicability. No significant differences were measured in the 18O/16O-ratios of distilled water after applying the extraction procedure and/or storing the samples for 6 month compared to fresh water. For mass spectrometric analysis of the 18O/16O-ratio in water, the usual standard deviation is 0.1& (Stosch, 1999). The standard deviation in our experiment of 0.12& lies within the range of 0.08–0.13& described by Midwood et al. (1992) for water samples. We conclude that the glass vessels used are suitable for storage of samples for isotopic analysis. Applying careful handling during meat preparation and extraction of liquids the brass vessel
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can be used for rapid extraction of meat juices. Faster extraction of smaller samples (e.g. 5–10 min for 5 g) with a handheld device is conceivable. Sampling in the field without further transportation of meat would thus be possible. The meat juice gain in our study corresponds to the known cooking loss of 23–50% (Honikel, 1987; Ternes, 1998). Higher cooking losses with higher temperatures, as described by Honikel and Hamm (1994) were found for the total meat juice yield. Pouring liquids from the brass vessel was not possible for samples heated at 60 °C due to less destruction of muscle protein structure at lower temperatures and thus higher water holding capacity (Ternes, 1998). At 90 °C, the maximum liquid yield from pork is 60% of the water in the tissue. Thus, in all further experiments 90 °C was used for meat juice extraction. Diced meat was preferred as minced samples gave less extract yield and allowed no pouring. Other authors have extracted water by freeze drying minced meat (Boner & Fo¨rstel, 2004) and this method is used in vine and fruit juice origin analysis (Roßmann, 1989; Schmidt, 1986). However, it is not suitable for in field extraction and it is relatively slow. Also in sublimation, almost all water molecules must be removed from the meat and transferred to the sampling device to prevent isotopic effects since the heavy oxygen isotopes are the last to leave the tissue. Hegerding et al. (2002) heated 100 g meat samples in evacuated PE/PP-bags for approx. 15 min in boiling water. The liquid was extracted via syringe and filter (0.45 lm). The authors did not describe the size of the samples. Meat juice originates mostly from the superficial parts of the sample when the volume of the sample is relatively large and/or the surface too small. This could be the case when samples of 100 g are not cut into smaller pieces. The amount of liquid might be sufficient to perform a mass spectrometric analysis, but the information may be faulty as changes in the isotopic distribution due to storage or manipulation affect the surface first (Thiem, Lu¨pke, & Seifert, 2004). Short cooking times lead to little depths of protein coagulation and, therefore, to a small amount of extract which comes mainly from superficial parts of the meat. The heating in boiling water and puncture of the bag with a needle can also cause cross contamination with oxygen isotopes of the heating water. With respect to these risks, we use a dry heating procedure and no filter to clean the extracts from proteins. Air contacting the meat juice during the pouring from the brass vessel could be reduced by using a closed device. The following sampling device could, in principle be used outside the laboratory: A freshly cut meat sample (e.g., 5 g) is placed in a small container. This can be heated up to 90 °C and then cooled by Peltier elements. A stamp would press the meat juice from the container through a filter and store the required amount of extract
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in an evacuated glass vessel. This is already filled with CO2 to reduce laboratory time. To avoid cross contamination all meat contacting parts must be cleanable or be disposable. Meat juice yield from German pork samples was significantly higher than from German beef and all British meats. British samples were purchased from local butchers and we have no information about the preceding storage conditions. German samples were wrapped to reduce meat juice loss soon after slaughter. The unknown procedures probably account for higher standard deviations for the total meat juice yield in the British samples compared to those for German meat. For pork, one additional factor may account for the observed differences in the meat juice yield since in our experiment we used German landrace. This breed is known to suffer from PSE (Breed Description, 2004). Another possible cause for inter species differences could be sought in the processing. Weight loss reduction and carcass cooling via water spray alter water contents and the 18O/16O-ratios of the water in the superficial meat (Weightloss Reduction, 2003). Pork is usually sold within a few days of death but beef may be stored up to 4 weeks (Fleischqualita¨t, 2003). During this time, the isotopic composition can be influenced as diffusion of water molecules between different water in the meat occurs (Honikel & Hamm, 1994). After open storage of pork a reduction of the meat juice yield after 10 h becomes obvious. This can be explained by increased water loss from the meatÕs surface via evaporation. Our findings correspond well with the observation of a limited independence of the cooking loss and preceding drip loss (Honikel & Hamm, 1994). Differing storage times or temperatures after slaughter can result in varying meat juice loss (Khan, 1977; Park, 1996). If the weight loss and the reduction in total extract yield is linear the storage time of meat could be easily estimated. Park (1996) states that water loss in food is linear down to the critical water content of 25%. The highest water loss in our study was 53.4% of the original weight (96 h of open storage at 21.5 °C), well above a water content of 25% in the meat. If the statement of Park (1996) is valid for meat the slope (m) should be constant for a given temperature. At 21.5 and 0 °C, m is smaller after 3 and 4 days, respectively, of open storage compared to the values for the first 10 h. At 0 °C, the change is very large. It can be concluded that in meat the loss of water (measured as weight loss) is not linear down to a water content of 25% in meat. Especially, cell walls and other structures (e.g., fat) might obstruct water evaporation and diffusion. While cooling down the water loss per hour is higher than at 0 °C. Here, not only does the weight of the carcass decrease (financial factor), but the isotopic composition of the
remaining water changes (analytical factor). It is not possible to correct for this analytical factor by determining the total extract yield and subsequent use of a simple linear equation. A simple correction factor is only applicable if the ratio of total yield to weight loss is constant. For the total yield the slope (m) changes moderately (21.5 °C) or is almost identical (0 °C) comparing the values of over 3 days of open storage to those over the first 10 hours. Therefore, the value of total yield is not a reliable tool to estimate loss or isotopic changes in meat water. Nevertheless, the relatively high total yield of extracts even after high weight losses allows comparison between different parts of the same meat sample. Determinations of the total extract yield allows only estimations of the magnitude of meat juice and/or water loss. Analysing the d18O value in meat water could provide useful data about an animal. Oxygen in meat water originates from drinking water, water and oxygen in food or oxygen in the air (Hegerding et al., 2002; Kornexl, Werner, Roßmann, & Schmidt, 1997). Most authors did not take into account possible postmortem changes in the 18O/16O-ratio. The main influence here is the evaporation of water. Due to isotope effects lighter molecules are lost more easily during evaporation and the remaining water becomes enriched in 18O (Thiem et al., 2004). The enrichment is directly correlated to the temperature and the area of the meat surface to the volume of the water. Variations between surface and central specimens of one meat piece may offer the chance to identify storage conditions. Water addition for illegal weight enhancement or water spray cooling could be determined because 18O is less abundant in tap water than it is in meat or milk (Kornexl et al., 1997; Thiem et al., 2004). Meat juice yield in poultry was not significantly different between slaughtered and deceased animals. In our study, it allowed no information about differences in the distribution of body water in the two groups even though differences in the 18O/16O-ratios were significant. Animals which were found dead (deceased) were enriched in 18O in the meat water (Thiem, 2003). The extract is a mixture of water, proteins and other compounds of meat. During the equilibration process, oxygen isotopes of the water equilibrate with oxygen isotopes in the measuring gas (CO2). Thus, the determination of the water contents of the (measured) extract was necessary for calculation of the 18O/16O-ratio. For the water contents of extracts, the results show a constant value across all studied species and even after significant weight loss of the pork sample after open storage. It can be assumed that possible influences of non-water-molecules to the isotope exchange reaction are also almost constant. The value of 93.2% as water contents of the extracts from meat can be used to simplify further calculations.
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Acknowledgement Special thanks to Prof. Neumann, University of Veterinary Medicine, Hannover, for providing poultry samples.
References Boner, M., & Fo¨rstel, H. (2004). Stable isotope variation as a tool to trace the authenticity of beef. Analytical and Bioanalytical Chemistry, 378(2), 301–310. Breed Description. www.tiho-hannover.de/einricht/zucht/eaap/descript/ 920.htm; (from 30.05.2004) Bronstein, S., & Semendjajew, K. A. (1991). Taschenbuch der Mathematik. 25. Aufl. B.G. Teubner Verlagsgesellschaft, Stuttgart, Leipzig, (pp. 687) Craig, H. (1957). Isotopic standards for carbon and oxygen and correction factors for mass spectrometry analysis of carbon dioxide. Geochimica et Cosmochimica Acta, 12, 133–148. Epstein, S., & Mayeda, T. (1953). Variation of O18 content of water from natural sources. Geochimica Cosmochimica Acta, 4, 213–224. Fleischqualita¨t: Ministerium fu¨r Erna¨hrung und La¨ndlichen Raum Baden – Wu¨rttemberg. www.infodienstlr.bwl.de/ernaehrung/lebensmittel/waren/fisch_fleisch_eier/qualitaet.htm (from 14.07.2003) Fo¨rstel, H., & Hu¨tzen, H. (1982). Variation des Verha¨ltnisses der stabilen Sauerstoff-Isotopen im Grundwasser der Bundesrepublik Deutschland. Ju¨l-1784, Mai Fo¨rstel, H., & Lickfett, J. (2002). Mit isotopem Fingerabdruck den Lebensmitteln auf der Spur. Bioworld, 1, 26–27. Hegerding, L., Seidler, D., Danneel, H.-J., Gessler, A., & Nowak, B. (2002). Sauerstoffisotopen-Verha¨ltnisanalyse zur Herkunftsbestimmung von Rindfleisch. Die Fleischwirtschaft, 4, 95–100. Honikel, K. O. (1987). Wasserbindungsvermo¨gen von Fleisch. Fleischwirtschaft, 67(4), 418–428. Honikel, K. O., & Hamm, R. (1994). Quality attributes and their measurement in meat, poultry and fish products. In A. M. Pearson & T. R. Dutson (Eds.). Advances in meat research series (9S, pp. S.125–S.150). St. Edmunds: Verlag Chapman & Hall.
341
IAEA/WMO: Global Network of Isotopes in Precipitation. (2001). The GNIP database. Available from http://isohis.iaea.org. Khan, A. W. (1977). Some factors causing variation in the texture of similar muscles from comparable animals. Meat Science, 1, 169–176. Kornexl, B. E., Werner, T., Roßmann, A., & Schmidt, H. L. (1997). Measurement of stable isotope abundances in milk and milk ingredience – a possible tool for origin assignment and quality control. Zeitschrift fu¨r Lebensmittel Untersuchung und Forschung, 205, 19–24. Midwood, A. J., Haggarty, P., Milne, E., & McGaw, B. A. (1992). Factors affecting the analysis of 18O-enriched aqueous samples when using CO2-equilibration in vacutainersä. Applied Radiation and Isotopes, 43(11), 1341–1347. Park, Y. W. (1996). In L. M. L. Nollet (Ed.). Handbook of food analysis: Physical characterisation and nutrient analysis (Vol. 1, pp. 60–74). New York: Marcel Dekker. Roß mann, A. (1989). Isotopenverteilung in Glucose und sekunda¨ren Naturstoffen und ihre Bedeutung zur Herkunftsbestimmung bei Lebensmitteln. Dissertation, Mu¨nchen-Weihenstephan, Techn. Univ Schmidt, H. L. (1986). Food quality control and studies on human nutrition by mass spectrometric and nuclear magnetic resonance isotope ration determination. Zeitschrift fu¨r Analytische Chemie Fresenius, 324, 760–766. Stosch, H. G. (1999). Einfu¨hrung in die Isotopengeochemie. Based on lectures at the Mineralogisch–Petrograpischen Institut of the University of Cologne between 1988 and 1994, revised and expanded (p. 186) Ternes, W. (1998). Naturwissenschaftliche Grundlagen der Lebensmittelzubereitung. unvera¨nderter Nachdruck der 2. Aufl. BehrÕs Verlag Hamburg (pp. 281–296) Thiem, I. (2003). 18O/16O-Isotopenverha¨ltnis in Fleisch zur Herkunftsanalytik bei Rind, Schwein und Geflu¨gel: Natu¨rliche Variation und Simulation von Sto¨rgro¨ß en. Dissertation, University of Veterinary Medicine, Hannover Thiem, I., Lu¨pke, M., & Seifert, H. (2004). Influencing factors to the 18 O/16O-ratio in meat juices. Isotopes in Environmental and Health Studies, 40, 4. Weightloss reduction. (2003). Societe dÕInnovation et de Recherche pour lÕIndustrie et lÕAgro-Alimentaire. www.sirial.fr/brumisation_angl.htm (from 14.07.2003).
Extraction of meat juices for isotopic analysis Ines Thiem *, Matthias Lu¨pke, Hermann Seifert Unit General Radiology and Medical Physics, University of Veterinary Medicine, Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany Received 11 November 2004; received in revised form 5 February 2005; accepted 8 April 2005
Abstract So far no standard procedure exists to obtain water of meat for isotopic 18O/16O-water analysis. Fast extraction via heating the tissues is possible when considering certain boundary conditions. A specially designed vessel was tested with water and was then used for meat juice extraction. The reproducibility (r) of d18O-values was 0.12&. Meat samples of six different species were analysed. Water of pork samples was extracted after open storage. Here, decreases in meat weight correspond to decreases in extract yield and to an increase in the 18O/16O-ratio. The mean water contents in extracts was almost constant [93.2 ± 0.05 wt% (p > 0.05)]. The technique offers an opportunity to develop an automatic, mobile extraction device and to obtain extracts with no further influences on their quality. This method could also be useful for the determination of meat quality attributes as cooking loss or drip without evaporative losses. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Meat juice; Extraction; Oxygen isotope
1. Introduction Consumers demand food from defined sources. The need for reliable identification of its origin increases in times of global meat transfer and pandemic problems. Measurement of 18O/16O-ratios is already being used to determine the origin or adulteration of fruit juices (Roßmann, 1989). Oxygen in water had been considered as a useful parameter for origin analysis due to the known differences in the global and local distribution of 18O (and 16O) in precipitation and thus in ground water (Fo¨rstel & Hu¨tzen, 1982; IAEA/WMO, 2001). Some recent publications discussed the possibility of determining the 18O/16O-ratio in the water in
*
Corresponding author. Tel.: +49 511 856 7445; fax: +49 511 856 827445 E-mail address: [email protected] (I. Thiem). 0309-1740/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2005.04.023
meat for origin analysis (Boner & Fo¨rstel, 2004; Fo¨rstel & Lickfett, 2002; Hegerding, Seidler, Danneel, Gessler, & Nowak, 2002). The preparation of the water from meat varied between authors and the precision of the methods was not published. Additionally, the origin and the amount of water from within the meat samples varied. This paper describes a rapid extraction procedure for meat juices. We analysed the precision of the extraction using distilled water. After that, meat samples from different animal species were studied. Simulations of the meatÕs storage conditions were used to look for possible changes in the meat juice yield and in the water contents of these extracts. The 18O/16O-ratio in these extracts was determined to examine variations in the 18O/16O-ratio additional to geographical and seasonal differences (in beef; Boner & Fo¨rstel, 2004; Hegerding et al., 2002). The possibility of applying the 18O/16O-ratio for origin analysis of meat is discussed.
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2. Materials and methods
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18.5 °C. Each sample was extracted and the extracts were divided as described above.
2.1. Origin of samples 2.3. Extraction Samples of water, beef, pork and poultry were analysed. Distilled water for determination of the reproducibility was stored in a glass vessel (5 l) with a rubber seal (reservoir). British pork and beef (250 g of 10 animals each species) originated from local butchers. German beef (10 animals) originated from two abattoirs. The meat was packed immediately after slaughter. Pork of German landrace (14 animals) was cut from M. gluteus and M. biceps femoris from at least 1-cm depth, 2 h after the animalsÕ death. Avian muscle tissue originated from 31 broiler pullets, 6 Muscovy and 15 Peking ducks, 16 laying hens, 1 cock and 11 turkeys. The animals were slaughtered for health control of the stables population or deceased in the stable. Samples were prepared from the inner pectoral muscles. None of the tissue had been in contact with air or superficial contaminations. From ducks, only muscles of the hind legs were prepared as the pectoral muscles were too small for good comparison (longer preparation time, less weight). 2.2. Management of samples and extracts
Materials contacting meat or meat juices were cleaned, rinsed with distilled water and dried for 60 min at 90 °C. Before use, all materials were cooled to ambient temperature. New materials, especially rubber seals were degreased with ethanol, rinsed with distilled water and finally degassed for at least a week at 90 °C (Midwood, Haggarty, Milne, & McGaw, 1992). Rubber seals were used not more than four times. All meat samples were extracted using the principle of water release from meat during heating (modified, based on Hegerding et al., 2002; Honikel & Hamm, 1994). To prevent alterations in the 18O/16O-ratio, an air and vapour tight vessel was used for the extraction (Fig. 1). We chose a brass pipe (diameter 30 mm, length 120 mm, wall thickness 1.5 mm) supplied it with a lid at each end and each lid was equipped with a 3-mm deep, 35-mm diameter gap for a rubber seal. The seals were resistant to heat, water and water vapour. Each lid was fastened by three wing-bolts and nuts (soldered to the brass pipe). Before filling, the reactor was closed at one side. A meat sample (50 g) was filled into the
The aim of origin analysis via oxygen isotopes in water is to determine the natural differences between samples. To assure a good reproducibility, the water must not get lost during the extraction process. Therefore, the reliability of the experimental setup and the storage containers was tested with distilled water before meat extraction and analysis to ensure an unchanged 18 O/16O-composition. Five or 10 ml of distilled water was filled in glass vessels (1st hydrol. class; ScherfChroma, 16 ml volume; PE-lid with teflon/butyl-membrane) and stored for 6 months at 20 °C. These specimens were compared to 11 water samples, which were freshly taken from the reservoir. Five water samples were heated according to the procedure performed with meat and then they were stored for 6 months at 20 °C before isotopic measurement. Meat with no air contact was cut into cubes (1–2-cm length). Each meat sample was divided into batches of 50 g. These were extracted individually. Each extract was divided into two or three duplicates (5 g meat juice) for determination of the 18O/16O-ratio. After isotopic measurement, the water contents of the extracts were determined on three duplicates (1 g each). Ninety one pork samples (two individuals) of 50 g each were used to examine changes in the meatÕs properties under different storage conditions. Meat cubes (1–2-cm length) were arranged on 64 cm2, in a single circular layer. The samples were extracted after 11 different periods of storage at 0 and 21.5 °C, respectively, and after 37 periods at
Fig. 1. Dimensions of a brass vessel for heating up tissue for the extraction of 50 g meat.
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brass vessel. Immediately after that the second lid was fastened tightly. The vessel stayed in an oven (90 °C) for 60 min. Time and temperature were chosen according to Honikel and Hamm (1994) to assure breakdown of water binding proteins throughout the sample. After cooling to ambient temperature (60 min), the vessel was shaken to mix all the liquids separated from the meat pieces and condensed water on the vesselÕs walls. The extract was poured into glass vessels within 30 s after opening one lid. To determine the maximum meat juice yield, the remaining meat was compressed for 90 s using a stamp of 29.5-mm diameter (Fig. 2). Extracts were stored at 0 °C and warmed to ambient temperature before subsequent analysis. 2.4. Isotopic measurements Oxygen isotopes in the water molecules of the extracted meat juices were equilibrated over night with those in CO2 (Epstein & Mayeda, 1953). Each sample was measured three times (5 automated cycles per turn). All measurements were carried out on an Isotope Ratio
Mass Spectrometer (IRMS; MAT 251 Thermo Finnigan, Bremen). The internal standard was calibrated by measuring international standards (VSMOW, VSLAP and GIPS), which were obtained from the IAEA in Vienna (Austria). 3. Statistics The 18O/16O-ratio is defined as the difference to the international standard VSMOW (Vienna Standard Mean Ocean Water) the so called d18O-value in & (per mill; Craig, 1957; Schmidt, 1986). RSA and RST are the absolute isotope ratios of sample and standard; cj is an isotopeÕs concentration in the isotopic mixture of an element d ½‰ ¼
RSA RST 1000 ½‰; RST
cj with Rj ¼ Pn
i¼1 ci
. ð1Þ
Normal distribution was tested via v2-test. Deviations of normal distribution were recorded via the Kolmogoroff–Smirnoff-test (Lilliefors modification). Significance was tested by the bilateral StudentÕs t-test (p < 0.05) (Bronstein & Semendjajew, 1991). The 95% confidence interval c0.95 for determination of the expectation value l was calculated according to: c0.95 ¼ x
ta; n1 s pffiffiffi ; n
ð2Þ
where x is the mean value of samples as estimation of expectation value l; ta, n-1 is the factor of bilateral studentÕs t-distribution of normally distributed samples; s is the empirical standard deviation and n is the number of samples. For slope equations the regression r was determined. 4. Results
Fig. 2. Principle of increasing the yield of extracts by meat compression after heating, allowing the determination of the maximum extract yield.
Table 1 shows the comparison of water samples (after heating and/or storage in small containers) with water taken directly from the reservoir. There is no significant difference between the mean d18O-values of heated, not heated or freshly sampled water. The results for the meat juice yield at different heating temperatures are presented in Table 2. The amount of liquids poured from the reactor and the total amount are compared. These samples had only minimum air contact during preparation. It can be seen that higher heating temperatures result in significantly higher meat juice yields. At 60 °C the fast liquid yield (pouring meat juice from the brass vessel) was impossible. In the case of minced meat, no fast extraction was possible. The total amount of extract from minced meat was always less than the extract yield from diced samples.
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Table 1 d18O values of water samples which were heated and stored, stored or fresh Sample preparation
Volume of storage vessel
Number of samples, n
x of d18 O vs. VSMOW (&)
r (&)
Standard Heated and stored Stored without heating
5l 10 ml 5 ml 10 ml –
11 5 5 1 22
8.49 8.55 8.41 8.39 8.46
0.07 0.15 0.15 – 0.12
All samples
Mean value x and reproducibility r in &. No significant difference was found between the mean d18O-values.
Table 2 Meat juice yield (fast = poured within 30 s; total amount) by heating meat samples which had no previous air contact Temperature:
90 °C
Species:
Beef
Origin:
German
Structure:
Diced
Fast x (%) r (%)
60 °C Pork British
German
Diced
Minced
Diced
24.6c 8.0
23.9 4.8
25.4 –
Total yield x (%) r (%)
35.3d 6.3
35.8* 3.8
28.5* –
Number, n
24
25
2
Beef
Pork
British
British
Diced
Minced
Diced
Minced
Diced
Minced
33.7a,c 6.1
20.2a 3.6
19.6 –
– –
– –
– –
– –
42.7b,d 2.2
32.5*,b 4.3
28.0* –
22.4* 3.9
20.8* 5.1
21.7* 7.2
18.7* 9.1
20
15
3
32
6
27
5
Two final heating temperatures, different geographical origin, species and sample structure (minced or diced) were examined. Mean yield x and reproducibility r are given in wt% of the original meat weight. n represents the number of extracted meat samples. – means: no liquid via pouring was extractable. Asterisks and identical letters indicate significant differences between related results (p < 0.05). * A higher heating temperature results in a significantly higher yield (p < 0.05). a,b Significantly higher yields (afast extraction; btotal) from diced meat were found in extracts from German samples. c German pork (diced) showed significantly higher yields (cfast extraction; dtotal) than German beef (diced).
The following results were found for extracts from diced meat (at 90 °C): German pork samples had significantly higher meat juice yields than British ones (pork and beef, respectively). The mean liquid yield (poured and total, respectively) of German pork was significantly higher than it was for German beef.
For German pork samples stored for less than 10 h (Fig. 3), the corresponding mean total extract yield seemed to be similar to the average yield from nonstored meat (42.7 wt%). Applying a linear regression (y = mx + b) to the slopes, the decrease of the total extract yield during the first 10 h of storage was higher
Fig. 3. Meat juice yield from pork samples after open storage at 0, 18.5 or 21.5 °C in weight percent. The time axis is divided in two periods (0–10 and 15–150 h). Each symbol represents 1 meat sample (50 g at t = 0 min, cut in pieces). Extract yield decreases with increasing time of storage. This decrease is higher at ambient temperatures. For data to the slopes (m) see text.
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Fig. 4. Comparison of the weight loss and the total extract yield for pork samples (n = 11) during open storage at 21.5 °C. Each symbol represents 1 sample (50 g at t = 0 min, cut in pieces), the lines are linear ‘‘trends’’.
Fig. 5. 18O/16O-ratio of pork (2 animals) after up to 120 h of open storage at 0, 18.5 or 21.5 °C. 18O/16O-ratio shown as Dd 18O in &, meaning the difference between the 18O/16O-ratio after open storage to the mean d18O-value of the identical individual without storage. Number of samples per symbol: 2 at 0 °C and 18.5 °C; 4 or 6 at 21.5 °C. 0 °C intentionally left without line.
at ambient temperatures (21.5 °C: m = 0.48% per hour; 18.5 °C: m = 0.19% per hour) than it was at 0 °C (m = 0.10% per hour). After more than 3 days
of open storage, the slope of the change in the total extract yield at 21.5 °C was less steep (m = 0.39% per hour), while the slope at 0 °C stayed almost constant (m = 0.12% per hour). Fig. 4 shows a comparison of the weight loss of meat during open storage at 21.5 °C with the corresponding total extract yield. Comparing the ‘‘trend’’-equations of the weight loss (assuming a linear regression: y = mx + b) the slopes differ, depending on the storage temperature and the time period chosen. During the first 10 h of open storage, the weight loss was highest at 18.5 °C (1.32% per hour). At 0 °C, the slope (m) of the weight loss was 1.24% per hour, while it was only 0.88% per hour at 21.5 °C. After a longer storage time (3 and 4 days, respectively), the slopes (m) of the weight loss decreased to 0.6% per hour at 21.5 °C and 0.22% per hour at 0 °C, respectively. After open storage and extraction, the 18O/16O-ratios were determined (Fig. 5). The linear regressions of the first 10 h of open storage at different temperatures were compared (formulae 3, 4). Dd18 21.5 C :
m ¼ 0.402‰=h;
r ¼ 0.958
ð3Þ
Dd18 18.5 C :
m ¼ 0.277‰=h;
r ¼ 0.957
ð4Þ
To describe the slope of the 18O/16O-ratio in water of meat after storage the best mathematical match (up to 96 h) at 21.5 °C was a fourth grade polynomic formula (r = 0.995). Increasing storage times result in decreasing meat juice yield, increasing weight loss and increasing 18 O/16O-ratio values in the extracts and the slopes are steeper at higher temperatures. For poultry, the total meat juice yield is shown in Fig. 6. The amount of extract yield differs between the avian species. From hind leg muscles of ducks and hens, the highest amount was extracted. There was no significant difference in the mean extract yield between the pectoral muscles of slaughtered and deceased broiler chickens.
Fig. 6. Total meat juice yield by heating up poultry in weight percent. i represents the number of individuals, n the number of extracts. There are differences between the avian species. In chickens (cock, hens; broiler deceased/slaughtered) the extract yield is similar.
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Fig. 7. Water contents of extracts from different species in weight percent. Error bars represent reproducibility r; n, number of extract samples.
Normal distribution was found via the v2-test and Kolmogoroff–Smirnoff-test for all non-stored samples (poured and total meat juices yield). Fig. 7 shows the water contents of the extracts from the non-stored meat samples, extracted at 90 °C. There is no significant difference in the mean water contents between extracts from pork and beef. The mean water content of the extracts from pork, which were extracted after open storage of the meat, was 93.4%. The mean water content of all meat juice samples from poultry was 93.5%. Normal distribution was found for the water contents. After measuring 2184 meat juice samples, the 95% confidence interval c0.95 of the expectation value l of the water contents is 93.2 ± 0.05 wt%.
5. Discussion Samples of meat and water were used to establish a method for fast extraction of meat juice for 18O/16O-isotopic analysis. Some manipulations were performed to identify factors influencing the 18O/16O-ratio of water in meat extending former findings (Boner & Fo¨rstel, 2004; Fo¨rstel & Lickfett, 2002; Hegerding et al., 2002). Measuring the 18O/16O-ratio in meat water requires several preparation steps. All handling must prevent loss or addition of water molecules since undefined water exchange could alter the original isotopic ratio. Vessels for meat juice extraction and for storage of extracts were tested for applicability. No significant differences were measured in the 18O/16O-ratios of distilled water after applying the extraction procedure and/or storing the samples for 6 month compared to fresh water. For mass spectrometric analysis of the 18O/16O-ratio in water, the usual standard deviation is 0.1& (Stosch, 1999). The standard deviation in our experiment of 0.12& lies within the range of 0.08–0.13& described by Midwood et al. (1992) for water samples. We conclude that the glass vessels used are suitable for storage of samples for isotopic analysis. Applying careful handling during meat preparation and extraction of liquids the brass vessel
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can be used for rapid extraction of meat juices. Faster extraction of smaller samples (e.g. 5–10 min for 5 g) with a handheld device is conceivable. Sampling in the field without further transportation of meat would thus be possible. The meat juice gain in our study corresponds to the known cooking loss of 23–50% (Honikel, 1987; Ternes, 1998). Higher cooking losses with higher temperatures, as described by Honikel and Hamm (1994) were found for the total meat juice yield. Pouring liquids from the brass vessel was not possible for samples heated at 60 °C due to less destruction of muscle protein structure at lower temperatures and thus higher water holding capacity (Ternes, 1998). At 90 °C, the maximum liquid yield from pork is 60% of the water in the tissue. Thus, in all further experiments 90 °C was used for meat juice extraction. Diced meat was preferred as minced samples gave less extract yield and allowed no pouring. Other authors have extracted water by freeze drying minced meat (Boner & Fo¨rstel, 2004) and this method is used in vine and fruit juice origin analysis (Roßmann, 1989; Schmidt, 1986). However, it is not suitable for in field extraction and it is relatively slow. Also in sublimation, almost all water molecules must be removed from the meat and transferred to the sampling device to prevent isotopic effects since the heavy oxygen isotopes are the last to leave the tissue. Hegerding et al. (2002) heated 100 g meat samples in evacuated PE/PP-bags for approx. 15 min in boiling water. The liquid was extracted via syringe and filter (0.45 lm). The authors did not describe the size of the samples. Meat juice originates mostly from the superficial parts of the sample when the volume of the sample is relatively large and/or the surface too small. This could be the case when samples of 100 g are not cut into smaller pieces. The amount of liquid might be sufficient to perform a mass spectrometric analysis, but the information may be faulty as changes in the isotopic distribution due to storage or manipulation affect the surface first (Thiem, Lu¨pke, & Seifert, 2004). Short cooking times lead to little depths of protein coagulation and, therefore, to a small amount of extract which comes mainly from superficial parts of the meat. The heating in boiling water and puncture of the bag with a needle can also cause cross contamination with oxygen isotopes of the heating water. With respect to these risks, we use a dry heating procedure and no filter to clean the extracts from proteins. Air contacting the meat juice during the pouring from the brass vessel could be reduced by using a closed device. The following sampling device could, in principle be used outside the laboratory: A freshly cut meat sample (e.g., 5 g) is placed in a small container. This can be heated up to 90 °C and then cooled by Peltier elements. A stamp would press the meat juice from the container through a filter and store the required amount of extract
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in an evacuated glass vessel. This is already filled with CO2 to reduce laboratory time. To avoid cross contamination all meat contacting parts must be cleanable or be disposable. Meat juice yield from German pork samples was significantly higher than from German beef and all British meats. British samples were purchased from local butchers and we have no information about the preceding storage conditions. German samples were wrapped to reduce meat juice loss soon after slaughter. The unknown procedures probably account for higher standard deviations for the total meat juice yield in the British samples compared to those for German meat. For pork, one additional factor may account for the observed differences in the meat juice yield since in our experiment we used German landrace. This breed is known to suffer from PSE (Breed Description, 2004). Another possible cause for inter species differences could be sought in the processing. Weight loss reduction and carcass cooling via water spray alter water contents and the 18O/16O-ratios of the water in the superficial meat (Weightloss Reduction, 2003). Pork is usually sold within a few days of death but beef may be stored up to 4 weeks (Fleischqualita¨t, 2003). During this time, the isotopic composition can be influenced as diffusion of water molecules between different water in the meat occurs (Honikel & Hamm, 1994). After open storage of pork a reduction of the meat juice yield after 10 h becomes obvious. This can be explained by increased water loss from the meatÕs surface via evaporation. Our findings correspond well with the observation of a limited independence of the cooking loss and preceding drip loss (Honikel & Hamm, 1994). Differing storage times or temperatures after slaughter can result in varying meat juice loss (Khan, 1977; Park, 1996). If the weight loss and the reduction in total extract yield is linear the storage time of meat could be easily estimated. Park (1996) states that water loss in food is linear down to the critical water content of 25%. The highest water loss in our study was 53.4% of the original weight (96 h of open storage at 21.5 °C), well above a water content of 25% in the meat. If the statement of Park (1996) is valid for meat the slope (m) should be constant for a given temperature. At 21.5 and 0 °C, m is smaller after 3 and 4 days, respectively, of open storage compared to the values for the first 10 h. At 0 °C, the change is very large. It can be concluded that in meat the loss of water (measured as weight loss) is not linear down to a water content of 25% in meat. Especially, cell walls and other structures (e.g., fat) might obstruct water evaporation and diffusion. While cooling down the water loss per hour is higher than at 0 °C. Here, not only does the weight of the carcass decrease (financial factor), but the isotopic composition of the
remaining water changes (analytical factor). It is not possible to correct for this analytical factor by determining the total extract yield and subsequent use of a simple linear equation. A simple correction factor is only applicable if the ratio of total yield to weight loss is constant. For the total yield the slope (m) changes moderately (21.5 °C) or is almost identical (0 °C) comparing the values of over 3 days of open storage to those over the first 10 hours. Therefore, the value of total yield is not a reliable tool to estimate loss or isotopic changes in meat water. Nevertheless, the relatively high total yield of extracts even after high weight losses allows comparison between different parts of the same meat sample. Determinations of the total extract yield allows only estimations of the magnitude of meat juice and/or water loss. Analysing the d18O value in meat water could provide useful data about an animal. Oxygen in meat water originates from drinking water, water and oxygen in food or oxygen in the air (Hegerding et al., 2002; Kornexl, Werner, Roßmann, & Schmidt, 1997). Most authors did not take into account possible postmortem changes in the 18O/16O-ratio. The main influence here is the evaporation of water. Due to isotope effects lighter molecules are lost more easily during evaporation and the remaining water becomes enriched in 18O (Thiem et al., 2004). The enrichment is directly correlated to the temperature and the area of the meat surface to the volume of the water. Variations between surface and central specimens of one meat piece may offer the chance to identify storage conditions. Water addition for illegal weight enhancement or water spray cooling could be determined because 18O is less abundant in tap water than it is in meat or milk (Kornexl et al., 1997; Thiem et al., 2004). Meat juice yield in poultry was not significantly different between slaughtered and deceased animals. In our study, it allowed no information about differences in the distribution of body water in the two groups even though differences in the 18O/16O-ratios were significant. Animals which were found dead (deceased) were enriched in 18O in the meat water (Thiem, 2003). The extract is a mixture of water, proteins and other compounds of meat. During the equilibration process, oxygen isotopes of the water equilibrate with oxygen isotopes in the measuring gas (CO2). Thus, the determination of the water contents of the (measured) extract was necessary for calculation of the 18O/16O-ratio. For the water contents of extracts, the results show a constant value across all studied species and even after significant weight loss of the pork sample after open storage. It can be assumed that possible influences of non-water-molecules to the isotope exchange reaction are also almost constant. The value of 93.2% as water contents of the extracts from meat can be used to simplify further calculations.
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Acknowledgement Special thanks to Prof. Neumann, University of Veterinary Medicine, Hannover, for providing poultry samples.
References Boner, M., & Fo¨rstel, H. (2004). Stable isotope variation as a tool to trace the authenticity of beef. Analytical and Bioanalytical Chemistry, 378(2), 301–310. Breed Description. www.tiho-hannover.de/einricht/zucht/eaap/descript/ 920.htm; (from 30.05.2004) Bronstein, S., & Semendjajew, K. A. (1991). Taschenbuch der Mathematik. 25. Aufl. B.G. Teubner Verlagsgesellschaft, Stuttgart, Leipzig, (pp. 687) Craig, H. (1957). Isotopic standards for carbon and oxygen and correction factors for mass spectrometry analysis of carbon dioxide. Geochimica et Cosmochimica Acta, 12, 133–148. Epstein, S., & Mayeda, T. (1953). Variation of O18 content of water from natural sources. Geochimica Cosmochimica Acta, 4, 213–224. Fleischqualita¨t: Ministerium fu¨r Erna¨hrung und La¨ndlichen Raum Baden – Wu¨rttemberg. www.infodienstlr.bwl.de/ernaehrung/lebensmittel/waren/fisch_fleisch_eier/qualitaet.htm (from 14.07.2003) Fo¨rstel, H., & Hu¨tzen, H. (1982). Variation des Verha¨ltnisses der stabilen Sauerstoff-Isotopen im Grundwasser der Bundesrepublik Deutschland. Ju¨l-1784, Mai Fo¨rstel, H., & Lickfett, J. (2002). Mit isotopem Fingerabdruck den Lebensmitteln auf der Spur. Bioworld, 1, 26–27. Hegerding, L., Seidler, D., Danneel, H.-J., Gessler, A., & Nowak, B. (2002). Sauerstoffisotopen-Verha¨ltnisanalyse zur Herkunftsbestimmung von Rindfleisch. Die Fleischwirtschaft, 4, 95–100. Honikel, K. O. (1987). Wasserbindungsvermo¨gen von Fleisch. Fleischwirtschaft, 67(4), 418–428. Honikel, K. O., & Hamm, R. (1994). Quality attributes and their measurement in meat, poultry and fish products. In A. M. Pearson & T. R. Dutson (Eds.). Advances in meat research series (9S, pp. S.125–S.150). St. Edmunds: Verlag Chapman & Hall.
341
IAEA/WMO: Global Network of Isotopes in Precipitation. (2001). The GNIP database. Available from http://isohis.iaea.org. Khan, A. W. (1977). Some factors causing variation in the texture of similar muscles from comparable animals. Meat Science, 1, 169–176. Kornexl, B. E., Werner, T., Roßmann, A., & Schmidt, H. L. (1997). Measurement of stable isotope abundances in milk and milk ingredience – a possible tool for origin assignment and quality control. Zeitschrift fu¨r Lebensmittel Untersuchung und Forschung, 205, 19–24. Midwood, A. J., Haggarty, P., Milne, E., & McGaw, B. A. (1992). Factors affecting the analysis of 18O-enriched aqueous samples when using CO2-equilibration in vacutainersä. Applied Radiation and Isotopes, 43(11), 1341–1347. Park, Y. W. (1996). In L. M. L. Nollet (Ed.). Handbook of food analysis: Physical characterisation and nutrient analysis (Vol. 1, pp. 60–74). New York: Marcel Dekker. Roß mann, A. (1989). Isotopenverteilung in Glucose und sekunda¨ren Naturstoffen und ihre Bedeutung zur Herkunftsbestimmung bei Lebensmitteln. Dissertation, Mu¨nchen-Weihenstephan, Techn. Univ Schmidt, H. L. (1986). Food quality control and studies on human nutrition by mass spectrometric and nuclear magnetic resonance isotope ration determination. Zeitschrift fu¨r Analytische Chemie Fresenius, 324, 760–766. Stosch, H. G. (1999). Einfu¨hrung in die Isotopengeochemie. Based on lectures at the Mineralogisch–Petrograpischen Institut of the University of Cologne between 1988 and 1994, revised and expanded (p. 186) Ternes, W. (1998). Naturwissenschaftliche Grundlagen der Lebensmittelzubereitung. unvera¨nderter Nachdruck der 2. Aufl. BehrÕs Verlag Hamburg (pp. 281–296) Thiem, I. (2003). 18O/16O-Isotopenverha¨ltnis in Fleisch zur Herkunftsanalytik bei Rind, Schwein und Geflu¨gel: Natu¨rliche Variation und Simulation von Sto¨rgro¨ß en. Dissertation, University of Veterinary Medicine, Hannover Thiem, I., Lu¨pke, M., & Seifert, H. (2004). Influencing factors to the 18 O/16O-ratio in meat juices. Isotopes in Environmental and Health Studies, 40, 4. Weightloss reduction. (2003). Societe dÕInnovation et de Recherche pour lÕIndustrie et lÕAgro-Alimentaire. www.sirial.fr/brumisation_angl.htm (from 14.07.2003).