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Appl. Phys. B (2013) 113:159163 DOI 10.

1007/s00340-013-5677-x

Raman spectroscopic differentiation of beef and horse meat using a 671 nm microsystem diode laser
Halah Al Ebrahim Kay Sowoidnich Heinz-Detlef Kronfeldt

Received: 24 May 2013 / Accepted: 26 September 2013 / Published online: 26 October 2013 Springer-Verlag Berlin Heidelberg 2013

Abstract A non-invasive Raman spectroscopic approach for meat species identication and quality detection was successfully demonstrated for the two closely related species beef and horse. Fresh beef and horse muscles were cut and ice-stored at 5 C, and time-dependent Raman measurements were performed daily up to 12 days postmortem. Applying a 671 nm microsystem diode laser and a laser power of 50 mW, spectra were recorded with integration times of 14 s. A pronounced offset of the Raman spectra was observed between horse and beef, with high uorescence background for horse compared to beef for all days of storage. Principal components analysis was applied for data evaluation revealing a clear distinction between beef and horse meat which can be attributed to differences in the myoglobin content of both species. Furthermore, separations according to aging and spoilage for the two species could be identied simultaneously. Therefore, Raman spectroscopy might be an efcient test method for meat species identication in combination with spoilage detection.

1 Introduction Recently, many European countries suffer from the adulteration scandal of horse meat being sold as beef.

H. A. Ebrahim (&) K. Sowoidnich H.-D. Kronfeldt Institute of Optics and Atomic Physics, Technical University Berlin, Sekr. EW 0-1, Hardenbergstrasse 36, 10623 Berlin, Germany e-mail: hala.ph@hotmail.com K. Sowoidnich e-mail: kay.sowoidnich@alumni.tu-berlin.de H.-D. Kronfeldt e-mail: kf@physik.tu-berlin.de

Here, horse meat from illegally slaughtered animals was taken as lower-cost replacement for beef. Due to the similar appearance of beef and horse meat, especially in frozen or deboned state, it was quite difcult to uncover the deceit. The adulteration of meat species is a major concern for consumers, incorporating terms of quality, safety, economic loss, and food allergies. Another important point affecting consumers interests is the spoilage detection of meat products. Therefore, the authentication and quality determination of meat is of great importance. In that way, selected analytical methods, such as electrophoresis [1, 2], immunology [3, 4], and real-time polymerase chain reaction (PCR) [57], have been applied for meat species identication. However, most of these techniques are destructive, expensive, and time-consuming, making them unsuitable for online applications. Furthermore, these methods are not able to detect the quality of meat species concurrently. To overcome these drawbacks, there is an increasing demand for instantaneous, non-destructive, analytical methods to provide information of meat species identication and spoilage detection, simultaneously. Here, optical methods provide a great potential as the recorded spectra usually contain a large amount of valuable information about the investigated sample. This approach has been successfully applied for the differentiation of beef and kangaroo meat by VIS and NIR reectance spectroscopy [8] or the identication of cattle, llama, and horse meat using NIR reectance and transectance spectroscopy [9]. Additionally, the adulteration detection of mince meat with horse meat and beef fat by means of MIR spectroscopy was reported [10]. In a similar way, optical techniques have proven successful for the identication of spoiled meat. This has been demonstrated exemplary for pork [11] and beef [12] using Fourier transform infrared

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spectroscopy as well as applying diffuse reectance spectroscopy for investigations with chicken [13]. Raman spectroscopy is a further very promising technique to address the above mentioned issues as it provides detailed information about molecular vibrations inside the specimen. The obtained molecular ngerprint is thus a great benet for sample identication. This could be demonstrated for the distinction of the two poultry species chicken and turkey [14] as well as for the differentiation of beef, pork, chicken, and turkey [15]. Moreover, Raman spectroscopy has proven successfully as a viable tool for rapid detection of meat spoilage [1619]. Nevertheless, none of the previous studies aimed on a combined approach of spoilage identication and species determination which will be presented in this paper. Here, Raman spectra of fresh beef and horse muscles were recorded daily for a time period up to 12 days after slaughter. With respect to in situ investigations, the measurements were performed directly on the muscles. The statistical method of principal components analysis (PCA) was used for evaluation of the complex spectral dataset. To our knowledge, this is the rst study dealing with the adulteration detection of the two closely related species beef and horse combined with the determination of quality aspects for both meat species.

671 nm [20] as excitation light source. The laser power at the sample was set to 50 mW enabling integration times of 1 s to 4 s for one Raman spectrum. Briey, the excitation radiation is guided by two dielectric mirrors and a Raman edge lter to a lens with a focal length of 30 mm. This lens focuses the laser beam onto the meat sample and also collects the backscattered light from the specimen. A subsequent set of two Raman edge lters blocks the Rayleigh as well as the anti-Stokes scattering. Only the Raman Stokes radiation is transmitted and then focused into the spectrograph (Chromex 250IS) by a lens with a focal length of 50 mm. The Raman spectra are recorded with a CCD camera (EHRB 1340 9 400, Princeton Instruments) cooled down to -70 C. A more detailed description of the experimental setup can be found in [21]. 2.3 Reference analysis As reference analyses, the surface pH values of beef and horse slices were determined in parallel to the Raman experiments. The pH values were recorded using two selected types of pH strips (Merck), one covering the pH range from 5.2 to 7.2 (special indicator for pH measurements in meat, non-bleeding) and the other covering the pH region from 4.0 up to 7.0 (non-bleeding). Smell test was also applied daily on the meat slices as sensory reference analysis. Despite it is based on a subjective impression, the human nose is a very sensitive indicator for off-odors caused by metabolic products of microorganisms during meat spoilage. Therefore, this kind of analysis was applied concurrently to the pH-value determination to reveal additional information about the meat status. 2.4 Spectral data analysis To analyze the Raman spectra, principal components analysis (PCA) as a chemometrical method was utilized in MATLAB (MathWorks Inc. Natick, MA) applying PLSToolbox (Eigenvector Research Inc., Wenatchee, WA). Considering the spectral region from 700 to 1700 cm-1, the daily average of 140 Raman spectra each for beef and horse meat was calculated. After that, the whole dataset was normalized to the baseline intensity at 1520 cm-1 and the data were pre-processed using mean centering and SavitzkyGolay smoothing with second derivative computation. By using PCA, the spectra are then analyzed for variations on a statistical basis, resulting in the so-called loadings. These are spectral patterns indicating at which wavenumbers the most signicant differences between the spectra occur. The loadings are attributed to principal components (PCs) representing the variations in the dataset in a decreasing order, i.e., PC 1 describes the largest

2 Materials and methods 2.1 Sample preparation For our investigations, fresh muscles of beef and horse (sirloin steak) were purchased from local butcher shops in Germany. Beef meat samples were bought from a local abattoir in Kulmbach and were then transported to Technical University Berlin one-day postmortem under chilled conditions. Samples of horse meat were obtained from chterei Alfred Bredel in Berlin 2-day postmortem. Roschla The muscles were cut into 2-cm thick slices and packed separately in petri dishes. All slices were stored at 5 C for a period of 12 days in a laboratory refrigerator (Spezial-468, Philipp Kirsch, Germany). For each measuring day, two cylindrical subsamples with a diameter of 2 cm from each slice were cut and subsequently mounted in PVC tubes. For each investigated meat cylinder, 7 different positions were probed, detecting 10 single Raman spectra each. In total, 140 single Raman spectra from 14 sampled positions per meat species were obtained for each measuring day. 2.2 Raman measurements For meat investigations, a 180 geometry conguration was used incorporating a microsystem diode laser operating at

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variance, PC 2 the second largest variance, etc. [22]. Expressed mathematically, all principal components are orthogonal vectors in a multi-dimensional vector space and therefore they are linearly independent. By calculating the scalar product between loading and Raman spectrum, the spectra are scored with respect to the corresponding PC. This results in the scores, which indicate how well spectrum and PC match. Hence, an effective data reduction can be realized as every Raman spectrum with, e.g., 1340 data points are transformed into a single coordinate value according to each PC. For graphical representation, e.g., the scores of the Raman spectra for the most signicant principal components PC 1 and PC 2 are used. In such a two-dimensional plot, each spectrum is reduced to one single data point. Similar spectra are located adjacently, while spectra with low similarity are clearly separated from each other, i.e., a separation in different clusters occurs. The corresponding loadings can then be used to identify specic Raman bands which contribute to the obtained separation in the scores plot. This sometimes allows relating the discrimination of groups of spectra to physical/chemical properties or even a specic component.

uorescence interference for the rst days of storage, followed by an increase in the uorescence until the last day of storage. However, the Raman spectra of both animals exhibit the characteristic meat protein structure comprising vibrations of the polypeptide backbone (amide, CH bending, and CC stretch bands) as well as aromatic amino acid signals (phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp)) [25, 26]. 3.2 Statistical data analysis To illustrate the distribution and potential clustering of samples according to the spectral features of the two investigated meat species, principal components analysis as statistical method was applied. Figure 2 displays the corresponding scores plot of beef and horse Raman spectra for the rst (PC 1) and the second (PC 2) principal component explaining 79 and 18 % of the total variance in the dataset, respectively. As the sum of all remaining PCs describes only 3 % of the variation in the data, these higher PCs were excluded from further analysis. A clear differentiation between beef and horse meat is made by PC 1, with negative values for beef samples and positive values for horse samples. Furthermore, PC 2 provides information about aging and spoilage effects revealing three separate stages for each meat species. Negative score values indicate a rst stage for both species including the days 3 till 5 for beef and the days 3 to 7 for horse. Subsequently, the sign according to PC 2 changes from negative to positive. The corresponding clusters in Fig. 2 comprise the 6th day to the 9th day for beef and the 8th day till the 11th day of storage for horse.

3 Results 3.1 Spectra characterization Figure 1 displays a comparison of unprocessed Raman spectra obtained for fresh beef (a) and horse (b) meat 3 days after slaughter. The uorescence background of horse meat shows very high values compared to beef. This behavior was also observed for all days of storage. The pronounced difference in the uorescence can be explained by different myoglobin contents of both species [23]. In addition, the uorescence intensity in the spectra is in accordance with our previous results [17, 18, 24], i.e., low

Fig. 1 Raman spectra of fresh beef (a) and horse (b) meat, normalized to 5-s integration time

Fig. 2 Scored Raman spectra for PC 1 and PC 2 for beef and horse meat for 12 days of storage. PC 1 reveals meat species identication, while PC 2 displayed time-dependent changes for both species

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162 Table 1 Results of pH value measurements and smell test for beef Day 3 4 5 6 7 8 9 10 11 12 pH values (pH strips 4.07.0) 5.35.5 5.35.5 5.35.5 5.5 5.5 5.5 5.5 5.55.8 5.55.8 5.8 pH values (pH strips 5.27.2) 5.55.7 5.55.7 5.55.7 5.55.7 5.55.7 5.56.0 5.76.0 5.76.0 5.76.0 5.76.3 Smell Normal Normal Normal Light sour Light sour Sour Sour Strong sour Strong sour Strong sour

H. A. Ebrahim et al.

Fig. 3 Loadings 1 and 2 from PCA of time-dependent storage time of beef and horse meat. Loading 1 contains primarily information about myoglobin signals, and loading 2 displays Raman signature of protein structure in meat

Table 2 Results of pH value measurements and smell test for horse Day 3 4 5 6 7 8 9 10 11 12 pH values (pH strips 4.07.0) 5.35.5 5.35.5 5.35.5 5.35.5 5.35.5 5.5 5.5 5.5 5.55.8 5.8 pH values (pH strips 5.27.2) 5.55.7 5.55.7 5.55.7 5.55.7 5.56.0 5.76.0 5.76.0 5.76.0 5.76.0 5.96.3 Smell Normal Normal Normal Normal Light sour Sour Sour Sour Sour Strong sour

The last stage containing the days 1012 for beef as well as day 12 for horse shows high positive score values. The storage time-dependent separations by means of PC 2 represent specic stages of meat status and can be correlated with the determined pH values as well as with the results of the smell test listed in Table 1 for beef and Table 2 for horse. The days 35 for beef as well as the days 3 till 7 for horse correspond to edible meat samples exhibiting low pH values and a normal smell. The second stage (6th to 9th day for beef and 8th to 11th day for horse) contains those samples showing incipient and considerable spoilage. Here, a slight increase in the pH value accompanied by the pronounced development of unpleasant smells (light sour changing to sour) could be observed. The third stage with beef samples with an age of 1012 days as well as the 12 days old horse meat represents completely spoiled specimens. In this case, signicantly increased pH values and very strong off-odors were determined. Briey, the statistical analysis of the recorded Raman spectra applying PCA allowed for a discrimination of beef and horse meat. Furthermore, an identication of distinct

stages of meat spoilage could be realized. Here, both species revealed a slightly different temporal behavior as the stage of edible meat lasts only up to day 5 for beef, while this stage continues till the 7th day of storage for horse. To analyze the spectral features responsible for the obtained separations, the rst two loadings of the PCA are presented in Fig. 3. Loading 1 which is responsible for the animal-specic distinction is dominated by characteristic signals attributed to myoglobin pigments at 1613, 1561, 1543, 1521, 1449, 1399, 1355, 1337, 1315, 1124 , 1000, 949, and 754 cm-1 [27, 28]. Additionally, the strong amide I band at 1649 cm-1 as well as two signals from Tyr at 827 and 850 cm-1 can be recognized. In this way, the separation between the two meat species is mainly based on differences in the myoglobin content between beef and horse meat. This nding is in accordance with our previous study with beef, pork, and poultry [15] where the separation of the investigated species was also based on differences in myoglobin content to a large extent. Additionally, small differences in myoglobin amino acid composition of beef and horse [29] possibly also have a contribution to the obtained distinction. In a similar way as observed in our previous investigations [18, 21, 30], loading 2 shows a typical protein structure of meat representing the signal-to-backgroundratio of the Raman spectra affected by aging and spoilage effects during time-dependent storage. Here, typical protein Raman bands as amide I at 1650 cm-1, amide III at 1316 cm-1, and 1261 cm-1, CH bending bands at 1447 cm-1 and 1340 cm-1 can be recognized. Furthermore, a CC stretch band at 933 cm-1, as well as protein backbone bands at 1117 cm-1 and 1086 cm-1 is observable. Characteristic signals of aromatic amino acids are present at 1604 cm-1 (Trp, Phe, Tyr), 1553 cm-1 (Trp), 1205 cm-1 (Phe, Tyr), 1002 cm-1 (Phe), 852 cm-1 (Tyr), and 822 cm-1 (Tyr).

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4 Conclusion In this paper, time-dependent Raman measurements for a combined approach of meat species identication and the determination of spoilage effects were performed for beef and horse meat using a 671 nm microsystem diode laser. Compared to beef, the Raman spectra of horse meat showed a high uorescence background for all days of storage. A PCA revealed an unambiguous distinction between the two meat species according to PC 1, while PC 2 displayed a separation related to aging and spoilage processes for both meat species. Moreover, characteristic myoglobin signals could be identied in the corresponding loading 1 of the PCA, whereas loading 2 represents the signal-to-background-ratio of the meat Raman spectra affected by storage time. In conclusion, no horse meat was misclassied as beef indicating that Raman spectroscopy could be used to screen beef for horse meat deterioration and adulteration.

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