Meat Science 69 (2005) 243–248 www.elsevier.com/locate/meatsci
On-line determination of fatty acid composition in intramuscular fat of Iberian pork loin by NIRs with a remote reﬂectance ﬁbre optic probe
´ ´ ´ ´ ´a, ´ I. Gonzalez-Martın *, C. Gonzalez-Perez, N. Alvarez-Garcı J.M. Gonzalez-Cabrera
´ ´ ´ ´ ´ Departamento de Quımica Analıtica, Nutricion y Bromatologıa, Facultad de CC, University of Salamanca, Quımicas, C/Plaza de la Merced s/n, 37008 Salamanca, Spain Received 8 December 2003; received in revised form 24 June 2004; accepted 12 July 2004
Abstract A near infrared spectrometer equipped with a standard 210/210 bundle remote reﬂectance ﬁbre-optic probe, with a 5 · 5 cm quartz window type, was used for the determination of fatty acids in the Longissimus dorsi muscle of Iberian breed swine. The fatty acids C14:0, C16:0, C16:1, C17:0, C17:1, C18:0, C18:1, C18:2, C18:3, Rpolyunsaturated, Rmonounsaturated and Rsaturated were determined in samples of intramuscular fat from Iberian breed swine by direct application of the ﬁbre-optic probe onto the loin sample, with no treatment or manipulation of the sample. The regression method employed was modiﬁed partial least squares. The calibration results using the ﬁbre-optic probe for 74 loin samples had multiple correlation coeﬃcients (RSQ) for C14:0, C16:0, C16:1, C17:0, C17:1, C18:0, C18:1, C18:2, C18:3, Rpolyunsaturated, Rmonounsaturated and Rsaturated acid of 0.785, 0.798, 0.788, 0.825, 0.762, 0.765, 0.696, 0.859, 0.878, 0.807, 0.943, 0.858, respectively, and standard errors of prediction corrected for the same fatty acids (%) of 0.08, 0.63, 0.26, 0.02, 0.02, 0.51, 0,77, 0.64, 0.05, 1.06, 0.34, 0.70, respectively. The robustness of the method was checked by applying the ﬁbre-optic probe to unknown samples of Iberian breed pork loin in a slaughterhouse, using 15 samples for the external validation. Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Iberian-pork loin; Fatty acid; Intramuscular fat; NIR; Fibre-optic probe; Determination
1. Introduction Pork products from the Iberian breed of swine are widely accepted by consumers owing to the special characteristics of the swine from which they are obtained. In the present work, products from the Iberian swine, or the same crossed with animals of the Duroc breed, fed for two months on a range regimen, mainly acorns and grass (montanera swine) or over one month on a montanera regimen followed by another month with
Corresponding author. Tel./fax: +34 23 29448. ´ ´ E-mail address: firstname.lastname@example.org (I. Gonzalez-Martın).
commercial swine feed (recebo swine) or on an intensive regimen with commercial feed (feed swine) (Cabeza de Vaca, Esparrago, Fallola, & Vazquez, 1992; MAPA, 1984), were studied. The content of intramuscular lipids and the nature of the fatty acids of the Longissimus dorsi muscle from Iberian swine at the time of sacriﬁce depend on the genetic origin of the animals, their age, and their feeding regimen (such as the montanera diet). However, the determination of fatty acids in the muscle of Iberian breed swine is a long and tedious task, involving extraction of total lipids (Folch, Lees, & Stanley, 1957), and determination of fatty acid methyl esters by gas chromatography.
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A sample with an H statistic of P3. was used. samples with high residual values were eliminated.4. according to the method of Bligh and Dyer (1959) were used. the set of calibration samples is divided in groups. The solvent was removed under vacuum in a rotary evaporator. 2001) with NIRS and a ﬁbre-optic probe used directly on the carcasses of Iberian swine allowed the determination of fat and protein and the mineralogical composition ´ ´ ´n. installed on a Hewlett–Packard Pentium III computer. R6539-A. ´ ´ ´ The work of Gonzalez Martın. The samples were ground and homogenised with a Knife 1095 Mill Sample homogeniser from Foss Tecator. Rmonounsaturated and Rsaturated) in subcutaneous fat. Garcıa Olmo (1999). several scattering corrections and mathematical treatments were tested (standard normal variate. The results indicate how diﬀerent a sample spectrum is from the average sample of the set (Willians & Norris.244
´ ´ I. De-trending. Measurements were carried out in reﬂectance mode between 1100 and 2000 nm. 2. with a standard 1. Bares. No. ‘‘recebo’’ or ‘‘feed’’ regimens. C18:0. Gonzalez Martı Gonzalez ´ ´ ´ ´ Perez. Material and methods 2. performing 32 scans for both the reference and samples. SNV.5 m 210/210 bundle ﬁbre-optic probe. Assessment of the calibration model was performed by cross-validation. The probe employs a remote reﬂectance system and uses a ceramic plate as reference. by direct application of the probe to the Longissimus dorsi muscle with no prior sample treatment or manipulation. deﬁned as the diﬀerence between the laboratory value and the value predicted by the equation) for each constituent.
2. 2. NIR spectra were measured by applying the ﬁbre-optic probe to intact pork loin samples. 2. The loin samples were from animals that had been fed on the so-called ‘‘montanera’’. Gonzalez Perez. palmitic and ´ stearic acids in Iberian pork. using the T > 2. cut longitudinally to provide a slice measuring 8 · 12 · 2 cm. ´ 2003) have reported on-line analysis methods of fatty acids (C14:0. C18:2. which gave a standard error of prediction corrected (SEPC) and bias (mean of residuals. NIR spectroscopy A Foss NIRsystems 5000. Statistical analyses The Mahalanobis distance (H statistic) is calculated from principal component analysis scores.1.5 criterion. and conserved at À18 °C. using a Hewlett–Packard HP-5890A gas chromatograph. applied the method to classify pieces of fresh Iberian ham comparing the results with those obtained with gas chromatography. ´ ´ ´ ´ ´ Hernandez Mendez. of pork loin. The best one was selected for each constituent on the basis of the highest multiple correlation coeﬃcient (RSQ) and the lowest standard error of calibration and crossvalidation (SEC and SEV. C18:1. This oﬀers an on-line technique for the immediate determination of the fatty acid composition of intramuscular fat in the animals. / Meat Science 69 (2005) 243–248
NIRS methodology was used by De Pedro. The average spectrum was used for NIR analysis.3. Here we report the rapid determination of the fatty acid content of the lipids of the muscle tissue of Iberian swine using NIRS technology with a ﬁbre-optic probe. C18:3. In this method. Samples We examined 74 samples of pork loin (Longissimus dorsi muscle) from Iberian swine purchased from ´ ‘‘Ganaderos Salmantinos de Porcino Iberico S. Garrido. Additionally. Casillas. MSC. respectively). Ref. Gonzalez-Martın et al. Samples from the validation set were then analysed with these equations.0 standardised units from the mean spectrum is deﬁned as a global H outlier and is then eliminated from the calibration set. C20:1. allowing instantaneous classiﬁcation of Iberian pork as a function of the animalsÕ feeding regimen. Calibrations were performed by modiﬁed partial least squares regression (MPLS). Later. The model is repeated as many times as there are groups in such a way that all pass through the calibration set and the prediction set. DT. C16:0. Intramuscular fat and fatty acid analysis To extract total lipids from the Longissimus dorsi muscle.A’’ taken from the end of the loin of the animals. To optimise the accuracy of calibration. The window is of quartz. Hernandez Mendez.05. equipped with a ﬂame ionisation detector (FID). Rpolyunsaturated. The same process was followed for the ground and intact samples.
ide in methanol before analysis by gas chromatography.2. using one of them to check the results (prediction) and the remaining to construct the calibration model. with a 5 · 5 cm surface area. In this step. The software used was Win ISI 1. and y Murrai (1992) to predict the subcutaneous fat content of oleic. 1987). multiplicative scatter corrections. All analyses were performed in duplicate (ISO Norm 5508:1990). and Alvarez Garcıa (2002a. measuring reﬂectance in the IR zone close to 1100– 2000 nm. Fatty acid methyl esters (FAMEs) of muscle were prepared by reaction with a solution of potassium hydrox-
. and Alvarez Garcıa (2002b. linoleic. Spectra were recorded at intervals of 2 nm. ﬁrst derivative and second derivative). 50 g of sample previously ground and homogenised with chloroform/methanol (1/2).
13 5.46 13.76 5.46 99.32 1.681 0.05 0. Chemical analyses and spectral information The chemical compositions of the pork loin samples used for calibration are shown in Table 1.30 9.031 0.54 99.007 0.59 0. / Meat Science 69 (2005) 243–248
After the calibration equations for the ﬁbre-optic probe had been obtained.008 0.094 1.´ ´ I.943 0. of principal components 7 7 7 9 9 7 8 7 9 7 9 8 8 7 9 Probability explained (%) 99.788 0.A’’ Company in Salamanca (Spain).09 0. Results and discussion 3.46 99. The calibration equations obtained in the development of the procedure were applied and the predicted values were compared with the laboratory results obtained later.36 99.74 1.017 0.64 3.45 57.762 0.30 23. 1.54 99. (b) Corrected spectrum using second derivative and DT for the fatty acid C18:2.859 0.31 0.662 0.62 Maximum 0.272 0.538 0. The causes of such high variability must be sought in the feeding regimen and genetics of the animals (the Iberian swine used in this work were Iberian swine of the subgenus ‘‘Sus mediterraneus’’ crossed with Duroc-Jersey. they were subjected to external validation by application to a set not involved in the calibration process (15 samples) checking the functioning of the ﬁbre-optic probe for instantaneous analysis at production level at the slaughterhouse of the ´ ‘‘Ganaderos Salmantinos de Porcino Iberica S.
Table 1 Statistical overview of chemical analysis (all units in %) N = 74 Fatty acid 12:0 14:0 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:1 SFA MUFA PUFA Minimum 0.807 0.05 0.71 58.47 64.51 0.55 41.01 0.94 49.763 1.58 0. using second derivative and DT.52 99.39 2. 72 samples were used.
Table 2 Calibration statistical descriptors for the NIR determination of fatty acids in intramuscular fat from loin Fatty acid 12:0 14:0 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:1 SFA MUFA PUFA N 69 70 69 71 68 72 73 67 71 67 71 71 70 69 71 Mathematical treatment None/2a derivada None2a derivada DT/2a derivada DT/2a derivada None/2a derivada DT/2a derivada Standar MSC/2a der DT/2a derivada DT/2a derivada DT/2a derivada SNV/2a derivada DT/2a derivada ´ Estandar MSC /2a deiv None/3a derivada DT/2a derivada RSQ 0.18 1. (a) NIR spectrum of the sample of loin measured with the ﬁbreoptic probe.14 1.035 0.21 11.043 0.52 99.05 0.28
.458 0639 0.13 2.76 31.070 1.61 0.05 1.30 99. The best of the diﬀerent mathematical treatments for each fatty acid was applied (Table 2).73 0.42 0. which is important when searching for calibrations equations to be used later in the prediction. Fig. The eﬀects of dispersion
Fig. Spectra were recorded in triplicate and the spectral average was taken.07 1.858 SEC 0.03 54.59 2. The spectra were recorded by direct application of the probe on intact pieces of Iberian pork loin.06 2.098 1.785 0.696 0.825 0.878 0.17 0.351 0.97
3.14 0.36 99.95 20.816 0.051 0.026 0.46 0.28 99.099 0.080 0.798 0. 1 shows the spectra of a sample of loin obtained with the ﬁbre-optic probe by direct application on the sample together with one of the mathematical treatments that proved optimum for the calibrations of the fatty acid C18:1 in the intramuscular fat of the loin.52 99.82 0.62 1.99 36.70 27.36 0.51 99.137 1. with Iberian genetics varying between 50% and 75%).79 SD 0.116 0.019 0.622 0.81 0.30 99.91 Mean 0.1. The results obtained for the content of the diﬀerent fatty acids revealed a broad range of variability.10 1. Gonzalez-Martın et al.23 0.054 0.10 0.76 9.146 0.249 No.13 8.472 0.743 SECV 0.934 1.78 1.287 1.19 0.28 99.16 0.765 0.83 5.09 52.
the 2310–2340 region corresponds to the combination bands of the C–H bond. & Fernandez-Cabanas. which is a fundamental constituent of fatty acid molecules. 2310 and 2340 nm (Willians & Norris. Correlation of the values obtained at the laboratory with respect to those predicted by NIR for measurement with a ﬁbre-optic probe of the diﬀerent fatty acids in Iberian pork loin samples.
1750. & Westerhaus. absorbs clearly at wavelengths close to 1200. i. Lister. DT or SNV–DT. The absorption in the 2150–2190 region at 1680 nm indicates the presence of cis double bonds in the molecules. Fearn. / Meat Science 69 (2005) 243–248
Fig. 2. Mac Dougall. MSC was ﬁrst used by Geladi. 1400.
were removed using MSC.e. and Martens (1985) and prevents the dispersion in the samples from imposing itself on the chemical signals. Dhanoa. and Hindle (1993) have attributed the absorption produced at a wavelength of 1210 nm the second overtone of the CH2 bond. and the absorption produced in the 1720–1760 region corresponds to the ﬁrst overtone of that bond (Shenk. 1987). Carrete. Moreover. Thus. the existence of unsaturated fatty ´ acids (Garrido-Varo.. 1992). Osborne.
. SNV. Gonzalez-Martın et al. The spectral information deﬁnes a series of characteristic absorption bands.246
´ ´ I. Workman. and Barnes (1995) indicate that SNV–DT were introduced not only for the reduction of multicollinearity but also to calculate spectral differences by reducing the confounding eﬀects of baseline shift and curvature. the C–H bond. 1998) in the samples analysed.
2. Fig.35 3.10 1. ‘‘recebo’’ and ‘‘feed’’).16–0. RMUFA.87–10.01 0. / Meat Science 69 (2005) 243–248
3.21 11. In later studies the method will be use to analyse the composition in fatty acids in the intramuscular fat of the loin by NIRS and a ﬁbre-optic probe in order to classify the animals on the basis of their feeding regimen in the fattening stage (‘‘acorn’’.24 10.24 2.81 28.95 0.87 SD 0.05 0. C17:1.06–13. The concentration ranges and standard deviations for each fatty acid that it is possible to determine in the intramuscular fat of Iberian pork loin are shown in Table 3. Table 4 shows the diﬀerences found between the reference
Table 3 Applicability of the calibration equations relating to fatty acids in intramuscular fat of Iberian pork loin Fatty acid 12:0 14:0 16:0 16:1 17:0 171 18:0 18:1 18:2 18:3 20:0 20:1 SFA MUFA PUFA Est. C18:2.´ ´ I. The predicted values gave validation errors that were combined into SEP(C). 3. 3.93 12.12 2.39 57.07 1.16 0.59 0.83 8. The procedure was as follows: spectra were recorded in triplicate and the spectral mean was taken. C18:2. 0.20 Diﬀerences (%) 9.3.71–9.50 0.26 0. C17:0.78 Mean 0. C18:3.39 17. C18:0.29 0. The results obtained in the determination of C12:0.18 0. C20:0 and C20:1 are acceptable.97 0. After the number of principal components had been calculated.61 21.98 36.88 62. RSFA and RMFA are very good.28 0. depending on the fatty acid to be determined. the calibration equations obtained during the work were applied and the predicted values were compared with those obtained later using gas chromatography. such that all pass through the calibration set and the prediction set. From this information it may be deduced that the NIRS technique. the NIRS technology with a ﬁbre-optic probe was used.09 1.17 1.47 0. establishing a limit value of H = 3.06 52.15 0.85 5.83–1. The numbers of remaining samples (N) are shown in Table 2.80 2.37 13.
. Validation 3.47 1.73 48.11–1.85 2. C18:1. 5 are ta-
ken for the calibration set and one for the prediction set.96–4.78–54.90 7.91 2. between 7 and 9 components were used.80–59. The process is repeated as many times as there are sets. we validated the model used and checked its prediction capacity.04 1.97
Table 4 External validation for the determination of fatty acids in the intramuscular fat of Iberian breed swine Fatty acid C12:0 C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 SFA MUFA PUFA Range 0.69 26.09 7.29 23.19 2. 2 shows the correlation of the values obtained at the laboratory with respect to those predicted by NIR with a ﬁbre-optic probe for the fatty acids C14:0.81 0.53 0.18–41.69–25.04 0. Internal validation (prediction) Model evaluation was performed by cross-validation. C17:0.24 5.49 0. applying the probe directly onto the loin samples.15 4.06–0.94 0.13–0.3. which reveals the broad applicability of the calibration equations obtained.72 0.04 0. C16:0.62 14. Calculation of the statistical parameters of the calibration equations for each fatty acid is shown in Table 2. RSFA. C16:0. Regarding the number of principal components.04 0. C16:1.14 34.09 13.15–0. the detection of anomalous spectra was accomplished using the Mahalanobis distance (H statistic).0.00 Est. such that the spectra whose H distance was greater than this ﬁgure were considered diﬀerent from the spectral population and were discarded. the set of calibration samples is divided into a series of subsets: in our case 6. RMUFA.3. RPUFA in the intramuscular fat of Iberian pork loin. together with the statistical descriptors of prediction. which also reﬂects the best treatments.63 28.04 0. Gonzalez-Martın et al. C18:1.10 11.29 10.06 0.32 1. C17:1. Of these. min.90 49. max 0. External validation We checked the robustness of the method by applying the ﬁbre-optic probe to 15 new samples in a slaughterhouse.82 0.41 0.36 0.00 0. using a ﬁbre-optic probe.11–0. We consider that the results obtained in the determination of the fatty acids C14:0.67 0.01 5.48 1.38 11.59 3.33 14. C18:0. C18:3.54 1. This process was carried out for the validation of fatty acids in intact loin samples.11 1. and these accounted for 99. Determination of the fatty acid composition To calibrate fatty acids in the intramuscular fat of Iberian pork loin. C16:1.2.67
Diﬀerences between the reference method (gas chromatography) and the NIRS technique.57 54.25 57. is a good alternative for the determination of the content in fatty acids in samples of intramuscular fat from Iberian breed swine. Using this process.63 53.1. In this method.30% of the variance in the loin samples.33 42.77 19.69 5.21 0.17 1.
& ´ ´a. Lees. (1992).). Lister.. J. & Westerhaus. J. it may be concluded that the NIRS technique with a ﬁbre-optic probe is good for the determination of fatty acids in the intramuscular fat of Iberian pork loin. Willians. Hernandez Mendez. T. R. A. Mineral analysis (Fe. (1993). J. J. (1984). M. ´ ´ ´ ´ ´ ´ Gonzalez Martın. ´ ´ ´ ´ ´ ´ Gonzalez Martın... J. F. Determinacion de acidos grasos en ´ ´ grasa subcutanea de cerdo iberico mediante espectroscopia en el ´ infrarrojo cercano (NIRS) con sonda de ﬁbra optica.. In view of the present ﬁndings. Casillas. A. In D. F. H. Mac Dougall. Madrid: MAPA. NY: Ellis Horwood. C. use of the ﬁbreoptic probe enables determinations to be made simply by placing the probe on the sample itself.. J. Ciurczak (Eds... C. & Barnes. 765. & Stanley. Linearizacion and scatter-correction for near-infrared reﬂectance spectra of meat. S. S. I. ´ Garrido-Varo. (1957)..
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