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On-line determination of fatty acid composition in intramuscularfat of Iberian pork loin by NIRs with a remote reflectance fibre

optic probe

I. Gonzalez-Martn *, C. Gonzalez-Perez, N. Alvarez-Garca, J.M. Gonzalez-Cabrera

Departamento de Qumica Analtica, Nutricion y Bromatologa, Facultad de CC, University of Salamanca, Qumicas,

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 dorsimuscle 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, Rpolyun-

saturated, 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.

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 char-acteristics 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

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 dorsimuscle from Ibe-

rian swine at the time of sacrifice depend on the genetic

origin of the animals, their age, and their feeding regi-

men (such as the montanera diet). However, the determi-

nation 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.

0309-1740/$ - see front matter 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.meatsci.2004.07.003

* Corresponding author. Tel./fax: +34 23 29448.

E-mail address: [email protected] (I. Gonzalez-Martn).

www.elsevier.com/locate/meatsci

Meat Science 69 (2005) 243248

MEATSCIENCE
mailto:[email protected]:[email protected]

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NIRS methodology was used by De Pedro, Garrido,

Bares, Casillas, and y Murrai (1992) to predict the sub-

cutaneous fat content of oleic, linoleic, palmitic and

stearic acids in Iberian pork. Later, Garca Olmo

(1999), applied the method to classify pieces of fresh Ibe-

rian ham comparing the results with those obtained with

gas chromatography.The work of Gonzalez Martn, Gonzalez Perez,

Hernandez Mendez, and Alvarez Garca (2002a, 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

of pork loin. Additionally, Gonzalez Martn, Gonzalez

Perez, Hernandez Mendez, and Alvarez Garca (2002b,

2003) have reported on-line analysis methods of fatty

acids (C14:0, C16:0, C18:0, C18:1, C18:2, C18:3,

C20:1, Rpolyunsaturated, Rmonounsaturated and Rsat-

urated) in subcutaneous fat, allowing instantaneous

classification of Iberian pork as a function of the ani-

mals feeding regimen.

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,

by direct application of the probe to the Longissimus

dorsimuscle with no prior sample treatment or manipu-

lation. This offers an on-line technique for the immedi-

ate determination of the fatty acid composition of

intramuscular fat in the animals.

2. Material and methods

2.1. Samples

We examined 74 samples of pork loin (Longissimus

dorsi muscle) from Iberian swine purchased from

Ganaderos Salmantinos de Porcino Iberico S.A taken

from the end of the loin of the animals, cut longitudi-

nally to provide a slice measuring 8 12 2 cm. NIR

spectra were measured by applying the fibre-optic probe

to intact pork loin samples. The loin samples were from

animals that had been fed on the so-called montanera,

recebo or feed regimens. The samples were ground

and homogenised with a Knife 1095 Mill Sample

homogeniser from Foss Tecator, and conserved at

18 C.

2.2. Intramuscular fat and fatty acid analysis

To extract total lipids from the Longissimus dorsi

muscle, 50 g of sample previously ground and homoge-

nised with chloroform/methanol (1/2), according to the

method of Bligh and Dyer (1959) were used. The solvent

was removed under vacuum in a rotary evaporator.

Fatty acid methyl esters (FAMEs) of muscle were pre-

pared by reaction with a solution of potassium hydrox-

ide in methanol before analysis by gas chromatography,

using a HewlettPackard HP-5890A gas chromato-

graph, equipped with a flame ionisation detector

(FID). All analyses were performed in duplicate (ISO

Norm 5508:1990).

2.3. NIR spectroscopy

A Foss NIRsystems 5000, with a standard 1.5 m

210/210 bundle fibre-optic probe, Ref. No. R6539-A,

was used. The probe employs a remote reflectance sys-

tem and uses a ceramic plate as reference. The win-

dow is of quartz, with a 5 5 cm surface area,

measuring reflectance in the IR zone close to 1100

2000 nm.

Measurements were carried out in reflectance mode

between 1100 and 2000 nm. Spectra were recorded at

intervals of 2 nm, performing 32 scans for both the ref-

erence and samples. The average spectrum was used for

NIR analysis. The software used was Win ISI 1.05, in-

stalled on a HewlettPackard Pentium III computer.

2.4. Statistical analyses

The Mahalanobis distance (H statistic) is calculated

from principal component analysis scores. The results

indicate how different a sample spectrum is from the

average sample of the set (Willians & Norris, 1987). A

sample with an H statistic ofP3.0 standardised units

from the mean spectrum is defined as a global Houtlier

and is then eliminated from the calibration set.

Calibrations were performed by modified partial leastsquares regression (MPLS). To optimise the accuracy of

calibration, several scattering corrections and mathe-

matical treatments were tested (standard normal variate,

SNV; De-trending, DT; multiplicative scatter correc-

tions, MSC; first derivative and second derivative).

The best one was selected for each constituent on the ba-

sis of the highest multiple correlation coefficient (RSQ)

and the lowest standard error of calibration and cross-

validation (SEC and SEV, respectively). Assessment of

the calibration model was performed by cross-valida-

tion. In this method, the set of calibration samples is di-

vided in groups, using one of them to check the results

(prediction) and the remaining to construct the calibra-

tion model. 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. The same

process was followed for the ground and intact samples.

Samples from the validation set were then analysed

with these equations, which gave a standard error of

prediction corrected (SEPC) and bias (mean of residu-

als, defined as the difference between the laboratory va-

lue and the value predicted by the equation) for each

constituent. In this step, samples with high residual val-

ues were eliminated, using the T> 2.5 criterion.

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After the calibration equations for the fibre-optic

probe had been obtained, they were subjected to exter-

nal validation by application to a set not involved in

the calibration process (15 samples) checking the func-

tioning of the fibre-optic probe for instantaneous analy-

sis at production level at the slaughterhouse of the

Ganaderos Salmantinos de Porcino Iberica S.A Com-pany in Salamanca (Spain). The spectra were recorded

by direct application of the probe on intact pieces of Ibe-

rian pork loin. Spectra were recorded in triplicate and

the spectral average was taken. The calibration equa-

tions obtained in the development of the procedure were

applied and the predicted values were compared with the

laboratory results obtained later.

3. Results and discussion

3.1. Chemical analyses and spectral information

The chemical compositions of the pork loin samples

used for calibration are shown in Table 1, 72 samples

were used. The results obtained for the content of the

different fatty acids revealed a broad range of variabil-

ity, which is important when searching for calibrations

equations to be used later in the prediction. 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, with

Iberian genetics varying between 50% and 75%).

Fig. 1 shows the spectra of a sample of loin ob-tained 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, using second derivative and DT. The best

of the different mathematical treatments for each fatty

acid was applied (Table 2). The effects of dispersion

Table 1

Statistical overview of chemical analysis (all units in %) N= 74

Fatty acid Minimum Maximum Mean SD

12:0 0.05 0.10 0.07 0.01

14:0 0.95 1.70 1.30 0.18

16:0 20.59 27.13 23.64 1.58

16:1 2.59 5.51 3.73 0.61

17:0 0.09 0.31 0.16 0.0517:1 0.13 0.46 0.21 0.05

18:0 8.94 13.71 11.09 1.14

18:1 49.06 58.30 52.83 1.74

18:2 2.46 9.23 5.42 1.81

18:3 0.14 0.82 0.36 0.17

20:0 0.10 0.32 0.19 0.05

20:1 0.76 1.55 0.99 0.13

SFA 31.03 41.47 36.45 2.62

MUFA 54.39 64.76 57.76 1.78

PUFA 2.62 9.91 5.79 1.97

Fig. 1. (a) NIR spectrum of the sample of loin measured with the fibre-

optic probe. (b) Corrected spectrum using second derivative and DT

for the fatty acid C18:2.

Table 2

Calibration statistical descriptors for the NIR determination of fatty acids in intramuscular fat from loin

Fatty acid N Mathematical treatment RSQ SEC SECV No. of principal components Probability explained (%)

12:0 69 None/2a derivada 0.622 0.007 0.008 7 99.46

14:0 70 None2a derivada 0.785 0.080 0.137 7 99.51

16:0 69 DT/2a derivada 0.798 0.662 1.051 7 99.54

16:1 71 DT/2a derivada 0.788 0.272 0.472 9 99.28

17:0 68 None/2a derivada 0.825 0.017 0.026 9 99.30

17:1 72 DT/2a derivada 0.762 0.019 0.031 7 99.54

18:0 73 Standar MSC/2a der 0.765 0.538 0.934 8 99.52

18:1 67 DT/2a derivada 0.696 0.816 1.094 7 99.46

18:2 71 DT/2a derivada 0.859 0.681 1.146 9 99.28

18:3 67 DT/2a derivada 0.878 0.054 0.099 7 99.36

20:0 71 SNV/2a derivada 0.458 0.035 0.043 9 99.30

20:1 71 DT/2a derivada 0639 0.070 0.098 8 99.52

SFA 70 Estandar MSC /2a deiv 0.807 1.116 1.763 8 99.52

MUFA 69 None/3a derivada 0.943 0.351 1.287 7 99.36

PUFA 71 DT/2a derivada 0.858 0.743 1.249 9 99.28

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were removed using MSC, SNV, DT or SNVDT.

MSC was first used by Geladi, Mac Dougall, and

Martens (1985) and prevents the dispersion in the

samples from imposing itself on the chemical signals.

Dhanoa, Lister, and Barnes (1995) indicate that

SNVDT were introduced not only for the reduction

of multicollinearity but also to calculate spectral dif-

ferences by reducing the confounding effects of base-

line shift and curvature.

The spectral information defines a series of charac-

teristic absorption bands. Thus, the CH bond, which

is a fundamental constituent of fatty acid molecules,

absorbs clearly at wavelengths close to 1200, 1400,

1750, 2310 and 2340 nm (Willians & Norris, 1987).

Moreover, the 23102340 region corresponds to the

combination bands of the CH bond, and the absorp-

tion produced in the 17201760 region corresponds to

the first overtone of that bond (Shenk, Workman, &

Westerhaus, 1992). Osborne, Fearn, and Hindle

(1993) have attributed the absorption produced at a

wavelength of 1210 nm the second overtone of the

CH2 bond. The absorption in the 21502190 region

at 1680 nm indicates the presence of cis double bonds

in the molecules; i.e., the existence of unsaturated fatty

acids (Garrido-Varo, Carrete, & Fernandez-Cabanas,

1998) in the samples analysed.

Fig. 2. 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.


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3.2. Determination of the fatty acid composition

To calibrate fatty acids in the intramuscular fat of

Iberian pork loin, the NIRS technology with a fibre-op-

tic probe was used, applying the probe directly onto the

loin samples. Calculation of the statistical parameters of

the calibration equations for each fatty acid is shown inTable 2, which also reflects the best treatments. After the

number of principal components had been calculated,

the detection of anomalous spectra was accomplished

using the Mahalanobis distance (H statistic), establish-

ing a limit value ofH= 3.0, such that the spectra whose

H distance was greater than this figure were considered

different from the spectral population and were dis-

carded. The numbers of remaining samples (N) are

shown in Table 2.

Regarding the number of principal components, be-

tween 7 and 9 components were used, depending on

the fatty acid to be determined, and these accounted

for 99.30% of the variance in the loin samples.

We consider that the results obtained in the determi-

nation of the fatty acids C14:0, C16:0, C16:1, C17:0,

C17:1, C18:0, C18:1, C18:2, C18:3, RMUFA, RSFA

and RMFA are very good. The results obtained in the

determination of C12:0, C20:0 and C20:1 are acceptable.

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, which reveals the broad applicability of the cal-

ibration equations obtained.

3.3. Validation

3.3.1. Internal validation (prediction)

Model evaluation was performed by cross-validation.

In this method, the set of calibration samples is divided

into a series of subsets: in our case 6. Of these, 5 are ta-

ken for the calibration set and one for the prediction set.

The process is repeated as many times as there are sets,

such that all pass through the calibration set and the

prediction set. Using this process, we validated the model

used and checked its prediction capacity. This process

was carried out for the validation of fatty acids in intact

loin samples.The predicted values gave validation errors that were

combined into SEP(C). Fig. 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, C16:0, C16:1, C17:0, C17:1,

C18:0, C18:1, C18:2, C18:3, RSFA, RMUFA, RPUFA

in the intramuscular fat of Iberian pork loin, together

with the statistical descriptors of prediction. From this

information it may be deduced that the NIRS technique,

using a fibre-optic probe, is a good alternative for the

determination of the content in fatty acids in samples

of intramuscular fat from Iberian breed swine. In later

studies the method will be use to analyse the composi-

tion 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, recebo and feed).

3.3.2. External validation

We checked the robustness of the method by applying

the fibre-optic probe to 15 new samples in a slaughter-

house. The procedure was as follows: spectra were re-

corded in triplicate and the spectral mean was taken;

the calibration equations obtained during the work were

applied and the predicted values were compared withthose obtained later using gas chromatography. 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 Est. min. Est. max Mean SD

12:0 0.04 0.10 0.07 0.01

14:0 0.77 1.81 1.29 0.17

16:0 19.17 28.01 23.59 1.47

16:1 1.95 5.49 3.72 0.59

17:0 0.04 0.28 0.16 0.04

171 0.09 0.33 0.21 0.04

18:0 7.73 14.39 11.06 1.11

18:1 48.41 57.29 52.85 1.48

18:2 0.06 10.94 5.50 1.81

18:3 0.00 0.82 0.36 0.15

20:0 0.04 0.32 0.18 0.05

20:1 0.63 1.33 0.98 0.12

SFA 28.63 42.88 36.25 2.54

MUFA 53.29 62.10 57.69 1.47

PUFA 0.00 11.78 5.87 1.97

Table 4

External validation for the determination of fatty acids in the

intramuscular fat of Iberian breed swine

Fatty acid Range Differences (%)

C12:0 0.060.09 9.93

C14:0 1.111.61 12.15

C16:0 21.6925.80 4.38

C16:1 2.964.97 11.62

C17:0 0.110.21 14.83

C17:1 0.150.24 8.24

C18:0 10.0613.90 5.91

C18:1 49.7854.24 2.09

C18:2 2.719.53 13.69

C18:3 0.160.67 26.39

C20:0 0.130.26 17.90

C20:1 0.831.14 7.35

SFA 34.1841.57 3.85

MUFA 54.8059.19 2.37

PUFA 2.8710.20 13.67

Differences between the reference method (gas chromatography) and

the NIRS technique.

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method (gas chromatography) and the NIRS technique

in the external validation.

In view of the present findings, 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. Furthermore, use of the fibre-

optic probe enables determinations to be made simplyby placing the probe on the sample itself, with no previ-

ous treatment. This has evident advantages as regards

the possible speed of the analyses.

Acknowledgements

The authors thank the Company Ganaderos Sal-

mantinos de Porcino Iberica S.L, Salamanca (Spain),

for providing the samples and the participation of INIA,

Ref CAL02-053 thanks to which this work was possible.

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