Fluorescence spectroscopy coupled with factorial discriminant analysis technique to identify sheep milk from different feeding systems.

Transcription

1 Fluorescence spectroscopy coupled with factorial discriminant analysis technique to identify sheep milk from different feeding systems. HAMMAMI Moncef, Ph.D Associate professor Higher School of Agriculture of Mateur, Tunisia

2 1. Introduction

3 2. Materials and methods 2.1. Animals Forty five Sicilo-Sarde ewes three homogenous groups (15 animals each), Weight : 51.3 ± 4.9 Kg for the corn (control); 51.9 ± 4.9 Kg for the soy bean meal and 52 ± 5.4 Kg for the scotch bean meal. The litter size :1.47 ± 0.5 for (C) ; 1.4 ± 0.5 for (S) and 1.5 ± 0.5 for (F). The numbers of lactation were respectively, 2.4 ± 0.9, 2.6 ± 0.9 and 2.6 ± 0.8 for the control, soy bean and scotch bean groups.

4 2.2. Diets 2 weeks of adaptation, Collectif boxes in an environmentally controlled sheep fold (17 ± 2 C) The ewes were fed ad libitum with three iso-energetic diets: control, soybean rich and scotch bean rich- diets (Table 1) during 11 weeks lactation period. Table 1: Compositional data (g 100 g-1) of the feeding systems given to the Sicilo Sarde ewes during the eleven-week lactation period DM TN CF MM OM PDIE PDIN UFL Aot hay Control Soy bean meal Scotch bean meal

5 2.3.Sampling and preservation of milk The ewes were milked by hand once a day (at 2 p.m.). Individual milk yield was recorded one day a week during the whole suckling period (11 weeks). an aliquot of 100 ml was taken and kept in a freezer at - 20 C until analyses. milk samples were thawed during one night at + 4 C in a refrigerator. All the analyses were made in triplicate (n = 11 samples x 3 repetitions = 33 analyses).

6 Animals group Milk hand FGE, Séminaire Franco Milk sampling tunisien, 29 octobre 2015, Tunis Lactoscan

7 2.4. Physico-chemical analyses Ewe milk production and physico chemical analyses were determined at weekly intervals during eleven-week lactation period. The ph, fat, protein, lactose, ash and freezing point were determined by using LactoScan (Milkotronic LTD, Serial n 4696, Hungary) Fluorescence measurements Fluorescence spectra were recorded using a Fluoromax-2 spectrofluorimeter (Spex-Jobin Yvon, Longjumeau, France) mounted with a variable angle frontsurface accessory. The incidence angle of the excitation radiation was set at 56 to ensure that reflected light, scattered radiation and depolarisation phenomena were minimised. for each milk sample, three spectra were recorded.

8 emission and excitation wavelengths Fluorophores Excitation (nm) Émission (nm) Increment (nm) aromatic amino acids and nucleotides (AAA +NA) Tryptophan ,5 Riboflavin vitamin A

9 2.5.1 Synchronous Fluorescence Spectra SF spectra were collected in the nm excitation wavelength range using offsets of 80 nm ( = 80) between excitation and emission monochromators. Spectra were recorded in triplicate for each condition using different samples.

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11 2.6.Mathematical analysis of data In a first step, principal component analysis (PCA) was separately applied to each intrinsic probe in order to investigate differences among the milk samples. In a second step, Factorial Discriminant Analysis (FDA) was performed on the first 10 Principal Components (PCs) resulting from the PCA applied to the fluorescence spectral data. Finally, the first 10 PCs of the PCA performed on each data set were pooled into one matrix and this new table was analysed by FDA. Chemometric analyses were performed in MATLAB (The Mathworks Inc., Natic, MA).

12 3 - Results and Discussion 3.1. Physico-chemical analyses No significant difference (P 0.5) among the three feedingsystems was observed for all compositional parameters throughout the whole lactation period (11 weeks) Fluorescence measurements The recorded fluorescence spectra provided information regarding molecules containing conjugated double bounds. AAA+NA, tryptophan residues of proteins, vitamin A and riboflavin, in particular, are the best known fluorescent molecules in milk.

13 AAA+NA fluorescence spectra Fig. 1. Normalised fluorescence emission spectra of AAA+NA fluorescence spectra recorded following excitation at 380 nm on Sicilo- Sarde ewe s milk fed scotch bean after 1 ( ), 11 ( ), soybean after 1 ( ), 11 (.... ) or control after 1 ( ) and 11 ( ) weeks of lactation, respectively.

14 Tryptophan fluorescence spectra Fluorescence intensity (a.u.) Wavelength (nm) Fig.2. Normalised fluorescence emission spectra of tryptophan fluorescence spectra recorded following excitation at 380 nm on Sicilo- Sarde ewe s milk fed scotch bean after 1 ( ), 11 ( ), soybean after 1 ( ), 11 (.... ) or control after 1 ( ) and 11 ( ) weeks of lactation, respectively

15 riboflavin fluorescence spectra Fig. 3. Normalised fluorescence emission spectra of riboflavin fluorescence spectra recorded following excitation at 380 nm on Sicilo- Sarde ewe s milk fed scotch bean after 1 ( ), 11 ( ), soybean after 1 ( ), 11 (.... ) or control after 1 ( ) and 11 ( ) weeks of lactation, respectively

16 vitamin A Fluorescence spectra Fluorescence intensity (a.u.) Wavelength (nm) Fig. 4: Normalised fluorescence excitation spectra of vitamin A recorded following emission at 410 nm on Sicilo-Sarde ewe s milk fed scotch bean after 1 ( ), 11 ( ), soybean after 1 ( ), 11 (.... ) or control after 1 ( ) and 11 ( ) weeks of lactation, respectively.

17 Fluorescence intensity (a.u.) Wavelengths (nm) Fig. 5: Normalised synchronous fluorescence spectra (offset = 80 nm) recorded on Sicilo-Sarde ewe s milk fed scotch bean after 1 ( ), 11 ( ), soybean after 1 ( ), 11 (.. ) or control after 1 ( ) and 11 ( ) weeks of lactation, respectively.

18 3.3. Statistical Analyses In order to derive information from the spectral data sets, the similarity map of the two first PCs of the PCA performed on each intrinsic probe was drawn. As a result, there was no observed discrimination among milk samples originating from the various feeding systems. Fig. 6: Discriminant analysis similarity map of the leave-one cross validation data set determined by discriminant factors 1 (F1) and 2 (F2) for the factorial discriminant analysis (FDA) performed on the vitamin A spectra of Sicilo-Sarde ewe s milk fed scotch bean meal ( ), soy bean meal ( ) or control ( ).

19 F2 (21.78 %) In order to compare the results obtained from the SFS and those found with the different intrinsic probes, concatenation technique was applied to the AAA+NA, tryptophan, vitamin A and riboflavin spectra F1 (68.22 %) Fig. 7: Factorial discriminant analysis (FDA) was performed on the 40 concatenated PCs corresponding to the PCA performed on the emission spectra of aromatic amino acids and nucleic acids, tryptophan fluorescence spectra, riboflavin and vitamin A fluorescence spectra of Sicilo-Sarde ewe s milk fed scotch bean meal ( ), soy bean meal ( ) or control ( ).

20 4. Conclusion The present investigation demonstrated the ability of front face fluorescence spectroscopy (FFFS) to monitor changes in ewe s milk throughout the lactation period. When factorial discriminant analysis (FDA) was separately applied to each intrinsic probe or to the synchronous fluorescence (SF) spectra,, the discrimination among the three groups was not very clear. That was probably due to the overlapping of spectra. The concatenation technique of the aromatic amino acids and nucleic acids (AAA+NA), tryptophan, vitamin A and riboflavin acquired by using FFF technique allowed a good discrimination of ewe s milk from the different feeding system

21 It was concluded that the use of specific intrinsic probes (AAA+NA, tryptophan, vitamin A, riboflavin) provided a good discrimination among the three groups of milk collected from ewes fed scotch bean, soy bean and corn (control). The results obtained in this study demonstrated that front-face fluorescence spectroscopy in combination with chemometrics can be considered as a fast, non-destructive and innovative technique to differentiate between ewe s milk samples originating from different feeding systems. It is clear from the present study that the replacement of soybean by scotch bean (a local and economical diet) could be considered as a promising alternative feeding system for livestock in Tunisia. Milk samples of ewes fed scotch bean were less oxidised than those fed soybean meal.

22 THANK YOU FOR YOUR ATTENTION