Institute of Food Research Orange juice authentication by H NMR G n L G ll M i n C n nd Gwen Le Gall, Marion Cuny and Ian Colquhoun
Need for authentication Orange Juice consumption is increasing world wide and freshly squeezed juices (Not from Concentrate, NFC) is a growing g market in US, Canada and Europe Million SSE Gallons 8 U.S. consumption of orange juice by category 6 4 8 6 4 89 9 9 9 93 94 95 96 97 98 99 YEAR http://edis.ifas.ufl.edu/fe95#table_4 From Conc OJ (FCOJ) Reconst Conc OJ (RCOJ) (chilled) Not From Conc (NFC) (chilled) TOTAL
Need for authenticating orange juice Some techniques exist to detect fraud (HPLC, SNIF-NMR) NMR) but still need for more Aim: would it possible to easily implement a technique such as H Nuclear Magnetic Resonance (NMR) spectroscopy to address authentication issues?
Techniques for measuring wide ranges of metabolites H-NMR GC/MS LC/MS Mainly primary major metabolites, compounds polar & nonpolar: (fingerprinting) g) amino-acids acids, organic acids, sugars, fatty acids Secondary metabolites, aromatic cpds, phenolics, glycoalkaloids METABOLITES
Preparation ph adjust ml of juice to 4± 4.±.3, 3 add DO to supernatant (5- samples a day) Proton NMR: min recording per sample Le Gall G, Puaud M, Colquhoun IJ, Discrimination between orange juice and pulp wash by H- nuclear magnetic resonance spectroscopy: Identification of marker compounds JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 49 (): 58-588 FEB
dmp? asn asp citric H NMR of fruit juice malic OVERALL SPECTRA suc + gluc suc gluc gluc 8 6 4 3.5 ethanol malic gaba dmp dmp pro dmp pro gaba arg ala? val fatty acids sterol Mean Orange Juice Mean Grapefruit.6.4..8.6.4..8
Mean spectra Low field region formic tyr * tyr acid?? * * niacin niacin??? hesp hesp?? naringin? * polyphenols hesp * phlorin naringin Mean OJ Mean GF 9 8.5 8 7.5 7 6.5 6
Study on orange and grapefruit juice 93 juices bought in local supermarkets, Norwich,UK 58 orange juices 3 grapefruit juices 4 freshly squeezed fresh concentrates 9 freshly squeezed 4 fresh concentrates pasteurised concentrates pasteurised concentrates mixtures orange/grapefruit juice 3 freshly squeezed 7 pasteurised concentrates freshly squeezed Clementine juices Can we differentiate orange juice from grapefruit by H NMR? Markers? Orange juice from mixtures? Freshly squeezed from concentrate? Markers?
Approaches for data analysis univariate, integrated multivariate, integrated multivariate, whole trace sample tyr 5_4 8489 3_4 5398 3_4 4869 sample tyr phe c. putr 5_4 8489 59 4937 3_4 5398 468 968 3_4 4869 57 89 sample measurements
Data reduction by PCA, whole measurements traces PCs z z z 3 samp ples sam mples z z z 3 scores z n z n
Principal Component Analysis on H NMR spectra of fruit juices x -3 Normalised covariance x -3 Normalised covariance 3 3 PC C % var PC C - - - - -3-3 -. -.5.5. PC 79% var -. -.5.5. PC grapefruit mixtures fresh squeezed (NFC) orange clementine fresh concentrate (RCOJ) pasteurised concentrate (FCOJ)
Origin of the discrimination - grapefruit orange PC ethanol? polyphenols? malic pro naringin PC 9 8.5 8 7.5 7 6.5 6.5.5 malic suc unk 4.5 4.4 4.3 5.5 5 glucose 4.5 4 3.5 3 citric loading
Approaches for data analysis univariate, integrated multivariate, integrated multivariate, whole trace sample tyr 5_4 8489 3_4 5398 3_4 4869 sample tyr phe c. putr 5_4 8489 59 4937 3_4 5398 468 968 3_4 4869 57 89 sample measurements
Any markers? Anova was performed on 35 signals representing most of the metabolites detected by H NMR F value was significant for 3 signals 5 h d hi h F l t ti l 5 showed high F values = potential markers discriminating OJ/GF
ANOVA AND INTENSITY PLOTS ANOVA on niacin 8.8 NMR In ntensit ty 8 6 4 x -7 F 5.9. NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF Intensity plot on niacin MR In ntensit ty N 6.4 x -6.8.6.4. mixtures 9% 5%OJ NFC RCOJ FCOJ NFC RCOJ FCOJ Even if niacin is 4 fold-diff diff to the mean, mixtures do not separate from pure OJ OJ GF
Principal Component Analysis on H NMR spectra of fruit juices x -3 Normalised covariance x -3 zoom 3 3 PC % var - - -3 -. -.5.5. PC 79% var PC - -3 7% %OJ 6% 5%75% 9% 85% 7% 5% 7% -?% -4-4 6 PC x -3 grapefruit mixtures % of grapefruit in mixture orange clementine needs to be high (>4%) for mixture to be discriminated
PCA on parts of spectra PCA on low field region x -3. 6 zoom.5 4 PC -.5 PC -. -.55-85%OJ 7%OJ?% -. -.3 -. -.. PC - -5 5 PC x -3 grapefruit orange mixtures clementine PCA on high field region not as good (not shown)
Authenticity by liquid chromatography Pea ak Intensity 7 HESPERIDIN NARINGIN 6 5 4 3 Pure Orange Juice Pure Grapefruit Mixture (6% OJ)
Detection of hesperidin (OJ marker) and naringin (GF marker) Low field region naringin hesp hesp hesp naringin Mean OJ Mean GF 7.8 7.6 7.4 7. 8 7.5 7 6.5 6 HPLC markers seen by NMR but signals affected by ph 6.9 6.8 6.7 6.6 6.5
Peak alignment done by Matlab macro written in house Hesperidin (OJ) before 7.8 7.6 7.4 after Matlab macro 7. N i i (GF) Naringin 7.8 7.6 7.4 Matlab collation 7. 7.8 7.6 7.4 7. after before Matlab macro 6.9 6.8 6.7 6.6 6.5 6. 6.8 6.6 6.4
ANOVA HESPERIDIN 7.8 7.6 NMR Intensi ity 7.4 5 4 3 x -6 7. 6 ANOVA F 6.7 N MR In ntensit ty 6 Intensity plot 6 x -6 5 4 3 mixtures NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF
x ANOVA NARINGIN 5 5-7 6. 6.8 6.6 NMR In ntensit ty 7 6 5 4 3 x -6 6.4 F64.7 ANOVA NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF 7 x Intensity plot -6 6 5 4 3 - NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF
Discrimination according to process x -3 Normalised covariance 3 ethanol PC C - - loading -3 -. -.5.5. PC.5.5 grapefruit orange mixtures clementine fresh squeezed fresh concentrate Pasteurised concentrate
ANOVA according to process At least two signals seem to be potential markers for (not from concentrate) freshly squeezed juices NM MR Int tensity 8 6 4 x -4 ethanol F9.63 NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF ensity NM MR Int x -4.5 5.5.5? 3.33 F.94 prob-4 NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF
ANOVA according to process Half a dozen signals seem to show a trend in difference between NFC, RCOJ and FCOJ especially for grapefruit NM MR Int tensity x -3.6 b-glucose 4.64.8 F8.7.4. NM MR Inte ensity.5.5 x -3 sucrose 5.5 F65.3 NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF NFC RCOJ FCOJ NFC RCOJ FCOJ OJ GF
CONCLUSIONS H NMR spectra easy to obtain (same results for 4 MHz, not shown) Orange juice discriminated from grapefruit juice Some new markers but mixtures with <4% grapefruit not easily discriminated using the whole or part of NMR trace HPLC markers observed by NMR; mixtures down to % grapefruit discriminated by ANOVA on the most important of the two selected NMR signals (naringin) Potential markers (suc, gluc,?) to authenticate freshly squeezed (not from concentrate, NFC) juices Could the technique be extended to undeclared addition of other fruit juices in order to justify the cost of the NMR machine?