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. 2023 Jul 18;28(14):5488.
doi: 10.3390/molecules28145488.

A Study of Greek Graviera Cheese by NMR-Based Metabolomics

Evangelia Ralli  1 , Apostolos Spyros  1
Affiliations

A Study of Greek Graviera Cheese by NMR-Based Metabolomics

Evangelia Ralli et al. Molecules. .

Abstract

Graviera is a very popular yellow hard cheese produced in mainland Greece and the Aegean islands, and in three PDO (protected denomination of origin) locations. Apart from geographic location, type of milk and production practices are also factors that affect cheese composition, and make this dairy product unique in taste and aroma. In this work, 1H nuclear magnetic resonance (NMR) spectroscopy in combination with chemometrics has been used to determine the metabolite profile (40 compounds) of graviera cheese produced in different geographic locations, with emphasis on cheeses produced on the island of Crete. Organic acids and amino acids were the main components quantified in the polar cheese fraction, while the fatty acid (FA) composition of the lipid fraction was also obtained. Analysis of variance (Anova) of the dataset showed that γ-aminobutyric acid (GABA), conjugated linoleic acids (CLA) and linoleic acid differentiate gravieras produced in different areas of Crete, and that the total amino acid content was higher in cheeses produced in eastern Crete. Targeted discriminant analysis models classified gravieras produced in mainland Greece, Cyclades and Crete based on differences in 1,2-diglycerides, sterols, GABA and FA composition. Targeted and untargeted orthogonal partial least squares discriminant analysis (OPLS-DA) models were capable of differentiating gravieras produced in the island of Crete and hold promise as the basis for the authentication of PDO graviera products.

Keywords: NMR spectroscopy; amino acids; fatty acids; graviera cheese; metabolite profile; metabolomics; multivariate analysis.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Typical 1H NMR spectrum of the water extract of graviera cheese sample in D2O-TSP, 500 MHz. Some metabolites are highlighted.
Figure 2
Figure 2
Typical 1H NMR spectrum of chloroform extract of graviera cheese sample in CDCl3 at 500 MHz. UFA: unsaturated fatty acids, TG: triglycerides, LN: linolenic acid, LO: linoleic acid, FA: fatty acids.
Figure 3
Figure 3
Box plots of 1,2-diglycerides, γ-aminobutyric acid, lactate and total amino acids (TFAA) in gravieras produced in Crete as a function of geographic area from west (Chania) to east (Lasithi).
Figure 4
Figure 4
Box plots of caproleic acid, conjugated linoleic acid (CLA), 1,2-diglycerides and ω-3 polyunsaturated fatty acids (LN) in gravieras produced in Greece as a function of geographic area.
Figure 5
Figure 5
OPLS-DA model score plot of the first two predictive components, t1 and t2, (a) and scatter plot of loadings p and q (b) for the geographical origin of graviera samples based on their metabolite profile. R2X = 0.636, R2Y = 0.532, Q2 = 0.261. The size of metabolite symbols in the loading plot reflects their Variable importance Parameter (VIP) value in the model.
Figure 5
Figure 5
OPLS-DA model score plot of the first two predictive components, t1 and t2, (a) and scatter plot of loadings p and q (b) for the geographical origin of graviera samples based on their metabolite profile. R2X = 0.636, R2Y = 0.532, Q2 = 0.261. The size of metabolite symbols in the loading plot reflects their Variable importance Parameter (VIP) value in the model.
Figure 6
Figure 6
OPLS-DA model score plots of the predictive, t1, and the first orthogonal component, to1, from metabolite data (a) and NMR spectra buckets (b) for the differentiation of the origin of Greek gravieras (Cretan/non-Cretan).
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