The Use of Right Angle Fluorescence Spectroscopy to Distinguish the Botanical Origin of Greek Common Honey Varieties

The standardization of the botanical origin of honey reflects the commercial value and quality of honey. Nowadays, most consumers are looking for a unifloral honey. The aim of the present study was to develop a novel method for honey classification using chemometric models based on phenolic compounds analyzed with right angle fluorescence spectroscopy, coupled with stepwise linear discriminant analysis (LDA). The deconstructed spectrum from three-dimensional-emission excitation matrix (3D-EEM) spectra provided a correct classification score of 94.9% calibration and cross-validation at an excitation wavelength (λex) of 330 nm. Subsequently, a score of 81.4% and 79.7%, respectively, at an excitation wavelength (λex) of 360 nm was achieved. Each chemometric model confirmed its power through the external validation with a score of 82.1% for both. Differentiation could be correlated with hydroxycinnamic and hydroxybenzoic acids, which absorb in this region of the spectrum. Fluorescence spectroscopy constitutes a rapid and sensitive technique, which, when combined with the stepwise algorithm and LDA method, can be used as a reliable and predictive authentication tool for honey. This study indicates that the developed methodology is a promising technique for determination of the botanical origin of common Greek honey varieties. Our long-term ambition is to support producers and suppliers to remain in a competitive national and international market

Determination of chemical indicators and development of chemometric models as tools for identifying the authenticity of honey

The European Union's focus on research projects to promote and protect the iconic products of its member states has been strong in recent years. Greece is one of the regions of the Balkan Peninsula with intense vegetation and biodiversity, which contributes to the production of high-quality honey of great commercial and research interest. The present thesis aimed at the isolation, detection and identification of volatile and non-volatile fractions of endogenous Greek honeys through instrumental chemical analysis techniques and the use of the results for the development of chemometric authenticity models. The study focused on the optimization of volatile fraction extraction, the study of phenolic compounds from selected samples, the recording of spectroscopic data and the creation of a database that will address honey authenticity issues with the help of chemometrics. The study started with an extensive literature review, highlighting the limitations of traditional physicochemical methods for the detection of the botanical origin of honey and the potential of instrumental chemical analysis techniques. The review highlighted the need for accurate and reliable methods that standardize monofloral Greek honeys and do not compromise consumer confidence and the reputation of the honey industry. The sampling of 655 honey samples was carried out in the framework of two research projects in conjunction with professional beekeepers during the 2018 and 2021 beekeeping season. The samples were analyzed according to European and Greek legislation and stored according to good practice standards. In the development and optimization of the surface response methodology for volatile components, varieties with strong economic, export and research interest were selected. Of the at least 31 volatile compounds isolated from thyme honey using the SPME technique and GC-MS chromatography analysis, 10 were considered to be the most important. Similarly, of the at least 49 volatile compounds from the Erica honey, 13 were considered to be the most important contributors. In both cases, temperature, extraction time and water: honey ratio (v/m) were the most important factors with a positive effect on recovery for most volatile compounds. Equilibration time ensured qualitative and quantitative reproducibility, while sample volume was related to the isolation of minor compounds. Also, the effect of magnetic stirrer speed was found to be quite significant in the context of predictive models. In each case, a set of external validation tests was used to document the method. In the next part of the study, the use of GC-MS instrumental analysis in combination with the Stepwise-LDA chemometric technique helped to construct a tool for authentication of the four most commercial honeys in Greece. Fir, thyme, pine and citrus honeys were correctly classified at 85.5% with cross-validation of 82.3% and external validation of 88.5%. Also, the same chemical analysis was used to construct an gas-chromatographic database of 596 samples. In this way, the main volatile compounds that could give the "type" of honey were identified and compounds with a significant presence in more than one type of honey were detected, such as benzaldehyde, furfural, octane, nonanol, nonanal, 2-phenyl acetaldehyde, and some methyl esters. The analysis of phenolic constituents using LC-Qtof-MS on 82 pure varieties including honey from thyme, pine, fir, heather, cotton, chestnut, sunflower and orange showed the detection of at least 28 compounds. Of these compounds, 23 were found at high levels, while some could potentially be chemical indicators of botanical origin for some of them. FTIR spectroscopy recorded spectroscopic data and in combination with the Stepwise-LDA chemometric technique accurately (correct classification 93.5%, cross-validation 82.5%, external validation 84.6%) classified the most commercial honey samples based on their botanical origin. Raman spectroscopy, compared to traditional physicochemical analyses of honey, appeared clearly more powerful as a tool for botanical discrimination of fir, pine and thyme honeys. According to the results, the percentage of correct classification of honey using Raman was 95.3%, 90.6% and 84.3% in terms of correct classification, internal and external validation, respectively. Differentiation based on registration with right angle FS registration proved to be another reliable tool. A total of 87 honey samples from four botanical cultivars (21 thyme, 18 pine, 21 fir and 16 citrus) were classified with high classification rates based on two fluorometric ranges (λex = 330 and 360). In the first case (λex = 330), the percentage of correct classification, internal and external validation was 94.9%, 94.9% and 82.1%, respectively. While in the second case (λex = 360) it was 81.4%, 79.7% and 82.1%, respectively. In both cases, the results confirmed that the fluorescent phenolic compounds absorbing at these wavelengths are related to the botanical origin of the pure melliferous olives. In conclusion, this PhD study successfully developed chemometric authenticity models for Greek honey using both chromatographic and spectroscopic data. The developed chemometric models offer a practical tool for honey producers, regulatory authorities and quality control laboratories to verify the authenticity and identity of Greek honey. By applying these models, producers and traders can protect consumers and maintain the integrity of the honey industry. The study also highlights the importance of continued research and development in the field of honey authenticity in conjunction with instrumental chemical analysis.Ο προσανατολισμός της Ευρωπαϊκής Ένωσης σε ερευνητικά προγράμματα ανάδειξης και προστασίας των εμβληματικών προϊόντων των κρατών-μελών της είναι έντονος τα τελευταία χρόνια. Η Ελλάδα αποτελεί μία από τις περιοχές της βαλκανικής χερσονήσου με έντονη βλάστηση και βιοποικιλότητα, που συμβάλλει στην παραγωγή υψηλής ποιότητας μελιού με έντονο εμπορικό και ερευνητικό ενδιαφέρον. Η παρούσα διδακτορική διατριβή είχε ως στόχο τη απομόνωση, ανίχνευση και ταυτοποίηση των πτητικών και μη πτητικών κλασμάτων αμιγών ελληνικών μελιών μέσω τεχνικών ενόργανης χημικής ανάλυσης και την χρήση των αποτελεσμάτων για την ανάπτυξη χημειομετρικών μοντέλων αυθεντικότητας. Η μελέτη επικεντρώθηκε στην βελτιστοποίηση της παραλαβής του πτητικού κλάσματος, την μελέτη των φαινολικών συστατικών από επιλεγμένα δείγματα, την καταγραφή φασματοσκοπικών δεδομένων αλλά και την δημιουργία μιας βάσης δεδομένων που με την βοήθεια της χημειομετρίας θα αντιμετωπίσει ζητήματα αυθεντικότητας του μελιού. Η μελέτη ξεκίνησε με μια εκτενή βιβλιογραφική ανασκόπηση, επισημαίνοντας τους περιορισμούς των παραδοσιακών φυσικοχημικών μεθόδων για την ανίχνευση της βοτανικής προέλευσης του μελιού και τις δυνατότητες των τεχνικών ενόργανης χημικής ανάλυσης. Η ανασκόπηση υπογράμμισε την ανάγκη για ακριβείς και αξιόπιστες μεθόδους που θα τυποποιούν τα αμιγή ελληνικά μέλια και δεν θα θέτουν σε κίνδυνο την εμπιστοσύνη των καταναλωτών και τη φήμη της βιομηχανίας μελιού. Η δειγματοληψία των 655 δειγμάτων μελιών πραγματοποιήθηκε στο πλαίσιο δυο ερευνητικών προγραμμάτων σε συνδυασμό με επαγγελματίες μελισσοκόμους κατά την διάρκεια της μελισσοκομικής περιόδου 2018 έως 2021. Τα δείγματα υποβλήθηκαν σε ανάλυση με βάση την ευρωπαϊκή και ελληνική νομοθεσία ενώ αποθηκεύτηκαν με βάση τα πρότυπα ορθής πρακτικής.Στην ενότητα της ανάπτυξης και βελτιστοποίησης της μεθοδολογίας επιφανειακής απόκρισης των πτητικών συστατικών, επιλέχθηκαν ποικιλίες με έντονο οικονομικό, εξαγωγικό και ερευνητικό ενδιαφέρον. Από τις 31 τουλάχιστον πτητικές ενώσεις που απομονώθηκαν από το θυμαρόμελο με την χρήση της τεχνικής SPME και την ανάλυση με χρωματογραφία GC-MS οι 10 κρίθηκαν ως οι κυριότερες. Αντίστοιχα, από τις 49 τουλάχιστον πτητικές ενώσεις του ερεικόμελου οι 13 θεωρήθηκαν ότι συμβάλλουν σημαντικότερα. Και στις δυο περιπτώσεις η θερμοκρασία, ο χρόνος εκχύλισης και η αναλογία νερού: μέλι (v/m) ήταν οι πιο σημαντικοί παράγοντες με θετική επίδραση στην ανάκτηση για τις περισσότερες πτητικές ενώσεις. Ο χρόνος εξισορρόπησης εξασφάλιζε την ποιοτική και ποσοτική επαναληψιμότητα, ενώ ο όγκος του δείγματος σχετιζόταν με την απομόνωση δευτερευουσών ενώσεων. Επίσης, η επίδραση της ταχύτητας αναδευσης διαπιστώθηκε ότι είναι αρκετά σημαντικός στο πλαίσιο προγνωστικών μοντέλων. Σε κάθε περίπτωση, ένα σύνολο δοκιμών εξωτερικής επικύρωσης χρησιμοποιήθηκε για την τεκμηρίωση της μεθόδου. Στο επόμενο τμήμα της μελέτης, η χρήση της ενόργανης ανάλυσης GC-MS σε συνδυασμό με την χημειομετρική τεχνική Stepwise-LDA βοήθησε στην κατασκευή ενός εργαλείου για τον έλεγχο της αυθεντικότητας των τεσσάρων πιο εμπορικών μελιών της Ελλάδας. Τα μέλια ελάτου, θυμαριού, πεύκου και εσπεριδοειδών ταξινομήθηκαν ορθά σε ποσοστό 85,5% με διασταυρούμενη επικύρωση 82,3% και εξωτερική επικύρωση 88,5%. Επίσης, η ίδια χημική ανάλυση χρησιμοποιήθηκε για την κατασκευή μιας βάσης αεροχρωματογραφικών δεδομένων 596 δειγμάτων. Κατά αυτόν τον τρόπο, επισημάνθηκαν οι κυριότερες πτητικές ενώσεις που θα μπορούσαν να δώσουν τον «τύπο» του μελιού ενώ ανιχνεύτηκαν ενώσεις με σημαντική παρουσία σε παραπάνω από έναν τύπο μελιού όπως, η βενζαλδεΰδη, η φουρφουράλη, το οκτάνιο, το εννεάνιο, το ενδεκάνιο, η 1-εννεανόλη, η 2-φαινυλοακεταλδεϋδη, και κάποιοι μεθυλεστέρες. Η ανάλυση των φαινολικών συστατικών με τη χρήση LC-Qtof-MS σε 82 δείγματα αμιγών ποικιλιών συμπεριλαμβανομένων μελιών από θυμάρι, πεύκο, έλατο, ερείκη, βαμβάκι, κουμαριά, καστανιά, ηλίανθο και πορτοκαλιά έδειξε την ανίχνευση τουλάχιστον 28 ενώσεων. Από αυτές τις ενώσεις, οι 23 βρέθηκαν σε υψηλή περιεκτικότητα, ενώ κάποιες θα μπορούσαν να αποτελέσουν, ενδεχομένως, χημικούς δείκτες βοτανικής προέλευσης για μερικά από αυτά. Η φασματοσκοπία FTIR κατέγραψε φασματοσκοπικά δεδομένα ενώ σε συνδυασμό με την χημειομετρική τεχνική Stepwise-LDA ταξινόμησε με ακρίβεια (ορθή ταξινόμηση 93,5%, διασταυρούμενη επικύρωση 82,5%, εξωτερική επικύρωση 84,6%) τα πιο εμπορικά δείγματα μελιού με βάση τη βοτανικής τους προέλευση.Η φασματοσκοπία Raman έναντι των παραδοσιακών φυσικοχημικών αναλύσεων του μελιού, φάνηκε σαφώς πιο ισχυρό ως εργαλείο ως προς την βοτανική διάκριση των μελιών ελάτου, πεύκου και θυμαριού. Σύμφωνα με τα αποτελέσματα το ποσοστό ορθής κατάταξης των μελιών με την χρήση Raman ήταν 95,3%, 90,6% και 84,3% ως προς την ορθή κατάταξη, εσωτερική και εξωτερική επικύρωση, αντίστοιχα. Η διαφοροποίηση βάσει της καταγραφής με FS ορθής γωνίας αποδείχθηκε ένα ακόμα αξιόπιστο εργαλείο. Τα 87 συνολικά δείγματα μελιού από τέσσερις βοτανικές ποικιλίες (21 θυμάρι, 18 πεύκο, 21 έλατο και 16 εσπεριδοειδή) ταξινομήθηκαν με υψηλά ποσοστά κατάταξης με βάσει δύο φθορισμομετρικές περιοχές (λex = 330 και 360 nm). Στην πρώτη περίπτωση (λex= 330 nm) το ποσοστό ορθής κατάταξης, εσωτερικής και εξωτερικής επικύρωσης ήταν 94,9%, 94,9% και 82,1%, αντίστοιχα. Ενώ στην δεύτερη περίπτωση (λex= 360 nm) ήταν 81,4%, 79,7% και 82,1%, αντίστοιχα. Και στις δύο περιπτώσεις, τα αποτελέσματα επιβεβαίωσαν ότι οι φθορίζουσες φαινολικές ενώσεις που απορροφούν σε αυτά τα μήκη κύματος σχετίζονται με την βοτανική προέλευση των αμιγών μελιών. Συμπερασματικά, η παρούσα διδακτορική μελέτη ανέπτυξε με επιτυχία χημειομετρικά μοντέλα αυθεντικότητας για το ελληνικό μέλι χρησιμοποιώντας τόσο χρωματογραφικά όσο και φασματοσκοπικά δεδομένα. Τα χημειομετρικά μοντέλα που αναπτύχθηκαν προσφέρουν ένα πρακτικό εργαλείο για τους παραγωγούς μελιού, τις ρυθμιστικές αρχές και τα εργαστήρια ελέγχου ποιότητας για την επαλήθευση της γνησιότητας και της ταυτότητας του ελληνικού μελιού. Με την εφαρμογή αυτών των μοντέλων, οι παραγωγοί και έμποροι μπορούν να προστατεύσουν τους καταναλωτές και να διατηρήσουν την ακεραιότητα της βιομηχανίας μελιού. Η μελέτη υπογραμμίζει επίσης τη σημασία της συνεχιζόμενης έρευνας και ανάπτυξης στον τομέα της αυθεντικότητας του μελιού σε συνδυασμό με την ενόργανη χημική ανάλυση

Greek Honey Authentication: Botanical Approach

Honey is a functional, honeybee product with a useful role in human nutrition and several health benefits. Greece is a Mediterranean region with several types of monofloral honey. Today, Greek honey has acquired an important position in national and international markets. Due to this increased industrialization and globalization, quality control is a necessity. Mislabeling constitutes one of the most notable types of fraudulence, while most consumers are looking for authentic honey. Moreover, producers and suppliers are searching for rapid and analytical methodologies to secure Greek honey in a competitive environment. In this context, we aimed to describe the classical (melissopalynological, physicochemical) and analytical (chromatographic, spectrometric, and spectroscopic) methods for the standardization of the botanical origin of Greek honey

Current Methods for the Extraction and Analysis of Isothiocyanates and Indoles in Cruciferous Vegetables

Cruciferous vegetables are characterized by the presence of sulfur-containing secondary plant metabolites known as glucosinolates (GLS). The consumption of cruciferous vegetables such as broccoli, cabbage, rocket salad, and cauliflower has been related to the prevention of non-communicable diseases. Their beneficial effects are attributed to the enzymatic degradation products of GLS, e.g., isothiocyanates and indoles. Owing to these properties, there has been a shift in the last few years towards the research of these compounds and a wide range of methods for their extraction and analytical determination have been developed. The aim of this review is to present the sample preparation and extraction procedures of isothiocyanates and indoles from cruciferous vegetables and the analytical methods for their determination. The majority of the references that have been reviewed are from the last decade. Although efforts towards the application of eco-friendly non-conventional extraction methods have been made, the use of conventional solvent extraction is mainly applied. The major analytical techniques employed for the qualitative and quantitative analysis of isothiocyanates and indoles are high-performance liquid chromatography and gas chromatography coupled with or without mass spectrometry detection. Nevertheless, the analytical determination of isothiocyanates presents several problems due to their instability and the absence of chromophores, making the simultaneous determination of isothiocyanates and indoles a challenging task

Development of a UPLC-Q-ToF-MS Method for the Determination of Sulforaphane and Iberin in Cruciferous Vegetables

Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)-butane) and iberin (1-isothiocyanato-3-methylsulfinylpropane) have attracted widespread attention due to their anti-inflammatory and cancer-preventive properties. These isothiocyanates are products of the enzymatic hydrolysis of the glucosinolates glucoraphanin and glucoiberin, which are found only in the plants of the order Brassicales. Cruciferous vegetables, such as broccoli, cabbage and cauliflower, belong to the order Brassicales, specifically, in the Brassicaceae family. Our aim was to develop an efficient and accurate method for the simultaneous determination of sulforaphane and iberin in cruciferous vegetables using Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-ToF-MS). The method was applied for the quantitative determination of these compounds in a variety of cruciferous vegetables (green and purple broccoli, white and purple cabbage, radish, turnip, arugula, watercress and cauliflower). The results showed that green and purple broccoli contained the highest levels of sulforaphane (660.14 ± 34.29 to 210.11 ± 9.76 μg g−1 dry weight), while the highest concentration of iberin was detected in purple broccoli (144.98 ± 3.56 μg g−1 dry weight). The lowest concentrations of sulforaphane and iberin were measured in watercress and radish. The differences in the content of these compounds can be attributed to the variability among Brassicaceae species, geography, season and various environmental factors

Greek Honey Authentication: Botanical Approach

Honey is a functional, honeybee product with a useful role in human nutrition and several health benefits. Greece is a Mediterranean region with several types of monofloral honey. Today, Greek honey has acquired an important position in national and international markets. Due to this increased industrialization and globalization, quality control is a necessity. Mislabeling constitutes one of the most notable types of fraudulence, while most consumers are looking for authentic honey. Moreover, producers and suppliers are searching for rapid and analytical methodologies to secure Greek honey in a competitive environment. In this context, we aimed to describe the classical (melissopalynological, physicochemical) and analytical (chromatographic, spectrometric, and spectroscopic) methods for the standardization of the botanical origin of Greek honey

Unifloral Autumn Heather Honey from Indigenous Greek Erica manipuliflora Salisb.: SPME/GC-MS Characterization of the Volatile Fraction and Optimization of the Isolation Parameters

For long heather honey has been a special variety due to its unique organoleptic characteristics. This study aimed to characterize and optimize the isolation of the dominant volatile fraction of Greek autumn heather honey using solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS). The described approach pointed out 13 main volatile components more closely related to honey botanical origin, in terms of occurrence and relative abundance. These volatiles include phenolic compounds and norisoprenoids, with benzaldehyde, safranal and p-anisaldehyde present in higher amounts, while ethyl 4-methoxybenzoate is reported for the first time in honey. Then, an experimental design was developed based on five numeric factors and one categorical factor and evaluated the optimum conditions (temperature: 60 °C, equilibration time: 30 min extraction time: 15 min magnetic stirrer velocity: 100 rpm sample volume: 6 mL water: honey ratio: 1:3 (v/w)). Additionally, a validation test set reinforces the above methodology investigation. Honey is very complex and variable with respect to its volatile components given the high diversity of the floral source. As a result, customizing the isolation parameters for each honey is a good approach for streamlining the isolation volatile compounds. This study could provide a good basis for future recognition of monofloral autumn heather honey

The Use of SPME-GC-MS IR and Raman Techniques for Botanical and Geographical Authentication and Detection of Adulteration of Honey

The aim of this review is to describe the chromatographic, spectrometric, and spectroscopic techniques applied to honey for the determination of botanical and geographical origin and detection of adulteration. Based on the volatile profile of honey and using Solid Phase microextraction-Gas chromatography-Mass spectrometry (SPME-GC-MS) analytical technique, botanical and geographical characterization of honey can be successfully determined. In addition, the use of vibrational spectroscopic techniques, in particular, infrared (IR) and Raman spectroscopy, are discussed as a tool for the detection of honey adulteration and verification of its botanical and geographical origin. Manipulation of the obtained data regarding all the above-mentioned techniques was performed using chemometric analysis. This article reviews the literature between 2007 and 2020

Optimized Isolation of Safranal from Saffron by Solid-Phase Microextraction (SPME) and Rotatable Central Composite Design-Response Surface Methodology (RCCD-RSM)

Safranal is the main aroma component of saffron stigmas. It is also a great antioxidant with known pharmacological properties and is a potent indicator for the grading and authentication of saffron. In this study, the optimum extraction conditions of safranal from saffron stigmas were investigated using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and response surface methodology (RSM). A rotatable-central composite design was applied, and a linear regression model has been used for the model building. The optimized factors were as follows: sample weight (15 mg), water volume (4 mL), exposure time in the headspace (20 min), and extraction temperature (45 °C). All factors were found significant; however, extraction temperature and exposure time were the most important for the isolation of safranal. The obtained model was successfully validated with a test set of saffron samples analyzed under the optimum extraction conditions. The optimized SPME extraction conditions of safranal found in this study contribute to the efforts towards the detection of saffron authentication and adulteration

Optimized Isolation of Safranal from Saffron by Solid-Phase Microextraction (SPME) and Rotatable Central Composite Design-Response Surface Methodology (RCCD-RSM)

Safranal is the main aroma component of saffron stigmas. It is also a great antioxidant with known pharmacological properties and is a potent indicator for the grading and authentication of saffron. In this study, the optimum extraction conditions of safranal from saffron stigmas were investigated using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and response surface methodology (RSM). A rotatable-central composite design was applied, and a linear regression model has been used for the model building. The optimized factors were as follows: sample weight (15 mg), water volume (4 mL), exposure time in the headspace (20 min), and extraction temperature (45 °C). All factors were found significant; however, extraction temperature and exposure time were the most important for the isolation of safranal. The obtained model was successfully validated with a test set of saffron samples analyzed under the optimum extraction conditions. The optimized SPME extraction conditions of safranal found in this study contribute to the efforts towards the detection of saffron authentication and adulteration