Università degli studi G. d’Annunzio
Dottorato in Scienze Biomolecolari e Farmaceutiche Ciclo XXXIII
Universidad de Granada
Facultad de Ciencias
Departamento de Química Analítica Doctorado en Química
PhD Thesis
FOOD CONTAMINANTS DUE TO THERMAL PROCESS
Memoria presentada por SARA PANTALONE
Para optar al grado de:
Doctora en Química por la Universidad de Granada
Dirigida por
Dr. D. Nicola D’Alessandro Dra. Dª. Ana Mª Gómez Caravaca
Granada, 2021
Editor: Universidad de Granada. Tesis Doctorales Autor: Sara Pantalone
ISBN: 978-84-1117-219-6
URI:http://hdl.handle.net/10481/72467
Esta tesis doctoral en cotutela ha sido realizada gracias a una beca (doctorado industrial) financiado por el Ministerio italiano y llevada a cabo entre la Universidad de Granada, la Università “G. D’Annunzio” Chieti-Pescara (Italia) y la empresa Pizzoli SpA.
.
Ai miei genitori Patrizio e Patrizia
Che mi hanno sempre sostenuta
CONTENTS
LIST OF TABLES………...1
LIST OF FIGURES………...5
LIST OF ACRONYMS AND ABBREVIATIONS……...…………..11
SUMMARY/SOMMARIO/RESUMEN...15
OBJECTIVES/OBJETIVOS………...29
INTRODUCTION………...33
1. Thermal process contaminants in food………...35
2.1. Acrylamide………...35
2.2. AA occurrence in food………...36
2.3. Formation of AA in food………...38
2.4. Extraction and determination of AA in food………...40
2.5. AA mitigation strategies in potatoes-base food…...41
3.1. 3-MCPDs, 2-MCPDs and GDs………...42
3.2. Occurrence 3-MCPDs, 2-MCPDs and GDs of in oils...45
3.3. Formation of 3-MCPDs, 2-MCPDs and GDs…...46
3.4. Extraction and determination of 3-MCPDs, 2-MCPDs and GDs………...47
3.5. 3-MCPDs, 2-MCPDs and GDs control and mitigation strategies………...48
4. Mitigation strategies of food thermal processing contaminants based in the use of natural antioxidants………...49
4.1. Rosemary as antioxidant………...51
4.2. Olive and olive oils antioxidant………...…...52
4.3. Tannins as antioxidant………...…………...54
5. References………...58
CHAPTER 1:Review: Acrylamide in widely consumed foods...69
1.1. Introduction...71
1.1.1. AA: general information and chemical-physical
properties………...72
1.1.2. Production and presence of acrylamide in foods...77
1.2. Objective...83
1.3.1. AA toxicology...84
1.3.2. Biomarkers of exposure...87
1.3.3. AA and oxidative stress...88
1.3.4. Neurotoxicity...89
1.3.5. Genotoxicity, cytotoxicity and carcinogenesis...90
1.3.6. Effects on reproduction and development...93
1.4. European legislation on AA...96
1.5. Mitigation strategies for acrylamide in consumer foods...102
1.5.1. Fried potatoes...103
1.5.2. Bread, breakfast cereals and baked goods...110
1.5.3. Coffee and its substitutes...116
1.5.4. Baby foods...120
1.6. Conclusions...122
1.7. References...123
CHAPTER 2: Acrylamide mitigation in processed potato derivatives by addition of natural phenols from olive chain by- products...137
2.1. Introduction...139
2.2. Objective...143
2.3. Materials and Methods...144
2.3.1. Chemicals, reagents and samples...144
2.3.2. Synthesis of hydroxytyrosol...144
2.3.3. Synthesis of hydroxytyrosol acetate and tyrosyl
acetate...145
2.3.4. Determination of AA by GC-MS...146
2.3.5. Colorimetric assays by Agtron instrument...146
2.3.6. Sample model...147
2.3.7. Model system containing phenolic compounds...147
2.3.8. Extraction and derivatization of AA...149
2.3.9. Partition coefficient octanol/water of TyAc...150
2.3.10. Statistical analysis...151
2.4. Results and Discussion...152
2.4.1. Analytical parameters of the AA quantitation method...153
2.4.2. Sample model frying...153
2.4.3 AA mitigation by phenolic compounds addition...153
2.5. Conclusion...159
2.6. References...160
CHAPTER 3:Evaluation of the effects of intermittent fryings in French fries and frying oil on MCPDs, glycidols and acrylamide...167
3.1. Introduction...169
3.2. Objective...170
3.3. Material and Method...171
3.3.1. Standard and reagent...171
3.3.2. Samples...171
3.3.3. Sample preparation...172
3.3.3.1. Extraction of 3-MCPD, 2-MCPD and GD and
derivatization...172
3.3.3.2. Extraction of AA...173
3.3.4. Determination of DAG by GC-FID...173
3.3.5. Determination 3-MCPD, 2-MCPD and GD by GC-MS...174
3.3.6. Determination of AA by UHPLC-QqQ-MS...175
3.3.7. Statistical analyses...176
3.4. Results and Discussion ...177
3.4.1. Evaluation of DAG in oils...177
3.4.2. Determination of 3-MCPD, 2-MCPD and GD in oils...177
3.4.3. Evolution of 3-MCPD, 2-MCPD and GD in oils during frying...179
3.4.4 Evolution of 3-MCPD, 2MCPD and GD in French fries during frying...183
3.4.5 Evolution of AA in French fries during frying...185
3.4.6. Factorial ANOVA Univariate Analysis...187
3.5. Conclusions...189
3.6. References...191
CHAPTER 4:Tannins impregnation pre-treatment as valuable strategy of mitigation of acrylamide, MCPDs and glycidols in fried potatoes...195
4.1. Introduction...197
4.2. Objective...201
4.3. Material and method...202
4.3.1 Standards and reagents...202
4.3.2. Tannins...202
4.3.3. Samples...203
4.3.4. Determination of AA by UPLC-MS/MS...204
4.3.5. Determination of 3-MCPD, 2-MCPD and GD by GC- MS...204
4.3.6. Evaluation of Maillard reactions trend by UV-Vis spectrophotometry...205
4.3.7. Statistical analyses...205
4.4. Result and Discussion...206
4.4.1. Effect of tannins on formation of AA...206
4.4.2. Effect of tannins on Maillard reaction progress...208
4.4.3. Influence of tannins in the content of 3-MCPD, 2-MCPD and glycidol of frying oils and potatoes chips...209
4.4.4. Factorial ANOVA Univariate Analysis...212
4.5. Conclusion...213
4.6. References...214
CHAPTER 5: Evaluation of the effects of rosemary extracts used as pre-treatment and in frying oil in an industrial pilot system on acrylamide and oil degradation...219
5.1. Introduction...221
5.2. Objective...224
5.3. Materia and method...225
5.3.1. Standard and reagent...225
5.3.2. Frying experiments...225
5.3.3. Adding antioxidant to oil...226
5.3.4. French fries quality...226
5.3.4.1. Determination of AA...226
5.3.4.2. Colorimetric assays...227
5.3.5. Oil Quality...228
5.3.5.1. Total Polar Materials...228
5.4. Result and Discussion...229
5.4.1. Antioxidants in oil...231
5.5. Conclusion...235
5.6. References...236
CONCLUSIONS/CONCLUSIÒNES...239
ANNEX...245
Listof Tables
1
LIST OF TABLES
Sara Pantalone
Table 1.1. Acrylamide chemical-physical characteristics, as defined by the Environmental Protection Agency (EPA, 2010)...71
Table 1.2. Regulation of acrylamide levels in food, as defined by EU Regulation N°
2158/2017...97
Table 1.3. Further potential food sources of acrylamide, as defined by the recent EU recommendations (N° 2019/1888)...99
Table 1.4. Process strategies for mitigation of acrylamide in baked goods (Fooddrinkeurope, 2019 and other studies)...111
Table 3.1. MRM transitions to identify AA and AA- d3...176
Table 3.2. Factorial ANOVA (Univariate Results). Column O, significative effect of type of oil; column F, significative effect of number of fryings; column O*F, significative effect of type of oil and number of fryings...189
Table 4.1. Factorial ANOVA (Univariate Results). Column T, significative effect of type of tannin extract; column F, significative effect of number of fryings; column T*F, significative effect of type of tannin extract and number of fryings...215
Table 5.1. Correlation betwen AA in μg/Kg and Agtron value...233
LIST OF FIGURES
Figure 1 AA structure...36
Figure 2. Possible pathway of formation AA...40
Figure 3. Major steps of extraction and determination of acrylamide in processed foods...41
Figure 4. A)3-MCPD structure, B) 2-MCPD structure and C) GD structure...43
Figure 5. Mechanism of formation of 2-MCPD, 3-MCPD and GE, with the
acyloxonium ion as intermediate reaction...47
Figure 6. A) Carnosic acid. B) Carnosol...51
Figure 7. A) Tyrosol. B) Tyrosol acetate. C) Hydroxytyrosol. D) Hydroxytyrosol acetate.
C) Oleuropein...53
Figure 8. A) Structure of catechin monomer (flavan-3-ol). B) General structures of condensed tannins...54
Figure 9. A) Ellagic acid. B) Galic acid...54
Figure 10. A) Galloyl unit. B) Structure of Pentagalloylglucose. C) Structure of HHDP...56
Figure 1.1. Chemical structure of AA....71
Figure 1.2. Formation of AA via the Maillard reaction.
...76
Figure 1.3. AA contents in various food categories (data from EFSA, 2015)...78
Figure 1.4. Relative contributions of foods in the diet in terms of the exposure of the adult population to AA (data from EFSA, 2015)...79
Figure 1.5. Phase I metabolism of AA in the liver. ...83
Figure 1.6. The DNA adduct N7-glycidamide-guanine...89
Figure 1.7. Acceptable and non-acceptable final colour for French fries...104
Figure 1.8. Operational procedures required from food business operators for the control of AA levels in their final products...106
Figure 1.9. Various grades of surface colour of toasted bread...112
Figure 1.10. Colour change of coffee beans according to increasing roasting conditions...115
Figure 2.1. A) Dough of sample mode. B) Raw sample model. C) Simple model after frying...146
Figure 2.2. Structure of phenols used as inhibitors of AA formation in the potato flake model...147
Figure 2.3. Simple model preparade with different concentration of phenolic compound...147
Figure 2.4. Schematized experimental procedure...149
Figure 2.5. AA content in spiked dried potato flake samples with four different phenol concentrations: the different letters (a, b and c) indicate significantly different values (P
< 0.05)...155
Figure 2.6. Comparison between the log KO/W values of the phenols used (A) and the AA inhibition maximum values (B), these last obtained considering the four increasing
concentrations of each phenol. The X axis is the same for both hisograms and lighter bars indicate the orto-diphenols while darker bars indicate the single phenols...157
Figure 3.1. GC-Q-MS (Agilent Technologies) used for the analysis of 3-MCPD, 2- MCPD and GD...175
Figure 3.2. UHPLC-QqQ-MS (Waters Corporation) used for the analysis of AA...177
Figure 3.3. Concentration of 3-MCPD (blue), 2-MCPD (red) and GD (green) in EVOO, ROO, HOSO and RSO before start to fry. In EVOO the contaminant was not find...180
Figure 3.4. Concentration trend of 3-MCPD, 2MCPD and GD along fryings. EVOO is represent by the light green, ROO by the dark green, SOHO by the orange and RSO by the yellow. A) trend of 3-MCPD. B) trend of 2-MCPD. C) trend of GD...183
Figure 3.5. Concentration trend of 3-MCPD, 2MCPD and GD in French fries along fryings. A) trend of 3-MCPD. B) trend of 2-MCPD. C) trend of GD...185
Figure 3.6. Concentration trend of AA in French fries along fryings...188
Figure 4.1. Simple after the first friyng (t0). A) Negative control potatoes chips. B) Potatoes chips pre-treated with C, C) potatoes pre-treated with QBC, D) potatoes pre- treated with UT, E) potatoes pre-treated with U and finally F) potatoes pre-treated T80...207
Figure 4.2. Effect of tannins pre-treatment on the formation of AA in potato chips. The value was reported with its deviation standard. (NAT=untrated potatoes immerged in water; C= tannins C; QBC= tannins QBC; U= tannin U; UT= tannins UT; T80= tannins T80)...210
Figure 4.3. In the graphic is reported the trend of absorbance at 284 nm of potatoes chips pre-treatment with tannins with different structure for each frying. The value was reported with deviation standard...211
Figura 4.4. Average content (µg/kg) of 3-MCPDs, 2-MCPDs and GDs of the 4 fryings repeated in the day for each tannin extract in frying oils (A, C and E, respectively) and potato chips (B, D and F, respectively). Different letters in the same graph mean statistical significant values (p <0.05)...214
Figure 5.1. A) Most popular pre-fried and frozen potato product of Pizzoli S.p.a. B) Potatoes sticks immerged in antioxidant solution...230
Figure 5.2. Agtron E30-FP III………..231
Figure 5.3. On the right the untreated potatoes. On the left the potatoes treated with rosemary extract solutions, in particular, at the top potato treated with INOLENS 4 1g/L, at the bottom potato treated with INOLENS 4 10g/L...234
Figure 5.4. The histogram in red represent the control, it is the average agtron value of 6 fried potatoes without the addition of polyphenols. The average agtron value of 6 French fries previously treated with the same rosemary extract (INOLENS 4) at two different concentrations is shown in blue. By performing the t-test it was found that with both concentrations of rosemary extract was obtained a significant increase, p> 0.05, of the agtron value, therefore a reduction in color, compared to control. While there was no significant difference in to the two concentrations of extract used...235
Figure 5.5. The red line represents the amount of TPC detected by “testo 270” in the control fryer, without the addition of rosemary extracts (SyneROX). The Blue line represents the TPCs detected in the fryer with the addition of rosemary extracts...237
Figure 5.6. The averages of the agtron values of the three fried potatoes made each day of the fried potatoes in the fryer containing rosemary extract (SyneROX) are shown in blue. In red that of the fried potatoes in the deep fryer without adding rosemary extract...238
LIST OF
ACRONYMS AND
ABBREVIATIONS
2-MCPD 3-MCPD 3-MCPD d5 AA
AA d3 BHA BHT b.w.
C
CONTAM panel DAG
EFSA EVOO GC GD GE HVP HHDP HOSO HTy HTyAc IARC LC LOD LOP LOQ MAG MCPDs MCPDE MS NAT QBC
2-monochloropropandiol 3-monochloropropandiol
Deuterate 3-monochloropropandiol d5 Aclylamide
Deuterated Acrylamide d3 Butylated hydroxyanisole Butylated hydroxytoluene Body weight
Ellagitannins from chestnuts
EFSA Panel on Contaminants in the Food Chain Diacylglycerols
European Food Safety Authority Extra virgin olive oil
Gas chromatography Glycidol
Glycidol ester
Acid-hydrolyzed vegetable proteins Hexahydroxydiphenic acid
Hight oleic sunflower oils Hydroxytyrosol
Hydroxytyrosyl acetate
International Agency for Research on Cancer Liquid chromatography
Limit of Detection Lipid Oxidation Product Limit of Quantitation Monoacylglycerols Cloropropandiols
Monocloropropandiols estes Mass spectrometry
Untrated potatoes
Profisetinidine tannins from Schinopsis balansae
ROO RSO SCF T80 TAG TBHQ TDI TPM Ty TyAc USDA U UT
Refined olive oil Refined sunflower oil
Scientific Committee on Food Callotannins from Tara spinose Triacylglycerols
Tert-butylhydroquinone Tolerable daily intake Total Polar Materials Tyrosol
Tyrosyl acetate
United States Department of Agriculture Proanthocyanidins from grape skin Proanthocyanidins from grape seeds
SUMMARY/
RIASSUNTO/
RESUMEN
SUMMARY
The current report encompasses all the results found during the work carried out for the PhD Thesis entitled: Food contaminants due to thermal process.
This PhD project started from the collaboration of three institutions: University G.d'Annunzio of Chieti-Pescara (Italy), University of Granada (Spain), and Pizzoli S.p.a., a food company located in Bologna (Italy). This collaboration made possible to win a National Operative Program (PON) PhD scholarship in 2017. This PhD scholarship was entirely funded by the Italian Ministry of Education, University and Research.
The PhD topic was centered on the study of process contaminants in food, particularly contaminants that are generated during the heat treatments commonly used in various agri-food processes. Concretely, the research has been focused on the study of some food typologies, namely vegetable oils and potatoes-based food, that can contain compounds such as acrylamide (AA), glycidols (GD), together with their esterified form and 2-monochloropropanediols (2-MCPD) and 3-monochloropropanediols (3-MCPD) in their free or esterified form.
AA is considered genotoxic, neurotoxic and a “probable human carcinogen”. It is included in group 2A of the International Agency for Research on Cancer (IARC). The formation of AA occurs when starch-based foods are subjected to temperatures higher than 120 °C in an atmosphere with very low water content.
3-MCPD and GD have been classified as "possible human carcinogenic" (group 2B) and "probably human carcinogenic" (group 2A), respectively. Their formation is also related to high temperatures (> 140 ° C), such as certain steps of lipidic food processing like refining of oils.
The present Doctoral dissertation reports the extraction, the identification and the quantification of these food contaminants in potato-based foods and frying oils. At the same time, different mitigation strategies, consisting in the addition of single phenolic
compounds, tannin extracts or natural rosemary extracts, were tested to reduce the formation of the contaminants during the cooking phases.
The Thesis is divided in two main sections, the first one is the Introduction, which reports a brief description of the food contaminants considered, the occurrence, the formation, the extraction as well as the quantification techniques and the mitigation techniques. Then, an overview of natural extracts used to mitigate the presence of contaminants in food and their industries application, the antioxidant ability of rosemary, the characteristic olive phenols and the classification of tannins was outlined.
The second part includes the Experimental Section and it is divided into five chapters, general structured into six sections: introduction, objectives, materials and methods, results and discussion, conclusions and bibliography.
The first chapter includes a review with the aim of shedding light on the toxicological aspects of the AA, showing its regulatory evolution, and describing the most interesting mitigation techniques for each food category involved, with a focus on compliance with EU legislation in the various classes of consumer products of industrial origin in Europe. This chapter was developed following the reworked of our important bibliographic analysis which could lay the foundations for all the work carried out subsequently.
The second chapter includes the optimization of AA extraction and analysis by GC-MS in a potato-based model system. Besides, an AA mitigation strategy was evaluated, based on the addition of different concentrations of individual phenolic compounds from the olive chain byproducts (tyrosol (Ty), hydroxytyrosol (HTy), tyrosyl acetate (TyAc), hydroxytyrosyl acetate (HTyAc)), to the potato-based model system and, afterwards, to evaluate the AA mitigation after frying process. The experimental work was developed at the chemistry laboratory of the Department of Engineering and Geology of the University d’Annunzio of Chieti-Pescara (Italy).
The third chapter focuses on the evaluation of 3-MCPDs, 2-MCPDs, GD during prolonged frying of frozen pre-fried potatoes in different oils such as extra-virgin olive oil, refined olive oil, high oleic sunflower oil and refined sunflower oil, which differ in
the type of extraction and chemical composition. At the same time, the content of 3- MCPDs, 2-MCPDs, GD in potatoes after every frying was checked as well as the formation of AA using the four different oils. For that purpose, the determination of 3- MCPDs, 2-MCPDs and GD was carried out by GC-MS and the evaluation of AA was done by HPLC-QqQ-MS.
The fourth chapter concerns the use of five natural tannin extracts with different chemical structures (gallotannins, ellagitannins, proanthocyanidins and profisetinidine tannins) to evaluate the possible influence in the mitigation of 3-MCPDs, 2-MCPDs, GD, and AA in oil and potatoes during successive fryings. To that aim, sliced natural potatoes were submerged in aqueous solutions of the tannin extracts as a pre-frying treatment and they were fried in sunflower oil. The determination of 3-MCPDs, 2-MCPDs and GD in frying oils and potatoes was carried out by GC-MS and the AA analyses were done by HPLC-QqQ-MS. Indeed, the trend of the Maillard reaction was checked by measurement of the absorbance at 284 nm.
The third and the fourth chapters were developed during a fourteen months stay at the University of Granada. Thanks to long-term stays, it was possible to apply for the joint research doctoral thesis between the University G.d'Annunzio of Chieti-Pescara (Italy) that will allow obtaining a double PhD degree (PhD in Chemistry and PhD in Biopharmaceutical and Molecular Sciences).
AA is a great problem for food companies; thus, the fifth chapter of the thesis was dedicated to the work carried out entirely in Pizzoli S.p.a (Italy), an Italian company in the fresh and frozen potatoes sector. The goal was to use natural rosemary extracts, as antioxidants, to counteract the formation of AA on potato sticks coming from the industrial production process. Rosemary extracts are naturally resistant to low and high temperatures and, therefore, ideal for application in the industrial sector. Potatoes were treated with the rosemary extract at different concentrations and, after that, they were frozen to simulate the entire industrial process. Rosemary extract was also added directly to the frying oil to assess whether there was a reduction in the degradation of the oil, normally due to high temperatures, and to check if there was a decrease in the formation
of AA by the color evaluation of cooked French fries with Agtron, a spectrophotometer that allows to perform analysis on the sample as it is, without the need for processing.
RIASSUNTO
Il presente riassunto racchiude tutti i risultati riscontrati durante il lavoro svolto per la Tesi di Dottorato intitolata: Food contaminants due to thermal process.
Questo progetto di dottorato è nato dalla collaborazione di tre istituzioni:
Università G. d'Annunzio di Chieti-Pescara (Italia), Università di Granada (Spagna) e Pizzoli S.p.a., un'azienda alimentare con sede a Bologna (Italia). Questa collaborazione ha permesso di vincere nel 2018 una borsa di dottorato del Programma Operativo Nazionale (PON). Tale borsa di dottorato è stata interamente finanziata dal Ministero dell'Istruzione, dell'Università e della Ricerca.
L'argomento del dottorato è stato incentrato sullo studio dei contaminanti di processo negli alimenti, in particolare dei contaminanti che si generano durante i trattamenti termici comunemente utilizzati nei vari processi agroalimentari.
Concretamente, la ricerca si è focalizzata sullo studio di alcune tipologie di alimenti, ovvero oli vegetali e alimenti a base di patate, che possono contenere composti come l'acrilammide (AA), i glicidoli (GD), insieme alla loro forma esterificata e 2- monocloropropandioli (2-MCPD) e 3-monocloropropandioli (3-MCPD) nella loro forma libera o esterificata.
L'AA è considerata genotossica, neurotossica e un "probabile cancerogeno per l'uomo". È incluso nel gruppo 2A dell'Agenzia internazionale per la ricerca sul cancro (IARC). La formazione di AA si verifica quando gli alimenti a base di amido sono sottoposti a temperature superiori a 120 °C in un'ambiente con contenuto d'acqua molto basso.
3-MCPD e GD sono stati classificati rispettivamente come "possibile cancerogeno per l'uomo" (gruppo 2B) e "probabilmente cancerogeno per l'uomo" (gruppo 2A). La loro formazione è correlata anche alle alte temperature (>140 °C), come ad esempio alcune fasi della lavorazione dei grassi, come la raffinazione degli oli.
La presente Tesi di Dottorato descrive l'estrazione, l'identificazione e la quantificazione di questi contaminanti alimentari negli alimenti a base di patate e negli oli di frittura. Allo stesso tempo, sono state sperimentate diverse strategie di mitigazione, consistenti nell'aggiunta di singoli composti fenolici, estratti di tannino o estratti naturali di rosmarino, per ridurre la formazione dei contaminanti durante le fasi di cottura.
La Tesi è divisa in due sezioni principali, la prima è l'Introduzione, che riporta una breve descrizione dei contaminanti alimentari considerati, l’incidenza, la formazione, l'estrazione nonché le tecniche di quantificazione e mitigazione. Quindi, è stata delineata una panoramica degli estratti naturali utilizzati per mitigare la presenza di contaminanti negli alimenti e la loro applicazione nelle industrie, la capacità antiossidante del rosmarino, i caratteristici fenoli dell'oliva e la classificazione dei tannini.
La seconda parte comprende la Sezione Sperimentale ed è suddivisa in cinque capitoli, strutturati in generale in sei sezioni: introduzione, obiettivi, materiali e metodi, risultati e discussione, conclusioni e bibliografia. Il primo capitolo è una rielaborazione bibliografica, si articola in sette sezioni: introduzione, obiettivi, conclusioni e bibliografia, e le altre tre sezioni per gli argomenti di interesse trattati.
Il primo capitolo comprende una review con l'obiettivo di fare luce sugli aspetti tossicologici dell'AA, mostrandone l'evoluzione normativa, e descrivendo le tecniche di mitigazione più interessanti per ciascuna categoria alimentare coinvolta, con un focus sul rispetto della normativa comunitaria nelle varie classi dei prodotti di consumo di origine industriale in Europa. Questo capitolo è stato sviluppato a seguito della rielaborazione di una importante analisi bibliografica che è servita per gettare le basi per tutto il lavoro svolto successivamente.
Il secondo capitolo include l'ottimizzazione dell'estrazione e dell'analisi dell'AA mediante GC-MS in un sistema modello basato sulla patata. Inoltre, è stata valutata una strategia di mitigazione dell'AA, basata sull'aggiunta di diverse concentrazioni di singoli composti fenolici dai sottoprodotti della filiera della lavorazione delle olive (tirosolo (Ty), idrossitirosolo (HTy), tirosil acetato (TyAc), idrossitirosil acetato (HTyAc)) nel sistema modello basato sulla patata, per valutare la mitigazione dell'AA dopo il processo
di frittura. Il lavoro sperimentale è stato sviluppato presso i laboratori dell'Università di Chieti-Pescara (Italia).
Il terzo capitolo si concentra sulla valutazione di 3-MCPD, 2-MCPD e GD durante la frittura prolungata di patate pre-fritte congelate in diversi oli come olio extravergine di oliva, olio di oliva raffinato, olio di girasole alto oleico e olio di girasole raffinato, che differiscono per tipo di estrazione e composizione chimica. Allo stesso tempo, è stato controllato il contenuto di 3-MCPD, 2-MCPD e GD nelle patate dopo ogni frittura, nonché la formazione di AA utilizzando i quattro diversi oli. A tal fine, la determinazione di 3-MCPD, 2-MCPD e GD è stata effettuata mediante GC-MS e la valutazione di AA è stata effettuata mediante HPLC-QqQ-MS.
Il quarto capitolo riguarda l'uso di cinque estratti naturali di tannino con diverse strutture chimiche (gallotannini, ellagitannini, proantocianidine e tannini profisetinidinici) per valutare la possibile influenza nella mitigazione di 3-MCPD, 2- MCPD, GD e AA in olio e patate durante fritture successive. A tal fine, le patate al naturale affettate sono state immerse in soluzioni acquose di estratti di tannino come trattamento di pre-frittura e fritte in olio di semi di girasole. La determinazione di 3- MCPD, 2-MCPD e GD negli oli per friggere e nelle patate è stata effettuata mediante GC.MS e le analisi AA sono state eseguite mediante HPLC-QqQ-MS. L'andamento della reazione di Maillard è stato infatti verificato misurando l'assorbanza a 284 nm.
Il terzo e il quarto capitolo sono stati sviluppati durante un soggiorno di quattordici mesi all'Università di Granada. Grazie ai soggiorni di lunga durata, è stato possibile presentare domanda per la tesi di dottorato di ricerca congiunta tra l'Università G.d'Annunzio di Chieti-Pescara (Italia) che consentirà di ottenere un doppio titolo di dottorato (Dottorato in Chimica e Dottorato in Biofarmaceutica e Scienze Molecolari).
L'AA è un grande problema per le aziende alimentari, quindi il quinto capitolo della tesi è stato dedicato al lavoro svolto interamente in Pizzoli S.p.a (Italia), azienda italiana leader nel settore della lavorazione delle patate. L'obiettivo era quello di utilizzare estratti naturali di rosmarino, come antiossidanti, per contrastare la formazione di AA sui
bastoncini di patate provenienti dal processo di produzione industriale. Gli estratti di rosmarino sono naturalmente resistenti alle basse e alle alte temperature e, quindi, ideali per l'applicazione nel settore industriale. Le patate sono state trattate con l'estratto di rosmarino a diverse concentrazioni e, successivamente, sono state congelate per simulare l'intero processo industriale. L'estratto di rosmarino è stato aggiunto anche direttamente all'olio di frittura per valutare se c'era una riduzione della degradazione dell'olio, normalmente dovuta alle alte temperature, e per verificare se c'era una diminuzione della formazione di AA dalla valutazione del colore del francese cotto frigge con Agtron, uno spettrofotometro che permette di eseguire analisi sul campione così com'è, senza necessità di elaborazione.
RESUMEN
La presente memoria recoge todos los resultados encontrados durante el trabajo realizado en la Tesis Doctoral titulada: Food contaminants due to thermal process.
Este proyecto de doctorado partió de la colaboración de tres instituciones:
Universidad G.d'Annunzio de Chieti-Pescara (Italia), Universidad de Granada (España) y Pizzoli S.p.a., una empresa de alimentación ubicada en Bologna (Italia). Esta colaboración hizo posible ganar una beca de doctorado del Programa Operativo Nacional (PON) en 2017. Esta beca de doctorado fue financiada en su totalidad por el Ministerio de Educación, Universidad e Investigación de Italia.
El tema del doctorado se centró en el estudio de los contaminantes de proceso en los alimentos, en particular los contaminantes que se generan durante los tratamientos térmicos comúnmente utilizados en diversos procesos agroalimentarios. Concretamente, la investigación se ha centrado en el estudio de algunas tipologías de alimentos, como los aceites vegetales y alimentos a base de patatas, que pueden contener compuestos como acrilamida (AA), glicidoles (GD), junto con su forma esterificada y 2- monocloropropanodioles (2-MCPD) y 3-monocloropropanodioles (3-MCPD) en su forma libre o esterificada.
La AA se considera genotóxica, neurotóxica y un "probable carcinógeno humano". Se incluye en el grupo 2A de la Agencia Internacional para la Investigación del Cáncer (IARC). La formación de AA se produce cuando los alimentos a base de almidón se someten a temperaturas superiores a 120 °C en una atmósfera con muy bajo contenido de agua.
3-MCPD y GD se han clasificado como "posible cancerígeno humano" (grupo 2B) y "probablemente cancerígeno humano" (grupo 2A), respectivamente. Su formación también está relacionada con altas temperaturas (> 140 °C), y con ciertos pasos del procesado de alimentos lipídicos como el refinado de los aceites.
La presente tesis doctoral estudia la extracción, identificación y cuantificación de estos contaminantes alimentarios en alimentos a base de patata y aceites de fritura. Al mismo tiempo, se probaron diferentes estrategias de mitigación, que consisten en la adición de compuestos fenólicos simples, extractos de taninos o extractos naturales de romero, para reducir la formación de contaminantes durante las fases de cocinado.
La tesis se divide en dos secciones principales, la primera es la Introducción, la cual reporta una breve descripción de los contaminantes alimentarios considerados, la ocurrencia, la formación, la extracción así como las técnicas de cuantificación y las técnicas de mitigación. A continuación, se realizó un resumen de los diferentes extractos naturales utilizados para mitigar la presencia de contaminantes en los alimentos y su aplicación industrial, la capacidad antioxidante del romero, los fenoles característicos del olivo y la clasificación de los taninos.
La segunda parte incluye la Sección Experimental y está dividida en cinco capítulos, de carácter general estructurado en seis apartados: introducción, objetivos, materiales y métodos, resultados y discusión, conclusiones y bibliografía.
El primer capítulo incluye una revisión con el objetivo de arrojar luz sobre los aspectos toxicológicos de la AA, mostrar cómo ha evolucionado la normativa al respecto de este contaminante y describir las técnicas de mitigación más interesantes para cada categoría de alimentos involucrada, con un enfoque en el cumplimiento de la legislación de la UE en las distintas clases de productos de consumo de origen industrial en Europa.
Este capítulo se desarrolló a raíz de un importante análisis bibliográfico que pudiera sentar las bases de todo el trabajo realizado posteriormente.
El segundo capítulo incluye la optimización de la extracción y el análisis de AA por GC-MS en un sistema de modelo a base de patata. Además, se evaluó una estrategia de mitigación de AA, basada en la adición de diferentes concentraciones de compuestos fenólicos individuales provenientes de subproductos de la cadena del olivo (tirosol (Ty), hidroxitirosol (HTy), tirosol acetato (TyAc) e hidroxitirosol acetato (HTyAc)), sobre el sistema modelo y, posteriormente, evaluar la mitigación de la AA después del proceso de fritura. El trabajo experimental se desarrolló en los laboratorios de la Universidad de Chieti-Pescara (Italia).
El tercer capítulo se centra en la evaluación de 3-MCPD, 2-MCPD y GD durante la fritura prolongada de patatas congeladas prefritas en diferentes aceites como aceite de oliva virgen extra, aceite de oliva refinado, aceite de girasol alto oleico y aceite de girasol refinado, que difieren en el tipo de extracción y en su composición química. Al mismo tiempo, se verificó el contenido de 3-MCPD, 2-MCPD y GD en las patatas después de cada fritura, así como la formación de AA utilizando los cuatro aceites. Para ello, la determinación de 3-MCPDs, 2-MCPDs y GDs se realizó mediante GC-MS y la evaluación de AA se realizó mediante HPLC-QqQ-MS.
El cuarto capítulo se refiere al uso de cinco extractos de taninos naturales con diferentes estructuras químicas (galotaninos, elagitaninos, proantocianidinas y taninos de profisetinidina) para evaluar la posible influencia en la mitigación de 3-MCPD, 2-MCPD, GD y AA en aceite y patatas durante sucesivas frituras. Para ello, se sumergieron patatas naturales en rodajas en soluciones acuosas de los extractos de taninos como tratamiento previo a la fritura y se llevó a cabo la fritura en aceite de girasol. La determinación de 3- MCPD, 2-MCPD y GD en aceites de fritura y patatas se llevó a cabo mediante GC.MS y los análisis de AA se realizaron mediante HPLC-QqQ-MS. De hecho, la tendencia de la reacción de Maillard se comprobó midiendo la absorbancia a 284 nm.
El tercer y cuarto capítulos se desarrollaron durante una estancia de catorce meses en la Universidad de Granada. Gracias a esta estancia de larga duración, fue posible realizar el doctorado en cotutela entre la Universidad de Granada y la Universidad G.d'Annunzio de Chieti-Pescara (Italia) que permitirá obtener un doble doctorado (PhD en Química y PhD en Biofarmacéutica y Ciencias Moleculares).
La AA es un gran problema para las empresas alimentarias, por ello, el quinto capítulo de la tesis está dedicado al trabajo realizado íntegramente en Pizzoli S.p.a (Italia), empresa italiana líder en el sector de procesado de patatas. El objetivo fue utilizar extractos naturales de romero, como antioxidantes, para contrarrestar la formación de AA en las patatas fritas provenientes del proceso de producción industrial. Los extractos de romero son naturalmente resistentes a bajas y altas temperaturas y, por tanto, ideales para su aplicación en el sector industrial. Las patatas se trataron con el extracto de romero a
diferentes concentraciones y, posteriormente, se congelaron para simular todo el proceso industrial. El extracto de romero también se añadió directamente al aceite de freír para evaluar si había una reducción en la degradación del aceite, normalmente debido a las altas temperaturas, y para comprobar si había una disminución en la formación de AA mediante la evaluación del color de las patatas fritas con Agtron, un espectrofotómetro que permite realizar análisis sobre la muestra tal cual, sin necesidad de procesamiento.
OBJECTIVES
The main objective of this thesis was to use different chromatographic techniques that allow the development of methods for the determination of food contaminants such as AA, GD, 3-MCPD and 2-MCPD in different vegetable oils and potatoes and to evaluate different mitigation strategies of these contaminants during frying process.
To reach this global objective, different specific objectives have been established:
1. To identify and quantify AA using different chromatographic techniques such as gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled to mass spectrometry (LC-MS).
2. To identify and quantify GD, 3-MCPDs and 2-MCPDs by GC-MS.
3. To evaluate the best vegetable oil (between extra virgin olive oil, refined olive oil, high oleic sunflower oil and refined sunflower oil) to fry frozen pre-fried potatoes in terms of the concentrations of GD, 3-MCPDs and 2-MCPDs in frying oils and fried potatoes and AA in fried potatoes when multiple and successive fryings are carried out.
4. To check the effect of the use of single phenolic compounds from olive oil by- products on the formation of AA in a potato-based model system after frying with high oleic sunflower oil.
5. To test the possible mitigation effect of GD, 3-MCPDs, 2-MCPDs in fried potatoes and frying oils as well as AA in fried potatoes thanks to the addition of different tannin extracts to submerge potatoes before frying in high oleic sunflower oil.
6. To evaluate the possible reduction of AA formation after the treatment of potatoes with phenolic rosemary extracts and fried with high oleic sunflower oil.
7. To improve the technology transfer between University and industry.
OBJETIVOS
El objetivo principal de esta tesis fue utilizar diferentes técnicas cromatográficas que permitan el desarrollo de métodos para la determinación de contaminantes alimentarios como AA, GD, 3-MCPD y 2-MCPD en diferentes aceites vegetales y patatas y evaluar diferentes estrategias de mitigación de estos contaminantes durante el proceso de fritura.
Para alcanzar este objetivo global se han establecido diferentes objetivos específicos:
1. Identificar y cuantificar AA mediante diferentes técnicas cromatográficas como la cromatografía de gases acoplada a espectrometría de masas (GC-MS) y la cromatografía líquida acoplada a espectrometría de masas (LC-MS).
2. Identificar y cuantificar GD, 3-MCPD y 2-MCPD por GC-MS.
3. Evaluar el mejor aceite vegetal (entre aceite de oliva virgen extra, aceite de oliva refinado, aceite de girasol alto oleico y aceite de girasol refinado) para freír patatas prefritas congeladas en cuanto a las concentraciones de GD, 3-MCPD y 2-MCPD en aceites para freír y patatas fritas y AA en patatas fritas cuando se realizan frituras múltiples y sucesivas.
4. Verificar el efecto del uso de compuestos fenólicos simples de subproductos del aceite de oliva sobre la formación de AA en un sistema modelo a base de patata después de freír con aceite de girasol alto oleico.
5. Probar el posible efecto mitigador de GD, 3-MCPD, 2-MCPD en patatas fritas y aceites de fritura, así como AA en patatas fritas gracias a la adición de diferentes extractos de taninos para sumergir las patatas antes de freírlas en aceite de girasol alto oleico.
6. Evaluar la posible reducción de la formación de AA tras el tratamiento de patatas con extractos fenólicos de romero y fritas con aceite de girasol alto oleico.
7. Mejorar la transferencia de tecnología entre la Universidad y la industria.
INTRODUCTION
Food contaminants due to thermal process
1. Thermal process contaminants in food
Thermal process contaminants are substances that are formed after food thermal processing such as refining, drying, smoking, fermentation and some cooking methods, including baking, grilling and frying (Alexander et al., 2012). These processes are essential to make food edible, as well as to confer the typical food characteristics such as color, flavor and texture; thus, the formation of contaminants is unavoidable. Besides, these thermal processes can cause chemical changes that lead, not only to the loss of vitamins and other micronutrients, but to the formation of potential toxic substances that can create adverse effects on human health (Alexander et al., 2012). This category of substances includes AA (prop-2-enamide) and chlorpropandiols and their esters. In the last years, these substances have been studied extensively to try to understand the formation, the potential risk to health as well as to put in place methods for analysis and mitigation techniques, as they are subjected to controls and legislative limitations (EFSA 2013; EFSA 2015).
Therefore, the thesis has been focused on the study of potatoes-based food potentially contaminated with these compounds: AA, GD (2,3-Epoxy-1-propanol) or glycidol ester (GE), 2-MCPDs (2-chloro-1,2-propanediol), 3-MCPDs (3-chloro-1,2- propanediol) and their respective esters of fatty acids.
2.1. Acrylamide
AA is a white, odourless, crystalline, water-soluble solid (Figure 1A). AA is a potent neurotoxin that affects male reproduction, causes birth defects, is carcinogenic in laboratory animals (EFSA, 2015) and it has been classified as a probably carcinogenic to humans (group 2A) by the International Agency for Research on Cancer (IARC, 1994).
It is readily absorbed through the skin, by inhalation and by the gastro-intestinal tract.
AA in food was discovered in 2002 and was quickly found in a large number of foods cooked in low humidity condition (Lofsted et al., 2003). AA is a substance that forms in certain products, particularly in plant-based rich in saccharides, during cooking at high temperatures (frying, oven, grill) (Becalski et al., 2011). Investigations conducted by European Food Safety Authority (EFSA) on samples of various foods such as biscuits,
bread, breakfast cereals, coffee and its derivatives, potatoes, etc., showed a high concentration of AA in each of them (EFSA, 2013). AA is formed when asparagine, a natural amino acid, reacts with the saccharides present in foods through the glycation reaction (Mottram et al., 2002; Halford et al., 2010). Once ingested, AA is absorbed from the gastrointestinal tract, distributed to all organs and metabolized. Glycidamide (Figure 1B) is a major metabolite and is the likely cause of genetic mutations and cancers found in animal studies (Mei et al., 2010). In 2013, EFSA published its first comprehensive risk assessment of AA in food, establishing that this contaminant in food can increase the risk of cancer in consumers of all age groups (EFSA, 2013). In 2015, EFSA published a recommendation indicating the threshold values to be taken into due consideration for the different categories of foods (EFSA, 2015).
Figure 1 A) AA structure. B) Glycidamide structure.
2.2. AA occurrence in food
AA is a process contaminant, a substance harmful to health, as above mentioned, not initially contained in the food matrix, which is generated during heat treatments such as frying, roasting, and baking (Halford et al., 2012; Curtis et al., 2013). AA can be formed starting from molecules naturally present in food, concern in particular fried products based on potatoes, like fried potatoes, baked goods, roasted coffee, snacks and infant food (Gokmen, 2015).
Data on the presence of AA in food has been collected in Europe since 2003, first by the European Commission Joint Research Centre's Institute for Reference Materials and Measurements and, since 2007, by Member States, who have supplied data to be compiled by the EFSA. The data had been analyzed and published in a series of extremely important
reports (EFSA, 2009; EFSA, 2010; EFSA, 2011; EFSA, 2015), that informed the development of what the Commission calls its “risk management measures for AA. The highest levels of AA were found in vegetable chips and coffee substitutes: the average for chicory-based coffee substitutes was 2.942 μg/kg. Other popular foods, such as potato chips, French fries, breakfast cereals, biscuits, and rusks, had average values of hundreds of μg/kg. The 2011 and 2015 reports (EFSA, 2015; EFSA, 2011) also included estimates of the contribution of different foods to dietary intake in different Member States of the European Union; in fact, the contribution of a type of food depends on its levels of consumption as well as its AA content. For example, the highest AA content is present in vegetable chips with an average AA content of 1846 μg/kg, while bread has an average AA content < 50 μg/kg. Despite this, the daily intake of AA coming from bread is higher than that coming from vegetable chips, since bread is one of the most consumed foods in the world, while vegetable chips are consumed by a relatively small number of people and not everyday (EFSA, 2015).
Between 2007 and 2013 the European Union issued three recommendations about the presence of AA in food, based on a series of EFSA reports resulting from the analysis of data provided by Member States and food industries. In 2007, the European Commission issued Recommendation 2007/331/EU on monitoring AA levels in food in 2007, 2008 and 2009.
In 2010, the Commission Recommendation 2010/307/EU (European Commission, 2010) required Member States to continue collecting data on AA levels in food until further notice. EFSA report (EFSA, 2010), covering the period up to 2008, affirmed that a trend towards lower AA values was observed, even if it was limited to certain food typology; in particular the product categories ‘potato crisps’, ‘instant coffee’
and ‘substitute coffee’ showed statistically significantly higher levels of AA, at least compared with those of the year before (2007). On the other hand, ‘French fries’ and
‘fried potato products for home cooking’, ‘soft bread’, ‘bread not specified’, ‘infant biscuit’, ‘biscuit not specified’, ‘muesli and porridge’ and ‘other products not specified’
showed statistically significantly lower levels of AA (again vs 2007 data). This led to the Commission Regulation to issue a new regulation in 2010/2011 (n° 9681), which introduced the concept of guideline values for AA in food where it is requested to the Member State competent authorities to verify the AA contents.
In 2013 the Commission issued Recommendation 2013/647/EU (European Commission, 2013), which revised and, in many cases, reduced the Indicative Values.
Only in 2017 the European Commission issued a regulation (EU) 2017/2158 (European Commission, 2017) which entered into force on 11 April 2018. Regulation (EU) 2017/2158 used stronger language, stating that the panel's assessment "confirmed previous evaluations that AA in food potentially increases the risk of developing cancer for consumers in all age groups”. The regulation replaced guideline values with reference levels, which were below the corresponding guideline value for almost all types of product. This regulation also indicates some techniques to be adopted in companies to obtain the lowest desirable amount of AA.
In 2019 the European Commission publishes the latest recommendations (2019/1888 EU), which refer to regulation 2158/2017, regarding the monitoring of some foods not yet fully considered and which could contribute also significantly to the exposure of the population to the contaminant; for example for potato-based products:
duchess potato croquettes, potato and meat pie and potato and cheese pie; for baked goods: hamburger buns, croissants, fried donuts and pancakes; for cereal-based products:
rice or corn-based crackers. Also, other foods such as vegetable chips, nuts, roasted cocoa beans and coffee substitutes.
2.3. Formation of AA in food
After the discovery of the presence of AA in widely consumed foods (Tareke et al., 2002), it was reported that AA could be formed through the glycation reaction from free asparagine and monosaccharides, D-glucose ((3R,4S,5S,6R)-6-(hydroxymethyl) oxane-2,3,4,5-tetrol) and D-fructose (3S,4R,5R)-2-(hydroxymethyl)oxane-2,3,4,5-tetrol) (Stadler et al., 2002).
The glycation reaction involves a first phase of glycosylation in which the carbonyl group of the monosaccharides reacts with the amino group of the amino acid, leading to the formation of a Schiff base, a glycosylamine. The Schiff base is a key intermediate, which can lead to the formation of AA following 3 distinct pathways. The Schiff base is unstable and can reorganize itself in the so-called Amadori compounds, if the starting monosaccharides is an aldose (D-glucose), or in the Heyns-Carlson
compounds, if the starting monosaccharides is a ketose (D-fructose) (Stadler et al., 2002).
The reaction is influenced by pH and temperature: at high temperatures the Amadori compounds can enolyze into dicarbonyl compounds, which react with amino acids, if the amino acid is asparagine, oxidative demination and decarboxylation are obtained, which lead to the formation of Strecker and 3-aminopropionamide aldehydes, which can directly lose an ammonia molecule and form AA (Granvogl et al., 2004). AA can also be formed from Streker's aldehyde (Zyzak et al., 2003). Alternatively, the Schiff base may be apt to make decarboxylation either directly via the Schiff betaine or via the intermediary oxazolidine-5-one to generate the azomethine ylide, which furnishes the decarboxylated Amadori product after tautomerization (Yaylayan et al., 2003). The pathway is summarized in Figure 2 (Pantalone et al., 2021).
In the last step of the reaction, the aldehyde reacts with the free asparagine and, therefore, AA is formed. Some intermediate reactions, in turn, produce melanoidins that give flavor and aroma to food as we know them (Halford et al., 2011; Mottram, 2007).
This means also that the measures reducing the formation of AA can influence the characteristics that define the type of product and that allow to distinguish one brand from another, making the problem even more difficult to deal.
The fact that colour results from the AA-like pathways means that there is usually a strong correlation between the AA formation and food colour, at least before the caramelization will become dominant (at very high temperature). Therefore, colour can be used as an indicator of AA formation and has become an important quality control parameter (Food Standards Agency, 2017).
There are other minor pathways by which AA is formed in food. Complete information regarding the different pathways for the formation of AA is not yet fully known because food matrices are often much more complex systems than model systems (Claus et al., 2006; Perez Locas & Yaylayan, 2008; Gokmen, 2015).
Figure 2 Possible pathway of formation AA (Pantalone et al.,2021).
2.4. Extraction and determination of AA in food
AA is a compound having a molecular weight of 71 Da. It has a melting point of 84.5 ° C and a high boiling point (136 ° C to 3.3 kPa). AA is stable at room temperature but undergoes violent polymerization at its melting point or following exposure to UV rays. It is soluble in water and other polar solvents such as acetone, ethanol and methanol (Matoso et al., 2019).
Rosén and Hellenäs first reported the analysis of AA (Rosén & Hellenäs, 2002).
In general, the extraction of AA involves a phase of homogenization of the heat-treated food to be analyzed, the addition of the internal standard, degreasing, extraction, and purification (Figure 3). The analytical methods are mainly based on mass spectrometry (MS) as a key technique for a better identification of AA in processed foods, coupled with a chromatographic phase by LC (Morales et al., 2014) or by GC, this can only be performed after derivatization of the analyte (Zokaei et al., 2017).
Figure 3. Major steps of extraction and determination of AA in processed foods.
2.5. AA mitigation strategies in potatoes-base food
Several methods have now been suggested to reduce AA formation. Some are not real methods, rather the precautions that the manufacturing companies must keep under control to try to minimize the formation of AA; these precautions are reported in the AA Toolbox by the Food and Drink Europe Federation, and in the regulation (EU) 2158/2017.
Others are optional techniques, mainly focused on the use of additives that interfere with the formation of AA and reduce its formation. Actually, these are the techniques mainly
studied by researchers. The precautions proposed by the regulation (EU) 2158/2017 in the potato industry are related above all to a strategic choice of raw material, that is to use potatoes that naturally have lower amounts of amino acids and monosaccharides; in this way, the lower quantity of precursors will produce lower quantity of AA. This can be achieved thanks to the choice of cultivars and of fertilizers that allow a lower development of precursor of AA during the growth of the tubers (Sun et al., 2020). For example, the correct use of nitrogen and sulfur influences the formation of asparagine and other amino acids (Muttucumaru et al., 2013). Also the conservation of raw materials can affect the amount of AA precursors in a manner dependent on the temperature and the duration.
Storing potatoes at temperatures above 8°C allows to keep low the concentration of monosaccharides s; in fact, a 10 times lower quantity of AA has been observed in fried potatoes obtained from potatoes stored at 4-8°C (Sun et al., 2020). Another aspect to consider is the frying temperature. Given that the formation of AA is directly dependent from the increase in temperature, one possibility could be to fry the potatoes at a maximum temperature of 175°C (Fooddrinkeurope, 2019).
Between the other techniques reported that interfere with the glycation reaction and reduce the formation of AA, there are the addition of antioxidants extract and different phenolic compounds. Immerging potato slices in different solutions of proanthocyanidins at several concentrations, a maximal inhibition rate of 44.2% was observed (Qi et al., 2018). Furthermore, also immerging them in extracts solutions of oregano, thyme, cinnamon, a reduction on the formation of AA in fried potatoes bougainvillea and green tea, was obtained. The extracts from green tea, cinnamon and oregano reduced the AA level by 62%, 39% and 17%, respectively (Morales et al., 2014).
Using muscardine grape pomace extracts on potato chips model, a reduction of 60.3% of AA was obtained (Xu et al., 2015). So, we can conclude that pre-treating potatoes with antioxidants before frying produces beneficial effects such as a reduction in AA content.
3.1. 3-MCPDs, 2-MCPDs and GD
Another family of food contaminants reported by EFSA are fatty acid ester of GD and 3-MCPD and 2-MCPD, known as chloropropanols (EFSA, 2013). 3-MCPD has been categorized as ‘‘possible human carcinogens’’ (group 2B) by the IARC in 2012. GD has
been categorized “probably carcinogenic to humans’’ (group 2A) in 2000 by the IARC.
The molecular structure of 3-MCPD (Figure 4A) and 2-MCPD (Figure 4B) presents two alcohol groups and one chlorine in 3- and 2-position, respectively. GD (Figure 4C) is a glycerol molecule with an epoxide and an alcohol functional group. The responsible for their formation is the high temperature, above all, during the refining steps, often necessary for some lipid foods, which leads to the formation of the esterified forms of monochloropropanediols (MCPDE) and GE. It is possible to find these molecules in food both in their free and esterified form. MCPDE and GE are substantially hydrolysed to their free forms in the gastrointestinal tract and provoke toxicity as MCPDs and GD, respectively. The EFSA, in two studies published in 2013 and 2016 on 3-MCPD, showed that toxicity is the same in the esterified form as in the free form (EFSA, 2013; EFSA, 2016). Currently, high levels of GE and MCPDEs have be found especially in food containing palm oil, even if the problem is more extended since also food containing other type of vegetable fats, can contain high amounts of both GE and 3-MCPD, as reported by EFSA that evidenced a mean value of 4750 μg/kg of 3-MCPD in walnut oil while margarine showed a mean value of 1500 μg/kg of 3-MCPD (EFSA, 2013).
Figure 4 A)3-MCPD structure, B) 2-MCPD structure and C) GD structure.
In 2001 the Scientific Committee on Food (SCF) established the tolerable daily intake (TDI) of 2 µg/kg body weight (b.w.) for 3-MCPD, declaring it a non-genotoxic carcinogen (SCF, 2001). JECFA established a provisional maximum tolerable daily intake (PMTDI) of 2 µg/kg b.w. (JECFA, 2002, 2007). The European commission in the Regulation (EU) N. 1881/2006, which established the levels of numerous food contaminants, established that the maximum level of 3-MCPD in HVP and soy sauce was 20 µg/kg; while, the provisions for sampling and analysis for the official control of 3- MCPD levels are defined in Regulation (EU) no. 333/2007 of the Commission. Specific
methods are not listed in this regulation, but rather some criteria that must be met, such as Limit of Detection (LOD) and Limit of Quantitation (LOQ) of 5 and 10 µg/kg.
In 2013, EFSA published a report on the presence of 3-MCPD in food based on data collected in European Member States from 2009 to 2011, as well as a preliminary exposure assessment. European Commission issues a recommendation in 2014 (2014/661/EU) with the aim to obtain more data on the presence of 2- and 3-MCPD, 3- and 2-MCPD esters of fatty acids and GE in food, and a more accurate assessment of exposure. The Recommendation specified the type of food to be analyzed, the sampling procedures and the analytical methods.
Following a request from the European Commission, CONTAM panel published in 2016 the first scientific opinion on the risk for human health related the presence of 3- MCPD, 2-MCPD and their fatty acid ester, and GD fatty acid esters in food. CONTAM Panel established a TDI of 0.8 µg/kg b.w. per day for 3-MCPD, but not for 2-MCPD and GD. The same year the Joint FAO/WHO Expert Committee on Food Additives (JECFA) established a TDI for 3-MCPD of 0,4 µg/kg b.w. per day. For this reason, on 2018 the CONTAM Panel published an update of its scientific opinion on 3-MCPD, in particular focusing on developmental and reproductive toxicity. (EFSA, 2016) After the assessments, the CONTAM Panel derived a TDI of 2 µg/kg b.w. per day for 3-MCPD.
On 2018 the European Commission published the Regulation (EU) N. 2018/290 amending Regulation (EU) N. 1881/2006 as regards maximum levels of glycidyl fatty acid esters in vegetable oils and fats, infant formula, follow-on formula and foods for special medical purposes intended for infants and young children. Compared to the 2006 regulation, is established the limit of 1000 µg/kg for vegetable oils and fats placed on the market for the final consumer or for use as an ingredient in food for GD and GE; 500 µg/kg for vegetable oils and fats destined for the production of baby food and processed cereal-based food for infants and young children; 50 µg/kg for infant formula, follow-on formula and foods for special medical purposes intended for infants and young children powder; and 6.0 µg/kg for infant formula, follow-on formula and foods for special medical purposes intended for infants and young children liquid. On September 2020 the European Commission published the Regulation (EU) N. 2020/1322 amending Regulation (EU) N. 1881/2006 as regards maximum levels of 3-MCPD, 3-MCPD fatty acid esters and glycidyl fatty acid esters in certain foods. The new limits concern in
particular the sum of 3-MCPD and 3-MCPD fatty acid esters, expressed as 3-MCPD. This cannot exceed the limit of 1250 µg/kg in oils and fat from coconut, maize, rapeseed, sunflowers, soybean, palm kernel, olive oil and the mixtures of oils and fat from this category; 2500 µg/kg in pomace olive oils, fish oils and oils from other marine organisms and mixtures of oils and fats from this category, and the mixtures of oils and fats from the two categories; 750 µg/kg in vegetable oils and fats, fish oils and oils from other marine organisms destined for the production of baby food and processed cereal-based food for infants and young children; 125 µg/kg in infant formula, follow-on formula and foods for special medical purposes intended for infants and young children and young- child formula powder; 15 µg/kg for infant formula, follow-on formula and foods for special medical purposes intended for infants and young children liquid. This in order to exclude any possible health concerns as regards infants and young children after the publication of scientific opinion by CONTAM Panel in 2018 (EFSA, 2018).
3.2. Occurrence 3-MCPDs, 2-MCPDs and GD of in oils
MCPDs were first identified in the late 1970s in acid-hydrolyzed vegetable proteins (HVP), soy sauces and various food ingredients formed as a reaction product of hydrochloric acid with triacylglycerols, phospholipids and glycerol (Velisek et al., 1979).
Since 2004 the problem has assumed particular relevance when the esters of MCPDs were identified in numerous foods. Today is well known that GD and MCPDs are process contaminants mostly present in vegetable and animal oils ad fat, produced during heat treatments, particularly during the deodorization process, so these molecules are found in all refined oils and fats. Modern studies have shown that frying and cooking at high temperatures can potentially lead to the formation of these molecules in food that contain oil or fat and sodium chloride, naturally present or added (Hamlet et al., 2002). For example, Inagaki and Hi-rai in 2016 evaluated the formation of GD acid GE in meat samples cooked with different methods (Inagaki & Hi-rai et al., 2016). Traces of these molecules have also been found in biscuits, probably formed during exposure to high temperatures in cooking (MacMahon et al., 2013). Small amounts of 3-MCPD have also been found in commercially available fried snacks (Arisseto et al., 2015), as well as in food mainly based on oil and fat such as mayonnaise, margarine and infant formula; the
