Microwave-Assisted Extraction/UHPLC-Q-Orbitrap-MS-Based Lipidomic Workflow for Comprehensive Study of Lipids in Soft Cheese.

In this work, Microwave-Assisted Extraction (MAE) was proposed as an alternative and environmentally friendly technique in lipidomics to study the lipid fingerprint of soft cheeses, such as mozzarella. For method development, a first step concerning an evaluation of extraction solvents was carried out via testing three different mixtures, including methanol/ethyl acetate, isopropanol/ethyl acetate, and ethanol/ethyl acetate, at a 1:2 v/v ratio. The latter was chosen as a solvent mixture for subsequent method optimization. MAE conditions, in terms of solvent volume, time, and temperature, were explored to define their effects on extraction capability through a full factorial experimental design. The best compromise to extract more lipids at the same time was obtained with 24 mL g-1 for solvent-to-solid ratio, 65 °C for temperature, and 18 min for time. Lipid analyses were conducted by UHPLC-Q-Orbitrap-MS associated with multivariate statistics. The developed lipidomic workflow allowed for the extraction of over 400 lipids grouped into 18 different subclasses. The results confirmed that MAE is a suitable technique for lipid extraction in the omics approach with high efficiency, even using low-cost and less toxic solvents. Moreover, a comprehensive structure characterization of extracted lipids, in terms of fatty acid composition and regiochemistry, was carried out.

Headspace Solid-Phase Microextraction/Gas Chromatography-Mass Spectrometry and Chemometric Approach for the Study of Volatile Profile in X-ray Irradiated Surface-Ripened Cheeses.

X-ray irradiation is an emerging non-thermal technology that is used as a preservation and sanitization technique to inactivate pathogens and spoilage organisms, increasing the shelf life of products. In this work, two different types of surface-ripened cheeses, Brie and Camembert, produced with cow milk, were treated with X-rays at three dose levels, 2.0, 4.0 and 6.0 kGy, to evaluate the irradiation effects on the volatile profile using a volatolomic approach. The headspace solid-phase microextraction (HS-SPME) technique combined with gas chromatography-mass spectrometry (GC-MS) was used to extract and analyze the volatile fraction from these dairy matrices. The HS-SPME method was optimized by a central composite design in combination with a desirability optimization methodology. The Carboxen/PDMS fiber, 50 °C for extraction temperature and 60 min for time extraction were found to be the best parameter settings and were applied for this investigation. The obtained fingerprints demonstrated that the irradiation-induced changes are dose dependent. The X-ray irradiation produced many new volatiles not found in the non-irradiated samples, but it also varied the amount of some volatiles already present in the control. Specifically, aldehydes and hydrocarbons increased with the irradiation dose, whereas alcohols, carboxylic acids, esters, methyl esters, ketones, lactones and sulfur-containing compounds showed a non-linear dependence on the dose levels; indeed, they increased up to 4.0 kGy, and then decreased slightly at 6.0 kGy. This trend, more evident in the Camembert profile, is probably due to the fact that these compounds are involved in different oxidation mechanisms of lipids and proteins, which were induced by the radiation treatment. In these oxidative chemical changes, the production and degradation processes of the volatiles are competitive, but at higher doses, the decomposition reactions exceed those of formation. A principal component analysis and partial least square discriminant analysis were used to discriminate between the treated and untreated samples. Moreover, this study allowed for the identification of potential markers of X-ray treatment for the two cheeses, confirming this approach as a useful tool for the control of irradiated surface-ripened cheeses.

Novel Approaches in Food Preservation and Their Impact on the Food System.

The increase in consumer demand for safe, convenient, and fresh food with an extended shelf life is accompanied by an interest in the environmental impacts caused by the food industry [...].

Untargeted Lipidomics and Chemometric Tools for the Characterization and Discrimination of Irradiated Camembert Cheese Analyzed by UHPLC-Q-Orbitrap-MS.

In this work, an investigation using UHPLC-Q-Orbitrap-MS and multivariate statistics was conducted to obtain the lipid fingerprint of Camembert cheese and to explore its correlated variation with respect to X-ray irradiation treatment. A total of 479 lipids, categorized into 16 different lipid subclasses, were measured. Furthermore, the identification of oxidized lipids was carried out to better understand the possible phenomena of lipid oxidation related to this technological process. The results confirm that the lipidomic approach adopted is effective in implementing the knowledge of the effects of X-ray irradiation on food and evaluating its safety aspects. Furthermore, Partial Least Squares-Discriminant Analysis (PLS-DA) and Linear Discriminant Analysis (LDA) were applied showing high discriminating ability with excellent values of accuracy, specificity and sensitivity. Through the PLS-DA and LDA models, it was possible to select 40 and 24 lipids, respectively, including 3 ceramides (Cer), 1 hexosyl ceramide (HexCer), 1 lysophosphatidylcholine (LPC), 1 lysophosphatidylethanolamine (LPE), 3 phosphatidic acids (PA), 4 phosphatidylcholines (PC), 10 phosphatidylethanolamines (PE), 5 phosphatidylinositols (PI), 2 phosphatidylserines (PS), 3 diacylglycerols (DG) and 9 oxidized triacylglycerols (OxTG) as potential markers of treatment useful in food safety control plans.

Volatolomic approach by HS-SPME/GC-MS and chemometric evaluations for the discrimination of X-ray irradiated mozzarella cheese.

In this work, an untargeted screening of the volatile profile of X-ray irradiated mozzarella cheese was carried out to study the possible radio-induced modifications. A Central Composite Design (CCD) for Response Surface Methodology (RSM) was employed to optimise the HS-SPME analysis of volatile organic compounds (VOCs). The optimised HS-SPME conditions, in terms of sample amount (5.0 g), extraction temperature (50 °C) and extraction time (75 min), were used to analyse non-irradiated and irradiated samples at three dose levels, 1.0, 2.0, 3.0 kGy. Partial Least Squares-Discriminant Analysis (PLS-DA) and Linear Discriminant Analysis (LDA) were applied to explore the variation of volatile profile with respect to the X-ray irradiation treatment. Both methods highlighted a high discriminant capability with excellent values of accuracy, specificity and sensitivity, demonstrating the effectiveness of the volatolomic approach to evaluate the variations induced by the treatment and allowing to select a total of 35 VOCs as potential irradiation markers.

Optimizing accelerated solvent extraction combined with liquid chromatography-Orbitrap mass spectrometry for efficient lipid profile characterization of mozzarella cheese.

In this study, a novel Accelerated Solvent Extraction (ASE) procedure combined with UHPLC-Q-Orbitrap-MS was developed for detailed untargeted lipid profile of mozzarella cheese. Response Surface Methodology and Pareto front, using a Central Composite Design (CCD), were employed to define the optimised combination of extraction temperature, number of extraction cycles and mix of solvents. LipidSearch™ software was used for a reliable and accurate lipid identification. A total of 13 subclasses, including ceramides, diacylglycerols, triacylglycerols, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, bismethyl phosphatidic acids, cholesterol ester, zymosterol ester, hexosyl ceramides were measured. The elaboration of the CCD showed that the solvents ratio was the main factor affecting the extraction efficiency. The optimised ASE method, together with the Folch extraction, synergistically contributed to a complete characterization of lipid profile of mozzarella cheese, confirming ASE technique, associated with high resolution mass spectrometry detection, as an efficient tool for Lipidomics in food science.

Accurate glutamate monitoring in foodstuffs by a sensitive and interference-free glutamate oxidase based disposable amperometric biosensor.

L-Glutamate (L-Glu) is a well-known flavour enhancer that is present in several foodstuffs. Although L-Glu is generally recognized as safe, the use in foodstuffs remains controversial and then its fast and accurate monitoring represents an important issue. In this work a sensitive and interference-free disposable amperometric biosensor for glutamate monitoring in foodstuffs was developed. The biosensor was prepared by immobilizing glutamate oxidase through co-crosslinking with bovine serum albumin and glutaraldehyde onto a screen printed disposable platinum electrode modified with a permselective overoxidized polypyrrole film. The enzyme immobilization was optimized by using different experimental procedures. The optimized glutamate biosensor was integrated in a flow injection system and characterized in terms of linearity (0.005-1.0 mM, r2 = 0.992), limits of detection (1.8 µM) and quantitation (5.4 µM), repeatability (RSD < 3%) and stability of response under operational conditions (up to 50 h, over 400 analysis). The biosensor showed also excellent anti-interference characteristics towards the main electroactive interferents present in food matrices, and this allowed the application to the accurate monitoring of glutamate in different foodstuffs.

Volatile organic compound data of ready-to-cook tuna fish-burgers: Time evolution in function of different and/or combined mild preservation technologies and relevant statistical analysis.

Volatile organic compound (VOC) composition from ready-to-cook tuna fish-burgers, prepared with and without a protective microbial strain (Lactobacillus paracasei) and/or stored with modified atmosphere packaging (MAP, 5% O2 and 95% CO2), were extracted by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry (HS-SPME-GC-MS) during the burger shelf-life. The collected data showed volatile composition profiles in function of the mild preservation technologies employed and the storage time. Furthermore, statistical data treatment (principal component analysis and Pearson's coefficients) highlighted differences among samples and positive/negative correlations during the storage time. This paper is related to an article already published in LWT (Investigating the effects of mild preservation technology on perishable foods by volatolomics: The case study of ready-to-cook tuna-burgers" https://doi.org/10.1016/j.lwt.2019.108425).

Chromatographic determination of 12 dyes in meat products by HPLC-UV-DIODE array detection.

The use of food dyes in meat is regulated by the current European and non-European legislation, due to several food safety concerns. A reliable method for the quali-quantitative determination of 12 food dyes (Amaranth, Ponceau 4R, Carmine, Ponceau SX, Ponceau 3R, Allura Red AC, Carmoisine, Erythrosine, Sudan I, Sudan II, Sudan III and Sudan IV) in meat products, by high performance liquid chromatography coupled to UV diode array detection is presented. The extraction was accomplished by using acetonitrile, methanol, water, and ammonia, 50:40:9:1 (v/v/v/v) as the solvent and ultrasonic bath. The chromatographic separation was obtained with a C18 RP column eluted by a gradient of acetate buffer/acetonitrile. Good analytical performances characterized this method (Table 1), in terms of selectivity, sensitivity, accuracy and ruggedness. Both method precision (CV% range: 6%-15%) and recovery percentages (range: 86%-105%) resulted in compliance with Decision 2002/657/EC, and the expanded measurement uncertainties, estimated by a bottom-up approach, were in the range 6%-20%. All these results demonstrated that the procedure can be applied successfully for confirmation analyses of commercial meat products. •12 food dyes were determined in meat by new HPLC/UV-DAD method.•The analytical method was fully validated for accurate confirmation analyses.•Method accuracy, sensitivity, selectivity and ruggedness resulted satisfactory.

Volatolomics approach by HS-SPME-GC-MS and multivariate analysis to discriminate olive tree varieties infected by Xylella fastidiosa.

INTRODUCTION: Xylella fastidiosa (Xf) is a pathogenic bacterium that causes diseases in olive trees. Therefore, analytical methods for both the characterisation of the host/pathogen interaction and infection monitoring are needed. Volatile organic compounds (VOCs) are emitted by plants relate to their physiological state, therefore VOCs monitoring can assist in detecting stress or infection states before visible signs are present. OBJECTIVE: In this work, the headspace-solid phase microextraction-gaschromatography-mass spectrometry (HS-SPME-GC-MS) technique was used for the first time to highlight VOCs differences between healthy and Xf-infected olive trees. METHODOLOGY: VOCs from olive tree twig samples were extracted and analysed by HS-SPME-GC-MS, and hence identified by comparing the experimental linear retention indexes with the reference values and by MS data obtained from NIST library. Data were processed by principal component analysis (PCA) and analysis of variance (ANOVA). RESULTS: The HS-SPME step was optimised in terms of adsorbent phase and extraction time. HS-SPME-GC-MS technique was applied to the extraction and analysis of VOCs of healthy and Xf-infected olive trees. More than 100 compounds were identified and the differences between samples were evidenced by the multivariate analysis approach. The results showed the marked presence of methyl esters in Xf-infected samples, suggesting their probable involvement in the mechanism of diffusible signal factor. CONCLUSION: The proposed approach represents an easy and solvent-free method to evaluate the presence of Xf in olive trees, and to evidence volatiles produced by host/pathogen interactions that could be involved in the defensive mechanism of the olive tree and/or in the infective action of Xf.

Simultaneous determination of twelve dyes in meat products: Development and validation of an analytical method based on HPLC-UV-diode array detection.

The use of food dyes in meat is subject to regulations, due to food safety concerns. A reliable method for the determination of 12 food dyes (Amaranth, Ponceau 4R, Carmine, Ponceau SX, Ponceau 3R, Allura Red AC, Carmoisine, Erythrosine, Sudan I, Sudan II, Sudan III and Sudan IV) in meat products using high performance liquid chromatography coupled to UV-diode array detection was developed, optimized and fully validated. The extraction was accomplished using acetonitrile, methanol, water, ammonia, 50:40:9:1 (v/v/v/v) as the solvent, and an ultrasonic bath. Chromatographic separation was achieved using a C18 RP column and samples eluted with a gradient acetate-acetonitrile mobile phase. Good analytical performance was obtained, in terms of selectivity, sensitivity, accuracy and ruggedness. Both method precision (CV% range: 6.2%-18.0%) and recovery (range: 86.4%-105.0%) complied with Decision 657/2002/EC, suggesting the procedure could be applied successfully for analyses of meat products in the European Union.

Microbial cell-free extracts affect the biochemical characteristics and sensorial quality of sourdough bread.

This study aimed to improve the sensorial quality of sourdough wheat bread by the addition of cell-free enzyme extracts (CFEs) from Lactobacillus sanfranciscensis (SF), Hafnia alvei (HF) and Debaryomyces hansenii (DH). CFEs were suitable sources of peptidases, glutamate dehydrogenase and cystathionine γ-lyase. The concentration of free amino acids (FAA) in the sourdoughs containing CFEs was higher than the control sourdough, produced without addition of CFEs. The community-level catabolic profiles showed that the highest number of carbohydrates, polymers and carboxylic acids were consumed in the SF sourdough. Breads produced with CFEs were characterized by higher specific volume than the control. The use of CFEs impacted on the profile of volatile organic compounds. Overall, positive correlations were found between some key-aroma compounds and enzyme activities/precursor FAA. The SF bread, characterized by highest level of alcohols, received the highest score for aroma and sweetness in the sensory analysis.

Mass spectrometry hyphenated techniques for the analysis of volatiles and peptides in soft cheese: Useful tools for the shelf life optimization.

In order to assess the product quality and shelf life of an Italian soft cream cheese under different storage conditions, the volatile and peptide profiles evolution were tested. Volatiles were sampled directly from the head space of cheese packaging by solid-phase microextraction and analyzed by GC-MS. Peptide profiles were obtained by nanoLC-MS/MS, following a novel bioinformatics approach based on scoring distribution associated to the protein hits originating from the database search. In particular, a refined identification by focusing on selected time segments corresponding to the most intense peaks was carried out. A total of 40 compounds including acids, aldehydes, ketones, lactones, alcohols, esters, hydrocarbons, terpene, sulfur, and aromatic compounds were detected. Significant differences in their abundance during the storage in different packagings were observed, as well as an evolution of peptides mainly belonging to αS1-casein. The results demonstrated the usefulness of the above-mentioned hyphenated techniques for the determination of the soft cheese shelf life under different storage conditions.

Differential Expression of Durum Wheat Gluten Proteome under Water Stress during Grain Filling.

Environmental stress during grain filling may affect wheat protein composition, thus influencing its final quality. A proteomic approach was used to evaluate changes in storage protein composition under water stress of two Italian durum wheat (Triticum turgidum ssp. durum) cultivars, Ciccio and Svevo. The high-molecular-weight glutenin region increased progressively in both cultivars and under two water regimens. The L48-35 region, corresponding to low-molecular-weight (LMW) glutenin subunits, increased slightly during grain development and decreased under water stress in both cultivars. In particular, an s-type LMW related to superior technological quality was down-expressed in the early-mid period in Svevo and in the mid-late period in Ciccio. Finally, the L<35 region, corresponding to gliadin-like proteins, decreased slightly during grain development and increased under stress in both cultivars. Several α-gliadins, associated with immunological potential, increased their expression under water stress, especially in Svevo in the early-mid stage of grain filling.

The effect of in-amphorae aging on oenological parameters, phenolic profile and volatile composition of Minutolo white wine.

A wine was obtained from cryomacerated Minutolo grapes under reductive conditions and aged for 12months in glass container and in 3 types of amphorae. After aging, wines in glass containers showed the highest alcohol content, volatile acidity, dissolved oxygen, concentrations of aromatics, alcohols, and esters and by the lowest contents of enols and terpenes. They also showed the highest decrease of flavonoids, hydroxycinnamoyl tartaric acids, and procyanidins. Wines in raw amphorae showed the dramatic decrease of flavonoids and flavans reactive with vanillin. The highest antioxidant activity was exhibited by wines in engobe amphorae, while the lowest values were showed by the wines in glass containers and glazed amphorae. Caftaric acid and procyanidin B3 decreased in wine aged under glass while epicatechin mainly reduced in raw amphorae. According to the Principal Component Analysis, the wines resulted homogeneously grouped as a function of the type of container in which were aged.

Simultaneous and accurate real-time monitoring of glucose and ethanol in alcoholic drinks, must, and biomass by a dual-amperometric biosensor.

In this work the optimization and application of a dual-amperometric biosensor for simultaneous monitoring of glucose and ethanol content, as quality markers in drinks and alcoholic fermentation media, are described. The biosensor is based on glucose oxidase (GOD) and alcohol oxidase (AOD) immobilized by co-cross-linking with bovine serum albumin (BSA) and glutaraldehyde (GLU) both onto a dual gold electrode, modified with a permselective overoxidized polypyrrole film (PPYox). Response, rejection of interferents, and stability of the dual biosensor were optimized in terms of PPYox thickness, BSA, and enzyme loading. The biosensor was integrated in a flow injection system coupled with an at-line microdialysis fiber as a sampling tool. Flow rates inside and outside the fiber were optimized in terms of linear responses (0.01-1 and 0.01-1.5 M) and sensitivities (27.6 ± 0.4 and 31.0 ± 0.6 µA·M(-1)·cm(-2)) for glucose and ethanol. Excellent anti-interference characteristics, the total absence of "cross-talk", and good response stability under operational conditions allowed application of the dual biosensor in accurate real-time monitoring (at least 15 samples/h) of alcoholic drinks, white grape must, and woody biomass.