Liquid chromatographic strategies for separation of bioactive compounds in food matrices

Nowadays, there is an increasing attention for nutraceuticals and, in general, bioactive compounds naturally present in food. Indeed, the possibility of preserving human health and preventing disease (e.g., cardiovascular diseases, cancer etc.) by the intake of healthy food is attractive for both consumers and food industries. In turn, research in this field was also prompted significantly, with the aim of characterizing these bioactive compounds and ascribe to them a specific activity. The bioactive compounds can belong to several chemical classes. However, their chemical diversity and presence in complex matrices, such as food, make it challenging both their isolation and characterization. To tackle this issue, efficient separation systems are needed, which are mainly based on chromatography. In this context, this mini-review aims to provide the reader with an overview of the most relevant and recent approaches for the separation of the most common bioactive compounds in food, in particular polyphenols, phenols, carotenoids, and peptides, by liquid chromatography approaches. © 2018 by the authors

A rapid magnetic solid phase extraction method followed by liquid chromatography-tandem mass spectrometry analysis for the determination of mycotoxins in cereals

Mycotoxins can contaminate various food commodities, including cereals. Moreover, mycotoxins of different classes can co-contaminate food, increasing human health risk. Several analytical methods have been published in the literature dealing with mycotoxins determination in cereals. Nevertheless, in the present work, the aim was to propose an easy and effective system for the extraction of six of the main mycotoxins from corn meal and durum wheat flour, i.e., the main four aflatoxins, ochratoxin A, and the mycoestrogen zearalenone. The developed method exploited magnetic solid phase extraction (SPE), a technique that is attracting an increasing interest as an alternative to classical SPE. Therefore, the use of magnetic graphitized carbon black as a suitable extracting material was tested. The same magnetic material proved to be effective in the extraction of mycoestrogens from milk, but has never been applied to complex matrices as cereals. Ultra high-performance liquid chromatography tandem mass spectrometry was used for detection. Recoveries were > 60% in both cereals, even if the matrix effects were not negligible. The limits of quantification of the method results were comparable to those obtained by other two magnetic SPE-based methods applied to cereals, which were limited to one or two mycotoxins, whereas in this work the investigated mycotoxins belonged to three different chemical classes

Comprehensive identification of native medium-sized and short bioactive peptides in sea bass muscle

Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides. The medium-sized peptides were identified by de novo and combination of de novo and spectra matching to a protein sequence database, with up to 4077 peptides (2725 modified) identified by database search and 2665 peptides (223 modified) identified by de novo only; 102 short peptide sequences were identified (with 12 modified ones), and most of them had multiple reported bioactivities. The method can be extended to any peptide mixture, either endogenous or by protein hydrolysis, from other food matrices. © 2020 Elsevier Lt

Bioactive compounds analysis in food matrices and waste products

Food and nutrition research have grown exponentially over the past two decades, changing the way food is considered. In fact, it is no longer considered simply energy for the body but provides components with specific functions and nutritional properties, which include potential benefits, as well as possible harmful effects on health; among these, fatty acids, vitamins, phenolic acids, and flavonoids have been studied in recent years. In recent years, proteins, peptides and lipids have also played an important role in the study of components with high nutritional value, giving way to a new branch of the omic sciences, namely nutritional proteomics (nutriproteomics) and lipidomics. The study of bioactive peptides present in various food products represents a new field of research and field of application of proteomic analysis. Some peptides are able to trigger physiological activities that promote skin health, such as stimulating collagen synthesis, controlling angiogenesis and melanogenesis and modulating cell proliferation. Peptides are often classified as multifunctional peptides, i.e. they are capable of inducing more than one physiological activity. These characteristics make them suitable for use as therapeutic agents in the pharmaceutical field or as functional compounds, food additives, bio-preservatives in the field of nutraceuticals. These peptides can be released in different ways: they can be naturally present (i.e. endogenous) in matrices due to the presence of enzymes or bacteria, they can be released during gastrointestinal digestion, they can be formed following processes that occur in foods such as maturation, fermentation, storage or cooking, or by in vitro hydrolysis with the selection of a specific and appropriate enzyme. Except in the case in which the digestion is carried out in vitro by site-specific proteases, the obtained peptides can have a wide range of terminal amino acids and sizes. Of interest are both the bioactive peptides derived from enzymatic digestion, but also the short peptides naturally present within the matrix. Another very interesting class of molecules, not widely studied but specifically analyzed in this work are: seleno-amino acids. These compounds regulate many biological activities, and are important against various diseases. Another important goal is the study of waste. This type of product is normally seen as a problem for the economy and the environment, because it must be disposed of waste could be better used, though, not only as feed for animals but, for example, as a source of bioactive compounds. Therefore, the purpose of this doctoral project was to develop effective and innovative analytical strategies for the study of bioactive molecules and identification of compounds not abundant or not yet identified in different food matrices

High-Resolution Mass Spectrometry and Chemometrics for the Detailed Characterization of Short Endogenous Peptides in Milk By-Products

The process of cheese-making has long been part of human food culture and nowadays dairy represents a large sector of the food industry. Being the main byproduct of cheese-making, the revalorization of milk whey is nowadays one of the primary goals in alignment with the principles of the circular economy. In the present paper, a deep and detailed investigation of short endogenous peptides in milk and its byproducts (whole whey, skimmed whey, and whey permeate) was carried out by high-resolution mass spectrometry, with a dedicated suspect screening data acquisition and data analysis approach. A total of 79 short peptides was tentatively identified, including several sequences already known for their exerted biological activities. An unsupervised chemometric approach was then employed for highlighting the differences in the short peptide content among the four sets of samples. Whole and skimmed whey showed not merely a higher content of short bioactive peptides compared to whole milk, but also a peculiar composition of peptides that are likely generated during the process of cheese-making. The results clearly demonstrate that whey represents a valuable source of bioactive compounds and that the set-up of processes of revalorization of milk byproducts is a promising path in the obtention of high revenue-generating products from dairy industrial waste

Characterization of the Trans-Epithelial Transport of Green Tea (C. sinensis) Catechin Extracts with In Vitro Inhibitory Effect against the SARS-CoV-2 Papain-like Protease Activity

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 μg mL−1). The untargeted characterization revealed that (−)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 μg mL−1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG

Investigating the Short Peptidome Profile of Italian Dry-Cured Ham at Different Processing Times by High-Resolution Mass Spectrometry and Chemometrics

Short peptides have been spiking interest owing to their significant contribution to the taste and functional properties of dry-cured ham. In this study, a suspect screening approach based on high-resolution mass spectrometry was employed for the comprehensive characterization of the short endogenous peptidome in dry-cured ham samples at different processing stages (14, 22, and 34 months). After careful manual spectra interpretation, a chemometric approach based on principal component analysis was employed for highlighting the differences between the three sets of samples. A total of 236 short peptide sequences was tentatively identified, including 173 natural short peptides and 63 sequences containing non-proteinogenic amino acids, the highest number ever reported for endogenous sequences in dry-cured ham. Samples in the latest processing stages presented a generally higher abundance of dipeptides, indicating residual proteolytic activity. Moreover, the several annotated modified short peptides, mainly pyroglutamination and lactoyl conjugation, allowed hypothesizing several reactions occurring over time. For the first time, several lactoyl-dipeptides were tentatively identified in dry-cured ham samples with maximum concentration in the late processing stage samples. The presented results significantly contribute to the understanding of the reaction involving short peptides that affect the sensory and functional properties of dry-cured ham

Multielement characterization and antioxidant activity of Italian extra-virgin Olive oils

Food product safety and quality are closely related to the elemental composition of food. This study combined multielement analysis and chemometric tools to characterize 237 extra-virgin olive oil (EVOO) samples from 15 regions of Italy, and to verify the possibility of discriminating them according to different quality factors, such as varietal or geographical origin or whether they were organically or traditionally produced. Some elements have antioxidant properties, while others are toxic to humans or can promote oxidative degradation of EVOO samples. In particular, the antioxidant activity of oils’ hydrophilic fraction was estimated and the concentrations of 45 elements were determined by inductively coupled plasma mass spectrometry (ICP-MS). At first, univariate and multivariate analyses of variance were used to compare the element concentrations, and statistically significant differences were found among samples from different regions. Successively, discriminant classification approaches were used to build a model for EVOO authentication, considering, in turn, various possible categorizations. The results have indicated that chemometric methods coupled with ICP-MS have the potential to discriminate and characterize the different types of EVOO, and to provide “typical” elemental fingerprints of the various categories of samples