Study of milk contaminants and re-evaluation of whey: potential use of dairy by-product in functional food

Milk is the most widely consumed beverage in the world, not counting water, which provides high-quality nutrition to infants, children, and adults, due to its high content of micro- and macronutrients. Despite the benefits that milk provides from a nutritional point of view, it may also be a vehicle of food contaminants, such as mycotoxins and veterinary drug residues (VDs), due to nasty agricultural practices or improper usage of these drugs. In this sense, the first part of my project includes the studies regarding the topics mentioned above. Hence, a multi-residue method based on a QuEChERS extraction for the simultaneous determination of veterinary drug residues (n=61) and mycotoxins (n=46) in milk was developed by using ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC Q-Exactive Orbitrap HRMS). The recoveries were in a range of 72-93% at three spiking levels, with repeatability and reproducibility results expressed as relative standard deviations (RSDs) lower than 7% and 14%, respectively. The limits of quantification (LOQs) were in the range of 0.01-50 pg/mL. Matrix effects obtained were in the range of 75-97%. The results obtained from the study showed that none of the analyzed samples (n=56) were contaminated with mycotoxins, whereas the presence of up to seven veterinary drug residues in 50% of analyzed samples was detected (range of 0.007-4.53 ng/mL). None of the analyzed VDs had a concentration level higher than their permitted limits, except for benzylpenicillin procain (4.53 ng/mL). In the post-target screening, 53 contaminants were tentatively identified. Regarding the incidence of mycotoxins, AFM2 contamination was displayed in >70% of analyzed samples. A significant percentage (>80%) has been shown by betamethasone, prednisolone and oxfendazole. Special focus should be paid to the latter, considering that the maximum residue limit permitted in EC 37/2010 is really low, 0.3; 6; 10 μg/Kg, respectively. In addition, other eleven VDs identified in analyzed milk samples are prohibited in milk by the regulation in force (EC 37/2010). From the obtained values seems to be clear the necessity of a continuous monitoring of contaminants in the milk production chain. The second part of my Ph.D. project was based on the re-evaluation of whey, one of the highest polluting liquid wastes of the dairy industry. Environmental and economic problems associated with food waste have induced industries to reuse these materials in order to minimize food waste. In this sense, I have evaluated the bioactivity of two typologies of whey (freeze-dried cow’s and liquid goat’s whey) after subjecting these by-products to the fermentation process by lactic acid bacteria (Lactobacillus plantarum CECT 220, 221, 223, 748). Antifungal activity performed against 30 mycotoxigenic fungi belonging to the Penicillium, Aspergillus and Fusarium genera has shown a satisfying inhibitory effect. The value of MIC for Aspergillus and Fusarium genera was included in a range from 1.95 to 62.5 g/L whereas for Penicillium genera was included in a range from 3.9 to 125 g/L. The value of MFC was contained between 15.6 and 250 g/L for Aspergillus, 62.5 and 250 g/L for Penicillium, 3.9 and 250 g/L for Fusarium. The antifungal activity could be correlated to the presence of phenolic compounds identified in cell-free-supernatant. Based on previously performed experiments, mycotoxigenic strains of P. expansum and P. brevicompactum were employed for studying the shelf-life improvement of bread. A valuable increase in the shelf-life was obtained for pita bread prepared with fermented whey. In particular, after the inoculation of bread with Penicillium, a rise of 1-2 and 7-8 days for the two typologies of assayed whey was compared to a positive control. Through a natural contamination, an increase in the preservation period of bread (until the 20th day) was observed. Regarding the antimicrobial activity, by adding the 1% of freeze-dried fermented whey in bread preparation, a percentage reduction ranged between 12-23% was obtained, whereas with the replacement of 100% of water used in bread preparation with whey fermented by LAB, the percentage of reduction has the highest peak of increase ranged from 42 to 92% for the different typologies of test performed. In addition, concerning freeze-dried whey, I have evaluated the antioxidant, antihypertensive, and iron binding activity of the permeate, consisting of small bioactive compounds purified on Centricon Amicon with a cut-off of 3 kDa. The results highlighted a radical cation scavenging activity ranging from 1.415 to 2.083 mmol trolox equivalents TE/per kg of dry weight, a percentage of iron binding capacity ranging between 23–55% and a percentage of ACE inhibitory activity ranging between 67–85%. The optimal biological activity was obtained from whey fermented by L. plantarum 220 for all the assays performed, except for the iron chelating activity. These results confirm the interesting use of lactic acid bacteria as a source of new natural preservatives, which can contribute to the maintenance of food quality and safety. The obtained results support the hypothesis of using whey as a functional ingredient to improve food preservation and its potential use in nutraceutical formulation

Mycotoxins: An Under-evaluated Risk for Human Health

Mycotoxins are secondary toxic metabolites produced mainly by fungi belonging to the Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps genera. These moulds can colonize agricultural crops and produce mycotoxins during pre- and post-harvest practices, processing, and storage. Animals fed with feed contaminated with mycotoxins may be a natural and unwanted bioenhancer way to transfer mycotoxins, eventually metabolized, to animal-derived food addressed to humans. The natural occurrence of mycotoxins, also a low concentration, in food may cause adverse health effects in humans, rarely showing acute symptoms but the chronic exposure causes problems ranging from gastrointestinal and kidney disorders to immune deficiency and to develop some types of cancers. Human exposure to mycotoxins can happen by eating directly contaminated foods or through contaminated animal products. This alternative entry of mycotoxin into the human food chain is a signal of animals fed with contaminated feed. The exposure danger to mycotoxins can be monitored by following the biotransformation product occurrence in tissues and biological fluids, and these data are needed to evaluate their potential risk for humans, in particular for weak subpopulations like babies, children, old, or pressed by food security troubles. In this regard, the main aim of this volume is to evaluate the occurrence of mycotoxins and other contaminants in food, nutraceuticals, and biological fluids in order to ensure human safety. To guarantee effective consumer safety, reliable methods have been validated for the analysis of contaminants in various matrices. In addition, the risk associated with the assumption of contaminated food was assessed. Risk characterization is an indispensable aspect to safeguard public health, which helps to identify risks threatening consumers

The Nutraceutical Properties of “Pizza Napoletana Marinara TSG” a Traditional Food Rich in Bioaccessible Antioxidants

: Italian gastronomy experiences have ever-enhancing fame around the world. It is due to the linkage between taste and salubriousness commonly related to Mediterranean foods. The market proposes many types of pizza to suit all palates. The antioxidant potential of the “Pizza Napoletana marinara” included in the register of traditional specialties guaranteed (TSG) was determined in this work. ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) method evaluated the antioxidant activity of the pizza homogenized. In vitro digestion models estimated the intestinal and gastric bioaccessibility of the main antioxidant compounds (lycopene and phenolics). To our knowledge, this is the first study to provide the content, antioxidant potential, and bioaccessibility of the antioxidants (polyphenols and lycopene) contained in the traditional pizza “marinara TSG”. Our results showed that the “Pizza Napoletana marinara” had polyphenols concentration, lycopene level, antioxidant activity, and bioaccessibility of phenolic compounds and lycopene better than other similar pizzas. They confirmed the nutritional importance of traditional preparations and established the nutraceutical potential of “pizza marinara TSG” as a food rich in bio-accessible antioxidants

The Nutraceutical Properties of “Pizza Marinara TSG” a Traditional Food Rich in Bioaccessible Antioxidants

Italian gastronomy experiences have ever-enhancing fame around the world. It is due to the linkage between taste and salubriousness commonly related to Mediterranean foods. The market proposes many types of pizza to suit all palates. In this work, the antioxidant potential of the "pizza “marinara” included in the register of traditional specialties guaranteed (TSG) was determined. ABTS method evaluated the antioxidant activity of the pizza homogenized. In vitro digestion models estimated the intestinal and gastric bioaccessibility of the main antioxidant compounds (lycopene and phenolics). To our knowledge, this is the first study to provide the content, antioxidant potential, and bioaccessibility of the antioxidants (polyphenols and lycopene) contained in the traditional pizza “marinara TSG”. Our results showed that the "pizza marinara TSG" had polyphenols concentration, lycopene level, antioxidant activity, and bioaccessibility of phenolic compounds and lycopene better than other similar pizzas. They confirmed the nutritional importance of traditional preparations and established the functional potential of "pizza marinara TSG" as a food rich in bio-accessible antioxidants

The nutraceutical properties of "pizza marinara TSG" a traditional food rich in bioaccessible antioxidants

Italian gastronomy experiences have ever-enhancing fame around the world. It is due to the linkage between taste and salubriousness commonly related to Mediterranean foods. The market proposes many types of pizza to suit all palates. In this work, the antioxidant potential of the "pizza “marinara” included in the register of traditional specialties guaranteed (TSG) was determined. ABTS method evaluated the antioxidant activity of the pizza homogenized. In vitro digestion models estimated the intestinal and gastric bioaccessibility of the main antioxidant compounds (lycopene and phenolics). To our knowledge, this is the first study to provide the content, antioxidant potential, and bioaccessibility of the antioxidants (polyphenols and lycopene) contained in the traditional pizza “marinara TSG”. Our results showed that the "pizza marinara TSG" had polyphenols concentration, lycopene level, antioxidant activity, and bioaccessibility of phenolic compounds and lycopene better than other similar pizzas. They confirmed the nutritional importance of traditional preparations and established the functional potential of "pizza marinara TSG" as a food rich in bio-accessible antioxidants

Whey fermented by using Lactobacillus plantarum strains: A promising approach to increase the shelf life of pita bread

Nowadays, there is an increasing concern regarding the shelf life of food products, leading producers to research natural antimicrobial agents to use in food preparation. In this study, we evaluated the antifungal activity of Lactobacillus plantarum fermented whey and then added the whey during preparation of pita bread to study shelf-life improvement. The fermented whey showed a satisfactory inhibitory (antifungal) effect against Penicillium expansum and Penicillium brevicompactum strains: the minimum inhibitory and minimum fungicidal concentrations ranged from 3.9 to 39.0 g/L and from 62.5 to 250 g/L, respectively. Addition of fermented whey increased the shelf life of the pita bread. After inoculation of the bread surface with Penicillium, an increase in shelf life until d 8 was achieved compared with the positive control, whereas under natural contamination conditions, an extension of shelf life until d 19 was observed. In terms of antimicrobial activity, the greatest reduction (100%) in fungal growth was achieved when all of the water in the dough was replaced with fermented whey. An untrained sensory panel could not identify differences between bread produced with fermented whey and control pita breads. These results suggest the possibility of using fermented whey in food preservation

Urinary levels of enniatin B and its phase I metabolites: First human pilot biomonitoring study

Enniatins (Enns) are mycotoxins produced by Fusarium spp. and are widely distributed contaminants of cereals and derivate products. Among the different identified enniatins, Enn B is the most relevant analogue in cereals in Europe. Therefore, the aim of this study was to investigate for the first time the occurrence of Enn B and Enn B phase I metabolites in 300 human urine samples throughout an ultrahigh-performance liquid chromatography high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) methodology. Three different sample preparation procedures were evaluated and salting-out liquid-liquid extraction showed satisfactory validation results. Enn B was quantified in 83.7% of samples ranging from 0.006 to 0.391 ng/mL (average content: 0.016 ng/mL). In line with the in vitro observations with human liver microsomes, in the here analyzed samples the Enn B monooxygenated, N-demethylated and dioxygenated metabolites were tentatively found in 87.7%, 96.3% and 6.7% of samples. The data of this pilot biomonitoring survey indicate a frequent intake of enniatins in Italy, supporting further toxicological studies to provide better basis for understanding their potential effects in humans

Target analysis and retrospective screening of mycotoxins and pharmacologically active substances in milk using an ultra-high-performance liquid chromatography/high-resolution mass spectrometry approach

Milk is a nutritious food suitable for infants and adults, and it plays an important role in the human diet. However, it may also be a vehicle for food contaminants. In this report, we developed a method using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS; Thermo Fisher Scientific, Waltham, MA) for simultaneous identification of target pharmacologically active substances and mycotoxins in milk. We also used the Q-Orbitrap operating in full scan mode to identify other possible drugs and microbial metabolites that occurred in samples. Fifty-six commercially available milk samples from the Italian market were analyzed. Investigated analytes were extracted using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach. Method detection and quantification limits and performance criteria set by European regulations were fulfilled. Pharmacologically active substances were detected in 49% of samples (range 0.007–4.53 ng/mL), including nontarget mycotoxins. Retrospective analysis allowed us to identify other antibiotics and pharmacologically active substances, as well as nonregulated fungal/bacterial metabolites at a relatively high incidence. From the obtained values, the need for continuous monitoring of contaminants in the milk production chain is clear. This is the first study to assess the presence of pharmacologically active substances, mycotoxins, and other microbial metabolites in Italian milk samples using the UHPLC-Q-Orbitrap HRMS system

Analysis of Polyphenolic Compounds in Water-Based Extracts of Vicia faba L.: A Potential Innovative Source of Nutraceutical Ingredients

The water-based extract of broad bean hulls contains several bioactive molecules, including polyphenols well-known to exert antioxidant activity, which could justify its use in nutraceutical formulations. Hence, the current investigation aimed to establish the polyphenolic profile of water-based extracts from broad bean hulls through UHPLC-Q-Orbitrap HRMS analysis. The findings highlighted that p-coumaric acid, chlorogenic acid, and epicatechin were the most common compounds found in the tested extracts, being quantified at a mean concentration of 42.1, 32.6, and 31.2 mg/100 g, respectively. Moreover, broad bean hull extracts were encapsulated into a nutraceutical formulation, after which the antioxidant properties and the bioaccessibility of phenolic compounds during the simulated gastrointestinal (GI) process were investigated and compared with the digested non-encapsulated extract. The data highlighted that following the GI process, the capsules were able to preserve active compounds from the adverse effects of digestion, resulting in a greater antioxidant capacity and polyphenol bioaccessibility in the duodenal and colonic phases, compared with the non-encapsulated extract. Our results showed that the water extract from broad bean hulls may be considered a valuable source of natural polyphenolic compounds; in addition, the use of a gastric-resistant capsule could be a suitable alternative to transport these bioactive compounds to the target tissues

Effect of Phenolic Extract from Red Beans (Phaseolus vulgaris L.) on T-2 Toxin-Induced Cytotoxicity in HepG2 Cells

Red beans contain human bioactive compounds such as polyphenols. Several in vitro studies have proposed the natural compounds as an innovative strategy to modify the toxic effects produced by mycotoxins. Hence, in this work, a complete investigation of the polyphenolic fraction of red beans was performed using a Q-Orbitrap high-resolution mass spectrometry analysis. Notably, epicatechin and delphinidin were the most detected polyphenols found in red bean extracts (3.297 and 3.108 mg/Kg, respectively). Moreover, the red bean extract was evaluated against the T-2 toxin (T-2) induced cytotoxicity in hepatocarcinoma cells (HepG2) by direct treatment, simultaneous treatment, and pre-treatment assays. These data showed that T-2 affected the cell viability in a dose-dependent manner, as well as observing a cytotoxic effect and a significant increase in ROS production at 30 nM. The simultaneous treatment and the pre-treatment of HepG2 cells with red bean extract was not able to modify the cytotoxic T-2 effect. However, the simultaneous treatment of T-2 at 7.5 nM with the red bean extract showed a significant decrease in ROS production, with respect to the control. These results suggest that the red bean extract could modulate oxidative stress on HepG2 cells