Quali-quantitative study of the phenolic and polyphenolic compounds and their antioxidant capacity in vegetal matrix processed with different technologies

Food technologies today mean reducing agricultural food waste, improvement of food security, enhancement of food sensory properties, enlargement of food market and food economies. Food technologists must be high-skilled technicians with good scientific knowledge of food hygiene, food chemistry, industrial technologies and food engineering, sensory evaluation experience and analytical chemistry. Their role is to apply the modern vision of science in the field of human nutrition, rising up knowledge in food science. The present PhD project starts with the aim of studying and improving frozen fruits quality. Freezing process in very powerful in preserve initial raw material characteristics, but pre-treatment before the freezing process are necessary to improve quality, in particular to improve texture and enzymatic activity of frozen foods. Osmotic Dehydration (OD) and Vacuum Impregnation (VI), are useful techniques to modify fruits and vegetables composition and prepare them to freezing process. These techniques permit to introduce cryo-protective agent into the food matrices, without significant changes of the original structure, but cause a slight leaching of important intrinsic compounds. Phenolic and polyphenolic compounds for example in apples and nectarines treated with hypertonic solutions are slightly decreased, but the effect of concentration due to water removal driven out from the osmotic gradient, cause a final content of phenolic compounds similar to that of the raw material. In many experiment, a very important change in fruit composition regard the aroma profile. This occur in strawberries osmo-dehydrated under vacuum condition or under atmospheric pressure condition. The increment of some volatiles, probably due to fermentative metabolism induced by the osmotic stress of hypertonic treatment, induce a sensory profile modification of frozen fruits, that in some way result in a better acceptability of consumer, that prefer treated frozen fruits to untreated frozen fruits. Among different processes used, a very interesting result was obtained with the application of a osmotic pre-treatment driven out at refrigerated temperature for long time. The final quality of frozen strawberries was very high and a peculiar increment of phenolic profile was detected. This interesting phenomenon was probably due to induction of phenolic biological synthesis (for example as reaction to osmotic stress), or to hydrolysis of polymeric phenolic compounds. Aside this investigation in the cryo-stabilization and dehydrofreezing of fruits, deeper investigation in VI techniques were carried out, as studies of changes in vacuum impregnated prickly pear texture, and in use of VI and ultrasound (US) in aroma enrichment of fruit pieces. Moreover, to develop sensory evaluation tools and analytical chemistry determination (of volatiles and phenolic compounds), some researches were bring off and published in these fields. Specifically dealing with off-flavour development during storage of boiled potato, and capillary zonal electrophoresis (CZE) and high performance liquid chromatography (HPLC) determination of phenolic compounds.

Technological processes study for high value health ingredients and foods production

This PhD thesis describes set up of technological models for obtaining high health value foods and ingredients that preserve the final product characteristics as well as enrich with nutritional components. In particular, the main object of my research has been Virgin Olive Oil (VOO) and its important antioxidant compounds which differentiate it from all other vegetables oils. It is well known how the qualitative and quantitative presence of phenolic molecules extracted from olives during oil production is fundamental for its oxidative and nutritional quality. For this purpose, agronomic and technological conditions of its production have been investigated. It has also been examined how this fraction can be better preserved during storage. Moreover, its relation with VOO sensorial characteristics and its interaction with a protein in emulsion foods have also been studied. Finally, an experimental work was carried out to determine the antioxidative and heat resistance properties of a new antioxidant (EVS-OL) when used for high temperature frying such as is typically employed for the preparation of french fries. Results of the scientific research have been submitted for a publication and some data has already been published in national and international scientific journals.

Functional components and lipid quality assessment in vegetable foods: analytical and technological approaches

Lipids are important components that contribute very significantly to nutritional and technological quality of foods because they are the least stable macro-components in foods, due to high susceptibility to oxidation. When rancidity take place, it makes food unhealthy and unacceptable for consumers. Thus, the presence of antioxidants, naturally present of added to foods, is required to enhance shelf life of foods. Moreover, antioxidant like phenolic compounds play an important role in human health enhancing the functionality of foods. The aim of this PhD project was the study of lipid quality and lipid oxidation in different vegetable foods focusing on analytical and technological aspects in order to figure out the effects of lipid composition and bioactive compounds (phenolic compounds, omega-3 fatty acids and dietary fiber) addition on their shelf life. In addition, bioavailability and antioxidant effects of phenolic compounds in human and animals, respectively, were evaluated after consumption of vegetable foods. The first section of the work was focused on the evaluation of lipid quality impact on technological behaviour of vegetable foods. Because of that, cocoa butter with different melting point were evaluated by chromatographic techniques (GC, TLC) and the sample with the higher melting point showed the presence of fatty acids, triglycerides, 2-monoglycerides and FT-IR profile different from genuine cocoa butter, meaning an adding of foreign fat (lauric-fat) not allowed by the law. Looking at lipid quality of other vegetable foods, an accelerated shelf life test (OXITEST®), was used to evaluate of lipid stability to oxidation in tarallini snacks made up using different lipid matrices (sunflower oil, extravirgin olive oil and a blend of extravirgin olive oil and lard). The results showed a good ability of OXITEST® to discriminate between lipid unsaturation and different cooking times, without any samples fat extraction. In the second section, the role of bioactive compounds on cereal based food shelf life was studied in different bakeries by GC, spectrophotometric methods and capillary electrophoresis. It was examined the relationships between phenolic compounds, added with flour, and lipid oxidation of tarallini and frollini. Both products showed an increase in lipid oxidation during storage and antioxidant effects on lipid oxidation were not as expected. Furthermore, the influence of enrichment in polyunsaturated fatty acids on lipid oxidation of pasta was evaluated. The results proved that LC n-3 PUFA were not significantly implicated in the onset of oxidation in spaghetti stored under daylight and accelerated oxidation in a laboratory heater. The importance of phenolic compounds as antioxidant in humans and rats was also studied, by HPLC/MS in the latter section. For this purpose, apigenin and apigenin glycosides excretion was investigated in six women’s urine in a 24 hours study. After a single dose of steamed artichokes, both aglicone and glucuronide metabolites were recovered in 24 h urine. Moreover, the effect of whole grain durum wheat bread and whole grain Kamut® khorasan bread in rats were evaluated. Both cereals were good sources of antioxidants but Kamut® bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied.

Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin

The objectives of this PhD research were: i) to evaluate the use of bread making process to increase the content of β-glucans, resistant starch, fructans, dietary fibers and phenolic compounds of kamut khorasan and wheat breads made with flours obtained from kernels at different maturation stage (at milky stage and fully ripe) and ii) to study the impact of whole grains consumption in the human gut. The fermentation and the stages of kernel development or maturation had a great impact on the amount of resistant starch, fructans and β-glucans as well as their interactions resulted highly statistically significant. The amount of fructans was high in kamut bread (2.1g/100g) at the fully ripe stage compared to wheat during industrial fermentation (baker’s yeast). The sourdough increases the content of polyphenols more than industrial fermentation especially in bread made by flour at milky stage. From the analysis of volatile compounds it resulted that the sensors of electronic nose perceived more aromatic compound in kamut products, as well as the SPME-GC-MS, thus we can assume that kamut is more aromatic than wheat, so using it in sourdough process can be a successful approach to improve the bread taste and flavor. The determination of whole grain biormakers such as alkylresorcinols and others using FIE-MS AND GC-tof-MS is a valuable alternative for further metabolic investigations. The decrease of N-acetyl-glucosamine and 3-methyl-hexanedioic acid in kamut faecal samples suggests that kamut can have a role in modulating mucus production/degradation or even gut inflammation. This work gives a new approach to the innovation strategies in bakery functional foods, that can help to choose the right or best combination between stages of kernel maturation-fermentation process and baking temperature.

Country-of-Origin Labeling for Processed Foods

The country-of-origin is the “nationality” of a food when it goes through customs in a foreign country, and is a “brand” when the food is for sale in a foreign market. My research on country-of-origin labeling (COOL) started from a case study on the extra virgin olive oil exported from Italy to China; the result shows that asymmetric and imperfect origin information may lead to market inefficiency, even market failure in emerging countries. Then, I used the Delphi method to conduct qualitative and systematic research on COOL; the panel of experts in food labeling and food policy was composed of 19 members in 13 countries; the most important consensus is that multiple countries of origin marking can provide accurate information about the origin of a food produced by two or more countries, avoiding misinformation for consumers. Moreover, I enhanced the research on COOL by analyzing the rules of origin and drafting a guideline for the standardization of origin marking. Finally, from the perspective of information economics I estimated the potential effect of the multiple countries of origin labeling on the business models of international trade, and analyzed the regulatory options for mandatory or voluntary COOL of main ingredients. This research provides valuable insights for the formulation of COOL policy.

Application of Pulsed Electric Fields for the Modulation of Chemico-Physical Properties of Different Foods

Pulsed electric field technology is one of the most attractive new non-thermal technology thanks to its lower energy consumption and short treatment times. It consists of an electric treatment of short duration (from several ns to several ms) with electric field strengths from 0.1 to 80 kV/cm that lead to an increase in the permeability of the cell membrane. In this PhD thesis, PEF technology was investigated with the aim of improving mass transfer in plant and animal foods by using it alone or in combination with conventional food processes. Different methods of evaluating electroporation for optimizing PEF processing parameters were investigated. In this respect, the degree of membrane permeabilization in plant and animal food matrices was investigated using electrical impedance spectroscopy, current-voltage measurements and magnetic resonance imaging. The research findings provided useful insights and calls for critical choice of electroporation assessment methods for the selection of adequate PEF treatment conditions. It was outlined that the effect of electroporation is highly dependent on the properties of the food matrix and secondary phenomena occurring in the cell structure undergoing PEF treatment, such as the water re-distribution in the tissue due to the exchange of fluids between intra- and extra-cellular environments. This study also confirmed the great potential of combining PEF technology with conventional food processes, with the main purpose of improving the quality of the food material and accelerating the kinetics of mass transfers, in both plant and animal tissues. Consistent reduction of acrylamide formation in potato crisps was achieved by monitoring key PEF process parameters and subsequent manufacturing steps. Kiwifruit snacks showed a significant reduction in drying kinetics when pre-treated with PEF, while their quality was well maintained. Finally, the research results showed that PEF pre-treatments can shorten the brine process as well as the rehydration kinetics of fish muscles.

Microbiome and resitome and foods of animal origin

To ensure food safety and to prevent food-borne illnesses, rapid and accurate detection of pathogenic agents is essential. It has already been demonstrated that shotgun metagenomic sequencing can be used to detect pathogens and their antibiotic resistance genes in food. In the studies presented in this thesis, the application shotgun metagenomic sequencing has been applied to investigate both the microbiome and resistome of foods of animal origin in order to assess advantages and disadvantages of shotgun metagenomic sequencing in comparison to the cultural methods. In the first study, it has been shown that shotgun metagenomics can be applied to detect microorganisms experimentally spiked in cold-smoked salmon. Nevertheless, a direct correlation between cell concentration of each spiked microorganism and number of corresponding reads cannot be established yet. In the second and third studies, the microbiomes and resistomes characterizing caeca and the corresponding carcasses of the birds reared in the conventional and antibiotic free farms were compared. The results highlighted the need to reduce sources of microbial contamination and antimicrobial resistance not only at the farm level but also at the post-harvest one. In the fourth study, it has been demonstrated that testing a single aliquot of a food homogenate is representative of the whole homogenate because biological replicates displayed overlapping taxonomic and functional composition. All in all, the results obtained confirmed that the application of shotgun metagenomic sequencing represents a powerful tool that can be used in the identification of both spoilage and pathogenic microorganism, and their resistome in foods of animal origin. However, a robust relationship between sequence read abundance and concentration of colony-forming unit must be still established.

The frozen elephant trunk technique for the treatment of complex aortic arch and descending thoracic aorta pathologies: long term outcomes and distal reintervention

Background: The frozen elephant trunk(FET) technique is one of the last evolution in the treatment of complex pathologies of the aortic arch and the descending thoracic aorta.Materials and methods: Between January 2007 and March 2021, a total of 396 patients underwent total aortic arch replacements with the FET technique in our centre.The main indications were thoracic aortic aneurysm(n=104,28.2%), chronic aortic dissection(n=224,53.4%) and acute aortic dissection(n=68, 18.4%). We divided the population in two groups according the position of the distal anastomosis (zone 2 vs zone 3) and the length of the stent graft (< 150 mm vs > 150 mm): conservative group (Zone 2 anastomosis + stent length < 150mm, n. 140 pts) and aggressive group (zone 3 anastomosis + stent length > 150mm, n. 141). Results: The overall 30-day mortality rate was 13%(48/369); the risk factor analysis showed that an aggressive approach was neither a risk factor for major complication (permanent dialysis, tracheostomy, bowel malperfusion and permanent paraplegia) neither for 30-day mortality. The survival rate at 1, 5,10 and 15 years was 87.7%,75%,61.3% and 58.4% respectively. During the follow up, an aortic reintervention was performed in 122 patients (38%), 5 patients received a non-aortic cardiac surgery. Freedom from aortic reintervention at 1-,5- and 10-year was 77%,54% and 44% respectively. The freedom from aortic reintervention was higher in the ‘aggressive’ group (62.5%vs40.0% at 5 years, log-rank=0.056). An aggressive approach was not protective for aortic reintervention at follow up and for death at follow up. Conclusions: The FET technique represents a feasible and efficient option in the treatment of complex thoracic aortic pathologies. An aortic reintervention after FET is very common and the decision-making approach should consider and balance the higher risk of an aggressive approach in terms of post-operative complication versus the higher risk of a second aortic reintervention at follow-up.

Selection of candidate probiotic strains from human microbial niches, for the development of tailored foods designed for specific consumers

My PhD project was intended, throughout the selection of probiotics from human milk and healthy vaginal environment, for the development of tailored fermented foods. According to this aim, several activities were carried out. The first one, concerning the isolation of Lactobacillus and Bifidobacterium strains from human milk to find new probiotic candidates to be included in food products showed promising results. Probiotics have been also proposed to improve female genital health and microbial strains isolated and connected with healthy vaginal ecosystem could be used to prevent or treat vaginal dysbiosis. In this context vaginal lactobacilli previously characterized for their technological features and antagonistic activity against several female uro-genital pathogens were investigated for their metabolic aptitude and additional probiotic features, showing interesting results hypothesizing their inclusion in foods. In addition, in order to preserve vaginal strains viability during food processing/digestion it was also evaluated the potential of microencapsulation by spray-drying. In this framework the results obtained were highly promising from the perspective of using encapsulated powders in food formulations. Another activity connected with the main idea to develop a food strategy for the administration of these vaginal strains was carried out. Lactobacillus crispatus BC4, was supplemented in a Squacquerone cheese, and its digestive fate was evaluated adopting SHIME® system. The results showed that during colonic fermentation, L. crispatus BC4 was metabolically active. Additionally, although probiotic delivery to humans has traditionally been associated with fermented dairy foods, recently the demand for non-dairy-alternatives as potential probiotics carrier is increasing. In this framework, my latest activity was connected with the development of fermented soy milks with encapsulated and non-encapsulated L. crispatus BC4 and L. gasseri BC9. The same fermented soy milks were also investigated for their nutritional qualities and after in vitro digestion for their specific functionality on post-menopausal fecal microbiota and protein bioaccessibility.