EXPLORING LACTOBACILLUS PARACASEI PROBIOSIS AND METABOLIC POTENTIAL

Probiotics have been used so far for the prevention and treatment of various medical conditions and to support general wellness: for this reason, they are currently the subject of significant microbiological and clinical research. In fact, a body of literature suggests that including probiotics in the diet can be a strategy to reduce host-related immune diseases and, in general, modulate the intestinal microbiota composition. Although the mechanisms of action of probiotics are still largely unknown, particularly at molecular level, it is well understood that they can act in different ways, through interaction with the other bacteria residing the same niche, as well as the host, at both local and systemic levels. The genus Lactobacillus, which has important industrial applications as fermented food starter and probiotic adjunct, is a taxonomically broad and heterogeneous group and includes the species Lactobacillus paracasei, which is generally associated with habitats rich in nutrients, such as dairy food and human ecosystems, like gut and vagina. The main purpose of my PhD activity, concerned the study of two L. paracasei strains, named DG and LPC-S01, isolated from two different ecological niches (human gut and vagina, respectively). They are already available on the market, included in two products as food supplements, but their characterization is still incomplete, particularly for LPC-S01. In order to deepen the knowledge about these strains, the first part of the work focused on exploring L. paracasei DG and LPC-S01 essential characteristics to define potential probiotics, by using L. paracasei Shirota as reference strain. The comparative genomic analyses evidenced that strain LPC-S01, a bacterium isolated from human vagina, but plausibly having its origin in the gut, resulted having the genetic features of a niche-generalist member of its species. Similarly, strain DG exhibited the potential ability to adapt to a wide range of environmental conditions if compared with other strains of dairy origin. In vitro tests conventionally used to evidence probiotic properties revealed that strains LPC-S01 and DG possessed comparable ability to resist to gastro-intestinal transit, as evidenced by tolerance to bile, and to decrease NF-\u3baB activation in Caco-2 cells, with respect to strain Shirota. Moreover, LPC-S01 displayed higher tolerance to gastric juice and higher capacity to adhere to Caco-2 epithelial cells (whereas Shirota showed inability to adhere on Caco-2-cells). The in vitro observations were confirmed by setting up a pilot intervention trial on healthy adult volunteers, that demonstrated that LPC-S01 and DG can transiently colonize the gastrointestinal tract of the host, persisting for at least 5 days after the end of a 7-days oral consumption (corresponding to an average of 7 evacuations). Thanks to the comparative genomic analysis on L. paracasei strains DG and LPC-S01, we identified two gene clusters putatively coding for exopolysaccharides biosynthesis related enzymes. Exopolysaccharides (EPSs), apart from their industrial applications, are found to be associated with many physiological functions, although their mechanism of action has not been fully clarified yet. In collaboration with Prof. Andy Laws, University of Huddersfield (United Kingdom), the second part of the work focused on the identification of the potential EPSs matrix from both strains DG and LPC-S01, and their structural characterization. Nonetheless, in the experimental conditions tested so far, only DG resulted able to synthetize EPSs. After performing its purification, we characterized DG derived EPS repeating unit by NMR spectroscopy based approaches. DG EPS structure resulted peculiar and unique compared to those identified in other lactic acid bacteria, prompting us to investigate its immunomodulatory potential, by using it as stimulus on phagocytes. Interestingly, THP-1 macrophages were highly responsive to the EPS stimulus, particularly through the activation of COX-2 expression. Moreover, COX-2 expression was also subjected to an additive effect due to the combination of EPS with the pro-inflammatory stimulus of lipopolysaccharides (LPS). This results strongly suggest a role of DG and its secreted polysaccharidic molecule in triggering stimulatory immune responses together with the activation of protective mechanisms of the intestinal mucosa. Starting by previous observations on other lactic acid bacteria, particularly on Lactococcus lactis, which demonstrated the ability to switch from fermentation to respiratory metabolism, and thanks to the identification of the operon cydABCD in L. paracasei, the third part of the work focused on evaluating if respiration was activated also in the strains under study. The respiratory metabolism, activated by the addition of heme and menaquinone, two essential co-factors not synthetized by the cell, typically results in two main advantages for the cell: increased biomass and long-term survival. Our preliminary data, however, indicated that only one of the two phenotypes occurred in L. paracasei, namely a very strong robustness achieved only upon addition of both heme and menaquinone, that resulted in the maintenance of viability for long periods of storage at 4\ub0 C (>200 days). The robustness phenotype was present also when the two co-factors were added during the storage time, and not during growth. This fact, along with (i) the detection of the added heme only outside the cells, (ii) the under regulation of cydA evaluated by gene expression analyses, and (iii) the decrease of ATP reservoir, suggested that the phenotype observed was not a consequence of a respiratory metabolism. Our data are instead more consistent with the presence of an external electron transfer, as already described for other intestinal bacteria. A deeper investigation is required to clarify the molecular basis of L. paracasei acquired resistance. However, our collected evidences, i.e. the robustness phenotype, can potentially be exploited in industrial applications to ameliorate the technological performances, but also in the niche colonized by the bacterium, where it can counteract environmental stresses and exert its probiotic potential by reducing and neutralizing damaging chemicals in the surrounding environment. In conclusion, this PhD work evidenced the multiple properties of two strains belonging to L. paracasei species to colonize the human gut (and potentially the vagina) upon oral administration, and to interact with the host immune system. Moreover, L. paracasei possesses very interesting metabolic abilities that may be exploited both in in vivo conditions and in the industrial processes

Validation study on new isothermal container for hot ready to eat food in catering establishments: Preliminary results

The production methods, in particular the cook-serve method, applied in high production catering establishments, even if inserted in a correct spatial design, it is affected by time-temperature flows characterized by inadequate designed moments during which critical points can be observed. The aim of this study was to evaluate, at a preliminary stage, how the new technologies can support the supply chain and production processes in catering establishments. To this end, a specific passive and active heat storage system was tested: "Polibox Smart Heater (R)" (PX SH), which makes an isothermal expanded polypropylene container an advanced maintainer for ready to eat food. The experimental design was divided into four different experiments, during which the functionality of the isothermal containers was evaluated, and thermal-tightness tests were carried out. The results showed that the PX SH containers, containing a heat accumulator, are temperature maintainers, which guarantee thermal stability (>65 degrees C), according to the current standard (UNI EN 12571:1999). In conclusion, the production chain of catering establishments should use innovative technologies such as PX SH, in order to benefit from performance consistent with the safety, hygiene and sensory criteria

From a food safety prospective: The role of earthworms as food and feed in assuring food security and in valuing food waste

The Sustainable Development Goals are a set of global goals that provide a framework for shared action. These goals also include the reduction of food waste and the definition of sustainable solutions to achieve food security. In this context, the aim of the study was to describe all phases of a pilot earthworm rearing project started in September 2017 and concluded in December 2017, together with a risk analysis carried out in order to evaluate if earthworms can represent a safe and sustainable protein source for human consumption and/or animal nutrition. The conversion rate, that in this study is more appropriately identified as the \u201cwaste reduction efficiency,\u201d was also calculated in order to define the extent to which earthworm rearing can contribute to the objective of reducing fruit and vegetable waste (FVW). The results showed that earthworms can bio-convert 3750 kg of FVW in three months producing 1050 kg of compost and 82 kg of fresh earthworms with minimal environmental impact showing good waste reduction efficiency. Moreover, the risk analysis conducted on earthworm rearing highlighted a microbiological hazard after the freeze-drying phase. The critical control point was therefore identified, and, in order to guarantee the total food safety of the finished product, corrective action was taken consisting in the implementation of heat treatment\u2014sterilization at 121\u25e6C for 20 min. The results of microbiological analyses carried out on the earthworm meal after the sterilization treatment showed that the treatment guarantees microbiological safety for the consumer and ensures a balanced approach in relation to two main topics\u2014public health and food-borne diseases. In conclusion, earthworm meal is a concentrate of valuable nutrients useful for human and animal nutrition and can also transform fruit and vegetable waste into a resource

The sustainability of urban food systems : the case of mozzarella production in the city of Milan

Background: Urban food production in the developed world has recently been facing the challenge of feeding the world\u2019s cities adequately and on a sustainable basis, and innovative models of food production can help this transition. Nevertheless, this field of research largely focuses on primary food production\u2014so-called urban farming\u2014while the subsequent steps of the food chain have scarcely been investigated. In this context, this study aimed to provide a preliminary evaluation of urban food processing, taking mozzarella cheese production in the city of Milan as a case study. Methods: Five urban farmers participated in a direct survey aiming at identifying how sustainable this specific production chain can be considered. Results: Results demonstrated that the concept of urban food production is really valorized through measures able to enhance the global sustainability of the food production process only in some of the dairy micro- enterprises and that the economic dimension must be considered the variable most critically involved in hampering the global sustainability of this specific food chain. Conclusions: This research suggests the need to disseminate a global governance of urban supply chains and to support local activities, which represent a sensitive and strategic sector

Probiotics action on gliadin sequences relevant to gluten sensitivity

The Celiac disease in genetically predisposed individuals is mainly induced by specific repetitive sequences in gliadins (PQPYP). This autoimmune disease stems from the interaction between toxic sequences and lamina propria cells, that is relevant also to other forms of gluten sensitivity. Specific endo-esoprolinase were isolated from lactic acid bacteria, suggesting possible practical applications. The ability of some probiotics at removing "toxic" celiac sequences was investigated, at first by assessing the presence and level of endo- and eso-prolinase activity in some of the most popular probiotic bacteria. Significant activities were detected in Lactobacillus and Bifidum species, as well as in the probiotic Escherichia coli Niessle 1917. On the basis of prolinase data, we investigated by mass spectroscopy the removal of "toxic" sequences in gliadin. A complete disappearance of these sequences was observed only with Escherichia coli Niessle 1917. Among the Bifidus and Lactobacillus species, only B. bifidum MIMBb23SG and L. acidophilus LA5 showed a significant decrease in the "toxic" sequences. All together, this study suggests a potential use of lactic bacteria to lower gluten response in sensitive individuals, including celiacs and gluten-sensitive

FATTY ACIDS AND STEROLS IN SEEDS FROM WILD SPECIES OF BERBERIS IN ARGENTINE PATAGONIA

Abstract Seeds of Berberis heterophylla an

When Innovation meets Tradition : the case of " Riso &Rane" Rural District in Lombardy Region

Lombardy, with 87.393 hectares of rice is one of the leader region for this production in Italy and in the European Union (EU) too. This area is characterized by a strong connection with tradition both in terms of agricultural landscape and food culture. Nevertheless, during the last decade, farmers faced increasing competitiveness issues, mostly related to EU subsidies losses, market prices and the technical constraints of the traditional rice supply chain: provider of technical means, farmers, brokers and rice mill. In this scenario, the \u201cRiso e Rane\u201d Rural District (R&RD) supports farmers in improving competitiveness through innovation. The aim of the paper is to investigate the innovation in the rice supply chain related to the specific action of R&RD, that accounts for 60 farms. Starting from the direct survey carried out on the district productive structures, we investigate the farms' degree of innovation related to the adoption of a new model of supply chain. The case study areas is characterized by rice that represents the most important culture with 2.773 hectares (more than 58% of the district Utilized Agricultural Area (UAA)). In 2012, R&RD won a regional project titled \u201cBuono, Sano e Vicino\u201d with the aim to help local rice farmers developing an alternative supply chain in which the district grow into the local actor to increase farmers bargaining power and promotes new market strategies. To make this the attention was focused on one of the most important variety of Italian rice: Carnaroli. The main results of the study showed that the project was able to innovate the traditional supply chain in all the four innovation areas according to OECD (2005): product, process, market and organization. In conclusion, our results suggest that the R&RD is able to respond to farmers necessities in term of market competitiveness and to improve the sustainability of local food system

Detection of perfluoroalkyl acids and sulphonates in Italian eel samples by HPLC-HRMS Orbitrap

Perfluoroalkyl substances (PFASs) contain one or more carbon-bound hydrogens substituted by fluorine. Since the 1950s, these compounds have been used to manufacture fat- and water-resistant fabrics, paper and food containers, and to produce photographic films, firefighting foams, detergents and insecticides. The widespread use and global distribution of PFASs, have led to their accumulation in the environment. Food, particularly fish and other seafood, is considered the main route of human exposure to PFASs. Consequently, the European Food Safety Authority (EFSA) recommends that more data be collected, to build a database on the contamination levels of the individual PFASs in food, to evaluate a reliable chronic risk to the European consumers. This requires high-sensitivity analytical methods, to increase the number of quantifiable samples and, thereby, improve the credibility of exposure assessments. In this context, the aim of the present research is to develop and validate a sensitive and specific method based on high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) analysis, to monitor the presence of 16 PFASs in Italian eels (Anguilla anguilla) from the Italian Lake Garda. The detection limits (CCa) and detection capability (CCb) in the order of pg g1 , the recoveries between 80 and 101% and the other validation parameters fulfilled the requirements of Commission Decision 657/ 2002/EC. The identification and quantification of PFASs, up to 11 in the same sample, showed a similar distribution among 90 eels. Perfluorooctane sulphonic acid (PFOS) and perfluorobutanoic acid (PFBA) were the analytes more frequently found in the eel samples (94 and 82%, respectively)

An efficient continuous flow process for the synthesis of a non-conventional mixture of fructooligosaccharides

A sustainable and scalable process for the production of a new mixture of fructooligosaccharides (FOS) was developed using a continuous-flow approach based on an immobilized whole cells-packed bed reactor. The technological transfer from a classical batch system to an innovative flow environment allowed a significant improvement of the productivity. Moreover, the stability of this production system was ascertained by up to 7 days of continuous working. These results suggest the suitability of the proposed method for a large-scale production of the desired FOS mixture, in view of a foreseeable use as a novel prebiotic preparation

Prevalence of Anisakis Larvae in Different Fish Species in Southern Albania: Five-Year Monitoring (2016-2020)

Anisakidae are nematodes that commonly parasitize in the coelomic cavity and viscera of several fish species. They can be found in flesh, which is why they have an important economic and public health impact. The aim of the current work was to assess the presence and prevalence of Anisakis larvae in fish species caught in the coastal area of the Karaburun Peninsula in Vlora Bay (Albania). A total of 856 of wild teleosts and 219 specimens of farmed fish were collected over a 5-year period (from 2016 to 2020). The results showed that out of a total of 1075 analyzed samples, 361 (33.58%) were parasitized with L3 larvae. In particular, only Solea vulgaris returned negative results, while Sparus aurata, Dicentrarchus labrax, and Sardinella aurita showed the lowest prevalence (4.55%, 9.17%, and 10.53%, respectively) and mean abundance (0.84, 1.19, and 0.92, respectively). Conversely, Scomber japonicus and Scomber scombrus showed the highest prevalence (74.07% and 68.00%, respectively) and mean abundance (188.24 and 249.82, respectively). The data suggest that the coastal area of the Karaburun Peninsula (southern Albania) may be a high-risk area for zoonotic diseases, and the consumption of raw or undercooked fish caught in the Vlora district could result in the acquisition of human anisakiasis. For these reasons, it is necessary to improve the surveillance plan