Summary and concluding remarks

133 Nowadays, the market of food supplements is particularly rich and varied. In recent years, the spread of probiotics containing Lactobacillus or Bifidobacterium mostly in association with other bacteria, is considerably increased.

The demand for health by the population has undergone a sharp increase in recent years in both quantitative and qualitative terms, consumers are becoming in fact more knowledgeable and attentive to the issues of health and prevention.

An increasing number of functional foods are enriched with probiotics and for consumers is becoming increasingly important to get the information necessary to know how to make the best choices.

For these reasons it is necessary an intensive research in order to respond to the needs of the individuals in term of health and prevention.

During my PhD thesis I was in charge for the screening of new potential probiotic strains by combining conventional approach to newly designed platforms especially targeted towards health-promoting applications. I initially focused on the so-called "conventional screening”

aimed to the isolation of new bacterial strains of lactobacilli and bifidobacteria and to assess the safety of the same for use in human food (second signs of national and international regulatory bodies). This first part of the work, in addition to safety, was aimed to assess the efficacy of the bacterial strains: for use in humans is in fact important that the strains are able to survive in the acidic environment of the stomach and able to survive and multiply in the intestine even in the presence of bile salts.

Bacterial strains found to possess these features were then subjected to a more specific screening, depending on the application to which they will be destined. They will therefore be intended to a process of "selection funnel" aimed at the realization of a series of in vitro tests to assess their applicability as probiotics for the promotion of human health.

About that, it is important to emphasize that while some effects of probiotics, such as their ability to reduce intestinal disorders, are well documented; for other conditions are not available strong evidences that demonstrate the efficacy of the same. Moreover, the effects are

“strain-specific” and must not be extended to other probiotics of the same genus or species.

I'm so focused on new areas of application, and I assessed the applicability of the selected strains in food or pharmaceutical field.

134 A first activity of functional characterization of new probiotic strains was carried out in collaboration with the Department of Food Science of the University of Udine and was aimed to evaluate the potential use of new strains in the food supplementation of patients that follow a high-protein diets to reduce the risk of intestinal accumulation of biogenic amines (metabolic application). This activity has allowed me to identify a strain of Lactobacillus able to synthesize biogenic amines and one strain with alleged amino degradative activity that must be confirmed soon. I would like in fact to continue the characterization of this best suitable candidate as BA degraders, by quantifying BA in culture medium incubated with and without the strain, via high-performance liquid chromatography (HPLC)-based methods or alternative developed methods and therefore ranking the degradative activity expressed by this selected strain.

Another screening activity is derived from the collaboration between AAT Srl and the Neurological Institute "Carlo Besta", which had as its main objective the study of the immunoregulatory effects of new strains of lactobacilli and bifidobacteria. Individual strains or combinations of probiotics were evaluated and are resulted good promising candidates for the treatment of inflammatory and autoimmune diseases. Among these were taken into account the myasthenia gravis and multiple sclerosis, diseases of great interest because of their highly debilitating impact on daily life.

The treatment of dendritic cells with different combinations of probiotics led to a strong immune modulation and oral administration showed therapeutic properties, thereby improving the clinical symptoms in both models used for the study of the two diseases.

In particular, we focused on two newly identified strains: L. crispatus LMG P-9437 and B. breve LMG S-28195 and we studied the effect of specific combinations of these two strains with other probiotic strains already available on the market, in order to improve their immunomodulatory action. In the case of myasthenia gravis, we obtained a significant improvement of the symptoms of the disease when given the combination of bifidobacteria while treatment with the lactobacilli was not equally efficient. In the second model of autoimmune disease used we have not observed statistically significant differences between lactobacilli and bifidobacteria, the clinical manifestations are in fact significantly improved both in the case of administration of the lactobacilli which in the case of administration of the strains of bifidobacteria, although this second treatment proved slightly more efficient. These data could be a promising new therapeutic approach for the treatment of autoimmune diseases, the future development will

135 be in fact the translation of these experiments in the in vivo context, by testing strains in patients with these diseases.

Finally, strains were screened also for a food application. The activity was carried out in collaboration with the Institute of Geology and Agro-Food Engineering of the Catholic University of Piacenza and it had the aim of investigate the possibility of producing spray-dried probiotic formulations to be used together with commercial ice-cream bases. A commercial cream base was selected for the study and, according to its composition and literature, two different formulations were investigated (one with maltodextrins, widely used in the food industry and spray-drying processes and one with inulin, a prebiotic fibre with both beneficial effects for humans and probiotics preservation) and used for encapsulation the strain L.

paracasei LMG S-27487. The results show that the maltodextrins formulation could not be used for probiotics encapsulation (null process yield), while inulin formulation gave a lower yield (51

%). A 82 % death cell occurred during drying, but the cells survived in the ice-cream making process. So, these results represent an important starting point for the development of encapsulated multiple bioactive ingredients containing probiotics or other functional compounds.

Summarizing, in this study I showed that the adoption of logical criteria for the in vitro selection of probiotic bacteria can result in the isolation of strains able to exert different effects mainly on human health. However, as reported in the recent literature, it appears unlikely that a single probiotic bacteria strain is able to exert the most different functions but, on the contrary, it is more likely that these effects are strain specific as observed in my study.