Iovine, Andrea (2025) Microbial and plant polysaccharides as resource for nutraceuticals. [Tesi di dottorato]

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Abstract

Nowadays, the use of nutraceuticals is increasing, since their consistent consumption over time allows for the achievement of physical well-being to prevent numerous diseases. Nutraceuticals are foods or food supplements that provide beneficial and healing effects to the host organism, in addition to their nutritional properties. Among the main ingredients of nutraceuticals are probiotics and prebiotics. Probiotics are viable microorganisms that provide beneficial effects to the body by colonizing the gastrointestinal tract and include Bifidobacterium spp., lactic acid bacteria (LAB), and yeasts. Prebiotics, on the other hand, are substances that are not digestible by the host organism but by the microbiota, promoting their growth. The main sources of prebiotics are plants, which are ingested in the diet through the consumption of cereals, herbs, fruits, and vegetables. The beneficial effects of probiotics and prebiotics come from the substances they produce, and polysaccharides play a key role in this. Indeed, microbial and plant polysaccharides exert antioxidant, antimicrobial, antitumor, anti-inflammatory, and blood cholesterol and glucose regulation properties. This is because polysaccharides are a class of bioactive macromolecules with the highest structural variability, which, however, makes their structural elucidation difficult. Indeed, studies that relate the structure and the activity of the carbohydrates are often lacking or non-existent, resulting in biological function being attributed to products for which the structure is unknown or only preliminary information are available. Therefore, this Ph.D. topic focuses on the extraction, purification, and structural characterization of novel microbial and plant polysaccharides as potential ingredients for nutraceutical formulations. Specifically, from the probiotic species L. paracasei NPB01, L. rhamnosus GG, and L. Plantarum DSA_B, the cell wall glycans have been structurally elucidated to evaluate their possible involvement in beneficial activity of these probiotic species on the host, as well as for the EPS from S. bacillaris to candidate this yeast as a probiotic species. Similarly, plant polysaccharides from the species S. cardifolia and M. sylvestris were structurally elucidated with the final aim of evaluating their immunostimulatory activities for humans and animals. Lastly, an off-topic regard the study of the Gram (-) bacterial cell wall glycans, through the opportunity of having been able to structurally characterize the CPS from two strains of A. baumannii MRSN31468 and MRSN31468-pJJK10, corresponding to the wild-type and mutant strains, respectively. L. paracasei is a member of the human microbiota and is widely found in dairy products as well as in the lactic ferment for infants to promote the maturation and the strengthening of their immune system. These activities may be directed at the cell wall glycan; therefore, the L. paracasei cell wall polysaccharides were isolated and studied using NMR and GC-MS techniques. A polyglycerol phosphate based teichoic acid polymer, and two capsular polysaccharides have been identified, the first one (CPS-1) with a repeating unit consisting of glucose, galactose, glucosamine and rhamnose also in the branching, and the second one (CPS-2), with a lower molecular weight, with a repeating unit consisting of a galactosamine and several rhamnoses, and non-stoichiometrically branched to a rhamnose residues with a glucose, glucosamine and rhamnose trisaccharide, or glucose only, making the entire structure non-regular and heterogeneous. Although the structures of teichoic acid and CPS-2 have already been widely reported in the literature, the structures for CPS-1 isolated from L. paracasei represent a total novelty. Hence, they were biologically evaluated together with CPS-2 and the TA on Caco-2 human cells. The CPS-1 has antimicrobial activity, while the CPS-2 is anti-inflammatory. Lastly, the TA shows poorer bioactivity than the CPSs. L. rhamnosus is a Gram (+) bacterium, belonging to the LAB group, and is recognised as GRAS. It’s a transient of the human microbiota, and it ferments almost all dairy products. L. rhamnosus GG also has a tolerogenic action in children with cow’s milk allergy (CMA), as well as exhibits antimicrobial and antioxidant activities, and these properties can be mediated by the cell wall glycans. Therefore, the cell wall glycans from L. rhamnosus were extracted, purified, and a well-known lipoteichoic acid and CPS were found together with a novel oligosaccharide whose putative structure was elucidated via NMR and composed of a rhamnose and N-acetyl galactosamine backbone and branched with rhamnose and glucose, in a non-stoichiometric manner. Therefore, a NOESY spectrum will be necessary to correctly detect the primary sequence of the oligosaccharide, as well as mass analysis and molecular weight evaluation, to then associate it with the biological function. L. plantarum is a Gram-positive and mesophilic bacterium belonging to the LAB group. It is recognized as GRAS and is involved in various food industry processes as well as intestinal treatments. Most notably, L. plantarum is known for being a probiotic, exhibiting antimicrobial and antioxidant activities, and being part of the human microbiota. This activity can be linked to its cell wall polysaccharide, and therefore, the capsular polysaccharide was structurally elucidated using NMR and mass spectrometry. In detail, two extraction methods were used, both essential for determining the CPS structure. One method involved autoclaving, which rapidly broke down the CPS into its components, revealing a backbone composed of a repeating glucose unit and a phosphorylated galactose, with branching involving both glucose and galactose in pyranose and furanose forms. The other method used lysozyme digestion to obtain the intact CPS, although it was not clearly visible in NMR; thus, mild acidic hydrolysis was necessary to identify the individual components, which matched those from autoclaving. Therefore, the CPS from L. plantarum consists of a [-3)-Glc-α-(1→3)-Glc-β-(1→3)-Gal-β-(1-] backbone, branched via a phosphodiester bridge on the galactose with a [Galf-β-(1→3)-Glc-β-(1→3)-Gal-β-(1-] branch. From a biological perspective, the CPS’s branching exhibits antioxidant activity, in addition to the backbone’s antimicrobial effects. However, the intact CPS primarily shows antimicrobial activity, whereas when hydrolysed, it also demonstrates antioxidant properties. Simulated digestion experiments indicated that the CPS maintains its structure through different digestive conditions, reaching the intestinal tract mostly intact, with 85% hydrolysed during the oral phase and 15% remaining. S. bacillaris is a very famous yeast since involved in the wine fermentation processes together with S. cerevisiae. In fact, it’s isolated from the grapes and can live under the harsh, high ethanol concentration environments. However, it’s recently been discovered that S. bacillaris has also antimicrobial activity against S. aureus, as well as can regulate cholesterol and glucose in the blood. The scientific interest is in candidate S. bacillaris as a new probiotic organism, and hence to discover new molecules produced by S. bacillaris that have beneficial activities on the host. Therefore, the EPS was extracted and structurally characterized via NMR, resulting in a (1→6) mannose polysaccharide in different lengths. Therefore, biological assays will be necessary to evaluate the bioactivity and to candidate S. bacillaris as a new probiotic organism for nutraceuticals. S. cardifolia and M. sylvestris are two different plant species belonging to the Malvaceae and widely used as ingredients such as salads and soups, as well as widely used in Oriental traditional medicines for the treatment of Gastrointestinal or respiratory disease. Preliminary studies show that the aqueous extracts of S. cardifolia and M. sylvestris were very rich in polysaccharides and with a strong immunostimulant activity. Therefore, the plant polysaccharides were extracted and isolated from S. cardifolia ed M. sylvestris, respectively. S. cardifolia produces an arabinan with a non-stoichiometric repeating unit and with different lengths, so much so that the same glycan was discovered both in oligo and polysaccharide length. The arabinan polysaccharide shows a strong immunostimulant activity on the RAW 264.78 cells, stimulating the NO production, the pinocytosis, viability, and cell reproduction, as well as being screened in 12 human primary cell systems simulating various human tissue and disease characteristics. From M. sylvestris, two different Type II arabinogalactan polymers were extracted and structurally elucidated and both with a non-stoichiometric repeating unit and therefore called AG-1 and AG-2. AG-2 was found to be larger than AG-1 and with some differences in the sugar motifs, but both were constituted with a -(1→6) galactose backbone and branched with different arabinose oligosaccharides. Final and decisive biological tests are underway to clarify their immunomodulatory properties and to make them good ingredients for functional feeds. Lastly, A. baumannii corresponds to the off-topic project carried out during this Ph.D. and is not included in the Ph.D. aim, but was studied as an opportunity to improve the expertise also on the Gram (-) cell wall glycans study, as well as the Gram (+) ones. In fact, A. baumannii is a Gram (-) nosocomial pathogen, and in recent times, its antibiotic resistance is becoming an issue to be resolved. Therefore, the CPS structural study is crucial to discover new CPS-target therapies, and in fact, during the Ph.D., a little time was dedicated to the NMR study of both wild and mutant types of A. baumannii, which differ in a nonulosonic acid residue on the CPS repeating unit. Both CPs from A. baumannii were structurally elucidated, and both were composed of a glucose and galactosamine backbone, branched on the glucose moiety with a disaccharide of galactose and pseudaminic acid. In the wild type, the Pse residue was O-4 acetylated for 70% while in the mutant CPS, the Pse was substituted with the C-8 epimer for 84% and keeping the same O-4 acetylation degree.

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