EXOPOLYSACCHARIDES OF LACTIC ACID BACTERIA ISOLATED FROM LABNEH: CHARACTERIZATION, BIOACTIVITIES, AND EFFECTS ON GUT MICROBIOME AND FERMENTED MILK RHEOLOGY

Exopolysaccharides (EPS), produced by Lactic Acid Bacteria (LAB), are carbohydrate polymers with unique physicochemical properties and various biological activities widely used in food and pharmaceutical industries. This dissertation is aimed to (1) screen 42 new isolates from traditional yogurt-like products (Labneh) for their probiotic nature and identify the LAB strains using 16S rDNA sequencing; (2) optimize the conditions like pH, temperature, time, and quantity of sucrose for the growth of EPS produced by four selected LAB isolates; (3) purify and characterize EPS for the contents and investigate the bioactivities of the partially purified EPS; (4) study the effects of the EPS on the gut microbiome and the rheological properties of fermented skimmed bovine milk. The results showed that 10 out of 42 isolates had good probiotic potential and were identified using 16S rDNA sequencing. The LAB isolates had reductions of 4.5 to 8.5 Log10 CFU/ml after in vitro digestion. The LAB exhibited cholesterol-lowering (> 30%), pathogen-inhibiting properties, and hydrophobicity values of 7–86%, 18–87%, and 21–87% (for xylene, octane, and hexadecane, respectively). Resistance to lysozyme activity was also high in the selected LAB isolates. Out of 10, four LAB isolates Lactobacillus delbrueckii, Lacticaseibacillus rhamnosus, Enterococcus faecium, and Streptococcus thermophilus were used to produce EPS-LB3, EPS-MLB3, EPS-LB13 and EPS-MLB10, respectively. The optimum conditions to produce EPS were pH 6, at 43ºC for 48 h with 20% sucrose, and the phenol-sulfuric acid method revealed that the average EPS production was 261.4 ± 15.2 mg/L. The refractive index detector showed that the average molecular weight ranged from 1272.2 kDa to 3762.4 kDa, and the diode array detector revealed the presence of galactose, lactose, glucose, mannose, xylose, ribose, arabinose, and galacturonic acid in different concentrations when derivatized using Phenyl-3-Methyl-5-Pyrazolone (PMP). The scavenging rate of the partially purified EPS at 250 mg/L against 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), 2,2’-Azino-bis (3-ethylbenzene-thiazoline-6-sulphonic acid) radical (ABTS), superoxide dismutase, superoxide anion radical, hydrogen peroxide, hydroxyl radical, metal chelating activity, and inhibition of lipid peroxidation ranged from 10% to 88%. The antioxidant capacity for Ferric Reducing Antioxidant Power (FRAP), Reducing Power (RP), and Total Antioxidant Capacity (TAC) ranged between 714 to 2848 μg/mL, equivalent to ascorbic acid activity. The partially purified EPS inhibited the growth and biofilm formation of E. coli, S. typhimurium, S. aureus, and L. monocytogenes with an average inhibition of 53% to 74%. The average inhibition of amylase, glucosidase, cholesterol, and Angiotensin-Converting Enzyme (ACE) was 2% to 66%, and inhibition of Caco-2 and MCF-7 cancer cells was 9% to 83%. All the EPSs supported the growth of beneficial gut bacteria from Proteobacteria, Bacteroidetes, Firmicutes, and Acinetobacter in fecal fermentation with total Short Chain Fatty Acids (SCFA) production from 4530 to 6104 PPM. This study showed that the extracted EPS from the selected LABs has good bioactivities, prebiotic nature, and impact gut microbiota. Moreover, the EPS-producing bacteria improve the gelation time of fermented skimmed bovine milk. Further studies are required to investigate the EPS effect in vivo conditions and to use industrial waste for EPS production using the LAB.