افزايش مقياس توليد پكتيك اليگوساكاريدها توسط باكتري پروبيوتيك و بررسي خواص ضد سرطاني آنها

Scale up of the pectic-oligosaccharides production by a probiotic bacterium an‎d investigation of their anticancer¬ properties

زيست شناسي سلولي مولكولي و ميكروبيولوژي

Abstract Upcycling fruit waste into health-promoting ingredients is an urgent sustainability challenge. Degradation of pectin, which is abundant in fruit waste, into its oligosaccharide subunits, introduce an approach to eliminate a vast portion of food waste an‎d production of anticancer an‎d antioxidant compounds. In this work, a microbial degradation is described that converts apple pectin into bioactive pectic oligosaccharides (POS) using Bacillus subtilis HA1, a strain isolated from traditional yogurt. Acid an‎d bile tolerance, antibiotic resistance an‎d enterotoxin production of HA1 were examined to eva‎luate its safety an‎d resistance to digestive fluids. TLC, LC-ESI-MS, an‎d FTIR methods were then performed to detect the resulted oligosaccharides. Moreover, the antioxidant activity of the obtained POS was eva‎luated using DPPH radical scavenging method. Their anticancer activity was also checked through MTT an‎d flowcytometry tests. HA1 is γ-hemolytic, lecithinase-negative, an‎d free of nhe/hbl enterotoxin genes, yet endures acid/bile an‎d adheres to intestinal cells, confirming its safety an‎d probiotic aptitude. The bacterium cleaves pectin within 6 h of incubation, an‎d under optimum conditions (50 °C, pH 6), the extracellular pectinase showed a maximum activity of ≈18 IU/mL. TLC, LC–ESI–MS, an‎d FTIR verify the formation of low-methylated mono- to tri-galacturonic acids. Crude POS scavenge up to 90 % of DPPH radicals at 10 mg/mL, which is five-fold higher than untreated pectin. POS also act selec‎tively against tumor cells: MCF-7 breast cancer viability dro‎ps to 17 %, while healthy L-929 an‎d HUVEC cells remain ≥95 % viable. Flow cytometry an‎d qRT-PCR confirm apoptosis via Bax up-regulation an‎d galectin-3 suppression. To further scale up POS production, a fed-batch culture was carried out in a 5 L bioreactor using a Bacillus subtilis strain isolated from a commercial “Foods for Gut” product that contains this bacterium as its sole probiotic strain. By applying H₂O₂ stress (at a concentration of 0.5 mM) an‎d nitrogen starvation (removal of nitrogen sources from the growth medium), 3.6 g/L of POS was produced from 5 g/L of pectin. In addition, the intrinsic ability of Bacillus to secrete a mixture of extracellular enzymes enabled the bacterium to grow efficiently on the white portion of pomelo (pomelo albedo) treated with 0.5% edible yeast extract. Incorporation of this fermented material into yogurt resulted in a probiotic product with bioactive functionality. Altogether, probiotic can‎didate strains belonging to the B. subtilis afford a safe, eco-friendly route to high-value POS with potent antioxidant an‎d anticancer activities, opening ways for functional foods, nutraceuticals, an‎d sustainable pectin valorization. We introduce a novel approach to scale-up the production of POS from pectin using a probiotic bacterial strain, which is a safe method for Upcycling fruit waste into value-added compounds. Keywords: Pectic oligosaccharides (POS); probiotic; Fermenter; Anticancer; Antioxidant

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