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Kefiran, an exopolysaccharide produced by lactic acid bacteria, has received a great interest due to a variety of health claims. In this study, we aim to investigate the physicochemical and biological properties of Kefiran polysaccharide extracted from Portuguese kefir grains. The kefir growth rate was about 56% (w/w) at room temperature and the kefir pH after 24 h was about 4.6. The obtained yield of Kefiran polysaccharide extracted from the kefir grains was about 4.26% (w/w). The Kefiran structural features were showed in the ¹H nuclear magnetic resonance spectrum. The bands observed in the infrared spectrum confirmed that the Kefiran had a β-configuration; and the X-ray photoelectron spectroscopy analysis confirmed the structure and composition of Kefiran and revealed a C/O atomic ratio of 1.46. Moreover, Kefiran showed an average molecular weight (Mw) of 534 kDa and a number-average molecular …