Avena sativa (A. sativa) oats have recently made a comeback as suitable alternative raw materials for dairy substitutes due to their functional properties. Amylolytic and cellulolytic enzyme-assisted modifications of oats produce new products that are more appealing to consumers. However, the biochemical and functional alteration of products and extracts requires careful selection of raw materials, enzyme cocktails, and technological aspects. This study compares the biochemical composition of different A. sativa enzyme-assisted water extracts and evaluates their microbial growth using spontaneous fermentation and the antimicrobial properties of the ferment extracts. Fibre content, total phenolic content, and antioxidant activity were evaluated using traditional methodologies. The degradation of A. sativa flour was captured using scanning electron microscopy (SEM); moreover, sugar and oligosaccharide alteration were identified using HPLC and HPLC-SEC after INFOGEST in vitro digestion (IVD). Additionally, taste differentiation was performed using an electronic tongue with principal component analysis. The oat liquid extracts were continuously fermented using two ancient fermentation starters, birch sap and Tibetan kefir grains. Both starters contain lactic acid bacteria (LAB), which has major potential for use in bio-preservation. In fermented extracts, antimicrobial properties against Gram-positive Staphylococcus aureus and group A streptococci as well as Gram-negative opportunistic bacteria such as Escherichia coli and Pseudomonas aeruginosa were also determined. SEM images confirmed the successful incorporation of enzymes into the oat flour. The results indicate that using enzyme-assisted extraction significantly increased TPC and antioxidant activity in both the extract and residues. Additionally, carbohydrates with a molecular mass (MM) of over 70,000 kDa were reduced to 7000 kDa and lower after the incorporation of amylolytic and cellulolytic enzymes. The MM impacted the variation in microbial fermentation, which demonstrated favourable antimicrobial properties. The results demonstrated promising applications for developing functional products and components using bioprocessing as an innovative tool.

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