This study explored the potential of two forms of sourdough—native and lyophilised—obtained through the spontaneous fermentation of whole grain flours from ancient wheat varieties, for cookie production. The research involved evaluated the dough’s rheological properties through creep and recovery measurements and Mixolab analysis, assessing proximate composition, physical attributes, texture, colour, and sensory characteristics using the Rate-all-that-apply (RATA) method. The rheological analysis revealed that native sourdough significantly impacted dough behaviour, making it more challenging to process. Although differences were observed in the proximate composition, colour, and texture, these factors did not influence the samples as much as the rheological parameters. Sensory evaluation identified Khorasan lyophilised sourdough, along with its control sample, as the most promising, while modern wheat, spelt, and emmer exhibited potentially undesirable attributes. Based on these findings, it was concluded that lyophilised Khorasan sourdough was very favourable for cookie production and should be considered for further in-depth research and development. This suggests that the lyophilised forms of ancient wheats could offer valuable alternatives for cookie formulation, with implications for both the processing and sensory attributes of the final product.
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