In this study, the production of 4,6-α (4,6-α-GTase) and 4,3-α-glucanotransferase (4,3-α-GTase), expressed previously in Lactococcus lactis, was optimized and these enzymes were used to investigate glycemic index reduction and staling delay in bakery products. HP–SEC analysis showed that the relevant enzymes were able to produce oligosaccharides from potato starch or malto-oligosaccharides. Response Surface Methodology (RSM) was used to optimize enzyme synthesis and the highest enzyme activities of 15.63 ± 1.65 and 19.01 ± 1.75 U/mL were obtained at 1% glucose, pH 6, and 30 °C for 4,6-α-GTase and 4,3-α-GTase enzymes, respectively. SEM analysis showed that both enzymes reduced the size of the starch granules. These enzymes were purified by ultrafiltration and used to produce bread and bun at an enzyme activity of 4 U/g, resulting in a decrease in the specific volume of the bread. It was found that the estimated glycemic index (eGI) of bread formulated with 4,6-α-GTase decreased by 18.01%, and the eGI of bread prepared with 4,3-α-GTase decreased by 13.61%, indicating a potential delay in staling. No significant differences were observed in the sensory properties of the bakery products. This is the first study showing that 4,6-α-GTase and 4,3-α-GTase enzymes have potential in increasing health benefits and improving technological aspects regarding bakery products.

Although some lactic acid bacteria species harbor genes encoding α-glucanotransferases (4,6-α-GTase and 4,3-α-GTase), the technological and functional implications of these enzymes within sourdough ecosystems remain poorly understood. This study investigated the effects associated with α-GTase-positive and α-GTase-negative strains of Limosilactobacillus reuteri and L. fermentum on sourdough microbiota, bread quality, and starch digestibility. Starter culture inoculation steered the assembly of the sourdough microbiota, resulting in stable acidification and altered microbial community structures. High-throughput sequencing analysis showed that starter culture addition generally increased taxonomic richness, while Shannon and Simpson diversity indices varied according to strain genotype and inoculation level. Sourdoughs fermented with α-GTase-positive strains exhibited higher viscosity while maintaining shear-thinning behavior, which was putatively linked to in situ enzymatic synthesis. Additionally, inoculation with these strains improved bread specific volume, suggesting a possible structural modification of the starch matrix or the overlapping effects of fermentation metabolites. During storage, the 4,3-α-GTase-positive L. fermentum PFC282 strain delayed the increase in bread hardness, reflecting a possible mitigation of amylopectin retrogradation and indicating a potential anti-staling effect. In addition, the 4,6-α-GTase-positive L. reuteri PFC338 strain produced breads with lower estimated glycemic index values, suggesting a lower susceptibility to enzymatic starch hydrolysis likely due to the hypothesized alterations in structural linkages. Overall, the findings highlight the potential of α-GTase-positive sourdough starter cultures to modulate the technological and nutritional properties of bread.