Marsak, NimetAkan, EcemYavas, AdemErbay, Zafer2026-02-272026-02-2720251867-13061867-131410.1007/s12602-025-10876-whttp://dx.doi.org/10.1007/s12602-025-10876-whttps://hdl.handle.net/20.500.14669/4612This study evaluated the viability and functional properties of postbiotics derived from Bifidobacterium animalis subsp. lactis BB-12 following thermal and ultrasound processing. Sixteen processing conditions were tested, including heat treatments (65-95 degrees C for 5-90 min) and ultrasound amplitudes (98-320 mu m for 15-60 min). Postbiotics were assessed for antimicrobial activity, probiotic-stimulating effects on Lacticaseibacillus casei 431 and Bifidobacterium spp. cultures, and their capacity to enhance short-chain fatty acid (SCFA) production. Complete inactivation was achieved under all heat treatment conditions, whereas only 11 ultrasound conditions resulted in total loss of culturability, indicating greater microbial resistance at lower intensities or shorter exposures. Discrepancies between flow cytometry and colony counting in ultrasound-treated samples suggest the presence of viable but non-culturable cells (VBNC), highlighting the limitations of culture-based viability assessments, especially when assessing VBNC states. Postbiotics from heat-treatment showed significantly stronger probiotic-enhancing effects, with growth increases up to 2.9-fold for L. casei and 37.7-fold for Bifidobacterium spp. compared to controls, alongside greater antimicrobial activity, especially against Enterococcus faecalis. Both processing methods significantly increased SCFA levels (p < 0.05). In conclusion, it can be said that heat treatment was more effective than ultrasound in producing biologically active postbiotics from B. lactis BB-12. The enhanced functional properties observed in heat-inactivated preparations underscore thermal processing as a robust method for postbiotic production. These findings highlight the potential of heat-derived postbiotics as active bioingredients for microbiota-targeted functional foods and nutraceuticals. Further in vivo validation and standardization efforts are needed to fully demonstrate their therapeutic potential and support regulatory approval.eninfo:eu-repo/semantics/closedAccessPostbioticParaprobioticProbioticHeat treatmentUltrasoundShort chain fatty acidArticle; Early Access41369876WOS:001636050400001