Abstract: This study integrated a gelatin-sodium alginate composite hydrogel system with 3D bioprinting technology to construct a functional starter culture with a dual-layered spatial structure that allows for its fast and slow release, aiming to enhance the survival and metabolic stability of Lacticaseibacillus paracasei PC18 in blueberry juice, highly acidic and rich in polyphenols. The results showed that this dual-layered model achieved spatiotemporally controlled release of lactic acid bacteria: the outer CaCl2 layer promoted rapid initiation in the early fermentation stage, significantly increasing viable cell count; the inner calcium ethylenediaminetetraacetate (EDTA-Ca) layer continuously released calcium during the mid-to-late fermentation stages, maintaining microbial viability and metabolic stability, with the final viable bacterial count reaching 8.75 (lg(CFU/mL)), pH steadily decreasing to 3.82, and total acid accumulation reaching 177.74 mg/L. Furthermore, the starter effectively improved the retention and transformation of functional components: the total phenol concentration reached 1 158.02 mg/L at 48 h, the anthocyanin concentration peaked at 9.14 mg/L at 12 h, and the starter demonstrated stable antioxidant performance, scavenging 62.12% of 2,2’-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation and 69.87% of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical after 72 h of fermentation. This study confirms that the multi-calcium source 3D printing strategy synergistically optimizes the fermentation process, offering an effective solution to address the decline in microbial viability and insufficient metabolic regulation during acidic juice fermentation, thereby demonstrating potential for the development of functional fermented foods.

Key words: gelatin-sodium alginate; 3D printing; lactic acid bacteria; fast-slow release; blueberry juice fermentation