The suitability of Lactiplantibacillus plantarum (L. plantarum) as a functional starter culture in Nham fermentation was investigated, with a focus on evaluating both its probiotic attributes and fermentation capability. L. plantarum BCC 4352 (LpbBCC4352) exhibited colony-associated antimicrobial activity against Kocuria rhizophila, L. plantarum, Latilactobacillus sakei ssp. sakei, and Pediococcus pentosaceus, as well as the zoonotic Streptococcus suis. LpbBCC4352 exhibited impressive acid (pH 2.5) and bile resistance, coupled with notable survival rates in a simulated human digestive model. In addition, the strain is able to utilize fructo-oligosaccharides in simulated human colon conditions. It also displayed robust adhesion to human colon cell monolayers (Caco-2) and gastric mucin. Furthermore, it showed a promising cholesterol reduction ability in the fermentation medium. The safety of LpbBCC4352 for human consumption was confirmed through a hemolytic activity assay and antibiotic susceptibility testing. Moreover, using LpbBCC4352 as a starter culture not only enhanced the firmness of Nham but also ensured consumer satisfaction. The overall findings emphasize the potential use of LpbBCC4352 as a safe and effective functional starter culture, particularly in the production of Nham.

Heterologous protein secretion in filamentous fungi is often constrained by limitations in signal peptide recognition and intracellular trafficking. Aspergillus oryzae, a food-grade industrial fungus, has a robust native secretory system. However, its capacity for recombinant protein secretion remains suboptimal. Here, we developed a two-step, carrier-free engineering strategy to enhance protein secretion in A. oryzae. We identified endogenous signal peptides among highly secreted proteins using a green fluorescent protein (GFP) reporter. The oryzin signal peptide SPAoalp1 increased GFP secretion 5.50-fold compared with a no-signal-peptide control. We co-overexpressed Aosly1, a Sec1/Munc18 family protein that regulates soluble N-ethylmaleimide-sensitive factor attachment protein receptor–mediated vesicle trafficking, which, in combination with SPAoalp1, increased secretion approximately two-fold compared with SPAlp1 control and ten-fold with no-SP control. Applying the engineered platform for genetic improvement of heterologous bovine κ-casein increased secretion from 0.11 to 0.24 mg/L. Physiological optimization further increased secretion. The developed system provided initial evidence for secretion of a ~12 kDa band consistent with Aopafb transcription, with MIC₉₀ values of 4.56–8.24% (v/v) against two Candida albicans strains and 4.68% (v/v) against Aspergillus niger. The system offers a modular framework for engineering fungal secretion and expands the utility of A. oryzae for recombinant protein production.