Impact of long-term preservation techniques on microbiota, volatilome and proteolysis in tempeh produced via back-slopping

Back-slopping is widely used in many food fermentations to ensure process continuity and product consistency. The feasibility of using tempeh in back-slopping has not yet been adequately investigated. This study hypothesized the possibility of storing and reusing tempeh as a starter for back-slopping. Tempeh was frozen, dried and lyophilized for up to 6 months and then used in back-slopping. The microbiota, proteolytic profile and volatilome of tempeh produced by back-slopping (BS-tempeh) were analyzed using culture-based and metataxonomic approaches, HPLC and GC-MS, respectively. Standard plate count analyses revealed a predominance of Rhizopus spp., whereas Enterobacteriaceae, coliforms and other undesirable microorganisms remained below detection limits in all samples. The microbiota profile showed that BS-tempeh produced with frozen starter better preserved the microbiota, with α-diversity values comparable to the control at T0 (p = 0.054), whereas BS-tempeh obtained with dried starter increased α-diversity (T0, p = 0.0076; T3, p = 0,0061; T6, p = 0.015) and introduced low-abundance and potentially opportunistic taxa, including Klebsiella, Enterococcus and other Enterobacteriaceae. No statistically significant differences in proteolysis and amino acid profiles were observed among the samples (p > 0.05). Multivariate analysis of volatilome profile revealed that the fermentation process led to a convergence of the volatile profiles, regardless of starter preservation type. Overall, the results demonstrated that frozen tempeh offers the highest microbiological stability compared to drying and lyophilization, highlighting the potential application of tempeh back-slopping in artisanal production and small- to medium-scale industrial settings as an alternative or complement to commercial starters.