In cheese production, microorganisms are usually added at the beginning of
the process as primary starters to drive curd acidification, while secondary
microorganisms, with other pro-technological features important for cheese
ripening, are added as selected cultures. This research aimed to investigate
the possibilities of influencing and selecting the raw milk microbiota using
artisanal traditional methods, providing a simple method to produce a natural
supplementary culture. We investigated the production of an enriched raw milk
whey culture (eRWC), a natural adjunct microbial culture produced from mixing
an enriched raw milk (eRM) with a natural whey culture (NWC). The raw milk was
enriched by spontaneous fermentation for 21 d at 10°C. Three milk enrichment
protocols were tested: heat treatment before incubation, heat treatment plus salt
addition, and no treatment. The eRMs were then co-fermented with NWC (ratio of
1:10) at 38°C for 6 h (young eRWC) and 22 h (old eRWC). Microbial diversity during
cultures’ preparation was evaluated through the determination of colony forming
units on selective growth media, and next-generation sequencing (16S rRNA
gene amplicon sequencing). The enrichment step increased the streptococci and
lactobacilli but reduced microbial richness and diversity of the eRMs. Although
the lactic acid bacteria viable count was not significantly different between
the eRWCs, they harbored higher microbial richness and diversity than NWC.
Natural adjunct cultures were then tested in cheese making trials, following the
microbial development, and assessing the chemical quality of the 120 d ripened
cheeses. The use of eRWCs slowed the curd acidification in the first hours of
cheese making but the pH 24 h after production settled to equal values for all
the cheeses. Although the use of diverse eRWCs contributed to having a richer
and more diverse microbiota in the early stages of cheese making, their effect
decreased over time during ripening, showing an inferior effect to the raw milk
microbiota. Even if more research is needed, the optimization of such a tool could
be an alternative to the practice of isolating, geno-pheno-typing, and formulating
mixed-defined-strain adjunct cultures that require knowledge and facilities not
always available for artisanal cheese makers.