Coagulase-negative staphylococci (CNS) are a group of Staphylococcus characterized by the absence of coagulase production. Numerous studies have demonstrated that CNS exhibit favorable fermentation characteristics, enhancing sensory attributes such as color, aroma, and taste in fermented foods (Heo et al., 2020; Sánchez Mainar et al., 2017). CNS is widely distributed in fermented meat products, and its prevalence and biodiversity in traditionally fermented meat products have been largely revealed. Taking into consideration a traditional Mediterranean fermented sausage like salami from Italy, Spain, Croatia and Slovenia, CNS were the predominant bacteria with S. epidermidis, S. saprophyticus, S. equorum, and S. xylosus the most detected species (Barbieri et al., 2021). In Europe, meat products from southern European markets were reported to be dominated by S. xylosus and S. equorum, while meat products from northern European markets were dominated by S. carnosus (Van Reckem et al., 2019). On the other hand, S. sciuri and S. xylosus were the main top bacteria in traditional dry sausages from different regions of Northern China (Hu et al., 2020).

Proteins, one of main components of meat, are degraded by endogenous proteases and microbial proteases during meat fermentation, thereby producing small peptides and free amino acids (Wang et al., 2022; Yu et al., 2020). Peptides and amino acids are essential precursors in the production of major volatile compounds, including aldehydes, alcohols, acids, esters, and sulfur compounds, which contribute to the formation of characteristic flavors of fermented meat products. For example, branched-chain amino acids (leucine, isoleucine, and valine) are decomposed by enzymatic/non-enzymatic reactions into methyl-branched aldehydes, methyl-branched alcohols, and methyl-branched carboxylic acids (Chen et al., 2022a). Chen et al. (2024) reviewed the flavor contribution, formation pathways, and enhancement strategies of aroma-active compounds derived from branched-chain amino acids in fermented meat products, and revealed that these flavor compounds demonstrate high odor activity values and should be enhanced in the future research. 3-methylbutanal, as a catabolite of leucine with nutty/malty/chocolate like aroma, exhibits a notably low flavor threshold and relatively high abundance in fermented meat products (Chen et al., 2022c; Liu et al., 2025), thereby significantly contributing to the overall flavor of the products.

As to the synthesis of 3-methylbutanal, leucine catabolism pathway involved in bacteria is illustrated as follows (see Fig. 1), leucine firstly undergoes deamination catalyzed by branched-chain amino acid aminotransferase (Bc-AT), utilizing α-ketoglutaric acid (α-KG) as the amino acceptor to yield α-ketoisocaproate (α-KIC). Simultaneously, L-glutamic acid could transform into α-KG to keep deamination abovementioned going, a process facilitated by glutamic dehydrogenase (GDH) (Afzal et al., 2017). Then, α-KIC undergoes non-oxidative decarboxylation catalyzed by α-ketoacid decarboxylase (KADC), yielding 3-methylbutanal. On the other hand, α-KIC is catalyzed by α-ketoacid dehydrogenase (KADH) into isovaleryl-CoA, subsequently which undergoes conversion to 3-methylbutanoic acid through the actions of phosphotransferases (PTA) and acyl kinases (ACK) (Afzal et al., 2012; Chen et al., 2022a). Furthermore, the interconversion of 3-methylbutanal, 3-methylbutanoic acid, and 3-methylbutanol is facilitated by alcohol dehydrogenase (AlcDH) and aldehyde dehydrogenase (AldDH). These compounds can also form corresponding esters via acyltransferases or esterases, with some such as ethyl 3-methylbutyrate having very low flavor threshold, e.g., 0.01 μg/kg (Lu et al., 2021), and significantly imparting pleasant fruity flavor to fermented meat products.

Addition of starter culture is one of strategies for enhancing the levels of branched-chain flavor compounds. Studies have focused on leucine catabolism of lactic acid bacteria, including the investigation of metabolic routes responsible for the biosynthesis of 3-methyl butanal in Carnobacterium maltaromacticum cultured in TSB-YE (Afzal et al., 2012); the characterization of branched-chain amino acid transaminase in Lactobacillus fermentum and its application in myofibrillar proteins model (Liu et al., 2022); the characterization of keto acid decarboxylation and dehydrogenation pathways for the production of 3-methyl butanal in Lactococcus lactis cultured in an AA medium (Chen et al., 2022a), and so on. On the other hand, certain attention has been paid to leucine catabolism of CNS, which is commonly added in fermented meat products. Bosse et al. (2017) reported that the inoculation of S. carnosus LTH 3838 alone into cured loins increased the content of 3-methylbutanal. Zhao et al. (2022) also demonstrated that the sausages fermented with S. carnosus alone or S. carnosus in combination with P. pentosaceus significantly raised the production of 3-methylbutanal and 2-methylbutanoic acid compared to the non-inoculated control. Our research group has reviewed and summarized metabolic pathways of staphylococci involved in generating aromas of fermented sausages, including the important way of amino acids catabolism, especially branched-chain amino acids conversion (Chen et al., 2025a).

The means of enhancing leucine-derived catabolite levels through CNS starter culture has been regarded as a promising strategy to improve the flavor of fermented meat products, however, the role of CNS on the development of pleasant nutty aroma is revealed little from a mechanistic perspective. The present study aims to develop a starter culture with the ability of improving nutty aroma in the formulation of fermented sausage, through the way of evaluating the production capability of nutty note, safety and technological properties of 16 Staphylococci isolated strains from fermented meat products, and inoculating the strain with excellent performances to ferment sausage. Metagenomics and volatolomics profiling were integrated to reveal correlations between the microbiota and volatile flavor compounds, clarifying the effect of the inoculum on the quality of fermented sausage through metabolic pathways annotation and analysis of the inoculated group in contrast to non-inoculated control. These results will provide sufficient scientific support for developing functional starter culture for fermented sausage production.