Shotgun Metagenomics and Volatilome Profile of the Microbiota of Fermented Sausages

Abstract

Changes in the microbial gene content and abundance can be analyzed to detect shifts in the microbiota composition due to the use of a starter culture in the food fermentation process, with the consequent shift of key metabolic pathways directly connected with product acceptance. Meat fermentation is a complex process involving microbes that metabolize the main components in meat. The breakdown of carbohydrates, proteins, and lipids can lead to the formation of volatile organic compounds (VOCs) that can drastically affect the organoleptic characteristics of the final products. The present meta-analysis, performed with the shotgun DNA metagenomic approach, focuses on studying the microbiota and its gene content in an Italian fermented sausage produced by using a commercial starter culture (a mix of and), with the aim to discover the connections between the microbiota, microbiome, and the release of volatile metabolites during ripening. The inoculated fermentation with the starter culture limited the development of and reduced the microbial diversity compared to that from spontaneous fermentation. KEGG database genes associated with the reduction of acetaldehyde to ethanol (EC 1.1.1.1), acetyl phosphate to acetate (EC 2.7.2.1), and 2,3-butanediol to acetoin (EC 1.1.1.4) were most abundant in inoculated samples (I) compared to those in spontaneous fermentation samples (S). The volatilome profiles were highly consistent with the abundance of the genes; elevated acetic acid (1,173.85 μg/kg), ethyl acetate (251.58 μg/kg), and acetoin (1,100.19 μg/kg) were observed in the presence of the starters at the end of fermentation. Significant differences were found in the liking of samples based on flavor and odor, suggesting a higher preference by consumers for the spontaneous fermentation samples. Inoculated samples exhibited the lowest scores for the liking data, which were clearly associated with the highest concentration of acetic acid. We present an advance in the understanding of meat fermentation by coupling DNA sequencing metagenomics and metabolomics approaches to describe the microbial function during this process. Very few studies using this global approach have been dedicated to food, and none have examined sausage fermentation, underlying the originality of the study. The starter culture drastically affected the organoleptic properties of the products. This finding underlines the importance of starter culture selection that takes into consideration the functional characteristics of the microorganism to optimize production efficiency and product quality.

Portuguese cacholeira blood sausage: A first taste of its microbiota and volatile organic compounds

“…Moreover, L. sakei is able to utilize glucose, fructose and different hexoses as primary energy sources during the initial growth stage; hence, it is likely that sugar added to the analyzed cacholeira sausages might have selected for the massive growth of this microorganism. L. sakei produces lactate mainly deriving from the homolactic fermentation of hexoses via the glycolytic pathway, whereas acetate is produced through heterolactic fermentation of pentoses (Ferrocino et al, 2018). Moreover, as reported by Fadda et al (1999), L. sakei exerts proteinase and aminopeptidase activities on proteins of sarcoplasmic and myofibrillar origin (Fadda et al, 1999), using free amino acids and even nucleotides as energy sources (Ferrocino et al, 2018).…”

“…The notable adaptation of L. sakei to the meat environment is also suggested by of its previous detection in Italian, Belgian, French and Greek salami (Federici et al, 2014;Aquilanti et al, 2016;Cardinali et al, 2018;Janssens, Myter, De Vuyst & Leroy, 2012;Aquilanti et al, 2007), irrespective of their ripening time. As evidenced by Ferrocino et al (2018), in dry fermented sausages, carbohydrate metabolic pathways of L. sakei are responsible for the development of VOCs, such as acetate, acetoin, diacetyl, acetic acid and isobutyric acid that notably contribute to the definition of the typical aroma of these products. Moreover, L. sakei is able to utilize glucose, fructose and different hexoses as primary energy sources during the initial growth stage; hence, it is likely that sugar added to the analyzed cacholeira sausages might have selected for the massive growth of this microorganism.…”

Sausage fermentation and starter cultures in the era of molecular biology methods

“…specific organisms) and instead to understand the relationships between microorganisms and their activities and functionalities in a particular niche (Cocolin et al, 2017). To understand and characterize the composition and function of the microbiota in a food ecosystem the evolution of the massive sequencing technologies such as shotgun DNA-seq or RNA-seq can help (Ferrocino et al, 2018). In the field of food microbiota structure and function, the identification of enzymes, pathways and mechanisms and how these operate under specific conditions may perhaps be of superior value than determining the taxonomic composition of specific samples alone (Santiago-Rodriguez et al, 2016).…”

“…The organoleptic characteristics of the final products are influenced by volatile organic compounds (VOCs) that are produced through the breakdown of carbohydrates, proteins and lipids. Ferrocino et al (2018) focused on studying the microbiota development and functions in an Italian fermented sausage through a shotgun DNA metagenomic approach. For the first time an integrated analysis related to volatilome profile, microbiota, gene content and consumers acceptability was presented.…”

Proteome Response of Staphylococcus xylosus DSM 20266T to Anaerobiosis and Nitrite Exposure

“…Likewise, Ferreira et al (2013) reported that, during the anaerobic growth, S. aureus COL-S strain used glucose at a rate higher than in presence of oxygen; lactate was the main end-product, but minor amounts of acetate, ethanol and 2,3-butanediol were also formed. Moreover, a shotgun metagenomics of the microbiota of sausages fermented with Lactobacillus sakei and S. xylosus revealed genes associated with the reduction of acetaldehyde to ethanol and acetyl phosphate to acetate and 2,3-butanediol to acetoin ( Ferrocino et al, 2018 ). In S. xylosus DSM 20266T the absence of oxygen prevents carbon flow through the TCA cycle, decreasing the concentrations of biosynthetic intermediates (such α-ketoglutarate, oxaloacetate) and consequently amino acids biosynthesis, such as glutamate/glutamine and aspartate/asparagine ( Figure 3 ).…”