The Impact of Different Inoculation Schemes on the Microbiota, Physicochemical and Sensory Characteristics of Greek Kopanisti Cheese throughout Production and Ripening.

Kopanisti is a Greek PDO cheese, which is traditionally produced by the addition of an amount of over-mature Kopanisti, called Mana Kopanisti, to initiate cheese ripening. The aim of this study was the production of four types of Kopanisti cheese (A-D) using pasteurized cow milk, and a combination of the following starters/adjuncts in order to test their ability to be used in Kopanisti cheese production: A: Lactococcus lactis subsp. lactis and Lacticaseibacillus paracasei, B: L. lactis and Lc. paracasei/Mana Kopanisti, C: L. lactis and Lc. paracasei/Ligilactobacillus acidipiscis and Loigolactobacillus rennini, D: Lig. acidipiscis and Loig. rennini. Throughout production and ripening, classical microbiological, metataxonomics and physicochemical analyses were employed, while the final products (Day 35) were subjected to sensory analysis as well. Most interestingly, beta-diversity analysis of the metataxonomics data revealed the clusters constructed among the Kopanisti types based on the different inoculation schemes. On day 35, Kopanisti A-C types clustered together due to their similar 16S microbiota, while Kopanisti D was highly differentiated. On the contrary, ITS data clustered Kopanisti B and C together, while Kopanisti A and D were grouped seperately. Finally, based on the sensory evaluation, Kopanisti C appeared to have the most suitable bacteria cocktail for the Kopanisti cheese production. Therefore, not only were the conventional starters used, but also the Lig. acidipiscis and Loig. rennini strains could be used in a standardized Kopanisti cheese production that could lead to final products of high quality and safety.

Omics Approaches to Assess Flavor Development in Cheese.

Cheese is characterized by a rich and complex microbiota that plays a vital role during both production and ripening, contributing significantly to the safety, quality, and sensory characteristics of the final product. In this context, it is vital to explore the microbiota composition and understand its dynamics and evolution during cheese manufacturing and ripening. Application of high-throughput DNA sequencing technologies have facilitated the more accurate identification of the cheese microbiome, detailed study of its potential functionality, and its contribution to the development of specific organoleptic properties. These technologies include amplicon sequencing, whole-metagenome shotgun sequencing, metatranscriptomics, and, most recently, metabolomics. In recent years, however, the application of multiple meta-omics approaches along with data integration analysis, which was enabled by advanced computational and bioinformatics tools, paved the way to better comprehension of the cheese ripening process, revealing significant associations between the cheese microbiota and metabolites, as well as their impact on cheese flavor and quality.

Comparison of the Microbiome of Artisanal Homemade and Industrial Feta Cheese through Amplicon Sequencing and Shotgun Metagenomics.

Feta is the most renowned protected designation of origin (PDO) white brined cheese produced in Greece. The fine organoleptic characteristics and the quality of Feta rely on, among other factors, its overall microbial ecosystem. In this study, we employed 16S rDNA and internal transcribed spacer (ITS) amplicon sequencing, as well as shotgun metagenomics, to investigate the microbiome of artisanal homemade and industrial Feta cheese samples from different regions of Greece, which has very rarely been investigated. 16S rDNA data suggested the prevalence of the Lactococcus genus in the homemade samples, while Streptococcus and Lactobacillus genera prevailed in the industrial control samples. Species identification deriving from shotgun metagenomics corroborated these findings, as Lactococcus lactis dominated two homemade samples while Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were found to be dominating one industrial sample. ITS data revealed a complex diversity of the yeast population among the samples analyzed. Debaryomyces, Kluyveromyces, Cutaneotrichosporon, Pichia, Candida, and Rhodotorula were the major genera identified, which were distributed in a rather arbitrary manner among the different samples. Furthermore, a number of potential metagenome-assembled genomes (MAGs) could be detected among assembled shotgun bins. The overall analysis of the shotgun metagenomics supported the presence of different foodborne pathogens in homemade samples (e.g., Staphylococcus aureus, Listeria monocytogenes, Enterobacter cloacae, and Streptococcus suis), but with low to very low abundances. Concluding, the combination of both amplicon sequencing and shotgun metagenomics allowed us to obtain an in-depth profile of the artisanal homemade Feta cheese microbiome.

Microbiome Research as an Effective Driver of Success Stories in Agrifood Systems - A Selection of Case Studies.

Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that showcase the importance of microbiome research in advancing the agrifood system. The selected case studies describe products, methodologies, applications, tools, and processes that created an economic and societal impact. Additionally, they cover a broad range of fields within the agrifood chain: the management of diseases and putative pathogens; the use of microorganism as soil fertilizers and plant strengtheners; the investigation of the microbial dynamics occurring during food fermentation; the presence of microorganisms and/or genes associated with hazards for animal and human health (e.g., mycotoxins, spoilage agents, or pathogens) in feeds, foods, and their processing environments; applications to improve HACCP systems; and the identification of novel probiotics and prebiotics to improve the animal gut microbiome or to prevent chronic non-communicable diseases in humans (e.g., obesity complications). The microbiomes of soil, plants, and animals are pivotal for ensuring human and environmental health and this review highlights the impact that microbiome applications have with this regard.

Zooming Into the Microbiota of Home-Made and Industrial Kefir Produced in Greece Using Classical Microbiological and Amplicon-Based Metagenomics Analyses.

Kefir is a high nutritional fermented dairy beverage associated with a wide range of health benefits. It constitutes a unique symbiotic association, comprising mainly lactic acid bacteria, yeasts, and occasionally acetic acid bacteria, which is strongly influenced by the geographical origin of the grains, the type of milk used, and the manufacture technology applied. Until recently, kefir microbiota has been almost exclusively studied by culture-dependent techniques. However, high-throughput sequencing, alongside omics approaches, has revolutionized the study of food microbial communities. In the present study, the bacterial, and yeast/fungal microbiota of four home-made samples (both grains and drinks), deriving from well spread geographical regions of Greece, and four industrial beverages, was elucidated by culture-dependent and -independent analyses. In all samples, classical microbiological analysis revealed varying populations of LAB and yeasts, ranging from 5.32 to 9.60 log CFU mL-1 or g-1, and 2.49 to 7.80 log CFU mL-1 or g-1, respectively, while in two industrial samples no yeasts were detected. Listeria monocytogenes, Salmonella spp. and Staphylococcus spp. were absent from all the samples analyzed, whereas Enterobacteriaceae were detected in one of them. From a total of 123 isolates, including 91 bacteria and 32 yeasts, Lentilactobacillus kefiri, Leuconostoc mesenteroides, and Lactococcus lactis as well as Kluvyeromyces marxianus and Saccharomyces cerevisiae were the mostly identified bacterial and yeast species, respectively, in the home-made samples. On the contrary, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lacticaseibacillus rhamnosus along with Debaryomyces hansenii and K. marxianus were the main bacterial and yeast species, respectively, isolated from the industrial beverages. In agreement with the identification results obtained from the culture-dependent approaches, amplicon-based metagenomics analysis revealed that the most abundant bacterial genera in almost all home-made samples (both grains and drinks) were Lactobacillus and Lactococcus, while Saccharomyces, Kazachstania, and Kluvyeromyces were the predominant yeasts/fungi. On the other hand, Streptococcus, Lactobacillus, and Lactococcus as well as Kluvyeromyces and Debaryomyces dominated the bacterial and yeast/fungal microbiota, respectively, in the industrial beverages. This is the first report on the microbiota of kefir produced in Greece by a holistic approach combining classical microbiological, molecular, and amplicon-based metagenomics analyses.

Lactobacillus kefiranofaciens: From Isolation and Taxonomy to Probiotic Properties and Applications.

One of the main lactic acid bacterial species found in the kefir grain ecosystem worldwide is Lactobacillus kefiranofaciens, exhibiting strong auto-aggregation capacity and, therefore, being involved in the mechanism of grain formation. Its occurrence and dominance in kefir grains of various types of milk and geographical origins have been verified by culture-dependent and independent approaches using multiple growth media and regions of the 16S rRNA gene, respectively, highlighting the importance of their combination for its taxonomic identification. L. kefiranofaciens comprises two subspecies, namely kefiranofaciens and kefirgranum, but only the first one is responsible for the production of kefiran, the water-soluble polysaccharide, which is a basic component of the kefir grain and famous for its technological as well as health-promoting properties. L. kefiranofaciens, although very demanding concerning its growth conditions, can be involved in mechanisms affecting intestinal health, immunomodulation, control of blood lipid levels, hypertension, antimicrobial action, and protection against diabetes and tumors. These valuable bio-functional properties place it among the most exquisite candidates for probiotic use as a starter culture in the production of health-beneficial dairy foods, such as the kefir beverage.

Unraveling the Microbiota of Natural Black cv. Kalamata Fermented Olives through 16S and ITS Metataxonomic Analysis.

Kalamata natural black olives are one of the most economically important Greek varieties. The microbial ecology of table olives is highly influenced by the co-existence of bacteria and yeasts/fungi, as well as the physicochemical parameters throughout the fermentation. Therefore, the aim of this study was the identification of bacterial and yeast/fungal microbiota of both olives and brines obtained from 29 cv. Kalamata olive samples industrially fermented in the two main producing geographical regions of Greece, namely Aitoloakarnania and Messinia/Lakonia. The potential microbial biogeography association between certain taxa and geographical area was also assessed. The dominant bacterial family identified in olive and brine samples from both regions was Lactobacillaceae, presenting, however, higher average abundances in the samples from Aitoloakarnania compared to Messinia/Lakonia. At the genus level, Lactobacillus, Celerinatantimonas, Propionibacterium and Pseudomonas were the most abundant. In addition, the yeasts/fungal communities were less diverse compared to those of bacteria, with Pichiaceae being the dominant family and Pichia, Ogataea, and Saccharomyces being the most abundant genera. To the best of our knowledge, this is the first report on the microbiota of both olives and brines of cv. Kalamata black olives fermented on an industrial scale between two geographical regions of Greece using metagenomics analysis.

Correction to: Microbiome definition re-visited: old concepts and new challenges.

An amendment to this paper has been published and can be accessed via the original article.

Microbiome definition re-visited: old concepts and new challenges.

The field of microbiome research has evolved rapidly over the past few decades and has become a topic of great scientific and public interest. As a result of this rapid growth in interest covering different fields, we are lacking a clear commonly agreed definition of the term "microbiome." Moreover, a consensus on best practices in microbiome research is missing. Recently, a panel of international experts discussed the current gaps in the frame of the European-funded MicrobiomeSupport project. The meeting brought together about 40 leaders from diverse microbiome areas, while more than a hundred experts from all over the world took part in an online survey accompanying the workshop. This article excerpts the outcomes of the workshop and the corresponding online survey embedded in a short historical introduction and future outlook. We propose a definition of microbiome based on the compact, clear, and comprehensive description of the term provided by Whipps et al. in 1988, amended with a set of novel recommendations considering the latest technological developments and research findings. We clearly separate the terms microbiome and microbiota and provide a comprehensive discussion considering the composition of microbiota, the heterogeneity and dynamics of microbiomes in time and space, the stability and resilience of microbial networks, the definition of core microbiomes, and functionally relevant keystone species as well as co-evolutionary principles of microbe-host and inter-species interactions within the microbiome. These broad definitions together with the suggested unifying concepts will help to improve standardization of microbiome studies in the future, and could be the starting point for an integrated assessment of data resulting in a more rapid transfer of knowledge from basic science into practice. Furthermore, microbiome standards are important for solving new challenges associated with anthropogenic-driven changes in the field of planetary health, for which the understanding of microbiomes might play a key role. Video Abstract.

Whole-genome sequence data and analysis of Lactobacillus delbrueckii subsp. lactis ACA-DC 178 isolated from Greek Kasseri cheese.

Lactobacillus delbrueckii subsp. lactis is employed in the production of various types of cheese. Here, we report the complete genome sequence of L. lactis ACA-DC 178 isolated from Greek Kasseri cheese. The chromosome of ACA-DC 178 contains 2,050,316 bp with a GC content of 49.6%. A total of 2,112 genes were identified in the genome sequence including 1,752 protein-coding genes, 239 putative pseudogenes, 94 tRNA and 27 rRNA genes. According to the COG annotation, about 80% of the protein-coding genes (1,417 proteins) were assigned to at least one functional category. Approximately the 1/3 of these proteins were distributed among three categories, namely replication, recombination and repair (category L: 10.6%), translation, ribosomal structure and biogenesis (category J: 7.5%) and amino acid transport and metabolism (category E: 7.2%). Fourteen integrated GIs with a total of 159 genes were found in ACA-DC 178 genome. Several of these genes encode proteins associated with exopolysaccharide biosynthesis, amino acid transport and subunits of restriction-modification systems. One large CRISPR array of 3,197 bp containing 52 spacers, several of which are identical to phage sequences having hosts in the genus Lactobacillus, was also identified. The annotated genome sequence of L. lactis ACA-DC 178 is deposited at the European Nucleotide Archive under the accession number LS991409. Raw reads are deposited in the Sequence Read Archive (SRR8866601-3).

Comparative Genomics of Streptococcus thermophilus Support Important Traits Concerning the Evolution, Biology and Technological Properties of the Species.

Streptococcus thermophilus is a major starter for the dairy industry with great economic importance. In this study we analyzed 23 fully sequenced genomes of S. thermophilus to highlight novel aspects of the evolution, biology and technological properties of this species. Pan/core genome analysis revealed that the species has an important number of conserved genes and that the pan genome is probably going to be closed soon. According to whole genome phylogeny and average nucleotide identity (ANI) analysis, most S. thermophilus strains were grouped in two major clusters (i.e., clusters A and B). More specifically, cluster A includes strains with chromosomes above 1.83 Mbp, while cluster B includes chromosomes below this threshold. This observation suggests that strains belonging to the two clusters may be differentiated by gene gain or gene loss events. Furthermore, certain strains of cluster A could be further subdivided in subgroups, i.e., subgroup I (ASCC 1275, DGCC 7710, KLDS SM, MN-BM-A02, and ND07), II (MN-BM-A01 and MN-ZLW-002), III (LMD-9 and SMQ-301), and IV (APC151 and ND03). In cluster B certain strains formed one distinct subgroup, i.e., subgroup I (CNRZ1066, CS8, EPS, and S9). Clusters and subgroups observed for S. thermophilus indicate the existence of lineages within the species, an observation which was further supported to a variable degree by the distribution and/or the architecture of several genomic traits. These would include exopolysaccharide (EPS) gene clusters, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-CRISPR associated (Cas) systems, as well as restriction-modification (R-M) systems and genomic islands (GIs). Of note, the histidine biosynthetic cluster was found present in all cluster A strains (plus strain NCTC12958T) but was absent from all strains in cluster B. Other loci related to lactose/galactose catabolism and urea metabolism, aminopeptidases, the majority of amino acid and peptide transporters, as well as amino acid biosynthetic pathways were found to be conserved in all strains suggesting their central role for the species. Our study highlights the necessity of sequencing and analyzing more S. thermophilus complete genomes to further elucidate important aspects of strain diversity within this starter culture that may be related to its application in the dairy industry.

Comparative Genomics of Lactobacillus acidipiscis ACA-DC 1533 Isolated From Traditional Greek Kopanisti Cheese Against Species Within the Lactobacillus salivarius Clade.

Lactobacillus acidipiscis belongs to the Lactobacillus salivarius clade and it is found in a variety of fermented foods. Strain ACA-DC 1533 was isolated from traditional Greek Kopanisti cheese and among the available L. acidipiscis genomes it is the only one with a fully sequenced chromosome. L. acidipiscis strains exhibited a high degree of conservation at the genome level. Investigation of the distribution of prophages and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) among the three strains suggests the potential existence of lineages within the species. Based on the presence/absence patterns of these genomic traits, strain ACA-DC 1533 seems to be more related to strain JCM 10692T than strain KCTC 13900. Interestingly, strains ACA-DC 1533 and JCM 10692T which lack CRISPRs, carry two similar prophages. In contrast, strain KCTC 13900 seems to have acquired immunity to these prophages according to the sequences of spacers in its CRISPRs. Nonetheless, strain KCTC 13900 has a prophage that is absent from strains ACA-DC 1533 and JCM 10692T. Furthermore, comparative genomic analysis was performed among L. acidipiscis ACA-DC 1533, L. salivarius UCC118 and Lactobacillus ruminis ATCC 27782. The chromosomes of the three species lack long-range synteny. Important differences were also determined in the number of glycobiome related proteins, proteolytic enzymes, transporters, insertion sequences and regulatory proteins. Moreover, no obvious genomic traits supporting a probiotic potential of L. acidipiscis ACA-DC 1533 were detected when compared to the probiotic L. salivarius UCC118. However, the existence of more than one glycine-betaine transporter within the genome of ACA-DC 1533 may explain the ability of L. acidipiscis to grow in fermented foods containing high salt concentrations. Finally, in silico analysis of the L. acidipiscis ACA-DC 1533 genome revealed pathways that could underpin the production of major volatile compounds during the catabolism of amino acids that may contribute to the typical piquant flavors of Kopanisti cheese.

Probiotic Features of Lactic Acid Bacteria Isolated from a Diverse Pool of Traditional Greek Dairy Products Regarding Specific Strain-Host Interactions.

The increased consumers' interest on the positive role of food in wellbeing and health underscores the need to determine new probiotic microorganisms. Triggered by the fact that artisanal food products can be a valuable source of novel probiotic strains, 106 lactic acid bacteria, all isolated from traditional Greek dairy products, namely Feta, Kasseri, Xynotyri, Graviera, Formaela, Galotyri, and Kefalotyri cheeses as well as yogurt and milk, were studied for probiotic properties. Based on their survival at pH 2.5 and their stability in the presence of bile salts, 20 strains were selected for further analysis. These strains exhibited diverse susceptibility to commonly used antibiotics, while none was hemolytic. Seven out of the 20 strains produced functional bile salt hydrolases in vitro. The only antimicrobial activity detected of Streptococcus thermophilus ACA-DC 26 against the oral pathogen Streptococcus mutans LMG 14558T was attributed to compound(s) of proteinaceous nature. Two Lactobacillus plantarum strains, namely ACA-DC 2640 and ACA-DC 4039, displayed the highest adhesion according to a collagen-based microplate assay and by using ΗΤ-29 and Caco-2 cells. Co-cultivation of THP-1 cells with selected strains indicated a tendency for anti-inflammatory modulation by Lactobacillus plantarum ACA-DC 2640 as well as Streptococcus thermophilus ACA-DC 26 and ACA-DC 170, as shown by an increase in IL10 mRNA levels. Moreover, milk cell-free supernatants of Lactobacillus plantarum ACA-DC 2640 and ACA-DC 4039 exhibited strong angiotensin I-converting enzyme inhibition. To conclude, new isolates presenting interesting probiotic features were described and should be further investigated as health-promoting factors.

Complete Genome Sequence of the Yogurt Isolate Lactobacillus delbrueckii subsp. bulgaricus ACA-DC 87.

Lactobacillus delbrueckii subsp. bulgaricus is widely used in the production of yogurt and cheese. In this study, we present the complete genome sequence of L. delbrueckii subsp. bulgaricus ACA-DC 87 isolated from traditional Greek yogurt. Whole-genome analysis may reveal desirable technological traits of the strain for dairy fermentations.

Complete Genome Sequence of the Sourdough Isolate Lactobacillus zymae ACA-DC 3411.

Lactobacillus zymae is a Gram-positive lactic acid bacterium belonging to the Lactobacillus brevis clade. Here, we report the first complete genome sequence of L. zymae ACA-DC 3411, which was isolated from traditional Greek wheat sourdough. Whole-genome analysis may reveal adaptive traits of strain ACA-DC 3411 in the sourdough ecosystem.

The complete genome sequence of the yogurt isolate Streptococcus thermophilus ACA-DC 2.

Streptococcus thermophilus ACA-DC 2 is a newly sequenced strain isolated from traditional Greek yogurt. Among the 14 fully sequenced strains of S. thermophilus currently deposited in the NCBI database, the ACA-DC 2 strain has the smallest chromosome, containing 1,731,838 bp. The annotation of its genome revealed the presence of 1,850 genes, including 1,556 protein-coding genes, 70 RNA genes and 224 potential pseudogenes. A large number of pseudogenes were identified. This was also accompanied by the absence of pathogenic features suggesting evolution of strain ACA-DC 2 through genome decay processes, most probably due to adaptation to the milk ecosystem. Analysis revealed the existence of one complete lactose-galactose operon, several proteolytic enzymes, one exopolysaccharide cluster, stress response genes and four putative antimicrobial peptides. Interestingly, one CRISPR-cas system and one orphan CRISPR, both carrying only one spacer, were predicted indicating low activity or inactivation of the cas proteins. Nevertheless, four putative restriction-modification systems were determined that may compensate any deficiencies of the CRISPR-cas system. Furthermore, whole genome phylogeny indicated three distinct clades within S. thermophilus. Comparative analysis among selected strains representative for each clade, including strain ACA-DC 2, revealed a high degree of conservation at the genomic scale, but also strain specific regions. Unique genes and genomic islands of strain ACA-DC 2 contained a number of genes potentially acquired through horizontal gene transfer events, that could be related to important technological properties for dairy starters. Our study suggests genomic traits in strain ACA-DC 2 compatible to the production of dairy fermented foods.

Whole-Genome Sequence of the Cheese Isolate Lactobacillus rennini ACA-DC 565.

In this study, we present the first complete genome sequence of Lactobacillus rennini ACA-DC 565, a strain isolated from a traditional Greek overripened Kopanisti cheese called Mana. Although the species has been associated with cheese spoilage, the strain ACA-DC 565 may contribute to the intense organoleptic characteristics of Mana cheese.

Complete Genome Sequence of the Dairy Isolate Lactobacillus acidipiscis ACA-DC 1533.

Lactobacillus acidipiscis is a Gram-positive lactic acid bacterium belonging to the Lactobacillus salivarius clade. Here, we present the first complete genome sequence of L. acidipiscis isolated from traditional Greek Kopanisti cheese. Strain ACA-DC 1533 may play a key role in the strong organoleptic characteristics of Kopanisti cheese.

Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches.

Over the past decades the food industry has been revolutionized toward the production of functional foods due to an increasing awareness of the consumers on the positive role of food in wellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer's health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut-brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms.

Engineered strains of Streptococcus macedonicus towards an osmotic stress resistant phenotype retain their ability to produce the bacteriocin macedocin under hyperosmotic conditions.

Streptococcus macedonicus ACA-DC 198 produces the bacteriocin macedocin in milk only under low NaCl concentrations (<1.0%w/v). The thermosensitive plasmid pGh9:ISS1 was employed to generate osmotic stress resistant (osmr) mutants of S. macedonicus. Three osmr mutants showing integration of the vector in unique chromosomal sites were identified and the disrupted loci were characterized. Interestingly, the mutants were able to grow and to produce macedocin at considerably higher concentrations of NaCl compared to the wild-type (up to 4.0%w/v). The production of macedocin under hyperosmotic conditions solely by the osmr mutants was validated by the well diffusion assay and by mass spectrometry analysis. RT-PCR experiments demonstrated that the macedocin biosynthetic regulon was transcribed at high salt concentrations only in the mutants. Mutant osmr3, the most robust mutant, was converted in its markerless derivative (osmr3f). Co-culture of S. macedonicus with spores of Clostridium tyrobutyricum in milk demonstrated that only the osmr3f mutant and not the wild-type inhibited the growth of the spores under hyperosmotic conditions (i.e., 2.5%w/v NaCl) due to the production of macedocin. Our study shows how genetic manipulation of a strain towards a stress resistant phenotype could improve bacteriocin production under conditions of the same stress.