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The postharvest treatment and processing of fresh coffee cherries can impact the quality of the unroasted green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through two different wet and dry methods to monitor differences in the microbial community structure and in substrate and metabolite profiles. The changes were followed throughout the postharvest processing chain, from harvest to drying, by implementing up-to-date techniques, encompassing multiple-step metagenomic DNA extraction, high-throughput sequencing, and multiphasic metabolite target analysis. During wet processing, a cohort of lactic acid bacteria (i.e., Leuconostoc , Lactococcus , and Lactobacillus ) was the most commonly identified microbial group, along with enterobacteria and yeasts ( Pichia and Starmerella ). Several of the metabolites associated with lactic acid bacterial metabolism (e.g., lactic acid, acetic acid, and mannitol) produced in the mucilage were also found in the endosperm. During dry processing, acetic acid bacteria (i.e., Acetobacter and Gluconobacter ) were most abundant, along with Pichia and non- Pichia ( Candida , Starmerella , and Saccharomycopsis ) yeasts. Accumulation of associated metabolites (e.g., gluconic acid and sugar alcohols) took place in the drying outer layers of the coffee cherries. Consequently, both wet and dry processing methods significantly influenced the microbial community structures and hence the composition of the final green coffee beans. This systematic approach to dissecting the coffee ecosystem contributes to a deeper understanding of coffee processing and might constitute a state-of-the-art framework for the further analysis and subsequent control of this complex biotechnological process. IMPORTANCE Coffee production is a long process, starting with the harvest of coffee cherries and the on-farm drying of their beans. In a later stage, the dried green coffee beans are roasted and ground in order to brew a cup of coffee. The on-farm, postharvest processing method applied can impact the quality of the green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through wet and dry processing in four distinct variations. The microorganisms present and the chemical profiles of the coffee beans were analyzed throughout the postharvest processing chain. The up-to-date techniques implemented facilitated the investigation of differences related to the method applied. For instance, different microbial groups were associated with wet and dry processing methods. Additionally, metabolites associated with the respective microorganisms accumulated on the final green coffee beans.

This study describes the genetic analysis of the riboflavin (vitamin B 2 ) biosynthetic ( rib ) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification.

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA , folB , folKE , folP , and folC . The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC , encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA , encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis , Streptococcus thermophilus , and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum , were not able to produce folate. Folate production was further investigated in L . lactis as a model organism for metabolic engineering and in S . thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S . thermophilus excreted more folate than L . lactis , presumably as a result of differences in the number of glutamyl residues of the folate produced. In S . thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L . lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S . thermophilus , but pH had no effect on folate excretion by L . lactis . Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p -aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.