Synthetic ecology of the human gut microbiota.

Despite recent advances in sequencing and culturing, a deep knowledge of the wiring and functioning of the human gut ecosystem and its microbiota as a community is still missing. A holistic mechanistic understanding will require study of the gut microbiota as an interactive and spatially organized biological system, which is difficult to do in complex natural communities. Synthetic gut microbial ecosystems can function as model systems to further current understanding of the composition, stability and functional activities of the microbiota. In this Review, we provide an overview of the current synthetic ecology strategies that can be used towards a more comprehensive understanding of the human gut ecosystem. Such approaches that integrate in vitro experiments using cultured isolates with mathematical modelling will enable the ultimate goal: translating mechanistic and ecological knowledge into novel and effective therapies.

Design of synthetic microbial consortia for gut microbiota modulation.

The use of and interest in probiotics to modulate the human intestinal microbiota have strongly increased in recent years. However, most of the current probiotic products have been limited to single-strain formulations of easily culturable food-grade microorganisms and often resulted in mixed results or limited effects on host health. Therefore, a revision of current probiotic strategies by using synthetic human-derived microbial multispecies consortia is necessary. In light of this ongoing evolution of the field, novel approaches are needed to design and assemble bacterial cocktails targeted to restore dysbiotic states in microbiota-associated diseases. This review discusses the steps in the process for identifying effective targets, predicting putative multistrain communities, assembling ecosystems in silico and in vitro and monitoring stability and outputs before in vivo trials.

Characterization and Degradation of Pectic Polysaccharides in Cocoa Pulp.

Microbial fermentation of the viscous pulp surrounding cocoa beans is a crucial step in chocolate production. During this process, the pulp is degraded, after which the beans are dried and shipped to factories for further processing. Despite its central role in chocolate production, pulp degradation, which is assumed to be a result of pectin breakdown, has not been thoroughly investigated. Therefore, this study provides a comprehensive physicochemical analysis of cocoa pulp, focusing on pectic polysaccharides, and the factors influencing its degradation. Detailed analysis reveals that pectin in cocoa pulp largely consists of weakly bound substances, and that both temperature and enzyme activity play a role in its degradation. Furthermore, this study shows that pulp degradation by an indigenous yeast fully relies on the presence of a single gene (PGU1), encoding for an endopolygalacturonase. Apart from their basic scientific value, these new insights could propel the selection of microbial starter cultures for more efficient pulp degradation.

Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production.

Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerant Saccharomyces cerevisiae cultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several new S. cerevisiae hybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomyces species that produce very large amounts of fruity esters (Pichia kluyveri and Cyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated with S. cerevisiae starter cultures and, to a lesser extent, in batches inoculated with P. kluyveri and Cyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most short-chain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor.

Aseptic artificial fermentation of cocoa beans can be fashioned to replicate the peptide profile of commercial cocoa bean fermentations.

The fermentation of cocoa beans is essential for the generation of flavour precursors that are required later on to form the flavour components of chocolate. From the many different precursors that are generated, oligopeptides and free amino acids comprise a significant proportion as some of them form Maillard reaction products during the roasting process. Therefore, the diversity of peptides is an important contributing factor to the quality of a fermentation which is in turn controlled by proteolytic activity within the cocoa bean, and is driven by changes in the presence of fermentation by-products as a result of microbial activity outside the bean. Being able to control proteolytic activity within the bean using only the presence of fermentation by-products would prove a valuable tool in the study of these proteases and the processing of cocoa storage proteins. Thus, this tool would help elucidate key mechanisms that generate the components responsible for flavour. In this study, we describe an artificial fermentation system, free from microbial activity, which is able to replicate proteolytic degradation of protein as well as to generate similar peptide fragments as seen during a commercial fermentation. It was also found that acidification is a main contributor to protein degradation.

Biochemical fate of vicilin storage protein during fermentation and drying of cocoa beans.

Key cocoa-specific aroma precursors are generated during the fermentation of cocoa beans via the proteolysis of the vicilin-like globulin. Previous studies had shown that degradation of this particular 566 amino acid-long storage protein leads to three distinct subunits with different molecular masses. Although oligopeptides generated from the proteolysis of vicilin-like globulin have been studied previously, changes occurring to vicilin at different stages of fermentation have not yet been explored in detail. The aim of this study was to investigate the fate of vicilin protein from the non-fermented stage up to the dried cocoa beans. Our results showed a remarkable shift in the electrophoretic mobility of vicilin towards higher pI during the onset of fermentation. The pI-shifted subunit was found susceptible to further degradation into a lower-molecular-weight vicilin subunit. The observed pI shift correlated with, but did not depend on protein phosphorylation. Glycosylation of some but not all vicilin subunits occurred at different stages of the fermentation process. Peptides generated from vicilin throughout fermentation were analyzed by UHPLC-ESI-MS/MS revealing an initial increase and subsequent decrease in the diversity of peptides with an increasing degree of fermentation. We furthermore describe the rate of degradation of different vicilin subunits. The detected diversity and dynamics of vicilin peptides will help to define biochemical markers of distinct steps of the fermentation process.

Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations.

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.

Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is an ultra-high resolution mass spectrometry technique used mainly in analysis of unresolved complex mixtures comprising tens of thousands of analytes. For the first time, it was used to analyze samples of raw fermented cocoa beans originating from Cameroon and Ivory Coast. The direct infusion mass spectra of the raw fermented cocoa bean extracts showed 10091 and 10911 peaks, resp., rating cocoa among the most complex organic mixtures ever analyzed. Automated molecular formula calculations could assign 2995 and 2968 of the peaks, resp. to formulae containing only C, H, O, N≤3 and S≤1 atoms. The formulae were separated into four groups depending on their heteroatom content and the intensities of the groups were compared in class plots, showing the highest population in the CHON species, but the highest abundance in the CHO species. Elemental ratios obtained from the molecular formulae were plotted in an intensity coded three-dimensional modification of the van Krevelen diagram. For the CHO species, the van Krevelen diagram showed that most of the intensity belongs to the lipid, polyphenol and carbohydrate regions of the plot. The biggest difference was observed in the CHON group, assigned as peptide degradation products, where the Ivorian beans showed greater variety and molecular diversity and higher total intensity of the nitrogen containing compounds, in accordance with the fact that the Ivorian beans show generally higher nitrogen content than the Cameroon beans. FTICR-MS proves capable not only for high-throughput comparison of major classes of metabolites from cocoa samples from different origins, but also can give insight into the different molecular formulae comprising these compound classes.

Detailed analysis of the microbial population in Malaysian spontaneous cocoa pulp fermentations reveals a core and variable microbiota.

The fermentation of cocoa pulp is one of the few remaining large-scale spontaneous microbial processes in today's food industry. The microbiota involved in cocoa pulp fermentations is complex and variable, which leads to inconsistent production efficiency and cocoa quality. Despite intensive research in the field, a detailed and comprehensive analysis of the microbiota is still lacking, especially for the expanding Asian production region. Here, we report a large-scale, comprehensive analysis of four spontaneous Malaysian cocoa pulp fermentations across two time points in the harvest season and two fermentation methods. Our results show that the cocoa microbiota consists of a "core" and a "variable" part. The bacterial populations show a remarkable consistency, with only two dominant species, Lactobacillus fermentum and Acetobacter pasteurianus. The fungal diversity is much larger, with four dominant species occurring in all fermentations ("core" yeasts), and a large number of yeasts that only occur in lower numbers and specific fermentations ("variable" yeasts). Despite this diversity, a clear pattern emerges, with early dominance of apiculate yeasts and late dominance of Saccharomyces cerevisiae. Our results provide new insights into the microbial diversity in Malaysian cocoa pulp fermentations and pave the way for the selection of starter cultures to increase efficiency and consistency.

Identification of a five-oxidoreductase-gene cluster from Acetobacter pasteurianus conferring ethanol-dependent acidification in Escherichia coli.

Acetobacter pasteurianus, a Gram-negative bacterium belonging to the α-divison of Proteobacteria, produces acetic acid through ethanol oxidation. A genomic bank of A. pasteurianus 386B DNA was cloned in the low-copy cosmid pRG930Cm vector and the resulting clones were screened for the production of protease using the skimmed-milk agar assay whereby a clearing zone around the inoculated spots indicates casein degradation. Several positive clones were selected and restriction analysis revealed that many contained the same inserts. One clone was further analyzed and the cosmid DNA subjected to in vitro transposon insertion. After electroporation, several clones having lost the capacity to cause casein degradation were isolated and the sequence of the transposon-flanking regions analyzed. The majority of insertions mapped to one gene encoding an NAD(P)(+)-dependent aldehyde dehydrogenase (ALDH) of the PNTB superfamily, whereas one insert was found upstream in a gene encoding an ethanol dehydrogenase. Addition of phenol red to the medium confirmed the ethanol-dependent acidification around the inoculated spots of the clones without transposon insertion, suggesting that casein degradation is due to the production of acetic acid as a result of the combined activities of the alcohol dehydrogenase and ALDH. Quantitative data and pH measurements confirmed a significant acidification, and the presence of acetic acid.

Mannitol production by heterofermentative Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in free and controlled pH batch fermentations.

Certain lactic acid bacteria, especially heterofermentative strains, are capable to produce mannitol under adequate culture conditions. In this study, mannitol production by Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in modified MRS medium containing a mixture of fructose and glucose in a 6.5:1.0 ratio was investigated during batch fermentations with free pH and constant pH 6.0 and 5.0. Mannitol production and yields were higher under constant pH conditions compared with fermentations with free pH, the increase being more pronounced in the case of the L. fermentum strain. Maximum mannitol production and yields from fructose for L. reuteri CRL 1101 (122 mM and 75.7 mol%, respectively) and L. fermentum CRL 573 (312 mM and 93.5 mol%, respectively) were found at pH 5.0. Interestingly, depending on the pH conditions, fructose was used only as an alternative external electron acceptor or as both electron acceptor and energy source in the case of the L. reuteri strain. In contrast, L. fermentum CRL 573 used fructose both as electron acceptor and carbon source simultaneously, independently of the pH value, which strongly affected mannitol production by this strain. Studies on the metabolism of these relevant mannitol-producing lactobacilli provide important knowledge to either produce mannitol to be used as food additive or to produce it in situ during fermented food production.

Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria.

Spontaneous organic cocoa bean box fermentations were carried out on two different farms in Brazil. Physical parameters, microbial growth, bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the fermented dry cocoa beans. The main end-products of the catabolism of the pulp substrates (glucose, fructose, and citric acid) by yeasts, LAB, and AAB were ethanol, lactic acid, mannitol, and/or acetic acid. Lactobacillus fermentum and Acetobacter pasteurianus were the predominating bacterial species of the fermentations as revealed through (GTG)(5)-PCR fingerprinting of isolates and PCR-DGGE of 16S rRNA gene PCR amplicons of DNA directly extracted from fermentation samples. Fructobacillus pseudoficulneus, Lactobacillus plantarum, and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Also, three novel LAB species were found. This study emphasized the possible participation of Enterobacteriaceae in the cocoa bean fermentation process. Tatumella ptyseos and Tatumella citrea were the prevailing enterobacterial species in the beginning of the fermentations as revealed by 16S rRNA gene-PCR-DGGE. Finally, it turned out that control over a restricted bacterial species diversity during fermentation through an ideal post-harvest handling of the cocoa beans will allow the production of high-quality cocoa and chocolates produced thereof, independent of the fermentation method or farm.

Adaptation of Lactobacillus plantarum IMDO 130201, a wheat sourdough isolate, to growth in wheat sourdough simulation medium at different pH values through differential gene expression.

Sourdough is a very competitive and challenging environment for microorganisms. Usually, a stable microbiota composed of lactic acid bacteria (LAB) and yeasts dominates this ecosystem. Although sourdough is rich in carbohydrates, thus providing an ideal environment for microorganisms to grow, its low pH presents a particular challenge. The nature of the adaptation to this low pH was investigated for Lactobacillus plantarum IMDO 130201, an isolate from a laboratory wheat sourdough fermentation. Batch fermentations were carried out in wheat sourdough simulation medium, and total RNA was isolated from mid-exponential-growth-phase cultures, followed by differential gene expression analysis using a LAB functional gene microarray. At low pH values, an increased expression of genes involved in peptide and amino acid metabolism was found as well as that of genes involved in plantaricin production and lipoteichoic acid biosynthesis. The results highlight cellular mechanisms that allow L. plantarum to function at a low environmental pH.

Dynamics and species diversity of communities of lactic acid bacteria and acetic acid bacteria during spontaneous cocoa bean fermentation in vessels.

To speed up research on the usefulness and selection of bacterial starter cultures for cocoa bean fermentation, a benchmark cocoa bean fermentation process under natural fermentation conditions was developed successfully. Therefore, spontaneous fermentations of cocoa pulp-bean mass in vessels on a 20 kg scale were tried out in triplicate. The community dynamics and kinetics of these fermentations were studied through a multiphasic approach. Microbiological analysis revealed a limited bacterial species diversity and targeted community dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation, as was the case during cocoa bean fermentations processes carried out in the field. LAB isolates belonged to two main (GTG)(5)-PCR clusters, namely Lactobacillus plantarum and Lactobacillus fermentum, with Fructobacillus pseudofilculneus occurring occasionally; one main (GTG)(5)-PCR cluster, composed of Acetobacter pasteurianus, was found among the AAB isolates, besides minor clusters of Acetobacter ghanensis and Acetobacter senegalensis. 16S rRNA-PCR-DGGE revealed that L. plantarum and L. fermentum dominated the fermentations from day two until the end and Acetobacter was the only AAB species present at the end of the fermentations. Also, species of Tatumella and Pantoea were detected culture-independently at the beginning of the fermentations. Further, it was shown through metabolite target analyses that similar substrate consumption and metabolite production kinetics occurred in the vessels compared to spontaneous cocoa bean fermentation processes. Current drawbacks of the vessel fermentations encompassed an insufficient mixing of the cocoa pulp-bean mass and retarded yeast growth.

Influence of temperature and backslopping time on the microbiota of a type I propagated laboratory wheat sourdough fermentation.

Sourdough fermentation is a cereal fermentation that is characterized by the formation of stable yeast/lactic acid bacteria (LAB) associations. It is a unique process among food fermentations in that the LAB that mostly dominate these fermentations are heterofermentative. In the present study, four wheat sourdough fermentations were carried out under different conditions of temperature and backslopping time to determine their effect on the composition of the microbiota of the final sourdoughs. A substantial effect of temperature was observed. A fermentation with 10 backsloppings (once every 24 h) at 23°C resulted in a microbiota composed of Leuconostoc citreum as the dominant species, whereas fermentations at 30 and 37°C with backslopping every 24 h resulted in ecosystems dominated by Lactobacillus fermentum. Longer backslopping times (every 48 h at 30°C) resulted in a combination of Lactobacillus fermentum and Lactobacillus plantarum. Residual maltose remained present in all fermentations, except those with longer backslopping times, and ornithine was found in almost all fermentations, indicating enhanced sourdough-typical LAB activity. The sourdough-typical species Lactobacillus sanfranciscensis was not found. Finally, a nonflour origin for this species was hypothesized.

Wickerhamomyces anomalus in the sourdough microbial ecosystem.

We previously found that Wickerhamomyces anomalus (formerly Hansenula anomala, Pichia anomala) was the second most frequently isolated yeast in Belgian artisan bakery sourdoughs and that the yeast dominated laboratory sourdough fermentations. Such findings are of interest in terms of the advantage of W. anomalus over other commonly encountered sourdough yeasts and its potential introduction into the sourdough ecosystem. Here, we provide a brief overview of current knowledge on yeast ecology and diversity in sourdough in the context of the potential natural habitat of W. anomalus. Insight into the population structure of W. anomalus was obtained by comparing internal transcribed spacer rDNA sequences of selected sourdough isolates with publicly available database sequences.

Metatranscriptome analysis for insight into whole-ecosystem gene expression during spontaneous wheat and spelt sourdough fermentations.

Lactic acid bacteria (LAB) are of industrial importance in the production of fermented foods, including sourdough-derived products. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as extended shelf-life, microbial safety, improved texture, and enhanced organoleptic properties. Triggered by the considerable amount of LAB genomic information that became available during the last decade, transcriptome and, by extension, metatranscriptome studies have become one of the most appropriate research approaches to study whole-ecosystem gene expression in more detail. In this study, microarray analyses were performed using RNA sampled during four 10-day spontaneous sourdough fermentations carried out in the laboratory with an in-house-developed LAB functional gene microarray. For data analysis, a new algorithm was developed to calculate a net expression profile for each of the represented genes, allowing use of the microarray analysis beyond the species level. In addition, metabolite target analyses were performed on the sourdough samples to relate gene expression with metabolite production. The results revealed the activation of different key metabolic pathways, the ability to use carbohydrates other than glucose (e.g., starch and maltose), and the conversion of amino acids as a contribution to redox equilibrium and flavor compound generation in LAB during sourdough fermentation.

Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library.

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation.

Yeast species composition differs between artisan bakery and spontaneous laboratory sourdoughs.

Sourdough fermentations are characterized by the combined activity of lactic acid bacteria and yeasts. An investigation of the microbial composition of 21 artisan sourdoughs from 11 different Belgian bakeries yielded 127 yeast isolates. Also, 12 spontaneous 10-day laboratory sourdough fermentations with daily backslopping were performed with rye, wheat, and spelt flour, resulting in the isolation of 217 yeast colonies. The isolates were grouped according to PCR-fingerprints obtained with the primer M13. Representative isolates of each M13 fingerprint group were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences, and partial actin gene sequences, leading to the detection of six species. The dominant species in the bakery sourdoughs were Saccharomyces cerevisiae and Wickerhamomyces anomalus (formerly Pichia anomala), while the dominant species in the laboratory sourdough fermentations were W. anomalus and Candida glabrata. The presence of S. cerevisiae in the bakery sourdoughs might be due to contamination of the bakery environment with commercial bakers yeast, while the yeasts in the laboratory sourdoughs, which were carried out under aseptic conditions with flour as the only nonsterile component, could only have come from the flour used.

Development and validation of a species-independent functional gene microarray that targets lactic acid bacteria.

During the last few years, genome-related information has become available for many microorganisms, including important food-related bacteria. Lactic acid bacteria (LAB) are important industrially in the production of fermented foods such as dairy products, sausages, sourdoughs, and vegetables. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as flavor and texture. In the present study, a species-independent functional gene microarray was developed that targets 406 genes that play key roles in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in the stress response. Also, genes linked to negative traits, such as antibiotic resistance and virulence, are represented. As LAB ecosystems contain a variety of species, there was a more global focus on these specific functional properties. Thus, an algorithm was used to design gene-specific oligonucleotides that preferably hybridize with multiple LAB species, thereby allowing controlled cross-hybridization. For proof of concept, the microarray composed of 2,269 30-mer oligonucleotides focused on LAB species that are prevalent in sourdough ecosystems. Validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides, showed that there were wide ranges in intensity and high reproducibility between microarrays.