Expanding the reach of probiotics through social enterprises.

The rapid rise in microbiome and probiotic science has led to estimates of product creation and sales exceeding $50 billion within five years. However, many people do not have access to affordable products, and regulatory agencies have stifled progress. The objective of a discussion group at the 2017 meeting of the International Scientific Association for Probiotics and Prebiotics was to identify mechanisms to confer the benefits of probiotics to a larger portion of the world's population. Three initiatives, built around fermented food, were discussed with different methods of targeting populations that face enormous challenges of malnutrition, infectious disease, poverty and violent conflict. As new candidate probiotic strains emerge, and the market diversifies towards more personalised interventions, manufacturing processes will need to evolve. Information dissemination through scientific channels and social media is projected to provide consumers and healthcare providers with rapid access to clinical results, and to identify the nearest location of sites making new and affordable probiotic food and supplements. This rapid translation of science to individual well-being will not only expand the beneficiaries of probiotics, but also fuel new social enterprises and economic business models.

Lactobacillus rhamnosus CNCM I-3690 and the commensal bacterium Faecalibacterium prausnitzii A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice.

Impaired gut barrier function has been reported in a wide range of diseases and syndromes and in some functional gastrointestinal disorders. In addition, there is increasing evidence that suggests the gut microbiota tightly regulates gut barrier function and recent studies demonstrate that probiotic bacteria can enhance barrier integrity. Here, we aimed to investigate the effects of Lactobacillus rhamnosus CNCM I-3690 on intestinal barrier function. In vitro results using a Caco-2 monolayer cells stimulated with TNF-α confirmed the anti-inflammatory nature of the strain CNCM I-3690 and pointed out a putative role for the protection of the epithelial function. Next, we tested the protective effects of L. rhamnosus CNCM I-3690 in a mouse model of increased colonic permeability. Most importantly, we compared its performance to that of the well-known beneficial human commensal bacterium Faecalibacterium prauznitzii A2-165. Increased colonic permeability was normalized by both strains to a similar degree. Modulation of apical tight junction proteins expression was then analyzed to decipher the mechanism underlying this effect. We showed that CNCM I-3690 partially restored the function of the intestinal barrier and increased the levels of tight junction proteins Occludin and E-cadherin. The results indicate L. rhamnosus CNCM I-3690 is as effective as the commensal anti-inflammatory bacterium F. prausnitzii to treat functional barrier abnormalities.

A high-throughput cheese manufacturing model for effective cheese starter culture screening.

Cheese making is a process in which enzymatic coagulation of milk is followed by protein separation, carbohydrate removal, and an extended bacterial fermentation. The number of variables in this complex process that influence cheese quality is so large that the developments of new manufacturing protocols are cumbersome. To reduce screening costs, several models have been developed to miniaturize the cheese manufacturing process. However, these models are not able to accommodate the throughputs required for systematic screening programs. Here, we describe a protocol that allows the parallel manufacturing of approximately 600 cheeses in individual cheese vats each with individual process specifications. Protocols for the production of miniaturized Gouda- and Cheddar-type cheeses have been developed. Starting with as little as 1.7 mL of milk, miniature cheeses of about 170 mg can be produced and they closely resemble conventionally produced cheese in terms of acidification profiles, moisture and salt contents, proteolysis, flavor profiles, and microstructure. Flavor profiling of miniature cheeses manufactured with and without mixed-strain adjunct starter cultures allowed the distinguishing of the different cheeses. Moreover, single-strain adjunct starter cultures engineered to overexpress important flavor-related enzymes revealed effects similar to those described in industrial cheese. Benchmarking against industrial cheese produced from the same raw materials established a good correlation between their proteolytic degradation products and their flavor profiles. These miniature cheeses, referred to as microcheeses, open new possibilities to study many aspects of cheese production, which will not only accelerate product development but also allow a more systematic approach to investigate the complex biochemistry and microbiology of cheese making.

Development of a high throughput screening method to test flavour-forming capabilities of anaerobic micro-organisms.

AIM: Development of a fast, automated and reliable screening method for screening of large collections of bacterial strains with minimal handling time. METHODS AND RESULTS: The method is based on the injection of a small headspace sample (100 microl) from culture vials (2 ml) in 96-well format directly into the mass spectrometry (MS). A special sample tray has been developed for liquid media, and anaerobically grown cultures. In principle, all volatile components can be measured, but a representative mass fragment has to be obtained in the MS. Representative masses for 3-methylbutanal, 2-methylpropanal and benzaldehyde are 58, 72 and 105, respectively. In 1 day over 1500 samples could be analysed and the coefficient of variation for the response was <5%. CONCLUSION: Screening of 72 strains belonging to the genus Lactococcus in quadruple on the production of the key-flavour compound 3-methylbutanal illustrated the effectiveness of the method. Furthermore, knowledge of the biochemistry and physiology of 3-methylbutanal formation was used to optimize the composition of the growth medium to enhance 3-methylbutanal production, and thereby improve the screening. SIGNIFICANCE AND IMPACT OF THE STUDY: A commonly used method to control flavour formation in fermented food products is the selection of bacterial strains, which are able to produce the desired flavour compounds. As large collections of strains are available for such screenings, studying biodiversity of micro-organisms on the level of metabolic routes is strongly facilitated by highly automated high throughput screening methods for measuring enzyme activities or production of metabolites. Therefore, this method will be a useful tool for selecting flavour-producing strains and for enhancing starter culture development.

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases.

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases.

Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar.

Highly enantioselective and regioselective biocatalytic azidolysis of aromatic epoxides.

[figure: see text] The halohydrin dehalogenase from Agrobacterium radiobacter AD1 catalyzed the highly enantioselective and beta-regioselective azidolysis of (substituted) styrene oxides. By means of kinetic resolutions the remaining epoxide and the formed azido alcohol could be obtained in very high ee. In a large scale conversion, the decrease in yield and selectivity due to the uncatalyzed chemical side reaction could be overcome by slow addition of azide.

Formation and detoxification of reactive intermediates in the metabolism of chlorinated ethenes.

Short-chain halogenated aliphatics, such as chlorinated ethenes, constitute a large group of priority pollutants. This paper gives an overview on the chemical and physical properties of chlorinated aliphatics that are critical in determining their toxicological characteristics and recalcitrance to biodegradation. The toxic effects and principle metabolic pathways of halogenated ethenes in mammals are briefly discussed. Furthermore, the bacterial degradation of halogenated compounds is reviewed and it is described how product toxicity may explain why most chlorinated ethenes are only degraded cometabolically under aerobic conditions. The cometabolic degradation of chlorinated ethenes by oxygenase-producing microorganisms has been extensively studied. The physiology and bioremediation potential of methanotrophs has been well characterized and an overview of the available data on these organisms is presented. The sensitivity of methanotrophs to product toxicity is a major limitation for the transformation of chlorinated ethenes by these organisms. Most toxic effects arise from the inability to detoxify the reactive chlorinated epoxyethanes occurring as primary metabolites. Therefore, the last part of this review focuses on the metabolic reactions and enzymes that are involved in the detoxification of epoxides in mammals. A key role is played by glutathione S-transferases. Furthermore, an overview is presented on the current knowledge about bacterial enzymes involved in the metabolism of epoxides. Such enzymes might be useful for detoxifying chlorinated ethene epoxides and an example of a glutathione S-transferase with activity for dichloroepoxyethane is highlighted.

Purification and characterization of a surface-binding protein from Lactobacillus fermentum RC-14 that inhibits adhesion of Enterococcus faecalis 1131.

Lactobacilli have been shown to be important in the maintenance of the healthy urogenital flora. One strain, Lactobacillus fermentum RC-14, releases surface-active components which can inhibit adhesion of uropathogenic bacteria. Using a quantitative method for determining inhibition of adhesion, a protein with high anti-adhesive properties against Enterococcus faecalis 1131 was purified. The N-terminal sequence of the 29-kDa protein was identical to that of a collagen-binding protein from Lactobacillus reuteri NCIB 11951, and exhibited close homology with a basic surface protein from L. fermentum BR11. The results suggest that this anti-adhesive cell surface protein of Lactobacillus could protect against uropathogens by preventing their adhesion. the Federation of European Microbiological Societies.

Detoxification of reactive intermediates during microbial metabolism of halogenated compounds.

The reactivity and toxicity of metabolic intermediates that are generated by initial biotransformation reactions can be a major limiting factor for biodegradation of halogenated organic compounds. Recent work on the conversion of haloalkanes, chloroaromatics and chloroethenes indicates that microorganisms may become less sensitive to toxic effects either by using novel pathways that circumvent the generation of reactive intermediates or by producing modified enzymes that decrease the toxicity of such compounds.

Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways.

The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1, 3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1, 3-dichloropropene and 1,2-dibromoethane.

Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45.

The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in Rhodococcus sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione S-transferase with activity towards 1,2-epoxy-2-methyl-3-butene (isoI) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase (isoH). Furthermore, a gene encoding a second glutathione S-transferase was identified (isoJ). The isoJ gene was overexpressed in Escherichia coli and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1, 2-epoxy-2-methyl-3-butene. Downstream of isoJ, six genes (isoABCDEF) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from Xanthobacter sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene (isoG) showed significant similarity with that of alpha-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism.

Trichloroethene degradation in a two-step system by methylosinus trichosporium OB3b. Optimization of system performance: use of formate and methane.

The breakdown of dissolved TCE in a two-step bioremediation system is described. In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE. A special design allowed both for the addition of air (uG,s = 0.01-0. 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor. In two-step systems presented thus far, only formate was used (excess, 20 mM). We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results. Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate. A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition. Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system. The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application. The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor.

Degradation of 1,2-dibromoethane by Mycobacterium sp. strain GP1.

The newly isolated bacterial strain GP1 can utilize 1, 2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a dhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the hheB gene found in Corynebacterium sp. strain N-1074.

Purification of a glutathione S-transferase and a glutathione conjugate-specific dehydrogenase involved in isoprene metabolism in Rhodococcus sp. strain AD45.

A glutathione S-transferase (GST) with activity toward 1, 2-epoxy-2-methyl-3-butene (isoprene monoxide) and cis-1, 2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1, 2-dichloroepoxyethane, with Vmax values of 66 and 2.4 micromol min-1 mg of protein-1 and Km values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM. 1H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD+-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD+ and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.

A glutathione S-transferase with activity towards cis-1, 2-dichloroepoxyethane is involved in isoprene utilization by Rhodococcus sp. strain AD45.

Rhodococcus sp. strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene). Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene, cis-1, 2-dichloroethene to cis-1,2-dichloroepoxyethane, and trans-1, 2-dichloroethene to trans-1,2-dichloroepoxyethane. Isoprene-grown cells also degraded cis-1,2-dichloroepoxyethane and trans-1, 2-dichloroepoxyethane. All organic chlorine was liberated as chloride during degradation of cis-1,2-dichloroepoxyethane. A glutathione (GSH)-dependent activity towards 3, 4-epoxy-3-methyl-1-butene, epoxypropane, cis-1,2-dichloroepoxyethane, and trans-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene. The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane. A conjugate of GSH and 1, 2-epoxyhexane was detected in cell extracts of cells exposed to 1, 2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity. The results indicate that a GSH S-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.

Bacterial degradation of 3-chloroacrylic acid and the characterization of cis- and trans-specific dehalogenases.

A coryneform bacterium that is able to utilize cis- and trans-3-chloroacrylic acid as sole carbon source for growth was isolated from freshwater sediment. The organism was found to produce two inducible dehalogenases, one specific for the cis- and the other for trans-3-chloroacrylic acid. Both dehalogenases were purified to homogeneity from cells induced for dehalogenase synthesis with 3-chlorocrotonic acid. The enzymes produced muconic acid semialdehyde (3-oxopropionic acid) from their respective 3-chloroacrylic acid substrate. No other substrates were found. The cis-3-chloroacrylic acid dehalogenase consisted of two polypeptide chains of a molecular weight 16.2 kDa. Trans-3-chloroacrylic acid dehalogenase was a protein with subunits of 7.4 and 8.7 kDa. The subunit and amino acid compositions and the N-terminal amino acid sequences of the enzymes indicate that they are not closely related.