A selection process based on the robustness of anti-Listeria monocytogenes activity reveals two strains of Carnobacterium maltaromaticum with biopreservation properties in cheese.

Biopreservation is an approach consisting of using microorganisms as protective cultures and/or their metabolites to optimize the microbiological quality and shelf life of food by ensuring safety or reducing food waste. Biopreservation strain selection pipelines mainly focus on inhibition strength to identify strains of interest. However, in addition to inhibition strength, inhibition activity must be able to be expressed despite significant variations in food matrix properties. In this study, the anti-Listeria monocytogenes EGDelux properties of a collection of 77 Carnobacterium maltaromaticum strains were investigated by high throughput competition assays under varying conditions of co-culture inoculation level, time interval between inoculation with C. maltaromaticum and L. monocytogenes, pH, and NaCl, resulting in 1309 different combinations of C. maltaromaticum strains and culture conditions. This screening led to the selection of two candidate strains with potent and robust anti-L. monocytogenes activities. Deferred growth inhibition assays followed by halo measurements, and liquid co-culture followed by colony counting, revealed that these two strains exhibit a wide anti-Listeria spectrum. Challenge tests in Camembert and Saint-Nectaire cheese revealed both strains were able to inhibit a cocktail of five strains of L. monocytogenes with high potency and high reproducibility. These results highlight the importance of including the robustness criterion in addition to potency when designing a strain selection process for biopreservation applications.

Serial cultures in invert emulsion and monophase systems for microbial community shaping and propagation.

BACKGROUND: Microbial communities harbor important biotechnological potential in diverse domains, however, the engineering and propagation of such communities still face both knowledge and know-how gaps. More specifically, culturing tools are needed to propagate and shape microbial communities, to obtain desired properties, and to exploit them. Previous work suggested that micro-confinement and segregation of microorganisms using invert (water-in-oil, w/o) emulsion broth can shape communities during propagation, by alleviating biotic interactions and inducing physiological changes in cultured bacteria. The present work aimed at evaluating invert emulsion and simple broth monophasic cultures for the propagation and shaping of bacterial communities derived from raw milk in a serial propagation design. RESULTS: The monophasic setup resulted in stable community structures during serial propagation, whereas the invert emulsion system resulted in only transiently stable structures. In addition, different communities with different taxonomic compositions could be obtained from a single inoculum. Furthermore, the implementation of invert emulsion systems has allowed for the enrichment of less abundant microorganisms and consequently facilitated their isolation on culture agar plates. CONCLUSIONS: The monophasic system enables communities to be propagated in a stable manner, whereas the invert emulsion system allowed for the isolation of less abundant microorganisms and the generation of diverse taxonomic compositions from a single inoculum.

Comparison of Electrostatic Spray Drying, Spray Drying, and Freeze Drying for Lacticaseibacillus rhamnosus GG Dehydration.

Spray drying (SD) is extensively used to encapsulate lactic acid bacteria in large-scale industrial applications; however, bacteria combat several harms that reduce their viability. In this study, a novel technique called electrostatic spray drying (ESD) was used to explore the benefits and disadvantages of using electrostatic charge and lower temperatures in the system. Freeze drying (FD) was used as a reference. The effect of different encapsulation agents, like maltodextrin, arabic gum, and skim milk, on the viability of Lacticaseibacillus rhamnosus GG (LGG) was investigated. The initial cell concentration, particle size distribution, aspect ratio, sphericity, scanning-electron-microscopy images, moisture content, water activity, glass transition, rehydration abilities, and survival during storage were compared. Skim milk was proven to be the best protectant for LGG, regardless of the drying process or storage time. A huge reduction in cell numbers (4.49 ± 0.06 log CFU/g) was observed with maltodextrin using SD; meanwhile, it was protected with minimum loss (8.64 ± 0.62 log CFU/g) with ESD. In general, ESD preserved more LGG cells during processing compared to SD, and provided better stability than FD and SD during storage, regardless of the applied voltage. The ESD product analysis demonstrated an efficient LGG preservation, close to FD; therefore, ESD presented to be a promising and scalable substitute for SD and FD.

Invert emulsions alleviate biotic interactions in bacterial mixed culture.

The large application potential of microbiomes has led to a great need for mixed culture methods. However, microbial interactions can compromise the maintenance of biodiversity during cultivation in a reactor. In particular, competition among species can lead to a strong disequilibrium in favor of the fittest microorganism. In this study, an invert emulsion system was designed by dispersing culture medium in a mixture of sunflower oil and the surfactant PGPR. Confocal laser scanning microscopy revealed that this system allowed to segregate microorganisms in independent droplets. Granulomorphometric analysis showed that the invert emulsion remains stable during at least 24 h, and that the introduction of bacteria did not have a significant impact on the structure of the invert emulsion. A two-strain antagonistic model demonstrated that this invert emulsion system allows the propagation of two strains without the exclusion of the less-fit bacterium. The monitoring of single-strain cultures of bacteria representative of a cheese microbiota revealed that all but Brevibacterium linens were able to grow. A consortium consisting of Lactococcus lactis subsp. lactis biovar diacetylactis, Streptococcus thermophilus, Leuconostoc mesenteroides, Staphylococcus xylosus, Lactiplantibacillus plantarum and Carnobacterium maltaromaticum was successfully cultivated without detectable biotic interactions. Metabarcoding analysis revealed that the system allowed a better maintenance of alpha diversity and produced a propagated bacterial consortium characterized by a structure closer to the initial state compared to non-emulsified medium. This culture system could be an important tool in the field of microbial community engineering.

Mining Biosynthetic Gene Clusters in Carnobacterium maltaromaticum by Interference Competition Network and Genome Analysis.

Carnobacterium maltaromaticum is a non-starter lactic acid bacterium (LAB) of interest in the dairy industry for biopreservation. This study investigated the interference competition network and the specialized metabolites biosynthetic gene clusters (BGCs) content in this LAB in order to explore the relationship between the antimicrobial properties and the genome content. Network analysis revealed that the potency of inhibition tended to increase when the inhibition spectrum broadened, but also that several strains exhibited a high potency and narrow spectrum of inhibition. The C. maltaromaticum strains with potent anti-L. monocytogenes were characterized by high potency and a wide intraspecific spectrum. Genome mining of 29 strains revealed the presence of 12 bacteriocin BGCs: four of class I and eight of class II, among which seven belong to class IIa and one to class IIc. Overall, eight bacteriocins and one nonribosomal peptide synthetase and polyketide synthase (NRPS-PKS) BGCs were newly described. The comparison of the antimicrobial properties resulting from the analysis of the network and the BGC genome content allowed us to delineate candidate BGCs responsible for anti-L. monocytogenes and anti-C. maltaromaticum activity. However, it also highlighted that genome analysis is not suitable in the current state of the databases for the prediction of genes involved in the antimicrobial activity of strains with a narrow anti-C. maltaromaticum activity.

Contribution of omics to biopreservation: Toward food microbiome engineering.

Biopreservation is a sustainable approach to improve food safety and maintain or extend food shelf life by using beneficial microorganisms or their metabolites. Over the past 20 years, omics techniques have revolutionised food microbiology including biopreservation. A range of methods including genomics, transcriptomics, proteomics, metabolomics and meta-omics derivatives have highlighted the potential of biopreservation to improve the microbial safety of various foods. This review shows how these approaches have contributed to the selection of biopreservation agents, to a better understanding of the mechanisms of action and of their efficiency and impact within the food ecosystem. It also presents the potential of combining omics with complementary approaches to take into account better the complexity of food microbiomes at multiple scales, from the cell to the community levels, and their spatial, physicochemical and microbiological heterogeneity. The latest advances in biopreservation through omics have emphasised the importance of considering food as a complex and dynamic microbiome that requires integrated engineering strategies to increase the rate of innovation production in order to meet the safety, environmental and economic challenges of the agri-food sector.

Identification of Potential Citrate Metabolism Pathways in Carnobacterium maltaromaticum.

In the present study, we describe the identification of potential citrate metabolism pathways for the lactic acid bacterium (LAB) Carnobacterium maltaromaticum. A phenotypic assay indicated that four of six C. maltaromaticum strains showed weak (Cm 6-1 and ATCC 35586) or even delayed (Cm 3-1 and Cm 5-1) citrate utilization activity. The remaining two strains, Cm 4-1 and Cm 1-2 gave negative results. Additional analysis showed no or very limited utilization of citrate in media containing 1% glucose and 22 or 30 mM citrate and inoculated with Cm 6-1 or ATCC 35586. Two potential pathways of citrate metabolism were identified by bioinformatics analyses in C. maltaromaticum including either oxaloacetate (pathway 1) or tricarboxylic compounds such as isocitrate and α-ketoglutarate (pathway 2) as intermediates. Genes encoding pathway 1 were present in two out of six strains while pathway 2 included genes present in all six strains. The two potential citrate metabolism pathways in C. maltaromaticum may potentially affect the sensory profiles of milk and soft cheeses subjected to growth with this species.

Impact of Lacticaseibacillus rhamnosus GG on the Emulsion Stability of Raw Milk.

Lactic acid bacteria (LAB) have been studied for several decades to understand and determine their mechanism and interaction within the matrix into which they are introduced. This study aimed to determine the spatial distribution of Lacticaseibacillus rhamnosus GG (LGG) in a dairy matrix and to decipher its behaviour towards milk components, especially fat globules. Two strains of this widely studied bacterium with expected probiotic effects were used: LGG WT with pili on the cell surface and its pili-depleted mutant-LGG ΔspaCBA-in order to determine the involvement of these filamentous proteins. In this work, it was shown that LGG ΔspaCBA was able to limit creaming with a greater impact than the wild-type counterpart. Moreover, confocal imaging evidenced a preferential microbial distribution as aggregates for LGG WT, while the pili-depleted strain tended to be homogenously distributed and found as individual chains. The observed differences in creaming are attributed to the indirect implication of SpaCBA pili. Indeed, the bacteria-to-bacteria interaction surpassed the bacteria-to-matrix interaction, reducing the bacterial surface exposed to raw milk. Conversely, LGG ΔspaCBA may form a physical barrier responsible for preventing milk fat globules from rising to the surface.

Assessment of Pb(II), Cd(II), and Al(III) Removal Capacity of Bacteria from Food and Gut Ecological Niches: Insights into Biodiversity to Limit Intestinal Biodisponibility of Toxic Metals.

Toxic metals (such as lead, cadmium, and, to a lesser extent, aluminum) are detrimental to health when ingested in food or water or when inhaled. By interacting with heavy metals, gut and food-derived microbes can actively and/or passively modulate (by adsorption and/or sequestration) the bioavailability of these toxins inside the gut. This "intestinal bioremediation" involves the selection of safe microbes specifically able to immobilize metals. We used inductively coupled plasma mass spectrometry to investigate the in vitro ability of 225 bacteria to remove the potentially harmful trace elements lead, cadmium, and aluminum. Interspecies and intraspecies comparisons were performed among the Firmicutes (mostly lactic acid bacteria, including Lactobacillus spp., with some Lactococcus, Pediococcus, and Carnobacterium representatives), Actinobacteria, and Proteobacteria. The removal of a mixture of lead and cadmium was also investigated. Although the objective of the study was not to elucidate the mechanisms of heavy metal removal for each strain and each metal, we nevertheless identified promising candidate bacteria as probiotics for the intestinal bioremediation of Pb(II) and Cd(II).

Engineering Pseudomonas putida KT2440 for the production of isobutanol.

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback-resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical YIso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non-growing conditions.

Shaving and breaking bacterial chains with a viscous flow.

Some food and ferment manufacturing steps such as spray-drying result in the application of viscous stresses to bacteria. This study explores how a viscous flow impacts both bacterial adhesion functionality and bacterial cell organization using a combined experimental and modeling approach. As a model organism we study Lactobacillus rhamnosus GG (LGG) "wild type" (WT), known to feature strong adhesive affinities towards beta-lactoglobulin thanks to pili produced by the bacteria on cell surfaces, along with three cell-surface mutant strains. Applying repeated flows with high shear-rates reduces bacterial adhesive abilities up to 20% for LGG WT. Bacterial chains are also broken by this process, into 2-cell chains at low industrial shear rates, and into single cells at very high shear rates. To rationalize the experimental observations we study numerically and analytically the Stokes equations describing viscous fluid flow around a chain of elastically connected spheroidal cell bodies. In this model setting we examine qualitatively the relationship between surface traction (force per unit area), a proxy for pili removal rate, and bacterial chain length (number of cells). Longer chains result in higher maximal surface tractions, particularly at the chain extremities, while inner cells enjoy a small protection from surface tractions due to hydrodynamic interactions with their neighbors. Chain rupture therefore may act as a mechanism to preserve surface adhesive functionality in bacteria.

Nested structure of intraspecific competition network in Carnobacterium maltaromaticum.

While competition targeting food-borne pathogens is being widely documented, few studies have focused on competition among non-pathogenic food bacteria. Carnobacterium maltaromaticum is a genetically diverse lactic acid bacterium known for comprising several bacteriocinogenic strains with bioprotective potentialities against the food-borne pathogen Listeria monocytogenes. The aim of our study is to examine the network properties of competition among a collection of 73 strains of C. maltaromaticum and to characterize their individual interaction potential. The performed high-throughput competition assays, investigating 5 329 pairwise interactions, showed that intraspecific competition was major in C. maltaromaticum with approximately 56% of the sender strains antagonizing at least one receiver strain. A high diversity of inhibitory and sensitivity spectra was identified along with a majority of narrow inhibitory as well as sensitivity spectra. Through network analysis approach, we determined the highly nested architecture of C. maltaromaticum competition network, thus showing that competition in this species is determined by both the spectrum width of the inhibitory activity of sender strains and the spectrum width of the sensitivity of receiver strains. This study provides knowledge of the competition network in C. maltaromaticum that could be used in rational assembly of compatible microbial strains for the design of mixed starter cultures.

A Fast, Efficient and Easy to Implement Method to Purify Bacterial Pili From Lacticaseibacillus rhamnosus GG Based on Multimodal Chromatography.

Pili are polymeric proteins located at the cell surface of bacteria. These filamentous proteins play a pivotal role in bacterial adhesion with the surrounding environment. They are found both in Gram-negative and Gram-positive bacteria but differ in their structural organization. Purifying these high molecular weight proteins is challenging and has certainly slowed down their characterization. Here, we propose a chromatography-based protocol, mainly relying on multimodal chromatography (core bead technology using Capto Core 700 resin), to purify sortase-dependent SpaCBA pili from the probiotic strain Lacticaseibacillus rhamnosus GG (LGG). Contrary to previously published methods, this purification protocol does not require specific antibodies nor complex laboratory equipment, including for the multimodal chromatography step, and provides high degree of protein purity. No other proteins were detectable by SDS-PAGE and the 260/280 nm ratio (∼0.6) of the UV spectrum confirmed the absence of any other co-purified macromolecules. One can obtain ∼50 µg of purified pili, starting from 1 L culture at OD600nm ≈ 1, in 2-3 working days. This simple protocol could be useful to numerous laboratories to purify pili from LGG easily. Therefore, the present work should boost specific studies dedicated to LGG SpaCBA pili and the characterization of the interactions occurring with their protein partners at the molecular level. Moreover, this straightforward purification process might be extended to the purification of sortase-dependant pili from other Gram-positive bacteria.

Adhesive Interactions Between Lactic Acid Bacteria and ß-Lactoglobulin: Specificity and Impact on Bacterial Location in Whey Protein Isolate.

In the last decade, there has been an increasing interest in the potential health effects associated with the consumption of lactic acid bacteria (LAB) in foods. Some of these bacteria such as Lactobacillus rhamnosus GG (LGG) are known to adhere to milk components, which may impact their distribution and protection within dairy matrices and therefore is likely to modulate the efficiency of their delivery. However, the adhesive behavior of most LAB, as well as its effect on food structuration and on the final bacterial distribution within the food matrix remain very poorly studied. Using a recently developed high-throughput approach, we have screened a collection of 73 LAB strains for their adhesive behavior toward the major whey protein ß-lactoglobulin. Adhesion was then studied by genomics in relation to common bacterial surface characteristics such as pili and adhesion-related domain containing proteins. Representative adhesive and non-adhesive strains have been studied in further depth through biophysical measurement using atomic force microscopy (AFM) and a relation with bacterial distribution in whey protein isolate (WPI) solution has been established. AFM measurements have revealed that bacterial adhesion to ß-lactoglobulin is highly specific and cannot be predicted accurately using only genomic information. Non-adhesive strains were found to remain homogeneously distributed in solution whereas adhesive strains gathered in flocs. These findings show that several LAB strains are able to adhere to ß-lactoglobulin, whereas this had only been previously observed on LGG. We also show that these adhesive interactions present similar characteristics and are likely to impact bacterial location and distribution in dairy matrices containing ß-lactoglobulin. This may help with designing more efficient dairy food matrices for optimized LAB delivery.

Occurrence and Dynamism of Lactic Acid Bacteria in Distinct Ecological Niches: A Multifaceted Functional Health Perspective.

Lactic acid bacteria (LAB) are representative members of multiple ecosystems on earth, displaying dynamic interactions within animal and plant kingdoms in respect with other microbes. This highly heterogeneous phylogenetic group has coevolved with plants, invertebrates, and vertebrates, establishing either mutualism, symbiosis, commensalism, or even parasitism-like behavior with their hosts. Depending on their location and environment conditions, LAB can be dominant or sometimes in minority within ecosystems. Whatever their origins and relative abundance in specific anatomic sites, LAB exhibit multifaceted ecological and functional properties. While some resident LAB permanently inhabit distinct animal mucosal cavities, others are provided by food and may transiently occupy the gastrointestinal tract. It is admitted that the overall gut microbiome has a deep impact on health and diseases. Here, we examined the presence and the physiological role of LAB in the healthy human and several animal microbiome. Moreover, we also highlighted some dysbiotic states and related consequences for health, considering both the resident and the so-called "transionts" microorganisms. Whether LAB-related health effects act collectively or follow a strain-specificity dogma is also addressed. Besides the highly suggested contribution of LAB to interplay with immune, metabolic, and even brain-axis regulation, the possible involvement of LAB in xenobiotic detoxification processes and metal equilibrium is also tackled. Recent technological developments such as functional metagenomics, metabolomics, high-content screening and design in vitro and in vivo experimental models now open new horizons for LAB as markers applied for disease diagnosis, susceptibility, and follow-up. Moreover, identification of general and more specific molecular mechanisms based on antioxidant, antimicrobial, anti-inflammatory, and detoxifying properties of LAB currently extends their selection and promising use, either as probiotics, in traditional and functional foods, for dedicated treatments and mostly for maintenance of normobiosis and homeostasis.

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes.

This article describes a method for high-throughput competition assays using a bioluminescent strain of L. monocytogenes. This method is based on the use of the luminescent indicator strain L. monocytogenes EGDelux. The luminescence of this strain is correlated to growth, which make it suitable to monitor the growth of L. monocytogenes in mixed cultures. To this aim, luminescence kinetics were converted into a single numerical value, called the Luminescence Disturbance Indicator (LDI), which takes into account growth inhibition phenomena resulting in latency increase, decrease in the luminescence rate, or reduction of the maximum luminescence. The LDI allows to automatically and simultaneously handle multiple competition assays which are required for high-throughput screening (HTS) approaches. The method was applied to screen a collection of 1810 strains isolated from raw cow's milk in order to identify non-acidifying strains with anti-L. monocytogenes bioprotection properties. This method was also successfully used to identify anti-L. monocytogenes candidates within a collection of Lactococcus piscium, a species where antagonism was previously described as non-diffusible and requiring cell-to-cell contact. In conclusion, bioluminescent L. monocytogenes can be used in HTS to identify strains with anti-L. monocytogenes bioprotection properties, irrespectively of the inhibition mechanism.

Adhesive interactions between milk fat globule membrane and Lactobacillus rhamnosus GG inhibit bacterial attachment to Caco-2 TC7 intestinal cell.

Milk is the most popular matrix for the delivery of lactic acid bacteria, but little is known about how milk impacts bacterial functionality. Here, the adhesion mechanisms of Lactobacillus rhamnosus GG (LGG) surface mutants to a milk component, the milk fat globule membrane (MFGM), were compared using atomic force microscopy (AFM). AFM results revealed the key adhesive role of the LGG SpaCBA pilus in relation to MFGM. A LGG mutant without exopolysaccharides but with highly exposed pili improved the number of adhesive events between LGG and MFGM compared to LGG wild type (WT). In contrast, the number of adhesive events decreased significantly for a LGG mutant without SpaCBA pili. Moreover, the presence of MFGM in the dairy matrix was found to decrease significantly the bacterial attachment ability to Caco-2 TC7 cells. This work thus demonstrated a possible competition between LGG adhesion to MFGM and to epithelial intestinal cells. This competition could negatively impact the adhesion capacity of LGG to intestinal cells in vivo, but requires further substantiation.

Use of imaging techniques to identify efficient controlled release systems of Lactobacillus rhamnosus GG during in vitro digestion.

Matrix composition plays a crucial role in the controlled release of viable and functional bacteria in the intestine. Imaging tools such as electronic and confocal microscopies were used in this work to investigate the influence of matrix composition on matrix integrity and porosity, bacterial spatial distribution and viability during simulated in vitro digestion. L. rhamnosus GG was encapsulated in matrices having different casein/whey protein ratios. The formulation with a casein/whey ratio of 60/40 presented a porous weak gel structure that resulted in its fast disintegration in gastric media showing the presence of dead bacteria in the intestine. For the formulation with a casein/whey ratio of 100/0, the matrix was dense with a strong gel structure. At the end of the intestine, total disintegration of microparticles was not achieved and bacteria were still embedded in the matrix instead of being liberated. Only the intermediate formulation (casein/whey-80/20) permitted a good bacterial protection in the stomach and release of viable bacteria during intestinal digestion.

Comparative Genomic Analysis Reveals Ecological Differentiation in the Genus Carnobacterium.

Lactic acid bacteria (LAB) differ in their ability to colonize food and animal-associated habitats: while some species are specialized and colonize a limited number of habitats, other are generalist and are able to colonize multiple animal-linked habitats. In the current study, Carnobacterium was used as a model genus to elucidate the genetic basis of these colonization differences. Analyses of 16S rRNA gene meta-barcoding data showed that C. maltaromaticum followed by C. divergens are the most prevalent species in foods derived from animals (meat, fish, dairy products), and in the gut. According to phylogenetic analyses, these two animal-adapted species belong to one of two deeply branched lineages. The second lineage contains species isolated from habitats where contact with animal is rare. Genome analyses revealed that members of the animal-adapted lineage harbor a larger secretome than members of the other lineage. The predicted cell-surface proteome is highly diversified in C. maltaromaticum and C. divergens with genes involved in adaptation to the animal milieu such as those encoding biopolymer hydrolytic enzymes, a heme uptake system, and biopolymer-binding adhesins. These species also exhibit genes for gut adaptation and respiration. In contrast, Carnobacterium species belonging to the second lineage encode a poorly diversified cell-surface proteome, lack genes for gut adaptation and are unable to respire. These results shed light on the important genomics traits required for adaptation to animal-linked habitats in generalist Carnobacterium.

Draft Genome Sequence of Carnobacterium divergens V41, a Bacteriocin-Producing Strain.

In this study, we present the draft genome sequence of Carnobacterium divergens V41. This strain was previously reported as producing divercin V41, a bacteriocin of interest for food biopreservation. Its genome revealed also the presence of a gene cluster putatively involved in polyketide production, which is unique in lactic acid bacteria.