Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

Introduction and objective: p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). Material and methods: This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. Results and discussion: No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Unlocking the Potential of Probiotics: A Comprehensive Review on Research, Production, and Regulation of Probiotics.

This review provides a comprehensive overview of the current state of probiotic research, covering a wide range of topics, including strain identification, functional characterization, preclinical and clinical evaluations, mechanisms of action, therapeutic applications, manufacturing considerations, and future directions. The screening process for potential probiotics involves phenotypic and genomic analysis to identify strains with health-promoting properties while excluding those with any factor that could be harmful to the host. In vitro assays for evaluating probiotic traits such as acid tolerance, bile metabolism, adhesion properties, and antimicrobial effects are described. The review highlights promising findings from in vivo studies on probiotic mitigation of inflammatory bowel diseases, chemotherapy-induced mucositis, dysbiosis, obesity, diabetes, and bone health, primarily through immunomodulation and modulation of the local microbiota in human and animal models. Clinical studies demonstrating beneficial modulation of metabolic diseases and human central nervous system function are also presented. Manufacturing processes significantly impact the growth, viability, and properties of probiotics, and the composition of the product matrix and supplementation with prebiotics or other strains can modify their effects. The lack of regulatory oversight raises concerns about the quality, safety, and labeling accuracy of commercial probiotics, particularly for vulnerable populations. Advancements in multi-omics approaches, especially probiogenomics, will provide a deeper understanding of the mechanisms behind probiotic functionality, allowing for personalized and targeted probiotic therapies. However, it is crucial to simultaneously focus on improving manufacturing practices, implementing quality control standards, and establishing regulatory oversight to ensure the safety and efficacy of probiotic products in the face of increasing therapeutic applications.

Propionibacterium freudenreichii CIRM-BIA 129 mitigates colitis through S layer protein B-dependent epithelial strengthening.

The growing incidence of human diseases involving inflammation and increased gut permeability makes the quest for protective functional foods more crucial than ever. Propionibacterium freudenreichii (P. freudenreichii) is a beneficial bacterium used in the dairy and probiotic industries. Selected strains exert anti-inflammatory effects, and the present work addresses whether the P. freudenreichii CIRM-BIA129, consumed daily in a preventive way, could protect mice from acute colitis induced by dextran sodium sulfate (DSS), and more precisely, whether it could protect from intestinal epithelial breakdown induced by inflammation. P. freudenreichii CIRM-BIA129 mitigated colitis severity and inhibited DSS-induced permeability. It limited crypt length reduction and promoted the expression of zonula occludens-1 (ZO-1), without reducing interleukin-1ß mRNA (il-1ß) expression. In vitro, P. freudenreichii CIRM-BIA129 prevented the disruption of a Caco-2 monolayer induced by proinflammatory cytokines. It increased transepithelial electrical resistance (TEER) and inhibited permeability induced by inflammation, along with an increased ZO-1 expression. Extracellular vesicles (EVs) from P. freudenreichii CIRM-BIA129, carrying the surface layer protein (SlpB), reproduced the protective effect of P. freudenreichii CIRM-BIA129. A mutant strain deleted for slpB (ΔslpB), or EVs from this mutant strain, had lost their protective effects and worsened both DSS-induced colitis and inflammation in vivo. These results shown that P. freudenreichii CIRM-BIA129 daily consumption has the potential to greatly alleviate colitis symptoms and, particularly, to counter intestinal epithelial permeability induced by inflammation by restoring ZO-1 expression through mechanisms involving S-layer protein B. They open new avenues for the use of probiotic dairy propionibacteria and/or postbiotic fractions thereof, in the context of gut permeability.NEW & NOTEWORTHY Propionibacterium freudenreichii reduces dextran sodium sulfate (DSS)-induced intestinal permeability in vivo. P. freudenreichii does not inhibit inflammation but damages linked to inflammation. P. freudenreichii inhibits intestinal epithelial breakdown through S-layer protein B. The protective effects of P. freudenreichii depend on S-layer protein B. Extracellular vesicles from P. freudenreichii CB 129 mimic the protective effect of the probiotic.

Comprehensive probiogenomics analysis of the commensal Escherichia coli CEC15 as a potential probiotic strain.

BACKGROUND: Probiotics have gained attention for their potential maintaining gut and immune homeostasis. They have been found to confer protection against pathogen colonization, possess immunomodulatory effects, enhance gut barrier functionality, and mitigate inflammation. However, a thorough understanding of the unique mechanisms of effects triggered by individual strains is necessary to optimize their therapeutic efficacy. Probiogenomics, involving high-throughput techniques, can help identify uncharacterized strains and aid in the rational selection of new probiotics. This study evaluates the potential of the Escherichia coli CEC15 strain as a probiotic through in silico, in vitro, and in vivo analyses, comparing it to the well-known probiotic reference E. coli Nissle 1917. Genomic analysis was conducted to identify traits with potential beneficial activity and to assess the safety of each strain (genomic islands, bacteriocin production, antibiotic resistance, production of proteins involved in host homeostasis, and proteins with adhesive properties). In vitro studies assessed survival in gastrointestinal simulated conditions and adhesion to cultured human intestinal cells. Safety was evaluated in BALB/c mice, monitoring the impact of E. coli consumption on clinical signs, intestinal architecture, intestinal permeability, and fecal microbiota. Additionally, the protective effects of both strains were assessed in a murine model of 5-FU-induced mucositis. RESULTS: CEC15 mitigates inflammation, reinforces intestinal barrier, and modulates intestinal microbiota. In silico analysis revealed fewer pathogenicity-related traits in CEC15, when compared to Nissle 1917, with fewer toxin-associated genes and no gene suggesting the production of colibactin (a genotoxic agent). Most predicted antibiotic-resistance genes were neither associated with actual resistance, nor with transposable elements. The genome of CEC15 strain encodes proteins related to stress tolerance and to adhesion, in line with its better survival during digestion and higher adhesion to intestinal cells, when compared to Nissle 1917. Moreover, CEC15 exhibited beneficial effects on mice and their intestinal microbiota, both in healthy animals and against 5FU-induced intestinal mucositis. CONCLUSIONS: These findings suggest that the CEC15 strain holds promise as a probiotic, as it could modulate the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings may have implications for the treatment of gastrointestinal disorders, particularly some forms of diarrhea.

Different culture media and purification methods unveil the core proteome of Propionibacterium freudenreichii-derived extracellular vesicles.

Bacterial extracellular vesicles (EVs) are natural lipidic nanoparticles implicated in intercellular communication. Although EV research focused mainly on pathogens, the interest in probiotic-derived EVs is now rising. One example is Propionibacterium freudenreichii, which produces EVs with anti-inflammatory effects on human epithelial cells. Our previous study with P. freudenreichii showed that EVs purified by size exclusion chromatography (SEC) displayed variations in protein content according to bacterial growth conditions. Considering these content variations, we hypothesized that a comparative proteomic analysis of EVs recovered in different conditions would elucidate whether a representative vesicular proteome existed, possibly providing a robust proteome dataset for further analysis. Therefore, P. freudenreichii was grown in two culture media, and EVs were purified by sucrose density gradient ultracentrifugation (UC). Microscopic and size characterization confirmed EV purification, while shotgun proteomics unveiled that they carried a diverse set of proteins. A comparative analysis of the protein content of UC- and SEC-derived EVs, isolated from cultures either in UF (cow milk ultrafiltrate medium) or YEL (laboratory yeast extract lactate medium), showed that EVs from all these conditions shared 308 proteins. This EV core proteome was notably enriched in proteins related to immunomodulation. Moreover, it showed distinctive features, including highly interacting proteins, compositional biases for some specific amino acids, and other biochemical parameters. Overall, this work broadens the toolset for the purification of P. freudenreichii-derived EVs, identifies a representative vesicular proteome, and enumerates conserved features in vesicular proteins. These results hold the potential for providing candidate biomarkers of purification quality, and insights into the mechanisms of EV biogenesis and cargo sorting.

Development of innate immune memory by non-immune cells during Staphylococcus aureus infection depends on reactive oxygen species.

Introduction: The mechanisms underlying innate immune memory (trained immunity) comprise epigenetic reprogramming of transcriptional pathways associated with alterations of intracellular metabolism. While the mechanisms of innate immune memory carried out by immune cells are well characterized, such processes in non-immune cells, are poorly understood. The opportunistic pathogen, Staphylococcus aureus, is responsible for a multitude of human diseases, including pneumonia, endocarditis and osteomyelitis, as well as animal infections, including chronic cattle mastitis that are extremely difficult to treat. An induction of innate immune memory may be considered as a therapeutic alternative to fight S. aureus infection. Methods: In the current work, we demonstrated the development of innate immune memory in non-immune cells during S. aureus infection employing a combination of techniques including Enzyme-linked immunosorbent assay (ELISA), microscopic analysis, and cytometry. Results: We observed that training of human osteoblast-like MG-63 cells and lung epithelial A549 cells with ß-glucan increased IL-6 and IL-8 production upon a stimulation with S. aureus, concomitant with histones modifications. IL-6 and IL-8 production was positively correlated with an acetylation of histone 3 at lysine 27 (H3K27), thus suggesting epigenetic reprogramming in these cells. An addition of the ROS scavenger N-Acetylcysteine, NAC, prior to ß-glucan pretreatment followed by an exposure to S. aureus, resulted in decreased IL-6 and IL-8 production, thereby supporting the involvement of ROS in the induction of innate immune memory. Exposure of cells to Lactococcus lactis resulted in increased IL-6 and IL-8 production by MG-63 and A549 cells upon a stimulation with S. aureus that was correlated with H3K27 acetylation, suggesting the ability of this beneficial bacterium to induce innate immune memory. Discussion: This work improves our understanding of innate immune memory in non-immune cells in the context of S. aureus infection. In addition to known inducers, probiotics may represent good candidates for the induction of innate immune memory. Our findings may help the development of alternative therapeutic approaches for the prevention of S. aureus infection.

Development of innovative fermented products by exploiting the diversity of immunomodulatory properties and fermentative activity of lactic and propionic acid bacteria.

Many consumers nowadays demand plant-based milk analogs for reasons related to lifestyle, health, diet and sustainability. This has led to the increasing development of new products, fermented or not. The objective of the present study was to develop a plant-based fermented product (based on soy milk analog or on hemp milk analog), as well as mixes, using lactic acid bacteria (LAB) and propionic acid bacteria (PAB) strains, as well as consortia thereof. We screened a collection of 104 strains, from nine LAB species and two PAB species, based on their ability to ferment plant or milk carbohydrates, to acidify goat milk, soy milk analog and hemp milk analog, as well as to hydrolyze proteins isolated from these three products. Strains were also screened for their immunomodulatory ability to induce secretion of two interleukins, i.e., IL-10 and IL-12, in human Peripheral Blood Mononuclear Cells. We selected five strains: Lactobacillus delbrueckii subsp. lactis Bioprox1585, Lactobacillus acidophilus Bioprox6307, Lactococcus lactis Bioprox7116, Streptococcus thermophilus CIRM-BIA251, and Acidipropionibacterium acidipropionici CIRM-BIA2003. We then assembled them in 26 different bacterial consortia. Goat milk and soy milk analog fermented by each of the five strains or by the 26 consortia were tested in vitro, for their ability to modulate inflammation in cultured Human Epithelial Intestinal Cells (HEIC) stimulated by pro-inflammatory Lipopolysaccharides (LPS) from Escherichia coli. Plant-based milk analogs, fermented by one consortium composed of L.delbrueckii subsp. lactis Bioprox1585, Lc.lactis Bioprox7116, and A.acidipropionici CIRM-BIA2003, reduced the secretion of the proinflammatory cytokine IL-8 in HIECs. Such innovative fermented vegetable products thus open perspectives as functional foods targeting gut inflammation.

Dairy starters and fermented dairy products modulate gut mucosal immunity.

The gut microbiota plays a crucial role in the regulation of mucosal immunity and of the function of the intestinal barrier. Dysbiosis is accordingly associated with rupture of mucosal immune homeostasis, leading to inflammatory intestinal diseases. In this context, probiotic bacteria, including a new generation of intestinal probiotics, can maintain intestinal homeostasis and promote health. Surprisingly, little is known about the impact of fermented dairy products in this context, while they represent our main source of live and active bacteria. Indeed, they provide, through our daily diet, a high number of bacteria whose effect on mucosal immunity deserves attention. Among bacteria ingested in fermented dairy products, Streptococcus thermophilus, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii are on top, as they are ingested in high concentrations (close to 109 per gram of product) in fermented milks or cheeses. This review gives an overview of the potential immunomodulatory effects of these main dairy starters. It further explores studies dealing with fermented dairy products containing theses starters, in a context of inflammation.

Data from a proteomic comparative analysis highlight differential adaptation of Lactobacillus delbrueckii subsp. bulgaricus to cow milk versus to soy milk environments.

The article presents a proteomic dataset generated by a comparative analysis, using gel-free nanoLC-MS/MS, of the cellular proteome of Lactobacillus delbrueckii subsp. bulgaricus, a yogurt starter, when cultivated in soy milk versus in cow milk. The CIRM-BIA1592 strain was cultivated in the aqueous phase of soy milk, or of cow milk. Whole-cell proteins were extracted, trypsinolyzed and analyzed by nano LC-MS/MS, prior to identification and to classification by function using the X!Tandem pipeline software and the proteomic data from NCBI.nlm.nigh.gov. Quantification of the proteins was moreover performed to evidence changes in their expression, depending on the culture medium. Data are available via ProteomeXchange with the identifier PXD033905 (http://www.proteomexchange.org/). This article is related to the research article entitled "The stressing life of Lactobacillus delbrueckii subsp. bulgaricus in soy milk", by G.Jan et al. in Food Microbiology, 2022. This proteomic differential analysis indeed revealed major modulation of the stress proteome, with many stress proteins upregulated in the soy environment.

Inflammatory bowel disease therapeutic strategies by modulation of the microbiota: how and when to introduce pre-, pro-, syn-, or postbiotics?

Inflammatory bowel diseases (IBD), a heterogeneous group of inflammatory conditions that encompass both ulcerative colitis and Crohn's disease, represent a major public health concern. The etiology of IBD is not yet fully understood and no cure is available, with current treatments only showing long-term effectiveness in a minority of patients. A need to increase our knowledge on IBD pathophysiology is growing, to define preventive measures, to improve disease outcome, and to develop new effective and lasting treatments. IBD pathogenesis is sustained by aberrant immune responses, associated with alterations of the intestinal epithelial barrier (IEB), modifications of the enteric nervous system, and changes in microbiota composition. Currently, most of the treatments target the inflammation and the immune system, but holistic approaches targeting lifestyle and diet improvements are emerging. As dysbiosis is involved in IBD pathogenesis, pre-, pro-, syn-, and postbiotics are used/tested to reduce the inflammation or strengthen the IEB. The present review will resume these works, pointing out the stage of life, the duration, and the environmental conditions that should go along with microbiota or microbiota-derived treatments.

A Unique Enhancement of Propionibacterium freudenreichii's Ability to Remove Pb(II) from Aqueous Solution by Tween 80 Treatment.

Microbial agents have promise for the bioremediation of Pb(II)-polluted environments and wastewater, the biodecontamination of foods, and the alleviation of toxicity in living organisms. The dairy bacterium Propionibacterium freudenreichii is poorly able to remove Pb(II) from aqueous solution at 25 ppm, ranging from 0 to 10% of initial concentration. Here, we report on an original strong enhancement of this activity (ranging from 75% to 93%, p < 0.01) following the addition of a polysorbate detergent (Tween® 80) during or either shortly after the growth of a P. freudenreichii culture. We evaluated the optimal Tween® 80 concentration for pretreatment conditions, documented the role of other detergents, and explored the possible mechanisms involved. Our results reveal a novel, environmentally friendly, low-cost pretreatment procedure for enhancing the selective removal of lead from water by probiotic-documented bacteria.

The stressing life of Lactobacillus delbrueckii subsp. bulgaricus in soy milk.

Lactobacillus delbrueckii subsp. bulgaricus is a beneficial lactic acid bacterium and constitutes one of the most used, and thus consumed, dairy starters, worldwide. This homofermentative bacterium was the first lactobacillus described and is involved in the fermentation of yogurt and of diverse other fermented products, including cheeses. It has a long history of safe use, as well as documented probiotic lato sensu effects, including alleviation of lactose intolerance. Plant-based fermented products presently experience a considerable development, as a result of evolution of consumers' habits, in a general context of food transition. This requires research and development, and thus scientific knowledge, to allow such transition, including the development of fermented soy milks. These last indeed offer an alternative source of live and active bacteria. The yogurt starters L. delbrueckii subsp. bulgaricus, together with Streptococcus thermophilus, have been implemented to generate yogurt-type fermented soy milks worldwide. While the adaptation of these starters to the dairy environment has been extensively studied, little is known about L. delbrueckii adaptation to the soy environment. We therefore investigated its adaptation to soy milk and compared it to cow's milk. Surprisingly, it did not grow in soy milk, neither alone, nor in co-culture with S. thermophilus. Acidification of soy milk was however faster in the presence of both species. In order to deepen such adaptation, we then compared L. delbrueckii growth and survival in soy milk ultrafiltrate (SUF, the aqueous phase of soy milk) and compared it to cow's milk ultrafiltrate (MUF, the aqueous phase of cow milk). This comparison revealed major differences in terms of cell morphology and proteome composition. Lactobacilli appeared deformed and segmented in soy. Major differences in both the surface and the cellular proteome indicated upregulation of stress proteins, yet downregulation of cell cycle and division machinery. Altogether, these results suggest that soy milk may be a stressing environment for the yogurt starter L. delbrueckii subsp. bulgaricus.

Lyophilized Symbiotic Mitigates Mucositis Induced by 5-Fluorouracil.

Mucositis is an adverse effect of cancer chemotherapies using 5-Fluorouracil (5-FU). It is characterized by mucosal inflammation, pain, diarrhea, and weight loss. Some studies reported promising healing effects of probiotic strains, when associated with prebiotics, as adjuvant treatment of mucositis. We developed a lyophilized symbiotic product, containing skimmed milk, supplemented with whey protein isolate (WPI) and with fructooligosaccharides (FOS), and fermented by Lactobacillus casei BL23, Lactiplantibacillus plantarum B7, and Lacticaseibacillus rhamnosus B1. In a mice 5-FU mucositis model, this symbiotic lyophilized formulation was able to reduce weight loss and intestinal permeability. This last was determined in vivo by quantifying blood radioactivity after oral administration of 99mTc-DTPA. Finally, histological damages caused by 5-FU-induced mucositis were monitored. Consumption of the symbiotic formulation caused a reduced score of inflammation in the duodenum, ileum, and colon. In addition, it decreased levels of pro-inflammatory cytokines IL-1ß, IL-6, IL-17, and TNF-α in the mice ileum. The symbiotic product developed in this work thus represents a promising adjuvant treatment of mucositis.

Improving the drying of Propionibacterium freudenreichii starter cultures.

Propionibacterium freudenreichii is a beneficial food-grade actinobacterium, widely implemented, and thus consumed, in various food products. As the main application, P. freudenreichii is used as a cheese-ripening starter, mostly in hard type cheeses. Indeed, during manufacture of "Swiss-type" cheeses (or opened-body cheeses), the technological process favors propionibacteria growth, as well as the corresponding propionic fermentation. This leads to the characteristic flavor of these cheeses, through the release of short chain fatty acids and through lipolysis, as well as to their specific texture. To fulfil this ripening, massive amounts of propionibacteria are industrially produced, dried and stored, prior to cheese making. Furthermore, P. freudenreichii is commercialized in various probiotic food supplements aiming at preserving intestinal health and comfort, in line with its ability to produce beneficial metabolites (short chain fatty acids, vitamins), as well as immunomodulatory compounds. Other industrial applications of P. freudenreichii include the production of food-grade vitamins of the B group, of trehalose, of conjugated linoleic acid, and of biopreservatives. For these different applications, maintaining survival and activity of propionibacteria during production, drying, storage and finally implementation, is crucial. More widely, maintaining live and active probiotic bacteria represents a challenge as the market for probiotic products increases. Probiotic bacteria are, for a bulk majority, freeze-dried, but spray drying is also more and more considered. Indeed, this process is both continuous and more cost-efficient, as it utilizes less energy compared to freeze-drying; on the other hand, it exposes bacteria to higher heat and oxidative stresses. Apart from process optimization and strain selection, it is possible to enhance the resistance of bacteria by taking advantage of their adaptation capacity. Indeed, P. freudenreichii stress tolerance can be boosted by different pretreatments applied before the drying step, thus considerably increasing its final survival. In particular, adaptation to hyperosmotic conditions improves stress tolerance, while the presence of osmoprotectants may mitigate this improvement. Thermal adaptation also modulates tolerance towards these technological challenges. The composition of the growth medium, including the ratio between the carbohydrates provided and the non-protein nitrogen, plays a key role in driving the accumulation of osmoprotectants. This, in turn, determines P. freudenreichii tolerance towards different stresses, and overall towards both freeze-drying and spray-drying. As an example, the accumulation of trehalose enhances its spray-drying survival, while the accumulation of glycine betaine enhances its freeze-drying survival. Growth of propionibacteria in hyperconcentrated whey was used to trigger multiple stress tolerance acquisition, underpinned by overexpression of key stress protein, accumulation of cytoplasmic storage compounds, and leading to enhanced spray-drying survival. A simplified process, from cultivation to atomization, was developed by using whey as a 2-in-1 medium in which propionibacteria were grown, protected and dried with minimal cell death. This innovative process was then subjected to scaling up at the industrial level. In this aim, a gentle multi-stage drying process offering mild drying conditions by coupling spray drying with belt drying, led to final probiotic survival close to 100% when stress tolerance acquisition was previously implemented. Such innovation opens new avenues for the efficient, cost-effective and sustainable development of new probiotic production technologies, as well as probiotic application in the context of food and feed. KEY POINTS: • Propionibacteria acquire multi-stress tolerance when grown in hyper-concentrated whey. • Spray drying of osmo-adapted probiotic bacteria is possible with limited cell death. • A two-in-one drying method is developed to grow and dry probiotic bacteria in the same matrix.

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).

Therapeutic Effects of Probiotic Minas Frescal Cheese on the Attenuation of Ulcerative Colitis in a Murine Model.

Inflammatory bowel diseases (IBDs) constitute disturbances of gastrointestinal tract that cause irreversible changes in the structure and function of tissues. Ulcerative colitis (UC), the most frequent IBD in the population, is characterized by prominent inflammation of the human colon. Functional foods containing probiotic bacteria have been studied as adjuvants to the treatment or prevention of IBDs. The selected probiotic strain Lactococcus lactis NCDO 2118 (L. lactis NCDO 2118) exhibits immunomodulatory effects, with promising results in UC mouse model induced by dextran sodium sulfate (DSS). Additionally, cheese is a dairy food that presents high nutritional value, besides being a good delivery system that can be used to improve survival and enhance the therapeutic effects of probiotic bacteria in the host. Therefore, this work investigated the probiotic therapeutic effects of an experimental Minas Frescal cheese containing L. lactis NCDO 2118 in DSS-induced colitis in mice. During colitis induction, mice that consumed the probiotic cheese exhibited reduced in the severity of colitis, with attenuated weight loss, lower disease activity index, limited shortening of the colon length, and reduced histopathological score. Moreover, probiotic cheese administration increased gene expression of tight junctions' proteins zo-1, zo-2, ocln, and cln-1 in the colon and increase IL-10 release in the spleen and lymph nodes. In this way, this work demonstrates that consumption of probiotic Minas Frescal cheese, containing L. lactis NCDO 2118, prevents the inflammatory process during DSS-induced colitis in mice, opening perspectives for the development of new probiotic functional foods for personalized nutrition in the context of IBD.

SlpB Protein Enhances the Probiotic Potential of L. lactis NCDO 2118 in Colitis Mice Model.

Bacteria used in the production of fermented food products have been investigated for their potential role as modulators of inflammation in gastrointestinal tract disorders such as inflammatory bowel diseases (IBD) that cause irreversible changes in the structure and function of gut tissues. Ulcerative colitis (UC) is the most prevalent IBD in the population of Western countries, and it is marked by symptoms such as weight loss, rectal bleeding, diarrhea, shortening of the colon, and destruction of the epithelial layer. The strain Propionibacterium freudenreichii CIRM-BIA 129 recently revealed promising immunomodulatory properties that greatly rely on surface-layer proteins (Slp), notably SlpB. We, thus, cloned the sequence encoding the SlpB protein into the pXIES-SEC expression and secretion vector, and expressed the propionibacterial protein in the lactic acid bacterium Lactococcus lactis NCDO 2118. The probiotic potential of L. lactis NCDO 2118 harboring pXIES-SEC:slpB (L. lactis-SlpB) was evaluated in a UC-mice model induced by Dextran Sulfate Sodium (DSS). During colitis induction, mice receiving L. lactis-SlpB exhibited reduced severity of colitis, with lower weight loss, lower disease activity index, limited shortening of the colon length, and reduced histopathological score, with significant differences, compared with the DSS group and the group treated with L. lactis NCDO 2118 wild-type strain. Moreover, L. lactis-SlpB administration increased the expression of genes encoding tight junction proteins zo-1, cln-1, cln-5, ocln, and muc-2 in the colon, increased IL-10 and TGF-ß, and decreased IL-17, TNF-α, and IL-12 cytokines in the colon. Therefore, this work demonstrates that SlpB recombinant protein is able to increase the probiotic potential of the L. lactis strain to alleviate DSS-induced colitis in mice. This opens perspectives for the development of new approaches to enhance the probiotic potential of strains by the addition of SlpB protein.

Environmental conditions modulate the protein content and immunomodulatory activity of extracellular vesicles produced by the probiotic Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions.Importance Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems, and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

Differential Adaptation of Propionibacterium freudenreichii CIRM-BIA129 to Cow's Milk Versus Soymilk Environments Modulates Its Stress Tolerance and Proteome.

Propionibacterium freudenreichii is a beneficial bacterium that modulates the gut microbiota, motility and inflammation. It is traditionally consumed within various fermented dairy products. Changes to consumer habits in the context of food transition are, however, driving the demand for non-dairy fermented foods, resulting in a considerable development of plant-based fermented products that require greater scientific knowledge. Fermented soymilks, in particular, offer an alternative source of live probiotics. While the adaptation of lactic acid bacteria (LAB) to such vegetable substrates is well documented, little is known about that of propionibacteria. We therefore investigated the adaptation of Propionibacterium freudenreichii to soymilk by comparison to cow's milk. P. freudenreichii grew in cow's milk but not in soymilk, but it did grow in soymilk when co-cultured with the lactic acid bacterium Lactobacillus plantarum. When grown in soymilk ultrafiltrate (SUF, the aqueous phase of soymilk), P. freudenreichii cells appeared thinner and rectangular-shaped, while they were thicker and more rounded in cow's milk utltrafiltrate (MUF, the aqueous phase of cow milk). The amount of extractable surface proteins (SlpA, SlpB, SlpD, SlpE) was furthermore reduced in SUF, when compared to MUF. This included the SlpB protein, previously shown to modulate adhesion and immunomodulation in P. freudenreichii. Tolerance toward an acid and toward a bile salts challenge were enhanced in SUF. By contrast, tolerance toward an oxidative and a thermal challenge were enhanced in MUF. A whole-cell proteomic approach further identified differential expression of 35 proteins involved in amino acid transport and metabolism (including amino acid dehydrogenase, amino acid transporter), 32 proteins involved in carbohydrate transport and metabolism (including glycosyltransferase, PTS), indicating metabolic adaptation to the substrate. The culture medium also modulated the amount of stress proteins involved in stress remediation: GroEL, OpuCA, CysK, DnaJ, GrpE, in line with the modulation of stress tolerance. Changing the fermented substrate may thus significantly affect the fermentative and probiotic properties of dairy propionibacteria. This needs to be considered when developing new fermented functional foods.

Extracellular Vesicles Produced by the Probiotic Propionibacterium freudenreichii CIRM-BIA 129 Mitigate Inflammation by Modulating the NF-κB Pathway.

Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered to be a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. Among probiotic Gram-positive bacteria, some Propionibacterium freudenreichii strains exhibit anti-inflammatory activity, notably via surface proteins such as the surface-layer protein B (SlpB). We have hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host. In order to demonstrate their production in this species, EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, and their physicochemical characterization, using transmission electron microscopy and nanoparticle tracking analysis (NTA), revealed shapes and sizes typical of EVs. Proteomic characterization showed that EVs contain a broad range of proteins, including immunomodulatory proteins such as SlpB. In silico protein-protein interaction predictions indicated that EV proteins could interact with host proteins, including the immunomodulatory transcription factor NF-κB. This potential interaction has a functional significance because EVs modulate inflammatory responses, as shown by IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells. Indeed, EVs displayed an anti-inflammatory effect by modulating the NF-κB pathway; this was dependent on their concentration and on the proinflammatory inducer (LPS-specific). Moreover, while this anti-inflammatory effect partly depended on SlpB, it was not abolished by EV surface proteolysis, suggesting possible intracellular sites of action for EVs. This is the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. This study has enhanced our understanding of the mechanisms associated with the probiotic activity of P. freudenreichii and identified opportunities to employ bacterial-derived EVs for the development of bioactive products with therapeutic effects.