Dairy Propionibacteria: Probiotic Properties and Their Molecular Bases

1. Introduction

Dairy propionibacteria are a taxonomically heterogeneous group of actinomycetes, i.e. high G+C Gram-positive bacteria, associated to food and feed environments that belong to the genera Propionibacterium and Acidipropionibacterium. There two genera were previously both classified as Propionibacterium that comprised both food associated species and cutaneous species of clinical significance. The currently recognized species associated to food products are Propionibacterium freudenreichii, Acidipropionibacterium acidipropionici, A. jensenii, A. thoenii and the less frequently isolated P. cyclohexanicum, A. microaerophilum, A. damnosum, A. olivae, A. virtanenii, and A. timonense. The species P. freudenreichii was previously separated into the subspecies freudenreichii and shermanii on the basis of lactose fermentation and nitrate reducing ability, a distinction no more valid since whole genome sequencing highlighted that the first trait was encoded by an integrative-conjugative element (ICE) acquired by horizontal gene transfer (HGT), while the second trait was lost in intra-species groups following a frameshift mutation [1,2,3,4].

Dairy propionibacteria are rod shaped, anaerobic or microaerophilic aerotolerant for their endowment of enzymes that protect against oxidative stress such as a superoxide dismutase, a catalase and a cytochrome bd oxidase. Moreover, they express heme biosynthesis proteins and functional electron transport chains and some strains are able to grow in aerobic conditions. Their central metabolism leads to the production of propionate, acetate and CO₂ from lactate through the Wood-Werkman cycle, involving succinate decarboxylation, and they can utilize different carbohydrates, including mannose, arabinose, and xylose, and glycerol as carbon sources [5,6,7]. P. freudenreichii typically produces SCFAs mixtures in which the amount of propionate produced is typically at least double compared to acetate [8,9].

These bacteria have been isolated from milk, acid whey, different traditional cheeses, feed flour and silage mixed or not with grains, soil, fermented vegetables, barley grains, goat rumen, chicken intestine and fecal samples from breast-fed preterm infants [9,10,11,12,13,14,15]. Moreover, one study indicated that they could survive the transit in the gastrointestinal tract when supplied in cheese [16]. Dairy propionibacteria, mainly P. freudenreichii, have an essential role in the Swiss Type cheese technology in which they allow to obtain the distinctive “eyes”, given by CO₂ accumulation during ripening, and the production of flavors. In other cheese types these bacteria are unwanted since they can cause defects such as anomalous blowing and colors so their presence in milk should be kept under control. However, only a few studies considered their distribution in milk and reported that their presence is very frequent, ranging between 50% and 95.6%/97% [17,18,19]. Carafa et al. reported an increase of the percentage of samples positive for dairy propionibacteria in milk from cows led to Alpine pastures compared to the milk of cows reared in permanent farms [20].

The relevance of dairy propionibacteria as probiotic organisms, i.e. “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [21] on human and animal health was documented by a body of research that dates back to the eighties according to a short review on the first findings from in vivo trials published in 1995. Until that time propionibacteria had been used as growth promoters for farmed animals such as calves, piglets and hens, mostly combined with lactic acid bacteria but also as pure cultures. Some P. freudenreichii were effective in increasing weight gain, decreasing feed conversion ratio and improving intestinal health. Though not adhering, P. freudenreichii showed a good survival rate in simulated gastrointestinal transit. Mixed cultures of dairy propionibacteria with bifidobacteria and lactic acid bacteria had shown anticholesterolemic and β-galactosidase activity and fermented milk containing two strains of P. freudenreichii and Lactobacillus acidophilus improved the overall health status in elderly people in a small-scale clinical trial. Other properties of propionibacteria were the capacity to stimulate the development of bifidobacteria and lactobacilli. At that time dairy propionibacteria were already industrially exploited to produce vitamin B12 and propionic acid [22].

Dairy propionibacteria are among the bacterial species involved in dairy technology and able to exert health promoting effects in vivo [23]. According to the latest review article regarding their probiotic properties published in 2017, P. freudenreichii and A. acidipropionici transiently survive and maintain metabolic activity in the gastrointestinal tract, and adhere to intestinal cells. These two species of dairy propionibacteria favor the increase of intestinal bifidobacteria by producing the bifidogenic molecules 1,4-dihydroxy-2-naphtoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), and also induce a decrease of toxin producing Bacteroides and Clostridium difficile. Moreover, P. freudenreichii selected strains exerted immunomodulation with anti-inflammatory effects in human cell-lines and in mice with induced colitis or mice fed on a high fat diet (HFD). The in vivo beneficial effects of P. freudenreichii were shown to be mediated by surface-layer proteins (Slps). In addition, P. freudenreichii favored the apoptosis in human cancer cell lines and in a mice tumor model for the ability to produce short chain fatty acids (SCFAs) propionate and acetate [24].

Currently, P. freudenreichii and A. acidipropionici are included in the list of microbial species with qualified presumption of safety (QPS) status for use in food and feed of the European Food Safety Authority (EFSA). Therefore, based on the taxonomic identification, body of knowledge and potential safety concerns, representatives of these species must not undergo a full safety evaluation process and must only satisfy the requirement of not harboring “any acquired antimicrobial resistance genes to clinically relevant antimicrobials” before use in food and feed [25]. In addition, the species P. freudenreichii, A. acidipropionici, A. jensenii and A. thoenii are included in the list of the microbial species with safety demonstration by the International Dairy Federation (FIL-IDF) [26].

This descriptive review summarizes the state-of-the-art of knowledge on the beneficial effects of dairy propionibacteria on disease prevention and mitigation and their molecular bases to obtain a complete and up-to-date overview of their probiotic potential. To this end, scientific articles regarding the evaluation of the probiotic properties of dairy propionibacteria were sought in the databases GoogleScholar (https://scholar.google.com/schhp?hl=it, accessed on 25 January 2025) and Embase (https://www-embase-com.bibliosan.idm.oclc.org/, accessed on 25 January 2025) by relevance sorting with the search strings “Propionibacterium probiotic” or “Acidipropionibacterium probiotic”. For the GoogleScholar search results 80 pages were screened to select pertinent titles since two subsequent pages did not contain any further items relevant to the study. In Embase 705 sources were retrieved and evaluated for pertinence. The articles finally selected after elimination of duplicates, abstracts screening and critical judgment of originality and novelty are commented in this review. Among articles regarding probiotic mixtures, only those indicating distinct actions of propionibacteria were considered.

2. Molecular Traits of Dairy Propionibacteria Relevant for Probiotic Activities

Dairy propionibacteria are rather understudied because of technical difficulties in their isolation, due to the long incubation times of up to seven days, poor selectivity of the isolation media [17,19], and difficulties in the outcomes of genetic tests determined their high G+C genome content higher than 67% [27]. Indeed, for polymerase chain reaction (PCR) tests and sequencing the addition of reagents that facilitate strand and secondary structure dissociation are needed for these bacteria [28]. Whole genome sequences became available rather recently for these bacteria with 125, 19, 11 and 1 annotated sequences for P. freudenreichii, A. jensenii, A. acidipropionici, and A. thoenii, respectively, available in GenBank [https://www.ncbi.nlm.nih.gov/nuccore, accessed on 24 January 2025].

A recent study used the P. freudenreichii species as a representative of high G + C genome content organisms to evaluate the performance of different long-read sequencing methods in providing complete sequence data, and highlighted the necessity to adopt appropriate sequencing strategies to obtain optimum completeness and contiguity, correct assemblies, and correct gene content determination from these bacteria. For three P. freudenreichii strains, TL19, TL29 and TL110, the genome dimension varied even for the same strain depending on the sequencing technique and assembling method adopted and ranged between 2,497,808 bp and 2,566,603 bp, while the number of coding sequences (CDS) was comprised between 2158 and 3236 [29].

The largest study on the genome features of dairy propionibacteria was carried out for 20 strains of P. freudenreichii isolated from milk, barley and cheese by using the long read sequencing with the PacBio RS II sequencing platform. Eight strains showed an additional genome deriving from duplication driven by transposable elements. The presence of these transposable elements, ICEs and prophages, the number of accessory and unique genes varied among the strains and inversions or other rearrangements were observed though the level of co-linearity was high. The composition of the accessory genome highlighted variability in probiotic properties, stress tolerance and safety. Variable genomic islands encode a pilus locus, rhamnose utilization, heat shock proteins, immunity related clustered regularly interspaced short palindromic repeats Cas (CRISPR-Cas) systems, restriction/modification systems, resistance to heavy metals and putative antibiotic resistance determinants. Some of these regions were probably acquired by horizontal gene transfer from A. acidipropionici. Putative plasmids were detected only in two strains but these lack a replication origin. Among the S-layer protein genes involved in the anti-inflammatory properties of P. freudenreichii, slpA and another slp gene were present in all the strains, but on genomic islands in some of them, the S-layer protein precursor ctc, slpE, and slpB were present in 13, 12 and 2 strains, respectively [2].

All the strains harbored the gene cluster for the production of vitamin B12 that comprises 33 genes and only in one strain some genes of the cluster showed mutations. The three riboswitches that regulate this gene cluster were also conserved. Pilus genes were found in all the strains and encode putative subunits of fimbriae anchored to the cell surface, fimbriae major subunits and a putative sortase C. However, the number of genes and gene sequence varied and only one strain showed pilus appendages in electron microscopy observation and exhibited mucus binding capacity [2].

The production of exopolysaccharides (EPS), which is commonly considered a trait involved in adhesion and immunomodulation [30], was analyzed in 68 P. freudenreichii strains and found to vary. PCR tests showed that all the strains carried a glucosyltransferase gtf gene homologous to the ttf gene responsible for EPS production in Streptococcus pneumoniae. However, only 24 strains agglutinated when exposed to a P. freudenreichii EPS specific antiserum. Optical and electron microscopy showed that in some of the agglutinating strains a capsule was present. The Gtf protein was highly conserved among strains with changes only in six amino acid positions. Its disruption in the P. freudenreichii type strain hindered EPS production and its heterologous introduction in Lactococcus lactis IL1403 conferred a polysaccharide production capacity to this strain, thus showing that gtf is the only gene required for EPS production in P. freudenreichii. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) highlighted that different levels of EPS production were correlated with the gtf transcription level. This was between 18 and 264 higher in agglutinating and a number of gtf transcript copies of at least 10⁷/100 ng of RNA conferred the capsular phenotype. The presence of two putative transposase genes 194 upstream to the gtf coding region could be responsible for the overexpression of gtf in the agglutinating strains [31].

It was later reported that the presence of a capsule hinders the immunomodulation effects of P. freudenreichii by masking the surface molecules responsible for the immune response induced. About 30% of the strains of this species produce EPS constituted by (1→3,1→2)-β-D-glucan. Strains with an inactivated gtfF gene showed a better biofilm formation capacity than the respective wild types but not enhanced adhesion to Caco-2 cells. The wild type strains were able to induce the anti-inflammatory interleukin IL-10 production by these cells but this property was enhanced in the ΔgtfF mutants. The presence of the EPS did not increase the survival of P. freudenreichii in the mouse gastrointestinal tract [32].

A study on the cell surface proteins obtained by guanidine hydrochloride extraction from P. freudenreichii CIRM BIA 129 (ITG P20) indicated the presence of the internalin A (InlA), S-layer proteins SlpA, SlpB and SlpE, the large surface protein A LspA, and proteins involved in adhesion (BopA), penicillin binding, solute binding, resuscitation (RpfB factor) and cytoplasmic proteins among which heat shock proteins and translation/elongation factors. Shaving with trypsin retrieved a subset of the proteins non-covalently bound to the cell surface among which the S-layer proteins. SlpB was the most abundant protein present in the guanidine hydrochloride extracts and its molecular mass was experimentally determined to be 54,147 Da. The surface protein extract induced a dose-dependent release of the immunomodulatory cytokines IL-10 and IL-6 by peripheral blood mononuclear cells (PBMCs) [33].

Mono-dimensional electrophoresis (1-DE) of proteins secreted by 27 strains of P. freudenreichii and A. acidipropionici showed different patterns for strains of dairy origin and those of cereal origin. Among the secreted proteins and with variability at the strain level a 27-kDa transglycosylase was in common between the two genera. Moreover, SlpA, RpfB, and a NlpC/P60 family peptidase were the most abundant secreted proteins. In addition, stress-response proteins and central metabolism enzymes were detected with differences in identity among the strains. Beyond the above listed proteins, two-dimensional electrophoresis (2-DE) followed by fluorescent staining allowed to identify a d-alanyl-d-alanine carboxypeptidase involved in peptidoglycan synthesis, and the lipoprotein OppA, involved in oligopeptide transport, more abundant in the dairy strains, InlA, and a metallo-endopeptidase M23B. Proteins involved in adhesion, such as a fimbrillin subunit FimB, were detected for the cereal strain P. freudenreichii JS14. This strain showed the capacity to adhere to a hydrophobic material and bovine serum albumin (BSA), while the dairy strain P. freudenreichii JS22 showed minimal adhesion and a clumping phenotype. None of the strains studied adhered to mucus [34].

Among the surface proteins of P. freudenreichii JS14 SlpB and the aminopeptidase N (PepN) were specifically identified [34]. The gene encoding the latter protein is present in the genomes of all the dairy propionibacteria type strains but A. microaerophilum was found to harbor two paralogs of pepN [35] and the genome sequences made available later show the same also for A. acidipropionici [https://www.ncbi.nlm.nih.gov/nuccore, accessed on 20 February 2025]. For P. freudenreichii JS22 a putative arabinose isomerase AraI, the Clp family ATPase ClpB and LspA predominated among the secreted proteins [34].

A P. freudenreichii CIRM BIA 129 slpB deletion mutant (ΔslpB) showed reduced cell surface electronegativity and reduced hydrophobicity determined as adhesion capacity to hydrocarbon solvents xylol, chloroform, and ethyl acetate. Moreover, the ΔslpB mutant showed reduced tolerance to acidic, bile salts and heat stress. Proteome analysis identified 27 proteins expressed only in the mutant strain and involved in metabolism, replication, recombination and repair, while a carboxylic ester hydrolase was expressed only in the wild type strain. The downregulated proteins in the ΔslpB mutant included an exported enolase known to be involved in immunomodulation and adhesion, and the surface-layer (S-layer) proteins SlpA, SlpD and SlpE and InlA [36].

The anti-inflammatory properties of P. freudenreichii were first demonstrated for the strain JS that reduced the expression of the pro-inflammatory interleukin IL-8 in Caco-2 cells infected with Helicobacter pilori and increased the expression of the anti-inflammatory cytokine IL-10 in PBMC. This effect was found to be strain-dependent since only P. freudenreichii CIRM BIA 129 among the 23 strains studied by Deutsch et al. [32] induced this cytokine at levels comparable to the anti-inflammatory probiotic Bifidobacterium longum BB536. Varying with the strain, the pro-inflammatory cytokines interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) were very weakly induced and no induction of interleukine IL-12 occurred. Shaving of P. freudenreichii cells with guanidine hydrochloride highlighted diversity in surface protein composition and a total number of 174 proteins unique for single strains. Proteins SlpA, SlpB, SlpE, LpsA containing the surface-layer homology (SLH) domain for anchorage to the cell wall, and a protein with glucosaminidase domain, were found in one or more strains [32].

Genomic, transcriptomic and surface proteome indicated respectively 158, 161 and 11 genes positively associated with IL-10 induction, with SlpE identified with all three data sets. Among fifteen genes with the strongest positive or negative association with IL-10 induction, the slpB, slpE, slpF, hsdM3, pep, htrA4, eno1 and pouf were successfully inactivated in different strains so it could be demonstrated that the inactivation of slpB, slpE and hsdM3 in P. freudenreichii CIRM BIA 129 induced a remarkably lower secretion of IL-10 by PBMCs, the inactivation of slpB, slpF and pouf 10925 reduced the secretion of tumor necrosis TNF-α and IFN-γ, and the inactivation of slpE reduced the secretion of TNF-α only. Inactivation of eno1, htrA4 and pouf 08235 in the weakly anti-inflammatory strain CIRM BIA 121 increased the secretion of IL-10, TNF-α and IL-6 [37].

The SlpB protein is also involved in adhesion of P. freudenreichii to human intestine epithelial cells HT29. This protein was detected in different amounts in four among seven P. freudenreichii strains tested for adhesion capacity. The P. freudenreichii CIRM BIA 129 strain showed highest adhesion rate and strain CIRM BIA 118, also expressing SlpB, showed about half its adhesion rate. The strain with the lowest adhesion rate did not express S-layer proteins. The P. freudenreichii strains were not internalized in the intestinal cells and scanning electron microscopy highlighted their localization on the brush border. The adhesion capacity was lost after enzymatic shaving with trypsin and after deletion of the slpB gene [38]. In HT29 cells P. freudenreichii CIRM BIA 129 or its S-layer proteins attenuated the upregulation of the pro-inflammatory interleukins IL-8 and IL-12 and TNF-α induction by the E. coli lipopolysaccharide (LPS). Moreover, the P. freudenreichii cells induced an upregulation of IL-10 both in presence and absence of LPS, while and its Slps stimulated the increase of the tumor growth factor-β (TGF- β) [39].

A ΔslpB mutant of the strain P. freudenreichii CIRM BIA 129 showed decreased adhesion compared to the wild type to intestinal epithelial barrier (IEB) in vitro models constituted by Caco-2 cell monolayers. Moreover, after inflammation induction, it did not reduce the trans-epithelial electrical resistance (TEER) and did not prevent the paracellular permeability to sulfonic acid-FITC, and the transcellular permeability to horse radish peroxidase (HRP) as the wild type strain. Western blotting highlighted that, differently from the wild type, it did not prevent the decrease of the zonula occludens 1 (ZO-1) protein expression. The organic acids produced by P. freudenreichii CIRM BIA 129 and its ΔslpB mutant did not induce significant changes on inflammation-induced permeability thus proving the essential role of SlpB in mitigating the inflammation induced effects [40].

Extracellular vesicles (EVs) are produced by cells of most organisms and are nanoparticles delimited by a lipid bilayer membrane that contain cell proteins, nucleic acids, and metabolites. EVs are implicated in the transfer of virulence and antibiotic resistance genes but also in the interaction of probiotic microorganisms with the host cells. Beneficial effects exerted by EVs were observed for the probiotic bacteria Lacticaseibacillus paracasei BL23 and rhamnosus GG, Lactiplantibacillus plantarum WCFS1 and Bifidobacterium longum KACC 91563. The EVs produced in ultrafiltered (UF) milk by P. freudenreichii CIRM BIA 129 were spherical with a diameter of about 84 nm, and found to contain 319 proteins representing 11% of the whole cell proteome. The proteins found in EVs were involved in energy metabolism, carbohydrate and amino acid transport, translation, ribosome structure and formation, cell envelope synthesis, protein turnover and signaling. Proteins involved in immunomodulation such as the enolase EnoI, the internalin InlA, the aconitase Acn, the glutamine synthetase Gln1, the glucose-6-phosphate isomerase Gpi, the triosephosphate isomerase Tpi, the surface-layer proteins SlpB and the chaperonin GroEL were detected in EVs [41].

The in silico prediction of protein-protein interactions of P. freudenreichii EV-associated proteins machine learning-based (interSPPI, http://zzdlab.com/intersppi/index.php, accessed on 30 March 2025) and homology-based (interolog), highlighted interactions with human proteins involved in metabolism, signal transduction, infectious diseases and immunity. Among these, the p105 subunit of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and members of its signaling pathway, such as adapters and Toll-like receptors, showed the highest number of interactions. A dose-dependent effect of P. freudenreichii EVs on the reduction of NF-κB activation was experimentally demonstrated in HT29/kb-seap-25 cells in which an inflammatory response was induced with the E. coli LPS but not in those treated with the inflammation inducers TNF-α and IL-1β. In addition, P. freudenreichii EVs induced a dose-dependent reduction of the pro-inflammatory cytokine IL-8 [41].

EVs produced in UF milk by P. freudenreichii CIRM BIA 129 more efficiently inhibited the nuclear factor NF-kB regulatory activity in inflammation induced by E. coli LPS in HT29 cells than those formed in yeast extract lactate medium (YEL). The two EV types shared 358 proteins but the UF milk EVs also contained proteins involved in carbohydrate and amino acid metabolism, DNA processing and immunomodulation targets in the NF-kB signaling pathway [42]. In addition, proteins involved in the production of secondary metabolites, metabolism in different environments, oxidative phosphorylation, peptidoglycan synthesis, ribosome composition, protein export, quorum sensing and proteins SlpB, SlpE, EnoI, aconitase Acn, GroEL2 and a membrane hypothetical lipoprotein, previously shown to participate to the immunomodulatory properties, were identified [43].

EVs produced by P. freudenreichii CIRM BIA 129 contain a correctly conformed SlpB protein. Those purified by size exclusion chromatography inhibited the increase in paracellular permeability to sulfonic acid-fluorescein isothiocyanate (FITC) in a cell monolayer, thus confirming the role of SlpB in mitigating the inflammation-induced changes [40].

3. In vivo Beneficial Effects of Dairy Propionibacteria

Dairy propionibacteria were shown to exert a multiplicity of health promoting effects in animal models for the prevention and treatment of different conditions including intestinal inflammations, colorectal cancer, bone diseases and infections. The best studied species was P. freudenreichii for which different strains were tested in vivo for the molecular mechanisms of action [14,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71].

The capacity to exert in vivo beneficial effects is determined by the ability of dairy propionibacteria to survive during transit in the gastrointestinal tract (GIT). In particular, A. jensenii 702 was fed to rats and retrieved from feces in high numbers [72] and the strain P. freudenreichii JS was recovered in high numbers from the feces of healthy subjects who consumed fruit juice enriched with whey containing this bacterium though gut colonization was transient [73]. Use of the in vitro dynamic DIDGI® digestion system allowed to observe that P. freudenreichii CIRM BIA 129 included in a cheese matrix did not lose viability during the gastric and small intestine phases and remained viable at high levels until the end of the process. After in vitro static digestion, sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) separation and Western blotting allowed to observe that SlpB remained intact until the end of digestion of cheese containing P. freudenreichii, while it was degraded after the gastric phase when the strain was supplied in UF milk. In the dynamic digestion system SlpB remained intact for longer in the duodenum phase when supplied with cheese compared to UF milk indicating that the assumption of P. freudenreichii with cheese favors the preservation of its probiotic properties [39].

The type strains of P. freudenreichii, A. acidipropionici, A. jensenii and A. thoenii adhered to normal cells of the ileal mucosa IPEC-J2 at rates comparable to L. reuteri 12002 used as positive control with P. freudenreichii showing the highest adhesion percentages enhanced in the presence of calcium. This finding was in accordance with some, though not all, results from previous studies. The A. acidipropionici strain showed the lowest viability loss at pH 2.5 since its decrease was negligible after 24 h, while the other species showed a viability loss of about 30% after 24 h. None of the strains showed a significant reduction after 3 h at this pH value [74]. However, literature data are not concordant, thus indicating strain-specificity for tolerance to low pH [75]. The tolerance to bile salts was also variable since high tolerance or high sensitivity was reported to 0.3% (w/v) of these compounds that could be explained with not uniform testing conditions [74]. Ibrahim et al. [74] observed that some strains were able to survive in presence of 1% bile salts for 48 h with little viability loss.

Growth promotion experiments in farm animals highlighted the beneficial effects of dairy propionibacteria on the health status and their capacity to maintain viability in GIT. Use of A. acidipropionici P169 as direct-fed microbial (DFM) in Holstein calves post-partum allowed to increase milk yield in a dose dependent manner and reduce weight loss [76]. A. jensenii 702 was used as DFM in calves since it was previously reported that dairy propionibacteria induce the increase propionate and butyrate in the rumen increasing feed conversion and growth rate and it was recovered from feces ant about 5 Log CFU/g during the treatment period when administered in daily doses of 9 Log CFU/kg. The group of calves treated with A. jensenii 702 showed a faster weight gain and the difference was statistically significant until 18 weeks after treatment. No adverse effects of the supplied propionibacteria emerged from hematological values [77]. The same strain was supplied to layer chicken in doses of 7 Log CFU per day and determined an egg weight increase of 4.2% compared to the control and no adverse effects. The A. jensenii strain was proven to survive the transit in the chicken GIT [78]. A. acidipropionici LET105 and LET107 isolated from chicken intestine were administered to 1 – 14 days aged chicks daily in a 6 Log CFU dose with no adverse effects or difference in growth rate. The group of animals receiving the propionibacteria showed a better development of the intestinal mucosa with increased crypt length, goblet cell number and mucus production [13].

3.1. Prevention and Mitigation of Intestinal Inflammatory Diseases

Inflammatory bowel diseases (IBD) conditions are accompanied by intestinal dysbiosis, i.e. a less diverse and more unstable intestinal microbiota than in healthy subjects and a reduction of microbial components with an immunomodulatory role [50]. Therefore, one of the expected functions of probiotics is the re-establishment of microbiota components with beneficial roles and the reduction of those with detrimental effects. Among dairy propionibacteria, A. jensenii 702 has been used in mixed commercial probiotic preparations since it favored the increase of bifidobacteria [79]. An in vitro study showed that P. freudenreichii W200 (Winclove Probiotics, Amsterdam, The Netherlands) was the only probiotic that did not increase Bacteroides-Prevotella spp. in fecal cultures and induced a higher production of lactate among the SCFAs compared to probiotic strains belonging to the species Bacillus coagulans, B. subtilis, Levilactobacillus reuteri and Lacticaseibacillus rhamnosus [80].

In murine colitis induced with sodium dextran sulfate (DSS), the P. freudenreichii DHNA metabolite attenuated the expression of the mucosal addressin cell adhesion molecule 1 (MAdCAM-1) that favors leucocyte infiltration, and determined a lower colon mucosa damage score reduction in the number of β7 integrin positive cells that mediate the adhesion of leucocytes to vascular endothelial cells, significantly suppressed mRNA levels of IL-1β, IL-6, and TNF-α, and significantly increased the number of lactobacilli and the amounts of SCFAs [45].

One mechanism of colitis inhibition observed for P. freudenreichii ET-3 involves the aryl hydrocarbon receptor (AhR) transcriptional factor that recognized xenobiotics and is involved in their detoxification by activating genes with a xenobiotic-responsive element (XRE) consensus sequence in the promoter. It was also reported that the activation of AhR-regulated pathways in gut by agents such as some probiotic bacteria, suppresses IBDs. A screening of metabolites produced by probiotic bacteria for AhR-regulated pathways activation identified the DHNA derived from P. freudenreichii ET-3 as a suitable substance able to increase the transcription level of the cytochrome P450 family 1 subfamily A member 1 (CYP1A1) induced by AhR in Caco2 cells without affecting their viability. This effect occurred also in vivo, mainly in the upper intestine of mice [46]. The Ahr-induced anti-microbial C-type lectins RegIII inhibit DSS-induced colitis and some members of this family increased in mice receiving DHNA. Finally, DHNA inhibited the production of IL-6 upon exposure to LPS in bone marrow macrophages without showing toxicity for these cells. Therefore, DHNA could represent a nontoxic Ahr activator to be exploited in colitis prevention [46].

The surface proteome of P. freudenreichii CIRM BIA 129 grown in a cheese comprising only this strain included as most abundant components identified by MS/MS the proteins SlpA, SlpB and SlpE, InlA, the chaperonins Hsp20, GroEL1 and groEL2 previously shown to be involved in immunomodulation by P. freudenreichii. Administration of the cheese to mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced colitis significantly reduced the body weight loss, disease scores and the inflammation marker IL-6 in blood. The attenuation of colon inflammation was indicated by the increased expression of Pparγ encoding the peroxisome proliferator-activated receptor γ. Attenuation of the oxidative stress induced by TNBS in the colon was indicated by the decreased expression of the gene cox2 for cytochrome c oxidase subunit 2. Moreover, the restoration of the intestinal barrier was indicated by an increased expression of the Zo1 gene for the zonula occludens-1, or occluding, a tight junction protein [47]. The increased expression of tight junction proteins counteracts the increased gut permeability occurring in IBDs such as ulcerative colitis (UC) and Crohn’s disease (CD) that can lead to inflammation with further damages induced by IFN-γ and TNF-α and reduced expression of ZO-1 and Claudin-2 [40].

When P. freudenreichii CIRM BIA129 was supplied to mice with TNBS induced colitis in a cheese co-fermented with Lactobacillus delbrueckii subsp. lactis it enhanced the protective effect of the lactobacillus by reducing weight loss and the inflammatory signs, with expression levels of IL-10 and ifnγ similar to healthy mice, and an increase of Pparγ expression. This cheese induced a decrease of oxidation markers cytochrome oxidase 2 (Cox2) and heme oxygenase 1 (Hmox) and protected the intestinal barrier by maintaining expression levels of Zo1 similar to those of healthy mice. The effect on the intestinal microbiota was an increase of Rikenellaceae and a decrease of Ruminococcaceae. Based on previous reports, the increase of Rikenellaceae was considered a consequence of the inhibition of the NF-kB mediated inflammatory response [48].

P. freudenreichii CIRM BIA 129 reached levels of 9/10 Log CFU/g as single strain in an experimental cheese and in industrial Emmental cheese in which also S. thermophilus and L. delbrueckii were present. Both P. freudenreichii cheeses mitigated the weight loss and the disease activity index (DAI) in mice with DSS-induced colitis. Moreover, the industrial Emmental cheese reduced mucosa alterations and cell infiltration, increased the expression of ZO-1 and reduced the level of IL-6 and IL-17. Both P. freudenreichii cheeses decreased immunoglobulin IgA production in the small intestine indicating a lower disturbance of the intestinal barrier. Therefore, beneficial effects on intestinal barrier maintenance and innate immunity were demonstrated [49].

P. freudenreichii CIRM BIA 129 was used to ferment UF, skim milk or whole milk and administered to mice with DSS-induced colitis. Both P. freudenreichii fermented milks alleviated colon shortening and the decrease of goblet cell number and crypt length. However, only the fermented whole milk kept the disease score significantly lower than the positive control. In addition, the P. freudenreichii fermented milk reduced the increase in gut permeability caused by DSS determined as passage of radiolabelled diethylenetriamine pentaacetic acid (DTPA) in blood. The fermented milk preparations, and whole milk at a higher extent, decreased the expression of pro-inflammatory genes il1b and il6 and of the nitric oxide synthase 2 nos2, while the peroxisome proliferator activated receptor gamma pparγ was upregulated [50].

P. freudenreichii CIRM BIA 129 administered to mice in the form of fermented milk ultrafiltrate (MUF) prior to colitis induction with DSS decreased the disease indexes, bleeding and stool consistency but not weight loss. Gut permeability, measured by administering fluorescein-5,6-sulfonic acid (sulfonic acid-FITC) and determining its concentration in plasma, was significantly lower in the P. freudenreichii group. The intracellular permeability, measured by determining the activity of administered HRP in plasma, was also reduced in the P. freudenreichii group. Histopathological observation showed that in the P. freudenreichii group crypt length was not decreased [40].

Different metabolites produced by dairy propionibacteria are able to alleviate the symptoms of IBDs and the propionate produced by the Swiss-type cheese isolate P. freudenreichii ET-3 in whey cultures was the first proven to alleviate colitis induced by TNBS in rats [44]. Moreover, the SCFAs acetate, butyrate and, above all, propionate in P. freudenreichii KCTC 1063 culture supernatant at 10% (v/v) concentration was not cytotoxic for LS 174T goblet cells and stimulated the expression of MUC2 as ascertained by RT-qPCR and enzyme linked immunosorbent assay (ELISA). The intestinal mucus layer represents a physical barrier against pathogens and harmful chemicals that also protects from IBD. It is constituted by highly glycosylated O-glycoproteins called mucins that are secreted by the epithelial goblet cells. Among twenty mucins, the gel-forming mucin MUC2 is the most abundant in the human intestine [51].

When administered to rats with DSS-induced colitis, P. freudenreichii KCTC 1063 and its culture supernatant determined, respectively, a histological disease score equal to the negative control with no induced disease and less damaged crypt structure with lower leucocyte infiltration degree compared to the disease positive control. Both P. freudenreichii treated groups showed a number of goblet cells similar to the healthy control. The expression of MUC2 was not significantly different from the healthy control or higher for live P. freudenreichii and culture supernatant, respectively, according to RT-qPCR and immunohistochemistry (IHC), respectively. The body weight, initially decreased in all groups, later increased more rapidly in those treated with living P. freudenreichii and its culture supernatant that experimented less severe diarrhea and bleeding and had a normal colon length. Moreover, these groups presented lower levels of TNF-α, IL-6, and IL-1β in the distal colon and higher propionate and butyrate SCFAs fecal contents. After administration for seven days at a level of 8 Log CFU, P. freudenreichii remained at levels of 5 Log CFU/g of feces until the end of the experiment at day 29, thus indicating gut colonization [51].

In the genesis of ulcerative colitis (UC) the intestinal microbiota plays an important role by inducing changes in the intestinal mucosa and modulating pro- or anti-inflammatory cytokines. In UC harmful bacteria exceed beneficial bacteria in this microbiota and the equilibrium can be restored by supplying probiotics. P. freudenreichii B1 was compared with Bifidobacterium bifidum H3-R2 and Clostridium butyricum C1-6 for the effects in DSS-induced colitis mice model. This strain decreased colon shortening and the DAI as the other two probiotics, induced a similar decrease of the pro-inflammatory cytokines IL-8, IL-1β and TNF-α and increase of IL-10 in the colon. However, expression of the tight junction proteins occludin ZO-1and claudin-1 was enhanced for P. freudenreichii and C. butyricum. P. freudenreichii B1 induced a decrease in the level of TLRs 2 and 4 and an increase of TLR-5 as the other two probiotics but also an enhanced expression of R-spondin-3 (Rspo3) active in the repair of damaged tissues. P. freudenreichii and B. bifidum more efficiently downregulated of RHO kinase ROCK-1 and Axin2 that inactivate the Wnt/β-catenin pathway for the regeneration of the intestinal epithelium. P. freudenreichii and C. butyricum better preserved the diversity of the intestinal microbiota, not significantly different from the positive control group gavaged daily with mesalazine (5-AS). The genuses Lactobacillus and Bifidobacterium were enriched in all the probiotic groups to different extents. P. freudenreichii induced the most pronounced effect on the increase of SCFAs whose amount is negatively correlated with the level of Escherichia-Shigella, Staphylococcus and Enterobacter genuses and proinflammatory indexes and positively correlated with anti-inflammatory molecules including IL-10 and TLR-5 [52].

Mucositis is a severe condition occurring in cancer patients treated with radiotherapy or chemotherapy and is characterized by the inflammation of the small bowel with degeneration of enterocytes and goblet cells, infiltration of leucocytes in the lamina propria, increased production of mucus, atrophic villi and hypoplastic crypts. Symptoms are diarrhea and weight loss and therapy with antimicrobials and anti-inflammatory agents may be ineffective and not well tolerated so treatment with probiotics should be considered an alternative. P. freudenreichii CIRM BIA 129 and its ΔSlpB mutant were evaluated in vitro and in vivo for anti-inflammatory activities and the effects in mice with mucositis induced by the chemotherapy drug 5-fluorouracile (5-FU). In HT29 cells exposed or not to E. coli LPS The P. freudenreichii wild type strain induced IL-10, as the purified SlpB protein also did, and inhibited IL-8 induction by LPS and ifnγ and tfna expression. Moreover, it induced the toll-like receptor gene tlr2 and repressed tlr4. The gene tlr9 was less expressed in LPS-treated cells in presence of the P. freudenreichii wild type [51]. The P. freudenreichii wild type significantly reduced weight loss and protected the intestinal mucosa with a reduction of leucocyte infiltration and ulceration, partially restored height of villi and lower reduction of granular density in the Paneth cells. This strain reduced the gut permeability induced by 5-FU, based on the extraintestinal translocation of radiolabeled 99mTc-DTPA. Indeed, this the expression of the claudin-1 cld1 gene was increased and the expression of IL-17a, IL-12 and IL-1β in the intestinal mucosa was reduced. These effects were not induced by the ΔslpB mutant [53].

Newborns are particularly susceptible to infections because of an immature immune system and an insufficient number of protective T cells. This risk is extremely high in preterm neonates who are exposed to inflammation induced by maternal pathogens that can lead to the deterioration of the intestinal microbiota and to the onset of neonatal necrotizing enterocolitis (NEC). The establishment of a protective intestinal microbiota stimulates the development of an immune system able to contrast enteric pathogens [53,54]. Breast milk reduces the risk of NEC by introducing beneficial bacteria that stimulate immune regulation. Indeed, a metagenome analysis of the intestinal microbiota of 20 preterm newborns fed with breast milk and 20 preterm newborns fed with formula milk, showed a higher microbial diversity and balance in the first group and presence of Actinobacteria among which the genus Propionibacterium, represented by the species P. freudenreichii, predominated and increased during time. These bacteria were isolated and one strain was obtained, Propionibacterium P. UF1, that showed highest genome identity with P. freudenreichii DSM 20271. When administered to C57BL/6 mice, this strain transiently colonized gut for about six days but its colonization lasted longer in germ-free mice [14].

The transfer of the intestinal microbiota of breast-fed newborns to mice did not increase IL-1β in colon dendritic cells (DCs), while the microbiota from formula fed newborns increased this pro-inflammatory cytokine. In addition, the microbiota from breast-fed infants increased T helper 17 (Th17) cells and anti-inflammatory regulatory T cells (Tregs) expressing IL-10. Similar effects were observed in mice receiving the microbiota of formula fed newborns and gavaged with P. freudenreichii P.UF1. Treatment with the Propionibacterium isolate decreased the level of γ-proteobacteria. This strain alone did not cause an increase of IL-1β in colon tissues and DCs, while it induced an increase of Th17 cells, in particular those IL-10⁺IFN-γ^–, and IL-10⁺ Tregs [14].

Dihydrolipoamide acetyltransferase (DlaT) was the main component of guanidine hydrochloride extracts from the surface of P. freudenreichii P.UF1 able to induce IL-10⁺ Th17 cells. This is a cytoplasmic enzyme of pyruvate decarboxylase complex. The major histocompatibility complex II (MHC II) was found to mediate the differentiation of Th17 cells from CD4⁺T cells induced by P. freudenreichii in MHC II^+/+ mice since this differentiation did not occur in MHC II^-/- mice. A ΔdlaT mutant of P. freudenreichii P.UF1 did not induce the differentiation of IL-10⁺ Th17 cells in the mouse colon and complementation of this deletion mutant with three peptides of DlaT restored the capacity to induce differentiation of Th17 cells and the induction of IL-10⁺ Tregs. The C-type lectin receptor (CLR) SIGNR1 recognizes bacteria from their surface composition and stimulate DCs to induce T-cell differentiation. SIGNR1 in the colon and DCs was upregulated in mice receiving P. freudenreichii P.UF1. The involvement of SIGNR1 in the attenuation of inflammation induced by P. freudenreichii P.UF1 was demonstrated by observing that mice in Signr1^–/– infected with a Listeria monocytogenes strain in which the act gene was inactivated to avoid its systemic spread, differentiation of Th17 cells and induction of IL-10⁺ Tregs did not occur, while it was observed in Signr^+/+ mice [14].

The colon microbiota of germ-free mice gavaged with P. freudenreichii P.UF1 was enriched with Lactobacillus and Ruminococcus genera, showing the ability of this strain to modify the intestinal microbiota and its metabolites. Since the intestinal microbiota of the mother influences the immunity system of the newborn, P. freudenreichii P.UF1 was administered to pregnant mice. The newborn from these mothers presented an increase in DCs expressing the transforming growth factor TGF-β and IL-10, of IL-10⁺Th17 and IL-10⁺ Tregs. In addition, weight and survival rate increased in the newborns submitted to NEC-like injury induced by gavage with a mixture of commensal bacterial from adults and exposure to hypoxia. In these newborns the transcription of the inflammatory inducible nitric oxide synthase iNOS, and interleukins Il-1b, Il-6, and Il-23, was reduced. Moreover, the innate lymphoid cells 3 (ILC3) expressing IL-17A and also IL-22, that protects from bacterial infections and mediates tissue repair, increased in the small intestine in newborn mice gavaged with P. freudenreichii P.UF1 [14].

P. freudenreichii P.UF1 was further tested for an effect in murine systemic infection with the strain L. monocytogenes 10403S with a mutation in the inlA gene that allowed to control the infection kinetics. Compared to mice gavaged with the ΔdlaT mutant of P. freudenreichii P.UF1 and later infected with L. monocytogenes, those receiving the wild type strain showed a reduction of IL-1β, IL-6, and IL-12/IL23p40 produced by DCs and Th1 cells producing IFNγ and an increase of Th17 cells and IL-10⁺ Treg cells. Moreover, the latter showed an increase of intestinal bifidogenic bacteria. Cloning of three DlaT peptides in the L. monocytogenes and subsequent infection of mice with this strain showed similar immunomodulatory effects as observed in mice receiving P. freudenreichii P.UF1, thus confirming that DlaT was responsible for the increase of Th17 cells and IL-10⁺ Treg cells. In mice receiving P. freudenreichii P.UF1 L. monocytogenes was cleared from feces and tissues. The same effects were not observed in Signr1^–/– mice. Therefore, it was demonstrated that P. freudenreichii P.UF1 mitigated the inflammation induced by the pathogen both locally and peripherally [14].

The role of the Th17 cells induced by P. freudenreichii P.UF1 in mitigating L. monocytogenes infection was demonstrated by neutralizing the IL-17A produced by these cells with specific antibodies injected in the mice peritoneum. In this case the pathogen persisted also in mice that received P. freudenreichii P.UF1. Moreover, infection mitigation by P. freudenreichii P.UF1 failed in mice defective in the recombination-activating gene required for the generation of T and B cells. In the induced Th17 cells the upregulation of pathways for the production of extracellular matrix components involved in tissue healing and genes involved in immune regulation was observed by transcriptome analysis. Principal coordinate analysis (PCoA) of the intestinal microbiota showed that mice receiving P. freudenreichii P.UF1 had higher numbers of Lactobacillaceae and Clostridiaceae able to produce SCFAs and lower levels of Prevotella species. Moreover, vitamins B2, B5 and B9 were significantly increased, while the proinflammatory molecules prostaglandine E1 and 20-Hydroxyleukotriene E4 decreased [54].

Table 1 summarizes the effects exerted by P. freudenreichii strains in vivo in intestinal inflammation models.

3.2. Immunomodulation

P. freudenreichii CIRM BIA 129 was selected among other ten strains for the anti-inflammatory effects in vitro. UF milk fermented by this strain administered to piglets improved food intake and growth rate. Moreover, it induced a decrease of IL-8 and TNF-α in the colon mucosa [8]. Supply of 11 Log CFU of P. freudenreichii CIRM BIA 129 in culture or in cheese determined the presence of this bacterium at about 7 Log CFU/g and the increase of bifidobacteria in piglet feces. After 14 days of administration total SCFAs and branched chain fatty acids (BCFAs) were significantly higher in the animals receiving P. freudenreichii. Slps from this bacterium increased IL-10 and reduced TNFα and IFNγ production in piglet PBMC and mesenteric lymph node immune cells (MLNC) in which inflammation was induced with E. coli LPS or concanavalin A. In MLNC cells P. freudenreichii supplied with cheese enhanced the expression of the regulators GATA binding protein 3 (GATA3) and forkhead box P3 (FoxP3) that have a role in immune tolerance [81,82].

Regardless of the method of administration, P. freudenreichii increased the ratio between anti-inflammatory Treg and pro-inflammatory Th17 cells. Ex vivo stimulation with LPS of PBMC from piglets treated with P. freudenreichii showed an increased secretion of IL-10, while stimulation with concanavalin A induced also an increased IFNγ secretion only in MLNC cells from the group treated with P. freudenreichii supplied in cheese. Importantly, IFNγ is responsible for the intestinal immune response against pathogens by macrophage activation and increase of Th1 cells [81]. The same strain was used to ferment sweet milk whey to obtain a functional food from a by-product. Supply of the P. freudenreichii fermented whey to piglets for 14 days did not influence growth or food intake but determined a significant increase of bifidobacteria in feces. MLNCs isolated after the treatment showed an upregulation of T-box expressed in T cells (T-bet) regulator of the Th1 response [83].

A. jensenii 702 isolated from raw cow milk in Australia was tested as adjuvant for an oral vaccine constituted by Mycobacterium tuberculosis H37Rv culture filtrate administered to rats. The A. jensenii strain survived the passage through the gastrointestinal tract and was recovered from feces during the whole trial. This strain induced an activation of spleen T-cells higher than the cholera toxin which is a strong mucosal adjuvant. The IFNγ levels, 2 to 3 Log higher than IL-4 in the A. jensenii groups, indicated a Th1 response that is effective in protection from tuberculosis [55].

The Caenorabditis elegans worm is used as an animal model to study the effects of different agents on innate immunity and ageing. Feeding on a P. freudenreichii KCTC 1063 cell lawn prolonged by 13% its lifespan compared to conventional feeding on E. coli OP50, increased the body movements that depend on muscular strength and decreased the accumulation of lipid granules indicators of aging. The immunity related pathways of DNA binding ligand (DBL)/TGF-β, P38 mitogen-activated protein kinase (MAPK) involved in innate immunity, Daf-2/DAF-16 involved in the response to insulin/insulin-like growth factor-1 (IGF-1) signalling (IIS) and the lys-7 and lys-8 genes for antimicrobial peptides were upregulated. Use of C. elegans mutants defective in innate immunity and life span extension related genes highlighted that P. freudenreichii regulates the p38 MAPK and TGF-β pathways. Moreover, activation of the p38 MAPK pathway by P. freudenreichii led to increased resistance to the infection by Salmonella Typhimurium [56].

P. freudenreichii JS induced an increase in the number of natural killer (NK) cells in the liver but also their decrease in the spleen with an increase of IFN-γ production in NK cells from the spleen in mice. Moreover, this strain induced serum IL-6 levels significantly higher than the control, a lower than control Th1/Th2 average ratio in the spleen, a %Th17 cells in CD4+T cells higher than the control both in liver and spleen, a % Treg cells in CD4+T cells higher than the control in the spleen, and a %Treg cells/%Th17 cells lower than the control in both liver and spleen, indicating a pro-inflammatory effect for most parameters [84].

In allergic reactions Th2 cells are induced and produce cytokines that promote the proliferation of B cells and immunoglobulin class switching to IgE that induce the release of histamine and other inflammatory substances from mast cells and basophils. A new exposure to the allergen induces the production of mediators that lead to the proliferation of Th2 cells and the activation of DCs towards the reduction of allergen-specific Treg cells and activation of mast cells. The intestinal microbiota is involved in the balance between the inflammatory reactions and immune tolerance by influencing innate and adaptive immune responses, as demonstrated with experiments in germ-free mice. SCFAs produced by components of the intestinal microbiota stimulate the production of ILC that distribute in peripheral tissues and promote homeostasis during inflammation [57].

P. freudenreichii CIRM BIA 129 administered at a daily dose of 9 Log CFU to mice with food allergy sensitized with orally supplied wheat gliadins prevented the increase in body temperature and in gliadin-specific IgE and IgG1 in serum, while it induced an increase of gliadin-specific IgG2. The level of mucosal mast cell protease-1 (mMCP-1) activation marker decreased in this group. Moreover, the levels of Th2 cells infiltrated in mesenteric lymph nodes were not significantly different from the control and the associated cytokines IL-5 and IL-13 were lower than in the food allergy controls. Treg cells that regulate Th1 and Th2 responses increased in the group treated with P. freudenreichii CIRM-BIA 129. Therefore, it was concluded that this bacterial strain exerted an anti-inflammatory effect by limiting the Th2 response. In addition, it prevented an increase of ILC2 in Peyer’s patches and significantly reduced the intestinal paracellular and transcellular permeability determined by measuring the translocation of fluorescein-5.6 sulfonic acid and HRP, respectively. Indeed, the expression of ZO-1 was unaltered. Co-cultures of P. freudenreichii CIRM-BIA129 with human PBMCs from healthy volunteers showed an increase of TGF-β lvels. Furthermore, this strain increased IL-10 and decreased IFN-γ production by monocyte-derived DCs (MoDCs). The role of SlpB in these effects was demonstrated by the comparison with the ΔSlpB P. freudenreichii CIRM-BIA129 mutant strain [57].

Potential protective effects of P. freudenreichii on the respiratory system from infections was suggested by the ability of strain PF-24 to induce the oxidative burst in bovine alveolar lavage cells (BAL) represented mainly by macrophages more intensely than other probiotics. Flow cytometry coupled with use of fluorescent antibodies showed that this strain also induced a higher percentage of cells expressing CD14 necessary for the recognition of Gram-negative bacteria and an increase, though not significant, of cells expressing the CD205 dendritic cell marker that modulate the immune response to microbial infections [85], thus showing a general capacity to enhance leukocyte functions [86].

Table 2 summarizes the effects on immune response observed in vivo for dairy propionibacteria.

3.3. Effects on Obesity

A high fat diet (HFD) predisposes to chronic conditions such as obesity preceded by low level intestinal inflammation and insulin resistance, type 2 diabetes, cardiovascular diseases and fatty liver disease. The effects of P. freudenreichii on ApoE*3Leiden transgenic mice receiving a HFD were demonstrated for the strain JS. These mice express a mutated human APOE3 gene associated with human dys-beta-lipoproteinaemia and also harbor a human APOC1 gene and a human promoter that determine high levels of lipoproteins and triglycerides in plasma. In these mice P. freudenreichii JS reduced the weight gain and the increase of gonadal adipose tissue compared to groups receiving L. rhamnosus GG, a HFD diet comprising also fibers or only the HFD. Moreover, in the P. freudenreichii group the level in plasma of the adhesion molecule VCAM-1, a marker of vascular inflammation, mast cell numbers and TNF-α level was lower. Like L. rhamnosus GG, also P. freudenreichii JS decreased ALT values and left the adiponectin levels unaltered [58].

Live and heat-killed P. freudenreichii MJ2 exerted anti-obesity effects and reduction of insulin resistance in mice in which obesity was induced with an HFD. It was at first shown that preadipocytes 3T3-L1 exposed to MDI (3-isobutyl-1-methylxanthine, dexamethasone, and insulin) and incubated with heat-killed P. freudenreichii MJ2 (hkMJ2) even at numbers as high as 8 Log CFU/ml did not lose viability, as determined the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then it was observed that hkMJ2 reduced fat accumulation in a dose-dependent manner and more efficiently of an anti-obesity L. plantarum KACC15357 probiotic. Moreover, hkMJ2 induced an increase in the expression of the transmembrane protein preadipocyte factor-1 (Pref-1), that favors the maintenance of the preadipocyte stage, and a decrease in the expression of genes encoding the adipogenic proteins PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and acetyl-CoA carboxylase (ACC) [59].

Treatment of HFD induced obese mice with live P. freudenreichii MJ2, hkMJ2 and L. plantarum KACC15357 led to decreases in body weight of 31%, 22.8%, and 18.5%, respectively, and a decrease of the food efficiency ratio. P. freudenreichii MJ2, hkMJ2 decreased the expression of PPARγ, FAS, SCD-1, and ACC in epididymal white adipose tissue (eWAT) and increased the expression of the lipolytic enzymes adipose tissue triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and carnitine palmitoyltransferase 1α (CPT-1α), and the enzyme involved in β-oxidation of fatty acids peroxisomal acyl-coenzyme A oxidase 1 (ACOX1). These findings were confirmed by Western blot analysis. Moreover, treatment with P. freudenreichii MJ2 and hkMJ2 decreased the adipocyte size in eWAT [59].

The effects on serum lipids were a significant decrease of low-density lipoprotein cholesterol (LDL) in the P. freudenreichii MJ2 group and the increase of the ratio high-density lipoprotein cholesterol (HDL)/total cholesterol (TCHO) in all probiotic groups. The fasting glucose levels were decreased in the P. freudenreichii MJ2 group and insulin levels decreased for all the probiotic treated groups with decreased homeostasis model assessment insulin resistance (HOMA-IR) scores. Staining with hematoxylin and eosin showed a significantly decreased lipid accumulation in hepatocytes of these groups and significantly lower levels of serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) indexes of hepatic damage [59].

Obesity is caused by a remodeling of the adipose tissue induced by the imbalance between energy intake and expenditure. In this condition the adipocytes originating from mesenchymal stem cells (MSCs) become hypertrophic and hyperplastic so molecules that impair this process could be useful to treat the condition. Preadipocytes 3T3-L1, in which lipid accumulation was induced with MDI, were treated with hkMJ2, cell wall (CW) and cytoplasmic (Cyto) fractions of the P. freudenreichii MJ2 cells during differentiation to adipocytes and a reduction of lipid levels was observed in cells treated with hkMJ2 and the CW fraction and the separated surface protein component (SP) but not with the CW fraction deprived of the SP fraction. The fractions CW and SP caused a reduction in the expression level of the lipogenesis factor Fas [60].

The main protein components of the SP fraction of P. freudenreichii MJ2 were chaperones, carbon metabolism proteins and ribosomal proteins. SP components were separated by Q fast, anion exchange and size exclusion chromatography and it was found that the protein fraction with molecular weight above 35 kDa was probably implied in the reduction of lipid accumulation. Among the proteins included in this fraction the chaperonin 60 (Cpn60), otherwise designated heat shock protein 60 (Hsp60) or GroEL, was expressed as recombinant protein, tested on 3T3-L1 cells and found to reduce adipogenesis at a dose-dependent extent. The observed effect was most likely attributable to the decreased expression of genes Pparγ, Cepba, Fas, and Scd1. Indeed, the terminal differentiation stage from MSC to adipocytes is mediated by the master regulators PPARγ and C/EBPα that induce the lipogenesis genes FAS and SCD1. The expression levels of the respective coding genes and the content of lipid droplets were progressively reduced during eight days of adipocyte differentiation for the reduced PPARγ translocation to the nucleus and binding to the promoters of the target gene [60].

Since Pparγ and Cebpa genes are positively regulated by C/EBPβ in MDI-induced adipogenesis, the level of C/EBPβ in the nucleus and its binding to the Pparg and Cebpa promoters was determined and found to decrease after 24 h of exposure to the P. freudenreichii GroEL as determined by Western blot and chromatin immunoprecipitation (ChIP), respectively. In addition, the upregulation of genes GATA2 and GATA3, encoding adipocyte differentiation suppressors that inhibit the expression of Cebpa and Pparg by binding to C/EBPβ, was observed. Therefore, the mechanism of adipogenesis inhibition by the GroEL protein of P. freudenreichii MJ2 was elucidated [60].

Cholesterol lowering ability is a trait of some probiotics that could help to prevent cardiovascular diseases. A. acidipropionici C03B-STR isolated from goat milk degraded cholesterol in vitro thus lowering its uptake by Caco-2 cells [87]. Cholesterol lowering activity was demonstrated in vivo in mice and cows for strains of dairy propionibacteria. One mechanism for cholesterol lowering by these bacteria is bile salt deconjugation and co-precipitation of cholesterol with the deconjugated bile salts followed by excretion. Of seven strains tested, all were able to deconjugate sodium glycocholate and two strains of P. freudenreichii and one P. jensenii also deconjugated sodium taurocholate. In the broth medium containing oxgall, cholesterol precipitation occurred mostly for one P. freudenreichii strain, one P. jensenii strain and one P. thoenii strain [88].

A. acidipropionici OB7439 was found to increase the SCFA levels in plasma, cecum and feces of HFD obese C57BL/6J mice. Moreover, administration of this strain in high levels induced an increase of insulin secretion and consequent suppression of blood glucose rise after its oral administration. These effects were not observed for A. acidipropionici JCM6427, thus proving strain-specific. In addition, mice fed with A. acidipropionici OB7439 showed a lower weight, lower white adipose tissue and liver weight gain than the control at 16 weeks of administration. Plasma triglycerides and total cholesterol levels were also reduced. Based on previous research, the observed effects were attributed to the increase of propionate intake. The mRNA levels of Tnfα, macrophage marker F4/80, the fibrosis marker ColIα, and Fas and Chrebp for fatty acid synthesis decreased in the liver, while those for the peroxixome proliferator-activated receptor α Ppara for lipid metabolism increased. Energy regulation via the G protein-coupled receptor GPR41 was involved in the observed effects, as demonstrated by the lack of similar outcomes in GPR41^-/- mice [61].

Table 3 summarizes the effects exerted in vivo by dairy propionibacteria in obesity animal models.

3.4. Anti-Cancer Effects

Colorectal cancer (CRC) is the third most prevalent cancer type worldwide mainly in developed countries but increasing also in developing countries with risk factors that are both genetic and linked to dietary habits, smoking and alcohol consumption. A rise of CRC incidence in young persons aged 20-50 is in course [62]. The first anticarcinogenic activity reported for strains of dairy propionibacteria regarded one A. acicipropionici strain and one P. freudenreichii strain isolated from Swiss type cheese that were found to decrease the β-glucuronidase activity, that releases toxic and carcinogenic compounds by hydrolyzing the glucuronic acid conjugates formed in the liver, in mice feces. The effect persisted also one week after that treatment with propionibacteria ceased [63].

It was demonstrated that the anticarcinogenic effect exerted by the propionibacteria A. acidipropionici CNRZ80, P. freudenreichii ITG18, and SI41 in vitro was attributable to the production of SCFAs able to induce the apoptosis of cancer cells such as Caco-2, HeLa, and HT29. A medium fermented by P. freudenreichii CIRM BIA 138 induced death in HT29 colon cancer cells, and an optimal SCFA concentration was 0.75 g L⁻¹ of acetate and 2.7 g L⁻¹ of propionate [89]. Lan et al. showed that propionate induced the death of HT29 colon cancer cells by apoptosis or necrosis in the pH range 6.0-7.5 or at pH 5.5, respectively [90]. Moreover, P. freudenreichii TL133, able to survive and be metabolically active during transit in the gastrointestinal tract, exerted this effect also in the colon of rats exposed to the genotoxic agent 1,2-dimethylhydrazine (DMH). This chemical induced apoptosis in all crypt zones and in different colon regions but also an increase of the proliferative index. The administration of P. freudenreichii further increased the number of apoptotic cells, mostly in the middle crypt zone, but decreased the proliferative index [64].

TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF superfamily, selectively kills cancer cells. To increase the effectiveness of TRAIL on CRC cells, synergistic treatments with chemotherapy agents or IFN-γ are required. As observed by DNA microarray analysis, culture supernatants or fermentation products from P. freudenreichii TL133 (CIRM BIA 138) induced 2180 genes in HT29 cells and co-treatment with TRAIL increased the number of induced genes to more than three thousand. The genes induced by all the treatments included those encoding apoptosis pathways and immune response elements such as nucleotide-binding oligomerization domain (NOD)-like receptors and interactions between cytokines and receptors. These were more numerous for the association TRAIL-fermentation products. Importantly, culture supernatants or fermentation products alone induced the TRAIL death receptor TRAIL-R2/DR5 and the presence of these proteins in the HT29 cell membrane was confirmed by flow cytometry combined with antagonistic mouse monoclonal antibodies [64].

The exposure of HT29 cells to the combination of TRAIL with P. freudenreichii culture supernatants or fermentation products induced 57% cell mortality while healthy epithelial colon cells HIEC were not sensitive to this treatment. The treatment with P. freudenreichii culture supernatants or fermentation products combined or not with TRAIL induced the acetylation of histone H3, thus indicating a histone deacetylase inhibition activity. In addition, the expression of the apoptosis inhibitors FLIPL and XIAP decreased. Milk and milk ultrafiltrate fermented by P. freudenreichii CIRM BIA 138 induce the death of HT29 cells in a similar manner. The fermented milk ultrafiltrate induced the caspases 3, -8 and -9. Apoptosis was confirmed by flow cytometry combined with propidium iodide staining, by the translocation of phosphatidylserine to the external layer of the cell membrane, the decrease of the mitochondrial membrane potential ΔΨm, accumulation of superoxide anion O₂^- and release of cytochrome c into the cytoplasm. The changes induced in HT29 cells by TRAIL and P. freudenreiichii metabolites indicated that the extrinsic apoptotic pathway mediated by the activation of death receptors and caspase 8, and the intrinsic pathway, involving mitochondria and caspase 9, were activated [64].

Lectins are proteins or glycoproteins that bind to cell surface carbohydrates and are abundant in foods of plant origin. Concanavalin A, the lectin present in Jack bean, binds to cells in the intestinal epithelium causing alterations that reduce nutrient adsorption and impair mucus secretion with an increase of mechanical stress [65]. Moreover, the lectins concanavalin A and peanut agglutinin stimulate the proliferation of epithelial cells in rat intestine and colon cancer cell lines and could have cancerogenic effects beyond an anti-nutritional function [91]. A. acidipropionici CRL 1198, a dairy strain able to survive in the gastrointestinal tract, when supplied together with concanavalin A, mitigated some detrimental effects observed with the administration of concanavalin A in a mouse model such as epithelial cell proliferation in the intestine, structural alterations of microvilli and the increase of enterobacteria and enterococci [65].

The lectins wheat germ agglutinin, the agglutinin from Artocarpus integrifolia , soybean agglutinin, Ulex europaeus agglutinin and concanavalin A were tested on cancer cell lines such as HCT-15, LoVo, SW837 and HT29 and all except the wheat germ agglutinin induced cell proliferation with a more pronounced effect of the Artocarpus integrifolia agglutinin and concanavalin A [91]. A. acidipropionici CRL 1198 was incubated in media containing these two lectins and the derived supernatants reduced the cell proliferation effect of lectins on SW837 cells indicating binding of these compounds by the propionibacteria. The adhesion capacity to cells of A. acidipropionici was decreased in presence of lectins and was restored after addition of sugars able to bind to the lectins (haptens). This was an indication of the lectin binding capacity of A. acidipropionici mediated by cell surface carbohydrates [91].

Previous evidence that the SCFAs acetate, propionate and butyrate favored the arrest of the cell cycle, inhibition of histone deacetylase and apoptosis in cancer gastric cells led to testing the effect of milk ultrafiltrate fermented by P. freudenreichii CIRM BIA 138 on HGT-1 cells. Fluorescence microscopy with Hoechst 33342 staining showed typical apoptotic nuclei and bodies in these cells exposed to the fermented milk with a higher degree of DNA fragmentation compared to the positive control with campothecin apoptosis induction. The fraction of cells in sub-G1 phase after treatment with the fermented milk wascomparable to that observed with campothecin. In the fermented milk treated cells the combination of Annexin labelled with Fluorescein isothiocyanate (Annexin V-FITC) staining and flow cytometry demonstrated the translocation of phosphatidylserine to the outer cell membrane leaflet in up to 80% cells, indicating apoptosis, while the number of necrotic cells was very low according to staining with 7-Amino Actinomycin D (7-AAD). Apoptotic mitochondrial modifications were indicated by the decrease in ΔΨm membrane potential based on DiOC(6)3 and JC-1 probes fluorescence, accumulation of O₂ ^– indicator of the production of reactive oxygen species (ROS) measured with dihydroethidium (DHE) and cytochrome c release into the cytoplasm determined by immunoblotting. Western blotting and determination of enzymatic activity demonstrated the activation of caspases 3, 8 and 9 in HGT-1 cells exposed to the fermented milk and to a mixture of propionate/acetate 2:1. The P. freudenreichii fermented milk doubled the cell killing potential of campothecin [92].

P. freudenreichii DSM 20271 showed the capacity to produce SCFAs in the culture medium used for CRC derived RKO cells, most probably stimulated by the lactate produced by these cells from glucose fermentation (warbourg effect). Culture broths of the P. freudenreichii DSM 20271 strain inhibited the proliferation of CRC cells at different extents depending on the growth medium and previous adaptation to simulated digestive stress. Flow cytometry coupled with propidium iodide staining demonstrated the accumulation of CRC cells with cell-cycle arrested at the apoptotic sub-G1 phase or G2/M phase indicating the inhibition of their proliferation [9].

The mitochondrial activity assay with 3--2,5-difeniltetrazolium bromide (MTT assay), used to investigate cell viability, showed that P. freudenreichii DSM 20271 inhibited the proliferation of HCT116 CRC cells in a dose-dependent manner in vitro. In vivo effects exerted by this strain included the mitigation of oxidative stress induced with azoxymethane (AOM) in the colon of rats as determined by measuring the concentration of malondialdehyde (MDA). Moreover, this strain reduced the formation of aberrant crypt foci induced by AOM to less than half and reduced the abnormalities observed in crypts at cell and tissue level. Supplementation with P. freudenreichii preserved the diversity of the intestinal microbiota compared to treatment with AOM alone. The enriched microbial groups were different from the control not treated with AOM and included lactobacilli and bifidobacteria [62].

To date, clinical studies on the indirect anti-cancer effects of dairy propionibacteria regarded only the association P. freudenreichii JS/Lacticaseibacillus rhamnosus LC705 (Valio, Finland) both originated from cheese and commonly used in the production of semi-hard cheese [93]. In one study this strain association was shown to decrease the risk of hepatocellular carcinoma (HCC) in Chinese young men exposed to aflatoxin B1 known to potentiate the liver cancer risk associated with hepatitis B virus (HBV) infection [94]. This strain association was previously shown to bind aflatoxin B1 and to decrease the level of this mycotoxin in the feces of healthy volunteers after two or three weeks of dietary supplementation with a preparation containing the bacteria [95]. In the double-blind trial 90 subjects were equally distributed in the intervention or the placebo group and the two groups did not significantly differ in the fecal concentration of aflatoxin B₁-N⁷-guanine (AFB-N⁷-guanine) marker of aflatoxin B1 exposure. Probiotic administration led to a significant decrease of this marker in the urine, up to 55%, after 5 weeks but this effect disappeared after the end of the intervention [94].

Another double-blind, placebo-controlled trial with the same bacterial association involving 38 men aged 24-55 years examined the effects on the activities of β-glucosidases, β-glucuronidases and β-galactosidases, produced mainly by clostridia and Bacteroides, that release potentially carcinogenic compounds and urease that is responsible for the formation of toxic and mutagenic substances from ammonia. The assumption of the two bacterial strains did not cause adverse effects and a 10% decrease of the β-glucosidase activity occurred in the intervention group and the decrease was negatively correlated with the number of propionibacteria. A 13% decrease of urease activity was also observed in the intervention group that was not correlated with the number of lactobacilli or propionibacteria [93].

Table 4 summarizes the effects exerted in vivo by dairy propionibacteria on cancer prevention in animal models.

3.5. Effects of Propionibacterium Freudenreichii on Bone Health

Rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of joints and can extend to most organs and the nervous system. This condition involves bone loss caused by enhanced osteoclast differentiation and the available treatments do not allow a definitive recovery [66,67]. The apoptosis regulator RANKL, ligand of the RANK receptor activator of NF-kB, is one of the factors stimulating osteoclast differentiation from macrophages [66,68]. Osteoclasts are multinucleated cells with a major role in bone-destruction. These create an acidic microenvironment in which they secrete proteins involved in bone destruction such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (Ctsk), and calcitonin receptor (Calcr). Moreover, these cells produce pro-inflammatory cytokines and chemokines [66]. Osteoprotegerin (OPG) is a receptor that binds to RANKL and inhibits osteoclastogenesis by hindering the RANK-RANKL interaction. Heat-killed P. freudenreichii MJ2, an isolate from raw milk, inhibited osteoclastogenesis and mitigated collagen-induced arthritis (CIA) in a mouse model by increasing the OPG/RANKL expression ratio [68].

This effect was mediated by P. freudenreichii MJ2 surface proteins extracted with guanidine hydrochloride. This substance did not disrupt cells, that remained viable, so the extraction of cytoplasmic proteins was considered to be negligible. The surface proteins were tested at concentrations of 2.5, 5, and 10 μg/mL on murine macrophages RAW 264.7 that can differentiate in osteoclasts and the latter concentration was selected for further experiments. Each addition of 10 μg/mL P. freudenreichii MJ2 surface proteins at different days significantly inhibited osteoclast differentiation. Moreover, TRAP+ osteoclasts decreased after 4 days of treatment with P. freudenreichii MJ2 surface proteins in a dose-dependent manner as well as the expression of osteoclastogenic genes induced by RANKL and encoding RANK, c-fos, NFATc1, and NF-κB with consequent lower levels of the genes regulated by the NFATc1 master regulator of osteoclast differentiation [66].

The transcriptome of cells treated with P. freudenreichii MJ2 surface proteins comprised 888 upregulated or downregulated genes among which lcn2 (lipocalin 2), with immunity function, showed the highest expression level with 2426-fold upregulation. Moreover, 128 genes involved in osteoclast differentiation were differentially expressed. STRING protein-protein interaction and functional enrichment analysis of the genes with higher degrees of differential expression highlighted that Lcn2 directly interacts with the tumor necrosis factor (Tnf) which in turn interacts with the RANK gene and the Nfatc1 gene thus influencing the expression of downstream genes Cst1, Ctsk, Mitf, and TRAP. Therefore, it was hypothesized that lcn2 might be involved in the inhibitory mechanism of P. freudenreichii MJ2 surface proteins on osteoclast differentiation. Inhibition of lcn2 expression by small interfering RNA (siRNA) silencing led to TRAP activity and TRAP(+) increase and formation of the F-actin ring responsible for bone resorption also in cells treated with P. freudenreichii MJ2 surface proteins, suggesting that these inhibit osteoclast differentiation by upregulating lcn2. Protein expression analysis showed that the effect was due to the downregulation of c-fos, and NFATc1 with its downstream genes. TRAP activity in osteoclasts was decreased also by surface proteins from P. freudenreichii MJ2 cells treated at 100°C for 30 min or the same proteins separated with trypsin from the cell surface. In the latter case TRAP activity inhibition was slightly enhanced, thus showing that the effect does not necessitate of entire proteins. Liquid chromatography tandem mass spectrometry (LC-MS/MS) separation of trypsin treated surface proteins highlighted that chaperonins and heat shock proteins were the main components of the surface protein extracts [66].

It was investigated if the EVs from P. freudenreichii MJ2 were involved in the inhibition of osteoclastogenesis in a murine model of rheumatoid arthritis. This strain produced spherical EVs with average diameter of 171 nm and containing 585 proteins involved in metabolism, cell structure composition and binding of various molecules such as ATP, nucleotides, ions, carbohydrates, cyclic and heterocyclic compounds. The effect of P. freudenreichii EVs on RAW 264.7 murine macrophages differentiation in osteoclasts when exposed to RANKL were investigated. The EVs were not toxic to the RAW 264.7 cells even at a number of 8 Log CFU/mL of P. freudenreichii so this level was used in the in vitro and in vivo experiments. RAW 264.7 cells exposed to P. freudenreichii EVs showed a decreased differentiation into TRAP osteoclasts and TRAP activity was significantly decreased compared to the control. Therefore, it was concluded that osteoclast differentiation induced by exposure to RANKL was inhibited. Experiments with fluorescent antibodies coupled with microscopy showed reduced binding of RANKL to RANK in EV-treated cells [67].

P. freudenreichii EVs significantly decreased the arthritis score in CIA mice that presented reduced bone erosion and synovial inflammation, a lower number of TRAP(+) osteoclasts and collagen-specific IgGs. In addition, the levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-17 decreased, while the anti-inflammatory IL-10 increased in serum. The in vivo data confirmed the decreased expression of genes associated to osteoclastogenesis and indicated an increase of the OPG/RANKL ratio. EVs administration did not increase aspartate transaminase (AST) and alanine transaminase (ALT) levels, indicating the absence of hepatotoxicity [67].

4. Antimicrobial Properties of Dairy Propionibacteria

P. freudenreichii JS, commercialized by Valio (Finland), a strain with a high capacity to adhere to intestinal mucus, prevented the adhesion of Staphylococcus aureus RN4220 by 39% and significantly reduced the viability of adhered S. aureus to 72% by producing non-bacteriocin antimicrobial substances [96]. One pathogen inhibiting substance produced by P. freudenreichii PTCC 1674 is a surfactant/emulsifier of lipopeptide nature showing slight inhibitory activity and prevented surface adhesion of E. coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus strains at different extents in culture plates [97].

Dairy propionibacteria were reported to inhibit Escherichia coli and Shigella sonnei strains, but not Listeria monocytogenes, in vitro for an effect of the low pH of the growth medium [74]. Two P. freudenreichii dairy strains B3523 and B4327 of dairy origin showed high percentages of adhesion to Budgerigar Abdominal Tumor Cells (BATCs) and their cell-free culture supernatants inhibited MDR S. Heidelberg, E. coli O157:H7 and L. monocytogenes. The P. freudenrichii strains did not invade BATCs and were not hemolytic [98].

The strain P. freudenreichii B3523 exhibited inhibitory activity towards three Salmonella serotypes, S. Enteriditis, S. typhimurium and S. Heidelberg, reducing pathogen motility, adhesion and invasion of avian epithelial cells, and multiplication in the cecal contents of turkeys. The P. freudenreichii strain was able to adhere to the epithelial cells and its cell-free extracts inhibited growth and motility of S. Heidelberg [99]. When this strain was supplied at about 10 Log CFU daily to turkey poults of 2 to 12 weeks of age challenged with the multidrug resistant (MDR) Salmonella Heidelberg GT2011, that caused a foodborne outbreak from turkey meat in the United States in 2011 [70], it induced a reduction in the levels of the pathogen in cecal content of 1 to 2 Log CFU/g. This effect was linked to the changes induced by P. freudenreichii in the intestinal microbiota composition of growing poults such as the increase of actinobacteria and the genus Subdoligranulum and the decrease of Streptococcus after 2 days of treatment but not later. In finishing turkeys, supplementation with P. freudenreichii induced an increase of lactobacilli and Ruminococcaceae after 2 days and an increase of the genera Lactococcus, Erysipelatoclostridium, Leuconostoc and Butyricicoccus after 7 days, while the Salmonella Heidelberg group showed a higher abundance of Turicibacter and Streptococcus in different groups and sampling times. Similar results were obtained for finishing turkeys in a separate experiment. The genuses enriched in the P. freudenreichii treatment groups are responsible for the formation of SCFAs that promote gut health, whereas the genus Streptococcus comprises opportunistic pathogens and the genus Turicibacter was associated to intestinal inflammatory diseases so the results indicated a protective effect of P. freudenreichii against intestinal dysbiosis induced by the pathogen Salmonella Heidelberg [69].

It was later reported that in treatments of turkeys with P. freudenreichii B3523 the dissemination of S. Heidelberg in liver and spleen was reduced, depending on the age of animals and treatment time, from to 20% - 60% [70]. The same P. freudenreichii strain was used to treat turkeys challenged with drug resistant field turkey isolates of S. Agona, S. Saintpaul, and S. Reading and proved equally effective with a Salmonella vaccine in the reduction of the pathogen in cecum, and even more effective when combined with the vaccine. All the liver and spleen samples from animals receiving the vaccine and P. freudenreichii were negative for Salmonella despite the fact that P. freudenreichii alone showed a relatively low percentage of Salmonella negative organ samples. However, the reason for this higher efficacy in comparison with the vaccine alone and the vaccine combined with a Ligilactobacillus salivarius strain was not investigated [71].

A. acidipropionici Q4, isolated from a Swiss-type cheese made in Argentina adhered to HT29 cells and prevented the adhesion of an E. coli and a S. Enteriditis strain mediated by its cell surface proteins (CSP) that separately exerted similar effects [100]. A. jensenii В-6085, and A. thoenii В-6082 showed moderate inhibitory activity against the intestinal pathogens E. coli ATCC 25922, S. enterica ATCC 14028, S. aureus ATCC 25923, P. aeruginosa B6643, P. vulgaris ATCC 63, and L. monocytogenes ATCC 7644 and the strain P. freudenreichii B-11921 showed a weaker activity compared to the Acidipropionibacterium spp. against L. monocytogenes [101].

P. freudenreichii DSM 20271 and SS10 (Optim PropioniBacter, Laboratoire Optim, Bionoto sprl., Belgium), and A. acidipropionici DSM 20272 showed very slight inhibiting effects against wound pathogens in vitro, confirming previous results for these species. However, moderate antimicrobial activities had been previously reported for Propionibacterium jensenii B-6085 and Propionibacterium thoenii B-6082 [102].

The cheese isolates P. freudenreichii AS2 and AS51, and the malt isolate A. virtanenii JS278 exerted anti-quorum sensing (QS) activity and inhibition of QS-dependent biofilm formation toward Chromobacterium violaceum ATCC 31532 with P. freudenreichii exerting the most pronounced effect. This activity depended on the SCFAs acetic and propionic acid produced that inhibited the synthesis of the auto-inducer (AI) acyl-homoserine lactone (AHL) as demonstrated by the comparison with a deletion mutant for the gene cvil for QS-dependent biofilm formation [103].

5. Production of Beneficial Metabolites

The role of A. jensenii 702 to correct B12 vitamin deficiency in vivo was demonstrated in rats fed with a diet deficient in B12 vitamin for three months. Supplementation with 10 Log CFU of the bacterium daily for two months restored the B12 vitamin levels before administration of the B12 vitamin deficient diet. After three months of A. jensenii 702 administration the B12 vitamin levels were comparable to those obtained by direct supplementation of the vitamin showing that its deficiency can be corrected by supplying the bacterium through fermented food [104].

A contrasting result was obtained with the generally recognized as safe (GRAS) certified strain P. freudenreichi ATCC 6207. This was added in 8 Log CFU/g to yogurt administered to 30 volunteers with 18 to 50 years of age at the KEM Hospital, Pune, India, in a double-blind placebo trial aimed to the prevention of vitamin B12 deficiency. No significant differences were found in vitamin B12 concentration in venous blood samples in the P. freudenreichii group compared to the placebo group, though the strain is a producer of vitamin B12. The causes of the missing beneficial effect were not investigated [105].

Four P. freudenreichii isolates from goat milk, selected on the basis of growth capacity and tolerance to gastrointestinal and technological stresses, were used to ferment the Scotta dairy product from two industries. The formation of vitamin B9 vitamers, cobalamin-derivatives and folates was demonstrated, though at extent that differed between the two dairies [15]. A P. freudenreichii PS-4 strain (Christian Hansen, Denmark) was used for in situ production of up to 6.4 mg/g of fat conjugated linoleic acid (CLA), a substance with antioxidant, anti-cholesterolemic, anti-inflammatory and anti-carcinogenic properties, in a yogurt supplemented with inulin [106].

Studies on fermented food products of both plant and animal origin such as sourdough bread, tofu and fermented milk have demonstrated the potential of dairy propionibacteria as single strains or combinations to enrich the diet with vitamin B12, SCFAs, folate and bioactive peptides [107,108,109,110,111,112,113]. Moreover, A. acidipropionioci LET 120, a strain with high β-galactosidase activity, produces prebiotics from lactose and lactulose, namely oligosaccharides from lactose (GOS) and lactulose (OsLu). P. freudenreichii strain DSM 4902 was utilized to produce vitamin B12 on spent beer yeast [114,115].

6. Safety of Dairy Propionibacteria

The only known virulence factor of dairy propionibacteria is β-hemolytic activity expressed by red pigmented strains of the species A. jensenii and A. thoenii that produce granadaene. Despite adverse events caused by granadaene producing strains were never reported, it is preferable that these bacteria are not present in food. No detrimental activities such as biogenic amine formation in cheese were reported for dairy propionibacteria. [116,117]. Granadaene production is encoded by the cyl gene cluster comprising the genes acpC, cylZ, cylA, cylB, cylE, homologous to those found in Streptococcus agalactiae and essential for hemolytic activity, in A. thoenii, A. vitanenii and A. jensenii. The link between the red pigmentation and hemolytic activity was confirmed for A. jensenii, A. thoenii and A. virtanenii strains and not pigmented strains, including P. freudenreichii strains, did not show any hemolytic activity. A conserved region in the cylG gene was selected for PCR-based diagnosis of hemolytic activity in dairy propionibacteria [117]. The safety of the pigmented Acidipropionibacterium strains has not yet been assessed in vivo.

No safety issues were reported for the strains tested as probiotics. In particular, A. jensenii 702 was fed to rats for 81 days and did not cause adverse effects, it did not affect body and organ weight and faecal β-glucuronidase activity. Moreover, extra-intestinal translocation was not observed [71]. The culture medium of P. freudenreichii ET-3, approved for use in form of tablets produced by Meiji and able to selectively stimulate the growth of bifidobacteria in human gut, when supplied to rats in high amounts of 6 g per kg per day did not cause any clinical sign and toxicity for different organs at macroscopic and microscopic level and no alterations of hematological and chemical values were observed. Moreover, the preparation was not mutagenic for Salmonella and E. coli indicators and for Chinese hamster lung cells [118]. Clinical safety assessment was carried out for participants receiving the culture medium for one week and no statistically different hematological and chemistry parameters were found compared to the placebo group and the baseline. In participants receiving the culture medium for 13 weeks there was a significant decrease of total blood protein levels, white blood cells, hemoglobin, mean corpuscular hemoglobin concentration and increased medium red cell volume and urine pH from the baseline but the values remained in the normal ranges. No gastrointestinal symptoms could be attributed to the P. freudenreichii culture medium. Therefore, no adverse effects were attributed to its dietary supplementation [119]. P. freudenreichii JS used in combination with L. rhamnosus GG did not induce gastrointestinal or respiratory disorders or infections in randomised, double-blind, placebo-controlled clinical studies carried out with 1,909 healthy participants [120].

Regarding the aspect of antibiotic resistance, it was reported that P. freudenreichii strains showed intrinsic resistance to aminoglycosides, quinolones, including levofloxacin, oxacillin, metronidazole, and kanamycin, and no transferable antibiotic resistance was described based on plasmid-curing experiments [116]. Phenotypic resistance tests carried out by disc diffusion for the antibiotics ampicillin, benzylpenicillin, carbenicillin, polymyxin, streptomycin, gentamicin, clotrimazole, chloramphenicol, tetracycline, neomycin, and kanamycin indicated for P. freudenreichii B-11921 moderate resistance to gentamicin and neomycin, sensitivity to polimixin and resistance to all the other antibiotics, for A. acidipropionici В-5723 moderate resistance to benzylpenicillin, gentamicin, and clotrimazole, sensitivity to polymyxin and tetracycline and resistance to all the other antibiotics, for A. jensenii В-6085 moderate resistance to carbenicillin, polymyxin, and tetracycline and high resistance to all the other antibiotics, and for A. thoenii В-6082 moderate resistance to streptomycin, clotrimazole, and chloramphenicol and resistance to all the other antibiotics [101]. However, the genetic bases of resistance were not investigated.

Testing with the broth microdilution method according to the ISO 10932:2010 norm [121] indicated that 47 dairy propionibacteria isolated from goat milk, cheese and rumen in the of Sardinia island, Italy, were resistant to amoxicillin. For the species P. freudenreichii, 71%, 48%, 24% and 13% of the isolates were resistant to spectinomycin, ciprofloxacin, erythromycin, and tetracycline, respectively, while four isolates were resistant to kanamycin. Among the 18 Acidipropionibacterium strains screened seven were resistant to tetracycline, three to ciprofloxacin, two to kanamycin and one to clindamycin according to the cut off values fixed for bifidobacteria and non-enterococcal lactic acid bacteria [122]. Four P. freudenreichii strains and one A. acidipropionici strain showed multiple antibiotic resistances and only three strains of P. freudenreichii and six Acidipropionici spp. strains did not show any antibiotic resistance [15].

Mutations G2294A and G2295A in the 23S rRNA that could determine resistance to macrolide antibiotics were observed in the P. freudenreichii strain T82 but these are non-transferable [123]. Proteins related to antibiotic resistance, namely a mitomycin radical oxidase, a protein with tetracyclin repressor domain and a puromycin resistance protein Pur8 were found to be encoded by a genomic island in P. freudenreichii JS8 [2].

Phenotypic resistance of some Propionibacterium and Acidipropionibacterium strains was reported towards vancomycin and ciprofloxacin, and reversible resistance was observed for tetracycline [74].

The lectin binding strain A. acidipropionici LET103 intended for use as avian growth promoter in a multi-strain preparation, did not show any expression of virulence factors or antibiotic resistance [124].

7. Discussion

Based on the studies consulted for this review, dairy propionibacteria can have a wide spectrum of applications as probiotics with proven benefits in IBDs, obesity, rheumatoid arthritis, allergy and infections in animal models [14,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71]. Despite these bacteria were less investigated than lactobacilli and bifidobacteria, they also demonstrated multiple disease mitigation activities often associated to the activation of anti-inflammatory components of the immune system. The capacity to produce vitamin B12, folate and CLA represents an additional benefit that could derive from supplementation of substrates or foods fermented by of dairy propionibacteria.

A fact that should be considered in favor of conducting new studies on the dietary supplementation of dairy propionibacteria is the total absence of infection case reports with their involvement, differently than found for lactobacilli [125], bifidobacteria [126] and the widely used probiotic Saccharomyces boulardii [127,128]. This might depend on their less frequent use in living probiotic supplementation for humans, that was limited to the strain P. freudenreichii JS [93]. However, it must be considered that the frequent occurrence of dairy propionibacteria in cheeses, naturally or intentionally added, represents an exposure source for which adverse effects were never reported. Another evidence in favor of the incapability of dairy propionibacteria to be harmful is the absence of adverse effects and extraintestinal translocation in farm animals and in animal disease models [71]. Moreover, studies aimed at evaluating the toxicity of dairy propionibacteria did not report any adverse effect and those regarding survival in the gastrointestinal tract never indicated long persistence after the administration period that could be a consequence of transient adhesion to intestinal epithelial cells or mucus. On the other hand, the inability of P. freudenreichii to invade intestinal epithelial cells was demonstrated in vitro [38,98]. All these indications should encourage the execution of clinical trials to explore the possible beneficial effects of dairy propionibacteria for humans.

Studies on P. freudenreichii ET-3 supplied as culture medium concentrate and not as living probiotic demonstrated that the production of postbiotics from dairy propionibacteria is also a possibility. A further indication supporting this opportunity are the beneficial effects obtained by using the purified immunomodulatory proteins or EVs that were comparable with those of the living bacteria in animal hosts [14,40,44,47,48,49,50,53,57,59,60].

The molecular basis of dairy propionibacteria interactions with the immune system of host cells and tissues were clarified for strains of the species P. freudenreichii able to induce host responses that led to inflammation mitigation and tissue integrity protection and were found to be strain-dependent. In particular, the gene encoding SlpB, one of the proteins with highest immunomodulatory effect produced by P. freudenreichii, was found in the genome of only two among 20 P. freudenreichii strains included in a genome sequencing and comparison study [2]. Other proteins involved in the probiotic effects of P. freudenreichii were the central metabolism protein DlaT and the chaperonin GroEL [14,57]. These proteins are known to belong to the core genome of bacteria and, in the case of P. freudenreichii, they function as mediators of probiotic activities only in some strains. This could be explained with their overexpression determined by the presence of more than one gene copy or to rearrangements in the regulatory regions that lead to increased expression. The genome duplication observed in some P. freudenreichii strains could be at the origin of the increased expression of some intracellular proteins found to be more abundant in cell surface protein extracts or EVs [2].

The identification of the protein mediators of the probiotic activities offers the possibility to design screening tests targeted on their encoding genes for the selection of new P. freudenreichii strains to be tested for probiotic activities. Similarly, tests aimed at determining the gtf gene expression level could allow to exclude the strains whose surface proteins are likely to be shielded by a polysaccharidic capsule [31].

In one P. freudenreichii strain among 20 studied a pilus structure was found to be expressed and promote binding to mucus [2]. Since this character is known as a main factor favoring adhesion and, if overexpressed, even to increase the capability of probiotics to cause infections [125], its occurrence in dairy propionibacteria and its involvement in persistence in the gastrointestinal tract should be evaluated.

No molecular investigations were carried out for the Acidipropionibacterium genus, despite the health-promoting effects observed in animal trials and in farm animals. Therefore, this research field should be explored for a possible expanded application of different dairy propionibacteria or derived culture products as food and feed supplements. From a one health perspective, the studies on the inhibition of Salmonella infections in turkeys demonstrated that use of dairy propionibacteria can reduce the risk of human exposure to zoonotic pathogens by decreasing the infection levels in farm animals [69,70].

A research area that must still be explored for a safe use of dairy propionibacteria as probiotics is the study of the genetic bases for the antibiotic resistance phenotypes observed in some studies and the definition of ecological cut off (ECOFF) values of antibiotic resistance by examining a large number of isolates. This would permit a more reliable distinction between intrinsic and acquired resistance than it is currently possible. Indeed, despite no transferable antibiotic resistance was reported for these species, up to date studies specifically focused on this aspect should be carried out considering the increasing trend of antibiotic resistance genetic determinants spread among bacteria.