On the basis of the GRAS status of dairy propionibacteria and the positive results obtained by us and other authors, further studies are encouraged in order to select the appropriate strains for developing new functional foods that include these bacteria for human and animal nutrition.

It should be emphasized that much of the health benefits described above could be related to the ability of propionibacteria to remain in high numbers in the gastrointestinal tract by surviving the adverse environmental conditions and adhering to the intestinal mucosa.

With regard to animals’ health it has been reported that dairy propionibacteria directly fed to farm animals increased weight gain, food efficiency and health of many animals like chickens, laying hens, piglets and cows. With a wider insight, propionibacteria may be assayed as probiotics for other ruminants like goats and sheep since their milk-derived products are highly appreciated by consumers.

Different studies have also described the ability of dairy propionibacteria to bind and remove toxic compounds from different environments such as the gut and food. Some of them have focused in the removal of mycotoxins, like Aflatoxin B and Fusarium sp. toxins by in vitro, ex vivo and in vivo assays whereas others have reported the binding of cyanotoxins and some heavy metals like cadmium and lead. It has been proposed that probiotic microorganisms may reduce by binding, the availability of free toxic compounds within the intestinal tract which reduces in turn, their negative effects. In this respect, in recent years we have been investigating the potential of dairy propionibacteria to protect the intestinal mucosa from the toxic and antinutritional effects of some common dietary substances like the plant lectins from the Leguminosae family. By in vitro and in vivo studies we have determined that certain strains are able to bind and remove different dietary lectins from media, preventing their cytotoxic effects on intestinal epithelial cells. Daily ingestion of P. acidipropionici CRL 1198, a dairy strain studied in our laboratory, at the same time than concanavalin A prevented the deleterious effects caused by this lectin on some morphological and physiological parameters related to intestinal functionality in mice. Propionibacteria reduced the incidence of colonic lesions, the enlargement of organs, the disruption of brush border membranes and the decrease of their disaccharidase activities. Since consumption of suitable propionibacteria may be an effective tool to avoid lectin-epithelia interactions, further investigations on their potential as probiotic detoxifying agents are actually ongoing

Nowadays there are clear evidences that propionibacteria used alone or combined with other microorganisms can exert beneficial effects in the host. Dairy propionibacteria have proven to posses many promising properties such as the production of nutraceuticals like vitamin B 2 , B 12 , K and conjugated linoleic acid, and their health promoting effects could be attributed to one or more of the following modes of action: i) influence on gut microbial composition and exclusion of pathogens; ii) modulation of the metabolic activities of the microbiota and host, and iii) immunomodulation. The most documented probiotic effects for propionibacteria within these categories include: bifidogenic effect in the human gut, improvement of nutrients utilization, hypocholesterolemic effect and anticarcinogenic potential immune system stimulation.

However, the ability of dairy propionibacteria to improve the health of humans and animals by being used as dietary microbial adjuncts has been extensively investigated. In this sense, our research group has been studying for the last two decades the probiotic properties of dairy propionibacteria isolated from different ecological niches. In the present article the current evidences supporting the potential of dairy propionibacteria to be used as probiotics are reviewed focusing in a less studied mechanism such as the protection of the intestinal mucosa by the binding of dietary toxic compounds.

The genus Propionibacterium has been historically divided, based on habitat of origin, into “dairy” and “cutaneous” microorganisms which mainly inhabit dairy/silage environments and the skin/intestine of human and animals, respectively. Dairy propionibacteria are generally recognized as safe microorganisms whereas members of the cutaneous group have shown to be opportunistic pathogens in compromised hosts. In consequence, the economic relevance of propionibacteria derives mainly from the industrial application of dairy species as cheese starters and as biological producers of propionic acid and other metabolites like exopolysaccharides and bacteriocins to be used as thickeners and foods preservers, respectively.

Although the great bulk of evidence concerns lactobacilli and bifidobacteria, since they are members of the resident microbiota in the gastrointestinal tract, other less conventional genera like Saccharomyces, Streptococcus, Enterococcus, Pediococcus, Leuconostoc and Propionibacterium have also been considered.

Probiotics are live microorganisms that confer health benefits to the host when administered in adequate amounts. In the last decades there has been a great interest from food and pharmaceutical industries to develop products containing probiotics due to the great demands of healthy foods and alternatives to conventional chemotherapy.

It is also known that dairy propionibacteria survive for many months at room temperature even under conditions of carbon starvation, being the majority of the strains non-lytic [ 2 ]. This long-term survival in stationary phase or dormant phase could be the consequence of a multi-tolerance response that involves the synthesis and accumulation of polyP, glycogen, trehalose and the over-expression of molecular protein chaperones. Besides, a gene encoding an Rpf (resuscitation promoting factor) protein which is essential for the growth of dormant cells from actinobacteria has been described in the genome of P freudenreichii and is probably involved in long-term survival of propionibacteria [ 40 ].

It has been reported that propionibacteria are able to withstand osmotic stress by accumulation of compatible solutes like glycine betaine and trehalose [ 52 ]. Trehalose is a non-reducing disaccharide that can be used by bacteria as a carbon and energy source and also can be accumulated as a compatible solute. All dairy propionibacteria are able, in a strain dependent manner, to synthesize and accumulate trehalose from glucose and pyruvate [ 53 ]. Both processes are enhanced at stationary phase and under oxidative, osmotic, and acid stress conditions [ 54 ]. Trehalose is commonly synthesised via the trehalose-6-phosphate synthase/phosphatase (OtsA–OtsB) pathway and catabolised by trehalose synthase (TreS). The genes otsA, otsB, and treS were identified in P. freudenreichii by Cardoso et al., 2007 [ 55 ] and Falentin et al 2010 [ 40 ].

Propionibacteria is also able to synthesize glycogen and all the genes related to glycogen metabolism were identified in the genome of the strain P.freudenreichii CIRM-BIA1T [ 40 ]. Some of these genes were also found in P. acnes. These enzymes seem to be involved in intracellular accumulation and hydrolysis of glycogen as neither P. freudenreichii nor P. acnes are able to ferment extracellular glycogen

In addition, long term survival of propionibacteria on adverse environments could be due to the accumulation of storage compounds, compatible solutes, and the induction of a multi-tolerance response under carbon starvation [ 40 ]. In contrast to other bacteria that use ATP, P. freudenreichii accumulates inorganic polyphosphate (polyP) as energy reserve. Short chains of PolyP are synthesized when bacteria grow on glucose whereas long chains are accumulated when the main carbon source is lactate. The synthesis of PolyP is catalysed by polyphosphate kinase (PPK) that transfers the terminal phosphate of ATP to polyP. It is proposed that PolyPs enable microorganisms to tolerate adverse conditions since ppk mutants are unable to survive during stationary phase [ 51 ]. The genes encoding for polyP or pyrophosphate (instead of ATP) using enzymes were found in the genome of P.freudenreichii CIRM-BIA1T [ 40 ].

Stress tolerance and cross-protection in strains of Propionibacterium freudenreichii were examined after exposure to heat, acid, bile and osmotic stresses. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts. Furthermore, acid pretreatment sensitized cells to bile salts challenge and vice versa. Heat and acid responses did not present significant cross-protection and no cross-protection of salt-adapted cells against heat stress was observed for these propionibacteria [ 48 - 50 ].

With respect to thermotolerance, the over-expression of constitutive stress-related molecular chaperones and ATP-dependent proteases as well as the induction of the dihydroxyacetone kinase locus (dhaKL, PFREUD_07980 and PFREUD_07990) by stress and starvation seems to be related to survival to thermal stress by difference to thermosensitive strains [ 40 , 50 ].

The stress adaptation proteins were particularly investigated in P. freudenreichii and its genome, finding out that they are differentially expressed depending on the strain and the stress [ 40 , 48 - 50 ]. Acid and bile stresses, induce the synthesis of the following proteins: pyruvate-flavodoxin oxidoreductase and succinate dehydrogenase which are involved in electron transport and ATP synthesis, as well as glutamate decarboxylase and aspartate ammonia-lyase, which are involved in intracellular pH homeostasis. Bile also induces oxidative stress so that survival and activity within the gut depend on remediation of oxidative damages. P. freudenreichii possesses an arsenal of genes for disulfide-reduction and elimination of reactive oxygen species. Moreover, in response to bile salts, P. freudenreichii overexpresses the iron/manganese superoxide dismutase, Glutathione-S-transferase, two cysteine synthases and S-adenosylmethionine synthetase [ 40 ]. The occurrence of a sodium/bile acid symporter (PFREUD_14830) reflects adaptation to the gut environment. Other inducible proteins involved in protection and repair of DNA damages include Ssb protein which is involved in DNA recombination and repair, as well as Dps which protects DNA against oxidative stress are stress-induced in P. freudenreichii [ 49 ].

Interestingly, the cell machinery involved in general stress adaptation in P. freudenreichii was shown to be encoded by multicopy stress-induced genes [ 40 ]. The redundancy and inducibility of this chaperone and protease machinery is in agreement with the ability of P. freudenreichii to adapt rapidly and efficiently to various unfavorable conditions [ 48 - 50 ].

In this sense, the manufacture of a swiss type cheese represents for microbial starters successive stresses like acidification of the curd, heating during cooking, osmotic stress due to brining, and low temperature (4 to 12 °C) during cheese ripening. The transit through the digestive tract also suppose stressful conditions for bacteria such as gastric acidity and the presence of other aggressive intestinal fluids like bile and pancreatic enzymes.

It is known that propionibacteria are able to adapt and survive to different stresses like industrial processes and the gastrointestinal transit, as well as to remain active for long periods of time in such adverse environments [ 43 ].

Although P. freudenreichii subspecies shermanii is able to ferment lactose, dairy propionibacteria show poor growth in milk, as they do not possess proteases capable of hydrolyzing milk caseins [ 46 ]. Some proteinases have been described for Propionibacterium, one cell wall associated and one intracellular or membrane bound but their activities are weak. By contrast, different peptidases such as aminopeptidases, proline iminopeptidase, proline imidopeptidase, X-prolyl-dipeptidyl-amino-peptidase, endopeptidases and carboxypeptidase, have been described. and characterized. Amino acids, especially aspartic acid, alanine, serine and glycine, are degraded by Propionibacterium, with variations among species and strain [ 47 ]. On the other side, cutaneous propionibacteria, have the ability to hydrolyze different proteins, like gelatin and fibronectin, and to promote damages and inflammation of the host tissues.

Propionibacteria are also mesophilic microorganisms, with optimal growth conditions at 30 ºC and pH 6.8. However, they grow in a temperature range between 15 a 40 ºC and tolerate pH variations between 5.1 and 8.5 [ 1 , 2 ]. Their nutritional requirements are low and almost the same for all the species. Dairy propionibacteria like P. freudenreichii are able to synthesize all amino acids [ 40 ]. They can grow in a minimal medium containing ammonium as the sole nitrogen source, but a higher growth is observed in media containing amino acids [ 45 ].

Depending on the strains, the substrate used, and the environmental conditions, propionibacteria modulate the proportions of pyruvate either reduced to propionate, or oxidised to acetate and CO 2 , to maintain the redox balance [ 43 ]. In this way the oxidation of glucose and lactic acid leads to a molar ratio of propionate:acetate of 2:1 whereas the oxidation of glycerol leads to the formation of propionate only. The co-metabolism of aspartate/asparagine and lactate has also been reported [ 44 ]. During lactate fermentation, aspartate is deaminated to fumarate by an aspartate ammonia lyase; fumarate is then converted to succinate, with a concomitant production of NAD and ATP. Cells using this pathway convert less pyruvate to propionate and oxidised more pyruvate to acetate+CO 2 .

Then, oxaloacetate is reduced to succinate, via malate and fumarate in two NADH requiring reactions. Succinate is then converted to propionate via methylmalonyl-CoA intermediates (succinyl-CoA and propionyl-CoA); the carboxyl group removed from methylmalonyl-CoA is transferred to pyruvate to yield oxaloacetate, thus completing one cycle. Methylmalonyl-CoA is also regenerated from succinyl-CoA during propionate production, thus creating the second of the two transcarboxylase cycles, and can react with a new molecule of pyruvate. All the reactions of this cycle are reversible. It must be emphasized that the Wood Werkman cycle used by propionibacteria to produce propionate is coupled to oxidative phosphorylation and yields more ATP than in the other bacteria producing propionic acid [ 42 , 43 ].

The most important reaction of this cycle is transcarboxylation that transfers a carboxyl group from methyllmalonyl-CoA to pyruvate to form oxaloacetate and propionyl-CoA, without ATP consumption. The enzyme catalyzing this reaction is a methylmalonyl-CoA carboxytransferase that has been fully characterized and its structure resolved [34; 40].

The production of propionic acid by these bacteria involves a complex metabolic cycle with several reactions in which substrates are metabolized to pyruvate via glycolysis, pentose phosphate or the Entner-Doudoroff pathways, generating ATP and reduced co-enzymes. Pyruvate is then oxidised to acetate and CO 2 or reduced to propionate. The latter transformation occurs via the Wood-Werkman cycle or transcarboxilase cycle which represents the key component of the central carbon metabolic pathway in propionibacteria [ 41 ].

Propionibacteria are heterotrophic microorganisms that mean they need an organic carbon source to grow and posses a fermentative metabolism [ 41 - 43 ]. They degrade carbohydrates like glucose, galactose, lactose, fructose and other sugars; poliols like glycerol; erythritol and others; and organic acids such as lactic and gluconic acids producing propionic, acetic and CO 2 as the main fermentation end-products [ 1 ].

In relation to probiotic functionality, it has been identified the complete biosynthesis pathway for a bifidogenic compound (DHNA) as well as the sequences corresponding to a high number of surface proteins involved in the interactions with the host (like adhesion and immunomodulation). By comparative genomics with P. acnes, no pathogenicity factors were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of this species.

With respect to technological application in dairy industries, various pathways for formation of cheese flavor compounds were identified in the genome of this strain such as the enzymes involved in the production of propionic acid, volatile branched chain fatty acids from amino acid degradation, and free fatty acids and esters from lipids catabolism.

The genome sequence also showed that P. freudenreichii possesses a complete enzymatic machinery for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) and genes involved in the metabolism of carbon sources, immunity against phages, chaperones for stress resistance, and storage of inorganic polyphosphate, glycogen and compatible solutes such as trehalose that confer these bacteria a long survival in stationary phase [ 40 ]. Although propionibacteria are usually described as anaerobes, all the genes encoding enzymes required for aerobic respiration such as NADH dehydrogenase, succinate dehydrogenase, cytochrome bd complex, ATPase and the complete pathway for heme synthesis have been identified in the genome of P. freudenreichii [ 40 ].

P. freudenreichii subsp. shermanii CIRM-BIAl T is able to metabolize lactose, although this trait is strain-dependent, since the Lac genes may have been acquired through a horizontal transfer event mediated by phage infection. In this sense it should be emphasized that the presence of the enzyme β-galactosidase should be the only feature that allows these bacteria to adapt to dairy niches like cheeses.

The genome of the type strain, P. freudenreichii subsp. shermanii strain CIRM-BIAl T, was sequenced with an 11-fold coverage [ 40 ]. It consists of a circular chromosome of 2,616,384 base pairs (bp) with 67% GC content, 2 rRNA operons and 45 tRNAs. The chromosome is predicted to contain 2439 protein-coding genes and also contains 22 different insertion sequences that represent 3.47% (in base pairs) of the genome. Insertion sequences and transposable elements may promote genome plasticity and induce phenotypic changes that contribute to bacterial adaptation to different environments; being particular for propionibacteria the synthesis of capsular EPS and the ability to ferment lactose [ 40 ].

Up to few years ago, the only completely sequenced and publicly available genome within the genus Propionibacterium was that of the commensal cutaneous species P. acnes [ 39 ]. However, in the year 2010, the complete genome of a species that belongs to the taxonomic group of dairy propionibacteria was described for the first time.

The members of the genus Propionibacterium possess a circular-shaped chromosome like most bacteria that varies in size between 2.3 and 3.2 Mb depending on the different species [ 35 ]. The G+C content in their DNA is in the range of 53-68 mol% and although they generally do not possess plasmids their existence has been reported in strains of P. acidipropionici, P. freudenreichii and P. jensenii [ 36 ]. In fact, it has been informed that between 10 and 30% of P. freudenreichii strains possess one or two cryptic plasmids [ 37 ]. The presence of two types of bacteriophages has also been described for propionibacteria. One of them, the bacteriophage B22, belongs to the Group B1 of Bradley classification, whereas the other, bacteriophage B5, would be the first infectious filamentous virus described in a Gram positive bacterium [ 38 ].

Finally, a real-time PCR method, based on the transcription of the enzyme transcarboxylase involved in propionic fermentation, was successfully used to detect a strain of P. freudenreichii in the intestinal ecosystem [ 34 ] and would be a valuable tool for monitoring survival and metabolic activity of propionibacteria in different environments.

Recently, a multicolor fluorescent in situ hybridization (FISH) assay targeting the 16S rRNA [ 30 ] or 23S rRNA [ 31 ] of P.acnes was developed and used to detect this bacterium in blood samples and tissues of patients with prostate cancer, respectively. A FISH protocol and oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria were developed in our laboratory [ 32 ] and successfully used for enumeration of P. acidipropionici in cecal samples of mice fed with a strain of this species [ 33 ].

Molecular methods are a valuable alternative to plating assays, being far more specific, and unhindered by the presence of non-target microorganisms. Different fingerprinting methods such as SDS-PAGE of whole cell proteins [ 23 ], 16s rDNA targeted PCR-RFLP [ 24 ], ribotyping [ 25 ], 16S-23S ribosomal spacer amplification and restriction [ 26 ], Pulsed-Field Gel Electrophoresis [ 27 ], Conventional Gel Electrophoresis Restriction Endonuclease Analysis (CGE-REA) and Randomly Amplified Polymorphic DNA-PCR [ 28 ] have been used for detection and accurate identification of dairy propionibacteria from various environments like milk, cheese, whey and flour. Genus and species-specific primers targeted to the genes encoding 16S rRNA for PCR-based assays were also designed for detection of dairy propionibacteria [ 29 ].

Isolation and enumeration of propionibacteria can be made by microbial culture and molecular methods [ 19 ]. Various agarized media with different degrees of selectivity have been used for detection and enumeration of classical propionibacteria in dairy environments, animal and human fecal samples. Among them it could be mentioned YELA [ 20 ], Pal Propiobac® medium, which contains glycerol, lithium lactate and antibiotics [ 21 ] or others including lithium chloride and sodium lactate in concentrations high enough to limit the growth of accompanying bacteria [ 22 ]. In all cases, incubations are made in anaerobiosis with an atmosphere of 10–20% CO 2 . Although these media may be successful for the isolation of classical and cutaneous strains of Propionibacterium, they have limitations for selective enumeration of bacteria in very complex ecosystems like intestinal microbiota. Furthermore, plate count methods for propionibacteria are time consuming since long incubation periods for at least 6 days are needed to obtain typical colonies and enumerations may be underestimated due to aggregation of bacteria in the diluents used, and/or growth inhibition by the selective agents used.

From a safety point of view, classical species have a long history of safe application on industrial processes whereas members of the cutaneous group are commonly considered opportunistic pathogens in compromised hosts. In consequence, the economic relevance of propionibacteria derives mainly from the industrial application of dairy species as cheese starters and as biological producers of propionic acid and other metabolites with a more recent interest on their usage as health promoters.

On the other side, cutaneous species are found mainly in the human skin, but have been isolated also from the intestine of humans, chicken and pigs [ 1 , 2 , 18 ], being best represented by the acne bacillus, Propionibacterium acnes.

Classical propionibacteria include among their main habitats: raw milk and cheese [ 1 , 2 ] but have been obtained also from silages and vegetables for human consumption [ 15 ], and from ruminal content and feces of cows and calves [ 16 ]. Furthermore, they are not limited to the gastrointestinal tract of ruminants being also isolated from the intestine of pigs and laying hens [ 17 ].

At present, the genus Propionibacterium is classified as Actinobacteria with a high G+C content, that make them more related to corynebacteria and mycobacteria than lactic acid bacteria. The current taxonomic position of propionibacteria is the following [ 2 ]: Phylum Actinobacteria; Class Actinobacteria; Subclass Actinobacteridae; Order Actinomycetales; Suborder Propionibacterineae; Family Propionibacteriaceae; Genus Propionibacterium.

In the last two decades six new species were isolated: P. cyclohexanicum was obtained from spoiled orange juice [ 10 ]; P. microaerophilum was isolated from olive mill wastewater [ 11 ]; P. australiense came from granulomatous bovine lesions [ 12 ] and P. acidifaciens from human carious dentine [ 13 ]. Recently, a new species isolated from human humerus, P. humerusii, has been proposed [ 14 ].

In 1972, Johnson and Cummins [ 6 ], classified strains with several common features into eight homology groups based on DNA-DNA hybridization and peptidoglycan characteristics. This study was the basis for the classification of propionibacteria into “dairy or classical” and “cutaneous” groups included in the 8th edition of Bergey’s Manual of Determinative Bacteriology (1974). Four dairy species were recognized in this edition: P. freudenreichii and their three subespecies (freudenreichii, shermanii y globosum), P. thoenii, P. jensenii and P.acidipropionici whereas other four species that inhabit the human skin were ascribed to the cutaneous propionibacteria: P. acnes, P. avidum, P. lymphophylum and P.granulosum. The same scheme was followed in the first edition of Bergey’s Manual of Systematic Bacteriology [ 1 ]. In 1988, on the basis of 16S rRNA sequences, the species Arachnia propionica was reclassified as Propionibacterium propionicus [ 7 ]. Then, in Bergey’s Manual 9th edition (1994), the classification of previous edition was maintained but the subspecies P. freudenreichii subsp. globosum was removed without justification. Other species like P. inoccuum and P lymphophilum were then also reclassified as Propioniferax innocua [ 8 ] and Propionimicrobium lymphophilum [ 9 ], respectively.

By the beginning of the XX th century, E. Von Freudenreich and Sigurd Orla-Jensen (1906) [ 4 ] isolated the bacteria responsible for the “eyes” formation in Emmental cheese and some years later, the name Propionibacterium was suggested by Orla-Jensen [ 5 ] for referring to bacteria that produced large amounts of propionic acid. Although several strains were isolated during the following years these microorganisms were not included in the Bergey’s Manual of Determinative Bacteriology till the third edition published in 1930. Since then, new species were described on the basis of their morphological and biochemical characteristics such as their typical pattern of Chinese characters, propionic acid production, and carbohydrate fermentation profile.

Although in 1861, Louis Pasteur demonstrated that propionic fermentation was due to the biochemical activity of microorganisms, the first studies about the morphology and physiology of propionibacteria were carried out by Albert Fitz (1879) [ 3 ], who observed that organisms from cheeses with “eyes” ferment lactate to propionic and acetic acids and liberate carbon dioxide.

Propionibacteria are Gram positive, catalase positive, high G+C%, non spore forming and non motile pleomorphic bacteria [ 1 , 2 ]. In general, microorganisms of the genus Propionibacterium are anaerobic to slightly aerotolerant and morphologically heterogeneous including rod-shaped and filamentous branched cells that may occur singly, in pairs forming a V or a Y shape, or arranged in “Chinese characters”. They have a peculiar metabolism leading to the formation of propionic acid as main end-product of fermentation.

Trehalose has been proposed as a healthy sugar substitute in foods because of its anticariogenic and dietetic properties. As described in paragraphs above, propionibacteria synthesize trehalose as a reserve compound and as a stress-response metabolite [ 52 - 55 ]. With respect to the production of this sugar in situ in food products, it has been observed that P. freudenreichii ssp. shermanii NIZO B365 produces high levels of trehalose in skim milk [ 54 ].

In dairy propionibacteria (P.freudenreichii subsp. shermanii), the single gene gtf encoding for a β-d-glucan synthase that is responsible for the synthesis of surface polysaccharide has been identified [ 40 , 116 ] and the EPS produced was also characterized. Both homopolysaccharide [ 116 , 117 ] and heteropolymers [ 118 ] were described and it has been reported that production of EPS by propionibacteria is a strain-dependent property (due to an IS element in the gtf promoting sequence) that is influenced by the medium composition and the fermentation conditions [ 119 , 120 ]. Further studies are needed to elucidate the role of these polymers and their potential applications.

Exopolysaccharides-producing bacteria and their secreted EPS are important biological thickeners for food industry. Besides, some health promoting properties such as immunomodulation and cholesterol lowering activities have been ascribed to EPS [ 115 ].

In another study, Hojo et al. [ 114 ] assessed the concentration of MK-9 (4H) in commercial propionibacteria-fermented cheeses finding out a positive correlation between the increase in propionibacteria and the generation of MK-9 (4H) in cheese. Due to their high MK-9 (4H) concentrations (200 to 650 ng/g), Emmental and Jarlsberg cheeses should be a meaningful source of vitamin K and potential protectors against osteoporosis.

It has been reported that Propionibacterium freudenreichii produces large amounts of tetrahydromenaquinone-9 (MK-9 (4H)) and the precursor 1,4-dyhidroxy-2-naphtoicacid (DHNA) which is a known bifidogenic factor [ 110 - 112 ]. In order to improve the production of these metabolites, different laboratory culture protocols that could be applied to an industrial scale have been assayed finding out that DHNA production is markedly influenced by carbon source limitation and the oxygen supply. An improvement in DHNA production could be obtained by a cultivation method that combines anaerobic fed-batch and aerobic batch cultures [ 112 , 113 ].

Vitamin K (a group of 2-methyl-1,4-naphthoquinone derivatives), is an essential cofactor for the formation of γ-carboxyglutamic acid-containing proteins that bind calcium ions and are involved in blood coagulation and tissue calcification. Its deficiency has been associated with low bone density and increased risk of fractures from osteoporosis and intracranial hemorrhage in newborns [ 109 ]. Vitamin K1 or phylloquinone is present in plants, and vitamin K2, also called menaquinone, is produced in animals and bacteria that live in the intestine.

Vitamin B2, also known as riboflavin, is the central component of the cofactors FAD and FMN, and is therefore required by all flavoproteins. As such, vitamin B2 is required for a wide variety of cellular reactions and is involved in vital metabolic processes in the body. It has been reported that P. freudenreichii NIZO2336, a mutant strain that produces larger amounts of riboflavin than the parental strain, improved riboflavin content of yogurt and riboflavin status of rats fed with this product [ 106 ].

In consequence dairy propionibacteria are the preferred microorganisms for the industrial production of this vitamin and many efforts have been made to improve the production process by using genetic engineering [ 102 , 103 ] and other biotechnological strategies like fermentation manipulations [ 104 , 105 ].

The pathway of vitamin B12 synthesis in Propionibacterium freudenreichii has been completely elucidated [ 40 , 102 ]. This microorganism synthesizes cobalamin as a cofactor for propionic acid fermentation [ 41 ] and is the only bacteria, among B12 producers that possess the GRAS status of the United States Food and Drug Administration.

Vitamin B12 also called cobalamin, is an essential nutrient for the human body that plays a key role in the normal functioning of the brain and nervous system, the formation of blood and also the metabolism of every cell, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. Its deficiency leads to a serious physiological disorder called pernicious anemia.

By varying the source of LA for conjugation and the fermentation conditions it has been observed that P. freudenreichii convert free LA to mainly extracellular CLA with a high efficiency (50-90%), being the optimal conditions that favor the accumulation of CLA also determined [ 97 , 101 ]. Besides, it has been observed that CLA formation and growth of dairy propionibacteria in fermented milks were enhanced in the presence of yogurt microorganisms whereas organoleptic attributes obtained with yogurt starter cultures were not affected by co-cultures with the propionibacteria [ 100 ].

In this regard, several studies have shown the potential of propionibacteria for producing CLA enriched products. Both growing and resting cells of dairy (P. freudenreichii) [ 96 , 97 ] and cutaneous propionibacteria (P. acnes) [ 98 ] produce cis-9, trans-11 and trans-10, cis-12, the major isomers with biological activity, on different growth media: culture broths [ 97 ], lipid containing plant materials [ 99 ], milk and ripening cheese [ 100 ].

Many health benefits have been attributed to consumption of CLA-containing foods such as anticarcinogenic, antiatherogenic, antidiabetogenic and antioxidative properties, immune system modulation and reduction of body fat gain [ 94 ]. CLA-isomers are formed by biohydrogenation of LA in the rumen and through conversion of vaccenic acid by Δ9-destaurase in the mammary gland so that ruminant meats and milk-derived products are main dietary sources of CLA. However, some microorganisms like Bifidobacterium, Lactobacillus, Enterococcus and Propionibacterium posses a linoleic acid isomerase that allow them to form CLA as a detoxification mechanism [ 95 ]. In consequence, they have been intended, either as starter or adjunct cultures, to increase the CLA level and nutritional value of some fermented products like yoghurt and cheese.

Propionibacteria are able to produce many biological compounds that enhance the human health so they can be used as “nutraceuticals cell factories” for food enrichment. In this regard, propionibacteria have already been considered as rich sources of conjugated linoleic acid, vitamins, exopolysaccharides and trehalose.

Although the ability of dairy propionibacteria to produce bacteriocins in situ in food products or inside the intestine has not been demonstrated yet, they have a potential application as safe biopreservatives. In this respect, some efforts have been made to improve the production processes [ 90 ] since the slow growth, late bacteriocin synthesis and low production represent limitations for the practical application of bacteriocin-producer propionibacteria.

Different bacteriocins produced by both dairy and cutaneous propionibacteria have been reported and characterized. Among them it could be mentioned: Propionicin PLG-1 and GBZ-1 produced by P. thoenii 127 [ 81 ]; Jenseniin G isolated from P. thoenii P126 [ 82 ]; Propionicins SM1 and SM2 produced by P. jensenii DF1 [ 83 ]; Propionicin T1 synthesized by P. thoenii 419 and LMG2792 [ 84 ]; Thoenicin 447 isolated from P. thoenii 447 [ 85 ]; Acnecin produced by a strain of P. acnes [ 86 ] and several other propionicins [ 87 - 89 ].

Both starters and naturally occurring bacteria on food have the ability to produce bacteriocins. Hence, they may have potentially important applications as food biopreservatives or bacteriocin-producer probiotics to inhibit intestinal pathogens [ 80 ]. However, only nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis, has attained the GRAS status of the FDA for use in certain foods.

Bacteriocins are antimicrobial peptides or proteins encoded by plasmid or chromosomal DNA of a wide range of Gram positive and negative bacteria. They have an antagonistic activity against species genetically related to the producer strain, but many of them exhibit a rather wide spectrum of activity and inhibit the growth of spoilage and pathogenic bacteria belonging to other genera [ 79 ].

Propionic acid, produced in vivo in the gut by viable bacteria, is also a desired healthy metabolite, as it is related to many probiotic properties of propionibacteria (as will be described below). In this respect, it has been demonstrated that SCFA favours the colonic recovery of water and electrolytes counteracting the osmotic diarrhea induced by lactose and/or other unabsorbed carbohydrates [ 76 ]. Besides, they exert anticarcinogenic effects by inducing apoptosis of neoplastic cells but not of healthy mucosa [ 77 ]. Finally, SCFA may exert hypocholesterolemic effects, since propionate lowers blood glucose and alters lipid metabolism by suppressing cholesterol synthesis in the liver and intestine [ 78 ].

Two commercial products that include propionibacteria or their metabolites aimed for controlling spoilage microorganisms are currently available at market. Microgard™ is a food grade biopreservative obtained by fermentation of skim milk with Propionibacterium shermanii that is active against some fungi and Gram negative bacteria, but not against Gram positive ones [ 73 ]. The other product named BioProfit, contains viable cells of P. freudenreichii subsp shermanii strain JS and is effective for inhibiting yeasts growth in dairy products, Bacillus spp. in sourdough bread [ 74 ]; and also used to preserve grain and produce good quality silages [ 75 ].

Most commercial propionic acid is produced by petrochemical processes since biosynthesis by microbial fermentation is limited by low productivity, low conversion efficiency, by-product formation (acetic acid and succinic acid) and end-product inhibition. However, different attempts have been made to improve biological production of propionic acid for industrial applications [ 68 ]. In this sense, it has been determined that the most appropriated species for bioproduction of propionic acid from carbohydrate-based feedstock, including glucose and whey lactose, is P.acidipropionici [ 69 , 70 ]. Since the use of glycerol as the principal carbon source enables the production of propionic acid without acetic acid, recent investigations have focused on the optimization of this particular homopropionic fermentation by propionibacteria [ 71 , 72 ].

Propionic acid and its salts, as well as Propionibacterium spp strains, are widely used as food and grain preservatives due to their antimicrobial activity at low pH. They are commonly incorporated in the food industry to prolong the shelf-life of many products by suppressing the growth of mold and spoilage microorganisms in bread and cakes, on the surface of cheeses, meats, fruits, vegetables, and tobacco.

Other dairy products such as yogurt and fermented milks seem to be less appropriated for delivery of propionibacteria due to their weak proteolitic activity, the presence of inhibitory substances and the low pH attained by lactic acid fermentation that do not allow their development. Currently, yogurt is used to deliver probiotic propionibacteria to the host’s intestine or to produce nutraceuticals, but in both cases inoculums higher than those used for cheese manufacturing are necessary.

It is important to emphasize that propionibacteria maintain metabolic activity up to the end of ripening, as shown by molecular methods [ 68 ] producing flavour compounds during growth in cheeses at 24 °C, and further cold storage [ 60 ].

In contrast, although it possesses diverse intracellular peptidases, P. freudenreichii has a limited role in secondary proteolysis, compared to starter and non-starter lactic acid bacteria (NSLAB), because it does not lyse in cheese [ 66 ].

P. freudenreichii also contributes in a great manner to cheese lipolysis by releasing free fatty acids from fat during cheese ripening. Two esterases, one extracellular and other surface-exposed seem to be involved in lipolysis of milk glycerides [ 63 , 64 ]. Furthermore, ten intracellular esterases were found in the P. freudenreichii genome that could be involved in the synthesis of the volatile esters associated with the fruity flavor of cheese [ 65 ].

Propionibacteria degrade branched-chain amino acids to branched-chain volatile compounds mainly 2-methylbutanoic acid and 3-methylbutanoic acid, which derive from isoleucine and leucine degradation, respectively [ 61 ]. These important flavour compounds are almost entirely produced in cheese by propionibacteria that synthesize them in closely related manner to that of cell membrane fatty acids [ 62 ].

P. freudenreichii greatly contributes to Swiss-type cheese flavour by producing compounds from three main pathways: lactate and aspartate fermentation, amino acid catabolism, and fat hydrolysis [ 59 ]. As described above, the end-products of propionic fermentation are considered as flavour compounds in cheese whereas the co-metabolism of aspartate leads to additional CO 2 production. However, strains with a high ability to metabolise aspartate can be associated with undesirable slits and cracks [ 60 ].

Interactions between microbiota and milk throughout ripening lead to biochemical changes that result in the development of the typical texture and flavor. During maturation in the cold room (15 °C) most of lactic starter lyse and release peptidases that produce free amino acids, which are precursors of many flavor compounds. The subsequent period of warm room ripening is characterized by a marked growth of propionibacteria that metabolize the lactate produced by the lactic acid bacteria into propionate, acetate and CO 2 . At the end of maturation that ranges from 6 weeks to 12 - 18 months in the hardest varieties, the number of propionibacteria reaches 10 8 - 10 9 cfu/g of cheese [ 41 , 57 ].

In swiss type cheeses, propionibacteria may be present either as contaminants of raw milk or as components of starter cultures. The typical starter for this variety includes Streptococcus thermophilus, Lactobacillus helveticus, Lactobacillus delbrueckii subsps. lactis or bulgaricus and Propionibacterium freudenreichii. During manufacture and early stages of ripening, the thermophilic bacteria develop at expense of lactose of milk being responsible for lactic acid production, and also contributing to casein hydrolysis during pressing of the cheese.

In these products propionibacteria are responsible for the typical sweet, nutty taste by production of acetic and propionic acids; aminoacids like proline and leucine but mainly for the characteristic “eyes” formation by releasing of CO 2 [ 56 - 57 ]. However, propionibacteria can also be used in the manufacture of various cheeses without eyes just to enhance flavour formation [ 58 ].

The main industrial application of the genus Propionibacterium is the usage of “classical propionibacteria” as dairy starters for the manufacture or Swiss type cheeses. This denomination refers to cheese varieties, such as Sbrinz, Emmental, Gruyère, Compté, Appenzeller and others riddled with holes and made with raw or pasteurized milk (depending on the variety).

4. Probiotic application of dairy propionibacteria

Since the last decades, there has been an increasing interest from food and pharmaceutical industries to develop healthy foods and therapeutic alternatives to conventional antibiotic treatments in response to consumers’ demands of natural products. Probiotics are “live microorganisms that confer health benefits to the host when administered in adequate amounts” [121]. In this respect, the great bulk of evidence concerning the beneficial effects of microorganisms both in human and animal health refers to lactic acid bacteria and bifidobacteria as they are common inhabitants of the gastrointestinal tract. However, in recent years several potential probiotic properties of propionibacteria have been reported and many studies on this subject have been published. In the following sections, safety aspects and the major health benefits ascribed to dairy propionibacteria are reviewed.

4.1. Safety and persistence in the gut Strains selected on the basis of their potential beneficial effects by in vitro tests, must demonstrate their safety both in humans and animals, before they could be incorporated as probiotics, either in food or pharmaceutical products. In this sense, dairy propionibacteria have a long history of safe use in human diet and animal feed. P. freudenreichii is widespread consumed in Swiss type cheeses in which they are present in concentrations close to 109 bacteria/g. Besides, classical propionibacteria have been isolated from soil, silage, vegetables, raw milk, secondary flora of cheese and other naturally fermented food. Therefore, it could be considered that they would arrive to the gut of different organisms, including the man, at least once in their lives. At present, no cases of sickness or toxicity after the ingestion of dairy propionibacteria have been reported [122] neither for humans (for a review of human trials see [123]) nor for animals [124-126]. In fact, it has been reported that propionibacteria did not translocate to blood, liver or spleen and no adverse effects on body weight gain and general health status was observed after short [124, 127] and long terms [125] administration of strains of Propionibacterium acidipropionici, P. freudenreichii and P. jensenii, respectively. Most studies have been performed with strains of P. freudenreichii since it is the traditional component of cheese starters being this species granted the Generally Recognized As Safe (GRAS) status from the US Food and Drug Administration. Furthermore, P. freudenreichii belongs with P. acidipropionici, to the list of agents recommended for Qualified Presumption of Safety (QPS) by the European Food Safety Authority [122, 128]. On the other side, most strains isolated from humans and animals belong to the “cutaneous group” [18, 129] and their use as probiotics is discouraged since they are potential pathogens. However, propionibacteria isolated from the intestine of animals and identified by molecular tools as dairy species, were not associated to pathogenesis. Besides safety, other criteria to take into account in the selection of strains for dietary adjuncts are the absence of antibiotic resistances (due to the risk of spreading any resistance to intestinal microbiota) and virulence factors. Dairy propionibacteria have natural resistance to some antibiotics and this resistance does not appear to be encoded by plasmids or other mobile genetic elements [36, 122, 130]. By comparative genomics, no virulence factors found in P. acnes or in other pathogenic species were identified in P. freudenreichii, although some P. thoenii and P. jensenii strains have β-haemolytic activity [40, 122]. In order to exert their beneficial effects in the host, it is generally accepted that ingested microorganisms must survive the hostile environmental conditions of the gastrointestinal tract represented by the low pH of the stomach and intestinal fluids such as bile and pancreatic enzymes. Many studies have demonstrated by in vitro assays the ability of dairy propionibacteria to survive and tolerate the gastrointestinal conditions [130-134]. This tolerance could be improved by a pre-adaptation of the microorganisms to the adverse conditions of the gut by a brief exposure to the stressful conditions at a non-lethal level [48, 135]. Both acid and bile tolerance have shown to be strain-dependent properties. In previous studies [131, 132] we observed that dairy propionibacteira developed in a medium containing bile (0 – 0.5%) behaved as “bile-tolerant” and “non bile-tolerant” strains and that there were differences among P. freudenreichii and P. acidipropionici strains in their tolerance to pancreatic enzymes when subjected to sequential digestion with artificial gastric and intestinal fluids. It has also been demonstrated that the vehicle used for delivery of probiotics is important for digestive stress tolerance since cells included in food matrices like milk or cheese had better tolerance to acid challenge than free cultures [132]. Similar results were obtained by Huang and Adams [134], by protecting propionibacteria from acid and bile stresses with a soymilk and cereal beverage, and Leverrier et al. [136], who used yoghurt-type fermented milk. Survival of propionibacteria during gastrointestinal transit has also been reported in vivo in rats [125, 126]; mice [124, 137] and humans [127, 130, 133]. Furthermore, Herve et al. [34], demonstrated that propionibacteria remain metabolically active since the P. freudenreichii-specific transcarboxylase mRNA was detected in human faeces. In most studies, a high level of propionibacteria was detected in intestinal contents and feces during the feeding period but this concentration gradually declined and returned to the initial levels a few weeks after consumption ceased. Besides surviving the gastrointestinal digestion, intended probiotics must remain in high levels in the intestine avoiding normal washout by peristaltic contractions of the gut. Therefore, microorganisms with a short generation time and/or the ability to adhere to the mucosa would have an extended survival in the body of the host. Bacterial adhesion to intestinal cells and mucus is generally considered as the initial step in the colonization of the gut and has been related to many of the health effects of probiotics, as it prolongs the time that beneficial bacteria can influence the gastrointestinal microbiota and immune system [138]. Since propionibacteria grow slowly in natural environments and culture media, adhesion ability becomes an important property in the selection of strains for probiotic purposes. Dairy propionibacteria have demonstrated to adhere to immobilized mucus [139]; to isolated mouse intestinal epithelial cells [140,141], to human intestinal cell lines [142-144] and in vivo to intestinal cells as was assessed by counting the adhering propionibacteria on the mucosa by a plate count method [124, 125, 137, 145]. In previous studies, we have correlated the in vitro and in vivo abilities of dairy Propionibacterium strains to adhere to intestinal epithelial cells and observed by scanning electron microscopy, that P.acidipropionici CRL 1198 adheres well to IEC or the mucus layer covering them [141]. Microscopic examination revealed two adhesion patterns in propionibacteria: autoaggregating strains adhere in clusters, with adhesion being mediated by only a few bacteria, whereas nonautoaggregating propionibacteria adhere individually making contact with each epithelial cell with the entire bacterial surface [140]. Besides, the adhesion of propionibacrteria of different dairy species such as P. freudenreichii subsp. shermanii JS, P. jensenii 702 and P. acidipropionici Q4 to Caco-2, C2BBe1 and HT29 cells respectively, was clearly stated [142-144]. Interactions with the host gut mucosa are also suggested by the analysis of the genome of P. freudenreichii that revealed the presence of genes encoding for a high number of surface proteins involved in adhesion and present in other probiotic bacteria [40]. To date, the ability of dairy propionibacteria (used alone or combined with other microorganisms) to improve the health of humans and animals by being used as dietary microbial adjuncts has been extensively investigated. Their health promoting effects could be attributed to one or more of the following modes of action: i) immunomodulation; ii) influence on gut microbial composition and exclusion of pathogens; and iii) modulation of the metabolic activities of the microbiota and host. Main evidences obtained by in vitro and in vivo studies supporting the potential of dairy propionibacteria to be used as probiotics are summarized below.

4.2. Propionibacteria for improving animal health Nowadays, the usage of probiotics as an alternative to antibiotics to enhance the growth and health of domestic animals is a growing practice. With this aim, different bacterial genera have been isolated from the intestine of farm animals and pets and employed as probiotics, such as Lactobacillus, Bifidobacterium and Enterococcus [146]. To date, most animal studies have been performed with ruminants (cows, calves, steers), chicken, pigs, and to a lesser extent with horses and pets. In this sense, it has been reported that dairy propionibacteria administered alone or combined with other microorganisms increase the weight gain, feed efficiency and health of different animals such as laying hens and broilers [147], pigs [148] and calves [149, 150]. Propionibacteria are natural inhabitants of the rumen microbiota. In consequence, they have been used as direct-fed microbial (DFM) feed additives in ruminant nutrition with strain-dependant results on animal performances. One desired effect for ruminant probiotics is an improvement in propionate production as it is considered the major precursor for hepatic gluconeogenesis that provides substrate for lactose synthesis in lactating dairy cows. Various strains of Propionibacterium have increased the molar proportion of ruminal propionate when fed to ruminants [151, 152]. In this respect, many researches have been done with the dairy strain Propionibacterium acidipropionici P169. It has been reported that, when administered to beef cattle, this microorganism was able to increase hepatic glucose production via enhanced ruminal propionate production and absorption, whereas directly fed to early lactating dairy cows, it tended to improve milk proteins content and energetic efficiency during early lactation, without affecting the reproductive function [152-154]. In general, these authors concluded that strain P169 might have potential as an effective direct-fed microorganism to increase milk production in dairy cows. In other studies, the supplementation of lactating dairy cows with a DFM product containing a mixture of L. acidophilus and P. freudenreichii improved milk and protein yield, and apparent digestibility of crude protein, neutral detergent fiber, and acid detergent fiber, so that it could be used to enhance the performance of cows subject to heat stress during hot weather [155]. With respect to calves, a preparation called Proma, which is a blended culture of lactic acid bacteria plus P. freudenreichii and a DFM product containing P. jensenii 702 showed to be effective to improve weight gain during pre-weaning and weaning periods [149, 150]. Propionibacteria have also been assayed as health and growth promoters in monogastric animals like pigs, with positive results. Mantere-Alhonen [148] was the first to achieve growth promotion in piglets fed with different species of propionibacteria being P freudenreichii ssp shermanii the most effective probiotic among the species tested. When propionibacteria were fed to piglets in a daily concentration of 2 x 109 cfu/g, the weight gain was 9.2-14.5% higher, the fodder demand was 7.2-46.1% lower than the control group and the animals had less diarrhoea. In bigger swine, the effects were even more evident. Cutaenous propionibacteria have also been used to improve the health of swine. Propionibacterium avidum KP-40 showed to be a potent immunomodulator that stimulated granulopoiesis as well as a faster body weight gain in pregnant swine and their offspring [156]. The usefulness of the prophylactic application of this strain, against porcine microbial infections was tested in swine finding out that propionibacteria application caused positive immunoregulation of porcine innate immune system effectors, non-specific activation of lymphocytes and antibody production that resulted in milder clinical symptoms, faster recovery and a larger body weight gain [157, 158]. In chicken, both undefined and defined “Nurmi Cultures” have been used to establish an intestinal flora that will prevent colonization by pathogenic bacteria in young animals. These formulas have shown to be effective for the protection against species of Salmonella and other avian pathogens; for immune system stimulation in newborn chicks, and also had growth promoting effects [159, 160]. The most frequently assayed bacteria as avian probiotics were several species of lactic acid bacteria [146, 159, 160]. Propionibacteria have not been widely studied in this ecological niche. However, some authors demonstrated the presence of this bacterial group in the ileum and cecum of chickens [161], and cecal Nurmi cultures characterized by microbiological and PCR-DGGE techniques, evidenced the presence of Propionibacterium propionicus [147]. In recent studies, the occurrence of Propionibacterium in different segments of the gastrointestinal tract of laying hens was demonstrated. Bacteria from this genus were evidenced in 27% of the animals sampled. Half of these isolates were identified by genus and species specific PCR as P. acidipropionici, belonging the others to the propionibacteria cutaneus group. This report represents the first evidence of dairy propionibacteria as inhabitants of the gastrointestinal tract of chickens. Some preliminary studies on the probiotic properties of these strains, suggest their potential application as probiotic to prevent intestinal infections in poultry [17].

4.3. Probiotic properties for human application Inmunomodulation: One of the most promoted properties of probiotics is their ability to regulate in a positive manner the innate and adaptive responses of the human immune system. It is well-documented that cutaneous propionibacteria are potent immunomodulators, since they have been tested in several assays both in humans and rodents used as animal models [162]. Administration of cutaneous propionibacteria (P. avidum, P. granulosum, P. acnes) have shown to be beneficial in the treatment of neoplastic and infectious diseases [163-165]. Besides, dead Propionibacterium acnes or a polysaccharide extracted from its cell wall have proven to be effective in the induction of macrophages with an antitumor effect [166] and in modulating an experimental immunization against Trypanosoma cruzi [167]. With respect to the immunomodulatory properties of dairy propionibacteria, many researches have been done in vitro and in vivo with the strain P. freudenreichii subsp. shermanii JS. It has been reported that this microorganism stimulated the proliferative activity of B and T lymphocytes depending on doses administration and treatment duration in mice [168]. Regarding to cytokine production, P. freudenreichii JS was able to induce TNF-α and IL-10 production in human PBMCs [169] and inhibited the H. pylori-induced IL-8 and PGE2 release in human intestinal epithelial cells [170]. Other dairy P. freudenreichii strains also showed promising immunomodulatory properties by strongly inducing the synthesis of anti-inflammatory IL-10 by human PBMCs and could be helpful in the treatment of inflammatory conditions or diseases [171]. Further beneficial results with P. freudenreichii JS were obtained with different randomised, placebo-controlled, double-blind trials in humans such as: reduction in the serum level of C-reactive protein (an inflammation marker) [172]; induction of IL-4 and IFN-gamma production in PBMCs of infants with cow’s milk allergy [173]; prevention of IgE-associated allergy in caesarean-delivered children [174] and increase in the resistance to respiratory infections during the first two years of life [175]. With respect to other dairy species, an increase in the phagocytic activity of peritoneal macrophages and the phagocytic function of the reticuloendothelial system was observed in mice fed with Propionibacterium acidipropionici CRL 1198 [124]. In addition, administration of this strain prior to infection of mice with Salmonella Typhimurium led to an increase of the anti-Salmonella IgA level and the number of IgA producing cells [176]. Dairy propionibacteria may also act as safe adjuvant for development of oral vaccines. Adams et al [177] found that Propionibacterium jensenii 702 co-administered orally with soluble Mycobacterium tuberculosis antigens to mice stimulate T-cell proliferation of splenic lymphocytes in a significant manner so that the strain PJ702 could act as a potential living vaccine vector to be used against mucosal transmitted diseases.

4.4. Gut microbial modulation Stimulation of bifidobacteria: It is well-documented that propionibacteria can modulate gut microbiota in a positive manner by enhancing bifidobacterial growth. This property has been demonstrated both in vitro [110, 111, 178, 179], and in vivo [127, 180-182] and the bifidogenic growth stimulators (BGS) involved in this effect were identified. The active compounds that were present in supernatants of P. freudenreichii, P. jensenii and P. acidipropionici were purified and identified as 2-amino-3-carboxy-1,4-naphtoquinone (ACNQ) [110, 178] and 1,4-dihydroxy-2-naphtoic acid (DHNA) a precursor of menaquinone (vitamin K2) biosynthesis [111]. It has been proposed that these compounds serve as electron transfer mediators for NADP regeneration in bifidobacteria [183], thus favoring growth. The bifidogenic effect of selected strains of P. freudenreichii [127, 180-182] or purified BGS [184] was assessed in independent studies performed on human volunteers. As a general result, increased fecal bifidobacterial populations were observed even after some days after stopping the consumption of propionibacteria. Besides a reduced colonic transit time and a reduction in the numbers of clostridia were evidenced in some studies. Inhibition of pathogens: There are several reports on the ability of dairy propionibacteria to inhibit exogenous and opportunistic pathogens. In vitro studies have demonstrated that P. freudenreichii strain JS was able to inhibit, alone or combined with other probiotics the adhesion of different pathogens including H.pylori to intestinal mucus and Caco2 cell line also improving the epithelial barrier function [170, 185]. Other dairy species like P. acidipropionici strain Q4 was able to prevent the adhesion of Salmonella enteritidis and Escherichia coli to HT29 cells [144] whereas P. acidipropionici CRL 1198 regulates in vitro the growth of Bacteroides and Clostridium in cecal homogenates of mice supplemented with propionibacteria and/or inulin [33]. Mice consuming this strain delivered in water, milk or cheese showed a decrease in the number of anaerobes and coliforms in the caecal content one week after feeding [124, 137, 145]. P. acidipropionici CRL 1198 also prevented tissue colonization by Salmonella Typhimurium in mice [176]. In humans, propionibacteria have been used in combination with Lactobacillus spp. and Bifidobacterium spp. in the treatment of intestinal disorders and regulation of gut flora and motility. It has been demonstrated that the consumption of probiotic mixtures containing Propionibacterium freudenreichii JS reduced oral Candida in elderly [186] and gastric inflammation of the mucosa caused by H.pylori in the host. [187]. Besides, infants and children fed with Propiono-Acido-Bifido (PAB) milk [188] or milk containing P. freudenreichii subsp. shermanii and L. acidophilus [189], showed a reduction in coliforms with an increase in lactobacilli and bifidobacteria population. Alleviation of IBD: It has been demonstrated that consumption of either isolated BGS or P. freudenreichii strains ameliorate experimental colitis in mice and human ulcerative colitis [171, 189-192]. The mechanism proposed for this effect was restoring of microbiota intestinal balance and suppressing inflammatory lymphocyte infiltration. In this respect, it has been proposed that some surface compounds should be involved in immunomodulatory effects of propionibacteria since removal of surface layer proteins decreased the in vitro induction of anti-inflammatory cytokines [171]. By their side, Michel et al. [193] demonstrated that colonic infusion with P. acidipropionici reduced the severity of TNBS induced colitis in rats whereas Kajander et al [194] reported that the multispecies probiotic mixture containing Propionibacterium freudenreichii JS was effective in alleviating irritable bowel syndrome symptoms.

4.5. Modulation of the host and resident microbiota metabolism Lactose malabsorption: The ability of probiotics to alleviate lactose intolerance by supplying β-galactosidase for the intraintestinal hydrolysis of lactose has been widely reported for LAB and bifidobacteria [196]. However there are no clinical reports on this property for dairy propionibacteria. Several evidences suggest the potential of Propionibacterium acidipropionici strains on this subject: they have high β-galactosidase activity that remain unaltered in the conditions of the human’s intestine, and cells are permeabilized by bile, which in turn may favour the hydrolysis of lactose within the intestine [131, 132]. Besides, the manufacture conditions of Swiss-type cheese did not decrease the synthesis and activity of the β-galactosidase of these propionibacteria [197]. When mice were fed with P.acidipropionici CRL 1198 included in milk or cheese, the β-galactosidase levels in the small bowel and the propionic acid concentration in the caecum were significantly increased. High SCFA concentration in the colon could counteract diarrhea induced by non-digested carbohydrates [137]. Hypocholesterolemic properties: The reduction of cholesterol has been assessed for many probiotics with conflicting results. Somkuti and Johnson [198] evidenced the ability of P. freudenreichii cells to remove by surface adsorption up to 70% of the cholesterol from the medium, whereas Perez Chaia et al [124] demonstrated, in an animal study, that P. acidipropionici CRL 1198 was able to reverse the hyperlipemic effect of a diet with a high lipid content. However, the mechanisms underlying this beneficial effect were not determined in this investigation. Antimutagenic properties: Vorobjeva [199] demonstrated the antimutagenic activity (AM) of Propionibacterium freudenreichii against the mutations induced by 4-nitro-quinoline and N-nitro-N-nitrosoguanidine (transition mutations), and by 9-aminoacridine and 2-nitrofluorene (frame-shift mutations). This AM activity was exerted by live and dead cells and by the cultured media. The active compound responsible for this activity was identified as a cysteine synthase which is induced by some stress factors. Anticarcinogenic properties: Several in vitro and in vivo studies (mainly in animal models) have suggested the potential of probiotics to prevent have suggested the potential of probiotics to prevent colon cancer as evidenced by colon cancer as evidenced by a decrease in the incidence and magnitude of tumours and preneoplastic lesions [200]. Among the mechanisms involved it could be mentioned: inhibition of enzyme activities that convert procarcinogens into carcinogens, control of harmful bacteria, antigenotoxicity, production of active metabolites and immunomodulation. Regarding propionibacteria, it has been demonstrated that P.acidipropionici CRL1198 fed to mice was able to modulate the metabolism of the resident microbiota as it prevented the induction of azoreductase, nitroreductase and β-glucuronidase activities caused by a cooked red-meat supplemented diet. Furthermore, feeding with propionibacteria resulted in a remarkable reduction of β-glucuronidase activity and slight reductions of azo and nitroreductase activities [201]. In humans, independent researches have shown that consumption of P. freudenreichii subsp. shermanii JS decreased to different extents fecal azoreductase activity in elderly subjects, β-glucosidase and urease in healthy young men and β-glucuronidase activity of irritable bowel syndrome patients [202, 203]. Other studies have reported that dairy propionibacteria kill human colorectal adenocarcinoma cells in vitro through apoptosis via their metabolites, propionate and acetate [204, 205]. In addition, consumption of P. freudenreichii TL133 by human microbiota associated rats significantly increased the number of apoptotic cells in the colon of 1,2- dimethylhydrazine treated rats but have no effect on healthy colonic mucosa [77]. The authors suggest that dairy PAB may help in the elimination of damaged cells by apoptosis within the colon epithelium after genotoxic insult. Long term studies assessing the protective role of PAB against colon cancer are still missing.