IngredientesPropionibacterium freudenreichii belongs to the domain Bacteria, phylum Actinomycetota, class Actinomycetes, order Propionibacteriales, family Propionibacteriaceae, and genus Propionibacterium. The genus Propionibacterium belongs to the class of high-GC actinobacteria, and all species of this genus produce propionic acid as a major metabolic end-product. Taxonomical studies clearly distinguish two groups of propionibacteria: cutaneous and dairy. P. freudenreichii belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes.
P. freudenreichii is currently divided into two subspecies, according to the phenotype for lactose fermentation and nitrate reduction (lac+/nit− and lac−/nit+ for P. freudenreichii subsp. shermanii and subsp. freudenreichii, respectively). However, the existence of unclassifiable strains (lac+/nit+ and lac−/nit−) has also been reported. The formal taxonomic naming of Propionibacterium freudenreichii occurred in 1928 by microbiologist Cornelis B. van Niel, who classified it within the genus Propionibacterium based on its metabolic properties, particularly its ability to ferment carbohydrates to propionic acid under anaerobic conditions.
Propionibacteria are Gram-positive, anaerobic to aerotolerant, non-motile microorganisms with high GC content. The cells most commonly take the shape of pleomorphic rods (0.2–1.5 μm × 1–5 μm) but can also be coccoid, bifid, branched, or filamentous, with a length that can reach as long as 20 μm. When grown on solid media, colonies can appear smooth, convex, or rough. The genome of P. freudenreichii CIRM-BIA1T consists of a circular chromosome of 2,616,384 base pairs (bp) with 67% GC content.
Over a century ago, P. freudenreichii was the first dairy species isolated from Emmental cheese. Propionibacterium freudenreichii is a gram-positive, non-motile bacterium that plays an important role in the creation of Emmental cheese, and to some extent, Jarlsberg cheese, Leerdammer, and Maasdam cheese. Its concentration in Swiss-type cheeses is higher than in any other cheese. Although dairy propionibacteria originate principally from the dairy environment, they can also be isolated from silage, soil, rumen, and other habitats.
P. freudenreichii is widely cultivated and consumed by humans in fermented dairy products such as Swiss-type cheese and in food probiotic supplements. It is widely implemented both in the manufacture of fermented dairy products such as Swiss-type cheeses and in the production of probiotic food complements, under the form of freeze-dried powders. A notable commercial preparation, the ET-3 culture (from Meiji Dairies Corporation), is provided as a freeze-dried pale orange–white powder. It is prepared by adding sodium ascorbate to ET-3 culture medium, followed by sterilization and freeze-drying. An estimated one billion living cells of P. freudenreichii are present in one gram of Emmental cheese.
Propionibacterium freudenreichii was first discovered and isolated in the early 20th century by Eduard von Freudenreich and Sigurd Orla-Jensen. They discovered the bacterium while studying propionic acid fermentation in Emmental cheese. Its genus is named after propionic acid, which this bacterium produces, and the species epithet freudenreichii is named after von Freudenreich.
Due to its long documented use in cheese, and in particular in Swiss-type cheeses, P. freudenreichii has an established history as a food microorganism. When Emmental cheese is being produced, P. freudenreichii ferments lactate to form acetate, propionate, and carbon dioxide (3 C3H6O3 → 2 C2H5CO2 + C2H3O2 + CO2). The products of this fermentation contribute to the nutty and sweet flavors of the cheese, and the carbon dioxide byproduct is responsible for forming the characteristic holes, or "eyes," in the cheese. Cheesemakers control the size of the holes by changing the acidity, temperature, and curing time of the mixture.
P. freudenreichii is a dairy-associated bacterium, traditionally used in the production of Swiss-type cheeses and also for industrial-scale production of vitamin B12. This "Generally Recognized As Safe" (GRAS) species is traditionally used as a starter for Swiss-type cheeses where it is responsible for holes and aroma production, as a vitamin B12 and propionic acid producer in white biotechnologies, and as a probiotic for use in humans and animals because of its bifidogenic and anti-inflammatory properties.
A cell-free product of whey fermentation using P. freudenreichii ET-3 (7025) has been used as a food ingredient in Japan, where it promoted the growth of Bifidobacteria through the action of 1,4-dihydroxy-2-naphthoic acid (DHNA), although its safety through traditional toxicity testing was not formally evaluated until relatively recently.
Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short-chain fatty acids (propionic and acetic acids) as well as vitamin B12. Various pathways for formation of cheese flavor compounds and metabolic end-products have been identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched-chain fatty acids, and esterases involved in the formation of free fatty acids and esters. Propionic acid (propionate) and acetic acid (acetate) are the primary short-chain fatty acid (SCFA) end-products of P. freudenreichii fermentation and are considered central to several proposed health mechanisms.
Propionibacterium freudenreichii is a food-grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. The P. freudenreichii genome has a fusion enzyme BluB/CobT2 implicated in production of the active form of vitamin B12. Understanding the mechanisms affecting the synthesis of different forms of cobalamin is important in the context of strain selection. Thirty genes are implicated in the biosynthesis of vitamin B12 in P. freudenreichii.
Research has demonstrated that cereal products can be naturally fortified with active B12 to a nutritionally relevant level by fermenting with P. freudenreichii. Vitamin B12 is naturally present only in foods of animal origin and in certain fermented plant foods or fortified products. According to a review by the WHO, B12 and folate deficiencies may be public health problems worldwide, particularly in developing countries due to inadequate consumption of animal-based foods; in wealthier countries, vegetarians, vegans, and the elderly are at risk of deficiency.
During manufacturing, P. freudenreichii is responsible for the production of the beneficial short-chain fatty acids acetate and propionate, the B9 (folate) and B12 (cobalamin) vitamins, and the bifidogenic compounds DHNA (1,4-dihydroxy-2-naphthoic acid) and ACNQ (2-amino-3-carboxy-1,4-naphthoquinone). In the food industry, P. freudenreichii is also used as a vitamin producer, with certain strains having the ability to produce vitamins such as riboflavin and cobalamin (vitamin B12). Along with being an excellent source of trehalose and vitamins B12, B9, and K, propionibacteria have been reported to boost the immune system and remove mycotoxins from the digestive tract.
Propionibacteria produce the short-chain fatty acids (SCFAs) acetate and propionate, and other beneficial metabolites such as vitamin B9 and B12, as well as 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), which were described as bifidogenic growth stimulators. The modulation of gut microbiota depends on the production of the DHNA and ACNQ molecules, which favor bifidobacteria at the expense of pathobiont Bacteroides.
Cell wall-related proteins, S-layer type proteins, moonlighting proteins, and proteins related to interactions with the host have been identified as important actors in immunomodulation of P. freudenreichii strain CIRM-BIA 129. Specifically, studies reported the role of surface-layer protein B (SlpB) from this strain in bacterial adhesion to intestinal HT-29 cells and immunomodulation, as well as that of large surface layer protein A (LspA) from strain P. UF1 in the regulation of colonic dendritic cells during inflammation via SIGNR1 binding.
Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, and bacterial EVs are associated with several processes including survival, competition, pathogenesis, and immunomodulation. Among probiotic Gram-positive bacteria, some P. freudenreichii strains exhibit anti-inflammatory activity notably via surface proteins such as SlpB. Evidence suggests that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs that export immunomodulatory proteins to interact with the host.
P. freudenreichii produces a bifidogenic compound that aids in stimulating bifidobacterial growth. In vivo experiments indicate that P. freudenreichii consumption results in modulation of the gut microbiota, including enhancement of the bifidobacterial population and decrease in Clostridium and Bacteroides. Modulation of gut content enzymatic activity was also reported, including enhancement of beta-galactosidase activity and decrease in beta-glucuronidase and azoreductase activities.
P. freudenreichii is known to adhere to intestinal epithelial cells and mucus and to modulate important functions of the gut mucosa, including cell proliferation and immune response. Removal of propionibacteria surface-layer (S-layer) proteins, which are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain, suppressed the induction of anti-inflammatory cytokines. In in vitro assays, P. freudenreichii wild type reduced expression of IL-8 and TNF-α cytokines in LPS-stimulated HT-29 cells; a mutant strain lacking the SlpB protein failed to do so.
In 2012, Cousin and collaborators demonstrated that dairy propionibacteria induce production of the regulatory cytokine IL-10 ex vivo in porcine colonic mucosa explants and decrease production of pro-inflammatory cytokines such as IL-8 and tumor necrosis factor-α (TNFα) in the gut mucosa of piglets after lipopolysaccharide (LPS) stimulation.
Dairy gram-positive propionibacteria, as the probiotic species P. freudenreichii, were shown to induce apoptosis of colon cancer cells via the intrinsic apoptotic death pathway. These propionibacteria induce apoptosis via the production of SCFAs not only in vitro but also in vivo in human microbiota-associated rats. Interestingly, P. freudenreichii enhances apoptosis and lowers proliferation only in the context of carcinogenesis, and not in healthy conditions. Notably, P. freudenreichii does not display cytotoxicity or inhibit the proliferation of normal colon cells such as Human Epithelial Intestinal Cells (HIEC).
The promising probiotic potential of P. freudenreichii relies largely on the active release of beneficial metabolites within the gut as well as the expression of key surface proteins involved in immunomodulation. Adaptation to the colon environment is requisite for the active release of propionibacterial beneficial metabolites and constitutes a bottleneck for metabolic activity in vivo. To trigger beneficial effects in the digestive tract, it is crucial for propionibacteria to be consumed alive, which closely depends on the drying technology used.
P. freudenreichii ET-3 culture, a cell-free product of whey fermentation, has been shown to promote the growth of Bifidobacteria through the action of 1,4-dihydroxy-2-naphthoic acid (DHNA), and therefore has potential use in the food and supplement industries. In humans, P. freudenreichii relieved constipation in 41 healthy young women. Human volunteer studies have been conducted to evaluate the bifidogenic effects of P. freudenreichii consumption, with evidence that bifidobacterial populations in the colon are selectively stimulated. Select strains of P. freudenreichii can modulate the gut microbiota in the colitis context and in healthy conditions.
Strength of evidence: The bifidogenic effect is one of the better-characterized activities of P. freudenreichii, supported by both human volunteer studies and mechanistic in vitro/in vivo work. However, the available human studies are generally small and not fully powered randomized controlled trials; larger and more rigorously controlled trials are needed to confirm clinical significance.
Whey fermented by P. freudenreichii was shown to alleviate symptoms of ulcerative colitis in humans. P. freudenreichii was also orally administered to human patients in a pilot study, being promising for the treatment of ulcerative colitis. In this human pilot study, P. freudenreichii improved the symptoms of mild to moderate ulcerative colitis. In preclinical work, P. freudenreichii also accelerated healing in rats with colitis.
One study evaluated whether milk whey culture with P. freudenreichii ET-3 (which has reported bifidogenic activity) was effective in colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rats. Colitis was induced by intracolonic injection of TNBS; milk whey culture was then administered orally at doses of 1 and 3 g/kg twice a day for 9 days. On the 10th day, rats were sacrificed and ulcer size measured. Milk whey culture significantly accelerated the healing of the colitis in a dose-dependent manner, whereas the culture medium alone did not.
DHNA attenuated inflammation through the modulation of intestinal bacterial microbiota and suppression of lymphocyte infiltration. P. freudenreichii strains, isolated or associated with other probiotic bacteria, have also been shown to attenuate colitis induced by trinitrobenzene sulfonic acid (TNBS) in BALB/c mice.
Strength of evidence: The evidence for P. freudenreichii in IBD/colitis includes one small human pilot study and multiple animal studies showing consistent beneficial effects. The human pilot study is described as a pilot study with limited power; it does not constitute high-level evidence. The preclinical data are supportive but cannot be directly extrapolated to human efficacy.
One rat study assessed the impact of P. freudenreichii against colorectal cancer (CRC) induced by azoxymethane (AOM). The experiment was performed using four groups of SD rats: normal control, AOM group, PF group (1 × 109 CFU/mL), and standard drug control (5-fluorouracil, 35 mg/kg). Methylene blue staining of colon tissues showed that administration of PF significantly reduced the formation of colonic aberrant crypt foci (ACF) compared to the AOM control group.
In vitro, P. freudenreichii was found to initiate apoptosis of HGT-1 human gastric cancer cells by acting on the mitochondria of the colorectal cancer cells through the metabolite propionate. Furthermore, several studies have demonstrated that P. freudenreichii exhibits inhibitory effects on various colon carcinoma cell lines, including HT-29, HeLa, and CaCo-2 cells. P. freudenreichii subsp. shermanii induces apoptosis of colon cancer cells, which is attributed to the production of propionate and acetate.
A first milk fermented exclusively by P. freudenreichii was obtained and shown to induce apoptosis in HGT-1 human gastric cancer cells.
Strength of evidence: Clinical studies are lacking in this area, and it is unclear whether the cell culture and animal results will be at all relevant to human cancers. All cancer-related evidence for P. freudenreichii currently remains at the in vitro and animal preclinical stage. No human clinical trials have been published evaluating P. freudenreichii as an oncological intervention.
P. freudenreichii CIRM-BIA 129 is a probiotic bacterium that exerts immunomodulatory effects. This strain possesses extractable surface proteins including SlpB, which are involved in anti-inflammatory effect and adhesion to epithelial cells. Investigators examined the impact of slpB gene mutation on immunomodulation in vitro and in vivo. This work demonstrated that a P. freudenreichii mutant lacking SlpB loses its ability to regulate pro-inflammatory cytokines in LPS-stimulated HT-29 cells and to alleviate 5-FU–induced mucositis. Clinical guidelines for the management of mucositis have recently added a suggestion for the use of probiotics.
Strength of evidence: Evidence for mucositis mitigation derives from in vitro and animal studies demonstrating mechanistic plausibility (SlpB-dependent immunomodulation). No clinical data in humans have been published specifically for P. freudenreichii in this context.
One study investigated the potential impact of P. freudenreichii CIRM-BIA129, a recognized immunomodulatory probiotic bacterium, on food allergy development in a murine model. Preventive effects of this probiotic were evaluated in the context of an induced wheat gliadin allergy. Following sensitization using gliadins, clinical and immunological parameters were monitored following an oral challenge with wheat gliadin.
Strength of evidence: Preclinical only (murine model). No human data are available. The study provides mechanistic hypothesis generation, not clinical evidence.
P. freudenreichii is a producer of short-chain fatty acids and vitamin B12 and is a promising organism for in situ fortification of foods with B12 vitamin since it is generally recognized as safe and is able to synthesize the biologically active form of the vitamin. A content of 12–37 μg/kg of active B12 was produced in bread-type matrices; this content increased 10-fold with cobalt supplementation and reached 940–1,480 μg/kg with both cobalt and DMBI. With riboflavin and nicotinamide supplementation, B12 production in cobalt-supplemented medium increased to 712 μg/kg.
Strength of evidence: The capacity of P. freudenreichii to produce and deliver biologically active vitamin B12 is well documented at the food science level. Research is ongoing to optimize production conditions for practical fortification applications, particularly for plant-based foods. Clinical outcomes of B12 fortification via P. freudenreichii-fermented foods specifically in humans have not yet been widely reported in controlled trials.
P. freudenreichii is widely implemented both in the manufacture of fermented dairy products such as Swiss-type cheeses and in the production of probiotic food complements, under the form of freeze-dried powders.
The following dosages are reported in specific studies and are cited directly from source materials:
P. freudenreichii has the GRAS (Generally Recognized As Safe) status in accordance with a long and documented history of safe use in food. P. freudenreichii and P. acidipropionici have also been listed in the QPS (Qualified Presumption of Safety) list by the European food safety authority.
By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii.
In a 4-week oral toxicity study, administration of 6,000 mg/kg body weight/day P. freudenreichii ET-3 culture was without compound-related adverse effects on clinical signs, body weights, food consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and gross and microscopic findings in male and female Sprague-Dawley rats.
In the 13-week human study, changes in hematological parameters (total protein, white blood cell count, hemoglobin, mean corpuscular hemoglobin concentration) that were observed from baseline were deemed not to be due to P. freudenreichii ET-3 culture supplementation given that all parameters remained within normal ranges and were not consistent with any clinically meaningful effect.
The genus Propionibacterium comprises both cutaneous species (including the well-known P. acnes), which may act as opportunistic pathogens, and dairy species, which have no reported adverse effects. The dairy species Propionibacterium freudenreichii and Propionibacterium acidipropionici are clearly distinct from cutaneous species. This distinction is clinically important: P. freudenreichii should not be confused with the cutaneous propionibacteria associated with acne or opportunistic infections.
P. freudenreichii has received the GRAS status for its use in cheese in healthy populations. The safety of its consumption by cancer patients with compromised immunity and mucosal barrier has been specifically flagged as requiring further investigation.
A bottleneck in the production of probiotic supplements is the limited survival of P. freudenreichii during the drying process and storage. Protective pre-treatments have been applied, but may confer enhanced resistance only in a strain-dependent manner, and very little information was published on P. freudenreichii adaptation to freeze-drying until recently.
The P. freudenreichii ET-3 culture test article used in formal safety studies is provided in a whey-based medium as a freeze-dried powder. This is a consideration for individuals with cow's milk protein allergy or lactose intolerance, as the preparation matrix may contain dairy-derived components.
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Propionibacterium freudenreichii subsp. shermanii was a key component of the landmark human RCT (El-Nezami et al., Am J Clin Nutr 2006) that found a 5-week probiotic intervention in young Chinese men significantly reduced urinary aflatoxin biomarkers and decreased liver cancer risk. It is one of the few probiotic bacteria with direct human clinical evidence for mycotoxin reduction when used in combination with Lactobacillus rhamnosus.
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