Propionibacterium freudenreichii: A Probiotic With Remarkable and Promising Properties

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Introduction to Propionibacteria and Propionibacterium freudenreichii

Propionibacterium freudenreichii belongs to the dairy group of the genus Propionibacterium.  Propionibacteria were first described at the end of the 19th century by E. von Freudenreich and S. Orla-Jensen, who were studying propionic acid fermentation in Emmental cheese (Swiss cheese), leading to propose the genus Propionibacterium.  1  Propionibacteria belongs to the phylum of firmicutes. They are characterised as gram-positive, non-sporing, non-motile pleomorphic rods. They are anaerobic to aerotolerant and generally catalase positive.

The genus Propionibacterium is divided in two groups based on habitat of origin:

classical or dairy propionibacteria (mainly isolated from dairy products such as cheese)

  • Propionibacterium acidipropionici
  • Propionibacterium microaerophilum
  • Propionibacterium cyclohexanicum
  • Propionibacterium freudenreichii subspecies freudenreichii
  • Propionibacterium jensenii
  • Propionibacterium freudenreichii subspecies shermanii
  • Propionibacterium thoenii

cutaneous propionibacteria (typically found on skin, also named “acnes group”)

  • Propionibacterium acidifaciens 
  • Propionibacterium acnes 
  • Propionibacterium australiense
  • Propionibacterium avidum
  • Propionibacterium granulosum
  • Propionibacterium propionicum

The dairy propionibacteria species are considered safe whereas cutaneous Propionibacterium species are pathogens.

The dairy propionibacteria do not normally belong to the human microbiota but can be isolated from various habitats including raw milk, dairy products, soil and fermenting food.  Dairy propionibacteria are used in the dairy industry as starter cultures for ripening Swiss cheeses, particularly in the creation of Emmental cheese, and to some extent, Jarlsberg cheese, Leerdammer and Maasdam cheese.  2  Industrial applications of Propionibacterium freudenreichii include production of vitamin B12 (cobalamin), propionic acid, trehalose and conjugated linoleic acid.  

Criteria for the Use of Propionibacterium freudenreichii

Dairy propionibacteria offer good prospects for their use as human probiotics due to the following qualities:  3

  • safety
  • gastrointestinal transit survival
  • adherence to intestinal cells and mucosa

Safety

Consumption of propionibacteria is considered safe since it is contained in Emmental cheese which is consumed worldwide.  It is estimated that Emmental cheese contains up to 1,000,000,000 (109) propionibacteria per gram.

In addition to its widespread consumption from Emmental cheese, Propionibacterium freudenreichii has received the “Generally Recognised As Safe” (GRAS) status and has been granted “Qualified presumption of safety” (QPS) status from the European food safety authority.

Lastly, there are no indication of side effects from consuming dairy propionibacteria that have been reported in any of the human trials.

Gastrointestinal transit survival

Dairy propionibacteria have shown good constitutional survival under digestive stress and their ability to survive low pH conditions of the stomach and exposure to bile.  4  Promising results of gut survival of dairy propionibacteria has been demonstrated in vivo in humans.  5

It is also evident that dairy propionibacteria has strong survival potential due to its role in the cheese making process, which imposes certain technological stressors on the bacteria. 

High levels of propionibacteria has been detected in feces which provides further proof that it can survive gastrointestinal transit.  Once consumption of the dairy propionibacteria ceased, levels in the feces declined.

Adherence to intestinal cells and mucosa

Dairy propionibacteria are able to adhere to immobilised mucus.  6  7  When Lactobacillus rhamnosus GG was added to Propionibacterium freudenreichii ssp. shermanii JS the adhesion to intestinal mucus of Propionibacterium freudenreichii ssp. shermanii JS increased from 1.9 to 2.3%.  8 

Dairy propionibacteria also adheres in vitro to human intestinal epithelial cell lines.  9  10

Mechanism of Propionibacterium freudenreichii

Propionibacterium freudenreichii ferments and converts lactate to form:

  • propionic acid (which favors the growth of bifidobacteria)  11 
  • acetic acid
  • carbon dioxide

In effect, Propionibacterium freudenreichii functions as a prebiotic in the colon by generating short-chain fatty acids (SCFA).  SCFA’s are powerful protectors of the large intestine since they:  12

  • protect the intestinal lining
  • down-regulate NF-κB (nuclear factor-kappa B)
  • support absorption of calcium, magnesium and potassium

Propionibactera also have profound probiotic effects.  These effects include:

  • production of bacteriocins  13
  • secretion of anti-fungal compounds  14  
  • secretion of anti-viral compounds  15
  • synthesis of vitamin B8 (biotin), B9 (folic acid) and B12 (cobalamin)  16  17
  • production of trehalose  18
  • production of conjugated linoleic acid  19
  • growth stimulation of other beneficial bacteria, like bifidobacteria  20
  • creation of a high level of β-galactosidase (lactase) activity  21
  • inhibition of beta-glucuronidase activities 22
  • reduction in pathogen adhesion to immobilised mucus  23
  • ability to aggregate with pathogenic bacteria  24
  • inhibition of Helicobacter pylori adhesion to a human intestinal epithelial cell line  25
  • ability to survive during gastric digestion

Propionibacterium freudenreichii produces a Bifidogenic Growth Stimulator (BGS) named ACNQ which selectively enhances the utilization of Oligosaccharides by Bifidobacteria.  A placebo-controlled study of the effects of BGS on fecal flora and stool frequency was carried out in healthy human subjects. A drink with the sterilized ET-3 culture was administered once a day for 7 days. Bifidobacterium percentage in the fecal flora and stool frequency were significantly increased by administration of the P. freudenreichii culture. The ACNQ exhibited growth stimulation of bifidobacteria at an extremely low concentration and enhanced the activities of NADH oxidase and NADH peroxidase in bifidobacteria. These ACNQ-mediated reactions seem to play roles in NAD (P) +-regeneration processes and seem to be responsible for the growth stimulation of bifidobacteria.   26  27 

Propionibacterium freudenreichii seems to grow and perform in conjuction with another probiotic called Lactobacillus helveticus, even though Propionibacterium freudenreichii does not need Lactobacillus helveticus to work and flourish.  Lactobacillus helveticus releases amino acids and peptides that Propionibacterium freudenrechii utilize.  28 

Bifidobacterium is one of the most researched genus of the human microbiota that is used for probiotic purposes. The primary interest in Propionibacterium freudenreichii in the scientific research is its potential to enhance the indigenous bifidobacteria population. 29

The in vivo experiements with Propionibacterium freudenreichii resulted in modulation of the gastrointestinal microbiota by decereasing Clostridium and Bacteroides.  30

Propionibacterium freudenreichii produces Trehalose in the colon which has been observed to reduce the level of enterohaemorrhagic Escherichia coli O157:H7.  31  

Propionibacterium freudenreichii as a Chemopreventive Agent

Propionibacterium freudenreichii demonstrates great promise in acting as a chemopreventive and chemoprotective agent.  These attributes are accomplished through a number of mechanisms:

  • antimutagenic properties preventing mutations caused by various mutagenic agents  32
  • ability to bind, in vitro, carinogenic compounds:
    • mycotoxins  33
    • cyanotoxin microcystin-LR  37
    • plant lectins – concanavilin and jacalin  38
  • ability to bind to heavy metals
    • cadmium  39
    • lead  40
  • induces NKG2D ligand expression on human-activated T lymphocytes and cancer cells  41
  • ability to lower and inhibit  beta-glucuronidase which is a risk factor for carcinogenesis  42

A number of research studies have shown that Propionibacterium freudenreichii is effective therapeutically in various cancers, especially colon cancer.  Table 1 lists the various cancers that have been treated with Propionibacterium freudenreichii:

Table 1: Propionibacterium freudenreichii Effects on Various Cancers

CancerAbstractReference
Breast cancer
To determine whether the purified 9c,11t conjugated linoleic acid (CLA) isomer, the main dietary isomer, is biologically active on mammary tumor growth, we carried out a dietary intervention study designed to compare its effects with those of a mixture of CLA isomers on the incidence and growth of autochthonous mammary tumors induced by methylnitrosourea in rats.1
Colon cancer
Furthermore, propionibacteria were able to decrease the proliferation index in the distal colon after treatment with DMH (P 2
Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria3
The human probiotic Propionibacterium freudenreichii kills colorectal adenocarcinoma cells through apoptosis in vitro via its metabolites, the short chain fatty acids (SCFA), acetate and propionate.4
The bacterial enzymes beta-glucosidase, beta-glucuronidase, and urease may contribute to the development of colon cancer by generating carcinogens. A reduction in the activity of these enzymes by certain lactic acid bacteria is considered to be beneficial. This study examined fecal beta-glucosidase, beta-glucuronidase, and urease activities during administration of Lactobacillus rhamnosus LC705 (LC705) together with Propionibacterium freudenreichii ssp shermanii JS (PJS).5
Stomach cancer
This study investigated the potential for butyrate and propionate to alter cell viability, cell cycle regulation and intracellular protective mechanisms in a human gastric cancer cell line (Kato III). Kato III cells were incubated with butyrate or propionate for 24, 48 and 72 hr.6

Propionibacterium Effects on Various Disease and Conditions

Propionibacterium freudenreichii and other strains of propionibacterium have demonstrated promising properties in treating and preventing a number of conditions and diseases, especially those of the colon such as Irritable Bowel Syndrome and Colitis.  Table 2 lists the various conditions and diseases that have been studied using Propionibacterium freudenreichii and other strains of propionibacterium:

Table 2: Propionibacterium freudenreichii Effects on Various Disease and Conditions

DiseaseAbstractReference(s)
Allergies
In a double-blinded, placebo-controlled study we randomized 1223 mothers with infants at high risk for allergy to receive a probiotic mixture (2 lactobacilli, bifidobacteria, and propionibacteria) or placebo during the last month of pregnancy and their infants to receive it from birth until age 6 months.1
Colitis
Experimental studies have shown that luminal antigens are involved in chronic intestinal inflammatory disorders. Bifidogenic growth stimulator (BGS) is a prebiotic preparation produced by Propionibacterium freudenreichii isolated from Swiss cheese. Previously BGS was shown to act in the colon as a growth stimulator of Bifidobacteria. This study investigated the efficacy and safety of BGS in the treatment of ulcerative colitis.2
Milk whey culture with Propionibacterium freudenreichii is a prebiotic preparation isolated from Swiss cheese. It selectively stimulates the growth of Bifidobacteria through the action of its component 1, 4-dihydroxy-2-naphthoic acid. Recent reports have shown that this foodstuff ameliorates experimental colitis and human ulcerative colitis. We discuss the characteristics and the therapeutic application of this foodstuff for patients with ulcerative colitis.3
The anti-inflammatory mechanism of prebiotics has recently been shown to have an impact on the host immune system. DHNA from Propionibacterium freudenreichii is known to promote the proliferation of Bifidobacterium and can ameliorate colitis, although its mode of action remains unknown.4
1.4-Dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms.5
Colonic infusion with Propionibacterium acidipropionici reduces severity of chemically-induced colitis in rats6
This study aimed to evaluate whether milk whey culture with Propinibacterium freudenreichii ET-3 (milk whey culture), which has been reported to have Bifidogenic activity, is effective on the colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rats.7
Inhibits Candida
R eport that a cheese containing a mixture of probiotics ( L. rhamnosus GG, L. rhamnosus LC705 and P. freudenreichii ssp. shermanii JS) reduced the risk of high yeast counts, especially Candida sp., in the mouth of elderly people.8
Constipation
The effects of fermented milk whey containing novel bifidogenic growth stimulator (BGS) produced by Propionibacterium on fecal microflora, putrefactive metabolite, defecation frequency and fecal properties were studied in 18 senile volunteers (64-102 yr age) needed serious nursing-care taking enteral nutrition by tube feeding. The test powder food containing 0.4 g/day of freeze-dried BGS were given for 4 weeks. During BGS intake, defecation frequency and fecal quantity were increased significantly, the ratio of color of dark brown and strong odor of feces were decreased significantly.9
Some relief from constipation may be observed with the combination of L. rhamnosus/P. freudenreichii. This probiotic combination also reduced fecal enzyme activity. The tested probiotics did not affect the mucosal barrier.10
Immunomodulation
Immunomodulatory properties of 10 dairy propionibacteria, analyzed on human peripheral blood mononuclear cells (PBMCs), revealed a highly strain-dependent induction of anti-inflammatory cytokine interleukin 10 (IL-10). Two selected strains of Propionibacterium freudenreichii showed a protective effect against two models of colitis in mice, suggesting a probiotic potential predicted by immune-based selection criteria for these cheese starter bacteria.11
The development of a dairy Propionibacterium and its establishment in the gut were studied. Mice fed a conventional diet received a suspension of propionibacteria in skim milk provided in their water bottles for 7 d. Counts of propionibacteria in faeces and intestinal sections indicated that the strain used reached significant levels in the gut during treatment.12
It was observed that Lactobacillus casei was able to stimulate phagocytosis both by the cell wall and the peptidoglycan, whereas it did not produce changes in IgA. L. acidophilus, on the other hand, produced an increase in the levels of IgA without modifying phagocytosis. Propionibacterium acidipropionici only showed immunostimulating activity with the cell wall, but not with the peptidoglycan.13
Immunomodulation by probiotics is a subject of growing interest, but the knowledge of dose response and time profile relationships is minimal. In this study we examined the effects of Lactobacillus rhamnosus GG (LGG) and Propionibacterium freudenreichii subsp. shermanii JS (PJS) on the proliferative activity of murine lymphocytes ex vivo.14
The efficacy of Propionibacterium jensenii 702 to stimulate a cell-mediated response to orally administered soluble Mycobacterium tuberculosis antigens using a mouse model15
Immunomodulatory properties of 10 dairy propionibacteria, analyzed on human peripheral blood mononuclear cells (PBMCs), revealed a highly strain-dependent induction of anti-inflammatory cytokine interleukin 10 (IL-10). Two selected strains of Propionibacterium freudenreichii showed a protective effect against two models of colitis in mice, suggesting a probiotic potential predicted by immune-based selection criteria for these cheese starter bacteria.16
Feeding synbiotics to newborn infants was safe and seemed to increase resistance to respiratory infections during the first 2 years of life.17
Inhibits H. Pylori
The aims of this study were (i) to evaluate the effect of recommended antimicrobial treatment of Helicobacter pylori infection, consisting of clarithromycin, amoxicillin and lansoprazole, on intestinal microbiota and (ii) to determine the ability of a probiotic combination containing Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 to prevent treatment-induced alterations in the intestinal microbiota.18
We characterize four probiotics and their combination in terms of pathogen adhesion, barrier function, cell death, and inflammatory response in Helicobacter pylori-infected epithelial cells. H. pylori-infected Caco-2 cells were pretreated with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii Js, Bifidobacterium breve Bb99, or all four organisms in combination.19
Inflammation and IBD
P robiotic intervention with P. freudenreichii subsp. shermanii JS in healthy adults led to a reduction in the serum level of C-reactive protein (CRP) compared to a placebo control.20
All tested bacteria induced TNF-alpha production. The best inducers of Th1 type cytokines IL-12 and IFN-gamma were Streptococcus and Leuconostoc strains. All Bifidobacterium and Propionibacterium strains induced higher IL-10 production than other studied bacteria.21
Effects of probiotic Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS supplementation on intestinal and systemic markers of inflammation in ApoE*3Leiden mice consuming a high-fat diet22
After the screening of microorganism culture, the culture of Propionibacterium freudenreichii ET-3 in the milk whey (milk whey culture) was found to stimulate the growth of our own Bifidobacteria in the colon but not the growth of other microorganisms.23
Our aim was to determine whether IBS-associated bacterial alterations were reduced during multispecies probiotic intervention consisting of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention has previously been shown to successfully alleviate gastrointestinal symptoms of IBS.24
To investigate the effects of multispecies probiotic supplementation (Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium animalis ssp. lactis Bb12) on abdominal symptoms, quality of life, intestinal microbiota and inflammatory markers in irritable bowel syndrome.25
To investigate the mode of action of a multispecies probiotic consisting of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 by monitoring its effects on intestinal microbiota and markers of microbial activity.26
I rritable bowel syndrome (IBS) is one of the most common diagnoses in gastroenterology, but current therapies are inefficient. Recent clinical trials suggest beneficial effects of certain probiotics in IBS. Because of the heterogeneity of IBS a probiotic combination may be more efficient than a single strain. We screened for optimal strains, and developed a multispecies probiotic combination consisting of L. rhamnosus GG, L. rhamnosus Lc705, P. freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99.27