Jenseniin G, a heat-stable bacteriocin produced by Propionibacterium jensenii P126.

The genus Propionibacterium includes cutaneous species typically found on human skin and the dairy or classical species (Propionibacterium freudenreichii, P. jensenii, P. thoenii, and P. acidipropionici) used industrially for the production of Swiss cheese and propionic acid. Grinstead (1989, M.S. thesis, Iowa State University, Ames) has previously observed that some dairy propionibacteria inhibit other species in the classical grouping. We further investigated the inhibitor(s) produced by P. jensenii P126 (ATCC 4872). An antagonist(s) from anaerobic agar cultures of P126 strongly inhibited two closely related strains of propionibacteria, P. acidipropionici P5 and P. jensenii P54, and Lactobacillus bulgaricus NCDO 1489, Lactobacillus delbrueckii subsp. lactis ATCC 4797, Lactococcus cremoris NCDO 799, and Lactococcus lactis subsp. lactis C2. The inhibitor, designated jenseniin G, was active at pH 7.0; inactivated by treatment with pronase E, proteinase K, and type 14 protease; insensitive to catalase; and stable to freezing, cold storage (4 degrees C, 3 days), and heat (100 degrees C, 15 min). Classification of the inhibitor as a bacteriocin is supported by its proteinaceous nature and its bactericidal activity against L. delbrueckii subsp. lactis ATCC 4797. The lack of detectable plasmids suggests a chromosomal location for the determinant(s) of jenseniin G.     

Prevalence of the genes encoding propionicin T1 and protease-activated antimicrobial peptide and their expression in classical propionibacteria

The purpose of this study was to investigate the frequency of production of the bacteriocin propionicin T1 and the protease-activated antimicrobial peptide (PAMP) and their corresponding genes in 64 isolates of classical propionibacteria. This study revealed that these genes are widespread in Propionibacterium jensenii and Propionibacterium thoenii but absent from the remaining species of classical propionibacteria that were studied. The pro-PAMP-encoding gene (pamA) was found in 63% of the P. jensenii strains and 61% of the P. thoenii strains, and all of these strains displayed PAMP activity. The propionicin T1-encoding gene (pctA) was present in 89% of the P. thoenii strains and 54% of the P. jensenii strains. All P. thoenii strains containing the pctA gene exhibited antimicrobial activity corresponding to propionicin T1 activity, whereas only 38% of the pctA-containing P. jensenii strains displayed this activity. Sequencing of the pctA genes revealed the existence of two allelic variants that differed in a single nucleotide in six strains of P. jensenii; in these strains the glycine at position 55 of propionicin T1 was replaced by an aspartate residue (A variant). No strains harboring the A variant showed any antimicrobial activity against propionicin T1-sensitive bacteria. An open reading frame (orf2) located immediately downstream from the pctA gene was absent in three strains containing the G variant of propionicin T1. Two of these strains showed low antimicrobial activity, while the third strain showed no antimicrobial activity at all. The protein encoded by orf2 showed strong homology to ABC transporters, and it has been proposed previously that this protein is involved in the producer immunity against propionicin T1. The limited antimicrobial activity exhibited by the strains lacking orf2 further suggests that this putative ABC transporter plays an important role in propionicin T1 activity.     

Evaluation of recA gene as a phylogenetic marker in the classification of dairy propionibacteria

The aim of this study was to investigate the validity of recA gene as a molecular marker for the reliable discrimination and classification of dairy propionibacteria and the closely related species. Regions of the recA gene, varying in size between 613 and 677 nucleotides, were sequenced for Propionibacterium acidipropionici, P. cyclohexanicum, P. freudenreichii, P. jensenii, P. microaerophilum and P. thoenii using degenerate consensus primers constructed by aligning recA sequences of some actinobacteria. The 16S rRNA encoding genes for the type and reference strains of the species P. acidipropionici, P. jensenii and P. thoenii were also sequenced to remove ambiguous positions present in the current database reports, such to improve the classification scheme of reference. As found for other bacterial species, recA sequences permitted a better distinction among the dairy propionibacteria considered than 16S rRNA gene. However, the topology of phylogenetic trees constructed on the recA gene regions sequenced and their putative translations appeared rather different and less statistically valid than the 16S rRNA gene tree. In addition, the possibility of designing PCR-based identification and detection tests on the new recA sequences was demonstrated by assessing specific amplification protocols for P. cyclohexanicum and P. microaerophilum.     

Acid-bile, antibiotic resistance and inhibitory properties of propionibacteria isolated from Turkish traditional home-made cheeses

In this study, a total of 29 Propionibacterium spp. were isolated from traditional home-made Turkish cheese samples. As a result of the identification, isolates were identified as Propionibacterium freudenreichii subsp. freudenreichii (15 strains), Propionibacterium jensenii (12), and Propionibacterium thoenii (2). All isolates and 5 reference strains were examined for their abilities to survive at pH 2.0, 3.0, 4.0, 5.0 and in the presence of 0.06, 0.15 and 0.30% bile salts, their influence on the growth of food-borne and spoilage bacteria, as well as their sensitivity against 11 selected antibiotics. Only seven propionibacteria strains survived in both the acidic and bile salt environments. Propionibacterium spp. strains strongly inhibited growth of the Escherichia coli ATCC 11229 and Shigella sonnei Mu:57 strains (91%). All propionibacteria strains were sensitive to a majority of the antibiotics used in the investigations. Overall, dairy propionibacteria showed high antibacterial activity, resistance to pH 4.0, 5.0, high resistance to bile salts and will provide an alternative source to Lactobacillus and Bifidobacterium as probiotic culture.     

Cultural characteristics and fatty acid composition of propionibacteria

The cultural characteristics and cellular fatty acid composition of 40 strains representing 7 species of Propionibacterium and of 9 cultures of anaerobic corynebacteria were studied. The cultures were characterized by means of 23 separate cultural and biochemical tests. Cultures of the two genera differed consistently in only two reactions; the propionibacteria did not produce indole or liquefy gelatin, whereas the anaerobic corynebacteria were consistently positive with these tests. The fatty acids were extracted from whole cells and examined as methyl esters by gas-liquid chromatography. The most abundant acid in the seven Propionibacterium species was a C(15)-saturated branched-chain acid which was present in both the iso-and anteiso-form. Based on a comparison of the relative abundance of these isomers (i-C(15) and a-C(15)), the species were separated into two groups. P. freudenreichii and P. shermanii (group one) were similar and contained the a-C(15) isomer as the predominant acid. The i-C(15) isomer was the most abundant acid in the second group (P. arabinosum, P. jensenii, P. pentosaceum, P. thoenii, and P. zeae). The fatty acid profiles of the anaerobic corynebacteria were somewhat similar to those of the second group of propionibacteria, but were distinct from the profiles of P. freudenreichii and P. shermanii. The addition of branched-chain amino acids (l-leucine and l-isoleucine) to the growth medium increased the synthesis of the specific fatty acid(s) structurally related to the added amino acid.     

Biochemical and genetic characterization of propionicin T1, a new bacteriocin from Propionibacterium thoenii

A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.     

Detection of the bacteriocin propionicin PLG-1 with polyvalent anti-PLG-1 antiserum

Polyclonal antibodies against the bacteriocin propionicin PLG-1 were produced in rabbits at high titer (256,000 to 512,000, as determined by indirect enzyme-linked immunosorbent assay [ELISA]). Anti-PLG-1 antiserum neutralized the antimicrobial activity of PLG-1 preparations in a well diffusion assay. Cross-reacting protein was detected using an indirect ELISA of the culture supernatant from a fed-batch fermentation of the producer strain Propionibacterium thoenii P127 within the first 24 h of incubation, but bacteriocin activity was not detected in the same culture until 217 h of incubation. Culture supernatants from 156 strains of classical dairy propionibacteria were tested by indirect ELISA at 5 and 12 days of incubation for production of cross-reacting protein and by well diffusion assay for bacteriocin activity. Cross-reacting protein was detected in 52 strains: all of the tested strains of P. thoenii, most of the strains of Propionibacterium jensenii, and a minority of the Propionibacterium acidipropionici and Propionibacterium freudenreichii strains. Of these 52 strains, only 4 strains of P. thoenii showed bacteriocin activity in a well diffusion assay. Eight bacteriocin-negative mutants of strain P127 were negative in both ELISA and well diffusion assays. Western blot analysis showed that three protein bands bound anti-PLG-1 antibodies in culture supernatants: a 9.1-kDa band that is assumed to be the PLG-1 monomer and 16.2- and 27.5-kDa bands that may be precursors, multimers, or complexes of PLG-1.     

New medium for isolating propionibacteria and its application to assay of normal flora of human facial skin.

The conditions for isolation and cultivation of Propionibacterium acnes and related propionibacteria were studied in detail. Triton X-100 added to the diluent inhibited the growth of propionibacteria in concentrations of 0.05 to 0.1%. However, such was not the case with Tween 80; rather, growth of the bacteria was further enhanced by this agent. Consequently, Tween 80 was considered to be a suitable surfactant for addition to the diluent for isolation of propionibacteria. A new medium for isolating propionibacteria from human skin was developed. Comparative studies with colonies of P. acnes, Propionibacterium granulosum, and Staphylococcus epidermidis showed morphological differences among the colonies; thus, the medium was very useful for differentiating and identifying species of the microbes. The new medium was used for studies on the distribution of propionibacteria on the foreheads of 30 Japanese volunteers. Among 447 strains of P. acnes and 86 strains of P. granulosum isolated from the volunteers, all strains of the former were positive for indole, nitrate, milk, and gelatin hydrolysis, whereas all strains of the latter were negative for all of the tests.     

Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria, and strains of Arachnia propionica

Eighty strains of anaerobic coryneforms were compared with 29 strains of classical propionibacteria and 8 strains of Arachnia propionica by cell wall analysis, deoxyribonucleic acid (DNA) base compositions, and nucleotide sequence similarities. The anaerobic coryneforms have DNA base compositions in the range of 58 to 64% guanine + cytosine (GC) and show at least three homology groups. The largest group corresponds to organisms identified as Propionibacterium acnes and shows about 50% homology to strains in the P. avidum homology group. The third group, P. granulosum, shows low levels of similarities to the other two. All strains of anaerobic coryneforms have some combination of galactose, glucose, or mannose as cell wall sugars, and most have alanine (ala), glutamic acid (glu), glycine (gly), and l-alpha-epsilon-diaminopimelic acid (l-DAP) as amino acids of peptidoglycan. However, a few strains in the P. acnes and P. avidum homology groups have meso-DAP and minimal amounts of glycine. Two serological types, based on cell wall antigens, were found in the P. acnes homology group. One type had galactose, glucose, and mannose as cell wall sugars, the other glucose and mannose only. The classical propionibacteria have DNA base compositions in the range of 65 to 68% GC and show four homology groups which correspond closely to van Niel's classification as given in the 7th edition of Bergey's Manual. The P. jensenii group showed about 50% homology to the P. thoenii group and about 30 to 35% to the P. acidi-propionici group. The P. freudenreichii strains showed a rather lower level of similarity (8 to 25%) to the other homology groups. Most of the strains of classical propionibacteria also have some combination of galactose, glucose, or mannose as cell wall sugars and ala, glu, gly, and l-DAP as peptidoglycan amino acids, but P. shermanii and P. freudenreichii strains, which form a single homology group, have galactose, mannose, and rhamnose as cell wall sugars and ala, glu, and meso-DAP in their peptidoglycan. There is a rather low level of DNA homology (10 to 20%) between the anaerobic coryneforms and classical propionibacteria. However, the strains of A. propionica which have a GC content of 64 to 65% and form a single homology group, show no homology to either of the other two major groups.     

A mixed culture of Propionibacterium jensenii and Lactobacillus paracasei subsp. paracasei inhibits food spoilage yeasts

Screening for antimicrobial features of 197 propionibacteria and tests with several antifungal lactobacilli led to the development of three protective cultures containing Propionibacterium jensenii SM11 and Lactobacillus paracasei subsp. paracasei strain SM20, SM29 or SM63. These cultures showed inhibitory activities (up to 5 orders of magnitude) against yeasts in dairy products such as yoghurt or cheese surface at refrigerator temperatures (6 degrees C) without an influence on the quality properties of the food. Initial cell numbers of 5 x 10(7) cells/g of propionibacteria and 1 x 10(8) cells/g of lactobacilli were the optimal concentrations to yield a total inhibition of the spoilage yeasts (Candida pulcherrima, Candida magnoliae, Candida parapsilosis and Zygosaccharomyces bailii).     

Antifungal compounds from cultures of dairy propionibacteria type strains

Antifungal compounds from cultures of five type strains of dairy propionibacteria, as well as from the cultivation medium, were studied. Cell-free supernatants and medium were fractionated by C(18) solid phase extraction. The aqueous 95% acetonitrile fractions were analyzed by GC-MS or subjected to reversed-phase HPLC, to identify, quantify or isolate antifungal substances. The resulting HPLC fractions were screened for antifungal activity against the mold Aspergillus fumigatus and the yeast Rhodotorula mucilaginosa. Active fractions were further separated by HPLC and the structures of the compounds were determined by spectroscopic and chromatographic methods. All five strains produced 3-phenyllactic acid, at concentrations ranging from 1.0 microg mL(-1) (Propionibacterium freudenreichii ssp. shermanii) to 15.1 microg mL(-1) (Propionibacterium thoenii), and at L/D -ratios ranging from 2 : 3 (Propionibacterium acidipropionici) to 9 : 1 (Propionibacterium freudenreichii). A number of active compounds found in cultures of propionibacteria were also present in noninoculated growth medium: two antifungal diketopiperazines, cyclo(L-Phe-L-Pro) and cyclo(L-Ile-L-Pro), and seven antifungal linear peptides. Three of the linear peptides corresponded to sequences found in the medium component casein, suggesting their origin from this component, whereas the diketopiperazines were suggested to be formed from medium peptides by heat treatment.     

Use of DNA quantification to measure growth and autolysis of Lactococcus and Propionibacterium spp. in mixed populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.     

Superoxide radicals and antiradical defence of propionic acid bacteria

Superoxide dismutase activity was demonstrated for 6 strains of 3 propionibacteria species. Rather high level of superoxide dismutase activity found in propionibacteria was in accordance with high level of catalase activity reported for propionibacteria previously. Both these activities were shown to have cytozolic localization. For the first time peroxidase activity was detected in gel-fractionated crude cell extracts of propionibacteria. The ability to produce superoxide radicals in NADH-dependent oxidation system was revealed for three strains of the bacteria. The level of superoxide production by the membrane particles of the propionic acid bacteria correlated with the levels of superoxide dismutase and catalase activities and was the lowest for Propionibacterium shermanii. The ability to perform monovalent oxygen reduction during succinate oxidation was not revealed. The intact cells of P. globosum, P. vannielii, P. shermanii apparently did not excrete superoxide radicals into culture fluid during respiration.     

Systematics of theLactobacillus population on rat intestinal mucosa with special reference toLactobacillus reuteri

The systematics of theLactobacillus population of the intestines of 88 different rats was studied; 80 rats had been fed on fermented oat-meal soup (Molin et al. 1992). One-hundred-twenty-twoLactobacillus strains from the intestinal mucosa were phenotypically classified together with twenty-eight reference strains ofLactobacillus andLeuconostoc, using 49 unit characters. Data were examined using Jaccard coefficient, and unweighted pair group algorithm with arithmetic averages. Two major and eleven minor clusters were defined at the 76% S_J-similarity level: Cluster 1 included thirty isolates which could not be identified further, but had resemblance to the type strains ofL. jensenii, L. gasseri, L. crispatus, and to some extent toL. acidophilus. Cluster 12 including fifty-four intestinal isolates was identified asL. reuteri; and so was cluster 13 (five isolates). Isolates of the major clusters were found in all parts of the intestines. The genomic homogeneity of theL. reuteri isolates was scrutinized by endonuclease restriction analysis of the chromosomal DNA, and the isolates could be divided into six genomic strains.     

Production of conjugated linoleic acid by dairy starter cultures

Nineteen different strains of lactobacilli, lactococci, streptococci and propionibacteria commonly used as dairy starter cultures were tested for their ability to produce conjugated linoleic acid (CLA) from free linoleic acid in vitro. Two strains of Propionibacterium freudenreichii ssp. freudenreichii and one strain of P. freudenreichii ssp. sheramnii were found to be capable of converting free linoleic acid to extracellular CLA. The highest level of CLA formed in the media was 265 μg ml⁻¹. Of the different isomers, cis- and trans-9,11-octadecadienoic acid represented more than 70% of the total CLA formed. The inhibitory effect of linoleic acid on the growth of the bacteria and its conversion to CLA in different media by propionibacteria are discussed.     

Phylogenetic analysis of some ll-diaminopimelic acid-containing coryneform bacteria from human skin: description of Propionibacterium innocuum sp. nov.

A new species, Propionibacterium innocuum, is proposed to accommodate strains of coryneform bacteria from human skin with phenotypic characters similar to those of the classical propionibacteria but differing in exhibiting primarily aerobic respiration and possessing a unique cell wall composition in which LL-diaminopimelic acid and arabinose occur together. The partial 16S rRNA sequence confirms an affinity with the genus Pro-pionibacterium and indicates that the species represents a distinct line within the genus. The type strain of Propionibacterium innocuum is NCTC 11082.     

An in vitro model for investigating intestinal adhesion of potential dairy propionibacteria probiotic strains using cell line C2BBe1

AIMS: The purposes of this study were to screen the adhesion properties of dairy propionibacteria strains and evaluate whether C2BBe1 could be used in the screening of potential probiotic strains. METHODS AND RESULTS: Thirteen dairy propionibacteria strains and two control strains, Lactobacillus acidophilus MJLA1 and Bifidobacterium lactis BDBB2, were tested for adhesion to C2BBe1. Electron microscopic observations demonstrated that the control strains, L. acidophilus MJLA1 and B. lactis BDBB2, had similar adhesive ability to C2BBe1 as had been previously shown to Caco-2. Only one of the 13 strains of dairy propionibacteria, strain P. jensenii 702, demonstrated adhesion to C2BBe1. CONCLUSIONS: C2BBe1 can provide an alternative to Caco-2 for assessing in vitro adhesion properties of probiotic strains. Adhesion properties of dairy propionibacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: C2BBe1 is highly suitable for application in bacterial adhesion studies, and was used successfully to select a new potential probiotic.