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.     

费氏丙酸杆菌两个亚种的分离与鉴定

用亨格特（Hungate）厌氧技术从不同奶制品分离出3株丙酸杆菌Propionibacterium,编号为PTC－1,PTC－2和PTC－3。细胞呈多形态,杆状,革兰氏阳性,不形成芽孢,不运动,菌落在PYG深层洋菜中呈双凸透镜状,白至土黄色,从葡萄糖、乳糖等8种碳水化合物发酵产酸,葡萄糖发酵产物包括大量丙酸和乙酸,少量异丁酸,琥珀酸和CO₂。厌氧至耐氧。PTC－1的DNA的GC百分含量测定值为67.8mol％（Tm）。三株菌的特性很接近,只在硝酸盐还原和牛奶凝固特性有差     

Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii

Several promoters from Propionibacterium freudenreichii subsp. shermanii were isolated using a promoter probe vector, pCVE1, containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene. Three of four promoters isolated exhibiting a strong activity in Escherichia coli also expressed a strong activity in P. freudenreichii subsp. shermanii IFO12426. Using two promoters with a strong activity and a previously constructed shuttle vector, pPK705, shuttling between E. coli and Propionibacterium. we constructed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first intermediate in the synthesis of porphyrins, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombinant P freudenreichii subsp. shermanii increased about 70-fold that in the strain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibitor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant P. freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of 1 mM levulinic acid and 30 mM glycine. The construction of an efficient expression vector will facilitate genetic studies of a vitamin B12 producer, Propionibacterium.     

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.     

Microfluidic electroporation for delivery of small molecules and genes into cells using a common DC power supply

The end-product profile of the glucose fermentation by Propionibacterium freudenreichii ET-3 changed on an electrochemical treatment, in which the culture vessel was filled with a carbon felt anode. Acetate and propionate were produced as final end products in a molar ratio of 2:3 without any electrochemical treatments at the point of the consumption of lactate as an intermediate of the glucose fermentation. The ratio was changed to 1:1 at the point of the lactate consumption by the electrochemical incubation at an electrode potential of 0.4 V versus Ag|AgCl for 100 h. During further electrochemical incubation, propionate was oxidized to acetate as a final end-product in the microbe-containing anode chamber. 1,4-Dihydroxy-2-naphthoic acid produced by P. freudenreichii ET-3 itself would receive electrons from the metabolic pathway and serve as an electron transfer mediator from the microbial cells to the electrode.     

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.     

First evidence of lysogeny in Propionibacterium freudenreichii subsp. shermanii

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.     

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.     

Anti-stress activity of Propionibacterium freudenreichii: identification of a reactivative protein

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.     

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.     

Biochemical properties of Streptococcus macedonicus strains isolated from Greek Kasseri cheese

A total of 32 Streptococcus macedonicus strains, isolated from Greek Kasseri cheese, were screened for biochemical properties of technological importance in milk fermentation processing, such as acid production, proteolytic and lipolytic activity, citrate metabolism, exopolysaccharide production, antimicrobial activity and biogenic amines production. All strains were found to be moderate acidifiers in milk. Only four strains could hydrolyse milk casein, while 11 strains showed lipolytic activity against tributyrin. Using amino acid derivatives of 4-nitroaniline as substrates, the highest peptidase activities were determined against phenylalanine- and glycine-proline-4-nitroanilide. Using fatty acid derivatives of 4-nitrophenol, it was shown that all strains exhibited esterase activities up to caprylate, with highest values against butyrate and caproate. Only one showed activity up to palmitate; this was also the most active strain against tributyrin. Five of the 32 strains could metabolize citrate but none of them produced exopolysaccharides. Nine strains displayed antimicrobial activity towards Clostridium tyrobutyricum, while no antimicrobial activity was detected against Listeria innocua and Propionibacterium freudenreichii subsp. shermanii. Finally, none was able to decarboxylize ornithine, histidine or lysine, and only four strains produced tyramine from tyrosine.     

Production of Propionibacterium shermanii biomass and vitamin B12 on spent media

AIMS: The propionibacteria are commercially important due to their use in the cheese industry, and there is a growing interest for their probiotic effects. Stimulatory effects of lactic acid bacteria (LAB) on propionic acid bacteria have been observed. This study was designed to examine the possibility of using spent media previously used to grow LAB for the production of biomass and metabolites of Propionibacterium freudenreichii subsp. shermanii. METHODS AND RESULTS: Seventeen MRS and vegetable juice media were prefermented by various LAB and evaluated for their ability to subsequently support the growth of Propionibacterium, using automated spectrophotometry (AS). Growth of Propionibacterium in spent media was strongly affected by the LAB strain used to produce the spent medium. The native MRS medium (not prefermented) yielded the highest optical density values followed by prefermented media by Lactobacillus acidophilus, Bifidobacterium longum and Lactococcus lactis. Prefermented cabbage juice enabled good growth of Propionibacterium. For the production of organic acids and vitamin B12, cells of Propionibacterium were concentrated and immobilized in alginate beads in the aim of accelerating the bioconversions. More propionic acid was obtained in spent media than in native MRS. The concentration of vitamin B12 was higher in media fermented with free cells than those with immobilized cultures; with the free cells, its concentration varied from 900 to 1800 ng ml(-1) of media. CONCLUSIONS: It was demonstrated that spent media could be recycled for the production of Propionibacterium and metabolites, depending on the LAB strain that was previously grown. Media remediation is needed to improve the production of vitamin B12, especially with immobilized cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an option for recycling of spent media generated by producers of LAB or producers of fermented vegetables. The propionic fermentation may result in three commercial products: biomass, vitamin B12 or organic acids, which may be used as starters, supplements or food preservatives. It is an attractive process from economical and environmental standpoints.     

Isolation and identification of a new bifidogenic growth stimulator produced by Propionibacterium freudenreichii ET-3

We have found a new growth stimulator for bifidobacteria in the culture broth of Propionibacterium freudenreichii ET-3. The bifidogenic growth stimulator (BGS) was purified by Diaion HP-20 column chromatography and preparative HPLC. Spectroscopic methods including 1H-NMR, UV, and LC-ESI-MS experiments indicated that the chemical structure of the bifidogenic growth stimulator was 1,4-dihydroxy-2-naphthoic acid (DHNA). Approximately 10 mg/L of DHNA was found to be produced in the culture broth of P. freudenreichii ET-3.     

Efficient transformation system for Propionibacterium freudenreichii based on a novel vector

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per microg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (> or =10(8) colonies per microg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.     

Determination of aspartase activity in dairy Propionibacterium strains

Propionic acid bacteria (PAB) are important as starter cultures for the dairy industry in the manufacture of Swiss-type cheeses, in which they are involved in the formation of eyes and are responsible for the typical flavour and aroma. These characteristics are mainly due to the classical propionic acid fermentation, but also the conversion of aspartate to fumarate and ammonia by the enzyme aspartase and the subsequent reduction of fumarate to succinate, which occur in dairy Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii starter strains. Additionally, the metabolism of free amino acids may be partly responsible for secondary fermentation and the subsequent split defects in cheese matrix. Here a method for aspartase activity was established and a number of dairy propionibacteria belonging to P. freudenreichii ssp. shermanii and freudenreichii were screened for this enzyme activity. A wide range of aspartase activity could be found in PAB isolates originating from cheese. The majority, i.e. 70% of the 100 isolates tested, showed very low levels of aspartate activity.     

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.     

Cloning and characterization of the gene encoding glutamate 1-semialdehyde 2,1-aminomutase, which is involved in delta-aminolevulinic acid synthesis in Propionibacterium freudenreichii.

The gene from Propionibacterium freudenreichii that encodes glutamate 1-semialdehyde 2,1-aminomutase (EC 5.4.3.8), which is involved in the C5 pathway for synthesis of delta-aminolevulinic acid (ALA), a precursor in heme and cobalamin biosynthesis, was cloned onto a multicopy plasmid, pUC18, via complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of fragments from the initial 3.3-kb chromosomal fragment allowed the isolation of a 1.9-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.9-kb DNA fragment revealed an open reading frame (ORF) that was located downstream from a potential ribosome-binding site. The ORF encoded a polypeptide of 441 amino acid residues, and the deduced molecular mass of this polypeptide is 45,932 Da. A high G+C content (70 mol%) of the codons of the ORF was found and was consistent with the taxonomic features of Propionibacterium species. The amino acid sequence showed a high degree of homology with those of the HemL proteins from other organisms, and a putative binding site for pyridoxal 5'-phosphate was conserved, with the exception of a single substitution of phenylalanine for leucine. These results suggest that ALA is synthesized via the C5 pathway in a producer of vitamin B12, P. freudenreichii.     

Accumulation of Intracellular Glycogen and Trehalose by Propionibacterium freudenreichii under Conditions Mimicking Cheese Ripening in the Cold

Seven Propionibacterium freudenreichii strains exhibited similar responses when placed at 4°C. They slowed down cell machinery, displayed cold stress responses, and rerouted their carbon metabolism toward trehalose and glycogen synthesis, both accumulated in cells. These results highlight the molecular basis of long-term survival of P. freudenreichii in the cold.