Strains Of Dairy Propionibacteria Research Articles

Dairy propionibacteria as direct-fed microbials: in vitro effect on acid metabolism of Streptococcus bovis and Megasphaera elsdenii

Ruminal acidosis caused by accumulation of lactic acid, a decrease of pH in the rumen and subsequent imbalance of the rumen fermentation process, affects the health and productivity of dairy cows and beef cattle. Direct-fed microbials have potential for use in the control and prevention of ruminal acidosis. This study investigated the interaction between five strains of dairy propionibacteria, Megasphaera elsdenii and Streptococcus bovis in various co-culture combinations in a simulated rumen environment comprising unmodified rumen digesta supplemented with excess glucose. While suppression of lactic acid accumulation by both the dairy propionibacteria and M. elsdenii in the presence of S. bovis in the simulated rumen conditions was evident, propionibacteria were found to be more effective than M. elsdenii in controlling lactic acid levels.

Potential influence of dairy propionibacteria on the growth and acid metabolism of Streptococcus bovis and Megasphaera elsdenii.

Ruminal acidosis is a prevalent disorder among dairy cows and feedlot cattle, which can significantly impair their health and productivity. This study, involving seven different strains of dairy propionibacteria, represents an in vitro investigation of the feasibility of using these organisms as direct-fed microbials to control lactic acid acumulation in the rumen. Interactions between the propionibacteria, Streptococcus bovis and Megasphaera elsdenii were evaluated in terms of effects on lactic, acetic and propionic acid metabolism, following co-incubation. Spot resistance tests showed slight but varying degrees of growth inhibition by S. bovis among the propionibacteria, while no inhibition was observed between M. elsdenii and the different strains of dairy propionibacteria. In the co-culture experiments comprising S. bovis in nutrient broth, significant differences in pH and the levels of production of lactic, acetic and propionic acid, were observed between treatments following inoculation with various propionibacteria and/or M. elsdenii. In general, lactic acid concentrations at the end of the incubation were significantly lower in the cultures containing propionibacteria compared with cultures comprising either S. bovis only or S. bovis + M. elsdenii, although efficacy of lactate metabolism varied between species and strains. Moreover,the accumulation of acetic and propionic acid in the combined cultures, but not in the solo S. bovis culture, indicated that these compounds were produced as a result of the metabolism of lactic acid by the propionibacteria and M. elsdenii.

Study on bioactivity of cell-free filtrates from dairy propionibacteria

In this paper, the bioactivity of cell-free extracts obtained by dairy propionibacteria strains was investigated. Probiotic bifidobacteria and lactobacilli were used as microbial targets. The extracellular filtrates were added as ingredient (1% v/v) into the growth medium; the effect of cell-free filtrates was evaluated through viable count of microbial targets on appropriate media, monitoring the response of target microorganisms both in growth and death phase. The Gompertz equation was used to model the experimental data. Kinetics and time parameters were estimated in order to quantify the effectiveness of cell-free filtrates effect.To emphasize the results about the bioactivity of cell-free extract, supporting a complete picture of response, a new approach was developed: Probiotic Stability Time was calculated.This temporal parameter, defined as the time over that the cell load preserve a living value upper than 107 cfu ml−1, was very useful to evaluate the probiotic capability and effectiveness.A stimulant effect was registered on growth and a positive one was recordered on survival of both bifidobacteria and lactobacilli strains, and the results obtained suggest that a prebiotic activity by dairy propionibacteria cell-free filtrates could be supposed.The cell-free filtrate obtained from Propionibacterium freudenreichii subsp. shermanii was the most effective, in our experimental conditions.Although bifidobacteria were the most sensitive to the effect of cell-free filtrates, lactobacilli have been showed a similar probiotic stability time, showing a high sensitivity to the filtrates.This paper is the first report of a positive bioactivity by propionibacteria cell-free filtrates on lactobacilli.

Relationship between gastrointestinal tolerance and exopolysaccharide production of propionibacteria strains under different pH and bile conditions

In this study, we report the effects of exposure to simulated gastric and intestinal juices on the survival of six strains of dairy propionibacteria. Gastrointestinal tolerance of propionibacteria strains was highly variable, depending on the strain and pH (P < 0.05). Exopolysaccharides (EPS) production of six propionibacteria strains under different pH conditions and bile concentrations was also studied. A positive correlation was obtained between the EPS production quantity of the strains and tolerance to simulated gastric juices at pH 2.0, 3.0 (P < 0.05). This investigation showed that high EPS production may be important in the selection of probiotic strains for resistance to upper gastrointestinal system conditions.

Research on Some Factors Influencing Acid and Exopolysaccharide Produced by Dairy Propionibacterium Strains Isolated from Traditional Homemade Turkish Cheeses

In this study, a total of 32 isolated strains and 5 reference strains of dairy propionibacteria were analyzed for acid and exopolysaccharide (EPS) production in skim milk and yeast extract-lactate broth (YEL) media in order to investigate the physiological background and preservative role of acid and EPS. The effects of final culture pH and optical density on acid and EPS production were also determined. On average, all strains produced more acid and reached lower final pH values in skim milk than in YEL medium. While the correlations obtained between the acid produced by propionibacterium strains and their final culture pH in skim milk medium were significant (P < 0.01), no correlations were found between optical density, final pH, and produced acid in YEL medium. Sixteen isolated and five reference strains of propionibacteria were tested further for the ability to produce propionic and acetic acids. On average, Propionibacterium freudenreichii subsp. shermanii and P. freudenreichii subsp. freudenreichii strains produced higher amounts of propionic and acetic acids than did Propionibacterium jensenii in YEL medium. The acid produced by these strains may be used as a preservative in the food industry for replacement or reduction of the increasing use of chemical additives. The EPS production by propionibacterium strains during growth in YEL medium was 72 to 168 mg/liter, while in skim milk it was 94 to 359 mg/liter. The monomer compositions of the EPSs formed by the six selected dairy propionibacteria strains were analyzed. The EPSs may have applications as food grade additives and viscosity-stabilizing agents.

Investigating hydrophobicity and the effect of exopolysaccharide on aggregation properties of dairy propionibacteria isolated from Turkish homemade cheeses.

Propionic acid bacteria have been used widely as starter cultures. However, their potential as probiotics has received little attention. The ability to auto- and coaggregate is a desirable property for probiotics in health-promoting foods. Therefore, in the current study, we assessed the effect of exopolysaccharides produced by dairy propionibacteria strains on the aggregative and hydrophobicity properties. All propionibacteria strains tested showed auto- and coaggregation ability with Escherichia coli ATCC 11229, but the results were strain specific and dependent on exopolysaccharides production and incubation conditions. In addition, propionibacteria strains tested were determined to be highly hydrophilic. Our results indicate that the ability to autoaggregate, together with cell-surface hydrophobicity and coaggregation abilities with an E. coli strain, can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use.

Aggregation and Hydrophobicity Properties of 6 Dairy Propionibacteria Strains Isolated from Homemade Turkish Cheeses

In the present study, 6 dairy Propionibacterium strains were assessed with regard to their hydrophobic characteristics and their autoaggregation and coaggregation abilities since these traits have been shown to be indicative of adherence in other microorganisms. Aggregation assays and bacterial adhesion to hydrocarbons demonstrated significant differences in cell surface properties among the tested propionibacteria strains. Almost all strains appeared relatively hydrophilic, which showed low affinity for p-xylene. Four of the tested strains showed the strong adhesion to ethyl acetate, a basic solvent, in comparison with microbial adhesion to chloroform, an acidic solvent, which demonstrated the particularity of propionibacteria to have an important electron donor and acidic character. Also, these strains simultaneously showed affinity to 3 hydrocarbons, suggesting a high complexity of the cell surface. All propionibacteria strains tested showed autoaggregation and coaggregation ability with the Escherichia coli ATTC 11229, but the results were strain-specific and dependent on incubation conditions. Anaerobic incubation conditions were determined as the best condition for aggregation abilities of propionibacteria strains. A relationship was obtained between aggregation abilities (auto- and coaggregation) and a correlation between adhesion to hydrocarbon (chloroform) and autoaggregation was possible. Our results indicate that the ability to autoaggregation together with cell surface hydrophobicity and coaggregation abilities with E. coli strain can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use. Autoaggregation, cell surface hydrophobicity, and coaggregation abilities with E. coli of the selected dairy propionibacteria strains could be used as probiotic in foods after in vivo studies.

Promising Immunomodulatory Effects of Selected Strains of Dairy Propionibacteria as Evidenced In Vitro and In Vivo

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.

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.

In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria

This study aimed to assess the transit tolerance of potential probiotic dairy propionibacteria strains in human upper gastrointestinal tract in vitro, and to evaluate the effect of food addition on viability of these strains in simulated pH 2.0 gastric juices. The transit tolerance of 13 dairy propionibacteria strains was determined at 37 °C by exposing washed cell suspensions to simulated gastric juices at pH values at 2.0, 3.0, and 4.0, and simulated small intestinal juices (pH 8.0) with or without 0.3% bile salts. The viability of dairy propionibacteria in pH 2.0 simulated gastric juice with So-Good™ original soymilk or Up & Go® liquid breakfast was also determined. The simulated gastric transit tolerance of dairy propionibacteria was strain-dependent and pH-dependent. All tested strains were tolerant to simulated small intestinal transit. The addition of So-Good™ original soymilk or Up & Go® liquid breakfast greatly enhanced the survival of dairy propionibacteria strains in pH 2.0 simulated gastric juices. Dairy propionibacteria strains demonstrate high tolerance to simulated human upper gastrointestinal tract conditions and offer a relatively overlooked, yet alternative source for novel probiotics besides Lactobacillus and Bifidobacterium.

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

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. 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. 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. C2BBe1 is highly suitable for application in bacterial adhesion studies, and was used successfully to select a new potential probiotic.

Viability and β-Galactosidase Activity of Dairy Propionibacteria Subjected to Digestion by Artificial Gastric and Intestinal Fluids

An important criterion to consider in the selection of strains for dietary adjuncts is the ability of the microorganisms to survive the severe conditions of acidity and bile concentrations usually found in the gastrointestinal tract. In the present work, we report the effects of digestions by artificial gastric and intestinal fluids on β-galactosidase activity and survival of four strains of dairy propionibacteria previously selected by their bile tolerance and β-galactosidase activity. The strains were exposed to artificial gastric juice at pH values between 2 and 7 and then subjected to artificial intestinal digestion. Both viability and β-galactosidase activity were seriously affected at pH 2. Skim milk and Emmental cheese juice exerted a protective effect on the parameters tested. The trypsin present in the intestinal fluid inactivated the enzyme β-galactosidase in strains of Propionibacterium freudenreichii but not in Propionibacterium acidipropionici. Moreover, the presence of bile salts enhanced the β-galactosidase activity of these strains by permeabilization of the cells during the first hour of exposure. The intestinal transit rate confirmed the permanence of the bacteria in the intestine for long enough to be permeabilized. These results suggest that P. acidipropionici would be a good source of β-galactosidase activity in the intestine. We also propose a practical and effective in vitro method as a tool of screening and selection of potential probiotic bacteria.

Rapid identification of dairy Propionibacterium species by restriction analysis of the insertion within the 23S rRNA gene

An amplified sequence of approximately 350 bases from the 23S rRNA gene of each bacteria was used to identify 37 strains of dairy propionibacteria. After generation of the fragment by PCR (polymerase chain reaction), it was eut with the restriction endonuclease MspI. The resulting electrophoretic pattern showed a distinct profile for each of the Propionibacterium species. Purification of DNA was not necessary and the analyses could also be performed with crude bacterial extract. A collection of anaerobie bacteria commonly found in milk and milk products was also submitted to the analysis. None yielded the same pattern as propionibacteria. The analysis of the propionibacteria was compared to that performed by electrophoresis of the soluble cell-free protein extracts and that of c1assical microbiological tests. Our method gave identical results and was less time consuming (results could be obtained within a single day and were also easier to read) than both the c1assical tests and protein electrophoresis. © Inra/Elsevier, Paris.

Strain variability of the cell-free proteolytic activity of dairy propionibacteria towards β-casein peptides

Strain selection of dairy propionibacteria is based on COz' acetate and propionate production. However, another criterion such as proteolytic activity could be of industrial inter- est for selection of flavor ripening agents. Given that the propionibacteria are variously able to auto- lyse and that proteolytic activity is mainly intracellularly located, the proteolytic potential was eva- luated on the cell-free extracts of 37 dairy propionibacteria strains. The substrate used to detect proteolytic activity was a tryptic/chymotryptic hydrolysate of ~-casein. The evaluation was performed at a temperature of 30 C and pH of 5.7. The increase in the free NHz groups over a 24 h period was monitored as weil as the evolution of the reversed-phase chromatographie profiles. In addition, for 14 strains, the free amino acids released from the ~-casein peptides were determined. Ali the strains were able to hydrolyze ~-casein peptides. Difference of proteolytic activity was observed among and within the four dairy propionibacteria species. This variability was expressed more in terms of nature of the products released (peptides and free amino acids) than in terms of degree of proteolytic activity. From the results, a strain selection system may be developed using as substrate the tryptic/chymotryptic ~-casein hydrolysate and as criteria the degree of hydrolysis at 24 h and the amount and the nature of free ami no acids released by cell- free ex tracts. Such a selection will give useful data for cheesemakers, especially if characteriza- tion of autolytic ability in the cheese environment is also determined. © InralElsevier, Paris. propionibacteria / proteolysis / peptide / Cree amino acid / selection

16S-23S Ribosomal spacer polymorphism in dairy propionibacteria

The 16S-23S spacer region was amplified from 67 strains of dairy propionibacteria using primers G1 and L1. All species are readily distinguishable from the different molecular weights of the amplified bands and sequence polymorphisms among the most similar groups can be enhanced by Hinf I restriction.

DNA Fingerprinting of Dairy Propionibacteria Strainsby Pulsed-Field Gel Electrophoresis

Restriction endonuclease patterns generated by Pulsed-Field Gel Electrophoresis (PFGE) were used to compare 96 strains of dairy propionibacteria originating from dairy products, international and industrial collections; endonucleases XbaI and SspI gave satisfactory restriction patterns. However, whereas XbaI can be used for Propionibacterium freudenreichii, SspI seems more suitable for the three other species: P. acidipropionici, P. thoenii, and P. jensenii. It is a convenient method to differentiate the dairy propionibacteria from closely related bacteria and from others usually present in dairy products. We observed a considerable restriction fragment length polymorphism among the Propionibacterium chromosomes and especially for P. freudenreichii: among 48 strains we detected 40 different patterns. This species is the most commonly encountered in the Swiss-type cheeses and is the only Propionibacterium species used as a cheese starter. Conversely, the species P. acidipropionici is not very diverse: among nine strains we observed only four different patterns, two of which were closely related. This is probably because this species is not used as a starter in cheese manufacture and consequently is poorly represented in collections. When strains come from geographical different isolates, their patterns are always different with very few common bands. The presence of numerous identical strains was due to the fact that they were present at the same time in the national collections, research laboratory collections, and in the industrial ones.

Autolysis of 57 strains of dairy propionibacteria

Dairy propionibacteria contribute to proteolysis and lipolysis of the curd in Swiss-type cheese technology by the release of intracellular enzymes. Autolysis of 57 strains of dairy propionibacteria has been compared in 0.1 molli potassium phosphate buffer (pH 6.2 ) and the rate and extent of autoIysis appeared to be strain-dependent. Two distinct clusters were observed: cluster A containing 8 strains highly prone to Iysis and cluster B containing ail the other strains. Genomic fingerprinting of the 8 strains constituting c1uster A revealed 7 distinct profiles. Growth of 4 of these 7 strains showed a spontaneous autolysis in the growth medium just after the maximal growth, without any stationary phase.

Identification of dairy Propionibacterium species by rRNA gene restriction patterns

A total of 78 strains of dairy propionibacteria, 4 reference strains of Propionibacterium and 8 related bacteria were characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns (ribotyping). The patterns were obtained after cleavage of total DNA with either BamHl or Clal restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16+23S rRNA from Escherichia coli.The four different species of dairy propionibacteria, P. freudenreichii, P. jensenii, P. thoenii and P. acidipropionici, gave different restriction patterns with species-specific fragments. Moreover, ribotyping allowed the differentiation of P. freudenreichii subsp. Freudenreichii from P. freudenreichii subsp. shermanii. The patterns of dairy propionibacteria were different from those of closely related bacteria and other bacteria used in the dairy industry.

Isolation and Characterization of Mutants of Propionibacterium Strains

Procedures were developed to isolate and characterize mutants of strains of dairy propionibacteria. These procedures included the construction of minimal defined media to support growth of the strains, optimization of conditions of exposure of the strains to nitrosoguanidine, and identification of the phenotypes of the mutants that were generated. The minimal defined medium contained inorganic salts, adenosine, three vitamins, and sodium lactate as the carbon source, with cysteine, methionine, or cysteine plus methionine added as required by some strains. For mutagenesis, cells were exposed to either 100, 200, or 1000μg/ml nitrosoguanidine, depending on the sensitivity of the strain, for 60min at 35°C. At least nine stable mutants were isolated and characterized for each of the five strains under study. The most frequent mutations generated were requirements for arginine, histidine, methionine, and uracil and alteration in pigment production.