Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits

AIMS: To determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. METHODS AND RESULTS: Twenty-eight of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin with respect to type strain Lactobacillus reuteri CECT 925T were identified as Lact. reuteri. They were able to survive at pH 3 and subsequent exposure to cholic acid or oxgall, and presented bile salt hydrolase and bacteriocin-like activities. CONCLUSIONS: Reuterin production is a frequently found trait among lactobacilli isolated from pig faeces. Selected Lact. reuteri isolates were able to survive at conditions likely to be encountered throughout the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: High yields of reuterin may be obtained from selected isolates of Lact. reuteri. Probiotic characteristics of isolates studied in the present work suggest their application in food and feed.     

Metabolism by bifidobacteria and lactic acid bacteria of polysaccharides from wheat and rye, and exopolysaccharides produced by Lactobacillus sanfranciscensis

AIMS: The metabolism by bifidobacteria of exopolysaccharide (EPS) produced by Lactobacillus sanfranciscensis was investigated. To evaluate the significance of the EPS produced by Lact. sanfranciscensis during dough fermentation on the overall prebiotic properties of bread, metabolism by bifidobacteria of water-soluble polysaccharides (WSP) from wheat and rye was investigated. METHODS AND RESULTS: Polyglucose and polyfructan contained in WSP from wheat and rye were metabolized by bifidobacteria. In contrast, WSP isolated from fermented doughs were not metabolized by bifidobacteria. The arabioxylan fraction of WSP was metabolized neither by bifidobacteria nor by lactobacilli. All the bifidobacteria tested were able to metabolize fructan from Lact. sanfranciscensis. The kinetics of EPS metabolism by various bifidobacteria were characterized by diauxic utilization of fructose and EPS. CONCLUSIONS: Bifidobacteria metabolize fructan from Lact. sanfranciscensis. Polyfructan and the starch fractions from wheat and rye, which possess a bifidogenic effect, were degraded by cereal enzymes during dough fermentation, while the EPS were retained. SIGNIFICANCE AND IMPACT OF THE STUDY: EPS produced by sourdough lactic acid bacteria will improve the nutritional properties of sourdough fermented products.     

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.     

Aggregation-promoting factor in pig intestinal Lactobacillus strains

V. KMET, M.L. CALLEGARI, V. BOTTAZZI AND L. MORELLI. 1995. Autoaggregation was frequently encountered among intestinal lactobacilli isolated from weaned pigs. The aggregation mechanism was shown to be mediated by the production of a proteinaceous aggregation-promoting factor in two strains of Lactobacillus reuteri . A 32 kDa aggregation-promoting protein was detected in these strains by cross-reaction with rabbit polyclonal antibodies for Aggregation-Promoting Factor produced by the human isolate Lact. plantarum 4B2. Coaggregation reactions of Lact. reuteri strains with pathogenic and non-pathogenic Escherichia, coli were detected.     

Succinate production and citrate catabolism by Cheddar cheese nonstarter lactobacilli

AIMS: To identify strains of Cheddar cheese nonstarter lactobacilli that synthesize succinate from common precursors and characterize the biochemical pathways utilized. METHODS AND RESULTS: Whole cell incubations of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus zeae and Lactobacillus rhamnosus, were used to identify strains that accumulated succinate from citrate, l-lactate, aspartic acid or isocitrate. In vivo 13C-nuclear magnetic resonance spectroscopy (13C-NMR) identified the biochemical pathway involved at pH 7.0, and under conditions more representative of the cheese ripening environment (pH 5.1/4% NaCl/13 degrees C). Enzyme assays on cell-free extracts were used to support the pathway suggested by 13C-NMR. CONCLUSIONS: The Lact. plantarum strains studied synthesize succinate from citrate by the reductive tricarboxylic acid (TCA) cycle at either pH 7.0 or pH 5.1/4% NaCl/13 degrees C. Lactobacillus casei, Lact. zeae and Lact. rhamnosus strains lack one or more enzymatic activities present in this pathway, and do not accumulate succinate from any of the four precursors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of Lact. plantarum strains to milk during cheese manufacture may increase the accumulation of the flavour enhancer succinate.     

Combined use of Lactobacillus reuteri and soygerm powder as food supplement

AIMS: The survival of Lactobacillus reuteri when challenged with glycodeoxycholic acid (GDCA), deoxycholic acid (DCA) and soygerm powder was investigated. Moreover, the impact of Lact. reuteri on the bioavailability of isoflavones present in soygerm powder was examined. METHODS AND RESULTS: The strain experienced a die-off when adding 2 or 3 mmol l-1 bile salts, with more pronounced effects in the case of DCA. By means of a haemolysis test it was shown that toxicity could be due to membrane damage. When 4 g l-1 soygerm powder was added, the Lactobacillus strain survived the bile salt burden better (P < or = 0.05) and the membrane damage in the haemolysis test decreased (P < or = 0.05). The Lact. strain cleaved beta-glycosidic isoflavones during fermentation of milk supplemented with soygerm powder. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The interactions between the Lactobacillus strain and soygerm powder suggest that combining both in fermented milk can exhibit advantageous probiotic effects. The relevance of the combination of the strain and the soygerm powder should be studied under more relevant physiological conditions.     

Glutamate dehydrogenase activity in lactobacilli and the use of glutamate dehydrogenase-producing adjunct Lactobacillus spp. cultures in the manufacture of cheddar cheese

AIMS: The study was undertaken to investigate the occurrence of glutamate dehydrogenase activity in different species of lactobacilli, and to determine, in a series of cheese-making trials, the effects of glutamate dehydrogenase-producing adjunct cultures on sensory attribute development during the maturation of cheddar cheese. METHODS AND RESULTS: The presence of dehydrogenase activity with glutamate as substrate was monitored in cell lysates of >100 strains from 30 different species of lactobacilli using a qualitative colorimetric plate screening assay. Activity was detectable in 25 of the 29 representative species obtained from culture collections and in 12 of the 13 non-starter species isolated from cheese. There were pronounced interspecies and strain differences in the occurrence, level and pyridine nucleotide specificity of the glutamate dehydrogenase activity detected. Among the non-starter lactobacilli the highest frequency of enzyme occurrence and activity was detected in the Lactobacillus plantarum isolates. The establishment of glutamate dehydrogenase-producing adjunct strains in the predominant population of lactobacilli in the cheese curd affected the formation of a number of volatile compounds in ripening cheddar cheese, while the presence of Lact. plantarum strains, in particular, was associated with an intensification and acceleration of aroma and flavour development during the maturation period. CONCLUSIONS: Glutamate dehydrogenase formation by lactobacilli is a strain-dependent metabolic attribute, and adjunct cultures expressing the activity that are able to proliferate during cheese ripening have a positive impact on the rate of development and the intensity of cheddar cheese aroma and flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been demonstrated that some strains of glutamate dehydrogenase-producing lactobacilli have potential use as adjunct cultures to accelerate and intensify aroma and flavour formation during the manufacture of cheddar and, by analogy, other similar varieties of cheese. The importance of phenotypic discriminative monitoring of the dominant lactobacilli present during ripening to confirm adjunct establishment and population complexity was highlighted as was the requirement to establish the metabolic attributes of the non-starter population in uninoculated control cheeses in comparative trials.     

Amino acid catabolism in cheese-related bacteria: selection and study of the effects of pH, temperature and NaCl by quadratic response surface methodology

AIMS: To screen the cystathionine lyase and L-methionine aminotransferase activities of cheese-related bacteria (lactococci, non-starter lactobacilli and smear bacteria) and to determine the individual and interactive effects of temperature, pH and NaCl concentration on selected enzyme activities. METHODS AND RESULTS: A subcellular fractionation protocol and specific enzyme assays were used, and a quadratic response surface methodology was applied. The majority of the strains, 21 of 33, had detectable cystathionine lyase activity which differed in the specificity. Aminotransferase activity on L-methionine was observed in only three strains. The cystathionine lyase activities of Lactobacillus reuteri DSM20016, Lactococcus lactis subsp. cremoris MG1363, Brevibacterium linens 10 and Corynebacterium ammoniagenes 8 and the L-methionine aminotransferase activity of Lact. reuteri DSM20016 had temperature and pH optima of 30-45 degrees C, and 7.5-8.0, respectively. As shown by the quadratic response surface methodology these enzymes retained activities in the range of temperature, pH and NaCl concentration which characterized the cheeses from which the bacteria originated. CONCLUSION: The enzyme activities may have a role in flavour development during cheese ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge about the amino acid catabolic enzymes in order to improve cheese ripening.     

Glutathione reductase from Lactobacillus sanfranciscensis DSM20451T: contribution to oxygen tolerance and thiol exchange reactions in wheat sourdoughs

The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451(T) on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451(T)DeltagshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451(T)DeltagshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 microM to 10.5 microM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451(T)DeltagshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 microM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451(T)DeltagshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.     

New convenient defined media for [(35)S]methionine labelling and proteomic analyses of probiotic lactobacilli

AIMS: To develop experimental conditions for efficient protein radiolabelling and two-dimensional (2D) polyacrylamide gel electrophoresis for investigation of stress proteomes of probiotic Lactobacillus spp. METHODS AND RESULTS: Three chemically defined media (CDM) optimized from a commercial medium supported rapid growth of the probiotic Lactobacillus rhamnosus E97800, Lactobacillus brevis ATCC 8287 and Lactobacillus reuteri E97849, and a broad range of other lactic acid bacteria. These CDM allowed efficient protein radiolabelling, requiring as little as 200 mul of logarithmic culture and pulse-chase labelling of 20 min to detect c. 300 distinct protein spots in a mini-scale 2D-gel. Proteins including DnaK, GroEL and ClpATPases were identified from the 2D-gels by immunoblotting. CONCLUSIONS: Radiolabelling coupled with 2D gel electrophoresis provides a sensitive means to monitor changes in protein synthesis rates in probistic lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient tools for proteomic analyses of probiotic Lactobacillus were developed and applied for stress-response studies.     

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

AIMS: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. METHODS AND RESULTS: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8.10-10.11 log10 cfu ml(-1), depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l(-1) total fermentable sugars) and free amino nitrogen (approx. 80 mg l(-1)). The consumption of sugars during the exponential phase (10-12 h) resulted in the accumulation of lactic acid (1.06-1.99 g l(-1)) and acetic acid (0.29-0.59 g l(-1)), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17-43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3.40 and 3.77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7.20-9.43 log10 cfu ml(-1)) than in the malt medium. This could be attributed to the low sugar content (3-4 g l(-1) total fermentable sugar for each medium) and the low free amino nitrogen concentration (15.3-26.6 mg l(-1)). In all fermentations, the microbial growth ceased at pH values (3.73-4.88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. CONCLUSIONS: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. SIGNIFICANCE AND IMPACT OF THE STUDY: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria.     

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.     

Proteolytic activity and reduction of gliadin-like fractions by sourdough lactobacilli

AIMS: To characterize the peptide hydrolase system of Lactobacillus plantarum CRL 759 and CRL 778 and evaluate their proteolytic activity in reducing gliadin-like fractions. METHODS AND RESULTS: The intracellular peptide hydrolase system of Lact. plantarum CRL 759 and CRL 778 involves amino-, di- (DP), tri- (TP) and endopeptidase activities. These peptidases are metalloenzymes inhibited by EDTA and 1,10-phenanthroline and stimulated by Co2+. DP and TP activities of Lact. plantarum CRL 759 and CRL 778, respectively, were completely inhibited by Cu2+. Lactobacillus plantarum CRL 778 showed the highest proteolytic activity and amino acids release in fermented dough. The synthetic 31-43 alpha-gliadin fragment was hydrolysed to 36% and 73% by Lact. plantarum CRL 778 and CRL 759 respectively. CONCLUSIONS: Lactobacillus plantarum CRL 759 and CRL 778 have an active proteolytic system, which is responsible for the high amino acid release during sourdough fermentation and the hydrolysis of the 31-43 alpha-gliadin-like fragment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new information of use when obtaining sourdough starters for bread making. Moreover, knowledge regarding lactobacilli capable of reducing the level of gliadin-like fractions, a toxic peptide for coeliac patients, has a beneficial health impact.     

Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance

Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.     

Development of intestinal lactobacilli in normal piglets

The development of intestinal Lactobacillus spp. in 18 normal piglets from six litters was investigated from 1 to 45 d after birth. Faecal Lactobacillus strains isolated from piglets and their dams were identified at species and biovar levels on the basis of carbohydrate fermentation. A definite pattern was observed in the changes of four species and 20 biovars of lactobacilli appearing in faeces, with growth of the piglets. Lactobacillus reuteri colonized the animals on the first day of birth. The Lact. acidophilus group appeared 1 week after birth and maintained high levels in the intestine. Biovars B, C and E of Lact. reuteri were recovered as major biovars on the first to fourteenth days after birth. In the third and fourth weeks after birth, however, biovars F and G of Lact. reuteri were mainly recovered from piglet faeces. By the seventh week, biovars B, C and E predominated again in the intestine. No change in the composition of species and biovars of the genus Lactobacillus in the dams was detected.     

Non-dairy lactic fermentations: the cereal world*

Sourdough is the foremost cereal fermentation performed in a variety of technologies with almost any cereal. The lactobacilli studied most intensely include Lactobacillus sanfranciscensis, L. reuteri and L. pontis isolated from traditional and modern rye and wheat fermentations. Molecular biology tools are available for their rapid identification and monitoring throughout a process. The currently available insight on their metabolism can be used to explain their prevalence in this environment and their interactions. Key genes of the sugar degradation pathway were cloned and characterised from L. sanfranciscensis. In addition some strains were found to have special properties including the production of antagonistic compounds or the adhesion to human intestinal cells.     

Extracellular homopolysaccharides and oligosaccharides from intestinal lactobacilli

AIMS: To characterize lactobacilli isolated from the intestines of ducks or pigs with respect to the production of extracellular homopolysaccharides (HoPS) and oligosaccharides. METHODS AND RESULTS: Lactobacillus strains of duck or pig origin were screened for HoPS synthesis and >25% of the isolates produced fructans or glucans from sucrose. Glucan-forming strains were found within the species Lactobacillus reuteri and Lactobacillus animalis and fructan-forming strains were found within Lactobacillus mucosae, Lactobacillus crispatus and Lactobacillus acidophilus. The glucan-forming strains of L. reuteri but not L. animalis produced glucose-oligosaccharides in additon to the respective polymers, and two fructan-forming strains of L. acidophilus produced kestose. Genes coding for glycosyltransferases were detected by PCR and partially characterized by sequence analysis. CONCLUSIONS: A large proportion of lactobacilli from intestinal habitats produce HoPS from sucrose and polysaccharide formation is generally associated with the formation of glucose- and fructose oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of the metabolic potential of intestinal lactobacilli contributes to the understanding of the molecular basis of autochthony in intestinal habitats. Moreover, this is the first report of glucose-oligosaccharide production during growth of lactobacilli, and one novel fructosyltransferase and one novel glucansucrase were partially characterized on the genetic level.     

Use of folic acid casei medium reveals trimethoprim susceptibility of Lactobacillus species

AIM: Lactobacilli have been reported to have intrinsic resistance to trimethoprim. The susceptibility of lactobacilli to trimethoprim on different media was investigated in order to search for a phenotypic test method that could indicate the presence of acquired resistance genes. METHODS AND RESULTS: Strains of Lactobacillus acidophilus, Lact. paracasei, Lact. rhamnosus and Lact. plantarum were susceptibility tested with E-tests on folic acid casei medium (FACM), MRS and defined medium 1. The effects of addition or removal of nucleosides and thymidine phosphorylase were investigated. E-tests on FACM yielded reproducible minimal inhibitory concentrations (MICs) for trimethoprim but addition of nucleosides was necessary for growth of Lact. acidophilus. MICs for the tested strains were 0.125-0.19, 0.25-3 and 0.064-0.19 microg ml(-1) for Lact. paracasei, Lact. rhamnosus and Lact. plantarum, respectively. With the addition of deoxyuridine and deoxyadenosine to FACM the MICs of Lact. acidophilus were 0.064-1 microg ml(-1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacilli do not have intrinsic resistance to trimethoprim. The results show that trimethoprim susceptibility testing of the tested Lactobacillus species is possible and indicate that transferable resistance genes are absent in all the tested strains.     

Protease,peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine

AIMS: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. METHODS AND RESULTS: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected strains could be used as adjunct starters to make high quality Feta cheese.