Michel J. Desmazeaud

Isolation and characterization of exocellular polysaccharide produced byLactobacillus bulgaricus

SummaryLactobacillus bulgaricus strains grown on skim milk produce a viscosifying exocellular watersoluble heteropolysaccharide composed of galactose, glucose and rhamnose in an approximately molar ratio of 4:1:1. The molecular weight is approximately 500.000.

Factors Affecting Exocellular Polysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus Grown in a Chemically Defined Medium

ABSTRACT We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains ofLactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp.bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricusCNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricusstrains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.

Evidence for two bacteriocins produced by Carnobacterium piscicola and Carnobacterium divergens isolated from fish and active against Listeria monocytogenes

Lactic acid bacteria (LAB) isolated from fish products (fresh fish, smoked and marinated fish, fish intestinal tract) were screened for bacteriocin production and immunity in conditions eliminating the effects of organic acids and hydrogen peroxide. Twenty-two isolates which were found to produce bacteriocin-like compounds were identified as Carnobacteria, Lactococci and Enterococci on the basis of morphological examination, gas production from glucose, growth temperatures, configuration of lactic acid, carbohydrates fermentation and deamination of arginine. Two Carnobacteria named V1 and V41 were selected for further studies and identified by DNA-DNA hybridization as Carnobacterium piscicola and Carnobacterium divergens , respectively. Their respective bacteriocins named piscicocin V1 and divercin V41 were heat-resistant and sensitive to various proteolytic enzymes. These bacteriocins were active against Listeria monocytogenes and exhibited a different spectrum of activity against LAB. Both bacteriocins had a bactericidal and non-bacteriolytic mode of action. Maximum production of piscicocin V1 and divercin V41 in Man Rogosa Sharpe (MRS) medium broth occurred at the beginning of the stationary phase and was higher at 20°C than at 30°C. When the cultures were maintained at pH 6.5, bacteriocin production was significantly increased.

Exocellular polysaccharide production byStreptococcus thermophilus

SummaryStreptococcus thermophilus strains grown on skim milk produce exocellular polysaccharide, essentially composed of galactose and glucose. Small amounts of xylose, arabinose, rhamnose and mannose are identified also. A relationship exists between viscosity of the culture medium and the amount of polymer produced. However, the thickness producing trait in the strains studied is highly unstable.

Peptide hydrolases of Lactobacillus casei: isolation and general properties of various peptidase activities.

Discovery of an endopeptidase by gel chromatography and separation of 3 exopeptidases (a dipeptidase, an aminopeptidase and a specific carboxypeptidase) from Lactobacillus casei NCDO 151 by affinity chromatography is described. The 3 exopeptidases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline but were reactivated with Co2+ and Mn2+. The pH optima for aminopeptidase, dipeptidase and carboxypeptidase activities were 6.5, 7.6 and 7.2, respectively. Maximum activity was obtained at 45 degrees C for the aminopeptidase, at 30 degrees C for the dipeptidase and at 40 degrees C for the carboxypeptidase. The substrate specificities of the 3 enzymes were also studied. The properties of these 3 enzymes are compared with those of other bacteria.

Inventaire des différentes activités peptidasiques intracellulaires de Streptococcus thermophilus: Purification et propriétés d'une dipeptide-hydrolase et d'une aminopeptidase

Summary Besides a proteolytic activity, four peptidases were isolated from S. thermophilus. The purified dipeptidase has a molecular weight of about 50 000 daltons. It was maximally active around pH 7.50 and 50°C, its apparent energy of activation was 4 500 cal./mole. The dipeptidase was most stable between pH 7.0 and 9.0, and for temperatures less than or equal to 50°C. E.D.T.A. or O-phenanthroline induced a very significant reduction in enzyme activity, but metal ions such as Co++, Zn++, Mn++, Mg++ or Ca++ can reactivate it partially after the inactivation by a chelating agent such as E.D.T.A. Iodoacetate, p-C.M.B. or D.F.P. induced a slight reduction in enzyme activity. Unsubstituted dipeptides only were hydrolysed by the enzyme. The dipeptidase exhibited a specificity toward dipeptides with a large and hydrophobic amino acid residue at the NH2-terminal end; the second amino acid residue (at the COOH-terminal end) had also an effect on enzymic activity. A homogeneous aminopeptidase — with regard to disc electrophoresis — was obtained. The molecular weight of this enzyme was found to be 62 000 daltons. It was maximally active around pH 6.40 and 35°C; its apparent energy of activation was 7 300 cal./mole. Aminopeptidase was most stable between pH 5.80 and 9.80 and at a temperature less than 40°C. E.D.T.A. or O-phenanthroline induced a complete inactivation of the enzyme, but metal ions such as Co++ and Mn++ can reactivate it completely; metal ions such as Ca++ and Zn++ or Mg++ partially after the inactivation by a chelating agent such as E.D.T.A. Zn++ 5.10−3 M and alcohols induced a significant reduction in enzyme activity. This enzyme can be precisely defined as an α-amino-acyl-peptide hydrolase EC.3.4.1. since only NH2-terminal end amino-acid residues were released from oligopeptides (comprising less than eleven amino-acid residues). Leucine-p-nitroanilide and amino-acid amides (except phenylalanine amide) were hydrolysed. Lys-Tyr and Leu-Leu were the best dipeptide substrates. Dipeptides with a glycyl or a histidyl residue at the NH2-terminal end were not hydrolysed as also dipeptides with a prolyl or phenylalanyl residue.

Acceleration of cheese ripening with liposome-entrapped proteinase

SummaryRulactine, a proteinase used for the acceleration of cheese ripening, was entrapped in three types of liposomes and these were added to Saint-Paulin cheese type manufacturing milk. Enzyme entrapment rates ranged from 3 to 9% according to the type of liposomes and liposome retention rates in cheese curd from 35 to 65%. An electrophoretic study of protein breakdown in the cheeses gave correlative data.

Phenylalanine and tyrosine catabolism in some cheese coryneform bacteria

Abstract 23 cheese coryneform bacteria (14 orange, 3 white, and 6 yellow-pigmented) were examined for five enzymes of two branch-point steps in the catabolic pathways of L -phenylalanine and L -tyrosine. Orange cheese coryneforms (‘Brevibacterium linens’) catabolized th amino acids by transamination and the benzene ring was cleaved by 3, 4-dihydroxyphenylacetate-2, 3-dioxygenase. Both enzymes appear to be inducible. Yellow and white strains possessed non-inducible low activity of aminotransferase and lacked completely benzene ring cleavage enzymes.

Utilization of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase

The first step of the utilization of the aromatic amino acids as sole nitrogen sources by Brevibacterium linens strain 47 was found to be a transamination. The deaminated metabolites of the amino acids were detected in culture supernatants, and the enzyme activity was identified in cell free extracts. The cells contained increased aromatic amino acid aminotransferase activities on growth on the aromatic amino acids as sole nitrogen sources. Two aromatic aminotransferases (AT-I and AT-II) were separated upon diethylaminoethyl-Trisacryl M column chromatography of cell free extracts. Only AT-I was responsible for the increased level of aromatic amino acid aminotransferase activity of ‘induced’ cells. The results suggested a catabolic role of AT-I in vivo.

Enzymatic methods for determining populations of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in pure and mixed cultures

SummaryStreptococcus cremoris AM2 is characterized by an aminopeptidase and Leuconostoc lactis CNRZ 1091 by an α-galactosidase and a citrate lyase. These strains were grown in pure or mixed cultures, in presence or absence of citrate (15 mM) and at controlled or uncontrolled pH. Cell populations and the activities of the enzymes were measured during microbial growth. Linear correlations were established between the population of S. cremoris AM2 and aminopeptidase activity, and between that of L. lactis CNRZ 1091 and the activities of α-galactosidase and citrate lyase. These correlations held regardless of whether the culture was pure or mixed and if the pH was controlled or not. The presence of citrate did not change citrate lyase and aminopeptidase activities, but inhibited the synthesis of the α-galactosidase and not its activity. The linear relationships permit the determination of bacterial populations in less than 2 h without counting but by measuring enzyme activities.