A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris.

The main autolysin PA49.5, an enzyme that hydrolyzes or destroys the components of a biological endogenous cell or a tissue, was purified 3045 times from the homogenate of a whole cell extract of Lactococcus lactis subsp. cremoris ATCC 9596 (Mc5), with a recovery yield of 52%. The purification of the protein was carried out through a micro-purification technique using SDS-BigCHAP polyacrylamide gel electrophoresis and concentrated with a Microcon-10 filtration system. SDS-polyacrylamide gel electrophoresis of the purified enzyme confirmed the presence of only one band having a molecular weight of 49.5 kDa. In view of its insolubility, PA49.5 contained in the cell extract precipitate was solubilized in the presence of 0.1% (w/v) of BigCHAP, a non-ionic detergent. Higher concentrations of this detergent completely inhibited the activity of solubilized PA49.5 or prevented its solubilization. The optimal pH and temperature for PA49.5 enzymatic activity are 7.5 and 45 degrees C respectively. In addition 0.1% or less of PA49.5 significantly increased Mc5 lysis. We observed 55% more lysis with 0.25 mug of purified PA49.5 compared to the control. Gas chromatography analysis of the components of the crude cell extract, of the precipitate and of the supernatant indicates the presence of at least 6 fatty acids. The long-chained fatty acids (e.g. C18:0 and C18:3) detected represent 81.65% of the precipitate from which PA49.5 was purified. Of these two acids, the C18:0 (stearic acid) alone represents 47.40% of the precipitate. Mc5 releases proteins at the beginning (major peak) and at the end (moderate peak) of the exponential stage of growth. Analysis by denaturing polyacrylamide gel electrophoresis with Mc5 cell walls incorporated as the autolysin's substrate identified a band corresponding to PA49.5 in the second peak of protein secretion.

Inhibitory Effect of Organic Acids upon Meat Spoilage Bacteria.

The relative ability of acetic, benzoic, citric, lactic, propionic, and sorbic acids to inhibit the growth of six common meat spoilage bacteria ( Brochothrix thermosphacta , Carnobacterium piscicola , Lactobacillus curvatus , Lactobacillus sake , Pseudomonas fiuorescens , and Serratia liquefaciens ) was compared under otherwise optimum conditions (BHI or MRS broths; 20°C). Because of their low solubility in the growth media, benzoic and sorbic acids could only be used in low concentrations (below 0.15% [wt/vol]) and did not efficiently inhibit bacterial growth. All other acids totally inhibited growth at concentrations ranging from 0.1 % to 1% (wt/vol). On a weight basis, acetic acid was found to be the most inhibitory, followed by propionic. lactic, and citric acid, while the order of efficiency was reversed (citric> lactic> propionic> acetic) when the acid concentrations were expressed on a molar basis or when the acid effectiveness was evaluated relative to the concentration of undissociated molecules. Overall, the lactobacilli were the bacteria most resistant to the action of organic acids, followed by P. fiuorescens and S. liquefaciens , while B. thermosphacta and C. piscicola were considerably more sensitive.

A Study of the Microbiological Quality of Goat Milk in Quebec.

The microbiological quality of goat milk collected at Quebec farms was observed over a one-year period. Microbial counts increased during the summer period. Only one farm had a total aerobic count of >105 CFU/ml in >50% of samples taken from storage tanks. Coliform counts were <103/ml even when the total aerobic count was 106/ml. No relationship between somatic cell counts and bacterial counts was found. Exponential bacterial growth began after a lag of at least 3 d at 0-4°C. Good hygiene, rapid cooling and refrigeration, as well as frequent collection by refrigerated vehicles, ensured the production of good quality milk.