Determination of the essential nutrient requirements of wine-related bacteria from the genera Oenococcus and Lactobacillus.

Wine lactic acid bacteria (LAB) are responsible for the malolactic fermentation (MLF) in wine production. Wine LAB have fastidious nutrient requirements but their auxotrophies remain little studied. The ability of specific wine nutrients to meet the nutritional requirements of wine LAB, and thus support MLF, remains unclear. This work investigated the essential growth requirements of four strains of wine LAB from the genera Oenococcus and Lactobacillus using the single omission technique with a suitable chemically defined medium. For the determination of auxotrophies, at least 3 (and up to 15) subcultures in deficient media were made, and intra- and extracellular nutrient carry over was reduced by small inoculation rates and washing cells 3 times between transfers. This careful methodology revealed more auxotrophies than those described for wine LAB in the literature. The essential bacterial nutrient requirements were found to be strain specific. 10 compounds were essential for all wine LAB tested, the carbon and phosphate source, manganese, as well as several amino acids (proline, arginine and the branched amino acids valine, leucine and isoleucine) and vitamins (nicotinic acid and pantothenic acids). Nucleotides were not essential for any of the bacteria studied. The two Oenococcus oeni strains revealed a larger number of auxotrophies (18 and 21) and had a higher degree of nutritional similarity (86%) defined as percentage of common requirements per maximum total requirements. The two Lactobacillus strains only had 11 and 14 auxotrophies and the similarity was 79%, but both were auxotroph for riboflavin, which was not needed by the O. oeni strains. Data on the common requirements may be used to further study the ability of wines or commercial nutrients to support MLF and to consider the microbiological stability of finished wines. The results indicate that absence of riboflavin in oenological nutrient preparations may allow to create a specific advantage for indigenous or inoculated O. oeni, which are generally desired for MLF.

Growth and enumeration of the meat spoilage bacterium Brochothrix thermosphacta.

Brochothrix thermosphacta is a common meat spoilage bacterium. The morphology of this bacterium changes from coccobacilli and short rods to chains during growth, which may give a false estimation in numbers using some enumeration techniques. Methods for the quantification of this bacterium have been compared. Turbidimetric readings showed good agreement with cell dry weight indicating that the former provides a good measure of the change in cell mass during growth. The turbidimetric method also correlated well with bacterial numbers determined by plate counts, flow cytometry and manual counts (by microscope) over a limited range of 10(7)-10(9) cells/ml. Flow cytometry and manual counts gave a linear relationship over a wider range of 10(5)-10(9) cells/ml. The sensitivity of analysis, growth rates and lag time attained using these methods were also compared. As a consequence of changes in bacterial cell size during growth, turbidimetry over-estimated the growth rate. The plate count method proved unable to detect the difference between bacteria existing as chains or single cells. The sensitivity of analysis and the calculated growth related parameters were similar for flow cytometry and manual counts. This suggests that flow cytometry is capable of counting individual cells in a chain. Further investigation showed that passage of B. thermosphacta cells through the flow cytometer resulted in the breakage of chains into single cells. The reliability, low error and rapidity of this technique make it attractive for bacterial enumeration, something which has been demonstrated using B. thermosphacta, a bacterium which exhibits complex morphologies.

Interactions between pairs of bacteriocins from lactic bacteria.

Activity of pairs of crude extracts of lactic acid bacteria (LAB) containing different bacteriocins (nisin, pediocin AcH, lacticin 481, lactacin F, and lactacin B) was measured against 10 different indicator strains. Experiments were carried out both in liquid and on solid media. Both synergisms and antagonisms were observed. Lacticin 481 produced mainly antagonistic effects whereas pediocin AcH produced mainly synergistic effects. The use of more than one LAB bacteriocin as a combination biopreservative might be envisaged.