Identification of lactic acid bacteria isolated from South African fortified wines

[摘要] ENGLISH SUMMARY: During the primary fermentation of wine, grape must is fermented to ethanol, CO2 and flavourcompounds by Saccharomyces cerevisiae. During the secondary fermentation malolacticbacteria converts L-malic acid to L-Iactic acid and CO2 and increases the wine pH by as muchas 0.03 units. Only certain species are capable of performing malolactic fermentation (MLF)and include Oenococcus oeni and members of the genera Lactobacillus and Pediococcus.Malolactic fermentation often takes place in the presence of 8% (v/v) or higher ethanol, a pHof3.2-3.8 and S02leveis of30-50 mg rl. In wines with a pH of3.75 and higher Pediococcusspp. often dominate and increases the wine pH to unacceptable levels. However, MLF can beof benefit to high acid wines, especially wines produced in cold viticultural regions.Fortification of wines with alcohol before primary fermentation is completed prevents theyeast from utilizing all sugars. The high level of sugars that remain renders the wine sweetand fruity, but also susceptible to bacterial spoilage. The high level of alcohol usuallyprevents microbial spoilage in these wines, but certain strains of Lactobacillus hilgardii,Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus collinoides, Lactobacillus maliand Lactobacillus buchneri are able to tolerate alcohol levels as high as 18-20% (v/v). Thesestrains are also known for their high acid tolerance (PH 3-4). It is interesting to note that thesespecies are all obligately heterofermentative, i.e. classified as Lactobacillus phenotypic GroupIII.Thirty-two strains, all Gram-positive, catalase negative rods, were isolated from a sweetSouth African fortified wine during its submerged-culture flor sherry production process; 27strains were isolated at the onset of this process and five strains from the mid-productionphase. No bacteria were isolated from the final phase of the production process. Thirtystrains were also isolated from a spoiled bottle of the same wine.Concluded from the morphology of the-cells, their gram-reaction and catalase activity, allstrains were identified as members of the genus Lactobacillus. Further separation was madebased on results obtained by C02 production from glucose and gluconate fermentation. Basedon these reactions, 35 strains were classified as facultative heterofermentative (Group II), and27 as obligately heterofermentative (Group III). Identification to species level was performedby numerical analysis of total soluble cell protein pa~ (SDS-PAGE) and 16S rRNAsequencing. Based on results obtained by numerical analysis of total soluble cell protein patterns, theGroup II lactobacilli were identified as members of the species Lactobacillus plantarum,Lactobacillus casei subsp. casei, Lactobacillus zeae and Lactobacillus casei subsp. alactosus.Sequencing of the 16S rRNA of representative strains from the different protein profileclusters confirmed the identity of the Latt. plantarum and Lact. casei strains.The Group ill lactobacilli grouped into four phenotypic clusters according to numericalanalysis of total soluble cell protein patterns. None of the wine strains were phenotypicallyclosely related to the reference strains included in this study. However, 16S rRNAsequencing analysis indicated that the strains from one of the phenotypic groups are membersof Lact.buchneri. Strains from the remaining three phenotypic groups were identified asLactobacillus vermiforme.Lact. casei was the oitly species isolated from the production-phase fortified wine. Theapparent absence of strains of Lact. casei from the final phase in production indicates that thespecies is not a potentially serious spoilage organism in the fortified wines studied. Strains ofLact. buchneri, Lact. casei, Lact. zeae, Lact. plantarum and Lact. vermiforme were isolatedfrom the spoiled fortified wine. This is the first report of the presence of Lact. casei, Lact.zeae and Lact. plantarum in spoiled fortified wines, their low numbers suggesting a minorrole as spoilage organisms.One of the Lact. plantarum strains produced a bacteriocin with a broad spectrum ofantimicrobial activity, also active against malolactic bacteria. It might thus very well be thatthis strain prevented the growth of other bacteria in the wine. This finding merits furtherresearch.

[发布机构] Stellenbosch University