Identification of lactic acid bacteria isolated from South African fortified wines

Stratiotis, Amalia Lilian (1999-12)

Thesis (M.Sc) -- University of Stellenbosch, 1999.

Thesis

ENGLISH SUMMARY: During the primary fermentation of wine, grape must is fermented to ethanol, CO2 and flavour compounds by Saccharomyces cerevisiae. During the secondary fermentation malolactic bacteria converts L-malic acid to L-Iactic acid and CO2 and increases the wine pH by as much as 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 pH of3.2-3.8 and S02leveis of30-50 mg rl. In wines with a pH of3.75 and higher Pediococcus spp. often dominate and increases the wine pH to unacceptable levels. However, MLF can be of benefit to high acid wines, especially wines produced in cold viticultural regions. Fortification of wines with alcohol before primary fermentation is completed prevents the yeast from utilizing all sugars. The high level of sugars that remain renders the wine sweet and fruity, but also susceptible to bacterial spoilage. The high level of alcohol usually prevents microbial spoilage in these wines, but certain strains of Lactobacillus hilgardii, Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus collinoides, Lactobacillus mali and Lactobacillus buchneri are able to tolerate alcohol levels as high as 18-20% (v/v). These strains are also known for their high acid tolerance (PH 3-4). It is interesting to note that these species are all obligately heterofermentative, i.e. classified as Lactobacillus phenotypic Group III. Thirty-two strains, all Gram-positive, catalase negative rods, were isolated from a sweet South African fortified wine during its submerged-culture flor sherry production process; 27 strains were isolated at the onset of this process and five strains from the mid-production phase. No bacteria were isolated from the final phase of the production process. Thirty strains were also isolated from a spoiled bottle of the same wine. Concluded from the morphology of the-cells, their gram-reaction and catalase activity, all strains were identified as members of the genus Lactobacillus. Further separation was made based on results obtained by C02 production from glucose and gluconate fermentation. Based on these reactions, 35 strains were classified as facultative heterofermentative (Group II), and 27 as obligately heterofermentative (Group III). Identification to species level was performed by numerical analysis of total soluble cell protein pa~ (SDS-PAGE) and 16S rRNA sequencing. Based on results obtained by numerical analysis of total soluble cell protein patterns, the Group 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 profile clusters confirmed the identity of the Latt. plantarum and Lact. casei strains. The Group ill lactobacilli grouped into four phenotypic clusters according to numerical analysis of total soluble cell protein patterns. None of the wine strains were phenotypically closely related to the reference strains included in this study. However, 16S rRNA sequencing analysis indicated that the strains from one of the phenotypic groups are members of Lact.buchneri. Strains from the remaining three phenotypic groups were identified as Lactobacillus vermiforme. Lact. casei was the oitly species isolated from the production-phase fortified wine. The apparent absence of strains of Lact. casei from the final phase in production indicates that the species is not a potentially serious spoilage organism in the fortified wines studied. Strains of Lact. buchneri, Lact. casei, Lact. zeae, Lact. plantarum and Lact. vermiforme were isolated from 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 minor role as spoilage organisms. One of the Lact. plantarum strains produced a bacteriocin with a broad spectrum of antimicrobial activity, also active against malolactic bacteria. It might thus very well be that this strain prevented the growth of other bacteria in the wine. This finding merits further research.

AFRIKAANSE OPSOMMING: Tydens die primere fermentasie van wyn word mos deur Saccharomyces cerevisiae na etanol, CO2 en geurkomponente gefermenteer. Tydens In sekondere fermentasie skakel appelmelksuurbakteriee L-appelsuur na L-melksuur en CO2 om en verhoog die wyn se pH met soveel as 0.03 eenhede. Slegs sekere spesies kan appel-melksuurgisting (AMG) teweegbring en sluit in Oenococcus oeni en lede van die genera Lactobacillus en Pediococcus. Appelmelksuurgisting vind dikwels plaas in die die teenwoordigheid van 8% (v/v) of selfs hoer alkoholvlakke, In pH van 3.2-3.8 en SO2-vlakke van 30-50 mg l⁻¹. In wyn met 'n pH van 3.75 en hoer mag Pediococcus spp. domineer en die wyn se pH tot onaanvaarbare hoe vlakke laat styg. Appel-melksuurgisting mag egter tot voordeel wees vir wyne met 'n hoe suurinhoud, veral wyne wat in koue streke geproduseer word. Fortifisering van wyn met alkohol voordat primere fermentasie voltooi is verhoed dat die wyngis die suikers volledig fermenteer. Die hoe vlakke van suikers wat oorbly maak die wyn soet en vrugtig, maar ook vatbaar vir bakteriese bederf. Die hoe alkoholvlakke verhoed gewoonlik mikrobiese bederf, maar sommige stamme van Lactobacillus hilgardii, Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus collinoides, Lactobacillus mali en Lactobacillus buchneri kan alkoholvlakke van tot so veel as 18-20% (v/v) weerstaan. Hierdie stamme is ook bekend vir hul hoe suurtoleransie (PH 3-4). Interessant genoeg is al hierdie spesies obligaat heterofermentatief en sorteer onder fenotipiese Groep III van Lactobacillus. Twee-en-dertig stamme, almal Gram-positiewe katalase negatiewe stafies, is van 'n Suid-Afrikaanse soet gefortifiseerde wyn geisoleer wat volgens die ondergedompelde-kultuur flor sjerrie produksieproses gemaak is. Sewe-en-twintig stamme was voor die aanvang van die fermentasieproses geisoleer en vyf stamme tydens die middel van die fermentasieproses. Geen bakteriee is van die finale fase van die produksieproses geYsoleer rue. Dertig stamme is van 'n bederfde bottel van dieselfde tipe wyn geisoleer. Afgelei van die morfologie van die selle, hul gram-reaksie en katalase aktiwiteit, behoort al die stamme tot die genus Lactobacillus. Verdere onderskeid is gemaak gebasseer op CO2 produksie vanaf die fermentasie van glukose en glukonaat. Gebasseer op hierdie reaksies, is 35 stamme as fakultatief heterofermentatief(Groep II) en 27 as verpligtend heterofermentatief (Groep III) geklassifiseer. Identifikasie tot op spesievlak is m.b.v. numeriese analise van totale oplosbare selproteien-bandpatrone (SDS-PAGE) en 16S rRNA-volgordebepaling gemaak. Gegrond op die resultate verkry met numeriese analise van totale oplosbare selproteien bandpatrone, is die Groep III lactobacilli as lede van die spesies Lactobacillus plantarum, Lactobacillus casei subsp. casei, Lactobacillus zeae en Lactobacillus casei subsp. alactosus geklassifiseer. Nukleotiedopeenvolgorde van die 16S rRNA van verteenwoordigende starnme van die onderskeie proteienprofiel-groepe het die identiteit van die Lact. plantarum- en Lact. casei-stamme bevestig. Die Groep III lactobacilli het, na aanleiding van numeriese analise van totale oplosbare selproteien-bandpatrone in vier fenotipiese groepe gesorteer. Nie een van die wynstamme is fenotipies naverwant aan die verwysingsstarnme wat in hierdie studie ingesluit is nie. Resultate verkry met 16S rRNA-volgordebepaling het egter getoon dat stamme van een van die fenotipiese groepe wel lid is van Lact. buchneri. Stamme van die oorblywende drie fenotipiese groepe is as Lactobacillus vermiforme geidentifiseer. Lact. case; was die enigste spesie wat van die ptoduksie-fase van die gefortifiseerde wyn geisoleer is. Die skeinbare afwesigheid van Lact. casei van die finale fase van produksie dui daarop dat die spesie nie 'n potensiele bederforganisme van die betrokke gefortifiseerde wyn is nie. Stamme van Lact. buchneri, Lact. casei, Lact. zeae, Lact. plantarum en Lact. vermiforme is van die bederfde gefortifiseerde wyn geisoleer. Hierdie is die eerste verslag van die teenwoordigheid van Lact. casei, Lact. zeae en Lact. plantarum in bederfde gefortifiseerde wyne. Die relatief lae selgetalle van hierdie twee spesies suggereer dat hulle 'n klein rol as bederforganismes speel. Een van die Lact. plantarum-stamme het 'n bakteriosien met 'n bree spektrum van antimikrobiese aktiwiteit, ook aktief teen appel-melksuurbakteriee, geproduseer. Dit mag dus wel wees dat hierdie starn die groei van ander bakteriee in die wyn inhibeer. Hierdie bevinding regverdig verdere navorsing.

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