Identification of bacteria isolated from malt, with the emphasis on lactic acid bacteria and their influence on brewer's yeast

Booysen, Clifford (2001-12)

Thesis (MScAgric.)--University of Stellenbosch, 2001.

Thesis

ENGLISH ABSTRACT: Changes in the bacterial population throughout the malting process of two barley cultivars, i.e. Clipper (local cultivar) and Prisma (imported cultivar), malted at Southern Associated Maltsters (SAM), Caledon, South Africa, were studied. Samples were taken from four individual runs of each cultivar at ten different stages, i.e. dry barley before steep, water from the first steep water-stand, barley after draining the first steep, water from the second steep water-stand, barley from the second steep water-stand, barley after draining of the second steep, barley from the first, second and third days of germination in the germination vessels (GV), and malt after kilning. Emphasis was placed on the taxonomy and composition of the lactic acid bacteria (LAB) isolated from the ten different phases. The LAB were identified to species level by using numerical analysis of total soluble cell protein patterns, RAPD-PCR banding patterns and 16S rRNA sequencing. The Gram-negative bacteria were identified to genus level by using the API 20E system and included Citrobacter spp., Enterobacter spp., Pantoea spp., Proteus spp., Seratia spp., Kluyvera spp., Klebsiella spp., Vibrio spp. and Escherichia coli. The number of viable bacteria throughout the malting process of the two cultivars did not differ significantly, although the LAB counts in the barley before steep and on the kilned malt were higher in Prisma than in Clipper. Leuconostoc argentinum, Leuconostoc laetis and Weissella confusa were the most predominant in both cultivars. A few strains of Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis and Lactobacillus rhamnosus were also isolated. Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, whilst W paramesenteroides and Le. laetis were absent in the Clipper cultivar. Kilned malt of the Clipper cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Le. lactis. The effect of these bacteria on the fermenting ability of the brewer's yeast Saccharomyces cerevisiae SAB 05, was also studied. Fermentations were conducted in wort prepared from Clipper and Prisma malt. Yeast in combination with the different bacteria were used in the fermentation studies. Wort with only yeast was used as control. Emphasis was placed on the effect the bacteria has on the gravity, pH, yeast- and bacterial- counts and the different volatile aroma compounds produced throughout the fermentations. The presence of LAB and Gram-negative bacteria had no effect on the yeast to reduce the gravity of the fermenting wort, whilst the LAB caused a decrease in the pH of the fermentations in both Clipper and Prisma wort. The cell numbers of the Gram-negative bacteria decreased throughout the fermentations, whilst the LAB cell numbers remained constant. Comparisons could be drawn between the volatile aroma compounds produced in the control fermentation and fermentations with yeast and Gram-negative bacteria, yeast and Lactobacillus spp. and yeast and Weissella spp. Leuconostoc spp. had a much greater influence on the aromatic composition of fermented malt, with much more clear variations between Prisma and Clipper. No major differences were recorded in the aroma profiles of Prisma and Clipper malt fermented in the presence and absence of Lactococcus spp. The Gram-negative bacteria had no significant effect on the volatile aroma compounds produced by the yeast, whilst the LAB had a definite effect on aroma composition in both cultivars. The levels of four of the five principle aroma compounds, present in beer, were in the acceptable concentration range on the fmal day of fermentation. The compounds with the highest concentrations were iso-amyl alcohol, acetic acid and acetoin, with acetic acid being present in the highest concentration in all the fermentations.

AFRIKAANSE OPSOMMING: Veranderinge in die bakteriese populasie van die gars kultivars, Clipper (plaaslik) en Prisma (ingevoer), vermout by Southern Associated Maltsters (SAM), Caledon, Suid Afrika, is ondersoek. Monsters is van vier individuele lopies van elke kultivar en tydens tien verskillende fases van die vermoutingsproses geneem. Die tien verskillende stadia het die volgende ingesluit: Droë gars voor benatting, water van die eerste benattingsfase, gars nadat water van die eerste benattingsfase gedreineer is, water van die tweede benattingsfase, gars van die tweede benattingsfase, gars na die dreinering van water in die tweede benattings fase, gars na die eerste, tweede en derde dag van ontkieming binne die ontkiemingstenke, en mout na droging. Klem is geplaas op die taksonomie en samestelling van melksuurbakterieë (MSB) wat tydens die tien verskillende fases geïsoleer is. Die MSB is tot spesievlak geïdentifiseer deur gebruik te maak van numeriese analise van totale oplosbare selproteïen bandpatrone, RAPD-PKR bandpatrone en 16S rRNA volgorde-bepaling. Gram-negatiewe bakterieë is tot op genusvlak geïdentifiseer deur gebruik te maak van die API 20E toetssisteem. Spesies van die genera Citrobacter, Enterobacter, Pantoea, Proteus, Seratia, Kluyvera, Klebsiella, Vibrio asook Escherichia coli is geïdentifiseer. Tydens die vermoutingsproses van die twee kultivars is geen beduidende verskille in die lewensvatbare bakterietellings gevind nie, alhoewel die MSB-tellings in die gars voor benatting en mout na droging in Prisma hoër was as in Clipper. Leuconostoc argentinum, Leuconostoc laetis en Weissella confusa het die meeste voorgekom in beide kultivars. Kleiner hoeveelhede van Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis en Lactobacillus rhamnosus is ook geïsoleer. Lb. casei en Lb. rhamnosus het nie in die Prisma-kultivar voorgekom nie, terwyl W paramesenteroides en Le. laetis nie in die Clipper-kultivar teenwoordig was nie. Le. argentinum het meestal in die gedroogde mout van die Clipper-kultivar voorgekom, terwyl Le. laetis meestal in die Prisma-kultivar waargeneem is. Die effek van hierdie bakterieë op die fermentasievermoë van die brouersgis Saccharomyces cerevisiae SAB 05 is ook bestudeer. Die fermentasies is in Clipper- en Prisma- wort gedoen. Vir die fermentasiestudies is gis in kombinasie met verskillende bakterieë gebruik. Wort met slegs gis het as kontrole gedien. Klem is geplaas op die effek van die bakterieë op die digtheid, pH, gis- en bakterietellings en die verskillende vlugtige komponente wat tydens die fermentasies geproduseer is. Die teenwoordigheid van MSB en Gram-negatiewe bakterieë het geen effek gehad op die vermoë van die gis om die digtheid van die gefermenteerde wort te verlaag nie. Die MSB het wel 'n verlaging van die pH in beide Clipper- en Prisma- wort teweeggebring. Tydens die fermentasie het die Gramnegatiewe bakterietellings verminder, terwyl die MSB-tellings konstant gebly het. 'n Verband is gevind tussen vlugtige komponente geproduseer in die kontrole-fermentasie en fermentasies met gis en Gram-negatiewe bakterieë, gis en Lactobacillus spp. en gis en Weissella spp. Leuconostoc spp. het groter veskille in die samestelling van die gefermenteerde wort teweeg gebring met duidelike verskille tussen Clipper en Prisma. Die teenwoordigheid van Lactococcus spp. het nie groot verskille in die samestelling van die gefermenteerde wort getoon nie. Op die laaste dag van die fermentasies was die vlakke van vier uit die vyfbelangrikste vlugtige aroma komponente wat in bier voorkom in die kontrole fermentasies in aanvaarbare konsentrasies teenwoordig. Die Gramnegatiewe bakterieë het geen beduidende invloed gehad op die vlugtige aroma komponente wat deur die gis geproduseer is nie, terwyl die MSB 'n besliste effek in die aroma-samestelling van beide die kultivars gehad het. Die komponente met die hoogste konsentrasies was, isoamiel-alkohol, asynsuur en asetoin. Asynsuur was in al die fermentasies in die hoogste konsentrasie teenwoordig.

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