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    An immobilized cell bioreactor for the malolactic fermentation of wine : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University
    (Massey University, 1991) Janssen, Denise E
    Malolactic fermentation using immobilized cells of Leuconostoc oenos was investigated in order to improve this fermentation at an industrial-scale. Three strains of bacteria were investigated in some detail, and one was chosen for further work. A satisfactory growth medium for the strain of bacteria used was found to be an apple juice broth. The effect, on both the growth and malic acid bioconversion for Leuconostoc oenos strain 1070, of having 6% v/v ethanol in the growth media was tested and found to cause a longer lag phase, and be slightly beneficial, respectively. Oak chips were decided on as the immobilization media, in preference to bone char, and a synthetic, apple-juice based wine was used to determine operation parameters for a continuous culture bioreactor. Temperature, pH, ethanol concentration, SO2, malic acid concentrations, anaerobic conditions and dilution rate were investigated and it was shown that lower malic acid concentrations, and also an interaction between low pH, high temperature and high ethanol concentration affected the malic acid bioconversion adversely. Increasing the dilution rate above 0.35 h·1 caused a 30% drop in the bioconversion rate. The pH level had no effect on bioconversion if the temperature was kept at 21°C or lower. Decreasing the temperature, increasing the ethanol concentration above 10% v/v and increasing SO2 levels all caused a slight drop in bioconversion rates while strict anaerobic growth and bioconversion conditions caused an increase. The bioconversion rates ranged between 20 and 100 mg malic acid consumed/lO0ml oak chips/hour. An industrial prototype bioreactor was built and used at Villa Maria Wineries, Auckland, during the 1991 vintage and successfully processed 200 litres of Chardonnay-style wine in 2 days. The bioconversion rate was between 25 and 30 mg malic acid consumed/l00ml oak chips/hour. Informal taste tests showed satisfactory malolactic characteristics in the treated wine.
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    Fluoride inhibition of wine yeasts : a thesis presented in partial fulfilment of the requirements for the degress of Master of Science in Microbiology at Massey University
    (Massey University, 1997) Clayton, Miranda Gaye
    Stuck or slowed fermentations are costly in time and money to winemakers. There are many variables that can interrupt fermentation. One of the lesser known factors is the effect of fluoride on grape juice fermentations. Winemakers in California have had problems with slow or stuck fermentations with grapes that have been treated with the insecticide Cryolite, which contains fluoride. A selection of 6 yeasts, 3 commercial strains and 3 natural strains, commonly associated with winemaking were used in this study. Preliminary experiments investigated a wide range of fluoride challenge with different pH and cell densities on solid and liquid media. The effectiveness of fluoride was compared between sodium fluoride and Cryolite, as the fluoride source. The effect of fluoride was more potent with sodium fluoride, as the fluoride source. The minimum inhibitory concentration of fluoride for the yeast strains was recorded. The most sensitive commercial yeast was Saccharomyces cerevisiae RS1, the most resistant commercial yeast was Saccharomyces bayanus RS2. The most sensitive yeast overall was Hansenula saturnus AWRI-354. The next stage examined the effect of fluoride on the selected yeast in small scale grape juice fermentations. Within this investigation the effect of different media sources and heat treatments was included. Fluoride concentrations reflected levels of fluoride found in grape musts and wines. During this study we found that the effect of fluoride on yeasts is increased with lower pH and lower cell densities. The effect of fluoride on yeast growth and fermentation was also strain dependent.
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    Maloethanolic deacidification of high acid juices during wine yeast alcoholic fermentation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1996) Ryan, Frances Alison
    Malic acid is a major acid found in grapes. In countries with cooler climates, such as New Zealand, this acid is not fully respired from grapes and will impart a sour taste on grape juices. Therefore steps must be taken to ensure deacidification of the juice or wine occurs. Deacidification is the process whereby the acidity of a juice or wine is lowered by physical, chemical or biological means. Biological methods of deacidification such as malolactic fermentation and maloethanolic fermentation involve the degradation of malic acid to other products. Malolactic fermentation is the bacterial conversion of malic acid to lactic acid and carbon dioxide and is commonly used in New Zealand wineries. Maloethanolic fermentation is the simultaneous conversion of grape sugars and malic acid to ethanol by specialized yeast strains and is the focus of this investigation. This research examines several commercially available yeast strains (Lallemand Lalvin strains 71B, ACID-, D432 and reference strains EC1118 and Red Star Montrachet (M)) and Schizosaccharomyces strain 442, for their ability to degrade malic acid during grape juice fermentation under New Zealand conditions. A Simulated Grape Juice medium was used to mimic these conditions, as well as commercial Chardonnay and Sauvignon Blanc juices. Strains 71B and D432 consistently degraded the greatest percentage of malic acid under all conditions and parameters investigated in this research. Respectively, these strains degraded malic acid by 36% and 22% of the initial concentration (7.0g/L) in industrial Chardonnay juice fermentations and by 47% and 36% of the initial concentration (3.7g/L) in industrial Sauvignon Blanc fermentations. Furthermore, in Sauvignon Blanc wines, a significant (P=0.05) difference was found between the wine made with strain 71B and all other wines. However, in Chardonnay wines, a significant difference was found between the wine made with strain D432 and all other wines. In addition, molecular genetic techniques (CHEF chromosomal banding pattern polymorphisms) were utilised to confirm yeast strain identity. from industrial fermentations. From this, it was concluded that all strains inoculated into the commercial juices were dominant at the most vigorous stage of fermentation. Factors influencing malic acid degradation were investigated in Simulated Grape Juice fermentations. These included initial concentrations of malic acid and nitrogen and the initial pH level of the juice. It was found that strains 71B and D432 degraded the greatest percentage of malic acid when the initial malic acid concentration of the juice was high (7.5g/L), the initial nitrogen concentration was low (463mg/L with proline) and an initial pH of pH 3.5. These results indicate that there is an interaction between yeast and grape variety/maturity, and that proper selection of yeast strain can be used as a tool for deacidification.