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Item Dominant lactic acid bacteria and yeasts in rice sourdough produced in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology, Massey University, Albany, New Zealand(Massey University, 2018) Yang, QiweiMost gluten free (GF) products on the market are described as bland with poor mouth feel and are considered low quality in terms of texture due to lack of gluten, which has positive effects on the texture and appearance of cereal bakery products. The application of sourdough is a recent development in improving the quality of GF bread due to its efficiency and low-cost. This study aims to understand the fermentation of GF rice flour mix used to improve the quality of rice sourdough bread. Rice sourdough samples from three stages of fermentation mother sourdough (MSD), dough before proofing (DBP) and dough after proofing (DAP) and sourdough bread were characterised for their acidity, soluble sugars and organic acids content and total free amino acid content. Sourdough breads were also tested for their texture and colour. Yeasts and LAB colonies were enumerated from sourdough samples and isolates of LAB and yeasts were identified using API test kits (API 50 CHL for LAB and API 32 C for yeasts) and sequenced using 16S metagenetics for LAB and ITS region for yeasts. Due to the metabolic activities of sourdough lactic acid bacteria (LAB) and yeasts, dough acidity increased significantly (p>0.05) and total free amino acid content decreased during fermentation. Compared to unleavened rice bread, the final rice sourdough bread had a softer, more elastic, less crumbly and chewier crumb and its crust colour was more similar to unleavened wheat bread. Mean LAB counts in MSD, DBP and DAP were 8.6 log CFU/g, 7.9 log CFU/g and 8.5 log CFU/g, respectively; while yeast counts were 5.4 log CFU/g, 6.4 log CFU/g, and 6.7 log CFU/g, respectively. LAB counts increased significantly (p<0.05) during proofing but yeasts did not exhibit significant growth (p>0.05). Dominant LAB and yeasts responsible for the fermentation of rice sourdough were of the genus Lactobacillus and S. cerevisiae. LAB isolates were identified as Lactobacillus plantarum CIP 102980 and Lactobacillus fermentarum DSM 10667 and yeast colonies as S. cerevisiae CBS 1171.Item Citric acid production by the yeasts Candida guilliermondii and Yarrowia lipolytica : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology and Bioprocess Engineering at Massey University(Massey University, 1993) Thomson, Karen RobertsThe aim of this thesis was to investigate the relationships, for a citric acid producing strain of yeast, among the growth rate, sugar uptake rate and the citric acid production rate, and to investigate the hypothesis that citric acid production occurs when the growth rate slows, but the sugar uptake rate is maintained. As previous experimental work in the Department of Process and Environmental Technology (formerly Biotechnology Department) of Massey University had been performed in shake flask cultures only, it was desired to scale-up the culture into a 21 laboratory scale batch culture, and then into a chemostat culture. The first yeast investigated, Yarrowia lipolytica IMK2, failed to successfully scale-up, so further investigations were performed using the yeast Candida guil!iermondii IMK1. Experiments were performed in shake flask culture to investigate the effect of using mixed carbon sources to adjust the carbon uptake rate, and hence the citric acid production rate, but no effect was noticed with the mixtures tested. Batch fermenter experiments were performed to investigate the effect of the culture pH, and the aeration rate, on citric acid production. The aeration rate was not observed to have an effect on the culture in the range tested (0.06 - 0.333 vvm), but the culture pH was observed to have an effect, with the maximum production occurring at pH 4.3, and no citric acid production occurring below pH 3.5. Chemostat culture experiments were performed to investigate the effect of culture pH and the specific growth rate on citric acid production. The specific growth rate was observed to have a significant effect, with the specific citric acid production rate increasing as the growth rate decreased. The effect of the culture pH was found to vary with the growth rate, with the maximum production rate and yield occurring at pH 3.8, and a growth rate of 0.02 h-1 • From cultures where the glucose was exhausted from the medium, and therefore glucose was a limiting nutrient, the specific citric acid production rate was observed to decrease as the glucose uptake rate decreased. Thus, it could be concluded that the specific citric acid production rate increased as the growth rate decreased, provided that the sugar uptake rate remained high.Item Aspects of cell death and autolysis in Saccharomyces cerevisae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1987) Hornblow, Susan ChristineThe kinetics of cell death and autolysis of twenty two haploid yeast strains were examined over a period of eight months in wine and synthetic media. Eight distinct patterns of cell death were observed using methylene blue staining and sample plating for viable cells. The rate of death was both yeast strain dependent and influenced by environmental factors such as temperature, nutrient supply and the presence or absence of ethanol. The activity of extracellular killer yeast toxin concentrated by ultrafiltration was examined under various environmental conditions. Toxin activity was pH and temperature dependent. Concentrations of ethanol greater than 2% completely inhibited killer toxin activity. A difference of 12 hours was detected between a yeast ce71 becoming incapable of reproduction as the result of killer toxin action and this inability becoming discernible by methylene blue staining. A maximum kill of 97 - 99% was obtained independent of cell or toxin concentration. Toxin induced death was accompanied by the release of arginine and lysine. A bioassay was developed to quantify the amounts of arginine and lysine released.Item An examination of the putative glucose tolerance factor activity of amino acid and peptide fractions isolated from brewer's yeast : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University(Massey University, 1985) Jackson, Timothy GrahamThe first report of the possible existance of a glucose tolerance factor (GTF) was made by Mertz and Schwarz (1955) who noticed that a dietary additive, termed factor 3, isolated from an enzymatic casein hydrolysate (Schwarz (1952)), maintained normal glucose removal rates in diabetic like rats. These rats were the subject of a study on the development of dietary necrotic liver degeneration. The immediate cause of death,,in these rats, could be demonstrated to be severe hypoglycaemia (Mertz and Schwarz (1955)) that initially manifested itself, during the latent period of degeneration, as impairment of excess blood glucose removal. The diet used to induce the development of necrotic liver degeneration was a semi-purified, vitamin E-free, ration of 30% Torula yeast which also represented the sole protein source. The vitamin E prevented the development of necrotic liver degeneration but did not affect the removal of excess blood glucose. In 1957, Schwarz and Mertz reported that the factor 3, in itself, was not responsible for the maintainance of normal glucose removal rates but rather that it contained an active fraction separable by fractionation procedures involving evaporation, in vacuo, of a NaCl-containing, factor 3 concentrate. The NaCl was removed by filtration and the GTF activity was found to be present in the separated salt fraction, from which it could be removed by treatment with 65% ethanol. A further claim was made that this separated substance, now termed the glucose tolerance factor (GTF), not only prevented but cured impairment of glucose removal when administered in the diet and that the initial glucose impairment observed was not a symptom of necrotic liver degeneration but a result of a dietary deficiency. GTF prepara tions were reported (Mertz and Schwarz (1959)) to be routinely obtained from brewer's yeast as well as acid hydrolysates of dried, defatted, pork kidney powder. [From Introduction]Item Factors affecting the activity of baker's compressed and active dry yeast : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University(Massey University, 1980) Espie, Stephen AngusFactors affecting the Activity of Baker's Compressed and Active Dry Yeast. Parameters important in the production of Baker's Yeast were correlated with the product's final activity. Activity was a measure of the gas evolved in a fermenting dough, expressed as mMCO2/hr/g yeast solids. The drying of Compressed Yeast to Active Dry Yeast was optimised in terms of the drying air. A tunnel tray drier was used to dry yeast to a 9% moisture content (dry weight). At 40°C. the optimum drying humidity was found to be 30-32% relative humidity. The leavening ability of yeast dried at 17% and 45% relative humidity decreased. A drying additive, 2% glyceryl monostearate, halved the drying time to 4 hours. Equations were developed to describe these observations as a function of relative humidity, drying time and additive concentration. The equilibrium relative humidity of stored dried yeast was found to be 32% at 20°C. Fermentation parameters were correlated with the activity of Compressed Yeast using an experimental design. Growth temperatures varied from 28°C. to 37°C., initial pH from 4 to 6, glucose concentrations from 0.5% to 3%, nitrogen concentrations from 0.3% to 1.2% and dissolved oxygen varied as either agitated or standing cultures. Factors significantly affecting cell yield and yeast activity were growth temperature, dissolved oxygen and glucose concentrations. Maximal yeast activity occurred at 0.5% glucose concentration, 28°C. and non-agitated conditions. A model was developed to describe yeast activity as a function of these variables. The observed optimal conditions for cell yield were similar to those for yeast activity except for the dissolved oxygen level. Maximum yeast activity of Compressed Yeast occurred in non-agitated fermentations, compared with cell yield which required agitated conditions to achieve the greatest cell yield. A rapid screening test for evaluating dried yeast was incorporated into the yeast activity analysis. This involved monitoring foam production during rehydration.Item Citric acid production from yeasts : comparison of a parent and a mutant strain of Candida guilliermondii, and subsequent reversion of the mutant : a thesis presented in fulfilment of the requirements for the degree of Master of Philosophy in Food Technology at Massey University(Massey University, 1997) Nutter, Anne-MarieCitric acid production from yeasts has been studied widely owing to the short duration of fermentation, the broad choice of carbon source and the better yields obtained when compared to the currently used submerged or surface fermentation with Aspergillus niger. In this work two strains of Candida guilliermondii were compared for their citric acid-producing capabilities, these being parent strain Candida guilliermondii NRRL Y-448, and mutant strain Candida guilliermondii IMK1. The mutant was previously selected for its ability to produce much higher concentrations of citric acid than the parent. These strains were grown under various nutrient limitations to determine if nutrient limitation had an effect on the amount of citric acid produced. Several differences were observed between the non-citric acid-producing parent and the citric acid-producing mutant. The mutant generally consumed less glucose (g.g-1), produced less biomass (g.L-1) and produced much higher levels of citric acid – the best production (7.34 g.g-1) seen from the culture grown under phosphorus-limited (0.15 mM) conditions. Upon assessment of enzyme activities it was found that the mutant also exhibited reduced activity of the enzyme NAD-ICDH (NAD-dependent isocitrate dehydrogenase), a recognised control point for the over-production of citric acid. NAD-ICDH is inhibited by increased concentrations of ATP - these are associated with the accumulation of citric acid in the cell in the stationary phase of growth. This reduction in NAD-ICDH activity correlated with a dramatic increase in the activity of NADP-ICDH (NADP-specific isocitrate dehydrogenase), the activity of which was thought to compensate for the loss of activity of NAD-ICDH. However, in a subsequent experiment, the mutant was found to have reverted - losing its ability to produce citric acid. This loss of productivity occurred before the levels of adenine nucleotides in the cell could be assessed, meaning that the suggested inhibition of NAD-ICDH by elevated levels of ATP could not be confirmed. Upon analysis of the revertant, it was found that glucose consumption (grams per gram of cells) had increased, as had the production of biomass (g.L-1). Even though the revertant failed to consume as much glucose as the parent, in many instances it produced higher levels of biomass. Upon analysis of enzyme activity, it was found that the activity of NAD-ICDH had increased, so reducing the accumulation of citric and isocitric acids. The activity of NADP-ICDH had decreased somewhat, but activity of this enzyme remained at significant levels. It is proposed that the activity of NADP-ICDH in the revertant was responsible for the increased efficiency of biomass production. In conclusion, it is suggested that overproduction of citric acid in Candida guilliermondii IMK1 was due to the consumption of lowered levels of glucose combined with the reduced activity of the enzyme NAD-ICDH, which it is speculated was due to elevated concentrations of ATP in the cell.Item Citric acid production by immobilized cells of the yeast Candida guilliermondii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology and Bioprocess Engineering at Massey University(Massey University, 1995) Tisnadjaja, DjadjatThe feasibility of using cells of Candida guilliermondii immobilized onto sawdust particles for production of citric acid was investigated. C. guilliermondii IMK1 from a stock culture (Department of Process and Environmental Technology, Massey University, Palmerston North, New Zealand) was reisolated for further study including strain improvement work by induced mutation using UV light. A mutant strain DT2 was isolated which produced a citric acid concentration of 9.2 g/l (yield 25 % (w/w)) in shake flask culture, using a defined medium containing 36 (g/l) glucose, compared with 4.9 (g/l) citric acid produced (yield 14 % (w/w)) by the parent strain. Experiments in a laboratory scale batch fermenter, in which a higher concentration of citric acid (11.7 g/1) was achieved, proved that citric acid production using the mutant strain C. guilliermondii DT2, could be scaled up successfully from shake flask to a 2 1 fermenter. This mutant was used throughout subsequent experiments. Sawdust was selected, as the most appropriate support material to immobilize the mutant strain C. guilliermondii DT2 via the adsorption method. Experiments using different concentrations of nitrogen nutrient in defined medium using cells of C. guilliermondii DT2 immobilized onto sawdust particles, in repeated batch shake flask culture, demonstrated a marked effect of the nitrogen concentration on citric acid production. Thus, an overall productivity of 0.11 (g/l.h) was obtained using a defined medium containing 0.53 (g/l) ammonium chloride, compared to overall productivities of 0.04 (g/l.h) and 0.01 (g/l.h) using defined media containing 0.1 (g/l) and no ammonium chloride, respectively. No significant effect of nitrogen concentration on citric acid yield was observed in this investigation. In contrast, similar experiments, in repeated batch shake flask culture, for the effect of phosphate concentration on citric acid production showed no effect of phosphate on either the production rate or yield of citric acid. In bubble column culture experiments, using cells of C.guilliermondii DT2 immobilized onto sawdust, the importance of pH control in citric acid production was demonstrated. In addition, it was demonstrated that the activity of immobilized cells which have lost the ability to produce citric acid can be revived by supplying medium containing sufficient concentrations of nitrogen and phosphate. Reduction of the nitrogen concentration in the medium from 0.53 (g/l) to 0.05 (g/l), provided that the reactor was well established, showed no significant influence on citric acid productivity, but significantly improved the citric acid yield. The highest productivity of around 0.21 - 0.24 (g/l.h) at a dilution rate of 0.21 h-1, accompanied by a citric acid yield of about 10 - 11% (w/w), was reached and maintained for more than 140 hours of stable operation. Overall, it was concludcd that cells of C. guilliermondii were succesfully immobilized onto sawdust particles, and the immobilized cell reactor produced citric acid at a higher rate compared to a free cell system. In particular, a high rate of citric acid production in a bubble column reactor, operated in continuous mode, was achieved.Item 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 GayeStuck 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.Item Investigations on the hexose-phosphorylating enzymes in the pentose-fermenting yeast, Pachysolen tannophilus : a thesis presented in partial fulfilment of the requirements for the degree in Doctor of Philosophy in Microbiology at Massey University(Massey University, 1988) Wedlock, David NeilMutants of Pachysolentannophilus, resistant to2- deoxyglucose, the toxic analogue of D-glucose, have been isolated and characterised. Their growth characteristics on hexose and pentosesugars, resistance to 2-deoxyglucose and cellular hexose-phosphorylating activities were investigated. Loss of hexose-ATP-kinase activity was found to correlate with loss of ability to grow on hexose sugars and increased resistance to 2-deoxyglucose. The growth of these mutants on D-xylose was not affected. A further series of fructose-negative and glucose-negative mutants were isolated by selecting for increased resistance To 2-deoxyglucose and by UV mutagenesis. Mutants, defective in each of the three hexose-phosphorylating enzymes found to be present in this yeast, were completely negative for growth on D-glucose,but could slowly convert this sugar to D-fructose. The conversion of D-glucose to D-fructose was hypothesised to be catalysed by the enzymes xylosereductase and xylitoldehydrogenase and experiments were conducted to investigate this possibility. Cell-free extracts from the wild type strain and several of The glucose-negative mutants were chromatographed on DEAE cellulose. The results of hexokinaseassays and anion exchange chromatography confirmed the existence of three hexose-phosphorylating enzymes in P.tannophilus. Two hexokinases which phosphorylated both D-glucose and Dfructose, exhibited F/Gratiosof1.3/1.0and3.0/1.0, while a glucokinase specific for D-glucose was also present.These enzymes were referred to ashexokinaseA and Bandglucokinase. Examination of the hexose-ATP-kinase profiles on DEAE- cellulose glucose, glucokinase of the wild type extract from cells grown on DO -xylose and glycerol indicated that the andhexokinaseB were constitutive, while hexokinaseA was inducible. Glucose repression ofxylosereductase and xylitol dehydrogenase was found to require an active hexokinaseA enzyme. This enzyme was purified from a glucokinase defective mutant by DEAE-cellulose chromatography, followed by affinity chromatography on CibacronBlueF3G-ASepharose (BlueSepharose) and examined further. The Km values for D-glucose and D-fructose were 0.36 and 2.28mM respectively. An estimated Vmaxfructose/Vmaxglucose was 1.5/1.0. When incubated with D-xylose in the presence of MgCl2 and ATP, the enzyme was inactivated. A strain of Pachysolentannophilus, defective in all three hexose-phosphorylating enzymes, was transformed with a plasmid carrying the cloned PII hexokinase gene from Saccharomycescerevisiae.The gene was expressed and the presence of the enzyme within the cells was demonstrated by DEAE-cellulose chromatography of a cell-free extract. As part of the overall plan to attempt genetic improvement in P.tannophilus, two superior ethanol producing mutants were hybridised and the segregants made available for fermentation trials at the Forest Research Institute. Hexose-negative mutants able to fermen D-xyloseinthe presence of D-glucose were selected for and subjected to fermentation trials. Several of these mutants produced promising concentrations and yields of ethanol from the fermentation of D-xylose, both as a sole carbon source and in a mixture of D-glucoseandD-xylose.Item Heterologous protein production in Kluyveromyces lactis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology and Bioprocess Engineering at Massey University, Palmerston North, New Zealand(Massey University, 1993) Russell, Carolyn MargaretIn this study, the recombinant yeast Kluyveromyces lactis CBS 683 : pCR1 was investigated as a model system for the production of a heterologous protein in a whey-based medium. The plasmid pCR1 has been constructed to express a wheat α-amylase enzyme in K lactis strains. The construct is based on the vector pCXJ-kan1, which has been derived from pKD1, a native plasmid of K lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Construct pCR1 produces an α-amylase from DNA isolated from a wheat cDNA clone which is controlled by a Saccharomyces cerevisiae PG K promoter. An electroporation method using a Bio-Rad Gene Pulser has been optimized for introducing heterologous DNA into K lactis yeasts. Selection of transform ants can be made using either the biosynthetic marker URAA or the G418 resistance gene, depending on whether the yeast is an auxotrophic mutant or a wild-type strain, respectively. Transformation was optimal at 4500 V cm-1, 25 J.LF, and oo n with 0.2 J.Lg plasmid DNA. Transformation efficiencies were comparable to those obtained using a PS1 0 Electropulsator, and were in the range 104-105 transformants per 107 cells per J.Lg DNA. Twenty-nine Kluyveromyces strains were examined for efficiency of transformation and fermentation performance on rich glucose and rich lactose media under high and low aeration in batch culture. Of these, K. lactis CBS 141 and CBS 683 were chosen for recombinant studies. The transformed yeasts K lactis CBS 141 : pCR1 and CBS 683 : pCR1 were qualitatively shown to produce an active α-amylase enzyme. The α-amylase was produced at a low level but could be measured using a modified starch-iodine assay. A typical yield of 6 U mr1 was obtained for batch growth of K Jactis CBS 683 : pCR1 in a rich lactose medium, where one unit is the amount of enzyme that will hydrolyze 0.1 mg starch in 30 minutes at 40°C when 4.0 mg starch is present. Both batch and continuous cultivation were used to investigate growth of the recombinant yeasts and, in particular, plasmid stability and protein production were examined. Three methods for measuring the stability of plasmid pCR1 in recombinant K /actis were statistically analyzed and compared, and two, the plate ratio and clearing zones methods, were chosen for use in the fermentation studies. Initial batch fermentation studies indicated plasmid pCR1 to be extremely unstable in K lactis CBS 141 : pCR1 and so only K. lactis CBS 683 : pCR1 was investigated further. Plasmid instability was also high in this latter yeast, with 50 - 60 % of cells becoming plasmid-free after 1 0 generations of non-selective growth in high aeration batch culture using a whey-based medium. In batch culture the stability of the plasmid pCXJ-kan1 was much higher, with minimal plasmid loss detected, and this indicated that the low stability of the plasmid pCR1 was probably due to the PGK-α-amylase DNA insert. The stability of plasmid pCR1 was shown to improve by using low aeration conditions, selective medium, or a growth temperature of 20°C in both batch and continuous culture. The use of a selective medium and a lower temperature also allowed the level of α-amylase to be maintained for an increased fermentation time, and the latter also gave an increased specific yield of α-amylase in continuous culture. Thus, this study has demonstrated the successful production of a wheat α- amylase from a K. lactis strain grown in a whey-based medium.