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    Characterisation of wine malolactic bacteria and acetic acid from fructose metabolism : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology, at Massey University
    (Massey University, 1993) Van Duivenboden, Robert John
    Twenty-four strains of wine Lactic Acid Bacteria from the genera Leuconostoc, Lactobacillus and Pediococcus were characterised with respect to their growth responses to ethanol, temperature, pH, ability to degrade wine organic acids and utilisation of carbon sources. A novel single broth culture (HFA) was developed for the determination of heterofermentation, mannitol formation and ammonia production. Some strains of Leuconostoc oenos were found to produce ammonia from arginine. The implications of this are discussed. The production of mannitol from fructose by heterofem1entative strains indicated potential acetic acid (volatile acid) spoilage risk for wrnes. To investigate this risk, semi-synthetic media were devised to simulate "stuck" yeast alcoholic fem1entation and the spoilage potential was evaluated under conditions of pH, substrate availability and ethanol concentration. Acetic acid production was analysed in the media by HPLC and found to occur at high levels from growth in the presence of fructose, but not glucose. The production was not affected by low pH or ethanol concentrations, or their combined effect. This indicated that acetic acid spoilage could occur under wine conditions. Other mechanisms of acetic acid production relative to this experiment are discussed. Erythritol and glycerol were detected in fermentation media but not quantified by HPLC. Their presence supported evidence of the activity of a novel glucose fem1entation pathway in Le. oenos.
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    Growth and metabolism of lactic acid bacteria in a model wine system and a red wine with emphasis on carbohydrate metabolism : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Technology (Food Technology) in the Faculty of Technology at Massey University, New Zealand
    (Massey University, 1990) Liu, Shao-Quan
    Studies were conducted to investigate the application of capillary gas­ liquid chromatography in analysis of wine carbohydrates, and the growth and metabolism of wine lactic acid bacteria in a synthetic model wine system. 1. Analysis of carbohydrates in wine using capillary gas-liquid chromatography Wine carbohydrates were analysed by capillary gas liquid chromatography of their acetate and aldononitrile acetate derivatives. A wide range of aldoses, polyols and disaccharides (30 compounds) were analysed in 55 minutes, using a single injection. All the derivatives were well­ separated except for ribose and rhamnose, which almost co-eluted. The method recovered spiked carbohydrates at 86 to 110% and had adequate reliability. This technique may be applied routinely to the analysis of other alcoholic and non-alcoholic beverages. 2. Growth and metabolism of wine lactic acid bacteria Malic acid and pH values had determinative effects on the growth of wine lactic acid bacteria. Malic acid stimulated the growth rate and cell population of - 122 and 252 at pH 4 and allowed their growth at pH 3.2. The absence of malic acid at pH 3.2 inhibited the growth of ­ oenos 122 and 252. The stimulatory effect of malic acid on growth was more striking at pH 3.2. This effect was not caused by the pH increases resulting from malic acid degradation. Malic acid had only a small stimulation on the growth rate of - plantarum 49 and f. parvulus 93 at pH 4 and their growth was suppressed at pH 3.5, irrespective of malic acid. These results imply that pH 3.5 is a critical value for the bacteriological stability of wine after malolactic fermentation. This study confirmed that sugars served as the main growth substrates for wine lactic acid bacteria and polyols did not act as growth substrates, with the exception of mannitol. Glucose and trehalose were the preferred substrates for all the bacteria tested. The significance of trehalose in relation to yeast autolysis in induction of malolactic fermentation was discussed. Wine lactic acid bacteria varied in the ability to utilise substrates. Malic acid, citric acid and arginine did not serve as single energy sources. Malolactic fermentation had a profound impact on substrate utilization by - oenos 122 and 252, yet seemed not to affect the substrate utilization of - plantarum 49 and f. parvulus 93. The presence of malic acid resulted in an increased utilization of sugars by k• oenos 122 and 252, and decreased utilization of arabinose by k• oenos 252. Trehalose utilization by - oenos 252 was not influenced by malolactic fermentation. The increased utilization of sugars may be the biological functions of malolactic fermentation. pH exerted a marked effect on the metabolism of k• oenos 122 and 252. More sugars were utilized at pH 4 and above than at pH 3.31 and below. k• oenos 122 attacked only a very minor amount of glucose and a portion of malic and citric acids at pH below 3.31. k• oenos 252 also used only a small quantity of sugars except for glucose, which was used completely, but degraded all malic and citric acid at pH below 3.42. These results strongly suggest that the degradation of malic acid, citric acid and arginine required the presence of fermentable sugars. This implies that the absence of fermentable sugars in wine may prevent malolactic fermentation. These results also justify the benefits of malolactic fermentation at low pH values (below 3.3). The role of wine lactic acid bacteria in formation of biogenic amines was clarified. - olantarum 49 was the only organism which reduced the levels of tyrosine and phenylalanine dramatically, indicating that this bacterium may be a potential producer of tyramine and phenylethylamine. - parvulus 93 did not markedly decrease the levels of any amino acids. Arginine was catabolised only by - 122 and 252 with the formation of ornithine and ammonia. Arginine was not degraded at low pH values (below 3.5), suggesting that arginine may not play any role in energy supply at low pH values. - oenos 122 and 252 did not significantly reduce the concentrations of other amino acids. The role of malolactic fermentation may lie in energy generation. Two potential energy-yielding mechanisms of malolactic reaction were proposed: ATP production through pyruvic acid cleavage (substrate level phosphorylation, pseudo-malolactic fermentation) and chemiosmotic ATP synthesis via formation of extra lactic acid (non-substrate level phosphorylation, real malolactic fermentation). It is speculated that ­ oenos 122 may employ the pyruvic acid cleavage pathway and generation of superfluous lactic acid may be adopted by - oenos 252, - plantarum 49 and - parvulus 93. The biological function of the extra lactic acid could be accounted for by the chemiosmotic theory that postulates energy (ATP) production through efflux of metabolic end-products (e.g., lactic acid) The origin of the superfluous lactic acid remains to be investigated. These findings suggest that the criteria for selection of starter cultures be redefined.
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    Carbohydrate effects on the inducement of the arginine deiminase pathway enzymes in wine lactic acid bacteria : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1998) Church, Peter R F
    Characterised by a fermentative sugar metabolism resulting in lactic acid as a major end product, the lactic acid bacteria (LAB) may be isolated from a broad range of sources. Dairy products, fermented vegetables, meats and baking products such as sourdough bread involve these organisms in a consistent and intentional manner in present times, no matter how accidental or fortuitous their initial involvement may have been. Alcoholic beverages such as beer, cider and, most pertinently here, wine are also affected by the presence of particular LAB. As conditions differ between nutrient environments so do the LAB found in wine differ to those isolated elsewhere - being both ethanol tolerant to the degree where growth is capable in 10% v/v ethanol and aciduric, able to maintain an active presence at acidic levels as great as pH 4 or less. This ability to remain viable during the primary yeast fermentation of juice into wine leads to these LAB being of no small practical interest in the wine industry. The process of malolactic fermentation (MLF) involves the wine LAB altering the law materials present in the juice and wine further, increasing the intricacies of the winemaking and final product. Primarily encouraged due to its effect of reducing wine acidity, MLF also alters flavour and aroma in what is generally thought to be an advantageous manner when applied correctly. Another factor thought to be of significance is an increase in biological stability. Found, for example, among the lactobacilli, pediococci and leuconostocs, the wine LAB are classed as either homofermentative or heterofermentative. Homofermenters commonly produce two moles of lactic acid per mole of glucose fermented, while heterofermenters form one mole each of lactic acid and carbon dioxide and varied quantities of ethanol and acetic acid from one mole of glucose. Natural or chance occurrences of wine LAB, whether as part of the microbiological community on the raw materials or from other sources - such as inoculation from contaminated equipment - were the original manner in which these organisms were introduced into the vinification equation. With the predilection towards quality control, standardisation and safety in the present day, the use of pure microbial starter cultures to initiate MLF has become increasingly widespread. In order to optimise the manipulation of wine LAB in both the laboratory and industry a thorough insight into their physiology and metabolism is an obvious necessity. Certain areas of interest have undergone more intensive study than others, with, for example, the catabolism of carbohydrates in both wine (Davis et al., 1986) and model wine systems (Liu et al., 1995a) having had a considerable amount of research compared to less primary sources of energy such as nitrogen metabolism.
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    The wine industry and the consumer : a study of the production, distribution and consumption of wine in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Ph. D. in Economics at Massey University
    (Massey University, 1984) Serrallach, Josephine
    This thesis is an investigation into production, distribution, and consumption of wine in New Zealand, with particular consideration of the effects of the wine industry's structure and behaviour on consumer interest. Government protection policies have greatly influenced expansion and structural development of the industry. Protection of the existing local industry was mainly effected by reducing opportunities for competition in two ways: implementation of import control regulations which have restricted competition from overseas wines, and introduction of licensing laws which have favoured control of the wine trade by a limited number of licence holders. Investigation into industry ownership patterns showed the presence of vertical and horizontal integration at various stages of the marketing system. Mergers and takeovers have resulted in the formation of a powerful group of companies which control wine marketing. These companies are usually associated with breweries, large distribution companies, and companies with overseas connections. In addition, concentration ratios calculated at the production level provided evidence that the wine industry is highly concentrated. In 1981 six firms controlled about 87% of the market. The degree of concentration and the ownership patterns of the New Zealand wine industry are indicators of its oligopolistic structure and market power. This market power is not altered by the presence of overseas wines in New Zealand as the volume of imports is very small (5% of total sales in 1978-1979). Limited competition and oligopolistic industry structure have contributed to the industry's behaviour in determining quality of products, prices and output. Lenient regulations governing wine-making have also affected wine quality. Adulteration of some New Zealand wines, evident from statistical data, was subsequently substantiated by the Consumer Institute analysis of local wines. Statistical data on industry production indicated that when the industry faced over-supply, output was reduced and prices were maintained. Legislation for the protection of consumer interest in New Zealand has been mainly directed at prevention of malpractice that may result from monopolies, mergers and takeovers, and at control of certain trade practices judged to be against the public interest (Commerce Act 1975). Legislation has also aimed to improve the availability of information to consumers and to regulate quality standards (Food and Drug Regulations 1973, Amendment No. 5, 1980/73). A consumer study was carried out in Palmerston North to relate the effects of industry structure and government intervention to actual consumer requirements and consumer perceptions of the products available in the New Zealand market. A survey, which involved a randomly selected sample of 237 respondents, helped to identify three main groups: 32% of the adult population surveyed were regular consumers of wine, 38% used wine occasionally, and the remaining 30% did not drink. Survey results indicated a reduction in the growth of wine consumption. Consumer preferences and consumer satisfaction with quality and prices of New Zealand wines, were also evaluated from survey results. Findings from the consumer survey showed that a group of wine users with similar socio-economic status (mainly professional people) was responsible for more than half of the total wine consumption. To provide an in-depth analysis of the perceptions and preferences of this particular group of regular consumers, a pilot study based on the application of multidimensional scaling was carried out. Results of this study indicated that consumers considered some New Zealand wines to be close substitutes for some overseas wines (e.g., a New Zealand Riesling for the German wine "Blue Nun"). New Zealand sparkling wines, however, were not considered to be good substitutes for overseas sparkling wines. No wines in the sample were closely identified with any of the consumer preferences (expressed as ideal points in a multidimensional scaling representation). This indicated that the wines included in the sample did not totally fulfil the requirements of the selected group of regular consumers, either in terns of quality or price. Research findings from both industry and consumer studies suggested that consumers had been deprived of the benefits of competition. Furthermore, if consumer interest is to be protected, a more competitive system has to be developed through liberalisation of import policies and licensing laws.
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    Arginine metabolism in malolactic wine lactic acid bacteria and its oenological implications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1993) Liu, Shao-Quan
    L-Arginine is a major amino acid found in grapes and wine which is degraded by some wine lactic acid bacteria (LAB). The mechanism of this degradation and its oenological implications were examined in this research. It was found that wine LAB able to degrade arginine do so by means of the arginine deiminase pathway, demonstrated by measuring the enzyme activitie since l l-free extracts: arginine deiminase, ornithinetrans carbamylase and carbamate kinase. These enzymes we represent in most heterofermentative lactobacilli and leuconostocs, but were absent in homofermentative lactobacilli and pediococci. The presence of arginine increased the activities of arginine deiminase pathway enzymes in heterofermenters, but failed to induce these enzymes in homofermenters even under conditions of low glucose concentration (1g/L). Glucose did not repress arginine utilisatio n but fructose appeared to do so, as fructose and arginine were metabolised sequentially, with arginine being metabolised mainly after utilisation of the fructose. D etailed studies o n Leuconostoc oenos OENO, Lactobacillus b uchneri CUC-3 and Lactobacillus brevis 250 showed that arginine was converted stoichiometrically to ammonia and ornithine as the major end-products and that arginine catabolism could supply energy (ATP) to support growth. lt was also demonstrated that citrulline was excreted during arginine catabolism by both the lactobacilli and the leuconostoc. Some of the excreted citrulline was reassimilated and catabolised after arginine depletion by the lactobacilli, but not by the leuconostoc. The implication of citrulline excretion for the wine industry was explored by studying the formation of the carcinogen ethyl carbamate (urethane) in a synthetic wine and a white wine, since citrulline is a known precursor of ethyl carbamate. During growth of Le. oenos OENO and Lb. buchneri CUC-3 in the synthetic wine and wine, significant amounts of ethyl carbamate were found i n the two wine types upon heat treatment of samples. The formatio n of ethyl carbamate correlated well with arginine degradation and citrulline excretion. Citrulline excretion during arginine degradation is of concern to the winemaker, since the reaction of citrulline and ethanol to form ethyl carbamate has been shown by other workers to occur even at normal wine storage temperatures. Winemakers, therefore , should avoid using argininedegrading LAB starter cultures for inducing malolactic fermentation (MLF). In addition, spontaneous MLF in wine by undefined LAB strains should be discouraged, as this may lead to formation of ethyl carbamate precursors. Ammonia detection with Nessler's reagent provides a simple, rapid test to assess arginine degradation by wine LAB in a complex medium, but is useful only for strains showing strong ammonia formation . The more sensitive enzymatic determination of ammonia is required for strains showing weak ammonia formation.