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Item Impact of protectant uptake on the shelf-life of dried Lacticaseibacillus rhamnosus(Elsevier, 2022-01) Priour S; Welman A; Singh H; Ellis ATo improve the survival of dried probiotics, it is advised to expose the bacteria to protectants prior to processing, allowing equilibration of internal solutes. However, optimal conditions for this exposure remain unclear. This study examined solute uptake by Lacticaseibacillus rhamnosus HN001 (formally known as Lactobacillus rhamnosus HN001) at 4 °C and 20 °C, over exposure times of 0–240 min. The cells were exposed to hyperosmotic solutions of glucose and sucrose, two potential protective sugars, which are metabolisable and have different molecular weights. Sugar uptake was analysed through HPLC, while the impact on cell viability after freeze-drying was examined at 30 °C and 40 °C. The interactions between cell biomolecules and sugars were examined using Nano DSC. Results showed that the sugars were rapidly taken up by the cells, independent of temperature. At 20 °C, glucose was readily metabolised, eventually resulting in loss of cell viability during storage. Conversely, the Nano DSC study revealed interactions between the cells and sucrose, potentially providing some explanation as to the stability of the cells. In conclusion, sugar type and exposure temperature were shown to exert a significant effect on the viability of Lacticaseibacillus rhamnosus. Nano DSC is a promising technique to understand the protectant and cells’ interactions.Item An exploratory analysis of the factors impacting on Chinese consumer trust in lactic acid bacteria preserved beef and its mediation impact on purchase intention : a thesis presented in partial fulfillment of the requirement of the degree Masters in Agricommerce, Massey University, Palmerston North, New Zealand(Massey University, 2019) Chen, JinyaEvery year, worldwide, millions of people die and many are hospitalized due to food-borne diseases and illnesses caused by the consumption of contaminated food. Food safety has continued to be a concern for consumers, the food industry, and regulatory agencies. In China, there is almost a constant stream of reports about various food safety issues. Chinese consumers are concerned about the need for healthier and safer food. The development of science has provided more opportunities and possibilities to change the way we live. However, consumers’ overall confidence in Chinese food is not high and they are increasingly skeptical about new food. This research focuses on a new and not yet launched biological food, Lactic Acid Bacteria preserved vacuum-sealed chilled beef (LAB beef), as an example to examine what factors would have a significant correlation with consumers’ trust in this product and to examine if trust is the key factor impacting on consumers’ purchase intention. In order to complete the study objectives, a self-completed social survey was conducted in Shanghai City and Chengdu City, totaling 514 respondents. The analysis methods used included a measure of correlation, Gamma, principal component analysis and structural equation modeling. SPSS, Excel and Amos software were used. One outcome of this research was the finding that a number of socio-demographic factors were not strongly correlated with consumer trust in LAB beef, unlike some previous research that found such relationships with trust in new food technologies. Personal beef consumption habits, consumers’ past purchase experience with current used beef, products, product knowledge and food safety concerns based on their awareness, experience and media exposure were found to be important in establishing trust in LAB beef. The second outcome of this research is the confirmation of the importance of trust in determining consumers’ willingness to buy LAB beef, as well as the confirmation of the mediation effect of trust in explaining the underlying causal relationship between a number of independent variables and the dependent variable, willingness to buy.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 A comparative study of an aminopeptidase from lactic acid bacteria: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1992) Midwinter, Robyn GillianAminopeptidase enzymes from the proteolytic systems of S.salivarius subsp.thermophilus Lactococcus lactis subsp.cremoris and Lactococcus lactis subsp.lactis have been investigated. An aminopeptidase was purified to near homogeneity from a crude cell free extract of S.thermophilus 5109. The enzyme had a native molecular weight of approximately 96kDa determined by gel-filtration, and a subunit molecular weight of 98kDa, determined by denaturing polyacrylamide gel electrophoresis, showing the native enzyme to be a monomer. The aminopeptidase activity was optimal at pH 7.0 and 35°C. The enzyme was inactivated by p-chloromercuribenzoic acid, iodoacetic acid,the chelating agents EDTA and 1,10-phenanthroline and the divalent cations Cu2+, Zn2+ and Co2+. The aminopeptidase was not inhibited by the serine protease inhibitor PMSF and only minor inhibition occured with the inhibitor No:-p-tosyl-L-lysine chloromethyl ketone (TLCK). The aminopeptidase was capable of hydrolysing several amino-acyl amido methyl coumarin (AMC) and p-nitroanilide (pNA) derivatives, particularly those of lysine, arginine and leucine. The enzyme showed greatest activity with lysyl derivatives (and is therefore referred to in this thesis as a lys-aminopeptidase). The enzyme was able to degrade several oligopeptides by progressive cleavage of the peptide bond but did not hydrolyse peptides containing a proline or aspartic acid residue in the second position. The aminopeptidase activity was dependent on the size of the peptide in that generally only peptides with more than three amino acids were degraded. The aminopeptidase had no endopeptidase or dipeptidase activity. Five different amino-acyl p-nitroanilides derivatives and two amido methyl coumarin derivatives were used to determine the kinetic parameters of the aminopeptidase. The Km values obtained for all the substrates tested were similar, with the exception of ala-pNA, for which the Km value was significantly higher. On the basis of the distribution of activity between different cell-fractions the lys aminopeptidase appears to be localised intracellularly. An aminopeptidase was also partially purified from cell-free extracts from Lactococcus lactis subsp.cremoris AM2 and Lactococcus lactis subsp.lactis ML3. The aminopeptidase from L.cremoris AM2 was shown to have a molecular weight of 106kDa and was a monomer. It showed optimal activity at a pH of 7.0 and 450c. The aminopeptidase activity was inhibited by metal-chelators, SH group inhibitors and TLCK. The aminopeptidase hydrolysed lysyl-, arginyl- and leucyl-p-nitroanilide derivatives, but had little or no activity with other pNA substrates. The aminopeptidase from L.lactis ML3 had a molecular weight of 100-105kDa and was monomeric. The optimal activity for the aminopeptidase was at pH of 7.0 and 40°C. The enzyme was inactivated by metal-chelators, sulphydryl inhibitors and by TLCK. Like the aminopeptidases from the other two strains the ML3 aminopeptidase was very specific hydrolysing lysyl-, leucyl- and arginyl-pNA but with very little or no activity with other amino-acyl derivatives.Item 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 JohnTwenty-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.Item 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-QuanStudies 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.Item 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 EMalolactic 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.Item A study of aminopeptidases from lactic streptococci : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1989) Lloyd, Richard JohnTwo arninopeptidase enzymes from the proteolytic system of Streptococcus lactis 4760 have been studied. An X-Prolyl dipeptidyl arninopeptidase has been purified and characterised. The enzyme has a native molecular weight of approximately 150 kDa determined by gel filtration, and a subunit molecular weight of 83 000, determined by denaturing polyacrylarnide gel electrophoresis, showing the native enzyme to be a dimer. It is inhibited by phenyl methyl sulphonyl fluoride and is active over a pH range of 6 - 9. A range of X-Prolyl-amido methyl coumarin (X-Pro-AMC) derivatives with different aminoacyl residues in the X position have been used to define the steady state kinetic parameters. The Km and kcat values obtained with all of the X-Pro-AMC substrates tested were similar, with the exception of Glu-Pro-AMC, which gave a somewhat higher Km value. The action of the enzyme in degrading small peptides has been studied. It was found to be capable of removing X-Proline residues from peptides, except where two proline residues are situated in consecutive positions. A Lysyl-arninopeptidase has been partially purified and its characteristics studied. This enzyme has been shown to have a native molecular weight of approximately 78 000. It hydrolyses lysyl-, arginyl-, and leucyl-arnido methyl coumarin derivatives, but has little or no activity with other arninoacyl-AMC substrates. It also catalyses the removal of lysine and arginine residues from the amino-terminus of short peptides. The partially purified arninopeptidase preparation also has endopeptidase activity which is probably due to contamination by a separate enzyme. The individual and combined effects of these two enzymes on -casein-derived oligopeptides (produced by proteolytic action of the S.lactis proteinase) have been studied. These results indicate that these enzymes may be important in degradation of some casein-derived peptides during cheese ripening, while other peptides are resistant to hydrolysis.Item Peptide metabolism in the lactococci and its regulation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1994) Moore, Ian LintonAspects of peptide metabolism in the lactococci have been investigated to increase the understanding of how these nutritionally fastidious bacteria, which have a central role in the dairy manufacturing industry, are able to grow in a complex medium such as milk. Peptide metabolism by lactococci in milk encompasses the processes by which large oligopeptides, produced from milk-caseins by the extracellular activity of the cell wall-associated proteinase, are converted into an intracellular pool of metabolisable amino acids. This involves the activities of both membrane-bound transport systems and peptidases. Early research into lactococcal peptide utilisation has proposed significant differences between Lactococcus lactis strains with respect to the mechanisms by which these bacteria utilise peptides in their environment. More recent studies of the lactococcal peptide carrier systems, based on intensive studies of only a single strain, have proposed a major role for a carrier system capable of transporting di- and tripeptides, and a subsidiary role for another system transporting oligopeptides containing four or more residues. Yet to date, peptidases with an extracellular location capable of degrading the large casein-peptides into smaller peptides have not been isolated. This current study has attempted to investigate more fully the in vivo activity of the oligopeptide transport system, and to assess whether it may have a more fundamental role in peptide utilisation than previous work has suggested. For this study a model series of homologous peptides of increasing size from the dipeptide Val-Gly to the octapeptide Val-Gly7 , all based on the essential amino acid valine, was used. The larger peptides in this series, Val-Gly3 , Val-Gly4 and Val-Gly7 , were synthesised for this work. The ability of Lactococcus lactis subsp. cremoris E8 8 to transport these peptides, and to grow in a chemically defined medium where they constitute the sole source of the essential amino acid valine, was studied . Preliminary peptide uptake studies were also performed using oligopeptides derived from a proteolytic cleavage of β-casein. The collective results of these studies suggest that the upper size limit, and the relative activity of this transport system, may be sufficient to permit this strain to utilise relatively large casein-derived oligopeptides without the need for hydrolysis into smaller peptides and free amino acids. A comparative study of peptide transport by a number of Lactococcus lactis strains was undertaken to investigate previously published observations indicating significant differences in the mechanisms of peptide uptake between lactococcal strains. While the results of this comparative study are consistent with the general model proposing two separate peptide carrier systems, they have revealed that significant differences can exist between strains in the relative activities and possible substrate specificities of these transport systems consistent with previous work that the lactococci have only two peptide carrier systems. These observations imply the need for caution in extrapolating the results obtained from the study of a single strain to lactococci as a whole. In contrast to the finding of significant strain differences with respect to the relative rates of peptide transport, a comparative study of the relative activity of six different intracellular peptidases showed relatively few differences in peptidase activity between strains. An investigation was also carried out to assess whether the peptidases and transport systems involved in the utilisation of peptides were nutritionally regulated. No clear evidence was obtained for the significant induction of either the intracellular peptidase complement or the di-/tripeptide transport system. An attempt was also made to isolate a mutant of Lactococcus lactis subsp. cremoris E8 unable to utilise dipeptides, to assess whether the di-/tripeptide transport system or the intracellular dipeptidase of this strain were essential to casein utilisation. This attempt was not successful.Item 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 FCharacterised 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.