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    Mathematical modelling of salt diffusion in dry-salted cheese : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Engineering at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Keck, Meghan Emily
    Control of salt uptake into cheese curds is vital to the production of safe, functional, and consistent cheese products. Development of mathematical models describing the mechanisms affecting salt uptake, namely the Fickian diffusion of salt into curds and osmotic pressure differential induced whey expulsion, are necessary to control curd properties, optimize salt uptake, and reduce final product inconsistency in commercial cheesemaking plants. Novel image analysis techniques were developed to assess whey expulsion behaviour in individual model renneted skim milk gels under different brining conditions. Whey expulsion results were combined with salt uptake data to develop mechanistically-derived mathematical models of the simultaneous whey expulsion and salt uptake under brining conditions. Whey expulsion data was combined with gel meso-structural properties to estimate the pressure gradients driving the whey expulsion behaviour. Finally, the simultaneous salt uptake and whey expulsion models were used to model salt and whey transport in model renneted gels treated under different dry salting conditions and compared to samples collected from an industrial cheesemaking plant. This work developed new techniques to evaluate whey expulsion in individual curds, demonstrated the importance of accounting for whey expulsion in the evaluation of salt uptake and modelling, and applied mathematical models describing simultaneous salt uptake and whey expulsion to milk gels exposed to brining and dry salting conditions.
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    The effects of pH-stat long-term lactic acid bacterial activity prior to curd formation on the development of cheese structure in a fat free model cheese : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Food Technology at Massey University, Manawatu, New Zealand
    (Massey University, 2018) Khodamoradi Dashtaki, Abbas
    Cheese ripening is an important step in most cheese production practices during which a tasteless fresh cheese is converted to a tasty and flavourful product with specific textural attributes. However, the complexity of its composition (pH, solubilisation of calcium from the colloidal casein proteins, salt concentration, acid production rate, indigenous and added enzymes, residual activity of the enzymes etc.) coupled with the length of time associated with manufacturing which in some cases can be in excess of two years has made it a complicated area of study. The influences of the composition and process contributors are confounded and it is impossible to connect the impact of one particular parameter on cheese making steps and quality attributes. pH has proven to be an important influential factor to influence the extent of effects of other parameters with significant influence on other ruling parameters in milk, curd and cheese. Proteolysis during ripening is the most important physicochemical pathway to define the quality of cheese. One of the major factors governing cheese ripening reactions is the starter bacteria. This study has aimed to characterise the effects of starter bacteria activity on curd formation and resultant cheese textural attributes of the long fermented cheesemilk. By developing a pH-stat system, long fermentations carried out to assess the proteolytic activity of selected starter lactic acid bacteria (LAB) on a milk based medium before rennet addition. It was attempted to assess the degree of hydrolysis of cheesemilk through extended bacterial fermentation, conducted under pH-stat conditions, prior to curd formation. The effects of the bacterial activity on casein proteins during pH-stat long term (PSLT) fermentations were evaluated by assessing proteolysis index from pH4.6 soluble nitrogen as a fraction of total nitrogen (pH4.6SN/TN). The proteolysis of proteins during PSLT was further assessed by doing reverse-phase high performance liquid chromatography (RP-HPLC) on 70%Ethanol soluble (70%EtOHS) and insoluble (70%EtOHI) fractions of pH4.6 soluble fraction of the samples. The effects of PLST fermentation on formation of small-size peptides were assessed by quadrupole time-of-flight mass spectrometry (Q-ToF MS) on the 70%EtOHS fraction. The effect of PLST fermentation on ‘depth of proteolysis’ during cheese ripening were assessed by analysing the quantity of free amino acid (FAA) formed in resultant cheese after 12 months storage at 4°C. The impact of PLST fermentation on gel formation attributes were assessed by doing dynamic law amplitude oscillatory rheometry (DLAOR). The consequent effects on resultant cheese texture were evaluated using texture profile analysis (TPA). The impact of PSLT on microstructure were assessed by confocal laser scanning microscopy (CLSM). The results provided evidence for the adequacy of developed fermentation to conduct PSLT with reproducible results. High correlation between the parameters of the PSLT fermentation system were obtained. The proteolysis index measured from the PSLT fermentations with different durations showed evidences on the significance of LAB proteolytic system on cheese milk prior to curd formation. The proteolysis index for the longest fermentation prior to curd formation was 5% which was comparable to day one cheese proteolysis index, in presence of rennet, in most cheeses varieties. Peptide profiling of the 70%EtOHS and 70%EtOHI sub-fractions of pH4.6S showed significant (p<0.05) effects arising from PSLT fermentations. Analysis of FAA of ripened cheese also showed a significant increase (p<0.05) in the samples with longer PSLT (20 times increase in total free amino acids compared to non-fermented treatment) fermentations. The differences in gelation behaviour of the sample and textural attributes of cheese and microstructure of final cheese were connected to the extent of proteolytic activity of LAB during PSLT fermentations. The hardness of cheese significantly (p<0.05) decreased (up to ~60%) by increasing fermentation duration over the studied timescale.
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    Substrate specificity and structural investigation into PepO and PepW : two peptidases from Lactobacillus rhamnosus : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Yates, Karen Maree
    The proteolytic systems of lactic acid bacteria have important roles in the maturation and flavour development of cheese. Lactic acid bacteria pepetidases contribute to the taste of cheese through the production of low-molecular weight peptides and free amino acids. Although some lactic acid bacteria peptidases have been structurally and enzymatically characterised for their substrate specificity, there are some that are yet to be completely biochemically characterised. The aim of the present study was to investigate the substrate specificity and three-dimensional structure of two peptidases that could potentially be used as a tool to modify and control cheese bitterness and possibly other flavour attributes from Lactobacillus rhamnosus, PepO and PepW. The pepW gene was successfully cloned from L. rhamnosus into an E. coli expression system. Recombinant PepW was purified to homogeneity and was shown to exist as a hexamer of 50 kDa subunits. Recombinant PepO was expressed from a previously established L. lactis expression system and purified to homogeneity. PepO was shown to exist as a 70 kDa monomer, and function as a metallopeptidase. Pepo and PepW were shown to selectively hydrolyse chymosin-derived bovine β- and κ-casein peptides, and casein peptides extracted from Cheddar cheese. One conclusive PepO cleavage site that had not been previously characterised was identified. This was the β-casein peptide bond between Leu₆-Asn₇. Several possible PepO and PepW cleavage sites in αs₁-, β- and κ- casein were identified, suggesting that PepO has a broad endopeptidase activity, whilst PepW has a specific exopeptidase activity. Pepo and PepW crystals were successfully grown for structure determination by x-ray crystallography. Native data sets were collected for both PepO and PepW, and derivative data were collected for PepO. Structure determination was attempted using Multiple Isomorphous Replacement and Molecular Replacement techniques. Results from the substrate specificity and structural investigation of the L. rhamnosus peptidases, PepO and PepW, are presented in this thesis.
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    Influence of the structure of rennet-induced gels on the cheesemaking process and cheese composition : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in food technology, Institute of Food, Nutrition and Human Health, Massey University
    (Massey University, 2002) Chiang, Mei-Li
    The purpose of this study was to investigate the effects of some selected processing conditions on the formation, structure and properties of rennet-induced milk gels and the impact of these conditions on the cheesemaking process and final cheese composition. The effect of pH, temperature, calcium chloride addition, rennet concentration and protein concentration on rennet gels were determined using rheology, permeability measurement and confocal microscopy. In rennet gels formed at pH 5.8, 6.2 and 6.5, the storage modulus (G') increased with a decrease in pH while the gelation time (GT) increased with pH increase. The permeability coefficient (B) increased with a decrease in pH. Rennet gels made at 25, 32 and 40°C showed that the G' values increased while gelation time decreased with an increase in temperature, with a maximum G' at 32°C. The B values increased with an increase in temperature. The rennet gels made with zero, 0.01% and 0.02% CaCl addition showed a slight increase in G' with CaC addition, but no significant differences in B values. For gels made with 40, 80 and 120μl rennet addition/l, the G' values increased slightly with an increase in rennet concentration, but there were no significant differences in B values. For rennet gels with different protein contents (range from 3.45 to 5.10%), the G' values increased whereas B values decreased with increasing protein content. All the confocal micrographs corresponded well with the results from the permeability and rheological analyses. Two rennet gel systems were developed in this study. "High syneresis" gel systems were made using low pH, high temperature and normal protein concentration and "low syneresis" systems with high pH, low temperature and high protein concentration. These two systems were used in cheese manufacture in a pilot plant. It was expected that 'high syneresis' gel system would expel more whey and result in low moisture cheese, while the 'low syneresis' system would yield cheese with higher moisture content. It was found that the cheese produced from high syneresis system had higher moisture content than the cheeses made from low syneresis system. It was opposite to what was expected. The set to cut time and acid production (starter levels) are thought to be the reasons for the outcome of the first pilot plant trial. The process conditions were redesigned to investigate the effect of gel firmness and pH on the composition of cheese. Similar results were found in the second pilot plant trial. The factors involved in the cheesemaking process are much more complex than what has been investigated in the present study. Therefore, further investigations on the other factors affecting cheese moisture content are recommended (e.g. syneretic power).