Chemical and Biomolecular Engineering - Theses
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ItemCharacterisation of buffalo milk, yoghurt and cheese( 2014)Buffalo provide the second largest source of milk in the world after bovine animals. Knowledge and past research on buffalo milk and its product properties, however, is limited, particularly research relevant to Australian manufacturing conditions. This thesis asked four key questions: whether the chemical composition and physicochemical properties of buffalo milk, particularly the microstructure and proteomics of the milk fat globule membrane, significantly differ to bovine milk; whether these differences in milk are translated into the differences in the properties of the resulting yoghurt and cheese products; how to improve the quality of buffalo products; and how the properties of the buffalo cheese change over storage time and vary when produced by different producers. Techniques employed in this thesis included: confocal laser scanning microscopy (CLSM) and cryo-scanning electron microscopy (cryo-SEM) for microstructural investigation, controlled-strain and controlled-stress rheometers for rheological examination, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) for mass and protein identification. Other techniques such as the inductively coupled plasma atomic emission spectroscopy (ICP-OES), high performance liquid chromatography (HPLC), light scattering and colorimetric methods were also used for the characterisation of chemical composition and physicochemical properties of buffalo milk and buffalo milk products under conditions relevant to an Australian manufacturing setting. This work showed that Australian buffalo milk had a richer composition than Australian bovine milk, including a higher concentration of fat, protein, total solids and calcium, consistent with other international studies. High performance liquid chromatography analysis showed that the organic acid profile was significantly different between the two milk types, with a lower concentration of orotic and uric acids in buffalo milk of particular note. Buffalo milk had larger fat globules with a broader size distribution. Confocal laser scanning microscopy observation showed a heterogeneous distribution of phospholipids with the occurrence of non-fluorescent domains that were hypothesized to be rich in sphingomyelin. These domains had various sizes and shapes at room temperature. The domains became larger and more irregular at 4oC and were smaller and more circular at 40oC or 60oC. Using a combination of both in gel digestion and in solution digestion methods, followed by liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis, 184 proteins were identified within the buffalo milk fat globule membrane. This is the largest profile of buffalo milk fat globule membrane proteomics to date. Further quantitative comparisons revealed that the buffalo milk fat globule membrane contained more xanthine dehydrogenase, platelet glycoprotein 4, heat shock cognate and cacineurin B homologous protein but less lactadherin and polymeric immunoglobulin receptor than the bovine counterpart. These differences in the properties of the two milk types affect the processing required during manufacturing and properties of the resulting products. The production of buffalo yoghurt does not require the addition of milk powder or thickener, as occurs for bovine yoghurt, due to the initial higher total solids content of buffalo milk. Yoghurt produced from buffalo milk, however, exhibited a significantly higher level of syneresis (17-20% w/w vs. 1-3% w/w) and poorer rheological properties compared to bovine yoghurt. Buffalo yoghurt was more susceptible to deformation and less able to recover the original network structure after deformation. These properties could be linked to the porous microstructure consisting of large fat globules that tended to disrupt the protein network. An optimisation of the process parameters was therefore performed to improve the quality of buffalo yoghurt, especially to reduce the syneresis. This optimisation initially considered the effect of varying fermentation temperature. Buffalo yoghurt was fermented at three different temperatures: 37oC, 40oC and 43oC. Buffalo yoghurt fermented at 37oC or 40oC required a longer fermentation and gelation time than at 43oC but exhibited a less porous microstructure with reduced syneresis (from 17-20% w/w to 14-16% w/w). The storage modulus was higher at lower temperatures but other rheological properties including the thixotropy, flow behaviour index and consistent coefficient were not improved by decreasing the fermentation temperature. The limited improvement in syneresis and rheological properties of buffalo yoghurt at different fermentation temperatures indicated that further optimisation of buffalo yoghurt production was required. Homogenisation has been reported to improve the quality of bovine yoghurt, including syneresis, texture and rheological properties. The effect of homogenisation on the properties of buffalo yoghurt, however, has not been explored systematically, despite the significantly higher fat content and larger fat globules in buffalo milk. In this experiment, buffalo yoghurt was produced from either milk homogenised at 80 bar or 160 bar. It was shown that homogenised buffalo yoghurt exhibited an improved microstructure consisting of a highly interconnected protein network with thick protein strands and small embedded fat globules. These structural changes resulted in a significant decrease in syneresis and thixotropy and led to a considerable increase in the storage modulus, gel firmness and flow behavior index. While both homogenisation pressures were effective, a higher homogenisation pressure of 160 bar resulted in a lower gel firmness and storage modulus, possibly due to the presence of bigger fat-protein clusters within the milk. These results suggest that a homogenisation pressure of 80 bar could be optimal for improving the quality of buffalo yoghurt and reducing syneresis. Traditional or high moisture buffalo Mozzarella cheese has long been produced but most of the studies in the literature to date have focused on low moisture bovine Mozzarella cheese. In this project, the microstructure and functional properties of laboratory prepared and commercially purchased high moisture buffalo Mozzarella cheeses were studied and compared to commercially purchased high moisture bovine Mozzarella cheeses. Laboratory cheeses were produced at an average yield of approximately 19% and the quality was stable during seven days at cold storage. The whey collected during buffalo cheese production was rich in calcium, lactose and contained an unidentified trisaccharide. Buffalo and bovine Mozzarella cheeses obtained from different producers were found to be significantly different in their chemical composition, organic acid profile and microstructure but had similar hardness and meltability. The buffalo cheeses exhibited a significantly higher ratio of fat/protein and larger fat patches with a less dense protein network within the microstructure compared to the bovine cheeses. These results reflect the effects of processing conditions and the milk types employed by different producers on the resulting cheese properties. They also demonstrate the potential application of buffalo cheese whey as a good source of prebiotics, sugars and minerals. These differences in the microstructure and chemical composition could also be used to identify the milk species of origin in commercial cheese products. In summary, buffalo milk exhibited significant differences from bovine milk, which in turn affected the properties of yoghurt and cheese. Buffalo yoghurt, prepared using the current industrial standard approach, exhibited a high degree of syneresis, a porous microstructure and poorer rheological properties than bovine yoghurt. Lowering the fermentation temperature and the utilisation of homogenisation lowered the syneresis and improved the microstructure and rheological properties of buffalo yoghurt. Buffalo Mozzarella cheese showed large variations and significant differences in microstructure and physicochemical properties compared to bovine Mozzarella cheeses. These results answer the four key questions posed in this thesis. The results presented are useful for buffalo farmers and manufacturers seeking to better understand and control buffalo milk quality and the properties of milk products, as well as the broader community of dairy researchers.