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Thermophilic enzymes and their impact on milk powder during storage

Milk powder quality and storage life can be compromised by functionality and flavour defects. These defects can be the result of chemical or biochemical reactions, such as the Maillard (browning) reaction, proteolysis and lipolysis. The New Zealand dairy industry has set specifications for thermophile numbers in milk powders that have been used for many years as an indicator of microbial quality. However, any correlation between the numbers of these bacteria and product quality is anecdotal, and there is little supportive scientific evidence. This research has set out to: (1) survey the cause of the problem for off-flavours in milk powder storage, mainly whole milk powders (WMPs) because they are the largest powder-product, and most likely to have problems associated with flavour; (2) develop assay methods for detecting possibly very low levels of protease and lipase in the WMPs; and finally (3) determine enzymatic effects in the WMPs during storage. The study has focused on the detection of protease and lipase activities. Several sensitive and commonly used assay methods were applied in reconstituted milk made from different batches of commercial WMPs. Both protease and lipase activities were detected, and the lipase activity was quantified at 0.7-1.1 U/g powder (pNP caproate unit), but the level of protease activity is yet to be accurately determined. A lipase was semi-purified from a WMP using Phenyl Sepharose hydrophobic chromatography. The enzyme is most active at 60°C under the conditions used, and has similar substrate specificity to the lipases produced by seven Bacilli isolated from a milk powder production stream. Although the source of the powder lipase is yet to be identified, data suggest that it is most likely from a thermophilic bacterium. Preliminary studies on the characteristics of the proteases and the lipases produced by the seven Bacilli showed that the proteases differ between isolates in term of pH optima and heat-stabilities, so do the lipases. The lipases are more heat-stable than the proteases in a buffer system. Under the protection of milk proteins, these enzymes can survive the heat-treatments applied in the milk powder manufacture process, and retain activities in the powder. Further studies on WMPs spiked with thermophilic proteases and lipases, or taken directly from the commercial process have confirmed that the proteases and the lipases are active in the powder (at moisture < 5%) during storage. Proteolysis was observed as the increase of 1% trifluoroacetic acid (TFA) soluble protein fragments and the decrease of solubility, and lipolysis was shown as the release of free fatty acids (FFA). The lipases showed the same specificities in a buffer system and in the powder. The levels of enzymatic products increase with increasing storage temperatures and time. Proteolysis resulted in poor solubility of WMP within one week of storage at 37°C. Lipolysis resulted in the levels of FFA, especially short chain butyric and caproic acids, exceeding the organoleptic threshold values under the same storage conditions. Nevertheless, none of the defects observed showed any correlation with thermophile numbers - one of the current specifications for quality control of WMP. Therefore, it is not thermophiles per se that affect milk powder quality during storage, it is the heat-stable enzymes, from whatever source.
Type of thesis
The University of Waikato
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