Lipase activity in Swedish raw milk

Bovine milk contains several enzymes which can affect the quality of milk. One of these enzymes are lipases which hydrolyse the triacylglycerides in milk. In this process off-flavours, odour and product defects are generated. By controlling the enzymes activity, milk quality can be increased and therefore milk and dairy products can be stored for a longer time. This is desirable since the world demand for long shelf-life milk and milk products is increasing. To monitor the quality of raw milk and control the enzyme activity, a well-functioning method to detect lipase activity is desired. Thus, the aim of this thesis was to investigate and improve an existing method to determine lipolytic activity which is based on a fluorescent approach. With this method, a sensitive fluorescent measurement of the lipase activity directly in the natural milk environment is possible. Once the method was evolved, raw milk samples from different regions in Sweden and from different origins, i.e. farm and dairy, were investigated and evaluated for possible differences in lipase activity. No significant difference was found between samples from farm and dairy origins. Between the different regions a significant difference was discovered. The lowest lipase activity in milk was found in milk from the south (Skånemejerier) followed by milk from the mid region of Sweden (Arla) and the highest lipase activity was seen in milk from the north (Norrmejerier). The lipase activity was also correlated to other previously measured properties of the milk. A significant negative correlation to some long chain fatty acids could be seen. This indicates that long chain fatty acids inhibit lipase activity in the milk which has previously been observed in the adipose tissue of rats and in goats milk. The long chain fatty acid content in milk can be influenced by the feed. Thus, the results of this thesis indicate that the raw milk quality could be improved by increasing the amount of long chain fatty acids in the milk through the feed and thereby reduce lipase activity.Lipases are enzymes that split the fat in milk. This reaction is called lipolysis and results in the release of free fatty acids. These free fatty acids give off-flavours, odour, and product defects to the milk. By controlling the enzyme activity, milk quality can therefore be increased and the shelf life of milk and dairy products can be extended. This is desirable since the world’s demand for long shelf-life milk and milk products is increasing. Hence, it is useful to have a well-functioning method which can detect lipase activity to monitor the quality of raw milk. There are some existing methods to measure the lipase activity. However, the optimal assay for routinely measurements of dairy products for predictive purposes is still not devised. One method was published that measures the lipase activity in the natural milk environment, which has a limited number of experimental steps and has a high sensitivity. However, previous attempts to repeat this method failed. Therefore, the scope of this project was to further investigate that method and improve it. The theory behind this method is to add a substrate to the milk and let it incubate for a specified time. This substrate consists of a fatty acid that is attached to a molecule called 4-methylumbelliferyl. This compound is non-fluorescent unless a lipase splits off the fatty acid to yield one molecule of highly fluorescent 4-methylumbellifery and one molecule fatty acid. Thus, if there is more lipase activity present, more fatty acids are split off and the fluorescence is higher. A fluorescence measurement is not possible in a turbid media like milk and therefore the turbidity needs to be decreased before a fluorescence measurement can be carried out. In milk, the fat droplets and proteins that are present in form of casein micelles cause the turbidity. The basic procedure to remove turbidity is a defatting step, followed by the addition of two solutions to remove the turbidity of the milk by unfolding the protein and make it go into solution. Furthermore, these added solutions stop the enzyme conversion by lowering the pH and thereby denature the lipase. Fluorescence intensity is highly dependent on pH and thus the last solution also rises the pH to a near neutral pH where acceptable fluorescence intensities are seen. By adjusting the pH as it was specified in the previously published method, the method to detect the lipolytic activity showed to be working. However, the variation between data was high and thus further investigation on optimal pH and experimental procedures finally resulted in a lower variation. Also, the centrifugation step and incubation temperature were optimized to attain a more reliable method. It was possible to reduce the limit of detection and quantification of lipase activity and thereby not just attain a more robust but also more sensitive method in comparison to the previously published method. The newly developed method was validated by incubating the substrate in the milk for different times and checked for a linear relation. Also, a comparison of a fresh milk and the same fresh milk which was frozen for different times showed that there is no difference in lipase activity between a fresh and frozen sample. This is important since the measurements carried out later were done on frozen samples. With the developed method, raw milk samples from different regions of Sweden were examined and evaluated for possible differences. The samples were taken both on dairy and farm level. No significant difference between these origins could be seen. However, all three regions were significantly different from each other - the milk from Skånemejerier (south of Sweden) exhibited the lowest lipase activity followed by milk from Arla (mid Sweden) and the highest lipase activity was detected in milk from Norrmejerier (north of Sweden). The lipase activity results were also correlated to other properties of the milk measured previously. These properties include the fat content, amount of fatty acids, and bacterial count of the milk. However, none of the properties had a significant correlation except for some long chain fatty acids which showed a negative correlation to the enzyme activity. This indicates that a high amount of certain long chain fatty acids reduces the lipolytic activity. This effect has been seen in the adipose tissue of rats and in goat’s milk before. Since the amount of long chain fatty acids in the milk can be influenced by the feed, a hypothesis is that the lipase activity could be reduced by changing the feed of the cows