«Lars Wiking Faculty of Natural Resources and Agricultural Sciences Department of Food Science Uppsala Doctoral thesis Swedish University of ...»
Milk Fat Globule Stability
Lipolysis with Special Reference to Automatic Milking
Faculty of Natural Resources and Agricultural Sciences
Department of Food Science
Swedish University of Agricultural Sciences
Acta Universitatis Agriculturae Sueciae
© 2005 Lars Wiking, Uppsala
Tryck: SLU Service/Repro, Uppsala 2
Wiking, L. 2005. Milk Fat Globule Stability - Lipolysis with Special Reference to Automatic Milking Systems.
ISSN 1652-6880, ISBN 91-576-7048-X The implementation of automatic milking systems (AMS) initially caused a lowering of the milk quality regarding free fatty acids (FFA). A high level of FFA increases the risk of rancid flavour in dairy products. The objective of the present thesis was to evaluate different factors in AMS which could potentially cause an increased level of FFA. The stability of the MFG was studied by using model systems.
A correlation (r2=0.54) was found between the average diameter of the milk fat globule (MFG) and the diurnal fat yield of cows. The activity of a MFG membrane enzyme was found to decline with increasing average MFG size. The results of this work indicate that the MFGs grow larger when the fat synthesis increases, probably because of a limitation in the production of MFG membrane.
This new, obtained knowledge was used to produce milk with various average diameters of MFGs. Three groups of cows were fed concentrates with different fatty acid compositions; one high in saturated fat, another high in unsaturated fat and the last one stimulating high de novo synthesis. The feedings resulted in milk with fat contents of 5.0,
3.7 and 4.0%, respectively. The MFGs were significantly larger in the milk with the highest fat content. All three types of milk were pumped at various shear rates and temperatures.
Afterwards, measurement of particle size distribution showed that the highest coalescence of MFGs in the milk occurred with the largest MFGs. Moreover, the MFGs were more unstable at a pumping temperature of 31°C compared with lower temperatures. Likewise, an increase was found in the FFA content for milk with the largest MFGs, indicating that milk with a high fat content is more unstable when subjected to mechanical stress. The results recommended to cool raw milk to 5°C before pumping it from the milking unit to the milk bulk tank.
The effect of milking frequency on FFA was studied because cows are more frequently milked in AMS. The level of FFA was significantly higher (1.49 meq./100g fat) in milk from the udder half milked four times daily compared with the milk from the udder half milked twice daily (1.14 meq./100g fat). This is ascribed to the fact that milk from the udder half milked four times daily contained MFGs with a significantly larger average diameter. The results are of great importance for further understanding of the mechanisms behind the increased content of FFA which is frequently observed in AMS.
Keywords: milk fat globule, milk fat globule membrane, fatty acid composition, free fatty acids, pumping, automatic milking systems, γ-glutamyl transpeptidase, xanthine oxidase, milking frequency Author´s address: Lars Wiking, Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark. Email: email@example.com Contents
Introduction, 10 Milk fat globule, 10 Synthesis of the milk fat, 10 Composition of the milk fat globule, 11 Composition of the milk fat globule membrane, 12 Lipolysis in milk, 15 Lipase, 15 Lipolytic rancid flavour, 16 Changes induced to milk fat globules during different treatments, 17 Influence of mechanical treatment on MFG stability, 17 Crystallisation of fat, 18
Materials and methods, 21 Animals and feed, 21 Pumping experiments, 21 Milk fat quality, 22 Determination of size distribution of milk fat globules, 22 Analysis of free fatty acids, 22 Fatty acid composition, 23 Assays for activity of milk fat globule membrane enzymes, 23 Analysis of xanthine oxidase activity in the milk serum, 23 Analysis of γ-glutamyl transpeptidase activity in milk, 23 Milk fat crystallisation, 24 Determination of liquid fat by nuclear magnetic resonance, 24 Results and discussion, 25 Influence of feed composition on milk fat globle size, 25 Influence of MFG size and fat contenten on MFG stability, 27 Influence of temperature on milk fat globule stability, 28 The effect of increased milking frequency on lipolysis in milk, 30 Conclusions, 32 References, 33 Acknowledgements/Tak, 39 Appendix Papers I-IV I. Wiking, L., Stagsted J., Björck, L. & Nielsen, J.H. (2004). Milk fat globule size is affected by fat production in dairy cows.
International Dairy Journal 14: 909-913 II. Wiking, L., Björck, L. & Nielsen, J.H. (2003). The influence of feed on stability of fat globules during pumping of raw milk.
International Dairy Journal 13: 799-803 III. Wiking, L., Bertram, H.C., Björck, L. & Nielsen. J. H. Evaluation of cooling strategies for pumping of milk - Impact of fatty acid composition on free fatty acid levels (submitted to Journal of Dairy Research) IV. Wiking, L., Nielsen, J.H., Båvius, A-K., Edvardsson, A. & Svennersten-Sjauna, K. Impact of milking frequencies on the level of free fatty acids in milk, fat globule size and fatty acid composition (manuscript to be submitted) Reprints are published by kind permission of the journals concerned List of abbreviations ADV Acid degree value BDI Bureau of Dairy Industry CD 36 Cluster of differentiation 36 FFA Free fatty acids FID Free induction decay GC Gas chromatography LPL Lipoprotein lipase MFG Milk fat globule MFGM Milk fat globule membrane MUC1 Mucin 1 NMR Nuclear magnetic resonance PAS Periodic acid schiff Background Automatic milking systems have been commercially available since 1992, and the numbers have grown rapidly during the last years. At present, 400 and 260 dairy farms in use automatic milking systems in Denmark and Sweden, respectively (personal communication, B. Everitt. Swedish Dairy Association). This corresponds approximately to 7 % and 5%, of the total bulk milk in Denmark and Sweden, respectively. In automatic milking systems the cows are milked by assistance of robots. By sensor technology, the robot finds the teats, and clean them before cluster attachment. The cows voluntarily attend the milking unit, and the cows are offered concentrate feed during milking.
This thesis was initiated due to reports in the beginning of implementation of automatic milking systems that the levels of free fatty acids (FFA) were higher in automatic milking systems compared to conventional milking systems (Justesen & Rasmussen, 2000; Klungel, Slaghuis & Hogeveen, 2000). With the steadily growing number of automatic milking systems, the quality of dairy products can be impaired in the future. The focus of this thesis has thus been how changes in the milking technology could affect the formation of FFA in milk.
Compared with conventional milking (i.e. milking twice daily), milk is through the whole day continuously pumped to the milk bulk tank at the farm which involves new cooling strategies. Furthermore, automatic milking often requires harsher mechanical treatment of the milk due to longer distances between milking unit and milk bulk tank and continuous pumping of smaller amounts of milk. Another factor altered from conventional milking is an increased milking frequency due to the fact that cows have free access to the milking unit.
This thesis contains an introduction that reviews present knowledge of the milk fat globule, material and methods and a discussion of the results obtained and finally conclusions. The appendix consists of four papers which together form the basis of this thesis.
Milk fat globule It has been known for more than 300 years that fat exists as globules in milk (Leewenhoeck, 1674). The fat globule size distribution is shown in Figure 1. The diameter of the milk fat globule (MFG) ranges from 0.1-12 µm with an average of around 4.5µm. The size distribution depends on breed as shown in Figure 1.
Figure 1. Milk fat globule size distribution from Danish Holsteins and Jersey cows.
Synthesis of the milk fat The MFG is formed in the secretory cells of the mammary gland. Precursors of milk lipid globules are formed at the endoplasmic reticulum and are transported through the cytosol as small droplets of triglycerides covered by a non-bilayer of polar phospholipids and proteins. During transport the droplets grow in size, apparently due to droplet-droplet fusion (Dylewski et al. 1984; Deeney et al.1985). At the apical plasma membrane, the droplets are secreted from the epithelial cell. During secretion, the droplets are covered by the plasma membrane and finally pinched off into the lumen of alveolus.
The precursors of milk lipid globules have a group of polypeptides on the surface in common with the membrane of the endoplasmic reticulum (Deeney et al., 1985). However, it is still unknown where in the endoplasmic reticulum network the lipid droplets are formed (Mather & Keenan, 1998). Another unknown mechanism is how the lipid droplets are transported to the apical plasma membrane of the cell.
Furthermore, there are two different theories of how the fat droplets are secreted.
One theory is that the lipid droplets reach the apical region of the cell, where they are secreted and covered by cellular membranes. The lipid droplets are gradually coated with plasma membrane until a narrow neck of membrane and cytoplasm remains. At the point when the membrane in the neck fuses together, the fat globule is secreted and expelled into the alveolar lumen (Mather & Keenan, 1998).
Another possible theory for the secretion of the lipid droplet suggested by 10 Wooding (1971 & 1973) is that the lipid droplets also associate with secretory vesicles in the apical cytoplasm. Likewise casein should be covered by a secretory vesicle and the content of such may then be released from the apical surface by exocytosis. The first described mechanism is the most accepted (Mather & Keenan, 1998). The hormones prolactin and oxytocin, affect the release of the lipid globules and is thought to affect the final size of the MFG (OllivierBousquet, 2002). The composition of the outer coat of the milk fat globule membrane (MFGM) is to a great extent similar to the apical plasma membrane of the secretory cells.
Composition of the milk fat globule Many studies and reviews have dealt with the composition of fatty acids in milk (Glass, Jenness & Lohse, 1969; Bitman & Wood, 1990; Jensen, Ferris, LammiKeefe, 1991; Bitman et al. 1995; Jensen, 2002). Several lipid classes are present in milk as shown in Table 1.
The composition of the fatty acids in milk fat is given in Table 2. The composition of fatty acids in milk is affected by feed and breed. The fatty acids containing from 4 to 14 carbon atoms are synthesized from the acetate and β-hydroxy butrate which are products of the fermentation of carbohydrates in the rumen. This pathway is called de novo synthesis. Some of the palmitic acid (C16:0) is also synthesized de novo. Long chain fatty acids, i.e. those containing 16 or more carbon atoms, are provided to the glands from the blood stream and originate directly from the diet or from the adipose tissue. Palmitic (C16:0) and stearic (C18:0) acids pass through the rumen unchanged while unsaturated fatty acids are subjected to biohydrogenation by the reducing environment caused by the microorganisms in the rumen, resulting mainly in stearic acid together with a smaller amount of oleic acid (C18:1). (Børsting, Hermansen & Weisbjerg, 2003) Furthermore, stearic acid derived from the diet is partly converted to oleic acid by stearoyl-CoA desaturase, in the intestines and the mammary tissue. Unsaturated lipid supplements are often protected/encapsulated to avoid biohydrogenation in the rumen. Moreover, high amounts of unsaturated lipids in the rumen result in incomplete biohydrogenation, so some of the linoleic acid (C18:2) and linolenic acid (C18:3) is transformed into conjugated linoleic acids (CLA). Specific isomers
Jersey cows produce a higher proportion of de novo fatty acids C4-16, C18 and lesser C18:1 compared with Holsteins cows (Beaulieu & Palmquist, 1995;
DePeters et al. 1995; Morales et al., 2001; White et al. 2001). The lower proportion of C18:1 in Jersey compared to Holstein milk is due to a lower mammary activity of stearoyl-CoA desaturase in Jerseys (Beaulieu & Palmquist, 1995; Drackley et al. 2001).
Several studies have been carried out to increase the proportions of unsaturated lipids in milk, in order to obtain “healthier” milk fat and softer butter (Banks, Clapperton & Morag, 1976; Urquhart, Cadden & Jelen, 1984; Grummer, 1991;
Goodridge, Ingalls & Crow, 2001; McName, 2002; Gonzalez et al. 2003). The main polyunsaturated fatty acids in milk are linolenic acid C18:3 (omega –3 fatty acid) and linoleic acid C18:2 (omega –6 fatty acid). Goodridge, Ingalls & Crow (2001) managed to increase the proportion from 4.8% to 10.3 % by feeding high amounts of protected Linola seed (containing a high level of linoleic acid) and to increase the proportion of linolenic acid from 0.8% to 6.4% by offering a high amount of protected flaxseed. The disadvantage with high levels of unsaturated fatty acid is the susceptibility to oxidation which produces an oxidative rancid flavour in dairy products.
Composition of the milk fat globule membrane The quantitative composition of the MFGM has been studied by several methods.
Therefore, some discrepancies exist between results. Estimates of the composition of MFGM found in the literature are summarised in Table 3. The major proteins found in the MFGM are MUC1, xanthine dehydrogenase/oxidase, PAS III, CD 36, butyrophilin, adipophilin, PAS 6, PAS 7 and fatty-acid binding protein. Three of 12 the proteins are described in this section. Furthermore, around 25 different enzymes are found associated with the MFGM.