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«Lars Wiking Faculty of Natural Resources and Agricultural Sciences Department of Food Science Uppsala Doctoral thesis Swedish University of ...»

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It was observed that the average diameter of MFGs was positively correlated with the concentration of C16:0, C16:1, C18:0 and C18:1 (paper I). No significant correlation between average diameter of MFGs and C4-14 or C18:2+C18:3 was found. This indicates that the fatty acid in milk originating from the diet C16:0, C16:1, C18:0 and C18:1 affects the size of the MFGs. This was confirmed by the feeding experiments (paper II and III) where diets rich in C16 and C18 resulted in milk with larger MFG diameter. In the study presented in paper II, the milk with the largest diameter of MGF contained higher concentration of C16:0, C16:1 and C18:0 (g fatty acid/100g milk) than the two other milk types. Concerning C18:1, milk from the unsaturated fat diet contained slightly more than milk from the saturated diet i.e. 1.17 versus 1.10 g fatty acid/100g milk. In the experiment reported in paper III, the concentrations of C16:0 and C16:1 were highest in the milk with the largest MFG which occurred in milk from the cows fed saturated fat diet compared with the milk coming from the cows administrated the high de novo diet. Milk from the high de novo diet contained slightly more C18:1 than milk from the saturated diet, 0.69 versus 0.60 g fatty acid/100g milk and marked more C18:0. Recently, Briard et al. (2003) found, in agreement with paper I, that large MFGs contain more C18:0 than small MFGs. In contrast they found that small globules contained more C12:0, C14:0 and C16:1. However, in the present study, the average diameter is correlated to the content of fatty acids in the milk (paper I), whereas in Briard et al. (2003) the MFG was separated into small and large sizes, and analysed for fatty acid composition (wt%).

26 The knowledge, obtained in these studies, makes it feasible to design the distribution of MFG through feeding. In the following section it will be showed and discussed that the size of the MFG has an impact on its stability during pumping. Recently, studies of Michalski et al. (2002, 2003 & 2004) have shown that the size of native MFG affects the firmness of milk gels, camembert and emmental cheeses. In theses studies, they separated MFGs in size fractions by microfiltration. By designing the distribution of MFG through feeding instead of by microfiltration, processing will appear more careful and products more natural.

Furthermore, the awareness of variations in fatty acid composition between small and large MFGs can be applied to development of dairy products with new texture behaviour.

Influence of MFG size and fat content on MFG stability Pumping is one of the factors inducing lipolysis of milk fat. Already, when the milk is transported from the cows to the farm bulk tank, it is pumped. The majority of FFA in milk is accumulated at farm level (Anderson, 1983). Furthermore, harsh mechanical treatment of milk can result in coalescence of MFGs which means that two or more MFGs share common membrane or fat touches fat, thus they can not be disrupted by shaking (Jennens & Walstra, 1984). This can lead to un-dissolved clumps of milk fat on the surface of unhomogenised drinking milk products which is unwanted by the consumers. Coalescence of MFGs is detected by an increase in the average diameter from the particle size distribution of MFGs.

The highest degree of coalescence of the MFGs during pumping occurred in milk with the highest fat content and MFGs with the largest average diameter (paper II and III). By comparing the milk from the saturated diets in the two studies, the level of coalescence was clearly higher in the experiment presented in paper II than in the other experiment (paper III). Despite, that the inherent average diameter of MFGs was larger in the experiment presented in paper III, the level of coalescence at 31˚C was lower than in the other experiment (paper II). That fact suggests that the fat content in addition with MFG size affect the stability of MFG regarding coalescence, since the fat content was highest in the study presented in paper II.

The accumulation of FFA was dependent on temperature and wall shear rate.

However, at temperatures lower than 31˚C, the formation of FFA is highest in the milk with the larges MFGs and the highest fat content (paper II and III), both inherently and after pumping. The level of FFA can not directly be compared in the two experiments (paper II and III) because the milk was stored for 24 and 2 h after pumping, respectively. At 31°C, there was no significant difference after pumping with the highest shear rate between the FFA content in milk with various fat content and diameter of MFG.

The emulsion stability of homogenised cream upon shearing is decreasing with increasing diameter of MFGs (Hinrichs, 1994). Furthermore, Hinrichs (1994) reported that the fat content is crucial for shearing of raw cream (16-45% fat).

Likewise, the presents studies demonstrated clearly an impact of fat content in raw 27 milk, although the studies were performed with raw milk with fat percentages only varying between 3.7-5.0%. Moreover, the fat content and diameter of MFG were regulated trough feeding of the cows. Since pumping of raw milk always takes place in all milk production, the knowledge of the feed-induced MFG stability is very important. The results clearly show that milk producers should be careful by feeding with saturated fat supplement as it increase the risk for lowering the milk quality, especially regarding FFA.

Influence of temperature on milk fat globule stability The chosen temperatures of milk during pumping was 4-5, 20 and 31 ˚C (paper II and III). These temperatures range from the milk leaving the udder to cool storage temperatures. Milk was cooled direct after milking to the relevant temperature for pumping. Furthermore, the shear rates used in the experiments (paper II and III) were within the ranges occurring in the dairy industry.

Significant coalescence of MFGs only occured at 31 ˚C and only in milk with the highest fat content and largest average MFG diameter (paper II and III). The coalescence of MFG at 31 °C already begins at a shear rate of 365 s-1. At 4-5 ˚C the MFGs were resistant to coalescence upon pumping. It could indicate that a high proportion of the milk fat need to be in liquid phase for initiating coalescence of MFGs. However, it is difficult to explain, why significant coalescence only occurs at 31 °C and only in the milk from the saturated fat diet based on the present work, since the proportion of liquid fat is assumed to vary in the milk from cows offered the saturated fat diet (paper II and III). In the experiment presented in paper III, the content of unsaturated fatty acid (C16:1, C18:1, C18:2 and C18:3) in the milk from cows fed the high de novo diet was lower than in the milk from cows fed the saturated fat diet in the experiment presented in paper II. Hence, the larger average diameter of MFG and higher fat content may explain the higher coalescence of MFGs in milk from cows fed the saturated fat diet, but the temperature dependence is not elucidated.

The large difference in the ratio of liquid fat between cream from the high de novo and saturated fat diet was demonstrated by NMR studies (paper III). Moreover, the effect of temperature and incubation time on liquid fat in the two milk types was demonstrated. The results of liquid fat in cream were explained by the fatty acid composition of the milk. Mulder & Walstra (1974) reported that partial coalescence only occurs when part of the fat is present as crystals. Our results shows that 14.8 % solid fat in the MFG was sufficient to cause coalescence upon pumping with a shear rate of 565s-1. Whereas levels of solid fat in MFG 26.6 % and at 3.4% did not cause coalescence of MFGs. By using much higher shear rates than in the present study, Hinrichs & Kessler (1997) observed that increased solid fat content in raw cream increased the level of the critical shear rate that caused destabilisation of the MFGs.

The accumulation of FFA in milk upon pumping was highest at 20˚C for milk with the highest fat content and largest average diameter of MFG (paper II). The milk from the unsaturated fat and the high de novo diet reached the same level of FFA 28 at 20 and 31˚C. This is in agreement with other studies reporting that the maximum formation of FFA upon agitation is at a temperature of ~15 °C and again after ~30 °C, (Fitz-Gerald, 1974; Deeth & Fitz-Gerald, 1977; Bhavadasan, Abraham & Ganguli, 1981; Hisserich & Reuter, 1984). The fatty acid composition of milk could be responsible for the high level of FFA at 20˚C for milk with the highest fat content and largest average diameter of MFG. At 5˚C, there was no significant increase in FFA content in milk upon pumping at various shear rates (paper II). However, the formation of FFA significantly increased upon pumping, when milk was cooled to 4˚C followed by 60 min incubation before pumping (paper III) as shown in Figure 5. Further research is needed to understand this observation. Assumptions can be made that the transition of polymorphic crystal forms of milk fat during cooling affect the susceptibility to lipolysis, or the growth of crystal size could have impact on the stability of the MFGM. Likewise, the longer incubation time at 4˚C could be expected to increase the attachment of LPL to the MFG and cause elevated levels of FFA during pumping. Backman & Wilcox (1990b) found that immediate cooling of raw milk after milking increased the level of FFA compared with 1h delayed cooling of the raw milk. However, this observation is only relevant if the raw milk is not subjected to mechanical treatment.

–  –  –

Figure 5. The effect of incubation time before pumping on the level of FFA in raw milk after pumping.

The milk was from cows fed the saturated fat diet and the high de novo diet, respectively.

29 The results show that the relationship between coalescence of MFGs and formation of FFA in milk subjected to pumping is complex (paper II and III). At low temperatures (4-5 ˚C), no coalescence of MFGs was detected, whereas the content of FFA significantly increased upon pumping, when milk was stored 60 min at 4˚C before pumping (paper III). Coalescence of MFG was only observed in milk from cows fed the saturated fat supplement diet at 31 ˚C (paper II and III). At the same temperature, the milk from the unsaturated fat and the high de novo diet accumulated the highest FFA content upon pumping (paper II), and in these two milk types no coalescence of MFGs was demonstrated at any of the used temperatures. Our results indicate that the formation of FFA begins before coalescence of MFGs occurs. One exception is otherwise when raw milk is subjected to pumping at 20 ˚C.

In order to detect other changes on the MFGM in milk subjected to pumping, the activity of xanthine oxidase in milk serum was determined (paper II). No effect of pumping on xanthine oxidase activity was detected, suggesting that xanthine oxidase is not released from MFGM to the serum phase upon mechanical treatments. In contrast, Back & Reuter (1973) reported that xanthine oxidase is released to milk serum when milk is subjected to shear forces. As expected, cooling of the milk released the xanthine oxidase from the MFGM.

The present results clearly suggest that cooling the milk to 4-5 °C stabilised the MFG upon mechanical treatment, resulting in lower formation of FFA and lower risk of coalescence of MFGs. By transferring the obtained knowledge to milking systems, it suggested that the milk cooling should be placed as close to the udder as possible. Thereby the transportation of warm milk would be reduced, leading to lower levels of FFA.

The effect of increased milking frequency on lipolysis in milk The introduction of automatic milking systems has made it relevant to study the effects of increased milking frequency i.e. milking more than twice daily, since the cows have free access to the milking unit. Studies have reported the average milking frequency in automatic milking systems to be between 2.4-2.6 daily (Svennersten-Sjaunja; Berglund & Petterson, 2000; Hogeveen et al., 2001;

Petterson & Wiktorsson, 2004). An increase in milking frequency results in higher milk production per cow (Stelwagen, 2001). However, it also affects the milk quality.

In the present study cows were milked 4 times daily on one udder half and twice daily on the opposite udder half (paper IV). The level of FFA was significantly higher (1.49 meq/100 g 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./100 g fat). Similar results have been found by Klei et al. 1999 and Slaghuis et al. (2004).

In order to study the possible mechanisms behind the increased FFA content in milk upon increased milking frequency, the average diameter of MFG, fatty acid composition and activity of γ-glutamyl transpeptidase in the milk were 30 determined. Milk from the udder half milked four times per day contained MFGs with a significantly larger average diameter (d(4,3)) compared with milk from the udder half milked twice times. Furthermore, the 90% percentiles of MFG size distribution significantly increased upon increased milking frequencies which indicates that transfer to larger globules occur for medium or larger fat globules of the distribution.

In the other experiment (Paper II), it was found that MFGs with the largest average diameter were inherently unstable, resulting in a high level of FFA in milk even without subjecting to pumping. Likewise, the significantly larger average diameter of MFGs in milk upon more frequent milking results in elevated levels of FFA (paper IV). This indicates that MFGs with a large diameter are more susceptible to spontaneous lipolysis. Even though larger globules have a smaller surface area, in total. The surface potential is lower for large MFGs than for small globules, presumably increasing the amount of LPL attaching the MFG.

There was no significant effect on the activity of γ-glutamyl transpeptidase upon milking frequencies (paper IV), indicating that the production of MFGM is sufficient to cover the secreted MFG, when the milking frequency is increased.

Furthermore, the results clearly indicate that the de novo synthesis of milk fat is not affected by milking frequencies, since the proportion of C4-C14 in milk is invariant between two and four times daily milking (paper IV).

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