The rheology of gel formed during the California Mastitis Test

Abstract

One of the most costly diseases in the dairy industry is mastitis, which is an inflammation of the mammary gland. Mastitis influences the quality of milk and therefore reduces financial returns to both the farmer and the processor. Early detection of mastitis typically reduces treatment cost and a significant amount of research has been done in this field. Currently, the three major methods for mastitis detection are: • The Foss Analysis, which physically counts each cell and is performed off-site. • The Whiteside Test, which is based on a direct relationship between the number of the blood cells and the intensity of a gel formed between NaOH and cells. It was developed for on-site mastitis detection, but is no longer used routinely. • The California Mastitis Test (CMT), which can be done on-site, but is only a quantitative indication of the severity of the infection. The California Mastitis Test has previously been adapted to determine the somatic cell count (SCC) in infected milk by correlating viscosity to cell count. Although highly successful, some uncertainty exists regarding the rheology of the gel formed during the test as well as factors that may influence the accuracy of the test. In this thesis, studies were undertaken on the rheology of the gel formed during the California Mastitis Test in order to develop an understanding of the mechanism of gel formation and how various factors influence the rheology of the gel. Basic biochemistry and physico-chemistry of the gel has been reviewed and it was found that the CMT gel is a DNA/histone/surfactant complex, which forms when SDS is introduced into infected milk with elevated somatic cell counts. Based on literature and some initial experimentation it was found that the gel is a time- and sheardependent, non-Newtonian fluid. Since the reliability of the CMT hinges on the correlation between viscosity and SCC, this study investigated specific factors that may influence gelation, these were: iii • rheology • testing conditions, such as time delay prior to viscosity testing, shear rate and temperature • surfactant type and concentration • milk composition, including fat content, somatic cell count and protein content. It was found that when using capillary viscometry a linear relationship exists between the relative viscosity of the gel and the SCC. The surfactant concentration determines the slope of this linear relationship and it was found that at least 3% SDS is necessary for accurate results. Using more than 3% SDS resulted in more scatter in the data. It was also found that a linear relationship exists between the maximum apparent viscosity and SCC. Either capillary or Brookfield viscometry can be used, however, Brookfield viscometry was found to be more sensitive at the lower SCC range. It was found that the combination of surfactant concentration and SCC influenced the rheology of the gel. The lower the SCC the more SDS was required for gel formation. It was found that when using 1% SDS the critical SCC was 79 k cell/ml, while using 3% SDS this was lowered to 59 k cell/ml. It was found that above the critical SCC the gel is a non-Newtonian rheopectic fluid. Dependent on shear rate, the gel shows rheodestructive behaviour. With a delay time, the peak viscosity of the gel formed faster with longer delay times. However, more than 30 seconds delay had no additional influence on gel formation. It was found that the shear rate or spindle speed influences both the time to reach the peak viscosity as well as the magnitude of this maximum. Higher shear rates shortened the time to reach the maximum apparent viscosity as well as the maximum viscosity. This is likely due to physical breakdown of the gel which is accelerated due to increased shear. Different surfactants have different effects on raw milk. Both acetic acid and Triton- 114 were found to be ineffective as CMT reagents. Acetic acid only denatures proteins and the increased viscosity is due to the precipitation of casein. Triton-114 cannot lyse nuclei walls and therefore gel formation was prohibited due to no DNA/histone complexes being released. Mixing SDS with Triton-114 was found to be less effective than SDS alone either due to the nucleus not being lysed, or because iv of interaction effects between SDS and Triton-114, reducing the available SDS for gelation. Lastly it was concluded that protein and fat content only contributes to the viscosity of milk by changing the solids content of milk and neither of these affects gelation during the CMT. Also, temperature only has a small influence on the relative viscosity and this influence could be neglected if the CMT is done around room temperature.