Proven Microfluidizer^® Technology
Cell Disruption Peer Reviews

Processing

Miller Brewing Company supplied the yeast (Saccharomyces cerevisiae) as a frozen cake. The yeast was resuspended in phosphate-buffered saline at 11% solids, then processed using a Microfluidizer^® processor and a French press at the same pressure for a direct comparison.

Conclusion

In this study, the Microfluidizer^® processor was faster and easier to use. After a similar number of passes, it achieved a cell rupture rate more than 80% higher than the comparison method. Its gentle cell disruption also delivered up to 2.5 times the yield of solubilized protein.

PUBLICATION SUMMARY #2

Characterization of E. coli Cell Disintegrates from a Bead Mill and High Pressure Homogenizers by Irene Agerkvist and Sven-Olof Enfors

This publication compares the performance of three cell disruption methods: Microfluidizer processor, Bead Mill and High Pressure Homogenizer (HPH) across a range of parameters:

Viscosity

Cell lysate viscosity differed significantly between disruption methods. After one pass through the high-pressure homogenizer (HPH), viscosity was very high but fell rapidly with additional passes. By comparison, the Microfluidizer^® processor produced a low-viscosity lysate after just one pass, with further reductions after subsequent passes.

Filtration

In this study, the cell lysate produced by the Microfluidizer^® processor had the shortest filtration times and separated more effectively during centrifugation than the lysate from the high-pressure homogenizer (HPH). Additional passes through the HPH reduced separation performance.

Protein Release

The reason for disrupting the cells in the first place. The Microfluidizer technology gave the highest overall protein release, effectively plateauing at around 5 passes.

Particle Size Distribution

In this study, changing the residence time in the bead mill did not affect particle size distribution. The bead mill produced two comparable peaks, representing large and medium-sized particles. The high-pressure homogenizer (HPH) produced a medium-sized particle peak and a second peak of smaller particles associated with viscosity and filtration problems. By comparison, the Microfluidizer^® processor produced a single medium-sized particle peak, resulting in low viscosity and better separation.

Temperature

High temperatures can denature proteins, affecting product recovery. In this study, Agerkvist and Enfors reported significantly higher processing temperatures with the high-pressure homogenizer (HPH) than with the Microfluidizer^® processor, which delivered the highest yield of β-galactosidase.

However, exit temperatures of 40–50°C are not necessarily unacceptable: protein denaturation depends on both temperature and exposure time. The reported residence time in the Microfluidizer^® processor was just 25–40 milliseconds, much shorter than in the HPH.

The HPH heats the sample higher & longer – hence the increased denaturation that can be seen in the yield data.

Conclusion

Microfluidizer^® technology achieved the highest protein and enzyme release of the three methods, approaching 100%, and produced a cell lysate that was easier to purify. The other methods produced smaller particles and broader particle size distributions, which complicated downstream processing. Smaller particles were harder to separate from proteins during centrifugation, while the broader size distributions contributed to filter blockage.

PUBLICATION SUMMARY #3

Evaluation of the Microfluidizer for Cell Disruption Purification of Cytochrome C Oxidase from Microfluidizer Processed Yeast by E. Llera-Santos, E.A. Davis, S. Ackerman and W.G. Hagar

Publication Summary #1 describes the most efficient operating conditions identified for disrupting yeast (Saccharomyces cerevisiae).

This third study examined the isolation and activity of the temperature-sensitive enzyme cytochrome c oxidase (EC 1.9.3.1). Solubilized protein was measured using the Bio-Rad Standard Assay, while enzyme activity was measured using a standard spectrophotometric method.

Processing

The study used a 10-liter, semi-industrial batch of baker’s yeast in culture medium. The suspension was cooled to 4–6°C, then processed with a Microfluidizer^® processor and a high-pressure homogenizer, both operating at 10,000 psi. The temperature-sensitive enzyme cytochrome c oxidase was isolated, and its activity was measured using standard procedures.

Conclusion

The Microfluidizer^® processor yielded approximately 3 times as much sub-mitochondrial protein (SMP) compared to the HPH, despite slightly less initial breakage, indicating loss or damage of fewer particles during processing.

PUBLICATION SUMMARY #4

Research report by Dr. Paul Sallis, University of Kent, England

This report shows the effect of the Microfluidizer technology on protein recovery from three different bacterial: Agrobacterium Sp., C. Glutamincum, and B. Lichenformulis.

Experiment #1

In Experiment 1A (Figure 2), the amount of protein recovered in the extract increased with each pass through the Microfluidizer^® processor. Agrobacterium sp. cells were processed at a lower pressure and released less protein, even after many more passes. This suggests that pressure and the number of passes should be optimized for each cell type.

Experiment 1B (Figure 3) compared the Microfluidizer^® processor with a French press. Under the conditions tested, the Microfluidizer^® processor achieved six times the protein recovery of the French press, which showed product losses after the first pass.

Experiment #2

The results in the table below show how Microfluidizer^® technology can increase protein recovery while maintaining enzyme specific activity. In these experiments, the M-120E processor operated at 15,000 psi. Additional passes increased the amount of enzyme recovered without changing its specific activity, increasing the total amount of usable enzyme.

The results also highlight the importance of selecting appropriate processing conditions. Under the conditions tested, the M-110Y processor operating at 20,000–23,000 psi released as much protein in one cycle as the M-120E operating at 15,000 psi over 21 passes.

PUBLICATION SUMMARY #5

Research report by Maurice Gaucher, University of Calgary, Canada

The following data compares three mechanical disruption techniques- the Microfluidizer technology, the Braun Cell Homogenizer and the APV/ Gaulin Homogenizer.

Experiment #1

m-Hydroxybenzylalcohol dehydrogenase activity in cell free extracts as produced by three different methods of disruption.

Experiment 1 shows that all three methods appear to be similar in their ability to disrupt cells as determined by protein release (mg. protein/g. dry cells). However, the Microfluidizer technology and Braun Bottle are associated with higher levels of enzymatic activity (approximately 40% higher) as well as efficient product temperature control. Given that the Braun Bottle method is only suitable for small scale preparations (10-20 ml), the method of choice for larger preparations would be the Microfluidizer technology.

Experiment #2

6-Methylsalicylic Acid Synthetase activity in cell free extracts as produced by three different methods of cell disruption.

In Experiment 2, extracts produced using Microfluidizer^® technology showed 100–200% higher enzyme activity than those produced using the Braun bottle method or APV/Gaulin homogenizers under the conditions tested.

¹ Temperature was increased immediately after cell disruption
² One unit of activity is the amount of enzyme required to catalyze the formation of 1 µmole of NADP 1 per minute at 30°C
³ One unit of activity is amount of enzyme required to catalyze the formulation of 1 µmole of 6-methyl-salicylic acid per minute at 30°C

PUBLICATION SUMMARY #6

Evaluation of the Microfluidizer for Cell Disruption of Yeast and Chlorella· by E Llera-Santos, C.D. Copple, E.A Davis and W G. Hagar

This application presents a difficult extraction for photosynthetic pigments since it is resistant to standard extraction methods which involve grinding with a mortar and pestle.

A 2,6 dichlorophenol indophenol (DCPIP) reduction assay was used to evaluate the functionality of the isolated thylakoid membranes. The assay dye is blue in its oxidized state and acts as a “sink" for the electron flow of Photosystem II; thus, the reduction of the dye is a measure of the integrity of the chloroplast preparation.