|
 APV CELL DISRUPTION BY HOMOGENIZATION APV Homogenizers Gaulin Rannie APV From its very genesis
APV has maintained an impeccable reputation for excellence world-wide. That
standard is perpetuated in the field of homogenization and high pressure pumps.
It's a tradition that dates back to our invention of the homogenizer in the
early 1900's. That desire to excel
in serving our customers is demonstrated today through innovative design,
precision engineering, quality manufactur-ing and attention to detail within
the APV Homogenizer Division. This new division
created by the unifying of APV Gaulin and APV Rannie introduced a new era in
the industry. With more than 150 years of combined experience, APV provides our
customers with the greatest resource for homogenization technology ever
assembled. While maintaining the distinguishing characteristics and unique
designs of the GAULIN and RANNIE products,
APV is now able to offer a much greater range of equipment and service from a
single source. TheAPV Homogenizers is
ready to meet the challenges facing us in this exciting, technologically
advanced world, and we look forward to serving you. World Leader in Homogenization Technology CELL DISRUPTION Recent developments in
biotechnology and genetic engineering have produced many products from
microorganisms. Many of these prod-ucts (proteins and enzymes) are
intracellular and need to be released from the interior of the cell. Cell
disruption (CD) techniques for breaking cell walls have been studied for many
years. Some of these methods are suitable only for small batches and can
adversely affect enzymes and proteins. One of the methods,
which can be used, for small batches and for production batches is the cell
disruption homogenizer. This report discusses the development of the CD
homogenizer and other disrup-tion techniques. Also, the effects of pressure,
homogenizing valve design and multiple passes on cell disruption are discussed. Introduction In the downstream
processing of fermentation products, the apparatus used for cell disruption has
become very important with regard to the efficiency of the overall process.
Recent advances in recombinant DNA technology have brought about the
development of new means for the production of useful enzymes and proteins 1. Most fermentation and recovery operations require the
breakage of the product micro-organism to release these enzymes and proteins
for separation and purification. Along with these biotechnological techniques
has come the need to disrupt cells in large scale and in an efficient and
economical manner. Even though CD
techniques have been investigated for the past forty years, only recently has
there been a need to apply these methods on a large scale. When considering the
requirements of large-scale, efficient cell disruption, many methods fall short
and only a few CD techniques can be applied to the rigorous demands of today's
biotech-nology operations. One device that fulfils these requirements is the CD
homogenizer, and the purpose of this report is to describe the CD homogenizer
and its use in downstream processing. At this point it is
important to define the term "homogenizee'. In recent years the term has
been applied to many different types of emulsifica-tion devices, but the
original name "homogenizee' was used by Auguste Gaulin in France to
describe the machine he invented at the turn of this century for the processing
of milk. Auguste Gaulin's machine consisted of a positive-displacement pump
forcing product through a restricted orifice (homogenizing valve), which is
adjustable so that pressure can be varied 2. With a homogenizing
valve designed to maximise cell disruption and with a special pump design for
biotech applications, the machine is called a "CD homogenizer". History The archives of APV
Gaulin, Inc. contain an interesting history of the development of the CD
homogenizer. The first mention of cell disruption in the archives relates to
beer fermentation. A patent specification from 1932 3 proposed using high pressure drop through an orifice for
disrupting yeast cells used in the fermentation of beer. During the
fermentation process, the yeast cells in the broth take up vitamins. When the
yeast cells are removed from the broth, the vitamins are also removed. However,
if the yeast cells are disrupted at the appropriate time in the fermentation,
then the cells would release the vitamins back into the beer, producing a
vitamin-enriched beer. Inves-tigation by Gaulin at that time did not reveal any
benefits from this process. From 1951 to 1953, the
Gaulin laboratory ran many tests on yeast to determine the effect different
homogenizing valve configurations had on the efficiency of cell disruption. At
that time the evaluation tests were simple procedures involving centrifuging
the disrupted samples and visually examining the type and amount of various
layers formed in the centrifuge tube. The conclusion was that a knife-edge-type
valve was best for efficient disruption. At that time, this valve was called a
"high impact valve". In 1962 the Company
worked with a brewery to test the effect homogenization had on brewer's yeast
and fermentation time. The idea was that an increase in fermentation rate would
be accomplished by uniformly distributing the yeast in the broth by breaking up
clusters of yeast cells or by releasing nutrients from disrupted yeast to
furnish food for fresh yeast. However, these tests did not demonstrate any
improvement in the fermentation process, despite some early claims that the
fermentation time was reduced. By 1963 Gaulin had a
homogenizing valve available specifically for cell disruption. This special
valve and seat with a knife-edge configuration on the seat is now designated a
CR valve. The National Institute of Health used the Gaulin homogenizer and the
CR valve in 1967 for disruption of E. coli, Salmonella species, clostridium and
bacillus species, fungus-Neurospora crassa and Aerobacter yeast. By the early 1970s
work was being done by many research centers using the Gaulin homogenizer for
cell disruption. Although there were a number of techniques for disrupting very
small quantities of microor-ganisms, the homogenizer was a useful tool for disrupting
large batches of slurry from
one liter to several liters. The concern was not with the efficiency of cell
disruption but rathe rwith being able to process larger quantities of slurry
than could be treated with other disrupting methods available at that time. The early sales of
homogenizers were mostly to research labs and universities. By 1973 Gaulin had
sold 66 homogenizers for cell disruption. Of these machines, 62 were lab units
and only four were small pilot lab or production units. Of the 24 units sold in
the United States, 18 were for universities. The advent of
recombinant DNA techniques in the 1980s brought many new products from
micro-organisms, which now necessitated not only that larger quantities of
cells be disrupted but that it be done continu-ously and efficiently in a
processing system downstream from the fermenter. These demands initiated a
re-examination at Gaulin of the CR valve and the homogenizer pump design for
biotech applications. From this research the old design CR valve was replaced
by the CD valve, and a modified pump design was developed for special biotech
applications (Figure 1). To date, Gaulin has sold over200 machines for cell
disruption. Cell Disruption Single-cell organisms
(microorganisms) consist of a semipermeable, tough, rigid, outer cell wall
surrounding the protoplasmic (cytoplasmic) membrane and cytoplasm. The
cytoplasm is made up of nucleic acids, proteins, carbohydrates, lipids,
enzymes, inorganic ions, vitamins, pigments, inclusion bodies and about 80%
water. In orderto isolate and extract any of these substances from inside the
cell, it is necessary to break the cell wall and protoplasmic membrane. In some
cases the cells may excrete the desired substance; but, in most cases, the cell
wall must be disrupted to release these substances. Methods for Cell
Disruption Over the years many
different techniques have been developed to disrupt cells. One of
the early references to cell disruption 4 describes the use of a
pressure vessel with discharge through a needle valve. The slurry is placed in
the vessel and a plunger in the vessel is used to bring the pressure up to
20,000 psi (137.9 MPa). The material in the vessel is released to atmosphere
through the needle valve. The pressure change causes disruption of the cells.
This type of device was the precursor to the modern-day French press. This
apparatus is limited to small sample sizes but can reach high pressures.
However, no special technology is involved in the design of the needle valve.
One problem associated with this technique is the large temperature rise in the
sample after passing through the valve. Duerre and Ribi 5 cooled the valve and removed heatfrom the product but
still found degradation of protein when operated in the range of 25,000 to
55,000 psi (172.4 to 379.3 MPa). Wimpenny 6 found that Gram
negative rod-shaped bacteria and Mycobacteria were the easiest cells to
disrupt, and the Gram positive cocci and the alga chlorella were the hardest to
break. Garver and Epstein 7 used hand grinding to break cells and scaled that up by
mixing glass beads with a cell slurry and processed the mix through a colloid
mill. After 15 minutes, 99% of E. coli cells were ruptured; and, after 20
minutes, 99% of baker's yeast was ruptured. Rodgers and Hughes 8disrupted cells
with glass beads also. In 1961 and 1972 Hughes 9,10 reported on the use of an ultrasonic probe, generating liquid cavitation at
20,000 Hz, for disrupting cells. The formation and collapse of cavitation
bubbles can produce large tem-perature and pressure gradients in proximity to
the collapsing bubbles. The theory was that the cells were disrupted due to the
shearing forces from turbulent eddies produced by the collapsing bubbles. The
amount of disruption depended on the type of organism and the time of
treatment. A one-hour treatment of E. coli produced a significant amount of
disruption. Tannenbaum and Miller 11 in 1967 made one of the first references to the use of
the Gaulin homogenizer for cell disruption. The purpose of using the
homogenizer for cell disruption was to release protein from cells that were
being used in feeding experiments with rats. Com-pared to feedings with whole
(unbroken) cells, the disrupted cells increased protein digestibility and net
protein utilisation and increased total body lipids. Other authors also described
the benefits of using cell disruption to improve
the digestibility of cells from single cell protein (SCP) fermentation 12-15 .
Even though much attention was focused on SCP as a potential food source,
activity in this project declined because of increased petroleum prices, lack
of taste or visual appeal and lack of approval from health authorities for
animal or human consumption of SCP 16. Wiseman 17 and Zetelaki'll in 1969 reviewed some of the different
methods used for cell disruption and reported on the relative efficien-cies of
the various techniques. By 1971 Jakoby (19) indicated that the two devices used
for large-scale cell disruption were the bead mill and the high pressure
homogenizer. Follows, et al.20and
Hetherington, et al .21 reported on detailed studies using the Gaulin
homogenizer for disruption of baker's yeast. Follows used the homogenizer in a
recycle mode to extract seven enzymes and protein from the cell slurries.
Hetherington produced the most detailed study, up to that time, on the use of
the high pressure homogenizer for cell disruption. A first-order equation was
derived from the data relating pressure and number of passes to yield. The
other results of this test were: 1 . Protein release is
temperature-dependent (higher is better). 2. Protein release is independent of yeast
concentration. 3. Protein release is
pressure-dependent (higher is better). 4. A knife-edge seat is better
than a standard seat. Whitworth 22,23 also
used the Gaulin homogenizer for cell disruption. In these tests it was found
that the extent of disruption was dependent on the operating pressure and the
number of passes through the homog-enizer. The homogenizer operating at high pressure
did not denature protein. In a report by
Cunningham et al . 24 , different techniques for cell
disruption of SCP were discussed and evaluated. The conclusion was that the
high-pressure homogenizer is the most feasible apparatus for scale-up of all the
methods studied. Also, as mentioned earlier, cell disruption is a necessary
step for the release and solubilization of intracellular proteins. If the whole
cells are ingested, the tough cell wall may allow the cells to pass through the
digestive tract intact with no utilisation of the cell protein25. At this point it would
be useful to review all of the methods used for cell disruption (Figure 2). As
can be seen from the list, there are many non- mechanical methods for cell
disruption. These techniques have been described in the literature, and they
will not be reviewed here in detail26. However, it can be said that some of the
problems associated with these methods include high cost of ingredients,
denaturing of proteins, destruction of enzyme products, small-scale batches and
lack of scale -up to continuous operation. Some of the items
listed as mechanical methods, such as sonification and the French press, have
been covered here already. Freeze pressing involves pressurising a chamber of
frozen slurry until a phase change occurs, allowing the slurry to pass through
a fixed orifice. Ice crystals in the mix may contribute to the grinding and
disrupting of the ce 1JS27. Decompression simply
involves placing slurry into a pressure vessel, charging with nitrogen gas to
the desired pressure and then releasing the pressure while either retaining the
slurry in the vessel or ejecting it through an orifice. Most of these
mechanical methods, except for two, have limitations with respect to batch size
and scale-up. The only two that can accommodate large slurry batches are the high-speed
bead mill and the homogenizer. Wet milling methods
include the use of high-speed bead mills. The slurry is pumped through a
chamber containing beads and agitator discs 28. The discs run at speeds of
1500to 2000 rpm, and cell disruption is caused by grinding between the beads,
collisions between the beads and the organisms, and shear forces due to
velocity gradients caused by the beads' movement. The beads are loaded into the
chamber at 80 to 85% of the free volume of the chamber. Glass beads at a
diameter greater than 0.5mm are best for yeast and beads at a diameter less Figure 2 CELL DISRUPTION METHODS NON-MECHANICAL METHODS CHEMICAL TREATMENT Acid Base Solvent Detergent ENZYME LYSIS Lytic Enzymes Phage Infection Autolysis PHYSICAL TREATMENT Freeze-Thaw Osmotic Shock Heating and Drying MECHANICAL METHODS HIGH PRESSURE HOMOGENIZATION WET MILLING SONIFICATION PRESSURE EXTRUSION French Press Freeze Pressing DECOMPRESSION (PRESSURE CHAMBER) TREATMENT WITH GRINDING PARTICLES than 0.5mm are best
for bacteria. Figure 3 lists some of the parameters that affect the disruption
efficiency of a bead mill 29. Some of the problems
associated with bead mills include large temperature rise, poor scale-up and
contamination of the product by bead material. A technique for cell
disruption now being investigated involves the use of supercritical fluids and
explosive decompression. A supercritical fluid is a gas that is pressurised
above its critical pressure, giving it unique properties of solvation and transport.
This approach to cell disruption may be beneficial in preserving the released
cell constituents because thermal and mechanical effects are minimised 30 Figure 3 PROCESS VARIABLES OF A BEAD MILL Agitator Speed Proportion of Beads Bead Size Cell Suspension Concentration Cell Suspension Flow Rate Agitator Disc Design Of the different
methods for cell disruption discussed here, the homog-enizer satisfies most of
the requirements for large-scale cell disruption. The homogenizer can be
operated at high pressure; the efficiency of disruption is good; it can be
placed in a continuous system and special designs are available specifically
for biotechnology applications 31. Homogenizer As previously
mentioned, the homogenizer consists of a positive -displacement pump and a
homogenizing valve. The pump delivers a relatively constant
flow of liquid, regardless of the pressure set on the homogenizing valve. Of
course, there will be some pumping loss depending on the efficiency of the pump
and the viscosity of the product, and the horsepower of the motor and pressure
rating of the pump limits the pressure. Figure 4 shows flow
through the homogenizing valve. In the jargon of the industry, the words
"homogenizing valve" mean the combination of the valve, seat and
impact ring. The pump forces liquid through the homogenizing valve assembly.
The valve is pushed towards the seat by the action of a handwheel or hydraulic
valve actuator, which reduces the flow area between the valve and seat. As the flow
area is reduced, the pressure increases, because the flow rate is constant. The
liquid is under pressure from the discharge manifold of the pump up to the
homogenizing valve. As the liquid passes through the homogenizing valve, the
velocity increases rapidly while the pressure decreases rapidly. An example
will illustrate how dynamic this process is. At 15,000 psi (103.4 MPa) the
velocity of the liquid goes from about 6.1 m/s to 337 m/s in a distance of
about.254 mm in 7.5 microseconds. The pressure drops from 15,000 psi (103.4
MPa) to atmospheric pressure also in this time. This intense energy change
produces the effect called homogenization. For a mixture of two immiscible
liquids and a surfactant, homogenization will produce an emulsion. For a disper- sion, homogenization
will break up solid agglomerates and disperse particles uniformly throughout a
liquid. In cell disruption the cell wall will be broken open. The actual
mechanism of homogenization is not completely defined because of the difficulty
of studying a phenomenon that occurs so rapidly and at high pressure. There are
many theories of homogenization of emulsions including cavitation, turbulence
and shear 2 , but no one theory has been absolutely proven. Some researchers
have proposed mechanisms for cell disruption such as turbulence 32 and
impingement33,34 . Experimentation by this author has determined that the
mechanism of cell disruption is different from that of emulsification, and this
type of information is useful in the proprietary design considerations of
homogenizing valves used for cell disruption. Obviously,
homogenizing valve design for cell disruption is best studied by disrupting
cell slurries with different valve configurations. TheAPV Homogenizers
laboratory in Wilmington, Massachusetts, uses baker's yeast for the
micro-organism, because it is inexpensive, easily obtained, non-toxic and has a
relatively tough cell wall compared to an organism such as E. coli. Test Methods The test procedure
used by this author involves the following. First, a slurry of 10% yeast (dry
cell weight) in water is prepared 35. The slurry is kept cold, because
homogenization will add heat to the broth. With water the temperature rise
through a homogenizer is 3.0*F (1.7*C) per 1000 psi (6.9 MPa) pressure. This
temperature rise is inversely proportional to the heat capacity of the liquid
and is independent of the type of homogenizer or valve 36. A batch of this slurry
is run through the homogenizer, and samples are collected at different pressure
settings. The collected samples are immediately cooled. Then the samples are
diluted and centrifuged. Also, a portion of each original sample is diluted
with 2N NaOH, heated at 90*C and then centrifuged. These samples treated with
caustic are the controls for each test condition and contain 100% of the
soluble protein. After centrifuging, the samples are again diluted, and the
amount of soluble protein is determined using a protein assay reagent from Bio-Rad
Laboratories. The amount of protein in the sample is divided by the amount of
protein determined from the caustic treatment. This ratio gives the percent of
soluble protein released from the cell for each condition. All assays are done
in triplicate. Because these analyses
are performed using commercially obtained baker's yeast, variations are
encountered even though the yeast is fresh and the operating conditions are the
same. Figure 5 shows three curves indicating the percent of protein released at
different homogenizing pressures. The homogenizer and the cell disruption valve
were the same in each case, but the batches were processed in different years.
Therefore, the variations are most likely due to the yeast. Kula 28 comments on the fact that the mechanical strength of a
microorganism depends on the growth conditions and history of the biomass. This
means that each test done on a batch of cell slurry must be judged
independently of other batches. The only way to relate results from different
batches would be to run a reference sample for each batch. The reference sample
might be one run with the same homogenizer, valve and pressure. This reference
point could be used to interconnect different batches. However, if the organism
is grown by the lab performing the CD test and growth is rigidly controlled, then
the batches would be more consistent than those made from
commercially-pre-pared organisms 37. Valve Design and
Operating Conditions Using this method for
protein analysis, this author has run many tests at flow rates from 5 gph (19
lph) up to 500 gph (1892 lph) evaluating many different designs and
configurations of the cell disruption homogenizing valve. For example, Figure 6
shows the difference in efficiency between the CD design and the CR design.
Obviously, these results show that the CD design is more efficient than the CR
design. The results from tests on various designs have allowed Gaulin to
develop an efficient CD valve, which can be scaled up to production flow rates. Along with design
considerations, the effect of pressure has also been studied. Pressure profile
studies have shown that high pressure, 10,000 to 15,000 psi (69 to 103.4 MPa),
is a benefit for cell disruption. Figure 7 Pressure profile of soluble protein released from
Saccharomyces cerevisiae (baker's yeast) from three different yeast batches
pro-cessed in different years but using the same style CD valve and
homogenizer. shows the type of
results obtained when going to high pressure. It is apparent that the degree of
disruption levels off above 12,000 psi (82.8 MPa). No particular theory has
been developed to explain this trend even though it has been reported in the
literature (35,38). The use of high
pressure is an alternative to multiple passes. The effect of multiple passes
though the homogenizer has been described by many researchers 21 . The important consideration to make when analyzing
these results is that multiple passing should be done in discrete steps.
Recycling back to the original feed sample means that processed material is
mixed into unprocessed material. Eventually some of the cells will have been
homogenized several times, and some will not have been homogenized at all 39. To be certain that all the sample sees that at least one
pass would require a longer processing time than one discrete pass. Therefore,
multiple-pass tests should be done without recycling. An understanding of
the effects of valve design, pressure and multiple passes can be put to use in
processing cell slurries. It has been demonstrated here that the CD valve
design is more efficient than the CR design, and high pressure is more
effective than low or moderate pressures. Therefore, the combination of these
conditions can make cell disruption more efficient. Masucci 35 found that using the CID valve at high pressure
eliminated multiple passing at lower pressure with the CR valve. For example,
one pass at 14,000 psi (96.6 MPa) with a CD valve released as much protein from
Leuconostoc mesenteroides as did four passes at 8000 psi (55.2 MPa) with the CR
valve. In his thesis on cell
disruption Sanchez 1 found that using the
Gaulin 30CD homogenizer with a CD valve gave slightly better yield after one
pass at 15,000 psi (103.4 MPa) than two passes at 8000 psi (55.2 MPa) for E.
coli. The percent of protein release was 79% for 15,000 psi (103.4 MPa) and 74%
for two passes at 8000 psi (55.2 MPa). One pass at 8000 psi (55.2 MPa) released
61 % protein. Sanchez also found
that, over the flow rates investigated, there were no significant differences
in the amount of solids removed by centrifu-gation for slurries processed at
8000 psi (55.2 MPa), 12,000 psi (82.8 Comparison of cell disruption efficiency for the CD and
CR valve using baker's yeast. Pressure profile for the 30CD showing a levelling off in
yields about 12,000 psi (82.8 MPa). MPa) or 15,000 psi
(103.4 MPa). This finding is surprising, because high pressure and multiple
passing will increase cell debris and reduce particle size. The assumption is
that this cell debris will make downstream separation more difficult. However,
Sanchez did not find this to be the case. Along with design
changes in the homogenizing valve for efficient disruption, the configuration
of the pump cylinder must be considered to accommodate high operating pressures
and biotech containment. The 30CD homogenizer is an example of a special design
for biotech application 31 40. The 30CD pump uses
counterbored O-rings instead of non-counterbored flat gaskets to lessen the
possibility of aerosoling product if a seal is breached. The counterbore means
that the escaping liquid must change direction resulting in visible drips as
opposed to an invisible mist. Also, the O-ring seal does not require the
clamping forces that a gasket does to seal properly. The pumping chamber is
made up of hemispherical ports for the suction and discharge channels. These
hemispherical ports replace intersecting bores which lower pressure pumps
possess. The inter-secting bores are potential areas of failure, if subjected
to the excessive stresses occurring at high pressures. Double seals for the
plungers are part of the 30CD design. This sealing arrangement allows for
containment of plunger cooling water that might become contaminated by product
passing by the plunger packing. Of course, if extremely hazardous pathogenic
micro-organisms are being processed, then the homogenizer should be contained
in a secondary cabinet. Some other operating parameters to consider when
selecting and installing a homogenizer are the f low rate, pressure
requirements and the piping arrangement. The flow rate and maximum operating
pressure will determine the size of the homog-enizer, because these conditions
determine the horsepower of the motor and the size of the pumping chamber
including the plunger diameter. The infeed pressure or
Net Positive Suction Head (NPSH) available to the suction manifold of the
homogenizer must satisfy the NPSH required by the
homogenizer, so that it is not "starved". Inadequate infeed pressure
could result in damage to the pump chamber. Exces-sive entrained gases can also
cause damage to the pump and result in poor operation of the homogenizer. The viscosity of the
slurry may have some effect on the efficiency of disruption, but very little
information on viscosity effects has been given in published literature.
Considering that a high concentration of yeast does not affect efficiency of
cell disruption and that the viscosity of the slurry most likely increases with
the amount of yeast, it can be assumed that any effect of viscosity would be
small. However, it is known that after the first pass through the CD
homogenizer, the release of nucleic acids from an E. coli slurry can increase
the viscosity of the slurry. The slurry will now have a greater viscosity for
the second pass through the homogenizer. It has been reported that the use of a
second-stage valve on the homogenizer reduces the amount of viscosity produced
after the first pass 1. Conclusions Cell disruption
techniques have been studied for the past forty years; however, many of these
methods cause loss of product and are not suitable for efficient processing of
large batches. The CD homogenizer is suitable for efficient disruption of small
or large batches of various microorganisms. The CD homogenizer consists of a
pump designed for the biotech environment, usually with high pressure
capabilities, and a homogenizing valve developed to maximize cell disruption. References 1 S. A. Ruiz,
"Studies on Cell Disruption and Cell Debris Removal in Downstream
Bioprocessing, WS Thesis, Massachusetts Institute of Technology, May 1989. 2. W. D. Pandolfe and R. R.
Kinney, "Recent Developments in the Understanding of Homogenization
Parameters," Paper delivered at the Summer National Meeting of the
American Institute of Chemical Engi-neers, Denver, Colorado, August 1983. 3. F. Lux, Patent
Specification 367,063; 1932. 4. H. W. Milner, N. L.
Lawrence and C. S. French, "Colloidal Dispersion of Chloroplast Material," Science 111 (June 1950) 633-634. 5. J. A. Duerre and E. Ribi, "Enzymes Released from
Escherichia coli with the Aid of a Servall Cell Fractionator," Appl. Microbiol. 11 (1963)
467. 6. J. W. T. Wimpenny, Process Biochem. (July 1967) 41-44. 7. J. C. Garver and R. L. Epstein, "Method for
Rupturing Large Quanti-ties of Microorganisms," Appl. Microbiol. 7 (September 1959) 318-319. 8. A. Rodgers and D. E. Hughes, "The Disintentegration
of Microorgan-isms by Shaking with Glass Beads, " J Biochem. Microbio
Tech. & Erg. 2 (1960) 49-70. 9. D. E. Hughes, "The Disintegration of Bacteria and
Other Microorgan-isms by the M.S.E.-Mullard Ultrasonic Disintegrator," J. Biochem. Microbio.
Tech. and Erg. 3 (1961) 405. 10. D. E. Hughes and W. L. Nyborg, "Cell Disruption
by Ultrasound," Science 138 (1962) 108. 11. S. R. Tannenbaum and S. A. Miller, "Effect of
Cell Fragmentation on Nutritive Value of Bacillus megaterium Protein," Nature 214 (1967)
1261-1262. 12. G. Hedenskog, H. Mogren and L. Enebo," A Method for
Obtaining Protein Concentrates from Microorganisms," Biotech. and Bioeng.
12 (1970) 947-959. 13. G. R. Lawford, et al, "Production of High Quality
Edible Protein from Candida Yeast Grown in Continuous Culture, "Biotech. and
Bioeng. 21 (1979) 1163-1174. 14. P. Dunnill and M. D. Lilly, Single Cell Protein 11, ed. S. R. Tannenbaum and D. Wang (Mass.: MIT Press, 1975) 179-207. 15. C. A. Batt and A. J. Sinskey, "Use of Biotechnology
in the Production of Single-Cell Protein, " Food Technology (February 1984) 18-21. 16. R. J. Zanetti, "Breathing new life into single-cell
protein," Chemical Engineering (Feb. 6, 1984) 18-21. 17. A. Wiseman, "Enzyme for Breakage of
Micro-organisms," Process Biochem. 63 (May 1969) 63-65. 18. K. Zetelaki, "Disruption of Mycelia for Enzymes, "Process
Biochern. 19 (Dec. 1969) 19-27. 19. W. B. Jakoby, "Enzyme Purification and Related
Techniques," in Methods in Enzynmology22 (New York: Academic Press, 1971) 482-487. 20. M. Follows et al, "Release of Enzymes from Baker's
Yeast by Disrup-tion in an Industrial Homogenizer, "Biotech and Bioeng. 13 (1971)
549-560. 21. P. J. Hetherington et al, "Release of Protein from
Baker's Yeast by Disruption in an Industrial Homogenizer," (inst. Chem.
Eng., 1971),
142-148. 22. D. A. Whitworth, "Assessment of an Industrial
Homogenizer for Protein and Enzyme Solubilization from Spent Brewery
Yeast," Comptes Rendus des Travaux du Laboratoire Carisberg (Report of
Work at Carisberg Lab. 40 (Danish Scientific
Press, 1974), 19 23. D. A. Whitworth, "Hydrocarbon Fermentation: Protein
and Enzyme Solubilization from C. lipolyticva Using an Industrial
Homogenizer," Biotech. and Bioeng. 16 (1974) 1399-1406. 24. S. D. Cunningham et al, "Rupture and Protein
Extraction of Petro-leum-Grown Yeast, " J. of Food Sci. 40 (1975) 732-735. 25. C. Lee et al, "Disintegration of Dried Yeast Cells
and Its Effect on Protein Extractability, Sedimentation Property and
Viscosity of the Cell Suspension," Biotech.
and Bideng. 21 (1979) 1-17. 26. C. R. Engler, "Disruption of Microbial
Cells," in Comprehensive
Biotechnology, vol. 2, ed. M. Moo-Young (Oxford: Pergamon Press, 1985),
305-324. 27. L. Edebo, "Disintegration of Cells by Extrusion
Under Pressure," Enzyme Technology, ed.
R. M. Lafferty (Berlin: Springer Veriag, 1983) 93-114. 28. M.-R. Kula and H. Schutte, "Purification of
Proteins and the Disruption of Microbial Cells," Biotechnology Progress 3 (1987) 31. 29. A. Wiseman, D. J. King and M. A. Winkler, "The
isolation and purifica-tion of protein and peptide products," Yeast Biotechnology, ed. D. R. Berry, 1.
Russell and G. G. Stewart (London: Alien and Onwin, 1987), 433-470. 30. T. P. Castor and G. T. Hong, paper presented at the
199th ACS National Meeting, Boston, April 1990. 31. W. D. Pandolfe, "The Cell Disruption
Homogenizer," Proceeding of the DT11/HSE/SCI Symposium on Large-Scale
Bioprocessing Safety, (Itevenage, England: Warne Spring Lab., 1989). 32. M. S. Doulah, T. H. Hammond and J. S. G. Brookman,
"A Hydro-dynamic Mechanism for the Disintegration of Saccharomyces
cerevisiae in an Industrial Hornogenizer," Biotechnol. and Bideng, 17 (1975) 845-858. 33. C. R. Engler and C. W. Robinson, "New Method of
Measuring Cell-wall Rupture," Biotech. and Bioeng 21 (1979) 1861. 34. C. R. Engler and C. W. Robinson, "Disruption of
Candida Utilis Cells in High Pressure Flow Devices, " Biotech. and Bioeng. 23 (1981) 765. 35. S. F. Masucci, 1mproving the Efficiency of High
Pressure Homogeniz-ers for Cell Disruption Applications," BS Thesis, M.
I.T., Feb. 1985. 36. H. R. Mitten, Jr., and H. L. Preu, "Temperature
Increase Due to Homogenization," J. Dairy
Sci. 42 (1959) 1880. 37. C. R.Engler, "Disruption of Microorganisms in
High Pressure Flow Devices" PhD Thesis, University of Waterloo, 1979. 38. E. Keshavarz, M. Hoare and P. Dunnill , Biochemical
engineering aspects of cell disruption," Separations for Biotechnology, ed. M. S. Verall and M. J. Hudson
(England: Soc. for Chem. Ind. by Ellis Horwood, 1987) 62-79. 39. A. Leviton, A. and M. J. Pallansch, "Continuous
Recycling in the Homogenization of Relatively Small Samples," J. Dairy Sci. 42 (1959) 20-27. 40. B. Wilkinson and J. M. Bristol, U. S. Patent
4,773,833 (1988).
ASESORIA:
Si requiere de mayor información le pedimos se comunique a nuestro Departamento
Técnico. Donde con el mayor gusto un Ingeniero especializado
le atenderá de una manera rápida y sencilla. |
| |
| Contáctenos | Curriculum | Presentación | Página principal |
 |
Agrégame a tus favoritos |
|
NOTA: Toda la información
contenida en este artículo, es de buena fe. Y es el resultado de varios
años de investigación, experiencia y trabajo directamente en las diferentes plantas
productoras . |
|
ATTE.: GRUPO INDUSTRIAL AISA, S.A. DE C.V. http://www.oocities.org/grupoindustrialaisa |