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Applied and Environmental Microbiology, October 1999, p. 4464-4469, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effects of Combined Shear and Thermal Forces on
Destruction of Microbacterium lacticum
S.
Bulut,*
W. M.
Waites, and
J. R.
Mitchell
Division of Food Sciences, School of
Biological Sciences, University of Nottingham, Sutton Bonington
Campus, Nr. Loughborough, Leicestershire LE12 5RD, England, United
Kingdom
Received 2 February 1999/Accepted 9 July 1999
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ABSTRACT |
A twin-screw extruder and a rotational rheometer were used to
generate shear forces in concentrated gelatin inoculated with a
heat-resistant isolate of a vegetative bacterial species,
Microbacterium lacticum. Shear forces in the extruder were
mainly controlled by varying the water feed rate. The water content of
the extrudates changed between 19 and 45% (wet weight basis). Higher
shear forces generated at low water contents and the calculated die
wall shear stress correlated strongly with bacterial destruction. No
surviving microorganisms could be detected at the highest wall shear
stress of 409 kPa, giving log reduction of 5.3 (minimum detection
level, 2 × 104 CFU/sample). The mean residence time
of the microorganism in the extruder was 49 to 58 s, and the
maximum temperature measured in the end of the die was 73°C. The
D75°C of the microorganism in gelatin at 65%
water content was 20 min. It is concluded that the physical forces
generated in the reverse screw element and the extruder die rather than
heat played a major part in cell destruction. In a rotational
rheometer, after shearing of a mix of microorganisms with gelatin at
65% (wt/wt) moisture content for 4 min at a shear stress of 2.8 kPa
and a temperature of 75°C, the number of surviving microorganisms in
the sheared sample was 5.2 × 106 CFU/g of sample
compared with 1.4 × 108 CFU/g of sample in the
nonsheared control. The relative effectiveness of physical forces in
the killing of bacteria and destruction of starch granules is discussed.
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INTRODUCTION |
There have been extensive studies on
the use of extrusion processing to reduce microbial counts in food
ingredients (2, 4, 7, 10, 16, 17). Likimani et al.
(12) proposed a methodology for determining the destruction
of bacterial spores in a Brabender single-screw Plasticorder extruder
under varying processing conditions. The D value
("extrusion D value" or "extrusion death rate" as
proposed by the authors) for extrusion was calculated on the basis of
average time at a mass temperature of >95°C. They stated that
destruction of spores in the extruder was a function of mass
temperature and residence time at constant moisture levels (18%) in a
corn-soybean (70%/30% [wt/wt]) mixture. Increasing mass temperature
resulted in greater lethality, while increasing screw speed, which
reduced residence time, resulted in lesser lethality.
Using the same system as mentioned above, Likimani and Sofos
(11) examined the potential for injury of Bacillus
globigii spores during extrusion cooking. Their results showed
that extrusion processing at low barrel temperatures (80 to 100°C)
resulted in injury to spores of B. globigii, whereas at
higher temperatures spore injury was not detectable.
In an extrusion process, both mechanical and thermal energy are applied
to the material. When the process is applied to starch-containing systems, it is clearly recognized that both forms of energy can play an
important role in starch conversion (18). In this context, starch conversion refers to the loss of ordered crystalline regions in
the granule and more severe damage including polysaccharide degradation
and loss of granule integrity (5). Attempts have been made
to deconvolute the influence of the two forms of energy by calculating
the expected effect for heat alone and comparing it with the total
extent of conversion (19). Studies using a capillary
rheometer have allowed an estimate of the shear forces required to
destroy the ordered structure within the granule. The amount of
residual order in this case can be measured by differential scanning
calorimetry. It has been shown that, for waxy maize starch, granule
destruction by mechanical forces can occur at much lower temperatures
than those required to gelatinize or melt the starch granule under
conditions where no mechanical energy is applied. The minimum shear
stress required to induce starch conversion decreases strongly with
increasing temperature (23).
In this paper, we present some results on the application of a similar
approach to the destruction of microorganisms by extrusion. The
objective of the work was to quantify the shear stress required to
promote microbial killing under thermal conditions which were mild
compared with those required to kill the microorganism. There were two
longer-term objectives of the study. Firstly, if the relationship
between physical forces and heat in the destruction of microorganisms
could be better understood, then it might be possible to devise
processes where a combination of the two energy forms could be used to
kill microorganisms more efficiently and at lower temperatures than
would be the case with heat alone. Secondly, the susceptibility of
microorganisms to physical forces might provide a useful way of
differentiating between microorganisms and possibly even fractionating
them by removal of the more susceptible in a mixture by the application
of physical forces.
The approach employed was to extrude the microorganisms in a gelatin
carrier through a narrow-slit die. Microbacterium lacticum, isolated from pasteurized liquid whole egg, was selected, as it has
been reported to have a high thermal stability (14).
Gelatin, when plasticized by low amounts of water, will form a melt at temperatures below that required to inactivate this organism, and
subsequent enumeration is facilitated by the way in which gelatin can
be solubilized at relatively low temperatures.
The result of the extrusion experiments was supported by using a
rheometer equipped with cone and plate geometry to shear samples
inoculated with M. lacticum at lower viscosities.
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MATERIALS AND METHODS |
Microorganism growth and enumeration.
The JP2/1/1 strain of
M. lacticum, which is a gram-positive, nonsporing
heat-resistant bacterium forming small round yellow colonies, was used
as a test organism. This strain was originally isolated from
pasteurized liquid whole egg (14).
Brain heart infusion (BHI) broth (Oxoid) was used for growth of liquid
cultures, and BHI agar plates were used for viable counts.
Phosphate-buffered saline (PBS), pH 7.3 (Oxoid), was used as the
dilution medium.
Sterile universal tubes containing 10 ml of BHI broth were inoculated
with 20 µl of liquid inoculum produced from a single
colony taken
from BHI agar. The inoculated tubes were incubated
at 30°C in a
shaker operating at 200 rpm. The growth curve of
the microorganism
obtained by reading the optical density of the
liquid culture at 600 nm
showed that the microorganism reached
the stationary phase after about
15 h. In our experiments, 18-h
cultures were used for inoculation
into the extruder and preparation
of the gelatin mix with the
microorganism for shearing in the
rheometer.
For enumeration, the Miles-Misra (
13) counting method was
applied, and the BHI plates were incubated at 30°C for 48 to 72
h.
Carrier medium.
As the carrier medium, for extrusion
experiments limed hide 225 bloom gelatin (Rousselot) was used without
any treatment. For the heat resistance experiments and the experiments
done with the rheometer, gelatin sieved through a 250-µm-pore-size
laboratory sieve was used.
Moisture determination.
Extruded gelatin samples were cut
into small pieces and dried at 70°C in a vacuum oven overnight.
Powdered gelatin samples were dried directly in the vacuum oven overnight.
Heat resistance tests.
The heat resistance of the
microorganism was tested in gelatin and PBS. In order to test the heat
resistance of the microorganism in gelatin, the liquid culture of the
microorganism was mixed with gelatin to give a final moisture content
of 65% (wt/wt). For this purpose, 3 g of gelatin with a known
amount of moisture content was mixed with a calculated amount of
culture broth diluted to 50% with PBS (pH 7.3) in a sterile universal
tube. This mix was kept in a 60°C water bath for 17 min in order to
melt the gelatin. The pH of this mix was measured as 5.8 to 6.0 with a paper pH indicator. The mix was transferred into sterile universal tubes of known weight in 1- to 2-ml amounts by using sterile syringes. After the weight of tubes containing the mix was recorded, the tubes
were immersed in a second water bath adjusted to the test temperature.
A heating-up time of 3 min was applied, and then the first tube was
taken from the water bath as the time zero sample. A dilution medium of
20 ml at 55°C was added to the tube and mixed with a WhirliMixer
(Fisons). The weights of the tubes with the dilution medium were
recorded, and serial dilutions were made for viable counting at 10- to
15-min intervals. Calculations were made on a weight basis in order to
plot the survival curves.
In order to understand the effect of gelatin on the survival of
M. lacticum, a heat resistance test was also carried out in
PBS at pH 5.9 in the absence of gelatin. A culture (3 ml) of
M. lacticum in BHI broth was added to 15 ml of PBS at pH 5.8 in a
sterile universal tube. The pH of this mix was measured as 5.9.
A
sample (1 ml) of this mix was added to screw-cap Eppendorf tubes
and
immersed in a 60°C water bath for 17 min before being transferred
to
another water bath adjusted to the test temperature. As previously,
a
3-min heating-up time was applied. Samples were taken from the
water
bath at 10- to 15-min intervals, and serial dilutions were
made for
viable
counts.
Extrusion.
Extrusion was carried out in a Clextral BC-21
self-wiping and corotating twin-screw extruder (Firminy, France). The
400-mm extruder barrel has a 16:1 length-to-diameter ratio and consists of four modules each 100 mm long. The temperatures of the last three
modules are controlled by electrical heating and water-cooling systems.
A slit die of 2 mm in height, 20 mm in width, and 14.5 mm in length was
used. A transducer port located next to the feed port on the extruder
barrel was modified to hold the hypodermic needle of a syringe. All
inoculations were carried out from this port by using sterile syringes.
A schematic diagram of the extruder showing the screw configuration and
the inoculation point is given in Fig. 1.
The set heater temperatures in the last three zones of the extruder
barrel varied in order to achieve stable extrusion but never exceeded
45°C. The solid gelatin was added in the form of granules to the feed
port at an addition rate of 3 kg · h
1 with a
volumetric feeder (K-TRON, type T20). Distilled water was continuously
added with a piston pump (DKM-Clextral, type TO/2). The rate of
addition was varied to give the desired die pressure. Both the feeder
and the water pump were calibrated prior to the extrusion in order to
avoid fluctuations during the operation. The torque was continuously
monitored, and the pressure and the temperature immediately before the
die were measured with a combined pressure-temperature transducer. The
temperature of the extrudate at the die exit (maximum temperature) was
measured by carefully inserting a thermocouple insertion probe
connected to a handheld digital thermometer (Digitron, 3202K) into the
die. The specific mechanical energy (SME) was calculated from the
following relationship:
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FIG. 1.
Schematic diagram of Clextral BC-21 twin-screw extruder
showing screw configuration and inoculation point.
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Residence time distribution analysis.
The dye tracer method
was used for the residence time distribution analysis. For this
purpose, erythrosin B
(C20H6I4O5Na2) (Sigma) was used as a tracer. The mean, minimum, and maximum residence times of the material inside the extruder were determined as explained in the literature (6, 9, 15, 20).
Inoculation of the microorganism into the extruder.
At a
steady-state operation of the extruder, two blank samples were put into
sterile stomacher bags and sealed. In order to avoid increasing the
water content of the material by liquid inoculum, which in turn may
reduce the local shear stress around the bacterial cell, the water pump
was switched off from the control panel momentarily, and depending on
extrusion moisture level, 3 to 5 ml of culture containing 1 × 109 to 8 × 109 CFU/ml was injected by a
sterile syringe within 5 s. The remainder of the inoculation
culture was kept in ice for determination of the exact inoculation
level. As soon as the inoculation of the microorganism was finished,
the water pump was switched on. The samples were collected every
15 s from the beginning of injection until 4 min after the start
of the operation. Samples were put into sterile stomacher bags, sealed,
and kept in ice until counted.
Sampling and enumeration of the surviving microorganisms after
extrusion.
For enumeration of viable cells after extrusion,
samples (1 g) were removed from the middle section of each extrudate
into sterile universal tubes and 19 ml of PBS was added to each tube. After the samples were melted in a 60°C shaker water bath for 45 min,
serial dilutions were made and the Miles-Misra (13) counting
method was applied for viable counts.
As a control, 200 µl of culture was added to a universal tube
containing 1 g of blank sample, and following the addition of
19 ml of PBS, the tube was kept in the 60°C water bath for 45
min. The
numbers of microorganisms in the inoculation culture
and in the
heat-treated control sample were determined by viable
counts. The
reduction in the numbers of the microorganisms in
the control sample
was considered when calculating the overall
reduction.
In order to validate the sampling method, 1-g samples taken from the
middle section of each extruded sample and the remaining
whole samples
of extrudates were analyzed for viable count. For
whole-sample
analysis, sterile 1-liter Erlenmeyer flasks were
used. According to
sample weights, 300 to 500 ml of PBS was added
to the flasks and the
samples were melted as explained above.
The weight basis was used to
calculate the number of surviving
cells.
Experiments with the rotational rheometer.
A
controlled-shear-rate rotational rheometer (Bohlin VOR) with cone and
plate geometry (2.5° cone angle, 30-mm cone diameter) was used for
shearing of the gelatin mix of M. lacticum. A thermal cover,
as shown in Fig. 2, was used in order to prevent the heat loss during
shearing of the samples. A digital thermometer (Digitron T202KC) was
used to monitor the temperature of the sheared sample during the
operation of the rheometer. As shown in Fig.
2, in order to avoid any interference by
thermocouple 2 with the flow pattern, the temperature of the sheared
sample inoculated with the microorganism was monitored indirectly by
thermocouple 1. Experiments done in the absence of the microorganism
showed that the temperature of the sheared sample measured by
thermocouple 2 was 1.5 to 1.7°C higher than the temperature measured
by thermocouple 1. When quoting the temperature of the sheared sample,
this correction was taken into account.
The gelatin mix of
M. lacticum was prepared at 65% (wt/wt)
moisture content level as explained for the heat resistance test
in
gelatin. The inoculation level of the bacterium in the mix
was between
1 × 10
8 and 8 × 10
8 cells/g of
sample. A sample of the mix (1.5 ml) was added to
a screw-cap Eppendorf
tube with a syringe under sterile conditions.
This tube containing the
sample was placed in a water bath adjusted
to the plate temperature of
the rheometer. After 3 min of heating
up, 0.5 ml of sample was removed
to the plate of the rheometer.
The remainder of the sample in the tube
was used as a control,
and the tube was submerged in the water bath
after a thermocouple
to monitor the temperature of the control sample
was mounted.
The temperatures of the sheared sample and the control
unsheared
sample were recorded at the start of shearing and every
minute
thereafter. At the beginning of shearing, the temperature of the
sheared sample (monitored by thermocouple 1 [Fig.
2]) and the
temperature of the control sample were 3 to 5°C lower than the
set
operation temperature of the rheometer. During the operation,
the
temperature of the samples increased gradually. During the
operation
time of the rheometer, the temperature of the control
sample was kept
higher (by 2 to 4°C) than the temperature measured
by thermocouple 1 (Fig.
2). This resulted in a higher (0.6 to
2.1°C) heat exposure of
the control samples than of the sheared
samples. As soon as the
shearing of the sample was finished, the
control sample was taken from
the water bath and the sheared sample
was recovered with a sterile
spatula. The recovered sample and
a sample taken with a sterile syringe
from the control sample
were added to sterile universal tubes of known
weights. Dilution
medium (15 to 20 ml) was added to each tube in the
proportion
of the sample weights, and the tubes were plunged into a
60°C
water bath for 10 min in order to melt the samples. Serial
dilutions
were prepared from both tubes for viable counts. Calculations
were made on a weight basis, and the reduction in the numbers
of
microorganisms due to shear was obtained by subtracting the
count
reduction in the control sample due to heat from the overall
count
reduction in the sheared sample. The results were expressed
in terms of
log
reduction.
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RESULTS |
Heat resistance tests.
Survival curves of M. lacticum at different temperatures in PBS and gelatin (Fig.
3) showed that heating in gelatin
increased the killing but that at 60, 65, and 70°C even in gelatin
there was less than 1 log kill in 1 h, while at 75°C there was
less than 1 log kill in 45 min in PBS and less than 3 log kills in 45 min in gelatin. Payne et al. (14) reported that the
microorganism shows almost no killing at 75°C in PBS, pH 7.1. They
also found that the D80°C value of the
microorganism was 9.5 min in the same medium. Compared to these
D values, our lower D values in PBS (Table
1) are probably due to the lower pH of
the medium employed. In addition, the heating time of 17 min at 60°C
and heating-up time at the test temperature also reduced the
D values.
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FIG. 3.
Survival curves of M. lacticum at different
temperatures in gelatin (moisture content, 65% [wt/wt]; pH 5.8 to
6.0) (A) and PBS (pH 5.9) (B).  , 60°C;  , 65°C;  , 70°C;
×, 75°C.
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TABLE 1.
D value of M. lacticum at different
temperatures in PBS (pH 5.9) and in 65% (wt/wt)-moisture-content
gelatin (pH 5.8 to 6.0)
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Sampling for extrusion experiments.
The whole-sample analysis
showed that there was no significant difference between the total count
obtained by 1-g-sample analysis and that obtained by the whole-sample
analysis. For example, in the analysis done for the experiment where
0.9 log reduction was obtained (Table 2)
the difference between the total counts obtained was less than 24%,
which resulted in only 0.1 log difference in terms of log reduction.
Also, residence time distribution analysis showed that the 1-g sample
taken from the middle section of each extrudate provided an accurate
representation of the whole extrudate and that the calculations made
based on this sampling method recovered more than 90% of the dye
introduced to the extruder.
Residence time distribution analysis.
The calculated mean
residence time was 58, 52, and 49 s for screw speeds of 120, 200, and 300 rpm, respectively. The maximum residence time ranged from 155 to 170 s, increasing with decreasing screw speed. The increase in
residence time with decreasing screw speed has been previously shown
for twin-screw extruders (9, 15).
Although the correlation between die wall shear stress and killing
suggests that bacterial destruction occurs within the extruder
die, it
also seems possible that some killing will occur within
the reverse
screw element before the die. Residence time measurements
made by using
radioactive tracers (
3) show that the material
spends a
major proportion of the total residence time in this
zone, whereas in
our experiments the mean residence time in the
die, calculated from the
die dimensions and throughput, was 1.3
s.
Microbial inactivation during extrusion.
The wall shear stress
(
w), the wall shear rate
(
w), and the apparent viscosity at the
die wall (
w) were estimated from the
expressions
where
P is the pressure drop across the slit die,
C is half of the slit thickness,
L is the slit
length,
Q is the volumetric
flow rate, and
w is
the slit width (
8).
The expression for the wall shear rate and hence the viscosity assumes
Newtonian behavior. It has been reported elsewhere
that this is the
case for gelatin solutions for concentrations
up to 29% (wt/wt) (dry
weight basis) (
22), although no information
is available for
the high concentrations used in the
extruder.
The log reduction of
M. lacticum (Tables
2 and
3) shows a strong correlation with die
wall shear stress, viscosity, and
maximum temperature and a strong
negative correlation with the
moisture content. These relationships are
self-consistent since
a reduction in water content will result in an
increase in viscosity
and shear stress. The maximum temperature will
increase with viscosity
due to increased conversion of mechanical
energy to heat. Interestingly,
the correlation coefficient between log
reduction of
M. lacticum and SME was relatively low. The
torque on the screws and hence
the SME were related to the consistency
of the product over the
whole screw length. This relatively low
correlation suggests that
the conditions at the die and possibly at the
end of the screws
(reverse screw element) are most important for
bacterial destruction.
The relationship between count reduction and the wall shear stress and
the maximum die exit temperature are shown in Fig.
4. The temperature measured at the
entrance to the die did not
exceed 50°C for any experiment. The
strong nonlinear relationship
between the count reduction and the
calculated die wall shear
stress, the fact that the highest temperature
recorded in the
die end was below 75°C, and the observation that
substantial count
reduction was obtained at lower temperatures suggest
that physical
forces around the reverse screw element and the die play
a major
part in the measured destruction of the cells. Within the limit
of detection (2 × 10
4 CFU/sample), no surviving
microorganisms could be detected at
the highest wall shear stress of
409 kPa, giving at least 5.3
log reduction.
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FIG. 4.
The dependence of log reduction of M. lacticum on die wall shear stress (A) and maximum extrudate
temperature measured at the die outlet (B). (Refer to Table 2 for the
other parameters of each experiment.)  , no residual microorganisms
detected.
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It was observed that there was little increase (e.g., from 1.3 × 10
7 to 1.5 × 10
7 CFU/total samples) in
the number of surviving microorganisms
on BHI agar plates, when the
incubation time increased from 60
to 90 h. No resuscitation media
and/or techniques were used to
discriminate between the inactivated and
the injured
cells.
Because the temperature and the shear stress distribution within an
extruder are difficult to define, further experiments
were carried out
with a rheometer equipped with cone and plate
geometry where the shear
stress and shear rate throughout the
sample should be constant. Because
of mechanical limitations,
lower sample viscosities than would be
possible within an extruder
had to be
used.
Rheometer experiments.
In the rheometer, the count reduction
of M. lacticum relative to the control increased with
increasing shear rate and temperature (Fig.
5). Shearing of the microorganism with
gelatin at 65% (wt/wt) moisture content for 4 min at a shear stress of
2.8 kPa and a temperature of 75°C resulted in a maximum killing of
1.4 log cycle (Fig. 5), where the number of surviving microorganisms in
the sheared sample was 5.2 × 106 CFU/g of sample
compared with 1.4 × 108 CFU/g of sample in the
nonsheared control.
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FIG. 5.
Log reduction of M. lacticum in 65%
(wt/wt)-moisture-content gelatin compared to that in the unsheared
control, at increasing shear rates at 60°C (), 70°C (), and
75°C (). Shearing time was 4 min.
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Figure
6 displays the count reduction
with time at a temperature of 75°C and a shear rate of 804 s
1. The curve in this figure intersects the
y
axis at a negative
value, probably because the control samples
experienced slightly
higher temperatures than the sheared samples.
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FIG. 6.
Dependence of log reduction of M. lacticum in
65% (wt/wt)-moisture-content gelatin on the time of shearing in the
rheometer at 75°C and a shear rate of 804 s1.
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DISCUSSION |
The extrusion results suggest that bacterial destruction takes
place within the reverse screw element and the extruder die region
where the highest shear forces are generated. The flow patterns in the
die are complex since the die is relatively short and fully developed
flow does not occur. This and the fact that the wall shear stress was
calculated only from an entrance pressure mean that the calculated
stress should be regarded only as an estimate. Nevertheless, it is of
interest to compare the values found with those required to "kill"
a starch granule. "Total destruction," defined as no viable
microorganisms above the limit of detection in this study, occurred at
a wall shear stress on the order of 400 kPa. Zheng and Wang
(23), using a combination of extrusion and capillary
rheometer, found that the minimum shear stress required to convert
starch ranged from 101 to 103 kPa, the value
decreasing as the temperature increased. In their case, a mean shear
stress taken as two-thirds of the wall shear stress was used. At a
temperature of 50°C, the shear stress for a rate constant of starch
granule destruction approaching the value required for complete
destruction for a 1-s pass through the die was 600 kPa. However, at the
moisture content employed, the peak differential scanning calorimetry
temperature for the melting endotherm was 148°C.
It therefore appears that at the same shear stress, temperatures closer
to the temperature required for thermal destruction of ordered
structures are required for a microorganism compared with a starch
granule. There are three possible reasons for the greater stability of
microorganisms compared with the starch granule. A factor of
significance may be the coupling between the particles in the carrier
medium. We have demonstrated elsewhere (21) that the level
of starch conversion at the same mechanical energy input is much lower
when the starch is in a gelatin medium than with the binary starch
water system. It was suggested that this was due to the poor transfer
of stress between the gelatin carrier and the starch granules. Another
factor that can explain the relatively modest destruction of the
bacteria compared with that of starch may be the particle size. The
linear size of a maize starch granule is an order of magnitude greater
than that of a microorganism. By using arguments analogous to those
used to explain the breakup of emulsion droplets in a shear field
(1), it has been shown that the stress required for
destruction scales with the dimension of the particle. Thirdly, the
nature of the particle itself is different. Microorganisms have
physical properties different from those of starch granules, and this
may be important.
The maximum shear stress in the rheometer was about 3.0 kPa. This is
more than 2 orders of magnitude lower than that required for the
maximum detectable killing in the extruder. It is therefore not
surprising that there is a maximum killing on the order of only 1 log
reduction in the rheometer compared with a 5 log reduction in the
extruder. It is clear from the rheometer experiments that, in addition
to shear stress, important factors are time and temperature. It would
be possible to combine these three factors into "shear D
value," which would be defined as the time for a decimal reduction at
a specific temperature and shear rate. For example, from the data in
Fig. 6 the shear D value for M. lacticum at
75°C and a shear rate of 804 s1 would be on the order
of 3.5 min. A comparison between the shear D value and the
D value for heat alone might provide some interesting information about the structure of microorganisms. Finally, we consider
that it would be interesting to compare the results with a different
carrier medium and determine whether elongational stresses have a
greater effect than shear stresses.
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ACKNOWLEDGMENTS |
This work was supported by The Higher Education Council of Turkey
and a United Kingdom ORS award.
John Payne (BBSRC Institute of Food Research, Norwich, United Kingdom)
is thanked for supplying M. lacticum.
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FOOTNOTES |
*
Corresponding author. Mailing address: Division of Food
Sciences, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Nr. Loughborough, Leicestershire LE12 5RD,
England, United Kingdom. Phone: 44 115 9516197. Fax: 44 115 9516142. E-mail: scxsab{at}szn1.agric.nottingham.ac.uk.
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Bouvier, J. M., and M. Gelus.
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Applied and Environmental Microbiology, October 1999, p. 4464-4469, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
