Inactivation of Lactobacillus plantarum by Pulsed-Microwave

Irradiation
JUNG-KUE SHIN and YU-RYANG PYUN

ABSTRACT
Suspensions of Lactobacillus plantarum cells were subjected to either
conventional heating, continuous microwave (CW) or pulsed microwave
(PW) irradiation at 507C for 30 min. Samples exposed to PW showed
greater reductions (2 ; 4 log) in survival counts than those treated with
either conventional heating or CW irradiation. As exposure time in-
creased, PW resulted in a remarkable increase in 260 nm-absorbing com-
pounds that leaked into the suspending menstruum, as compared to CW
or conventional heating, indicating that PW irradiated cells were the
most injured. The growth of PW irradiated cells was delayed about 24h
and the final acidity of the culture broth was about 60 ; 80% that of
other cells treated with conventional heating or CW irradiation.

Key Words: pulsed microwave, nonthermal pasteurization, Lactobacillus

INTRODUCTION
Fig. 1—Schematic of pulsed microwave irradiation.
WITH INCREASED INTEREST in minimally processed foods, non-
thermal preservation methods are being developed. The design
Table 1—Growth characteristics of L. plantarum after conventional or mi-
and application of nonthermal food preservation processes have crowave heating at 50&C for 30 min
included utilization of electronic or magnetic fields, microwave
Lag time Final cell mass Final Total acidity
radiation and high isobaric pressure, chemical agents and lytic (h) (g/L) pH (%)
enzymes (Chipley, 1980; Mertens and Knorr, 1992; Miyakawa, Unheated 0 1.8 3.84 2.15
1996). Conventional heating 10 1.7 3.85 1.96
Microwaves interact with water in food products and generate CW irradiation 22 1.8 3.93 1.76
heat. Production of heat occurs primarily when ions accelerate PW irradiation 24 1.5 4.20 1.41
and collide or dipoles rotate and line up in the rapidly alternating
electric field. Microwave energy has been used to pasteurize or
sterilize food at lower temperatures and shorter times than nec- more deeply than continuous wave irradiation with the same
essary with conventional heating. The mechanism for the lethal carrier frequency (Lin, 1989; Shizuki, 1991).
effects of microwave radiation on microorganisms is not fully Our objective was to examine the effectiveness of a pasteur-
understood. Many studies have shown that heat generated by ization system using PW. A comparative study on the inacti-
microwaves in food systems exerts most of the killing effect. vation of Lactobacillus plantarum using two types of microwave
Although some scientists have hypothesized that heat may not and conventional heating systems was carried out.
be the only effect, they have not presented evidence of non-
thermal killing effects. Destruction of microorganisms by mi-
crowaves at temperatures lower than the thermal destruction MATERIALS & METHODS
point of microorganisms has been reported by Cunningham
Culture preparation
(1978), Dreyfuss and Chipley (1980) and Khalil and Villota
(1985, 1989). Lactobacillus plantarum isolated from fermented Kimchi was used as
The biological effects of pulsed microwave (PW) are not fully a test organism. Two or three loopfuls of growth from the slant were
understood but the possibility of using PW for pasteurizing inoculated into a 100 mL flask containing 50 mL of Lactobacilli MRS
foods with reduced heat has been proposed (Boon and Kok, Broth Dehydrated (Difco) and then incubated at 377C for 24h. Activated
preculture (3 mL) was again inoculated into a 500 mL flask containing
1987; Mudgett, 1989; Mertens and Knorr; 1992, Shizuki, 1991). 200 mL of MRS broth and incubated for 24h. The final culture broth
Microwave pulses are described by the duty cycle, which is (late-log phase, ca. 108 ; 109 CFU/mL) was subjected to various treat-
defined as the ratio of pulse width to the period (Buffer, 1992). ments. A subset of this culture was maintained on MRS agar slants at
A duty cycle of 1.0 describes a continuous wave. The average 47C and used at various stages of the study.
power is given by the product of peak power duty cycle. Note
that for short pulses with a low pulse repetition rate, the average
power can be very low, when although the peak power may be Microwave source
in megawatts or gigawatts. Therefore, when the shortest pulse
which can be conveniently generated is used, a very high peak The CW irradiation was performed in a household microwave oven
power results. This pulse modulation increases the nonthermal (Samsung model RE-610 TCW, 650W, Korea). The PW irradiation sys-
effects of microwaves on biological materials with reduced heat tem consists of a modified 2450MHz microwave source (Samsung model
generation. Moreover, pulse modulated radiation may penetrate RE-610 TCW, 650W, Korea), pulse generator and pulse driver (Fig. 1).
The generator was adjusted to deliver repetitive rectangular pulse trains
at 500 pulses/sec. The duty ratio was 1/10, equivalent to a pulse width
The authors are affiliated with the Dept. of Food & Biotechnology of 0.2 ms and a period or time base of 2 ms (Fig. 2). The pulse repetition
and Bioproducts Research Center, Yonsei Univ., Seoul, 120-749, frequency was monitored with a frequency counter and an oscilloscope
Korea. (Fig. 1).

Volume 62, No. 1, 1997—JOURNAL OF FOOD SCIENCE—163

PULSED-MICROWAVE INACTIVATION OF L. PLANTARUM . . .
Fig. 2—Modulation characteristics of the microwave (pulse with
a repetition rate of 500 pps).
Fig. 3—Survival of L. plantarum exposed to different heating on
MRSS medium.
Thermal treatments
100 mL of culture broth of L. plantarum, which contained 108 ; 109
cells/mL, was sublethally heated at 507C for 30 min using microwave
irradiation or conventional heating.
Conventional. For conventional heating, 3 mL of cell suspensions in
glass tubes (diameter 5 mm 3 length 150 mm) were heated by immer-
sion in a shaking water bath (Julabo, model SW-21C) maintained at 50
5 0.17C. During treatment, the glass tubes were removed at designated
time intervals and immediately cooled in an ice-water bath for micro-
biological analysis. Temperatures in the glass tubes were measured by a
Teflon-coated thermocouple and recorded by a data logger linked to a
microcomputer.
Microwave. For the CW or PW irradiation, a Pyrex cylindrical glass
container of the culture broth was placed at the center of the oven and
exposed to CW or PW. For maintaining the sample temperature at 50
5 27C, cooling water at 2157C was circulated through the jacket and
coil of the container under CW and 57C water was used during PW
irradiation. Temperatures of cooling water and come-up time from room
temperature to 507C were determined. During microwave irradiation, 5
mL aliquots of the suspensions were removed at designated times and
immediately cooled in an ice-water bath for microbiological analysis.
The temperature of the suspension was measured with a T type copper-
constantan, Teflon-coated thermocouple (Omega Eng. Co.).
Microbiological analysis
Viable cell counts. Cells were separated by centrifugation from the
treated samples, washed with sterile peptone water and then serially di-
luted for plate counts on MRS agar and MRS plus 4% sodium chloride
(MRSS) agar. Because injured cells are unable to form colonies on media
containing high levels of NaCl, this MRSS agar was provided for count-
ing only the noninjured cells, (Mitchell and Moyle, 1956, Khalil and
Villota, 1985). The plates were incubated at 377C for 48h and then col-
164—JOURNAL OF FOOD SCIENCE—Volume 62, No. 1, 1997
Fig. 4—Colony forming of L. plantarum on different media as re-
lated to type heating at 50&C.
Fig. 5—Amount of 260 nm-absorbing material leaked into cell
suspension medium during sublethal heating of L. plantarum at
50&C.
onies were counted. Each sample was spread on three plates and an
average was calculated.
Recovery of injured cells. Following heat treatments at 507C for 30
min, 3 mL portions of cell suspensions were reinoculated into flasks
containing 250 mL of fresh MRS medium and allowed to recover at
377C for 18h. Monitoring recovery from sublethal heat injury was ac-
complished by differential plate counting on MRS agar and MRSS agar.
Samples were removed for plating at 1-h intervals during recovery.
Growth and lactic acid production. 3 mL samples of the cell sus-
pensions after each treatment at 507C for 30 min were inoculated into
flasks containing 250 mL of fresh MRS medium, followed by incubation
at 377C for 60h. Samples (5 mL) were removed at designated time in-
tervals for measuring cell mass and acidity.
Assays
Measurement of leaking materials. The culture broth was centri-
fuged and the pellet resuspended in 0.85% NaCl solution. This suspen-
sion (ca 108 ; 109 CFU/mL) was then used in conventional and
microwave heating experiments. During treatment, sample (5 mL) of cell
suspensions were removed at designated time intervals and centrifuged.
Absorbance of supernatant was measured at 260 nm with a Hewlett
Packard model 8452A Diode Array Spectrophotometer.
Acidity. The samples were centrifuged. Supernatants of centrifuged
samples were diluted with double their volume of CO2-free distilled wa-
ter and then titrated with 0.1N NaOH to pH 8.3. Total acidity was cal-
culated by the AOAC method.

Fig. 6—Recovering growth of L. plantarum on two media incu-
bated at 37&C after exposure to PW irradiation for 30 min at 50&C.
RESULTS & DISCUSSION
Inactivation of L. plantarum
We compared the effectiveness of three sublethal injury treat-
ments for the inactivation of L. plantarum. Suspensions of 109
cells/mL were exposed to CW, PW and conventional heating at
507C for 30 min and viability determined on MRSS agar (Fig.
3). Differences in come-up time from room temperature in the
three heating modes were not significant. Come-up times were
in the range 58 ; 83s for microwave and 43 ; 55s for con-
ventionally heated samples. Judging from the survivor curve,
exposure to a sublethal temperature of 507C by conventional
treatment had virtually no effect on viability of cells. However,
four log cycle reductions of viable cells occurred when suspen-
sions were exposed to a sublethal 507C by CW irradiation. PW
irradiation also to 507C yielded lower viable counts by another
two to three orders of magnitude. Results indicated that PW
irradiation was more effective than conventional heat treatment
or CW irradiation.
Destruction of microorganisms by microwaves at tempera-
tures lower than the thermal destruction point of has been re-
ported. Olsen (1965) used 2450 MHz CW irradiation and
reported a great reduction of viability of Aspergillus niger, Rhi-
zopus nigricans, and Penicillum sp. For a given level of kill, a
Fig. 7—Growth (a) and acid production (b) of L. plantarum after exposure to conventional heating, CW and PW at 50&C for 30 min.n
unheated;▫ conventional heating;¶ CW irradiation;C PW irradiation.
Volume 62, No. 1, 1997—JOURNAL OF FOOD SCIENCE—165
lower temperature was recorded in the CW treatment than with
conventional treatment. Amannus (1979) studied the effects of
CW (2450 MHz) irradiation and conventional heating upon sev-
eral microbial genera. The theory of nonthermal effects of mi-
crowave irradiation was questioned, but at relatively low
temperatures, microwave irradiation was more lethal to micro-
organisms. Boon and Kok (1987) described in a patent appli-
cation the alternate application of microwave pulses and cooling
to achieve biochemical and chemical reactions. They indicated
the procedure enabled such reactions, including inactivation of
microorganisms and enzymes, at a much higher reaction rate
while limiting the increase in temperature of the treated material.
The proposed mechanism was that high power pulsed micro-
wave irradiation was directly absorbed by molecules, in contrast
to conventional heating where molecules are brought to a higher
energy level through collision with other molecules. Thus, some
molecules would be at a higher energy level or temperature than
others during the short microwave pulse. Consequently, such
molecules would have higher reaction rates than expected based
on average product temperatures and thus there would be selec-
tive heating on a molecular level (Mertens and Knorr, 1992).
Injury to L. plantarum
The extent of injury is frequently measured by differential
growth on a specific medium that contains sodium chloride. Col-
ony forming ability of the treated cells on MRS and MRSS
media was compared (Fig. 4). Cells treated by conventional
heating showed essentially the same results for both MRS and
MRSS media regarding numbers of viable cells. However, when
samples were exposed to PW, a significant reduction of CFU
on MRSS medium was observed, indicating that the cells be-
came salt sensitive. PW-irradiated cells appeared to undergo rel-
atively higher injury compared to conventionally heated cells.
Another indication of heat damage to bacteria is the leakage
of cell components into the heating menstruum. When cells are
subjected to sublethal stress, a loss in membrane integrity oc-
curs. Thus amino acids, peptides, membrane lipids and ions
many be found in the extracellular environment as a response
to injury (Moat, 1961; Smith et al., 1982). Among components
often leaking out of the cells during sublethal heating, 260 nm-
absorbing materials have been commonly repeated (Khalil and
Villota, 1989; Scopes, 1994). For conventional heating, there
was no increase in the amount of material that leaked out of the
cells (Fig. 5). For microwave irradiation, as exposure time in-
creased, absorbance at 260 nm increased gradually. And the
PULSED-MICROWAVE INACTIVATION OF L. PLANTARUM . . .
difference in the absorbance between PW and CW irradiation
became larger. This indicated that the integrity of the L. plan-
tarum cell membrane was more damaged by microwave irra-
diations in comparison to conventional heating, and to a great
extent by PW. Barnes and Hu (1977) hypothesized that non-
thermal effects of microwave irradiation could result from ion
shifts across membranes and reorientation of long chain mole-
cules. It has also been hypothesized that the energy from mi-
crowave treatment may disrupt the membrane and/or the
sub-cellular structure, thus liberating lipids (Ke et al., 1978).
Furthermore, the lipid components of the cell membrane may
be overheated during microwave irradiation and cause greater
injury (Khalil and Villota, 1988). Lin (1978) proposed that the
absorption of high power PW radiation could produce thermoe-
lastic waves in biological tissues. Such pressure and displace-
ment may cause physical damage to cell membranes and
cytoplasm (Lin et al., 1988).
Recovery of injured cells
Cells injured by sublethal heating (507C for 30 min) could be
repaired after incubation under optimal conditions. This have
been demonstrated by their regaining normal growth curves and
salt tolerance. The initial CFU values on MRSS medium were
one log lower that those on MRS medium (Fig. 6). For the initial
stage of recovering growth, the viable population increased at a
slow rate and differences in viable cells between the two media
were maintained. After 10h recovery, growth rates began to in-
crease, with the CFU in MRSS broth showing a more rapid
increase. The growth in MRSS broth reached almost the same
level as MRS broth after 17h recovery, and salt tolerance was
regained (Fig. 6). It appeared that injured cells required a min-
imum of 10h for repair and adaptation. Thus the PW irradiation
apparently injured the cells, slowing their rate of division and
inhibiting some undetermined metabolic processes in the early
stages of cell growth.
Lactic acid production of injured cells
Another phase of the study was to investigate the effects of
heat on metabolic activity. The effect of sublethal microwave
irradiation on growth and total acid production by L. plantarum
was compared (Fig. 7) by growth profiles of L. plantarum after
exposure to the different heating modes. Unheated cells grew
rapidly without lag and reached the stationary phase after 24h
incubation. Conventionally and CW irradiated cells showed 10h
and 22h lag periods, respectively, and then nearly the same ex-
ponential growth as unheated cells. The lag phase, which often
indicates the repair and adaptation period, was almost two time
longer for the microwave injured cells. With PW irradiation,
however, the growth rate in the exponential period was slower
than those of the conventional and CW irradiation and did not
reach the same level of cell mass as the control after 60h in-
cubation.
Acid production was restored during recovery (Fig. 7). The
onset of acid production at a detectable level for conventionally
and both types of microwave treated cells, respectively, was 10
and 20h. Thus, nearly the same repair periods were required for
initiation of acid production as for starting exponential growth
after the lag phase. Conventionally heated organisms regained
production rates almost identical to the unheated culture by 50h.
166—JOURNAL OF FOOD SCIENCE—Volume 62, No. 1, 1997
However, microwave stressed cells constantly produced a lower
concentration, particularly PW irradiated cells.
Growth of PW irradiated cells was delayed about 24h (total),
and acid production after 60h incubation reached about 60 ;
80% of the other treatments. Results indicate that PW irradiation
was more effective than CW irradiation for inactivation of L.
plantarum, and provides an effective alternate for conventional
pasteurization.
CONCLUSIONS
COMPARATIVE STUDY of the inactivation of L. plantarum using
microwave and conventional heating at 507C for 30 min indi-
cated significant nonthermal effects of pulsed microwave heat-
ing. Pulsed microwave treatment appeared to induce irreversible
damage to membrane structures with an increase of permeability
in cell components. The growth of cells treated with PW was
delayed about 24h and acid production after 60h incubation
reached about 60 ; 80% that of the other treatments.
REFERENCES
Amannus, I. 1979. The effect of microwave and conventional cooking upon
food-born bacteria inoculated in foods. M.A. Thegis. Mankato State Univ.,
Mankato, MN.
Barnes, F.S. and Hu, C.L.J. 1977. Model for some nonthermal effects of ratio
and microwave fields on biological membranes. IEEE Trans. Microwave
Theory Tech. 25: 42.
Boon, M.E. and Kok, L.P. 1987. Werkwijze voor het laten verlopen van(bio-
) chemisthe of (micro-) biolgische reakties, onder gebruikmaking van mi-
crogolven, daarvan gebruikmakende werkwijzen en inrichting daarvan.
Terinzagelegging 8702338, Octrooiraad Neverland.
Buffer, R.B. 1992. In Microwave Cooking and Processing, p. 23. AVI Pub-
lishing Co., New York.
Chipley, J.R. 1980. Effects of microwave irradiation on microorganisms. Adv.
Appl. Microbiol. 26: 129–144.
Cunningham, F.E. 1978. The effect of brief microwave treatment on num-
bers of bacteria in fresh chicken patties. Poult. Sci. 57: 296–297.
Dreyfuss, M.S. and Chipley, J.R. 1980. Comparison of effects of sublethal
microwave radiation and conventional heating on the metabolic activity of
Staphylococcus aureus. Appl. Environ. Microbiol. 39: 13–16.
Ke, P.J. Linke, A.B., and Ackm, R.G. 1978. Acceleration of lipid oxidation
in frozen mackerel fillet by pretreatment with microwave heating. J. Food
Sci. 43: 38–40.
Khalil, H. and Villota, R. 1985. A comparative study on the thermal inac-
tivation of Bacillus stearothermophilus spores in microwave and conven-
tional heating. Proc. Fourth Intern. Congr. Eng. Food. Applied Science
Publishers, Essex, England.
Khalil, H. and Villota, R. 1989. The effect of microwave sublethal heating
on the ribonucleic acids of Staphylococcus aureus. J. Food Protection.
52(8): 544–548.
Lin, J.C. 1978. Microwave auditory effects and applications. C.C. Thoma-
omas, Springfield, IL.
Lin, J.C., Su, J.L., and Wang, Y.J. 1988. Microwave-induced thermoelastic
pressure wave propagation in the cat brain. Bioelectromagnetics 9: 141–
147.
Mertens, B. and Knorr, D. 1992. Developments of nonthermal processes for
food preservation. Food Technol. 46(5): 124–133.
Mitchell, P. and Moyle, J. 1956. Osmotic structure and function in bacteria.
Society of General Microbiology.
Miyakawa, S. 1996. Recent Trends of Sterilizing Technology, in Up-to-Date
Food Processing, p. 38–43, Health Industry News Co., Japan.
Moat, W.A. 1961. Chemical changes in bacteria heated in the milk as related
to lose of stainability. J. Dairy Sci. 44: 1431–1439.
Mudgett, R.E. 1989. Microwave food processing. Food Technol. 43(1): 117.
Olsen, C.M. 1965. Microwave inhibit bread molds. Food Eng. 37: 51–53.
Scopes, R.K. 1994. Protein Purification Principles and Practice. Springer-
Verlag.
Shizuki, H. 1991. Application Handbooks of New Sterilization Technology.
Science Forum, Japan.
Smith, J.L., Buchanan, R.L. and Palumbo, S.A. 1982. Effects of food envi-
ronment on Staphylococcal enterotoxin synthesis. J. Food Prot. 46: 545–
555.
Ms received 9/16/95; revised 7/15/96; accepted 7/20/96.
This study was supported in part by a research grant from Bioproducts Research Center
of Yonsei University.