Beruflich Dokumente
Kultur Dokumente
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.
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).
Thermal treatments Fig. 5—Amount of 260 nm-absorbing material leaked into cell
suspension medium during sublethal heating of L. plantarum at
100 mL of culture broth of L. plantarum, which contained 108 ; 109 50&C.
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- onies were counted. Each sample was spread on three plates and an
sion in a shaking water bath (Julabo, model SW-21C) maintained at 50 average was calculated.
5 0.17C. During treatment, the glass tubes were removed at designated Recovery of injured cells. Following heat treatments at 507C for 30
time intervals and immediately cooled in an ice-water bath for micro- min, 3 mL portions of cell suspensions were reinoculated into flasks
biological analysis. Temperatures in the glass tubes were measured by a containing 250 mL of fresh MRS medium and allowed to recover at
Teflon-coated thermocouple and recorded by a data logger linked to a 377C for 18h. Monitoring recovery from sublethal heat injury was ac-
microcomputer. complished by differential plate counting on MRS agar and MRSS agar.
Microwave. For the CW or PW irradiation, a Pyrex cylindrical glass Samples were removed for plating at 1-h intervals during recovery.
container of the culture broth was placed at the center of the oven and Growth and lactic acid production. 3 mL samples of the cell sus-
exposed to CW or PW. For maintaining the sample temperature at 50 pensions after each treatment at 507C for 30 min were inoculated into
5 27C, cooling water at 2157C was circulated through the jacket and flasks containing 250 mL of fresh MRS medium, followed by incubation
coil of the container under CW and 57C water was used during PW at 377C for 60h. Samples (5 mL) were removed at designated time in-
irradiation. Temperatures of cooling water and come-up time from room tervals for measuring cell mass and acidity.
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- Assays
constantan, Teflon-coated thermocouple (Omega Eng. Co.).
Measurement of leaking materials. The culture broth was centri-
fuged and the pellet resuspended in 0.85% NaCl solution. This suspen-
Microbiological analysis sion (ca 108 ; 109 CFU/mL) was then used in conventional and
microwave heating experiments. During treatment, sample (5 mL) of cell
Viable cell counts. Cells were separated by centrifugation from the suspensions were removed at designated time intervals and centrifuged.
treated samples, washed with sterile peptone water and then serially di- Absorbance of supernatant was measured at 260 nm with a Hewlett
luted for plate counts on MRS agar and MRS plus 4% sodium chloride Packard model 8452A Diode Array Spectrophotometer.
(MRSS) agar. Because injured cells are unable to form colonies on media Acidity. The samples were centrifuged. Supernatants of centrifuged
containing high levels of NaCl, this MRSS agar was provided for count- samples were diluted with double their volume of CO2-free distilled wa-
ing only the noninjured cells, (Mitchell and Moyle, 1956, Khalil and ter and then titrated with 0.1N NaOH to pH 8.3. Total acidity was cal-
Villota, 1985). The plates were incubated at 377C for 48h and then col- culated by the AOAC method.
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.
difference in the absorbance between PW and CW irradiation However, microwave stressed cells constantly produced a lower
became larger. This indicated that the integrity of the L. plan- concentration, particularly PW irradiated cells.
tarum cell membrane was more damaged by microwave irra- Growth of PW irradiated cells was delayed about 24h (total),
diations in comparison to conventional heating, and to a great and acid production after 60h incubation reached about 60 ;
extent by PW. Barnes and Hu (1977) hypothesized that non- 80% of the other treatments. Results indicate that PW irradiation
thermal effects of microwave irradiation could result from ion was more effective than CW irradiation for inactivation of L.
shifts across membranes and reorientation of long chain mole- plantarum, and provides an effective alternate for conventional
cules. It has also been hypothesized that the energy from mi- pasteurization.
crowave treatment may disrupt the membrane and/or the
sub-cellular structure, thus liberating lipids (Ke et al., 1978). CONCLUSIONS
Furthermore, the lipid components of the cell membrane may
be overheated during microwave irradiation and cause greater COMPARATIVE STUDY of the inactivation of L. plantarum using
injury (Khalil and Villota, 1988). Lin (1978) proposed that the microwave and conventional heating at 507C for 30 min indi-
absorption of high power PW radiation could produce thermoe- cated significant nonthermal effects of pulsed microwave heat-
lastic waves in biological tissues. Such pressure and displace- ing. Pulsed microwave treatment appeared to induce irreversible
ment may cause physical damage to cell membranes and damage to membrane structures with an increase of permeability
cytoplasm (Lin et al., 1988). 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.
Recovery of injured cells
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This study was supported in part by a research grant from Bioproducts Research Center
after the lag phase. Conventionally heated organisms regained of Yonsei University.
production rates almost identical to the unheated culture by 50h.