Damage%20of%20yeast%20cells%20induced%20by%20pulsed%20light%20irradiation

International Journal of Food Microbiology 85 (2003) 151 – 158 www.elsevier.com/locate/ijfoodmicro

Damage of yeast cells induced by pulsed light irradiation Kazuko Takeshita a,*, Junko Shibato a, Takashi Sameshima a, Sakae Fukunaga b, Seiichiro Isobe c, Keizo Arihara d, Makoto Itoh d b

a Basic Research Department, PRIMA Meat Packers, Ltd., 635 Nakamukaihara, Tsuchiura, Ibaraki 300-0841, Japan Industrial Machine and Plant Development Center, Ishikawajima-Harima Heavy Industries Co., Ltd., 1 Shin-nakahara-cho, Isogo, Yokohama, Kanagawa 235-8501, Japan c National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan d Faculty of Animal Science, Kitasato University, 35-1 Higashi Nijyusanban-cho, Towada, Aomori 034-8628, Japan

Received 8 April 2002; received in revised form 4 October 2002; accepted 21 October 2002

Abstract DNA damage, such as formation of single strand breaks and pyrimidine dimers was induced in yeast cells after irradiation by pulsed light, which were essentially the same as observed with continuous ultraviolet (UV) light. The UV-induced DNA damage is slightly higher than seen with pulsed light. However, increased concentration of eluted protein and structural change in the irradiated yeast cells were observed only in the case of pulsed light. A difference in the inactivation effect between pulsed light and UV light was found and this suggested cell membrane damage induced by pulsed light irradiation. It is proposed that pulsed light can be used as an effective sterilizing method for the yeast Saccharomyces cerevisiae.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Saccharomyces cerevisiae; Cell damage; DNA damage; Pulsed light; Sterilization

1. Introduction A new sterilization technique/technology based on the use of pulsed light, which has been developed by PurePulse Technologies (San Diego, CA, USA), is suggested to have great potential in the development of a new method of sterilization. It is now possible to apply this new technology to sterilize air, water, and packaging surfaces in situations where light can be easily accessible. The pulsed light consists of * Corresponding author. Tel.: +81-298-42-4333; fax: +81-29842-5216.

E-mail address: Kazuko.Takeshita@primaham.co.jp (K. Takeshita).

intense flashes of broad-spectrum white light containing wavelengths from 200 nm in the ultraviolet (UV) to 1000 nm in the near-infrared region (Fig. 1).

The pulsed light distribution is almost similar to that of sunlight, except that the contents of UV region under 320 nm are very rich in the pulsed light. Each pulse has 90,000 times the intensity of sunlight at sea level and each pulse lasts only a few hundred millionths of a second with the result that this system can produce very high peak power pulsed light (Fig. 2b).

Pulsed light is effective for killing bacteria, fungi, virus, oocyst, and the killing effect is much higher in much shorter time than with continuous UV treatment (Dunn et al., 1995; Dunn, 1998). The killing effects of

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(02)00509-3

152

K. Takeshita et al. / International Journal of Food Microbiology 85 (2003) 151–158

no killing effect if a filter, which can remove the UV wavelength region under 320 nm, is used (Takeshita et al., 2002). However, in the case of the pulsed light method, it appears that both the visible and infrared regions, combined together with the high peak power of pulsed light also works for killing microorganisms.

In this study, we observed Saccharomyces cerevisiae (an unicellular eukaryote and simple model organism, which shows morphological alterations typical for apoptosis or cell death) cell and DNA damage induced by pulsed light and continuous UV light, and tried to clarify the difference of the mechanism between the killing effect of pulsed light and that of continuous UV light.

Fig. 1. Comparison of pulsed light and sunlight wavelength.

pulsed light are caused by the rich, broad-spectrum UV content, the short duration, and the high peak power of the pulsed light (Dunn et al., 1995). In fact, the UV region is a very important factor for pulsed light sterilization and the pulsed light contains around 25% in the UV region. It was confirmed that there is

2. Materials and methods 2.1. Pulsed light equipment The pulsed light system is composed of a power supply unit and a flash lamp (QUARTZ 340 mm wide area lamp, PPT P/N266-007, PurePulse Technologies) (Fig. 2a) (Dunn et al., 1995). Except in the case of experiments for peak power (Section 3.5), the peak power, output energy of one shot, was 3997 kW (Type A, Table 1).

2.2. Cultivation of yeast S. cerevisiae IFO2347 was obtained from Institute for Fermentation (Osaka, Japan). The yeast was cultured on agar plates and stored at 80 jC in 10% skimmed milk. Yeast cells were precultured on malt extract agar (Difco, Detroit, MI, USA) plates for 24 h at 28 jC, then one colony from the agar plate was inoculated in 10 ml of malt extract liquid medium (Difco) and cultured for 24 h at 28 jC. Table 1 Power supply performance

Type A Type B Type C Fig. 2. The schematic of pulsed light experimental apparatus (a), and the image of pulsed light and continuous light (b).

Tp [As]

P [kW]

e [J/cm2]

240 55 110

3997 4655 2473

0.70 0.23 0.23

Tp = Pulse width (FMWH).

P = output energy of one shot (peak power). e = energy (at 25 mm from flash lamp).

K. Takeshita et al. / International Journal of Food Microbiology 85 (2003) 151–158

2.3. Irradiations The yeast cells were centrifuged (4000 g, 10 jC, 10 min), washed with sterile distilled water, and resuspended in sterile 0.067 M potassium phosphate buffer (pH 7.0). The 5 ml test suspensions (106 –107 cells/ml) were irradiated with pulsed light or UV light (100W (QGL100-2X) 3 lamps, IWASAKI ELECTRIC, Tokyo, Japan) in a 110 mm diameter watch glass surrounded by ice. The pulsed light energy (0.23 and 0.7 J/cm2/flash) on a surface was determined by a calorimeter (Model ED-500 L, Gentec Electro-Optics, Quebec, Canada) with a 1 cm2 aperture, and measured as incident light energy at the wavelength of 200 – 1100 nm per unit area, J/cm2. The UV light energy (60 mW/cm2/s) on a surface was determined by a UV meter (Model UVP 254-01, IWASAKI ELECTRIC) and measured as light energy at the wavelength of 254 nm per unit area per 1 s, W/cm2/s. The number of flashes and total energy was in the range of 1– 5 flashes and 0.23 –0.7 J/cm2 per flash (pulsed light), respectively. The experiments were done in duplicate.

2.4. Viability of yeast Cell viabilities of untreated and treated samples were determined by serial dilutions in 0.5% salt solution, and subsequently by plating on malt extract medium solidified with 1.5% agar. Each of the plates was incubated for 48 h at 28 jC and the colony number was counted.

153

of 140 Al of 5 M NaCl and 70 Al of 25% N-lauroylsarcosine (pH 5.3). A 175 Al solution containing TrisHCl (pH 7.5) and 2 mM EDTAwas added to each lysate and mixed gently with equilibrated phenol. After extraction with phenol, the aqueous phase was collected by centrifugation, and followed by chloroformisoamyl alcohol (24:1) extraction. DNA concentration was about 100 Ag/ml (in TE buffer).

2.6. UVendonuclease treatment UVendonuclease was used for detecting cyclobutane pyrimidine dimers induced during DNA damage.

UVendonuclease was partially purified from Micrococcus luteus IFO3333 (Institute for Fermentation) according to the procedure of Carrier and Setlow (1970). A preparation corresponding to fraction II was used. Samples of genomic DNA (6 Al) from S. cerevisiae cells were incubated for 30 min at 37 jC with 3 Al of partially purified UVendonuclease (fraction II) in the reaction mixture (total volume 15 Al) containing 5 Ag/ml Calf thymus DNA, 0.05 M potassium phosphate buffer (pH 6.5), 0.001 M EDTA, and 0.01 M h-mercaptoethanol. The reaction was stopped by adding 1.5 Al of alkaline buffer (5 M NaOH, 50% glycerol).

2.7. Alkaline treatment For detecting single-strand breaks induced during DNA damage, alkaline treatment of DNA (6 Al) was done by adding 0.6 Al alkaline buffer (final concentration 0.5 M NaOH, 5% glycerol).

2.5. DNA extraction 2.8. Electrophoresis Genomic DNA from S. cerevisiae was isolated by the method of Resnick et al. (1987) with modifications.

Cell suspensions were centrifuged at 4000 g for 10 min at 10 jC and the supernatant was discarded. The yeast cells (around 2 108 cells) were suspended in 100 Al of solution containing 0.2 M Tris-HCl (pH 9.1), 0.1 M EDTA, 1.2 M Sorbitol, 0.1 M h-mercaptoethanol, and kept for 10 min at 0 jC. The cell suspension was washed with 1ml of SCE (1 M sorbitol, 0.1 M Sodium citrate (pH 5.8), 0.06 M EDTA) solution and resuspended in the 300 Al of SCE solution. The cells were spheroplasted by incubating with 50 Al of Lyticase (400 Units, Sigma, St. Louis, MO, USA) for 60 min at 30 jC. The spheroplasts were lysed by addition

Electrophoresis loading buffer (1 Al of 300 mM NaOH, 6 mM EDTA, 0.25% xylene cyanol FF) was added to each of the samples of genomic DNA prepared above and electrophoresed for 40 min at 100 V in 0.8% agarose L03 (Takara Shuzo, Tokyo, Japan) gel in running buffer (0.045 M Tris-borate, 0.001 M EDTA). The gel was then stained with ethidium bromide.

2.9. Determination of protein concentration Irradiated or unirradiated cell suspensions were centrifuged at 4000 g for 10 min at 10 jC and the

154

K. Takeshita et al. / International Journal of Food Microbiology 85 (2003) 151–158

supernatant was collected. Concentration of elute protein from yeast cells in the supernatants were measured using a spectrophotometer and a BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA).

cerevisiae cell viability was reduced from an initial concentration of 7 106 cfu/ml to 10 cfu/ml. In the case of continuous UV light, the S. cerevisiae cell viability was reduced from an initial concentration of 7 106 cfu/ml to 10 cfu/ml with 60 mW/cm2/s and 7 s

2.10. Transmission electron microscopy Transmission electron microscopy of yeast cells was performed using a previously described method (Kawai et al., 1999) with modifications. Irradiated and unirradiated yeast cell suspensions were centrifuged immediately after treatment at 4000 g for 1 min at 10 jC and the supernatant was discarded.

The yeast cells were washed 3 times with sterile distilled water. The yeast cells were fixed with 2.5% glutaraldehyde (TAAB Laboratories Equipment, Berkshire, UK) in 0.1 M PB buffer (pH 7.2) for 2 h at 4 jC. The fixed cells were washed twice with 0.1 M PB buffer (pH 7.2), and washed with sterile distilled water. Three percent potassium permanganate in distilled water was added to the microcentrifuge tube to resuspend the yeast cell pellet and post-fixed for 90 min at room temperature.

After fixation, yeast cells were washed 3 to 4 times with sterile distilled water and embedded in 2% agarose, and stained with 2% uranyl acetate at 4 jC. Dehydration was performed with an acetone series for 10 min each, 50%, 70%, 80%, 90%, 95% and 99.5% acetone twice, and 15 min with absolute acetone 3 times. The specimens were embedded in a Quetol 653 Mixture and incubated at 60 jC for 2 to 3 days. Ultrathin sections were made with Ultramicrotome UCT (LEICA Microsystems Lithography, Cambridge, UK). The sections were stained with 6% uranyl acetate for 10 min and 0.4% lead citrate for 5 min. A JEM-1010 (JEOL, Tokyo, Japan) transmission electron microscope operating at 100 kV was used for subsequent electron microscopy experiments.

3. Results 3.1. S. cerevisiae viability After five flashes of pulsed light treatment (five flashes at 0.7 J/cm2/flash, total: 3.5 J/cm2), the S.

Fig. 3. S. cerevisiae viability after pulsed light (a) and UV light (b) irradiation. Test suspension OD660 3.8 ( , E) used for DNA experiment, and OD660 1.5 (o, D) used for TEM. Error bars are the average of three experiments.

.

K. Takeshita et al. / International Journal of Food Microbiology 85 (2003) 151–158

155

irradiation time (Fig. 3a,b). Experiments were repeated at least 5 times.

3.2. Electrophoresis Fig. 4 shows the electrophoresis pattern of genomic DNA from unirradiated and irradiated yeast cells. The non-treated DNA band patterns are shown in Fig. 4a. The DNA samples in Fig. 4b were alkaline treated and Fig. 4c were UVendonuclease and alkaline treated prior to electrophoresis. Comparing the electrophoresed DNA band patterns in Fig.

Fig. 5. Eluted protein concentration (Ag/ml) and yeast cell viability (log cfu/ml). (a) Pulsed light at 0.7 J/cm2/flash, (b) UV light at 60 mW/cm2/s. , E: cell viability (log cfu/ml), o, D: concentration of eluted protein (Ag/ml).

.

Fig. 4. Electrophoresis of genomic DNA extracted from yeast cells.

Lanes 2, 3, 4, and 6, 7, 8 show genomic DNA extracted from yeast cells irradiated by pulsed light (Type A condition; pulse width 240 As/flash, peak power 3997 kW/flash) and from yeast cells irradiated

by UV light, respectively. Lanes 1 and 5 show genomic DNA extracted from unirradiated yeast cells. (a) Non-treatment (control), (b) alkaline treatment, (c) UVendonuclease and alkaline treatment.

Lane 2; 1 flash at 0.7 J/cm2/flash, lane 3; three flashes at 0.7 J/cm2/ flash, lane 4; five flashes at 0.7 J/cm2/flash. Lane 6; 2 s at 60 mW/ cm2/s, lane 7; 5 s at 60 mW/cm2/s, and lane 8; 7 s at 60 mW/cm2/s.

4a– c, it can be seen that single-strand breaks and cyclobutane dimers are present. In the case of alkaline and UVendonuclease treatments, presence of smeared bands in both the pulsed light (lanes 2– 4) and UV-treated (lanes 6 –8) cells, which are especially clear after UV-treatment (both Fig. 4b and c), indicates that single strand breaks (Fig. 4b) and cyclobutane dimers (Fig. 4c) had indeed occurred in double stranded DNA. Although DNA damage was

156

K. Takeshita et al. / International Journal of Food Microbiology 85 (2003) 151–158

detected in both the DNA from pulsed light and UV irradiated yeast cells, it should be noted that the UVinduced DNA damage is slightly higher than that seen under pulsed light.

pulsed light irradiation was higher than observed under UV irradiation.

3.3. Concentration of eluted protein

The transmission electron micrographs (TEMs) of unirradiated and irradiated yeast cells are shown in Fig. 6. After two flashes of pulsed light irradiation (two flashes at 0.7 J/cm2/flash, total: 1.4 J/cm2), distinct structural changes in the yeast cells were observed. Approximately 30% of yeast cells showed

S. cerevisiae cell viability and concentration of eluted protein from yeast cells after pulsed light and UV irradiation is shown in Fig. 5. As can be observed from this result, protein elution from yeast cells after

3.4. Transmission electron microscopy

Fig. 6. TEM of S. cerevisiae. (a) Unirradiated, (b) irradiated with two flashes pulsed light at 0.7 J/cm2/flash and three flashes at 0.7 J/cm2/flash (c), (d) irradiated with UV light (3 s at 60 mW/cm2/s). Bar corresponds to 0.5 Am. N; nucleus, Vc; vacuole. Arrowheads indicate damaged/ broken membranes. Magnification 40,000.

K. Takeshita et al. / International Journal of Food Microbiology 85 (2003) 151–158

raised and expanded vacuoles and cell membrane distortion, as determined by visually checking the cell sections. The vacuole (Vc) and regions of cell membrane distortion/damage are visible in the TEMs, and indicated by arrowheads. After three flashes of pulsed light treatment (three flashes at 0.7 J/cm2/flash, total: 2.1 J/cm2), vacuole expanding and cell membrane distortion were observed in 50% of the yeast cells,

157

and the shape of yeast cell was changed to circular. On the other hand, after irradiation with UV light, the yeast cell structure was almost the same as in the case of unirradiated cells.

3.5. Peak power S. cerevisiae cell viability and concentration of eluted protein from yeast cells after irradiation with different peak power conditions of pulsed light are shown in Fig. 7. If compared at the same killing level, protein elution from yeast cells after high peak power pulsed light (Type B, Table 1) irradiation was higher than the low peak power condition (Type C, Table 1).

4. Discussion

Fig. 7. Eluted protein concentration (Ag/ml) and yeast cell viability (log cfu/ml). Detected after high peak power pulsed light at 0.23 J/ cm2/flash under Type B condition (pulse width; 55 As/flash, peak power; 4655 kW/flash) (a) and low peak power pulsed light at 0.23 J/ cm2/flash under Type C condition (pulse width; 110 As/flash, peak power; 2473 kW/flash) (b). , E: cell viability (log cfu/ml), o, D: concentration of eluted protein (Ag/ml).

.

This new sterilization technology, using very intense, broad-spectrum and high peak power pulsed light, is effective for highly contaminating, highly damaging and persistent agents such as fungal spores, viruses and oocysts, which have resistance against UV light (Dunn, 1998). It is known that near UV (320 – 400 nm) radiation also induces DNA damage in bacteria, such as formation of single strand breaks and pyrimidine dimers (Tyrrell, 1973; Rosenstein and Ducore, 1983). We expected that DNA damages induced by pulsed light would be much higher than the case of continuous UV light, and that this might be one of the reasons that give an advantage to the pulsed light killing effect. From the present results, it can be clearly seen that the UV-induced DNA damage is a little higher than that observed with pulsed light, although the killing level of irradiated yeast cells remained almost the same. It is likely that the efficiency killing effect of pulsed light does not depend only on DNA damage.

This unexpected result prompted us to look at other possible factors in the high efficiency of killing by pulsed light. One way was to look for damages to other essential macromolecular components of the cell, such as proteins, and organelles. We first checked the concentration of eluted protein from irradiated yeast cells, and found considerable differences between the pulsed light irradiated and the UV irradiated ones. It was further reasoned that if a higher level of proteins were eluted after irradiation, it could

158

K. Takeshita et al. / International Journal of Food Microbiology 85 (2003) 151–158

be an indication of potential cell membrane damage that was being induced by irradiation. From the TEMs of yeast cells after irradiation with pulsed light, it was clear that structural change in yeast cells occurred after pulsed light irradiation. These structural changes observed in the TEMs, such as expanded vacuoles, cell membrane distortion and change in the cell shape strongly suggested that cell membrane damage was induced by pulsed light irradiation. Investigations into near UV radiation affected membrane function damage have been previously reported (Ito and Ito, 1983; Arami et al., 1997). However, to date, there are no reports on the structural changes in yeast cell after UV or near UV irradiation, although some structural changes have been observed under the electric field treatment (Harrison et al., 1997).

The next question asked was, what/which factors of pulsed light caused cell damage? We focused on the peak power effect of pulsed light. Peak power is one of the important factors in pulsed light, which differentiates pulsed light from continuous UV light. It was impossible to change only the peak power condition without changing the pulsed light flash energy for the pulsed light equipment used in this work. So we searched for the conditions for peak power experiments, in which different peak power could be combined with same flash energy condition. The results revealed that under high peak power conditions, the killing effect and concentration of eluted protein were higher than under low peak power conditions. It seems that high peak power conditions were effective in inducing cell membrane damage, but we will need more detailed experiments for clarifying the precise effects of peak power on cell damage. As high peak power conditions includes a richer (150%) UV region under 300 nm, it might be possible that the content of UV region under 300 nm may also work to induce the cell membrane damage. However, this also needs clarification in future experiments.

On the basis of the results obtained with yeast (S. cerevisiae) in the present study, it is proposed that pulsed light may be an effective alternative to UV irradiation as a sterilizing technique for killing microorganisms.

Acknowledgements This investigation was supported in part by a grant from Japanese Research and Development Association for Application of Electronic Technology in Food Industry, Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan. We greatly appreciate the valuable advice and discussion of Dr. Akinori Noguchi, Japan International Research Center for Agricultural Science, MAFF.

References Arami, S., Hada, M., Tada, M., 1997. Near-UV-induced absorbance change and photochemical decomposition of ergosterol in the plasma membrane of the yeast Saccharomyces cerevisiae. Microbiology 143, 1665 – 1671.

Carrier, W.L., Setlow, R.B., 1970. Endonuclease from Micrococcus luteus, which has activity toward ultraviolet-irradiated deoxyribonucleic acid: purification and properties. J. Bacteriol. 102, 178 – 186.

Dunn, J., 1998. Pure bright pulsed light sterilization and food processing. Bull. Jpn. Food Pack. Assoc. (Shoku-Hou-Ken) 77, 15 – 24 (in Japanese).

Dunn, J., Ott, T., Clark, W., 1995. Pulsed-light treatment of food and packaging. Food Technol. 49 (9), 95 – 98.

Harrison, S.L., Barbosa-Canovas, G.V., Swanson, B.G., 1997. Saccharomyces cerevisiae structural changes induced by pulsed electric field treatment. Lebensm.-Wiss. Technol. 30, 236 – 240.

Ito, A., Ito, T., 1983. Possible involvement of membrane damage in the inactivation by broad-band near-UV radiation in Saccharomyces cerevisiae cells. Photochem. Photobiol. 37, 395 – 401.

Kawai, R., Fujita, K., Iwahashi, H., Komatsu, Y., 1999. Direct evidence for the intracellular localization of Hsp104 in Saccharomyces cerevisiae by immunoelectron microscopy. Cell Stress Chaperones 4, 46 – 53.

Resnick, M.A., Westmoreland, J., Amaya, E., Bloom, K., 1987.

UV-induced damage and repair in centromere DNA of yeast.

MGG, Mol. Gen. Genet. 210, 16 – 22.

Rosenstein, B.S., Ducore, J.M., 1983. Induction of DNA strand breaks in normal human fibroblasts exposed to monochromatic ultraviolet and visible wavelengths in the 240 – 546 nm range.

Photochem. Photobiol. 38, 51 – 55.

Takeshita, K., Yamanaka, H., Sameshima, T., Fukunaga, S., Isobe, S., Arihara, K., Itoh, M., 2002. Sterilization effect of pulsed light on various microorganisms. Bokin Bobai 30, 277 – 284 (in Japanese).

Tyrrell, R.M., 1973. Induction of pyrimidine dimers in bacterial DNA by 365 nm radiation. Photochem. Photobiol. 17, 69 – 73.