Microwave Heating Inactivates Shiga Toxin (Stx2) in Reconstituted Fat

Mar 26, 2014 - observations suggest that microwave heating has the potential to destroy the Shiga toxin in liquid food. KEYWORDS: milk, Shiga toxin 2 ...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JAFC

Microwave Heating Inactivates Shiga Toxin (Stx2) in Reconstituted Fat-Free Milk and Adversely Affects the Nutritional Value of Cell Culture Medium Reuven Rasooly,*,† Bradley Hernlem,† Xiaohua He,† and Mendel Friedman‡ †

Foodborne Toxin Detection and Prevention Unit and ‡Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, California 94710, United States ABSTRACT: Microwave exposure is a convenient and widely used method for defrosting, heating, and cooking numerous foods. Microwave cooking is also reported to kill pathogenic microorganisms that often contaminate food. In this study, we tested whether microwaves would inactivate the toxicity of Shiga toxin 2 (Stx2) added to 5% reconstituted fat-free milk administered to monkey kidney Vero cells. Heating of milk spiked with Stx2 in a microwave oven using a 10% duty cycle (cycle period of 30 s) for a total of 165 kJ energy or thermal heating (pasteurization), widely used to kill pathogenic bacteria, did not destroy the biological effect of the toxin in the Vero cells. However, conventional heating of milk to 95 °C for 5 min or at an increased microwave energy of 198 kJ reduced the Stx2 activity. Gel electrophoresis showed that exposure of the protein toxin to high-energy microwaves resulted in the degradation of its original structure. In addition, two independent assays showed that exposure of the cell culture medium to microwave energy of 198 kJ completely destroyed the nutritional value of the culture medium used to grow the Vero cells, possibly by damaging susceptible essential nutrients present in the medium. These observations suggest that microwave heating has the potential to destroy the Shiga toxin in liquid food. KEYWORDS: milk, Shiga toxin 2 (Stx2), microwave heating, inactivation, cell culture, food safety

■

INTRODUCTION Numerous foodborne diseases result from ingesting foods that are contaminated with microbial and plant toxins. Naturally occurring food toxicants can adversely affect the nutritional quality and safety of foods. To improve the quality and food safety of our food supply, research is needed to define conditions that inactivate or inhibit toxin production to minimize the levels of active toxic compounds in foods. Shiga toxins Stx1 and Stx2 are produced by enterohemorrhagic strains of Escherichia coli (EHEC). These pathogens are of major importance for food safety, causing foodborne illnesses ranging from mild diarrhea to a life-threatening complication known as hemolytic uremic syndrome (HUS). It has been reported that orally ingested Stx2 alone, without EHEC colonization of the distal small intestine and the colon, causes histopathological changes in the kidney, spleen, and thymus and mortality in mice.1 Rasooly et al.2 discovered that freshly prepared juice from locally purchased apples inhibits the biological activity of Stx2. Other investigators reported that the probiotic bacteria Lactobacillus plantarum isolated from a fermented milk beverage called Kefir protected Vero cells against the cytotoxicity of Stx2 present in supernatants of E. coli O157:H7 bacteria.3 It was also shown that heating in a microwave oven reduced the colony number of EHEC. The reduction depended on the duration and power level of the microwaves.4 Microwaves act by causing polar molecules in food, such as water, to rotate rapidly, thus energizing neighboring food molecules, which raises their temperature. To our knowledge, no studies have examined the effect of microwave heating on the biological activity of Shiga toxin produced by the foodborne pathogen E. coli. To help answer this question, we spiked milk This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society

with Stx2, exposed it to microwave treatment, and measured the toxin’s inhibition effects on protein synthesis in Vero cells and the degradation of the protein toxin by gel electrophoresis. In the course of this study, we discovered that microwave heating that inactivates Stx2 also adversely affects the nutritional properties of the cell culture medium.

■

MATERIALS AND METHODS

Materials. Shiga toxin (Stx2), 50% pure, was obtained from Toxin Technology (Sarasota, FL, USA). Human Embryonic Kidney 293 cells (HEK293) (ATCC CRL-1573) and Vero African Green Monkey adult kidney cells (ATCC CCL-81) were obtained from the American Type Culture Collection (Manassas, VA, USA). A General Electric model Je1160wd microwave oven was used (Louisville, KY, USA). Preparation of Reconstituted Milk. Nonfat dry milk from Nestlé Carnation (Vevey, Switzerland) was dissolved in sterile distilled water to form a 5.0% solution. Effect of Microwave Treatment on Stx2 Activity. To determine the effect of microwave treatments on Stx2 activity, Stx2 (20 μL) at a concentration of 0.5 mg/mL was added to the milk (180 μL) in a borosilicate glass culture test tube (Fisher, 10 × 75 mm) and microwaved using the following two treatments: (1) 25 min, 10% power duty cycle (cycle period of 30 s) for a total of 165 kJ energy and 65 °C at the end of this cycle, and (2) 20 min, 20% duty cycle for 198 kJ and 78 °C at the end of this cycle. The cooled samples (2 μL) were then incubated with Dulbecco’s Modified Eagle’s medium (DMEM, 98 μL) containing Vero cells (final concentration, 1 μg/mL). The microwave-heated solutions were removed from the microwave oven, and the amount of energy absorbed by the samples was Received: Revised: Accepted: Published: 3301

January 16, 2014 March 13, 2014 March 17, 2014 March 26, 2014 dx.doi.org/10.1021/jf500278a | J. Agric. Food Chem. 2014, 62, 3301−3305

Journal of Agricultural and Food Chemistry

■

estimated by inserting a preheated thermometer. When the thermometer was preheated to 65 and 78 °C, the temperature remained steady in the respective samples (165 and 198 kJ energy), the two lowest levels (settings) on the microwave oven. The amount of energy required to produce the corresponding temperature rise in the samples from room temperature (20 °C) is accounted for by an approximate 0.024% absorption of the total input energy, assuming the specific heat of the sample to be equal to that of water. Cell Culture. Vero cells and Human Embryonic Kidney 293 cells (HEK293) were maintained in DMEM containing 10% fetal bovine serum (FBS) and 100 units/mL of both penicillin and streptomycin. Cells were trypsinized when ready to harvest. Generation of Adenoviral Vectors That Express the GFP Gene. To visualize the effect of Shiga toxin on living cells, we measured changes in green fluorescent protein (GFP) expression levels. GFP gene was isolated from the Green Lantern vector (BRL) by digestion with the Not I restriction enzyme. The 750-bp fragment was purified from the gel using a Qiagen kit and was subcloned into the Not I site of the adenoviral shuttle plasmid between the Cytomegalovirus immediate-early promoter (CMV) and the polyadenylation signal from bovine growth hormone. The plasmid pJM17 containing the full length of the adenovirus genome including a 4.4-kb sequence of antibiotics resistance gene were cotransfected in HEK293 cells with the shuttle plasmid containing the GFP gene flanked by the adenovirus E1 sequences. After 10 days, the cytopathic effect appeared, and the transfected cells became round and detached from the plate. The cells were then analyzed by fluorescence microscopy to detect GFP gene expression. Individual plaques of Ad-GFP were amplified.1,2 Plaque Assays for Purification and Titration of the Adenovirus. Plaque assays depend on the ability of the adenovirus to propagate in HEK293 cells. Six 35-mm tissue culture plates were seeded with HEK293 cells. The cells were incubated at 37 °C in a 5% CO2 incubator until they were 90% confluent. Serial dilutions were made in the DMEM supplemented with 2% FBS. The diluted virus was added to the cells. After 2 h, the medium was removed and replaced with the Modified Eagle’s medium (MEM) and 1% seaplaque agarose (FMC). The agar overlay was added to keep the virus localized after the cells had lysed. After 5 days, plaques were visible, and after 7 days, they were counted for titer determination. Quantifying Stx2 Activity. Vero cells were plated on black 96well plates (Greiner 655090 obtained from Sigma) at 1 × 104 cells in 100 μL of medium per well. Cells were incubated overnight to allow time for cells to attach to the plate. Spiked samples were added to each well, and the plates were then incubated for 48 h at 37 °C in a 5% CO2 incubator. The cells were then transduced with Ad-GFP at a multiplicity of infection (MOI) of 100 for 48 h. The medium was removed, and the cells were washed three times with pH 7.4 phosphate-buffered saline (PBS). Quantification of fluorescence emission by the cells expressing GFP was measured using a 528/20 nm emission filter and 485/20 nm excitation filter in a Synergy HT Multi-Detection Microplate Reader (BioTek, Winooki, VT, USA). Visualized Stx2 by Gel Electrophoresis after Exposure to Microwaves. The electrophoresis equipment, buffers, gels, and SeeBlue Plus2 Protein Standard were purchased from Invitrogen (Carlsbad, CA, USA). Samples of Stx2 in phosphate-buffered saline (PBS, pH 7.4) were exposed to 165 and 198 kJ microwave treatments. The cooled Stx2 samples (2 μg) were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 4−12% NuPAGE Novex Bis-Tris mini gels following the manufacturer’s protocol. To visualize proteins directly after gel electrophoresis, the gels were stained with Coomassie Blue G-250 (Bio-Rad, Hercules, CA, USA). Statistical Analysis. Statistical analysis was performed with SigmaStat 3.5 for Windows (Systat Software, San Jose, CA, USA). Multiple comparisons among transduced treated cells were made. One-way analysis of variance (ANOVA) was used to compare transduced treated cells (containing increasing concentrations of toxin or milk) to transduced untreated cells. The experiments were repeated at least three times, and results with P < 0.05 were considered statistically significant.

Article

RESULTS Low Level of Microwave Energy Does Not Inactivate the Stx2 Toxin in Milk. To test whether microwave heating inactivates the Stx2 toxin produced by toxigenic E. coli, we generated adenoviral vectors that encode and express the green fluorescent protein (GFP) gene (Ad-GFP) and used the GFP fluorescent intensity in transduced Vero cells for quantitative measurement of biological effects and cell viability. Reduced fluorescent intensity of the cells is the result of inhibition of protein synthesis by the toxin and is compared with cells starved in phosphate-buffered saline (PBS, pH 7.4) that lacks essential nutrients. Samples of milk spiked with Stx2 were tested for Stx2 activity after exposure to 165 or 198 kJ microwave treatments. Figure 1 shows that the toxin was

Figure 1. Microwave heating at 165 kJ does not reduce Shiga toxin inhibition of GFP expression. (A) Milk was spiked with 1 ng/mL or 100 pg/mL of Shiga toxin and treated with a microwave energy of 165 or 198 kJ. (B) Unheated milk containing Stx2 (1 ng/mL) was thermally treated at 95 °C for 5 min or pasteurized at 63 °C for 30 min. PBS buffer was used as a control, representing medium devoid of essential nutrients. Five microliters of spiked milk with 95 μL of medium was added to Vero cells. GFP expression was quantified fluorometrically after 48 h of incubation with the treated samples. Error bars represent standard errors.

inactivated neither by the low microwave radiation heating at 165 kJ nor by heat pasteurization. However, thermal heating at 95 °C for 5 min and the higher level of microwave radiation did reduce the activity of the toxin. Degradation of Stx2 after Exposure to Microwave Treatment. Our results showed that heating of Stx2 with microwaves at 165 kJ did not inactivate the toxin. However, 3302

dx.doi.org/10.1021/jf500278a | J. Agric. Food Chem. 2014, 62, 3301−3305

Journal of Agricultural and Food Chemistry

Article

exposure to the higher energy level of 198 kJ did inactivate the toxin. We then used SDS-PAGE to analyze the effect of exposure to the microwave treatment on Stx2 A and B subunits. As shown in Figure 2, microwave heating at 165 kJ did not

Figure 3. Microwave heating reduces the nutritional value of the cell culture medium as measured by reduced GFP expression in Vero cells. Samples of cell culture medium were treated with microwave energy at 198 or 165 kJ or by overnight heating at 100 °C. Untreated or treated medium or PBS was added to Vero cells. After incubation of treated culture medium for 24, 48, or 72 h with transduced Vero cells, GFP expression was quantified fluorometrically. Error bars represent standard errors. Note that the cell culture medium was microwaved in these experiments.

Figure 2. SDS-PAGE gel comparison of Stx2 samples (2 μg) treated at three microwave energy levels. The gel (NuPAGE Novex Bis Tris 4− 12%) was stained with coomassie. Lane 1 shows the protein standard; lane 2, Stx2 treated at 165 kJ; lane 3, Stx2 treated at 198 kJ.

degrade the toxin. However, the microwave energy level of 198 kJ did degrade Stx2 and cut the A subunit into smaller sizes, with one fragment equal to a molecular weight of about 14 kD. Microwave Heating at 198 kJ Destroys the Nutritional Value of Vero Cell Culture Medium. Our results showed that, under the tested conditions, Stx2 is inactivated by microwave heating at 198 kJ. To test whether microwave heating has the potential to reduce or destroy nutrients in culture medium used to grow the Vero cells, we quantitatively measured the biological effects of microwave energy on the cell culture medium. As shown in Figure 3, heating of the cell culture medium with a microwave energy of 165 kJ increased the cellular GFP fluorescent intensity comparable to that of the control untreated culture medium. Exposure to the higher energy level of 198 kJ resulted in decreased GFP fluorescent intensity. The intensity was reduced to the same level as exhibited by the PBS medium devoid of essential nutrients or the medium treated overnight at 100 °C. These results suggest that microwave heating at an energy level of >165 kJ caused a loss of the nutritional value of the growth medium, as indicated by its inability to support the growth of Vero cells. These results were further confirmed by quantifying the fluorescent product of the cleaved substrate (glycyl-phenylalanyl-amino fluorocoumarin, GF-AFC) (Figure 4). This fluorogenic substrate enters cells and is cleaved by the protease activity of live cells, releasing AFC and generating a fluorescent signal proportional to the number of living cells. By contrast,

Figure 4. Microwave heating at energies of 198 kJ or higher lowers the nutrient value of the cell culture medium. Samples of cell culture medium were treated with microwave energy at 198 or 165 kJ or by overnight heating at 100 °C and then incubated for 72 h with Vero cells, followed by cleavage of the peptide GF-AFC by the live cell protease, releasing AFC and generating a fluorescent signal. The resultant fluorescence was measured by a fluorescence plate reader (excitation at 355 nm and emission at 523 nm). Error bars represent standard errors.

cells with compromised or disrupted cell membranes (i.e., dead cells) lose their protease activity and are unable to cleave the GF-AFC substrate. The absence of a fluorescent product usually formed in normal growing cells suggests that the cells have lost their viability. Figure 3 confirms by a second independent assay that medium treated at the higher levels of microwave energy or by overnight heating at 100 °C had a reduced ability to maintain the viability of the cells

■

DISCUSSION Kindle et al.5 found that microwave heating reduced the activities of Pseudomonas aeruginosa, E. coli, Enterobacter sakazakii, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans, Mycobacterium terrae, and bacteria in poliomyelitis vaccine suspended in five milk-type infant formulas. In another study, Santos Ferreira et al.6 found that microwave heating of 3303

dx.doi.org/10.1021/jf500278a | J. Agric. Food Chem. 2014, 62, 3301−3305

Journal of Agricultural and Food Chemistry

Article

different foods to microwave-cooking-induced destruction suggests that microwave destruction of these vitamins is a likely cause of the observed loss of nutritional value in the culture medium used to grow the Vero cells.

contaminated human milk prevented transmission of Chagas disease in mice caused by the protozoan Trypanosoma cruzi trypomastigotes. The cited results indicate that pasteurization and microwave heating can be used to eliminate pathogenic bacteria such as E. coli from contaminated milk and milk-based products. There seem to be no reported studies on the effect of microwave heating on the biological activity of Stx2 or any other toxin in milk. Because high levels (306 ng/mL in milk and 452 ng/mL in meat)7−9 of Shiga toxin have been observed in foods inoculated with E. coli, there is a need for such studies. It has been reported that a malfunction at a milk production plant, prior to pasteurization, contributed to an outbreak of hemolytic uremic syndrome that was linked to drinking the subsequently pasteurized milk in which no live bacteria were found.10 This finding suggests that the toxin produced and secreted by the bacteria was not inactivated by pasteurization and might be responsible for the illness We previously reported that oral ingestion of Stx2 toxin alone without E. coli bacteria that produce the toxin causes histopathological changes in kidney, spleen, and thymus tissues and mortality in mice.1 This finding demonstrated the need for new approaches to inactivate both the bacteria and the toxin produced by the bacteria. None of the cited studies examined the use of microwave radiation to inactivate Stx2 and other toxins produced by bacteria, although they do report effects beyond the mere elimination of the microbial pathogen. In principle, microwave-induced inactivation of the toxin can occur by at least two pathways: (1) changes in the native integrity to a denatured form might inactivate the protein toxin by preventing molecular interaction with cell-membrane receptor sites of their host Vero cells and (b) the protein toxin with an AB5 structure in which five subunits are attached to a straight chain can undergo degradation (cleavage) to smaller fragments. Figure 2 shows that the toxin seems to undergo degradation into smaller fragments and that the extent of degradation increases with the energy of the microwaves from 165 to 198 kJ. These results imply that the high-energy microwave radiation has the potential to protect consumers against adverse effects of Stx2 and possibly other toxins in contaminated water, milk, and possibly other foods. In the present study, we demonstrated that exposure of the milk solution to a higher level of microwave heating or to 95 °C for 5 min reduced Stx2 activity. In additional experiments, we found that heating reconstituted fat-free milk internally by higher microwave energy also inactivates Stx2 in milk. The use of microwave heating to inactivate the Shiga toxin complements published studies on the inhibition of the toxin’s biological activity by naturally occurring compounds, reviewed in Friedman and Rasooly.11 Exposure of food to microwave cooking is a much simpler process than the use of wet chemistry to inhibit the biological activity of Shiga and possibly other microbial and plant toxins that contaminate food.12 We also observed that the microwave heating destroyed the nutritional value of the culture medium used to grow the Vero cells. Our findings with the Shiga toxin and culture medium suggest the need to determine in detail the impact of microwave cooking on susceptible essential nutrients. Finally, allthugh it is difficult to translate our results in culture medium and in milk to other food categories exposed to microwave radiation, the reported high susceptibility of vitamin C (ascorbic acid)4,12,13 and vitamin B1 (thiamin)14,15 in

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: 510-559-6478. Fax: 510-559-6429. E-mail: reuven. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Daphne Tamar and Sharon Abigail for helpful suggestions.

■

REFERENCES

(1) Rasooly, R.; Do, P. M.; Griffey, S. M.; Vilches-Moure, J. G.; Friedman, M. Ingested Shiga toxin 2 (Stx2) causes histopathological changes in kidney, spleen and thymus tissues and mortality in mice. J. Agric. Food Chem. 2010, 58, 9281−9286. (2) Rasooly, R.; Do, P. M.; Levin, C. E.; Friedman, M. Inhibition of Shiga toxin 2 (Stx2) in apple juices and its resistance to pasteurization. J. Food Sci. 2010, 75, M296−M301. (3) Kakisu, E.; Abraham, A. G.; Farinati, C. T.; Ibarra, C.; De Antoni, G. L. Lactobacillus plantarum isolated from kefir protects Vero cells from cytotoxicity by type-II Shiga toxin from Escherichia coli O157:H7. J. Dairy Res. 2013, 80, 64−71. (4) Chandrasekaran, S.; Ramanathan, S.; Basak, T. Microwave food processing-A review. Food Res. Int. 2013, 52, 243−261. (5) Kindle, G.; Busse, A.; Kampa, D.; Meyer-König, U.; Daschner, F. D. Killing activity of microwaves in milk. J. Hosp. Infect. 1996, 33, 273− 278. (6) Santos Ferreira, C.; Amato Neto, V.; Gakiya, E.; Bezerra, R. C.; Rodríguez Alarcón, R. S. Microwave treatment of human milk to prevent transmission of Chagas disease. Rev. Inst. Med. Trop. Sao Paulo 2003, 45, 41−42. (7) Weeratna, R. D.; Doyle, M. P. Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food. Appl. Environ. Microbiol. 1991, 57, 2951−2955. (8) Cobbold, R. N.; Davis, M. A.; Rice, D. H.; Szymanski, M.; Tarr, P. I.; Besser, T. E.; Hancock, D. D. Associations between bovine, human, and raw milk, and beef isolates of non-O157 Shiga toxigenic Escherichia coli within a restricted geographic area of the United States. J. Food Prot. 2008, 71, 1023−1027. (9) Farrokh, C.; Jordan, K.; Auvray, F.; Glass, K.; Oppegaard, H.; Raynaud, S.; Thevenot, D.; Condron, R.; De Reu, K.; Govaris, A.; Heggum, K.; Heyndrickx, M.; Hummerjohann, J.; Lindsay, D.; Miszczycha, S.; Moussiegt, S.; Verstraete, K.; Cerf, O. Review of Shiga-toxin-producing Escherichia coli (STEC) and their significance in dairy production. Int. J. Food Microbiol. 2013, 162, 190−212. (10) Goh, S.; Newman, C.; Knowles, M.; Bolton, F. J.; Hollyoak, V.; Richards, S.; Daley, P.; Counter, D.; Smith, H. R.; Keppie, N. E. coli O157 phage type 21/28 outbreak in North Cumbria associated with pasteurized milk. Epidemiol. Infect. 2002, 129, 451−457. (11) Friedman, M.; Rasooly, R. Review of the inhibition of biological activities of food-related selected toxins by natural compounds. Toxins 2013, 5, 743−775. (12) Galgano, F.; Favati, F.; Caruso, M.; Pietrafesa, A.; Natella, B. The influence of processing and preservation on the retention of health-promoting compounds in broccoli. J. Food Sci. 2007, 72, S130− 135. (13) Friedman, M.; Gumbmann, M. R.; Ziderman, I. I. Nutritional value and safety in mice of proteins and their admixtures with carbohydrates and vitamin C after heating. J. Nutr. 1987, 117, 508− 518.

3304

dx.doi.org/10.1021/jf500278a | J. Agric. Food Chem. 2014, 62, 3301−3305

Journal of Agricultural and Food Chemistry

Article

(14) Khatoon, N.; Prakash, J. Nutritional quality of microwavecooked and pressure-cooked legumes. Int. J. Food Sci. Nutr. 2004, 55, 441−448. (15) Khatoon, N.; Prakash, J. Nutritional quality of microwave and pressure cooked rice (Oryza sativa) varieties. Food Sci. Tech. Int. 2006, 12, 297−305.

3305

dx.doi.org/10.1021/jf500278a | J. Agric. Food Chem. 2014, 62, 3301−3305