Effect of Gamma Radiation on Escherichia coli, Salmonella enterica Typhimurium and Aspergillus niger in Peppers

Abstract

Two widely used spices, Chili pepper (Capsicum frutescens L. var. conoides Bailey) and Sichuan pepper (Zanthoxylum bungeanum Maxim.) inoculated with Escherichia coli, Salmonella enterica Typhimurium and Aspergillus niger were irradiated with gamma ray. All inoculated samples were irradiated with a series of dosages. The efficacy of irradiation to inactivate pathogens was investigated. The results showed that irradiation at doses 4.00 kGy and 5.00 kGy was appropriate for eliminating almost all E. coli and S. Typhimurium in two peppers, respectively. A. niger was undetectable in chili and Sichuan pepper at doses 1.50 kGy and 1.00 kGy radiation. The D₁₀-values of E. coli, S. Typhimurium and A. niger in Sichuan pepper were 0.81, 0.93 and 0.20 kGy , and 0.82, 0.69 and 0.49 kGy in chili pepper. The results indicated that irradiation at appropriate doses is a promising approach for producing safe and pathogen-free peppers for consumers.

Introduction

Chili pepper and Sichuan pepper are important spices which extensively used as natural flavor in food, and they are especially in great demand in tranditional Chinese cuisine. Besides, they were mixed with other spices, and added directly into many ready-to-eat foods. However, these spices are highly susceptible to microbial contamination in the process of growing, harvesting, processing and transporting, etc. These peppers are usually used after dehydrating, which prolongs storage time but could not prevent the growth of microbes (Darfour et al., 2014). In recent years, prevalence of pathogens in spices, such as toxigenic fungi and bacteria, have been reported in many studies (Hammami et al., 2014; Zweifel and Stephan, 2012; Van Doren et al., 2013; Kong et al., 2014; El Mahgubi et al., 2013). The consumption of pathogen-contaminated spices can lead to foodborne diseases that could be serious enough to require hospitalization and even death (Van Doren et al., 2013).

Most E. coli strains are part of the normal intestinal flora but some strains, such as diarrhoeic E. coli, can cause enteric infections. E. coli is known as a common cause of foodborne illness. It massively exists in human and animal excreta which can be extensively used for manure in agriculture, thus it is regarded as an important source of contamination of the crops (Kim et al., 2012). Salmonella is recognized as being one of the most common bacterial causes of food-borne desease worldwide. Moreover, it is also the most common bacterial pathogen associated with product recalls and outbreaks in spices. In the United States, a Salmonella outbreak relating to white pepper gave rise to at least 85 confirmed cases (Zweifel and Stephan, 2012). S. Typhimurium was one of the most frequently reported serotypes which has been detected in spices (Zweifel and Stephan, 2012), and could cause symptoms that include fever, diarrhea, and vomiting etc. (Ban and Kang, 2014). A. niger is widely distributed in the soil, air and liable to cause mould of the spices, particularly it could produce mycotoxins that may be harmful to human health. In a study of fourteen spice samples, chili powder showed the highest fungal contamination, and A. niger took a great proportion in the isolated fungi (Hammami et al., 2014).

In recent years, gamma irradiation has been proved as an effective method to eliminate microbes and widely used in the food processing around the world. It can avoid secondary pollution by packing food into final packaging before irradiation and food can be distributed into market promptly after treatment (Roberts, 2014). Compared with some other sterilization methods, such as heat treatment and chemical fumigation, irradiation has significant advantages like non-thermal process and no chemical residues that ensure the quality and safety of spices (Roberts, 2014).

Little was known about the effect of gamma irradiation on pathogens in chili pepper and Sichuan pepper. Therefore, the objective of this study was to examine the effect of gamma irradiation dose for inactivating pathogens including E. coli, S. Typhimurium and A. niger inoculated in dried chili pepper and dried Sichuan pepper and to evaluate the sufficient dosages among different samples and pathogens.

Materials and Methods

Sample preparation    Dried chili pepper and dried Sichuan pepper were purchased from a supermarket (Chengdu, China). Each 15 grams of samples were packed in sterile ziplock bags (16 cm in length × 11 cm in width × 0.11 mm in thickness) for different experimental conditions, and each 5 grams of samples were packed in sterile ziplock bags (12 cm in length × 8 cm in width × 0.11 mm in thickness) used as controls to measure background microflora in every dose.

Strains and medium    Three strains, E. coli Z1, S. Typhimurium ATCC 14028 and A. niger Q5 were used in this study. E. coli Z1 isolated from pepper was obtained from the laboratory of microbiology in Sichuan Agricultural University. A. niger Q5 was donated by college of life science of Sichuan Agricultural University. E. coli and S. Typhimurium were stored at −80°C in tryptone soy broth (TSB, Hangzhou microbial reagent co., Hangzhou, Zhejiang, China) containing 15% glycerol and A. niger was stored at 4°C in potato dextrose agar slant medium (PDA, Hangzhou microbial reagent co., Zhejiang) until use.

Preparation of inoculum    The E. coli and S. Typhimurium were recovered by streaking onto trypticase soy agar (TSA, Hangzhou microbial reagent co., Zhejiang) plates and incubated at 37°C for 18 h. Subsequently, each pathogen was cultured separately in 50 mL tryptic soy broth in a 250-mL flask with agitation at 180 rpm on a rotary shaker (Forma, USA) for 18 – 24 h at 37°C to obtain an initial population level of 10⁸–10⁹ CFU/mL. A. niger was cultured on PDA plates for 3 days at 28°C until good sporulation was observed. The spores were harvested and suspended homogeneously in sterile normal saline. Then, the spores were counted with a hemocytometer and expressed the level of conidia approximately 5 log/mL. After preparation, three inocula were kept in a refrigerator (4°C) and used within 2 h.

Inoculation of test strains    Pepper samples were irradiated using a Cobalt-60 gamma irradiator (Russian) at doses 4 kGy to remove the background microflora. Dried chili pepper and dried Sichuan pepper were inoculated with each inoculum of three different pathogens. One and a half milliliter of inoculum was added to each 15 g sample using a sterile ziplock bag. The bag was then closed and shaken for 1 min to assist in uniform distribution. The inoculated samples labelled with irradiation doses were stored in 4°C until they were used. The initial level of E. coli and S. Typhimurium in samples was approximately 10⁶–10⁷ CFU/g and A. niger was 10³–10⁴ CFU/g .

Irradiation    All samples were irradiated using a Cobalt-60 gamma irradiator at room temperature at Biotechnology and Nuclear Technology Research Institute of Sichuan Academy of Agriculture Sciences with doses of 0, 0.25, 0.5, 1.00, 1.50, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00 and 8.00 kGy. The dose rate was 8.456 kGy/h . The samples were kept in a cooler during transport and stored in a refrigerator (4°C) before following analysis.

Enumeration    After irradiation, the stomacher bags were aseptically opened, 5 g of each inoculated samples and non-inoculated samples were aseptically added into asterile plastic tube. Then 45 mL of sterile 0.1% peptone water was added into inoculated and non-inoculated samples. The samples were homogenized by a vortex machine at least 2 min. Serial dilutions were made depending on the radiation dose applied. Aliquots (0.2 mL) of the diluent were surface plated onto TSA and PDA plates to determine the total count. Subsequently, all TSA media (E. coli and S. Typhimurium) were incubated at 37°C for 18 – 24 h, while PDA media (A. niger) incubated at 28°C for 3 – 5 days. Viable colonies were counted and confirmed by three independent experiments.

Statistical analysis    Colony counts were transformed into log values for data analysis. The D₁₀-value for each pathogen was determined using Excel's linear regression analysis by calculating the negative reciprocal of the survival slope (Rajkowski et al., 2006; Kim et al., 2010; Mustapha et al., 2014). To avoid possible shoulder effect, the zero-dose values were excluded from the calculation and at least four detectable data points were used to calculate the slopes and D₁₀-values (Rajkowski et al., 2006; Waje et al., 2009; Kundu et al., 2013). However, due to the limited detectable data, two to three data points were used to calculate D₁₀-values of A. niger. The triplicate data were presented as means ± SD (standard deviation) using Microsoft Excel Office 2010 (Microsoft Corp., Redmond, WA).

Results

Effect of irradiation on background microbes in peppers    In non-inoculated samples, initial populations of the bacteria in dried chili pepper and dried Sichuan pepper were at a level of 4.47 log CFU/g and 3.96 log CFU/g, but fungi were not detected. The inactivation effect of gamma irradiation on bacteria in dried chili pepper and dried Sichuan pepper was shown in Table 1. Bacterial counts were decreased significantly with the increase of doses, and reached under the detection limit in dried chili pepper and in dried Sichuan pepper after radiation at dose 3 kGy. As shown in Table 1, although initial value of dried chili pepper was higher, lower values were observed than those of dried Sichuan pepper at other doses. The D₁₀-values (Table 2) was 1.13 kGy for dried chili pepper and 1.32 kGy for dried Sichuan pepper.

Table 1. Effect of gamma radiation on the growth (Log CFU/g) of background microbes, Escherichia coli, Salmonella enterica Typhimurium and Aspergillus niger in peppers

Peppers	Pathogens	Irradiation dose (kGy)
		0	0.25	0.50	1.00	1.50	2.00	3.00	4.00	5.00–8.00
	BMa	4.47 ± 0.11b	2.61 ± 0.10	1.75 ± 0.33	1.06 ± 0.1	0.96 ± 0.12	0.90 ± 0.17	NDc	ND	ND
Chili	E. coli	7.15 ± 0.02	4.89 ± 0.19	2.93 ± 0.03	2.01 ± 0.01	1.40 ± 0.00	1.02 ± 0.24	1.00 ± 0.59	ND	ND
pepper	S. enterica	7.26 ± 0.08	6.21 ± 0.06	5.57 ± 0.09	5.05 ± 0.15	4.13 ± 0.02	1.98 ± 0.17	1.38 ± 0.00	1.18 ± 0.00	ND
	A. niger	3.68 ± 0.03	2.67 ± 0.03	2.13 ± 0.23	1.12 ± 0.17	ND	ND	ND	ND	ND
	BMa	3.96 ± 0.24	3.08 ± 0.07	2.54 ± 0.05	2.40 ± 0.14	2.26 ± 0.17	1.69 ± 0.07	ND	ND	ND
Sichuan	E. coli	6.22 ± 0.03	4.24 ± 0.09	3.79 ± 0.08	3.32 ± 0.15	2.82 ± 0.08	1.72 ± 0.05	0.88 ± 0.28	ND	ND
pepper	S.enterica	6.40 ± 0.05	4.71 ± 0.19	4.28 ± 0.29	3.90 ± 0.13	3.02 ± 0.10	2.00 ± 0.16	1.24 ± 0.08	0.95 ± 0.00	ND
	A. niger	3.39 ± 0.03	2.53 ± 0.15	1.26 ± 0.13	ND	ND	ND	ND	ND	ND

^a  BM: background microbes

^b  Values are means of triplicate samples ± standard deviation (SD).

^c  ND, not detected.

E. coli, E. coli Z1; S. enterica, S. Typhimurium ATCC 14028; A. niger, A. niger Q5.

Table 2. D₁₀-values (kGy) of pathogens inoculated in two peppers

Pathogens	Chili pepper	Sichuan pepper
BMa	1.13	1.32
Escherichia coli Z1	0.82	0.81
Salmonella enterica Typhimurium ATCC 14028	0.69	0.93
Aspergillus niger Q5	0.49b	0.20c

^a  BM: background microbes

^b  Three detectable data points were included.

^c  Two detectable data points were included.

Effect of irradiation on E. coli in peppers    E. coli inoculated in dried chili pepper were initially 7.15 log CFU/g, much higher than that in dried Sichuan pepper of 6.22 log CFU/g. The effect of gamma irradiation on E. coli in dried chili and Sichuan pepper was shown in Table 1. A dose of 3 kGy reduced counts of E. coli in dried chili pepper by approximately 6 log units. This dose also reduced approximately 5 log units of E. coli in dried Sichuan pepper. The D₁₀-values (Table 2) of dried chili pepper and dried Sichuan pepper were 0.82 kGy and 0.81 kGy separately. No surviving colony was detected in both types after radiation at dose 4 kGy revealed that this dosage was good for reducing almost all E. coli.

Effect of irradiation on S. Typhimurium in peppers    The initial populations of S. Typhimurium inoculated in dried chili and Sichuan pepper were 7.26 log CFU/g and 6.4 log CFU/g, respectively. Table 1 showed the inactivation effect of gamma irradiation on S. Typhimurium. After 5.00 kGy treatment, S. Typhimurium in samples was below the limit of detection in both peppers. The D₁₀-value (Table 2) of dried Sichuan pepper was equal to 0.93 kGy, higher than that of dried chili pepper (0.69 kGy).

Effect of irradiation on A. niger in peppers    The effect of gamma irradiation on dried chili pepper and dried Sichuan peppers inoculated with A. niger was shown in Table 1. The initial level of A. niger in samples was relatively lower, around 3 – 4 log CFU/g. After 1.00 kGy irradiation, A. niger in dried Sichuan pepper can not be detected and dried chili pepper was reduced 3.5 log CFU/g. However, A. niger was undetectable in dried chili pepper after radiation at dose 1.50 kGy. The D₁₀-values (Table 2) of dried chili pepper and dried Sichuan pepper were 0.49 kGy and 0.20 kGy.

Discussion

Spices are indispensable part of our daily diet, however, people paid little attention to the contamination of spices which may lead to foodborne diseases. Therefore, decontamination technologies should be applied for spices to limit the quantities of pathogens. Some studies have investigated the sterilization effect of gamma irradiation on spices, and the irradiation sensitivity varied from type of spices inoculated with various pathogens (Kirkin et al., 2014; Song et al., 2014). In addition, a certain number of dosages were used to get an acceptable level that is likely to completely eliminate the pathogens (Mohácsi-Farkas et al., 2014). However, little was known about the radiation survival of E. coli, S. Typhimurium and A. niger in dried chili and Sichuan peppers.

Through calculating of the D₁₀-values, the relative irradiation resistance of pathogens and the factors that could influence the survival of pathogens can be compared. In our study, the D₁₀-values for E. coli in both peppers were 0.82 and 0.81 kGy, which was in accord with the reported D₁₀-value in a range of 0.59 – 1.26 kGy for E. coli O157:H7 in red pepper (Song et al., 2014). They also found that the D₁₀-value was in a range of 0.55 – 0.83 kGy for S. Typhimurium inoculated in black pepper. Likewise, the D₁₀-values for S. Typhimurium in two peppers in our study were 0.93 and 0.69 kGy. In previous studies, however, radiation sensitive of pathogens inoculated in some food samples differed. Rajkowski et al. (2006) found the D₁₀-values for Salmonella DT 104 in three ground pork products with the gamma process were in a range of 0.54 – 0.66 kGy (Rajkowski et al., 2006) and the D₁₀-value of S. Typhimurium in chicken breast meat was in a range of 0.51 – 0.58 kGy (Kudra et al., 2011). Moreover, in three ready-to-eat food, mean D₁₀-values of Salmonella spp. and E. coli O157:H7 were 0.61 kGy and 0.36 kGy, respectively (Sommers and Boyd, 2006). A. niger inoculated in two peppers have the lowest D₁₀-value (0.49 and 0.20 kGy) among three pathogens. Kim et al. (2010) also obtained the D₁₀-values of fungi (Botrytis cinerea, Penicillium expansum, Rhizopus stolonifer var. stolonifer and Monilinia fructicola) in peach pulp which ranged from 0.14 to 0.23 kGy. The D₁₀-values of pathogens were relatively low in these different food samples, though inoculated with the same pathogens. Different chemical and physics properties of the food may give rise to the varied resistances (Monk et al., 1995) and a lower water content in spices lead to higher resistances (Waje et al., 2009).

Radiation resistance of pathogens inoculated in peppers pointed toward the trend: S. Typhimurium (0.93 kGy) > E .coli (0.82 and 0.81 kGy) > A. niger (0.49 and 0.20 kGy) except for the D₁₀-value of S. Typhimurium (0.69 kGy) in chili pepper. The order of radiation resistance in this study was in accord with the other studies. Sommers and Boyd (2006) examined varied radiation resistances of foodborne pathogens inoculated into several ready-to-eat food products, finding the D₁₀-value of Salmonella spp. was higher than E. coli O157:H7 (Sommers and Boyd, 2006). Meanwhile, an investigation has found that E. coli was not resistant to irradiation that can be controlled under a dosage to inhibit total number of colonies (Ma et al., 2013). Similar result was found that in black pepper and red pepper D₁₀-value of S. Typhimurium was larger than E. coli O157:H7 (Song et al., 2014). Besides, the D₁₀-value of E. coli and A. niger were higher in dried chili pepper than dried Sichuan pepper. However, S. Typhimurium was found a higher D₁₀-value in dried Sichuan pepper than dried chili pepper. It may be due to the effect of antimicrobial activity of spices. Hammami et al. (2014) noted that the formulation of some mixed spices represented an inhibiting factor against fungal growth.

In this study, 5 kGy and 4 kGy were observed as the most effective dosage for eliminating S. Typhimurium and E. coli. Similar result revealed that 5 kGy was effective for reducing almost all Salmonella Enteritidis (Rodrigues et al., 2011). In 1981, the WHO/IAEA/FAO announced that irradiation of food up to an overall average dose of 10 kGy presented no toxicological hazard and introduced no special nutritional or microbiological problems (Roberts, 2014). In previous studies, it was confirmed that spices with a dosage up to 10 kGy is safe for human to consume without obviously physicochemical properties changing (Darfour et al., 2014). In poached chicken meal, a lower dose of 2 kGy reduced counts of E. coli, and no colonies were detected (Adu-Gyamfi et al., 2008). Due to a low initial population, A. niger was undetectable in two kind of samples with relatively low dosages (1 and 1.5 kGy). The result was in accordance with those demonstration that fungal load reduced by 1—2 log in ground and whole chili samples (Iqbal et al., 2013). It was documented that no surviving fungi (Botrytis cinerea, Rhizopus stolonifer var. stolonifer and Monilinia fructicola) was detected in peach pulp with 1 kGy treatment (Kim et al., 2010). Thus, spices can maintain a good store quality by inhibiting pathogens with sufficient irradiation dosages.

Conclusion

In conclusion, the results confirmed that gamma irradiation was an effective way to inactivate E. coli, S. Typhimurium and A. niger in dried chili and Sichuan pepper. The total viable E. coli and S. Typhimurium in both samples were below the detectable levels at doses 4.00 kGy and 5.00 kGy treatment. A. niger decreased to undetectable level at dose 1.00 kGy and 1.50 kGy in dried Sichuan and chili pepper. The D₁₀-values varied depending on the type of pathogens and peppers used. A. niger has the least resistance to irradiation in two peppers, meanwhile, S. Typhimurium was the most resistance to irradiation in Sichuan pepper. Low levels of radiation by gamma ray can assure the inactivation of E. coli, Salmonella and A. niger in peppers, which makes for a safer product for the consumer.

Acknowledgements    This work was supported by the National Natural Science Foundation of China (31400066), Scientific Backbone Research Project and Postdoctoral Science Foundation of Chengdu University of Technology . We are grateful to Qi Wu for the donation of A. niger Q5.

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