APC)

Food Control 14 (2003) 489–494 www.elsevier.com/locate/foodcont

Screening of gamma irradiated spices in Korea by using a microbiological method (DEFT/APC) K.N. Oh a, S.Y. Lee a, H.J. Lee a, K.E. Kim b, J.S. Yang b

b,*

a Graduate School of Food and Nutrition, Chung-Ang University, 221, HukSuk-Dong, DongJak-Ku, Seoul 156-070, South Korea Korea Atomic Energy Research Institute, Detection Lab. of Irradiated foods, 150 Duckjindong, Yusong-Ku, Taejon 305-353, South Korea

Received 19 May 2002; received in revised form 9 September 2002; accepted 10 September 2002

Abstract Irradiation can be used to control the microbiological spoilage and pathogen microorganisms of foods. Microbiological changes associated with food irradiation include the reduction or elimination of spoilage and pathogenic microorganisms, insect disinfestation and parasite disinfection. The effects on the microbial populations of irradiated foods have been used to develop detection methods for irradiated foods. The method used in this study was direct epifluorescent filter technique/aerobic plate count (DEFT/ APC), which is based on the difference between DEFT counts and APC counts. The samples were imported spices and domestic spices produced in Korea. These samples were irradiated at 1.0, 3.0, 5.0, 7.0, and 10 kGy to reduce the spoilage organisms. Irradiation doses of 3.0 kGy or over eliminated viable microorganisms effectively, and the logDEFT=APC ratio gradually increased with dose increment in all the samples. It could be suggested that if the logDEFT=APC ratio is 2.5 or over for peppers in Korea, the samples can be suspected as irradiated at a dose level of at least 3.0 kGy. The DEFT/APC method could be used for screening for the irradiation treatment of spices produced in Korea. Ó 2003 Elsevier Ltd. All rights reserved.

1. Introduction Decontamination of spices that are used as ingredients in processed foods is necessary in order to reduce the numbers of spoilage microorganisms. Spices can be contaminated with 103 –108 /g microorganisms such as bacteria and molds (Hammerton & Benos, 1996). Dried spices and herbs are often heavily contaminated with microbes originating from different sources. The microbial characteristics of spices depend on their origin and the nature of the processing, transport and storage technique. The most effective means of decontaminating spices is irradiation treatment with an absorbed radiation dose of 5.0–10 kGy. Appropriate techniques for the detection of irradiated foods and the determination of the applied dose requirements are needed to guarantee the proper consumer information, to prevent misuse of the technology and to facilitate the trade of irradiated foods (Schreiber, Helle, & B€ ogl, 1993). The characteristics of the microbial population of irradiated foods

*

Corresponding author. Tel./fax: +82-42-868-8064. E-mail address: [email protected] (J.S. Yang).

0956-7135/03/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0956-7135(02)00108-1

have been used for developing detection methods for irradiated foods (Delincee, 1998). A microbiological method based on the use of the direct epifluorescent filter technique (DEFT) and the conventional aerobic plate count (APC) has been used for the detection of irradiated spices (Boisen, Skovgaard, Ewalds, Olsson, & Wirtanen, 1992; Wirtanen, Sj€ oberg, Boisen, & Alnko, 1993). The DEFT count enumerates the total number of contaminating microorganisms, irrespective of viability in untreated or treated spice samples. The APC indicates the number of viable microorganisms capable of forming colonies on an agar plate and is expressed as colony forming units (cfu). If samples are irradiated, most viable microorganisms are killed and the ratio of DEFT/ APC increases. The quotient of the two counts is used for assessing whether the sample is irradiated or not. In this study, the DEFT/APC method was used to detect or screen whether the imported and domestic spices in Korea were irradiated or not. In the literature search, few DEFT/APC studies were found on the detection of the irradiated spices. Because the DEFT/APC criterion to screen for irradiated foods maybe different in various countries, the suggestion and establishment of detection criterion in each country are very important.

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Therefore, the aim of this study was to establish the criteria to identify or screen the irradiated spices in Korea by applying the microbiological method (DEFT/ APC).

2. Materials and methods

2.4. Calculation The DEFT count (X ) per gram of spices was calculated by using the mean number of DEFT units per microscope field (N =n), the dilution factor (DF) of the sample, and the microscope factor (MF) (Wirtanen et al., 1993) X ¼ DEFT count=g ¼ ðN  MF  DFÞ=n

2.1. Samples Samples used for the experiment were both imported and domestic spices in Korea. Imported spices were whole black pepper (Malaysia), powdered black pepper (Malaysia), powdered white pepper (Malaysia), marjoram (America), and thyme (America). Domestic spices were red pepper powder, garlic powder, onion powder and ginger powder produced in Korea. Before the experiment, these samples were confirmed not to have any pretreatment to improve. All the samples were received from Dongbang food master company in Chungbuk, Korea. The samples were packed in sterilized tubes and labeled each with the irradiation dose. 2.2. Irradiation The radiation source was 60 Co package irradiator (point source, AECL, IR-79, Nordion, Canada). The applied doses were 0, 1.0, 3.0, 5.0, 7.0, and 10 kGy, and the total absorbed dose was identified by ceric cerous dosimeter (
5%). The temperature during the radiation was 15
0:5 °C. Each sample was weighed to 10 g, packed in sterilized corning tube in triplicate packages for irradiation treatment. 2.3. Analysis Samples were irradiated in 60 Co research facilities and tested immediately after irradiation. Unirradiated and irradiated spices of 10 g were added to peptone saline diluent (pH 7.2; 8.5 g sodium chloride (Sigma Chemical Co.) and 1.0 g peptone (Difco Becton Dickinson)/1000 ml), diluted by 10 times or 20 times and stirred vigorously. The solution was then filtered through a fast filter paper (Whatman No. 4). The filtered solution was diluted with peptone saline diluent by logarithmic dilution series (101 –103 times). Each diluted solution was transferred to the filtration manifold tower for DEFT. The total viable count (APC) was determined after the filtration through a fast filter paper (Whatman No. 4). 0.2 ml aliquots of suitable dilutions were spread on Plate Count Agar (Difco Becton Dickinson). The plates were incubated upside down at 30°
1 °C for 72 h and then counted (Betts, 1993; Wirtanen & Sj€ oberg, 1993; Wirtanen et al., 1993). All the experiments were conducted in triplicate.

ð1Þ

The DEFT count was then converted to a logarithmic value. The difference between the DEFT count and APC count is then obtained by subtracting the APC count (logarithmic value) from the DEFT count (logarithmic value).

3. Results and discussion The results of both non-irradiated and irradiated spices are presented in Tables 1 and 2, Figs. 1 and 2. Table 1 shows the results of the imported spices; Table 2 shows the results of the Korean spices. The logDEFT= APC ratio was determined for each spice. The logDEFT=APC ratio increased in general with irradiation dose increment because the logDEFT unit barely changed whereas the logAPC unit decreased gradually. The result of logDEFT=APC ratio for whole black pepper is shown in Table 1 and Fig. 1(A). The DEFT counts were nearly at the same level independent of the irradiation, but the APC counts decreased gradually with irradiation dose increment. Therefore, the logDEFT=APC ratio increased with the dose increment. The logDEFT=APC ratio of unirradiated and irradiated with 1.0 kGy were 1.14 and 2.38, respectively. The logDEFT=APC ratio of the sample irradiated at 3.0 kGy was about 2.5 and the logDEFT=APC ratio of the samples irradiated at 5.0 kGy or more was 2.5 or over. Table 1 The logarithmic microbial counts of the imported spices in Korea Irradiation dose (kGy)

Samples

0

1.0

3.0

5.0

7.0

10.0

log DEFT

6.92

6.88

6.90

6.83

6.85

6.92

log APC

5.77

4.50

4.41

4.18

3.93

2.92

log DEFT

6.20

6.19

6.21

6.25

6.22

6.29

log APC

4.22

3.52

3.30

2.91

2.26

2.62

log DEFT

5.31

5.46

5.36

5.50

5.2

5.43

log APC

4.10

3.71

3.19

3.00

2.75

2.49

Marjoram

log DEFT log APC

7.38 5.89

6.89 5.22

3.91 4.76

6.93 4.28

6.81 3.94

6.91 3.46

Thyme

log DEFT log APC

6.54 5.35

6.58 4.49

6.58 4.09

6.61 3.88

6.58 3.65

6.60 6.46

Whole black pepper Powdered black pepper Powdered white pepper

K.N. Oh et al. / Food Control 14 (2003) 489–494 Table 2 The logarithmic microbial counts of the domestic spices produced in Korea Irradiation dose (kGy)

Samples Red-pepper powder Garlic powder Onion powder Ginger powder

0

1.0

3.0

5.0

7.0

10.0

log DEFT

7.11

6.95

7.22

6.78

7.11

7.17

log APC log DEFT

6.01 5.94

4.55 5.96

4.75 5.86

3.68 5.94

3.23 5.95

2.98 5.87

log APC log DEFT

4.59 6.66

3.72 6.44

3.47 6.90

3.22 6.57

2.80 6.60

2.29 6.61

log APC log DEFT

3.90 5.71

3.37 5.53

2.91 5.76

2.52 5.75

2.20 5.69

2.00 5.72

log APC

4.52

3.17

3.19

2.51

2.19

1.83

The logDEFT=APC ratio for 10 kGy was about 4.0, resulting from very low viable counts. The logDEFT= APC ratio for irradiated black pepper was lower than the results of the other work (Wirtanen et al., 1993). The

491

reason for this probably comes from its protection against irradiation or the rather low original microbial load (Sj€ oberg, Wirtanen, & Alanko, 1993). The mechanisms of the protection against irradiation might be that spices had different water activity and environment. Also, depending on the number and types of microorganisms and the chemical composition of the spice, a radiation dose required may be varied to achieve sterility (reduction of total viable cell count less than 10/g) (Farkas, 1988). Wirtanen et al. (1993) reported that irradiation dose needed for the reduction of pathogenic bacteria was ranged from 3.0 to 7.0 kGy. They reported that spices irradiated spices at 5.0 kGy or over, showed 3.5 or over in the logDEFT=APC calculation. The logDEFT=APC ratio of the whole black pepper tested in this study was 2.5 or more with the irradiation doses of 5.0–10 kGy. The results for powdered black pepper for logDEFT=APC ratio is shown in Table 1 and Fig. 1(B). The logDEFT=APC ratio for unirradiated pepper was less than 2.5, while the irradiated samples even with

C

Fig. 1. Histogram of logDEFT=APC ratio for imported spices in Korea. (A) Whole black pepper, (B) powdered black pepper, (C) powdered white pepper, (D) marjoram, (E) thyme.

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K.N. Oh et al. / Food Control 14 (2003) 489–494

Fig. 2. Histogram of logDEFT=APC ratio for domestic spices. (A) Red-pepper powder, (B) garlic powder, (C) onion powder, (D) ginger powder.

1.0 kGy showed 2.5 or more logarithmic units. Although the DEFT counts were nearly at the same level in all samples regardless of irradiation, the APC counts decreased with the irradiation dose given. Powdered black pepper showed higher logarithmic units than whole black pepper. The logDEFT=APC counts for powdered white pepper of unirradiated and irradiated with 1.0 kGy was less than 2.0 (Table 1 and Fig. 1(C)). In the samples irradiated with 5.0 kGy, the difference between DEFT and APC counts was 2.5 or more logarithmic units. The DEFT counts were nearly at the same level regardless of the irradiation, but the APC counts decreased with the irradiation dose increment. The result of marjoram also showed an increase in logDEFT=APC ratio with the increase in dose (Table 1, Fig. 1(D)). The logDEFT=APC ratio of unirradiated and irradiated marjoram at 1.0 kGy was 1.48 and 1.57, respectively, which were below the 2.0 logarithmic units. The logDEFT=APC ratio of the sample irradiated at 3.0 kGy was 2.14 and the logDEFT=APC ratio for the samples irradiated with over 5.0 kGy were 2.5 or more. The marjoram irradiated with 10 kGy had few viable counts and the corresponding logDEFT=APC ratio was high. The logDEFT=APC ratio for the irradiated samples except 10 kGy was below 3.0. Boisen (1993) reported that cut marjoram showed a log difference higher than 4.0 at 5.0 kGy. However, the marjoram tested in this study had the 2.5 logarithmic units or more with 5.0 kGy irradiation. We could suggest that a DEFT/APC ratio of 2.0 logarithmic units could be a criterion of judging the sample as unirradiated or irradiated in

Korea. The type of spices may influence the irradiation effect and also the filtration procedure could be difficult in some cases depending on the type of sample. The filtration step could be improved by the pre-centrifugation (Sj€ oberg, Manninen, Pinnioja, Honkanen, & LatvaKala, 1991). Slow centrifugation reduces the number of larger particles in the sample solution but not the number of microbes, which makes it easier to filter the solution. In this study, we used a dilution procedure instead of pre-centrifugation. Twenty times dilutions were conducted to imported marjoram to ease the filtration. This process did not influence the number of microbes detected. The logDEFT=APC ratio for thyme also increased with the irradiation dose increment (Table 1, Fig. 1(E)). The logDEFT=APC ratio was 2.0 for the unirradiated thyme while the logDEFT=APC ratio was 2.0 for the irradiated samples. The logDEFT=APC ratio was about 2.5 or more for the samples irradiated with a dose 5.0 kGy. Hammerton and Benos (1996) reported that the most effective means of decontamination was the irradiation treatment with an absorbed radiation dose from 5.0 to 10 kGy. Also they found that for spices treated with a radiation dose of 5.0 kGy, the logDEFT=APC ratio was 4.0. In this study, the logDEFT=APC ratio was about 2.5 or more in the samples irradiated with 3.0 kGy or over. Therefore, we could suggest that if the difference between DEFT and APC count is at least 2.5 logarithmic units, the imported thyme has been irradiated with a dose level of at least 5.0 kGy or over. The result of Korean red-pepper powder for logDEFT=APC ratio is shown in Table 2 and Fig. 2(A).

K.N. Oh et al. / Food Control 14 (2003) 489–494

The DEFT counts were nearly at the same level independent of the irradiation, but the APC counts decreased with dose applied. Therefore, the logDEFT= APC ratio increased depending on the dose increment. The logDEFT=APC ratio was 2.0 for the unirradiated red-pepper powder while the logDEFT=APC ratio was 2.0 for the irradiated powders. Indeed, the logDEFT= APC ratio was 3.0 for the samples irradiated with a dose 5.0 kGy. The red-pepper powder irradiated with 10 kGy had few viable counts and the resulting logDEFT=APC ratio was very high (4.0). Boisen (1993) reported that if the log difference is 4.0, the spices could be suspected as the irradiated. In this study, the red-pepper powder showed 2.5 or more logarithmic value in the samples irradiated with 5.0 kGy or over. Therefore, the logDEFT=APC ratio 2.5 could be a criterion of the irradiated red-pepper powder with dose level of at least 5.0 kGy or over. The result of garlic powder for logDEFT=APC ratio is shown in Table 2 and Fig. 2(B). The garlic powder had slightly less units for logDEFT=APC than the redpepper powder had. Using doses of 5.0 kGy the difference between DEFT and APC log counts was 2.5 or above. Consequently, we could suggest that if the Korean garlic powder has 2.5 or more in the logDEFT= APC ratio, the garlic powder could be suspected as irradiated with a dose level of at least 5.0 kGy. Onion powder showed higher logarithmic units than red-pepper powder and garlic powder did (Table 2 and Fig. 2(C)). The logDEFT=APC ratio of onion powder irradiated with 1.0 kGy or more exceeded 3.0 logarithmic units. In this sample, the irradiated onion powders were obviously distinguished from the unirradiated powder. Comparison of the results from onion powder with those from the red-pepper powder and garlic powder suggested that the microflora associated with onion might be more susceptible to the irradiation (Sj€ oberg et al., 1993), or the onion powder had more antibacterial activity, which could be deduced from low level of aerobic microorganisms. Korean red-pepper powder and onion powder were differed to garlic powder, therefore to ease the filtration, 20 times dilutions at these spices were done. This process did not influence the number of microbes enumerated. The result of logDEFT=APC ratio for Korean ginger powder is shown in Table 2 and Fig. 2(D). The DEFT/ APC logarithmic units increased from the unirradiated to irradiated sample. The logDEFT=APC ratio was 2.0 for the unirradiated ginger powder while the logDEFT=APC ratio was 2.0 for the irradiated powders. The logDEFT=APC ratio was 2.5 or more for the samples irradiated with a dose 3.0 kGy. In the previous researches carried out by Oh, Lee, and Yang (2002a, 2002b), they found that cereal grains and beans had logDEFT=APC ratio ranged from 2.0 to 3.0 at 0.5 kGy or over.

493

In general, spices may contain initial microbial levels of with 105 –108 , before application of any hygiene treatment. If the foodstuffs were gamma irradiated, the level of viable microorganisms decreased to below 104 . Therefore, the ratio was at least 2.0. In addition, the logDEFT=APC ratio would vary because the total and viable microorganisms are different because of the degree of initial contamination. As a result, the suggested logarithmic DEFT/APC ratio of 2.0 as a criterion of samples irradiated 3.0 kGy or 5.0 kGy in this study was not absolute criterion value. It is considered that the DEFT/APC method could be used for the pre-screening test. The spices experimented in this study had total microorganisms with 106 –105 in the initial status. The viable microorganisms were decreased to the level of 103 for the c-irradiation and the logDEFT=APC ratio was 2.5 or more in the samples irradiated with 3.0 kGy or more.

4. Conclusions The DEFT/APC method can be used for screening the irradiated spices. For the imported spices (whole black pepper, powdered white pepper, marjoram, and thyme) except powdered black pepper and marjoram, a logDEFT=APC ratio 2.5 could be indicative of irradiation treatment with a dose level of at least 3.0 kGy or over. The Korean red-pepper powder and garlic powder showed 2.5 or more logarithmic units in the irradiated samples with 5.0 kGy or more, while the onion power and ginger powder showed 2.5 or more units in the irradiated samples with 3.0 kGy or more. Spices irradiated with a dose 5.0 kGy were clearly identified in all the samples. This method reveals the microbiological characteristics of the product at the time of analysis (APC count) and also provides the information about history of the product (DEFT count). The combined DEFT/APC method can be used for screening purposes and also provides information on the microbiological characteristics of the spices before and after radiation processing.

Acknowledgement This work was supported by Mid-and-Long-term Nuclear R&D Programs of Korea Ministry of Science and Technology and we greatly appreciate it.

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