Food Control 15 (2004) 441–445

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Isolation of enteric pathogens in the fermentation process

of Kimchi (Korean fermented vegetables) and its radicidation
by gamma irradiation
Dong-Ho Kim a, Hyun-Pa Song a, Hong-Sun Yook b, Yung-Gi Ryu c,
Myung-Woo Byun a,*
a
Team for Radiation Food Science and Biotechnology, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong-gu, Daejeon 305-600,
South Korea
b
Department of Food and Nutrition, Chungnam National University, Yuseong-gu, Daejeon 305-764, South Korea
c
Department of Food Science, Yonsei University, Seodaemun-gu, Seoul 120-749, South Korea
Received 19 August 2002; received in revised form 1 July 2003; accepted 3 July 2003

Abstract
The isolation of enteric bacteria in the fermentation process of Kimchi and its radicidation by gamma irradiation were inves-
tigated. Viable cell numbers of enteric bacteria were 104 CFU g 1 at the initiation of the Kimchi fermentation process, gradually
reducing during the fermentation period, and not detected after 10 days. The enteric bacteria in the early fermentation period of
Kimchi were eliminated by 2–3 kGy of gamma irradiation, but Lactobacillus spp. survived and fermentation was maintained. The
D10 values of total enteric group and Latobacillus spp. were about 0.32 and 0.87 kGy, respectively. The three typical enteric bacteria
were identified presumptively, as Enterobacter agglomerans, Salmonella typhimurium and Alcaligenes xylosoxydans, and the D10
values were 0.38, 0.54 and 0.47 kGy, respectively.
Ó 2003 Elsevier Ltd. All rights reserved.

Keywords: Kimchi; Fermentation; Irradiation; Enteric bactera

1. Introduction & Park, 1994). The widespread procedure of Kimchi

Kimchi is a famous Korean traditional fermented
vegetable food and it has been placed as an important
portion of the diet and nutrition in Korea (Lee, 1997;
Steinkraus, 1997). Recently, Kimchi become popular in
the Western world due to its characteristic taste and
several physiological effects including antioxidant, anti-
mutagenesis, and fibrinolytic activity (Lee, 1997), which
originate from various raw materials and secondary
metabolites of fermentative microorganisms (Caplice &
Fitzgerald, 1999; Svanberg & Lorri, 1997). The major
raw materials of Kimchi are oriental cabbage or radish.
Various vegetables (onion, green onion, carrot, cucum-
ber, leaf mustard, etc.), spices (hot pepper, garlic, ginger,
sesame, pine nuts, etc.) and non-vegetable ingredients
(fermented anchovy, fermented shrimp, boiled starch,
sugar, oyster, etc.) are used as minor additives (Cheigh
*
Corresponding author. Tel.: +82-42-868-8060; fax: +82-42-868-
8043.
E-mail address: mwbyun@kaeri.re.kr (M.-W. Byun).
0956-7135/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0956-7135(03)00119-1
processing includes; (1) brining of cabbage, (2) blending
brined cabbage with spices and ingredients, (3) aging
and fermentation. Kimchi fermentation is initiated by
various microbes originating from the raw materials and
environment. The microflora is gradually simplified and
finally lactic acid bacteria (Lactobacillus, Leuconostoc,
Pediococcus, etc.) and yeast (Hansenula, Pichia, De-
baryomyces, etc.) become dominant (Cheigh & Park,
1994).
The characteristics of the fermentative microorgan-
isms were well established, but the studies of pathogens
were rarely performed in spite of a high possibility of
pathogenic microbial contamination in the Kimchi.
There are a number of reports isolating Aeromonas
(McMahon & Wilson, 2001), Escherichia (Nguyen-the &
Carlin, 1994), Salmonella (Doyle, 1990) and Listeria
(Robertson, Johannessen, Gjerde, & Loncarevic, 2002)
from raw vegetables, indicating that the raw vegetables
may harbor potential food-borne pathogens.
It is presumed that acids or bacteriocins produced
from fermentative bacteria may eliminate pathogenic
442 D.-H. Kim et al. / Food Control 15 (2004) 441–445
bacteria during fermentation (Caplice & Fitzgerald,
1999; Mah et al., 2001). However, the bacteriocin-pro-
ducing microbes lagged in the growth at the early fer-
mentation stage and antibacterial activity of the
bacteriocin may therefore be limited. The activities of
bacteriocins are considered to be species specific (Tagg,
Dajani, & Wannamaker, 1976) and lactic acid bacterial
bacteriocins are generally less active against Gram
negative species (Nettles & Barefoot, 1993). This may
allow some pathogens to remain in Kimchi. Therefore,
characterization of pathogenic microorganisms during
the Kimchi fermentation process and development of
proper decontamination methods are required. Re-
cently, significant beneficial effect has been reported
when the gamma irradiation technology was applied to
Korean traditional fermented foods such as fermented
soybean paste (Byun, Kim, Yook, Cha, & Kim, 2001)
and soy sauce (Kim, Yook, & Byun, 2001). Therefore, in
the present study, the characterization of some enteric
pathogens isolated from early fermentative stage of
Kimchi was investigated and a proper radiation dose for
radicidation was defined using gamma irradiation.
2. Materials and methods
2.1. Kimchi preparation
Kimchi preparation followed the method of Lee,
Shin, Lee, Kim, and Cheigh (2001). Oriental cabbages,
cut into half heads, were dipped in 15% salt solution for
4 h. The pieces of cabbage were washed with tap water,
drained and then mixed with spices and additives. The
recipe of spices and additives was; sliced radish 5 g, red
pepper powder 3 g, garlic 1 g, green onion 2.5 g, fer-
mented anchovy sauce 2 g and ginger 0.5 g per 100 g of
the brined cabbage. Each half head of blended cabbage
was anaerobically packed in polyethylene vinyl bags and
used as a sample. Kimchi fermentation was carried out
at 10 °C for 20 days.
2.2. Microbiological evaluation
A piece of Kimchi was homogenized using a lab
blender (Hanil, FM 680T, Seoul, Korea) for 60 s, and
then filtered (No. 2, Whatmann, Kent, England). Each
filtrate (0.1 ml) was smeared in triplicate on SS agar
(Salmonella/Shigella agar, Difco Lab, Detroit, MI,
USA) plate, the selective media for enteric bacteria
(Busse, 1995). The plates were incubated at 37 °C for 48
h, colony forming units (CFU) per ml were counted, and
typical dominant colonies were selected and identified.
The isolates were identified presumptively by physio-
logical and biochemical tests, based on API 20 E and
API 20 NE kit (API System, bioMerieux SA, France).
Fatty acid analysis of the cell membranes by Microbial
Identification System (MIDI; Microbial ID, Inc., New-
ark, Delaware, USA) was performed for certification.
The isolates, identified by the MIDI system, were cul-
tured in nutrient broth (Difco Lab.) at 37 °C for 24 h
and radiation sensitivity was determined. The total
counts of Lactobacillus spp. were determined on MRS
agar plates.
2.3. Gamma irradiation
Each half head of blended cabbage, packed in a
polyethylene vinyl bag, was irradiated in a cobalt-60
gamma irradiator (point source, AECL, IR-79, Nor-
dion, Canada) at doses of 1, 3 and 5 kGy. The source
strength was approximately 100 kCi with a dose rate of
70 Gy min 1 at 15 ± 0.5 °C and the actual doses were
within 2% of the target dose. Non-irradiated controls
were also prepared. Broth cultures of the isolates were
transferred in 5 ml conical tubes and irradiated at doses
of 1, 3 and 5 kGy.
2.4. Calculation of radiation sensitivity
Viable counts of irradiated and non-irradiated sam-
ples on SS agar plates were examined and radiation
sensitivities were calculated as D10 value. The D10 value
is the expression of the radiation dose needed to reduce
the number of microorganisms by 10-fold and was cal-
culated from the equation of the survival plot (FAO/
IAEA/WHO, 1999).
2.5. Statistical analysis
Two-way analyses of variance (ANOVA) was used to
determine the effect of irradiation dose on the microbi-
ological changes by SAS software (SAS, 1989). Student-
Newman–KeulÕs multiple range test was used to
compare differences among means at P < 0:05 (Steel &
Torrie, 1980).
3. Results and discussion
3.1. Changes of microflora in kimchi fermentation
Viable cell counts of the Lactobacillus spp. group,
which are representative of fermentative microorgan-
isms in the Kimchi processing, were 105 CFU g 1 early
in the fermentation process, reaching 109 CFU g 1 after
10 days (Fig. 1), and at this point the content of lactic
acid reached up to 1.2% (data not shown). Viable cells
on the SS agar plate were 104 CFU g 1 at initial fer-
mentation process (Fig. 2). Therefore, it was considered
that the potential of pathogenic contamination is fairly
high in the Kimchi fermentation process. However, the
cell counts of SS agar plate were gradually reduced, and
 D.-H. Kim et al. / Food Control 15 (2004) 441–445
Fig. 1. Growth of Lactobacillus during the fermentation period of
gamma irradiated and non-irradiated Kimchi. Symbols are d, 0 kGy;
j, 1 kGy; N, 2 kGy; r, 3 kGy.
Fig. 2. Growth of enteric microbes on SS agar plate during the fer-
mentation period of gamma irradiated and non-irradiated Kimchi.
Symbols are d, 0 kGy; j, 1 kGy; N, 2 kGy; r, 3 kGy.
not detected after 10 days (Fig. 2). It is interpreted from
previous studies (Alakomi et al., 2000; Cheigh et al.,
2002) that the bacteriocins and/or organic acid, pro-
duced from various fermentative microbes, induce
spontaneous inactivation of pathogenic microorganisms
during Kimchi fermentation. However, Kimchi is con-
sumed not only after fermentation but also early in the
process, before fermentation is complete. So, although
pathogenic microbes were eliminated spontaneously
during the fermentation period, it was considered that
elimination of pathogenic microbes in the early stage of
fermentation is required.
3.2. Gamma irradiation of Kimchi
The viable cells detectable on SS agar plate were
eliminated >5 log reduction by gamma irradiation with
doses of 2–3 kGy, and Lactobacillus group numbers
were reduced by three decimal reductions with a dose of
3 kGy (Fig. 3). The D10 values of total enteric group and
Latobacillus group were 0.32 and 0.87 kGy, respectively
(Fig. 3). These survival rates are similar to those of
previous reports against Salmonella (Niemira, Sommers,
443
Fig. 3. Effect of gamma irradiation on the viability of Lactobacillus
and enteric microbes. Symbols are d, Lactobacillus group; j, enteric
microbes on SS agar plate.
& Boyd, 2001) and Lactobacillus group (Kim, Jo, Yook,
Park, & Byun, 2002).
Changes in microbial growth during fermentation
period were observed. The population of total enteric
group in the 1 and 2 kGy irradiated Kimchi rapidly
decreased and was not detected after 1–2 day (Fig. 2). It
was considered that the decrease in the microbial pop-
ulation was due to the post-irradiation effect where
surviving cells that had been damaged by gamma irra-
diation gradually died, not adapting to the surrounding
environment during storage (Byun et al., 2001). The
growth rates of the Lactobacillus group in the 1–3 kGy
irradiated samples lagged, following 1–4 decimal re-
ductions, till the mid-stage of fermentation, but gradu-
ally reached maximally in the stationary stage (Fig. 1).
The result showed that gamma irradiation was not only
effective in sanitizing the early fermentation stage of
Kimchi but also in maintaining the fermentation pro-
cess.
3.3. Identification of the isolates from SS agar plate
The three typical and most abundant colonies on the
SS agar plates were isolated. Typical growth appear-
ances of the isolates were dark Chinese yellow and me-
tallic sheen (S-1), pink with double halo of clean and
pink (S-3), and orange (S-4) colony. The presumptive
identifications of S-1, S-3 and S-4 by physiological and
biochemical tests based on API kit (API System, bio-
Merieux SA, France) were Enterobacter spp., Salmonella
typhimurium and Alcaligenes spp., respectively (Data not
shown). Microbial Identification System confirmed that
S-1 was Enterobacter agglomerans, S-3 was Salmonella
typhimurium and S-4 was Acaligenes xylosoxydans (Ta-
ble 1). Alcaligenes spp. and Enterobacter spp. are com-
mon in the mammalian intestine and in soil and aquatic
environments. Salmonella typhimurium is an aerogenic
and group B serotype strain, which commonly causes
444 D.-H. Kim et al. / Food Control 15 (2004) 441–445

Table 1
Fatty acid compositions of three enteric bacterial strains isolated from early fermentation stage of Kimchi and presumptive identification
Fatty acid (relative %) Isolates
S-1 S-3 S-4
12:0 3.47 5.39 0.89
12:0 2OH 2.13
13:0 0.21
14:0 6.80 8.60 5.11
14:0 3OH/16:1 iso I 7.91 6.87
15:0 1.33
16:1 iso I/14:0 3OH 9.07
16:1 w7c/15 iso 2OH 18.05 27.90 37.77
16:0 29.63 22.87 30.96
17:0 cyclo 9.89 7.70 6.83
17:0 0.46 0.48
18:1 w7c/w9t/w12t 23.62 15.59 7.52
18:0 1.45
Presumptive identification Enterobacter agglomerans Salmonella typhimurium Alcaligenes xylosoxydans

gastro-enteritis. These results showed that the intake of
non-fermented Kimchi may cause intestinal sickness or
lead to secondary contamination. Normal shelf life of
Kimchi is 10–15 days even in refrigerated condition, and
usually non-fermented Kimchi is packaged for extension
of selling period in international trading (Lee et al.,
2001). In this case, detection of pathogenic microbes can
be a reason of rejection at the quarantine requirement.
So, decontamination in the early stage of fermentation
might be desirable. More than 90% of the pathogenic
contaminants originated from red pepper powder (data
not shown), the major spice of Kimchi, so that radici-
dation of red pepper powder (Byun, 1985) may be ef-
fective and more easily applied to reduce pathogens in
Kimchi, too. The D10 values of Enterobacter agglomer-
ans, Salmonella typhimurium and Alcaligenes xylosoxy-
dans were 0.38, 0.54 and 0.47 kGy, respectively (Fig. 4),
and these results were similar to previous studies of
foodborne pathogens (Grant & Patterson, 1992; Nie-
mira et al., 2001).
4. Conclusion
The potential of pathogenic contamination is fairly
high in the early fermentation stage of Kimchi pro-
cessing and although enteric microbes were eliminated
spontaneously during fermentation period, decontami-
nation of pathogenic microbes in the early fermentation
stage is required. The enteric microorganisms were
eliminated by 2–3 kGy of gamma irradiation and fer-
mentation was just delayed. So, it was considered that
gamma irradiation was not only effective in sanitizing
the early fermentation stage of Kimchi but also in
maintaining fermentation process.

Acknowledgement

This work was supported by Korea Research Foun-

dation Grant (KRF 2002-042-001118).

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