IJERD – International Journal of Environmental and Rural Development (2022) 13-1

Research article
erd

Microbial Behavior in Cambodian Homemade-style Pickles
SHUKI MURAMATSU*
Faculty of Health and Food Sciences, Showa Women’s University, Tokyo, Japan
Email: s-muramatsu@swu.ac.jp

SOKLY SORM
Graduate School of Agriculture, Tokyo University of Agriculture, Tokyo, Japan

MASATAKA UCHINO
Faculty of Life Sciences, Tokyo University of Agriculture, Tokyo, Japan

MOTOE SEKIDO
Department of Food and Nutrition, Yamanashi Gakuin Junior College, Tokyo, JapanYOSHIKI MURAMATSU
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, JapanDAIKI OKA
Faculty of Applied Biosciences, Tokyo University of Agriculture, Tokyo, JapanYURI TANIOKA
Faculty of International Agriculture and Food Studies, Tokyo University of Agriculture, Tokyo,Japan

TAKAHIKO NAKAMURA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, JapanTORU NAKAJIMA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, JapanMARI ARIMITSU
Extension Center, Institute of Environmental Rehabilitation and Conservation, Tokyo, Japan

MACHITO MIHARA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan
Received 26 January 2022

Accepted 2 May 2022(*Corresponding Author)

Abstract Pickled products made using vegetables are popular in Cambodia. Many types ofhomemade pickles are sold at wet markets. Our previous study showed that pickles areseriously contaminated by microbes, including food poisoning bacteria. Such contamination
may depend on several factors. The objectives of this study were to investigate the levels ofcontaminating microbes and their growth patterns in pickles prepared according totraditional Cambodian recipes and to suggest an improved method to ensure the microbialsafety of homemade pickles. Three kinds of pickles, namely cucumber pickles with orwithout fish sauce and bok choy pickles, were prepared following the methods used bylocal people. Five sample bottles for each kind of pickle were prepared to observe changes
in their characteristics over time. The Brix values, salt concentrations, and pH of thesamples were measured. Next, we conducted microbiological examination of the samples
by testing the presence of total viable bacteria, coliforms, and fungi. More than 5.00 log10
CFU/g of total bacteria were detected in all pickle samples at day 0; moreover, the numberof bacteria increased until day 4. A similar concentration of coliforms was observed. Somesamples tested positive for fungi. In conclusion, all samples were contaminated by harmfulⒸ ISERD

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microorganisms that may cause food spoilage. The results of this study revealed thatmicrobial growth occurred in all tested pickles, resulting in a high risk of food spoilage andfood poisoning. We suggest that an additional step of pasteurization would help provide
microbiologically safe products. Moreover, use of food additives that do not alter the tasteof pickles may allow maintenance of low levels of microorganisms.
Keywords pickles, microorganisms, contamination, microbial quality

INTRODUCTION
Pickled products made using vegetables are popular in Cambodia. Many types of homemade
pickles are sold at wet markets. In our previous study, we showed that pickles are seriously
contaminated by microbes, including food poisoning bacteria (Muramatsu, 2020a, b). Despite theseriousness of this issue for local people in Cambodia, there are few reports on the food safety ofhomemade products. In particular, lack of surveys on food poisoning cases in the local population
makes it hard to grasp the current state of food safety in Cambodia.
Food safety is a pivotal issue for all countries. Indeed, food safety and/or food sanitation isindispensable for human quality of life and promotes not only good health but also economic
growth (Fung, 2018). Nevertheless, the World Health Organization (WHO) reported approximately
600 million cases of foodborne illness and more than 400,000 cases of death from foodborne illnessin 2010 (WHO, 2015). Foodborne illness is a serious threat in both developing and developedcountries. Most outbreaks of illnesses associated with food consumed at home have been underdiagnosed and/or under-reported (Redmond and Griffith, 2003; Keegan, 2009; Vrbova, 2012). In
particular, it is estimated that less than 1% of such cases are reported in developing countries(Satcher, 2000). Especially in developing country, food poisoning incidents are underestimated(Bhaskaran et al., 2020; Rusnan et al., 2020; Le et al., 2021). Thus, investigating only food poisoning
incidents that have been reported is not sufficient to ensure public health.
Contamination of homemade-style pickles may have several causes. For instance, microbial
behavior during the preparation of pickled products is plausibly associated with food contamination.
Therefore, in this study, we focused on microbial quality of homemade pickles and highlight
existing issues in cooking methods and types of ingredients and seasoning used for theirpreparation.
In this study, three kinds of pickles, namely cucumber pickles with and without fish sauce, andbok choy pickles were prepared following the methods used by local people. The samples preparedin this study showed a highly risk to food poisoning. We aimed to investigate the microbiologicalquality of homemade pickles and suggest an improved method to obtain safe homemade products.
OBJECTIVE
The objectives of this study were 1) to determine the microbial quality and nutritional properties ofhomemade-style pickles, and 2) to suggest an improved method to obtain safe homemade products.
METHODOLOGY
Materials and Pickle Production Process
Three kinds of pickles were prepared according to recipes used by local Cambodians. The
ingredients and seasonings of the pickles are shown in Table 1. Sugar, salt, soy sauce, and fishsauce were purchased in Cambodia and used for seasoning. The other materials used werepurchased in Japan. Bok choy was used instead of mustard because of unavailability of the latter.
Bok choy belongs to the Brassica genus similar to mustard. Cucumbers were washed with tap
water and pat dried with a paper towel. The cucumbers used for sample A were rubbed with salt,
compressed using a weight, and left to stand for 60 minutes. Then, the salt on the surface of theⒸ ISERD170

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cucumbers was wiped off with a paper towel. The cucumbers were cut to fit into a glass bottle andused until the stem end and the tip. For bok choy, the end of each bunch was first cut off, andleaves were separated from the stalks. The leaves were then washed with tap water and pat dried
with a paper towel. Garlic and a small onion were peeled and sliced. A piece of chili was sliced
into rings. After placing the mixed ingredients and liquid seasoning into glass bottles, these werekept at 30°C for 4 days until the products were ready (Fig. 1). Since the original homemade-styleprocess includes keeping the mixture under the sun for 4 days, in this study, the pickles were kept
at 30°C under conditions close to those of the traditional method. Five samples for each kind ofpickle were prepared in order to observe changes in their characteristics over time. A mixed sample
was kept at 30°C for 30 minutes and used as the sample at day 0. Another sample was kept at 30°C
for 20-24 hours and considered the sample at day 1, and samples at days 2-4 were prepared in asimilar manner. The samples were stored at 4°C for subsequent analysis.
Table 1 Ingredients and seasonings used in the three kinds of pickles
Sample A
Cucumber withfish sauce
500
80.0
70.0
48.0
48.0
300
1551

Ingredients andseasonings
Cucumber (g)Bok choy (g)Steamed rice (g)Sugar (g)Salt (g)Soy sauce (mL)Fish sauce (mL)
Hot water (mL)
Water (mL)
Garlic (g)Small onion (g)Chili (piece)

Sample B
Cucumber without
fish sauce
500
17.540018.75
1Sample C
Bok choy500
18.06.0
16.0
400-Fig. 1 Appearance of pickles at day 0 and day 4(a) sample; cucumber with fish sauce, (b) sample B; cucumber without fish sauce,and (c) sample C; bok choy

Measurement of Food Properties
Since the sugar and salt contents and pH of foods affect microbial growth, their values can be good
indicators of the preservability of food. Therefore, we measured the Brix sugar content (soluble
solids content), salt content, and pH of the pickled liquid of the samples using a Brix refractometer(Atago, Tokyo, Japan), a salt meter (Horiba, Kyoto, Japan), and a pH meter (Horiba), respectively,for all prepared samples.
Detection of Microbes in the SamplesⒸ ISERD

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After collecting all samples, several microbial detection assays were performed. Ten grams of eachsample containing pickled ingredients and seasoning liquid was mixed with 90 mL of Maximum
Recovery Diluent (Merck KGaA, Darmstadt, Germany) solution in a sterilized storage bag. To
obtain homogeneous suspensions, samples were homogenized using a Stomacher blender. Afterstomaching, 1 mL of sample suspension was mixed with 9 mL of Maximum Recovery Diluent
solution in a sterile tube. A series of diluted samples was used for the detection of total viablebacteria, coliforms, and fungi. Standard agar medium (AS ONE, Osaka, Japan) was used for thedetection of total viable bacteria. Desoxycholate agar medium (AS ONE) was used for thedetection of coliforms. Solid plates were incubated at 35 ℃ for 48 hours. For the detection of fungi,plates with Potato Dextrose Agar medium (AS ONE) were incubated at 30 ℃ for 5 days.
RESULTS AND DISCUSSION
Nutritional Properties of Pickles Prepared According Traditional Recipes
The nutritional properties of the final pickled products are shown in Table 2. Sample A showed thehighest Brix value and salt concentration. The measured pH values varied slightly among replicates.
The pH value could be affected by the microbes present in the samples; therefore, differences in theidentity of contaminating microorganisms would result in variations in pH.
Table 2 Nutritional properties of final pickled products
Measured
itemBrixpHSalt (%)

Sample A
Cucumber withfish sauce
11.2
4.55
5.1

Sample B
Cucumberwithout fish sauce
4.4
4.5
2.3

Sample C
Bok choy
4.7
4.88
2.4

Fig. 2 Changes in chemical properties of the three kinds of pickles(a) salt concentration and (b) pH
The Brix values of all pickles did not change much from day 0, just after pickling, to day 4,
and were approximately constant (data not shown). The lack of decreasing Brix values indicatesthat the microorganisms in samples A and C were unlikely to utilize sugar. During the 4-day
storage period, the salt concentrations in samples B (cucumber without fish sauce) and C (bokchoy) decreased slightly, whereas that in sample A (cucumber with fish sauce) remained almost
constant (Fig. 2(a)). All samples first displayed pH values of approximately 6, but these gradually
decreased over time (Fig. 2(b)). Decreasing pH values indicated that the microbes in the samples
produced acids. Clostridium botulinum, which can trigger deadly diseases, can grow at a minimum
pH of 4.6. Improper home canning of vegetables and improperly fermented and processed foods
have caused outbreaks in many countries (Matthews, 2017). In particular, pickles in sealedcontainers with pH higher than 4.6 seem to have considerable potential to cause severe foodpoisoning. Moreover, the Brix value and salt concentration of pickles are presumably not sufficientⒸ ISERD

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to inhibit the growth of most bacteria and yeasts; therefore, molds grow regularly in pickles(Matthews, 2017).
Microbial Behavior in Homemade Pickles
A total of five samples (from day 0 to day 4) for each of the three kinds of pickles were used toobserve changes in microbial composition over time. We tested the presence of total viable bacteria,
coliforms, and fungi (Fig. 3). More than 5.00 log10 CFU/g of total bacteria were detected in all
pickle samples at day 0; also, the number of bacteria increased until day 4 (Fig. 3(a)). Coliforms
were detected at a level similar to that of total viable bacteria. The initial number of coliforms insamples B and C was almost 5.00 log10, and sample A also tested positive for these bacteria. The
concentration of coliforms increased until day 4 (Fig. 3(b)). Coliforms are considered indicators offecal contamination; however, they have been recently acknowledged to be able to grow in nonfecal sites such as food, water, and waste (Matthews, 2017). Therefore, contamination by coliforms,
as shown in Fig. 3(b), does not directly imply insanitary conditions. Izumi (2010) reported thatvegetables dipped in tap water showed a total bacterial count of approximately 5 log10. Thissuggests that only washing vegetables with tap water does not eliminate harmful microorganisms.

Fig. 3 Changes in the number of microbes in the three kinds of pickles(a) total viable bacteria, (b) coliforms, and (c) fungiColony observation on agar plates revealed that many samples were contaminated bydangerous microorganisms that could cause food spoilage. Thus, none of the prepared pickles weresafe to eat. Fungi were not detected on day 0 in sample A, and some samples tested negative orpositive for fungi on days 1-4. Conversely, samples B and C showed fungal contamination on days
0, 1, and 4. We believe that continuous growth was not observed because each sample formicrobial counting was placed in an independent bottle in order to prevent contamination of thesample once opened for analysis. The microflora of vegetables is generally composed by 80%
bacteria and 20% fungi (Izumi, 2008). This observation suggests that bacteria remained in all
samples prepared by washing with tap water, but fungi were eliminated from some samples. The
results of this study suggest that microbial growth occurred in all three kinds of pickles, resulting ina high risk of food spoilage and food poisoning. Therefore, the current production process needs tobe modified to improve microbial quality. The addition of a pasteurization step and/or the use ofⒸ ISERD173

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food additives is an effective and practicable solution. Similar cucumber pickles containing asimilar amount of salt, after storage at 30°C for 2 days, were reported to carry 8 log10 CFU/g oftotal viable bacteria and maintain this initial concentration for 8 days at 4°C (Miyao, 2004).
Moreover, some trials of pickle production showed that the products were of uneven quality. Thisimplies that the traditional methods and ingredients used in this study do not allow spontaneousdesirable fermentation into safe products.
In this study, pickles were prepared following homemade-style recipes and analyzed for theirmicrobial quality. We suggest that pasteurization is required to obtain microbiologically safeproducts. Moreover, the use of food additives such as glycine, which does not alter the taste ofpickles, sorbic acid, or alcohol during preparation seems to be effective in controlling theconcentration of microorganisms.
CONCLUSION
In this study, three kinds of pickles were prepared following local traditional methods. To observechanges over time, five pickle samples were prepared for days 0 to 4. The Brix value, salt
concentration, and pH of the pickling liquids were measured, and microbial detection assays wereperformed. More than 5.00 log10 CFU/g of total bacteria was detected in all the samples at day 0;
also, the number of bacteria increased until day 4. Coliforms showed concentrations similar to
those of total viable bacteria. Some samples were positive for fungi. In conclusion, all samples
were contaminated by harmful microorganisms that could cause food spoilage.
The results of this study revealed that microbial growth occurs in all pickles, posing high risk
to food spoilage and food poisoning. We suggest that pasteurization would help obtainmicrobiologically safe products. Moreover, use of food additives that do not alter the taste ofpickles may allow maintenance of low levels of microorganisms.
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