IJERD – International Journal of Environmental and Rural Development (2023) 14-1

Research article
erd

articleAssessment of Antibacterial Activity of Lactic Acid Research
Bacteria
Isolated from Fermented Foods against Escherichia coli
O157:H7 and Proteus penneri and Their Potential as Starter
Cultures

SINETH CHEV*
Faculty of Agro-Industry, Royal University of Agriculture, Phnom Penh, Cambodia
Email: chevsineth9999@gmail.com

SIVENG MENG
Faculty of Agro-Industry, Royal University of Agriculture, Phnom Penh, Cambodia

BORARIN BUNTONGDivision of Research and Extension, Royal University of Agriculture, Phnom Penh, Cambodia

CHINDA CHHE
National Institute of Science, Technology, and Innovation, Phnom Penh City, Cambodia

RITHY CHRUN
Faculty of Agro-Industry, Royal University of Agriculture, Phnom Penh, Cambodia
Received 15 January 2023

Accepted 15 May 2023(*Corresponding Author)

Abstract Fermented fish and vegetables produced by spontaneous fermentation are the mostwell-known traditional foods in Cambodia. Even though fermented foods provide healthbenefits related to nutrition, probiotics, and postbiotics, some of them are not safe due to thesurvival of bacteria harmful to humans. The purpose of this research is to examine in vitro theantibacterial activity of lactic acid bacteria (LAB) isolated from fermented fish and vegetables
against E. coli O157:H7 and Proteus penneri, histamine forming LAB, and the survival of
LAB in low pH levels and high salt concentration using pH and salt tolerance tests. LAB was
isolated from 134 naturally fermented fish and vegetables from four different provinces andidentified using the Biolog GEN III MicroStation semi-automated system. The antagonistic
properties of these isolates against E. coli O157:H7, and Proteus penneri were examined using
a co-culture method at 24, 48, and 72 hours of incubation times. As a result, 36 strains of LAB
were identified from a total of 134 samples. Seven LAB species out of 36 strains can survive
at pH 3 for 6 hours, while four of these seven LAB can tolerate pH 2. Lactobacillus plantarum,
Lactococcus lactis, Lactobacillus gasseri, Leuconostoc lactis, and Leuconostoc gilidum havea significant capacity to inhibit E. coli O157:H7 and Proteus penneri growth when compared
to control. These five LAB do not produce histamine and can tolerate up to a 10% salt
concentration. All the results demonstrate that Lactobacillus plantarum, Lactobacillus lactis,
Lactobacillus gasseri, Leuconostoc lactis, and Leuconostoc gilidum have great potential for
use as starter cultures for suppressing pathogenic bacteria growth in fermented fish andvegetables.
Keywords fermented fish and vegetables, E. coli 0157:H7, lactic acid bacteria,
antimicrobial activity, pH and NaCl tolerance

INTRODUCTION
The nutritional balance and food security of traditional fermented foods have made them popularthroughout Asia. Across several Asian nations, methods for preserving crops, vegetables, meat, andfishery products seem to be well-developed. Moreover, Food preservation was the prime purpose ofⒸ ISERD

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fermentation, which was achieved by the synthesis of inhibitory metabolites including organic acid,ethanol, and antimicrobials combined with decreased water activity (Anal, 2019).
Cambodia is a kingdom in Southeast Asia that borders Vietnam, Laos, and Thailand. There aremany varieties of fresh fish and vegetables caused by the Tonle Sap Lake in the north of Cambodia
is recognized for its extensive river system and favorable climate for agriculture(LeGrand et al.,2020). Mostly in the traditional Cambodian diet, fermented foods play a significant role. There aredifferent fermented foods ready to eat (Chrun et al., 2017). It is well known that five groups offermented fish and vegetables including Pra hok (Fish paste), Pha’ork (Fermented fish), Trey
proheum (Salt Fish), Mam (Fermented Fish), and Chruk (Fermented vegetable) (LeGrand et al.,2020) and have their benefit in term nutritional function such as vitamins, proteins, essential fatty
acids, and amino acids (Nuraida, 2015). In addition, at the same time add value to agricultural
products (Chrun et al., 2017).
Microbiological research on Asian fermented foods reveals that the identification of 68 samplesof fermented vegetables from a local market in Cambodia contained Enterobacter spp. in 24% of thesamples (Chrun et al., 2017). Another study that examined 13 samples of fermented fish in Cambodia
discovered that the majority of the microbial communities were gram-positive cocci and rods suchas Bacillus, Clostridium, Staphylococcus, and Tetragenococcus (Chuon et al., 2014). In the case ofVietnam was found that unprocessed fish and poultry are likely to be contaminated with Salmonella
and in the absence of proper kitchen hygiene and may contaminate processed foods. Raw poultry
samples were found high levels of contamination with E. coli (45%), Campylobacter jejuni (28.3%),
and Salmonella (8.3%) and high-risk food classification. Additionally, raw fish, meat, and vegetables
all contained E. coli at rates of 21.3%, 6.6%, and 18.5%, respectively. This article confirmed theimportance of hygienic working practices when preparing food. (Thi et al., 2006). The purpose ofthis research is to examine in vitro the antibacterial activity of lactic acid bacteria (LAB) isolated
from fermented fish and vegetables against E. coli O157:H7 and Proteus penneri, histamine forming
LAB and and the survival of LAB in low pH level and high salt concentration using pH and salt
tolerance tests.
OBJECTIVE
The objective of this research is to examine in vitro the antibacterial activity of lactic acid bacteria(LAB) isolated from fermented fish and vegetables against E. coli O157:H7 and Proteus penneri,histamine forming LAB and the survival of LAB in low pH levels and high salt concentration using
pH and salt tolerance tests.
METHODOLOGY
Sample Collection Methods
Fermented fish and vegetable samples are divided into five groups such as Pra hok (Fish paste),
Pha’ork (Fermented fish), Mam (Fermented fish), Salted fish (trey proheum) and Chruk (Fermented
vegetable). All samples were collected from 4 provinces such as Kompong Thom, Siem Reap,
Kandal, and Kompong Cham province. All samples are purchased from small-scale producers andlocal markets. There are 134 samples in total. For fish paste (Pra hok, n=17), Fermented fish (Pha’ork,n=30), Fermented fish (Mam, n=11), and Fermented vegetables (Chruk, n=76). All samples weresealed in plastic sample bags and stored in cool boxes during transportation to the laboratory which
was less than 24 hours. After the arrival, all samples were immediately studied for their identification.
Sample Identification
Pure colonies were conducted with several tests before using Biolog System (Semi-Auto
Microstation) for identification. These tests helped to avoid misidentification and save a lot of money
since the Biolog System is costly. Most important tests such as Gram staining, Catalase, and OxidaseⒸ ISERD

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are alternatives yet also help to give information on bacterial characteristics. The suspected pure
LAB were sub-cultured two times to ensure the purification of the colony on MRS agar before culture
on BUG Agar before identification with the Biolog system. By using GENIII-Microplate (BIOLOG)
within protocol-C, inoculate pure colony from BUG Agar into Inoculating Fluid-C (IF-C) (provided
by BIOLOG) within 92 to 95% Turbidity by using a Turbidity Meter (BIOLOG). Using an electric
multi-dispense micropipette (SARTORIOUS), dispense 100 microliters in each microplate well thenincubate the plate at 30oc for 20 to 24 hours and using Microstation (BIOLOG) accompany with
Microlog Software (BIOLOG) for interpreting the ID result of identification(Al-Dhabaan & Bakhali,2017).
Antimicrobial Activities by Co-culture Method
10% of LAB suspension (9 Log CFU/ml) was inoculated with 6 Log CFU/ml of indicator E. coli
0157:H7 and Proteus penneri in each tube of MRS broth. This experiment was performed underaerobic conditions at 30 0C in the water bath for 72 hours. Every 24 hours, the presence of indicators
was determined by streaking on selective Agar and incubating at 350C (Balouiri et al., 2016).
pH and Sodium Chloride Tolerance
LAB against E. coli 0157:H7 and Proteus penneri by co-culture method have been conducted with
pH and sodium chloride tolerance. Selected LAB isolates were grown in MRS broth at 30°C
overnight before being sub-cultured in new MRS broth by using HCl (0.1M) adjusted pH to 2 and 3
and used to adjust the salt content in MRS broth (5%, 6%, 7%, 8%, 9%, and 10%). Following an
incubation period of 24 hours at 30°C (Dimic et al., 2015). After this, Streak Method on MRS Agar
was used to confirm the tolerance of each LAB (Balouiri et al., 2016).
Determine the Concentration of Histamine Produced by Lactic Acid Bacteria
LAB was refreshed on MRS Agar at 30 0C for 24 hours. Inoculate one loop full colony of both Lactic
acid bacteria in TSB broth with histidine and incubate at 30 ℃ for 24 hours centrifuge 6000 rpm/min
for 15 min. Suspend 1 ml of supernatant in 9 ml of sterile saline for dilution 10-fold. After dilutionthat supernatant was used in the enzymatic histamine assay (Leszczyocha and Pytasz, 2018).
Histamine concentration was calculated as follows.
Histamine concentration (mg/L = ppm) = (Es - Eb) ÷ (Estd - Ec) × 4 × 25 × dilution factor
RESULTS AND DISCUSSION
The LAB species isolates were examined based on selective agar media called MRS and sub-cultured
for few times until obtained a pure culture. The later suspected colony was Gram-positive, clusteredcocci or bacilli, catalase-positive, and oxidase positive. LAB was later identified by using Biolog
semi-auto system as shown in Fig. 1. Of 134 samples, 98 samples were rejected due to the absenceof a suspected colony. group of Mam (n=11) isolated as L. lactis and P. parvulus (27%). There are
12% of group Prahok (n=17) isolated as P. parvulus and P. pentosacues. Other groups of Pha’ork(n=30) detected 10% were L. garvieae, P. parvulus, and P. pentosaceus. And 37% of group Chruk(n=76) isolated as Lactobacillus, P. parvulus, P. pentosaceus, L. alimentarius, L. mali, L. gasseri, P.
acidilactici, L. acidophilusBGA, L. lactis, P. sanguinis, Leu. gelidum, Tetra. solitarius, L. garvieae,
L. reuteris, L. plantarum, Leu. citrem, L. bifermentans, and L. salivarius. We found the relevance of
the identification LAB by the API 50 CHL system from fermented food in Cambodia, thus three
species of lactic acid bacteria were found, including Lactobacillus acidophilus and Lactobacillus
plantarum. strains Y'11b,2, Y'11e,2, and Y'85,1. (Sophakphokea1 et al., 2021). According to (Ly etal., 2022) to study dentiﬁcation, Classiﬁcation and Screening for γ-Amino-butyric Acid Productionin Lactic Acid Bacteria from Cambodian Fermented Foods by using Matrix-assisted laserⒸ ISERD

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desorption/ionizing time-of-flight mass spectrometry (MALDI-TOF MS) and partial 16S rDNA
sequencing were used to identify 68 LAB. And the result shows as one Lactobacillus futsaii, two
Lactobacillus namurensis, and three Lactobacillus plantarum strains were found in fermented foodin Cambodia.

Fig. 1 Isolated LAB from fermented fish and vegetables in Cambodia
Table 1 Result of antimicrobial activities by co-culture method
SpeciesnTime
E. coli 0157:H7
24 h+++
Leuconostoc lactis
3
48 h
++
72 h
+
24 h
++
Lactobacillus gasseri
3
48 h
72 h
24 h
+++
Lactobacillus plantarum
3
48 h
++
72 h
+
24 h
++
Lactococcus lactis
3
48 h
72 h
24 h
+++
Leuconostoc gelidum
3
48 h
+
72 h
+
24 h
++
Lactococcus garvieae
3
48 h
+
72 h
+
24 h
+++
Lactobacillus mali
3
48 h
++
72 h
++
“+” represent to positive or resistance and “-” represent to negative or susceptible

Proteus penneri
+++
+
+++
+
++
+
+
+++
++
++
+
+++
+
-

According to the results shown in Table 1. Lactic acid bacteria are able to against E. coli
0157:H7 such as Lactobacillus gasseri and Lactococcus lactis. Moreover, Lactic acid bacteria able
to against Proteus penneri were Lactobacillus mali, Lactococcus garvieae, Leuconostoc gelidum,
Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc lactis. Hassanzadazar et al. (2012)
studied about antibacterial activity of Lactic Acid Bacteria against Escherichia coli, Listeria
monocytogenesis, Bacillus cereus, and Salmonella enteritidis, and reported that lactic acid showed
the strongest inhibitory activity against gram-positive bacteria than gram-negative bacteria. Similarly,
Muzikowski (2009) also reported some species of Lactobacilli against different gram-positive andgram-negative bacteria were determined by agar-well diffusion assay. It also shows the antagonistic
effect of the antibacterial agent on the growth of other gram-positive and gram-negative pathogenicⒸ ISERD

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microorganisms which E. coli, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus
and Bacillus cereus.
The pH tolerance is an important characteristic of species that hoped to affect the gastrointestinal
tract. Moreover, tolerance to salt was an important cause of cell membrane structure can become
damaged by salt. A study by Azam et al. (2017) focused on the isolation and characterization of
Lactobacillus spp. from kefir samples in Malaysia mentions that Lactobacillus spp. isolated fromkefir E was unable to survive in all pH conditions (pH 2, 3, and 4). All isolates tested did not survive
pH 2.0, most of the isolated Lactobacillus spp. from kefir samples were able to tolerate the moderate
pH levels of pH 3.0, pH 4.0, and all the isolated Lactobacillus spp. from kefir samples survived at
0.3-0.5% bile concentration after incubation. The Lactobacillus spp. from kefir H conferred thehighest survival rate at 0.3% and 0.5% bile concentration, with a survival rate of 96.89 ± 0.02% and96.84 ± 0.02%, respectively. Subsequently, the survival rate in bile salt condition was followed byisolated Lactobacillus spp. from kefir G and kefir C. (Hassanzadazar et al., 2012) research was done
into the Investigation of antibacterial, acid, and bile tolerance properties of lactobacilli isolated from
Koozeh cheese. The screening process involved 28 different Lactobacillus species that were obtained
from Koozeh cheese, a typical cheese. A control pH of 7.5 was used while the acid tolerance test wasinvestigated at pH 2.0 and 3.0 with 5 N HCl. Results showed that only one out of twenty-eight isolates
have the ability to tolerate acid and bile salts. The ability to produce histamine of LAB was acceptable
due to the concentration of histamine higher than 200 ppm will cause the disease in a human(Leszczyocha and Pytasz, 2018, Food Safety Authority of Ireland, 2005).
Table 2 Result of salt, pH tolerance test, and Histamine of identified bacteria fromfermented samples
pH and salt tolerance
Bacteria species

Salt (%)
pH
5
6
7
8
9
10
2
3
Lactococcus garvieae
+
+
+
+
+
+
0%
14%
Lactobacillus mali
+
+
+
+
+
+
21% 50%
Leuconostoc lactis
+
+
+
+
+
+
10% 13%
Lactobacillus gasseri
+
+
+
+
+
+
12% 18%Lactobacillus plantarum
+
+
+
+
+
+
32% 52%
Lactococcus lactis
+
+
+
+
+
+
26% 44%
Leuconostoc gelidum
+
+
+
+
+
+
0%
20%
“+” represent to positive or resistance and “-” represent to negative or susceptible

Histamine
concentration
ppm
2.35
4.70
6.79
7.05
7.8
10.1
3.9

CONCLUSION
Seven Lactic acid bacteria out of 36 species, such as Lactobacillus mali, Lactococcus garvieae,
Leuconostoc gelidum, Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc lactis wereisolated and identification by using Biolog System (Semi-Auto Microstation) were found fromfermented fish and vegetables. according to this study two species of LAB inhibition against E. coli
0157:H7 and all these seven species inhibitions against Proteus penneri. The identified bacteria
isolated were subjected to different salt concentrations (5%, 6%, 7%, 8%, 9%, and 10%). Bacteria
species Lactococcus garvieae, Lactobacillus mali, Leuconostoc lactis, Lactobacillus plantarum,
Lactococcus lactis, Leuconostoc gelidum can tolerate with salt up to 10% and tolerate either pH3 inrate (14%, 50%, 13%, 18%, 52%, 44%, and 20%) nor pH2 in rate (21%, 10%, 12%, 32%, and 26%).
In contrast, Lactococcus garvieae and Leuconostoc gelidum weren’t tolerated with pH2. On the other
hand, these seven species LAB were not histamine-producing bacteria due to the level of histamine
concentration lower than 10 ppm. All the results demonstrate that Lactobacillus plantarum,
Lactococus lactis, Lactobacillus gasseri, Leuconostoc lactis, and Leuconostoc gelidum have a great
potential for use as starter cultures for suppressing pathogenic bacteria growth in fermented food.

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ACKNOWLEDGEMENTS
This work was supported by World Bank under Higher Education Improvement Project (HEIP), IDA
Credit No. 6221-KH, Sub-project Partnership Program to Enhance Agro-Industry Education andResearch Project SGA#02, Faculty of Agro-Industry, Royal University of Agriculture.
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