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

Research article
erd

Determination of Histamine Level and Its Correlation with
Viable Bacterial Count in Cambodian Fermented FishLENGHEANG CHOENG
Faculty of Chemical and Food Engineering, Institute of Technology of Cambodia,
Phnom Penh, Cambodia

CHANTHOL PENG*
Research and Innovation Center / Faculty of Chemical and Food Engineering,
Institute of Technology of Cambodia, Phnom Penh, Cambodia
Email: peng@itc.edu.kh

LEANGEY SET
Research and Innovation Center / Faculty of Chemical and Food Engineering,
Institute of Technology of Cambodia, Phnom Penh, Cambodia

SEYHA DOEURN
Research and Innovation Center / Faculty of Chemical and Food Engineering,
Institute of Technology of Cambodia, Phnom Penh, Cambodia
Received 15 February 2023

Accepted 12 May 2023(*Corresponding Author)

Abstract Nem trey (NT), Sangvak (SV) (minced fish), and Nem sbek chrouk (NC) (mincedfish mixed with pork skin) are fermented fish products popular in Cambodia; NC and NT are
ready for direct consumption after two or three days of fermentation, while SV must be
additionally grilled before consuming. However, food safety standards for these products
including control of biogenic amines such as histamine are neglected, which could pose ahealth problem. Biogenic amines are nitrogenous organic chemical compounds mainly
produced from the decarboxylation of amino acid, histidine, by the activity of certain bacteria
associated with fish products processed under poor hygiene and unstable temperature control.
This study aimed to determine the histamine level in NT, SV, and NC products and studied its
correlation with the viable bacterial count (BVC). Twenty-six samples, NT (n=12), SV (n=5),
and NC (n=9), were collected from different producers from Battambang (BB) and Kratie (KT)provinces. A histamine test kit utilizing a colorimetric enzymatic assay, was used to determine
histamine level. The BVC was done using the spread plate technique on Luria Bertani agar
media incubated at 37oC for 24h. For KT province, NT and NC contained BVC (log10 CFU/g)in the range of 5.74±0.02 to 6.57±0.01 and 5.52±0.01 to 5.98±0.01, while histamine (mg/kg)
ranged from 0 to 156.43±0.13 and 28.76±0.71 to 73.87±0.13, respectively. SV, NT, and NC
from BB province contained the BVC (log10 CFU/g) ranging from 6.02±0.16 to 6.56±0.15,
5.97±0.15 to 6.04±0.15, and 5.69±1.18 to 6.12±0.11, while histamine (mg/kg) ranging from
6.10±0.44 to 54.52±0.00, 0 to 10.90±0.14, and 0 to 38.54±0.00, respectively. The BVC andhistamine showed a significant correlation, with a value of 0.448 (p-value < 0.05). Therefore,
raw materials, processing, and storage conditions along the production chain should be
evaluated in further studies to ensure safety of the fermented fish products.
Keywords biogenic amine, viable bacterial count, fermented fish, food safety

INTRODUCTION
Fish and fishery products have long been associated with people's socioeconomic lives. To preservefish, fermentation is one of the oldest and most cost-effective methods (Uochoi, 2019). Fishfermentation is practiced in many parts of the world, but the most popular practice is found inSoutheast Asia, including Cambodia. Generally, the common fermented fish product in CambodiaⒸ ISERD

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includes prahok (fish paste), teuk trey (fish sauce), trey praherm; trey proma (salted fish), and mamtrey, paork, and nem (other fermented fish) that have played an essential part of the typical diet inCambodia involved to fermented fish (Ly et al., 2018, 2020). Among these types of Cambodian
fermented fish products, Nem such as Nem trey (NT), Sangvak (SV) (minced fish), and Nem sbek
chrouk (NC) (minced fish mixed with pork skin) are the fermented fish products that usually take
two to three days for fermentation. These ready-to-eat appetizers, except for Sangvak, which needs
to be grilled before consumption, are popular among Cambodians.
Potentially pathogenic bacteria such as Bacillus, Clostridium, and Staphylococcus were foundin traditional Cambodian fermented fish products (Chuon et al., 2014; Ly et al., 2018). So, fermented
fish products can contain microorganisms that affect human health. Chemical compounds andmicrobiological contamination can cause acute poisoning or long-term disorders such as cancer(Ruiz-Capillas and Herrero, 2019). Another study reported that many bacterial isolates from
Cambodian fermented fish products were from the non-lactic acid bacteria (LAB) genera, including
Staphylococcus, Bacillus, Clostridium, Virgibacillus, Kocuria, Clostridium, Lisinibacillus,
Psychrobacter, Lantibacillus, and Micrococcus (Chuon et al., 2014).
Next to microorganisms, a toxic chemical substance such as biogenic amines (BAs) is the mostcommon compound associated with fermented fish (Visciano et al., 2012). Biogenic amines are basic
nitrogenous chemicals with various structures that may be found in various foods, including fish andfishery products, meat and animal products, cheeses, wine, beer, and other fermented meals andbeverages (Durak-Dados et al., 2020). Histamine, tyramine, putrescine, cadaverine, and bphenylethylamine are the most significant BAs in foods and drinks, both qualitatively andquantitatively, as a result of the decarboxylation of histidine, tyrosine, ornithine, lysine, andphenylalanine, respectively (Spano et al., 2010). Fish and fish products show the highest levels of
BAs in previous studies (Linares et al., 2011; Durak-Dados et al., 2020). Among all types of biogenic
amines, histamine (HIS) is the most concern BAs related to food safety (EFSA, 2011) as histamine
has been shown to cause severe symptomatology such as skin rashes, headache, nausea, diarrhea,
and variations in blood pressure (Botello-Morte et al., 2022). The synthesis of histamine in mackerel
and other marine fish carrying high levels of endogenous histidine is linked to the action of microflora
rather than the activity of a decarboxylase found natively in fish (Durak-Dados et al., 2020).
Histamine consumption of 8-40 mg, 40-100 mg, and more than 100 mg may generate mild, moderate,and severe poisoning, respectively (Sahu et al., 2016). European Union limited the histamine levelin food products by 100 mg/kg on average, no one sample exceeds 200 mg/kg, and no more than two
samples may each have a value of more than 100 ppm, but less than 200 ppm for nine samples tested(EFSA, 2007).
Microorganisms, known as factors, that affect the formation of biogenic amines and histamine
formation, have been reported. The genus Photobacterium, Aeromonas hydrophila, andEnterobacteria such as Morganella morganii, Enterobacter aerogenes, Raoultella planticola, andKlebsiella oxytoca are involved in the accumulation of histamine in fish and seafood products(Fernández-No et al., 2010; Küley et al., 2013). Some fungi (yeast and molds) are implicated in BAs
formation, although their significance is debatable in several ways (Tristezza et al., 2013). Poor raw
material quality and improper handling are two reasons for including BAs in fermented fish (RuizCapillas and Herrero, 2019).
OBJECTIVE
According to the relation between histamine level and microorganisms, this study aimed to determine
the histamine level and identify the correlation between histamine level and viable bacterial count in
Cambodian fermented fish, Nem, collected from two provinces, including Battambang and Kratie.
This study provides insight into predicting the safety of the fermented fish product and can be a
supporting document for standard quality development for the fermented fish product in Cambodia.
METHODOLOGYⒸ ISERD

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Samples Collection
Twenty-six samples were collected from local producers in two provinces in Cambodia, Kratie (KT)and Battambang (BB) provinces. From KT, there were 11 samples collected, and separated into three
types of samples such as fermented fish packaged by banana leaves, Nem Trey (KTNTB) threesamples; fermented fish packaged by plastic, Nem Trey (KTNTP) four samples; and fermented fishmixed with pork skin, Nem Sbek Chrouk (KTNCB) four samples were collected. Whereas for BB
province, 15 Nem samples were collected including Songvak (BTBSV) five samples, Nem Trey
(BTBNT) five samples and fermented fish with pork skin, Nem Sbek Chrouk (BTBNC) five samples.
Table 1 Description of collected samples

BTBSV

Main
ingredient
Fish
Fish and pork
skin
Fish

Nem trey (packaged by banana leave) (n=3)

KTNTB

Fish

Nem trey (packaged in plastic) (n=4)

KTNTP

Nem sbek chrouk (packaged by banana leave) (n=4)

KTNCB

Fish
Fish and pork
skin

Province

Type and number of samples

Battambang

Nem trey (fermented fish) (n=5)
Nem sbek chrouk (fermented fish mixed with porkskin) (n=5)
Songvak (fermented fish) (n=5)

KratieSample
code
BTBNT
BTBNC

Viable Bacterial Count Analysis
The enumeration of viable bacterial count (BVC) was done using the spread plate technique. For
each Nem product, a homogenized sample of 10g was diluted with 90 mL of phosphate-bufferedsaline (PBS). Appropriate dilutions of the samples were prepared using the same diluent. Then 0.1mL
aliquots of each dilution were applied to Luria Bertani agar media and spread on its surface until
completely absorbed. Plates were then incubated at 37℃ for 24 hours. The colonies counting of BVC
were presented as logarithms colony-forming units per gram (log10 CFU/g).
Histamine Concentration Analysis
The sample preparation and extraction followed the manufacturer's instructions for the extraction kit(Kikkoman Biochemifa, Tokyo, Japan). 10 g of each Nem sample was first homogenized, and 1 g of
the homogenized sample was diluted with 24 ml of sample treatment buffer (EDTA) in a centrifuged
tube. The tube was vortexed for 30 mins and then boiled for 20 mins. The tube was cold by placing
it on an ice box (until < 20℃). The tube was vortexed again and put in an ice box to separate thephase. The tube was centrifuged 10 000 x g for 5 min; then the supernatant was collected. 11 ml ofdeionized water was added to the colorimetric reagent vial. 6 ml of buffer vial was added to the
enzyme reagent vial. The concentration of histamine (mg/kg or ppm) (HIS) was calculated by Eq.
(1).
𝐻𝐼𝑆 = (𝐸𝑠 − 𝐸𝑏) ÷ (𝐸𝑠𝑡𝑑 − 𝐸𝑐) × 100(1)

The absorbance of the sample, Es, was measured by mixing extracted sample solution,colorimetric reagent, and enzyme solution. Eb stands for absorbance of sample blank obtained bymixing extracted sample solution, colorimetric reagent, and buffer. The absorbance of standard
solution, Estd, by mixing standard histamine solution, colorimetric reagent, and enzyme solution.
The last absorbance of reagent blank, Ec, got by mixing Distilled water, colorimetric reagent, andbuffer. Absorbance was measured using a spectrophotometer (UV-1280, Shimadzu, Japan) with a 1
cm optical wavelength of 470 nm.

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Statistical Analysis
Results units of the quantitative bacterial viable count were log10 CFU/g. The histamine content andviable bacterial count were analyzed with statistical analyses using the Statistical Package for theSocial Sciences (SPSS, Version 20.0.0 for Windows, 2018; IBM Co., Somers, NY, USA). Data wereanalyzed for the degree of variation by calculating the result’s mean and standard deviations (SDs).
The significance of differences was evaluated using analysis of variance (ANOVA). A p-value ofless than 0.05 was considered statistically significant. The correlation value was determined using
the Pearson Correlation coefficient.
RESULT AND DISCUSSION
Evaluation of Viable Bacterial Count and Histamine Level
Figures 1 and 2 described the result of HIS and BVC, respectively. For KT province, Nem trey
packaged by banana leaves and plastic (KTNTB and KTNTP) contained HIS (mg/kg) in the range
from 6.35±0.48 to 156.43±0.13, while BVC (log10 CFU/g) showed a range from 5.74±0.02 to
6.57±0.01. However, Nem sbak chrouk described the HIS (mg/kg) in the range 228.76±0.71 to
73.87±0.13. At the same time, BVC (log10 CFU/g) presented from 5.52±0.01 to 5.98±0.10. On theother hand, from BB province, NT showed the range of HIS (mg/kg) from 0 to 10.90±0.14, while
BVC (log10 CFU/g) represented from 5.97±0.15 to 6.04±0.15. NC showed a range of HIS (mg/kg)
and BVC (log10 CFU/g) from 0 to 38.54±0.00 and 5.69±1.18 to 6.12±0.11, respectively. For
Songvak, the data represented 6.10±0.44 to 54.52±0.00 and 6.02±0.16 to 6.56±0.15 for HIS(mg/kg) and BVC (log10 CFU/g), separately.

Fig. 1 Viable bacterial count in all samples
The result of all samples was compared with maximum limits. The US Food and Drug
Administration (FDA) set the maximum of histamine in fish and fish products at 50 mg/kg (Ly et al.,2020), while European Food Safety Authority set the limit of histamine in fishery products at 200
mg/kg (EFSA, 2007). Seven in twenty-six samples exceed 50 ppm, and no one above 200 mg/kg.
The safety of these fermented products should be slightly concerning. Previous studies on
Cambodian fermented fish showed that the histamine concentration loaded in products ranged from32 to 840 mg/kg (Ly et al., 2020). This result showed higher histamine levels, but it might becomparable with a concentration of histamine studied in this paper as the Nem products requiredⒸ ISERD

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HIS (mg/kg)

only a few days to consume, while other fermented fish mentioned in that previous study were othertypes of fermented products that require a longer time to consume (Paork Chav, Paork Chau, and
Mam Trey). The Malaysian fermented fish, Budu, detected 187 mg/kg of histamine concentration(Saaid et al., 2009). Histamine was found at a high concentration in Feseekh, Egyptian fermented
fish (521 mg/kg) (Rabie et al., 2011).
The microbial ranged from 5.52 to 6.32 log10 CFU/g. The total cell counts identified on otherCambodian fish products were 5 to 7 log10 CFU/g for Prahok (fermented fish paste), 5 to 8 log10
CFU/g for Kapi (fermented fish), and 2 to 6 for Toeuk Trey (fermented fish sauce) (Chuon et al.,2014). The viable plate count found 6 log10 CFU/g in the Indonesian salt-fermented fish product(Kusmarwati et al., 2020). This information may be compared with each other for the same type of
products (fermented fish products).
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0

a
b

c
d
e
f

g
h

n

q

i

p

j

k

l

m

o
s

r

r

p

r

C1 C2 C3 C4 C5 NT1 NT2 NT3 NT4 NT5 SV1 SV2 SV3 SV4 SV5 CB1 CB2 CB3 CB4 TB1 TB2 TB4 TP1 TP2 TP3 TP4
BN TBN TBN TBN TBN TB TB TB TB TB TB TB TB TB TB TN TN TN TN TN TN TN TN TN TN TNT
B B B B B B B B B B B B B B B K K K K K K K K K K K
Samples CodeFig. 2 Histamine content in all samples
As determined by One-way ANOVA and LSD tests in SPSS 2018. The significant differencesof all samples are shown in script on the bar chart in Fig 1 and 2 for BVC and HIS, respectively.
Statistically significant differences (p<0.05) were found among the defined concentration ofhistamine, except BTBSV2, KTNTP1, and BTBNT5 showed non-significant differences (p>0.05)
and pair of BTBNT2 and BTBSV5 found that non-significant differences also (p>0.05). However,for the viable bacterial count, statistically significant differences (p<0.05) were found, but some of
the group samples were described that they are non-significant differences (p>0.05). Moreover,comparing histamine levels with 50 ppm showed non-significant differences (p>0.05). In contrast,
the significance differences (p<0.05) were found in analyzing 200 mg/kg histamine levels using the
One Sample T-test. Table 2 showed the significant moderate correlation between histamine
concentration and bacterial viable count value r=0.448, p<0.05, n=26 was found using Pearson
Correlation. Sample form Kratie showed a non-significant different (p>0.05) between banana leave
packaging and plastic packaging through Paired Samples T-test.
Table 2 Correlation between BVC and HIS by Pearson Correlation

Histamine content

Viable bacterial count
0.448
0.022
26

Pearson Correlation
Significant
Number of samples

The histamine concentration and microbial loaded in these Nem samples were correlated.
Therefore, to prevent the increase of histamine levels in Nem products, the elimination of microbial
through applied raw material quality, good hygiene production, and cold processing line play animportant factor in reducing histamine in food.
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CONCLUSION
The histamine level, a formation chemical concerning food safety, in Cambodian fermented fish
products, was studied on 26 samples collected from 2 different provinces in Cambodia. The range ofhistamine concentration was detected from 0 to 156.43±0.13 mg/kg, with seven samples exceeding
the maximum limit of 50 mg/kg set by the FDA. Microbial loads of each product were also
determined, and the results ranged from 5.52±0.01 to 6.32±0.07 log10 CFU/g. The correlation
between histamine and bacterial concentration was significantly correlated with a moderatecorrelation on the positive side (r=0.48). This result is important for setting limits of food safety
criteria for local fermented products. It is important to note that to reduce the risk of the formationof histamine concentration of the Nem product, reduction or elimination of certain bacteria species
through the hygienic practice through food supply chains such as raw material, operating, andtemperature controlling shall be applied.
ACKNOWLEDGEMENTS
The work was funded by Cambodia Higher Education Improvement Project (Credit No. 6221-KH)
through sub-project HEIP-ITC-SGA#17.
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