Food and Dairy Microbiology

BIOGENIC AMINE
CONTENTS AND MICROBIAL
CHARACTERISTICS OF
CAMBODIAN
FERMENTED FOODS

Table of Contents
 Table Of Content
FOOD AND DAIRY MICROBIOLOGY

01. 02.
INTRODUCTION MATERIALS/METHODS

05.
POTENTIAL

03. 04.
RESEARCH TOPIC

RESULTS & DISCUSSION CONCLUSION Contact Us

INTRODUCTION
Cambodia is a tropical country with two monsoon seasons (dry
and rainy).
Spoilage is mainly caused by microorganisms, thus
fermentation is one of the methods for food preservation.
Intrinsic factors: pH, aw, composition
Extrinsic factors: temperature, relative humidity,
atmospheric gases
Smallholder farmers, often women, produce fermented
products that are sold in wet markets by mostly female
retailers.
Fermented foods are generally not labelled with an
appropriate shelf-life and usually stored at room temperature.
Fermented fishery products - cooked
Fish paste and sauce - raw
Fermented vegetables - Ready-to-eat (RTE)
Microorganisms detected in fermented vegetables:
Escherichia coli, Cronobacter sakazakii, Enterobacter
spp., opportunistic non-Enterobacteriaceae,
Staphylococcus spp., and Listeria spp.

Microorganisms detected in fermented fishery: Bacillus,

Clostridium, and Staphylococcus

Chemical substances: Biogenic Amines (BAs) and

Biotoxins

BAs are low molecular weight organic molecules,

formed by microbial decarboxylation of their precursor
amino acids or by transamination of aldehydes and
ketones by amino acid transaminases.
Most common BAs: Histamine (HIS), Tyramine (TYR),
Putrescine (PUT), and Cadaverine (CAD)
Low level of BAs in foods are not considered as a
serious risk.
High level of BAs in foods:
Hypotension (HIS, PUT, and CAD)

Hypertension (TYR)
Nausea, Headache, rash, dizziness, cardiac
palpitation, and intracerebral hemorrhage
and death
BAs - used to evaluate the hygienic quality of
foods
Baseline surveillance data
Physicochemical Properties (pH, aw, and salt content)
Presence of microorganisms (spoilage and pathogenic bacteria)
Concentrations of BAs (HIS, TYR, PUT, and CAD) - 57 Cambodian
fermented food sample

Purpose - to give an overview of the

quality of Cambodian fermented
foods, to correlate physicochemical
parameters with BA contents, and to
describe the prevailing habits and
practices of Cambodians in dealing
with this type of food.
 ENTEROBACTERIACEAE PSEUDOMONADACEAE PHOTOBACTERIUM

ENTEROPATHOGENIC VIBRIO STAPHYLOCOCCUS AUREUS ENTEROCOCCUS

LACTOCOCCUS LACTIS BACILLUS SPP. CLOSTRIDIUM SPP.

METHODS 01 Sample Collection

& MATERIALS 02 Physicochemical

Properties Analysis

03 Microbial Analysis

04 Determination of
Biogenic Amines

05 Statistical Analysis
Table 1.
 Sample A total of 57 samples of fermented products
were randomly purchased from different wet
Collection markets in Phnom, Pehn.

FERMENTED FISHERY: FERMENTED VEGETABLES:

7 fish sauce 3 salted fermented radish
12 fish paste 3 sweet fermented radish
6 shrimp paste 3 fermented melon
7 fermented fish 3 fermwnted mustard
3 sour fermented fish 3 fermented papaya
4 salted fish

After collection, the samples were transported to The

Department of Food Science and Technology to
receive pre treatment and storage at an optimum
temperature of 4° C for further laboratory analyses.
The samples are analyzed within the typical shelf-life
of the products.
 Physicochemical
2.1 Determination
of pH and Properties Analysis
Water activity

FERMENTED FISHERY: FERMENTED VEGETABLES:

The pH value was determined by

penetrating the spear tip of the Blueline 21
pH electrode and later measured using a
digital pH meter.
The aw value was measured using the
digital water activity meter Rotronic
Hygropalm HP23-AW-A after equilibration
◦
at room temperature (~25 C).
 Physicochemical
2.2. Determination
of Salt Content Properties Analysis
The salt content was analyzed by potentiometric
FERMENTED

precipitation
FISHERY: titration
FERMENTED of
VEGETABLES:
chloride-ions with the 877 Titrino plus-Titrator equipped with a calomel electrode.

The salt content of the original samples was calculated as

The samples were prepared into three ratios % (w/w) NaCl = V × M × 0.0584 × 100/m
depending on the expected salt contents:

where:
a) 1 to 10 g ± 0.01 g of the sample V is volume
b) 200 g ± 0.01 g of sample M is molarity of the AgNO3 standard solution
c) 50 g ± 0.01 g of homogenized sample

The initial sample weight is m where:
2mL of AgNO3 was added before being titrated m = a × c/b.
with 0.1 M AgNO3. The test was conducted twice.
 Microbal
Analysis

•Lactic Acid Bacteria (LAB) were anaerobically grown on DeMan Rogosa Sharpe agar at 30
for 72 hours.
◦C
•Enterobacteriaceae were enumerated using the pour plate method with an additional overlay
◦
on Violet Red Bile Dextrose agar and an incubation at 37 C for 24 hours.
•Pseudomonas spp. were detected by plating appropriate dilutions on Cephalothin-Sodium
◦
Fusidate-Cetrimide agar and incubation at 25 C for 44 hours.
 Microbal
Analysis
2.3 Microbiological
Analysis

•Yeasts and molds were determined using the spread plate method on Dichloran
◦
Glycerol agar. The plates were incubated at 25 C for 5–7 d and counted on the 5th
and 7th day of incubation. Colonies confirmed via methylene blue staining and
microscopy.
◦
•Halophilic and halotolerant bacteria - after incubation at 30 C for 2–4 d on Tryptone
Soya agar supplemented with 10% NaCl.
•Staphylococcus aureus - Baird Parker agar which was incubated at 37 ◦ C for 24
hours, evaluated for another 24 hours of incubation and colonies confirmed via Gram-
stain and DNase agar.
•Bacillus cereus - by spreading dilutions on Mannitol Yolk Polymyxin agar and
incubating plates at 30 ◦ C for 18–48 hours, evaluated after 18 h and 48 h of
incubation. Colonies confirmed by endospore staining.
 Microbal
Analysis

•E. coli was enumerated by pour plating on Tryptone Bile Glucuronic medium with an
◦
incubation at 44 C for 18–24 hours.
•Sulfite-reducing Clostridium spp. (SRC) - by pour plating with an additional overlay on
Sulfite-Cycloserin agar and anaerobically incubated at 37 ◦ C for 20 h. Confirmation
tests using Lactose-Gelatine medium and Motility-Nitrate medium.
•Salmonella spp. - VIDASUP Salmonella(SPT)system, whereasthat of Listeria spp. and
L. monocytogenes was tested by the VIDAS LDUO system.

2.3 Microbiological Analysis

 Determination of
Biogenic Amines (BAs)

•The concentrations of BAs were analyzed by reverse-phase HPLC.

•Briefly, 1g of the homogenized sample was extracted overnight with 5mL of 0.4M
perchloricacid. Then, the sample was centrifuged at 5000 rpm for 10 min. For
derivatization, 80 µL of 2MNaOH, 120 µL of saturated sodium bicarbonate solution
and 400 µL of derivatization reagent (1% dansyl chloride in acetone; prepared daily)
were added to 400 µL of sample solution. The sample was mixed and incubated for 45
min at 40 ◦ C. Afterwards, 60 µL of 1 M ammoniasolution was added, mixed, and
incubated in the dark for 60 min at room temperature. Finally, 940 µL acetonitrile was
added. The sample was mixed and centrifuged for 10 min at 13,400 rpm. A RP-18
column with a LiChroCART 4-4 Guard Column (RP-18, 5 µm) and a manu-CART NT
cartridge holder was used for separation. The flow rate was 1 mL/min, the column
temperature was 40 ◦ C, and the injection volume was 20 µL. The mobile phase A
consisted of 0.1 M ammonium acetate and the mobile phase B was 100% acetonitrile.
 Determination of
Biogenic Amines (BAs)

The following gradient was used for the separation:

time = 0 min, 50% A and 50%B;

time=19min, 10% A and 90% B;
time=20min, 50% A and 50% B;
time = 28 min, 50% A and50% B.
 Determination of
2.4. Determination Biogenic Amines (BAs)
of Biogenic Amines
(BAs)

•The detection was performed by UV–vis at a wavelength of

254nm. Heptylamine was used as an internal standard that was
well separated from other compounds. The specificity of the
method was checked using standard mixtures of 12 BAchemicals,
which included spermine tetrahydrochloride, spermidine
trihydrochloride, ethanolamine, isopropylamine, histamine
dihydrochloride, putrescine (1,4-diaminobutan dihydrochloride),
methylamine hydrochloride, agmatine sulfate, cadaverine (1,5-
diaminopentan dihydrochloride), tyramine hydrochloride,
dimethylamine hydrochloride, and pyrrolidine. Except for the last
four chemicals, which were from Sigma-Aldrich all chemicals were
from Fluka.
 Determination of
2.4. Determination Biogenic Amines (BAs)
of Biogenic Amines
(BAs)

•Standard stock solutions of BAs were prepared at 500 mg/L in 0.01

M perchloric acid. The stock solutions were diluted with 0.4 M
perchloric acid to obtain series of working standard solutions (0.25, 1,
5, 10, and 15 mg/L). The derivatization procedure was the same as for
the samples. All compounds were separated and could be identified
by their retention times. The linearity of the method was tested by
analyzing the series of working standard solutions. The correlation
coefficients for the linear regression lines were better than 0.99 for all
compounds. The limit of detection of all BAs ranged between 0.5 and
1.5 ppm, and the limit of quantification ranged between 1.5 and 4.8
ppm. PUT, CAD,HIS, and TYR were analyzed in duplicate.
Statistical
Analysis
Unit of Measurement: colony-forming units/gram (cfu/g)
Statistical Package for the Social Sciences - an analysis software
Mean and Standard deviation
Analysis of Variance (ANOVA)
Least-Squares Difference (LSD)
Pearson Correlation Coefficient
RESULTS &
DISCUSSION
RESULTS
Table 2. Physicochemical characteristics (pH, aw, % NaCl) of fermented food products.
RESULTS
Table 3. Microbial profiles found in fermented food products.

The counts of all other microorganisms were <10^2 cfu/g.

RESULTS
Table 4. Contents of biogenic amines in fermented food products.

 4.1. Physicochemical Characteristics

DISCUSSION in Fermented Foods

Fermented Fishery Products

pH values are in good range similar to other Southeast Asian countries
Salt concentration are consistent to previous data
aw values are comparable to Thai shrimp paste and Indonesian fermented
fish
Fermented Vegetable Products
pH values is the same with previous study (3.6 - 5.5)
The Chaipov brey was found to have the highest salt value (25%)
Lowest aw values were determined in this sample
Growth of microorganisms in foods are mainly influenced by the aw and pH
 4.2. Microbiological Parameters in
DISCUSSION Fermented Foods

Microorganisms associated with fermented foods are commonly present on

the external surface and in the pre- and post-harvest environment of raw
materials.
Bacillus spp. and Clostridium spp., are comparable to the result in Chuon et
al’s study.
no C. perfringens could be confirmed within this study
It is known that >105 cfu/g B. cereus group members are potentially harmful
for human consumption.
 4.2. Microbiological Parameters in
DISCUSSION Fermented Foods

Moreover, no S. aureus could be quantified (<102 cfu/g) in the tested

samples, which is in contradiction to a previous study. S. aureus is
uniquely resistant to adverse conditions such as low aw values
(0.83), high salt contents, and pH stress.
most strains can grow over an aw and pH range of 0.83 to >0.99
and 4.5–9.3,respectively.
11 of all 57 food products tested (19.3%) had an aw value in the
range specified above,all trasork chav (fermented melon), spey
chrourk (fermented mustard),and mam lahong (fermented papaya),
and one chaipov paem (sweet fermented radish) product had a pH <
4.5. Overall, S. aureus could only have grown in one sample.
 4.2. Microbiological Parameters in
DISCUSSION Fermented Foods

no representatives of L. monocytogenes as well as of other Listeria

species were verified, which may be due to the low aw (<0.9) of most
food samples tested (90%).
less than 102 cfu/g of Pseudomonas spp., Enterobacteriaceae, and E.
coli were detected
no Salmonella spp. could be determined using the VIDAS system.
Forty-three samples (~75%) of all fermented products tested in this
study contained more than 10% salt (Table 3). LAB were only present
in high numbers in samples with less than 10% salt
 4.2. Microbiological Parameters in
DISCUSSION Fermented Foods

The unfavorable conditions for bacterial growth (high salt content, a low
pH or aw) may result in higher yeasts and mold numbers. These
microorganisms are quite salt-tolerant . As recommended by the European
Food Safety Authority (EFSA), the accepted limit for molds in foods is
<103 cfu/g . As shown in Table 3, all 57 fermented food products were
acceptable regarding molds. It has been reported that <106 cfu/g of yeasts
are acceptable in RTE foods placed on the market . An unsatisfactorily
higher yeasts count (>106 cfu/g) was only found in one paork chav
(fermented fish) sample, which could lead to spoilage by acid and gas
production . However, the limit was just exceeded marginally (Table 3).
Detected counts of the investigated microorganisms in this study are
satisfactory. The fermented foods tested are suitable for human
consumption regarding the microbiological quality.
 4.3. Formation of BAs in Fermented
DISCUSSION Foods

pH and aw values, nutrients, and bacteria, as well as extrinsic influences

including storage time, temperature, and production methods large
range of BA concentrations within the same studied food type as well
as between the fermented fishery items.
According to Shalaby (1996), BA levels vary not just among different
dietary kinds but even within a single variation. Vegetables that had
been fermented, however, showed no noticeable difference during this
investigation.
teuktrey, paork chou, and mam trey had greater HIS concentrations. Six
kapi has low HIS and TYR concentrations (shrimp paste)
Accordingly, HIS has been found in all fermented spey chrourk
(fermented mustard) and mam lahong (fermented papaya) samples, but
only in one trasork chav (fermented melon) and in no chaipov
(fermented radish)
 4.3. Formation of BAs in Fermented
DISCUSSION Foods

TYR has been detected in more fermented vegetable samples than HIS.
TYR and CAD have been described in few vegetables in relatively low
concentrations

In contrast, it has been reported that PUT is the most common BA found
in food of plant origin. It is particularly abundant in vegetables [64,65]
and fermented products. PUT was the only one that was verified in all
fermented vegetable samples with relatively high values.
The action of microbial decarboxylases is known to allow many genera,
species, and strains of Gram-positive and Gram-negative bacteria to
make BAs, though [66]. The families Enterobacteriaceae,
Pseudomonadaceae, and the genera Photobacterium, Vibrio, and
Staphylococcus were identified as HIS-producing bacteria in particular,
as well as halophilic and halotolerant bacteria, LAB, Bacillus spp., and
Costridium spp.
 4.3. Formation of BAs in Fermented
DISCUSSION Foods

Enterobacteriaceae were also reported to produce PUT, CAD, and to a

lesser extent TYR.
TYR should be mainly formed by LAB (Lactobacillus, Enterococcus) during
fermentation. Next to strains of the genera Clostridium, Pseudomonas,
and Staphylococcus, LAB (Enterococcus, Lactococcus) are also involved in
the production of PUT.
main factors affecting microbial activities in food are temperature, salt
concentration, and pH
 4.3. Formation of BAs in Fermented
DISCUSSION Foods

Enterobacteriaceae were also reported to produce PUT, CAD, and to a

lesser extent TYR.
TYR should be mainly formed by LAB (Lactobacillus, Enterococcus)
during fermentation. Next to strains of the genera Clostridium,
Pseudomonas, and Staphylococcus, LAB (Enterococcus, Lactococcus)
are also involved in the production of PUT.

main factors affecting microbial activities in food are temperature, salt

concentration, and pH
 4.3. Formation of BAs in Fermented
DISCUSSION Foods

Hence, it contributed to higher BA contents (Table 4). This effect

could be confirmed within this study, as weak and moderate negative
correlations (r = −0.22 and −0.57) were respectively found between
the total BA contents and pH values for fermented fishery and
fermented vegetable products.
In general, increasing salt concentration contribute to the reduction of
BA accumulation in foods, mainly reducing the metabolic activities of
decarboxylase-positive microorganisms as it may have been the case
for the fermented vegetables.
 4.3. Formation of BAs in Fermented
DISCUSSION Foods

the rate of BA accumulation decreases with the decrease of aw values

due to the water loss. Correspondingly, a positive correlation should
be observed between total BA contents and aw values. In fact, weak
and strong positive relationships were determined for fermented
fishery (r = 0.22) and vegetable (r = 0.79) products, respectively
higher BA amounts could be expected for these products in this
country. higher BA values were detected in fermented vegetables with
lower salt contents (2–5%).
 4.4 BAs and Food Safety
DISCUSSION

 4.4 BAs and Food Safety

DISCUSSION

US Food and Drug Administration (FDA) - 50 ppm

FAO/WHO - 200 ppm
EFSA - 400 ppm
Codex Alimentarius Commission and EFSA - 400 ppm
Teukrey (fish sauce) - safe accdg. to EFSA and CAC
One park chou (sour fermented fish), one mam trey (fermented
fish), and two paork chav (fermented fish) products - not safe for
human consumption
TYR values - 600 ppm or more, recommended by EFSA
One park chav (fermented fish) sample - may pose health risk
According to Prester et. al., dietary value of up to 600 ppm of TYR is
acceptable but more than 1080 ppm are toxic for adults
 4.4 BAs and Food Safety
DISCUSSION

Acute toxicity level for TYR is greater than 2000 ppm and CAD is
greater than 2000 ppm
Oral toxicity level of PUT is 2000 ppm

PUT AND CAD CONTENT

Low levels of BAs in the tested fermented vegetables

 4.5 BAs and Food Quality
DISCUSSION

PUT & CAD - spoilage indicators and spoilage index, thus is used as
quality indicator
BAI = HIS + TYR + PUT + CAD
Satisfactory to fresh products
50 ppm for freshwater fish
No record for fermented fishery products
500 ppm for fermented food (Bologna sausages)
About one-third (31%) fermented fishery products had BAI
less than 500 ppm and the remaining two-thirds (69%) had a
higher BAI
about 13% (2/15) had a BAI higher than 300 ppm of
fermented vegetables
CONCLUSION
CONCLUSION

The presence of microorganisms in the examined fermented samples

presented no health risk since pathogenic and spoilage microorganisms
were in acceptable ranges.
CONCLUSION

Sour Fermented Fish Fermented

Fish Fermented Fish Fermented Fish
Paork Chou Mam Trey Paork Chav Paork Chav
Trey Sleuk Reusey Trey Bondol Ampor Trey Por Trey Chhkork
BIOGENEIC AMINE INTAKE

FERMENTED FISH 69%

FERMENTED VEGETABLE 13%
RECOMMENDATION

The production process, distribution, and domestic handling of

fermented products in Cambodia should be re-evaluated in order to
minimize the content of BAs and microbiological contamination.

Further research is required to establish preservation techniques that

could be applied on an industrial and small-scale in Cambodia.
POTENTIAL RESEARCH/
THESIS TOPIC

Food and Dairy Microbiology

BIOGENIC AMINE
CONCENTRATIONS IN THE
PHILIPPINES

FERMENTED FOOD PRODUCTS

AND IT'S HEALTH IMPLICATIONS
Barron, Quennie Joyce F. Enciso, Kyla Joice D.
Ysip, Jevoh Bernal, Florence May A. Veloria, Adrianne Einar
OBJECTIVES

GENERAL OBJECTIVES
The study aims to determine the biogenic amine concentrations in
fermented food products in the Philippines.

SPECIFIC OBJECTIVES
1. To determine and monitor whether the concentrations of the biogenic
amines present in the fermented food products are within the appropriate
range.
2. To determine the health implications caused by high concentrations of
biogenic amines in the Philippines' fermented food products.
RATIONALE

The production of fermented food products from fishery to

vegetable varieties is a significant part of the Philippines' culture and local
livelihood. These food products contain specific amounts of biogenic
amines, nitrogenous and organic compounds formed from microbial
decarboxylation of amino acids. Although biogenic amines are usually
found in fermented goods, high concentrations of these compounds pose
a risk to health upon consumption due to their toxicological effects that
may trigger symptoms, including headaches, nausea, and skin irritations.
This study aims to determine the concentrations of biogenic amines
present in the Philippines' fermented food products and their implications
for the health of its consumers.
Food and Dairy Microbiology

BIOGENIC AMINE
CONTENTS AND MICROBIAL
CHARACTERISTICS OF
CAMBODIAN
FERMENTED FOODS

QUIZ
1. What are the low molecular weight organic molecules,
formed by microbial decarboxylation of their precursor
amino acids or by transamination of aldehydes and ketones
by amino acid transaminases and when it is consumed in
high amounts it may result to potential health risk?

A Biogenetic Amide
B Biogenetic Amines
C Biogenic Amide
D Biogenic Amines
2. What are the 4 most common BAs?

A Hypotension, HIS, PUT, and CAD

B
Hypertension, TYR, Intracerebral
Hemorrhage and Cardiac palpitation

C
Histamine, Tyramine, Putrescine
and Cadaverine

D Histidine, Thymine, Putrescine

and Cadaverine
3. Growth of microorganisms in foods are mainly influenced
by the _______, _______, the addition of salt has an
inhibitory effect on the growth of microorganisms due to
its impact on the aw value.

A Aw, pH
B Salt content, temperature
C Aw, storage
D Salt content, storage
4. ____ may be reliable indicator of food safety but not of
food quality. While the ____ and ___ are used as spoilage
index.

A PUT : HIS and CAD

B HIS : TYR and CAD
C CAD : TYR and CAD
D HIS : PUT and TYR
5. What is the maximum BAI content for fermented food
(Bologna sausages)?

A 500 ppm
B 5000 ppm
C 1000 ppm
D 2000 ppm
5. What is the maximum BAI content for fermented food
(Bologna sausages)?

A 500 ppm
B 5000 ppm
C 1000 ppm
D 2000 ppm
1. What are the low molecular weight organic molecules,
formed by microbial decarboxylation of their precursor
amino acids or by transamination of aldehydes and ketones
by amino acid transaminases and when it is consumed in
high amounts it may result to potential health risk?

A Biogenetic Amide
B Biogenetic Amines
C Biogenic Amide
D Biogenic Amines
2. What are the 4 most common BAs?

A Hypotension, HIS, PUT, and CAD

B
Hypertension, TYR, Intracerebral
Hemorrhage and Cardiac palpitation

C
Histamine, Tyramine, Putrescine
and Cadaverine

D Histidine, Thymine, Putrescine

and Cadaverine
3. Growth of microorganisms in foods are mainly influenced
by the _______, _______, the addition of salt has an
inhibitory effect on the growth of microorganisms due to
its impact on the aw value.

A Aw, pH
B Salt content, temperature
C Aw, storage
D Salt content, storage
4. ____ may be reliable indicator of food safety but not of
food quality. While the ____ and ___ are used as spoilage
index.

A PUT : HIS and CAD

B HIS : TYR and CAD
C CAD : TYR and CAD
D HIS : PUT and TYR
Food and Dairy Microbiology

BIOGENIC AMINE
CONTENTS AND MICROBIAL
CHARACTERISTICS OF
CAMBODIAN
FERMENTED FOODS

Table of Contents