SURVEY AND ISOLASTION OF HISTAMINE PRODUCING BACTERIA FROM FAYOUM CITY, EGYPT

SURVEY AND ISOLASTION OF HISTAMINE
PRODUCING BACTERIA FROM
FAYOUM CITY, EGYPT
Gehad H.S. Hassan ; Kh. M. Atalla ;
O. A. Seoudi and Y.F. Abdelaliem
Agric. Microbiology Dept., Fac. of Agric., Fayoum University, Egypt.
ABSTRACT
Histamine food poisoning is found to be associated with consumption
of foods containing unusually high levels of histamine. Fish belonging to
scombroid and non-scombroid fish groups may also cause histamine
poisoning. In this study, histamine forming bacteria in the commercial food
samples of local markets of Fayoum region were investigated. A total of 185
unique isolates were selected on the basis of colony morphology, 28 of
which were found to be prominent histamine producers on Niven’s medium.
The percentage of histamine producing bacteria was 15.13% of the total
bacterial load. The dominant microflora was found to be Gram positive
bacilli, Gram negative bacilli and cocci. Out of the 185 isolates, 28 were
from salted fish, 17 from sardine, 13 from mullet, 12 from herring, 24 from
mish, 16 from raw milk, 15 from fermented milk, 21 from rumi cheese, 14
from white cheese, 11 from palm paste and 14 isolates were from pickle.
Results indicated that, regarding to fish samples, the highest content of
histamine was obtained from mullet followed by salted fish and sardine
products by 0.359, 0.316 and 0.259 mg/100g, respectively. Meanwhile,
herring fish sample recoded the minimum histamine levels. For dairy
samples, the highest content of histamine was obtained from rumi cheese
and the lowest histamine concentration recorded in raw milk being 0.379
and 0.133 mg/100g, respectively. Moreover, histamine concentration was
0.119 and 0.110 mg/100g, recorded from pickles and date palm paste,
respectively.
INTRODUCTION
The bacterial spoilage of scombroid and nonscombroid fishes and
certain other foods have high levels of free histidine in their tissues is
sometimes accompanied by the formation of high levels of histamine in the
edible tissues of their types of food (Frank et al., 1981). The formation of
histamine is particularly noteworthy because the presence of high levels of
histamine in spoiled fish and certain other foods have been associated with
outbreaks of food poisoning also known as scombroid fish poisoning
(Arnold and Brown 1978). Histamine is generated from histidine during
spoilage by bacteria that possess the requisite enzyme histidine
decarboxylase (Omura et al., 1978 and Yoshinaga and Frank, 1982).
Egypt. J. of Appl. Sci., 35 (11) 2020 103-116
Histamine, 4-(2-aminoethyl) imidazole, is a primary amine arising from the
decarboxylation of the amino acid, L-histidine.
The biogenic amines are well known for their implication in serious
human intoxications, associated with the consumption of spoiled food. Even
though the isolation and characterization of bacterial strains producing
biogenic amines in general and particularly histamine knew a great
evolution, the methods used for their detection in foods depend on many
parameters, such as the nature of food and the bacterial flora. The early
methods used were based on the measure of the carbon dioxide produced
with the decarboxylation of amino acids. This process is inaccurate and is
now abandoned. Thereafter, several authors proposed the use of selective
culture media, in order to obtain a rapid selection of the biogenic amines
producing bacteria, such as histamine Niven's agar medium supplemented
with L-histidine (Niven et al., 1981).
Many different bacterial species are known to possess histidine
decarboxylase (Arnold and Brown, 1978 and Yoshinaga and Frank,
1982). However, only Proteus morganii (Sakabe, 1973), Klebsiella
pneumoniae (Taylor et al., 1979), and Hafnia alvei (Havelka, 1967) have
been isolated from fish incriminated in scombroid fish poisoning incidents.
The purpose of this study was to survey, isolate and identify histamine
producing bacteria, as well as quantification of histamine content in certain
food sources sold from the local markets of Fayoum region, Egypt.
MATERIALS AND METHODS
Samples and raw materials
Samples used included fishes such as Salted Fish, Mullet, Sardine
and Herring fish, as well as dairy products like Mesh, Rumi cheese, and
Raw Milk, Fermented Milk and White cheese. Otherwise, Pickles and Date
Palm Paste were purchased from different retail and wholesale markets in
Fayoum Governorate. Ten gram of each sample was mixed with 90 ml of
sterile physiological saline (0.85% (w/v) NaCl), homogenized in a
stomacher for 2 min and then further diluted in physiological saline at 1:10
dilutions then serial dilutions were made from 10-1 to 10-5. The diluted
sample solutions were spread on Niven's medium agar (Niven et al., 1981
and Mah et al., 2001) to qualitatively detect histamine produce ability of the
bacterial isolates.
Enumeration of histamine producing bacteria
For total aerobic histamine producing bacterial count, 1.0 ml portion
of each dilution was poured onto petri dishes and then 15 ml of Niven's agar
medium containing 0.5 % NaCl at 45 °C was added and gently mixed. The
poured plates were allowed to solidify and incubated at 37 °C for 48 hours.
Bacterial counts from the different samples were expressed as colony
forming units (CFU/g). To confirm histamine production, 1.0 ml of each
dilution was spread on histamine Niven's agar medium supplemented with
104 Egypt. J. of Appl. Sci., 35 (11) 2020
L-histidine. After incubation of plates for 48 hours at 37°C, colonies with
blue or purple color were picked and further streaked on Niven's agar
medium to obtain pure isolates.
Detection of histamine producing bacteria (HPB)
Histamine producing bacteria (HPB) detection was done by Niven's
methods (Niven et al., 1981). Histamine-producing bacteria were inoculated
on agar plates containing Niven's medium (0.5% tryptone, 0.5% yeast
extract, 2.0% L-histidine-monohydrochloride was purchased from Sigma
(Sigma Aldrich), 0.5% NaCl, 0.1% CaCO3, 2.0% agar and 0.006% bromo
cresol purple, at pH 5.3) which was sterilized at 121°C for 10 min. The
plates were incubated at 37°C for 48 hrs. aerobically (Lopes –Sabater et
al., 1996). Purple zone appeared around colony is an indicator of histamine
producing bacteria. When the indicator showed an increase of pH on agar
plates containing Niven's medium. To determine histamine qualitative assay
of isolates, the purple zone diameter (mm) was measured. The colonies of
these zones were transferred to new plates, and the morphology of the
colonies was observed under a microscope following Gram staining.
Histamine food quantification
Quantification of histamine was carried out using colorimetric
method reported by Patange et al., (2005). In this method, 1 ml of the
muscle extract was taken into a glass-stoppered test tube and diluted to 2 ml
with saline and 0.5 g of salt mixture containing 6.25 g of anhydrous sodium
sulfate to 1 g trisodium phosphate monohydrate was added. The tubes were
stoppered and thoroughly shaken. 2 ml of n-butanol was then added and the
tubes shaken vigorously for 1 min and allowed to stand for 2 min and then
shaken briefly to break the protein gel. The tubes were further shaken
vigorously for few seconds and then centrifuged at 3100 rpm for 10 min.
The upper butanol layer (only 1 ml) was transferred into a clean and dry test
tube and evaporated to dryness in a stream of nitrogen. The residue was
dissolved in 1 ml of distilled water. In a clean tube 5 ml of 1.1% sodium
carbonate solution was taken and 2 ml of the chilled reagent, pphenyldiazonium
sulfonate was added slowly and mixed. It was then added
to the tube containing 1 ml solution of the residue collected in the extraction
process. The absorbance of the color produced was measured immediately
after 5 min at 496 nm. The concentration of histamine in sample was
obtained from the standard curve for the corresponding absorbance
measured at 496 nm. The histamine concentration in sample was estimated
using the following formula.
Where: A is the value of histamine obtained in (μg/ml) from the standard
curve.
Egypt. J. of Appl. Sci., 35 (11) 2020 105
Determination of histamine production
For histamine determination, Gram positive and Gram negative
bacterial isolates were streaked on triplicates on tryptic soy agar (TSA)
plates containing 2% NaCl supplemented with 2% L-histidine and incubated
at 37°C for 24 h. Representative colony from each of the plate was
inoculated into 9 ml of tryptic soy broth (TSB) with 2% NaCl concentration,
2% histidine and 0.0005% pyridoxal-HCl (pH 5.8) and incubated at 37°C
for 24h. 1 ml sample of each TSB+ suspension was transferred into a tube of
fresh TSB + media and incubated at 37°C for 48 h. 3 ml subsample of this
final culture was transferred into polypropylene centrifuge tubes and
centrifuged at 1100 rpm for 20 min (Joshi and Bhoir , 2011). Supernatants
were diluted 1 to 10 in saline solution and quantification of histamine was
carried out using colorimetric method reported by Patange et al., (2005).
RESULTS AND DISCUSSION
Histamine contents of tested food samples
Biogenic amines content expressed as histamine (mg/100g) in
different Egyptian food samples, which were obtained from retail markets of
Fayoum city shown in Table (1). Obtained results indicated that, there were
differences in the contents of biogenic amines in different food samples.
Histamine concentration of tested food types was among 0.110 to 0.379
mg/100 g. In related to fish samples, the highest content of histamine was
obtained from mullet followed by salted fish and sardine products being
0.359, 0.316 and 0.259 mg/100g, respectively. Meanwhile, herring fish
sample recoded the minimum histamine levels. For dairy samples, the
highest content of histamine was obtained from rumi cheese and the lowest
histamine concentration recorded in raw milk was 0.379 and 0.133 mg/100g,
respectively. As shown in Table (1) the pickle histamine concentration was
0.119 mg/100g, while date palm paste histamine concentration recorded
0.11 mg/100g.
The differences in the biogenic amines concentrations of food
samples could be due to the hygienic quality of raw material, manufacturing
practices, the specific bacteria, ripening period and the type of culture. The
biogenic amines concentration may be used as a quality index for these
kinds of products. Handling of raw materials and production technology for
fermented foods and fish products are relatively primitive in Egypt. These
results indicate that the natural fermentation process used for dairy products
and other food samples may result in accumulation of high levels of
biogenic amines. The brand specific variation suggests that different
environmental conditions have some effect on histamine content. Although
the natural fermentation process and storing conditions used in the
preparation of these products (i.e. fish and dairy products) probably did not
involve in growth of any major biogenic amines-producing bacteria. The
106 Egypt. J. of Appl. Sci., 35 (11) 2020
lack of quality control in their production and the use of natural fermentation
make selection of desirable organism’s difficult (Bodmer et al., 1999).
Determination of the exact toxicity threshold of biogenic amines in
individuals is extremely difficult, since the toxic dose is strongly dependent
on the efficiency of the detoxification mechanisms of each individual.
Although the toxicity of biogenic amines to man is a controversial subject,
ingestion of from 70 to 1000 mg histamine will usually cause clinical
symptoms intoxication (Henry, 1960).
Food Drug Administration (FDA) has established a hazard action
concentration for histamine in tuna fish of 50 mg of histamine/100 g
(Stratton et al., 1991). Moreover, it must be noted that smaller amounts of
biogenic amines may cause poisoning particularly if the person is
vulnerable, because of the inhibition of the biogenic amines detoxification
mechanism in the body due to reasons such as personal predisposition,
gastrointestinal diseases, the use of certain medicines and alcohol intake and
the existence of other amines (Bodmer et al., 1999 and Joosten and Van-
Boekel 1988).
Fermentation of milk, a considerable increase of histamine content
often occurs, leading to contents of up to 7 μg/ml histamine in sour cream
and even slightly higher levels in yoghurt. Finally, in cheese production a
rather drastic increase of histamine content often occurs, leading to
maximum levels of histamine of up to 2500 ppm in aged cheese (Bodmer et
al., 1999).
Table (1): Histamine concentration in different types of food samples
(mg/100 g wet weight).
Sources Histamine concentrations (mg/100g)
Fish
Products
Salted fish 0.316
Sardine 0.259
Mullet 0.359
Herring fish 0.128
Dairy
Products
Mish 0.193
Raw milk 0.133
Fermented milk 0.207
Rumi cheese 0.379
White cheese 0.191
Pickles Pickle 0.119
Fruits Date palm paste 0.110
Microbiological profile
Total counts of bacteria
Histamine is a natural constituent of fermented foods generated by
microbial activity. Microbiological profile of different food sources i.e. fish,
dairy, pickles and fruits were studied. The average number of aerobic plate
count (APC) of histamine forming bacteria CFU/g in salted fish sample was
Egypt. J. of Appl. Sci., 35 (11) 2020 107
18.0×105, salted sardine sample was 10.7×105, mullet sample was 3.3×103,
herring fish sample was 10.2×105, mish sample was14.0×103, raw milk
sample was 10.6×104, fermented milk sample was 15.8×103, rumi cheese
sample was 11.4×104, white cheese sample was 14.3×103, date palm paste
sample was 10.1×103 and pickle sample was 14.0×104. In the present study,
a total of 185 isolates were obtained from 11 types of fish and dairy
products.
Isolation of histamine producing isolates
Different types of food sources were screened for the detection of
histamine-producing bacteria (HPB) as shown in Table (2). A total of 185
unique isolates were selected on the basis of colony morphology, 28 of
which were positive producers on Niven’s media. The dominant microflora
was found to be gram positive bacilli, gram negative bacilli and cocci. Out
of the 185 isolates, 28 were from salted fish, 17 from sardine, 13 from
mullet, 12 from herring fish, 24 from mish, 16 from raw milk, 15 from
fermented milk, 21 from rumi cheese, 14 from white cheese, 11 from date
palm paste and 14 isolates were from pickle. Presumptive colonies were
isolated from the Niven’s medium plates and screened for histamine
production.
Table (2): Microbiological profile of food sources i.e. fish, dairy,
pickles and fruits products.
Sources
Number of
isolates
Number of
histamine
producing
isolates
Percentage of
histamine
producing
isolates
Fish
Products
Salted fish 28 12 42.85
Sardine 17 5 29.41
Mullet 13 0 0.00
Herring fish 12 0 0.00
Dairy
Products
Mish 24 4 16.66
Raw milk 16 3 18.75
Fermented milk 15 2 13.33
Rumi cheese 21 1 4.76
White cheese 14 0 0.00
Pickles Pickle 14 0 0.00
Fruits Date palm paste 11 1 9.09
Total 185 28 15.13%
The percentage of histamine producing bacteria was 15.13% of the
total bacterial load. Similar results obtained by Koohdar et al., (2011) who
found that counts of mesophilic and psychrophilic producers of 45 samples
of skipjack were 7.2×106 and 2.9×106 CFU/g, respectively. The mean count
108 Egypt. J. of Appl. Sci., 35 (11) 2020
of histamine forming bacteria was about 2.8×10² CFU/g and it was 0.004%
and 0.009% of the total and psychrophilic bacterial loads, respectively.
Purple halo around Niven´s medium is an indicator of positive histamine
producing bacteria HPB (Chong et al., 2011).
Based on positive/negative Niven’s media test, among the isolates
obtained previously, 12 isolates were associated with salted fish, 5 were
associated with sardine, 4 were associated with mish, 3 were associated with
raw milk, 2 were associated with fermented milk, and only one was
associated with rumi cheese or date palm paste were positive for histamine
formation. On the other hand, the isolates obtained from mullet, herring,
white cheese and pickles were negative for histamine production.
Histamine qualitative assay
One of the aims of this study was to isolate and characterize the
histamine forming bacteria in traditional Egyptian foods included fish, dairy
and date palm past. In all cases of fish products, the histamine-producing
isolates were obtained from muscles. As mentioned previously in Table (1)
a total of 185 isolates were tested to verify their ability to produce histamine
based on Niven's positive test. Only 28 tentative histamine-producing
isolates were initially obtained from the Niven agar (NA) plates and
confirmed as histamine producer as indicated by halo-purple zone assay, as
shown in Table (3). The decarboxylating Niven agar medium is due to the
corresponding precursor amino acids (histidine monohydrochloride). The
pH of the medium was adjusted to 5.3, and it was sterilized at 121°C for 10
min. The plates were incubated at 37°C for 7 days under anaerobic or
aerobic conditions. During this time, halos formed around colonies on
histamine medium on a yellowish background were measured.
Quantitative determination of histamine
Out of 28 Niven's positive isolates were tested to verify their ability
to produce histamine. Only 11 isolates were chosen as the strongest
histamine producers in addition to reference strain Enterobacter
aerogenes when determined by colorimetric assay of histamine. As
presented in Table (4) among salted fish isolates His 18 was the most
efficient histamine producer by (97.00 μg/ml) and equal to Enterobacter
aerogenes. As well, histamine isolate obtained from fermented milk with
code His 8 showed the highest histamine producer by (94.00 μg/ml) in
comparison with the other histamine isolates of dairy products.
Meanwhile, the lowest histamine producing isolates by (90.00 μg/ml)
were obtained from raw milk and date palm past.
Egypt. J. of Appl. Sci., 35 (11) 2020 109
Table (3): Histamine qualitative assay by histamine producing
isolates after 48 hours.
Code of isolates Sources of isolates Histamine producer
(zone diameter mm)
1 Date palm Paste 30
2 Rumi Cheese 25
3 Salted Fish 25
4 Raw Milk 20
5 Fermented Milk 15
6 Salted Fish 20
7 Salted Fish 25
8 Mish 30
9 Raw Milk 15
10 Fermented Milk 25
11 Salted Fish 20
12 Raw Milk 15
13 Sardine 20
14 Salted Fish 20
15 Sardine 20
16 Sardine 15
17 Sardine 15
18 Salted Fish 15
19 Salted Fish 10
20 Sardine 10
21 Mish 15
22 Mish 15
23 Mish 10
24 Salted Fish 15
25 Sardine 10
26 Salted Fish 15
27 Salted Fish 10
28 Salted Fish 10
Table (4): Histamine concentration produced by different isolates.
Isolates Source Histamine Concentration
(μg/ml)
Enterobacter aerogenes* Reference strain 97.00
His 1 Date palm Paste 90.00
His 8 Mish 93.00
His 9 Raw Milk 91.00
His 10 Fermented Milk 94.00
His 11 Salted Fish 93.00
His 12 Raw Milk 90.00
His 13 Sardine 91.00
His 15 Sardine 92.50
His 16 Sardine 91.00
His 18 Salted Fish 97.00
His 19 Salted Fish 93.00
* reference strain
Koohdar et al., (2011) found that fourteen bacterial strains with
histidine decarboxylase activity were isolated and then tested for their ability
to produce histamine, of which 8 strains (57.14%) of these tentative isolates
showed positive results. Sixteen of the total 45 samples of frozen skipjacks
contained less than 20 μg/ml histamine, but these amounts were 20-50
μg/ml and more than 50 μg/ml in 10 and 19 samples, respectively.
110 Egypt. J. of Appl. Sci., 35 (11) 2020
There were clear differences in histamine contents in samples with
different counts of histamine forming bacteria, so that the samples with high
counts of histamine forming bacteria had relatively higher levels of
histamine than other samples.
Identification of the most two histamine bacterial isolates using
16SrDNA gene
The highly talented of the two histamine bacterial isolates with code
of His 10 and His 18 were selected to identification based on their
pronounced results. The 16SrDNA gene sequence of isolates were
determined and compared with sequences in Gene-Bank database. A
phylogenetic tree based on 16SrDNA sequences (Fig 1 and 2) revealed
affiliation of His 10 to Escherichia coli and His 18 to Pseudomonas otitidis.
Nucleotide of 16SrDNA of His 10
GCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTC
TGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGT
AGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCT
TCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAG
TAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGT
CTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCA
GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGG
CGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTT
CGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGT
TAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGG
CTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAG
CGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTT
GTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGC
ATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAAT
TCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATAC
CGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGG
TGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAG
TCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAG
GCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGA
GTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCC
CGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCG
AAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGA
TGAGAATGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGG
CTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCA
ACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGG
AACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGG
GATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACA
Egypt. J. of Appl. Sci., 35 (11) 2020 111
CGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGA
GCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTC
TGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGA
TCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACC
GCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAG
CTTAACC
Nucleotide of 16SrDNA of His 18
GCAGTCGAGCGCAGGAATCGACGGAACCCTTCGGGGGGAAGT
CGACGGAATGAGCGGCGGACGGGTGAGTAACACGTAAAGAAC
CTGCCCTCAGGTCTGGGATAACCACGAGAAATCGGGGCTAAT
ACCGGATGGGTCATCGGACCGCATGGTCCGAGGATGAAAGGC
GCTTCGGCGTCGCCTGGGGATGGCTTTGCGGTGCATTAGCTAG
TTGGTGGGGTAATGGCCCACCAAGGCGACGATGCATAGCCGA
CCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCC
AGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGG
ACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGCC
TTCGGGTCGTAAAGTTCTGTTGTAAGGGAAGAACAAGTGCCGC
AGGCAATGGCGGCACCTTGACGGTACCTTGCGAGAAAGCCAC
GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA
AGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGG
CCTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGG
GCCATTGGAAACTGGGAGGCTTGAGTATAGGAGAGAAGAGTG
GAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGA
ACACCAGTGGCGAAGGCGACTCTTTGGCCTATAACTGACGCTG
AGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
TAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTT
CCGCCCTTCAGTGCTGAAGCTAACGCATTAAGCACTCCGCCTG
GGGAGTACGGTCGCAAGGCTGAAACTCAAAGGAATTGACGGG
GACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAA
CGCGAAGAACCTTACCAACTCTTGACATCCCCCTGACCGGTAC
AGAGATGTACCTTCCCCTTCGGGGGCAGGGGTGACAGGTGGT
GCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT
CCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCACCATTCAG
TTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAG
GTGGGGATGACGTCAAATCATCATGCCCCTTATGAGTTGGGCT
ACACACGTGCTACAATGGACGGTACAAAGGGCAGCGAAGCCG
CGAGGTGGAGCCAATCCCAGAAAGCCGTTCTCAGTTCGGATTG
CAGGCTGCAACTCGCCTGCATGAAGTCGGAATCGCTAGTAATC
GCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGTCTTGTAC
ACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCG
GTGAGGTAACCTTA
112 Egypt. J. of Appl. Sci., 35 (11) 2020
Figure (1): Tree showing the estimated phylogenetic relationships of His 10:
Figure (2): Tree showing the estimated phylogenetic relationships of His 18:
CONCLUSION:
It could be concluded that, histamine concentration of tested food
samples was among 0.110 to 0.379 mg/100 g. The highest histamine content
0.379, 0.359 and 0.316 mg/100g were obtained from rumi cheese, mullet
and salted fish, respectively, whereas pickles and date palm paste associated
with the lowest histamine food content, as compared to the other tested food
samples. The use of Niven agar aided considerably in the isolation and
identification of the histamine-producing bacteria, the histamine-producing
bacteria isolated from fish products was 12 out of 28 from salted fish
followed by 5 out of 17 form sardine and 4 out of 24 from mish followed by
3 out of 16 from raw milk as dairy products were tentative histamine
producers. It is worth to note that, salted fish as a popular food in Fayoum
city pronounced to be the higher food source containing histamine
producing isolates with 12 isolates.
REFERENCES:
Arnold, S. H. and W. D Brown (1978). Histamine (?) toxicity from fish
products. Adv. Food Res., 24: 113-54.
LR134239 Escherichia coli strain NCTC9100
LR134296.Escherichia coli strain NCTC9041
CP046000.Escherichia coli strain 1916D18
AP019675. Escherichia coli GSH8M-2
FAY-G1
4 3 2 1 0
FAYG-2
JQ768370. Pseudomonas otitidis strain B1
MK177190.Pseudomonas guezennei strain DU13
MF092809.Pseudomonas stutzeri strain MHFRK03
4 3 2 1 0
Egypt. J. of Appl. Sci., 35 (11) 2020 113
Bodmer, S.; C. Imark and M. Kneubühl (1999). Biogenic amines in
foods: histamine and food processing. Inflammation Research,
48 (6): 296-300.
Chong, C. Y.; F. AbuBakar ; A. R. Russly ; B. Jamilah and N. A.
Mahyudin (2011). The effects of food processing on biogenic
amines formation. Int. Food Res. J., 18 (3): 867-876.
Frank, H. A.; D. H. Yoshinaga and W. K. Nip (1981). Histamine
formation and honeycombing during decomposition of skipjack
tuna, Katsuwonus pelamis, at elevated temperatures. Mar. Fish.
Rev., 43(10):9-14.
Havelka, B. (1967). Role of the Hajnia bacteria in the rise of histamine
in tuna fish meat. Cesk. Hyg., 12: 343-352.
Henry, M. (1960). Dosage biologique de l'histamine dans les aliments,
Ann. Fals. Eexp. Chim., 53: 24–33.
Joosten, H. M. L. J. and M. A. J. S. Van-Boekel (1988). Condition
allowing the formation of biogenic amines in cheese. A study of
the kinetics of histamine formation in an infected Gouda cheese,
Neth.Milk. Dairy. J., 42: 3–24.
Joshi, P. A. and V. S. Bhoir (2011). Study of histamine forming bacteria
in commercial fish samples of kalyan city.Int J.Cur. Sci. Res.,1
(2):39 – 42.
Koohdar, V. A.; V.Razavilar; A. A.Motalebi; F. Mosakhani and T.
Valinassab (2011). Isolation and identification of histamineforming
bacteria in frozen skipjack tuna (Katsuwonus pelamis).
Iranian Journal of Fisheries Sciences, 10 (4): 678-688.
Lopes – Sabater, E. I.; J. J. Rodriguez-Jerez; M. Hernandez- Herreo
and M.T. Morana-Ventura (1996). Incadence of histamine –
forming bacteria and histamine content in Scombroid fish
species from retail markets in the Barcelona area. International
Journal of Food Microbiology, 28 (3): 411- 418.
Mah, J. H.; K. S. Kim ; J. H. Park ; M. W. Byun ; Y. B. Kim and
H.J. Hwang (2001). Bacteriocin with a broad antimicrobial
spectrum, produced by Bacillus sp. isolated from kimchi.
Journal of Microbiology and Biotechnology, 11: 577–584.
Niven C. F. ; M. B. Jeffreg and D. A. Corlett (1981). Differential
plating medium for quantitative detection of histamine
producing bacteria. Applied Environmental Microbiology, 41:
321–322.
114 Egypt. J. of Appl. Sci., 35 (11) 2020
Omura, Y.; R. J. Price and H. S. Olcott (1978). Histamine-forming
bacteria isolated from spoiled skipjack tuna and jack mackerel.
J. Food Sci., 43: 1779-1781.
Patange, S. B.; M. K. Mukundan and K. A. Kumar (2005). A simple
and rapid method for colorimetric determination of histamine in
fish flesh. Food Control, 16: 465–472.
Sakabe, Y. (1973). Studies on allergy like food poisoning. Histamine
production by proteus morganii. J. Nara Med. Assoc., 24: 248-
256.
Stratton, J. E.; Hutkins ; R. W. and S. L. Taylor (1991). Biogenic
amines in cheese and other fermented foods: J. Food Prot., 54:
460–470.
Taylor, S. L.; N. A. Woychik and L. Lieber (1979). Histamine
production by Klebsiella pneumoniae and an incident of
scombroid fish poisoning. Appl. Environ. Microbiol., 37: 274-
278.
Yoshinaga, D. H. and H. A. Frank (1982). Histamine producing
bacteria in decomposing skipjack tuna (Katsuwonus pelamis).
Applied and Environmental Microbiology, 44: 447-452.
حصر وعزل البکتيريا المنتجة لمهستامين من مدينة الفيوم , مصر
جهاد حمدي سيد حسن , خالد محمد عطالله , اسامة عبدالتواب سعودي ,
ياسر فتحي عبدالعميم
قسم الميکروبيولوجيا الز ا رعية - کمية الز ا رعة - جامعة الفيوم – مصر
أجريت هذه الد ا رسة بهدف حصر مصادر الأغذية ذات المحتوى العالي من الأمينات
الحيوية )الهستامين( والشائعة بمحافظة الفيوم ، وکذلک تم عزل البکتيريا التى لها المقدرة عمى
انتاج الهستامين من تمک المصادر ، مثل منتجات الأسماک )السمک المممح ، السردين ، سمک
الفسيخ ، السمک المدخن( ومنتجات الألبان )المش ، المبن الخام ، المبن المتخمر ، الجبن
الرومي ، الجبن الأبيض( ومنتج من المخملات وآخر من عجوه نخيل البمح.
وکانت النتائج المتحصل عميها کالأتي:
0.310 ، 0.359 ، الجبن الرومي وسمک الفسيخ والسمک المممح سجمت 0.379
مميج ا رم هستامين لکل 100 ج ا رم عمى التوالي. وقد تم عزل 82 سلالة البکتيرية منتجة
لمهستامين من هذه المصاد ا رلمختبرة من أصل 521 عزلة بکتيرية. وکانت اکثر العزلات البکتيرية
Egypt. J. of Appl. Sci., 35 (11) 2020 115
المنتجة لمهستامين التى تم الحصول عميها من السمک المممح والسردين بواقع 58 و 1 عزلات
His البکتيرية عمى الترتيب. وقد تم اختيار وتعريف أقوى ع زلتين منتجة لمهستامين تحت کود
وکانت .Enterobacter aerogens بالمقارنة بالسلالة البکتيرية المرجعية His 18 ، 10
والعزلة البکتيرية ذات ، Escherichia coli هي His تعريف العزلة البکتيرية ذات الکود 10
. Pseudomonas otitidis هي His الکود 18
116 Egypt. J. of Appl. Sci., 35 (11) 2020