Author name:
صلاح ناجي عزيز الخيون
Supervisor name:
قيثار رشيد مجيد | طالب احمد جايد
Abstract:
ELISA technique was used to estimate of histamine in muscles of several types of local fresh fish, including : Tenualosa ilisha, Megalaspis cordyla , Chirocentrus dorab and Scomberoides commersonianus in addition of the shrimp Metapenaeus affinis , all samples of fish and shrimp were collects from Fao markets in Basarah city .Results showed the percentages of histamine in all types of fish, as well as shrimp were : 4.483 and 3.953 and 3.353 and 3.16 and 2.03 mg/kg respectively.Accumulation of histamine in fish after refrigerating for three days was also determinated. Results showed that the values of histamine accumulate were increased within preservation period .On the third day of histamine accumulate of C. dorab , T. ilisha , M. cordyla , S. commersonianus and shrimp were 4.56 , 3.99 , 3.43 , 3.19 and 2.01 mg / kg, respectively. While , on the first day were 4.01 , 3.65 , 3.03 , 2.98 and 1.89 mg/kg respectively .ELISA technique was also used to estimate histamine in Ripening cheese such as cheddar , Alkuda , Almoszerla and Oloadam cheese and smoked Hajdu . They were 4.267 , 2.433, 1.167 , 3.667 and 1.833 mg / 100 g , respectively.Many bacteria were isolated isolated from fishes and shrimp, they identified according to : growth characteristics on selective media, morphological, and microscopic examinations in addition to biochemical tests.Fifty three isolates of bacteria were found, they were : Klebsiella pneumonia sub. pneumonia , Klebsiella oxytoca , Morganella morganii sub. Morganii, Enterobacter aerogenes, Enterobacter cloacae , Cronobacter sakazakii ,AEnterobacter taylorae, Citrobacter freundii , Citrobacter amalonaticus , Proteus marina, Proteus vulgaris , Proteus mirabilis, Hafnia alvei , Escherichia coli, Serratia marcescens, Serratia fonticola , Vibrio vulnificus, Vibrio diazotrophicus , Vibrio hollisae , Vibrio alginolyticus, ,Vibrio cholera, Vibrio parahaernolyticus, Vibrio fluvialis, Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus salivarius, Pseudomonas aeruginosa, Pseudomonas fluorescens, Staphylococcus saprophyticus, Staphylococcus aureus , Staphylococcus intermedius , Staphylococcus epidermidis, Bacillus pumilus , Bacillus licheniformis , Bacillus firmus , Bacillus subtilis, Bacillus alvei , Aeromonas. caviae , Aeromonas veronii bv.veronii , Aeromonas eucrenophila, A. sobria , Aeromonas veronii and Aeromonas encheleia .Isolation and identification of Lactobacillus from all samples of ripening cheese were done. Six species of Lactobacillus were found and as a follow : Lactobacillus plantarum , Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum and Lactobacillus paracasei .DNA was extracted from all types of G+ and G - bacteria which isolated from fish and shrimp in addition to Lactobacillus bacteria that isolated from ripening cheeses . the DNA of all bacteria was good and pure .Polymerase chain reaction (PCR) technique was used for identification all isolates of Lactobacillus which were isolated from fish and shrimp, as well as ripening cheese depending on the bacterial 16S rRNA gene sequences of bacteria with using different types of diagnosed primers, genes had been identified for : Lactobacillus acidophillus , Lactobacillus plantarum , Lactobacillus delbrueckii , Lactobacillus fermentium , Lactobcillus helveticus , Lactobcillus crispatus ,BLactobcillus gasseri, Lactobcillus reuteri , Lactobcillus rhamnosus, Lactobcillus paracasei, Lactobacillus johnsonii , Lactobacillus salivarius and Lactobacillus casei .Nine primers were in RAPD technique to find out the genetic link between different strains of Lactobacillus bacteria, the results showed that the number and location of randomized bands were differed in some bacterial species but others was fitted and produced a variety genetic of bacterial strains, in particular a number of bands as possible to get a variety of different genetic profiles among Lactobacillus isolates .The results appeared different styles of bands and presence of specific bands in Lactobacillus bacteria in all primers . The results obtained from RAPD technique showed that the total number of bands were 630 bands, the total number of bands which formed genetically was 127 and the percentage of total proportion of genetic variation among the isolates was 20.15% .The results found of genetic primers scheme that the genetic distance was as close as possible between bacteria L.fermentum and L.rhamnosus and was a part of the one genetic group, but L.helveticus was close as possible genetically to this group , and then the L.johnsonii and L.acidophilus was more genetically distant to this group. Also it found that the genetic distance was very near between L.plantarum and L.paracasei and were a part of a one hereditary group and found also that L.reuteri closest genetically to this group, then followed by more genetically L.delbrueckii . However, L.acidophilus was also more genetically distant to this group .CThe results revealed that L.salivarius and L.crispatus were a one hereditary group and the genetic distance for this group was very close to the L.gasseri and it was farther genetically for genetic group, which includes L.fermentum and L.rhamnosus , also found that the bacteria L.casei showed far genetic distance between them and the groups of Lactobacillus .All fifty three isolates of bacteria which isolated from fish , shrimp and ripening cheeses that producing and did not producing histamine subjected to amplified genes which responsible of the histamine production by using the primers HIS1 - F/HIS1 - R ; JV16HC/ JV17HC ; Hdc - f/ Hdc - r ; 106/107 and UNI - L/ UNI - R . Amplification results showed that all primers used to amplified genes of histamine gene different genes sizes depending on the types of bacteria when were positive or negative to Gram stain and their OrigenIt was found the size of the gene was 350 base pairs , was found in G+ bacteria which isolated from fish , shrimp and cheese ripening . These bacteria included : L.acidophilus ,L.helveticus ,L.crispatus, L.gasseri ,L.casei , L.reuteri , L.fermentum,L.rhamnosus, L.paracasei, L.johnsonii, L.salivarius L.plantarum , L.delbrueckii , S.aureus , S. saprophyticus , S.epidermidis , S. intermedius , Bacillus pumilus, B.subtilis, B.licheniformis, B.firmus and B. alvei .May same results were found when amplified HISI - R/HISI - F primer , the size of histamine gene most positive Gram bacteria stain .High size of histamine gene 709 base pair appeared when amplified Hdc - r/ Hdc - f primer .This gene was found in G - bacteria which isolated from fish and shrimp . These bacteria included : Pseudomonas aeruginosa , Pseudomonas fluorescens, Klebsiella pneumonia , Morganella morganii , Enterobacter aerogenes, KlebsiellaDoxytoca, Hafnia alvei, Enterobacter cloacae, Vibrio alginolyticus, Cronobacter sakazakii, Escherichia coli , Enterobacter taylorae, Proteus vulgaris, Proteus mirabilis, Vibrio vulnificus , Vibrio diazotrophicus, Vibrio hollisae , Vibrio fluvialis , Serratia marcescens and Serratia fonticola .However , the size of histamine gene was 530 base pairs , when amplified 106 / 107 primer , the gene was found in most G - bacteria .On the other hand , the UNI - L/UNI - R primer did not amplify in all isolates of bacteria which isolated from fish , shrimp and ripening cheeses .Extraction of plasmids from all isolated bacteria was done . Results showed that isolates have different types of plasmids bands (small and large size) and when the electric deportation of the genetic material of plasmids on gel agarose while some other isolates appeared possess one plasmid.In this study , PCR technique was not detected the presence of histamine genes in plasmid of bacteria , because not occurred any amplify for any type of primers used . This indicates the presence of histamine genes on the genome of the bacteria and not on plasmids of bacteria .
