الفهرس 
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Abstract
Protein molecules in fish are destroyed by bacterial enzymes and chemical processes into BAs via particular types of bacteria that contain enzymes capable of decarboxylating and converting amino acids freely available in the tissues of some fish as decarboxylating of histidine into HIS. Due to inadequate storage conditions and high temperature, these fish develop excessive levels of HIS, which is referred to as HIS-poisoning. The analysis of BAs in fish and fish products is gaining worldwide attention due to their close association with their quality, safety, and trade, which may have some harmful effects on humans.
In this study, TAMB, TAPB, HIS-forming bacteria, and the presence of certain food-borne bacterial pathogens were examined in five fish and fish products (chilled mackerel, chilled sardine, smoked herring, fermented anchovies, and fermented feseekh). Fermented feseekh contained significant counts (P < 0.05) of mesophilic and HIS-forming bacteria at 6.09 and 3.12 log10 CFU/g, respectively. Also, four pathogenic bacterial genera were isolated and identified from tested samples on their selective media: Staphylococcus, Salmonella, Vibrio and Pseudomonas. from the bacterial evaluation of fish and fish products, it is clear that most of the tested samples contained huge numbers of bacterial loads, in addition to the presence of some pathogenic bacteria that may pose a serious threat to the health of consumers.
All obtained samples were also screened for BA formation (HIS, CAD, and TYR) using TLC. The findings revealed that 65 of the 125 samples tested positive for BAs. There were fifteen samples of mackerel, sardines, and feseekh, as well as ten samples of anchovies and smoked herring. The levels of BAs in these samples were measured using HPLC, and the findings revealed that fermented feseekh had the greatest concentration of total BAs (355.8 mg kg−1), followed by fermented anchovies (192.2 mg kg−1). The highest level (77.10 mg kg−1) of HIS was detected in chilled mackerel samples, followed by fermented feseekh that contained 70.75 mg kg−1. Out of 65 tested samples, 16 (22.61%) samples had HIS levels greater than the FDA guideline of 50 mg kg-1. Using the Pearson correlation coefficient, a positive correlation was found between the levels of BAs in fish and changes in chemical attributes (pH and TVB-N).
Ninety-five bacterial isolates were isolated from all fish and fish products. These isolates were tested for their ability to produce HIS using TLC. The results showed that out of 95 bacterial isolates, 20 isolates were found capable of producing HIS at different levels. Some of them also produced CAD and TYR in varying quantities via the actions of their individual decarboxylase enzymes on various amino acids present in the culture medium. Through measuring the amount of HIS, it was found that isolates H6, H13, H64 and H79 are the most HIS-forming bacteria. Using 16S rDNA sequencing, such isolates were identified as Klebsiella pneumoniae MS-H1, Bacillus sp., MS-H2, Pseudomonas fluorescens MS-H3, and Pantoae agglomerance MS-H4 under accession numbers MW405912.1, MZ570589.1, MZ798200, and MZ796281, respectively. The highest HIS-forming bacterial strain was K. pneumoniae MS-H1, producing 970 g/ml.
Even under similar conditions, radiation resistance of microorganisms varies from genus to genus as well as across species within the same genus. The radiation resistance of bacteria is measured by the so-called D10-value. Knowing this value aids in predicting the amount of radiation required to eradicate the bacterial species. Therefore, the D10-values were estimated for both the identified HIS-producing bacteria and pathogenic bacteria isolated from fish and fish products under study. According to the results obtained, D10-value ranged from 0.17 to 1.03 and from 0.27 to 1.12 kGy in HIS-producing bacterial strains after being injected into tryptone soy broth and mackerel meat, respectively. K. pneumoniae MS-H1 showed a high sensitivity to radiation. It was killed by a radiation dose of 1.5 kGy, whereas Bacillus sp., MS-H2 was highly resistant, with D10-values of 1.03 and 1.12 kGy in TSB and mackerel muscles, respectively.
Also, D10-values of food-borne pathogenic bacteria were determined. The findings revealed that the D10-values for Staphylococcus aureus, Salmonella sp., Vibrio sp., and Pseudomonas aeruginosa varied from (0.41 to 0.46 kGy), (0.44 to 0.50 kGy), (0.055 to 0.077 kGy), and (0.28 to 0.31 kGy), respectively indicating that Vibrio sp. were the most radiosensitive among all tested bacteria.
Different irradiation doses (0.0, 2.5, 5, 10, and 15 kGy) were used to investigate the effect of irradiation on the degradation of BAs in liquid medium. The results showed that a 5.0 kGy irradiation dose decreased HIS, CAD, TYR, and total BAs by 72.98, 34.77, 87.34, and 55.10%, respectively. Similarly, irradiation at 10 kGy resulted in 82.14–88.1% breakdown of HIS and TYR. All BAs were completely broken down by irradiation at dose of 15 kGy.
The most prolific strain of K. pneumoniae MS-H1 (970 g mL−1), was chosen to evaluate the effect of -irradiation on the production of HIS. Samples of mackerel fish were artificially inoculated with this bacteria, irradiated at different doses (0.0, 1.0, 2.0, and 3.0 kGy), and stored at 4 °C for 12 days. The results proved that the highest production of HIS was recorded in the non-irradiated samples after 12 days of storage at a level of 958 µg mL−1, and a radiation dose of 1.0 kGy led to a slight decrease in the production of HIS. At a radiation dose of 2.0 kGy, the level of HIS decreased to only 1.6 μg mL−1 after 12 days of storage, whereas no HIS levels were reported at 3.0 kGy.
The antibacterial activity of eight medicinal plant EOs (cinnamon, tea tree, rosemary, thyme, clove, marimia, black seed, and black pepper) against HIS-producing bacteria was tested. Out of the tested oils, only EOs of clove, rosemary, and black seed were proven to be effective against the HIS-forming bacteria. Clove oil was found to be the most effective in comparison with rosemary and black seed oils against HIS-forming bacteria, with inhibition zones ranging from 18.5 ± 0.50 mm to 26.02 ± 0.73 mm. The MIC (the lowest concentration that has the ability to inhibit the growth of bacterial cells) ranged from 0.125 to 0.25 mg L−1 in each of clove oil and black seed oil, and from 0.25 to 0.50 mg L−1 in rosemary oil.
GC-MS analyses were used to identify the major compounds in these EOs and the results showed the presence of 11, 14, and 16 major compounds in the clove, rosemary, and black seed oils, respectively. Eugenol (55.46%) and caryophyllene (27.88%) were the main components of clove oil, while isopropyl myristate was found as a major component (68.44%) of rosemary oil, followed by camphor (7.65%). On the other hand, 9, 12-octadecadienoic acid (21.08%), palmitic acid (11.21%), and o-cymene (5.29%) were the major compounds in black seed oil.
The impact of EOs treatment on the formation of HIS by K. pneumoniae MS-H1 in mackerel fish was investigated. K. pneumoniae MS-H1 was inoculated into radiation-sterilized mackerel fish, which were individually treated with 250, 500, and 250 g L−1 for clove, rosemary, and black seed oils, respectively, and kept at 4 °C for 12 days. The highest HIS level (969.3 μg mL−1) was recorded in the control sample after 12 days of storage. The lowest concentration of HIS produced in mackerel fish after 12 days of storage was recorded for clove oil (591 μg mL−1), indicating that clove oil was the most effective in reducing the formation of HIS in mackerel fish.
As a result of the previous findings, an applied experiment was conducted to study the effects of the combination treatment of 250 g L−1 clove oil and 2.0 kGy irradiation dose on the natural microbial load, food-borne pathogenic bacteria, BA formation, chemical quality attributes, and sensory properties of mackerel fish during storage at 4 °C for 12 days. It was found that treatment of mackerel samples with 250 g L−1 clove oil followed by irradiation at a dose of 2.0 kGy significantly decreased TAMB, TAPB, and HIS-forming bacteria at zero time of storage. After 8 days of storage, the counts of TAMB and TAPB were below the detection level (<10 CFU/g), while HIS-forming bacteria was under the detection limit after 4 days of storage. Concerning food-borne pathogenic bacteria, this combination treatment reduced the counts of all tested pathogens to below the detection limit (<100 CFU/g) throughout the storage period.
BAs levels of non-treated samples gradually increased during cold storage period indicating that refrigeration at 4 °C did not impede the development of BAs in seafood. BA levels in treated samples were lower than those in non-treated ones all over the storage period, indicating that combination treatment resulted in reducing BA formation, particularly HIS, to less than the acceptable limit (50 mg kg−1).
In regard to the effect of combination treatment on the biochemical attributes of mackerel fish (pH, TBAS, TVB-N, and TMA). The results revealed that combination treatment slightly decreased pH values in both non-treated and treated samples considerably, reaching the lowest values of 6.31 and 6.24 after 8 days of storage. The pH values of both lots increased after 12 days of storage, with a slight increase in the treated ones. TBAS values increased in both non-treated and treated samples, and there were no significant differences between TBAS values of non-treated and treated samples throughout the storage period. During storage time, TVB-N increased in both non-treated and treated samples, but the rate of increase was lower in treated samples. TVB-N levels in non-treated samples exceeded the acceptable limits of TVB-N (30 mg/100 g) after 12 days of storage. However, the TNB-N levels in treated samples were still below the rejection level throughout the storage period. Similarly, TMA levels in non-treated samples surpassed the acceptance level (10-15 mg/100 g) after 12 days of storage, but they were below the rejection limit in treated samples during all storage periods. Combination treatment of clove oil (250 g L−1) and a radiation dose of 2.0 kGy preserved the organoleptic properties of mackerel fish for 12 days of storage through inhibiting the development of spoilage microorganisms.