|Year : 2018 | Volume
| Issue : 3 | Page : 190-200
Studies on the microbial decontamination of Egyptian bee pollen by γ radiation
AS Hosny1, FM Sabbah2, ZE EL-Bazza3
1 Prof. of Microbiology and Immunology Department, Faculty of Pharmacy, Cairo University, Egypt
2 Bachelor of Pharmaceutical Science, Atomic Energy Authority, Cairo, Egypt
3 Prof. of Pharmaceutical Microbiology, Drug Radiation Research Department National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt
|Date of Submission||29-Apr-2018|
|Date of Acceptance||11-Jul-2018|
|Date of Web Publication||07-Dec-2018|
Dr. F M Sabbah
Bachelor of Pharmaceutical Science
Source of Support: None, Conflict of Interest: None
Objective Bee pollen are used as health food ingredients, but may be subjected to microbial contamination; γ radiation technology can offer the process of microbial decontamination as a means of achieving microbiological safety limits.
Materials and methods Thirty bee pollen samples were collected from Egyptian markets. Detection of contamination and the microbial counts were carried out on nutrient and Sabouraud’s agar media for bacteria and fungi, respectively, using the aerobic plate technique. Identification of bacteria was carried out by biochemical methods and analytical profile index. Moreover, identification of fungi was carried out morphologically and microscopically.
Results and conclusion The order of bacterial contamination was Gram-positive rods>Gram-negative rods>Gram-positive cocci. The order of fungal contamination is Penicillium spp.>Aspergillus flavus>Aspergillus niger>Aspergillus ochracueus. Only three strains of A. flavus could produce aflatoxin B1. The microbial counts of bee pollen samples decreased with increasing γ radiation doses. The most radio-resistant bacteria that were isolated at 5.0 kGy were identified as Bacillus megaterium, Bacillus pumilis and Bacillus subtilis. The most radio-resistant fungi were identified as Penicillium chrysogenum, Penicillium expansum and Penicillium corylophilum. Using of γ radiation can decrease the bioburden in bee pollen, and eliminate pathogenic microorganisms including fungi, which can produce carcinogenic aflatoxins.
Keywords: aflatoxins, bee pollen, γ radiation, micro-organisms
|How to cite this article:|
Hosny A S, Sabbah F M, EL-Bazza Z E. Studies on the microbial decontamination of Egyptian bee pollen by γ radiation. Egypt Pharmaceut J 2018;17:190-200
|How to cite this URL:|
Hosny A S, Sabbah F M, EL-Bazza Z E. Studies on the microbial decontamination of Egyptian bee pollen by γ radiation. Egypt Pharmaceut J [serial online] 2018 [cited 2021 Jun 13];17:190-200. Available from: http://www.epj.eg.net/text.asp?2018/17/3/190/247070
| Introduction|| |
Bee pollen are produced by flowering plants and collected by bees. Pollen is considered as the primary food source of bees . Bee pollen results from adhesion of flower pollen, nectar and/or honey and the salivary substances of bees . Pollen contains very variable and important components. It contains all of the essential amino acids. It also has vitamins A, D, E, K, C and bioflavonoids as well as B-complex, especially B5 and niacin . It can be used as an antioxidant, anti-inflammatory, antimicrobial agent, and the great therapeutic action is of clinical value because of its antiprostatic effect . Fresh bee pollen contains about 20–30 g of water per 100 g, which is a very high humidity that helps in the growth of micro-organisms, such as bacteria and yeast . Molds, yeast, aerobic and spore-forming bacteria were found in pollen grains. Moreover, Aeromonas hydrophilia, Salmonella More Details spp., Clostridium spp., Staphylococcua aureus and Streptococcus faecalis, were detected in pollen samples . Twenty-one fungal species formed 13 genera of microscopic fungi in the pollen samples, while the highest number of mold species was classified in the genera of Mucor, Rhizopus, Aspergillus, Alternaria and Paecilomyces . Aflatoxin-producing fungi are wide spread worldwide and can produce these toxic compounds either before or after harvest . Aflatoxins are hepatotoxic, teratogenic, mutagenic and carcinogenic. Aflatoxins are produced mainly by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 is listed as a group I carcinogen by the International agency for research on cancer .
γ Radiation is a type of electromagnetic radiation, and it is a cold method for sterilization. The γ radiation process does not create any residuals or impart radioactivity. Complete penetration can be achieved depending on the thickness of the material. It saves energy without the need for chemicals or heat . Irradiation of food using ionizing radiation is used to prevent spoilage and pathogenic microorganisms and also to guarantee the hygienic quality of foodstuffs . Microorganisms resistant are different to ionizing radiation. Bacterial spores are more resistant than yeast, molds and vegetative cells of bacteria .
The aim of the present study was to document the microbial contamination of bee pollen, evaluate the effectiveness of γ irradiation on microbial decontamination, identify the most radio-resistant contaminants and suggest the radiation decontamination doses, as well as detection of aflatoxins produced by the contaminated fungi.
| Materials and methods|| |
Bee pollen samples
A total of 30 bee pollen samples were purchased from the markets of different localities in Egypt. The samples were packed in sterile closed glass jars.
A total of 95 microbial contaminants isolated from bee pollen samples (60 bacterial and 35 fungal isolates) were used in the present study. The contaminants were purified and maintained on nutrient agar for bacteria and on Sabouraud’s agar for fungi. All cultures were stored at 4°C and subcultured monthly on the same medium.
Media and ingredients of media
Nutrient broth, Sabrouaud’s agar, potato dextrose agar and Czapek’s Dox agar were the products of Oxoid (Wade Road Basingstoke, Hampshire, RG24 8PW, United Kingdom). MacConkey agar was the product of LAB. Agar–agar was the product of Adwic (El Nasr Pharmaceutical Chemicals Co, Abu Zaabal, Center Khanka, Qaliubiya, Egypt). Yeast extract was the product of BBL. Peptone was the product of Oxoid.
Methyl alcohol was the product of Piochem (6th of October 6th Zone, Giza, Egypt), dimethyl sulphoxide was the product of Loba Chemie (Jehangir Villa, 107, Wode House Road, Colaba, Mumbai, Maharashtra 400005, India). Other chemicals used in the present study were of reagent grade.
Standard aflatoxins B1, B2, G1 and G2 were provided by the applied science division, Milton Roy Company, laboratory group (State College, Pennsylvania, USA).
Silica gel D for thin-layer chromatography was obtained from Riedel-De Haen (AG, Sleez, Hannover, Germany). Silica gel for column chromatography (0.05–0.2 mm) was obtained from ICN Pharmaceuticals (Eschwege, West Germany).
Microbial evaluation of the tested bee pollen samples
Samples were analysed after purchasing. The samples were kept at room temperature and were not incubated, refrigerated, or freezed before analysis. Surfaces of the containers were disinfected with 70% ethanol before opening the containers in a laminar air flow cabinet.
For detection of contamination of bee pollen samples, aliquots of 1 g of each tested sample were suspended in test tubes containing 9 ml of the sterile diluent (0.1% peptone, 0.1% tween 80, 0.89% NaCl in H2O), and the test tubes were shaken well on vortex (type paramix II number 65, West Germany). For bacterial detection, 0.1 ml was taken from each test tube and streaked on nutrient agar plates. The plates were incubated at 35±2°C for 24 h. For fungal detection, 1 ml was taken from each test tube and mixed with the melted Sabouraud’s agar in sterile plates and incubated at 28±2°C for up to 5 days. After incubation, the microbial growth was noticed.
Total microbial counts
The contaminated bee pollen samples were exposed to the aerobic plate count technique according to Kacaniova et al. . On nutrient agar plates for bacteria and on Sabouraud’s agar plates for fungi, the microbial counts were recorded as cfu/g.
Isolation of the microbial contaminants
According to the morphological characters of the microbial isolates, they were separated on nutrient agar for bacteria and on Sabouraud’s agar for fungi, purified and then kept on slants of the same medium, at 4°C for further investigations.
γ Irradiation studies
γ Irradiation facility
Cobalt-60 (6°Co) 220 Gamma Cell, Canada Co Ltd, located at the National Center for Radiation Research and Technology (Nasr City, Cairo, Egypt), have been utilized as radiation resources. The dose rate was 1.7 kGy/h at the time of experiments.
Determination of the microbial counts in irradiated bee pollen sample
Heavily contaminated bee pollen samples (18) were selected for this study. Aliquots of 1 g of each selected sample of bee pollen in sterile test tubes were exposed to increasing doses of γ radiation from 0.0 to 20.0 kGy, and nonirradiated samples were used as control. After irradiation, each sample was suspended in 9 ml peptone tween saline. The test tubes were shaken well on the vortex. From each irradiated sample, 0.1 ml was inoculated on nutrient agar in triplicates in a sterile plate for bacteria, and 1 ml was mixed with 20 ml Sabouraud’s agar in sterile plates for fungi. Thereafter, the inverted solidified plates were incubated at 35±2°C for 24 h for bacteria and at 28±2°C up to 5 days for fungi. After incubation, the bacterial and fungal counts were recorded as cfu/g, and log number of survivors was calculated. Histograms were constructed as log number of bacterial or fungal survivors against the radiation doses .
Determination of the microbial sublethal and lethal doses in bee pollen samples
One gram of each pollen sample (30 samples) in sterile test tubes was exposed to γ radiation doses of 0.0–15.0 kGy. After irradiation, each irradiated sample was suspended in 9 ml peptone tween saline. The test tubes were shaken well on the vortex. The microbial growths in each irradiated pollen sample were detected on nutrient agar and Sabouraud’s agar for bacteria and fungi, respectively, as mentioned before. The sublethal dose was considered as the highest radiation dose at which the least growth was detected. The lethal dose is the radiation dose at which no growth was detected .
Isolation of the most radio-resistant bacterial and fungal micro-organisms
The micro-organisms that could survive the highest radiation doses from the irradiated pollen samples (sublethal doses) were isolated and taken as the most radio-resistant isolates and hence subjected for studying the response to γ radiation.
Identification of the bacterial isolates
Identification of the bacterial isolates involved the following steps:
Examination with naked eye for morphological shape, size and color of colonies. All plates were examined, and morphologically dissimilar colonial types were cultured on MacConkey agar. Microscopical identification using Gram stain of the bacterial isolates was performed before and after exposure to γ radiation.
Identification of the most radio-resistant bacterial isolates
The identification of the most radio-resistant bacteria (MRB) was carried out according to  using the biochemical methods and confirmed at Vacsera (Egyptian company for production of vaccines) using Api system (Api CH 50 B) ‘analytical profile index’ (Biomerieux Inc., BioMérieux Marcy l’etoile, France).
Identification of fungal isolates
The fungal isolates were examined morphologically and microscopically on Czapek’s Dox agar and potato dextrose agar according to Moubasher  and Nyngesa et al. .
Identification of the most radio-resistant fungal isolates
The most radio-resistant fungal (MRF) isolates grown on Sabouraud’s agar and Czapek’s dox agar media were examined morphologically and microscopically according to Moubasher .
Study of the response of the radio-resistant bacterial strains to γ radiation
The test was carried out according to El-Bazza et al.  with some modifications in that the dense suspensions were γ irradiated and used for the study.
Study of the response of the radio-resistant fungal strains to γ radiation
The MRF strains from the irradiated pollen samples were used to study the response to γ radiation according to El-Bazza et al.  and El-Fouly et al. .
Construction of dose–response curves of the microbial isolates and calculation of D10 values
The survival curves were obtained by plotting the logarithm of the number of microbial survivors versus the radiation doses in kGy. The D10 values, which are the measure for the radiation resistance of the microorganisms to γ radiation, were read directly from the curves by finding the γ radiation dose, which reduces the microbial population by one logarithmic cycle.
Detection of aflatoxins
Aflatoxins were detected by using the fungi isolated (35) from bee pollen samples as follows:
Production of aflatoxins by the tested isolated fungal organisms and extraction of aflatoxins (if any) were carried out according to El-Bazza et al. . Purification of aflatoxins was carried out using silica gel column chromatography according to A.O.A.C. .
Detection of aflatoxins was carried out on thin-layer chromatography. The solvent system used was chloroform–acetone (90 : 10, v/v) according to Younis and Malik .
| Results|| |
In this study, the contamination of the tested bee pollen samples is summarized in [Table 1]; 29 (96.7%) samples were found to be contaminated with bacteria, 17 (56.7%) samples were contaminated with fungi and 16 (53.3%) samples were contaminated with bacteria and fungi.
Results reveal that the level of microbial contamination of the examined bee pollen samples differs between the different samples. The level of contamination of examined samples with bacteria ranges between 5.7×103 and 3.1×107, and a total of 60 bacterial isolates were isolated. The level of contamination of the samples with fungi ranges between 4.3×102 and 6.5×104, and 35 fungal isolates were isolated.
[Table 2] shows the evaluation of the bacteria contaminating the examined bee pollen sample. The order of contamination in bee pollen samples is Gram-positive rods>Gram-negative rods>Gram-positive cocci.
|Table 2 Evaluation of the bacteria contaminating the examined bee pollen samples|
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[Table 3] illustrates that a total of 35 fungal organisms were isolated on Sabouraud’s agar from the studied pollen samples, and the order of contamination was Penicillium spp.>A. flavus>Aspergillus niger>Aspergillus ochracueus.
|Table 3 Evaluation of the fungi contaminating the examined bee pollen samples|
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In our study, the detection of aflatoxin production by the fungi isolated from the tested pollen samples was carried out in Sabouraud’s broth supplemented with 0.5% yeast extract; the results of the thin-layer chromatographic plates show that only three isolates were aflatoxin B1 producers. Aflatoxin B2, G1 and G2 were not produced.
[Figure 1],[Figure 2],[Figure 3],[Figure 4] illustrate the results of exposure of the selected heavily contaminated bee pollen samples to γ radiation doses that ranged from 0.0 to 15.0 kGy; the results show that the counts of bacteria and fungi in irradiated samples decreased by increasing the γ radiation doses. A dose of 7.0 kGy showed decontamination of the microbial organisms.
|Figure 1 Effect of γ radiation on the total bacterial counts (cfu/g) in the heavily contaminated pollen samples (5, 6, 9, 10 and 11).|
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|Figure 2 Effect of γ radiation on the total bacterial counts (cfu/g) in the heavily contaminated pollen samples (13, 15, 17, 27 and 28).|
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|Figure 3 Effect of γ radiation on the total fungal counts (cfu/g) in the heavily contaminated pollen samples (1, 2, 3, 4 and 7).|
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|Figure 4 Effect of γ radiation on the total fungal counts (cfu/g) in the heavily contaminated pollen samples (11, 12, 15, 16 and 23).|
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The results of exposure of bee pollen samples to γ radiation doses that ranged from 0.0 to 25.0 kGy showed that 37 radio-resistant bacterial contaminants were isolated from 30 irradiated bee pollen samples at sublethal dose levels between 2.0 and 5.0 kGy. The radio-resistant bacteria were identified as Gram positive rods (34), two isolates as Gram negative rods and only one isolate as Gram positive cocci. Percentage of the radio-resistant bacteria are illustrated in [Figure 5]. In contrast, 19 radio-resistant fungal contaminants were isolated from 17 irradiated bee pollen samples at the sublethal dose levels of 1.5 and 2.0 kGy. The isolates were identified as Penicillium spp.
[Table 4] shows that the MRB that survived the highest dose level under treatment (5.0 kGy) were identified, using analytical profile index 50 CH system, as Bacillus megaterium, Bacillus subtilis and Bacillus pumilis. The MRF isolates that survived the highest dose level (2.0 kGy) were Penicillium chrysogenum, Penicillium expansum and Penicillium corylophilum ([Table 5]).
|Table 4 Identification of the most radio-resistant bacteria isolated from irradiated bee pollen samples|
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|Table 5 Identification of the most radio-resistant fungi isolated from irradiated bee pollen samples|
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In the present study, the MRB and MRF were selected for studying their response towards γ radiation through plotting their dose–response curves and calculation of the D10 values from the curves by finding the γ radiation doses that reduce the microbial population by one logarithmic cycle. The dose–response curves of B. megaterium, B. pumilis, B. subtilis and of Penicillium spp. were constructed. The curves showed exponential rate of death. The mean of the D10 values of B. megaterium isolated from samples number 1, 7 and 9 were calculated to be 3.5±0.00, 1.4±0.10 and 3.2±0.15, respectively. While, the mean of the D10 values of B. pumilis isolated from samples 13 and 19 were 2.4±0.17 and 1.5±0.00, respectively. The mean of the D10 values of B. subtilis isolated from sample numbers 6, 21, 24 and 28 were calculated to be 2.1±0.12, 1.5±0.00, 2.7±0.25 and 2.3±0.15, respectively. Furthermore, the mean of the D10 value of P. expansum isolated from sample number 4 was calculated to be 0.48±0.03. In contrast, the mean of the D10 values of P. chrysogenum isolated from sample number 1 were 0.64±0.01 and 0.48±0.05, while the mean of the D10 values of P. corylophilum isolated from samples number 4 and 21 were found to be 0.54±0.01 and 0.49±0.01, respectively ([Figure 6],[Figure 7],[Figure 8],[Figure 9]).
|Figure 6 Dose–response study for Bacillus megaterium isolated from irradiated pollen samples (no. 1, 7, 9).|
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|Figure 7 Dose–response study for Bacillus pumilus isolated from irradiated pollen samples (no. 13, 19).|
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|Figure 8 Dose–response study for Bacillus subtilis isolated from irradiated pollen samples (no. 6, 21, 24, 28).|
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|Figure 9 Dose–response study for Penicillium spp. (a) isolated from irradiated pollen samples (no. 1a, 1b, 4c,4d, 21e).|
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| Discussion|| |
Bee pollen has the image of being natural, healthy and clean. However, the products are produced in an environment, polluted by different sources of contamination. Thus, it is of utmost importance to bee keepers to localize and exclude the different contamination sources .
In the present study, the samples were contaminated with bacteria and/or fungi, and detection of the contamination differed between the different bee pollen samples from different localities.
According to Campos et al. , pollens should have the following microbial aspects: absence of Salmonella/10 g; absence of Staphylococcus and Escherichia More Details coli/1 g; total aerobic bacterial count could not exceed more than 105 cfu/g; total mould yeast count should be less than 5×104 cfu/g; and the maximum count of enterobacteria is 100 cfu/g.
The results obtained by Nogueira et al.  for the parameters that indicate commercial quality are in agreement with the Argentinian Food Code, which establishes the maximum of 1.5×l03 cfu/g for moulds and yeasts. In their study, moulds and yeasts were detected in 50% of the samples, while other researchers , found it in all the samples.
Comparing of Campos et al.  recommendations with our study, 44.8% of the bacteria contaminated bee pollen samples have acceptable criteria (<105 cfu/g), while 47.1% have the acceptable criteria for fungi (<1.5×l03 cfu/g), when comparing with the Argentinian food code. The highest contamination was seen in bee pollen with the Gram-positive rods and least contamination with the Gram-positive cocci.
Five species belonging to the genus Bacillus were isolated from pollen and bee bread. Of the 41 isolates, 33 were B. subtilis, which was the only species associated with all pollen and bee bread samples. Possibly because of some role of the organism in the elaboration of bee bread and/or because of the ability of the organism to survive in this particular environment. B. megaterium, B. licheniformis, B. pumilus, and B. circulaus were also isolated. Only B. subtilis was found in pollen from the flower. As the greatest number of Bacillus isolates and species was found in pollen from the trap, the foraging bees may have added these organisms to the pollen, when making a suitable mass to carry back to the hive .
There were 21 species of thirteen genera of microscopic fungi in the pollen samples. Most often the presence of the species Mucor, Fusarium spp., Rhizopus (Rhizopus arrhizus, Rhizopus nigricans) and Aspergillus was detected .
The aflatoxins are a group of mycotoxins produced by certain Aspergillus spp., in particular, A. parasiticus, A. flavus, A. nomius, and A. pseudotamarii. Three species of aflatoxin-producing fungi were present, and six pollen samples contained aflatoxins. The microbial and aflatoxin contents of pollen were strongly related to the previous handling and the methods of drying and storage used ,.
In the present study, aflatoxin B1 was detected in 8.6% of the tested fungi isolated from the pollen samples, while aflatoxin B2, G1 and G2 were not detected.
In other study, it was reported that 29% of the fungal isolates were A. flavus plus A. parasiticus produced the aflatoxin B1 in cultures. Aflatoxin B2 was detected in only 10% of the cultures. Aflatoxins G1 and G2 were not detected in cultures under the assayed conditions .
Irradiation of food using ionizing radiation (γ and x rays or electron beam) is used to inactivate both spoilage and pathogenic microorganisms and to guarantee the hygienic quality of several foodstuffs .
There was gradual decrease of the microbial counts in the selected heavily contaminated bee pollen samples irradiated at γ radiation doses of up to 7.0 kGy. This maximum dose was enough for decontamination of bacteria and fungi in the bee pollen samples under test.
It was found that 60Co γ radiation had an effective influence on the sterilization of the pine bee pollen and that the intensity of the bacterial contamination decreased with the increase of the radiation doses. The pine pollen without radiation treatment had a high number of bacterial contaminations. The pine pollen radiated with different doses had excellent results, especially with the doses between 6 and 12 kGy . Moreover, γ rays at 7.5 kGy reduced the total microbial loads in bee pollen below 102 cfu/g without affecting its physiochemical properties such as amino acid, fatty acid composition also; thiobarbituric acid value, mineral content and pigment were not significantly changed by γ ray . In contrast, exposure of bee pollen to 4 kGy was sufficient for microbial decontamination or reducing the count to less than 10 cfu/g .
In 1997, FAO/IAEA/WHO group’s study on high-dose irradiation examined and evaluated the results of safety studies carried out on foods irradiated on the dose range 25–60 kGy to achieve the intended technological objective. The conclusion for such foods are both safe to the consumer and nutritionally adequate ,.
Irradiation of foods up to an overall average dose of 10 kGy introduced no specific nutritional or microbiological problems . The irradiation dose of 10 kGy is the maximum dose allowed in food according to the Codex standards .
Exposure of the contaminated bee pollen samples to γ radiation led to isolation of radio-resistant bacteria that were identified as Gram-positive rods, Gram-negative rods and Gram-positive cocci, at a dose that ranged between 2.0 and 5.0 kGy, while the isolated radio-resistant fungal organisms were identified as Penicillium spp. at 1.5–2.0 kGy.
The study of the effect of γ radiation on micro-organisms isolated from contaminated samples showed that the lethal dose level of bacteria and fungi isolates ranged between 2 and 25 kGy .
Radiation resistance can be associated with the D10 value (the dose of γ radiation required to reduce a microbial population by 90%), and it is the measure for the radiation resistance of the micro-organisms to γ radiation ,.
In our study, the mean of the D10 values of the MRB organisms, B. subtilis (4), B. megaterium (3) and B. pumilus (2), which were isolated at 5.0 kGy from the irradiated bee pollen samples, were found to be from 1.5 to 2.7 and from 1.4 to 3.5 and from 1.5 to 2.4, respectively. However, for the fungal organisms, the D10 values were found to be 0.48 for P. expansum (1) and 0.48, 0.64 for P. chrysogenum (2), while it was 0.49, 0.54 for P. corylophilum (2). All the microbial strains showed an exponential rate of death.
The D10 values of Bacillus cereus and A. flavus were found to be 1.02 and 0.48, respectively , while the D10 values of four different Bacillus spp. ranged from 2.3 to 2.9 kGy . B. cereus strains exhibited exponential rate of death, and the D10 values were calculated to be 1.9 and 2.2 kGy .
Moreover, the D10 values of two Gram-positive spore-forming B. megaterium were calculated to be 1.7 and 1.8 kGy. Furthermore, D10 values of the most radio-resistant strains of B. sphaericus (2) were found to be 0.85 and 1.25 kGy, while two strains of B. pantothenticus were 0.4 and 0.5 kGy, and six strains of A. niger ranged between 0.7 and 1.0 kGy; the D10 value for P. chrysogenum was 0.95 kGy. The microbial strains exhibited exponential rate of death, while one strain of B. pantothenticus and one of A. niger exhibited a nonexponential rate of death. In contrast, the D10 values of Bacillus spp. strains ranged from 0.83 to 0.99 kGy ,,.
Our results and that obtained by other investigators show that the resistance and response of micro-organisms towards radiation differ between the different microorganisms and the different strains of the same micro-organisms. This may be attributed to species and number of micro-organisms, medium suspending the microorganisms, temperature, water activity, oxygen effect and the use of sensitizing compounds during the irradiation process ,.
| Conclusion|| |
γ Radiation technology can offer the process of microbial decontamination as a means of achieving microbiological safety limits. A dose level of 7.0 kGy was enough to inhibit the growth of micro-organisms on the bee pollen, and eliminate pathogenic micro-organisms including fungi, which may produce carcinogenic aflatoxins.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]