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العنوان
BACTERIAL PRODUCTION OF PIGMENTS /
المؤلف
AHMED, SOHILA GAMAL EL-SAYED.
هيئة الاعداد
باحث / سهيله جمال السيد أحمد
مشرف / محمود محمد زكى
مشرف / السيد أحمد صالح
مناقش / محمد عثمان عبد المنعم
تاريخ النشر
2021.
عدد الصفحات
165 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
الزراعية والعلوم البيولوجية (المتنوعة)
تاريخ الإجازة
1/1/2021
مكان الإجازة
جامعة عين شمس - كلية الزراعة - قسم ميكروبيولوجيا زراعية
الفهرس
Only 14 pages are availabe for public view

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Abstract

SUMMARY AND CONCLUSION
Pigment producing bacteria hold a promising potential to meet present-day challenges. They are preferred than other natural sources due to several reasons; produce colors of different shades, safe, fast growing, production being independent of seasons and cost effectiveness can be further enhanced by growing on low-cost substrates. Furthermore, natural pigments not only improve the marketability of the product but also add extra feathers like antimicrobial, antioxidant, anticancer, anti-inflammatory and used in textile dying.
The purpose of this research was to isolate and classify pigments producing bacteria and enhance the fermentation conditions for maximum pigment yield. Moreover, evaluate the selected pigments as antioxidants, cytotoxicity, and antibacterial effects against foodborne pathogenesis, in addition, an applied experiment was designed for the use of the pigment tested in the dyeing of garments.
The obtained results can be summarized as follows:
1- Forty-six local pigments producing bacterial isolates were isolated from different sources of soil (23 isolates), air (5 isolates), and food (18 isolates). Only 12 isolates produced extracellular pigments (fluorescence and blue green pigments) and 34 isolates produced intra-cellular pigments (red, brown, yellow, rose, and orange pigments).
2- Extraction of pigment using solvent extraction and ultrasonication are a significant phenomenon for opting the appropriate solvent for extracting high yields of pigments. Different solvents of ethanol, methanol, acetone, chloroform, ethyl acetate, and mixture of methanol and ethanol were used to extract the pigment from the inner cells. Ethyl acetate was more efficient than all other solvents tested in extracting most pigments and was therefore selected for further studies.
3- The maximum absorption of extracellular fluorescence and blue green pigments were observed at 400 nm and 520 nm. As well as, for intra-cellular red, brown, yellow, rose, and orange pigments were observed at 530 nm, 320 nm, 460 nm, 470 nm, and 440 nm, respectively.
4- The highest significant (p ≤ 0,05) fluorescence, blue green, red, brown, orange, yellow, and rose colours of pigments (O.D) values were noted by SF2, SG1, LRe6, SB1, AO2, WY12, and WRo5 isolates being 0.62, 1.42, 1.05, 1.11, 0.45, 0.98, and 0.40, respectively. In addition, the yield of extracellular pigments was 1.46 and 0.88 g achieved by both isolates of SG1 and SF2. Whereas intra-cellular pigments yield was 2.18, 2.09, 1.12, 1, and 1.30 g extracted from LRe6, SB1, AO2, WRo5, and WY12, respectively.
5- Pigments producing bacterial isolates were evaluated as antibacterial activity against 7 pathogenic bacterial strains using agar well diffusion method. The results revealed that:
- Both blue green and fluorescent extra-cellular pigments produced from SG1, and SF2 inhibited the growth of all the tested pathogenic bacteria with spectrum activity of 100%.
- Brown, yellow and rose intra-cellular pigments produced from SB1, WY12, and WRo5 were achieved 100% spectrum activity against all the tested pathogenic bacteria whereas red and orange pigments produced from LRe6 and AO2 were 85.7 and 42.9 % of spectrum activity, respectively.
6- The most efficient blue green, red, brown, yellow, rose, and orange pigments producing from SG1, SB1, LRe6, WY12, WRo5, and AO2 isolates were completely identified up to species according to cultural, morphological, and physiological characteristics as Pseudomonas aeruginosa, Azotobacter chroococcum, Serratia marcescens, Micrococcus luteus, Micrococcus roseus, and Micrococcus kristinae, respectively.
7- The antioxidant potential of bacterial synthesizing extra- and intra- cellular pigments was studied by estimating free radicals scavenging assay. The scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) exerted by extra-cellular pigment of blue green showed 70 % inhibition. Whereas DPPH scavenging activities exerted by intra-cellular pigments of rose, orange, red, brown and yellow gave 40, 50, 65, 72, and 80 % inhibition, respectively.
8- Allergy of the blue green, brown, red, yellow, rose, and orange pigments produced from most efficient strains of P. aeruginosa, A. chroococcum, S. marcescens, M. luteus, M. roseus, and M. kristinae showed no erythema or oedema on the ratsꞌ skin.
9- The cytotoxicity of the blue green, brown, red, yellow, rose, and orange pigments produced from most efficient strains of P. aeruginosa, A. chroococcum, S. marcescens, M. luteus, M. roseus, and M. kristinae was estimated using neutral red uptake (NRU) assay against human normal lung cell lines (Wi38) at different concentrations (25, 50, and 100 µg/ml) for brown and orange pigments, and (12.5, 25, and 50 µg/ml) for blue green, red, yellow, and rose pigments. Results appeared that orange pigment hadn’t any cytotoxicity. The IC50 values of brown, blue green, red, yellow, and rose pigments were 145, 47, 113.5, 593, 37.6 µg/ml, respectively.
10- The blue green, red, brown, yellow, rose, and orange pigments used as application of dying fabrics cotton. The result appeared that red pigment of S. marcescens dyed cotton fabric with pretty deep shade, followed by blue green of Pseudomonas aeruginosa, while the remainder four pigments (brown, yellow, rose, and orange) of A. chroococcum, M. luteus, M. roseus, and M. kristinae exhibited very poor dyeing abilities.
11- Molecular identification of the most efficient red pigment isolate (Serratia marcescens LRe6) by 16s DNA, and it was confirmed to belong to Serratia marcescens strain (NBRC 102204) 16s ribosomal RNA, partial sequence with similarity of 99.8 %.
12- Fourier transform infrared (FT-IR) spectroscopy of prodigiosin produced by S. marcescens LRe6 showed the presence of many functional chemical bonds from hydroxyl, amine, methyl, methylene, carboxylic, and aromatic rings.
13- Five oil sources (fish waste oil, Falafel waste oil, sesame oil, fenugreek, and peanut powder) were used to increase production of red pigment from S. marcescens LRe6 strain. The result showed that sesame oil at 1% concentration was more significant density (O.D) of red pigment than other sources, followed by peanut powder and fenugreek.
14- Statistical experimental designs for optimization of prodigiosin synthesis from S. marcescens LRe6 showed that:
- Nine various factors including sesame oil, peptone, beef extract, NaCl, temperature, pH, agitation speed, incubation period and inoculum size were selected for optimize production of pigment yield (O.D) by the tested strain using minimum run resolution IV (MRR-IV) design. The most significant variables were peptone, temperature, pH, and incubation period.
- Optimized the 4 significant factors using central composite split plot (CCSP) design, which revealed that the highest values of pigment density were 1.250.and respectively at 10 g/L peptone, pH 7, and incubated at 23°C for 72 hours.
15- from the above mentioned results, it could be concluded that the maximum yield (O.D 1.250) of prodigiosin red pigment produced by S. marcescens strain LRe6 could be obtained by using modified medium containing: sesame oil 10 ml, peptone 10 g/l, beef extract 5 g/l, NaCl 1 g/l and inoculum size 2% at pH 7 and temperature 23°C after 3 days of incubation, under submerged condition (200 rpm).