الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
β-glucan is one of the most promising bioactive compounds, it consists of a heterogenous polysaccharides. Glucan is recommended for use as an ingredient, additive, or supplement in animal feed. The manufacture of yeast origin beta (1, 3-1, 6)-glucan for food, feed, medicinal, and wastewater treatment applications is gaining biotechnological and industrial attention. In addition, some novel roles of glucan in cancer treatment have recently been described, ranging from glucans being a delivery platform for anticancer drugs to regulating trained immunity. The aim of this study was maximize the beta glucan polymer from yeast cultures by investigation the environmental and nutritional factors and characterized its properties by different techniques. Some of agro industrial wastes were used to design low cost medium using response surface methodology. Additionally, it was examined the potential use of ionizing radiation technology for modified yeast strains to improvement glucan production.
The obtained results can be summarized as follow:
1. Isolation of yeasts from different sources
About 13 different samples of different food stuff such as (apples, banana, figs, grapes, plums, and prunes), fermented juices of banana, guava, orange, and sugarcane as well as like dairy products of cottage cheese, cream, and processed cheese were used as isolation sources for yeast. A total of forty-nine yeast isolates were selected (rotten fruits, fresh juices and some dairy products). The highest number of yeast isolates was achieved by grapes and figs (seven isolates) which accounts for nearly 14% of total. Whereas the cream and banana juice samples gave the lowest number of isolates (one isolate) followed by orange and purnes sample (two isolates) represent 2% and 4% of total isolates number, respectively. selected yeasts were characteristically differentiated according to morphological phenotyping and conventional microscopic observation for choosing yeasts belonging to different genera.
2. Screening and genetically identification for the high beta-glucan producers
Yeast β-glucan production by collected isolates was categorized into three groups according to beta-glucan yield in comparison to the reference yeast strain; low yield (Less than 50 mg glucose/100ml) (15 isolates), moderate yield (50-100 mg glucose /100ml) (19 isoletes) and high yield (More than 100mg glucose/100ml) (16 isolates). The highest β-glucan production was achieved by 4F yeast isolate (350mg glucose/100ml) followed by 1Cc yeast isolate (150 mg glucose/100ml).
Analyzed genomes of both yeast isolates (1Cc & 4F) showed a high degree of similarity to Kluyveromyces lactis and Meyerozyma guilliermondii, and they were accessed in the Genbank with accession No. OP967932 and OP967929, respectively.
3. Identification of produced yeast beta-glucan by selected strains
Beta-glucans recovered from Kluyveromyces lactis NCCRT-2A and Meyerozyma guilliermondii NCCRT-18F were analyzed using FT-IR to detect the major functional groups. characteristic peaks appeared at 571.76 and 615 cm-1 represent the skeletal vibration of glucose pyranose moiety. The absorption peak at 1013.67 cm-1 revealed 1,3 -linked D-glucose molecules forming the linear backbone of β-glucan. Several peaks can be easily recognized between 1200 and 1400 cm-1 which mainly reflect carbonyl linkage. The band at 1640.59 cm-1 was related to the tightly bound water molecules in the β-glucan polymer.
4. Factors affecting yeast beta-glucan production
The highest beta glucan achieved by K. lactis NCRRT-2A in cultivation medium (basal medium) after 5 days at pH 6, 20oC under shaking condition with lactose and sodium nitrate as a carbon and nitrogen sources respectively, being 250 mg glucose/ 100ml. For Meyerozyma guilliermondii NCCRT-18F the highest beta glucan achieved after 3 days at pH 6, 20oC under shaking condition with maltose and ammonium sulfate as a carbon and nitrogen sources respectively, being 470 mg glucose/ 100ml.
5. β-glucan biosynthesis in non-conventional medium
Six fruit and vegetable waste extracts (FVWs) including banana peels, guava pulp, mango peels, orange peels, potato peels, sweet potato peels as well as whey for supporting β-glucan biosynthesis, by K. lactis NCRRT-2A and M. guilliermondii NCRRT-18F. Whey significantly supports the maximum β-glucan biosynthesis by K. lactis NCRRT-2A (355.5 mg/100 ml). Regarding M. guilliermondii NCRRT-18F, the water extract of potato peels has the maximum impact on enhancing β-glucan biosynthesis recorded (495 mg/100 ml).
6. Statistical optimization of β glucan biosynthesis on low cost cultivation medium
Plackett-Burman method offers a design of 20 runs to evaluate the influence of five nutritional variable which are N2, P, Mg sources, minerals solution and vitamins (as yeast extract) on β-glucan biosynthesis. The actual values of beta glucan ranged from 83.9 to 332.5 mg/100ml for K. lactis NCRRT-2A and from 190.1 to 662.6 mg/100ml for M. guilliermondii NCRRT-18F. ANOVA statistical analysis showed that there non-significant interaction between all variables under investigation for both K. lactis NCRRT-2A and M. guilliermondii NCRRT-18F at p-value 0.05 which recorded p-values ranged from 0.076 to 0.576 for the fromer starin and from 0.079 to 0.742 for the last strain.
7. characterization of produced yeast beta-glucan
The elements constituents were analyzed and the recovered samples were almost entirely composed of β-D-glucose with very low protein content indicating a high purity of the recovered 𝛽- glucan.
Thermal analysis for recovered yeast beta-glucan didn’t show any glass transition peaks. Meanwhile, a very weak endothermic peak was noticed in the β-glucan recovered from Kluyveromyces lactis NCCRT-2A and Meyerozyma guilliermondii NCCRT-18F at 8.8- 9oC and at 7.4 - 7.7oC; respectively, these peaks were followed by a broad endothermic peak representing the water evaporation. Thermo gravimetric analysis (TGA) graphs show no drastic difference between TGA patterns of yeast β-glucan recovered from K. lactis NCCRT-2A or M. guilliermondii NCCRT-18F.
8. Biosynthesis and characterization of beta-glucan nanoparticles
Yeast β-glucan nanoparticles (βGN) were synthesized from the β-glucan polymer using N-dimethylformamide as a stabilizer and characterized using UV–vis spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). UV-Vis spectra of βGN exhibited a broad peak (220-300nm) resembling the successful formation of nanoparticles. TEM micrograph of βGN showed spherical shape particles with smooth surfaces. The size of formed nanoparticles has a broad range; from smaller than 20 nm to larger than 90 nm. The average size of βGN was about 300 nm as determined by DLS. The quantitative variation of functional groups between β-glucan polymer and βGN was evaluated by FT-IR.
9. Cytotoxicity
The influence of yeast β-glucan at increasing concentrations (31.25 to 1000µg/ml) was studied on the toxicity of normal human amnion (Liver cells) and Liver cancer (HepG2) cell lines by MTT assay. The results revealed that the toxicity (%) of recovered β-glucan on normal human amnion (Liver cell) cells increased slightly with elevating concentration to 250 µg/ml whereas higher concentrations; 500 and 1000 µg/ml of β-glucan dramatically enhanced toxicity up to 54.42 and 89.42%; respectively. Additionally, it can be noticed that the IC50 of β-glucan was 478.7 µg/ml. The HepG2 carcinoma cells seemed to be damaged since changed to diploid or irregular shape and clumped together as compared to the negative control. While, a negative effect of βGN on the viability of Normal Homo amnion and Hepatic cancer (HepG2) cell lines up to 250 μg/ml. Then, the viability of HepG2 cell line declined gradually and reached to nearly 50% at 1000 μg/ml whereas no lethal effect was observed on the viability of normal cell lines.
10. Radiation
Gamma radiation showed a positive effect on β-glucan yield which increased gradually with radiation dose till reached the maximum at 1kGy being 219mg glucose/100ml by K. lactis NCRRT-2A. log (N/N0) diminished by incremental gamma dose and reached the lowest at 4 and 2 kGy being - 5.39 and - 4.04 for strains K. lactis NCRRT-2A and M. guilliermondii NCRRT-18F respectively. The D10 of gamma radiant was 0.8 KGy for the former strain and 0.446 kGy for the latest one.