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The Research on Extraction Technology of Agarose from Gracilaria lemaneiformis

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Tutor: WuChengYe; SuYongChang
School: Fujian Agriculture and Forestry University
Course: Aquatic Products Processing and Storage Engineering
Keywords: Gracilaria lemaneiformis,agar,agarose extraction and purification,response surfa
CLC: TS254.1
Type: Master's thesis
Year:  2013
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Abstract:
With the rapid development of biotechnology, agarose is more and moreattentioned by people as a kind of neutral polysaccharide, it has been widely used ingel electrophoresis, affinity chromatography, molecular sieve, immune and so on.Gracilaria lemaneiformis has been widely cultivated in fujian coastal as a typicalrepresentative of gracilaria algaehas. Using of Gracilaria lemaneiformis extractingagar and agarose can not only improve its added value and reduce the waste ofresources. In this study, we should optimize the agar extraction process usingGracilaria lemaneiformis as raw materials and compare the different extractionmethods of agarose. We selecte EDTA-Na2and DEAE-cellulose method as extractionand purification method of agarose, through the optimization of these two process, wedetermine the best extraction process and extract the high quality agarose. Then wedetermine the physical and chemical properties of agarose and analyse its structure.Main research contents are as follows:1Agar extraction process includes alkali treatment, acidification, bleaching,boiling gel, freezing and thawing, drying etc. Alkali treatment and bleaching processinfluence the quality of agar greatly. This study conducts the single factor experimentto select the appropriate factors and levels, with sulfate content and gel strength asevaluation index and alkali treatment concentration, temperature and time,concentration of bleaching as impacting factors. The concentration of sodiumhypochlorite is0.1%(in terms of available chlorine concentration). UsingBox-Behnken response surface method analyse the significance of alkali treatmentfactors and interactions, geting the optimal extraction process: the concentration ofalkali treatment is5%, alkali treatment time is2h, alkali treatment temperature is75℃, the sulfate content is1.72%under this condition.2Compare ten different extracting methods such as EDTA-Na2, DEAE-cellulose,alkali treatment, NaI, ammonium sulfate and so on in extraction principle, extractionprocess, product indicators (sulfate content, gel strength, whiteness, yield, EEO etc) ofagarose to selecte the extraction method, which process is simple and low sulfatecontent, high gel strength, high whiteness, high yield, low EEO. The final selection is EDTA-Na2and DEAE-cellulose method as the extraction and purification methods ofthe study by comprehensive consideration.3The preparation process of agarose include extraction and purification twosteps. Firstly, using EDTA-Na2method extracts agarose from agar and researchvarious influencing factors of EDTA-Na2concentration, extraction temperature andextraction time on the sulfate content and gel strength in extraction process withsingle factor experiment, then we should use response surface method to optimizeextraction process and determine the optimal process parameters: EDTA-Na2concentration is4.5g/L, extraction temperature is65℃, extraction time is3h.Based on forecast model, the sulfate content is0.48%and gel strength is1073.09g/cm2of agarose extracting by the optimal process. Then DEAE-cellulose method isused to purify the agarose, we should research the influencing factors ofDEAE-cellulose content, extraction temperature and extraction time on the sulfatecontent and gel strength with the single factor experiment, using response surfacemethod optimize the process parameters: the quantity of DEAE-cellulose added is55g, extraction temperature is80℃, extracting time is2.5h.4With the optimum process to extract the agarose products, through determiningthe physical and chemical indicators and analysing the structure of homemade agarosewe know: the sulfate content is0.17%, the freezing temperature is38.8℃, the meltingtemperature is87.8℃, EEO is0.027, whiteness is80.9, transparency is0.396, thedistance of crystal violet electrophoresis is9.64cm longer than the agarose maded inBIOWEST company, the ash content is0.554%;The agarose powders are observedunder the scanning electron microscopy (SEM), which shows it is loose macroporousstructure; The energy spectrum diagram shows that agarose contains only twoelements C, O. The genomic DNA electrophoresis shows that it has a goodelectrophoretic performance. Using infrared spectrum and nuclear magnetic resonance(NMR) analyse the structure of the agarose lastly, the results show that it is mainlycomposed of galactose and3,6-AG, it has low sulfate content.
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