Study On Preparation Of Gracilaria Lemaneiformis Agarose And Its By-products

?High gel strength agar was prepared by Gracilaria lemaneiformis in this study, and Gracilaria lemaneiformis agarose was prepared by modified DEAE-cellulose method, an ion chromatography method for the determination of sulfate content in agar and agarose was also established. The DEAE-cellulose was recovered from agarose preparation; the DEAE-cellulose was eluted with NaCl solution in order to prepare the agaropectin. Agaropectin oligosaccharides was prepared by the agaropectin degrading using hydrochloric acid, then the antioxidant activity of agaropectin oligosaccharides was evaluated in this article. After removing the filter aid from Gracilaria lemaneiformis residue, the Gracilaria lemaneiformis dietary fiber was prepared. In order to enhance the activity of dietary fiber, the Viscozyme L was used in the hydrolysis process to hydrolysis the dietary fiber.The main tasks and conclusions of this paper are as follows:1. In order to study the relationship between the condition of cold alkali processing on Gracilaria lemaneiformis and gel strength of agar, it was studied that the quantitative relation between the three factor : temperature (T), alkali concentration (M), processing time (t) and gel strength(GS) of agar from Gracilaria lemaneiformis by using orthogonal and uniform design. Regression equation , GS=27.72×T+19.69×M+2.75×t-488.62 ,P<0.0001, was fitted by multiple linear regressing. From the regression equation, among the three factors, the effect on the GS of agar was T>M>t. The validating test was carried under the three factors level, and the result showed that there was no significant difference between the test-value and the forecasting-value(P>0.05).Therefore, the regression equation could highly forecast the GS of agar from Gracilaria lemaneiformis, and it could provide scientific basis for optimizing process conditions, improve quality and reducing cost on the agar from Gracilaria lemaneiformis.2. The Gracilaria lemaneiformis agarose was prepared by KMnO₄-H₂C₂O₄ bleaching , ethanol immersion and DEAE-cellulose purification , respectively. Orthogonal test was carried out to optimize the process parameters, the physical and chemical index, electrophoretic performance of agarose was determined . The results showed that the optimum conditions of bleaching assay are concentration of KMnO₄ 0.10%, pH 6.0, bleaching time 5min, concentration of H₂C₂O₄ 0.30%, after bleaching assay, the whiteness (HW value) of agar is 82.98, the gel strength is 1083 g/cm²; the optimum conditions of ethanol immersion assay are solid-liquid ratio 1:40, concentration of ethanol 60%, treatment time 6h, after immersion by ethanol, the transparency of 1.0% agar(transmittance value) is 50.2%. The ash content, gel strength and sulfate group content of the agarose prepared by the optimal method are 0.25%, 1127g/cm², 0.24%, respectively. Gentian violet electrophoresis, electroendosmosis and genome DNA gel electrophoresis are also performed in this study, which is showed that the agarose prepared by modified DEAE-cellulose method was suitable for biochemistry and molecular biology electrophoresis.3. Using agarose as experimental materials, after degrading the organic components in agarose by ashing method, we use ultrapure water and 1.0mol/L hydrochloric acid solution to dissolve the ash, then detected by Ion Chromatography (IC) and barium sulfate turbidimetry (BST) respectively. When sample detected by IC, in the SO₄^2- content range of 1.0～15.0mg/L, the linearity is good with R²=0.9996.The precision of detecting results by IC is good with RSD=1.40%(n=6), which recovery of standard addition is from 86.33% to 97.40%; As well as sample detected by BST, in the SO₄^2- content range of 6.0—30.0mg/L, the linearity is good with R²=0.9991. The precision of detecting results by BST is good with RSD=3.51%(n=6), which recovery of standard addition is from 84.17% to 96.67%.The results show that both IC and BST could detect the sulfate content accurately.4. The DEAE-cellulose was eluted with NaCl solution in order to prepare the Gracilaria lemaneiformis agaropectin. Infrared spectrum analysis of Gracilaria lemaneiformis agar and agaropectin was performed, Gracilaria lemaneiformis agaropectin was degraded to prepare agaropectin oligosaccharides. After evaluating the antioxidant activity of agaropectin oligosaccharides in vitro, the results showed that both of the agar oligosaccharides and agaropectin oligosaccharides could scavenge the·O₂^- and DPPH free radical significantly. The inhibition rate of agaropectin oligosaccharides was higher than that of agar oligosaccharides, but lower than that of ascorbic acid. The IC₅₀ against·O₂^- and DPPH free radical were 0.5 mg/ml and 7 mg/L respectively. They are both excellent antioxidants, and playing a role to maintain health of body. The results of the infrared spectrum for agaropectin were as follows: characteristic absorption at 851.3 cm^-1 shows that there is S-O at C4 of D-galactose, characteristic absorption at 932.2 cm^-1and 1075.6 cm^-1 shows that there is 3,6-AG in the sample, furthermore the characteristic absorption at 932.2 cm^-1 also shows that there is 6-SO₄ in agaropectin. The strong absorption at 1255.2 cm^-1 shows that there are plenty of sulfate groups in agarpectin. 5. The filter aid of the Gracilaria lemaneiformis algae residue was removed by a centrifugal method, and the dietary fiber was prepared, the yield of dietary fiber is 21.4%. The expansive force and water retention ability of the dietary fiber were 3.37 ml/g and 285% respectively. Using the Viscozyme L to hydrolysis the dietary fiber in order to enhance the activity of dietary fiber. The optimum conditions of the hydrolysis process are solid-liquid ratio 1:30, enzyme amount 20 FBG/g, pH 4.5, enzymolysis time 2.5 h, enzymolysis temperature 55℃. Under these conditions, the yield of the soluble dietary fiber was 24.63%, the expansive force and water retention ability of the dietary fiber after activating were 10.25 ml/g and 542% respectively.