Enzymatic Preparation Of Agar Oligosaccharides And Its Physicochemical Properties

Many data indicate that degradation products of agarose show some specific functional activities.Up to now,effective means for agarose degradation are chemical hydrolysis and agarase hydrolysis.Based on the previous work,the agar oligosaccharides were prepared by the screened commercial cellulase.The process conditions were optimized,and the hydrolyzs were preliminary identified and studied.The main conclusions are summarized as follows:1)The conditions of cellulase enzymatic hydrolysis degradation of agarose was studied with the yield of reducing sugar as the index.Following single factor experiments,the enzymatic hydrolysis conditions of agarose were optimized by four factors(cellulase content,enzymolysis temperature,pH and enzymolysis time)Box-Behnken experiment design and response surface methodology(RSM),and a regression model was established for predicting the enzymatic hydrolysis yield at differents levels of cellulase conten,enzymolysis temperature,pH and enzymatic hydrolysis time.The optimum enzymolysis process:the substrate content was 0.2%,the cellulase content was 1909.3 U/mL,the enzymolysis temperature was 50.0?,the pH was 4.9 and the enzymolysis time was 24.2 h.Under these conditions,the enzymolysis rate of agarose reached(29.58%±0.26%).The results of thin layer chromatography(TLC)showed that the main products were agar oligosaccharides with polymerization degree of 2-6.2)The results of static rheological properties show that,agarose and agar oligosaccharides L1,L2 and L3 solutions are shear thinning pseudoplastic fluids whose flow characteristics conform to the Power Law(or Ostwald)model.The apparent viscosity and shear stress increase with the concentration increasing,the solution farther deviates from the Newtonian fluid and the stronger the pseudoplasticity by higher concentration.In addition to,the apparent viscosity and shear stress of agarose are much larger than that of agar oligosaccharides L1,L2 and L3.The decrease in apparent viscosity followed an Arrhenius formula.The addition of different saline ions ruduced the apparent viscosity of agarose and agar oligosaccharides L1,L2 and L3 solutions,when the concentration exceeds a certain degree,the apparent viscosity gradually stabilized.After the alcohol precipitation,agar oligosaccharide's components are smaller than the agarose hysteresis area,and showing better structural stability.The shear structure restoring force farther confirmed that the agarose and agar oligosaccharides L1,L2 and L3 solutions were all thixotropic system.The results of dynamic rheological properties show that in the range of linear viscoelastic zone,with the increase of frequency,in addition to the modulus of L3 increased greatly,showing a weak gel.The agarose,LI and L2 are mainly viscous and the system is relatively stable.3)Antioxidant activity of agar oligosaccharides L1,L2 and L3 was studied by different antioxidant systems.The results showed that agar oligosaccharides LI,L2 and L3 had good antioxidant ability in the experimental concentration range,and the scavenging ability was different in different antioxidant systems.Three agar oligosaccharides components of superoxide anion(O2-·)scavenging ability at the same level,IC50 of about 5.80 mg/mL.The scavenging results of thehydroxyl radical(·OH)show that L1 has the best effect of scavenging the ·OH,while L3 is the weakest.IC50 of L1 and L2 were about 8.40 mg/mL and 10.35 mg/mL,respectively.The maximum scavenging rate of L3 was close to 43.3 mg/mL in the experimental concentration range.The scavenging results of free radical DPPH showed that three agar oligosaccharides components are just the opposite of the activity sequence of the ·OH scavenging ability.the IC50 of the three agar oligosaccharides components about 6.76 mg/mL,10.00 mg/mL and 8.16 mg/mL,respectively.Three groups of O2-·?·OH and DPPH· free radical scavenging ability are weaker than antioxidant Vc.The scavenging results of the total antioxidant capacity show that L2 exhibits the strongest free radical ABTS·+ scavenging ability,and when the sugar concentration is about 6.0 mg/mL,the components L1 and L3 exhibit opposite antioxidant capacity,whereas the Trolox scavenging ABTS·+ ability is much stronger than agar oligosaccharides compositions.The IC50 of the three components was about 6.45 mg/mL,8.65 mg/mL and 7.29 mg/mL,respectively.4)The results of fourier transform infrared spectroscopy(FT-IR)showed that the components of agar oligosaccharides L1,L2 and L3 were very similar to agarose,indicating that the structure of the sugar ring did not change significantly before and after the modification,except that the glycosidic bonds fracture.The result of high performance liquid chromatography(HPLC)showed that the L1 component is mainly composed of degree of polymerization of 2-6 agar oligosaccharides and a small amount of agar oligosaccharides with a degree of polymerization of 6 or more.the L2 component is mainly composed of degree of polymerization of 2-6 agar oligosaccharides,among which the content of neoagarotetraose and neoagarohexaose is more.The L3 component mainly contain the neoagarobiose and neoagarotetraose,the former content of more.Indicating that different volume of ethanol alcohol precipitation,can get different degrees of polymerization of agar oligosaccharides mixtures.