Studies On Fractionation And Transformation Of Kelp Dealginate Residue Based On Novel Solvents

At present,China's annual output of kelp(Laminariajaponica)ranks first in the world.Besides being consumed as food,a remarkable amount of kelp is used to extract alginate to produce sodium alginate.The kelp dealginate residue(KR)accounts for one third of the dry weight of kelp,and the amount is quite large.Traditional treatment of KR by landfill not only occupies land,but also wastes this biomass resource.KR is rich in protein and cellulose.Studies on usages of this unique protein-cellulose biomass resource are important to both academia and practical application.Due to the shortcomings of traditional solvents,researchers pay more attention to the development and usage of novel sustainable,low toxicity and biodegradable solvents.GVL(gamma-valerolactone)and DES(deep eutectic solvent)show great potential in biomass field due to their excellent physiochemical properties,especially the strong solvent capacity.Hence,two novel "green"solvents,GVL/H2O and DES,were innovatively applied by us in the fractionation of KR biomass.The fractionation processes were investigated,and the fractionation rules of crude protein(CP)and crude cellulose(CC)were clarified.Moreover,the fractionation mechanisms of the two solvents for KR biomass were revealed.Furthermore,the CP was transformed into enzymatic hydrolysate with antioxidant activity by enzymolysis,and the effect of fractionation process on the KR protein enzymolysis was discussed.The CC was transformed into cellulose nanocrystals(CNCs)by DES in combination with ultrasonic or solid acid.The physiochemical properties of the prepared CNCs and the influence factors of the CNCs preparation processes were studied.Finally,antibacterial composite films with enhanced mechanical properties were prepared by mixing the prepared KR protein hydrolysate and CNC into sodium alginate.After the calcium containing ash was removed,remaining in the KR were two major components,cellulose and protein,the contents of which were 52.3%and 37.2%,respectively.The GVL/H2O system could effectively fractionate the KR biomass into CP and CC.The fractionation effect was affected by the content of GVL in solvent,fractionation temperature and time.The optimal CP purity and KR protein recovery were obtained at the GVL content of 50 wt%,temperature 140?and time 3 h.Under these conditions,the CP purity and KR protein recovery were 66.3%and 62.2%,respectively,while the CC purity and KR cellulose recovery were 79.7%and 92.4%,respectively.SDS-PAGE results showed that the KR protein was degraded in the fractionation process.Fourier transform infrared(FTIR)spectra analysis showed that the chemical structures of KR protein and cellulose were not changed by the fractionation process and no new functional groups were introduced.X-ray diffraction(XRD)characterization showed that the crystal forms of cellulose in KR and CC were both cellulose I.Scanning electron microscope(SEM)observation showed that the KR before fractionation were irregular compact particles with rough surface.With the removal of protein,the CC from fractionation gradually became porous,and exposed internal structure.Compared to the KR,the CP from fractionation was much smaller in size,and most of the CP particles were far less than 100 ?m.It was indicated that the fractionation of KR by GVL/H2O was a mild acid-driven process.In the fractionation process,some peptide bonds of protein could be cleaved,and the molecular weight of protein was reduced.Consequently,protein was removed from the KR and separated from CC.The 5 DESs,imidazole/choline chloride(IM/CCI),choline chloride/urea(CCl/U),choline chloride/glycerol(CC/G),imidazole/glycerol(IM/G)and sodium acetate/urea(NaAcO/U)all showed fractionation abilities to the protein in KR,whereas the IM/G behaved the best.The fractionation ability of the DESs to KR protein was not only determined by the apparent alkalinity and viscosity of the DESs,but also related to the hydrogen bond-forming ability of the DESs.The fractionation effect of DES IM/G was affected by the fractionation temperature and time.Optimal CP purity and KR protein recovery were obtained at 90? and 12 h.Under such conditions,the purities of CP and CC were 72.3%and 83.6%,respectively,while the recoveries of KR protein and cellulose were 70.2%and 91.1%,respectively.SDS-PAGE showed that the molecular integrity of protein in CP could be well maintained under proper fractionation conditions.Moreover,FTIR and solid 13C NMR analysis showed the fractionation process was mild,and the chemical structures of KR protein and cellulose were not changed.It could be inferred that the DES IM/G fractionated the CP and CC by dissolving the KR protein rather than degradation or derivatization.Further analysis indicated that the solubilization of KR protein in DES IM/G was realized by penetrating of DES IM/G into the KR protein and forming hydrogen bonds with peptide chains.The DES IM/G showed excellent recyclability in the fractionation of KR.Fractionation treatment by the DES improved the enzymolysis efficiency of KR protein.Under the same enzymolysis conditions,compared with the direct enzymolysis of KR,the initial reaction rate was increased from 2.3 mg/(mL·h)to 6.5 mg/(mL·h)when CP from fractionation was used as the substrate.Meanwhile the degree of hydrolysis and yield of the hydrolysate were increased from 13.8%and 46.2%to 20.9%and 81.0%,respectively.The improvement of enzymolysis efficiency of KR protein was closely related to the increase of protein content and specific surface area of CP,as well as the change of molecular structure of protein in CP.The composition and structure of KR protein hydrolysates were changed by the fractionation treatment.Compared with the hydrolysate from direct enzymolysis of KR(KRPH),in the primary structure,the content of 1-5 kDa in the hydrolysate of CP(DES-KRPH was higher,and the contents of antioxidant amino acids increased.In the secondary structure,the contents of a helix and ? turn in DES-KRPH decreased,while the contents of ? sheet and random coil increased.In the spatial structure,more aromatic amino acids were exposed to the surface of DES-KRPH.The change of composition and structure of DES-KRPH released more antioxidant amino acid residues and active fragments embedded in the KR protein molecules.The structure of DES-KRPH was stretched,reducing the steric hindrance of interacting with free radicals,and thus could capture free radicals more effectively.Antioxidant activity tests in vitro showed that at the concentrations of 5 mg/mL,the scavenging ratios of DES-KRPH on DPPH and hydroxyl radicals increased by 50.1%and 48.2%,respectively,compared with KRPH.KR cellulose was transformed into CNCs by DES choline chloride/Oxalic acid dihydrate in combination with ultrasonic or solid acid(phosphotungstic acid).The CNCs prepared by DES-ultrasonic(DUS-CNC)and DES/solid acid(DSA-CNC)were both rod-like in shape with high aspect ratio,polydispersity and negative surface potential.The zeta potentials of DUS-CNC and DSA-CNC were-45.5 mV and-48.6 mV,respectively.In addition to the newly attached oxalate functional groups,the DUS-CNC and DSA-CNC maintained the chemical structure of KR cellulose.The yield of DSA-CNC was affected by the phosphotungstic acid dosage,temperature,time and pulp-liquid ratio of CNC preparation process.The orthogonal assay showed that an optimal DSA-CNC yield of 62.3%could be obtained at the phosphotungstic acid dosage of 30 wt%,temperature 110?,time 150 min and pulp-liquid ratio of 1:40(w/w).The CNC prepared by DES could be used as reinforcing agent and the KR protein hydrolysate prepared via DES had antibacterial activity.Both of them were used to prepare antibacterial sodium alginate composite films with enhanced mechanical properties.The composite films with 1%DSA-CNC and 1%DES-KRPH loading had antibacterial activity,and the composite films were smooth and uniform without bubbles,soft and easy to fold;SEM analysis showed that the surface of the composite films was uniform,and the film matrix was orderly arranged;the tensile index(TI)and bursting index(BI)values of the composite films were 41.4 N·m/g and 267.3 kPa.m2/g,respectively,which were 19.0%and 12.6%higher than the composite films without CNC.With increasing CNC loading dosage,the light transmittance of the composite films decreased,and excessive addition of CNC led to the aggregation of CNC and the formation of CNC aggregates in the film matrix,resulting in decline of the TI and BI values of the composite films.The growth inhibition effect of the composite films on S.aureus(Gram-positive)was better than on E.coli(Gram-negative).Compared with the dosage of 1%,DES-KRPH at the dosage of 2%showed better inhibition effect on the two bacteria,while the inhibition effect decreased with increasing time.