Preparation And Toxicological Evaluation Of Cellulose Nanocrystals From The Basal Part Of Bamboo Shoot

Cellulose nanocrystals(CNC)are nano-sized cellulose prepared from natural cellulose with a diameter of 3-10 nm and an aspect ratio higher than 5.Compared with nanofibrillated cellulose(NFC),CNC has high crystallinity,small aspect ratio,large specific surface area and other advantages.It contains lots of negatively charged hydroxyl on the surface,which contributing to the significant surface activity,and thus can be used as a new type of dietary fiber,fat replacers and food stabilizer in a variety of food applications.The basal part of bamboo shoot is the inedible part of bamboo shoot,accounting for about 40% of the whole shoot.Every year in China,lots of basal parts of bamboo shoots are discarded as agricultural waste,causing waste and environmental pollution.Basal part of bamboo shoot is rich in cellulose,which is a cheap new material used for CNC production,and it is of great significance to improve the comprehensive utilization and economic value of bamboo shoots.Based on this,in this study,the basal part of bamboo shoot in Fujian province was used as raw material to optimize the production conditions of basal part of bamboo shoot CNC by sulfuric acid hydrolysis method,and its structure characterization and physicochemical properties were analyzed.Finally,the biological safety of CNC was verified by human colon cancer cells(caco-2)toxicology study.The main research results were as follows:(1)On the basis of the successful preparation of bamboo shoot NFC by the research group,this study further removed the amorphous region in the NFC by sulfuric acid hydrolysis to produce the CNC,and optimized the preparation conditions.The effects of sulfuric acid concentration,reaction temperature,ratio of material to liquid and reaction time on the yield and particle size of CNC were studied by means of single factor and orthogonal experiment.The optimized preparation conditions of CNC were as follows: sulfuric acid concentration of 61%,reaction temperature of 40℃,material/liquid ratio of 1:21(g/m L),reaction time of 120 min.The average yield of CNC was 47.34%.(2)Transmission electron microscopy(TEM)and atomic force microscopy(AFM)were used to observe the CNC prepared by sulfuric acid hydrolysis method.It was found that the CNC existed in a needle-like form,with a length mainly between 40-190 nm and a diameter mainly between 4-12 nm,and an aspect ratio of about 1:16.Zeta potential analysis results showed that the CNC has negative charge due to carrying sulfate,and its Zeta potential is-34.3 m V,making it able to maintain good stability in water.However,the result of thermogravimetric analysis showed that the thermal stability of CNC was lower than that of NFC due to the introduction of sulfate groups.After acid hydrolysis,CNC remained cellulose Ⅰ characteristics,and its crystallinity increased to 78.87% as part of the amorphous region was destroyed by acid solution.The results of solid nuclear magnetism further verified that sulfuric acid could reduce the particle size of CNC and improve its crystallinity by further hydrolyzing the amorphous region of fiber.The change of fiber structure leads to the change of fiber physicochemical properties and affects its application potential.Compared with IDF and NFC,CNC showed the highest water holding and swell capacities.The aqueous solution of CNC showed the optimal stability and can maintain the state of suspension after 24 h.(3)Human colon cancer cells(caco-2)were used as cell model to study the effects of different concentrations of CNC on cell morphology and viability by microscopy,MTT and flow cytometry,and the effects of different concentrations of CNC on apoptosis-related protein expression in cells were studied by immunofluorescence detection,Western blot and RT-PCR.Different concentrations of CNC(0 μg/m L,50 μg/m L,100 μg/m L,200 μg/m L)were added to the cell culture medium and cultured for 48 hours.According to the scanning electron microscopy(SEM)and the inverted microscope,the different concentrations of CNC had no effect on cell morphology,indicating that the addition of CNC would not cause damage and toxicity to cells.The results of MTT colorimetry showed that even the highest concentration of CNC(200 μg/m L)had no adverse effect on cell viability.The results of the growth period of caco-2 cells by flow cytometry showed that CNC had no inhibitory effect on the growth of caco-2 cells.Immunofluorescence,Western blot and RT-PCR were used to analyze the protein expression levels and RNA transcription on three apoptotic related genes: Bax,bcl-2 and caspass-3.It was found that CNC could increase the expression level of bcl-2 protein and inhibit the expression of pro-apoptotic Bax and the key apoptotic gene caspass-3,thus slowing down the apoptosis of cells and increasing the survival rate of cells.In summary,the experimental results obtained by various detection techniques all proved that the CNC had no toxic effect on caco-2 cells.