Isolation and characterization of cellulose nanocrystals from jackfruit peel

In the present work, sustainable nanomaterials, cellulose, and spherical cellulose nanocrystals (SCNCs) were isolated from the non-edible parts of jackfruit (Artocarpus heterophyllus). Of the three different methods tested, sodium chlorite treatment produced the highest yield of cellulose, 20.08 ± 0.05% w/w (dry weight). Peaks observed in CP/MAS 13C NMR spectrum and FTIR frequencies revealed the presence of α-cellulose and absence of other biomass fractions like hemicellulose and lignin. XRD analysis showed a high crystallinity of 83.42%. An appearance of a sharp endothermal peak at 323 °C in DSC and decomposition patterns between 310–420 °C of TGA confirms the presence of cellulose. Further, Sulphuric acid hydrolysis was employed to produce SCNCs and examined by TEM for the morphology and by HPLC for the presence of glucose.

isolation of cellulose. Bleaching and pulping are two essential steps for the effective isolation of cellulose from the lignocellulosic biomass 17 . Out of the three different methods tested for bleaching, sodium chlorite yielded the maximum holocellulose (hemicellulose + cellulose), 44.13 ± 0.22% w/w. Hence the sodium chlorite method was used for the isolation of cellulose for all the analysis and characterization. Sodium chlorite is an excellent bleaching agent which aids in the removal of lignin and produces holocellulose [18][19][20] . The other two bleaching methods tested had chlorine-free eco-friendly chemicals such as hydrogen peroxide, acetic acid, nitric acid, and formic acid. Chlorine-free methods have been tried previously for the isolation of cellulose from oil palm empty fruit bunches, maize straw, rice husk, and wheat straw [21][22][23][24] . After bleaching, alkali treatment using sodium hydroxide at mild conditions helps to remove hemicellulose and isolate cellulose 17 . Initially, the untreated jackfruit peel powder was brownish-white in appearance (Fig. 1A,B). The presence of lignin, hemicellulose, extractives such as wax, cutin, and pectin, would render such appearance. Figure 1C depicts the final amount of cellulose from the tested methods. The combination of ethanol, water extraction, acid bleaching, and alkaline treatment ensured the removal of extractives, hemicelluloses, lignin, and produced cellulose (Fig. 1D). Figure 2 represents a detailed scheme for the isolation of cellulose and SCNCs by sodium chlorite treatment followed by sulphuric acid hydrolysis. In the current study, 20.08% ± 0.05 cellulose, 24.04% ± 0.23 hemicellulose, 1.85% ± 0.01 lignin, 52.18% ± 0.03 extractives, and 1.85% processing waste were determined in the jack fruit peel. SEM, TEM and zeta potential analysis. The functional properties of cellulose and SCNCs were influenced by their morphological features which depend on the source and method of hydrolysis. Morphological examination by SEM analysis revealed that the jackfruit peel contains curled and soft-flat shaped cellulose with rough pits (Fig. 3A). The curled shape provides a high surface area which favors the preparation of various composites and also for hydrolysis. Figure 3B shows the self-assembled structures of cellulose fibers due to the strong interfibrillar attraction between the surface hydroxyl groups.
Nanocellulose was obtained by removing the amorphous cellulose and fragmentation of cellulose fibers by sulphuric acid hydrolysis (Fig. 3C). Acid hydrolysis also produces spherical cellulose nanocrystals (SCNCs) which are novel and unique 25 . Hydrolysis of the inner amorphous cellulose fibers leads to the formation of spherical nanocellulose. In the present work the combined use of a strong bleaching agent, acid and ultrasonication would have led to the hydrolysis of inner amorphous regions of cellulose fibers. Figure 3D shows a soft, non-fibrous structured, non-agglomerated spherical shaped cellulose nanocrystals (SCNCs) with 130 nm diameter indicating the surface engraving and degradation nature of the hydrolysis 26,27 . In the present study, 7% w/w SCNCs was obtained from the isolated cellulose of jackfruit peel. The produced SCNCs has an average zeta potential −11.6 mv and size of 346 nm, which is in good agreement with the literature 28,29 . Nanocellulose has been reported for its large size variation 26,27 . Individual crystals would tend to aggregate due to water evaporation leading to larger particle size. The presence of aggregated and individual SCNCs in the acid hydrolyzed cellulose suspension led the particle size discrepancy as determined by TEM and zeta size. NMR analysis. The solid-state NMR spectrum of the isolated cellulose was consistent with the literature 30 .
The solid-state CP/MAS 13 C NMR spectrum of cellulose (Fig. 4A)  www.nature.com/scientificreports www.nature.com/scientificreports/ ftiR analysis. The FTIR spectrum of the isolated cellulose displayed characteristic absorption patterns corresponding to the specific functional groups of α-cellulose (Fig. 4B) and was also in good agreement with the reported cellulose ( Table 2). The peak at 1641 cm −1 may be formed due to the bending mode of adsorbed water 22,31,32 . The peak at 1442 cm −1 may be due to CH 2 bending vibration 33 . The sharp transmittance peak around 1384 cm −1 represents a bending of OH groups 21 . The peak at 1174 and 1120 cm −1 corresponds to C-O asymmetric bridge stretching 21 . The peak at 1057 cm −1 may be due to C-O-C pyranose ring skeletal vibration 21 . The peak at 905 cm −1 is because of glycosidic linkages between sugar units 18 . The absence of shoulder peak at 1726 cm −1 indicates that there is no acetyl and uronic ester groups of the hemicelluloses or the ester linkage of lignin 30 .   34 . The major crystalline peak was observed at 22.6° with an intensity of 100%, confirms the presence of crystalline cellulose 22,31 . From Eq. (1), 83.42% crystallinity index (CI) was calculated for the isolated SCNCs. Similar CI was reported for celluloses of poplar wood chips (50%) 35 , Eucalyptus globulus (55.3) 36 and E. Benthamii (54%) 36 . Crystallite size is also supportive of the description of crystallinity of the cellulose. From Eqs (2, 3), the crystal size of the isolated cellulose was found to be 2.80 nm, and the interplanar distance was 0.21 nm. Similar results were found with the cellulose from S. japonica 33 . thermal analysis. Thermal stability of the isolated cellulose was determined using TGA (Fig. 5A). Three stages of thermal degradation in the form of weight loss were observed. An initial steady-state decrease in weight till 150 °C is due to the evaporation of water. A sudden weight loss in the second stage during 200-380 °C is due to cellulose depolymerization. The final stage of rapid depolymerization of carbon residues occurred after 380 °C. An initial weight loss of 7% was observed below 100 °C, and after that, no decomposition was recorded till 180 °C. Thus TGA confirms the thermal stability of isolated cellulose and the absence of hemicellulose and extractives. Approximately 85% of the weight was lost by drying at 300-350 °C. A linear region of weight loss arose from 312-350 °C, which is the major characteristic thermal property of cellulose 37 .
DSC was performed to determine the energy consumption property of the isolated cellulose (Fig. 5B). A characteristic sharp endothermal peak at 323.5 °C was recorded 38 . No peaks corresponding to the hemicellulose (150 °C) and lignin (380-500 °C) were noticed. Thus, TGA and DSC results confirm the purity and excellent thermal stability of the isolated cellulose from the jackfruit peel.
HpLc analysis. HPLC analysis of the SCNCs showed a single sharp peak corresponding to the standard glucose at 9.98 min (Fig. 5C). Approximately 67.5% w/w glucose was recovered from the SCNCs, and further optimization studies are under progress to enhance the hydrolysis and the yield of glucose. experimental Materials. Jackfruit peel was collected from the same region of Thanjavur local market, India, throughout the entire work to avoid variations in nutritional content. Sodium Chlorite (NaClO 2 ), Sodium hydroxide (NaOH), acetic acid (CH 3 COOH) was purchased from HIMEDIA Mumbai, India. Sulphuric acid (H 2 SO 4 ) was obtained from SIGMA-ALDRICH, India. All other chemicals used in the study were AR grade and procured from MERCK, India. www.nature.com/scientificreports www.nature.com/scientificreports/ compositional analysis. Jackfruit peels were washed first with tap water followed by distilled water to remove the adhered surface dust particles (Fig. 1A), and later air-dried for 48 h and milled. The dried powder was sieved with sieve numbers 80 (0.1 mm) and 20 (0.8 mm). Only those particles that passed through sieve number 20 and retained at sieve number 80 was used throughout the work (Fig. 1B). The amount of moisture, total solids, ash, and lignin were determined as per the NREL procedures 39,40 . The carbon, hydrogen, and nitrogen were determined using Elementar Vario EL III equipment, Germany.   www.nature.com/scientificreports www.nature.com/scientificreports/ isolation of cellulose. Three methods, namely sodium chlorite method, acetic acid plus nitric acid treatment and formic acid treatment, were screened for the maximum removal of non-cellulosic fragments and isolation of cellulose from the jackfruit peel powder.

Method 1: Sodium chlorite method.
Non-cellulosic fractions were completely removed, and cellulose was isolated as described elsewhere 34 . The powdered jackfruit peel was dewaxed with water followed by ethanol extraction in a Soxhlet apparatus for 8 h. The dewaxed powder was bleached using 1.5% w/v sodium chlorite in water (1:25 g/mL), pH 3.5 at 70 °C for 2 h. The bleaching process was repeated for four times till a white-colored holocellulose was formed and later subjected for alkali treatment for complete removal of the hemicellulose. Then the cellulose was suspended in water at 60 °C for 2 h to remove the excess sodium hydroxide. The pure cellulose obtained was filtered and dried. Method 2: Acetic acid plus nitric acid treatment. A slightly modified method suggested by Rehman et al., 2014 was followed for the isolation of cellulose 23 . The dewaxed biomass was treated with 5% w/v NaOH in water (1:30 g/mL) for 2 h to remove the hemicellulose. The treated biomass was bleached with a 10% v/v hydrogen peroxide at pH 11.8 for 12 h at 50 °C for 1:25 (g/mL) ratio. The bleached cellulose was purified further using 80% acetic acid (1:30 g/mL) and 65% nitric acid (1:4 g/mL). The purified cellulose was washed thrice with water and 95% ethanol and dried overnight at 50 °C.
Method 3: Formic acid treatment. The hemicellulose in the biomass was removed using 3% v/v (1:25 g/ ml) hydrogen peroxide and 10% w/v NaOH in water (1:25 g/mL) for 2 h at 65 °C. Later, 20% formic acid (1:10 g/ mL) and 10% hydrogen peroxide (1:10 g/mL) in a 1:1 ratio was added and incubated at 85 °C for 2 h. The bleaching process using formic acid was repeated for 4-5 times until a white-colored product was obtained 23 . isolation of nano-cellulose. The isolated cellulose was hydrolyzed using 65% H 2 SO 4 at 1:9 (g/mL) ratio, 37 °C for 1 h 23 . The hydrolysis was stopped by the addition of double the amount of cold water. The cellulose suspension was later centrifuged at 3000 rpm for 30 min. The suspension was subsequently dialyzed using a dialysis membrane with cut off 12000 Da to 14000 Da to remove the excess sulphuric acid. Dialysis was continued till pH 5.5 was reached. The dialyzed suspension was ultrasonicated (Labman) for 15 min in bath sonication to get a uniform suspension of nanocrystals. The aqueous suspension was subsequently concentrated using rotary evaporator and dried (Buchi Rotavapor R-210, Switzerland). characterization SEM analysis. SEM imaging of the isolated cellulose from jackfruit peel was studied using a JEOL JSM 6701 F microscope with an accelerating voltage of 6 kV. The sample was attached to an aluminum stub and was gold-sputtered to avoid electron charging effects. Zeta potential. Zeta potential and particle size analysis were measured using Malvern instruments, UK.
Following parameters were followed for zeta potential measurement; temperature: 25 °C, count rate: 75.9 kcps and zeta runs: 100. For particle size analysis the temperature was kept at 25 °C, count rate of 125.9 kcps and duration of 80 s. NMR analysis. CP/MAS 13 C spectrum of cellulose in D 2 O at room temperature (Bruker Avance HD 500 MHz spectrometer) was recorded at SAIF, IIT Madras, Chennai, India. The 13 C NMR was operated at 125.76 MHz. The Proton 90° pulse was 4.30 µs; contact time was 3300 µs, recycle delay was 5 s and acquisition time was 0.02 s. Typically, 1024 scans per spectrum were recorded. ftiR analysis. A sample mixture of isolated cellulose, 2 mg, and dry KBr, 200 mg was prepared, pressed into a 16-mm diameter mold and FTIR spectra were measured on a Spectrum one, (Perkin Elmer, USA) instrument in the diffuse reflectance mode. XRD analysis. XRD pattern was recorded using Bruker D8 advance, equipment. The isolated cellulose was scanned in the range of 2θ = 5° to 90° at a step time of 0.2 s/step at 25 °C with Cu kα radiation, λ = 0.1540 nm. The Crystallinity index (CI) of dried cellulose was determined using the following equation 41 where d is the interplanar distance between lattice planes, θ is the scattering angle in degrees, n is a positive integer and λ is the wavelength of the x-ray TGA analysis. To determine the thermal stability and decomposition pattern of isolated cellulose, Thermogravimetric analysis (TGA), was performed in an SDT Q600 instrument. For each measurement, approximately 5 mg of the sample was used. Patterns were recorded under a nitrogen atmosphere at a flow rate of 100 mL/ min by heating the material from room temperature to 500 °C at a heating rate of 20 °C/min. HpLc analysis. HPLC analysis was conducted to determine the presence of glucose and the absence of hemicellulose sugars in the SCNCs. Briefly, the depolymerized cellulose was dissolved in the mobile phase (80% acetonitrile and 20% water) and loaded into a spherisord amino column (4.6 × 250 mm) of Waters HPLC system. The system was equipped with a Waters 2535 quaternary gradient pump and Waters 2414 Refractive index detector. Isocratic elution was performed after injecting 10 µL of the sample at a mobile phase flow rate of 1 mL/min, a column temperature of 35 °C, and a detector temperature of 30 °C.

conclusion
In the present work, cellulose and SCNCs were successfully isolated from the non-edible jackfruit peel using sodium chlorite treatment followed by sulphuric acid hydrolysis. Approximately 44% w/w (on a dry weight basis) of holocellulose was isolated, of which 20% w/w was cellulose. The isolated cellulose was characterized for its morphology, functional, crystal, thermal properties by SEM, FTIR, NMR, XRD, DSC and TGA. The SCNCs were characterized for its size, surface charge and monomer analysis by TEM, Zeta potential, zeta size and HPLC. The isolated cellulose and SCNCs exhibited similar characteristics reported in the literature and was found to be devoid of hemicellulose and lignin. The present results indicated the significance of the valorization of jackfruit peel. Several potential applications of CNCs in the field of food, paper, paints, optics, pharmaceutics, environment remediation, composite synthesis, and so forth have been reported. Hence the isolated cellulose and SCNCs from jackfruit peel waste can be employed for such applications.

Data availability
All data generated or analyzed during this study are included in this published article.