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Extraction and characterisation of natural cellulose fibers from Kigelia africana
Journal Pre-proof Extraction and Characterisation of Natural Cellulose Fibers from Kigelia africana Manikandan Ilangovan, Vijaykumar Guna, B. Prajwal, Qiuran Jiang, Narendra Reddy
PII:
S0144-8617(20)30170-3
DOI:
https://doi.org/10.1016/j.carbpol.2020.115996
Reference:
CARP 115996
To appear in:
Carbohydrate Polymers
Received Date:
11 November 2019
Revised Date:
11 February 2020
Accepted Date:
11 February 2020
Please cite this article as: Ilangovan M, Guna V, Prajwal B, Jiang Q, Reddy N, Extraction and Characterisation of Natural Cellulose Fibers from Kigelia africana, Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2020.115996
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Extraction and Characterisation of Natural Cellulose Fibers from Kigelia africana Manikandan Ilangovan1,2, Vijaykumar Guna2,3, Prajwal B2, Qiuran Jiang4,5, Narendra Reddy2,* 1Department
of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan for Incubation, Innovation, Research and Consultancy, Jyothy Institute of Technology,
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2Centre
Thataguni Post, Bengaluru 560082, India
Technological University – Research Resource Centre, Jnana Sangama Belagavi-
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3Visvesvaraya
Laboratory of Textile Science &Technology, Ministry of Education, College of Textiles,
Donghua University, Shanghai, China
of Technical Textiles, College of Textiles, Donghua University, Shanghai, China
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5Department
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4Key
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590018, India
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* Corresponding Author: Narendra Reddy, Centre for Incubation, Innovation, Research and Consultancy, Jyothy Institute of Technology, Thataguni Post, Bengaluru 560082, India
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Phone: +919611233707 Email: [email protected]
Graphical abstract
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Highlights
Natural cellulosic fibers were extracted from Kigelia africana (sausage plant)
The fibers had high cellulosic content (70%) and good tensile properties
Sausage fibers possessed excellent thermal stability and moderate hydrophobicity
Sausage fibers showed decent activity against gram positive and negative bacteria
Storage boxes for dry food items were developed with good structural integrity
Abstract
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Kigelia africana also known as sausage plant, yields highly fibrous fruit with a hard shell. Many medicinal uses are reported for the extracts from the fruits, seeds and leaves of sausage trees. In this research, natural cellulose fibers were extracted from the fruit using NaOH and later bleached and characterized for their properties. Results revealed that significant amount of hemicellulose and lignin was lost after the alkali treatment and bleaching leading to a highly cellulosic fiber (up to 71%). Morphologically, surface of the fibers varied from rough to smooth 2
depending on the extent of treatment. The thermal stability, crystallinity and hydrophobicity increased after the treatment. Sausage fibers also possessed anti-microbial activity against common gram negative and gram positive bacteria. Overall, sausage fibers have properties similar to that of cotton and better than fibers obtained from many unconventional sources. With improved hydrophobicity and anti-bacterial properties, sausage fibers could be potentially
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Key words: Sausage fruit, Anti-bacterial, Bio-Product, Cellulosic Fibers
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applied in functional polymer composites.
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1.0 Introduction Among the different applications of agricultural residues, extraction of natural cellulose fibers is highly preferable since fibrous applications have a large market and offer higher value. Natural cellulose fibers have been extracted from several unconventional sources in an attempt to obtain fibers with unique properties (Guna et al., 2019b, 2019a; Ilangovan et al., 2018;
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Jebadurai et al., 2019; Kathirselvam et al., 2019; Liu et al., 2019). Fibers obtained from cotton
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stalks, rice and wheat straw, cornstalks and husks had properties similar to that of cotton, jute and linen and were found to be useful for textile, composites and other applications (Reddy and
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Yang, 2009a, 2009b, 2009c). Recent studies have also shown the possibility of extracting and
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demonstrated the unique features of natural cellulose fibers from aerial roots of banyan trees and wild plants such as Tridaxprocumbens and Catharanthusroseus (Ganapathy et al., 2019;
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Vijay et al., 2019; Vinod et al., 2019). Similarly, fibers obtained from tulsi stems or sabaigrass, which are considered as weeds, not only had good mechanical properties but also showed
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antimicrobial activity and hence were suggested for medical applications (Guna et al., 2019a, 2019b). In addition, sabai fibers also possessed high thermal and noise insulation comparable to industrial standards when used as reinforcement for composites, extending its applications
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beyond automotive to green building and interior applications (Guna et al., 2019b). Kigelia Africana also known as sausage tree is an evergreen plant that can reach up to
20m in height. Fruits of Kigelia Africana (sausage fruit) can grow up to 1m x 18cm, 30-100 cm long and can weigh up to 12 kg. Sausage fruits consist of a hard protective and inedible shell, that forms more than 80% of the weight of the fruit (Bello et al., 2016). In addition to use as food, the pulp, seeds and other components of the sausage fruit have been proven to have 4
medicinal properties. Extracts obtained from the sausage fruit and tree have shown antibacterial, antifungal, antioxidant, anti-inflammatory and central nervous system (CNS) stimulant activities (Akah, 1996; Bello et al., 2016).Ethanol and n-hexane extracts from the leaves and barks of Kigelia africana tree had good antibacterial activity against six common micro-organisms with inhibition level as high as 67% (Hussain et al., 2016). Extracts from the
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bark of the plant showed higher activity compared to the leaves or fruit extracts. Methanol extracts from the leaves are reported to contain flavonoid rich fractions that will aid in
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therapeutic treatment of Alzheimer’s disease (Falode et al., 2017). Other studies have shown
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that the sausage plant extracts can increase body growth and organ development (Micheli et al., 2019). Apart from phenolic components (saponin, tannin, terpenoid), sausage fruits were
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reported to contain minerals such as potassium and calcium, proteins and fat which are suitable
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for several food and non-food applications (Abass, Oseni, 2018). Although the extracts of the fruits and other parts of the sausage tree are studied for their medicinal properties, to the best
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of our knowledge, there are no studies conducted on the non-food applications of the fibers in the fruit. In this research, we have studied the structure and properties of the fibers in the sausage fruit before and after chemical treatments. Properties of the fibers have been compared to common natural cellulose fibers and also to those obtained from agricultural
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residues and biomass.
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2.0 Materials and Methods 2.1 Materials Sausage fruits each weighing about 5kg were collected from local farms near our campus in Bengaluru, Karnataka. Analytical grade sodium hydroxide, acetic acid, acetone and other chemicals required for the experiment were purchased from Hybrid Fine Chemicals,
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Bangalore, Karnataka.
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2.2 Fiber extraction
Untreated Sausage fruit fibers (SFF) were separated from the fruits manually. Initially
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the raw fruit were cut into sections, oven dried for 24 hours and then fed through a two roller
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mechanical crusher. Partially crushed fruits were collected and soaked in boiling water prior to
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the separation of fibrous part.
SFF was then treated with 0.2N NaOH solution for 90min at 100 o C. The untreated fiber
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to alkali ratio was maintained at 1:10. After treatment, the liquid containing dissolved hemicelluloses, lignin and other extractives was decanted and disposed. The fibers extracted through alkali treatment (ATF) were thoroughly washed until the pH of the fibers was neutral. The neutral pH fibers were later immersed in 10% acetic acid solution for 10 min and rinsed.
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Later, the fibers were dried in a hot air oven at 110oC for 3h and stored for further characterization. 2.3 Bleaching The alkali treated fibers (ATF) were further bleached using 7% sodium chlorite at 80oC for 1h. The fiber: sodium chlorite ratio was maintained at 1:5. After bleaching, the fibers were 6
washed multiple times using de-ionized water. The fibers were then immersed in 5% acetic acid to neutralize the alkali. The bleached fibers (BF) were then dried at 110oC for 3h and used for further analysis. 2.4 Fiber Characterization The percentage cellulose, hemicellulose, lignin and ash content of SFF, ATF and BF were
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determined according to the standard test methods as described in (Guna et al., 2019b). Each
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component was analyzed in triplicates and the average value was reported.
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The fibers were tested for their tensile strength on a Universal Tensile Tester (MTS Mechatronics, Ichalkaranji, India) according to the ASTM D 3822-14 standard. The cross-head
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speed and the gauge length was set at 10 mm/min and 25.4mm, respectively. Samples from
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three different sets of extraction were used for the characterization. Twenty samples from each set, i.e., a total of 60 samples were tested. The mean and standard deviation was reported.
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The morphology of the fibers was observed in a Scanning Electron Microscope (Hitachi Model SU 3500). Before observation, the fibers were sputter coated with gold-palladium in an Ion Beam coater for 60s. The coated samples were then analyzed at an operational voltage of
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15kV.
X-ray diffraction study was conducted on the powdered fiber samples (particle size:
200m) in a Bruker D8 Advanced Eco X-ray diffractometer equipped with Bragg-Brentano Focusing geometry. The analysis was done using a Cu-Kα radiation at a wavelength (λ = 1.54 Å) and the diffraction patterns were recorded at 2θ angles varying from 5o to 65o. The readings
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were analyzed in Origin Pro software and the peak intensities were identified. The Segal equation (Segal et al., 1959)was used to calculate the crystallinity index (Xc) as shown below:
𝑋𝑐 = (
𝐼002 − 𝐼𝑎𝑚 ) × 100% 𝐼002
Where, I002 and Iam are the peak intensities at the crystalline and amorphous region
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respectively. Size of cellulose crystallites in the fibers were calculated using the Scherrer
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equation and the data has been included in the supplementary file (Table S1).
FTIR was carried out to identify the type of bonding and components present in the
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fibers. The spectrum was recorded using Perkin Elmer/Spectrum 2 (Diamond UTAR) in 4000 to
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400 cm-1range at a resolution of 2 cm-1.
Wettability of liquids over the fiber surface was carried out using a contact angle
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apparatus (OCA 15EC, Data Physics Instruments, Germany). To measure the water contact angle (WCA) , 2 μL of water was dispensed using a micropipette over the fibers along the longitudinal
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axis. Images were then captured using a camera connected with a computer interface. Contact angle measurements were done on the liquid spread over the fiber surface. Ability of the extracted fibers to resist common bacteria was also studied using two
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gram positive (Staphylococcus aureus, Bacillus cereus) and two gram negative (Escherichia coli, Serratia marcescens) bacterial strains. The analysis was conducted according to the method described in (Guna et al., 2019a). Extracted fibers were used as reinforcements in gluten matrix to develop potential bioproducts. Products in the shape of packaging boxes were fabricated at 160 oC and 600 MPa
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pressure in a custom made hot press with box shape mold. The fibers: gluten ratio was kept at (90:10% w/w) for the process. Digital images of the boxes were included in the article. 3.0 Results and discussion 3.1 Composition of the fibers SFF is lignocellulosic with high percentage of lignin and lower concentration of
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hemicellulose. Untreated SFF have similar cellulose content as that of alkali extracted fibers
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from tulsi and turmeric stalks (Table 1). Cellulose content of the fibers increases after treating with alkali and further increases to about 72% after bleaching. Correspondingly, the
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hemicellulose and lignin contents are also altered (Table 1). As the cellulose content increases,
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the fibers also change color becoming brownish due to the alkali treatment but later turn white after bleaching (Figure.1). Higher cellulose content not only increases whiteness but should also
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provide the fibers with better tensile properties unless the cellulose in the fibers get damaged.
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Hemicellulose content of all the fibers is lower than the lignin content as is typically observed in non-traditional fibers (Guna et al., 2019a, 2019b). Lignin content is higher than cotton but lower than that found in flax, jute, coir and other common natural cellulose fibers (Reddy and Yang, 2009c; Vijay et al., 2019). Typically, the lignin content should decrease after
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bleaching but we observe a contrary effect in this study. Increase in the lignin content may be because of its recalcitrant form or higher concentration of lignin in the treated fibers compared to those removed during chemical treatment and washing. Ash content in the fibers decreased by about 65% from SFF to BF and were found to be up to 93% lower than that in turmeric and tulsi fibers, but comparable to cotton and cotton stalk fibers (Guna et al., 2019a; Ilangovan et
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al., 2018; Reddy and Yang, 2009c). Treating at stronger conditions may further increase the cellulose and reduce lignin and hemicellulose content., However, the fibers may become smaller and unusable for high value applications. Nevertheless, the fiber properties are equally influenced by the morphology of the individual fibers, crystallinity and orientation. Table 1 Compositional data of fibers obtained from sausage plant compared to that of other
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non-traditional cellulosic fibers (Guna et al., 2019a; Ilangovan et al., 2018)
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Sample
Turmeric
SFF
ATF
BF
Tulsi Fibers
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Fibers
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Composition 55.1±0.2
69.9±0.1
71.6±1.2
56.5±0.6
52.5±0.2
Hemicellulose (%)
9.34±0.08
9.01±0.5
9.81±0.4
12.4±0.2
18.5±1.3
Lignin (%)
11.7±0.2
10.00±0.4
13.5±1.1
14.8±1.3
14.3±0.19
Ash(wt.%)
1.85±0.1
1.3±0.1
0.64±0.03
10.2±1.2
9.61±0.19
Flavonoids μg/ml
160±0.1
145±0.2
ND
196±0.1
180±0.1
Phenol μg/ml
240±0.2
180±0.1
92±0.2
180±0.1
89±0.2
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Cellulose (%)
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ND – Not Detected
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Figure. 1 Schematic representation of the process used for fiber extraction from sausage fruit
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3.2 Morphology
Surface of untreated fibers is rough due to the presence of impurities (Figure. 2 a-c).
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Bundles of individual fibers of width ranging from 40 to 80 µm are observable (Figure. 2 a). On further magnifying, a thick coating of substances most likely hemicellulose, ash and other impurities can be seen (Figure. 2 b,c). Alkali treatment removes most of the surface substances
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resulting in fibers with smooth texture (Figure. 2 d,e). Some of the individual fibers get separated from the bundles which could disintegrate into fibrils if treatment conditions are too aggressive or not controlled. Individual fibers had widths of 50 µm and length more than 4550mm.Microfibrils arranged at an angle to the fiber-axis (micro fibrillar angle or MFA) are seen in ATF (Figure. 2 f). The MFA of ATF and BF were measured using ImageJ software and are reported in Table 2. Bleaching removes almost all of the impurities leading to a cleaner surface 11
in BF (Figure. 2 g, h). Not only fibers but individual fibrils can be observed on the surface of the BF. Further, the convolutions and distinct fiber taper typical to cellulosic fibers are also apparent in the ATF and BF (Figure. 2f, g, i). Morphological analysis indicates that BF have
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characteristics very similar to that of cotton.
Figure. 2 SEM images of: (a-c) SFF, (d-f) ATF, (g-i) BF
3.3 Tensile properties Images of the tensile experiment can be found in the supplementary file (Figure S2). The tensile properties increase successively after alkali treatment and further after bleaching (Table 12
2). An 8% increase in the strength of ATF and 28% increase in BF was observed compared to SFF. Similarly, the elongation and modulus in BF increased by about 44% and 24% respectively compared to SFF. Increase in the tensile properties can be directly related to the chemical treatment and specifically attributed to the following two reasons: the increase in cellulose content and reorganization of the cellulose fibers and crystals during the treatment. As a result
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of reorganization, the MFA increased from 13.5° in ATF to 16.4° in the BF. Typically, higher MFA leads to higher flexibility and hence higher elongation(Reddy and Yang, 2005)which was also
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observed in the case of sausage fibers. Tensile strength of sausage fruit fibers was comparable
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(Ilangovan et al., 2018; Reddy and Yang, 2009c).
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to that cotton and jute. The elongation was however, higher than jute and lower than cotton
Table 2 Tensile properties of untreated, alkali treated, and bleached sausage fruit fibers.
Sample
Elongation
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Young’s
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Strength (MPa) At break (%) modulus (GPa)
Microfibril angle (°)
379.28± 19.53
2.61 ± 0.74
15.68 ± 2.92
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Alkali treated Fiber
411.08± 14.56
3.68 ± 0.46
17.52 ± 1.72
13.54 ± 1.04
Bleached Fiber
484.01± 16.84
3.75± 0.564
19.47 ± 1.47
16.41 ± 1.48
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Untreated Fiber
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3.4 X- ray diffraction analysis (XRD) A gradual but distinct change in the crystal structure of the fiber occurs after alkali treatment and bleaching. Intensity of the main 002 cellulose I crystal peak at 22° increases sharply and becomes narrower after alkali treatment and even further after bleaching (Figure. 3). In the raw and alkali treated fibers, the 101 and 110 peaks are merged due to the presence
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of hemicellulose and impurities. The two peaks are distinct in the bleached fibers since they
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have high cellulose content. However, there is no change in the cellulose allomorph from I to II as observed after strong alkaline treatments (Song et al., 2015). Corresponding to the variations
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in peak intensities and positions, the % crystallinity of raw, alkali treated, and bleached fibers
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was 59, 60 and 70%, respectively. Bleached sausage fibers have crystallinity similar to cotton and linen (65 to 70%) and higher than fibers obtained from lignocellulosic crop residues (50 to
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60%) (Guna et al., 2019a; Ilangovan et al., 2018; Reddy and Yang, 2009c). Higher crystallinity would reduce the accessibility of the cellulose to chemicals and enzymes and hence chemical
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modifications and dyeing would be difficult. Therefore, the extent of treatment must be
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controlled depending up on the intended applications.
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Figure.3 Normalized XRD spectrum of untreated, alkali treated, and bleached sausage fibers
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3.5 Fourier Transform Infrared Spectroscopy
The FTIR spectrum of SFF, ATF, BF is given in Figure. 4. A typical vibration at the 1048
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cm-1 region (C-O group related to cellulose content) is prominent in BF compared to SFF and ATF. Similar behavior was observed in the OH stretch at 3424 cm-1 indicating higher cellulose content in the BF(Yang et al., 2007). The hemicellulose and pectin fingerprint region at the 1734
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cm-1 (C=O stretch) is visible only in the SFF which suggests successful removal after treatment with alkali and bleaching. A small peak at 1513 cm-1 (C=C group) relating to the lignin aromatic network is evident in the BF. Stronger peak at the C-H region (2922 cm-1) typical to aromatic compounds is also observed in the BF, suggesting higher lignin content than SFF and ATF. The FTIR spectra are in good accordance with the compositional data (Table 1).
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3.6 Changes in surface wettability
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Figure.4 FT-IR spectrum of untreated, alkali treated, and bleached sausage fibers
Treating the fibers with alkali and later bleaching, lead to a decrease in the contact angle
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(Figure. 5). SFF contains non-polar, hydrophobic surface impurties such as wax and pectin. Also, the heterogenous surface of the SFF results in air pockets trapped between the droplet and surface, there by increasing the contact angle (Le Phuong et al., 2019). After alkali treatment or bleaching, the non-polar impurities are gradually removed (as observed in Figure. 2). This
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removal leads to an increase in the polarity and surface energy of the fibers thereby turning them hydrophilic (Raharjo et al., 2018; Vijay et al., 2019). The smoother BF in comparison to SFF and ATF had over 20o and 15o lower WCA respectively, attributed to the higher cellulose (hydrophilic) content. However, all three fiber surfaces have contact angle less than 90°C which
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is typically treated as hydrophilic. Similar behavior was reported in the studies of Tridax
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procumbens , Cantala and hemp fibers (Raharjo et al., 2018; Vijay et al., 2019).
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Figure. 5 Digital image depicting the wettability of (a) SFF (b) ATF (c) BF
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3.7 Thermal degradation
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Thermal analysis shows that the degradation of the fibers follows a three step process (Figure. 6). The initial stage occurring between 80 to 90 °C leads to about 5% weight loss due to
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the removal of surface moisture. Degradation behavior of the fibers can then be divided into two regions (191-300°C) and (300 to 406°C). Weight loss at the former is possibly due to the
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degradation of mostly hemicellulose and partially lignin and at the latter due to the decomposition of cellulose (Kathirselvam et al., 2019). All three fibers are quite stable after the initial step, with 25% weight loss occurring only between 302 to 332 °C (Figure. 6). Interestingly,
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SFF show higher thermal stability than the alkali treated or bleached fibers in this region probably due to the presence of impurities with higher stability on the surface of the fibers. Bleached fibers show marginally slower rate of degradation compared to untreated and alkali treated fibers (Figure.6). Since all three fibers are stable below 300 °C, they are suitable for processing for composites and textiles without being damaged.
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Figure. 6 TGA/DTG profile of untreated, alkali treated, and bleached sausage fibers.
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3.8 Antibacterial activity
Natural cellulose fibers obtained from several non-conventional sources have shown
inherent antibacterial activity (Guna et al., 2019a, 2019b; Ilangovan et al., 2018). We hypothesized that fibers from sausage fruit will also potentially exhibit anti-microbial properties given its other proven medicinal applications. Having resistance to micro-organisms will be 18
useful in food packaging, medical and other fibrous applications. As hypothesized, sausage fruit fibers showed appreciable antimicrobial activity to common microorganisms. Generally speaking, with increased dosage, the inhibition activity increased due to the higher quantities of phytochemicals present. Phytochemicals play a significant role in the anti-bacterial activity of any material (Bandeira Junior et al., 2018; Oikeh et al., 2016). As seen in Table 1, phenols and
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flavonoids are higher in SFF compared to ATF and BF. Consequently, the percentage inhibition of SFF (54 - 97%) was higher at all dosages against all the four organisms compared to ATF and
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BF. As the concentration of phytochemicals decreased, the activity in ATF and BF reduced by up
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to 63%. This phenomenon was observed in other natural fibers from non-traditional sources such as tulsi, turmeric stalks and sabai grass as well (Guna et al., 2019a, 2019b; Ilangovan et al.,
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2018). The degree of inhibition was high in gram-positive bacteria than the gram-negative
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bacteria in all the fibers. This could be attributed to the outer membrane in the gram negative bacteria that acts as a barrier to foreign substances (Oikeh et al., 2016). Gram negative bacteria
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contains a typical preventive area known as the periplasmic space where enzymes break down foreign particles (even the anti-microbial agents) trying to access the organism (Cheruiyot et al., 2009; Holetz et al., 2002; Oikeh et al., 2016). Although, the presence of phenols and flavonoids improves the anti-bacterial activity, the exact mechanism of inhibition is different for different
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compounds (Abdulkhani et al., 2017). Thorough micro-biological studies are required to predict the inhibition mechanism of sausage fruit fibers. However, the fiber extraction conditions could be further adjusted for the optimum concentration of phytochemicals for specific applications.
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Table 3 Extent of inhibition (%) of four common bacteria by untreated, alkali-treated and bleached fiber extracted from the sausage tree fruit fibers at different dosages. Inhibition at 50 mg/ml dosage (%)
Inhibition at 100 mg/ml dosage (%)
Organism SFF
ATF
BF
SFF
ATF
BF
S aureus
91.5 ± 0.1
70.8 ± 0.2
57.6± 0.2
97.5 ± 0.4
86.4 ± 0.3
62.2± 0.4
Positive
B cereus
72.4 ± 0.2
69.6 ± 0.3
54.2 ± 0.4
92.2 ± 0.1
82.6 ± 0.2
68.4 ± 0.4
Gram
E coli
82.4 ± 0.4
61.9 ± 0.2
42.6 ± 0.4
88.4 ± 0.6
65.2 ± 0.4
48.4 ± 0.2
Negative
S marcescens
54.5 ± 0.3
39.4 ± 0.4
19.8 ± 0.6
42.7 ± 0.1
26.3 ± 0.2
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64.9 ± 0.3
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3.9 Potential to develop bio-products
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Sausage fruit fibers obtained had good mechanical properties which enables us to use them in developing various bio-products. As seen from Figure. 7, both untreated and bleached
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fibers were compression molded into boxes. Since sausage fruit fibers demonstrated antimicrobial properties, the products developed could be used in food applications such as packaging of dry fruit, ready-to-eat foods and sweets, replacing currently used synthetic containers. Further, they are can be used in developing bio composites with promising
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mechanical strength.
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Figure. 7 Boxes suitable for food and non-food applications molded from : Top and Bottom Row Left: SFF , Top and Bottom Row Middle: ATF, Top and Bottom Row
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Right: BF
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4.0 Conclusions
Sausage fruit is composed of lignocellulosic fibers with cellulose and lignin content of
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about 15 and 12%, respectively and generally considered non-edible for humans. However, the raw fibers and those after treating with alkali and subject to bleaching all have good mechanical properties, crystallinity and morphology. After bleaching, cellulose content in the fibers increased to 71% and the tensile strength was 484 MPa, similar to that of cotton. Further,
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sausage tree fibers have convolutions and tapered edges as seen in cotton. With thermal stability (up to 300 oC), high crystallinity (70%) and good elongation (3.75%), the fibers can be used for composites and non-woven applications. However, the fibers are coarser and also are shorter in length and hence unsuitable for typical textile applications. The new approach to
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develop sustainable bio-products using sausage fruit fibers shows promise to replace some of the non-biodegradable, synthetic polymer based products in the market.
Author credit statement
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Narendra Reddy and Vijaykumar Guna conceptualized and designed the research. MI, QJ and PS conducted and analyzed the data. MI, NR wrote the manuscript with help from VG.
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Acknowledgements
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The authors thank Azyme Biosciences Private Limited for their help in characterizing the anti-bacterial properties. The authors also acknowledge the financial support from the
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Department of Biotechnology, Government of India (for NR) and the Ministry of Education,
References
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Culture, Sports, Science and Technology - Japan (for MI).
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Abass, Oseni, O., 2018. In-vitro compositional investigations of antioxidants, phytochemicals, nutritional and minerals in the fruit of Kigelia africana (Lam.) Benth. Int. J. Contemp. Res. Rev. 9, 20259–20268. https://doi.org/10.15520/ijcrr/2018/9/08/585
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Abdulkhani, A., Hosseinzadeh, J., Ashori, A., Esmaeeli, H., 2017. Evaluation of the antibacterial activity of cellulose nanofibers/polylactic acid composites coated with ethanolic extract of propolis. Polym. Compos. 38, 13–19. https://doi.org/10.1002/pc.23554
Akah, P.A., 1996. Antidiarrheal Activity of Kigelia africana in Experimental Animals. J. Herbs. Spices Med. Plants 4, 31–38. https://doi.org/10.1300/J044v04n02_06
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Bandeira Junior, G., Sutili, F.J., Gressler, L.T., Ely, V.L., Silveira, B.P., Tasca, C., Reghelin, M., Matter, L.B., Vargas, A.P.C., Baldisserotto, B., 2018. Antibacterial potential of phytochemicals alone or in combination with antimicrobials against fish pathogenic bacteria. J. Appl. Microbiol. 125, 655–665. https://doi.org/10.1111/jam.13906 Bello, I., Shehu, M.W., Musa, M., Zaini Asmawi, M., Mahmud, R., 2016. Kigelia africana (Lam.)
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Benth. (Sausage tree): Phytochemistry and pharmacological review of a quintessential
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African traditional medicinal plant. J. Ethnopharmacol. 189, 253–276. https://doi.org/10.1016/j.jep.2016.05.049
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Cheruiyot, K.R., Olila, D., Kateregga, J., 2009. In-vitro antibacterial activity of selected medicinal
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plants from Longisa region of Bomet district, Kenya. Afr. Health Sci.
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Falode, J.A., Akinmoladun, A.C., Olaleye, M.T., Akindahunsi, A.A., 2017. Sausage tree ( Kigelia africana ) flavonoid extract is neuroprotective in AlCl 3 -induced experimental Alzheimer’s
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disease. Pathophysiology 24, 251–259. https://doi.org/10.1016/j.pathophys.2017.06.001 Ganapathy, T., Sathiskumar, R., Senthamaraikannan, P., Saravanakumar, S.S., Khan, A., 2019. Characterization of raw and alkali treated new natural cellulosic fibres extracted from the aerial roots of banyan tree. Int. J. Biol. Macromol. 138, 573–581.
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https://doi.org/10.1016/j.ijbiomac.2019.07.136
Guna, V., Ilangovan, M., Hu, C., Nagananda, G.S., Ananthaprasad, M.G., Venkatesh, K., Reddy, N., 2019a. Antimicrobial Natural Cellulose Fibers from Hyptis suaveolens for Potential Biomedical and Textiles Applications. J. Nat. Fibers 0, 1–10. https://doi.org/10.1080/15440478.2019.1658258 23
Guna, V., Ilangovan, M., K., A., C.V., A.K., C.V., S., S., Y., Nagananda, G.S.S., Venkatesh, K., Reddy, N., Adithya, K., Akshay, A.K., Srinivas, C. V., Yogesh, S., Nagananda, G.S.S., Venkatesh, K., Reddy, N., 2019b. Biofibers and biocomposites from sabai grass: A unique renewable resource. Carbohydr. Polym. 218, 243–249. https://doi.org/10.1016/j.carbpol.2019.04.085 Holetz, F.B., Pessini, G.L., Sanches, N.R., Cortez, D.A.G., Nakamura, C.V., Dias Filho, B.P., 2002.
of
Screening of some plants used in the Brazilian folk medicine for the treatment of infectious
ro
diseases. Mem. Inst. Oswaldo Cruz. https://doi.org/10.1590/S0074-02762002000700017 Hussain, T., Fatima, I., Rafay, M., Shabir, S., Akram, M., Bano, S., 2016. Evaluation of
-p
antibacterial and antioxidant activity of leaves, fruit and bark of kigelia Africana. Pakistan J.
re
Bot. 48, 277–283.
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Ilangovan, M., Guna, V., Hu, C., Nagananda, G.S., Reddy, N., 2018. Curcuma longa L. plant residue as a source for natural cellulose fibers with antimicrobial activity. Ind. Crops Prod.
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112, 556–560. https://doi.org/10.1016/j.indcrop.2017.12.042 Jebadurai, S.G., Raj, R.E., Sreenivasan, V.S., Binoj, J.S., 2019. Comprehensive characterization of natural cellulosic fiber from Coccinia grandis stem. Carbohydr. Polym. 207, 675–683.
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https://doi.org/10.1016/j.carbpol.2018.12.027 Kathirselvam, M., Kumaravel, A., Arthanarieswaran, V.P., Saravanakumar, S.S., 2019. Characterization of cellulose fibers in Thespesia populnea barks: Influence of alkali treatment. Carbohydr. Polym. 217, 178–189. https://doi.org/10.1016/j.carbpol.2019.04.063
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Le Phuong, H.A., Izzati Ayob, N.A., Blanford, C.F., Mohammad Rawi, N.F., Szekely, G., 2019. Nonwoven Membrane Supports from Renewable Resources: Bamboo Fiber Reinforced Poly(Lactic Acid) Composites. ACS Sustain. Chem. Eng. https://doi.org/10.1021/acssuschemeng.9b02516 Liu, Y., Lv, X., Bao, J., Xie, J., Tang, X., Che, J., Ma, Y., Tong, J., 2019. Characterization of silane
of
treated and untreated natural cellulosic fibre from corn stalk waste as potential
ro
reinforcement in polymer composites. Carbohydr. Polym. 218, 179–187. https://doi.org/10.1016/j.carbpol.2019.04.088
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Micheli, V., Sanogo, R., Mobilia, M.A., Occhiuto, F., 2019. Effects of Kigelia africana (Lam.)
re
Benth. fruits extract on the development and maturation of the reproductive system in
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immature male rats. Nat. Prod. Res. 1–5. https://doi.org/10.1080/14786419.2019.1579809 Oikeh, E.I., Omoregie, E.S., Oviasogie, F.E., Oriakhi, K., 2016. Phytochemical, antimicrobial, and
ur na
antioxidant activities of different citrus juice concentrates. Food Sci. Nutr. https://doi.org/10.1002/fsn3.268
Raharjo, W.W., Soenoko, R., Irawan, Y.S., Suprapto, A., 2018. The Influence of Chemical Treatments on Cantala Fiber Properties and Interfacial Bonding of Cantala Fiber/Recycled
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High Density Polyethylene (rHDPE). J. Nat. Fibers 15, 98–111. https://doi.org/10.1080/15440478.2017.1321512
Reddy, N., Yang, Y., 2009a. Natural cellulose fibers from soybean straw. Bioresour. Technol. 100, 3593–3598. https://doi.org/10.1016/j.biortech.2008.09.063
25
Reddy, N., Yang, Y., 2009b. Properties of natural cellulose fibers from hop stems. Carbohydr. Polym. 77, 898–902. https://doi.org/10.1016/j.carbpol.2009.03.013 Reddy, N., Yang, Y., 2009c. Properties and potential applications of natural cellulose fibers from the bark of cotton stalks. Bioresour. Technol. 100, 3563–3569. https://doi.org/10.1016/j.biortech.2009.02.047
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Reddy, N., Yang, Y., 2005. Structure and properties of high quality natural cellulose fibers from
ro
cornstalks. Polymer (Guildf). 46, 5494–5500.
-p
https://doi.org/10.1016/j.polymer.2005.04.073
Segal, L., Creely, J.J., Martin, A.E., Conrad, C.M., 1959. An Empirical Method for Estimating the
re
Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer. Text. Res. J. 29,
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786–794. https://doi.org/10.1177/004051755902901003 Song, Y., Zhang, J., Zhang, X., Tan, T., 2015. The correlation between cellulose allomorphs (I and
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II) and conversion after removal of hemicellulose and lignin of lignocellulose. Bioresour. Technol. 193, 164–170. https://doi.org/10.1016/j.biortech.2015.06.084 Vijay, R., Lenin Singaravelu, D., Vinod, A., Sanjay, M.R., Siengchin, S., Jawaid, M., Khan, A.,
Jo
Parameswaranpillai, J., 2019. Characterization of raw and alkali treated new natural cellulosic fibers from Tridax procumbens. Int. J. Biol. Macromol. 125, 99–108. https://doi.org/10.1016/j.ijbiomac.2018.12.056
Vinod, A., Vijay, R., Singaravelu, D.L., Sanjay, M.R., Siengchin, S., Moure, M.M., 2019. Characterization of untreated and alkali treated natural fibers extracted from the stem of
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Catharanthus roseus. Mater. Res. Express 6, 085406. https://doi.org/10.1088/20531591/ab22d9 Yang, H., Yan, R., Chen, H., Lee, D.H., Zheng, C., 2007. Characteristics of hemicellulose, cellulose
Jo
ur na
lP
re
-p
ro
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and lignin pyrolysis. Fuel 86, 1781–1788. https://doi.org/10.1016/j.fuel.2006.12.013
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PII:
S0144-8617(20)30170-3
DOI:
https://doi.org/10.1016/j.carbpol.2020.115996
Reference:
CARP 115996
To appear in:
Carbohydrate Polymers
Received Date:
11 November 2019
Revised Date:
11 February 2020
Accepted Date:
11 February 2020
Please cite this article as: Ilangovan M, Guna V, Prajwal B, Jiang Q, Reddy N, Extraction and Characterisation of Natural Cellulose Fibers from Kigelia africana, Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2020.115996
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Extraction and Characterisation of Natural Cellulose Fibers from Kigelia africana Manikandan Ilangovan1,2, Vijaykumar Guna2,3, Prajwal B2, Qiuran Jiang4,5, Narendra Reddy2,* 1Department
of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan for Incubation, Innovation, Research and Consultancy, Jyothy Institute of Technology,
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2Centre
Thataguni Post, Bengaluru 560082, India
Technological University – Research Resource Centre, Jnana Sangama Belagavi-
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3Visvesvaraya
Laboratory of Textile Science &Technology, Ministry of Education, College of Textiles,
Donghua University, Shanghai, China
of Technical Textiles, College of Textiles, Donghua University, Shanghai, China
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5Department
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4Key
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590018, India
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* Corresponding Author: Narendra Reddy, Centre for Incubation, Innovation, Research and Consultancy, Jyothy Institute of Technology, Thataguni Post, Bengaluru 560082, India
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Phone: +919611233707 Email: [email protected]
Graphical abstract
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Highlights
Natural cellulosic fibers were extracted from Kigelia africana (sausage plant)
The fibers had high cellulosic content (70%) and good tensile properties
Sausage fibers possessed excellent thermal stability and moderate hydrophobicity
Sausage fibers showed decent activity against gram positive and negative bacteria
Storage boxes for dry food items were developed with good structural integrity
Abstract
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Kigelia africana also known as sausage plant, yields highly fibrous fruit with a hard shell. Many medicinal uses are reported for the extracts from the fruits, seeds and leaves of sausage trees. In this research, natural cellulose fibers were extracted from the fruit using NaOH and later bleached and characterized for their properties. Results revealed that significant amount of hemicellulose and lignin was lost after the alkali treatment and bleaching leading to a highly cellulosic fiber (up to 71%). Morphologically, surface of the fibers varied from rough to smooth 2
depending on the extent of treatment. The thermal stability, crystallinity and hydrophobicity increased after the treatment. Sausage fibers also possessed anti-microbial activity against common gram negative and gram positive bacteria. Overall, sausage fibers have properties similar to that of cotton and better than fibers obtained from many unconventional sources. With improved hydrophobicity and anti-bacterial properties, sausage fibers could be potentially
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Key words: Sausage fruit, Anti-bacterial, Bio-Product, Cellulosic Fibers
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applied in functional polymer composites.
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1.0 Introduction Among the different applications of agricultural residues, extraction of natural cellulose fibers is highly preferable since fibrous applications have a large market and offer higher value. Natural cellulose fibers have been extracted from several unconventional sources in an attempt to obtain fibers with unique properties (Guna et al., 2019b, 2019a; Ilangovan et al., 2018;
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Jebadurai et al., 2019; Kathirselvam et al., 2019; Liu et al., 2019). Fibers obtained from cotton
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stalks, rice and wheat straw, cornstalks and husks had properties similar to that of cotton, jute and linen and were found to be useful for textile, composites and other applications (Reddy and
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Yang, 2009a, 2009b, 2009c). Recent studies have also shown the possibility of extracting and
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demonstrated the unique features of natural cellulose fibers from aerial roots of banyan trees and wild plants such as Tridaxprocumbens and Catharanthusroseus (Ganapathy et al., 2019;
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Vijay et al., 2019; Vinod et al., 2019). Similarly, fibers obtained from tulsi stems or sabaigrass, which are considered as weeds, not only had good mechanical properties but also showed
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antimicrobial activity and hence were suggested for medical applications (Guna et al., 2019a, 2019b). In addition, sabai fibers also possessed high thermal and noise insulation comparable to industrial standards when used as reinforcement for composites, extending its applications
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beyond automotive to green building and interior applications (Guna et al., 2019b). Kigelia Africana also known as sausage tree is an evergreen plant that can reach up to
20m in height. Fruits of Kigelia Africana (sausage fruit) can grow up to 1m x 18cm, 30-100 cm long and can weigh up to 12 kg. Sausage fruits consist of a hard protective and inedible shell, that forms more than 80% of the weight of the fruit (Bello et al., 2016). In addition to use as food, the pulp, seeds and other components of the sausage fruit have been proven to have 4
medicinal properties. Extracts obtained from the sausage fruit and tree have shown antibacterial, antifungal, antioxidant, anti-inflammatory and central nervous system (CNS) stimulant activities (Akah, 1996; Bello et al., 2016).Ethanol and n-hexane extracts from the leaves and barks of Kigelia africana tree had good antibacterial activity against six common micro-organisms with inhibition level as high as 67% (Hussain et al., 2016). Extracts from the
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bark of the plant showed higher activity compared to the leaves or fruit extracts. Methanol extracts from the leaves are reported to contain flavonoid rich fractions that will aid in
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therapeutic treatment of Alzheimer’s disease (Falode et al., 2017). Other studies have shown
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that the sausage plant extracts can increase body growth and organ development (Micheli et al., 2019). Apart from phenolic components (saponin, tannin, terpenoid), sausage fruits were
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reported to contain minerals such as potassium and calcium, proteins and fat which are suitable
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for several food and non-food applications (Abass, Oseni, 2018). Although the extracts of the fruits and other parts of the sausage tree are studied for their medicinal properties, to the best
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of our knowledge, there are no studies conducted on the non-food applications of the fibers in the fruit. In this research, we have studied the structure and properties of the fibers in the sausage fruit before and after chemical treatments. Properties of the fibers have been compared to common natural cellulose fibers and also to those obtained from agricultural
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residues and biomass.
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2.0 Materials and Methods 2.1 Materials Sausage fruits each weighing about 5kg were collected from local farms near our campus in Bengaluru, Karnataka. Analytical grade sodium hydroxide, acetic acid, acetone and other chemicals required for the experiment were purchased from Hybrid Fine Chemicals,
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Bangalore, Karnataka.
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2.2 Fiber extraction
Untreated Sausage fruit fibers (SFF) were separated from the fruits manually. Initially
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the raw fruit were cut into sections, oven dried for 24 hours and then fed through a two roller
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mechanical crusher. Partially crushed fruits were collected and soaked in boiling water prior to
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the separation of fibrous part.
SFF was then treated with 0.2N NaOH solution for 90min at 100 o C. The untreated fiber
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to alkali ratio was maintained at 1:10. After treatment, the liquid containing dissolved hemicelluloses, lignin and other extractives was decanted and disposed. The fibers extracted through alkali treatment (ATF) were thoroughly washed until the pH of the fibers was neutral. The neutral pH fibers were later immersed in 10% acetic acid solution for 10 min and rinsed.
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Later, the fibers were dried in a hot air oven at 110oC for 3h and stored for further characterization. 2.3 Bleaching The alkali treated fibers (ATF) were further bleached using 7% sodium chlorite at 80oC for 1h. The fiber: sodium chlorite ratio was maintained at 1:5. After bleaching, the fibers were 6
washed multiple times using de-ionized water. The fibers were then immersed in 5% acetic acid to neutralize the alkali. The bleached fibers (BF) were then dried at 110oC for 3h and used for further analysis. 2.4 Fiber Characterization The percentage cellulose, hemicellulose, lignin and ash content of SFF, ATF and BF were
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determined according to the standard test methods as described in (Guna et al., 2019b). Each
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component was analyzed in triplicates and the average value was reported.
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The fibers were tested for their tensile strength on a Universal Tensile Tester (MTS Mechatronics, Ichalkaranji, India) according to the ASTM D 3822-14 standard. The cross-head
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speed and the gauge length was set at 10 mm/min and 25.4mm, respectively. Samples from
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three different sets of extraction were used for the characterization. Twenty samples from each set, i.e., a total of 60 samples were tested. The mean and standard deviation was reported.
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The morphology of the fibers was observed in a Scanning Electron Microscope (Hitachi Model SU 3500). Before observation, the fibers were sputter coated with gold-palladium in an Ion Beam coater for 60s. The coated samples were then analyzed at an operational voltage of
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15kV.
X-ray diffraction study was conducted on the powdered fiber samples (particle size:
200m) in a Bruker D8 Advanced Eco X-ray diffractometer equipped with Bragg-Brentano Focusing geometry. The analysis was done using a Cu-Kα radiation at a wavelength (λ = 1.54 Å) and the diffraction patterns were recorded at 2θ angles varying from 5o to 65o. The readings
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were analyzed in Origin Pro software and the peak intensities were identified. The Segal equation (Segal et al., 1959)was used to calculate the crystallinity index (Xc) as shown below:
𝑋𝑐 = (
𝐼002 − 𝐼𝑎𝑚 ) × 100% 𝐼002
Where, I002 and Iam are the peak intensities at the crystalline and amorphous region
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respectively. Size of cellulose crystallites in the fibers were calculated using the Scherrer
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equation and the data has been included in the supplementary file (Table S1).
FTIR was carried out to identify the type of bonding and components present in the
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fibers. The spectrum was recorded using Perkin Elmer/Spectrum 2 (Diamond UTAR) in 4000 to
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400 cm-1range at a resolution of 2 cm-1.
Wettability of liquids over the fiber surface was carried out using a contact angle
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apparatus (OCA 15EC, Data Physics Instruments, Germany). To measure the water contact angle (WCA) , 2 μL of water was dispensed using a micropipette over the fibers along the longitudinal
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axis. Images were then captured using a camera connected with a computer interface. Contact angle measurements were done on the liquid spread over the fiber surface. Ability of the extracted fibers to resist common bacteria was also studied using two
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gram positive (Staphylococcus aureus, Bacillus cereus) and two gram negative (Escherichia coli, Serratia marcescens) bacterial strains. The analysis was conducted according to the method described in (Guna et al., 2019a). Extracted fibers were used as reinforcements in gluten matrix to develop potential bioproducts. Products in the shape of packaging boxes were fabricated at 160 oC and 600 MPa
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pressure in a custom made hot press with box shape mold. The fibers: gluten ratio was kept at (90:10% w/w) for the process. Digital images of the boxes were included in the article. 3.0 Results and discussion 3.1 Composition of the fibers SFF is lignocellulosic with high percentage of lignin and lower concentration of
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hemicellulose. Untreated SFF have similar cellulose content as that of alkali extracted fibers
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from tulsi and turmeric stalks (Table 1). Cellulose content of the fibers increases after treating with alkali and further increases to about 72% after bleaching. Correspondingly, the
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hemicellulose and lignin contents are also altered (Table 1). As the cellulose content increases,
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the fibers also change color becoming brownish due to the alkali treatment but later turn white after bleaching (Figure.1). Higher cellulose content not only increases whiteness but should also
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provide the fibers with better tensile properties unless the cellulose in the fibers get damaged.
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Hemicellulose content of all the fibers is lower than the lignin content as is typically observed in non-traditional fibers (Guna et al., 2019a, 2019b). Lignin content is higher than cotton but lower than that found in flax, jute, coir and other common natural cellulose fibers (Reddy and Yang, 2009c; Vijay et al., 2019). Typically, the lignin content should decrease after
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bleaching but we observe a contrary effect in this study. Increase in the lignin content may be because of its recalcitrant form or higher concentration of lignin in the treated fibers compared to those removed during chemical treatment and washing. Ash content in the fibers decreased by about 65% from SFF to BF and were found to be up to 93% lower than that in turmeric and tulsi fibers, but comparable to cotton and cotton stalk fibers (Guna et al., 2019a; Ilangovan et
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al., 2018; Reddy and Yang, 2009c). Treating at stronger conditions may further increase the cellulose and reduce lignin and hemicellulose content., However, the fibers may become smaller and unusable for high value applications. Nevertheless, the fiber properties are equally influenced by the morphology of the individual fibers, crystallinity and orientation. Table 1 Compositional data of fibers obtained from sausage plant compared to that of other
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non-traditional cellulosic fibers (Guna et al., 2019a; Ilangovan et al., 2018)
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Sample
Turmeric
SFF
ATF
BF
Tulsi Fibers
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Fibers
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Composition 55.1±0.2
69.9±0.1
71.6±1.2
56.5±0.6
52.5±0.2
Hemicellulose (%)
9.34±0.08
9.01±0.5
9.81±0.4
12.4±0.2
18.5±1.3
Lignin (%)
11.7±0.2
10.00±0.4
13.5±1.1
14.8±1.3
14.3±0.19
Ash(wt.%)
1.85±0.1
1.3±0.1
0.64±0.03
10.2±1.2
9.61±0.19
Flavonoids μg/ml
160±0.1
145±0.2
ND
196±0.1
180±0.1
Phenol μg/ml
240±0.2
180±0.1
92±0.2
180±0.1
89±0.2
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Cellulose (%)
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ND – Not Detected
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Figure. 1 Schematic representation of the process used for fiber extraction from sausage fruit
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3.2 Morphology
Surface of untreated fibers is rough due to the presence of impurities (Figure. 2 a-c).
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Bundles of individual fibers of width ranging from 40 to 80 µm are observable (Figure. 2 a). On further magnifying, a thick coating of substances most likely hemicellulose, ash and other impurities can be seen (Figure. 2 b,c). Alkali treatment removes most of the surface substances
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resulting in fibers with smooth texture (Figure. 2 d,e). Some of the individual fibers get separated from the bundles which could disintegrate into fibrils if treatment conditions are too aggressive or not controlled. Individual fibers had widths of 50 µm and length more than 4550mm.Microfibrils arranged at an angle to the fiber-axis (micro fibrillar angle or MFA) are seen in ATF (Figure. 2 f). The MFA of ATF and BF were measured using ImageJ software and are reported in Table 2. Bleaching removes almost all of the impurities leading to a cleaner surface 11
in BF (Figure. 2 g, h). Not only fibers but individual fibrils can be observed on the surface of the BF. Further, the convolutions and distinct fiber taper typical to cellulosic fibers are also apparent in the ATF and BF (Figure. 2f, g, i). Morphological analysis indicates that BF have
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characteristics very similar to that of cotton.
Figure. 2 SEM images of: (a-c) SFF, (d-f) ATF, (g-i) BF
3.3 Tensile properties Images of the tensile experiment can be found in the supplementary file (Figure S2). The tensile properties increase successively after alkali treatment and further after bleaching (Table 12
2). An 8% increase in the strength of ATF and 28% increase in BF was observed compared to SFF. Similarly, the elongation and modulus in BF increased by about 44% and 24% respectively compared to SFF. Increase in the tensile properties can be directly related to the chemical treatment and specifically attributed to the following two reasons: the increase in cellulose content and reorganization of the cellulose fibers and crystals during the treatment. As a result
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of reorganization, the MFA increased from 13.5° in ATF to 16.4° in the BF. Typically, higher MFA leads to higher flexibility and hence higher elongation(Reddy and Yang, 2005)which was also
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observed in the case of sausage fibers. Tensile strength of sausage fruit fibers was comparable
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(Ilangovan et al., 2018; Reddy and Yang, 2009c).
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to that cotton and jute. The elongation was however, higher than jute and lower than cotton
Table 2 Tensile properties of untreated, alkali treated, and bleached sausage fruit fibers.
Sample
Elongation
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Tensile
Young’s
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Strength (MPa) At break (%) modulus (GPa)
Microfibril angle (°)
379.28± 19.53
2.61 ± 0.74
15.68 ± 2.92
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Alkali treated Fiber
411.08± 14.56
3.68 ± 0.46
17.52 ± 1.72
13.54 ± 1.04
Bleached Fiber
484.01± 16.84
3.75± 0.564
19.47 ± 1.47
16.41 ± 1.48
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Untreated Fiber
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3.4 X- ray diffraction analysis (XRD) A gradual but distinct change in the crystal structure of the fiber occurs after alkali treatment and bleaching. Intensity of the main 002 cellulose I crystal peak at 22° increases sharply and becomes narrower after alkali treatment and even further after bleaching (Figure. 3). In the raw and alkali treated fibers, the 101 and 110 peaks are merged due to the presence
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of hemicellulose and impurities. The two peaks are distinct in the bleached fibers since they
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have high cellulose content. However, there is no change in the cellulose allomorph from I to II as observed after strong alkaline treatments (Song et al., 2015). Corresponding to the variations
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in peak intensities and positions, the % crystallinity of raw, alkali treated, and bleached fibers
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was 59, 60 and 70%, respectively. Bleached sausage fibers have crystallinity similar to cotton and linen (65 to 70%) and higher than fibers obtained from lignocellulosic crop residues (50 to
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60%) (Guna et al., 2019a; Ilangovan et al., 2018; Reddy and Yang, 2009c). Higher crystallinity would reduce the accessibility of the cellulose to chemicals and enzymes and hence chemical
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modifications and dyeing would be difficult. Therefore, the extent of treatment must be
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controlled depending up on the intended applications.
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Figure.3 Normalized XRD spectrum of untreated, alkali treated, and bleached sausage fibers
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3.5 Fourier Transform Infrared Spectroscopy
The FTIR spectrum of SFF, ATF, BF is given in Figure. 4. A typical vibration at the 1048
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cm-1 region (C-O group related to cellulose content) is prominent in BF compared to SFF and ATF. Similar behavior was observed in the OH stretch at 3424 cm-1 indicating higher cellulose content in the BF(Yang et al., 2007). The hemicellulose and pectin fingerprint region at the 1734
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cm-1 (C=O stretch) is visible only in the SFF which suggests successful removal after treatment with alkali and bleaching. A small peak at 1513 cm-1 (C=C group) relating to the lignin aromatic network is evident in the BF. Stronger peak at the C-H region (2922 cm-1) typical to aromatic compounds is also observed in the BF, suggesting higher lignin content than SFF and ATF. The FTIR spectra are in good accordance with the compositional data (Table 1).
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3.6 Changes in surface wettability
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Figure.4 FT-IR spectrum of untreated, alkali treated, and bleached sausage fibers
Treating the fibers with alkali and later bleaching, lead to a decrease in the contact angle
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(Figure. 5). SFF contains non-polar, hydrophobic surface impurties such as wax and pectin. Also, the heterogenous surface of the SFF results in air pockets trapped between the droplet and surface, there by increasing the contact angle (Le Phuong et al., 2019). After alkali treatment or bleaching, the non-polar impurities are gradually removed (as observed in Figure. 2). This
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removal leads to an increase in the polarity and surface energy of the fibers thereby turning them hydrophilic (Raharjo et al., 2018; Vijay et al., 2019). The smoother BF in comparison to SFF and ATF had over 20o and 15o lower WCA respectively, attributed to the higher cellulose (hydrophilic) content. However, all three fiber surfaces have contact angle less than 90°C which
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is typically treated as hydrophilic. Similar behavior was reported in the studies of Tridax
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procumbens , Cantala and hemp fibers (Raharjo et al., 2018; Vijay et al., 2019).
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Figure. 5 Digital image depicting the wettability of (a) SFF (b) ATF (c) BF
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3.7 Thermal degradation
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Thermal analysis shows that the degradation of the fibers follows a three step process (Figure. 6). The initial stage occurring between 80 to 90 °C leads to about 5% weight loss due to
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the removal of surface moisture. Degradation behavior of the fibers can then be divided into two regions (191-300°C) and (300 to 406°C). Weight loss at the former is possibly due to the
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degradation of mostly hemicellulose and partially lignin and at the latter due to the decomposition of cellulose (Kathirselvam et al., 2019). All three fibers are quite stable after the initial step, with 25% weight loss occurring only between 302 to 332 °C (Figure. 6). Interestingly,
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SFF show higher thermal stability than the alkali treated or bleached fibers in this region probably due to the presence of impurities with higher stability on the surface of the fibers. Bleached fibers show marginally slower rate of degradation compared to untreated and alkali treated fibers (Figure.6). Since all three fibers are stable below 300 °C, they are suitable for processing for composites and textiles without being damaged.
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Figure. 6 TGA/DTG profile of untreated, alkali treated, and bleached sausage fibers.
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3.8 Antibacterial activity
Natural cellulose fibers obtained from several non-conventional sources have shown
inherent antibacterial activity (Guna et al., 2019a, 2019b; Ilangovan et al., 2018). We hypothesized that fibers from sausage fruit will also potentially exhibit anti-microbial properties given its other proven medicinal applications. Having resistance to micro-organisms will be 18
useful in food packaging, medical and other fibrous applications. As hypothesized, sausage fruit fibers showed appreciable antimicrobial activity to common microorganisms. Generally speaking, with increased dosage, the inhibition activity increased due to the higher quantities of phytochemicals present. Phytochemicals play a significant role in the anti-bacterial activity of any material (Bandeira Junior et al., 2018; Oikeh et al., 2016). As seen in Table 1, phenols and
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flavonoids are higher in SFF compared to ATF and BF. Consequently, the percentage inhibition of SFF (54 - 97%) was higher at all dosages against all the four organisms compared to ATF and
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BF. As the concentration of phytochemicals decreased, the activity in ATF and BF reduced by up
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to 63%. This phenomenon was observed in other natural fibers from non-traditional sources such as tulsi, turmeric stalks and sabai grass as well (Guna et al., 2019a, 2019b; Ilangovan et al.,
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2018). The degree of inhibition was high in gram-positive bacteria than the gram-negative
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bacteria in all the fibers. This could be attributed to the outer membrane in the gram negative bacteria that acts as a barrier to foreign substances (Oikeh et al., 2016). Gram negative bacteria
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contains a typical preventive area known as the periplasmic space where enzymes break down foreign particles (even the anti-microbial agents) trying to access the organism (Cheruiyot et al., 2009; Holetz et al., 2002; Oikeh et al., 2016). Although, the presence of phenols and flavonoids improves the anti-bacterial activity, the exact mechanism of inhibition is different for different
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compounds (Abdulkhani et al., 2017). Thorough micro-biological studies are required to predict the inhibition mechanism of sausage fruit fibers. However, the fiber extraction conditions could be further adjusted for the optimum concentration of phytochemicals for specific applications.
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Table 3 Extent of inhibition (%) of four common bacteria by untreated, alkali-treated and bleached fiber extracted from the sausage tree fruit fibers at different dosages. Inhibition at 50 mg/ml dosage (%)
Inhibition at 100 mg/ml dosage (%)
Organism SFF
ATF
BF
SFF
ATF
BF
S aureus
91.5 ± 0.1
70.8 ± 0.2
57.6± 0.2
97.5 ± 0.4
86.4 ± 0.3
62.2± 0.4
Positive
B cereus
72.4 ± 0.2
69.6 ± 0.3
54.2 ± 0.4
92.2 ± 0.1
82.6 ± 0.2
68.4 ± 0.4
Gram
E coli
82.4 ± 0.4
61.9 ± 0.2
42.6 ± 0.4
88.4 ± 0.6
65.2 ± 0.4
48.4 ± 0.2
Negative
S marcescens
54.5 ± 0.3
39.4 ± 0.4
19.8 ± 0.6
42.7 ± 0.1
26.3 ± 0.2
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Gram
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64.9 ± 0.3
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3.9 Potential to develop bio-products
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Sausage fruit fibers obtained had good mechanical properties which enables us to use them in developing various bio-products. As seen from Figure. 7, both untreated and bleached
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fibers were compression molded into boxes. Since sausage fruit fibers demonstrated antimicrobial properties, the products developed could be used in food applications such as packaging of dry fruit, ready-to-eat foods and sweets, replacing currently used synthetic containers. Further, they are can be used in developing bio composites with promising
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mechanical strength.
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Figure. 7 Boxes suitable for food and non-food applications molded from : Top and Bottom Row Left: SFF , Top and Bottom Row Middle: ATF, Top and Bottom Row
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Right: BF
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4.0 Conclusions
Sausage fruit is composed of lignocellulosic fibers with cellulose and lignin content of
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about 15 and 12%, respectively and generally considered non-edible for humans. However, the raw fibers and those after treating with alkali and subject to bleaching all have good mechanical properties, crystallinity and morphology. After bleaching, cellulose content in the fibers increased to 71% and the tensile strength was 484 MPa, similar to that of cotton. Further,
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sausage tree fibers have convolutions and tapered edges as seen in cotton. With thermal stability (up to 300 oC), high crystallinity (70%) and good elongation (3.75%), the fibers can be used for composites and non-woven applications. However, the fibers are coarser and also are shorter in length and hence unsuitable for typical textile applications. The new approach to
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develop sustainable bio-products using sausage fruit fibers shows promise to replace some of the non-biodegradable, synthetic polymer based products in the market.
Author credit statement
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Narendra Reddy and Vijaykumar Guna conceptualized and designed the research. MI, QJ and PS conducted and analyzed the data. MI, NR wrote the manuscript with help from VG.
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Acknowledgements
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The authors thank Azyme Biosciences Private Limited for their help in characterizing the anti-bacterial properties. The authors also acknowledge the financial support from the
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Department of Biotechnology, Government of India (for NR) and the Ministry of Education,
References
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Culture, Sports, Science and Technology - Japan (for MI).
ur na
Abass, Oseni, O., 2018. In-vitro compositional investigations of antioxidants, phytochemicals, nutritional and minerals in the fruit of Kigelia africana (Lam.) Benth. Int. J. Contemp. Res. Rev. 9, 20259–20268. https://doi.org/10.15520/ijcrr/2018/9/08/585
Jo
Abdulkhani, A., Hosseinzadeh, J., Ashori, A., Esmaeeli, H., 2017. Evaluation of the antibacterial activity of cellulose nanofibers/polylactic acid composites coated with ethanolic extract of propolis. Polym. Compos. 38, 13–19. https://doi.org/10.1002/pc.23554
Akah, P.A., 1996. Antidiarrheal Activity of Kigelia africana in Experimental Animals. J. Herbs. Spices Med. Plants 4, 31–38. https://doi.org/10.1300/J044v04n02_06
22
Bandeira Junior, G., Sutili, F.J., Gressler, L.T., Ely, V.L., Silveira, B.P., Tasca, C., Reghelin, M., Matter, L.B., Vargas, A.P.C., Baldisserotto, B., 2018. Antibacterial potential of phytochemicals alone or in combination with antimicrobials against fish pathogenic bacteria. J. Appl. Microbiol. 125, 655–665. https://doi.org/10.1111/jam.13906 Bello, I., Shehu, M.W., Musa, M., Zaini Asmawi, M., Mahmud, R., 2016. Kigelia africana (Lam.)
of
Benth. (Sausage tree): Phytochemistry and pharmacological review of a quintessential
ro
African traditional medicinal plant. J. Ethnopharmacol. 189, 253–276. https://doi.org/10.1016/j.jep.2016.05.049
-p
Cheruiyot, K.R., Olila, D., Kateregga, J., 2009. In-vitro antibacterial activity of selected medicinal
re
plants from Longisa region of Bomet district, Kenya. Afr. Health Sci.
lP
Falode, J.A., Akinmoladun, A.C., Olaleye, M.T., Akindahunsi, A.A., 2017. Sausage tree ( Kigelia africana ) flavonoid extract is neuroprotective in AlCl 3 -induced experimental Alzheimer’s
ur na
disease. Pathophysiology 24, 251–259. https://doi.org/10.1016/j.pathophys.2017.06.001 Ganapathy, T., Sathiskumar, R., Senthamaraikannan, P., Saravanakumar, S.S., Khan, A., 2019. Characterization of raw and alkali treated new natural cellulosic fibres extracted from the aerial roots of banyan tree. Int. J. Biol. Macromol. 138, 573–581.
Jo
https://doi.org/10.1016/j.ijbiomac.2019.07.136
Guna, V., Ilangovan, M., Hu, C., Nagananda, G.S., Ananthaprasad, M.G., Venkatesh, K., Reddy, N., 2019a. Antimicrobial Natural Cellulose Fibers from Hyptis suaveolens for Potential Biomedical and Textiles Applications. J. Nat. Fibers 0, 1–10. https://doi.org/10.1080/15440478.2019.1658258 23
Guna, V., Ilangovan, M., K., A., C.V., A.K., C.V., S., S., Y., Nagananda, G.S.S., Venkatesh, K., Reddy, N., Adithya, K., Akshay, A.K., Srinivas, C. V., Yogesh, S., Nagananda, G.S.S., Venkatesh, K., Reddy, N., 2019b. Biofibers and biocomposites from sabai grass: A unique renewable resource. Carbohydr. Polym. 218, 243–249. https://doi.org/10.1016/j.carbpol.2019.04.085 Holetz, F.B., Pessini, G.L., Sanches, N.R., Cortez, D.A.G., Nakamura, C.V., Dias Filho, B.P., 2002.
of
Screening of some plants used in the Brazilian folk medicine for the treatment of infectious
ro
diseases. Mem. Inst. Oswaldo Cruz. https://doi.org/10.1590/S0074-02762002000700017 Hussain, T., Fatima, I., Rafay, M., Shabir, S., Akram, M., Bano, S., 2016. Evaluation of
-p
antibacterial and antioxidant activity of leaves, fruit and bark of kigelia Africana. Pakistan J.
re
Bot. 48, 277–283.
lP
Ilangovan, M., Guna, V., Hu, C., Nagananda, G.S., Reddy, N., 2018. Curcuma longa L. plant residue as a source for natural cellulose fibers with antimicrobial activity. Ind. Crops Prod.
ur na
112, 556–560. https://doi.org/10.1016/j.indcrop.2017.12.042 Jebadurai, S.G., Raj, R.E., Sreenivasan, V.S., Binoj, J.S., 2019. Comprehensive characterization of natural cellulosic fiber from Coccinia grandis stem. Carbohydr. Polym. 207, 675–683.
Jo
https://doi.org/10.1016/j.carbpol.2018.12.027 Kathirselvam, M., Kumaravel, A., Arthanarieswaran, V.P., Saravanakumar, S.S., 2019. Characterization of cellulose fibers in Thespesia populnea barks: Influence of alkali treatment. Carbohydr. Polym. 217, 178–189. https://doi.org/10.1016/j.carbpol.2019.04.063
24
Le Phuong, H.A., Izzati Ayob, N.A., Blanford, C.F., Mohammad Rawi, N.F., Szekely, G., 2019. Nonwoven Membrane Supports from Renewable Resources: Bamboo Fiber Reinforced Poly(Lactic Acid) Composites. ACS Sustain. Chem. Eng. https://doi.org/10.1021/acssuschemeng.9b02516 Liu, Y., Lv, X., Bao, J., Xie, J., Tang, X., Che, J., Ma, Y., Tong, J., 2019. Characterization of silane
of
treated and untreated natural cellulosic fibre from corn stalk waste as potential
ro
reinforcement in polymer composites. Carbohydr. Polym. 218, 179–187. https://doi.org/10.1016/j.carbpol.2019.04.088
-p
Micheli, V., Sanogo, R., Mobilia, M.A., Occhiuto, F., 2019. Effects of Kigelia africana (Lam.)
re
Benth. fruits extract on the development and maturation of the reproductive system in
lP
immature male rats. Nat. Prod. Res. 1–5. https://doi.org/10.1080/14786419.2019.1579809 Oikeh, E.I., Omoregie, E.S., Oviasogie, F.E., Oriakhi, K., 2016. Phytochemical, antimicrobial, and
ur na
antioxidant activities of different citrus juice concentrates. Food Sci. Nutr. https://doi.org/10.1002/fsn3.268
Raharjo, W.W., Soenoko, R., Irawan, Y.S., Suprapto, A., 2018. The Influence of Chemical Treatments on Cantala Fiber Properties and Interfacial Bonding of Cantala Fiber/Recycled
Jo
High Density Polyethylene (rHDPE). J. Nat. Fibers 15, 98–111. https://doi.org/10.1080/15440478.2017.1321512
Reddy, N., Yang, Y., 2009a. Natural cellulose fibers from soybean straw. Bioresour. Technol. 100, 3593–3598. https://doi.org/10.1016/j.biortech.2008.09.063
25
Reddy, N., Yang, Y., 2009b. Properties of natural cellulose fibers from hop stems. Carbohydr. Polym. 77, 898–902. https://doi.org/10.1016/j.carbpol.2009.03.013 Reddy, N., Yang, Y., 2009c. Properties and potential applications of natural cellulose fibers from the bark of cotton stalks. Bioresour. Technol. 100, 3563–3569. https://doi.org/10.1016/j.biortech.2009.02.047
of
Reddy, N., Yang, Y., 2005. Structure and properties of high quality natural cellulose fibers from
ro
cornstalks. Polymer (Guildf). 46, 5494–5500.
-p
https://doi.org/10.1016/j.polymer.2005.04.073
Segal, L., Creely, J.J., Martin, A.E., Conrad, C.M., 1959. An Empirical Method for Estimating the
re
Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer. Text. Res. J. 29,
lP
786–794. https://doi.org/10.1177/004051755902901003 Song, Y., Zhang, J., Zhang, X., Tan, T., 2015. The correlation between cellulose allomorphs (I and
ur na
II) and conversion after removal of hemicellulose and lignin of lignocellulose. Bioresour. Technol. 193, 164–170. https://doi.org/10.1016/j.biortech.2015.06.084 Vijay, R., Lenin Singaravelu, D., Vinod, A., Sanjay, M.R., Siengchin, S., Jawaid, M., Khan, A.,
Jo
Parameswaranpillai, J., 2019. Characterization of raw and alkali treated new natural cellulosic fibers from Tridax procumbens. Int. J. Biol. Macromol. 125, 99–108. https://doi.org/10.1016/j.ijbiomac.2018.12.056
Vinod, A., Vijay, R., Singaravelu, D.L., Sanjay, M.R., Siengchin, S., Moure, M.M., 2019. Characterization of untreated and alkali treated natural fibers extracted from the stem of
26
Catharanthus roseus. Mater. Res. Express 6, 085406. https://doi.org/10.1088/20531591/ab22d9 Yang, H., Yan, R., Chen, H., Lee, D.H., Zheng, C., 2007. Characteristics of hemicellulose, cellulose
Jo
ur na
lP
re
-p
ro
of
and lignin pyrolysis. Fuel 86, 1781–1788. https://doi.org/10.1016/j.fuel.2006.12.013
27