**2. Preparation of CNFs from crab and prawn shells, and mushrooms**

## **2.1. CNFs from crab shells**

using a series of chemical treatments followed by mechanical grinding. The width of the fibers was measured by field emission scanning electron microscopy (FE-SEM). The authors were first successfully reduced the size of extracted NFs from 50 to 15 nm from any natural resources.

Chitin is second most plentiful biomaterial [9] next to cellulose exists on earth with yearly production of 1011 tons. Chitin raw dried powder is manufactured from exoskeleton of sea food shellfish, crabs, shrimps, and insects and edible mushrooms of fungus species and sea weed algae. Chitin content in fish industrial waste is 8-33% which is thrown if not used. Thus our group is actively engaged in developing chitin research to make a number of products from atomized or fibrillated chitin in the form of chitin nanofibers (CNFs) and its derivatives [10-13]. Chitin obtained from its natural resources is highly crystalline and most of it is α-chitin

We have published a number of review articles [11, 14, 15] covering back ground of CNFs in detail, method of preparation, sources, composition, physical and chemical properties, characterization, their composites and derivatives preparations, surface modification. Com‐ mercialization of dry chitin powder and CNFs has also been described. For atomization or fibrillation of 1 wt.% wet chitin to CNFs three types of methods were used and compared. A very recently developed [10] Star Burst atomization system which employed high pressure water jet system where slurry of chitin in high acetic acid medium is introduced in chamber of Star Burst system machine where it is fibrilized into NFs of width (18.0-19.0 nm). Atomiza‐ taion occurred in this newly developed machine chamber by collision to ceramic ball that throws out fine fibrillated NFs at extremely high pressure of 245 MPa through an out let nozzle. The two other commonly used apparatus used for fibrillation are a blender and grinder. The advantages of Star Burst system over blender or grinder for fibrillation have been described in article [10] published recently. CNFs obtained by Star Burst system were studied thoroughly recording FE-SEM images of fibers obtained after a number of passes up to ten. The width of NFs decreased from 19.0 nm to 16.5 nm when number of passes increased from one to ten, respectively. Effect of number of passes on the CNFs properties was investigated by FT-IR, XRD profiles of chitin. In review article [11] molecular structure of chitin, hierarchical organ‐ ization on the surface of crab shell exoskeleton and isolation from crab and prawn shell has been described. Method of isolation of CNFs from crab or prawn shell using a number of chemical treatments followed by grinder treatment has been explained. The width of NFs was determined by FE-SEM recordings, NFs of 10-20 nm diameter with high aspect ratio were obtained after one pass. FE-SEM images were recorded of stepwise isolation of NFs, just after removing the matrix components and after one pass treatment in acetic acid and without acetic acid condition. Without grinding treatment the fibers were like accumulated ribbons, after one pass treatment without acid the fibers were not separated but in acidic condition after one pass the fibers separated due to repulsion among the positive charges generated on the surface of fibers in acidic conditions. Chitin NFs were modified to produce novel green materials into nano-whiskers of width 6.2 nm and length 250 nm when fibers were deacetylated by treating with 33% NaOH. This contribution discusses most recent advances in preparation, derivati‐ zation, characterization and applications of CNFs. Most of the work has been conducted in our

conformation though the contents of α- and βchitin depends on the source.

86 Advances in Nanofibers

laboratory and we have also discussed the results from other groups as well.

Commercial grade dried crab shell flakes of species *Paralithodes camtschaticus* (red king crab) were used as a raw starting material to isolate NFs. Flakes from red king crab shell are so cheap and abundant that they are used in fertilizer industry. Crab shells were crushed to powder and purified according to the well established method. 1 wt.% slurry of crab chitin was prepared by a series of chemical treatment described in a previous chapter [14]. In brief minerals were removed by HCl treatment, suspension was filtered and washed thoroughly with distilled water, removal of proteins was done by refluxing the suspension with NaOH, pigments and lipids were removed by ethanol. After completion of above the treatments, suspension was filtered washed with distilled water and kept wet for mechanical grinding for fibrillation, this wet slurry was made to a concentration of 1 wt.% and called chitin slurry. Chemical treatment loosened the tightly bonded fibrils bundles to larger extent apart from removal of minerals, proteins, pigments, and lipids as shown in Fig. 1a and b.

**Figure 1.** FE-SEM images of crab shell surface after removal of matrix from shell surface by chemical treatment with‐ out mechanical grinding at different magnification scales; a) 1000 nm; b) 100 nm. Reprinted with permission from ref. 1. Copyright 2009, American Chemical Society.

Bundles of NFs of width 30 nm are visible in micrographs without mechanical grinding. For fibrillation, 1 wt.% slurry was passed through a grinder of the model (MKCA6-3; Masuko Sangyo Co., Ltd.). After passing through the grinder, chitin slurry changed to highly viscous stable wet gel of CNFs. To record FE-SEM picture of sample, sheet of chitin material was prepared. Sheet was coated with 2 nm layer of platinum by an ion sputter coating before recording SEM micrographs. Chitin slurry was passed for one cycle through grinder at pH 7 and 3. As shown in Fig. 2a at neutral pH, fibers had width in wider range 10-100 nm. The bundles of embedded chitin-protein fibers were fibrillated successfully by grinding of wet chitin. It was easy to remove protein from water soaked chitin to isolate chitin fibrils. Authors [16] reported preparation method of CNFs from wet squid pen β-chitin at pH 3–4. In acidic condition at low pH cationization of C2 amino groups in β-chitin occurred resulted in more dispersed and stable phase because of electrostatic repulsion. Similar electrostatic phenomena occurred at low pH of 3 when amino groups cationized in preparation of CNFs from α-chitin obtained from crab shell in one grinding pass in acidic condition by our research group [1]. Fine fibrils in the narrow range of 10-20 nm were obtained as shown in Fig. 2b and c. Chitin slurry of 1 wt.% became a highly viscous gel phase after one cycle of grinding treatment is due to large surface area of NFs. Viscous gel phase formation is the indication that fibrillation was successful in one cycle of grinding and it was more facilitated in acidic medium as unbroken high aspect ratio NFs were visible in FE-SEM images.

micrograph of the black tiger prawn shell surface after removal of the matrix components is shown in Fig. 3. SEM picture shows exocuticle which is the main part of the prawn shells. It is important that uniform CNFs with an elaborate interwoven structure is clearly visible in the image. 1 wt.% chemically treated chitin suspension was crushed by a domestic blender and passed through a grinder for fibrillation without addition of acid. Chitin slurry obtained was high viscous gel after a single grinding treatment similar to that observed in CNFs from crab shell. Fig 4a and b are the SEM images of one pass NFs at different magnifications. The width of NFs was 10–20 nm. In crab shell NFs at neutral pH, width of the fibers was widely distributed in range 10-100 nm after single grinding pases. Thus preparation of NFs from prawn shells is much advantageous than crab shell. Using prawn shells, thin, homogeneous, uniformly distributed, well separated, and large aspect ratio CNFs were successfully prepared in neutral medium with much superiority over acidic crab shell preparations to apply for a number of industrial applications. The explanation we have given of fibrillation of prawn shell achieved at neutral pH unlike to crab shell that occurred at pH 3 is following. The outer most skeleton (exoskeleton) of prawn or crab shell is made up of two parts exocuticle and endocuticle. Exocuticle has a very fine interwoven plywood type structure, endocuticle is rather more coarse and has thick fibers as shown in Fig.1. 90% of crab shell is made up of these thicker endocuticular fibers [17]. Thus a low pH of 3 medium is used to obtain nanofibrils in crab shell. On the other hand the exoskeleton of prawn including black tiger prawn made up of mostly semitransparent soft shell of fine exocuticle as shown in Fig. 3, thus their fibrillation occurs easier than crab shell at neutral pH. The preparation for CNFs from prawn shells in neutral pH is also valid to other

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**Figure 3.** FE-SEM image of black tiger prawn after chemical treatment. Reproduced with permission from ref. 2. Copy‐

species of prawn as described in article [2].

right 2011, Elsevier.

**Figure 2.** FE-SEM pictures CNFs prepared from crab shell after one cycle of grinding at pH values; a) pH 7; b and c) pH 3; the scale is a and b) 400 nm, c) 200 nm. Reprinted with permission from ref. 1. Copyright 2009, American Chemical Society.

#### **2.2. CNFs from prawn shell**

In section 2.1, CNFs were isolated successfully from crab shells flakes in acidic medium with uniform width (10-20 nm) and high aspect ratio after series of chemical treatments followed by one pass of mechanical grinding. Acidic pool and excess acetic acid, however, in CNFs is a matter of worry for applications of NFs especially in pharmaceutical, cosmetic, biomedical indus‐ tries. Acidic contamination of NFs for above applications is an important issue to address from toxicity viewpoint. Removal of acid from NFs is difficult, complicated, and make products expensive. Therefore preparation of CNFs in normal condition of neutral pH is utmost and immediate necessity to apply them for above products. CNFs from prawn shells have been extracted under neutral conditions without addition of any acid. Fresh shells of prawn species *Penaeus monodon* (black tiger prawn) was used to prepare CNFs. Prawns are cultivated on large scale world wide and its shells are thrown as industrial waste. Chemical treatments to remove minerals, pigments and proteins and lipids are same for prawn [2] as described for isolation of chitin from crab shells. In brief, addition of NaOH and HCl removed proteins and minerals, respectively leaving the chitin and pigments in shell. Pigments were removed with ethanol extraction. Yield of dry chitin from prawn shells was 16.7%. Degree of deacetylation (DDA) of prepared samples determined by the content of C and N by elemental analysis was 7%. SEM micrograph of the black tiger prawn shell surface after removal of the matrix components is shown in Fig. 3. SEM picture shows exocuticle which is the main part of the prawn shells. It is important that uniform CNFs with an elaborate interwoven structure is clearly visible in the image. 1 wt.% chemically treated chitin suspension was crushed by a domestic blender and passed through a grinder for fibrillation without addition of acid. Chitin slurry obtained was high viscous gel after a single grinding treatment similar to that observed in CNFs from crab shell. Fig 4a and b are the SEM images of one pass NFs at different magnifications. The width of NFs was 10–20 nm. In crab shell NFs at neutral pH, width of the fibers was widely distributed in range 10-100 nm after single grinding pases. Thus preparation of NFs from prawn shells is much advantageous than crab shell. Using prawn shells, thin, homogeneous, uniformly distributed, well separated, and large aspect ratio CNFs were successfully prepared in neutral medium with much superiority over acidic crab shell preparations to apply for a number of industrial applications. The explanation we have given of fibrillation of prawn shell achieved at neutral pH unlike to crab shell that occurred at pH 3 is following. The outer most skeleton (exoskeleton) of prawn or crab shell is made up of two parts exocuticle and endocuticle. Exocuticle has a very fine interwoven plywood type structure, endocuticle is rather more coarse and has thick fibers as shown in Fig.1. 90% of crab shell is made up of these thicker endocuticular fibers [17]. Thus a low pH of 3 medium is used to obtain nanofibrils in crab shell. On the other hand the exoskeleton of prawn including black tiger prawn made up of mostly semitransparent soft shell of fine exocuticle as shown in Fig. 3, thus their fibrillation occurs easier than crab shell at neutral pH. The preparation for CNFs from prawn shells in neutral pH is also valid to other species of prawn as described in article [2].

condition at low pH cationization of C2 amino groups in β-chitin occurred resulted in more dispersed and stable phase because of electrostatic repulsion. Similar electrostatic phenomena occurred at low pH of 3 when amino groups cationized in preparation of CNFs from α-chitin obtained from crab shell in one grinding pass in acidic condition by our research group [1]. Fine fibrils in the narrow range of 10-20 nm were obtained as shown in Fig. 2b and c. Chitin slurry of 1 wt.% became a highly viscous gel phase after one cycle of grinding treatment is due to large surface area of NFs. Viscous gel phase formation is the indication that fibrillation was successful in one cycle of grinding and it was more facilitated in acidic medium as unbroken

**Figure 2.** FE-SEM pictures CNFs prepared from crab shell after one cycle of grinding at pH values; a) pH 7; b and c) pH 3; the scale is a and b) 400 nm, c) 200 nm. Reprinted with permission from ref. 1. Copyright 2009, American Chemical

In section 2.1, CNFs were isolated successfully from crab shells flakes in acidic medium with uniform width (10-20 nm) and high aspect ratio after series of chemical treatments followed by one pass of mechanical grinding. Acidic pool and excess acetic acid, however, in CNFs is a matter of worry for applications of NFs especially in pharmaceutical, cosmetic, biomedical indus‐ tries. Acidic contamination of NFs for above applications is an important issue to address from toxicity viewpoint. Removal of acid from NFs is difficult, complicated, and make products expensive. Therefore preparation of CNFs in normal condition of neutral pH is utmost and immediate necessity to apply them for above products. CNFs from prawn shells have been extracted under neutral conditions without addition of any acid. Fresh shells of prawn species *Penaeus monodon* (black tiger prawn) was used to prepare CNFs. Prawns are cultivated on large scale world wide and its shells are thrown as industrial waste. Chemical treatments to remove minerals, pigments and proteins and lipids are same for prawn [2] as described for isolation of chitin from crab shells. In brief, addition of NaOH and HCl removed proteins and minerals, respectively leaving the chitin and pigments in shell. Pigments were removed with ethanol extraction. Yield of dry chitin from prawn shells was 16.7%. Degree of deacetylation (DDA) of prepared samples determined by the content of C and N by elemental analysis was 7%. SEM

high aspect ratio NFs were visible in FE-SEM images.

Society.

88 Advances in Nanofibers

**2.2. CNFs from prawn shell**

**Figure 3.** FE-SEM image of black tiger prawn after chemical treatment. Reproduced with permission from ref. 2. Copy‐ right 2011, Elsevier.

due to over processing as can be seen from SEM pictures Fig. 6e and f.). Fiber thickness in one, five, and ten passes are 19.0, 18.0, and 16.5 nm, respectively. It is very noteworthy that advatage of very renetly developed advanced technology high pressure jet SBS can atomized chitin slurry with or wthout acetic acid in just five passes to give NFs of small diameter (18.0-18.2 nm) and with high aspect ratio. Increasing number of passes to ten that is considered over processing decreases the width of NFs to very smaller extent. So five passes are optimum with

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**Figure 5.** FE-SEM of CNFs; a) one pass, b and c) 5 passes, d and e) 10 passes prepared by SBS instrument without acetic

Fig. 7 shows the FT-IR spectra of chitin fibers treated by the Star Burst system after 1, 5, and 10 passes under both neutral and acidic conditions. All spectra of obtained of CNFs showed that spectral features are in excellent agreement with the spectrum of commercial chitin. In particular the OH stretching band at 3424 cm−1, NH stretching band at 3259 cm−1, amide band I at 1652 and 1621 cm−1, and amide II band at 1554 cm−1 of the CNFs are observed. These absorption peaks are especially characteristic of chitin. This suggests that original chemical structures of chitin were maintained even after 10 passes of Star Burst mechanical treatments with or without acidic pool. Fig. 8 are the XRD pattern of commercial chitin and processesed CNFs. X-ray diffraction profiles of CNFS processed by the Star Burst system after several passes under both neutral and acidic conditions. All diffraction patterns coincide closely with

acid. The scales are shown in the pictures. Reprinted from ref. 10.

**2.4. Characterization of CNFs by FT-IR and XRD recoding**

or wthout acetic acid medium.

**Figure 4.** FE-SEM image of black tiger prawn NFs after one pass in grinder at neutral pH; a) scale: 1000 nm, b) scale: 200 nm. Reproduced with permission from ref. 2. Copyright 2011, Elsevier.

#### **2.3. Nanofibrillation of dry chitin powder by Star Burst system**

Authors [10] used new fibrillation machine Star Burst system (SBS) developed by Sugino Machine Co., Ltd. to prepare NFs from commercially available dry α-chitin powder from crab shell with and without acetic acid medium. The working principle of SBS instrument has been described in introduction part of the review. Instrument uses high pressure water jet to atomized the chitin slurry into NFs. FE-SEM showed that NFs became thinner as the number of SB passes increased. Fibers were thinner in acidic medium than neutral conditions. NFs prepared in SBS were thinner than reported earlier [1] using grinder in acidic medium. XRD recording showed that SBS did not damage NFs and did not reduce crystallinity.

Fig. 5 shows FE-SEM micrographs of CNFs after SB treatments under a neutral aqueous condition. After one pass, the chitin was not fibrillated (Fig. 5(a)). Thick aggregates of CNFs were observed. There was a significant change in the morphology of CFs after the treatment with five and ten passes (Fig. 5b, c, d, and e). In five and ten passes the NFs are fibrillated as shown in Fig. 5c and e on maginification of 300 nm. The width of fibers in five passes is 18.2 and it reduced to 17.3 nm in ten passes. Thus SBS is powerful tools to give fibers of very thin diameter even without acetic acid solution pool. If we consider the atomization of CNFs in SBS with acetic acid then even in one pass fibrillation occurred as shown in Fig. 6a, b.). Fibrillation completed in five passes as can be seen from SEM pictures Fig. 6d, c.), while processing the fibers in ten passes the thickness of fibers decreases while aspect ratio reduced as fibers breaks due to over processing as can be seen from SEM pictures Fig. 6e and f.). Fiber thickness in one, five, and ten passes are 19.0, 18.0, and 16.5 nm, respectively. It is very noteworthy that advatage of very renetly developed advanced technology high pressure jet SBS can atomized chitin slurry with or wthout acetic acid in just five passes to give NFs of small diameter (18.0-18.2 nm) and with high aspect ratio. Increasing number of passes to ten that is considered over processing decreases the width of NFs to very smaller extent. So five passes are optimum with or wthout acetic acid medium.

**Figure 5.** FE-SEM of CNFs; a) one pass, b and c) 5 passes, d and e) 10 passes prepared by SBS instrument without acetic acid. The scales are shown in the pictures. Reprinted from ref. 10.

#### **2.4. Characterization of CNFs by FT-IR and XRD recoding**

**Figure 4.** FE-SEM image of black tiger prawn NFs after one pass in grinder at neutral pH; a) scale: 1000 nm, b) scale:

Authors [10] used new fibrillation machine Star Burst system (SBS) developed by Sugino Machine Co., Ltd. to prepare NFs from commercially available dry α-chitin powder from crab shell with and without acetic acid medium. The working principle of SBS instrument has been described in introduction part of the review. Instrument uses high pressure water jet to atomized the chitin slurry into NFs. FE-SEM showed that NFs became thinner as the number of SB passes increased. Fibers were thinner in acidic medium than neutral conditions. NFs prepared in SBS were thinner than reported earlier [1] using grinder in acidic medium. XRD

Fig. 5 shows FE-SEM micrographs of CNFs after SB treatments under a neutral aqueous condition. After one pass, the chitin was not fibrillated (Fig. 5(a)). Thick aggregates of CNFs were observed. There was a significant change in the morphology of CFs after the treatment with five and ten passes (Fig. 5b, c, d, and e). In five and ten passes the NFs are fibrillated as shown in Fig. 5c and e on maginification of 300 nm. The width of fibers in five passes is 18.2 and it reduced to 17.3 nm in ten passes. Thus SBS is powerful tools to give fibers of very thin diameter even without acetic acid solution pool. If we consider the atomization of CNFs in SBS with acetic acid then even in one pass fibrillation occurred as shown in Fig. 6a, b.). Fibrillation completed in five passes as can be seen from SEM pictures Fig. 6d, c.), while processing the fibers in ten passes the thickness of fibers decreases while aspect ratio reduced as fibers breaks

recording showed that SBS did not damage NFs and did not reduce crystallinity.

200 nm. Reproduced with permission from ref. 2. Copyright 2011, Elsevier.

90 Advances in Nanofibers

**2.3. Nanofibrillation of dry chitin powder by Star Burst system**

Fig. 7 shows the FT-IR spectra of chitin fibers treated by the Star Burst system after 1, 5, and 10 passes under both neutral and acidic conditions. All spectra of obtained of CNFs showed that spectral features are in excellent agreement with the spectrum of commercial chitin. In particular the OH stretching band at 3424 cm−1, NH stretching band at 3259 cm−1, amide band I at 1652 and 1621 cm−1, and amide II band at 1554 cm−1 of the CNFs are observed. These absorption peaks are especially characteristic of chitin. This suggests that original chemical structures of chitin were maintained even after 10 passes of Star Burst mechanical treatments with or without acidic pool. Fig. 8 are the XRD pattern of commercial chitin and processesed CNFs. X-ray diffraction profiles of CNFS processed by the Star Burst system after several passes under both neutral and acidic conditions. All diffraction patterns coincide closely with

**Figure 6.** FE-SEM of CNFs; a and b) one pass, c and d) 5 passes, e and f) 10 passes prepared by SBS instrument with acetic acid. The scales are shown in the pictures. Reprinted from ref. 10.

**Figure 7.** FT-IR spectra of chitin fibers after 1, 5, and 10 passes through Star Burst with and without acetic acid. Re‐

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**Figure 8.** X-ray diffraction profiles of chitin fibers after 1, 5, and 10 passes through Star Burst with and without acetic

printed from ref. 10.

acid. Reprinted from ref. 10.

original chitin powder. The four diffraction peaks of the CNFs observed at two θ= 9.2, 19.1, 20.9, and 23.1◦ corresponded to 020, 110, 120, and 130 planes, respectively [18]. They were typical antiparallel crystal patterns of α-chitin. Thus, the original crystalline structure was maintained after the purification process followed by the Star Burst treatments. Following are the relative crystalline indices of CNFs determined from X-ray diffraction profiles. Original chitin powder has a comparatively high crystallinity of 83.7%. After the Star Burst process under both acidic and neutral conditions, there was no significant difference in the relative degree of crystallinity after the various numbers of passes. This result indicates that at least 10 mechanical treatments with the SBS did not damage the CFs, even though the system used a super high pressure water jet operated at 245 MPa.

#### **2.5. Preparation of CNFs from edible mushrooms**

CNFs were isolated and charecterized [3] from cell wall of edible mushrooms by a number of chemical treatments to remove glucans, minerals, and proteins associated with mushrooms followed by grinding treatment in acidic medium. NFs width was in the range 20-28 nm depending on the type of mushroom used. The goal of extraction of CNFs from edible mushrooms of nano-sized scale fibers is to develop novel functional food materials. The detailed extraction method and final SEM images of NFs extracted and methods employed to characterize them have been described below. The mushrooms species *Pleuotus eryngii* (king trumpet mushroom), *Agaricus bisporus* (common mushroom), *Lentinula edodes* (shiitake), *Grifola frondosa* (maitake), and *Hypsizygus marmoreus* (buna-shimeji) commonly used for human

**Figure 7.** FT-IR spectra of chitin fibers after 1, 5, and 10 passes through Star Burst with and without acetic acid. Re‐ printed from ref. 10.

original chitin powder. The four diffraction peaks of the CNFs observed at two θ= 9.2, 19.1, 20.9, and 23.1◦ corresponded to 020, 110, 120, and 130 planes, respectively [18]. They were typical antiparallel crystal patterns of α-chitin. Thus, the original crystalline structure was maintained after the purification process followed by the Star Burst treatments. Following are the relative crystalline indices of CNFs determined from X-ray diffraction profiles. Original chitin powder has a comparatively high crystallinity of 83.7%. After the Star Burst process under both acidic and neutral conditions, there was no significant difference in the relative degree of crystallinity after the various numbers of passes. This result indicates that at least 10 mechanical treatments with the SBS did not damage the CFs, even though the system used a

**Figure 6.** FE-SEM of CNFs; a and b) one pass, c and d) 5 passes, e and f) 10 passes prepared by SBS instrument with

CNFs were isolated and charecterized [3] from cell wall of edible mushrooms by a number of chemical treatments to remove glucans, minerals, and proteins associated with mushrooms followed by grinding treatment in acidic medium. NFs width was in the range 20-28 nm depending on the type of mushroom used. The goal of extraction of CNFs from edible mushrooms of nano-sized scale fibers is to develop novel functional food materials. The detailed extraction method and final SEM images of NFs extracted and methods employed to characterize them have been described below. The mushrooms species *Pleuotus eryngii* (king trumpet mushroom), *Agaricus bisporus* (common mushroom), *Lentinula edodes* (shiitake), *Grifola frondosa* (maitake), and *Hypsizygus marmoreus* (buna-shimeji) commonly used for human

super high pressure water jet operated at 245 MPa.

acetic acid. The scales are shown in the pictures. Reprinted from ref. 10.

92 Advances in Nanofibers

**2.5. Preparation of CNFs from edible mushrooms**

**Figure 8.** X-ray diffraction profiles of chitin fibers after 1, 5, and 10 passes through Star Burst with and without acetic acid. Reprinted from ref. 10.

consumption were used in this study. The purification was done by a series of chemical treatments to remove associated compounds: proteins, pigments, glucans, and minerals according to the following procedure. Sodium hydroxide was used to dissolve, hydrolyze, and remove proteins and alkali soluble glucans. Hydorochloric acid was used to remove minerals. At this stage partial neutral saccharides and acid soluble protein compounds were also removed. The extraction step with sodium chlorite and acetic acid removed pigments from the sample. At final stage, the sample was treated with sodium hydroxide again to eliminate and remove the residual glucans including trace amount of proteins. After chemical treatment if the extracted mass allowed to dry, it causes strong hydrogen bonding between CNFs when all matrix substances are washed away which makes it difficult to fibrillate chitin to NFs. Thus the sample was kept wet after removal of the matrix for preparation of CNFs. The purified sample with 1 wt.% content of chitin was passed through a grinder for nano-fibrillation in acetic acid medium at pH 3. After grinder treatment, the chitin homogeneous stable dispersed slurry of chitin with high viscosity was obtained resulted due to high surface-to-volume ratio of NFs thus finally the sample was successfully fibrillated. Fig. 9 shows SEM images of CNFs from five mushrooms after removing matrix components and one pass though the grinder. The isolated chitins are well fibrillated and uniform. The width of the fibers was in the range 20-28 nm depending on the species of mushroom. The yield of CNFs contents in mushrooms was not so high as in crab or prawn shells, it was merely in the range 1.3-3.5 wt.% depending on the species of mushrooms.

FT-IR and XRD spectrometry were employed to characterized the CNFs from mushrooms. FT-IR spectra (Fig. 10) of commercially available chitin derived from crab shell and CNFs from 5 types of mushroom were compared for analysis. The major bands of the spectra of CNFs are in agreement with commercial chitin. Similarly XRD of commercially available chitin and the CNFs prepared from five types of mushrooms were compared (Fig. 11). The four diffraction bands of CNFs are typical crystal patterns of α-chitin. Thus, CNFs extracted from several types of mushroom maintained α-chitin crystalline structures after the removal of matrix substances followed by grinder treatment. However, in the case of *Hypsizygus marmoreus*, X-ray diffrac‐ togram (Fig. 11f) contains crystal patterns of cellulose (Fig. 11g). The diffraction peaks observed from 15° to 17°, and 22.5°, corresponding to the 110, 1–10, and 200 planes, respectively are

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**Figure 10.** FT-IR spectra of a) commercial chitin, and CNFs from mushroom source of species b) *Lentinula edodes*, c) *Pleuotus eryngii*, d) *Hypsizygus marmoreus,* e) *Grifola frondos,* and f) *Agaricus bisporus*. Reprinted from ref. 3.

**Figure 11.** XRD pattern of a) commercial chitin, and CNFs from mushroom source of species b) *Pleuotus eryngii*, c) *Agaricus bisporus*, d) *Lentinula edodes*, e) *Grifola frondos*, f) *Hypsizygus marmoreus,* and g) commercially available cel‐

typical for the cellulose I crystal.

lulose. Reprinted from ref. 3.

**Figure 9.** FE-SEM images of CNFs prepared from a) *Pleuotus eryngii*, b) *Agaricus bisporus*, c) *Lentinula edodes*, d) Grifo‐ *la frondosa*, e) *Hypsizygus marmoreus*. The scale bars are 200 nm. Reprinted from ref. 3.

FT-IR and XRD spectrometry were employed to characterized the CNFs from mushrooms. FT-IR spectra (Fig. 10) of commercially available chitin derived from crab shell and CNFs from 5 types of mushroom were compared for analysis. The major bands of the spectra of CNFs are in agreement with commercial chitin. Similarly XRD of commercially available chitin and the CNFs prepared from five types of mushrooms were compared (Fig. 11). The four diffraction bands of CNFs are typical crystal patterns of α-chitin. Thus, CNFs extracted from several types of mushroom maintained α-chitin crystalline structures after the removal of matrix substances followed by grinder treatment. However, in the case of *Hypsizygus marmoreus*, X-ray diffrac‐ togram (Fig. 11f) contains crystal patterns of cellulose (Fig. 11g). The diffraction peaks observed from 15° to 17°, and 22.5°, corresponding to the 110, 1–10, and 200 planes, respectively are typical for the cellulose I crystal.

consumption were used in this study. The purification was done by a series of chemical treatments to remove associated compounds: proteins, pigments, glucans, and minerals according to the following procedure. Sodium hydroxide was used to dissolve, hydrolyze, and remove proteins and alkali soluble glucans. Hydorochloric acid was used to remove minerals. At this stage partial neutral saccharides and acid soluble protein compounds were also removed. The extraction step with sodium chlorite and acetic acid removed pigments from the sample. At final stage, the sample was treated with sodium hydroxide again to eliminate and remove the residual glucans including trace amount of proteins. After chemical treatment if the extracted mass allowed to dry, it causes strong hydrogen bonding between CNFs when all matrix substances are washed away which makes it difficult to fibrillate chitin to NFs. Thus the sample was kept wet after removal of the matrix for preparation of CNFs. The purified sample with 1 wt.% content of chitin was passed through a grinder for nano-fibrillation in acetic acid medium at pH 3. After grinder treatment, the chitin homogeneous stable dispersed slurry of chitin with high viscosity was obtained resulted due to high surface-to-volume ratio of NFs thus finally the sample was successfully fibrillated. Fig. 9 shows SEM images of CNFs from five mushrooms after removing matrix components and one pass though the grinder. The isolated chitins are well fibrillated and uniform. The width of the fibers was in the range 20-28 nm depending on the species of mushroom. The yield of CNFs contents in mushrooms was not so high as in crab or prawn shells, it was merely in the range 1.3-3.5 wt.% depending

**Figure 9.** FE-SEM images of CNFs prepared from a) *Pleuotus eryngii*, b) *Agaricus bisporus*, c) *Lentinula edodes*, d) Grifo‐

*la frondosa*, e) *Hypsizygus marmoreus*. The scale bars are 200 nm. Reprinted from ref. 3.

on the species of mushrooms.

94 Advances in Nanofibers

**Figure 10.** FT-IR spectra of a) commercial chitin, and CNFs from mushroom source of species b) *Lentinula edodes*, c) *Pleuotus eryngii*, d) *Hypsizygus marmoreus,* e) *Grifola frondos,* and f) *Agaricus bisporus*. Reprinted from ref. 3.

**Figure 11.** XRD pattern of a) commercial chitin, and CNFs from mushroom source of species b) *Pleuotus eryngii*, c) *Agaricus bisporus*, d) *Lentinula edodes*, e) *Grifola frondos*, f) *Hypsizygus marmoreus,* and g) commercially available cel‐ lulose. Reprinted from ref. 3.
