A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus)

Materials Today: Proceedings 23 (2020) 119–122
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Materials Today: Proceedings
journal homepage: www.elsevier.com/locate/matpr
A study of chitosan and glucosamine isolated from Sri Lankan local
mushroom Schizophyllum commune and oyster mushroom
(Pleurotus ostreatus)
N.K. Kalutharage ⇑, D.L. Rathnasinghe
Department of Chemistry, University of Ruhuna, Matara, Sri Lanka
a r t i c l e
i n f o
Article history:
Received 14 March 2019
Received in revised form 28 July 2019
Accepted 30 July 2019
Available online 19 August 2019
Keywords:
Chitosan
Glucosamine
Mushroom
Schizophyllum commune
Pleurotus ostreatus
a b s t r a c t
Properties of chitosan extracted from local mushroom Schizophyllum commune and Pleurotus ostreatus
were compared. Percentage yield, solubility, moisture-content, ash-content, N-content, water-bindingcapacity
were
1.73 ± 0.05%/1.22 ± 0.01%,
7.38 ± 0.10%/3.41 ± 0.14%,
6.39 ± 0.20%/8.16 ± 0.42%,
8.19 ± 0.04%/1.63 ± 0.05%, 11.31%/3.02%, 387.13 ± 15.57%/402.57 ± 12.78% respectively for chitosan samples from S. commune followed by P. ostreatus. FBC were varied approximately 250–350% in coconut oil,
soy bean oil and sunflower oil. DD% of chitosan using FT-IR and conductometric titration were 53.10% and
60.68% respectively for two species. The yield and purity of glucosamine sample 95.70% and 0.97 ± 0.08%
and 58.14% and 0.52 ± 0.04% for Schizophyllum commune and Pleurotus ostreatus were respectively.
Ó 2019 Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the Advanced Materials for Clean Energy
and Health Applications (AMCEHA).
1. Introduction
Chitosan is an amino polysaccharide, composed of b-1-4-Dglucosamine linked to N-acetyl-D-glucosamine residues, synthesized from de-acetylation of chitin. Chitin is the second most abundant polysaccharide and naturally occurring substances on the
earth and it is structurally similar to the cellulose. Chitin occurs
in nature as ordered crystalline micro-fibrils forming structural
components in the exoskeleton of arthropods or in the cell walls
of fungi and yeast [1]. Glucosamine is the building unit of biopolymer chitosan. Glucosamine monomers are connected together by
1-4 b glycosidic bonds in both chitin and chitosan. Glucosamine
is a type of amino sugar and a prominent precursor in the biosynthesis of proteins and lipids [2].
Mushroom is a macro fungus with a distinctive fruiting body,
which can be either epigeous or hypogeous and large enough to seen
with naked eye and to be picked by hand [3] Mushroom cultivation is
environmental friendly and can use waste materials to generate
mushrooms, such as paddy straw, cotton waste; tree saw dust, sugar
cane bagasse, wild grasses and lingo cellulosic waste [4].
Chitosan has received considerable attention in recent years
in the literature as a renewable resource for their extensive
⇑ Corresponding author.
E-mail address: knishantha@chem.ruh.ac.lk (N.K. Kalutharage).
applications in different field. In particular the development of chitosan derivatives as useful polymeric material is an expanding field
in the science. Chitosan have wide range of applications due to its
antibacterial, non-toxicity and hypoallergenic properties. It is used
in biomedical industries, agriculture, genetic engineering, food
industry, environmental pollution control, water treatment, paper
manufacture, and photography etc. [5–7].
Glucosamine is a substance produced naturally in the body
from an amino acid (glutamine), and a sugar (glucose). Recently
glucosamine is widely applicable in different field including pharmaceuticals which is applicable in different way in food industry as
nutritional supplement, food and beverage, and dairy products and
in pet food industry. Glucosamine is widely used in biomedical
science to relieve osteoarthritis symptoms. And it can also be used
to treat cardiovascular disease, Neurological deficits, and Skin disorders due to its anti-inflammatory and anti-oxidant activities.
Glucosamine can survive cell by controlling production of inflammatory cytokine. Glucosamine is natural constituent of some glycosaminoglycans and in the proteoglycans found in cartilage,
intervertebral disc and synovial fluid. Glucosamine is used as glucosamine chloride and glucosamine sulfate by osteoarthritis
patients and it act as chrondro protector to relieving joint pain,
swelling and stiffness caused by arthritis.
Chitosan is
prepared by
hydrolysis
of acetamide
(ANHACOACH3) groups of chitin. This is reported to be normally
https://doi.org/10.1016/j.matpr.2019.07.713
2214-7853/Ó 2019 Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the Advanced Materials for Clean Energy and Health Applications (AMCEHA).
120
N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings 23 (2020) 119–122
conducted by severe alkaline hydrolysis treatment due to the
resistant of such groups imposed by the trans-arrangement of
the C2-C3 substituents of the sugar ring [8] In this study used
method is modified method rather than conventional method
and it is accurate, simple and cheap method.
Synthesization and characterization of chitosan and glucosamine derived from mushrooms have already been studies in
world using Pleurotus ostreatus. But Less number of research have
been done using Schizophyllum commune mushrooms. This study
aims to determine the most suitable mushroom type, which can
be used to isolate and extract high quality chitosan and glucosamine, out of mushrooms namely Pleurotus ostreatus and
Schizophyllum commune.
2. Materials and method
S. commune and P. ostreatus were collected from Deniyaya Pallegama and Beliata, Sri Lanka. All the mushrooms were washed
with running tap water to remove impurities and soluble organics.
They were chopped in to small pieces and sun dried for nearly two
days until completely drying. To obtain a uniform size fine particles, dried products were ground using a centrifugal grinding mill
and grounded particles were sieved using 500 mm sieve. Sieved
mushrooms particles were placed in to a beaker and stored at
ambient temperature in a dry place until using. This storage may
help for the partial autolysis to improve facilitate chemical extraction of chitosan and to improve the quality of the chitosan [9].
Dried, ground and sieved 100 g of each kind of mushroom powder was used to isolate and extraction of chitosan and glucosamine.
The following first five steps were used to isolate chitosan from
mushrooms and final step was used to extract glucosamine from
chitosan namely (i) pre-conditioning (ii) de-mineralization (iii)
de-proteinization (iv) de-coloration (v) de-acetylation and (vi)
de-polymerization [1].
Pre-conditioning step is followed to reduce the required chemical amount for next step and to remove loosely bound protein from
mushrooms before de-proteinization. The weight of each mushroom samples were measured using an analytical balance (Ohaus
corp. pine Brook, NJ. USA). This is the first step to the common procedure of the chitosan extraction [1,10]. Pre-conditioned mushrooms samples were de-mineralized using 1 M HCl solution. But
mushrooms do not have lot of minerals like crustacean shells. 1 M
NaOH is added to the de-mineralized mushrooms in order to remove
protein and sugars thus isolating the crude chitin. Samples were
allowed to soak for 24 h to remove the proteins [10]. In order to
remove pigments from chitin freshly prepared NaOCl is added as
bleaching agent [11]. NaOH 40% is added to remove acetyl group
from chitin producing chitosan in de-acetylation step [12]. Each
and every step product should be washed with water and stirred
well using magnetic stirrer in order to remove remaining chemicals
and impurities to increase efficiency of next step and to get better
mixing of mushrooms powder with chemicals. Chitosan polymer
should be hydrolyzed with acid breaking 1-4 b glycosidic bonds to
get glucosamine. The chitosan material was coarsely grinded using
mortar and pestle and hydrolyzed with concentrated HCl [13].
After isolation yield, solubility, moisture content, ash content, N
content, Water Binding Capacity, Fat Binding Capacity, DD% and
purity were determined. The weight of each chitosan samples were
measured after de-acetylation and the yield was calculated using
the equation [11].
Weight of Glucosamine sampleðg Þ
100%
Yieldð%Þ ¼
Weight of mushrooms sampleðg Þ
Yieldð%Þ ¼
Weight of Glucosamine sampleðg Þ
100%
Weight of mushrooms sampleðg Þ
Percentage moisture content of chitosan samples were calculated according to the following equation as described by AOAC
[14].
Moistureð%Þ ¼
Weight of chitosan sampleðgÞ Weight of dry chitosan sampleðgÞ
100%
weight of wet chitosan sampleðgÞ
Ash content of chitosan samples were determined using
standard ashing method [14] using following equation.
Ashð%Þ ¼
weight of residue or ashðg Þ
100%
weight of initial chitosan sampleðg Þ
N-content of chitosan samples was determined by Kjeldhal
method using Kjeldhal Nitrogen Analyzer. Percentage moisture
content of chitosan samples were calculated according to the
following equation as described by AOAC [14].
Nitrogenð%Þ ¼
ðVt VbÞ N 14:007
100%
W ðmgÞ
where, Vt = Volume of HCl (in mL) used for the titration of chitosan
samples, Vb = Volume of HCl (in mL) used for the titration of blank,
N = Normality of HCl, W = Weight of chitosan sample (mg).
Solubility of chitosan samples were tested following modified
literature method. 0.0500–0.1000 g were placed into previously
weighed centrifuge tubes and 10 mL of 1% acetic acid was added
and left for 30 min. The mixture was then centrifuged (Gallenkamp
Centrifuge 200) at 10,000 rom for 10 min. The supernatant liquid
was decanted. The un-dissolved particles were washed with distilled water (25 mL) and centrifuged (Gallenkamp Centrifuge
200) at 10,000 rpm. The supernatant liquid was removed and the
un-dissolved pellets dried at 60 °C for 24 h in an electric oven.
Finally, the amount of the residue was weighted and the percentage of solubility was determined.
Water Binding Capacity (WBC) and Fat Binding Capacity (FBC)
of chitosan were determined using a modified method [15]. For
the determination of FBC three types of oil were used [1], i.e., coconut oil, soybean oil, sunflower oil.
Chitosan samples prepared in the form of KBr disc were studied
for the degree of de-acetylation and compared with commercial
chitosan sample. The prepared chitosan KBr disc were re-scanned
using Fourier Transformed Infra-Red (FT-IR) spectroscopy instrument (Nicolet 380). The DD of the chitosan was calculated using
the baseline reported by Khan et al. [16] using following equation.
DD% ¼ 100 A1655
100
1:33
A3450
where, A1655 = absorbance at 1655 cm1 of the amide-I band as a
measure of the N-acetyl group content, A3450 = absorbance at
350 cm1 of the hydroxyl band as an internal standard to correct
Table 1
Physicochemical properties of Chitosan samples.
Physicochemical
properties
Schizophyllum commune
(%)
Pleurotus ostreatus
(%)
moisture content
ash content
solubility
water binding capacity
fat binding capacity
Coconut oil
Soy bean oil
Sunflower oil
N content
Degree of De-acetylation
FT- IR
Conductometric titration
6.39
8.19
7.38
387.13
8.16
1.63
3.41
402.57
313.27
306.99
295.86
3.02
279.00
260.15
271.98
11.31
53.10
54.5
60.68
59.87
N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings 23 (2020) 119–122
for disc thickness, The factor ‘1.33’ denoted the value of the ratio of
A1655/A3450 for fully N-acetylated chitosan.
During conductometric titration, chitosan samples (0.2000 g)
were dissolved in 0.05 M standardized HCl solution and titrated
with portions of 0.50 mL standardized NaOH (0.1 M) in 20 s time
interval and the value of the conductance with the corresponding
titrant volumes were measured and recorded [17]. The DD was calculated by the formula.
DD% ¼
½baseðV 2 V 1 Þ161
m
121
where, [base] = The concentration of NaOH solution, V2 & V1 = The
volume of NaOH used in titration (mL), 161 = Molar mass of the
monomer (C6H11O4N), m = Mass of the chitosan in mg.
Isolated chitosan was analysed by X-ray diffraction and compared with commercial available chitosan samples.
Percentages of purity of glucosamine samples were determined
by titrimetric method. The glucosamine samples (0.0200 g) were
dissolved in distilled water and titrated with standardized NaOH
(0.01 M) with phenolphthalein as an indicator.
Fig. 1. FT-IR spectrum of chitosan isolated from (A) Schizophyllum commune and (B) Pleurotus ostreatus.
Fig. 2. FT-IR spectrum of Glucosamine Hydrochloride isolated from (A) Schizophyllum commune and (B) Pleurotus ostreatus.
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N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings 23 (2020) 119–122
The extracted glucosamine samples of mushrooms were characterized by Fourier Transformed infra-Red (FT-IR) spectroscopy and
1
H NMR studies.
3. Results and discussion
The highest percentage of yield of chitosan and glucosamine
were obtained from Schizophyllum commune with the value of
1.73%, 0.97% and the lowest yield were obtained from Pleurotus
ostreatus with the value of 1.22%, 0.52% (Table 1).
A high quality grade chitosan should have less than 1% of ash
(Fig. 1). Some residual ash of chitosan may affect their solubility
consequently contributing to lower viscosity and can affect the
quality of final product such as N-content. Lower solubility values
indicate incomplete removal of protein and acetyl group. Since solubility of chitosan depends on the removal of acetyl group from
chitin. Due to the presence of protein residues Percentage of nitrogen content can be increased. Protein is bound by covalent bonds
forming stable complex with chitosan. Thus, it is very difficult to
achieve 100% de-proteinization. Even with complete deproteinization, nitrogen of the free amino (–NH2) group cannot
be estimated. The pre-conditioning and de-protenization steps
help to reduce protein content in chitosan. There are high amount
of protein content in the mushroom than crustacean shells and in
the de-protenization step weight loss of mushroom powder was
higher than other steps.
Highest percentage of purity of glucosamine sample was exhibited in the Pleurotus ostreatus glucosamine samples with the value
of 95.70% and lowest percentage of purity was observed in the
Schizophyllum commune glucosamine samples with the value of
58.14% (Fig. 2). Purity of glucosamine samples depend on the solvent evaporation time and washing with ethanol. Acid hydrolysis
of chitin with concentrated HCl for longer time leads to breakdown
of glucosamine and decreased recovery producing impurities.
4. Conclusion
Mushroom industry is one of the profitable small scale industry
in Sri Lanka without consuming high energy and cost. Mushroom
products have high demand as dietary supplement and medicinal
drug. Pharmaceutical industry needs to refine different types of
chitosan and glucosamine to meet the required standards. The
quality and the physico-chemical properties of chitosan and glucosamine vary widely with mushroom species and method of
preparation. According to the present study Pleurotus ostreatus is
the suitable mushroom species to extract pharmaceutical grade
and high quality chitosan and glucosamine.
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