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A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus)
Materials Today: Proceedings xxx (xxxx) xxx
Contents lists available at ScienceDirect
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 xxxx 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: [email protected] (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).
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713
2
N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings xxx (xxxx) xxx
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
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713
N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings xxx (xxxx) xxx
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
3
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.
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713
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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. References [1] R.G. Sanuja, N.K. Kalutharage, P.R.T. Cumaratunga, Sri Lanka J. Aquat. Sci. 22 (2017) 45–53. [2] M. Benavente, S. Arias, L. Moreno, J. Martinez, J. Pharm. Pharmacol. 3 (2015) 20–26. [3] S.T. Chang, Mushrooms as Functional Foods, John Wiley & Sons Inc, 2008. [4] J.C. Rajapakse, P. Rubasingha, N.N. Dissanayake, J. Agric. Sci. (2007) 82–85. [5] B.A. Cheba, Global J. Biotechnol. Biochem. 6 (2011) 149–153. [6] Q. Li, E.T. Dunn, E.W. Grandmaison, M.F.A. Goosen, J. Bioact. Compat. Polym. 7 (1992) 370–397. [7] P.K. Dutta, J. Dutta, V.S. Tripathi, J. Sci. Ind. Res. 63 (2004) 20–31. [8] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras, Curr. Chem. Biol. 3 (2009) 203–230. [9] Y.S. Puvvada, S. Vankayalapati, S. Sukhavasi, Int. Curr. Pharm. J. 1 (2012) 258– 263. [10] G.A. Sewwandi, S.U. Adikary, Trop. Agric. Res. 3 (2012) 272–276. [11] H.K. No, S.P. Meyers, J. Aquat. Food Prod. Technol. 21 (1992) 319–326. [12] M. Huang, E. Khor, L.Y. Lim, Pharm. Res. 21 (2004) 344–353. [13] L. Stryer, Biochemistry, W.H. Freeman and Company, New York, 1995. [14] K. Helrich, AOAC, Official Method of Analysis, AOAC Inc, Virginia, 1995. [15] D.J. Knorr, Food Sci. Technol. 47 (1982) 593–595. [16] T. Khan, K. Peh, H.S. Chang, J. Pharmacol. Pharm. Sci. 5 (2002) 205–212. [17] E.S. de Alvarenga, C.P. de Oliveira, C.R. Bellato, Carbohydr. Polym. 80 (2010) 1155–1160.
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713
Contents lists available at ScienceDirect
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 xxxx 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: [email protected] (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).
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713
2
N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings xxx (xxxx) xxx
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
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713
N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings xxx (xxxx) xxx
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
3
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.
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713
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N.K. Kalutharage, D.L. Rathnasinghe / Materials Today: Proceedings xxx (xxxx) xxx
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. References [1] R.G. Sanuja, N.K. Kalutharage, P.R.T. Cumaratunga, Sri Lanka J. Aquat. Sci. 22 (2017) 45–53. [2] M. Benavente, S. Arias, L. Moreno, J. Martinez, J. Pharm. Pharmacol. 3 (2015) 20–26. [3] S.T. Chang, Mushrooms as Functional Foods, John Wiley & Sons Inc, 2008. [4] J.C. Rajapakse, P. Rubasingha, N.N. Dissanayake, J. Agric. Sci. (2007) 82–85. [5] B.A. Cheba, Global J. Biotechnol. Biochem. 6 (2011) 149–153. [6] Q. Li, E.T. Dunn, E.W. Grandmaison, M.F.A. Goosen, J. Bioact. Compat. Polym. 7 (1992) 370–397. [7] P.K. Dutta, J. Dutta, V.S. Tripathi, J. Sci. Ind. Res. 63 (2004) 20–31. [8] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras, Curr. Chem. Biol. 3 (2009) 203–230. [9] Y.S. Puvvada, S. Vankayalapati, S. Sukhavasi, Int. Curr. Pharm. J. 1 (2012) 258– 263. [10] G.A. Sewwandi, S.U. Adikary, Trop. Agric. Res. 3 (2012) 272–276. [11] H.K. No, S.P. Meyers, J. Aquat. Food Prod. Technol. 21 (1992) 319–326. [12] M. Huang, E. Khor, L.Y. Lim, Pharm. Res. 21 (2004) 344–353. [13] L. Stryer, Biochemistry, W.H. Freeman and Company, New York, 1995. [14] K. Helrich, AOAC, Official Method of Analysis, AOAC Inc, Virginia, 1995. [15] D.J. Knorr, Food Sci. Technol. 47 (1982) 593–595. [16] T. Khan, K. Peh, H.S. Chang, J. Pharmacol. Pharm. Sci. 5 (2002) 205–212. [17] E.S. de Alvarenga, C.P. de Oliveira, C.R. Bellato, Carbohydr. Polym. 80 (2010) 1155–1160.
Please cite this article as: N. K. Kalutharage and D. L. Rathnasinghe, A study of chitosan and glucosamine isolated from Sri Lankan local mushroom Schizophyllum commune and oyster mushroom (Pleurotus ostreatus), Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.07.713