CHARACTERISATION AND SORPTION PROPERTIES TO HEAVY METALS (Cu2+, Pb2+) OF CHITOSAN COMPLEXES EXTRACTED FROM COCKROACHES OF RWANDA
Cockroaches of Rwanda are one of the most important sources of chitosan. In chitosan extraction from Cockroaches, conditions for deproteinization were: 8% NaOH at 95 Ã‚ÂºC for 1 h and demineralization involved treatment with 6.7 % HCl at room temperature. Yield of extract products was ..... Chitin-glucan insoluble complexes in the extracted products were identified by FTIR specusing reference sample of Aspergillus Niger. Using elementary analysis, the quantities of chitin-glucans and content of chitin extracted from the sample were estimated and their ratios for the 2 types of mushrooms were compared. The results showed that the two types of mushrooms contain chitin and its glucan complexes with some impurities and Amanita Phalloide mushrooms contain more chitin than Lactarius Subdulius mushrooms. The percentages of chitosan extracts from Amanita Phalloide and Lactarius Subdulius mushrooms were Â¦Â¦.., respectively.
SUPERVISED BY Prof. E. KOVALEVA
Among the novel families of biological macromolecules, whose relevance is becoming increasingly evident, are chitin and its main derivatives, chitosan. Chitin is a non-toxic, biodegradable polymer of high molecular weight. After cellulose, it is the second most abundant biopolymer on Earth found in the nature. Like cellulose, chitin is a fiber, and in addition, it presents exceptional chemical and biological qualities that can be used in many industrial and medical applications (Campbell N.A, 1996).
Chitin is a polysaccharide composed from N-acetyl-D-glucosamine units. Mainly found in invertebrates, insects, marine diatoms, algae, fungi, and yeasts. Recent investigations confirm the suitability of chitin and its derivatives in chemistry, biotechnology, medicine, veterinary, dentistry, agriculture, food processing, environmental protection, and textile production (Cousin M. A., 1984).
This biopolymer consists predominantly of high molecular weight composed of N-acetyl-D glucosamine residues in a (1-4) linkage. Chitin has been utilized as an adsorbent for a variety of substrates. Potential and usual applications of chitin, chitosan and their derivatives are estimated to be more than 2000. This wide range of applications includes biomedicine, food, biotechnology, agriculture and cosmetics. Chitin and chitin derivatives are biodegradable and biocompatible natural polymers that have been used in virtually every significant segment of the economy (e.g. water treatment, pulp and paper industry, biomedical devices and therapies, cosmetics, biotechnology, agriculture, food science and membrane technology) (Li et al., 1997). The traditional source of chitin is shellfish waste from shrimp, Antarctic Krill, crab and lobster processing ( Shahidi, 1991). It is present in amounts varying from trace quantities up to about 40% of the body weight of the organism. The crustacean waste is the most important chitin source for commercial use due to its high chitin content and ready availability (GagnÃƒÂ© and Simpson, 1993). However, chitin present in the crustacean waste is associated with proteins, minerals (mainly calcium carbonate) and lipids including pigments.
Chitin's properties as a flexible and strong material make it favorable as surgical thread. Its biodegradability means it wears away with time as the wound heals. Moreover, chitin has some unusual properties that accelerate healing of wounds in humans. Occupations associated with high environmental chitin levels, such as shellfish processors, are prone to high incidences of asthma. Recent studies have suggested that chitin may play a role in a possible pathway in human allergic disease. Specifically, mice treated with chitin develop an allergic response, characterized by a build-up of interleukin-4 expressing innate immune cells. Treatment with a chitinase enzyme abolishes the response (Campbell, 1996). Most recent studies point out that chitin is a good inductor for defense mechanisms in plants. It was recently tested as a fertilizer that can help plants develop healthy immune responses, and have a much better yield and life expectancy. Chitin can cause harmful effects on living organisms and many problems in different domains can be solved using this compound, and different methods can be used for extracting chitin-containing products.
Chitin can be used for obtaining chitosan. Chitosan possesses a large set of useful properties as compared to cellulose and its natural precursor chitin. This product is obtained by deacetylating chitin with a hot alkali solution (Muzzarwelli R.A.A, et.al, 1980).The complexes of chitosan with other different polysaccharides are also shown good properties (Grini.G, 2005). It can be used in the purification of waste water from the textile industries, and chitosan pervaporation membranes are used to separate ethanol of DMSO from water (Kurita.K, 1997). Chitosan is used as pharmaceutical compound and as additional material in system of transport of medicines, for treating wounds, in the structure of bio adsorbed surgical threads (Ravi Kumar et.al, (2004).
Resources of chitin for industrial processing are crustacean shells (Shahidi F. (1991) and fungal mycelia (Peter M. G., 2002). The chitin was found also in alternative sources, for instance, in the lowest plants â€œ mushrooms (Vetter J., 2007), insects such as bees and Colorado beetles and in the industrial wastes from manufacturing wine and beer. Therefore many problems in different sectors of economy of Rwanda can be solved using the above-mentioned compounds.
1.1 Problem statement
As known chitin and its glucan complexes can be found in different fungi (Peter, 2002), especially in mushrooms (Vetter, 2007) .There are a lot of sorts of mushrooms forests of Rwanda, which can be raw sources of these substances.
The present study is aimed to extraction of chitin and its glucan complexes from mushrooms of some sorts and their characterization by using elementary analysis and FTIR spectroscopy to determine the ratio of chitin and glucan parts in chitin-glucan complexes and content of chitin in the extract products.
1.2. Significance statement
The development of raw sources of chitin and its glucan complexes such as mushrooms, their extraction and characterization are very important for numerous applications of these natural materials in medicine, biotechnology, veterinary, dentistry, agriculture, food processing, environmental protection, and textile production as plant enhancers and protectors against different fungal infections. Also chitin can be used for obtaining chitosan. Chitin and chitosan are one of the most important key materials in the 21st century. Chitin, obtained chitosan and their glucan complexes can be modified additionally incorporating different functional groups, for instance, carboxyalkyl (or/and) sulfoethyl groups (Pestov, 2008 ) to produce new sorption materials.
CHAPTER 2.0 LITERATURE REVIEW
2.1 Definition of Chitin
Chitin is a versatile environmentally friendly modern material. It is a naturally occurring high molecular weight linear homopolysaccharide composed of N-acetyl-D glucosamine residues in a (1-4) linkage. Chitin is a plentiful biomass, which is widely distributed in nature as the skeletal structure of crustaceans, insects, mushrooms, and cell wall of fungi (Knorr, 1984). The method of chitin preparation can vary with different sources. The physical and chemical characteristics of chitin accordingly differ with species and preparation methods. These variations in preparation methods are likely to result in differences in the degree of deacetylation, the distribution of acetyl groups, the chain length and the conformational structure of chitin (Fig. 1).
Figure 1: Structure of the chitin molecule.
This structure is shown two of the N- acetylglucosamine units that repeat to form long chains in beta- (1,4)-linkage
2.2. Chitin in fungi
Chitin is a major component of the cell walls of fungal spores and mycelium such as the hyphal of Amanita Phalloide and Lacterius Subdulius. The chitin of fungi possesses principally the same structure as the chitin occurring in other organisms. However, a major difference results from the fact that fungal chitin is associated with other polysaccharides, which do not occur in the exoskeleton of arthropods. Furthermore, the occurrence of chitosan is apparently restricted to fungi (Peter, 2002).
Fungal cell walls contain proteins, lipids, and 80-% polysaccharides. Most common cell wall component is chitin (Bartnicki, 1984). However in some fungi cellulose or other glucans are present. Some other substances associated with the fungal cell wall in different members are cellulose-glycan; cellulose-glucan, cellulose -chitin, chitin- glucan, mannan-glucan, mannan-chitin and polygalactosamine galactan. (Bartnicki-Garcia, 1984) said that the oomycetous cell wall is mainly composed by cellulose beta-glucan, the chitin is absent. However, the research suggests the presence of chitin in the cell walls of some oomycetes . The presence of polysaccharides provides excellent properties of chitin as to sorption of heavy metals. The given property is well studied for Zygomycota, Ascomycota, Deuteromycota, mushrooms. The chitin content of food or row material can provide an estimate of fungal contamination.
In terms of its chemical structure, chitin and chitosan have very similar Chemical structure. Chitin exhibits structural similarity to cellulose and differs from it with the replacement of C-2 hydroxyl residues by acetamide groups (Kurita, 1997). Chitin can be transformed into chitosan that has free amino groups by removing acetyl groups (CH3-CO) from chitin molecules. Chitosan (deacetylated chitin) is insoluble in water, alkali and organic solvents, but soluble in most diluted acids with pH less than 6. When chitosan is dissolved in an acid solution, it becomes a cationic polymer due to the protonation of free amino groups on the C-2 position of pyranose ring. Its cationic properties in acidic solutions give it the ability to interact readily with negatively charged molecules such as fats, cholesterols, metal ions, and proteins.
Figure 2: Chitosan synthesis from chitin precursor
2.4 Biomedical applications of chitin and chitosan
Due to its high biocompatibility, chitosan has been employed in drug delivery systems, implantable and injectable systems such as orthopaedic and periodontal composites, wound healing management and scaffolds for tissue regeneration.
2.4.1 Wound Healing
Chitin and chitosan activate immunocytes and inflammatory cells such as PMN, macrophage, fibroblasts and angioendothelial cells. These effects are related to the DD of the samples, chitin presenting a weaker effect than chitosan. Chitosan oligomers have also exhibited wound-healing properties, it is suggested that their wound-healing properties are due to their ability to stimulate fibroblast production by affecting the fibroblast growth factor. Subsequent collagen production further facilitates the formation of connective tissue (Howling GI et al, 2001). Recently, the effects of chitin and chitosan oligomers and monomers on wound healing have been studied. This study shows that in addition to chitin and chitosan, their oligomers and monomers enhance wound healing acceleration. Wound break strength and collagenase activity of the chitosan group (D-glucosamine (GlcN), chito-oligosaccharide (COS), chitosan) were higher than the chitin group (Nacetyl- D-glucosamine (GlcNAc), chiti-oligosaccharide (NACOS), chitin).
Collagen fibres run perpendicular to the incisional line in the oligosaccharide group (NACOS, COS) and many activated fibroblasts were observed in the histological studies around the wound in the chitosan groups. The break strength was stronger and more activated fibroblasts were observed at higher DD. The potential use of chitin oligosaccharides (DP2, DP3, DP4, DP5 and DP7) in wound healing as well as their capacity against chronic bowel disease has been studied. For the first time, a mucin-stimulating effect of chitin oligomers DP3 and DP5 has been observed in an ex-vivo model (Deters A et al,2008) . The wound healing effect of chitin and chitosan oligomers and monomers is of great interest because in vivo lysozyme degrades chitin and chitosan to these smaller molecules.
2.4.2 Drug Delivery Systems
An important application of chitosans in industry is the development of drug delivery systems such as nanoparticles, hydrogels, microspheres, films and tablets. As a result of its cationic character, chitosan is able to react with polyanions giving rise to polyelectrolyte complexes. Pharmaceutical applications include nasal, ocular, oral, parenteral and transdermal drug delivery. Three main characteristics of chitosan to be considered are: Mw, degree of acetylation and purity. When chitosan chains become shorter (low Mw chitosan), it can be dissolved directly in water, which is particularly useful for specific applications in biomedical or cosmetic fields, when pH should stay at around 7.0.
In drug delivery, the selection of an ideal type of chitosan with certain characteristics is useful for developing sustained drug delivery systems, prolonging the duration of drug activity, improving therapeutic efficiency and reducing side effects. Kofuji suggested that the physicochemical characteristics of chitosan are important for the selection of the appropriate chitosan as a material for drug delivery vehicles (Kofuji K et al, 2005). Investigations have indicated that DD and Mw of chitosan have significantly affected the role of chitosan in therapeutic and intelligent drug delivery systems.
Mi studied the gelation properties of microspheres cross-linked with glutaraldehyde as it had significant effect on drug incorporation (Mi et al, 2000). Microspheres prepared with a high molecular weight chitosan gelled faster than those prepared with a low Mw because they have different activation energies of gelation. Chitosan with short chains have higher activation energy and need more time to interact with the other chains and to gelate with glutaraldehyde. Gupta and Jabrail studied the effect of degree of deacetylation and cross-linking on physical characteristics, swelling and release behaviour of centchroman loaded chitosan microspheres. The DD controls the degree of crystallinity and hydrophobicity in chitosan due to variations in hydrophobic interactions which control the loading and release characteristics of chitosan matrices. The DD also controls the degree of cross-linking of chitosan in the presence of any suitable cross-linker. The higher the DD is, the higher the number of free amino groups and therefore the degree of covalent cross-linking increases.
When analyzing the influence of cross-linking degree and degree of deacetylation on size and morphology of the microspheres, these authors reported that the size and the surface roughness decreased on increasing the degree of cross-linking and the degree of deacetylation. Zhang also reported that a high degree of chitosan deacetylation and narrow polymer Mw distribution were shown to be critical for the control of particle size distribution (Zhang H et al, 2004). A higher degree of cross-linking and a higher DD in chitosan increase the compactness of matrices and its hydrophobicity, thus controlling the degree of swelling and diffusivity of the drug entrapped in chitosan matrixes.
It was observed that a DD between 48-62% promotes maximal loading capacity, due to the size of the cross-links and pores formed. Regarding the release properties, a very low DD can induce burst release. In another study with chitosan microspheres loaded with centchroman and cross-linked with glutaraldehyde, Gupta and Jabrail observed that the lower Mw of chitosan employed, the lower sphericity of the microspheres obtained and these microspheres were larger in size than those prepared with medium-high Mw chitosan due to the poorer molecular packing and cross linking. These results are in agreement with those presented by Desai and Park, who studied the influence of Mw of chitosan on chitosan-TPP microspheres prepared by spray-drying (Desai KG et al, 2006). They observed that an increase of Mw also produced more spherical microspheres, with greater size homogeneity and a smoother surface.
In addition, they found that an increase in molecular weight gave bigger microspheres as a result. Gupta and Jabrail in their study also found that microspheres prepared with high Mw chitosan presented a very low degree of swelling and a high degree of cross linking, thus, those microspheres prepared with medium molecular weight chitosan that lead to less strong intermolecular interactions being more appropriate for sustained release(Gupta K et al,2007) . These results were also in agreement with Desai and Park who observed that the release rate of vitamin C was much lower as the Mw of chitosan used for preparing microspheres increased. They studied the release kinetics and found that it followed Fickâ„¢s law of diffusion. Low molecular weight chitosan leads to poor retention of centchroman in microspheres due to a high degree of swelling and a fragile network structure.
The microspheres with medium Mw chitosan showed an optimum loading efficiency (Gupta K et al, 2007). Microspheres with medium Mw chitosan are more efficient in releasing the centchroman in a controlled manner in comparison to low and high Mw chitosan microspheres. Desai and Park in the study of the influence of chitosan Mw on chitosan-TPP microspheres found that it does not affect the spray drying yield. However, it has influence on some parameters of the microspheres that have already been commented on. In addition they studied the influence on zeta potential and observed some differences that were not very significant. Chiou investigated the effect of post-coating PLLA microspheres with different chitosans on the initial burst and controlling the drug release of the microspheres . Without chitosan, 20% lidocaine was released within the first hour and the time of 50% release was 25 hours. This period was extended to 90 hours after coating with chitosan. They observed that when applying chitosan of the same molecular weight, High Mw Chitosan (640 kDa) microspheres cross linked with 0.2% TPP obtained by spray-drying. Efficacy of reducing the initial burst of drug release was higher for a lower degree of deacetylation. With chitosan in acetic acid solution, coating the microspheres with high molecular weight and high viscosity could most effectively reduce the initial burst and control drug release of PLLA microspheres. The study indicated that manipulating the viscosity of the chitosan solution was the most important factor in contributing to controlling the drug release of chitosan post-coated PLLA microspheres.
2.4.3 Gene Delivery
Due to its positive charge, chitosan has the ability to interact with negative molecules such as DNA. This property was used for the first time to prepare a non-viral vector for a gene delivery system by Mumper in 1995. The use of chitosan as non-viral vector for gene delivery offers several advantages compared to viral vectors. Mainly, chitosan does not produce endogenous recombination, oncogenic effects or immunological reactions. Moreover, chitosan/pDNA complexes can be easily prepared at low cost. The Mw of chitosan is a key parameter in the preparation of chitosan/pDNA complexes since transfection efficiency correlates strongly with chitosan Mw. High molecular weight chitosan renders very stable complexes but the transfection efficiency is very low. To improve transfection efficiency, recent studies have examined the use of low molecular weight chitosans and oligomers (Koping-Hoggard M, 2003) in gene delivery vectors. It appears that a fine balance must be achieved between extracellular DNA protection (better with high Mw) versus efficient intracellular unpackaging (better with low Mw) in order to obtain high levels of transfection. Lavertu studied several combinations of Mw and DA of chitosan finding two combinations of high transfection efficiency using a chitosan of 10 kDa and DD of 92 and 80%, respectively.
Kiang studied the effect of the degree of chitosan deacetylation on the efficiency of gene transfection in chito san-DNA nanoparticles (Kiang T, 2004). Highly deacetylated chitosan (above 80%) releases DNA very slowly. They suggest that the use of chitosan with a DD below 80% may facilitate the release of DNA since it lowers the charge density, may increase steric hindrance in complexion with DNA, and is known to accelerate degradation rate. They reported an increase in luciferase expression when the degree of deacetylation was decreased from 90% to 70%. Formulations with 62% and 70% deacetylation led to luciferase transgenic expression two orders of magnitude higher than chitosan with 90% deacetylation.
2.5 Production of Chitin and Chitosan
A variety of procedures have been developed over the years for the preparation of chitin and chitosan. Main sources of the commercial chitosan come from crustaceans such as crab, krill and crawfish primarily because large amounts of the crustacean exoskeleton is available as a byproduct of food processing (Methacanon et al., 2003).
Mushrooms mainly contain about 20~30% chitin on a dry basis. This proportion varies with species and with season. Thus, the method of chitin preparation can vary with different sources. Isolation of chitin from mushrooms consists of four basic steps including deproteinization (DP) for protein separation, demineralization (DM) for calcium carbonate separation, decoloration (DC) for pigments separation, and deacetylation (DA) for removal of acetyl groups.
2.6.1 General objectives
The project is aimed to extraction of chitin and its glucan complexes from mushrooms of Rwanda and characterization of the extracted products using elementary analysis and FTIR spectroscopy.
2.6.2 Specific objectives
1. Sampling of mushrooms of Rwanda as raw sources of chitin and its glucan complexes.
2. Extraction of chitin and its glucan complexes from mushrooms of type Amanita Phalloide and Lactarius Subdulius mushrooms.
3. Characterization of chitin and its glucan complexes using elementary analysis and FT-IR spectroscopy.
4. Comparative analysis of a ratio of chitin-glucans and chitin for different types mushrooms of Rwanda (Amanita Phalloide and Lactarius Subdulis mushrooms).
CHAPTER 3.METHODS AND MATERIALS
3.1 Work plan
1. Collecting and identification of Amanita Phalloide and Lactarius Subdulius mushrooms.
2. Pre-treatment of mushrooms for extraction.
3. Extraction of chitin and its glucan complexes from Amanita Phalloide and Lactarius Subdulius mushrooms.
4. Characterization of the extracted products using elementary analysis and FT-IR spectroscopy.
5. Determination of a ratio of chitin-glucans and chitin in different types of mushrooms and comparative analysis of the obtained results.
3.2 Research methods
3.2.1 Collecting samples
The following two types of mushrooms were gathered in forest : Amanita Phalloides (Fig.3) and Lacterius Subdulius (Fig.4) mushrooms.
3.2.2. Pre-treatment of mushrooms for extraction
The raw mushrooms bodies (approximately 10 ones) were cleaned separated into pileus and stipes and milled.
3.2.3 Extraction of chitin and its glucan complexes.
A raw biomass of mushrooms (150g) was successive treated twice with 8% solution of NaOH at 90Ã‚Âº for 1 h (deproteinization process). Then demineralization was carried out with 6.7 % solution of HCl at room temperature for 3 h. The extracted products was washed from fats and lipids, filtered and dried up to constant weight at 70 Ã‚Âº (Teslenko, 1995) (Fig.5).
18.104.22.168 Deproteinization process (DP)
Mushrooms contain about 30~40% protein on a dry basis. Mushrooms are usually ground and treated with dilute sodium hydroxide solution (8%) at elevated temperature (90Ã‚ÂºC) to extract the proteins present. Reaction time is 1 hour. Prolonged alkaline treatment under severe conditions causes depolymerization and deacetylation. Shahidi and Synowiecki (1991) conducted a study of extraction time of proteins from crab (2% KOH) and shrimp (1% KOH) shells at 90 Ã‚ÂºC with a solid to solution ratio of 1:20 (w/v). They found that a minimum period of 1hr was needed to extract over 90% of the proteins, and 2hr extraction time was required for removal of all proteins. To obtain uniformity in reaction, it is recommended to use relatively high ratios of solid to alkali. Below two tables with conditions for deproteinization in extracting chitin from two types of mushrooms studied (Table 1, Table 2)
Table 1: Condition used for deproteinization in preparation of chitin from Amanita Phalloide mushrooms
source Chemical type concentration Temperature (Ã‚ÂºC) Length of treatment Yield
150 g of Amanita Phalloide mushrooms (write) NaOH 8%(W/W) 90 Ã‚ÂºC 1hr,repeat tuice 16.6 g
Table 2: condition used for deproteinization in preparation of chitin from Lactarius Subdulius mushrooms
Source Chemical type concentration Temperature(Ã‚ÂºC) Length of treatment Yield
150 g of Lactarius Subdulius mushrooms brown) NaOH 8% (W/W) 90 Ã‚ÂºC 1hr, repeat tuice 17.6 g
22.214.171.124. Demineralization (DM)
Demineralization is conventionally accomplished by extraction with dilute hydrochloric acid (up to 6.7%) at room temperature with agitation to dissolve calcium carbonate as calcium chloride for 3 hrs. A variety of the demineralization methods have been reported in the literature. The reaction time varies depending on the different species and preparation methods. The use of HCl at a concentration of 6.7% to achieve demineralization has been reported. Some drastic treatments with highly concentrated acids may result in modification such as depolymerization and deacetylation of the native chitin (Synowiecki , 1991). Below two tables with conditions for deproteinization in extracting chitin from two types of mushrooms studied (Table 3, Table 4)
Table 3: Conditions used for demineralization in preparation of chitin from Amanita Phalloide mushrooms
source Chemical type concentration Temperature(Ã‚ÂºC) Length of treatment Yield
Deproteinized product from Amanita Phalloide mushrooms(write) HCl 6.7%(W/W) 25Ã‚ÂºC 3 Hrs 5g
Table 4: Conditions used for demineralization in preparation of chitin from Lactarius Subdulius mushrooms
source Chemical type concentration Temperature(Ã‚ÂºC) Length of treatment Yield
Deproteinized product from Lactarius Subdulius mushrooms(brown) HCl 6.7%(W/W) 25Ã‚ÂºC 3 Hrs 5,8g
3.2.3 Characterization of extracted products using FTIR spectroscopy and elementary analysis
Using FTIR spectroscopy chitin- glucan complexes was identified by comparing with literature data. The extracted products were analyzed using elementary analysis. The percentage of C, N, and H elements was determined.
3. 4 Equipment and materials
1. Perkin- Elmer CHN Elemental Analyzer (USA);
2. Perkin-Elmer FTIR-spectrophotometer.
3. Drying oven, Temperature range 50-300 Ã‚Âº.
4. Analytical Balances
1. Sodium hydroxide (NaOH);
2. Hydrochloric acid (HCl).
3. Potassium bromide (KBr) for FTIR.
Mushrooms from forest
- Flasks (Erlenmeyer conical and Volumetric);
- Filter paper;
- China dishes for oven.
CHAPTER 4. RESULTS AND DISCUSSION
4.1. Interpretation of FTIR spectra
Identification of isolated chitin-glucan complexes (ChGC) was carried out using FT-IR spectroscopy.
Figure 6: FTIR spectroscopy chitin-glucan spectra of Amanita Phalloide (a) and Aspergillus Niger (reference) (b) mushrooms, respectively.
Figure 7: FTIR spectroscopy chitin-glucan spectra of Lactarius Subdulius (a) and Aspergillus Niger (reference) (b) mushrooms, respectively
The below table explains very well the spectra of Amanita Phalloide and Lactarius Subdulius mushrooms, respectively. This table shows the type of mushrooms, the range of absorption bands, the absorption bands for each type of mushrooms and the cause of the absorption bands (assignment of functional groups).
Table 5: Interpretation of FTIR spectroscopy spectra of Amanita Phalloide and Lactallius Subdulius mushrooms
Mushroom type Range, cm-1 Absorption band, cm-1 Assignment
Amanita Phalloide 3580-3700 3694.1 & 3620,4
3250 Free OH
2850-3000 2919,1&2850,5 CH3,CH2&CH
2361,4 & 2341,8 Impurities
1560,2 Amide I
1000-1250 1029,7 C-N
Lactarius Subdulius 3580-3700 3270 H-bonded OH
2850-3000 2923,6 CH3,CH2&CH
1560,2 Amide I
1000-1250 1644,6 C-N
For the two types of mushrooms we could observe approximately the same type of spectra with small changes (Fig.6a,7a) compared with the reference spectrum (Fig 6b,7b). This indicates their similar structure and the changes are due to the presence of impurities.
The presence of chitin parts causes the absorption band at 1637, 4, 1560, 2 and 1644, 8 (Fig.6, a) in amanita phalloide mushrooms, which correspond to vibrations of amide groups Ã‚Â«amide IÃ‚Â»and Ã‚Â«amide IIÃ‚Â» respectively. For chitin in Lacterius Subdulius species, the values of the same absorption bands is present at 1644, 6 cm-1 (Fig 7, a ). The presence of absorption bands in the 3400-2800 and 1160-1020 cm-1 in the spectra of the studied mushrooms indicates the existence of glucan part. These absorption bands appear due to O-H, C-H, C-O and C-C vibrations which manifest in the spectra of both chitin chitin glucan complexes.
4.2. Elementary Analysis data interpretation
The elementary analysis principle consists of determination of percent composition of certain elements. In our case, we carried out this analysis in order to find the ratio of chitin and glucan parts in extracted chitin-glucan complexes. The results of elementary analysis for Amanita Phalloide and Lactarius Subdulius mushrooms are shown in Table 6. Elementary analysis of the obtained chitin-glucan complexes of the two species shows approximately the same percentage of C and H. It also shows that Amanita Phalloide has more N compared to that of Lactarius Subdulius species hence more chitin.
Table 6 & 7: Elemantary analysis resultrs of Amanita Phalloide and Lactallius Subdulius mushrooms
Mushroom type Element %
Phalloide C 43.16
Mushroom type Element %
Phalloide C 42.83
4.2.1. Chemical Formula of Chitin-Glucan Complexes. Chitin and Glucans parts.
The general chemical formula of chitin-glucan complexes, chitin and glucans are shown below.
The Chemical formula of wet chitin-glucan complexes is:
(C6H10O5) x(C6H11O4N)y(H2O)z for white mushrooms. (4.1)
and (C6H10O5) x(C6H13O5N)y(H2O)z for brown mushrooms (Pestov,2008) (4.2)
The Chemical formula of chitin in Amanita Phalloide mushrooms is: C6H11O4N)y (4.3)
and the chemical formula of chitin in Lactarius Subdulius is: (C6H13O5N)y (4.4)
The Chemical formula of glucans is: (C6H10O5)Y for both types of mushrooms. (4.5)
where x, y and z are indexes characterizing fractions of chitin, glucan parts and water, respectively. The main difference between chitin and glucan is the presence of nitrogen in chitin.
4.2.2. Dtermination of a percentage of chitin and glucans parts in chitin - glucan complexes
The formulas (4.1) and (4.2) were used for calculation of a percentage of chitin, glucan parts and water in the wet chitin glucan complexes of Amanta Phalloide mushrooms and Lactarius Subdulius mushrooms. The following formulas for calculation x, y and z were applied :
For chitin (C6H11O4N)y or (C6H13O5N)y depending on type of mushrooms and for glucan part (C6H10O5)Y, the formulas used to calculate the percentage of elements are shown below:
%C (total) =(% C in glucan part)Ãƒâ€”x + (% C in chitin part)Ãƒâ€”y (4.6)
% H (total) = (% H in glucan part) Ãƒâ€” x + (% H in chitin part) Ãƒâ€”y + (% H in water) Ãƒâ€”z (4.7)
% N (total) = (% N in chitin) Ãƒâ€”y (4.8)
The ratio of chitin part is equal to the molar mass of chitin part times index y over the total mass of chitin â€œ glucan complexes. (Give formula).
For glucan there is no nitrogen and the chemical formula of both types of mushrooms is the same.
The ratio of glucan part in chitin- glucan complexes is equal to molar mass of glucan part times index x over the total mass of chitin- glucan complexes. (Give formula).
The obtained percentages of chitin, glucans and water in Amanita Phalloide and Lactarius Subdulius are shown in the table below:
Table 8: Percentage of chitin, glucan and water in extracted chitin-glucan complexes of Amanita Phalloide and Lactallius Subdulius mushrooms.
Mushroom type % of chitin % of glucans % water
Amanita phalloide 41.04 57.81 1.40
Lactarius subdulius 23.96 74.93 1.09
Calculate a percentage of chitin in the sample.
For example, in the formula (C6H10O5)0.49(C8H13O5N)0.51(H2O)0.81 â€œ (49% of glucan, 51 % of chitin ) in chitin-glucan complexes, but there is water. Did you calculate y So,( x + y + z) -100% and Y- n %. You need to determine n. n will be percentage chitin in a sample.
Based on elementary analysis, it was shown that the obtained chitin-glucan complexes of Amanita Phalloide have more chitin compared to that of Lactarius subdulius species and they have less glucan than that of Lactarius Subdulius mushrooms. This means that the Amanita Phalloide is better than Lactarius Subdulius mushrooms because of high content of chitin that has more applications.
What about percentage of chitin in the samlples You need to calculate !!!
CHAPTER 5. CONCLUSION AND RECOMMENDATION
The chitin-glucan complexes of the two types of mushrooms were obtained by extraction. The wet chitin-glucan complexes yield were 5 g (in %) for Amanita Phalloide and 5.8 g (in %) for Lactarius subdulius. Chitin and glucan parts of these complexes were identified by FTIR spectroscopy.
Some insoluble impurities were found in the extracted chitin - glucan complexes. The percentage of chitin and glucans was estimated using elementary analysis and the comparative analysis showed that Amanita Phalloide has more chitin than Lactarius subdulius.
About content of chitin in the samples
The extracted products can be used for sorption of different metals such as copper and zinc from waste water. This type of our experiment should be continued in the future and the presence of insoluble impurities in the studied extracted chitin glucan complexes indicated the insufficiency of washing during chitin glucan complexes extraction.
The chitin-glucans should be analyzed by FTIR immediately after extraction for the purpose to repeat process in the case of finding impurities. This will help researchers to control their experiment and end up with the very precise results ( to obtain pure chitin glucan complexes).
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