Preparation of Chitosan from Agaricus bisporus Brown Stems and Studying some Its Physicochemical and Functional Properties

The stems of the brown Agaricus bisporus mushroom, which were obtained from the Al-Wadq mushroom farm / Baghdad, The chemical composition of the mushroom stems was estimated as the proportions of the mushroom components: moisture, protein, fat, ash, carbohydrates (91, 2.8, 0.25, 0.88, 5.07)% respectively. Chitin was extracted from the stemsof A. bisporus brown mushrooms by chemical method using 2 M sodium hydroxide solution and 2% acetic acid. The percentage of chitin extracted from mushroom stems was 13.8%. Chitosan was prepared from the stems of A. bisporus brown using a 50% sodium hydroxide solution at a temperature of 100°C for 3 hours, and the percentage of chitosan in mushroom stems was 10.5%. The chitosan under study was identified using Fourier Transform Infra-Red (FTIR). The degree of deacetylation of chitosan produced from the stems of A. bisporus brown was 83%. The viscosity of mushroom stems chitosan was 40cP. When the viscosity was estimated by dissolving chitosan in 1% acetic acid solution, the molecular weight of mushroom stems chitosan was 46.922 kDa. The stems chitosan was distinguished by its high solubility in 1% acetic acid solution, reaching 90%.


Introduction
Food fungi are among the distinguished organisms in the biology world, due to their direct association with human food, as these fungi belong to the kingdom Myceteae, as the equivalent of approximately 1.5 million species falls under this kingdom [1].Mushrooms have been a part of human diets and human medicine for centuries.Mushrooms currently consist of three main groups: cultivated edible, wild-harvested, and medicinal mushrooms.business valued at more than 75$ billion in 2017, while edible mushrooms constitute more than half of the economic value of the total global mushroom industry [2].There are more than 2000 species of edible mushrooms, and Agaricus bisporus is the most consumed species, which is found in two types, white and brown ، The name Agaricus bisporus Portobello began 2 to be used for the brown mushroom in the 1980s and is often shortened to Portobello, which is a variant of the common cultivated white mushroom.Portobello mushroom has a distinctive flavor and meaty texture.Mushroom stipe, also known as stems, is used in soups and Stock, while the cap is grilled for use in sandwiches or thickly sliced in salads or appetizers [3].Agaricus bisporus is a rich source for bioactive compounds with functional properties that have a positive effect on human health, as the mushroom A.bisporus contains multiple phenols, sugars, vitamins, carotenoids, and minerals and has beneficial and nutritional effects on one or more functions of the body and reduces the risk of diseases [4].Chitin is often found in the cell walls of most fungi, yeasts, crustacean shells, and the exoskeleton of some marine organisms.Chitosan is a derivative of chitin, second only to cellulose, as the most abundant biopolymer in nature.The difference between chitin and chitosan from a structural point of view lies in the position of carbon atom No. 2, as chitin contains an acetyl group in this site, while chitosan contains an amine group in the same site [5].Chitin is distinguished from chitosan by the degree of removal of acetyl groups, which is the balance between the two types (chitosan is rich in amine and chitin is rich in N-acetyl group).The degree of removal of acetyl groups that exceeds 50% makes chitosan highly soluble in acidic aqueous solutions with The pH is less than 6, which is due to the protonation of NH2 groups, and during the process of removing acetyl groups, acetyl groups are removed to obtain chitosan, but this process may affect the molecular weight of the resulting chitosan.The source of chitin, the degree of removal of acetyl groups, and the reaction conditions are the main determining factors for the molecular weight of chitosan [6].Chitosan and its derivatives have received great attention from academic and industrial circles, as chitosan has antifungal activity against plant pathogens [7].In addition to its use in the food industry [8][9][10][11].Chitosan is also used in the field of pharmacy and its use as anti-microbial coatings, as well as its use in the manufacture of wound dressings and removal of heavy metals [12][13][14][15].Accordingly, the aim of the research is to extract chitin from the stems of the brown A.bisporus and transform it by chemical treatment into chitosan, and then study some of its physicochemical and functional properties.

Sources of Mushroom Waste
The wastes of the Agaricus bisporus brown, strain Sylvan A 15, were obtained from Al-Wadq farm located in Baghdad -Iraq.The remaining waste in the soil on which the mushrooms grow were collected from the production halls after harvesting the mushrooms for the purpose of marketing them by the farm.

Chemical Composition of Agaricus Bisporus Brown stems
The chemical composition of the mushroom stemsAgaricus bisporus brown was estimated according to the standard methods mentioned in [16].The percentage of moisture, protein, fat and ash was estimated by three replicates, and the percentage of carbohydrates was calculated by calculating the difference based on the values of the chemical composition results from the following equation: Total carbohydrates (% fresh weight) = 100 -(moisture + protein + fat + ash) %

Deproteinization
The step of Deproteinization from mushroom stems powder was carried out according to the method mentioned by [17] with some modifications, as the mushroom stems powder was treated with sodium hydroxide solution with modification of the concentration of the solution and the mixing ratio.Sodium hydroxide solution was added at a concentration of 2 M to the powder of mushroom stems at a mixing ratio of 1: 30 (w/v).The mixture was heated with slow stirring for three hours at a temperature of 100 °C for the purpose of removing the protein.The mixture was filtered under vacuum and the precipitate, which represents the insoluble basic materials, was washed.(AIM) Alkalin Insoluble Materials With distilled water, the ions were removed several times until pH = 7 was reached, then the sample was dried.(AIM) was treated with 2% acetic acid solution, at a mixing ratio of 1:100 (w/h), and the sample was placed in a reverse condenser for 6 hours at a temperature of 95°C.The centrifugation process was carried out at a speed of 1000 x g at room temperature for 10 minutes.The obtained precipitate was washed with distilled water until reaching pH = 7, then lyophilizated to obtain chitin.

Decolourisation
The process of bleaching the obtained chitin was carried out by mixing it with acetone in a ratio of 1:10 (w/v) with stirring at a speed of 150 cycles/min at room temperature in a heating device with a magnetic stirrer, then filtering using Whatmann No.2 paper and leaving at the same temperature for 2 hours.Mixing chitin with sodium hypochlorite at a concentration of 0.3155% at a ratio of 1:10 (w/v) with stirring at 1500 xg for 15 minutes at room temperature, filtering using Whatmann No.2 paper, washing the precipitate with distilled water until the pH is reached.= 7 Then lyophilizated the chitin [18].

Extraction of the Ratio of Chitin
The percentage of chitin produced from the stems of the fungus A.bisporus brown was calculated by weighing the resulting chitin according to the following equation [19]: Chitin %=Chitin weight after lyophilization (g)/ The weight used from the original model × 100

Converting Chitin to Chitosan
The acetyl groups were removed from the extracted chitin by treating the latter with a 50% sodium hydroxide solution at a mixing ratio of 1:50 (w/v).The sample was placed in a reflux condenser with heating at a temperature of 100°C and a time interval of 3 hours.The sample was filtered.After cooling at room temperature using Whatman NO. 1, the filtrate was discarded and the precipitate was washed with distilled water several times until reaching a neutral pH = 7, and then the precipitate was lyophilized to obtain chitosan [20].The yield of chitosan resulting from the chitin of the stems of A.bisporus was calculated according to the following equation [19]: Chitosan yield % = dry weight of chitosan / dry weight of chitin × 100

Diagnosis of Chitosan using the FTIR Device
The chitosan prepared from the stems of the mushroom brown A. bisporus was detected using the FTIR device (Fourier Transform InfraRed Spectrophotometer), by mixing the dry form of chitosan with dry potassium bromide in a ratio of (1:5) using a ceramic mortar for 2 minutes and compressing the mixture using a hydraulic press of the FTIR device.At a pressure of 8 bar and for 60 seconds, the disk was placed in the FTIR device for the purpose of analysis, using a frequency ranging between 400 -4000 cm -1 [21].

Degree of Deacetylation (% DD)
The degree of removal of acetyl groups (DD) for chitosan was estimated based on the FTIR results, as the absorbance was calculated at wavelength A 1655 (1655), which represents the carbonyl group, to the absorbance at wavelength A 3450 (3450), which represents the absorbance of amine groups, which is considered as The internal standard, because it does not decompose and is not affected by the treatments that take place when extracting chitosan, the degree of removal of acetyl groups was calculated according to the equation mentioned [22].

Determination of Viscosity
A solution of chitosan was prepared at a concentration of 1% by dissolving the chitosan with a solution of 1% acetic acid with continuous stirring until the chitosan dissolved.The Brookfield Viscometer was used to measure the viscosity of the chitosan solution, as it used the spindle No. 5 with a number of turns of 100 rpm at 25 °C [23].

Determination of Solubility
The solubility of the chitosan under study was estimated according to the method mentioned by [24].A 1% concentration of chitosan solution was prepared by dissolving 0.1 g of the sample under study in 10 ml of 1% acetic acid solution, and placing the sample in a centrifuge tube of known weight.The tubes were placed in a shaking incubator.At a speed of 60 rpm for 48 hours at 25 °C, the sample was centrifuged at 10,000 xg for 15 minutes.The supernatants were discarded and the insoluble particles were washed with distilled water, then centrifuged again at the same speed.This process was repeated three times and the insoluble particles were dried at 105 °C for 24 hours.The percentage of solubility was calculated from the following equation: Solubility % = (chitosan + final weight of the tube) -(chitosan + primary weight of the tube)g (chitosan + primary weight of the tube) -(chitosan + primary weight of the tube ) g

Determination of Molecular Weight
The molecular weight of chitosan was estimated based on the viscosity measurement, as the Mark-Houwink-Sakurada equation (MHS) was used to calculate the molecular weight according to [25] ŋ = kMWa Since the values of (a, k) are constants k = 1.49x10-4 dl/gm, a = 0.79, ŋ = viscosity (cP), MW = molecular weight (Daltons)

Chemical Composition of Brown A.bisporus Stems Powder
The results show in Table (1) the chemical composition of brown A. bisporus mushroom stems powder, as the moisture content in the mushroom stems reached 91%. .This result is consistent with what was found by [4] when estimating the moisture content of brown A. bisporus, which amounted to 91.15% .While [26] mentioned that the percentage of moisture in the stems of Portobello mushrooms was 92.82%.The reason for this difference in moisture content in edible mushrooms is attributed to the dependence of the moisture content in the fungus on the mushroom cultivar and other criteria related to harvesting, growth and storage conditions.The percentage of protein in the stems of the brown A.bisporus mushroom was 2.8%, while the percentage of fat and ash was (0.25, 0.88)%, respectively, and these percentages of protein and fat were higher than what was found by [27], which indicated that the percentage of protein and fat in the brown mushroom was (1.29, 0.14)%, respectively, while the percentage of ash was lower, amounting to 0.95%.[26], mentioned that when studying the chemical composition of some types of edible fungi showed that the percentage of protein, fat and ash in the stems of the brown A.bisporus mushroom was (2.07, 0.27, 0.86)%, respectively.The reason for this discrepancy in proportions may be attributed to the difference in the stage of maturity of the mushroom, as the brown mushroom is often harvested at an early stage of maturity and before the stage of opening the hat, in order to meet the desire of the consumer, as well as the conditions of storage and marketing.The percentage of carbohydrates in the stems of the brown A.bisporus mushroom was 5.07%, and this percentage was close to the range mentioned by [28], which indicated that the total content of carbohydrates in the fruiting bodies of the white A.bisporus mushroom ranged between (4.5-4.6)% of the fresh weight of the mushroom.In a study conducted by [3], which included analyzing and evaluating the components of the distinctive taste in the A.bisporus brown (Portobello), it was found that the percentage of total sugars in the mushroom stems reached (20.64 -28.24)% relative to the dry weight of the mushroom.The results of the study show that the percentage of total carbohydrate in the stems of white mushrooms is higher than it is in the stems of brown mushrooms.The reason behind this variation may be the different type of mushroom as well as the different values of the components of the mushroom depending on the cultivation conditions, stage of maturity, methods and storage period.

The Yield of Chitin and Chitosan
Figure (1) shows the percentage of chitin and chitosan prepared from the conversion process of chitin.The percentage of chitin in the stems of the brown A.bisporus mushroom was 13.8%.This percentage is higher than what was found by [29] when estimating chitin in Lactarius vellereus And Phyllophora ribis Which amounted to (11.4,7.9)%, respectively.The reason for this difference in the percentage of chitin may be attributed to the different type of mushrooms and the method of preparation.In addition, the study under comparison relied on powdering the fruiting body and not powdering mushroom stems only, as [30] indicated that The percentage of chitin in the stems of the white A.bisporus mushroom reached (7-19)%, which is higher than what is found in the fruiting body as a whole and in the cap of the mushroom, which amounted to (3-9, 7-6)%, respectively.In a study conducted by [31], which included the extraction of chitin from powdered stems of the white A.bisporus mushroom, it was found that the percentage of chitin reached 22.5%, and this percentage is much higher than the value of chitin obtained in this study.The reason for the difference in the proportions of chitin may be attributed to The difference in the type of mushroom, in addition to the difference in the proportions of the components in the different edible fungi and thus the effect on the proportion of chitin.The proportion of chitosan prepared from the stems of the A.bisporus brown was 10.5% based on the dry weight of the mushroom stems powder Figure (1), which is equivalent to 76% based on the dry weight of the produced chitin, and this result is less than what was found [32] when preparing chitosan from Champignon mushroom wastes Although the study relied on the same type of strain Sylvan A-15, as the percentage of chitosan extracted from champignon mushroom stems was 17.6%, the method of preparation and extraction conditions may be behind the difference in the percentage, in addition to that it is possible that the stage of picking valid mushrooms For eating, period and storage conditions are matters that must be taken into account, as [33] pointed out the possibility of increasing the content of the components of the cell walls of the A.bisporus when stored in different conditions and temperatures.The method and conditions of extracting chitin and converting it into chitosan play an important role in the final outcome of the extraction rate.In a study by [34] which included the extraction and characterization of chitin and chitosan extracted from the Termitomyces titanicus .The study relied on the use of different concentrations of sodium hydroxide solution, as the study relied on concentrations (1, 2, 3, 4) molar concentrations of sodium hydroxide solution in the Deproteinization step.The study showed that the yield of chitin increased directly with the increase in the concentration percentage, reaching (9.77, 22.13, 38.04, 39.87)%, respectively.

Diagnosis of Chitosan using the FTIR Device
Table (2) shows the wave numbers of functional groups of chitosan extracted from Agaricus bisporus brown (Fig. 2) compared with the FTIR spectrum of the commercial chitosan as a standard (Fig. 3).
The active group that represents the hydroxyl stretching band appeared at the wave numbers (3452.58 and 3448.72)cm -1 for extracted chitosan and commercial chitosan, respectively.The absorbance at the wavenumbers (2918.30and 2877.79)cm -1 of the studied chitosan represents the Stretchy vibration of CH3 and CH2 groups, respectively.Amide bands are among the most important active groups whose absorption peak appeared at the wave number (1658.78 cm -1 ) for commercial chitosan.While this group did not appear in the chitosan under study, the reason behind the absence of this group may be attributed to the conditions of the removal process acetyl groups.The bands at wavenumbers (1579.70 and 1573.91)cm -1 of the extracted and commercial chitosan, respectively, refer to the N -H group in the second ΙΙ amide bond, while the bands that appeared absorbance peaks at the wavenumbers (1382.96and 1379.10)cm -1 of chitosan The extracted and commercial ones are in the order of the bending vibration of the group C -N.As for the band whose absorption peak appeared according to the wavenumber (894.90 and 896.90) cm -1 , it represents the glycosidic bond β- (1,4) in the extracted and commercial chitosan, respectively [35].The above results for functional groups agreed with what found [36] when diagnosing chitosan extracted from the stems of mushroom A. bisporus brown , and also agreed with [37] when diagnosing chitosan extracted from the Ganoderma lucidum mushroom.The results also agreed with what he found [38] when diagnosing chitosan produced from shrimp waste with an FTIR device.and [39] when characterizing chitosan extracted from crab shells.

Physiochemical and Functional Properties of Chitosan
Figure (4) shows the physical properties of chitosan extracted from the stems of the A.bisporus brown, as the rate of Degree of deacetylation for chitosan under study was 83%, and this percentage is within the range mentioned by [40], which indicated that the degree of deacetylation of chitosan extracted from yeast and fungi, depending on the results of the FTIR, ranged between (79 -87)%, while the percentage of deacetylation based on the Nuclear Magnetic Resonance method was (89 -70)%.[24] showed when estimating Degree of deacetylation from chitosan extracted from mushrooms, which amounted to 78.1%, while [35] indicated that the percentage of Degree of deacetylation from chitosan produced from A.bisporus reached 66.35%.The reason for this difference may be attributed to the Degree of deacetylation of chitin to produce chitosan depend on the different type of mushroom and extraction conditions in terms of approved concentrations, temperature and time, as well as the method of calculating the Degree of deacetylation based on the results of the FTIR device, which differs in the type and accuracy of sample preparation upon examination as well as the equation used in the estimate.The viscosity of chitosan reached 40 centipoise, Figure (4), and this percentage is less than what was found [41] when estimating the viscosity of chitosan produced from the stems of the white A. bisporus mushroom, as it reached 55 cP.The reason for the difference in the viscosity value may be attributed to the different type of mushrooms as well as extraction conditions.The solubility of chitosan reached 90% (Fig. 4), and this percentage is close to what was found by [24] when estimating the solubility of chitosan produced from Ugandan edible Mushrooms, which amounted to 86%.The molecular weight of the chitosan under study was 46.922 kDa, and this value is less than what was found by [24] when estimating the molecular weight of chitosan produced from the source of edible mushrooms, which amounted to 384 kDa, It was found [35] when estimating the molecular weight of chitosan extracted from the A.bisporus, which amounted to 37.3 kDa, and this percentage is less than the result of the molecular weight of chitosan under study, the difference in the value of the molecular weight of chitosan may be attributed to the difference in the fungal source adopted for the production of chitosan and the method and conditions used in extraction, as well as the dependence of the study under experiment on extracting chitosan from A.bisporus mycelium and not from mushroom stems.

Conclusions
The results of this research showed the possibility of benefiting from the wastes of mushroom farms in the extraction of some biologically active compounds, as it was possible to extract chitosan from the wastes of the fungus farm, represented by the stems.Thus, it is possible to benefit from recycling these wastes, as well as finding an alternative to the animal source in the production of chitosan

Figure 1 .
Figure 1.Percentage of chitin and chitosan prepared from stems of A.bisporus brown mushrooms.

Figure 2 .
Figure 2. Infrared spectrum of chitosan extracted from the stems of the A. bisporus brown.

Figure 4 .
Figure 4.The physicochemical properties of chitosan prepared from the stems of the A.bisporus brown.

Table 1 .
The chemical composition of brown A.bisporus stems powder.

Table 2 .
Wave numbers of functional groups of chitosan extracted from Agaricus bisporus brown and commercial chitosan.