Optimization of deproteinization modes of chitine-containing raw materials in producing chitosane from micellar mushrooms of Aspergillus species

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1 Academia Journal of Biotechnology 6(2): , February 208 DOI: 0.543/ajb ISSN Academia Publishing Research Paper Optimization of deproteinization modes of chitine-containing raw materials in producing chitosane from micellar mushrooms of Aspergillus species Accepted 6 th February, 208 ABSTRACT Oksana Pavlova *, Volga Tratsiakova 2 and Milena Stachelska 3 Department of Nutritional Sciences, Yanka Kupala State University of Grodno, Belarus. 2 Department of Chemistry and Chemical Technologies, Yanka Kupala State University of Grodno, Belarus. 3Lomza State University of Applied Sciences, Poland. This work aims to determine optimal deproteinization modes for chitinecontaining raw materials from micellar biomass of waste citric acid producer. In order to determine how the scores of the factors in the study influence the removal of the protein component from Aspergillus niger, micellar mushroom s biomass, a full multifactorial experiment on optimizing deproteinization modes for chitine-containing raw materials was conducted. In order to build an efficient mathematical model the importance of factors were analyzed (degrees of their influence on the function) and were able to determine and eliminate those with low importance. In order to analyze the importance of factors regression analysis was used to process the mathematical data from the experiment and the range of optimal scores of the factors determined (such as: concentration of sodium hydroxide, exposition and temperature) for obtaining maximum removal of the protein component. The results of the regression analysis proved that the combination of temperature and concentration of NaOH significantly influence the deproteinization of the micellar biomass. The highest values of protein content in washwater and accordingly the lowest values in samples of chitincontaining raw material can be observed at NaOH concentration of 2%, where the value of the protein content in washwater rises with the increase of temperature and reaches its maximum at 90 C. The time factor is not significant in any of the modifications. The authors obtained data on how the factors in the study range in their influence on the controlled variable and used this data to determine the optimal deproteinization mode for chitin-containing raw material. On the basis of the obtained results, we were able to compile the optimal mode for processing raw material, which includes NaOH with a concentration of 2% at a temperature of 90 C with the maximum time allowed by the technology of the process. Keywords: Chitin, chitin-glucan complex, chitosan. INTRODUCTION Analysis of scientific and technical literature shows that at present the main goal of industrial biotechnology in producing certain types of products is to develop technologies that would help eliminate food shortage, increase environmental sustainability and enhance nonwaste production. In order to address these important issues, we have to conduct a thorough study of the ways and methods of regulating metabolism in microbial cells, which would make provisions for directed biosynthesis of metabolites that are invaluable to national economy. LITERATURE REVIEW Chitin has unique and complex properties: it s non-toxic, biocompatible, adsorptive and film-forming. It can be used

2 Academia Journal of Biotechnology; Pavlova et al. 043 Table : Units of variation and concentration of environmental components. Environmental component Factor Level Low (-) Mid (0) High (+) Unit of variation (λ) NaOH (%) Х Temperature ( С) Х Exposure (h) Х3 2 3 in medical, cosmetic, food, agricultural and ecological industries. Economically, one of the most promising sources of chitin in the Republic of Belarus is the byproduct of microbiologically synthesized citric acid - the biomass of Aspergillus niger micellar mushroom, which grows in the process of deep cultivation of beet molasses (JSC Skidelsky Sugar Refinery) (ТУ ). The dry mycelium by-product has to meet the requirements for product specification # "Dry mycelium - byproduct of citric acid production" (ТУ ). The cell wall of A. niger micellar mushroom, which is the producer of citric acid and its by-product may contain up to 20 to 25% of chitin. About 250 to 250 g of chitin can be obtained from kg of citric acid producer. In this regard, great importance is placed on developing different ways of obtaining chitine, chitin-glucan complexes and chitosan from the micellar biomass of A. niger, which in its turn would solve the problem of finding secondary use for biotechnological by-products such as: used enzyme producents, antibiotics and organic acids. It would also satisfy the domestic market s demand for polyfunctional chitin-containing raw-materials, which can later serve as a basis for obtaining biologically active components, new generation foods and highly active sorbents for heavy metal ions. RESULTS AND DISCUSSION The chitin-glucan complex - chitosan - is extracted from the biomass of A. niger as a result of sequential three-step acid-base hydrolysis. The first stage of processing raw material with sodium hydroxide has the best effect on removing fats, proteins and carbohydrates, whereas the structure of the sorbent is left undamaged (Kotlyar, 200; Kanarskaya, 200). Using harsh thermal and chemical treatment for processing the biomass helps remove protein substances more efficiently. At the same time, choosing the optimal mode for deproteinizing chitin-containing raw material is an important biotechnological issue and solving this problem will reduce the aggressiveness of the chemical environment, while also reducing material costs. The aim of this work is to develop more efficient deproteinization modes for chitine-containing raw materials from micellar biomass of citric acid producer. When choosing the factors for carrying out the experiment, we followed the classical method of isolating the chitincontaining complex by means of acid-base hydrolysis (Kotlyar, 200). Besides that, we took into account how the factors comply with conditions of controllability (the ability to set and maintain the parameter value as constant), compatibility (should not cause technological disruption), independence (can be set for each one separately), and unambiguity (are not functions of each other). Table shows the operating conditions of the selected factors. The controlled variable - protein content - was analyzed in washwaters by means of spectrophotometric method (Dawson, 99). In a quartz cuvette ( cm) we measured the absorbance A (optical density) of the protein solution at 260 and 280 nm, respectively. In order to calculate the concentration of protein we used the formula: Protein concentration (mg/мl) =.45А А 260 The authors compiled the matrix of full factorial experiment, according to which we then prepared 3 3 variants of deproteinization modes for raw materials in order to determine the optimal mode for removing fat, protein and carbohydrate. The planning matrix for the three-factor experiment for assessing the effects of processing modes on the controlled variable (protein content mg/ml) and its results are reviewed in Table 2. Each experiment was repeated thrice. In order to build an efficient mathematical model we analyzed the importance of factors (degrees of their influence on the function) and were able to determine and eliminate those with low importance. In order to analyze the importance of factors regression analysis were used to process mathematical data from the experiment and determine the range of optimal values of the factors such as: concentration of sodium hydroxide, exposition, temperature for obtaining maximum removal of the protein component with the help of Statistic graphics program. Based on the results of the experiments, we acquired a regression equation that adequately describes the relationship between protein removal and variation of the factors in study. This equation is an empirical mathematical model describing the process of determining the optimal mode for deproteinization. After excluding insignificant factors from the equation, it acquires the following form: Y= А А В

3 Academia Journal of Biotechnology; Pavlova et al. 044 Table 2: The planning matrix for the three-factor experiment and its results. Environment option Factors in study Х Х2 Х3 NaOH (%) Temperature ( С) Exposure (h) Average protein content (mg/ml) ,29±0, ,23±0, ,27±0, ,26±0, ,27±0, ,29±0, ,32±0, ,29±0, ,3±0, ,24±0, ,25±0, ,27±0, ,20±0, ,27±0, ,27±0, ,25±0, ,25±0, ,27±0, ,29±0, ,24±0, ,22±0, ,23±0, ,24±0, ,28±0, ,22±0, ,22±0, ,25±0,03 3g gg Figure : Standardized Pareto map for protein content. Where: Y Value of protein concentration mg/ml; А Concentration of NaOH, (%); В Processing temperature, ( С); С Processing time, (h). Figure shows the effects of the influence of experimental factors on the function of the Pareto map. It can also be used to evaluate the significance of the coefficients that we obtained from the regression equation. The effects on the diagram are placed in accordance with the absolute degree of their value (large to small) and the direction the factor s action takes is also shown. If the column of the factor s standardized effect crosses the vertical line, then, the influence of the factor on the response function is statistically significant (P<0,05). As can be seen on Pareto map in Figure, the factors in study influence the degree of protein content in washwaters

4 Academia Journal of Biotechnology; Pavlova et al Figure 2: Response surface that reflects how the output of protein depend on the processing temperature and time with NaOH concentration at 4% ' Figure 3: Response surface that reflects how the output of protein depends on the concentration of NaOH and the processing time at 80 С temperatures. unidirectionally. They also have almost equal influence on the output parameter. The diagram confirms the significance of the coefficients of regression equations. The analysis also established a determination coefficient (R 2 = 0.75), that confirms the adequacy of the mathematical model obtained. The closer its value is to, the stronger the connection between the effective variable (in this case - output of dry seed material biomass) and the factors in study. The value of the coefficient of determination is sufficient. The more significant the share of the explained variation, the less the role of other factors, since the regression model well approximates the initial data and such a model can be used to predict the values of the effective variables. Figures 2 to 4 show the response surfaces that graphically reflect how the output parameter depends on processing modes. These surfaces allow us to select the ranges of variation for the technological factors that are responsible for maximum output of the protein in the washing waters. CONCLUSION Based on the results obtained, the authors determined the optimal mode for deproteinization: sodium hydrozide 2 to 3%, temperature at 80 to 90 С and processing time is 2

5 Academia Journal of Biotechnology; Pavlova et al Figure 4: Response surface that reflects how the output of protein depends on the processing temperature and concentration of NaOH at 2 h exposure. to 3 h. REFERENCES Dawson R, Elliott D, Elliot W, Mir M (99). Data for Biochemical Research: Translated from English Kanarskaya ZA (200). Acquisition and characteristics of chitin-glucan adsorbent from mushroom biomass: author s abstract. Ph.D: / Z.A. Kanarskaya; Kazan National Research Technological University Kazan. 20. Kotlyar MN (200). Methods of isolating and modifying the chitin-glucan complex from Aspergillusniger biomass: author s abstract. Ph.D: / M.N. Kotlyar; Kazan National Research Technological University Kazan. 23. ТУ Dry mycelium - by-product of citric acid production. Cite this article as: Pavlova O, Tratsiakova V, Stachelska M (208). Optimization of deproteinization modes of chitine-containing raw materials in producing chitosane from micellar mushrooms of Aspergillus species. Acad. J. Biotechnol. 6(2): Submit your manuscript at