Enhanced melanin pigment production from Dietzia schimae NM3 in cheese whey using Box-Behnken design


 Melanin is a natural, dark-brown complex molecular structure pigment formed by the oxidative polymerization of phenolic compounds. Microbial melanin pigment can be use in industrial fields, canned additives and preservatives. Optimization method is used to produce a quick and sufficient product with reliable and cost-effective processes. In this project, four factors (temperature, l-tyrosine, pH and CuSO4) affecting in melanin production by Dietzia schimae NM3 were optimized by response surface methodology with Box–Behnken design in inexpensive medium (whey powder). The anti-bacterial activity of D. schimae melanin was assayed by disk diffusion test. The optimal medium compositions were obtained in whey 5% (w/v), l-tyrosine 2.5 g/l, CuSO4 0.013 g/l, pH 10.5, and temperature 32 °C by maximum yield of 790 mg/l melanin pigment. The ANOVA results of RSM showed a significant P-value (0.0001), model F-value (78.84) and probability R² (0.98), with insignificant lack of fit (0.091). Melanin also showed antibacterial activity against gram-positive strains such as Bacillus cereus (20 mm), B. subtilis (18 mm), Streptococcus pyogenes (17 mm), Staphylococcus epidermidis (18 mm), and S. aureus (18 mm), which was comparable with amoxicillin (AMX) and cefotaxime (CTX) as control positives. We realized the ability of D. schimae melanin pigment as natural substances to be considered for industrial fields due to its biocompatibility and physicochemical properties.


Introduction
Melanin pigment is a dark or black brown polymer with an irregular complex structure, a hydrophilic and a negative charge produced by nitrogen oxidation or free nitrogen-containing diphenols and produced through oxidative polymerization of phenolic or indolic compounds in various organisms (Plonka and Grabacka 2006). Melanin biosynthesis begins with a series of enzymatic and nonenzymatic reactions by tyrosinase enzyme (El-Naggar and El-Ewasy 2017). Melanin is produced by a wide variety of microorganisms such as plants, fungi, yeast, algae and bacteria. Melanin have powerful antioxidant, anti-viral and antibacterial properties (Plonka and Grabacka 2006). Melanin are resistant to heat and chemicals (e.g., heavy metals and oxidizing agents) and biochemical agents (e.g., host defense against invasive germs) (Cordero et al. 2017). Melanin possesses anti-UV radiation property by absorbing the electromagnetic spectrum and preventing optical damage in living organisms. Melanin has been used in antifungal drugs (Kurian and Bhat 2014;Venil et al. 2013).
Optimizing the growth conditions for microorganisms, particularly the physiochemical parameters and nutrition in developing every type of pigment production is significant. An optimization method using "one factor at a time" is difficult and time consuming. The production of pigments is under the influence of the physical and chemical conditions governing the production system (El-Naggar and El-

Materials And Methods Preparation of inoculum and media
Dietzia schimae strain NM3(KP207685), which isolated previously as an actinobacterium which resisted to UV radiation, dryness, oxidant agents including hydrogen peroxide and mitomycin C, was

RSM based Optimization of Melanin Production by Dietzia schimae NM3
D. schimae NM3, inoculated in whey medium along with l-tyrosine as substrate. The dark brown pigment melanin was diffused after 3-4 days in whey broth medium. The optimization process showed appropriate result than to nutrient broth melanin production (450 mg/L). Maximum melanin production was 790 mg/L in large-scale fermentation (one liter) whey medium as an inexpensive medium.
The statistical methods can be considered as the part of the primary stages of every study. They pursue the goal to focus on the critical variables and to discover the most effective ones in the study.
Through this method, it is merely viable to gain the proper concentration for each factor separately.  Predicted and actual charts show that our data are normal. The more linear the data is and the nearer the middle line the data has a normal distribution. Vertical or curved lines indicate abnormal scattering. In this study, the dispersion is normal. Chart shows actual test data to match the expected is high Fig. 1.
The Fig. 2 is the scatter chart showing the relationship of the variables. The horizontal column (x) shows the coded value and the vertical column (y) shows the value of the product. The left side shows the lowest and the right side shows the highest value. From left to right the value of the variable increases. As the amount of the variable increases, the amount of product goes up to a point and then declines. The variable factor pH (A) had the most drop in Fig. 2.
The three-dimensional graphs are shown the overall impact of the factors relationship. In Fig. 3a two factors of temperature at 32 °C and cooper 0.013 g/L are constant and two factors of acidity and tyrosine are measured by their interaction. Blue shows the lowest product and red shows the highest product. Interactions between L-tyrosine and pH improved the yield of melanin by increasing the pH from 9 to 11 and decreasing L-tyrosine to 2.2-2.8 g/L.
The interactions of copper and temperature was shown in Fig. 3b. Two factors of acidity at 10.5 and Ltyrosine 2.5 g/L are constant and two factors of temperature and cooper were measured by their interactions. It was indicated that by increasing of temperature to 32 °C and Cu to 0.013 g/L, the melanin production was increased.
The interactions of copper and pH (Fig. 3c) indicated an increase in melanin production in the pH range of about 9 to 11 with a decrease in copper concentration to 0.013 g/L. The interactions of Ltyrosine and CuSO 4 (Fig. 3d), indicated an increase in melanin production depended on L-tyrosine 2.5 g/L than to copper. pH and temperature (Fig. 3e) indicated temperature and acidity factors have an equal effect on melanin production. the interaction of two factor temperature and L-tyrosine ( Fig. 3f), indicated the effect of factors action on constant acidity on melanin production. According to this image, the increase in temperature does not increase the production of melanin, but shows the increase in tyrosine up to 2.5 g/L effectively.
Melanin showed maximum production at fixed pH of 10.5, temperature (32 °C), and L-tyrosine 2.5 g/L.
In the other hand, CuSO 4 (0.013 g/L) was so effective than temperature, and L-tyrosine considered highly important factor in melanin production ( Table 2).
The optimal medium compositions were obtained in whey 5% (v/v), L-tyrosine 2.5 g/L, CuSO 4 0.013 g/L, pH 10.5, at temperature 32 °C by maximum yield of 790 mg/L melanin pigment production.
These interactions indicated design experiment increased the growth Dietzia schimae NM3 and melanin production in whey medium are significantly.

Discussion:
Melanins are important pigment that have many applicable in medicine, cosmetic and other fields. In several studies, the intracellular and extracellular melanin in the bacteria has been reported such as The predicted yield of melanin by optimal levels of the variable generated by the model was in close correlation with experimental value, which signifies the RSM methodology over traditional optimization approach. In addition, the increased melanin production was observed with the parameters optimized using RSM than the initially used conditions (Surwase et al. 2013). These results suggested that the developed model was very valid in the present study. Results showed that quality of model was adequately good and might describe real relationship among medium components.

Figure 1
Residual diagnostics of crossed model for melanin production: predicted (left) and actual (right) Figure 2 The graph shows melanin effect for RSM method in four factors include: pH (A), temperature (B), L-tyrosine (C), and CuSO4 (D).