Journal of Agricultural Science and Technology A 3 (2013) 948-954 Earlier title: Journal of Agricultural Science and Technology, ISSN

1939-1250

DAVID

PUBLISHING

Production of Lipase Using Cassava Peel and Sunflower Oil in Solid-State Fermentation: Preliminary Study
Caroline Branco Gerber1, Francieli Kaufmann1, Gabrieli Nicoletti1, Marlia Dalla Costa1 and Aniela Pinto Kempka2
1. Volunteer Scholarship Holder of the Scientific Initiation, Food Engineering Department, State University of Santa Catarina, Pinhalzinho 89870-000, Santa Catarina, Brazil 2. Food Engineering Department, State University of Santa Catarina, Pinhalzinho 89870-000, Santa Catarina, Brazil

Received: August 2, 2013 / Published: December 20, 2013. Abstract: Full use of residues from industrial processes is a fundamental necessity of contemporary society, since it avoids impacts to the environment by using residues as inputs for other products of high economic and social importance. In this study, lipase production of the crude enzymatic extracts obtained by Aspergillus niger using cassava peel as substrate and sunflower oil as an inductor was investigated. The optimized cultivation temperature and concentration of inductor were determined using the response surface methodology. The two variables studied exercised influence in the production of lipase in the 95% level of confidence. The response surface obtained indicated that the conditions that maximize lipase activity production were 30.5 C and initial concentration of sunflower oil was 2.5% (w/w). Through this analysis, it is evident that extremes in temperature and concentration of inductor tend to decrease lipase production, since low temperatures decrease metabolism and high temperatures may inactivate the lipase. Optimum lipase yield was 59.8 U/g of dry peel which was fermented for 60 h. Lipase production presents a peak of 61.3 U/g, at 72 h of fermentation. However, this value is statistically equal (p > 0.05) of the value of lipase activity obtained for 60 h and 84 h of fermentation. Key words: Cassava peel, lipase, production, solid-state fermentation, sunflower oil.

1. Introduction
Cassava, Manihot esculenta Crantz (Euphorbiaceae), is a perennial shrub belonging to the family of Euphorbiaceae. The genus Manihot comprises 98 species of which M. esculenta is the most widely cultivated member. The tuberous root of cassava is the fourth most important food source of carbohydrate in the tropics after rice, maize and sugar cane, due to its high starch content [1]. Production of cassava (Manihot esculenta Crantz) starch results in creation of around 10%-15% of the original root weight as solid waste [2], and maybe results in using fermentation processes. Bioprocessing based on
Corresponding author: Aniela Pinto Kempka, Ph.D., research fields: enzyme, bioprocess, food and residues. E-mail: aniela.kempka@udesc.br.

microbial conversion is an important approach for the production of value-added products from cassava [3], including the production of enzymes. Cassava peel is a byproduct of cassava processing either to food or other industrial products. This peel could make up to 10%-20% of the weight of the roots, thus indicating its enormous potential for biotechnological processes [4]. The major share of the industrial enzyme market is occupied by hydrolytic enzymes, such as proteases, amylases, esterases and lipases [5]. Lipases, triacylglycerol ester hydrolases (EC 3.1.1.3) are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoacylglycerols, glycerol and reactions of esterification, transesterification and

Production of Lipase Using Cassava Peel and Sunflower Oil in Solid-State Fermentation: Preliminary Study

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interesterification of lipids [6, 7]. Promising applications about lipases are found in organic chemical processing, detergent formulations, synthesis of biosurfactants, the oleochemical industry, the dairy industry, the agrochemical industry, paper manufacture, nutrition, cosmetics and pharmaceutical processing [8]. Microbial lipases are mostly extracellular and are greatly influenced by nutritional and physico-chemical factors, such as temperature, pH, nitrogen and carbon sources, presence of lipids, inorganic salts, agitation and dissolved oxygen concentration [5], and also may be used submerse fermentation or solid-state fermentation (SSF). When developing industrial fermentation, designing media and optimizing fermentation conditions are of critical importance, because these factors could strongly interfere with the yield of lipase production. Experimental design techniques present a more balanced alternative to the one-factor-at-a-time

the techniques to be used account for these interactions, so that a set of optimal process conditions can be determined [13]. Response surface methodology (RSM) is an effective statistical technique for the investigation of complex processes. The main advantage of RSM is the reduced number of experimental runs needed to provide sufficient information for statistically acceptable result. It is a faster and less expensive method for gathering research result than the classical method [14]. In this study, RSM was used to optimize lipase production by Aspergillus niger, using cassava peel as substrate and sunflower oil as inductor. A 22 central composite rotatable design (CCRD) was used to optimize fermentation conditions to verify the possibility of using cassava peel as substrate and sunflower oil as inductor.

2. Materials and Methods

2.1 Microbial Culture and Inoculum Preparation Aspergillus niger was obtained from Fundao Andr Tosello culture collection. The propagation of spores prior to fermentation was carried out for seven days at 27.5 C in a medium constituted by potato dextrose agar (PDA) 3.9% (w/v) and distilled water. Medium for inoculum production consisted of (w/v): 2% starch, 1% olive oil, 0.1% yeast extract, 0.025% MgSO47H2O, 0.05% KH2PO4, 0.5% CaCO3 and 1.5% agar. This medium was inoculated with a spore suspension and incubated at 27 C for seven days. The spores were collected adding 10 mL of a sterile 0.1% Tween 80 aqueous solution and glass beads to the fermented agar medium and kept at 4 C until use [11]. 2.2 Preparation of the Substrate and the SSF Cassava peel was used as substrate and was previously comminuted and dried in the drying oven (Solab) at the temperature of 105 C for 24 h and subsequently frozen for maintaining their physicochemical characteristics. Inducer was used as

approach for fermentation improvement [9]. SSF is defined as the fermentation process on moist solid substrate in the absence or near absence of free water. SSF can be used for the production of enzymes utilizing various substrates including solid wastes [10]. The use of agro-industrial residues as substrate in lipase production by SSF can significantly reduce the final price of enzyme, and also add value to low cost materials in the market [11]. Many studies have been undertaken to define the optimal culture and nutritional requirements for lipase production. These requirements are influenced by the type and concentration of the carbon and nitrogen sources, culture pH and growth temperature. Lipidic carbon sources generally seem to be essential to obtain a high lipase yield, although a few authors observed that the presence of fats and oils was not statistically significant for enzyme production [12]. It is noteworthy that during fermentation, the operating conditions interact and influence their respective effects on the response. It is important that

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Production of Lipase Using Cassava Peel and Sunflower Oil in Solid-State Fermentation: Preliminary Study

sunflower oil (Salada) (the vegetable oil was considered a supplementary carbon source since their composition is mainly constituted by lipids). The experiments for lipase production were carried out aseptically in conical reactors covered with hydrophobic fabric. Aqueous solution containing the supplements was added to the substrate and the resulting medium was then sterilized at 121 C for 20 min. The sterile medium was then inoculated with 108 spores/g dry substrate using spore suspension previously prepared. Cultivation was carried out in an incubator BOD (Solab). The moisture of the medium was set at 55% in accordance with Kempka et al. [11]. 2.3 RSM and Optimization of Lipase Production A factorial design with two factors and four axial points (22 CCRD) was carried out to evaluate the effect of incubation temperature and concentration of inductor (%, w/w) on lipase production. Table 1 shows the values of the variables and their respective levels coded (in brackets). Each assay was carried out in duplicate and a center point was performed in triplicate for experimental error evaluation. A five-level, two-factor CCRD was employed, requiring 10 experiments. The fractional factorial design consisted of three factorial points, two axial points and two central points. The data obtained were fitted to a second-order polynomial equation. The analysis was performed using software Statistica 10.0Statsoft Inc.. 2.4 Extraction and Determination of Lipase Enzyme Activity and Biomass Determination The fermented medium was weighed, added to 45 mL of 0.1 mol/L sodium phosphate buffer at pH 7.0 and incubated at 35 C and 200 rpm for 30 min for enzyme extraction. Following the extraction, the liquid fraction was separated by filtration and assayed for lipase activities. Lipase activity was determined by titration method. An emulsion of olive oil (10% w/v) and arabic gum (5% w/v) in sodium

phosphate buffer 0.1 mol/L and pH 7.0, was incubated with a sample of the enzymatic extract at 37 C and 160 rpm for 15 min. The reaction was stopped and the fat acids were extracted with a solution of acetone and ethanol (1:1). The fatty acids produced were titrated with NaOH (0.05 mol/L) [15]. One unit of lipase activity was defined as the amount of enzyme that produced 1 mol of fatty acids/min, under the assay conditions. 2.5 Physicochemical Characterization of Cassava Peel The physicochemical characterizations of cassava peel, such as pH, moisture, ash and starch were analyzed. For the determination of pH, 5 g of peel homogenized in 5 mL distilled water were used and preceded with the reading digital potentiometer (Quimis). For the moisture determination, the authors used the gravimetric method. For the determination of ash, incineration was used in a muffle at 550 C and for determination of starch, the authors used the method of acid digestion and determination of sugars reducers [16].

3. Results and Discussion

Physicochemical characterizations of cassava peel were obtained before drying, such as humidity value 72.26%, ash 2.27%, pH 6.0 and starch 56.5%, proving that cassava peel is an appropriate substrate for fermentation. The lipase production was possible in this way due to the supplementation of sunflower oil, characterized as an inducer in the process. 3.1 Optimization of Lipase Production Table 2 presents the matrix of the complete factorial design with the lipase activity obtained at 60 h of fermentation. The highest lipase production, showed higher amount of lipase activity occurred to the center point of the experiment, with mean lipase activity of 59.8 U/g, at a temperature of 30.5 C and the percentage of inductor 2.5 (w/w). Tukey test can verify

Production of Lipase Using Cassava Peel and Sunflower Oil in Solid-State Fermentation: Preliminary Study Table 1 Factorial design with two factors and four axial points (22) was carried out to evaluate the effect of incubation temperature and concentration of inductor (%, w/w) on lipase production using cassava peel and sunflower oil. Experiment E1 E2 E3 E4 E5 E6 E7 E8 E9 (C) E10 (C) Temperature (C) 25 (-1) 25 (-1) 36 (1) 36 (1) 22.7 (-1.41) 38.2 (1.41) 30.5 (0) 30.5 (0) 30.5 (0) 30.5 (0) Concentration of inductor (%, w/w) 1.0 (-1) 4.0 (1) 1.0 (-1) 4.0 (1) 2.5 (0) 2.5 (0) 0.4 (-1.41) 4.6 (1.41) 2.5 (0) 2.5 (0)

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may be related to the characteristics of that mode of cultivation, if compared to the submerged one. In the SSF, the final product is concentrated and fungi have the appropriate characteristics for the bioprocess such as tolerance to low water activities and production of enzymes by the hyphae [17]. The use of agro-industrial residues as the substrate could result in a reduction in the costs of lipase production, considering that the culture medium usually represents 25%-50% of the total production costs [18]. The production of lipase is mostly inducer dependent; in many cases, oils act as good inducers of the enzyme. The requirement of sugar as a carbon source in addition to lipids varies with the microorganism. In general, media supplemented with glucose along with triglycerides stimulate maximum lipase production in the case of fungi [9]. As an example, the lipase production by Yarrowia lipolytica resulted in activity of 69 U/g when using ground peanut as substrate [19]. The profiles of lipase production (in terms of lipase activity) for all experiments in the study are shown in Fig. 1. It can be seen that the lipase activity increased throughout the fermentation process, except for the experiments E1, E4 and E5 (Fig. 1a) that from the 48 h remained constant. All experiments of Fig. 1b show increased lipase activity over the 60 h of fermentation. The enzyme productivity was also determined in 60 h of fermentation being achieved 0.99 U/g h (central point). The value is higher that obtained by Kempka et al. [11], 0.74 U/g h in 72 h of fermentation using Penicillium verrucosum, by Gutarra et al. [20], 0.26 U/g h in 72 h of fermentation and by Mahadik et al. [21], 0.87 U/g in 144 h of fermentation using Aspergillus niger and wheat bran/sunflower oil as substrate. The statistical analysis of the results allowed fitting a coded empirical model relating lipase activity (equation) to temperature and inductor. The ANOVA analyses for lipase activity showed high correlation coefficient and a good performance of the F-test for regressions. Therefore, the equation is predictive of lipase

Table 2 Matrix of the experimental design (real values) with responses in terms of lipase activity for lipase production using cassava peel and sunflower oil. Temperature Inductor Lipase activity (U/g) (C) (%, w/w) standard deviation E1 25 1.0 18.87b 2.88 E2 25 4.0 28.49ab 0.84 E3 36 1.0 43.80de 0.97 E4 36 4.0 26.37ab 1.59 E5 22.7 2.5 31.18ac 1.61 E6 38.2 2.5 42.33cde 5.52 E7 30.5 0.4 48.33ef 2.36 E8 30.5 4.6 34.69acd 3.92 E9 (C) 30.5 2.5 58.87fg 4.21 E10 (C) 30.5 2.5 60.83g 1.18 Mean lipase activity followed by different letters differ significantly with 95% confidence (p < 0.05) by Tukey test. Experiment

that the result of the experiment E10 differs significantly (p < 0.05) from all the other experiments, and the experiment E9 differs from the others (p < 0.05) except for the experiment E7. In the experiments E7, E9 and E10, the same process was used (temperature being 30.5 C), differing in relation to the percentage of the inductor. Colla et al. [17] who studied the lipase production using soybean and rice husk obtained 22.5 U/g of lipase activity. Contesini et al. [18] obtained 33.03 U/g of lipase activity using wheat bran as substrate. In the two studies cited, the micro-organisms used were Aspergillus niger. The highest lipolytic activities obtained in the SSF

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Production of Lipase Using Cassava Peel and Sunflower Oil in Solid-State Fermentation: Preliminary Study

(a) (b) Fig. 1 Profiles lipase activity over 60 hours of fermentation using A. niger and cassava peel supplemented with sunflower oil (inductor).

production in the investigated range of factors.

LA -487.48 31.21T 49.08I 0.46T 2 5.27 I 2 0.82TI

where, LA denotes lipase activity (U/g dry substrate), T is the temperature, and I the perceptual inductor content. The graph of predicted and observed values for the empirical model shown in Fig. 2, shows a good correlation between theoretical values and those obtained in practice (R = 0.92). Biological processes are highly complex and the enzyme production depends on the interaction of several processes influencing microbial cellular metabolism [13]. Fig. 3 shows a Pareto Chart obtained from experimental results of the lipase activity. It is observed that both the temperature and the inductor have significant effect on lipase production. Separately, the temperature influenced significantly positive and the percentage of inductor has significant negative effect, which means the increase of temperature and decrease in the percentage of inductor lead to increased lipase activity. However, from the interaction between the variables, it can be seen that the significant effect was negative and quadratic terms also have significant negative effects. The 3D response surface plot is the graphical representations of the regression equation used to investigate the interaction among variables and to determine the optimum concentration of each factor

Fig. 2 Correlation between observed and predicted values by the mathematical model.

Fig. 3 Pareto chart obtained from experimental results of the lipase activity.

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for maximum lipase [13]. The 3D plots shown in Fig. 4 were based on the function of two variables at its optimum level. This surface indicates that the conditions that maximize lipase activity production are 30.5 C and inductor of 2.5% (w/w). Through this analysis it is evident that extremes in temperature and inductor tend to decrease lipase production, since low temperatures decrease metabolism and high temperatures may inactivate the lipase. Fig. 5 shows the lipase production for optimum cultivation conditions. Lipase production presents a peak of 61.3 U/g at 72 h of fermentation. However, this value (61.3 U/g) does not differ statistically (p > 0.05) by Tukey test with the values of lipase activity obtained for 60 h of fermentation and 84 h of fermentation. It can be concluded that the time of 60 h is sufficient to maximize the production of lipase by A. niger using cassava peel/sunflower oil (2.5% w/w) as a substrate and temperature of 30.5 C. In the study of Contesini et al. [18], all the experiments were carried out for 72 , 96 and 120 h. Maximum activity was reached after 96 h for all assays, and after this, a decrease occurred in activity. When Kaushik et al. [22] studied about lipase production using Aspergillus carneus and the methodology response surface, they obtained the concentration of sunflower oil 1%, temperature 37 C and incubation time of 96 h, in addition to other optimal conditions.
(b) Fig. 4 Response surface for lipase activity as a function of inductor and temperature.

(a)

4. Conclusions
The fungus Aspergillus niger showed a good performance in the enzyme production. Lipase activity values of 59.8 U/g at 60 h of fermentation and 61.3 U/g at 72 h of fermentation in the optimized operational conditions (T = 30.5 C, inductor = 2.5%) were obtained. The response surface methodology is an efficient methodology to identify the significant variables and to optimize factors using a minimum number of experiments for lipase production. More studies are needed to complement the present study.

Fig. 5 time.

Lipase production under optimal conditions along

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