Application of Response Surface Methodology for Optimization of Mushroom Sacchari cation using Crude Enzymatic Extract from Solid-State Fermentation of Pineapple Peels by Aspergillus niger KWM


 Mushrooms are a rich source of high value compounds. Efficient enzymatic degradation of mushroom cell-wall matrix is therefore critical in the recovery of cellular components in high yields. In the present study, the effect of reaction variables on mushroom saccharification using crude enzymatic extract was evaluated and optimized using the central composite design based on the response surface methodology. The crude extract displayed CMCase, Fpase and xylanase activities of 1.23Umg− 1 protein, 0.95Umg− 1 protein and 1.52 Umg− 1 protein. The model was validated by the analysis of variance with a coefficient of determination of 0.866 and F test value of 7.39, making the model valid at the 95 % confidence limit. The model achieved glucose yield of 1.490mg/mL at pH 6.5, temperature 50oC, enzyme load of 5% (v/v) and reaction time of 12h. The experiment using optimal model conditions yielded 1.582 mg/mL glucose which is 1.1 folds higher than the predicted model value. This study demonstrated potential of crude extract from solid-state fermentation of low-cost pineapple peels in mushroom processing.


Introduction
Edible mushrooms are nutritious food items rich in health promoting compounds. Traditionally, mushrooms were not only eaten for their nutritional value but also for treatment of various illnesses [1,2].
The choice of extraction techniques is important in maintaining quality of biologically active compounds from mushrooms for speci c applications in food, pharmaceutical and cosmetic industries. The conventional extraction techniques have involved the use of toxic and highly in ammable solvents which have adverse effects in the environment and the quality of the nal products [9]. Enzyme-based extraction technique is considered therefore, a viable alternative to the solvent-based extraction methods [10]. The use of enzymes in industrial processing does not cause adverse effect on the environment and the quality of the nal product [11]. However, despite many advantages, low product recovery, long extraction period and high cost of commercial enzymes are some of the major bottlenecks associated with enzymebased processing [12].
In an attempt to improve the commercial viability of industrial processing using enzymes, the use of low cost crude enzymatic extracts from the microbial fermentation of agro-waste materials is increasingly being adopted [13]. This is because crude extracts from the fermented lignocellulosic biomass contain enzyme mixtures capable of hydrolyzing many biomaterials [14]. In fact, in certain instances crude enzymatic extracts have proved to be more e cient compared to the commercial enzymes under optimized reaction conditions [15,16]. The traditional one-variable-at-a-time method has widely been used in many optimization processes; however, it certain limitations including need for a number of experiments to be performed and is also time consuming [17]. A response surface methodology which is a mathematical and statistical tool has been developed and is currently used in the modeling and optimizing reaction parameters to achieve maximum response [18][19][20]. In the present study, a central composite design based on the response surface methodology was used to evaluate and optimize effect of reaction parameters (medium pH, reaction time, reaction temperature and enzyme loading) on the glucose yield during sacchari cation of mushroom biomass using crude enzymatic extract.

Materials And Method
Microorganism and culture condition A stock culture of Aspergillus niger KWM, was propagated on potato dextrose agar at 30 °C for 5 days. The conidia from the sporulating plate cultures were suspended in 5 mL of sterile water and 1 mL (~1.0x10 7 spores/mL) of the spore suspension was transferred to a 250-mL Erlenmeyer ask containing 50 mL of the basal medium. The medium was composed of: The medium culture was incubated at 30 °C at an agitation speed of 120rpm in an orbital shaker (Gerhardt, GmbH, Germany) for 5 days.

Solid-state fermentation of pineapple peels
The pineapple (Ananas cosmosus, Linn.) peels were obtained from the local fruit vendor and dried in an electric oven to ~5% (w/w) moisture content. The dried peels were then milled using a laboratory blender (Sumeet Inc., Mumbai, India) into ne powder (mesh size~32). The dried powder (10g) was put into 250mL Erlenmeyer ask and moistened with basal salt solution and then was autoclaved at 121 o C for 30 min. The substrate was inoculated with 1 mL of spore suspension, and the culture medium was then incubated at 30 o C for 7 days. The substrate bed was suspended in 50mM phosphate buffer, pH 6.8 and agitated at 200rpm in an orbital shaker (Gerhardt, GmbH, Germany) for 1h. The mixture was centrifuged at 5000rpm for 10 min at 4•C. The total protein content of the supernatant was estimated by the method of Lowry et al. [21] using BSA as a standard.

Enzyme assays
The CMCase activity of the crude extract was determined by mixing 490µL of carboxymethyl cellulose (CMC) solution (1%, w/v CMC prepared in 50mM phosphate buffer, pH 6.0) and 10µL of crude enzymatic extract. Similarly, xylanase activity was determined by mixing solutions of Beechwood xylan prepared in citrate buffer. Filter paperase (Fpase) activity was determined by adding small pieces (~50mg) of Whatman lter paper no.1 to appropriately diluted crude extract. The mixtures were incubated at 50 o C for 60min. Glucose content of the CMC and lter paper degradation were determined using DNS method [22].
Similarly, the xylose content of the Beechwood xylan degradation was determined. The standard graphs were prepared using 0-500 μg of glucose for CMC and lter paper degradation. The standard graph of 0-500 μg xylose was used for xylan degradation. One unit of enzyme activity is de ned as the amount of enzyme that catalyzes the release of 1 µmol of reducing sugar equivalent per minute under the speci ed assay conditions.

Modeling reaction parameters for enzymatic sacchari cation
The Minitab software (Minitab, Inc., Penn, USA) was used to build and analyze the experimental design. The central composite design was used for evaluating the effect of reaction variables on mushroom sacchari cation. The response surface methodology was used for optimizing reaction variables to achieve maximum response (glucose yield).

Mushroom hydrolysis using crude enzymatic extract
Preliminary research was undertaken to identify key reaction variables that affect the enzymatic hydrolysis of lignocellulosic biomass including mushrooms [23]. In order to initiate enzymatic hydrolysis, 200 mg of powdered Pleurotus ostreatus was incubated in crude extract (prepared in 50mM phosphate buffer, pH 4.5-6.5), at temperatures 30-50 o C for up to 60h, as per the experimental design. Aliquot (1mL) was removed at predetermined time intervals and immediately 3mL of DNS reagent was added and heated at 80 o C for 5 min. The mixture was then centrifuged at 5000×g for 10 min. Time zero samples were collected before the addition of the crude extract and used as blank. The glucose concentration was determined using spectrophotometer at 540nm.

Statistical analysis
The F test was used to interpret the coefficients and the significance of each model term was determined by the analysis of variance (ANOVA). The student's t-test was performed to determine the signi cance of the regression coe cients. The contour plot and regression analysis were performed to establish the optimal conditions for mushroom sacchari cation using crude enzymatic extract for maximum glucose yield.

Results And Discussion
Enzyme activities of the crude extract The solid-state fermentation of pineapple peels using A.niger KWM yielded crude extract with protein content of 145mg mL -1 . This is indicated that the fungus released extra cellular enzymes onto the growth medium during fermentation. The choice of growth substrate is critical as it influences specific enzyme production during microbial fermentation of lignocellulosic biomass [24]. The pineapple peels used as fermentation medium influenced the enzymes mixtures capable of hydrolyzing pineapple peel substrate.
The crude extract displayed enzyme activities of 175UmL -1 , 138UmL -1 and 221UmL -1 for CMCase, Fpase and xylanase respectively. Most filamentous fungi have been reported to produce cellulase and xylanase enzymes with either low or high activities [25][26][27]; however, this study showed that solid-state fermentation of pineapple peels using A.niger KWM can potentially produce enzyme cocktail with relatively close cellulase and xylanase activities which is important in many industrial bioprocesses.
Optimizing mushroom saccharification parameters for higher glucose yield In the present study, the pH range of 4.5-6.5, reaction time ranging from 12-60h, temperature range of 30-50 o C and enzyme loading of 1-5% (v/v) were chosen as key independent variables that affect enzymatic saccharification of mushroom biomass. Table 1 illustrates the coded and real values for the experimental variables. Table 1 Coded and real values of reaction variables for central composite design A second-order polynomial model that describes the effect of variables on glucose yield from the enzymatic saccharification of mushroom is presented in Eq 1.
The CCD and their coded, experimental and predicted values are shown in Table 2 Experiments done under these conditions yielded slightly higher glucose (0.945mg/mL). The reaction at pH 5.5, temperature 40 o C and enzyme loading of 5% (v/v) for a period of 36h yielded 1.046mg/mL (run 24); this was 2.8 folds higher than the glucose yield predicted by the model. Table 2 Matrix of the CCD for the evaluation of the effect of independent variables on the glucose yield during mushroom saccharification The model expressed by equation (3) represents glucose yield (Y) as a function of pH (X 1 ), time (X 2 ), temperature (X 3 ), and enzyme loading (X 4 ).
The t and p-values for linear, quadratic and combined effects of the variables are given in Table 4     1.582mg/mL which is 1.1 folds higher than the predicted value. This confirms the validity of the model as a viable tool that may be used for modeling and optimizing reaction parameters for mushroom hydrolysis using crude enzymatic extract. The glucose yield is likely to even increase with mushroom pretreatment and optimization of other reaction parameters such as agitation speed during enzymatic processing [32,33]. Commercial enzymes have been used in mushroom processing to recover high value products. Ang and Ismail-Fitry [39] and Banjongsinsiri et al. [40] used commercial bromelain and papain enzymes respectively to enhance recovery of protein from mushroom. Similarly, Poojary et al [41] digested mushroom biomass with commercial enzymes to recover amino acids responsible for umami taste. The processing of mushrooms Page 10/19 using commercial enzymes of some mushroom species such as Shiitake have been patented [42]. However, for a long time, the cost of commercial enzymes has remained a major bottleneck in industrial bioprocessing. The application of crude enzymatic extracts in bioconversion processes has been therefore driven majorly by the need to make enzymebased processing more cost-effective and competitive. Mahamud & Gomes [43] applied crude enzymatic extract for saccharification of sugarcane bagasse in bioethanol production; crude extracts showed higher saccharification efficiency compared to their commercial counterparts. The use of crude enzymatic extract has also been extended in many industrial bioprocesses including fruit processing industries for fruit juice clarification [44,45]. Despite increasing research outputs in the applications of crude enzymatic extracts in industrial bioprocessing, the information on mushroom processing using crude enzymes is quite limited or probably not available.

Conclusions
The solid-state fermentation of pineapple peels using A.niger KWM produced enzyme mixtures capable of hydrolyzing mushroom cell-wall matrix. Besides, the solid-state fermentation of low-cost pineapple peels is potentially cost-effective strategy of obtaining enzyme for mushroom processing at an industrial scale; an approach that offers the promise of signi cant advances that can remarkably make mushroom processing using enzymes more competitive. The response surface methodology proved to be a reliable tool for predicting process performance of mushroom sacchari cation using crude enzymatic extracts.

Declarations
Funding This study did not have any grant funding Data Availability All data are fully available without restriction.

Compliance with ethical standards
Competing interests The authors declare that they have no competing interests.
Ethics Approval and Consent to Participate This article does not contain any studies with human participants or animals performed by any of the authors.
Consent for Publication This study does not contain any individual person's data.  Table 2 Std Order X 1 X 2 X 3 X 4 Glucose yield(mg/mL) Experimental Predicted      Optimization plot for enzymatic sacchari cation of mushroom