Comparison of Lipase Production by Enterococcus faecium MTCC 5695 and Pediococcus acidilactici MTCC 11361 Using Fish Waste as Substrate: Optimization of Culture Conditions by Response Surface Methodology

A medium using fish waste as substrate was designed for production of lipase by Enterococcus faecium MTCC 5695 and Pediococcus acidilactici MTCC 11361. Medium components and culture conditions (fish waste protein hydrolysate (FWPH) concentration, fish waste oil (FWO) concentration, pH, temperature, and fermentation time) which affect lipase production were screened using factorial (5 factors ∗ 2 levels) design of which FWPH concentration, FWO concentration, and fermentation time showed significance (P < 0.05). The levels of these factors were optimized further by Box-Behnken design using response surface methodology (RSM). Optimized conditions were found to be 5% v/v FWO, 0.15 mg/mL FWPH and 24 h of fermentation time for MTCC 5695, and 4% v/v FWO, 0.15 mg/mL FWPH and 24 h of fermentation for MTCC 11361, which were further validated. Under optimized conditions, MTCC 5695 and MTCC 11361 showed 3.15- (543.63 to 1715 U/mL) and 2.3- (214.74 to 493 U/mL) fold increase in lipase production, respectively, as compared to unoptimized conditions.


Introduction
Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are a class of serine hydrolases which catalyze the hydrolysis of triglycerides to glycerol and free fatty acids over oil-water interface [1]. In addition, lipases catalyze the hydrolysis and transesteri�cation of other esters as well as the synthesis of esters and exhibit enantioselective properties [2]. ese unique properties of lipases make them a very important enzyme of industrial signi�cance. Lipases are used in chemical processing, dairy industries for improvement of �avour, paper industries, oleochemical industries, pharmaceuticals, synthesis of surfactants, detergent industries, leather industries, and polymer synthesis [3,4].
Lipases are produced by plants, animals, and microbes but only microbial lipases are found to be industrially important since they are diversi�ed in their enzymatic properties and substrate speci�city [5]. Bacterial lipases that are of commercial importance are obtained from Achromobacter, Alcaligenes, Arthrobacter, Bacillus, Burkholderia, Chromobacterium, and Pseudomonas [6,7].
Lactic Acid Bacteria (LAB) are generally considered to be weakly lipolytic, as compared to other groups of microorganisms. e lipolytic activity by LAB plays an important role in the determination of special aroma of different cheeses [8,9]. Since they are considered as generally recognised as safe (GRAS), they are used extensively as starter cultures in food and feed industries [10]. Although there are reports on lactic acid bacterial lipase production [11][12][13], they are fewer in comparison to other microorganisms like Bacillus.
Most research is now focused on the use of waste residues generated by industries as inexpensive substrates for microbial growth and metabolite production. Fish processing industries generate around 63.6 million metric tons (MMT) in which 2.8 MMT of waste are generated in India alone [14]. ese wastes are a rich source of biomolecules such as lipids, proteins, chitin, collagen, minerals, and vitamins that can be recovered and utilized [15]. e lipids and proteins are extracted from the �sh wastes either by addition of enzymes or by fermentation with LAB [16]. Lactic acid bacterial fermentation has been used for recovery of oil from �sh viscera and also for extraction of proteins from shrimp waste and leather industry waste [15,17,18]. ese lipidic carbon and nitrogen rich sources can be used as ample substrates for lipase production by LAB. However, these carbon and nitrogen supplements used must be optimized for maximal lipase production.
e most challenging task in optimization is the presence of interactive effects of media components and culture conditions. Response Surface Methodology (RSM) is a collection of statistical and mathematical techniques useful for developing, improving, and optimizing processes in which a response of interest is in�uenced by several variables and the objective is to optimize this response [19]. It de�nes the effect of independent variables, alone or in combination, on the processes and generates a mathematical model that describes the process [20].
In the present study, �sh waste was used to design a medium for lipase production by Enterococcus faecium MTCC 5695 and Pediococcus acidilactici MTCC 11361. e signi�cant parameters (media components and cultural conditions) on lipase production were identi�ed using a factorial design and optimized using a Box-Behnken design. To the very best of our knowledge, there are no reports on the optimization of lipase production by LAB from �sh waste by RSM.

Substrates and Chemicals.
Fresh water �sh visceral waste devoid of air bladder was collected from local �sh markets in Mysore, India. Enterococcus faecium NCIM5335 (EF-35) used for extraction of �sh oil was obtained from institute collection centre. All microbiological media were procured from Hi-Media (M/s Hi-Media, Mumbai, India). Para-nitrophenyl acetate (p-NPA) and p-nitrophenol were obtained from SRL (SRL chemicals, Bangalore, India). All other chemicals, solvents, and reagents used in the study were of analytical grade, unless otherwise mentioned.

Extraction of FWO and FWPH from Fish
Waste. Extraction of FWO was done as per the procedure detailed in Rai et al. [15] with slight modi�cations. Fresh water �sh visceral waste devoid of air bladder was subjected to homogenization in a Waring blender (Stephen Mill, UM5 Universal, Hong �ong). e uniformly homogenized �sh viscera was steam cooked at 85 ∘ C for 10 minutes to inactivate the inherent enzymes and micro�ora, followed by fermentation for 72 hours using EF-35. e fermented mass was then centrifuged at 6000 rpm for 20 min. FWO separated out into the top layer followed by protein rich residue layer. e protein hydrolysate was extracted from the protein rich residue layer as per Bhaskar et al. [21] with few modi-�cations. e protein residue layer was extracted thrice with distilled water in the ratio 1 : 1 w/v. Protein extract obtained on centrifugation was subjected to lyophilisation to give FWPH, which was then dissolved in distilled water. e protein concentration was measured using Biuret's method [22]. Table 1.

Screening of Signi�cant Parameters ��ecting �ipase
Production by Factorial Design. e effect of pH (X1), temperature (X2, ∘ C), time (X3, h), FWPH concentration (X4, mg/mL), and FWO concentration (X5, %v/v) on lipase production by MTCC 5695 and MTCC 11361 was studied by a (5 factors * 2 levels) factorial design encompassing 32 runs (Table 2). Lipase activity ( was determined as the response (dependent variable) and speci�cally designated as and  Tables 2 and 4 in 250 mL Erlenmeyer �asks containing 100 mL media. e experiments were performed in triplicates. e pH and temperature were maintained at 6.0 and 43 ∘ C (central values generated through factorial design), respectively. As per the time intervals indicated in Tables 2 and 4, sample aliquots were collected and centrifuged at 10,000 rpm for 10 min. Cell pellet was collected and sonicated in phosphate buffer (pH 7.0) for complete lysis. e lysed cells were centrifuged and lipase assay was performed for the cell free extract.
Lipase activity was determined spectrophotometrically using p-NPA as the substrate as described by Wang et al. [23] with slight modi�cations. 300 L of cell supernatant and 900 L of acetonitrile : ethanol : phosphate buffer (pH 6.8) in ratio of 1 : 4 : 95 was mixed with 800 L of p-NPA (100 mM) in acetonitrile. is mixture was then incubated at 37 ∘ C for 15 minutes. e liberated p-nitrophenol was estimated at 408 nm. One unit of lipase activity is de�ned as the amount of enzyme required to liberate one μmol of p-nitrophenol per minute under the standard assay conditions. 2.6. Statistical Analysis. e screening and optimization experiments were designed by STATISTICA soware [24]. e data generated from the experiments were analyzed to obtain the optimized conditions by the same.

Selection of Substrate for Efficient Lipase Production by MTCC 5695 and MTCC 11361.
Fish waste contains a rich source of both lipids and proteins and thereby can be applied as an efficient substrate for microbial growth and production of various metabolites [16,25]. Henceforth, this study aims at the use of �sh waste as an effective alternative for the carbon and nitrogen sources present in media currently used for cultivation of LAB. In this study, the carbon and nitrogen sources in the commercial MRS medium were replaced with FWO and FWPH, respectively, as indicated in Table 1. FWO and FWPH helped in enhanced lipase production by both the organisms thereby acting as a replacement for carbon and nitrogen sources, respectively. FWO consists mainly of triacylglycerols that comprises a variety of fatty acids that act as a remarkable lipidic carbon source for induction of lipase production [15]. On the other hand, FWPH serves as a rich source of proteins aiding in the luxurious growth of organisms and metabolite production. Moreover, most of the protein supplements used for the cultivation of LAB are of bovine origin which makes it unsuitable for use in food industries [26,27].

Screening of Signi�cant �ndependent
e optimum levels of the signi�cant independent parameters were determined further by a Box-Behnken design and the insigni�cant independent parameters, that is, pH (X1) and temperature (X2) were maintained at the centre of their levels. . Similar studies stating the signi�cant in�uence of sun�ower oil and palm oil as inducible carbon sources on lipase production have been reported [28,29]. e response surface graph for 1 and 2 as a function of FWPH concentration and FWO concentration is presented in Figures 1(a) and 1(c), respectively. It was observed that lipase production increased with increase in FWPH concentration up to 0.16 mg/mL beyond which there was a decrease in case of both the organisms probably due to inhibition of enzyme activity in the presence of excess nitrogen. A possible mechanism may be that FWPH is a complex nitrogen source and the cells may produce more protease for the degradation of FWPH before its utilization. is might result in lower production and higher degradation of the lipase [30]. e lipase production increased with increase in FWO concentration for MTCC 5695, whereas lipase production increased with increase in FWO concentration up to 4% v/v T 5: (a) ANOVA beyond which there was a decrease for MTCC 11361. e decrease in lipase production by MTCC 11361 beyond 4% v/v of FWO concentration may be due to the reason that high concentrations of FWO have more incidence of long chain fatty acids which might have an inhibitory effect on lipase production [5]. However, MTCC 5695 was found to be more tolerant to FWO. e in�uence of time and FWO concentration on 1 and 2 is presented in Figures 1(b) and 1(d), respectively. e �gure clearly indicates that lipase production decreases with increase in time for MTCC 5695 however, a slight increase was observed aer 48 h for MTCC 11361. is may be probably due to the fact that MTCC 5695 and MTCC 11361 achieve maximum growth in 24 h aer which they enter the stationary phase resulting in a steady decline in lipase production. e optimized levels of variables (X1, X2, and X3) were determined using desirability pro�les for 1 and 2 (Figures  2(a) and 2(b)). e optimized factors for obtaining the highest 1 were 5% v/v FWO concentration, 0.15 mg/mL FWPH concentration at 24 h of fermentation whereas for 2 , 4% v/v FWO concentration, 0.15 mg/mL FWPH concentration at 24 h of fermentation were found to be optimum. e response variables 1 and 2 were assigned a desirability of 1.0 for the highest observed value ( 1 -1707 U/mL and 2 -487.22 U/mL) and a desirability of 0 for the lowest observed value ( 1 -437 U/mL and 2 -10.48 U/mL) to get the overall desirability. e desirability function to get the optimum lipase production was �tted by the least square method. e level of variable giving the highest desirability (1.0) was selected as the optimum level.

Optimization of Parameters for Lipase Production by
e regression equation for lipase activity of MTCC 5695 and MTCC 11361, as a function of the three independent variables (X1, X2, and X3) and their linear and quadratic interactions, is represented by the following: Coefficient of determination ( 2 ) is a measure of the strength of the linear relationship between the experimental and predicted values. 2 for the correlation between the observed and predicted lipase activities of MTCC 5695 and MTCC 11361 was 0.9808 and 0.94246, respectively. e model was validated by conducting experiments at 5% v/v FWO concentration, 0.15 mg/mL FWPH concentration at 24 h of fermentation for MTCC 5695 and 4% v/v FWO concentration, 0.15 mg/mL FWPH concentration at 24 h of fermentation for MTCC 11361. e experimental values of 1 (1715 U/mL) and 2 (493 U/mL) at these optimum conditions were quite close to the predicted values (1645.75 U/mL and 481.662 U/mL, resp.) which indicated that the model was highly signi�cant. A fold increase of 3.15 and 2.3 was obtained, respectively, in lipase production for MTCC 5695 and MTCC 11361 by optimization using RSM (i.e. lipase activity of 543.63 U/mL and 214.74 U/mL under unoptimized conditions, resp.). is fold increase is more than the fold increase obtained by Sharma et al. [31] wherein a 1.6-fold increase in lipase production was observed in Arthrobacter sp. BGCC#490 and Kumari et al. [32] obtained 1.4-fold in lipase production in Enterobacter aerogenes under optimized conditions. Liu et al. [1] reported a 5-fold increase in lipase production by Burkholderia sp.

Conclusion
Enterococcus faecium MTCC 5695 and Pediococcus acidilactici MTCC 11361 were found to be potential lipaseproducing strains using �sh waste substrates. RSM was found to be an efficient methodology for rapid optimization of in�uencing parameters and development of polynomial equation for lipase production. e signi�cance of this work is that it includes the use of an economical substrate for lipase production, which in turn diminishes the problem of waste disposal from �sh processing industries. Moreover, the optimized conditions obtained from this study can be used for large-scale cost-effective production of lipase from LAB.