Assessment of pectinase production by Bacillus mojavensis I4 using an economical substrate and its potential application in oil sesame extractionby Imen Ghazala, Nadhem Sayari, Molka Ben Romdhane, Semia Ellouz-Chaabouni, Anissa Haddar

J Food Sci Technol


Food Science



Assessment of pectinase production by Bacillus mojavensis

I4 using an economical substrate and its potential application in oil sesame extraction

Imen Ghazala1 & Nadhem Sayari1 & Molka Ben Romdhane1 &

Semia Ellouz-Chaabouni1,2 & Anissa Haddar1

Revised: 7 July 2015 /Accepted: 14 July 2015 # Association of Food Scientists & Technologists (India) 2015

Abstract Carrot (Daucus carota) peels, local agricultural waste product, is rich in lignocellulolytic material, including pectin which can act as an inducer of pectinase production.

Pectinolytic enzymes production by Bacillus mojavensis I4 was studied in liquid state fermentation using carrot peel as a substrate. Medium composition and culture conditions for the pectinase production by I4 were optimized using two statistical methods: Taguchi design was applied to find the key ingredients and conditions for the best yield of enzyme production and The Box-Behnken design was used to optimize the value of the four significant variables: carrot peels powder,

NH4Cl, inoculum size and incubation time. The optimal conditions for higher production of pectinase were carrot peels powder 6.5 %, NH4Cl 0.3 %, inoculum level 3 % and cultivation time 32 h. Under these conditions, the pectinase experimental yield (64.8 U/ml) closely matched the yield predicted by the statistical model (63.55 U/ml) with R2=0.963. The best pectinase activity was observed at the temperature of 60 °C and at pH 8.0. The enzyme retained more than 90 % of its activity after 24 h at pH ranging from 6.0 to 10.0. The enzyme preserved more than 85 % of its initial activity after 60 min of pre-incubation at 30–40 °C and more than 67 % at 50 °C. The extracellular juice of I4 was applied in the process of sesame seeds oil extraction. An improvement of 3 % on the oil yield was obtained. The findings demonstrated that the

B. mojavensis I4 has a promising potential for future use in a wide range of industrial and biotechnological applications.

Keywords Bacillus mojavensis I4 . Alkaline thermostable pectinase . Carrot peels powder . Culture medium optimization . Oil extraction


Pectinolytic enzymes that hydrolyze pectins have important applications in various industries. Based on pH requirement for optimum enzymatic activity, pectinases can be broadly classified into acidic and alkaline pectinases. Acidic pectinases are useful for the extraction, clarification, and liquefaction of fruit juices (Kaur et al. 2004) and wines (FavelaTorres et al. 2005). Alkaline pectinases are widely used the fabric industry, for the retting of plant fibers, biopreparation of cotton, and enzymatic polishing of jute/cotton blended fabrics, and in the pulp and paper industry, for solving problems pertaining to waste treatment, retention, and recovery processes (Favela-Torres et al. 2005; Sharma and Satyanarayana 2004). Bioprep 3000L (Novozymes), an alkaline pectinase with a standard activity of at least 3000 APSU (Alkaline

Pectinase Standard Unit), currently represents one of the most commonly commercialized pectinases.

The statistical design experiments have often been applied for the optimization and development of technically and economically viable bioprocesses. In this context, the wide scale industrial application of pectinases requires their costeffective production to make the process economically viable.

This can be achieved through the use of cheaply available agroindustrial residues, including wheat bran, oat bran, rice straw, and orange peels (Heerd et al. 2012; Kapoor et al. 2008; Sun et al. 2008). Large amounts of lignocellulosic * Anissa Haddar 1 Unité Enzymes et Bioconversion - Ecole Nationale d’Ingénieurs de

Sfax, B.P. 1173, 3038 Sfax, Tunisia 2 Unité de Service Commun Bioréacteur Couplé à L’Ultrafiltration Ecole Nationale d’Ingénieurs de Sfax, B.P. 1173, 3038 Sfax, Tunisia

J Food Sci Technol

DOI 10.1007/s13197-015-1964-3 wastes are annually generated from agro-industrial processing operations (Anuradha et al. 2007). The by-products from the fruit and vegetable industries may offer inexpensive and readily available resources from which several useful biological products can be derived (Chantaro et al. 2008). Carrot (Daucus carota) is a good source of natural antioxidants, especially carotenoids and phenolic compounds (Prakash et al. 2004). After processing, carrot residues, such as peels and pomace, are usually discarded or used as animal feed. Carrot by-products have, however high contents of beneficial substances, especially bioactive compounds with antioxidant activities (Zhang and Hamauzu 2004).

The present study aimed to investigate the feasibility of using carrot peels, a by-product from a ready-to-eat vegetable industry, as a carbon source to produce high-level of pectinase under submerged fermentation from a newly isolated strain of

Bacillus majavensis I4. To the authors’ knowledge, no data is currently available in the literature on the use of carrot peels powder in the fermentation medium for the production of pectinase. The Taguchi approach and Box-Behnken design were employed to identify and optimize the critical variables for enzyme production. The biochemical properties of the extracellular pectinases were determined, and their potential gain effects on sesame seeds oil extraction were also evaluated.

Materials and methods

Bacterial strain

The bacterial strain was isolated from a soil sample collected from Sfax city, Tunisia, and maintained on nutrient agar slants at 4 °C and also stored as glycerol stocks at −20 °C. The isolate was identified as B. mojavensis according to the methods described in Bergey’s Manual of determinative

Bacteriology and on the basis of the 16S rDNA sequence analysis. A phylogenetic tree was constructed and edited using

Molecular Evolutionary Genetics Analysis version 5. Genetic relationships were inferred from neighbor-joining nucleotide alignment after 1000 bootstrap replicates using the Tajima-Nei model.