Article pubs.acs.org/JAFC
Tamarind Kernel Powder: A Novel Agro-residue for the Production of Cellobiose Dehydrogenase under Submerged Fermentation by Termitomyces clypeatus Tanima Saha,*,†,∥ Soumya Sasmal,‡,∥ Shariful Alam,§ and Nirmalendu Das# †
Department of Molecular Biology and Biotechnology, University of Kalyani, Distt: Nadia, West Bengal 741 235, India Center of Innovative and Applied Bioprocessing, Mohali 160 071, India § Department of Mathematics, Bengal Engineering and Science University, Shibpur, Howrah 711 103, India # Department of Botany, Barasat Government College, Barasat, N-24 Pgs, Kolkata 700 124, India ‡
ABSTRACT: The study investigates the potential of substitution of the conventional carbohydrate nutrient (cellulose) in media with cheap agro-residues for cellobiose dehydrogenase production by Termitomyces clypeatus (CDHtc) under submerged conditions. Different agro-residues tested for enzyme production were characterized using FTIR and XRD analysis. As CDHtc production was highest with tamarind kernel powder (TKP), it was selected for process optimizations through shake-flask fermentations. The optimized parameters were then applied to batch cultures in a 5 L bioreactor that gave enzyme yield (57.4 U mL−1) similar to that obtained under shake-flask fermentations (57.05 U mL−1). The study also made an attempt to predict CDHtc production with respect to time of fermentation and mycelial growth. The specific growth rate and carrying capacity of the mycelia were also determined, and the values lie in the ranges of 0.024−0.027 h−1 and 7.2−7.1 mg mL−1, respectively. KEYWORDS: tamarind kernel powder, cellobiose dehydrogenase, Termitomyces clypeatus, agro-residue, fermentation, FTIR, XRD
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studied for their efficacy as novel drug-delivery agents.10,11 This powder is widely used as a substitute for starch and galactomannans.12 The refined powder is widely used as a thickening and gel-stabilizing agent in the food industry.13 It is used in the jute, textile, and paper industries as a sizing material.14 TKP has also been used for ethanol production using the thermotolerant Ascomycetous fungi Debaromyces hansenii.7 TKP and its derivatives have been used for adsorption of metal ions, as ion exchangers, and in effluent treatment processes.15−20 It has also been exploited for the production of different enzymes, such as pectinase, using some species of Aspergillus,21,22 xylanase, and cellulase from Termitomyces clypeatus.23 During saccharification of cellulose by the cellulosedegrading enzyme system, endo-1,4-β-glucanase produces cellobiose as the end product, which inhibits further action of both endo- and exoglucanases. Cellobiose dehydrogenase (CDH) produced by wood rotting fungi increases the efficiency of cellulose hydrolysis by oxidizing cellobiose to cellobiono-1,5lactone.24 Moreover, it also reduces many electron acceptors such as quinones, phenoxy, and cation radicals, Fe(III), cytochrome c, and molecular oxygen.25 CDH has several applications, as biosensors, in bioremediations, or for biocatalysts.26 An important application of the enzyme deals with the use of CDH for the production of lactobionic acid.27,28 Lactobionic acid (β-D-galactopyranosyl-(1→4)-D-gluconic acid) was first described and synthesized by Fischer and Meyer29
INTRODUCTION An appreciable advancement in the application of agricultural residues has been observed over the past few decades depending on the source of biomass feedstock in each country. The utilization of agricultural wastes as growth substrates to produce specific products is certainly economical and reduces environmental pollution. The agricultural wastes are composed of lignocellulosic materials, which can be degraded by the lignolytic and cellulolytic enzymes produced by various microbial organisms, in turn leading to the formation of several value-added products.1−5 The present study used tamarind kernel powder (TKP) as the sole substrate for the production of cellobiose dehydrgenase. TKP is produced from the seeds of Tamarindus indica, an abundantly grown tree of Southeast Asia, India, Mexico, and Costa Rica.6 The fruits of T. indica are known as tamarinds, which are consumed in large quantities throughout the year. TKP is obtained from the leftover inconsumable portions of the seed. Tamarind production in India is about 250,000 t per annum and that of TKP is about 20,000 t per annum.7 TKP includes all of the constituents found in the tamarind seed kernel: polysaccharide (composed of uronic acid and the neutral sugars arabinose, xylose, mannose, glucose, and galactose), protein, and other cellular debris from the tamarind kernel seed.8 The reported fermentable sugars, namely, glucose, xylose, and galactose units, are present in the ratio of 2.8:2.25:1.0.6 TKP has several drawbacks, such as obnoxious odor, dull color, and low solubility in cold water (in the range of 5−35 °C). The application of TKP is extensive and variable. It acts as an enhancer for solubilization of drugs such as celecoxib, which are poorly soluble in water.9 TKP derivatives have also been © 2014 American Chemical Society
Received: Revised: Accepted: Published: 3438
May 23, 2013 March 27, 2014 March 28, 2014 March 28, 2014 dx.doi.org/10.1021/jf405278y | J. Agric. Food Chem. 2014, 62, 3438−3445
Journal of Agricultural and Food Chemistry
Article
autoclaving. Yeast extract (0.2%) and peptone (0.2%) were added in the medium as and where mentioned. The fungus was cultured for up to 17 days at 30 °C under constant shaking of 150 rpm. Aliquots of the culture filtrate were collected at 1 day intervals and clarified by centrifugation at 21000g for 30 min to determine the enzyme activities. The culture medium was adjusted to its original volume with fresh medium after each collection. Enzyme Production in 5 L Stirred-Tank Bioreactor. Largescale enzyme production was carried out in a 5 L benchtop stirred-tank bioreactor (BioFlo 3000, New Brunswick Scientific Co., Inc., New Brunswick, NJ, USA ). The fermenter was equipped with an automatic computerized control system (Bio Command Software, New Brunswick Scientific Co.) for temperature, aeration, agitation, and pH. The temperature was maintained at 30 °C. Air was supplied at a continuous rate of 1.0 vvm. Agitation was limited to 150 rpm. The initial pH of the medium was adjusted to 7.0 before sterilization, and no pH control was imposed during growth. Dow Corning silicon antifoam (0.2% v/v) was used to check the frothing during the fermentation. Samples were withdrawn after every 24 h during 12 days of fermentation and centrifuged at 21000g for 30 min. The supernatants thus obtained were used as enzyme solutions for the assays. Enzyme Activity Assay. CDHtc activity was measured by following the decrease in absorbance of DCIP for 5 min at 520 nm, pH 5.0, and 25 °C with a Shimadzu UV−vis spectrophotometer (model 1700).43 The reaction mixture (1 mL) contained 20 mM cellobiose, 0.3 mM DCIP, 100 mM sodium acetate buffer (pH 5.0), and enzyme solution. One unit (U) of enzyme activity was defined as the amount of enzyme that reduced 1 μmol of DCIP per minute under the experimental conditions. All results presented are the mean of five replicates. The error bars in the figures indicate standard deviations. Measurement of Mycelial Growth. Mycelial growth was measured by determining the glucosamine concentration after acid hydrolysis of mycelia following the method of Desgranges et al.44 Mycelia after growth were collected through centrifugation at 21000g for 30 min and washed repeatedly to remove the media remaining with the mycelial pellets. Subsequently, the samples were freeze-dried and treated with 10 mL of 10 M HCl for 16 h at 20 °C. Hydrolysis was completed by autoclaving the acid-hydrolyzed mycelia at 121 °C for 2 h after the total volume of the incubation mixture had been adjusted to 50 mL with the addition of distilled water. The concentration of glucosamine−HCl released after hydrolysis of the mycelia was measured colorimetrically according to the method of Nilsson and Bjurman.45 Measurement of Crystallinity. The crystallinity of the agroresidue samples was determined by using a wide-angle X-ray diffractometer. The samples were transferred to the glass sample holder and analyzed under plateau conditions. The radiation was generated at a voltage of 40 kV and a current of 40 mA. The scan scope was set between 7° and 40° with a step size of 0.02°. Holding time was 1 s. The crystallinity index along with the intensity of the main crystalline plane (002) and the amorphous fraction was calculated using the intensity at ∼18°of 2θ for amorphous fraction and maximum intensity of (002) plane at ∼22.5° of 2θ for crystalline fraction as described by Segal et al.46 and Sasmal et al.47
through oxidiation of lactose with bromine under mild conditions. Since then, many chemical, electrocatalytic,30−32 and microbial or enzymatic processes33,34 for the production of lactobionic acid have been investigated. The major application of lactobionic acid is its use as a constituent in solutions that stabilize organs before transplantation. This is based on the excellent metal-chelating properties of the acid which reduce the oxidative damages, caused by certain metal ions, to tissues during the storage and preservation of organs.35 Additionally, the acid is also used for the stabilization of interferon preparations or for the improvement of solubility of macrolide antibiotics such as erythromycin.36 Furthermore, lactobionic acid can have its utility as a biodegradable cobuilder in washing powder, which can contain up to 40% lactobionic acid.37 The fatty acid derivatives of lactobionic acid can be used as biosurfactants. Lactobionic acid could have several possible applications in food technology. These include improvement in the taste perception of sour, elimination of bitterness, flavor enhancement, preservation of aroma freshness, and reduction of souring and ripening time of cheese and yogurt products.38 Moreover, its mineral salt complexes are used for the fortification of functional drinks with essential minerals,39 and its addition to functional food might be useful for its presumed prebiotic effect.40 T. clypeatus produces large quantities of endo-1,4-βglucanase, β-glucosidase, and CDH in cellulose-containing medium.41,42 The fungus was reported to be the highest producer of CDH.42 In the present investigation, several agroindustrial byproducts were tested as substrates for CDH production by T. clypeatus (CDHtc) during shake-flask fermentations and the suitability of TKP was evaluated to stimulate CDH production in a 5 L stirred-tank fermenter.
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MATERIALS AND METHODS
Microorganism. The stock cultures of T. clypeatus were maintained in PDA slants (20% potato extract, 2% dextrose, and 1.5% agar, pH 7.0) at 4 °C. Mycelia were transferred to fresh PDA slants for growth at 30 °C for 5 days. These slants were then stored at 4 °C for use in inoculum preparation. Agro-residues. Corncob, coconut coir, rice husk, wheat bran, ground nut shell, sugar cane bagasse, sawdust (from Tectona grandis), and TKP were collected from the local markets of Kolkata, India. Various media constituents including malt extract, yeast extract, peptone, and other salts including buffer components were purchased from Hi-Media, Bombay, India. Fine chemicals including 2,6dichlorophenol indophenol (DCIP), cellobiose, and other analytical reagents were obtained from Sigma-Aldrich, USA. Substrate Preparation. The substrates were washed thoroughly in distilled water and dried at 50 °C until use. Corncob, coconut coir, ground nut shell, and bagasse were crushed to a fine powder in a grinder and sieved (mesh size BSS 30) to get a homogeneous powder (diameter of particles
