APPENDIX

APPENDIX-I

LIST OF RESEARCH PAPERS PUBLISHED

Raghuwanshi, S., Misra, S., Saxena, R.K. (2012) Enzymatic treatment of black tea (CTC and Kangra orthodox) using Penicillium charlesii tannase to improve the quality of tea. Journal of Food Processing and Preservation. DOI: 10.1111/j.1745-4549.2012.00721.x

Raghuwanshi, S., Dutt, K., Gupta P., Misra, S., Saxena, R. K. (2011) Bacillus sphaericus: The highest bacterial tannase producer with potential for gallic acid synthesis. J. Biosci. Bioeng. 111(6): 635-640.

Raghuwanshi, S., Misra, S. and Saxena R. K. (2012). Enrichment of the feed through enzymatic treatment of wheat straw using Penicillium charlesii tannase and white-rot fungus (Accepted).

Misra, S., Raghuwanshi, S., Saxena, R.K. (2012) Evaluation of corncob hemicellulosic hydrolysate for xylitol production by adapted strain of Candida tropicalis. Carbohydrate Polymers. 10.1016/j.carbpol.2012.11.033.

Misra, S., Raghuwanshi, S., Saxena, R.K. (2012) Statistical approach to study the interactive effects of process parameters for enhanced xylitol production by Candida tropicalis and its potential for the synthesis of xylitol monoesters. Food Science and Technology International. (Accepted).

Misra, S., Raghuwanshi, S., Saxena R.K. (2012) Fermentation behavior of an osmotolerant yeast D. hansenii for xylitol production. Biotechnology Progress. DOI: 10.1002/btpr.1630.

Misra, S., Raghuwanshi, S., Pritesh, G., Saxena, R. K. (2012) Examine growth inhibition pattern and lactic acid production in Streptococcus mutans using different concentrations of xylitol produced from Candida tropicalis by fermentation. Anaerobe, DOI: 10.1016/j.anaerobe.2012.03.001. 18(3): 273–279.

Misra, S., Raghuwanshi, S., Gupta, P., Saxena, R.K. (2012) Efficient 1-5 regioselective acylation of primary hydroxyl groups of fermentative derived xylitol catalyzed by an immobilized Pseudomonas aeruginosa lipase. Biotechnol. Bioproc. Engg. DOI: 10.1007/s12257-011-0491-y. 17 (2): 398-406.

Misra, S., Raghuwanshi, S., Gupta, P., Dutt, K., Saxena, R. K. (2012) Fermentation behavior of osmophilic yeast Candida tropicalis isolated from the nectar of Hibiscus rosa sinensis flowers for xylitol production. Antonie Van Leeuwenhoek. 101 (2): 393-402.

Misra, S., Gupta, P., Raghuwanshi, S., Dutt, K., Saxena, R.K. (2011) Comparative study on different strategies involved for xylitol purification from culture media fermented by Candida tropicalis. Sep. Purifi. Technol. 78: 266-273.

Gupta, P., Dutt, K., Misra, S., Raghuwanshi, S., Saxena, R. K. (2009) Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde. Bioresource Technol. 100: 4074–4076.

Appendix

CONFERENCE ATTENDED / WORK PRESENTED

International conference on New Horizons in Biotechnology (NHBT-2011), November 21-24, 2011, Trivandrum, India. (Synthesis of gallic acid esters using Penicillium charlesii tannase: its potential as anti-tumorigenic compound).

Xylitol in prevention of dental caries. Swati Misra, Pritesh Gupta, Shailendra Raghuwanshi, Kakoli Dutt, R.K.Saxena (2011). Ist National Science Day Symposium held at University of Delhi South Campus, New Delhi, India and was awarded the best poster award.

Xylitol in prevention of dental caries. Swati Misra, Pritesh Gupta, Shailendra Raghuwanshi, Kakoli Dutt, R.K. Saxena (2010). 2nd International Conference on Drug Discovery and Therapy held at Dubai Men’s College, Dubai, U.A.E. (www.icddt.com/icddt-abstracts/PP-0300.htm).

Xylitol production from Candida tropicalis: its process optimization, scale up and synthesis of xylitol monoesters. Swati Misra, Pritesh Gupta, Shailendra Raghuwanshi and R.K. Saxena (2010). Society of Industrial Microbiology 60th Annual Meeting and Exhibition, San Francisco, CA (http://sim.confex.com/sim/2010/webprogram/paper 16480.html).

International Conference on New Frontiers in Biofuels. 18-20 January 2010, New Delhi, India (lipase mediated biodiesel production).

International conference on Green chemistry and Natural Products 06-10 December (2009) at the University of Delhi, India (Microbial cellulose production).

49th International annual conference of the association of Microbiologists of India to be held on 18th - 20th November (2008) at the University of Delhi, India. (Tannase from bacteria).

4th Indo-Italian conference Green chemistry and Natural Products 5-6 December (2008) at the University of Delhi, India.

1st Indo-Danish DU-SDU Seminar on” Emerging Trends in interfacial Areas of Chemical, Biological and Environmental Sciences”. Department of Chemistry, University of Delhi, New Delhi. India, 2008.

International conference on Green chemistry and Natural Products 26-27 November (2007) at the University of Delhi, India (Gallic acid production).

AWARDS

Senior Research Fellowship (SRF) awarded by Council of Scientific and Industrial Research (CSIR) (2012).

Selected in regional level (Northern zone) in i3 National fair held on 7th Nov. 2011 at University of Delhi South Campus, New Delhi organized by DST, Agilent Technologies and Confederation of Indian Industries.

Awarded financial assistance from Council of Scientific and Industrial Research

(CSIR) India, to attend a 2nd international conference on drug discovery and

therapy, Dubai (2010).

ENZYMATIC TREATMENT OF BLACK TEA (CTC AND KANGRAORTHODOX) USING PENICILLIUM CHARLESII TANNASE TOIMPROVE THE QUALITY OF TEAjfpp_721 1..9

SHAILENDRA RAGHUWANSHI, SWATI MISRA and RAJENDRA KUMAR SAXENA1

Department of Microbiology, University of Delhi South Campus Benito Juarez Road, New Delhi 110 021, India

1Corresponding author.TEL: +91-11-24116559;FAX: +91-11-24115270;EMAIL: rksmicrodu@gmail.com

Accepted for Publication March 3, 2012

doi:10.1111/j.1745-4549.2012.00721.x

ABSTRACT

Tannase mediated biotransformation of black tea (CTC and Kangra orthodox)was investigated. Black tea infusion treated with tannase showed that both epigal-locatechin (EGC) gallate and epicatechin (EC) gallate of tea catechins were hydro-lyzed by this enzyme into EGC and EC, respectively, accompanied by 11.2-fold(CTC tea) and 10.29-fold (Kangra orthodox tea) increase in gallic acid concentra-tion. The tannase treated tea infusion showed reduction in tea cream formationand an increase in antioxidant activity to 1.73- and 1.61-fold, respectively.However, there was no change in the content and concentration of volatile com-pounds. Moreover, the results also showed that there was an improvement in thequality of tannase treated black tea infusion in relation to color, brightness,strength and flavor as compared with control.

PRACTICAL APPLICATION

The tannase mediated biotransformation of tea infusion (CTC and Kangra ortho-dox) improves the quality of tea in relation to reduction in tea cream formation.The reduction will help to produce a turbidity free, cold water soluble instant teaor tea extract. Furthermore, on tannase treatment, an increase in an antioxidantactivity was also observed due to certain flavonoids present in tea that showpotential health benefits against cardiovascular diseases and cancer. There was nosignificant change in the volatile compounds that thereby resulted in an improvedcolor, brightness, strength and flavor of tea.

INTRODUCTION

Tea (Camellia sinensis) is the second most widely consumednonalcoholic beverage and is rich in polyphenolic com-pounds, known as tea flavonoids. Green tea contains severalpolyphenols, including epigallocatechin gallate (EGCG),EGC, epicatechin gallate (ECG) and EC (Suganuma et al.1999). These flavonoids or polyphenolic compounds (alsoknown as catechins) possess strong antioxidant properties(Cao and Ito 2004; Majchrzak et al. 2004). Moreover, thesecatechins have also shown antimutagenic and anticarcino-genic properties along with their role in preventing cardio-vascular diseases (Cao and Ito 2004).

It has already been established that the antioxidant activ-ity of phenolic compounds present in tea is mainly due to

their redox properties, which allow them to act as reducingagents, singlet-oxygen quenchers and metallic-ion chelators(Atoui et al. 2005; Macedo et al. 2011). Many clinical studiesand animal models have shown that despite the proven anti-oxidant capacity of tea polyphenols, these compounds,especially the polymers, esters and glycosides, are abundantbut are not always absorbed by oral administration.However, the functional effect of these compounds dependsnot only on the amount ingested but also on its bioavail-ability (Holst and Williamson 2008). Therefore, it would beworthwhile to investigate the enzymatic hydrolysis ofpolyphenols present in tea in order to make these com-pounds available, which are having great importance.

Tannin acylhydrolase, commonly referred to as tannase(E.C. 3.1.1.20), is an inducible enzyme produced by

Journal of Food Processing and Preservation ISSN 1745-4549

1Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.

Bacillus sphaericus: The highest bacterial tannase producer with potential for gallicacid synthesis

Shailendra Raghuwanshi, Kakoli Dutt, Pritesh Gupta, Swati Misra, and Rajendra Kumar Saxena⁎

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

Received 9 September 2010; accepted 16 February 2011Available online 12 March 2011

An indigenously isolated strain of Bacillus sphaericus was found to produce 1.21 IU/ml of tannase under unoptimizedconditions. Optimizing the process one variable at a time resulted in the production of 7.6 IU/ml of tannase in 48 h in thepresence of 1.5% tannic acid. A 9.26-fold increase in tannase production was achieved upon further optimization usingresponse surface methodology (RSM), a statistical approach. This increase led to a production level of 11.2 IU/ml in mediumcontaining 2.0% tannic acid, 2.5% galactose, 0.25% ammonium chloride, and 0.1% MgSO4 pH 6.0 incubated at 37°C and 100 rpmfor 48 h with a 2.0% inoculum level. Scaling up tannase production in a 30-l bioreactor resulted in the production of 16.54 IU/mlafter 36 h. Thus far, this tannase production is the highest reported in this bacterial strain. Partially purified tannase exhibited anoptimum pH of 5.0 with activity in the pH range of 3 to 8; 50°C was the optimal temperature for activity. Efficient conversion oftannic acid to purified gallic acid (90.80%) was achieved through crystallization.

© 2011, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Tannase; Bacillus sphaericus; Process optimization; Response surface methodology (RSM); Fermentor; Gallic acid crystallization]

Tannases (tannin acyl hydrolase EC 3.1.1.20) are hydrolyticenzymes that catalyze the hydrolysis of ester and depside bonds inhydrolysable tannins such as tannic acid and release glucose and gallicacid (1). This enzyme is used in the manufacturing of instant tea andthe production of gallic acid, a substrate for propyl gallate productionand trimethoprim synthesis (2,3). Tannase is also used in the beer andwine industries to remove chill haze formation in beer and wine (4).Additionally, it is used to reduce the antinutritional effects of poultryand animal feed along with food detanification and industrial effluenttreatment (5,6).Many fungi, such as Aspergillii, Penicillii, Fusaria, and Trichoderma

(7–10) as well as yeast like Candida sp., Pichia sp., and Saccharomycescerevisiae (11), have been reported to be tannase producers. On theother hand, few bacteria are known to produce tannase and includecertain species of Bacilli, Corynebacterium sp., Lactobacillus sp., andSerratia sp., (12–14). When tannase titers are compared betweenfungal and bacterial sources, the former are higher producers. In thisrespect, the literature on bacterial tannase is limited in comparison tofungal tannase.Realizing the immense potential of bacterial tannase made it

worthwhile to optimize the production process of this tannase to achievemaximumyields. Conventionalmethods for the optimization ofmediumand fermentation conditions involve varying one parameter at a timewhile keeping the others constant. The process is time consuming andexpensive. It also does not take into account the combined interactionsbetween various physicochemical parameters. To overcome this diffi-

culty and to evaluate and understand the interactions between differentphysiological and nutritional parameters, response surface methodologyhasbeenwidelyused (15,16). RSM is anefficient tool for theoptimizationof different physiochemical parameters. It has an advantage over thetraditional approach as a statistical optimization study is carried out todetermine the influence of variables on enzyme units and to optimizethese variables in order to achieve maximum yield under the bestpossible economic conditions (15,16). Here, face-centered centralcomposite design (FCCCD), a type of RSM, was employed to optimizeproduction conditions to obtain maximum tannase yields.Therefore, in the present investigation, we report the optimization

of the production process of tannase by Bacillus sphaericus isolatedfrom soil, which was followed by its biochemical characterization andapplication in gallic acid synthesis.

MATERIALS AND METHODS

Microorganisms and growth One hundred and fifty bacterial strains wereisolated from soil and were maintained on nutrient agar slants at 8–10°C with periodicsubculturing. Tannase production was carried out in bacterial minimal mediumcomposed of 0.5 g/l K2HPO4, 0.5 g/l KH2PO4, 0.5 g/l MgSO4, 1.0 g/l NH4Cl, 0.1 g/l CaCl2,and 5.0 g/l glucose (pH 6.0) supplemented with 1% tannic acid. An inoculum level(grown in nutrient broth with an OD600 of 0.6–0.8) of 2% was used, and incubation wascarried out at 37°C and 200 rpm for 48 h. Samples were withdrawn at regular 12-h intervals and were centrifuged at 10,000 rpm for 10 min in a refrigerated centrifugeat 4°C. The supernatant was the enzyme source and was assayed for tannase activity.

Screening of potent tannase producers Tannase producers were screenedusing both the tannic acid agar platemethod (7) and the visual detectionmethod (17). Forthe visual detection method, the bacterial isolates were grown individually in productionmedium for 48 h and centrifuged. The obtained cell-free culture broth was mixed with1.0 ml of a solution containing NaH2PO4 (33 mM, pH 5.0) and methyl gallate (20 mM).

Journal of Bioscience and BioengineeringVOL. 111 No. 6, 635–640, 2011

www.elsevier.com/locate/jbiosc

⁎ Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24115270.E-mail addresses: rksmicro@yahoo.co.in, rksmicro@hotmail.com (R.K. Saxena).

1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved.doi:10.1016/j.jbiosc.2011.02.008

Carbohydrate Polymers 92 (2013) 1596– 1601

Contents lists available at SciVerse ScienceDirect

Carbohydrate Polymers

jou rn al hom epa ge: www.elsev ier .com/ locate /carbpol

Evaluation of corncob hemicellulosic hydrolysate for xylitol production by

adapted strain of Candida tropicalis

Swati Misra, Shailendra Raghuwanshi, R.K. Saxena ∗

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110 021, India

a r t i c l e i n f o

Article history:Received 6 September 2012

Received in revised form 12 October 2012

Accepted 11 November 2012

Available online xxx

Keywords:Xylose

Corncob hemicellulosic hydrolysate

C. tropicalisXylitol

Immobilization

a b s t r a c t

A maximum xylose extraction of 21.98 g/L was obtained in hydrolysate with a solid to liquid ratio of 1:8

(w/v) at 1% H2SO4 and treated for 30 min. The optimized and treated corncob hemicellulosic hydrolysate

medium supplemented with (g/L) yeast extract 5.0, KH2PO4 2.0, MgSO4·7H2O 0.3 and methanol 10 mL

whose pH was adjusted to 4.5 acts as production medium. Under this condition; the adapted strain

of C. tropicalis resulted in 1.22-fold increase in xylitol yield and 1.70-fold enhancement in volumetric

productivity was obtained as compared to parent strain of C. tropicalis. On concentrating the hydrolysate

under vacuum using rotavapor proves to be efficient in terms of improved xylitol yield and productivity

over microwave assisted concentration using adapted strain of C. tropicalis. The immobilized cells of C.tropicalis resulted in more than 70% efficiency up to third cycle. The xylitol production could be scaled

up to 10 L fermentor.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The utilization of lignocellulosic wastes for industrial purposes

is very attractive and promising as these are inexpensive, renew-

able and widely available in nature. Hemicelluloses are a plant cell

wall polysaccharide and the third most abundant renewable poly-

mer in nature. The main component of the hemicellulosic fraction

is xylan, a heteropolysaccharide with homopolymeric backbone of

xylose units (Saha, 2003). In this context, extensive research has

been undertaken for bioconversion of hemicellulosic hydrolysate

derived carbohydrates, particularly xylose, into several value added

products (Chandel & Singh, 2011). It is a naturally occurring

five-carbon sugar alcohol with outstanding organoleptic and anti-

cariogenic properties (Mäkinen, 2000; Rao, Jyothi, Prakasham,

Sarma, & Rao, 2006). Besides this, it prevents osteoporosis and can

be used in diabetic food products which thereby make xylitol as

an attractive sucrose substitute. Due to its properties, xylitol has

become an attractive option in food and pharmaceutical industries.

The production of xylitol involves established commercial process

based on catalytic hydrogenation of highly purified xylose derived

from hydrolysates of hemicellulosic rich materials, a high produc-

tion cost process that uses elevated pressure and temperature, and

requires extensive xylose purification steps (Liaw, Chen, Chang, &

Chen, 2008). For these reasons, several researchers have explored

∗ Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24115270.

E-mail addresses: swati.misra11@gmail.com (S. Misra), rksmicrodu@gmail.com

(R.K. Saxena).

the alternative route, wherein the existence of xylose-fermenting

microorganisms i.e. bacteria, yeasts and fungi opened the possi-

bility to produce xylitol by fermentation using xylose present in

hydrolysates derived from agro-industrial lignocellulosic residues.

This alternative route proves to be interesting as it requires use of

mild conditions of pressure and temperature, and very little xylose

purification thus making the process economical (Tada, Horiuchi,

Kanno, & Kobayashi, 2004; Wang et al., 2011). The most studied

xylitol producers are yeasts, with strains of the genus Candida and

Debaromyces being the best natural producers (Sampaio et al., 2004;

West, 2009). Xylose fermenting yeasts reduces xylose to xylitol by

the NAD (P) H-dependent xylose reductase (XR) (Winkelhausen &

Kuzmanova, 1998).

The global process from the raw material (agro residues) to

the final product has the following sequential steps: reduction

of size, acid hydrolysis, neutralization, detoxification, fermenta-

tion, recovery and purification. The hemicellulosic fraction can

easily be hydrolyzed using dilute acids (Sarrouh, Santos, & Silva,

2007). Hydrolysis of hemicellulose yield sugars which are rapidly

degraded to fermentation inhibitors i.e. furfural, hydroxymethyl-

furfural and other condensation byproducts (Rao et al., 2006).

The amount of sugar released during hydrolysis is dependent on

the type of material and operating conditions including tempera-

ture, reaction time and acid concentration (Rahman, Choudhury,

Ahmad, & Kamaruddin, 2007). Nolleau, Preziosi Belloy, Delgenes,

and Navarro (1993) pointed out that xylose in higher amounts

favors xylitol production by yeasts. Thus, the hydrolysate needs

to be concentrated before being used as a culture medium. How-

ever, at the same time, the concentrations of by-products originally

0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.carbpol.2012.11.033

XML Template (2012) [26.11.2012–4:01pm] [1–14]{SAGE}FST/FST 462230.3d (FST) [PREPRINTER stage]

Article

Statistical approach to study the interactive effectsof process parameters for enhanced xylitol productionby Candida tropicalis and its potential for the synthesisof xylitol monoesters

Swati Misra, Shailendra Raghuwanshi and RK Saxena

AbstractPrevious results showed that an indigenously isolated yeast strain of Candida tropicalis was found to produce12.11 gl�1 of xylitol under unoptimized conditions in presence of 50gl�1 of xylose. In the present study,optimizing the process using one-variable at-a-time resulted in the production of 59.07 gl�1 of xylitol in 96 hin presence of 100gl�1 xylose. Further optimization using response surface methodology led to the produc-tion of 65.45 gl�1 in medium containing 100gl�1 xylose, 0.5% yeast extract, 0.03% MgSO4.7H2O and 0.2%KH2PO4, pH-4.5, 30

�C, 200 r/min for 96 h with 4% inoculum level. Addition of 1% methanol in responsesurface methodology optimized–medium led to the production of 67.12 gl�1. Scaling up in 10 L fermentorresulted in productivity of 0.80 g/lh�1 with yield of 0.68 gg�1. Efficient synthesis of xylitol esters was achievedwith butyric acid (50.32%) and caproic acid (38.36%) in 4 h using Pseudomonas aeruginosa lipase int-butanol: tetrahydrofuran (1:1 v/v).

KeywordsXylitol, Candida tropicalis, process optimization, response surface methodology, ester synthesis

Date received: 28 March 2012; revised: 13 August 2012

INTRODUCTION

In the last decade, demand for the bulk natural sweet-eners has increased among health-conscious consumerslooking for alternatives as against potentially danger-ous artificially manufactured sweeteners. Therefore, inthis context, the bulk natural sweetener which includespolyols or sugar alcohols has taken over the market asa safe sugar substitute. Amongst various sugar alco-hols, xylitol is a five-carbon sugar alcohol, havingsweetness which equals that of sucrose. It providesone-third fewer calories than sugar and has antiketo-genic and anticariogenic properties. Due to these prop-erties, xylitol can be applied to food, pharmaceutical

and odontological industries (Ahmet and Gurbuz,2006).

Currently, the entire world demand for its produc-tion has increased and is achieved by chemical catalyticreduction (hydrogenation) of D-xylose or xylose-richhemicellulose hydrolysates of substrate sources suchas birchwood and other hardwoods (Melaja et al.,1981; Silva et al., 2006; Prakasham et al., 2009).However, due to environmental concerns and conceptof sustainability, researches are directed towards themicrobial route for xylitol production by yeasts con-taining xylose reductase (EC 1.1.1.21), which in thepresence of NADPH as a cofactor catalyzes the reduc-tion of xylose to xylitol as first step in xylose

Department of Microbiology University of Delhi South Campus,New Delhi, India

Corresponding author:RK Saxena, Department of Microbiology, University of Delhi SouthCampus, Benito Juarez Road, New Delhi-110 021, India.Email: rksmicrodu@gmail.com

Food Science and Technology International 0(0) 1–14! The Author(s) 2012 Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/1082013212462230fst.sagepub.com

Clinical microbiology

Examine growth inhibition pattern and lactic acid production in Streptococcusmutans using different concentrations of xylitol produced from Candidatropicalis by fermentation

Swati Misra, Shailendra Raghuwanshi, Pritesh Gupta, R.K. Saxena*

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India

a r t i c l e i n f o

Article history:Received 30 June 2011Received in revised form3 February 2012Accepted 7 March 2012Available online 15 March 2012

Keywords:XylitolStreptococcus mutansPlate assaySpectrophotometric method

a b s t r a c t

Twenty clinical isolates of Streptococcus sp. were isolated from six clinical samples of dental caries onMSFA. Amongst these isolates, five clinical isolates were identified as Streptococcus mutans on the basis ofmorphological, biochemical and 16S rDNA sequencing. The isolated strains of S. mutans were exposed tofermented and purified xylitol (0.25e15.0%) and tested for its anti-microbial effects against controlmedium (Brain Heart Infusion without xylitol) after 12 h. The plate assay was developed using bro-mocresol green as an indicator dye in order to study the relative growth inhibition pattern of clinicalsample at different concentrations of an anti-microbial compound in a single petriplate. The morphologyof S. mutans cells in brain heart infusion (BHI) medium containing xylitol resulted in a diffused cell wallas observed using gram staining technique. The minimum inhibitory concentration (MIC) is 0.25% forS. mutans obtained from different clinical samples. The MIC50 and MIC90 is 5.0% and 10.0% xylitolrespectively of the selected S. mutans being designated as clinical isolate B (6). The zone of inhibition was72 mm and lactic acid production was 0.010 g/l at 10% xylitol concentration in Brain Heart Infusion Broth.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Dental caries is a localized and transmissible pathologicalinfectious process that ends up in the destruction of hard dentaltissue. Caries has plagued humans and it continues to be one of themost common human infectious disease [1]. The principle etio-logical agent of dental caries is Streptococcus mutans [1,2]. These aregram positive cocci, non motile facultative anaerobes whichmetabolize carbohydrates through the production of glucosyltransferase thereby forming water insoluble glucans and facilitatesadherence of the organism to dental plaque. Therefore, muchattention was focused on this bacterium as a target for theprevention of this disease using anti-plaque agents for regular useand it should not interfere with biologic processes occurring in themouth, be harmless to oral mucosa and should have low toxicity ifaccidentally swallowed and should be sugar free. In this respect,sugar substitutes like xylitol and sorbitol has been studied [3].

Xylitol is a naturally occurring sugar substitute with an anti-cariogenic property and has been a focus of scientific inquiry forseveral decades [4]. Consequently, many attempts have been madeto develop a rapid and sensitive method for S. mutans detection [5].

In this respect, a possible mechanism for xylitol’s remarkableefficacy was investigated [6,7] and several researchers have earlierreported the growth inhibition studies using commercial xylitol.Kontiokari et al. [8] demonstrated the growth inhibition pattern forsome otopathogenic bacteria with 1.0% and 5.0% xylitol.

The aim of the present study was to evaluate the antibacterialeffect of xylitol produced from Candida tropicalis by fermentationand to determine the concentration of xylitol that can significantlyinhibit the growth of S. mutans isolated from the clinical samplesusing plate assay method, spectrophotometric method and analysisof lactic acid production using HPLC.

2. Materials and methods

2.1. Media and fermentation conditions for xylitol production

The fermentation broth was prepared using 175.0 g/l ofcommercial xylose for adapted strain of C. tropicalis. The mediumwas supplemented with (g/l) yeast extract 5.0, KH2PO4 2.0,MgSO4.7H2O 0.3. The mediumwas inoculated with 5.0% of the seedinoculum and fermentation was carried out at 30 �C in 10 Lfermentor with 7.5 L working volume (New Brunswick Sci.Inc.Fermentor Bioflow IV, USA). The pH was controlled automaticallywith 1 N NaOH/1 N HCl using a pH controller at pH-4.5. Agitation

* Corresponding author. Tel.: þ91 11 24116559; fax: þ91 11 24115270.E-mail address: rksmicrodu@gmail.com (R.K. Saxena).

Contents lists available at SciVerse ScienceDirect

Anaerobe

journal homepage: www.elsevier .com/locate/anaerobe

1075-9964/$ e see front matter � 2012 Elsevier Ltd. All rights reserved.doi:10.1016/j.anaerobe.2012.03.001

Anaerobe 18 (2012) 273e279

ORIGINAL PAPER

Fermentation behavior of osmophilic yeast Candidatropicalis isolated from the nectar of Hibiscus rosa sinensisflowers for xylitol production

Swati Misra • Shailendra Raghuwanshi •

Pritesh Gupta • Kakoli Dutt • R. K. Saxena

Received: 5 July 2011 / Accepted: 16 September 2011 / Published online: 29 September 2011

� Springer Science+Business Media B.V. 2011

Abstract Eighteen yeast species belonging to seven

genera were isolated from ten samples of nectar from

Hibiscus rosa sinensis and investigated for xylitol

production using D-xylose as sole carbon source.

Amongst these isolates, no. 10 was selected as the best

xylitol producer and identified as Candida tropicalis

on the basis of morphological, biochemical and 26S

rDNA sequencing. C. tropicalis produced 12.11 gl-1

of xylitol in presence of 50 gl-1 of xylose in 72 h at

pH 5, 30�C and 200 rpm. The strain of C. tropicalis

obtained through xylose enrichment technique has

resulted in a yield of 0.5 gg-1 with a xylitol volumet-

ric productivity of 1.07 gl-1h-1 in the presence of

300 gl-1 of xylose through batch fermentation. This

organism has been reported for the first time from

Hibiscus rosa sinensis flowers. Realizing, the impor-

tance of this high valued compound, as a sugar

substitute, xylose enrichment technique was devel-

oped in order to utilize even higher concentrations of

xylose as substrate for maximum xylitol production.

Keywords Hibiscus rosa sinensis � Indigenousyeast � Xylitol � Cell adaptation

Introduction

Nectar is the most frequent form of floral reward that

animal pollinated plants provide for their mutualistic

counterparts (Simpson and Neff 1983). The presence

of yeasts in floral nectar is well evident from already

existing reports and publications (Boutroux 1884;

Ehlers and Olesen 1997; Kevan et al. 1988). Flower

nectar is believed to be an ideal habitat for yeasts

because of rich sugar content primarily in the form of

simple mono and disaccharide sugars which majorly

consists of glucose, fructose and sucrose and xylose as

the minor sugar. However, there are few reports from

the family proteaceae wherein xylose has been

regarded as the major sugar occurring in floral nectar

(Nicolson and Van Wyk 1998). Chemically, D-xylan-

opyranose (xylose) is a pentose sugar and its polymer

xylan is the major component of hemicellulose. After

glucose, xylose is the second most abundant plant

sugar. It is approximately 17% of the dry weight in

woody angiosperms and up to 31% in herbaceous

angiosperms (Jeffries and Shi 1999).

There are several new species of the genus Candida

which are reported to be associated with flowers and

insects including Candida hawaiiana and Candida

kipukae (Lachance et al. 2003), Candida picachoen-

olis and Candida pimensis (Suh et al. 2004), Candida

Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-011-9646-2) containssupplementary material, which is available to authorized users.

S. Misra � S. Raghuwanshi � P. Gupta �K. Dutt � R. K. Saxena (&)

Department of Microbiology, University of Delhi South

Campus, Benito Juarez Road, New Delhi 110 021, India

e-mail: rksmicrodu@gmail.com

123

Antonie van Leeuwenhoek (2012) 101:393–402

DOI 10.1007/s10482-011-9646-2

Fermentation Behavior of an Osmotolerant Yeast D. hansenii forXylitol Production

Swati Misra, Shailendra Raghuwanshi, and Rajendra K. SaxenaDept. of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110 021, India

DOI 10.1002/btpr.1630Published online in Wiley Online Library (wileyonlinelibrary.com).

Realizing the importance of xylitol as a high-valued compound that serves as a sugar sub-stitute, a new, one step thin layer chromatographic procedure for quick, reliable, and effi-cient determination of xylose and xylitol from their mixture was developed. Two hundredand twenty microorganisms from the laboratory stock cultures were screened for their abilityto produce xylitol from D-xylose. Amongst these, an indigenous yeast isolate no.139 (SM-139) was selected and identified as Debaryomyces hansenii on the basis of morphologicaland biochemical characteristics and (26S) D1/D2 r DNA region sequencing. Debaryomyceshansenii produced 9.33 gL�1 of xylitol in presence of 50.0 gL�1 of xylose in 84 h at pH 5.5,30�C, 200 rpm. In order to utilize even higher concentrations of xylose for maximum xylitolproduction, a xylose enrichment technique was developed. The strain of Debaryomyces han-senii was obtained through xylose enrichment technique in a statistically optimized mediumcontaining 0.3% yeast extract, 0.2% peptone, 0.03% MgSO4.7H2O along with 1% methanol.The culture was inoculated with 6% inoculum and incubated at 30�C and 250 rpm. A yieldof 0.6 gg�1 was obtained with a xylitol volumetric productivity of 0.65 g/L h�1 in the pres-ence of 200 gL�1 of xylose although up to 300 gL�1 of xylose could be tolerated throughbatch fermentation. Through this technique, even higher concentrations of xylose as sub-strate could be potentially utilized for maximum xylitol production. VVC 2012 American Insti-tute of Chemical Engineers Biotechnol. Prog., 000: 000–000, 2012Keywords: xylitol, thin layer chromatography, indigenous yeast, xylose enrichment method, HPLC

Introduction

Xylitol, a five carbon sugar alcohol, is a high value prod-uct, due principally to its sweetening power, noncariogenicproperties, and insulin-independent metabolism properties.Therefore, it justifies the growing interest in developing abetter method to determine xylitol using a thin layer chro-matographic (TLC) method. The use of TLC method is rela-tively fast (1–3 h), requires small amount of sample (5–10lL) and is easy to carry out with inexpensive semimicroequipment and materials.1

Xylitol is widely distributed in nature, particularly in cer-tain fruits and vegetables but its extraction is economicallyunfeasible.2,3 Xylitol is currently produced by chemicalreduction of D-xylose. However, this is believed to be anexpensive process as initially xylose has to be purified.Therefore, biological production of xylitol could be of eco-nomic interest as it is performed at mild temperature andpressure conditions. It requires lower energy; bioconversionis highly specific, resulting in higher yields and efficientproduct separation, lower purification costs, and cleanereffluents.4,5,6 The current literature includes process optimi-zation but less attention is paid to isolation of yeasts strains

capable of producing xylitol in higher yield and also todevelop rapid and efficient xylitol estimation method.

Selection of microorganisms that ferment D-xylose to xylitol isthe first step in developing a bioconversion process. Filamentousfungi, yeasts, and some bacteria are able to perform this conver-sion through the action of NAD(P) H-dependent enzyme xylosereductase (EC 1.1.1.21).7 There are few studies regarding xylitolproduction in bacteria 8,9 and filamentous fungi10 as the level ofproduction from bacteria and fungi is comparatively lower ascompared to yeasts.5 Therefore, most of the studies haveinvolved yeasts, with special emphasis on the genus Candida.11

In order to initially screen xylitol producing microorganismsfrom the existing laboratory stock cultures we have developeda new, one step TLC method for the separation of xylitol fromxylose that is simple, rapid, efficient, reliable, and is economi-cal as no special equipment or chemicals are required. Themicroorganisms were grown on xylose enriched culture broth.Thereafter, the best xylitol producers were analyzed by high-performance liquid chromatography (HPLC). The selectedyeast isolate was adapted to higher concentrations of xylosethrough the xylose enrichment technique and its batch fermen-tation behavior was studied upto 300.0 gL�1 of xylose.

Materials and Methods

Chemicals and reagents

The commercial xylose was purchased from Central DrugHouse (Mumbai, India). All other medium components and

Additional Supporting Information may be found in the online ver-sion of this article.

Correspondence concerning this article should be addressed to andR. K. Saxena at rksmicrodu@gmail.com.

VVC 2012 American Institute of Chemical Engineers 1

Biotechnology and Bioprocess Engineering 17: 398-406 (2012)

DOI 10.1007/s12257-011-0491-y

Efficient 1-5 Regioselective Acylation of Primary Hydroxyl Groups of

Fermentative Derived Xylitol Catalyzed by an Immobilized

Pseudomonas aeruginosa Lipase

Swati Misra, Shailendra Raghuwanshi, Pritesh Gupta, and R. K. Saxena

Received: 3 October 2011 / Revised: 24 November 2011 / Accepted: 7 December 2011

© The Korean Society for Biotechnology and Bioengineering and Springer 2012

Abstract The experiment described in this paper synthe-

sizes xylitol acylated products from fermentative derived

xylitol and acid anhydrides of various chain lengths in the

presence of Tetrahydrofuran (THF) and acetonitrile using

immobilized Pseudomonas aeruginosa (PL) lipase as a

biocatalyst (97% residual activity up to five cycles) at

37°C, 200 rpm. This study examines a number of different

acid anhydrides for their highly selective and efficient

lipase-catalyzed acylation of primary hydroxyl groups in

xylitol. Of those studied, the best results are obtained with

butanoic anhydride, 80.12% after 4 h in acetonitrile follow-

ed by vinyl acetate, which results in 77.79% conversion

after 8 h of incubation in THF as analyzed through high

performance liquid chromatography (HPLC).

Keywords: Pseudomonas lipase, acylating agent, xylitol

diacylated compounds

1. Introduction

In recent years, organic chemists have made increasing use

of biocatalysis as a synthetic tool in the preparation of ever

more challenging compounds [1]. This has the added

advantage of environmental friendliness [2]. Biocatalyst

involves stereo selective transformation, primarily for the

synthesis of enantiopure compounds that are of great

importance to the pharmaceutical industry [3,4]. Therefore,

organic synthesis is receiving much academic and pro-

fessional interest over conventional chemical synthesis.

Existing reports by several researchers state that hydrolytic

enzymes are used for specific substitution and trans-

formation of complex carbohydrates, lipids, and peptides at

low water activity without the need for the protection and

deprotection steps that are required in conventional chiral

and regioselective organic synthesis [5-7].

Among known biocatalysts, the synthetic utility of lipase

(triacylglycerol acyl hydrolase E.C. 3.1.1.3) is outstanding

as it catalyzes the hydrolysis and synthesis of long chain

acylglycerols under aqueous, microaqueous, and inorganic

solvents [8,9]. This enables lipase to carry out a variety of

different catalytic reactions with enantomeric selectivity,

hydrolysis, ester synthesis or transesterifications, which

have advantages over conventional, non-enzymatic pro-

cesses [10]. These lipase-catalyzed reactions are exploited

in various industries such as food and feed, detergents,

pharmaceuticals, leather, textiles, and paper [8,9]. Various

lipase-catalyzed acylation reactions have also been report-

ed for synthesis of xylitol diacylated compounds, which are

especially useful in polyol blends and are employed in the

preparation of self-extinguishing, fire-retardant, polyurethane

foams. Haveren, van Oostveen, Miccichè and Weijnen [11]

report the potential and versatility of isosorbide diesters

and acylated compound when used as substitutes for the

currently phthalate-based plasticizers for polyvinyl chloride

(PVC) and other resins. These substances are used as

biosurfactants and bioplasticizers.

Acylation of carbohydrate hydroxyl groups is one of the

most commonly used functional group protection techni-

ques in the synthesis of oligosaccharides. In carbohydrate

chemistry, per-O-acetylated sugars are inexpensive and

useful intermediates for the synthesis of naturally occurring

glycosides, oligosaccharides, and other glycoconjugates

Swati Misra, Shailendra Raghuwanshi, Pritesh Gupta, R. K. Saxena*

Department of Microbiology, University of Delhi South Campus, New

Delhi 110-021, India

Tel: +91-11-2411-6559; Fax: +91-11-2411-5270

E-mail: rksmicrodu@gmail.com

RESEARCH PAPER

Separation and Purification Technology 78 (2011) 266–273

Contents lists available at ScienceDirect

Separation and Purification Technology

journa l homepage: www.e lsev ier .com/ locate /seppur

Comparative study on different strategies involved for xylitol purification from

culture media fermented by Candida tropicalis

Swati Misra, Pritesh Gupta, Shailendra Raghuwanshi, Kakoli Dutt, R.K. Saxena ∗

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

a r t i c l e i n f o

Article history:Received 6 November 2010

Received in revised form 16 February 2011

Accepted 19 February 2011

Keywords:Xylitol

Fermentation broth

Activated charcoal treatment

Crystallization

Xylitol recovery

a b s t r a c t

Xylitol, a sugar substitute, is a high value product for pharmaceutical and food industries and its purifi-

cation being of commercial importance. In the present study, the purification of xylitol obtained through

Candida tropicalis by fermentation using synthetic xylose and corn cob hemicellulosic hydrolysate as sub-

strates were studied for liquid–liquid extraction (21.72 g/l xylitol extracted in 1:5 (v/v) of ethyl acetate)

and precipitation (67.44% xylitol recovery along with certain impurities). By this method xylitol recovery

is difficult and expensive for large scale processes. Therefore, activated charcoal treatment followed by

vaccum concentration and crystallization method for xylitol extraction was evaluated. The optimized

conditions obtained for activated charcoal treatment followed by vaccum concentration and crystalliza-

tion method were 15.0 g/l of charcoal concentration at 30 ◦C for 1 h with 10 times super saturation of

initial concentration and crystallization temperature of −20 ◦C for initiation and then at 8 ◦C yielding

43.97%. After 4 cycles of crystallization, 76.20% and 68.06% xylitol crystallization yield was obtained in

50 ml and 5.0 l of the synthetic xylose fermentation broth by adapted strain of C. tropicalis respectively.

The effect of solvents on the crystalline structure of xylitol showed prismatic structure in the presence

of ethanol and orthorhombic needles in the presence of tetrahydrofuran. The purity of the xylitol was

characterized using 13C and 1H nuclear magnetic resonance, mass spectroscopy, and optical rotation,

confirming 98.99% purity in a pure crystallized form.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Industries producing polyol sweeteners have registered a grow-

ing demand for the consumption of sugar-free and low heat value

products. Among these, xylitol is an important sugar substitute

with certain interesting physical and chemical properties which

make it a high value compound for pharmaceutical, odontological

and food industries. At present, large scale commercial production

of xylitol is by an expensive catalytic hydrogenation of d-xylose

from acid hydrolysis of lignocellulosics [1]. Hence, it is worthwhile

to explore an alternative process for the effective production of xyl-

itol using micro-organisms which make use of the semi synthetic

media [2,3] or detoxified hemicellulosic hydrolysate [4] in order to

reduce the manufacturing costs with minimal environmental and

energy issues [5].

The recovery and purification of the product exists as a very

complicated step in several industrial fermentative processes,

which majorly depend on the nature of the product as well as on

Abbreviations: NMR, nuclear magnetic resonance; OR, optical rotation; MS, mass

spectroscopy; HPLC, high performance liquid chromatography.∗ Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24115270.

E-mail address: rksmicro@hotmail.com (R.K. Saxena).

the complex composition of the fermentation broth [6]. In order

to recover a product which requires higher purity for commercial-

ization [7], it is often implied that important steps characterized

by costs even higher than the production process are used. How-

ever, in literature on polyols, very little information is available

about xylitol recovery [8–10] and mainly reports are related to

the obtainment and treatment of the hemicellulosic hydrolysate,

its fermentation and metabolic bioconversion [11,12]. Until now,

on industrial scale, the xylitol obtained is separated and purified

by chromatographic methods [13,14]. Jandera and Churacek [15]

used cation exchange resin columns for xylitol separations followed

by crystallization at low temperatures of the xylitol-rich solutions.

Whereas, Gurgel et al. [8] used both anion and cation exchange

resins to purify xylitol from sugarcane bagasse hydrolysate fermen-

tation broth and observed a xylitol loss of about 46–57%. However,

such techniques tend to be expensive for industrial scale processes.

In order to overcome this hurdle, an efficient and econom-

ically competitive strategy for xylitol purification and recovery

from fermented broth was developed. The purification of solutions

by liquid–liquid extraction and precipitation is used in numerous

industrial processes in order to recover dissolved substances or to

remove undesirable impurities. However, the most efficient strat-

egy used for xylitol purification and its extraction is the activated

charcoal treatment followed by vaccum concentration and crystal-

1383-5866/$ – see front matter © 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.seppur.2011.02.018

Characterization of cross-linked immobilized lipase from thermophilic mouldThermomyces lanuginosa using glutaraldehyde

Pritesh Gupta, Kakoli Dutt, Swati Misra, Shailendra Raghuwanshi, R.K. Saxena *

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India

a r t i c l e i n f o

Article history:Received 2 February 2009Received in revised form 26 March 2009Accepted 26 March 2009Available online 28 April 2009

Keywords:LipaseCross-linked enzyme aggregatesAmmonium sulphate

a b s t r a c t

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobi-lization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyceslanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was usedas precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM micropho-tographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the freelipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability ofthe CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residualactivity even after 10 cycles of repeated use.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Lipases arehighlyexploited inseveral industries due to theirbroadspecificity coupled with a high enantio and regio selectivity (Hasanet al., 2006). However, their use in commercial applications becomesdifficult due to the cost andproblems in facile separation and reuse. Inthis context, use of cross-linked enzyme aggregates (CLEAs) preparedby combining purification via precipitation and immobilization inone step has led to the development of a new family of immobilizedenzymes. These cross-linked enzyme aggregates form highly stableandactivebiocatalysts (López-Serranoet al., 2002). TheCLEA technol-ogy offers many advantages for industrial applications as it is simpleand amenable to rapid optimization, which translates to low costsand short time- to-market processes (Sheldon, 2007).

In the present investigation, lipase from Thermomyces lanuginosawas used to prepare cross-linked enzyme aggregates (CLEA). Elec-tronmicroscopic studies were carried out for studying the structureof the aggregates formed. These immobilized enzyme preparationsare preferred for easy recovery of the product and increased stabil-ity as was evaluated and confirmed for olive oil hydrolysis.

2. Method

2.1. Micro organism and production conditions

T. lanuginosa, a thermophilic mold was maintained on YPSS agarslants (% wv�1: Starch- 1.5, Yeast extract- 0.4, KH2PO4- 0.2, MgSO4-

0.05, Agar–agar- 2.0; pH- 7.0). Production of lipase was carried outin 2 L Erlenmeyer flasks containing 400 ml of optimized productionmedium (% wv�1: sunflower oil (emulsified with 2% gum acacia),8.0 ml; sorbitol, 0.3; CSL, 1.0; Tween-80, 0.4; CaCl2 � 2H2O, 0.10 g;MgSO4 � 7H2O, 0.50; KCl, 0.50, pH 9.0) inoculated with 5 � 106

spores/50 ml and incubated at 45 �C, 200 rpm for 96 h.The crude enzyme was obtained after filtration, centrifugation

of the culture broth and subsequent four fold concentration using10 kDa cellulose acetate membrane (Millipore). This concentratedculture filtrate was used as lipase source for immobilization viacross-linking with glutaraldehyde.

2.2. Synthesis of CLEA

2.2.1. Using ammonium sulphate as precipitantLipase (5.0 ml) was taken in 50 ml of centrifuge bottle. Ammo-

nium sulphate was added at a concentration of 60% saturation. Tothis mixture, 10 ml of Tris–HCl buffer containing ammonium sul-phate at 60% saturation was added along with 1 ml of glutaralde-hyde. This mixture was stirred at 4 ± 1 �C for 12–14 h and 10 mlof double distilled water was added to it. The mixture was centri-fuged at 5000 rpm for 5 min at 4 ± 1 �C. The supernatant was dec-anted and the residue was washed with double distilled watertwice. The final enzyme preparation was stored in 5 ml distilledwater at 4 ± 1 �C.

2.2.2. Using 1,2 dimethoxy ethane as precipitantLipase (5.0 ml) was taken in 50 ml centrifuge tube and 5.0 ml of

Tris–HCl buffer (50 mM, pH 9.0) was added. To the reaction mix-ture, 20 ml of 1,2 dimethoxy ethane and 1 ml of glutaraldehydewere added and stirred at 4 ± 1 �C for 12–14 h. 1,2 dimethoxy eth-

0960-8524/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2009.03.076

* Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24116427/24115270.

E-mail addresses: rksmicro@hotmail.com, rksmicro@yahoo.co.in (R.K. Saxena).

Bioresource Technology 100 (2009) 4074–4076

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech