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Biochemical Engineering Journal 50 (2010) 77–81

Contents lists available at ScienceDirect

Biochemical Engineering Journal

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

hort communication

ulti-objective process optimization and integration for the sequential andncreased production of biomass, lipase and endospores of a probiotic bacterium

ubhasish Das, Shailesh Kharkwal, Saurabh K. Pandey, Ramkrishna Sen ∗

epartment of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India

r t i c l e i n f o

rticle history:eceived 20 December 2009eceived in revised form 10 March 2010ccepted 13 March 2010

a b s t r a c t

The objective of this study was to substantially enhance the yields of lipase, biomass and spores froma sporogenous probiotic bacterium, Bacillus coagulans RK-02 by multivariate response surface modelingand genetic algorithm based optimization. The effect of temperature, agitation and aeration on timecourse of growth, lipase formation and sporulation of individual batch cultivation were also studied.The optimum conditions for three responses were found to be different from each other. Comparatively

eywords:acillus coagulans RK-02robioticiomasspore

lower temperature and higher agitation and aeration were needed for biomass and lipase productionthan that for maximizing sporulation rate. In the final validation experiment, three different optimalconditions for maximizing each of these responses, namely biomass, lipase and spore yields, in a stagewise manner were maintained respectively. This strategy produced 6.25 g L−1 biomass, 6 × 1012 sporesper gram of biomass, and maximum of 13.46 IU lipase. Such high yield of biomass, lipase and spore from

ver to

ipaseultivariate optimization

batch cultivation is first e

. Introduction

The term ‘Probiotics’ is no more unknown and uncommonven for the laymen, because of its frequent scores in the pre-criptions made by family doctors. ‘Probiotics are friendly bacteriahich when administered in adequate quantity confer beneficial

ffect on the host’ [1]. Out of several lactobacilli, bifidobacteria andther bacteria which are popular in current market as probiotics,acillus coagulans has established its superiority in pharmaceuti-al industries for its unique properties [2]. Firstly, B. coagulans issporogenous [3] lactic acid producing bacteria [4], which can be

tored at room temperature and therefore can be handled more eas-ly in different stages of processing and packaging. Secondly, it canroduce number of industrially important extracellular enzymes,hen nutrient in the culture broth is exhausted, before finally

porulation starts. One such enzyme is lipase, which is one of theost demanding enzymes worldwide. Lipases (EC 3.1.1.3) catalyze

ydrolysis of fatty acid esters of triglycerides to produce fatty acidnd glycerol. Among several applications of lipases, most popu-ar are as an ingredient in detergent, in paper and pulp industry,

iocatalysis of stereoselective transformation, increasing stabil-

ty of sol–gel matrices and enantioselectivity [5] and in biodieselreparation [6]. Lipase is produced and is stable in alkaline pH [7].

nterestingly Bacilli form spores at alkaline pH [8]. It was there-

∗ Corresponding author. Tel.: +91 3222 283752; fax: +91 3222 278707.E-mail address: rksen@yahoo.com (R. Sen).

369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2010.03.006

be reported.© 2010 Elsevier B.V. All rights reserved.

fore hypothesized that an insoluble carbon source namely edibleoil, if present in media then, upon exhaustion of primary source,i.e., glucose, cells might start producing lipase to use as alternativecarbon source for growth. Upon exhaustion of oil in media sporu-lation would be facilitated due to presence of favorable metaboliccondition in medium. Maximization of biomass in a spore form withextracellular production of lipase simultaneously in a batch culturecan therefore be very economical. In this paper these various goalshave been tried to achieve from same batch cultivation of a pro-biotic B. coagulans RK-02 by optimizing environmental conditions,using a previously optimized culture medium. Such multi-responseoptimization is not only difficult from mathematical point of view,but also is tricky for a biochemical engineer to design the exper-iments and finally implementing them into practice. This type ofwork is first of its kind to be reported for any esterase producingprobiotic bacteria.

2. Materials and methods

2.1. Culture media and fermenter

A locally isolated strain designated as B. coagulans RK02 [13],and a previously optimized medium containing 10 g L−1 peptone,

10 g L−1 glucose and mineral salts (NH4NO3 0.22, KH2PO4 0.14, NaCl0.01, MgSO4 0.6, CaCl2 0.04, FeSO4 0.02, K2HPO4 2.2, CH3COONa 5,50 �L trace element solution with the composition of trace ele-ments (g L−1): ZnSO4, 7H2O 23.2, MnSO4, 4H2O 17.8, H3BO3 5.6,CuSO4, 5H2O 10, Na2MO4·2H2O 3.9, CoCl2·6H2O 4.2, EDTA 10,

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iCl2·6H2O 0.04, KI 6.6) was used for all the batch cultivations,hich were performed in Bioengineering KLF2000 benchtop fer-enter (vessel volume 3.7 L). For inoculum preparation 200 mLlucose Yeast Extract Acetate (GYEA) medium (HiMedia Labora-

ories Pvt. Ltd., Mumbai, India) was used. 8 h old inoculum, whichas cultivated in shake flask with incubation temperature 37 ◦C and

gitation 180 rpm was added to sterilized medium in fermenter toake final volume 2 L. Total amount of oil added (0.5%, v/v) is kept

ow deliberately, compared to previous reports (generally 1% or 2%,/v) [9] for lipase producing media, so that oil is consumed early,o pave the way for spore formation.

.2. Spore count

Sample culture broth was heated at 75 ◦C for 30 min, followed bymmediately cooling down to 45 ◦C. This heat treatment activateshe spores, while the vegetative cells die. Number of spores per mln this heat-treated mixture was determined by plate counting.

.3. Assay of residual sugar in culture media

Anthrone sulfuric acid method [10] was used for the analysis ofesidual sugar.

.4. Microscopy of vegetative cells and endospores

For quality control gram staining was performed for eachample. This confirmed whether any contamination occurred.lympus fluorescent microscope (Model-IX51) was used to study

he morphologies at various stages of growth and sporulation.or spore staining, a 0.1% aqueous solution of auramine O (4,4′-midocarbonyl-bis-N,N-dimethylaniline, Sigma–Aldrich, USA) wassed [11].

.5. Assay of lipase activity

Lipase activity was assayed by the colorimetric method of Win-ler and Stuckmann, using pNPP (p-nitrophenyl palmitate) [12].

.6. Designing the experiments: justification for selection ofarameter range

There are a few reports on optimization of environmental con-itions for maximum production of biomass, spore and lipaseormation separately from B. coagulans. One such report said opti-

um condition for maximum biomass formation in GYEA mediumas: pH 6.65, temperature = 38.3 ◦C, agitation = 247 rpm, and aer-

tion = 1.05 vvm, whereas for maximum sporulation pH 6.27,emperature = 41.4 ◦C, agitation = 115 rpm, and aeration = 0.33 vvm13]. Another report stated that at 30 ◦C lipase production was max-mum in a culture of B. coagulans isolated from soil sample, whilebove 40 ◦C and bellow 20 ◦C lipase production was insignificant.H 7 was best for production [14]. For present study pH was notontrolled and was allowed to change freely according to culturalondition of the broth, because of two reasons—(a) to study theffect of temperature, agitation and aeration on physicochemicalroperties of broth over different time intervals and (b) not tobstruct normal metabolic activities of broth, so that normal sporeormation and product formation are not reduced. High value of

gitation or aeration could be detrimental to culture and resultingesponses may lead to misleading conclusion. Therefore upper lim-ts of both agitation and aeration were deliberately kept low. Theanges (−1.414 to +1.414 level) of temperature, agitation and aera-ion for experimental were chosen as 31.6–48.4 ◦C, 48.9–301.1 rpm,9.1–220.9 L h−1, respectively.

ing Journal 50 (2010) 77–81

2.6.1. Statistical methodKeeping all these informations in mind a central composite

design (CCD) with six centre point replications was designed withfollowing combination of environmental conditions were designedfor 23 + 2 × 3 + 6 = 20 experiments. This helped to keep the numberof experiments to lowest possible. The list of all possible combi-nations of different environmental conditions is given in Table 1.Coded values are related to independent variables in Table 1 byfollowing equation:

xi = Xi − X∗i

�X,

where xi and Xi are coded and natural values of ith variable respec-tively, X∗

iis centre point value (for temperature, X∗

i= 40 ◦C, for

agitation X∗i

= 175 rpm, for aeration X∗i

= 120 L h−1) and �X is stepchange value (for temperature, �X = 5 ◦C, for agitation �X = 75 rpm,for aeration �X = 60 L h−1) and responses yj = (Yj − Y∗

j)/Y∗

j, where

yj , Yj and Y∗j

denotes coded response, observed response and aver-age response respectively and j = 1, 2, 3 denote biomass, number ofspores per mL and lipase activity, respectively. The coded responseswere so used instead of original ones to remove model specificationerror or any multicollinearity, if exists at all. According to experi-mentally obtained response data response surfaces were generatedusing genetic algorithm with the help of MatLab 7.6. Multipleregression analysis by least square method would generate ANOVAdata for verification of the robustness of the models.

It was found after trying several different models a quadraticmodel would be best to describe each of the response surfaces interms of the three coded parameters:

yj = ˇ0 + ˇ1x1 + ˇ2x2 + ˇ3x3 + ˇ11x12 + ˇ22x22 + ˇ33x32

+ ˇ12x1x2 + ˇ23x2x3 + ˇ31x3x1 + ε (1)

In Eq. (1), ˇ0 and ˇi, (i = 1, 2, 3), are unknown parameters, andε represents the random error in yj . For three different responsesthree different equations would be obtained.

3. Results and discussion

The design setting in original variables along with multi-response data, are given in Table 1. All experiments were performedin triplicates and average values were plotted in graphs show-ing trends in time of production (Fig. 1). For all six central pointsvalues were kept same by putting average value to improve R2.However putting same value for all these six experiments were notmandatory, because doing so may sometime result in decreasedadjusted R2 and erroneous model fitting. However it was not thecase for this work. It was observed that rates of lipase produc-tion, biomass production and spore formation were differentlyaffected by temperature, agitation and aeration. More lipase wasproduced in lower temperature, and higher agitation and aera-tion, whereas for biomass formation slightly higher temperaturewould be needed. Spore formation was increased with increase intemperature and lowering of agitation and aeration (Table 1).

Effect of temperature, agitation, and aeration variation on timecourse of biomass, lipase activity, and number of spores per gram ofbiomass is given in Fig. 1. It was common for all batches that afterglucose was consumed totally (data not shown in figures) lipaseproduction started. When glucose was consumed rapidly biomassincreased exponentially. Thereafter lipase was formed to consume

oil in media. B. coagulans RK-02 produces exopolysaccharides in thestationary phase [15], which helps to emulsify oil and thereby oil istaken up from culture media by the bacteria, after the triglyceridesof fatty acids are hydrolyzed by lipase. When all carbon sourceswere exhausted sporulation started.

S. Das et al. / Biochemical Engineering Journal 50 (2010) 77–81 79

Table 1Experimental design and response values.

Std Temperature (◦C), X1 Agitation (rpm), X2 Aeration (L/h), X3 Biomass (g), Y1 No. of spores/g, Y2 Lipase activity (U/mL), Y3

1 35 100 60 1.92 6 × 1011 2.672 45 100 60 1.23 2.6 × 1012 1.383 35 250 60 2.65 3.7 × 1011 2.884 45 250 60 1.51 9 × 1011 1.435 35 100 180 2.49 2.8 × 1010 3.036 45 100 180 1.47 2 × 1011 1.527 35 250 180 2.76 1 × 1010 3.048 45 250 180 1.86 8 × 1010 1.579 31.6 175 120 2.14 6.4 × 109 2.5

10 48.4 175 120 1.12 2 × 1012 0.4311 40 48.9 120 1.63 3.3 × 1012 1.512 40 301.1 120 2.72 4.4 × 1011 2.6513 40 175 19.1 1.81 2.4 × 1012 1.7814 40 175 220.9 2.54 9 × 1010 2.7515 40 175 120 2.64 5.3 × 1012 2.6416 40 175 120 2.64 5.3 × 1012 2.6417 40 175 120 2.64 5.3 × 1012 2.6418 40 175 120 2.64 5.3 × 1012 2.6419 40 175 120 2.64 5.3 × 1012 2.6420 40 175 120 2.64 5.3 × 1012 2.64

Fig. 1. Time courses of biomass, lipase activity, spore concentration at (A) 31.6 ◦C, 175 rpm, 120 L h−1; (B) 40 ◦C, 175 rpm, 19.1 L h−1; (C) 48.6 ◦C, 175 rpm, 120 L h−1; (D) 40 ◦C,301 rpm, 120 L h−1.

Table 2Least squares fit and parameter estimates for biomass, spores and lipase.

Factor Y1 (biomass) Y2 (no. of spores/g) Y3 (lipase activity)

Coeffn Std. err. t-Stat p-Val Coeffn Std. err. t-Stat p-Val Coeffn Std. err. t-Stat p-Val

Intercept 0.208 0.026 8.088 0.000 1.368 0.093 14.764 0.000 0.171 0.057 2.996 0.013x1-temperature −1.466 0.137 −10.737 0.000 1.602 0.492 3.255 0.009 −2.402 0.303 −7.916 0.000x2-agitation 0.274 0.040 6.881 0.000 −0.524 0.143 −3.654 0.004 0.229 0.088 2.590 0.027x3-aeration 0.167 0.034 4.906 0.001 −0.525 0.123 −4.274 0.002 0.116 0.076 1.527 0.158x1x2 −0.352 0.416 −0.847 0.417 −1.633 1.499 −1.090 0.302 −0.125 0.924 −0.135 0.895x1x3 −0.082 0.357 −0.231 0.822 −2.040 1.285 −1.588 0.143 −0.214 0.792 −0.270 0.793x2x3 −0.093 0.104 −0.898 0.390 0.467 0.375 1.245 0.241 −0.052 0.231 −0.225 0.827x2

1 −10.279 1.065 −9.653 0.000 −46.587 3.836 −12.144 0.000 −9.997 2.366 −4.226 0.002x2

2 −0.393 0.090 −4.339 0.001 −3.212 0.326 −9.855 0.000 −0.519 0.201 −2.583 0.027x2

3 −0.288 0.066 −4.339 0.001 −2.753 0.239 −11.500 0.000 0.003 0.148 0.020 0.985

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A second order model was fitted to each of the three set ofesponse values. R2 values above 0.9, high F-values, high t-valueeither positive or negative) and p-values less than 0.05 implieshat the models for all three responses fitted well to experimentalata. A visual overview of interaction between the environmentalarameters can be compared with products of coefficient estimatesx1x2, x2x3 and x3x1 in Table 2). For 95% confidence interval wenalyzed the t-statistics of parameters. For 95% confidence inter-al, if t-statistics is in the interval of [−2, +2] then the coefficientould not be significant or we cannot reject the hypothesis that

orresponding coefficient is zero. In case of biomass t-statistics ofnteraction terms are in between this range. So coefficients of thesenteraction terms are not significant. Similarly in case of sporeshese interaction terms are not contributing significantly in the pre-iction of response variable. However since both adjusted R2 and2 are above 0.9 models were not changed.

In case of lipase only coefficient of temperature and agitationad a significant coefficient. Neither aeration nor the interactionerms are significant and therefore could be omitted to give bet-er fitting. Since for this model adjusted R2 was only 0.82, whichould be improved in the course of exploring better model. How-ver removing coefficients of x3, x2

3, x1x2, x2x3, and x3x1 decreased2 to 0.85. Keeping coefficient of x3 did increase adjusted R2 to 0.87nd R2 was 0.90 and t-statistic for coefficient of x3 was increasedrom 1.53 to 1.8. So the final equation for lipase activity would be:

ˆ3 = 0.172 − 0.3x1 + 0.982x2 + 0.058x3 − 0.156x21 − 0.095x2

2

Therefore lipase production had linear relationship with aer-tion. Although variation of aeration affected the response lesserhan the other terms did, its effect could not be ignored totally.

The optimum conditions and effects of temperature, agita-ion and aeration on biomass, spore concentration and lipase inarvests are shown in Fig. 2A–C. As discussed earlier the corre-ponding maxima of the three-response functions were obtainedt different locations within experimental regions. Actual val-es of all parameters are calculated by formula: actual value = av.alue or central value × (1 + xi). All the parameters and responsesave been presented in corresponding coded values. For biomassptimum condition was determined as: temperature 36.86 ◦C,gitation 238 rpm, and aeration 149 L h−1. The contour plot rep-esenting effect of agitation and aeration on biomass productionas drawn at x1 (temperature) = −0.627, i.e., 36.86 ◦C. Similarly

emperature–aeration contour plot was drawn at x2 (agita-

ion) = 0.828, i.e., 238 rpm and temperature–agitation contour plotas drawn at x3 (aeration) = 0.488, i.e., 149 L h−1. Optimum biomassas 0.34 (coded value), i.e. 2.92 g L−1 (Fig. 2A).

Optimum condition for maximum possible spore yield wasredicted to be 40.95 ◦C temperature, 158.3 rpm agitation and

ig. 2. Effects of temperature, agitation and aeration on (A) final biomass yield, (B) finaarameters while keeping the third at its optimum value fixed, are shown simultaneously.roduct yields.

ing Journal 50 (2010) 77–81

106 L h−1 aeration. Three contour plots was drawn at x1 (temper-ature) = 0.171, i.e., 40.95 ◦C; x2 (agitation) = −0.222, i.e., 158.3 rpm;x3 (aeration) = −0.223, i.e., 106 L h−1; optimum spore yield was 1.44(coded value), i.e., 3.23 × 1012 g−1 (Fig. 2B).

Optimum condition for maximum possible lipase yield waspredicted to be 35.2 ◦C temperature, 213.3 rpm agitation and220.9 L h−1 aeration. Three contour plots have been drawn atx1 (temperature) = −0.961, i.e., 35.2 ◦C; x2 (agitation) = 0.511, i.e.,213.3 rpm; x3 (aeration) = 1.68, i.e., 220.9 L h−1; optimum lipase was0.44 (coded value), i.e., 3.23 IU (Fig. 2C).

When all three-response functions would be made to achieveglobal maxima at the same time, they would practically fail toachieve the same values for individual maximum [16]. There-fore another strategy was applied to achieve maxima in thesame batch, by maintaining three different conditions for obtain-ing maximum biomass, lipase and spore concentration, one afteranother in same batch. 36.86 ◦C temperature, 237 rpm agitation,and 150 L h−1aeration were maintained for first 9 h till glucose isconsumed. Oil was added 0.05% initially as antifoaming agent, sothat bacteria can adapt to this different medium and more oxygenis available for each cell. Since glucose was found to be consumedwithin first 9 h of cultivation, therefore slightly before end-logphase (i.e., after 6 h) rest 9 ml oil was added, when glucose con-centration was just bellow 6 g L−1. Starting from 9th h to 22ndh 35.2 ◦C temperature, 213 rpm agitation, and 220 L h−1 aerationwere maintained, in order to implement predicted condition formaximum lipase production. At 22nd h frothing started in mediaand a drop in lipase level in media was also noted at 24th h. So40.85 ◦C temperature, 158.4 rpm agitation, 106 L h−1 aeration weremaintained till the end of the batch for maximizing spore formationrate in media. This strategy could not be applied for all batches ofstudy because of huge variation in parameter values and resultingvariation in metabolic activities. Following this strategy yields ofbiomass, spore and lipase activity were improved manifold. After36 h of fermentation, the respective values of biomass concentra-tion, lipase activity and spore yield were: 6.19 g L−1, 9.1 IU and6 × 1012 per gram of biomass. However, maximum lipase activity of13.46 IU was obtained at 18 h, and maximum biomass (6.25 g L−1)was obtained at 24th h (Fig. 3). Therefore biomass yield coeffi-cients upon glucose as substrate was YX/S = 0.625 and lipase yieldwas YP/X = 2153.6 U g−1 biomass. These findings are very interest-ing and can be exploited for developing a continuous fermentationstrategy using chemostat with cell recycle mode of reactor oper-ation wherein the clarified broth is withdrawn and processed for

lipase production and the biomass is recycled back to the reactorfor high cell density biomass and spores.

Though several companies are now marketing B. coagulans asprobiotic product, process details of production unit are generallynot brought into general reader’s domain. However [17] reported

l spore yield and (C) final lipase yield. Three contour plots, each with two varyingThree contours in each case meet each other to optimum point denoting maximum

S. Das et al. / Biochemical Engineeri

Fig. 3. Time course of (a) biomass, lipase activity, spore concentration and (b) pHa9t1

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[16] A.I. Khuri, J.A. Cornell, Response surfaces: design and analyses, Dekker, NewYork, 1987, pp. 30–31, 291.

nd D.O. at temperature of 36.86 ◦C, agitation 237 rpm and aeration 149 L h−1 for firsth, followed by temperature of 35.2 ◦C, agitation 213 rpm and aeration 221 L h−1 up

o 22nd h and for the rest of the time afterwards temperature 40.85 ◦C, agitation58 rpm and aeration 107 L h−1.

biomass yield 3.1 g L−1 in steady state in continuous cultivation13]. Reported maximum biomass of 4.3 g L−1 and 9 × 1011 sporeser gram after 33 h. There are plenty of reports on lipase fromicroorganisms other than B. coagulans, but there is scarcity of data

n process optimization of lipase from B. coagulans source in batchultivation. One report by [7] says that lipase activity of culturefter 48 h of cultivation of B. coagulans BTS-3 was 1.16 U mL−1 and14] reported 0.2034 U mL−1 lipase Therefore this result study hasutsmarted all other reports so far in terms of biomass, lipase andpore yield and faster rate of production at the same time from a. coagulans cultivation as far as available literature is concerned.here are other high lipase yielding strains of different microorgan-sms that yielded either less or similar yield of lipase. Only a fewtudies have been reported to yield more lipase than the presenttudy.

. Conclusion

A multivariate process optimization and integration strategyas effectively employed for the simultaneous optimization of the

ritically influencing process parameters for the enhanced produc-ion of probiotic biomass, endospores and extracellular lipase by. coagulans RK-02. In the validation protocol, maintaining threeifferent optimal environmental conditions each for maximizingiomass, lipase, and spore concentration respectively with slightlyodified strategy resulted in more than two times increase in

iomass, about four times more lipase and about twice sporeoncentration than that predicted for individual responses. Con-idering an incubation period of 36 h, these values are significantly

igher than those reported in literature. To the best of our knowl-dge this is the first time process optimization and integrationrocedure was employed to simultaneously enhance biomass sporend an important enzyme like lipase simultaneously enhanced byprobiotic culture.

[

ng Journal 50 (2010) 77–81 81

Acknowledgements

SD acknowledges the financial assistance received from theCSIR, Govt. of India through a sponsored project [Grant No.:37(1230)/05/EMR-II]. SD and SK acknowledge Mr. Prakash Sahaand L. Vineet Kishore for some technical assistance in instrumenthandling.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bej.2010.03.006.

References

[1] G. Reid, J. Jass, M.T. Sebulsky, McCormick, Potential uses of probiotics in clinicalpractice, Clin. Microbiol. Rev. 16.4 (2003) 658–672.

[2] H.A. Hong, L.H. Duc, S.M. Cutting, The use of bacterial spore formers as probi-otics, FEMS Microbiol. 29 (2005) 813–835.

[3] P.S. Bora, V. Puri, A.K. Bansal, Physicochemical properties and excipient com-patibility studies of probiotic Bacillus coagulans spores, Sci. Pharm. 77 (2009)625–637.

[4] V. Heribane, E. Sturdik, L. Zalibera, P. Matus, Process and metabolic character-istics of Bacillus coagulans as a lactic acid producer, Lett. Appl. Microbiol. 16(1993) 243–246.

[5] K.-E. Jaeger, M.T. Reetz, Microbial lipases from versatile tools for biotechnology,Trends Microbiol. 16 (1998) 396–403.

[6] J. Huang, Y. Liu, S. Wang, Silanized palygorskite for lipase immobilization, J.Mol. Catal. B: Enzym. 57 (2009) 10–15.

[7] S. Kumar, K. Kikon, A. Upadhyay, S.S. Kanwar, R. Gupta, Production, purifica-tion, and characterization of lipase from thermophilic and alkaliphilic Bacilluscoagulans BTS-3, Protein Exp. Purif. 41 (2005) 38–44.

[8] J.-H. Cho, Y.-B. Kim, E.-K. Kim, Optimization of culture medium for Bacillusspecies by statistical optimization design method, Korean J. Chem. Eng. 26.3(2009) 754–759.

[9] S.S. Kanwar, I.A. Ghazi, S.S. Chimni, G.K. Joshi, G.V. Rao, R.K. Kaushal, R. Gupta,V. Punj, Purification and properties of a novel extra-cellular thermotolerantmetallolipase of Bacillus coagulans MTCC-6375 isolate, Protein Expr. Purif. 46(2006) 421–428.

10] E.W. Yemm, A.J. Willis, Estimation of carbohydrates in plant extracts byanthrone, J. Biochem. 57 (1954) 508–514.

11] J.W. Bartholomew, M.W. Lechtman, H. Finkelstein, Differential spore and lipidstaining at room temperature by use of fluorescent dye, J. Bacteriol. 90.4 (1965)1146–1147.

12] U.K. Winkler, M. Stuckmann, Glycogen, hyaluronate and some other polysac-charides greatly enhance the formation of exolipase by Serratia marcescens, J.Bacteriol. 138 (1979) 663–670.

13] R. Sen, K.S. Babu, Modelling and optimization of the process conditions forbiomass production and sporulation of a probiotic culture, Proc. Biochem. 40(2005) 2531–2538.

14] M.P.P. Kumar, A.K. Valsa, Optimization of culture media and cultural conditionsfor the production of extracellular lipase by Bacillus coagulans, Ind. J. Biotechnol.6 (2007) 114–117.

15] V.P. Kodali, S. Das, R. Sen, An exopolysaccharide from a probiotic: biosynthe-sis dynamics, composition and emulsifying activity, Food Res. Int. 42 (2009)695–699.

17] T. Payot, Z. Chemaly, M. Fick, Lactic acid production by Bacilluscoagulans—kinetic studies and optimization of culture medium for batchand continuous fermentations, Enzyme Microbial. Technol. 24 (3–4) (1999)191–199.