Acido Lactico SSF

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Process Biochemistry 40 (2005) 681690

Direct fermentation of starch to l (+ ) lactic acid in SSF by Lactobacillusamylophilus GV6 using wheat bran as support and substrate:

medium optimization using RSM

B.J. Naveena a, Md. Altaf a, K. Bhadrayya b, S.S. Madhavendra c, Gopal Reddy a,

a Department of Microbiology, Osmania University, Hyderabad 500007, AP, Indiab Swaroop Tech. Consultancy, Secunderabad 500003, AP, India

c Indian Institute of Chemical Technology (IICT), Hyderabad 500007, AP, India

Received 6 November 2003; received in revised form 2 January 2004; accepted 30 January 2004

Abstract

Wheat bran is an underexploited cheap byproduct obtained during the milling process of wheat. The ability of Lactobacillus amylophilusGV6 to hydrolyze raw starch in wheat bran to produce l (+ ) lactic acid was studied in solid state fermentation (SSF), opening a novel methodfor the utilization of these agricultural byproducts. l (+ ) Lactic acid production by L. amylophilus has been reported by various groups fromprocessed and unprocessed starch but there are no reports available from natural starches in SSF. All amylolytic wild strains reported sofar have high yield efciencies at low substrate concentrations whereas at high substrate concentrations lactic acid production was low. L.amylophilus GV6 showed high yield efciency at high substrate concentration in SSF. To improve and optimize l (+ ) lactic acid productionby L. amylophilus GV6, response surface methodology (RSM) using central composite rotatable design was adopted in SSF. MINITAB-13was used for planning the experiments, data analysis, contour diagrams and response optimizations. The optimum media composition wasobtained as peptone, 0.9%; yeast extract, 0.88%; tri-ammonium citrate, 0.379%; NaH 2PO4 2H2O, 0.769% and Tween-80, 0.30 ml. Under

these conditions a maximum of 36g of lactic acid was produced per 100 g of wheat branhaving 54 g of starch. The organismshowed 90% yieldefciency based on substrate consumed. Successful optimization of the selected ingredients, led to 100% increase in lactic acid production,i.e. from 18 to 36 g. Due to its high potentiality in conversion of starch to l (+ ) lactic acid, L. amylophilus GV6 can be exploited industriallyfor developing a novel technology using inexpensive renewable resources. 2004 Elsevier Ltd. All rights reserved.

Keywords: Lactobacillus amylophilus ; Wheat bran; Lactic acid; RSM; SSF

1. Introduction

Lactic acid fermentation has received extensive attentionfor a long time [1,2] . Lactic acid has wide applicationsin food, pharmaceutical, leather, textile industries and as achemical feed stock. It has two enantiomers l (+ ) and d ( )of which l (+ ) isomer is used by human metabolism due tothe presence of l lactate dehydrogenase and is preferred forfood. In view of recent developments l (+ ) lactic acid is ingreat demand due to its use as starting material to producebiodegradable polymers used in medical, industrial and con-sumer products [36] . The lactic acid market is expected to

Corresponding author. Tel.: + 91-4027682246; fax: + 91-4027090020. E-mail addresses: [email protected] (B.J. Naveena),

[email protected] (G. Reddy).

grow 8.6% annually and in US alone its demand is expectedto be 49,600MT in future [7,8] .

Lactic acid is produced by chemical synthesis and mi-crobial fermentation. Chemical synthesis results in racemicmixture of lactic acid whereas specic stereo isomeric formcan be obtained by microbial fermentation [9,10] . Renedsugars, although expensive are the most commonly usedsubstrates for producing lactic acid by fermentation [2].Lactic acid is also produced from cheaper substrate likestarch in two-step process of saccharication followed by Lactobacillus fermentation [10]. Use of a microorganismwhich directly ferments starch to l (+ ) lactic acid wouldeliminate saccharication cost and Lactobacillus amy-lophilus GV6 is one such organism having high productionefciency in submerged fermentation as reported earlier[1113] .

0032-9592/$ see front matter 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.procbio.2004.01.045


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682 B.J. Naveena et al. / Process Biochemistry 40 (2005) 681690

Solid-state fermentation (SSF) technology has gained re-newed attention from industry, due to its advantages andreported to be the most appropriate process for developingcountries [14,15] . Use of SSF in direct conversion of starchto l (+ ) lactic acid is not yet exploited. It is known that mini-mal preprocessing and supplementing of inorganic nutrients

to inexpensive substrates, originated from waste materials(starch or cellulosic substances), would offer great advan-tage to increase lactic acid production [1618] . A number of cheaply available raw substrates (brans) have been screenedfor single step production of lactic acid by L. amylophilusGV6 in SSF. Wheat bran was selected as the best substrateand support in SSF in laboratory level fermentations [19].

In the present study, to achieve success in faster yieldimprovements, the media components peptone, yeast ex-tract, tri-ammonium citrate, NaH 2PO4 2H2O and Tween-80were evaluated for lactic acid production in SSF and sta-tistically optimized to enhance productivity. Each variablewas tested based on our prior experience over a range andxed in central composite rotatable design of RSM. The roleof each variable, their interactions and statistical analysis toobtain predicted yields of lactic acid was explained by ap-plying second-order polynomial model. The data was ana-lyzed statistically and response surface contour plots wereconstructed which indicated the possibility of enhancementin the production of lactic acid. The analysis was done usingMINITAB-13.

2. Materials and methods

2.1. Microorganism

Lactobacillus amylophilus GV6, a facultatively anaero-bic, amylolytic lactic acid producing strain, isolated, char-acterized and maintained in this laboratory was used in thepresent SSF studies [1113] .

2.2. Fermentation media

SSF was carried out anaerobically at 37 C, pH 6.75 in120 ml serum vials with prereduced and sterilized mediumcomposed of 10 g of wheat bran and appropriate volumeof moistening liquid (83%). The moistening liquid containssodium acetate 0.2%, magnesium sulphate 0.05%, calciumcarbonate 8%, and the ve nutrients (peptone, yeast extract,tri-ammonium citrate, NaH 2PO4 2H2O and Tween-80) se-lected for optimizations according to the requirement of ex-perimental design. N 2 is used as head space gas. Lactic acidextraction is done after 5 days of incubation.

2.3. Substratemicrobe interaction by scanning electronmicroscope

Fermented and unfermented wheat bran samples werexed in 4% gluteraldehyde in phosphate buffer (pH 6.9

and 0.1M), dehydrated in alcohol and these dehydratedsamples were placed on aluminium stubs gold coated inHUS-GB vacuum evaporator, observed in Hitachi S-520scanning electron microscope (SEM).

2.4. Extraction and estimation of lactic acid

Lactic acid formed after fermentation was extractedfrom fermented bran with 50 ml distilled water by vigor-ous shaking and ltering through cheesecloth after allow-ing it to stand for 3 h. The extract was cold centrifuged(8000 rpm for 20 min) and supernatant was taken for es-timating the lactic acid. Total lactic acid was estimatedaccording to the method of Kimberley and Tailor [20].The concentration of lactic acid is expressed as gramper 10g of the fermented bran. Total starch content inbran was determined by standard acid hydrolysis method[21].

2.5. Response surface methodology (RSM)

The central composite rotatable design [22] was used todevelop mathematical models that relate the variables in theexperiments and optimize them for lactic acid production.According to this design, 32 experiments were performed.Details of response surface methodology can be found inNeter et al. [23]. The matrix for this design along with ob-served results are shown in Table 1 .

2.6. Statistical analysis

RSM is used to study the variables independently fortheir interactions and quadratic effects. MINITAB-13 wasused for analysis of observed data, contour diagrams, re-gression coefcients, t -statistics and response optimizations.Five variables were considered for application of RSM. Thebehaviour of the system was explained by the followingsecond-order polynomial equation.

Y = B0 + B i X i + B iiX2i + B ijX iX j (1)

in which Y is predicted response which is a dependentvariable, i.e. lactic acid production; B0 is an off set term(constant); Bi is linear effect; Bij is quadratic effect wheni = j and interaction effect when i < j ; Bii is a squaredterm; X i is ith variable, which are called as independentvariables.

The second-order polynomial equations were used to esti-mate the response of the dependent variable, i.e. lactic acid.Later an experiment was run using the optimum values forvariables given by response optimization for conrmationof the predicted value and the lactic acid production wasconrmed.


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Table 1Central composite rotatable design for optimization of ve nutrients (each at ve levels) and mathematically predicted values and experimental valuesfor the production of l (+ ) lactic acid by Lactobacillus amylophilus GV6 in SSF

Run Peptone ( x1) Yeast extract ( x2) Triammoniumcitrate ( x3)

NaH2PO4 2H2O ( x4) Tween-80 ( x5) Lactic acid production gram per 10g fb a

Experimental values Predicted values

1 ( 1) 0.875 ( 1) 0.875 ( 1) 0.4 ( 1) 0.55 (1) 0.25 2.90290 2.78052 (1) 1.625 ( 1) 0.875 ( 1) 0.4 ( 1) 0.55 ( 1) 0.15 1.90190 1.73693 ( 1) 0.875 (1) 1.625 ( 1) 0.4 ( 1) 0.55 ( 1) 0.15 2.23210 2.09494 (1) 1.625 (1) 1.625 ( 1) 0.4 ( 1) 0.55 (2) 0.25 1.42890 1.48215 ( 1) 0.875 ( 1) 0.875 (1) 0.8 ( 1) 0.55 ( 1) 0.15 1.78200 1.63896 (1) 1.625 ( 1) 0.875 (1) 0.8 ( 1) 0.55 (2) 0.25 1.42230 1.46937 ( 1) 0.875 (1) 1.625 (1) 0.8 ( 1) 0.55 (2) 0.25 1.80990 1.88498 (1) 1.625 (1) 1.625 (1) 0.8 ( 1) 0.55 ( 1) 0.15 1.47280 1.57739 ( 1) 0.875 ( 1) 0.875 ( 1) 0.4 (1) 1.45 ( 1) 0.15 1.02540 0.8389

10 (1) 1.625 ( 1) 0.875 ( 1) 0.4 (1) 1.45 (1) 0.25 1.68280 1.686511 ( 1) 0.875 (1) 1.625 ( 1) 0.4 (1) 1.45 (1) 0.25 1.55160 1.582912 (1) 1.625 (1) 1.625 ( 1) 0.4 (1) 1.45 ( 1) 0.15 2.72520 2.786113 ( 1) 0.875 ( 1) 0.875 (1) 0.8 (1) 1.45 (2) 0.25 2.00480 2.030114 (1) 1.625 ( 1) 0.875 (1) 0.8 (1) 1.45 ( 1) 0.15 1.71130 1.766115 ( 1) 0.875 (1) 1.625 (1) 0.8 (1) 1.45 ( 1) 0.15 1.57110 1.653716 (1) 1.625 (1) 1.625 (1) 0.8 (1) 1.45 (2) 0.25 1.62050 1.893317 ( 2) 0.500 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 0.88990 1.111718 (2) 2.000 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 1.32000 1.102919 (0) 1.250 ( 2) 0.500 (0) 0.6 (0) 1.00 (0) 0.20 1.63540 1.913120 (0) 1.250 (2) 2.000 (0) 0.6 (0) 1.00 (0) 0.20 2.45590 2.183121 (0) 1.250 (0) 1.250 ( 2) 0.2 (0) 1.00 (0) 0.20 1.79710 2.062722 (0) 1.250 (0) 1.250 (2) 1.0 (0) 1.00 (0) 0.20 2.07260 1.811923 (0) 1.250 (0) 1.250 (0) 0.6 (0) 0.10 (0) 0.20 1.54550 1.723724 (0) 1.250 (0) 1.250 (0) 0.6 (2) 1.90 (0) 0.20 1.80880 1.634925 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 ( 2) 0.10 2.10300 2.265626 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 (2) 0.30 2.58505 2.427127 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 1.93460 1.910928 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 1.90360 1.910929 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 1.80970 1.910930 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 2.05280 1.910931 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 1.85600 1.910932 (0) 1.250 (0) 1.250 (0) 0.6 (0) 1.00 (0) 0.20 1.91300 1.9109

Coded values are given in parenthesis. Real values are in percent w/w.a fb, fermented bran.

3. Results and discussion

3.1. Substratemicrobe interaction

An amylolytic strain, L. amylophilus GV6 is used forproduction of l (+ ) lactic acid in SSF using wheat bran assolid support and substrate having a particle size ranging1.53mm. Smaller particle size is generally used as it pro-vides larger surface area, for diffusion of nutrients, both atthe surface and in the pores of substrates having the sametortuosity [24] and wheat bran fullled the above require-ments in the present study. As the substrate and microbialinteractions are unique, chemical composition and physi-cal properties of the substrate should be taken into consid-eration. Growth characteristics, physiology and productionof metabolites by the organism are based on the extracel-lular enzymes in growth-associated metabolism. Interactionof the organism with wheat bran was observed using SEM.The surface structure of substrate before inoculation was

rough in texture with extensive compact and complex mesh(Fig. 1). After inoculation there was extensive growth of microbial cells seen adsorbed on to the wheat bran parti-cles and dissolution of mesh bres were observed due toextra cellular amylopullulanase production leaving behindrelatively smooth substrate particles ( Fig. 2). This observa-tion explains the conversion of raw starch to glucose, whichin turn is converted to l (+ ) lactic acid by L. amylophilusGV6. There are certain reports explaining the effect of sup-port for Lactobacillus sps. for production of lactic acid. In-ert support of inorganic porous materials was used for lacticacid production by immobilized cells of Lactobacillus rham-nosus [25]. Lactobacillus delbruckii (mutant DP3), Lacto-bacillus casei (ATCC 11443) and L. amylophilus (NRRLB-4437) produced more lactic acid using glucose as carbonsource in a trapped state on solid support (polypropylenechips) than in free form [26]. In the present study inexpen-sive wheat bran served both as solid support and substratesuccessfully.


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Fig. 1. Scanning electron microscope (SEM) photograph of unfermented wheat bran with compact starch bres.

3.2. Optimization for lactic acid production

Signicantly contributing variables analyzed from previ-ous experiments were used in RSM to check the best oper-ating parameters and decide optimum operating conditionsfor the fermentation process. The Students t distributionand corresponding values, along with parameter estimateare given in Table 2 . The probability ( P ) values were used asa tool to check the signicance of each of the coefcients.The results show that among the independent variables ( x1),two variables ( x2) yeast extract and ( x3) tri-ammonium cit-rate have signicant effect. Among the interactions( x1 x4)peptone and NaH 2PO4 2H2O ( x1 x5) peptone and Tween-80( x2 x4) yeast extract and NaH 2PO4 2H2O ( x2 x5) yeast

Fig. 2. Scanning electron microscope photograph of fermented wheat bran with bacterial cells showing the hydrolyzed starch bres.

extract and Tween-80 ( x3 x4) tri-ammonium citrate andNaH2PO4 2H2O are highly signicant ( Table 2). FromFig. 3 it is evident that due to dominating interaction effectsof peptone and NaH 2PO4 2H2O higher levels of these nutri-ents give higher yields of lactic acid. It is also observed thatdecrease in peptone and increase in Tween-80 concentrationsis giving higher yield of lactic acid ( Fig. 4). Fig. 5 showshigher yield of lactic acid at both higher and lower levels of yeast extract and NaH 2PO4 2H2O whereas yield is promi-nent at higher levels of these variables. Maximum lactic acidproduction is observed at lower yeast extract concentrationwith increase in Tween-80 concentration and vice versa dueto interaction effect as shown in Fig. 6. At higher concen-trations of both tri-ammonium citrate and NaH 2PO4 2H2O


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Table 2Coefcients and t -values for l (+ ) lactic acid production in SSF using central composite rotatable design

Variable Designate Coefcients Standard errorof coefcients

t -value Probability

Constant I 1.9109 0.10649 17.944 0.000Peptone x1 0.0022 0.05450 0.041 0.968Yeast extract x2 0.0675 0.0545 1.238 0.241(NH4)3citrate x3 0.0627 0.05450 1.151 0.274NaH2PO4 2H2O x4 0.0222 0.05450 0.738 0.476Tween-80 x5 0.0402 0.05450 0.788 0.476Peptone peptone x21 0.2009 0.04930 4.076 0.002Yeast extract yeast extract x22 0.0343 0.04930 0.695 0.502(NH4)3 citrate (NH4)3 citrate x23 0.0066 0.04930 0.133 0.897NaH2PO4 2H2O NaH2PO4 2H2O x24 0.0579 0.04930 1.174 0.265Tween-80 Tween-80 x25 0.1088 0.04930 2.208 0.049Peptone yeast extract x1 x2 0.0675 0.06675 1.011 0.334Peptone (NH4)3 citrate x1 x3 0.0605 0.06675 0.906 0.384Peptone NaH2PO4 .2H2O x1 x4 0.2555 0.06675 3.828 0.003Peptone Tween-80 x1 x5 0.2072 0.06675 3.104 0.010Yeast extract (NH4)3 citrate x2 x3 0.0544 0.06675 0.815 0.432Yeast extract NaH2PO4 2H2O x2 x4 0.1318 0.06675 1.975 0.074Yeast extract Tween-80 x2 x5 0.1989 0.06675 2.980 0.013(NH4)3 citrate NaH2PO4 2H2O x3 x4 0.1188 0.06675 1.780 0.103(NH4)3 citrate Tween-80 x3 x5 0.0399 0.06675 0.598 0.562NaH2PO4 2H2O Tween-80 x4 x5 0.0218 0.06675 0.326 0.750

S , 0.2670; R2 , 6.7%.

lactic acid production was observed to be high ( Fig. 7).Fig. 8 is showing the effect of Tween-80 and tri-ammoniumcitrate on lactic acid production. Either lower or higherconcentrations of tri-ammonium citrate and Tween-80 isfavouring higher yield of lactic acid. Overall it is observedthat Tween-80 is inuencing the production of lactic acidto maximum extent. At higher concentrations of Tween-80the organism might be utilizing certain nutrients present insolid substrate whereas, when concentration of Tween-80 isreduced it is dependent on supplemented nutrients. It is re-ported that use of Tween-80, a surfactant, increased the pro-

Fig. 3. Response surface showing the effect of peptone and NaH 2PO4 2H2O concentrations on lactic acid production.

duction of enzymes [27,28] considerably in turn increasingthe biomass and this may lead to increase in lactic acid pro-duction. NaH 2PO4 2H2O is also observed to have inuenceon lactic acid production as seen in Figs. 3, 5, and 7 wherethe organism requires inorganic phosphate for higher yieldsof lactic acid. It is reported that inorganic phosphate haseffect on lactic acid production by Lactobacillus helvictus

[29].By applying the multiple regression analysis on experi-

mental data, a second-order polynomial model (1) explainsthe role of each variable and their second-order interactions


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Fig. 4. Response surface showing the effect of peptone and Tween-80 concentrations on lactic acid production.

Fig. 5. Response surface showing the effect of yeast extract and NaH 2PO4 2H2O concentrations on lactic acid production.

Fig. 6. Response surface showing the effect of yeast extract and Tween-80 concentrations on lactic acid production.


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Fig. 7. Response surface showing the effect of (NH 4)3 citrate and NaH 2PO4 2H2O concentrations on lactic acid production.

in producing lactic acid.

Y = 1.9109 0.0022x1+ 0.0675x2 0.0627x3 0.0222x4+ 0.0403x5+ 0.0675x1x2 0.0605x1x3 + 0.2555x1x4 0.2072x1x5 0.0544x2x3+ 0.1318x2x4 0.1989x2x5 + 0.1188 x3x4 + 0.0399x3x5 0.0218x4x5 0.2009x21 + 0.0343x

22 + 0.0066x

23

0.0579x24 + 0.1089x25 (2)

The quadratic model in Eq. (2) with 20 terms contains

5 linear terms, 5 quadratic terms and 10, two-factor inter-actions. Out of these, insignicant terms (on the basis of P -values which are more than 0.05) are neglected ( Table 2 ).

Fig. 8. Response surface showing the effect of (NH 4)3 citrate and Tween-80 concentrations on lactic acid production.

The model equation was modied to reduced tted model(3).

Y = 1.9109 0.2009x21 + 0.1088x25 + 0.2555x1x4

0.2072x1x5 0.1989x2x5 (3)

This reduced tted model is considerably simpler and tsthe data almost as well the model (2) with all terms. Henceit can be used for further exploration and validation.

Analysis of variance (ANOVA) wasdone by MINITAB-13.ANOVA ( Table 3 ) indicates that the effect of interactions of variables and their quadratic effects are highly signicant

and the regression between independent variables and output is also quite signicant. The observed and predictedexperimental results are given in Table 1 . The quadratic


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Table 3Analysis of variance for the production of l (+ ) lactic acid by Lactobacillus amylophilus GV6 in SSF

Serial number Source DF SS MS F -value P -value

1 Regression 20 5.1282 0.2564 3.60 0.0172 Linear 5 0.2547 0.0529 0.71 0.6263 Square 5 1.7933 0.3586 5.03 0.012

4 Interaction 10 3.0803 0.3080 4.32 0.0125 Residual error 11 0.7842 0.07126 Model 6 0.7501 0.1250 18.38 0.0037 Pure error 5 0.0340 0.00688 Total 31 5.9123

model in Eq. (2) was used to predict the output of lacticacid with planned parameters and compared with observedvalues. The coefcient of determination ( R2) for productionof lactic acid is 0.8670 ( Table 2 ). This observed variationin production of lactic acid (86.7%) can be explained bythe tted model Eq. (2). The multiple regression ( R) for theproduction of lactic acid is 0.9311. This value shows a goodagreement between experimental observations and predictedvalues. It can be seen that while the highest out put wasobserved at run 1 (2.9029g/10g of bacterial bran) ( Table 1 ).

The overlaid contour plot, response surface plot of yieldfor certain pairs of variables shown at Figs. 6, 8 and 9 in-dicated that there are regions where the out put could bemore than 3.000 g. By running the optimization programwith MINITAB-13 within the experimental range investi-gated, the following optimum values were obtained:peptone,0.9%; yeast extract, 0.88%; tri-ammonium citrate, 0.379%;

0.5 1.0 1.5 2.0

0.5

1.0

1.5

2.0

Peptone

Y E

Hold values: Tri-Amm: 0.6 Sod Di H: 1.0 Tween-80: 0.2

Contour Plot of Lactic acid(

Lactic ac id( 24

Lower BoundUpper Bound

White area: feasible r egion

Fig. 9. Response surface showing the effect of peptone and yeast extract (YE) concentrations on lactic acid production.

NaH2PO4 2H2O, 0.769% and Tween-80, 0.30 ml. The pre-dicted lactic acid production was 32 g/100g of wheat branhaving 54 g of starch. Hence the parameters given by re-sponse optimization were used for conrmation of the pre-dicted value of 32 g lactic acid/100 g of wheat bran. The or-ganism produced 36 g of l (+ ) lactic acid from 54 g of starchpresent in 100 g of wheat bran.

Lactobacillus amylophilus JCM 1125 produced 53.4 g/lof lactic acid using 100g/l, liqueed starch as reportedby Yumoto and Ikeda [10]. Lactobacillus plantarum NCIM2084 produced 72.9 g/l of lactic acid when provided with100g/l of liqueed starch [30]. L. amylophilus (NRRLB4437) produced 29g/l lactic acid from 45 g/l of cornstarch and L. amylovorous was used in conversion of 120 g/lliqueed starch to 92.5g/l lactic acid in submerged fermen-tation [31,32] . In the above reports, enzymatic hydrolysisof starch was done prior to Lactobacillus fermentation


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which is expensive. Wheat straw hemicellulose pretreat-ment through enzymes/acid released 1112g/l fermentablesugars and were used for production of lactic acid inco-culture fermentation using Lactobacillus brevis and Lac-tobacillus pentosus [33]. L. casei subsp. casei CFTRI 2022, L. helviticus CFTRI 2026 and Streptococcus thermophilus

CFTRI 2034 were used in SSF with sugarcane pressmud asa substrate containing 14% total sugars on dry weight basisand reported 6.14% lactic acid production [34]. At highstarch concentrations, the lactic acid production was low[5,10,31,32] . In the present study L. amylophilus GV6 wasfound to produce 36 g of lactic acid from a high concen-tration of raw starch (54g) present in 100 g of wheat bran.Wheat bran was utilized as both support and substrate by L. amylophilus GV6 in single step conversion of raw starchto l (+ ) lactic acid. Compared with other reported strains , L. amylophilus GV6 showed higher amylolytic and lacticacid production efciencies which ensured elimination of saccharication steps of raw starch. A detailed study wasmade to nd out the lactic acid production yield (i.e. gramof lactic acid produced per gram of starch utilized). Theorganism could produce 90% lactic acid yield, which iscomparable with that of submerged fermentation reportedearlier for this organism [13]. Expensive pure starch couldbe replaced with cheaper underutilized abundantly availablebyproduct like wheat bran. The replacement of pure starchwith wheat bran will bring down the cost of substrate 100times and will make the whole process more economicalin terms of substrate costs. When compared to sucrose andhydrolyzed starch, the substrate costs will be still loweredto many folds.

Successful application of RSM to optimize the selectedingredients led to two-fold (100%) increase in lactic acidproduction. L. amylophilus GV6 is found to be the mostefcient in production of l (+ ) lactic acid both in submergedand solid state fermentations at high substrate concentrationsas per the available reports. Also there are no reports on useof amylolytic Lactobacillus in SSF. Thus, L. amylophilusGV6 with its 90% lactic acid yield efciency with cheaprenewable natural starchy substrates could turn out to be anovel strain to develop a potential technology.

Acknowledgements

BJN thanks the Council of Scientic and Industrial Re-search (CSIR) Government of India, New Delhi, for provid-ing Senior Research Fellowship (SRF) to carryout this work.

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