一篇典型的响应曲面法优化培养基的文献

Process Biochemistry 40(2005)
681–690
Direct fermentation of starch to l (+)lactic acid in SSF by Lactobacillus
amylophilus GV6using 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 ,India
b Swaroop Tech.Consultancy,Secunderabad 500003,AP ,India
c Indian Institute of Chemical Technology (IICT),Hyderaba
d 500007,AP ,India
Received 6November 2003;received in revised form 2January 2004;accepted 30January 2004
Abstract
Wheat bran is an underexploited cheap byproduct obtained during the milling process of wheat.The ability of Lactobacillus amylophilus GV6to hydrolyze raw starch in wheat bran to produce l (+)lactic acid was studied in solid state fermentation (SSF),opening a novel method for the utilization of these agricultural byproducts.l (+)Lactic acid production by L.amylophilus has been reported by various groups from processed and unprocessed starch but there are no reports available from natural starches in SSF.All amylolytic wild strains reported so far have high yield efficiencies at low substrate concentrations whereas at high substrate concentrations lactic acid production was low.L.amylophilus GV6showed high yield efficiency at high substrate concentration in SSF.To improve and optimize l (+)lactic acid production by 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 was obtained as peptone,0.9%;yeast extract,0.88%;tri-ammonium citrate,0.379%;NaH 2PO 4·2H 2O,0.769%and Tween-80,0.30ml.Under these conditions a maximum of 36g of lactic acid was produced per 100g of wheat bran having 54g of starch.The organism showed 90%yield efficiency based on substrate consumed.Successful optimization of the selected ingredients,led to 100%increase in lactic acid production,i.e.from 18to 36g.Due to its high potentiality in conversion of starch to l (+)lactic acid,L.amylophilus GV6can be exploited industrially for developing a novel technology using inexpensive renewable resources.©2004Elsevier Ltd.All rights reserved.
Keywords:Lactobacillus amylophilus ;Wheat bran;Lactic acid;RSM;SSF
1.Introduction
Lactic acid fermentation has received extensive attention for a long time [1,2].Lactic acid has wide applications in food,pharmaceutical,leather,textile industries and as a chemical feed stock.It has two enantiomers l (+)and d (−)of which l (+)isomer is used by human metabolism due to the presence of l lactate dehydrogenase and is preferred for food.In view of recent developments l (+)lactic acid is in great demand due to its use as starting material to produce biodegradable polymers used in medical,industrial and con-sumer products [3–6].The lactic acid market is expected to
∗
Corresponding author.Tel.:+91-4027682246;fax:+91-4027090020.grow 8.6%annually and in US alone its demand is expected to be 49,600MT in future [7,8].
Lactic acid is produced by chemical synthesis and mi-crobial fermentation.Chemical synthesis results in racemic mixture of lactic acid whereas specific stereo isomeric form can be obtained by microbial fermentation [9,10].Refined sugars,although expensive are the most commonly used substrates for producing lactic acid by fermentation [2].Lactic acid is also produced from cheaper substrate like starch in two-step process of saccharification followed by Lactobacillus fermentation [10].Use of a microorganism which directly ferments starch to l (+)lactic acid would eliminate saccharification cost and Lactobacillus amy-lophilus GV6is one such organism having high production efficiency in submerged fermentation as reported earlier
682 B.J.Naveena et al./Process Biochemistry40(2005)681–690
Solid-state fermentation(SSF)technology has gained re-
newed attention from industry,due to its advantages and
reported to be the most appropriate process for developing
countries[14,15].Use of SSF in direct conversion of starch
to 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[16–18].A number of
cheaply available raw substrates(brans)have been screened
for single step production of lactic acid by L.amylophilus
GV6in SSF.Wheat bran was selected as the best substrate
and support in SSF in laboratory level fermentations[19].
In the present study,to achieve success in faster yield
improvements,the media components peptone,yeast ex-
tract,tri-ammonium citrate,NaH2PO4·2H2O and Tween-80 were evaluated for lactic acid production in SSF and sta-
tistically optimized to enhance productivity.Each variable
was tested based on our prior experience over a range and
fixed in central composite rotatable design of RSM.The role
of each variable,their interactions and statistical analysis to
obtain 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 were
constructed which indicated the possibility of enhancement
in the production of lactic acid.The analysis was done using
MINITAB-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 the
present SSF studies[11–13].
2.2.Fermentation media
SSF was carried out anaerobically at37◦C,pH6.75in
120ml serum vials with prereduced and sterilized medium
composed of10g of wheat bran and appropriate volume
of moistening liquid(83%).The moistening liquid contains
sodium acetate0.2%,magnesium sulphate0.05%,calcium
carbonate8%,and thefive nutrients(peptone,yeast extract,
tri-ammonium citrate,NaH2PO4·2H2O and Tween-80)se-lected for optimizations according to the requirement of ex-perimental design.N2is used as head space ctic acid extraction is done after5days of incubation.
2.3.Substrate–microbe interaction by scanning electron microscope
Fermented and unfermented wheat bran samples were and0.1M),dehydrated in alcohol and these dehydrated samples were placed on aluminium stubs gold coated in HUS-GB vacuum evaporator,observed in Hitachi S-520 scanning electron microscope(SEM).
2.4.Extraction and estimation of lactic acid
Lactic acid formed after fermentation was extracted from fermented bran with50ml distilled water by vigor-ous shaking andfiltering through cheesecloth after allow-ing it to stand for3h.The extract was cold centrifuged (8000rpm for20min)and supernatant was taken for es-timating the lactic acid.Total lactic acid was estimated according to the method of Kimberley and Tailor[20]. The concentration of lactic acid is expressed as gram per10g of the fermented bran.Total starch content in bran was determined by standard acid hydrolysis method [21].
2.5.Response surface methodology(RSM)
The central composite rotatable design[22]was used to develop mathematical models that relate the variables in the experiments and optimize them for lactic acid production. According to this design,32experiments were performed. Details of response surface methodology can be found in Neter et al.[23].The matrix for this design along with ob-served results are shown in Table1.
2.6.Statistical analysis
RSM is used to study the variables independently for their interactions and quadratic effects.MINITAB-13was used for analysis of observed data,contour diagrams,re-gression coefficients,t-statistics and response optimizations. Five variables were considered for application of RSM.The behaviour of the system was explained by the following second-order polynomial equation.
Y=B0+
B i X i+
B ii X2i+
B ij X i X j(1)
in which Y is predicted response which is a dependent variable,ctic acid production;B0is an off set term (constant);B i is linear effect;B ij is quadratic effect when i=j and interaction effect when i<j;B ii is a squared term;X i is i th variable,which are called as independent variables.
The second-order polynomial equations were used to esti-mate the response of the dependent variable,ctic acid. Later an experiment was run using the optimum values for variables given by response optimization for confirmation of the predicted value and the lactic acid production was
B.J.Naveena et al./Process Biochemistry40(2005)681–690683 Table1
Central composite rotatable design for optimization offive nutrients(each atfive levels)and mathematically predicted values and experimental values for the production of l(+)lactic acid by Lactobacillus amylophilus GV6in SSF
Run Peptone(x1)Yeast extract(x2)Triammonium
citrate(x3)NaH2PO4·2H2O(x4)Tween-80(x5)Lactic acid production gram per10g fb a
Experimental values Predicted values
1(−1)0.875(−1)0.875(−1)0.4(−1)0.55(1)0.25 2.90290 2.7805 2(1)1.625(−1)0.875(−1)0.4(−1)0.55(−1)0.15 1.90190 1.7369 3(−1)0.875(1)1.625(−1)0.4(−1)0.55(−1)0.15 2.23210 2.0949 4(1)1.625(1)1.625(−1)0.4(−1)0.55(2)0.25 1.42890 1.4821 5(−1)0.875(−1)0.875(1)0.8(−1)0.55(−1)0.15 1.78200 1.6389 6(1)1.625(−1)0.875(1)0.8(−1)0.55(2)0.25 1.42230 1.4693 7(−1)0.875(1)1.625(1)0.8(−1)0.55(2)0.25 1.80990 1.8849 8(1)1.625(1)1.625(1)0.8(−1)0.55(−1)0.15 1.47280 1.5773 9(−1)0.875(−1)0.875(−1)0.4(1)1.45(−1)0.15 1.025400.8389 10(1)1.625(−1)0.875(−1)0.4(1)1.45(1)0.25 1.68280 1.6865 11(−1)0.875(1)1.625(−1)0.4(1)1.45(1)0.25 1.55160 1.5829 12(1)1.625(1)1.625(−1)0.4(1)1.45(−1)0.15 2.72520 2.7861 13(−1)0.875(−1)0.875(1)0.8(1)1.45(2)0.25 2.00480 2.0301 14(1)1.625(−1)0.875(1)0.8(1)1.45(−1)0.15 1.71130 1.7661 15(−1)0.875(1)1.625(1)0.8(1)1.45(−1)0.15 1.57110 1.6537 16(1)1.625(1)1.625(1)0.8(1)1.45(2)0.25 1.62050 1.8933 17(−2)0.500(0)1.250(0)0.6(0)1.00(0)0.200.88990 1.1117 18(2)2.000(0)1.250(0)0.6(0)1.00(0)0.20 1.32000 1.1029 19(0)1.250(−2)0.500(0)0.6(0)1.00(0)0.20 1.63540 1.9131 20(0)1.250(2)2.000(0)0.6(0)1.00(0)0.20 2.45590 2.1831 21(0)1.250(0)1.250(−2)0.2(0)1.00(0)0.20 1.79710 2.0627 22(0)1.250(0)1.250(2)1.0(0)1.00(0)0.20 2.07260 1.8119 23(0)1.250(0)1.250(0)0.6(0)0.10(0)0.20 1.54550 1.7237 24(0)1.250(0)1.250(0)0.6(2)1.90(0)0.20 1.80880 1.6349 25(0)1.250(0)1.250(0)0.6(0)1.00(−2)0.10 2.10300 2.2656 26(0)1.250(0)1.250(0)0.6(0)1.00(2)0.30 2.58505 2.4271 27(0)1.250(0)1.250(0)0.6(0)1.00(0)0.20 1.93460 1.9109 28(0)1.250(0)1.250(0)0.6(0)1.00(0)0.20 1.90360 1.9109 29(0)1.250(0)1.250(0)0.6(0)1.00(0)0.20 1.80970 1.9109 30(0)1.250(0)1.250(0)0.6(0)1.00(0)0.20 2.05280 1.9109 31(0)1.250(0)1.250(0)0.6(0)1.00(0)0.20 1.85600 1.9109 32(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.Substrate–microbe interaction
An amylolytic strain,L.amylophilus GV6is used for production of l(+)lactic acid in SSF using wheat bran as solid support and substrate having a particle size ranging 1.5–3mm.Smaller particle size is generally used as it pro-vides larger surface area,for diffusion of nutrients,both at the surface and in the pores of substrates having the same tortuosity[24]and wheat bran fulfilled the above require-ments in the present study.As the substrate and microbial interactions are unique,chemical composition and physi-cal properties of the substrate should be taken into consid-eration.Growth characteristics,physiology and production of metabolites by the organism are based on the extracel-lular enzymes in growth-associated metabolism.Interaction of the organism with wheat bran was observed using SEM.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 meshfibres were observed due to extra cellular amylopullulanase production leaving behind relatively smooth substrate particles(Fig.2).This observa-tion explains the conversion of raw starch to glucose,which in turn is converted to l(+)lactic acid by L.amylophilus GV6.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 lactic acid production by immobilized cells of Lactobacillus rham-nosus[25].Lactobacillus delbruckii(mutant DP3),Lacto-bacillus casei(ATCC11443)and L.amylophilus(NRRL B-4437)produced more lactic acid using glucose as carbon source in a trapped state on solid support(polypropylene chips)than in free form[26].In the present study inexpen-sive wheat bran served both as solid support and substrate
684 B.J.Naveena et al./Process Biochemistry 40(2005)
681–690
Fig.1.Scanning electron microscope (SEM)photograph of unfermented wheat bran with compact starch fibres.
3.2.Optimization for lactic acid production
Significantly contributing variables analyzed from previ-ous experiments were used in RSM to check the best oper-ating parameters and decide optimum operating conditions for the fermentation process.The Student’s ‘t ’distribution and corresponding values,along with parameter estimate are given in Table 2.The probability (P )values were used as a tool to check the significance of each of the coefficients.The results show that among the independent variables (x 1),two variables (x 2)yeast extract and (x 3)tri-ammonium cit-rate have significant effect.Among the interactions(x 1x 4)peptone and NaH 2PO 4·2H 2O (x 1x 5)peptone and Tween-80(x 2x 4)yeast extract and NaH 2PO 4·2H 2O (x 2x 5)
yeast
extract and Tween-80(x 3x 4)tri-ammonium citrate and NaH 2PO 4·2H 2O are highly significant (Table 2).From Fig.3it is evident that due to dominating interaction effects of peptone and NaH 2PO 4·2H 2O higher levels of these nutri-ents give higher yields of lactic acid.It is also observed that decrease in peptone and increase in Tween-80concentrations is giving higher yield of lactic acid (Fig.4).Fig.5shows higher yield of lactic acid at both higher and lower levels of yeast extract and NaH 2PO 4·2H 2O whereas yield is promi-nent at higher levels of these variables.Maximum lactic acid production is observed at lower yeast extract concentration with increase in Tween-80concentration and vice versa due to interaction effect as shown in Fig.6.At higher concen-trations of both tri-ammonium citrate and NaH 2PO 4·2H 2O
B.J.Naveena et al./Process Biochemistry 40(2005)681–690
685
Table 2
Coefficients and t -values for l (+)lactic acid production in SSF using central composite rotatable design Variable
Designate Coefficients Standard error of coefficients t -value Probability Constant I 1.91090.1064917.9440.000Peptone
x 1−0.00220.05450−0.0410.968Yeast extract x 20.06750.0545 1.2380.241(NH 4)3citrate x 3−0.06270.05450−1.1510.274NaH 2PO 4·2H 2O x 4−0.02220.054500.7380.476Tween-80
x 50.04020.054500.7880.476Peptone ×peptone
x 21−0.20090.04930−4.0760.002Yeast extract ×yeast extract x 220.03430.049300.6950.502(NH 4)3citrate ×(NH 4)3citrate x 230.00660.049300.1330.897NaH 2PO 4·2H 2O ×NaH 2PO 4·2H 2O x 24−0.05790.04930−1.1740.265Tween-80×Tween-80x 250.10880.04930 2.2080.049Peptone ×yeast extract x 1x 20.06750.06675 1.0110.334Peptone ×(NH 4)3citrate x 1x 3−0.06050.06675−0.9060.384Peptone ×NaH 2PO 4.2H 2O x 1x 40.25550.06675 3.8280.003Peptone ×Tween-80
x 1x 5−0.20720.06675−3.1040.010Yeast extract ×(NH 4)3citrate x 2x 3−0.05440.06675−0.8150.432Yeast extract ×NaH 2PO 4·2H 2O x 2x 40.13180.06675 1.9750.074Yeast extract ×Tween-80
x 2x 5−0.19890.06675−2.9800.013(NH 4)3citrate ×NaH 2PO 4·2H 2O x 3x 40.11880.06675 1.7800.103(NH 4)3citrate ×Tween-80x 3x 50.03990.066750.5980.562NaH 2PO 4·2H 2O ×Tween-80x 4x 5
−0.0218
0.06675
−0.326
0.750
S ,0.2670;R 2,6.7%.
lactic acid production was observed to be high (Fig.7).Fig.8is showing the effect of Tween-80and tri-ammonium citrate on lactic acid production.Either lower or higher concentrations of tri-ammonium citrate and Tween-80is favouring higher yield of lactic acid.Overall it is observed that Tween-80is influencing the production of lactic acid to maximum extent.At higher concentrations of Tween-80the organism might be utilizing certain nutrients present in solid substrate whereas,when concentration of Tween-80is reduced it is dependent on supplemented nutrients.It is re-ported that use of Tween-80,a surfactant,increased the
pro-duction of enzymes [27,28]considerably in turn increasing the biomass and this may lead to increase in lactic acid pro-duction.NaH 2PO 4·2H 2O is also observed to have influence on lactic acid production as seen in Figs.3,5,and 7where the organism requires inorganic phosphate for higher yields of lactic acid.It is reported that inorganic phosphate has effect on lactic acid production by Lactobacillus helvictus [29].
By applying the multiple regression analysis on experi-mental data,a second-order polynomial model (1)explains the role of each variable and their second-order interactions
686 B.J.Naveena et al./Process Biochemistry40(2005)681–690
Fig.4.Response surface showing the effect of peptone and Tween-80concentrations on lactic acid production.
Fig.5.Response surface showing the effect of yeast extract and NaH2PO4·2H2O concentrations on lactic acid production.
B.J.Naveena et al./Process Biochemistry 40(2005)681–690
687
Fig.7.Response surface showing the effect of (NH 4)3citrate and NaH 2PO 4·2H 2O concentrations on lactic acid production.
in producing lactic acid.
Y =1.9109−0.0022x 1+0.0675x 2−0.0627x 3−0.0222x 4
+0.0403x 5+0.0675x 1x 2−0.0605x 1x 3+0.2555x 1x 4−0.2072x 1x 5−0.0544x 2x 3+0.1318x 2x 4−0.1989x 2x 5+0.1188x 3x 4+0.0399x 3x 5
−0.0218x 4x 5−0.2009x 21+0.0343x 22+0.0066x 2
3−0.0579x 24+0.1089x 25
(2)
The quadratic model in Eq.(2)with 20terms contains
5linear terms,5quadratic terms and 10,two-factor inter-actions.Out of these,insignificant terms (on the basis of P -values which are more than 0.05)are neglected (Table 2
).
The model equation was modified to reduced fitted model
(3).
Y =1.9109−0.2009x 21+0.1088x 2
5+0.2555x 1x 4
−0.2072x 1x 5−0.1989x 2x 5
(3)
This reduced fitted model is considerably simpler and fits the data almost as well the model (2)with all terms.Hence it can be used for further exploration and validation.
Analysis of variance (ANOV A)was done by MINITAB-13.ANOV A (Table 3)indicates that the effect of interactions of variables and their quadratic effects are highly significant and the regression between independent variables and out put is also quite significant.The observed and predicted experimental results are given in Table 1.The quadratic
688 B.J.Naveena et al./Process Biochemistry 40(2005)681–690
Table 3
Analysis of variance for the production of l (+)lactic acid by Lactobacillus amylophilus GV6in SSF Serial number Source DF SS MS F -value P -value 1Regression 20 5.12820.2564 3.600.0172Linear 50.25470.05290.710.6263Square 5 1.79330.3586 5.030.0124Interaction 10 3.08030.3080 4.320.0125Residual error 110.78420.07126Model 60.75010.125018.38
0.003
7Pure error 50.03400.0068
8
Total
31
5.9123
model in Eq.(2)was used to predict the output of lactic acid with planned parameters and compared with observed values.The coefficient of determination (R 2)for production of lactic acid is 0.8670(Table 2).This observed variation in production of lactic acid (86.7%)can be explained by the fitted model Eq.(2).The multiple regression (R )for the production of lactic acid is 0.9311.This value shows a good agreement between experimental observations and predicted values.It can be seen that while the highest out put was observed at run 1(2.9029g/10g of bacterial bran)(Table 1).The overlaid contour plot,response surface plot of yield for certain pairs of variables shown at Figs.6,8and 9in-dicated that there are regions where the out put could be more than 3.000g.By running the optimization program with MINITAB-13within 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
1.01.5
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(
2
4
Lower Bound Upper Bound
White area:feasible region NaH 2PO 4·2H 2O,0.769%and Tween-80,0.30ml.The pre-dicted lactic acid production was 32g/100g of wheat bran having 54g of starch.Hence the parameters given by re-sponse optimization were used for confirmation of the pre-dicted value of 32g lactic acid/100g of wheat bran.The or-ganism produced 36g of l (+)lactic acid from 54g of starch present in 100g of wheat bran.
Lactobacillus amylophilus JCM 1125produced 53.4g/l of lactic acid using 100g/l,liquefied starch as reported by Yumoto and Ikeda [10].Lactobacillus plantarum NCIM 2084produced 72.9g/l of lactic acid when provided with 100g/l of liquefied starch [30].L.amylophilus (NRRL B4437)produced 29g/l lactic acid from 45g/l of corn starch and L.amylovorous was used in conversion of 120g/l liquefied starch to 92.5g/l lactic acid in submerged fermen-tation [31,32].In the above reports,enzymatic hydrolysis of starch was done prior to Lactobacillus fermentation
B.J.Naveena et al./Process Biochemistry40(2005)681–690689
which is expensive.Wheat straw hemicellulose pretreat-ment through enzymes/acid released11–12g/l fermentable sugars and were used for production of lactic acid in co-culture fermentation using Lactobacillus brevis and Lac-tobacillus pentosus[33].L.casei subsp.casei CFTRI2022, L.helviticus CFTRI2026and Streptococcus thermophilus CFTRI2034were used in SSF with sugarcane pressmud as a substrate containing14%total sugars on dry weight basis and reported6.14%lactic acid production[34].At high starch concentrations,the lactic acid production was low [5,10,31,32].In the present study L.amylophilus GV6was found to produce36g of lactic acid from a high concen-tration of raw starch(54g)present in100g of wheat bran. Wheat bran was utilized as both support and substrate by L.amylophilus GV6in single step conversion of raw starch to l(+)lactic pared with other reported strains, L.amylophilus GV6showed higher amylolytic and lactic acid production efficiencies which ensured elimination of saccharification steps of raw starch.A detailed study was made tofind out the lactic acid production yield(i.e.gram of lactic acid produced per gram of starch utilized).The organism could produce90%lactic acid yield,which is comparable with that of submerged fermentation reported earlier for this organism[13].Expensive pure starch could be replaced with cheaper underutilized abundantly available byproduct like wheat bran.The replacement of pure starch with wheat bran will bring down the cost of substrate100 times and will make the whole process more economical in terms of substrate costs.When compared to sucrose and hydrolyzed starch,the substrate costs will be still lowered to many folds.
Successful application of RSM to optimize the selected ingredients led to two-fold(100%)increase in lactic acid production.L.amylophilus GV6is found to be the most efficient in production of l(+)lactic acid both in submerged and solid state fermentations at high substrate concentrations as per the available reports.Also there are no reports on use of amylolytic Lactobacillus in SSF.Thus,L.amylophilus GV6with its90%lactic acid yield efficiency with cheap renewable natural starchy substrates could turn out to be a novel strain to develop a potential technology. Acknowledgements
BJN thanks the Council of Scientific and Industrial Re-search(CSIR)Government of India,New Delhi,for provid-ing Senior Research Fellowship(SRF)to carryout this work. References
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