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African Journal of Food Science Research ISSN 2375-0723 Vol. 8 (1), pp. 001-005, January, 2020. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 
 
 
 

Full Length Research Paper 

 

Response surface optimization of D(-)-lactic acid 
production from Lactobacillus SMI8 using corn steep 

liquor and yeast autolysate as nitrogen sources 
 

C. J. B. de Lima*, L. F. Coelho, K. C. Blanco and J. Contiero 

 
Department of Biochemistry and Microbiology, Institute of Biological Sciences, São Paulo State University, 

UNESP/Rio Claro, Av. 24-A, 1515 Bela Vista, CEP 13506-900, Rio Claro, SP, Brazil. 
 

Accepted 07 August, 2019 
 
The production of D(-) lactic acid from Lactobacillus LMI8 sp. was optimizated by central composite design, 
using two low-cost nitrogen sources: corn steep liquor (CSL) and yeast autolysate (YA). The fermentation 
were performed in 250 mL Erlenmeyer flasks containing 100 mL of production medium, maintained at 200 rpm 
and 37±1°C for 48 h. After data analysis by surface response method, it was revealed that Lactic acid 
production was significantly affected by the isolated CSL as well as the interaction between CSL and YA and 
the maximal production of D(-) lactic acid was 41.42 g/L – a value located at the central point, which 
corresponded to 15 g/L of CSL and 5 g/L of YA. 
 
Key words: D(-)-Lactic acid, Lactobacillus, Corn steep liquor, Yeast autolysate, Response surface methodology. 

 
INTRODUCTION 

 
Lactic acid is the common name given to 2-hydroxypro-
panoic acid (Datta et al., 1995), which is a versatile prod-uct 
applied in diverse fields, including as an acidulant in the food 
and pharmaceutical industries (Schepers et al., 2002). With 
the development and commercialization of biopolymers, 
poly(lactic acid) use has an increasing use in these new 
applications (Datta and Henry, 2006).  
The high chemical resistance of poly(lactic acid) is adv-

antageous for the manufacture of fibers, non- woven fab-rics, 
and films while the high heat resistance is suitable for producing 
the cutlery and deli trays/cups. Thus, micro-bial production of 
optically pure lactic acid has extensi-vely been studied because 
chemically synthesized lactic acid is racemic. Recently, the 
development of stere-ocomplex of poly(L-lactic acid) and 
poly(D-lactic acid) is one of the topics in the application of the 
poly(lactic acid) because its melting point (225ºC) is higher than 
that of poly(L-lactic acid) (177ºC) (Ikada et al., 1987; Tsuji and 
Fukui, 2003). 

Lactic acid bacteria make up a group of diverse 

microorganisms that are widely distributed in nature and 

associated with plants (kale, corn, barley), meat, milk 

products, porridge and silage. These Lactobacilli are mic-  
 
 
 
 
*Corresponding author. E-mail: cristian@rc.unesp.br. 

 

 
 
ro-organisms in great need of fermentable carbohyd-
rates, amino acids, B complex vitamins, nucleic acids and 
minerals for growth as well as other nutrients that are 
specific to each strain (Gomes and Malcata, 1999).  

A number of byproducts and raw materials from the food 
and/or agriculture industries have been employed to feed 
Lactobacilli due to their considerable availability and low cost, 
including cheese whey, corn steep liquor, corn syrup, distillery 
yeast and molasses (Moraes et al., 1991). In Brazil, lactic acid 
production from sugarcane and corn residuals has considerable 
economic appeal due to the abundance and low cost of these 
byproducts.  

Molasses, a by- product of the sugar manufacturing pro-cess, 
is used as an animal feed as well as for ethanol and yeast 
production (Focus, 1975). It can be also used for lactic acid 
production (El-Sherbiny et al., 1986; Tiwari et al., 1979). The 
most abundant sugar is sucrose, which raises the viscosity of 
the liquid at high concentration (Focus, 1975). Moreover, 
molasses and corn steep liquor are prominent culture media in 
fermentative processes due to the high content of sugars and 
nitrogen, respect-ively (Beaulieu et al., 1995).  

The aim of the present study was to optimize the pro-
duction medium of lactic acid by Lactobacillus sp. LMI8 

by employing a central composite design (CCD) and 
response surface method, using sugarcane molasses of 
low cost enriched with corn steep liquor and yeast 
autolysate as the culture medium. 



 
 
 

 
Table 1. Experimental range and levels of the independent variables (Xi, i = 1 and 2) used in 

central composite design.  
 

 
Independent Variables 

 Range and levels     
 

  

- -1 0 +1 + 
 

 

    
 

 Production medium optimization (g/L)        
 

 Corn steep liquor (CSL) X1 0 4.3 15 25.7 30.1  
 

 Yeast autolysate X2 1.47 2.5 5 7.5 8.52  
 

 

 
MATERIALS AND METHODS 
 
Microorganisms 
 
The Lactobacillus sp. LMI8 was isolated from a decantation tank of 
residuals from the flour industry (Plaza Starch Mill - Santa Maria-
SP, Brazil). The culture was stored in Man, Rogosa and Sharpe 
(MRS) medium with 20% (v/v) glycerol at -20ºC. 

 

Isolation of LMI8 culture 
 
The successive dilution method was employed and plating was 
performed using the “pour plate” method. Bacterial strain selection 
was based on the greatest production of the D(-) lactic acid isomer 
from the culture medium used in the fermentation process. 

 

Sugarcane molasses 
 
Sugarcane molasses was obtained from the Santa Lucia Sugar 
Processing Plant located in the region of Araras- SP, Brazil. The 
molasses was diluted and hydrolyzed at a ratio of 1 mL of H2SO4 
(20%) in 100 mL of molasses solution and heated for 20 min. After 
hydrolysis, the pH of the molasses was adjusted to 6.5 with 4.0 M of 
KOH. 

 
Medium and growth conditions 
 
The inoculum was prepared through the transference of 2 mL of 
stock culture to Erlenmeyer flasks containing 100 mL of growth 
medium (MRS) . The MRS growth medium was made up of 
peptone (10 g/L), yeast extract (5 g/L), meat extract (10 g/L), 
glucose (20 g/L), sodium acetate (5 g/L), ammonium citrate (2 g/L), 
K2HPO4 (5 g/L), Na2HPO4.2H2O (2 g/L), MgSO4.7H2O (0.1g/L) and 
MnSO4.4H2O (0.05g/L). The inoculated medium was incubated at 
37 ± 1ºC for 18 h in a refrigerated incubator (New Brunswick, USA) 
at 200 rpm. Initial pH of the medium was adjusted to 6.7. A total of 
10% (v/v) of the inoculum was transferred to 250-mL Erlenmeyer 
flasks containing 100 mL of production medium (modified MRS 
medium) agitated at 200 rpm in a refrigerated incubator at 37 ± 1ºC 
for 48 h. Calcium carbonate (100 g/L) was added to the medium in 
order to maintain the pH constant. The modified MRS medium 
consisted of the same salts as in the growth medium, with the 
addition of 70 g/L of molasses, corn steep liquor ranging from 0 to 
30.1 g/L and yeast autolysate ranging from 1.475 to 8.525 g/L. 

 
Central composite design (CCD) 
 
In order to study the influence of the corn steep liquor (CSL) and 
yeast autolysate (YA) in the synthesis of lactic acid by the 
Lactobacillus sp. LMI8 isolate, a central composite design (CCD) 

was used. For the two factors, this design was made up of a full 2
2
 

factorial design with its four cube points, augmented with three 

 

 
replications of the center points and the four star points, that is, 
points having an axial distance to the center of (± 1.41) for one 
factor, whereas the other factor is at level 0, resulting in a total of 11 
experiments. Thus, the influence of all experimental variables, 
factors and interaction effects on the response was investigated. 
The experimental design was determined using the Statistic 7.0 
software program. The independent variables, experimental range 
and levels investigated for the CCD is given in Table 1. In 
performing the regression equation, the test variables were coded 
according to the following equation 1: 
 

x i   
X i   X cp   

(1) 
 

 X i  

 

  
 

 
where xi is the coded value of an independent variable, Xi is the 

real value of an independent variable, Xcp is the real value of an 
independent variable at the center point, and Xi is the step change 

value. 

 

Analytical methods 
 
Substrate consumption and lactic acid concentrations (sample of 20 
L) were determined using a high performance liquid 
chromatography system (Waters Co., Milford, MA) equipped with a 
tunable UV detector set at 210 nm. An Aminex HPX-87H ion-
exchange column (300×7.8 mm, Bio-Rad, Hercules, CA) was eluted 
with 0.005 N of H2SO4 as a mobile phase at a flow rate of 0.6 
ml/min. Column temperature was maintained at 60ºC and a 
refraction index (RI) detector was used.  
D( -)Lactic acid concentrations were determined by an enzyme test 
kit (R-biopharm AG - Roche, Darmstadt, Germany), as reported 
elsewhere (Yun et al, 2003). Cell growth was measured by a UV-
160A spectrophotometer (Shimadzu Co., Tokyo, Japan) set at 650 
nm. 
 

 

RESULTS AND DISCUSSION 

 

Optimization of fermentative production of lactic acid 
 

The influence of corn steep liquor and yeast autolysate 
on the fermentation of sugarcane molasses was studied 
using CCD with three replicates at the center, totaling 11 
experiments. The results are displayed in Table 2.  
Analyzing Table 2, the best results of all the responses 
evaluated were obtained in the experiments located at 
the central point (Experiments 9, 10 and 11). Comparing 
Experiment 5 with Experiments 6, 9, 10 and 11, 
performed with the lowest and highest concentrations of 
CSL (0, 15 and 30 g/L) and equal concentrations of YA (5 



 
 
 

 
Table 2. Results of lactic acid production obtained in 

experiments employing the LMI8 isolate.  
 

 
Experiments 

CSL YA Lactic acid 
 

 
(g/L) (g/L) (g/L)  

  
 

 1 4.3 2.5 15.32 
 

 2 4.3 7.5 20.43 
 

 3 25.7 2.5 35.23 
 

 4 25.7 7.5 22.7 
 

 5 0 5 12.11 
 

 6 30.1 5 25.39 
 

 7 15 1.47 24.16 
 

 8 15 8.52 31.27 
 

 9 15 5 41.42 
 

 10 15 5 41.23 
 

 11 15 5 39.98 
 

 
Concentration of corn steep liquor (CSL); Concentration of 

yeast autolysate (YA). 
 

 
Table 3. Stationary point for lactic acid production and 

coded x1 and x2 values at the optimization point.  
 

 P0 Lactic acid Coordinates Lactic acid 

 1 -12.233 x1 0.139 

 2 -5.833 x2 -0.029 
 

 

g/L), it is evident that lactic acid production was directly 
proportional to the increase in CSL, reaching a maximal 
value of 25.39 (Experiments 6) and 41.42 g/L 
(Experiments 9), both in 32 h of fermentation. Moreover, 
the increase in the nitrogen concentration in the 
fermentative medium led to a reduction in fermentation 
time, reaching maximal lactic acid production. Wee et al. 
(2006) studied the effect of CSL on lactic acid production 
using different concentrations (15 to 60 g/L) and obtained 
the same final concentration of lactic acid in all 
experiments, but the time needed to achieve this 
concentration decreased significantly when using greater 
concentrations of CSL. Yu et al. (2007) used sugarcane 
molasses ranging from 12 to 60 g/L and CSL ranging 
from 24 to 56 g/L and report that the increase in 
molasses and CSL provided greater lactic acid production 
in a shorter fermentation time. Selmer-Olsen and 
Sorhaug (1998) and Altaf et al. (2006) report that peptone 
and yeast extract are the main nitrogen sources used for 
the production of lactic acid because yeast extract is an 
excellent source of B complex vitamins and is often used 
to provide these factors to the bacteriological culture 
media, which are often considered indispensable to 
obtaining faster growth and production rates of lactic acid 
by lactic bacteria. The high cost of yeast extract, 
however, has a negative impact when used in industrial 
processes (Hurok et al., 2005).  

The precision of the results of the experiments can be 

        
 
 

 

seen in the assessment of the results obtained at the 
central point of the design, where the difference between 
responses was close to 1%.  
The identification of significant parameters was per-
formed through testing the hypotheses using the Stu-
dent’s t-test. The maximal likelihood of error on the test 
was established as 5%. Thus, parameters with a level of 
significance greater than 5% were discounted. The adju-
sted empirical equation that represents the synthesis of 
lactic acid is expressed in equation 2: 
 
Lactic acid = 40.876 + 5.12X1 – 4.41X1X2 – 11.01X12 – 6.53X22 (2) 
 

In Experiment 5 (Table 2), there was lactic acid 
production in the absence of CSL. Despite being less 
significant within the fermentation process (Eq. 1), YA 
provided a good interaction with CSL, generating greater 
lactic acid production.  

The fitness of the model was checked by the deter-

mination coefficient (R
2
) and multiple correlation coeffi-

cients (R). In this case, the value of the R
2
 (0.96) for 

equation 2 indicates that the sample variation of 96.0% 
for lactic acid was attributed to the independent variables 
and only 4% of the total variation cannot be explained by 
the model. The value of the adjusted determination coeff-

icient (adjusted R
2
 = 0.935) is also high, which stresses 

the significance of the model. The high value of R (0.98) 
demonstrates a high degree of agreement between the 
experimental observations and predicted values. This 
correlation is also proven by the plot of predicted versus 
experimental values of lactic acid in Figure 1, as all the 
points cluster around the diagonal line, which means that 
no significant violations of the model were found.  

The effects of the independent variables and their inter-
actions on the formation of the product are illustrated in 
the analysis of the response surfaces (Figure 2) cons-
tructed from equation 2.  

Analyzing Figure 2, maximal lactic acid production 
occurred when nitrogen concentrations were near 15 g/L 
(CSL) and 5 g/L (YA). For greater and lower concen-
trations of CSL and YA, there was a reduction in resp-
onse, likely due to either a lack or excess of nitrogen in 
the fermented medium. According to Wood and Holzapfel 
(1995) the genus Lactobacillus has complex nutritional 
requirements. As lactic acid synthesis through fermen-
tation is associated cell growth, there is no formation of 
product if the medium does not have an adequate conc-
entration of nitrogen in order to promote this growth 
(Pritchard and Coolbear, 1993). On the other hand, high 
concentrations of nitrogen may lead to cell death and 
inhibition of the product.  

An algorithm carried out on the Maple 9.5 program 
(Waterloo Maple, Inc., Canada) was used to calculate the 

stationary point (P0) for synthesis of lactic acid. These 
values are displayed in Table 3. The values indicate that 
these responses have a maximal point, as they have 
equal and negative signs. Lactic acid production was 

41.52 g/L at the optimization point from the coded x1 and 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 1. Predicted vs. experimental values plot for lactic acid.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Response surface and contour curve representing the 

effects of corn steep liquor and yeast autolysate concentrations on 

lactic acid production. 
 

 

x2 values (Table 3).  
Comparing this value with lactic acid production values 

in experiments 9, 10 and 11 (Table 2), the results were 
practically the same, as the conditions of the maximal 
point were near to those of the central point. Thus, the 
corresponding values for the optimal concentrations of 
CSL and YA were 16.48 and 4.93 g/L, respectively. The 
model predicted a maximal response of 41.38 g/L of lactic 
acid at this point.  

Wee et al. (2006) produced 48.6 g/L of lactic acid 
using wood hydrolysate (equivalent to 50 g/L of glucose) 
supplemented with 60 g/L of CSL in 36 h of fermentation. 
Bustos et al. (2004) obtained maximal lactic acid 
production (58.9 g/L) in 96 h of culturing using 5 g/L of 
CSL together with 3.6 g/L of yeast extract and 10 g/L of 
peptone. Using a mathematical model furnished the 
response surface method, Hauly et al. (2003) determined 
maximal concentrations values tested for sugarcane 
molasses (100 g/L), yeast extract (20 g/L) and peptone (4 
g/L) as the best medium composition for the production of 
lactic acid. In practice, these conditions provided a 

 
 
 
 

 

production of 30.5 g/L of lactic acid. 
 

 

Conclusion 

 

The central composite design was proved to be a useful 
and applicable tool for determining the behavior of the 
variables studied in the production of lactic acid. The 
Lactobacillus sp. LMI8 strain using organic nitrogen 

source (corn steep liquor and yeast autolysate) achieved 
significant results regarding lactic acid production. Under 
optimal conditions (16.48 g/L of CSL and 4.93 g/L of YA) 
a lactic acid concentration of 41.52 g/L was obtained. 
 

 

ACKNOWLEDGEMENTS 

 

We thank the Fundação de Amparo à Pesquisa de 

Estado de São Paulo (FAPESP) for fellowships and 

financial support. 
 
 
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