219

Vol.53, n. 1: pp. 219-226, January-February 2010 BRAZILIAN ARCHIVES OF

ISSN 1516-8913 Printed in Brazil
BIOLOGY AND TECHNOLOGY
A N I N T E R N A T I O N A L J O U R N A L

Production of L(+) Lactic Acid using Lactobacillus casei

from Whey
Parmjit S. Panesar1*, John F. Kennedy2, Charles J. Knill3, and Maria Kosseva4
1
Department of Food Engineering and Technology; Sant Longowal Institute of Engineering and Technology;
Longowal 148 106; Punjab - India. 2,3Chembiotech Laboratories; Institute of Research and Development;
University of Birmingham Research Park; Vincent Drive; Birmingham B15 2SQ; UK. 4School of Chemical and
Bioprocess Engineering University College Dublin 4; Ireland

ABSTRACT

The aim of this work was to study the fermentation of whey for the production of L(+) lactic acid using
Lactobacillus casei. The effect of different process parameters such as pH of the medium, temperature, inoculum
size, age of inoculum, agitation and incubation time was monitored to enhance the lactose conversion in whey to
L(+) lactic acid. Fermentations were performed without any pH control. The optimization of the fermentation
conditions resulted in significant decrease in fermentation time, besides increase in lactose conversion to lactic
acid. The optimized process conditions resulted in high lactose conversion (95.62%) to L(+) lactic acid production
(33.73 g/L) after an incubation period of 36 h.

Key words: Whey, lactic acid, lactose utilization, lactic acid bacteria, L. casei

INTRODUCTION would a town with 55000 residents (Sienkiewicz

and Riedel, 1990).
Whey, the greenish translucent liquid obtained The production of these products in large
from milk after precipitation of casein, has been quantities leads to enormous quantities of whey as
viewed as one of the major disposal problems of a byproduct in the dairy industries, which
the dairy industry, because of the high volumes represents 85-95% of the milk volume and retains
produced and having a high biochemical oxygen 55% of milk nutrients. Among the most abundant
demand (Marwaha and Kennedy, 1988; Mawson, of these nutrients are lactose, soluble proteins,
1994). As a general rule, about nine litres of whey lipids and mineral salts (Marwaha and Kennedy,
is obtained for every kilogram of cheese produced. 1988; Mawson, 1994; Gonzalez-Siso, 1996). The
Thus, the volume of whey to be processed, availability of carbohydrate reservoir of lactose in
originating from just one typical large scale cheese whey and presence of other essential nutrients for
making operation can exceed 1 x 106 litres/day the growth of microorganisms makes the whey one
(Jelen, 2003). A dairy farm processing 100 t of of the potential substrate for the production of
milk per day produces approximately the same different bio-products through biotechnological
amount of organic products in its effluent, as means. The production of lactic acid production

*
Author for correspondence: [email protected]

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010
220 Panesar, P. S. et al.

through lactic acid bacteria could be an alternative Maintenance and cultivation of the culture
processing route for whey lactose utilization. The bacterial culture was revived on MRS (de
Of the total lactic acid produced worldwide every Mann Rogosa Sharpe) broth with pH 6.2+0.2. The
year, about 90% are made by lactic acid bacterial process of activation of the freeze dried culture
fermentation and the rest is produced synthetically was carried out on a regular basis by transferring
by the hydrolysis of lactonitrile (Hofvendahl and them after every 48 h up to three generations. The
Hahn-Hagerdal, 2000). Microbial fermentation has culture was maintained on MRS slopes (MRS
the advantage that by choosing a strain of lactic medium supplemented with 15.0 g/L agar) by
acid bacteria (LAB) producing only one of the subculturing, aseptically at fortnight intervals and
isomers, an optically pure product can be obtained, stored at 4°C, until further use.
whereas synthetic production always results in a
racemic mixture of lactic acid. The production of Preparation of starter culture
optically pure lactic acid is essential for the The bacterial culture was grown in 50 mL of MRS
polymer synthesis in which lactic acid is used medium in 250 mL Erlenmeyer flask. After
(Litchfield, 1996; Lunt, 1998). In addition, sterilization, the medium was inoculated with a
optically pure L(+) lactic acid is polymerized to a loopful of cells from agar slant and incubated at
high crystal polymer suitable for fiber and oriented 37°C for 24 h under stationary conditions.
film production and is expected to be useful in the
production of liquid crystal as well (Amass et al., Fermentation medium
1998). Moreover, L(+) lactic acid is used by Whey powder was procurred from Sigma-
human metabolism due to the presence of L-lactate Chemicals Company (USA) and was reconstituted
dehydrogenase and is preferred in foods as (6%, w/v) with water to prepare liquid whey
preservative as well as emulsifier (Litchfield, having lactose concentration of 4% (w/v). Whey
1996; Jarvi’s, 2001). clarification was carried through protein
Presently, starch or sugar containing substances precipitation induced by heating the whey at 90°C
are used for the production of lactic acid. for 20 min. Precipitated proteins were removed by
However, lactose rich dairy by-product whey can centrifugation at 4,000 rpm for 15 min. The treated
be low cost substrate for the production of lactic whey was supplemented with yeast extract
acid. The use of biotechnological techniques to (0.75%, w/v), manganese sulphate (20 mg/L), and
find the suitability of whey for lactic acid calcium carbonate (1.5%, w/v). The whey medium
production can serve dual purpose, i.e. production was sterilized at 121°C for 20 min. The
of valuable product, lactic acid and addressing to fermentation medium prepared in this way was
the whey disposal environmental pollution used for the production of lactic acid using
problem. Most of the work has been carried out on Lactobacillus cells.
the production of D(-) and DL mixture of lactic
acid. However, now a days, production of L(+) Optimization of process parameters
lactic acid has attracted more attention. In order to Different process parameters such as pH, inoculum
enhance the economics of the lactic acid age, inoculum size, temperature, agitation, and
fermentation process, it is necessary to increase incubation period were optimized by varying the
the lactic acid concentration in the medium respective parameters to enhance lactose
through optimization of fermentation conditions. utilization and lactic acid production from whey
The present work was, therefore, carried out to medium.
optimize the process conditions for efficient
lactose conversion in whey to L(+) lactic acid. Analytical techniques
The fermented broth was used for the
determination of lactic acid and residual lactose.
MATERIALS AND METHODS Lactic acid estimation was accomplished using
high performance liquid chromatograph (HPLC)
Micro-organism system following the method of Marsili et al.
Lactobacillus casei NBIMCC 1013 was procured (1981) with little modifications. Samples were
from National Bank for Industrial Micro- filtered through 0.20 µm membrane filters. A Bio-
organisms and Cell Cultures, Bulgaria. Rad Aminex HPX-87H column (300 x 7.8 mm)

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010
 Production of L(+) Lactic Acid using Lactobacillus casei from Whey 221

packed with a sulphonated divinyl benzene-styrene RESULTS AND DISCUSSION

copolymer was used for the separation of
compounds. The mobile phase (0.005 M H2SO4), Effect of pH
was fed at a flow rate of 0.6 mL/min and The effect of pH on lactic acid production was
temperature was kept 50°C. The concentration of evaluated by using fermentation medium having a
L(+)-lactate was estimated using an enzymatic kit pH range of 5.0-6.8 (Figs. 1 and 2). The maximum
(Boehringer Mannheim, Germany). The residual lactose conversion (95%, w/v) and lactic acid
lactose concentration was estimated following the production (33.48 g/L) was observed at pH 6.5.
procedure of White and Kennedy (1981). All
analyses were performed in triplicate and the mean
values are reported.

35
5
Lactic acid production (g/L)

5.5
30 6
6.5
25 6.8

20

15

10

0
0 12 24 36 48 60 72
Incubation time (h)

Figure 1 - Lactic acid production in whey by L. casei with pH as a function

100
5
90 5.5
Lactose utiliz ation (% , w /v)

6
80 6.5
6.8
70
60
50
40
30
20
10
0
0 12 24 36 48 60 72
Incubation time (h)

Figure 2 - Lactose utilization in whey by L. casei with pH as a function

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010
222 Panesar, P. S. et al.

However, at higher and lower pH levels, a 28 h old cultures. An increase in the lactose
decrease in the both the function was observed, utilization and lactic acid production was observed
with insignificant decrease at pH 6.0 and 6.8. A when bacterial culture of 16-20 h old was used
pH range of 6.0-6.5 has been reported optimal for (Fig. 3). The maximum lactose utilization and
lactic acid production using L. casei strain lactic acid production of 95.62% (w/v) and 33.71
(Krischke et al., 1991). However, pH 5.5 has been g/L, respectively was observed with 20 h old
used for lactic acid production using L. helveticus bacterial culture. Insignificant decrease in these
by Ghaly et al. (2004). functions was observed with 24 h old culture.
The hydrogen ion concentration of medium has the However, suppression in both the functions was
maximum influence on the microbial growth. The observed, when 28 h old growth was used. The
pH affects at least two aspects of microbial cells, low lactose conversion with inoculum age of 16 h
i.e. functioning of its enzymes and the transport of could be attributed to the fact that bacterial culture
nutrients into the cell. It limits the synthesis of might have not yet entered in log phase of growth.
metabolic enzymes responsible for the synthesis of The maximum lactose conversion observed with
new protoplasm. The pH values also affect the inoculum of 20 h, could be due to the exponential
RNA and protein synthesis. When micro- phase of the bacterial culture used as an inoculum.
organisms are grown on either side of their A 20 h old culture of L. helveticus for lactic acid
optimum pH range, there may be an increased lag production has been used by Roy et al. (1986).
phase. However, Gandhi et al. (2000) used 24 h old
From the above observations, a pH 6.5 was culture of Lactobacillus cultures for lactic acid
considered optimal for maximum lactic acid production. The use of 24 h old culture of L. casei
production. In the subsequent experiments, the pH has also been reported for lactic acid production
of the fermentation medium was adjusted to 6.5. (Krischke et al., 1991).
Since, 20 h bacterial culture displayed maximum
Effect of inoculum age lactic acid production, it was selected for further
To find the effect of inoculum age on lactic acid studies.
production, whey medium was inoculated with 16-

Lactic acid (g/L) Lactose utilization (%)

100
90
80
70
60
50
40
30
20
10
0
16 20 24 28
Inoculum age (h)

Figure 3 - Lactose conversion to lactic acid by L. casei in whey with inoculum age as a function.
Bars indicate the standard deviation from triplicate determinations.

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010
 Production of L(+) Lactic Acid using Lactobacillus casei from Whey 223

Effect of inoculum size The use of 2% (v/v) inoculum for the lactic acid
To study the influence of inoculum size on the production has been reported in earlier studies also
lactic acid production, different inoculum levels (Roy et al., 1986; Gandhi et al., 2000). However,
(1-5%, v/v) were added to the fermentation the higher inoculum (3%, v/v) has also been used
medium (Fig. 4). The lactose utilization and lactic for lactic acid production (Chiarini et al., 1992).
acid production increased with the increase in From the above observations, an inoculum of 2-
inoculum size up to 2% (v/v), thereafter no 4% (v/v) could be considered optimal for
improvement in both the functions was observed. achieving maximum lactic acid production using
The maximum lactic acid production of 33.72 g/L 20 h old bacterial culture, however, keeping in
was observed with 2-4% (v/v) inoculum of view the economics of the process, 2% (v/v)
bacterial culture. The low lactic acid production at inoculum size was used in the subsequent studies.
1% (v/v) inoculum level could be attributed to the
low density of starter culture.

Lactic acid (g/L) Lactose utilization (%)

100
90
80
70
60
50
40
30
20
10
0
1 2 3 4 5
Inoculum size (%, v/v)

Figure 4 - Lactose conversion to lactic acid by L. casei in whey with inoculum size as a function.
Bars indicate the standard deviation from triplicate determinations.

Effect of temperature The optimal temperature for growth of lactic acid

To find the optimum temperature for lactic acid bacteria varies between the genera from 20 to
production, whey medium after inoculation was 45°C (Wood et al., 1995; Dicks et al., 1995). In
incubated at a temperature range of 30-45°C. The fermentations using L. delbrueckii, and L.
lactose utilization and lactic acid production bulgaricus a temperature of 45°C, or higher may
increased with increase in the temperature up to be maintained (Buchta, 1983). L. helveticus, and L.
37°C; however, an insignificant decrease in the acidophilus can be used in a temperature range of
both the functions was found at 40°C (Fig. 5). 37-45°C. Krischke et al. (1991) used 37°C
Other tested temperatures displayed low values of temperature for lactic acid production using L.
lactose utilization and lactic acid production. The casei. However, a temperature of 28°C has also
maximum lactic acid production of 33.72 g/L was been reported optimal for L. casei in a separate
observed at 37°C. study (Nabi et al., 2004).
The temperature is also one of the important From the above observations, a temperature range
factors, which influences the activity of of 37-40°C was considered optimal for lactose
metabolic/cell enzymes. Enzymes are most active conversion to lactic acid using bacterial cells;
at optimum temperature and enzymatic reaction however, a temperature of 37°C was selected for
proceeds at maximum rate. However, below and further experimentation.
above optimal temperature reaction rate is
decreased which causes the problems in cell
metabolism.

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010
224 Panesar, P. S. et al.

Lactic acid (g/L) Lactose utilization (%)

100
90
80
70
60
50
40
30
20
10
0
30 37 40 45
Temperature (°C)

Figure 5 - Lactose conversion to lactic acid by L. casei in whey with temperature as a function.
Bars indicate the standard deviation from triplicate determinations

Effect of agitation stationery mode for lactic acid production. Gandhi

To study the effect of agitation on lactic acid et al. (2000) used stationary conditions for the
production by the bacterial culture, the cultivation lactic acid production using different lactobacilli
was carried under stationary condition (control) in cultures (L. delbrueckii subsp. bulgaricus, L.
a biological oxygen demand incubator and shaking acidophilus, L. casei etc.). However, stationary
condition (100 rpm) on an orbital shaker (Fig. 6). conditions for growth and agitation mode for
The agitation mode of cultivation did not support fermentation have also been reported in some
any increase in lactose conversion as compared to earlier studies (Roy et al., 1986).
the culture maintained under stationary condition, Since, no difference was observed for lactic acid
which could be attributed to the microaerophilic production with agitation, stationary mode was
nature of the bacteria. selected in further investigations.
The earlier studies have also supported the

Lactic acid (g/L) Lactose utilization (%)

100
90
80
70
60
50
40
30
20
10
0
0 100
Agitation (rpm)

Figure 6 - Lactose conversion to lactic acid by L. casei in whey with agitation as a function. Bars
indicate the standard deviation from triplicate determinations.

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010
 Production of L(+) Lactic Acid using Lactobacillus casei from Whey 225

Effect of incubation period The incubation period of 48 h has been generally

To find out the optimal incubation time for the used for lactic acid production using different
maximal lactose utilization and lactic acid lactobacilli cultures (Chiarni et al., 1992; Gandhi
production, the whey medium inoculated with et al., 2000; Kumar et al., 2001). Thus, reduction
bacterial culture was incubated for 48 h under the in the fermentation period along with high lactose
above optimized conditions. The samples were conversion to L(+) lactic acid are the advantages
drawn at specified time intervals and the results of the developed process.
obtained are presented in Fig. 7. As evident from From the observations made during the process
the results, an increase in lactose utilization and optimization studies, it could be concluded that
subsequent lactic acid production was found up to maximum lactose conversion to lactic acid was
36 h and thereafter no improvement in both the obtained with the process conditions of pH 6.5,
functions was observed. This could be attributed to temperature 37°C and inoculum size 2% (v/v) of
the growth of the culture reached to the stationary 20 h old culture under stationary conditions with
phase and as a consequence of metabolism, micro- an incubation of 36 h. The different optimal
organisms continuously change the characteristics conditions reported by various workers for
of the medium and the environment. A maximum maximum lactic acid production could be
lactic acid production of 33.73 g/L with lactose explained by the differences in the nature of the
utilization of 95.62% (w/v) was observed after 36 strains and medium composition used in their
h of incubation. The reduction in fermentation studies. The above optimized process parameters
period is additionally advantageous to improve the can be used in scale up studies in further
economics of the process. Therefore, an incubation investigations.
time of 36 h was considered optimal for maximum
lactose conversion to lactic acid.

35 Lactic acid
100
Lactic acid production (g/L)

Lactose utilization 90
30
80
25 70
20 60
50
15 40
10 30
20
5
10
0 0
0 6 12 18 24 30 36 40 44 48
Incubation time (h)

Figure 7 - Lactose conversion to lactic acid by L. casei in whey with incubation period as a
function. Bars indicate the standard deviation from triplicate determinations.

ACKNOWLEDGEMENTS a BOYSCAST fellowship. The authors are grateful

to Visualisation and Imaging Network (VIN), UK,
Dr. Parmjit S. Panesar acknowledges support from and National Bank for Industrial Micro-organisms
the Department of Science and Technology (DST), and Cell Cultures, Bulgaria for providing L. casei
Government of India, New Delhi, for the award of strain.

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010
226 Panesar, P. S. et al.

REFERENCES Kumar, S., Jha, Y.K. and Chauhan, G.S. (2001),

Process optimization for lactic acid production from
Amass, W., Amass, A. and Tighe, B. (1998), A review whey using Lactobacillus strains. J. Food Sci.
of biodegradable polymers: uses, current Technol., 38, 59-61.
developments in the synthesis and characterization of Litchfield, J.H. (1996), Microbial production of lactic
biodegradable polymers, blends of biodegradable acid. Adv. Appl. Microbiol., 42, 45-95.
polymers and recent advances in biodegradation Lunt, J. (1998), Large-scale production, properties and
studies. Polymer Intl., 47, 89-114. commercial applications of polylactic acid polymers.
Chiarini, L., Mara, L. and Tabacchioni, S. (1992), Polymer Degrad. Stability, 59, 145-152.
Influence of growth supplements on lactic acid Marsili, R.T., Ostapenko, H., Simmons, R.E. and
production in whey ultrafiltrate by Lactobacillus Green, D.E. (1981), High performance liquid
helveticus. Appl. Microbiol. Biotechnol., 36, 461-464. chromatographic determination of organic acids in
Gandhi, D.N., Patel, R.S., Wadhwa, B.K., Bansal, N., dairy products. J. Food Sci., 46, 52-57.
Kaur, M. and Kumar, G. (2000), Effect of agro-based Marwaha, S.S. and Kennedy, J.F. (1988), Review:
by-products on production of lactic acid in whey whey pollution problem and potential utilization. Intl.
permeate medium. J. Food Sci.Technol., 37, 292-295. J. Food Sci. Technol., 23, 323-336.
Ghaly, A.E., Tango, M.S.A., Mahmood, N.S. and Mawson, A.J. (1994), Bioconversions for whey
Avery, A.C. (2004), Batch propagation of utilization and waste abatement. Biores. Technol.,
Lactobacillus helveticus for production of lactic acid 47,195-203.
from lactose concentrated cheese whey with Nabi, B., Gh, R. and Baniardalan, P. (2004), Batch and
microaeration and nutrient supplementation. World J. continuous production of lactic acid from whey by
Microbiol. Biotechnol., 20, 65-75. immobilized lactobacillus. J. Environ. Studies, 30,
Hofvendahl, K. and Hahn-Hagerdal, B. (2000), Factors 47-53.
affecting the fermentative lactic acid production from Roy, D., Goulet, J. and LeDuy, A. (1986), Batch
renewable resources. Enzyme Microb. Technol., 26, fermentation of whey ultrafiltrate by Lactobacillus
87-107. helveticus for lactic acid production. Appl. Microbiol.
Jarvi’s, L. (2001), Lactic acid outlook up as poly lactide Biotechnol., 24, 206-213.
nears market. Chemical Market Reporter. Feb. 26, 14. Sienkiewicz, T. and Riedel C.-L. (1990), Whey and
Jelen, P. (2003), Whey processing, In: Roginski, H.; Whey Utilization. Th Mann, Germany.
Fuquay, J.W. and Fox, P.F. (eds.). Encylcopedia of White, C.A. and Kennedy, J.F. (1981), Manual and
Dairy Sciences, Vol. 4, London: Academic press. pp. automated spectrophotometric techniques for the
2739-2751 detection and assay of carbohydrates and related
Krischke, W., Schroder, M. and Trosch, W. (1991), molecules. In: Techniques in Carbohydrate
Continuous production of L-lactic acid from whey Metabolism. Vol. B3, B312, Elsevier/North-Holland
permeate by immobilized Lactobacillus casei subsp. Publishers Ltd., Ireland. pp. 12-13.
casei. Appl. Microbiol. Biotechnol., 34, 573-578.
Received: October 30, 2006;
Revised: August 14, 2007;
Accepted: May 13, 2009.

Braz. Arch. Biol. Technol. v.53 n.1: pp. 219-226, Jan/Feb 2010