PRODUCTION OF LACTIC ACID FROM SWEET MEAT INDUSTRY WASTE BY LACTOBACILLUS DELBRUKI

Text-only Preview

ANTARA GUHA* et al ISSN: 2319 - 1163
Volume: 2 Issue: 4 630 - 634

PRODUCTION OF LACTIC ACID FROM SWEET MEAT INDUSTRY
WASTE BY LACTOBACILLUS DELBRUKI

Antara Guha1, Soumitra Banerjee 2& Debabrata Bera3
1, 2, 3Food Technology, Techno India, Salt lake, Kolkata, India
[email protected]

Abstract
A large amount of whey is discharged from sweet meat industry, which is responsible for environmental pollution and a large amount
of whey protein and milk sugar are also wasted. This whey may be utilized for valuable lactic acid production. Lactobacillus delbruki
was used for lactic acid production from cow-milk whey. Lactic acid production was 12.22 gm/L at pH: 6.8, temperature: 420C,
Inculum volume: 4% , fermentation time: 24hr, medium volume: 125 mL in 250 mL Erlenmeyer flask, medium composition, whey
supplemented with peptone : 1.5%, glucose: 2.0% and ammonium chloride: 1.0%.

Keywords: Lactic acid, whey, Lactobacillus delbruki


Short title: Lactic acid production from whey

--------------------------------------------------------------------***-----------------------------------------------------------------------
1. INTRODUCTION:
Whey, the greenish translucent liquid obtained from milk after
precipitation of casein, has beenviewed as one of the major
Fermentative product Lactic acid is most widely used in food
disposal problems of the dairy industry, because of the high
industries
as
preservative,
acidulant
and
flavoring
volumesproduced and having a high biochemical oxygen
components. It is also used in pharmaceutical, cosmetic,
demand [5, 6]. As a general rule, about nine litres of whey is
textile and chemical industries for the production of lactate
obtained for every kilogram of cheese produced.
ester, propylene glycol, 2, 3-pentan dione, propionic acid,

acrylic acid, acetaldehyde and dilactide [1]. Currently lactic
Thus, the volume of whey to be processed, originating from
acid consumption rate is increasing for the production of
just one typical large scale cheesemaking operation can
biodegradable polylacted (PLA) polymer, which is known as
exceed 1 x 106 L/day. A dairy farm processing 100 t of milk
sustainable bio-plastic material [2]. Lactic acid naturally
per day produces approximately the same amount of organic
available in two form: d(-)-lactic acid and l(+)-lactic acid.
products in its effluent, as would a town with 55000 residents
Higher amount of d(-)-lactic acid are harmful to humans [3].
[7].
L(-) isomer of lactic acid is preferably used for food related

and pharmaceutical applications.
The production of these products in large quantities leads to

enormous quantities of whey as a byproduct in the dairy
Commercially lactic acid is produced by means of chemical
industries, which represents 85-95% of the milk volume and
synthesis or by fermentation. Racemic mixture of two optical
retains 55% of milk nutrients. Among the most abundant of
isomers of lactic acids are the result of chemical synthesis,
these nutrients are lactose, soluble proteins, lipids and mineral
where fermentation process can yield an optically pure form of
salts [5, 6]. The availability of carbohydrate reservoir of
lactic acid or racemate, depending on microorganisms,
lactose in whey and presence of other essential nutrients for
substrate and fermentation conditions for the production [4].
the growth of microorganisms makes the whey one of the

potential substrate for the production of different bio-products
Generally lactic acid is produced by hydrolysis of acetonitril
through biotechnological means [3]. Microbial fermentation
derivatives from acetaldehyde and hydrogen cyanide, which
has the advantage that by choosing a strain of lactic acid
are produced by petrochemical process. So, chemical synthesis
bacteria (LAB) producing only one of the isomers, an
may be limited due to limiting source of naturally available
optically pure product can be obtained, whereas synthetic
raw materials in future and negative feature for environmental
production always results in a racemic mixture of lactic acid.
pollution. Whereas renewable sources including lingo-
The production of optically pure lactic acid is essential for the
cellulose, starch, agricultural waste materials, sugars are
polymer synthesis in which lactic acid is used [8, 9, 10].
abundant substrates for fermentative production.
Moreover, L(+) lactic acid is used by human metabolism due
__________________________________________________________________________________________
IJRET | APR 2013, Available @ http://www.ijret.org/
630

ANTARA GUHA* et al ISSN: 2319 - 1163
Volume: 2 Issue: 4 630 - 634

to the presence of L-lactate dehydrogenase and is preferred in
parameters to enhance lactic acid production from whey
foods as preservative as well as emulsifier [8, 11].
medium.


Presently, starch or sugar containing substances are used for
2.7 Assay method:
the production of lactic acid. Whey is the attractive raw
material for lactic acid production for its low cost, low levels
After completion of fermentation the fermented broth was
of contaminants, fast fermentation rate, high lactic acid yield,
centrifuged at 10,000 rpm for 10 min to pellet out the bacterial
little or no by-product formation, ability to be fermented with
growth. Then the supernatant was used for estimation of lactic
little or no pretreatment, year-round availability and reduction
acid by titrimetric method [13].
of waste products.


3. RESULTS & DISCUSSION
The present paper deals with the production of lactic acid
3.1 Optimization of medium volume:
using whey supplemented with different components.

The effect of medium volume on lactic acid production was
2. MATERIALS& METHODS
performed by varying medium volume from 50 mL to 150 mL
(Fig. 1). The maximum lactic acid production (1.52 gm/L) was
2.1 Preparation of whey:
observed at volume 125 mL in 250 mL Erlenmeyer flask at pH
Calcium lactate was added at boiling condition to cuddle down
6.8 and 420C on 24 h.Oxygen requirement of microorganisms
protein portion from milk. Coagulated protein was separated
during growth and fermentation is indicated by medium
from whey by filtration technique.
volume in a specified flask volume. Because air is penetrated

by cotton and mixed in medium, mixing of air is dependant on
2.2 Microorganism:
available surface area, which is decreased with medium
volume in a same size flask.
Lyophilized culture of Lactobacillus delbrukii MTCC 911,

was procured from supplied by MTCC, Chandigarh.
3.2 Effect of pH

2.3 Maintenance and cultivation of the culture:
The effect of pH on lactic acid production was evaluated by
using fermentation medium having apH range of 5.5-7.0 (Fig.
The bacterial culture was revived on MRS (de Mann Rogosa
2). The maximum lactic acid production (1.53 gm/L) was
Sharpe) broth with pH 6.20.2. Theprocess of activation of the
observed at pH 6.8 on 24h.
freeze dried culture was carried out on a regular basis by

transferringthem after every 48 h up to three generations. The
However, at higher and lower pH levels, a decrease in the both
culture was maintained on MRS (MRSmedium supplemented
the function was observed, with significant decrease at pH 6.0
with 30.0 gm/L agar) by subculturing, aseptically at fortnight
and 7.0. A pH range of 6.0-6.5 has been reported optimal for
intervals and stored at 4C, until further use [12].
lactic acid production using L. caseistrain [14]. However, pH

5.5 has been used for lactic acid production using L.
2.4 Preparation of starter culture:
helveticusby [15]. From the above observations, a pH 6.8 was
considered optimal for maximum lactic acid production. In the
The bacterial culture was grown in 50 mL of MRS medium in
subsequent experiments, the pH of the fermentation medium
250 mL Erlenmeyer flask. Aftersterilization, the medium was
was adjusted to 6.8.
inoculated with a loopful of cells from agar stab and incubated

at37C for 24 h under stationary conditions[12].
3.3 Effect of inoculum size

2.5 Fermentation medium:
To study the influence of inoculum size on the lactic acid
production, different inoculum levels(1-5%, v/v) were added
Whey was supplemented with yeast extract (0.75%, w/v),
to the fermentation medium (Fig. 3). The lactic acid
manganese sulphate (20 mg/L), andcalcium carbonate (1.5%,
production increased with the increase in inoculum size up to
w/v) [12]. The whey medium was sterilized at 121C for 15
4% (v/v), thereafter no improvement in both the functions was
min. Thefermentation medium prepared in this way was used
observed. The maximum lactic acid production of 2.52 gm/L
for the production of lactic acid usingLactobacillus
was observed with 4% (v/v) inoculum of bacterial culture. The
delbrukiicells.
low lactic acid production at 1% (v/v) inoculum level could be

attributed to the low density of starter culture. The use of 2%
2.6 Optimization of process parameters:
(v/v) inoculum for the lactic acid production has been reported
Different process parameters such as pH, inoculum size,
in earlier studies also [16, 17]. However, the higher inoculum
temperature, composition of fermentation medium and
(3%, v/v) has also been used for lactic acid production [18].
incubation period were optimized by varying the respective
From the above observations, an inoculum of 4% (v/v) could
__________________________________________________________________________________________
IJRET | APR 2013, Available @ http://www.ijret.org/
631

ANTARA GUHA* et al ISSN: 2319 - 1163
Volume: 2 Issue: 4 630 - 634

be considered optimal for achieving maximum lactic acid
lactose conversion to lactic acid was obtained with the process
production using 24 h old inoculum, however, 4% (v/v)
conditions of pH 6.8, temperature 42C and inoculum size 4%
inoculum size was used in the subsequent studies.
(v/v) with an incubation of 24h. The different optimal

conditions reported by various workers for maximum lactic
3.4 Effect of temperature
acid production could be explained by the differences in the
nature of the strains and medium composition used in their
To find the optimum temperature for lactic acid production,
studies. The above optimized process parameters can be used
whey medium after inoculation wasincubated at a temperature
in scale up studies in further investigations.
range of 40-50C. Lactic acid production increased with

increase in the temperature up to 42C; however, an
3.6 Optimization of supplement concentration
insignificant decrease was found at 45C (Fig. 4). Other tested
temperatures displayed low values of lactose utilization and
Whey contains lesser amount of carbon and nitrogen sources.
lactic acid production. The maximum lactic acid production of
So the medium was enriched with different concentration of
2.53 gm/L was observed at 42C. The temperature is also one
peptone, glucose and ammonium chloride. Concentrations
of the important factors, which influences the activity of
were varied from 1.0-2.5% and other environmental
metabolic/cell enzymes. Enzymes are most active at optimum
conditions maintained constant. Productivity of lactic acid was
temperature and enzymatic reaction proceeds at maximum
increased with increase in peptone concentration from 1 to
rate. However, below and above optimal temperature reaction
1.5% and then remained about constant (8.53 gm/L). Similarly
rate is decreased which causes the problems in cell
it was also increased with glucose concentration from 1 to
metabolism The optimal temperature for growth of lactic acid
2.0%, then decreased (maximum production was 8.54 gm/L)
bacteria varies between the genera from 20 to 45C [19]. In
and productivity was about constant with ammonium chloride
fermentations using L. delbrueckii, and L.bulgaricusa
concentration (4.1 gm/L). After optimization of all of these
temperature of 45C, or higher may be maintained (Buchta,
parameters productivity was 12.22 gm/L at pH 6.8, inoculum
1983)[20]. L. helveticus, and L.acidophiluscan be used in a
volume 4%, peptone concentration 1.5%, glucose 2.0%,
temperature range of 37-45C. Krischke et al. [14] have used
ammonium chloride 1.0% and medium volume was taken as
37C temperature for lactic acid production using L.casei.
125 mL in 250 ml Earlenmeyer flask at 420C.
However, a temperature of 28C has also been reported

optimal for L. caseiin a separate study [21]. From the above
CONCLUSION
observations, a temperature of 42C was considered optimal
for lactose conversion to lactic acid using bacterial cells;
Whey is the waste byproduct of milk industry, which can be
however, a temperature of 42C was selected for further
used for lactic acid production. Lactic acid production was
experimentation.
12.22 gm/L at pH: 6.8, temperature:420C, Inoculum volume:

4% , fermentation time: 24h, medium volume: 125 mL in 250
3.5 Effect of incubation period
mL
Erlenmeyer
flask,
medium
composition
whey
supplemented with peptone : 1.5%, glucose: 2.0% and
To find out the optimal incubation time for the maximal lactic
ammonium chloride: 1.0%.
acid production, the whey medium inoculated with bacterial

culture was incubated for 48h under the above optimized
ACKNOWLEDGEMENT
conditions. The samples were drawn at specified time intervals
and the results obtained are presented in Fig. 5. As evident
The authors like to express their gratitude to the management
from the results, an increase in lactose utilization and
authority of Techno India, Salt lake, Kolkata for providing the
subsequent lactic acid production was found up to 24 h and
necessary infrastructure facilities to carry out this work.
thereafter no improvement in both the functions was observed.

This could be attributed to the growth of the culture reached to
REFERENCE
thestationary phase and as a consequence of metabolism,
1. Davison, B.B., Llanos, R.L., Cancilla, M.R., Redman, N.C.
microorganisms continuously change the characteristics of the
and Hillier, A.J. "Current research on the genetics of lactic
medium and the environment. A maximum lactic acid
acid production in lactic acid bacteria", Int. Dairy J.,5:763-
production of 2.58 gm/L was observed after 48 h of
784(1995).
incubation. The reduction in fermentation period is
2. Datta, R., Tsai, S.P., Bonsignor, P., Moon, S. and Frank, J.,
additionally advantageous to improve the economics of the
"Technological and economic potential of poly(lactic acid)
process. Therefore, an incubation time of 24 h was considered
and lactic acid derivatives", FEMS Microbiol. Rev., 16, pp.
optimal for maximum lactose conversion to lactic acid. The
221-231 (1995).
incubation period of 48 h has been generally used for lactic
3. Hofvendahl, K. and Hagerdal, B. H., "Factors Affecting the
acid production using different lactobacilli cultures [17, 18,
Fermantative Lactic Acid Production from Renewable
22]. From the observations made during the process
Resources". J. of Enzymes and Microbial Technol, 26, pp.87-
optimization studies, it could be concluded that maximum
107 (2000).
__________________________________________________________________________________________
IJRET | APR 2013, Available @ http://www.ijret.org/
632

ANTARA GUHA* et al ISSN: 2319 - 1163
Volume: 2 Issue: 4 630 - 634

4. Benninga, H., A History of Lactic Acid Making, Kluwer
21. Nabi, B., Gh. R. and Baniardalan, P., "Batch and
Academic Publishers, Dordrecht, Netherlands (1990).
continuous production of lactic acid from whey by
5. Marwaha, S.S. and Kennedy, J.F., "Review: whey pollution
immobilized lactobacillus", J. Environ. Studies,30, pp. 47-53
problem and potential utilization", Int.J. Food Sci. Technol.,
(2004).
23, pp. 323-336 (1988).
22. Kumar, S., Jha, Y.K. and Chauhan, G.S., "Process
6. Mawson, A.J., "Bioconversions for whey utilization and
optimization for lactic acid production from whey using
waste abatement", Biores. Technol.,47, pp. 195-203 (1994).
Lactobacillus strains". J. Food Sci.Technol.,38, pp. 59-
7. Sienkiewicz, T. and Riedel, C.-L., Whey and Whey
61(2001).
Utilization. Th Mann, Germany, (1990).

8. Litchfield, J.H., "Microbial production of lactic acid", Adv.
LIST OF FIGURES:
Appl. Microbiol.,42, pp.45-95 (1996).
9. Lunt, J., "Large-scale production, properties and
Fig 1: Effect of medium volume on lactic acid production
commercial applications of polylactic acid polymers",
Fig 2: Effect of pH on lactic acid production
Polymer Degrad. Stability, 59, pp. 145-152 (1998).
Fig 3: Effect of inoculum volume on lactic acid production
10. Amass, W., Amass, A. and Tighe, B., "A review of
Fig 4: Effect of temperature on lactic acid production
biodegradable polymers: uses, current developments in the
Fig 5: Effect of fermentation on lactic acid production
synthesis and characterization of biodegradable polymers,
Fig 6: Optimization of supplements concentration (Peptone,
blends of biodegradable polymers and recent advances in
glucose and ammonium chloride) for lactic acid production
biodegradation studies", Polymer Int, 47, pp. 89-114 (1998).
from whey.
11. Jarvi's, L., "Lactic acid outlook up as poly lactide nears

market", Chemical Market Reporter, 26, pp.14 (2001).
1.6
12. Panesar, P.S., Kennedy, J.F., Knill, C.J. and Kosseva, M.,
"Production of L(+) Lactic Acid
L)
/
1.4
usingLactobacillus caseifrom Whey"Braz. Arch. Biol.
gm
(
1.2
Technol., 53, pp. 219-226 (2010).
d
i
c

1
13. Aggarwal, S., "Isolation and characterization of starch
a

c
i

degrading lactic acid bacteria", Master degree dissertation,
t 0.8
c
a
l

Thapar Institute of Engg& Technology, India (2006).
0.6
of
14. Krischke, W., Schroder, M. and Trosch, W., "Continuous

nt
0.4
production of L-lactic acid from whey permeate by
ou
immobilized Lactobacillus caseisubspcasei", Appl. Microbiol.
m 0.2
A
Biotechnol.,34, pp. 573-578 (1991).
0
15. Ghaly, A.E., Tango, M.S.A., Mahmood, N.S. and Avery,
40
60
80
100
120
140
160
A.C., "Batch propagation of Lactobacillus helveticusfor
Medium volume (mL)
production of lactic acid from lactose concentrated cheese

whey with microaeration and nutrient supplementation" World

J. Microbiol. Biotechnol.,20, pp. 65-75 (2004).
Fig 1: Effect of medium volume on lactic acid production
16. Roy, D., Goulet, J. and LeDuy, A., "Batch fermentation of

whey ultrafiltrate by Lactobacillus helveticusfor lactic acid
production",
)
Appl. Microbiol. Biotechnol.,24, pp. 206-213
L
/
1.8
(1986).
m 1.6
g
(

17. Gandhi, D.N., Patel, R.S., Wadhwa, B.K., Bansal, N.,
1.4
d
i

Kaur, M. and Kumar, G., "Effect of agro-based by-products
c 1.2
a

on production of lactic acid in whey permeate medium", J.
c
i

1
t
c

Food Sci.Technol., 37, pp. 292-295 (2000).
a 0.8
l

18. Chiarini, L., Mara, L. and Tabacchioni, S., "Influence of
f 0.6
o

t

growth supplements on lactic acid
0.4
n
u

production in whey ultrafiltrate by Lactobacillus helveticus",
o 0.2
m
Appl. Microbiol. Biotechnol.,36, pp. 461-464 (1992).
0
A
19. Wood B.J.B., Holzapfel W.H., The Genera of Lactic Acid
5.5
6
6.5
7
7.5
Bacteria, Glasgow: Blackie Academic & Professional, USA,
pH
(1995).

20. Buchta, K. Lactic Acid: In Biotechnology. Germany:

VCH VerlagWeinheim(1983).
Fig 2: Effect of pH on lactic acid production

__________________________________________________________________________________________
IJRET | APR 2013, Available @ http://www.ijret.org/
633

ANTARA GUHA* et al ISSN: 2319 - 1163
Volume: 2 Issue: 4 630 - 634

)
3
9.75
Peptone
L
/

Glucose
L)
m
/ 8.75
g 2.5
Amonium chloride
(

gm
(

d
i

7.75
d
c
2
i
c

a

a
6.75
c
c
i
i
t 1.5
t
c
c
a
a
l
5.75
l


f

1
of
o


4.75
t
nt
n
ou
u 0.5
3.75
o
m
A

m
A

0
2.75
1
1.5
2
2.5
0
2
4
6
Concentration (%)
Inoculum volume (mL)




Fig 6: Optimization of supplement concentration (Peptone,
Fig 3: Effect of inoculum volume on lactic acid production.
glucose and ammonium chloride) for lactic acid production

from whey.

3
L)
/
2.5
gm
(
d
i

2
c
a

c
i
t
1.5
c
a
l

of

1
nt
ou
0.5
m
A

0
35
37
39
41
43
45
47
49
51
Temperature (0C)


Fig 4: Effect of temperature on lactic acid production

2.7
L)
/
2.5
gm
(

d
i
2.3
c
a

c
i
t
2.1
c
a
l

of
1.9
nt
ou 1.7
m
A

1.5
10
15
20
25
30
35
40
45
50
Time (hr)


Fig 5: Effect of fermentation time on lactic acid production

__________________________________________________________________________________________
IJRET | APR 2013, Available @ http://www.ijret.org/
634