Pre-inoculum preparation

Culture medium preparation

It will be used a MRS culture medium. The typical formula in the MRS medium is
shown in Table 1.
Table 1. Chemical composition of MRS culture medium.
Proteose peptone
Meat extract
Yeast extract
D(+)-Glucose
Sodium acetate
Trimmoniun citrate
Magnesium sulfate
Manganese sulfate
Dipotasium phosphate
Polysorbate 80

Component composition in g/l

10
8
4
20
5
2
0.2
0.05
2
1

1. The culture medium will be dissolve in distillated water to reach a concentration

of 52 g/l, through a hot plate the mixture should be heated up to achieve a
homogeneous solution. The solution cannot be boiled up.
2. The culture medium will be sterilized using an autoclave at 121 C and 1.5 bar
during 15 minutes. The pH of the culture medium into the range of 6.2 0.2 at
25 C.
Microorganism
3. For the pre-inoculum preparation, the bacteria lactobacillus casei will be used to
produce lactic acid. The microorganism is provided by the laboratory of food
engineering, the microorganism is cryo-conserved in Cryobank cryopearls at -20
C.
4. The microorganism activation is made by adding one cryopearl in the sterilized
and cooled (25 C) culture medium. It should be used 8 ml of the culture medium
to activate the lactobacillus casei.
5. The culture medium solution with the initial amount of the microorganism will
be maintained in an incubator at 37 C during 48 h. It is expected that the
microorganism final concentration will be around of 109 CFU/ml.
Inoculum preparation
6. The pre inoculum will be transferred to 40 ml of MRS medium at same
condition for the pre inoculum preparation. To obtain the starter culture of the

acid lactic fermentation. After sterilization, the medium will be inoculated in an

incubator at 37 C for 24 h under isothermal condition.
Fermentation medium
7. The inoculum will be transferred to 210 ml of the MRS medium at 52 g/l. Initial
glucose, acid acetic and microorganism concentration will be measured. The
cells concentration will be determined based on the cells dry-weight. From the
initial solution, 2 ml will be removed and centrifuged. The liquid phase is
separated by pipetting to measure the residual substrate and product by
chromatography. The glucose and acetic acid will be detected by a high
performance liquid chromatography (HPLC), using a Hitachi L series 2000
chromatographer with a column type CSep ORH801 and a refractive index
detector. The mobile phase that will be adopted for the carbohydrate
characterization and the used column-type is sulfuric acid in aqueous solution at
0.0025 N. Finally, the Eppendorf with the precipitate is submitted to a slow
drying process. The difference between the dried Eppendorf without precipitate
and the precipitate contained in the dried Eppendorf will determinate the cells
mass per volume sample.
8. The batch fermentation will make in 1 L bioreactor (Applikon e-z control)
during 18-30 h. The fermentation will be performed at 37 C, the temperature
control is made by an electric resistance jacket (applikon). The pH will maintain
in a pH range of 5-6, the pH control is made through a peristatic pump with a
solution of NaOH at 3 M. Each 3 h, a sample of 2 ml will be taken and
determine glucose, acetic acid and cell concentration with the procedure
mentioned above. Immediately the samples are taken, they will be store at 4 C to
end the fermentation.
Determination of kinetic parameters
9. The kinetic model will be based on three rate equations: biomass, lactic acid and
substrate. The cell growth is:
S
dX
X max X
dt
KS S
The product formation in lactic acid fermentation by growth and non-growth
contributions is:
dP
dX

X
dt
dt
The substrate consumption:
dS
1 dX
1 dP

mS X
dt
Y XS dt YPS dt
10. The kinetic parameters: max, KS, and will be determined. Then expression
can be linearized:

KS
1
1

max max S
Where,

ln X ln X o
t t0

Then, with the slope and the x-intersection max and KS can be determined. The
biomass yield is determined as:
X Xo
Y XS
So S
And product yield is:
YPS

P Po
So S

The -coefficient is calculates:

( P Po ) ( X X o )
Otherwise, is an empirical constant and it is calculated by fitting the product
rate equation. An minimization method with the objective function of error: (PPcal)2 can be used to determine as a constant. This applied to ms as well.