Material and methods

39

3.2.2.2.5 L/D-Lactic acid test

For the determination of D- and L- lactic acid, the UVmethod, cat. no. 11112821035 from R-Biopharm AG was used. In the presence of D-lactate dehydrogenase (D-LDH), D-lactic acid (D-lactate) is oxidized to pyruvate by nicotinamide-adenine dinucleotide (NAD). The oxidation of L-lactic acid requires the presence of the enzyme L-lactate dehydrogenase (L-LDH). The equilibrium of these reactions lies on the side of lactate. By trapping pyruvate in a subsequent reaction catalyzed by the enzyme glutamate-pyruvate transaminase (GPT) in the presence of L-glutamate, the equilibrium can be displaced in favour of pyruvate and NADH. The amount of NADH formed in the above reactions is stoichiometric to the amount of D-lactic acid and of L-lactic acid, respectively. The increase in NADH is determined by means of its light absorbance at 334, 340 or 365 nm.

3.2.3Crystallization experiments

3.2.3.1 Measurement of growth rate of sucrose crystals

A laboratory crystallization unit was built according to Wittenberg, (2001). The unit scheme is given in Figure 16. A double glass wall crystallizer was equipped with stirrer, automatic measurement devices for dry substance content (Refractometer IPR2, Schmidt and Haensch) and temperature as well as temperature control. Data processing was realized on a central computer unit. Dry substance and temperature were determined every 120 s. Batch isothermal crystallization experiments were carried at constant temperatures (60, 65 and 70 °C) by seeding syrup with a supersaturation of 1.15. The experiment was stopped, when the supersaturation had reached 1.05. Varying admixtures of different dextran fractions (T40, T500 and T2000) at different concentrations (500 – 5000 mg/kg DS) were used.

mSo wDS

mW.DE m#DE

Material and methods

40

	2
		M
		1
	Crystallizer	T
		Central Computer
	3
		T +/-
		Thermostat
	4	Control unit
		5

Figure 16: Pilot scheme of laboratory crystallization device

1- Thermometer	2 - Automatic Stirrer	3- Double glass wall 4- Cooling water
	5- Refractometer (IPR2, Schmidt and Haensch)

3.2.3.1.1 Required amount of dextran and seed

The amount of dextran mDE in mg added to the crystallizing charge was calculated according to Equation (3-2).

mDE = a b

(3-2)

with

a = mSo wDS /100

b = m#DE 100 /(100 − wW,DE )

Total mass of solution in (kg)

Dry substance content in (%)

Water content of dextran in %

Target dextran concentration in mg/kg DS

After reaching the required temperature, the solution in the crystallizer was seeded with sucrose crystals (size 200 µm) using a syringe. The amount of seed mSeed in g was calculated as follows:

Material and methods

41

				3
	mSeed =mMa		di		(3-3)
		wCry
		df
mMa	Mass of the massecuite in g
wCry	Final crystal content in massecuite
di	Initial size of the crystal (200 µm)
df	Final size of the crystal

A sample was taken before the seeding point to carry out the first image analysis for the evaluation of the solubilization process. Further on, samples for image analysis were taken every 30 minutes in order to follow the growth of crystals through the crystallization.

3.2.3.1.2 Calculation of the growth rate of sucrose crystals:

In the present work, crystallization was pursued basing on refractometric dry substance content and the temperature as:

			wDS,ML,t				wDS,ML,t +1
	m	=		m		−		m
			100 − w				100 − w
	S,Cry				W				W
			DS,ML,t				DS,ML,t +1
	Difference
mS,Cry	Crystallized sucrose mass
t	Time (min)
wW	Mass of water which can be calculated as follow:

the change of the

(3-4)

m	= m	So	−	(mSo wDS,ML )	(3-5)
W			100

ML Mother liquor

The following equation helps to calculate the sucrose crystal surfaces area:

	A = f	A	m	2 / 3 n	(3-6)
	Cry		Cry	Cry
A	Total crystal surfaces in m2
Cry
fA	Form factor (0.0423) according to Austmeyer, (1981)