3.2.1.2 Haze method

The determination of dextran in sugar solutions by a modified alcohol Haze method is conducted according to the ICUMSA method (1994). The test sample is dissolved in water. Soluble starch is destroyed by incubation with a suitable enzyme (Novo Termamyl 120L, Novo Industri A/S, Bagsvaerd, Denmark). Protein is removed by precipitation with trichloroacetic acid (TCA) followed by filtration with acid-washed kieselguhr. The dextran haze is produced by diluting an aliquot of the treated, filtered solution to twice the aliquot volume by the addition of ethanol. The turbidity of the dextran haze is measured by reading the absorbance in a spectrophotometer at a wavelength of 720 nm. The method is standardized against a commercially available dextran.

3.2.2Microbiological experiments

3.2.2.1 Isolation

Sugar beet, cossettes and raw juice were collected during the practical course (2005) in the Department of Food Engineering, Technical University Berlin. Samples (10 g) were token and diluted by sterilized distilled water to 100 g. The serial dilutions of collected samples (10-1 to 10-6) were made and 1 ml portions of the appropriate dilutions were pour-plated on MRS-Agar media (De Man et al., 1960). The cultivated microorganisms were incubated anaerobically for 48 hours at 30±1 °C for enumeration of microorganisms (Leuconostocs sp) (Beukes et al., 2001).

The MRS-broth (Bouillon) consists of (g/liter): Peptone from casein 10, meat extract 8.0, yeast extract 4.0, D (+)-glucose 20.0, di-potassiumhydrogenphosphate 2.0, Tween 80 1.0, di-ammoniumhydrogencitrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2 and Manganese sulfate 0.04. To provide MRS-Agar media, agar was added to the mixture.

3.2.2.2 Identification

3.2.2.2.1 Gas and acid formation

MRS-Bouillon with Durham tubes was used to perform this test with Lactic acid bacteria according to Back, (1994).

3.2.2.2.2 Catalase test

The catalase test involves adding of 5 % hydrogen peroxide (H2O2) to a culture sample or agar slant. If the bacteria in question produce catalase, they will convert the hydrogen peroxide and oxygen gas will be evolved. The evolution of gas causes bubbles (Figure 15) as an indicator of a positive test (Back, 1994).

Figure 15: Colony of bacteria mixed in a 5% solution of H2O2 demonstrating a positive and negative test for producing of catalase.

3.2.2.2.3 Gram characteristics (KOH-Test)

After the mixing of the bacterial colony with ca. 3 % potassium hydroxide on a glass slide, the KOH solution characteristically became very viscous and mucoid with gram-negative bacteria. The KOH test was only considered positive if stringing occurred within the first 30 s of mixing of the bacteria in KOH solution. Grampositive bacteria suspended in the KOH solution generally displayed no reaction (absence of stringing) (Halebian et al., 1981).

3.2.2.2.4 Identification by API 50 CHL test

API 50 CHL Medium, intended for the identification of the genus Lactobacillus and related organisms, is a ready-to-use medium, which enables the fermentation of 49 carbohydrates on the API 50 CHL strip to be studied.

A suspension is made in the medium with the microorganism to be tested and each tube of the strip is inoculated. During incubation, carbohydrates are fermented to acids, which produce a decrease in pH, detected by color change of the indicator. The results make up the biochemical profile of the strain and are used in its identification or typing.