Determination of hydroxymethylfurfural using HPLC
Fruit juice, NAB, beverages
5-Hydroxymethylfurfural (HMF) is separated using HPLC and reversed phases. This substance is measured with a UV detector.
HPLC analysis specifically detects 5-hydroxymethylfurfural.
A photometric method, B-590.59.111 Hydroxymethylfurfural - photometrisch, using barbituric acid and o-toluidine detects all aldehydes present in the sample. This method serves as an alternative for laboratories without HPLC.
Determination of hydroxymethylfurfural by photometric means.
5-Hydroxymethylfurfural (HMF) is detected in a color reaction with barbituric acid and p-toluidine. However, this detects all of the aldehydes present in the sample. The determination by HPLC specifically detects the HMF. As with other aldehydes, HMF reacts with barbituric acid and p-toluidine to create a reddish compound. HMF reacts with any sulfurous acid that is present, as do other aldehydes; in this case, it is, therefore, undetectable without prior treatment. If more than 10 mg/l of free sulfurous acid are present, this will bind to acetaldehyde. Subsequently, the determination can be performed. The presence of excess acetaldehyde does not interfere with the analysis.
Industrial yeasts from the brewing process.
Live, aerobic yeast cells possess dehydrogeneases which reduce the methylene blue absorbed into the cell to its colourless leuco form, giving the cells a pale blue colour. Dead or inactive cells lack dehydrogenase activity and turn an intense blue colour.
Detection of harmful yeasts and bacteria in clear, non-alcoholic beverages
All clear, non-alcoholic drinks with a pH value < 4.3.
The analysis of clear beverages, e.g. lemonades, fizzy drinks, as well as samples from intermediate stages such as rinsing water samples, should cover the relevant beverage pathogens, in particular fermentable yeasts, respiratory yeasts, moulds as well as lactic and acetic acid bacteria.
The corresponding procedure is described in the methods:
Anaerobic incubation is recommended for carbonated beverages in order to increase the selectivity of the analysis for this type of beverage.
Microscopy of yeast samples and process samples and beer that contain yeast prior to enrichment.
Dark-field microscopy of the sample.
The method describes the conditions for DNA extraction.
Laboratories in the beverage industry in general and the brewing industry in particular.
DNA extraction for bacteria is performed according to a standard protocol as developed by Marmur [1] using lysozyme. A commercially available chitinase/zymolyase is used instead of lysozyme for filamentous fungi/yeasts. As an alternative to enzymatic lysis, the cells can be digested using several "freeze and thaw" cycles. Some fungi can only be broken down sufficiently by liquid nitrogen treatment. This involves freezing the cell material directly in liquid nitrogen and then grinding it with a mortar. The culture is frozen three times in liquid nitrogen and then heated and incubated for several hours in a lysis buffer.
Commercially available kits for DNA extraction can also be used as an alternative.