AGRE-0014 New Methods for the Selection of Raw Materials and the Control and Monitoring of Microbiological Parameters within the Brewing Industry

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Biological Conversion : Biotechnology : ECLAIR Cluster III - Carbohydrates

Detection of wild yeast by flow cytometry

SUMMARY

This project aimed to develop new biological tests for use throughout the brewing process. As beer manufacture becomes increasingly industrialised in order to meet the needs of international markets, the industry has been seeking to improve process efficiency and maintain product quality. Beer manufacture is a complex procedure in which several highly variable raw materials are fermented by microorganisms (yeasts), which are themselves subject to variability from batch to batch. The main shortcoming of many existing analytical methods used in the industry is their lack of speed, sensitivity and specificity. Hence, in many cases they fail to provide the brewer with a decision making tool enabling rapid corrective action at the appropriate process stage. As described overleaf, this project has resulted in a wide range of such new techniques being adopted by the participating breweries, more details of which can be obtained from the coordinator.

INTRODUCTION

Brewing is becoming increasingly industrialised in order to meet the needs of international markets. As part of this process, the brewing industry has been seeking to improve process efficiency and maintain product quality. Beer manufacture is a complex procedure in which several highly variable raw materials are fermented by microorganisms (yeasts), which are themselves subject to variability from batch to batch. The main shortcoming of many existing analytical methods used in the industry is their lack of speed, sensitivity and specificity. Hence, in many cases they fail to provide the brewer with a decision making tool enabling rapid corrective action at the appropriate process stage.

OBJECTIVES

The objective of this project was thus to develop a complete range of new process control and quality assurance methods based on advanced biotechnology, immunochemistry and other novel analytical instrumentation. Therefore, the project addressed both the selection and characterisation of raw material for the brewing industry and the monitoring and control of key aspects of the brewing process itself. The work was performed in the following specific areas:

1. barley and malt selection and control;
2. yeast purity and activity control;
3. fermentation control;
4. plant hygiene control and
5. finished and intermediate product control.

It included measurement of biochemical markers, such as thionins, alpha-amylase, ß-amylase, ß-glucanase, gliadines, glutenins and microbiological parameters such as mould contaminants, which can affect the quality of barley and malt, as well as purity, contaminants and activity of yeasts, the detection of spoilage bacteria in intermediate and final products and hygiene measures linked to control of process plant environment and process rinse water. In all cases attempts were made to show correlations between these parameters and the quality of beer.

RESULTS AND THEIR APPLICATION

1. Raw materials. Samples of barley, malt and wort were used in immunoassays developed for the quantification of thionins, alpha-amylase, ß-amylase and ß-glucanase. These assays were tested by the partners for confirmation validation and for correlation between the barley and the corresponding malt and wort quality. Thionins were considered as a possible inhibitor of normal fermentation process. However, it was shown that they are lost during the malting process. alpha-Amylase and ß-glucanase in barley and malt did not correlate with beer quality. However, levels of ß-amylase in barley and malt did correlate with the beer quality. An ELISA microplate test is now used routinely by Central de Cervejas.
2. Gliadines, glutenins and barley cultivar identification. Central de Cervejas developed and tested sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of seed storage proteins, gliadines and glutenins for identification of barley cultivar and determination of varietal purity of barley deliveries within one hour. The method is performed on a microprocessor controlled electrophoresis unit with automatic densitometric analysis of the electrophoregrams. This technique is now used routinely.
3. Microflora testing of barley and malt. Barley samples from the 1990, 1991 and 1992 harvests were collected together with (where possible) corresponding malt from different European countries. These were analysed for moulds on three semi-selective media in order to measure the contamination by Penicillium, Eurotium, Alternaria, Cladosporium, Aspergillus and Fusarium including F. poae, F. equiseti, F. tricinctum, F. avenacum and F. culmorum. The weather had been quite dry and no major contamination by Fusarium was found. Hence, the main problem caused by Fusarium (beer gushing) was not encountered. Work on immunoassays for Fusarium was initiated. However, since a successful kit for the detection of mould contamination became available, activities in this area were stopped.
4. Yeast quality. Investigations included yeast identification by DNA fingerprinting using pulse field gel electrophoresis (PFGE) together with an automatic reading and pattern recognition system. The system has been extensively used and a database of DNA chromosome profiles has been established. Yeast purity control was achieved by detection of wild yeasts using flow cytometry at a level of 5 to 10 wild yeasts among 10^6 culture yeasts. Flow cytometry was also shown to be favourable to the EBC methylene blue method for measuring the viable yeasts in ferments. This technique was used to measure the effect of yeast exposure to acid washings, to ethanol treatment and wort high gravity effect.
5. Fermentation monitoring. An attempt was made to established a correlation between the fluorescence intensity (FI) obtained by viability staining and flow cytometry of yeasts and the fermentation quality. It was shown that the FI is highly correlated with the glycogen content and the esterase activity of the cells, and inversely proportional to their sterol content. The method was tested on pitching yeasts after 2, 3, 5 or 6 harvest in the fermentation vessels. Even if the activity and the viability of the yeasts were slightly different, FI could not be used to predict the quality of the fermentation.
6. Beer spoiling bacteria. Immunoassays were produced for detection of Lactobacilli and Pediococci, based on a cocktail of 13 monoclonal antibodies. However, these were not selective enough. Hence, an alternative assay for gram positive bacteria was developed for the detection and quantification of bacterial contaminants at the level of 10^4 among 10^8 yeasts by selective bacteria fluorescent staining and flow cytometry counting. A laser filter membrane scanning system for the detection of low level contamination was developed. At the end of this project, a final prototype was working at Chemunex and 8 industrial systems were foreseen for delivery during 1995. A new indirect immunofluorescence technique, using fluorescence signal amplification with fluorescent microbeads was also developed which detected Lactobacilli on filter membranes.
7. Detection and counting of dead and alive bacteria in beer. A new procedure was developed to measure total bacterial population in pasteurised and unpasteurised beer, based on epifluorescence microscopic counting of cells stained with two azo dyes INT (2-(p-iodophenyl)-3-(p-notrophenyl)-5-phenyl-tetrazolium chloride) and berberine sulphate.
8. Plant hygiene monitoring. This was investigated using ATP bioluminescence (Biotrace) for the control of plant and process hygienisation. This technique is now in routine use.

PARTICIPANTS

Alfred Jorgensen Laboratory for Fermentation Ltd, Copenhagen (Denmark), Tepral SA, Strasbourg (France) and the following brewers: ALKEN-MAES Brewery, Kontich (Belgium), BAVARIA BV, Lieshout (The Netherlands), Central de Cervejas, Lisboa (Portugal).