CHANGES IN HISTAMINE CONTENT IN BEER PRODUCTION PROCESS

Barbara Czerniejewska–Surma, Michał Alchimowicz
Food Quality Department, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

Raw materials, semi-products and residues of five successive Pilsner beer production processes of the same brand were examined for the histamine content. A high level of histamine was noted in raw materials, hop in particular. In spite of various levels of histamine in successive processes the changes noted in its content were correlated. The highest variability was noted for the fermentation process. It is highly probable for the microbiological contamination of raw materials, particularly yeasts, to be responsible for changes in histamine content to a greater extent, that the initial histamine level in raw materials.

Key words: histamine, beer, brewing.

INTRODUCTION

Beer enrolled tenaciously into cultural behaviour of many nations. Beer owes its popularity to sensory characteristics and low alcohol content compared to other alcoholic drinks. Equally essential is a long lasting tradition of beer production and consumption reaching as far as the beginning of the Earth civilisation. The essential stages in beer production, namely malting and alcoholic fermentation, have not been changed, simply, since the very beginning of its manufactoring. Progress in hygienic practices and increasing role of production hygiene are essential mostly because of a possible harmfull effects of beer consumption on human health. One of such health hazards being possible presence of biogenic amines [23]. Biogenic amines are normally present in cells where they play important physiological roles [14]. They are also formed by the microbes in aminoacids decarboxylation process.  Amines delivered in high quantities in food can be responsible for several negative reactions in humans. They, in between, can be psychoactive and vasoactive because, when in small quantities, they become neurotransmitters as well as vasoconstrictors – i.e. tyramine or vasodilators (histamine) [15]. However the biogenic amines activity is much broader and affects many systems, organs and tissues [22], for example the frequently described "scombroid poisoning" symptoms, caused by consumption of the histamine rich fish of the Scombridae and Scombereresocidae families. At present this term is no longer used because the simillar type of food poisoning symptoms can be triggered by high histamine content in other fish species or other food products [19]. Biogenic amines can act as food quality indicators and their content in product can indicate also the hygienic level of the technological process and, above all, level of the toxicological threat [6].

Quantification of the amines toxic to humans is difficult. It was noted for the enzymatic detoxification mechanisms (monoaminooxydase i diaminooxydase) to protect human organism succesfully against the amine dosis of 50 mg/kg of food [18]. However any disturbance to these mechanisms; taking drugs inhibiting aminooxydase activity, exposure to other substances affecting these mechanisms may increase susceptibility to such amines. According to others, 2 mg/l is considered as potentially toxic dose of histamine in beer [25] while due to [13] histamine level of 10 mg/l is being tolerated by the organism.

Ethanol is one of the substances inhibitory to monoaminooxydase (MAO) activity, that is why the biogenic amines content in alcoholic drinks is so essential for human health. Besides amines detoxication mechanisms is inhibited also by other biogenic amines [22]. Beer consumption by the patients treated with MAO inhibitors resulted in acute reactions of the organism. Besides some publications confirm strong allergic reactions to beer [4], which when biogenic amines, histamine included, present can constitute an additional risk to the consumer. There are many publications on the biogenic amines concentration in different types of alcoholic beverages [26]. The amines were studied, in between, in Brasilian beers [6], Spanish beers [9], Czech ones [15], Belgian's [18], Israeli [7], Italian [1] and Polish ones [24]. Ussually the histamine level was low, however the variability noted for the consecutive samples inspired us to look for the differences in the histamine level in the production process. Reports on the histamine content at succesive stages of the beer production process are scarce fragmentary. Analysis of the accessible data add some information on the histamine content at some of the beer production process phases. The most frequently tested were wort, young beer and final product [7], raw materials such as malt, hop and yeasts were also tested [15]. However there was no complex approach to changes in histamine content at the consecutive stages of the beer production process treated as a whole.

The subject of surveys was to estimate the histamine content in raw materials used, semi-products and residues obtained during brewing process and to determine the effects of successive stages of beer production process on the histamine content in the final product.

MATERIALS AND METHODS

Samples of the Pilsner light beer, including 4.2% alcohol, were collected from the local brewery directly from the production process where the beer is being produced in a traditional way at the automated, mostly closed production line.

The subject of surveys were: raw materials (1 – malt; 2 – setting yeasts; 3 – fragrant hop; 4 – bitterish hop; 5 – hop's extract, wastes; 6 – spent grains; 7 – sediment from the Whirlpool; 8 – post fermentation yeasts sediment; 9 – yeasts centrifuged after the fermentation process and semi-products; 10 – mash from the mash tun 1; 11 – mash from the mash tun 2; 12 – anterior wort when filtered 13 – anterior wort from the intermediate cask; 14 – pitched wort after Whirlpool; 15 – wort with yeasts after one day fermentation; 16 – after 4 days fermentation; 17 – after  6 days fermentation; 18 – beer after yeasts' centrifugation; 19 – beer after 7 days of seasoning; 20 – beer prior to pasterization; 21 – beer after pasterization and  22 – bottled beer.

The scheme of the beer production line, with points marking the sampling sites, are presented on Fig. 1. Samples were transported to the laboratory and tested after ~1 h. Prior to analysis they were kept at 3ºC. Five consecutive brewing cycles were tested (P1, P2, P3, P4, P5). Results are the average of three repetitions.

METHODS

Determination of histamine content was done spectrophotometrically according to [20], after separation of histamine from the sample with trichloroacetic acid, elimination of ballast substances with anionite SEPHADEX DEAE A-25, coupling reaction with p-nitrobenzendiazonic chloride and spectrophotometric measurement in the presence of ethyl acetate at the λ wave length = 500 nm at HELIOS α spectrophotometer (SPECTRONIC UNICAM).

Anionite and main reagents were of Merck origin. Other were supplied by the Polish Chemical Reagents Company – POCH SA.

STATYSTICAL ANALYSIS

Statistical analysis was done with Analysis ToolPack Microsoft Office® and statistical programme Statistica PLŽ for Windows 5.5 version.

Correlation coefficients were calculated; ANOVA test with post-hoc tests included. As to compare The histamine content at particular beer production stages were compared by the t tests. Level of signifficance (P < 0.05) for all variables noted.

RESULTS

Analysis of histamine content at different stages of the beer production process show no essential differences only for malt (Table 1). The other brewing stages, raw materials and wastes were more variable in histamine content in individual brewing processes. Despite the essential differences between different brewing processes high correlations in histamine contents were noted (Table 2). The least correlated, for individual brewing processes, were histamine changes during fermentation process (Table 3), which suggests the microbiological purity of raw materials to be of a great significance. According to previously noted data [3], raw materials were rich in histamine content. The highest histamine content was noted for hop, the hop' s extract in particular (Fig. 2). It is in agreement with data presented by other authors [15], however the histamine content noted in hop by others was quite low [12]. It proves the quality of hop to vary a lot.

When testing the brewing process, the highest – almost twofold higher- increase in histamine content in mash from the mash tun 1 compared to that for malt was noted for P5 brewing process. (Table 1). It could be due to favourable conditions for histamine-producing microbes present in malt. Additional role can be played also by the plant enzymes. Lower histamine content in mash tun 2, after subjecting the mash to boiling, seem to confirm these suggestions (Fig. 3). Further decrease in histamine content in filtration vat and in wort of the intermediate container, noted for the most of tested brewing processes confirm suggestion on both; the microbiological contamination of malt to be the main source of histamine in wort and enzymes activity being inactivated at the following steps of the brewing process. It is in agreement with others who pointed at microflora as the main source of histamine in malt   not the initial value of this amine in barley [8]. Addition of hop resulted in statistically essential increase of the histamine content in 4 tested brewing processes (Fig. 3). Lower histamine level in "Whirlpool", in P3 process can be due to discharging of a greater amount of histamine in waste after a centrifugation. This might confirm significantly higher histamine content in sediment compared to pitched wort. On the other hand conditions in the brewing gyle, inhibiting the histamine have to be considered as well. It is also worth mentionning that hop is added to wort in minute amounts (promilles) so even when rich in histamine it should not add to histamine increase at further production stages.

Addition of yeasts resulted in essential increase of histamine content compared to that in the initial wort. However fermentation process as such have more variable effects on histamine content at subsequent tested brewing phases. In general during fermentation process a decrese in histamine content was noted. While testing 11 fermentation processes, Izquierdo – Pulido, Marine – Font & Vidal – Carou [11] did not observed any changes in the histamine content in the processes under surveillance, however they did not tested wheather there were any changes between the particular stages of the analysed processes. It is worthy to say, that after yeasts' centrifugation, histamine content in 4 brewing processes was higher than during fermentation process.

Variability in histamine content noted for particular brewing processes could be due to differences in yeast's contaminating microflora. According to Leuschner, Heidel and Hammes [17] some microbes are able to degrade amines. These microbes might, to some extent, although under unfavourable environmental conditions, add to the noted changes. Relatively low correlation between changes in histamine contents in particular kettles (Table 3) seem to confirm a high variability of bacteria present during fermentation process.

Seasoning process resulted in further increase but filtration and carbonisation processes caused decrease in histamine content.  It might be explained also by a correction – done from time to time – in malt extract concentration by water addition [2].

Pasterization caused a drop in histamine content in most of the tested brewing processes. It should be noted for the histamine content of the bottled beer to differ visibly between the brewing processes tested. In one brewing cycle, only, the histamine level of the bottled beer is not lower compared to beer after the pasterization. Kalač et al. [15] note further increase in histamine content during storage of the bottled beer. It is suggested an increase noted to be due to contamination of bottled beer with lactic acid bacteria [16]. Such contamination is well documented for other fermented products [17,5].

Analysis of histamine content in wastes of the brewing process indicated to be the highest one for sediment after initial wort centrifugation (Fig. 4). Histamine quantity in this particular sediment (residue) was also higheer compared to initial wort (Table 1). Higher content can result from changes taking place while mashing the malt and/or adding hop of high initial histamine content.

When comparing post fermentation yeast's sediment and centrifuged yeasts the last one had higher content of histamine. It might be either due to uneven distribution of histamine in particular fraction removed from beer or its presence in microbes not sedimenting in fermentors but removed during centrifugation process. Formation of histamine in beer not only due to yeasts activity but also due to microbiological contamination of the yeasts was confirmed by Izquierdo – Pulido, Font – Fábregas and Vidal – Carou [10]. Because of practical usage of post fermentation yeasts sediment for agricultural purposes it has become subject of particular interest.

Post fermentation yeasts both from sediment and centrifuged ones had histamine content 2 to 3 times lower compared to initial yeasts culture. Decrease of histamine content in yeasts can suggest possible decomposition of histamine during fermentation process. On the other hand it is more possible for the histamine to switch from yeasts into beer. An indirect prove to that are results obtained for beers of various fermentation types showing great variability in histamine content  [18].

Up to date results prove histamine content in beers to show a great variability [6]. Noted differences were eplained by different brewery origin of the beer using various technological parameters  [15]. Some authors pointed out to higher effect of microbiological contamination than an initial histamine content in raw materials used for beer production [21].

Presented results proved the same type of beer from different lots may show essential differences in histamine content, which confirms the good manufacturing practice and hygienic conditions of brewing process to be of a great significance in that respect.

CONCLUSIONS

1. High histamine content in the raw materials did not affect the histamine content in the later stages of beer production.

2. Changes of histamine content in beer production processes were correlated with each other.

3. The highest variability of histamine content was noted for the fermentation process.

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Accepted for print: 16.02.2010

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