1995_influence of saccharomyces cerevisiae var. uvarum on histamine and tyramine formation during...

8/12/2019 1995_Influence of Saccharomyces Cerevisiae Var. Uvarum on Histamine and Tyramine Formation During Beer Fer

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Food Chemi stry 54 (1995) 51-54Copyright 0 1995 Else&r Science LimitedPrinted in Great Britain. All rights reserved0308-8 146/95/$9.50ELSEVIER

Influence of Saccharomyces cerevisiae var.uvarum on histamine and tyramine formationduring beer fermentation

M. Izquierdo-P do, J. Font-Fhbregas C. Vidal-CarouUni tat de Nut ri cid i Bromatol ogia, Facult at de FarmLicia, Uni versit at de Barcelona, Av . Joan XX II I, s/n. 08028 Barcelona, Spain

(Received 22 June 1994; revised version received and accepted 29 September 1994)

Data are provided on the effect of brewers yeast on the formation of histamineand tyramine during beer fermentation in pilot plant. Biogenic amines weredetermined by spectrofluorometric methods. Saccharomyces cerevisiae var. uvarum,a bottom yeast, did not produce histamine or tyramine during fermentation.Yeast recycling did not influence biogenic amines formation.

INTRODUCTIONToxic effects after consumption of food products withhigh contents of biogenic amines, such as histamineand tyramine, have been reported (Stratton et al.,1991). Among them, the best known are the histaminicintoxications (Taylor, 1985) and the hypertensive crisisdue to the interaction between these amines andmonoamine-oxidase inhibitor (MAOI) drugs (Lippman& Nash, 1990). Histaminic intoxications have notbeen described for beers; however, the appearance ofsymptoms of a severe hypertensive crisis were describedin a patient taking a MAO1 drug (tranylcypromine)after drinking 2.50 ml of an Irish beer (Murray et al.,1988). It has been strongly recommended that beersshould be included among the prohibited foodstuffs forpatients on MAOIs (DArcy, 1988).In addition to toxicological implications, biogenicamines have been proposed as indices of defective foodmanufacturing processes related with poor sanitaryconditions (Cerutti, 1989; Halasz et al., 1994). Ceruttiet al. (1989) suggested that it is possible to obtain beerswith low amine levels using good quality raw materialsand applying suitable technologies. Several authorshave concluded that these amines are mainly formedthrough the decarboxylation of their precursor aminoacids during beer fermentation (Narziss et al., 1984a,b;Cerutti et al., 1989; Izquierdo-Pulido et al., 1991).However, it is still not clear whether amine productionis a result of yeast fermentative activity, or whethercontaminant microorganisms, such as Lactobacillus andPediococcus, are the actual mediators (Zee et al., 1981;Halasz et al ., 1994). Literature reports of yeast effectson biogenic amines formation during brewing arescarce. Chen and Van Gheluwe (1979) and Zee et al.

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(1981) studied the influence of Succharomyces uvarum(a top or ale yeast); however, we are not aware ofresearch done with bottom or lager yeasts. The aim ofour work was to study the influence of Saccharomycescerevi siae var. uvarum, a bottom fermentative yeast, onthe production of amines during beer fermentation. Anadditional objective was to check whether yeast recy-cling (a normal brewing practice) played any role in thebiogenesis of those amines. The present study was donein a pilot plant using glass fermenters. Working on asmall scale allowed us to control different factors,including uniformity of sampling, and to work withpure yeast and sterile wort, avoiding possible microbialcontamination.

MATERIALS AND METHODS

Fifteen pilot-scale fermentations were conducted usingEBC-style glass fermenters (3 litres capacity, 150 cmhigh) recommended by the European Brewery Conven-tion (EBC, 1977). Samples were withdrawn using asterile syringe through a rubber stopper located 50 cmfrom the top of the fermenter. Glass fermenters werethoroughly cleaned and disinfected every time a newfermentation process started. Inocula of a pure Saccha-romy ces cerevi siae var. uvurum culture and sterile wortswere used.Preparation of yeast inoculum and wortAn initial yeast inoculum was propagated in 100 ml ofsterile wort in a conical flask in a gyratory shaker at250 rpm for 48 h at 20C. Afterwards, the content ofthe conical flask was poured into another flask with 3


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52 M. fzquierdo-P do, 1. Font-F bbregas, C. Vidal-Camulitres of sterile wort. After slow agitation for 3 days atroom temperature, a low temperature (4C) wasapplied on the flask to provoke yeast flocculation.Yeast was collected and kept at 4C. Worts were sterilizedat 121C for 15 min, then cooled to 10C and used im-mediately for fermentation.Pilot-scale fermentationsFermentation was started by adding a yeast inoculumto 3 litres of sterile wort and then the mixture wastransferred to a glass fermenter. The yeast inoculumemployed was calculated to obtain a final count of 20million cells per ml of wort. Fermenters were connectedto a refrigerated bath at a constant temperature (1OC).The yeast/wart ratio and the temperature were thesame as those used in an industrial brewery. Sampleswere taken every day during the 6 or 7 days that thefermentation lasted. One portion of sample was usedfor microbial analysis and the other portion was cen-trifuged to remove yeasts, filtered (0.22 pm filter) andthen frozen (-20C) until amine analysis were done.Conditions of yeast recyclingFive consecutive fermentations were carried out in glassfermenters with the same yeast but recycled for everynew fermentation. For the first fermentation, an inocu-lum of a fresh yeast was used. At the end, yeast was har-vested in aseptic conditions, recycIed and used for a newprocess. Yeast recycling was done with phosphoric acid(Hough, 1991). Two hundred ~1 of 75% phosphoric acidwere added to 100 ml of yeast. The yeast was brieflyshaken and kept at 4C at least 30 min before using foranother fermentation. Yeast was recycled up to fourtimes, as the cooperating brewery usually did. This pro-cess of 5 consecutive fermentations was repeated using afresh inoculum of the same pure yeast culture. Sampleswere drawn and manipulated as described above.Analytical methodsHistamine was determined according to a spectrofl-uorometric method (Vidal-Carou et al., 1989). Tyra-mine determination was carried out according toRivas-Gonzalo et al. (1979). Each analysis was alwayscarried out in duplicate.Microbial analysisYeast cell counting was done with a Thoma cell, afterdiluting samples with distilled water (EBC, 1977). Con-taminant flora, such as wild yeasts and lactic acid bac-teria, were monitored with WLN (Oxoid, Unipaph Ltd,Hampshire, UK) (Roecken & Schulte, 1986) and NBB(DBhler, Darmstadt, Germany) (Back, 1980; Dachs,1981) plates, respectively. WLN plates were incubatedat 37C for 3-5 days in aerobic conditions and NBBplates were incubated in anaerobic conditions at 28Cfor 10 days.

Statistical analysisData were analyzed by one-way ANOVA followed bytesting of specific mean differences using Fishers test.Significant differences were established at PI 0.05.Analyses were performed by means of the SPSS Statis-tical software package (SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSIONEffect of yeast on biogenic amine formationIn a previous work done in the same cooperating brew-ery, histamine and other amines, such as cadaverine,putrescine, agmatine and spermine, remained constantand even decreased, e.g. spermidine, during beer fer-mentation (Izquierdo-Pulido et al., 1994). Those aminesproceed mainly from raw materials and the mash pro-cess. Only tyramine formation was detected (final leveisranging from 9.50 to 30.10 mgl). Therefore this studyfocused on tyramine evohrtion. Histamine evolutionwas also included because of its important toxicologicalimplications. Furthermore, legal regulations or recom-mendations of maximum tolerable contents in foodshave been only proposed for this amine (Stratton et al.,1991).

No contaminant flora were detected in any of thefermentation samples. We observed that yeast followeda typical bottom-yeast growth-curve in all fermenta-tions. Yeast counts at the beginning of the fermenta-tion were about 20 X lo6 cells/ml, increasing up to 40X lo6 to 50 X lo6 cells/ml in 4 or 5 days of fermen-tation. Final yeast counts ranged from 25 X lo6 to 15X lo6 cells/ml.Initial levels of histamine in wort ranged from 0.45to 0.85 mg/litre and for tyramine from 1.35 to 2.90mg/litre. Evolution of histamine and tyramine duringfermentation is shown in Fig. 1. An ANOVA was car-ried out to test differences in amine contents among thedifferent days of fermentation. No significant differ-ences (P>O.O5) were found for both amines, indicating

l HirtaminsLb IT II T I IT 1I 9 I2 3 4 5 6 7 3

Fermentation dayFig. 1. Evolution of histamine and tyramine contents duringbeer fermentation using pure yeast and sterile wort. Data areaverages (k standard deviations) of 15 processes.


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Yeast end b geni e am ines i n beers 53Table 1. Evolution of histamiae and tyramine contents (mg/litre) during five consecutive fermentations carried out with the same

yeast but recycled for every new process

DaysOb I II III IV

HIS TYRd HIS TYR HIS TYR HIS TYR HIS TYR0 0.82 + 0.11 2.17 + 0.39 0.82 k 0.03 2.40 f 0.14 0.70 + 0.14 2.42 * 0.32 0.80 f 0.05 2.27 * 0.25 0.57 + 0.11 2.21 * 0.031 0.55 k 0.07 2.07 k 0.67 0.55 k 0.14 2.40 k 0.07 0.62 + 0.18 2.70 + 0.21 0.61 f 0.12 2.31 f 0.20 0.45 + 0.07 1.92 k 0.252 0.57 If 0.11 2.10 k 0.42 0.60 + 0.14 2.07 + 0.11 0.60 f 0.13 2.45 * 0.18 0.35 + 0.07 2.02 + 0.13 0.40 f 0.10 1.90 & 0.153 0.51 k 0.13 2.12 k 0.87 0.56 ? 0.08 2.17 + 0.20 0.40 + 0.08 2.14 + 0.16 0.40 + 0.21 I.85 * 0.42 0.45 z z .11 2.00 * 0.124 0.55 + 0.09 2.00 + 0.56 0.40 + 0.11 2.34 +. 0.32 0.45 + 0.07 2.40 f 0.28 0.30 k 0.07 1.72 f O-18 0.35 + 0.07 1.80 + 0.215 0.42 + 0.18 1.85 ? 0.78 0.47 + 0.14 2.40 k 0.21 0.35 + 0.07 2.05 f 0.07 0.42 + 0.18 1.62 f 0.39 0.45 t 0.08 2.50 * 0.306 0.40 + 0.20 2.17 + 0.39 0.42 f 0.18 2.35 + 0.21 0.45 f 0.21 2.00 + 064 0.45 + 0.14 1.65 f 0.14 0.40 + 0.11 2.35 * 0.23

Mean + SD are averages from two processes.Each amine determination was carried out in duplicate. bRecycling time; Histamine;vyramine.

that no amine formation or reduction occurred duringfermentation. Narziss et al. (1984a) previously observedthat histamine contents decreased during fermentation.Although a slight decrease in histamine contents wasobserved, this may have been due to unavoidable varia-tions in the analytical method rather than an actualdecrease. It would seem that the normal amounts ofhistamine and tyramine in beer are probably inheritedfrom brewing raw materials.

method to control contamination of yeast, at least on asmall scale (Hough, 1991), since contaminant micro-organisms were not detected.

Current research is underway to study amine productioncapability of common contaminant microorganismsencountered in breweries. In addition, the influence ofprecursor amino acid levels on biogenic amine forma-tion is also being studied.

Our results are consistent with the hypothesis thatbiogenic amine production is more related to contami-nant microorganism activity than to yeast activity. Zeeet ai. (1981), after following amine evolution duringsmall-plant fermentations with Succharomyces uvarum,pointed out that biogenic amines were not formed byyeast. It appears that amines in beers are related, tosome extent, to the hygienic conditions of yeast inocu-lum.

CONCLUSIONSOn the basis of our results, Saccharomyces cerevi siaevar. uvarum alone has no ability to produce biogenicamines during beer fermentation. On the other hand,yeast recycling for several fermentations does not infl-uence biogenic amine contents during fermentation.

Effect of yeast recycling on biogenic amine formation ACKNOWLEDGEMENTSNo literature has been found about the effect of yeastrecycling on amine formation. Our objective was tocheck if old yeast (yeast used for several fermentations)showed ability to form biogenic amines that was notshown by new cultures of yeast.

No contaminant flora were detected in any fermenta-tion process. Yeast followed a typical growth-curve inall fermentations. Evolution of histamine and tyraminecontents is summarized in Table 1. Initial levels of his-tamine in wort ranged from 0.50 to 0.70 mg/litre andfor tyramine from 1.90 to 2.90 mg/litre. No amine for-mation was detected in any fermentation. No higher orlower final amine contents (P > 0.50) were found in thefermentations with old yeast (after being recycled 3 or4 times) than in those carried out with new yeast.Final histamine levels always ranged between 0.30 and0.65 mg/litre and final tyramine levels between 1.55 and2.60 mg/litre regardless of the age of the yeast. Fromour data, yeast recycling had no influence on biogenicamine evolution.

The authors thank J. M. Carceller (Dept. of Microbiol-ogy) and M. Ferrer from S. A. DAMM (Barcelona,Spain) for their helpful assistance and valuable sugges-tions and S. A. DAMM for its co-operation. This pro-ject was supported by the Comissib Interdepartamentalde Recerca i Innovacib Tecnolbgica (CIRIT) de laGeneralitat de Catalunya, Spain.

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1X2-9.Cerutti, G. (1989). Nuovi indici di qualita per alimenti e be-vande. I1 Latte, 14, 382-4.Cerutti, G., Finoli, C. & Vecchio, A. (1989). Non-volatileamine biogenesis from wort to beer. Monatsschr. Brauwiss.,42, 246-8.

Haikara (1986) pointed out that as yeast becomesolder the number of microorganisms, either gram pos-itive or negative, increases considerably. Phosphoricacid treatment for recycling seemed to be a proper

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54 h4. Izqui erdo-Pul id o, J. Font- Fci bregas, C. Vi dal- Carou

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1995_influence of saccharomyces cerevisiae var. uvarum on histamine and tyramine formation during...

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