-

ReferInentation in and : a rigorous approach

called natural or farmer method is based G. Derdelinckx, B. Vanderhasselt, M. Maudoux and J. P. Dufour, on the alcoholic in the Louvai n-Ia -Neuve/Belg ium of residual sugars non fermented by the during the primary fermentation; the Bottle and or cask refermentation was a worldwide technology currently ap• presence of yeast in the bottle is mostly the plied to saturate before the existence of the artificial carbonation tech• consequence of the clarification technolo• niques. Actually, it is applied to several well known special Belgian, British and gy of the wine. In modem productions. all German of the ale type (Trappists, white beers, some abbey's beers and the fermentable sugars are metabolized by typical strong ales) (18, 19, 20). The purpose of this is to rigorously describe the technology, the flavour the yeast and refermentation needs the modifications and the possible troubles occurring by refermentation. addition of a solution containing yeast and sugar; this procedure is actually used in most of the industrial factories producing (bottle refermentation) and Introduction mentation is a usual practice in the sparkling wines (tank refermentation or production of sparkling wines. Bottles Charmat Method). A third system is based Although brewers have certainly prac• containing such type of wines are charac• The authors: Or. Guy Oerdelinckx, Ass. Prof.. B. ticed refermentation as long as winemak• terized by an internal pressure higher than Vanderhasselt. M. Maudoux. J. P. Oufour. Labora• ers we have to st3te that on a scientific 0.3 kg/cm2 (2 - 3 g CO/I). wire des Sciences et Technologies brassicoles. Uni• point of view, this technique is described Several methods can be used to produce versite CathQlique de Louvain. Place Croix du Sud. in more details for winery. Indeed. refer- sparkling wine (I). The oldest technique 2 Bte 7. B-1348 Louvain-Ia-Neuve, Belgium -

1111992 157

on the malolactic fermentation of wines, this procedure is used for the production of Table 1 Evolution of the aminoacid content of a refennented beer bottie(ruDde~·. green wines. Finally, an artificial tech• different conditions (pitching rate and dissolved ) .... ,,:, ,:;:-+-';. nique based on direct CO,-injection for carbonation exists too. - Pitching rate Total amino acids concentration (mg/L) . Historically, we can state that refermenta• and dissolved tion is certainly a very old phenomenon in oxygen fermented beverages; but before the use of 200 103 cells/m 1 1780 1765 as a it was impossible to keep and 2 ppm 02 the CO, in the bottle for a long time. Dom Perignon (1638 - 1715), cellarer at the 650 IQ3 cells/m! 1880 1856 1914 .1956 abbey of Hautvillers (France), discovered and 0 ppm 02 this property around 1700 and produced 650 IQ3 cells/ml 1917 1825 1950 the first bottle of refermented white wine and 2 ppm 02 called "Champagne". IQ,ooO 103 cells/ml 2625 2240 2720 . Regarding beer, bottle and keg or cask refermentation was a worldwide technolo• and 2 ppm 02

gy currently applied to saturate beer be• Beer characteristics: Pure malt; original gravity 22 Plato; alcoholic content &.5%.vol.; colour 12;' fore the existence of the artificial carbon• EBC; storage temperature 24°C ation techniques. Actually, it is applied to several well known special Belgian, Brit• ish and German beers of the ale type Fermenting wort can also be used as a shown in Tables 1 and 2 and developed (Trappists. white beers, some abbey's carbohydrate source for refermentation under 2.2. beers and typical strong ales) (18,19,20). and has the advantage for small The true quantity of sugar to use is The purpose of this paper is to rigorously plants that active yeast is already present calculated by following the empiric for• describe the technology. the flavour mod• in the pitching liquor. mula: ifications and the possible troubles occur• The quantity of sugar to use for refermen• ring by refermentation. tation depends on three major parameters: A = 2B - (OJ C + D) the desired CO, content of beer. the quantity of fermentable sugars remaining A: Quantity of fermentable sugar to be added to 1. Technology of referrnentation in beer and the CO, content of beer at the beer to reach saturation value B; start of refermentation. Considering the B: Wished CO, content of beer: The primary objective of refermentation is beer CO, content, we advise values C: Quantity onermentable sugars remaining in the saturation of the beverage: therefore. beer before refermemation (bearing a coeffi• between 5.0 g CO,1l and 8.0 COjI respec• only fermentable sugars and yeast are cient 0.3 since this sugar is generally maltotri• tively to avoid a lack of saturatIon and to ose which is not easily fermemable by yeast needed. Quantity and type of both factors prevent some overfoaming. Even though under re fermentation conditions); are dependent on the desired values (CO, 5.0 g CO,1l can be considered as the lower 0; CO,remaining in beer before bottling. content. flavour, taste). • limit especially if beer is dispensed cold, We have to mention that the original characteristics of beer such as alcohol sometimes values till 9.0 g CO,1l were 1.2. Addition of yeast ascertained without any trouble but such content, colour and ionic balance can high values cannot be recommended. Selecting a yeast to insure refermentation, influence the refermentation: external A parameter particularly important is the the brewer has to take two parameters into characteristics as beer temperature at the CO, content before bottling. This parame• account: yeast characteristics and yeast start of refermentation and the tempera• quantity. ture of the refermentation room also. All ter IS dependent of the fermentation and lagering characteristics of beer. Iffermen• these parameters will be studied hereafter. tation is carried out at room temperature Yeast characteristics (20°C) and without pressure the CO, 1.1. Addition of fermentable sugars Considering yeast characteristics, we concentration in beer is approximatively have to distinguish yeast type and physio• The fermentable sugars added to beer are 1.5 gll; if a pressure is exerted the CO, logical properties of the pitching yeast. generally glucose or sucrose. Commer• increases (for example 4.4 gll by an cially, they are available under different absolute pressure of 2.8 bar); such a Yeast type forms as granulated sugar with an extract technique can also be applied during content of about 100%; syrups are also lagering. Practical advantages are finan• Our scientific and industrial experience available with the following usual charac• cial (reduction of the refermentation time) allows us to suggest a classification of teristics: 66% of extract containing be• and sometimes qualitative (reduction of used by refermentation based on tween 25% and 90% of fermentable sug• the air content in the bottle neck if the practical considerations. Primarily, we ars, the other fractions are mostly unfer• bottler is not equipped with a vacuum can distinguish brewers who use for refer• men table carbohydrates which increase system). On the other hand we have to mentation the same yeast as for the main mouthfeel of beer. The syrups are dis• mention a weaker flavour enhancement by fermentation, thus of the type Saccharo• pensed by suppliers in heated tanks at comparison with a real bottle or keg myces cerevisiae var. cerevisiae; a second relatively high temperature or in contain• refermented beer. This seems linked to the category of brewers uses a typical brew• ers at ambient temperature but in that isoamyl acetate and ethyl caproate flavour er's yeast which is different from the yeast the user has to take care of the microbial unit increases observed in beers where used by the primary fermentation, this quality of the solution because contamina• most of the refermentation phase takes yeast is also of the type Saccharomyces tions can have severe consequences (see place in bottles or kegs (increase of CO, cerevisiae var. cerevisiae; a third category 3.3.). content of minimum 3.5 g CO"/l). This is of brewers uses for refermentation a non- 158 11/19921 ~;i 11t' -r -: 7 "" [. ~. ., 106 =€ 8- 6 _. ::!. ~ 6 '" ~ '0 Inoculation r.llC in ceHS/ml.

107 ~

lO 11 ;; 10.' -, 10' >-

5,10 .1 -10' SalUf:Jliun of beer.-\; M_.~_.' Y~~l l,:ontcntof beer A

S;J1U(;l!lOn or t-.:<:r B: •••..••• ' Yca.'il COOlcnl of heel B

12 16 20 o I' 16 :!O 24 Time elapsed sio..::cbouling (days) Time: dapsed ~in(,;cbouling (days) Fig. 2 Evolution of the beer saturation in terms of time Fig. 1 Evolution of the beer saturation in terms of time elapsed since bottling of two different beers (strong and elapsed since bottling of a beer inoculated before refer• normal) pitched before bottle refermentation with the mentation with different quantities of yeast cells same quantity of yeast cells Beer characteristics: original gravity 12 Plato; alcoholic Characteristics of beer A: original gravity 19 Plato; alcoholic content 5.1 % v/v; bitterness 20 EBU; total acidity 17 ml content 8.1 % v/v; bitterness 29 EBU; total acidity 29 ml NAOH 0.1 N/1 00 ml; colour 8.5 EBC; storage temperature 24°C NaOH 0.1 N/1 00 ml; colour 11.5 EBC; storage temperature 24°C. Characteristics of beer B: original gravity 11.5 Plato; alcoholic content 8.1% v/v; bitterness 29 EBU; total acidity specific brewer's yeast as Saccharomyces dependence and 29 ml NaOH 0.1 N/1 00 ml; colour 11.5 EBC; storage temper• ature 24°C cerevisiae vaT. pastorianus known to that studies trying brewers as baker's yeast. to classify brewer's It stands to reason that the easiest way to yeast according to this characteristic are sterilization to reduce the formation of perform refermentation is by using the still going on. some Maillard compounds which pos• same yeast as in the main fermentation. When the cell wall is a pure physical sibly hamper yeast growth; pH and Depending on their flavouring character• barrier. the cell membrane is a chemical other physicochemical characteristics istics, these yeasts can be very aromatic or barrier consisting of a double layer of are the usual values). neutral and alcohol sensitive or not. phospholipids enclosing an hydrophobic ~ Wort oxygenated with pure oxygen Aromatic brewer's yeasts of the type liquid structure formed essentiall y by fatty just before pitching (till 30 ppm to saccharomyces cerevisiae \'01', cerel'isiae acids (saturated and unsaturated) and ster• allow an optimal fatty acid synthesis). are often alcohol sensitive while neutral ols. This membrane also called plasma• --, Wort stirred during propagation (in a yeasts seem more alcohol tolerant: note lemme is responsible for the regulated propagator equipped with baffles or a that this phenomenon is amplified by uptake of nutrients, excretion of metabo• vibromixer to move the yeast with temperature (see Part 3: Troubles by refer• lites and is the site of cell wall synthesis regard to the medium and to exert a mentation). and secretion of extracellular enzymes. type of physical cleaning of the cell As indicated, another possibility is the use The efficiency of yeast activity depends wall). of baker's yeast and some brewers per• strongly on the fluidity of this chemical form refermentation in this way; it is an barrier, it needs to be not too rigid or too Using such propagation parameters, a easy technology but it may be mentioned fluid, in other words a just equilibrium high cell concentration can be reached (up that the flavor obtained cannot be com• between saturated and unsaturated fatty to 200 106 cells/m!). Based on this princi• pared with the one obtained using an acids. ple, se'veral manufacturers build discon• aromatic brewer's yeast (see Part 2: Fla• The chemical composition of cell mem• tinuous, semi-continuous and continuous vouring factors affected by refermenta• brane regarding lipids in terms of temper• yeast propagation plants for use in main tion). ature was described by Ohno and Taka• fermentation and/or by refermentation in On the other hand, physiological proper• hashi (9). bottles and kegs. ties as viability, fermentation efficiency On a practical point of view, pitching yeast ,As described earlier, wort after two days and surface properties of yeast are also of for 'refermentation has to possess an ideal fermentation can also be used as pitching solution for refermentation of unsaturated big importance. membrane permeability at the selected refermentation temperature. Ideally, it beers; the best is to use wort of pale beer Physiological and swface properties means that the utilized yeast has to be (colour lower than 15 EBC). of the pitching yeast developed at the same temperature as the A third possibility is to utilize yeast of refermentation temperature to prevent previous main , stored in Yeast is an eucaryotic cell and is thus stress conditions as lack or excess of cold cellars or fridges (4°C) and stirred for characterized by a cell wall and a cell membrane fluidity. 5 minutes twice a day to prevent tempera- , membrane (4). ture increase and loss of viability due to i! Our experience taught us that the best way Cell wall composition is typical and influ• excretion (5). Their use necessitates abso- i: of proceeding is to use yeast in propaga• ences the sedimentation, adhesion and lutely an evaluation of yeast cells viability ~i flocculation properties of yeast. Indeed, tion on wort, which is well oxygenated and stirred. More precisely it signifies: (Methylene blue method) and never yeast ~ the external structure presenting only aged more than 2 weeks may be used i: mannan sites or mannan and protein sites o Yeast propagated on sterilized hopped is partly responsible for the physical be• wort (original gravity: 12° Plato to means that freshly well aerated wort has to havior of yeast in the bottle or keg. Let us avoid an inbitory effect due to alcohol; be added to yeast (minimum 3 of / mention that flocculation is under genetic colour: not more than 20 EBC after freshly aerated wort for I of stored'

without reconditioning. Reconditioning I,', Brauwelc InternatlMal .~;'i:"''';~S:'ri?~

1111992 159 yeast) 12 hours (or I night) before addition a too rapid refermentation can have as sensItivity to alcohol. strong dark beer to the unsaturated beer. consequence a bad CO, retention to beer containing growth inhibitors, a refermen• If no pure yeast (without microbial con• inducing overfoaming by opening during tation room temperature higher than 30°C tamination) is available. then the brewer the first weeks after bottling. can also have heavy consequences be• can take extreme measures by applying an The refermentation speed in terms of yeast cause it increases yeast sensitivity to acidic washing of the yeast but this prac• quantity added to an identic unsaturated alcohol and inhibits the metabolism. Such tice can not be recommended (10). The beer is shown in Figure I. An example of a situation can have catastrophic conse• consequence of such a yeast treatment refermentation velocity in terms of beer quences because yeast whose refermenta• before bottle refermentation is a bad adhe• type is shown in Figure 2. tion activity has be~n storped by a warm sion of yeast at the bottom of the bottle. the In conclusion. a value of 100000 cells/ml temperature stress does not start again and cells forming a dusty layerprobably due to is advisable for beers characterized by a the beer frequently remains unsaturated the destruction of external sites of the cell low colour « 12° EBC) and a normal and thus unsalable. wal!. alcoholic content (5.0% vol) and 500000 cells/ml for beers with a dark colour (> 35° 1.3. A typical technology: Yeast quanrity and temperature of EBC). brewed with special (torrefied malt refermentation of true gueuze refermenration or barley) and with an high alcoholic Refermentation by bottle of a mixture of The yeast quantity to use in refermentation content (> 7.0% vol). Iambics is a well known technique used by has to be expressed in celts/m!. The needed Regarding the refermentation tempera• most true gueuze producers. Indeed, quantity can be transformed later on in ture. we recommend bottling or kegging gueuze is also called the "Champagne of volume or weight for practical reasons but beer at 22°C and storing it in a room at a beers" because this beverage is obtained by each pitching of unsaturated beer yeast constant temperature of 24°C. In most by mixing in the right proportion (estab• counting by microscope is a must. cases Iow temperatures (between 12°C Iished by tasting) of Iambics *) of different Moreover. this technique allows a subjec• and 17°C) have to be avoided because they ages (1. 2 and 3 years old). Normally, no tive evaluation of the yeast by appreciat• strongly affect the refermentation speed. yeast is added and the part of beer pro• ing the buds. the external form of yeast. the Let us mention that for some top ferment• duced during the bottling year plays the viability (Methylene blue colouring) and ing yeast strains. a stress due to a too low role of "pitching beer". even sometimes by displaying an impor• temperature « Isac) can inhibit yeast This technology was well studied on a tant contamination. development which never starts again. the scientifical point of view by van Oevelen Practically. the quantity of yeast to intro• consequence is unsaleable beer or some• and Verachterr (16. ]7, 18). duce into the unsaturated beer is depend• times saleable beer containing fermenta• .) beer obtained by spontaneous fermentation in oak ent on the beer type and on the wished ble sugars. casks of a brew produces by malted barley (60%). fermentation velocity. Let us mention that Depending on yeast and beer type as yeast wheat (40'/'<) and aged hop (3 years and more)

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160 11/1992

2. Beer flavour modifications 2.1. Evolution of beer flavour contains also amino acids, the yeast devel• during the lag phase opment has to start immediately to avoid a Bottle and keg refermentation have to be possible contaminant development. An• - considered as a succession of several The lag phase is a critical period during other reason is the presence of oxygen in phases which strongly influence the qual• refermentation in bottles or kegs, conse• beer by bottling, which has to be removed ity of the final beer. Regarding yeast, we quently the brewer has to reduce this phase as soon as possible to prevent chemical distinguish successively: a lag phase, a to a maximum. Just before bottling or oxidation. growth phase, a fermentation phase, an kegging, a solution containing easily fer• Since only events with negative influence excretion phase and finally an autolysis mentable sugars, generally glucose or on beer quality and flavour can arise sucrose is added to the beer. Since beer phase. during the lag phase, ideally this period has to be inexistent, therefore the brewer has to use yeast during the growth phase. Table 2 Evolution of the flavour profile of a strong top fermented beer, referment• ed in bottle, during a period of one year 2.2. Evolution of beer flavour during Compound11.81322100.65104.8196.33.150.8333.111.8337.7188.70.381.7916.773.39597.4After280.380.86105.3125537.341.5179.30.4124290.840.8916.463.612323.07250.470.370.813.53.11274417.020.863.121.09I3days1239.242.335.338.2186.2184.399.5After15.450.9117.483.113393.180.453.9After30.696.3260.8911.48303133.23.08I91.2182.36monthAfter35.440.9monthsyearBeermonths15.68' Threshold*A 200125705080330.90.680.23 the yeast growth and fermentation phases EthylDiacetylIsoamylEthylBeta-fenylcaproatecaprylatealcoholacetateethonal FUFU Start DMSIsobutanolEthyl acetate OtherHigherEsterscompounds(mgIL)alcohols (mgIL)(J.tg/L)Total FU The multiplication phase is the most im• portant one regarding such beer character• istics as smell, taste and pressure in the bottle. Generally the growth phase goes on for two to four days depending on the yeast concentration, on the dissolved oxygen by filling and on the beer type. We know by experience that yeast growth is quite nonexistent if concentrations higher than 1,500,000 cells/ml are used; on the other hand growing ratios higher than 4 were never established.

Amino acids and higher alcohols

During the growth phase, yeast takes up all the oxygen present in beer; if freshly

Beer characteristics: Original gravity: 21 PlatO: alcohol content: 9.2% vo!: colour: 13 EBC. The beer was pitched propagated yeast is used, it takes only a for refermentation with 200 10' cells/m! of yeast in the growth phase. *: the FU (Flavour Unit) is defined as the few hours. Later on budding and absorp• concentration of the aromatic compound divided by its threshold value as described by Meilgaard., MBAA Tech- tion of aminoacids and fermentable sugars start. It is the fermentation phase; several aromatic compounds are then produced. Although the modifications expressed in Table 3 Evolution of the flavour profile of normal top fermented beer, refermented in bottle, terms of concentration (mg/l) seem weak during a period of one year they can strongly affect beer flavour; because of this, changes are to be consid• 20.40.711.6125.7151.110.880.292.451.7075·6.7331.538'1.06125.8After12.00362.081.7127298.800.351.790.830.87264433233.1390.2913328.21632373922.2I2.6729.20.6510.8912.416.931.59After0.790.251.880.898.000.33After27.55.620.17103119.91.691.0924.2After6monthmonthsBeerBmonths8.61Threshold*200331250.230.90.68708050 . CompoundIQ 31.3days1 year ered in. Flavour Units (FU). Aromatic EthylIsoarncaproatecaprylateyI acetate FUDiacetyl StartIsoamylBeta-fenylalcoholethanolFU FU. Higher alcohol3 (mgIL) EthylacetateIsobutanolDMS Esters (mgIL) Other compoundsTotalFU (JliIL) compounds produced by the yeast's me• tabolism during refermentation are the same as those formed during the main fermentation. An example of Ct.-amino uptake during the fermentation phase is shown in Table 1. Considering the four classes of amino acids, a decrease varying between 12% (first family), 18% (second family) and 6% (third family) is observed. Regarding the consequences of these changes on the aroma profile by the possible higher alcohols synthesis, we can assess that no significative increase of flavour can be justified by this way even if the changes are expressed in Flavour Units. In consequence, the increase of isoamyl alcohol observed by refermenta• tion of some beers (see Tables 2 and 3) is Beer characteristics: Original gravity: 12 Plato; alcohol content: 5.2% vol; colour: 9 EBC. The beer was pitched for refermentation with 200 1()3cells/ml of yeast in the growth phase. *: the FU (flavour due to another reason (probably synthesis unit) is defined as the concentration of the aromatic compound divided by its threshold value as via the pathway of Genevois). described by Meilgaard. MBAA Technical Quarterly, 1975. 12, 151. On the other hand we have to note diacetyl synthesis during these phases (Tables 2 i.. "':':K Brau.ell int67Uti1Hla1 ~

11/1992 161

beer by bottling to the bottle neck after capping. Figure 3 4 '"..,..,u enhance the reduc• .-.8~'"" points out that at maximum 30% of that "' f B~cr b

162 11/1992

dissolved oxygen led by adding fresh active yeast to the 10 not higher than 2 beer and by verifying if fermentation Dt~.\ol\<;uo'ygo.:lIl0 h1.:cr hy tll1tlllllg mg/l: takes place by measuring the attenua• :::J the absence (l rrm tion. Another measure to perform is the -- .:!I'pm of air in the bottle quantification of yeast viability by the --0-- :'ppm neck. ---- IOpplll "Methylen blue colouring test": if most of the cells have died (more than El'olution 50% blue cells) the origin of the of hitterness problem is probably a yeast inhibition = - by extreme temperatures during refer• ~ ;: It is well known that mentation (higher than 28°C or lower bitterness decreas• than !4°C), such problems often arise es during the ageing in beers characterized by a high alco• {J III le l.l 11, I x of beers. This holic content and/or brewed using same phenomenon torrefied grains. Practically this can be occurs in bottle re• Fig. 5 Valuation by tasters in terms of time of a refer• avoided using a normal temperature in fermented beers as mented beer bottled under the same conditions except the refermentation rooms or, if no dissolved oxygen shown in Figure 6. other solution exists, by working with As we can observe, Beer characteristics: original gravity 19° Plato; alcoholic a more resistant yeast. a loss of 25% of content 8.1 % v/v; bitterness 29 EBU; total acidity 29 m! o An escape of CO, pressure by a badly NaOH 0.1 N/100 ml; colour 11.5 EBC; storage temperature bitterness units is 24°C sealed bottle or keg. Beer characteris• established 2 years tics are then normal regarding attenua• after bottling. tion and viable yeast cells into the bottle. Practically, the different sealing Viability afyeast cells und autolysis 3. Physicochemical and microbial equipments have to be checked. Determination of viability (Methylen blue troubles by refermentation coloration) also gives an idea of the ageing 3.2. Beer overfoaming of beer; by experience we are able to assert Problems which occur during beer refer• by opening of the bottle that a count of more than 50% of blue cells mentation are often very damaging as they Three causes can explain this phenome• indicates an aged beer. As shown in Table frequently lead to the beer becoming non: -t. several factors as yeast physiology by unsaleable. The defaults arising during bottling and yeast quantity. affect the refermentation and affecting finished beer Excess of fermentahle sugars in the percentage of dead cells in the bottle after can be classitied according to three major hottle. The origin is often a wrong I year. practical statements: calculation of the quantity of sugar added to beer before refermentation The loss of \'iability is accompanied by Lack of pressure in the bottle and/or (see I .1.). It is mostl y the consequence yea.'t autolysis which intluences the lla• residual fennentable sugar: vour profile of beer. The major come• of a bad tinal attenuation at the end of Overfoaming by bottle opening; the main fermentation which induces quence of yeast autolysis is the liberation Abnonnal aspect of the yeast layer at into the beer of hydrolytic enzymes of the the bottom of the bottle and/or micro• an excess of fermentable sugars and vacuole content: these influence impor• bial contamination. consequently an oversaturation (more tant beer characteristics as t1avour and than 9 g COe/l). It can be detected by foam stability. Aroma is strongly affected 3.1. Lack of pres• by an increase of volatile fatty acids with sure into the bottle 30 a low threshold value as (isovaleric acid. ~ Three causes can be '" .D-

measuring the CO" by titration for ,:::~::~:;;;,:q-'r-?:~?:~.i1::~~:;:?~~~ example (not by pressure since this Table 4 Evolution of the percentage of dead cells a refermented beer bOttled under;~} method also measures the other gases different conditions (pitching rate, yeast growth phase and dissolved oxygen»;f,;~\,~t;: present such as nitrogen and oxygen encapped in the bottle neck). On the other hand, the use by some brewers of amyloglucosidases during the main ::~~~~: by ~g P,=nUge of:,7~iRJ~}~~1~ Inoculum 1 Inoculum 2 InoculumJ::,:;JnoculuIri;2t;,o fermentation can also be responsible -., ,', ~, " ..', ...'-.",- --' -', , ''':'',,," for overcarbonation as generally speaking these enzymes are not inacti• 200 103 cells/ml6% 1%1% vated before bottling or kegging. and 20 ppmppm°202 :J Presence of Saccharomyces cerevisiae 10,000650 103103cells/mlcells/m! var. diastaticus. This type of yeast is able to ferment starch and thus the beer polysaccharides~ it thus produces CO, in excess. Moreover, the superattenua~ tion can induce such high pressure values that the bottle explodes. 34% ,59%,', ::J Presence of heterofermentative lactic

acid bacteria strains. Several lactic . ' .- .'-""', -_: ',:,::~;~.>";;. ~,-,,::\ _t't'~:~::";.?··_ acid bacteria which are able to grow in Inoculum I: Yeast cells in the log. phase; Inoculum 2: Yeast cells inthestationiuyphaSe;'t.:t~'~;4C;~":c" beer belong to the heterofermentative Beer characteristics: Pure malt; original gravity 22 Plato; alcoholic content 8.5%voL;' colOUl"J2>~c".": EBC; storage temperature 24°C .. ;';:'r2., class and thus metabolize sugar (glu• cose) by forming lactic acid, ethanol and CO,. Theoretically, heterofermen• most part the consequence of beer and Granular structures in the bottle tative lactic acid bacteria as Lacto• yeast characteristics; actually research is bacilllus breves should be able to Granular structures being like polyhe• being carried out on flocculence and the drons are sometimes in the bottles. Their induce an excess of CO, into the beer, composition of the medium to try to but the authors could never confirm it understand the involved mechanisms. On origin is not yet known but their presence is often linked to a former or actual beer on an industrial scale: this is probably a practical point of view both types ofIayer due to the environmental conditions contamination by lactic acid bacteria never cause problems in tasting or evalua• (even Lactobacillus sp. as Pediococcus such as the high pressure in the keg or tion. Let us mention that the dusty charac• the bottle. the alcohol content, the low sp.). teristics can be the consequence of an quantity offermentable sugars and the acidic washing of yeast before use (see presence of growth inhibitors as iso• 1.2.) or of a calcium deficiency; on an Yeast adhearing to the bottle sides alpha-acids. industrial scale, we were able to achieve Yeast adhering to the bottle's sides form• Presence of gushing inducers. This is yeast sticking at the bottom of the bottle by ing a fine layer occurs mostly during the the consequence of either malt con• increasing the calcium concentration of refermentation phase and is a temporary tamination by moulds as Fusarium and the beer up to 50 mgIL (expressed as CaH). phenomenon; it is generally removed by a Aspergillus (these have to be detected slight agitation of the bottle, even by on malt before use in the and shifting the bottle-rack. The reasons why the contaminated malt has to be reject• Cloudy structures in the bottle such a problem arises are not yet known ed) or of the presence of crystals such but are ceitainly linked with surface prop• as oxalate (this can be detected by erties of yeast and . examining the beer by microscope to Big or small cloudy structures can appear display the oxalate crystals; to prevent in the bottle; they can occur either by the . their formation, a correct wort boiling, use of unclean yeast dirtied by colloidal 4. Conclusion a sufficient calcium concentration - 50 matter (this is often the consequence of an mgIL - and an evaporation ratio of 5%/ insufficient boiling or hot wort decanting) Beer refermentation is a practice generally hour are necessary). or by the presence of unusual microorgan• applied to the production of typical special isms during the main fermentation phase beers. It has to be considered as a particu• 3.3. Abnormal aspect of the yeast (7). A drastic cleaning of vessels and lar technique where the brewer has to take at the bottom or into the bottle piping is then necessary, the consequence several parameters into account. is catastrophic because beer with such Indeed as described, pitching rate for The aspect of a layer of yeast after refer• characteristics is generally unsalable. mentation can be of four different types: refermentation, yeast viability, quantity of D thin layer (compact or dusty) on the A cloudy appearence can also be the added sugar, bottling or kegging condi• bottom of the bottle; consequence of the use of alginates as tions, temperature at the start of refermen• foam stabilizer in refermented beer. It [J cloudy structures in the bottle; tation, physicochemical characteristics of seems that alginates prevent yeast floccu• beer, storage temperature, microbiologi• i:::J granular structures in the bottle; ., yeast adhering to the bottle's sides. lation and sticking at the bottom of the cal quality, dissolved oxygen, air in the bottle. Moreover, in most of cases, a bottle neck are all parameters which influ• Thin layer (compact or dusty) cloudy structure appears later on due to the ence strongly the beer characteristics. on the bottom of the hottle slow hydrolysis of the alginates in beer The brewer has to understand the impor• and the poor solubility of the liberated tance and control continuously and pre• This layer can have a compact or a dusty structures (mannuronic acid and other by• cisely these parameters in order to keep aspect. These particularities are for the products). constant the quality of his beer. , ~t.

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164 11/1992

5. Bibliography (7) Melotte. L. and Derdelinckx, G., confi• (14) Ussiglio-Tomasset, L., Bosia, P. D., di dential research. Stefano, R. and Castino, M., Annali (1) Bidan. P., Les vins mousseux, Centre de (8) Molnar, 1., Oura, E. and Suomalainen, H., dell'Istituto sperimentale per l'Eno-logia documentation internationale des indus• Acta Alimentana, 1981,10,27. Asti, 1983,14,91. tries utilisatrices de produits agricoles, (9) Ohno, T. and Takahashi, R., Report of the (15) Van de Meersche, J., Devreux, A. and Massy, France, 1975, chapter I. Research Laboratories of Kirin Brewery, Masschelein, C. A., European Brewery (2) Derdelinckx, G., Cerevisia, 1987,3, 153. 1983,26, 15 and 25. Convention, Proceedings of the 17th Con• (3) Derdelinckx, G., Vanderhasselt, B. and (10) Simpson, W. J., Journal of the Institute of gress, Ed. EBC, Zoeterwoude, 1979,787. Alvarez, P., J. De Clerck Chair Ill, Ed. J. Brewing, 1987,93,405. (16) Van Oevelen, D., de L'escaille, F. and De Clerck, Louvain-Ia-Neuve, 1988, 11th (11) Suarez, M. A., Polo, M. C and Llaguno, Verachtert, H., Journal of the Institute of lecture. C, Connaissance de la Vigne et du Vin, Brewing, 82, 322. (4) Dufour, J. P. and Malcorps, P., Louvain 1979,13,199. (17) Van Oevelen, D., Spaepen, M., Timmer• Brewing Letters, 1990, 1, 3. (12) Steiner, K. and Lanzlinger, U., European mans, P. and Verachtert,H., Journal of the (5) Martens, F. B., Egberts, G. T. C, Kem• Brewery Convention, Proceedings of the Institute of Brewing, 1977, 83, 356. pers. J., Robles de Medina, M. H. L. and 22nd Congress, Ed. Irl Press, Oxford, (18) Verachtert, H. and Dawoud, E., Louvain Welten, H. G. J., E. B. C Symposium on 1989,585. Brewing Letters, 1/2, IS. brewers' yeast. Monograph XII, Verlag (13)Takahashi, Y., Takahashi, S. Ujiie, E, (19) Wackerbauer, K. and Methner, F.-l., Hans Carl, Niirnberg, 1986,95. Sakuma, S., Shimazu, T and Kojima, Brauwelt international, 1988, no. 3, 382. (6) Melis, M. and Derdelinckx, G., Louvain K., MBAA Technical Quarterly, (20) Wackerbauer, K. and Methner, F.-l., Brewing Letters, 1989,3/4,38. 1991,28,60. Brauwelt international, 1989, no.l, 68 .•