28th October 2018
It is not easy to “live” in the beer
In principle, lactic acid bacteria (LAB) and many other bacteria and generally most microorganisms, do not have it easy to survive in beer or other alcoholic beverages such as wine. This is one of the main reasons why wines and beers have been from ancient times the safest ways to drink hygienically something similar to water and that it was not contaminated, apart from boiled waters, such as tea and other herbal infusions.
The reasons for the difficult survival of microorganisms in beer are ethanol, the pH quite acidic (around 4), the lack of nutrients due to the fact that the yeasts have assimilated them, the little dissolved oxygen, the high concentration of carbon dioxide (0.5% by weight / volume) and the presence of humulone derived compounds (Figure 1) of hops: iso-alpha-acids, up to 50 ppm, which are microbiocides. All these obstacles make it very difficult for any microorganism to thrive. The most susceptible beers of unwanted microbial growth are those where some of the mentioned obstacles are dampened: beers with a higher pH of 4.5, or with little ethanol or little CO2, or with added sugars – which are nutrients -, or with little amount of compounds derived from hops (Vriesekoop et al 2012).
Figure 1. Humulone (left) of the hop is degraded during beer elaboration to isohumulone (right) and other iso-alpha-acids, which are compounds bitter and microbiocides (Wikipedia; Sakamoto & Konings 2003)
The acid pH of the beer (slightly higher than the wine) inhibits many of the best-known pathogens (Figure 2). And the cases we see that could grow at this pH near 4 are inhibited by other factors such as ethanol.
Figure 2. Range of acid pH for the growth of various bacteria, compared to the typical beer pH (Menz et al 2009).
The “bad” lactic acid bacteria of beer
Despite what we have just seen, some bacteria, particularly some LAB, have been able to adapt evolutionarily to the strict beer conditions, and they can survive and spoil them. In particular, the most frequent harmful species against the quality of beers are Lactobacillus brevis and Pediococcus damnosus (Figure 3). The first is the most frequent, and it can give tastes and undesired aromas, as well as turbidity to the final product. P. damnosus has the advantage of growing at low temperatures, and it can also produce undesired aromas, such as diacetyl (Vriesekoop et al 2012). Some Pediococcus and Lactobacillus may adhere to yeast, inducing them to sediment, which delays fermentation (Suzuki 2011).
Figure 3. Lactobacillus brevis (left) and Pediococcus damnosus (right) at the electronic scanning microscope.
Some Pediococcus may also be responsible for the appearance of biological amines in some beers, at risk for the consumer. Amines in a certain concentration are toxic, they may be present in some fermented foods such as cheese, cold meat and alcoholic beverages such as wines and beers, and are produced by decarboxylation of amino acids by LAB. The level of tyramine and other amines has been used as a measure of quality in some Belgian beers made with LAB (Loret et al 2005).
Apart from these LAB, other bacteria related to problems of beer contamination are acetic acid bacteria such as Acetobacter, typically associated with oxygen intake in packaging or distribution. Other harmful bacteria are some enterobacteria, such as Shimwellia pseudoproteus or Citrobacter freundii, which proliferate in the early stages of fermentation, and produce butanediol, acetaldehyde and other unwanted aromatic compounds (Vriesekoop et al 2012). Other harmful bacteria for beer, especially when bottled, are Pectinatus and Megasphaera, which are strict anaerobes, of the clostridial family, and can produce hydrogen sulphide and short chain fatty acids, all of them unpleasant (Suzuki 2011 ).
The “good” lactic acid bacteria of beer
LAB are well known for being some of the microbes that most benefits contribute to the food production, on the one hand as an economic means of preserving food, and on the other hand to improve their quality and organoleptic characteristics. That’s why they are the main agents of fermented foods, along with yeasts. We have seen some of the LAB’s food benefits in other posts in this blog: prehistoric cheeses, or breast milk microbiota, and even wine bacteria.
Therefore, LAB also have a good role in the production of beers: in particular, as we will see below, in the production of acidified malt, and in some peculiar styles of beer such as the Belgian Lambic and the Berliner Weissbier.
As you know, malt is the raw material for making beer. The cereal is subjected to the malting process, where cereal grains, mainly barley, are germinated, the enzymes hydrolyse the starch into sugars, and all of this is then heated obtaining the must, the substrate solution which will be fermented by the yeasts ferment, producing ethanol and carbon dioxide.
The acidification of the malt, that is, with a lower pH, has the advantages of activating many important enzymes in malting, giving a lower viscosity to the malt and therefore to the final beer. Although adding mineral acids or commercial lactic acid can achieve acidification, it is often recommended or legislated a biological acidification, which is achieved by adding LAB. The use of LAB starter cultures is a relatively new process and in addition to the commented benefits on the quality of the malt, it has been shown to also inhibit unwanted molds that are a real problem in malting and that can give mycotoxins. The compounds produced by LAB that can inhibit the fungi are the same lactic acid and the consequent pH drop, bacteriocins, hydrogen peroxide, and other compounds not well known as perhaps some peptides (Lowe & Arendt 2004).
The most commonly LAB strains used to acidify malt are Lactobacillus amylolyticus previously isolated from the same malt. These strains are moderately thermophile, resistant to compounds derived from humulone, and they have the advantage of being amylolytic in addition to producing lactic acid, which lowers the pH (Vriersekoop et al 2012).
Beers with LAB participating in the fermentation, such as Lambic and Berliner Weissbier styles, belong to the type of spontaneous fermentation beers. The other types of controlled fermentation beers are the best-known Ale and Lager, both inoculated with specific yeasts. Ale beers are those of high fermentation, where Saccharomyces cerevisiae yeast used tends to remain on the surface and the fermentation temperature is above 15-20ºC. Lager ones are those of low fermentation, originally from Bavaria, where yeast S. pastorianus (S. carlsbergensis) tends to settle at the bottom of the fermenter and the temperature is between 7 and 13ºC.
Belgian Lambic beer
Traditional Belgian beers (in Dutch lambiek or lambik) are known for their sensorial characteristics due to LAB activity. They are traditional in Brussels itself and in the neighbouring region of Pajottenland, in the Zenne river valley, in the Flemish Brabant on the SW of the Belgian capital. One of the villages in this valley is Lembeek, which could be the origin of the name of this beer.
These beers of spontaneous fermentation represent the oldest style of making beer in the developed world, for some centuries. For a few years now (since around 2008), similar beers are made in the USA, called “American coolship ales” (Ray 2014).
Lambic beer is made with barley malt and a minimum of 30% of non-malted wheat. The cones of a special hops, completely dried and aged for 3 years, are added to the must. They are added not for their aroma or bitterness, but rather as antimicrobial, to prevent above all, the growth of gram-positive pathogenic bacteria in the fermentation broth.
Also to avoid these contaminants and to promote the microbiota typical of the Lambic fermentation, these beers are brewed only between October and May, since in summer there are too many harmful microorganisms in the air that could spoil the beer, and it is necessary to lower the temperature after boiling. Boiling of the must is done intensively, with an evaporation of 30%.
After boiling, the broth is left in open deposits, and in this way the microorganisms of the air present in the fermentation rooms of the brewery (usually at the top of the building) are acquired, and of the outside air, since the tradition says that the windows must be left open. It is assumed that the captured microbes are specific to the Zenne Valley. These open deposits are the koelschip in Dutch (coolship in English), like swimming pools (Figure 4). Being well open, with a lot of surface (about 6 x 6 m) and shallow depth (about 50 cm), they favour the collection of microbes from the room and from the outside. Another purpose of this form is the fastest cooling of boiled broth to start fermentation. They can be made of wood, copper, or stainless steel more recently.
Figure 4. Koelschip (in Dutch) or coolship in English, the open deposits, as swimming-pools, where the Lambic beer process begins (Brasserie Cantillon, Brussels).
The “inoculated” broth in this spontaneous way is left only one night in the coolship, and on the following day this must is pumped into fermentation tanks where there will stay a year, during which the sugar content will go down, up to about 30 g/L. Then it is transferred to oak barrels, previously used for sherry or port, and there it can be left for another two years, at temperatures of 15-25ºC. Some barrels are the same used since 100 years ago. The final product is a cloudy beer, with a pale yellow, very little CO2, dry, acidic, with about 6-8º of ethanol. It reminds a bit like the sherry and especially the cider, and with a slightly bitter taste (Jackson 1999).
In this long process of fermentation, up to 3 years, of course there is a diversity in the composition of the microbial population. In a first phase there is a certain predominance of Kloeckera yeasts and especially enterobacteria during the first month. After 2 months, Pediococcus damnosus and Saccharomyces spp. predominate, and alcoholic fermentation begins. After 6 months of fermentation the predominant yeast is Dekkera bruxellensis (Spitaels et al 2014), or what is the same, Brettanomyces (Kumara & Verachtert 1991), of which Dekkera is the sexual form.
Figure 5. Species of isolates in MRS and VRBG agar media, for lactic acid bacteria and enterobacteria respectively, during the process of making a Lambic beer. The number of isolates is given between brackets (Spitaels et al 2014).
We see (Figure 5) as in particular after 2 months the predominant bacterium is the LAB P. damnosus. It was appointed in the first studies as “P. cerevisiae“, but this name was finally not admitted because it included other species. The count of these in MRS is 104UFC per mL until the end of fermentation. Acidification seems to be rapidly taking place in the transition from the first stage to that of maturation, coinciding with the growth of P. damnosus, which produces lactic acid, although Dekkera/Brettanomyces and acetic acid bacteria also contribute to the acidification (Spitaels et to 2014).
In other trials with the American coolship ales (ACA) of Lambic style, Lactobacillus spp. have also been found, and in a metagenomic study (Bukolich et al 2012) of these ACA, DNA of several Lactobacillales has been detected. At the end of the process, a predominance of Pediococcus (Figure 6, panel C) was also observed. In the same figure in panel A we observe how the predominant unicellular fungus is also Dekkera/Brettanomyces.
Figure 6. TRFLP analysis (polymorphisms of lengths of PCR-amplified terminal restriction fragments) of total DNA extracted from the fermentation samples of ACA beers (similar to Lambic) during 3 years, using primers for: ITS1/ITS4 of 26S rDNA for yeasts (panel A), 16S rDNA for bacteria (panel B), and specific ones for LAB (panel C). Samples marked with * did not give amplification (Bukolich et al 2012).
Lambic derived beers: Gueuze, Faro, fruity and others
The basic Lambic, which is difficult to purchase, is only found in a few Brussels cafes and the production area. In fact, Lambic is the basis for elaborating the others, much more common to consume:
The Faro is a Lambic sweetened with brown sugar and sometimes with spices.
The fruity Lambic are those that have been added whole fruits or fruit syrup. They can be with bitter cherry (kriek), which are the most traditional, or with raspberry, peach, grapes, strawberry, and sometimes also apple or pineapple or apricot or other.
And finally, the Gueuze, which are sparkling and easy to find. They are made by mixing young Lambics (from 6 months to 1 year) with other more mature ones (2-3 years) in thick glass bottles similar to those of champagne or cava and left for a second fermentation with the remaining sugars from the young Lambic. This would have been begun by a mayor of Lembeek in 1870 that owned a brewery and applied the fermentation techniques in the bottle that had been successful in the Champagne some years before (Cervesa en català 2012). The word Gueuze can have the same etymological origin as gist(yeast in Flemish) and it could also refer to the fact that it produces bubbles of CO2, that is, gas (Jackson 1999). However, another historical version would be that this beer was called “Lambic de chez le gueux” (Welsh from poor people) because the mentioned mayor of Lembeek had similar socialist ideas to those of the “Parti des Gueus” founded by the Calvinists from Flanders in the 16th century to fight against the Spanish empire. And since beer is feminine in French, the gueuxfeminine is gueuze, here it is.
In this refermentation in the bottle the populations of Dekkera/Brettanomycesand LAB are maintained, although other unicellular fungi such as Candida, Hansenula, Pichia or Cryptococcus (Verachtert & Debourg 1999) appear in limited numbers.
Figure 7. Several beer Gueuze and fruity Lambic, mostly Belgian (from www.swanbournecellars.com.au/).
The Berliner Weissbier (Figure 8) is another beer relatively similar to Lambic ones. It is also brewed with an important part of wheat must, it is cloudy, acidic and with 3% ethanol. It is traditional in Berlin and the north of Germany, made from the s. XVI and the most popular alcoholic beverage in Berlin until the end of the s. XIX. It was called the “northern champagne” by the Napoleon’s soldiers. Spontaneous fermentation of must involves a mixture of Dekkera/Brettanomyces, Saccharomycesand hetero-fermentative Lactobacillus.
Figure 8. Berliner Weisse beer (from G-LO, @boozedancing wordpress).
Beers similar to Lambic brewed in Spain
In the same way that the commented American Coolship Ales, Lambic style beers are also made in many other countries and, in the case of Spain, coinciding with the boom of artisanal beers, they are also elaborated, especially the fruity Lambic ones. According to the Birrapedia website, 6 of these are currently being processed, all of which are cherries. Two of them are made in Lleida, one in Barcelona, one in Alicante, one in the Jerte valley, and another in Asturias.
Resistance of lactic acid bacteria from beer to hop compounds
Lactobacillus and Pediococcus, both bad and good we have seen, and other contaminating bacteria of beers, have the ability to withstand hop compounds, which, as we have seen, are natural microbiocides. This resistance can be due to various defence systems, both active and passive (Sakamoto & Konings 2003). The active systems include efflux pumps, such as HorA and HorC, which carry the iso-alpha-acids (Figure 1) out the cell. HorA does it with ATP consumption, and HorC using the proton driving force (Figure 9). The corresponding genes horA and horC were originally found in L. brevis, but later they were also found in L. lindneri, L. paracollinoides and in the best known P. damnosus(Suzuki et al., 2006).
Curiously, HorA shows a resemblance of 54% to OmrA, a membrane transporter of Oenococcus oeni, related to the tolerance of this bacterium from wine to ethanol and other stressors (Bourdineaud et al 2004) (See some more about O. oeni in my post on the bacteria of the vine and the wine). Therefore, it is probable that HorA also has functions of exclusion of other compounds aside from those of the hops. It has been seen that these horAand horC resistance genes and their flanking regions are well preserved and have sequences almost identical to the different species that have them. Therefore, it is very likely that some have been acquired from others by means of horizontal gene transfer, by plasmids or transposons, as is usual in many other bacteria (Suzuki 2011).
Figure 9. Mechanisms of resistance to hop compounds in Lactobacillus brevis (Suzuki 2011).
As we see in Figure 9, protons are pumped out by an ATPase, and the consumption of ATPs is compensated by forming it thanks to the consumption of substrates such as citrate, malate, pyruvate or arginine. Another mechanism of resistance, passive in this case, is the modification of the composition of membrane fatty acids, with the addition of more saturated ones, such as C16:0, which reduces the membrane fluidity and makes it difficult the entrance of the hop compounds. This also reminds us of the changes in membrane of O. oeni related to the resistance to ethanol (Margalef-Català et al 2016). The cell wall also changes its composition in the presence of the hop alpha-iso-acids, increasing the amount of high molecular weight lipoteichoic acid, which would also be a barrier. We also see (Figure 9) how hop compounds can lower the intracellular levels of Mn2+, and then a greater synthesis of Mn-dependent proteins is observed, and a greater capture of Mn2+ from outside. Finally, cells of L. brevis reduce their size when they are in beer (Figure 10), probably in order to decrease the extracellular surface, thus minimizing the effect of external toxic compounds (Suzuki 2011).
Figure 10. Effects of beer adaptation (left) in the size of Lactobacillus brevis cells compared to well grown cells in rich media MRS (right). The bars are 5 mm (Suzuki 2011).
All these mechanisms have been studied in L. brevis strains harmful to beer, but it is assumed that the resistance of beneficial bacteria from Lambic and others would be due to the same mechanisms, since they are of the same bacterial species.
As a conclusion to all said, we see that LAB have outstanding roles as beneficial in various aspects of brewery and malting, despite their most known role of harmful in the processing of the most common beers.
Birrapedia (seen 18 august 2018) Cervezas de tipo Fruit Lambic elaboradas en España. https://birrapedia.com/cervezas/del-tipo-fruit-lambic-elaboradas-en-espana
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