Fermented beverages

17 Fermented beverages

I Introduction

A Definitions Fermentation is any process during which the enzymes of a microorganism convert an energy source to

one or more chemical products, which may be for foods or for industrial or pharmaceutical use. Yeasts are by far the most important microorganisms used for liquid fermentations, though bacteria are used in some milk-based products such as yoghurts.

The most important yeast fermentation product is alcohol, and this chapter will discuss the major product of such fermentations, beer, and wine. Solid fermentations, i.e. of fruit and vegetables, are treated under other chapters.

B Important properties Beer, wine, and other alcoholic beverages usually contain >4% alcohol, which gives the products their

character. Products from grapes (wines) have an acidic pH. Desirable pH values for white wine are 3.0–3.4 and for red wines 3.3–3.7 (Rankine, 1989). Together with the alcohol content, these low pH levels provide stability against most microorganisms. Products from apples (cider) are also of acid pH. Products from cereals (beers) are generally of higher pH, 4.0–4.5, and here the addition of carbon dioxide provides additional microbial stability. Products from honey (mead) are also of higher pH.

C Methods of processing Beer. Barley is the main substrate for production of beer, though wheat is also used. Barley is treated

to produce malt by “steeping”, in which a sufficient amount of water is added to induce germination of the barley. Enzymes produced during germination break down the starch in the kernels into small polysaccharides, making it fermentable by yeasts. After this process is complete, the barley is kiln dried to produce malt. Wort is produced from the dry malt by the addition of hops and sometimes adjuncts (starch in the form of rice, maize, etc.), and boiling in water.

The wort is then cooled, oxygenated, and fermented using specific yeast species and strains, usually Saccharomyces cerevisiae or Sacc. pasteurianus, which produce lager beer or ale. An example of a fermentation chart for a lager beer showing the temperature and the decrease in pH and fermentable extract ( ◦ Plato) over time is given in Figure 17.1 [professional brewers often use the Plato ( ◦ P) scale, instead of specific gravity, as a measure of the sugar levels in wort and beer]. A specific gravity (SG) of 1.004 is equivalent to 1 ◦ Plato (1% sucrose) and 1.040 to 10 ◦ Plato (10% sucrose). Hence, each Plato degree accounts for 0.004 SG. To convert SG to Plato, divide the digits to the right of the decimal point by 4. For example, 1.044 is 11 ◦ Plato and 1.054 is 13.5 ◦ Plato). After fermentation and maturation (low temperature and secondary fermentation), the beer is sterile filtered or plate pasteurized before bottling or canning, or pasteurized in tunnels after filling and sealing.

Wines. The grape juice (“must”) is extracted from the grapes by crushing and often maceration of the grapes. A starter culture, usually Sacc. cerevisiae, may be added, or fermentation may rely on naturally

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Fermentation Diagram

Figure 17.1 Fermentation chart for lager beer: changes in temperature ( ◦ C), pH, and fermentable extract (%P.)

occurring yeasts present in the grapes. Greater control of the fermentation process is possible if starter cultures are used. In the production of white wines, the skin of the grapes is removed from the must before fermentation; while red wines are produced by fermentation with the skins present. Controlled temperatures are sometimes used, especially for white wine production.

Traditionally, SO 2 has been used to control undesirable microorganisms as well as unwanted color deterioration (browning) due to enzyme activity. However, the increasing focus on health issues has lead to a reduction in the use of SO 2 . Fermentation may involve microorganisms from the grapes or winery environment as well as starter cultures, usually Saccharomyces spp. The purpose of using a starter culture is to establish a controlled dominance of the culture yeast over the native microflora.

To produce wines of lower acidity, the primary fermentation is often followed by malolactic fer- mentation, where L-malic acid is converted into l-lactic acid. The microorganisms responsible for the

malolactic fermentation are lactic acid bacteria (LAB) dominated by several species of Lactobacillus spp. (Lactobacillus casei, Lb. plantarum, Lb. sake, Lb. brevis, Lb. fructivorans, etc.), Pediococcus spp. (Pediococcus parvulus, Ped. pentosaceus, and Ped. damnosus) and Leuconostoc species. Because of the detrimental effect on shelf life of pasteurization, sterile filtration is usually the preferred method for stabilizing the wine microbiologically before bottling.

Fortified wines, sherries, and distilled products. Sherries are produced by using special alcohol- tolerant yeast strains. The introduction of oxygen during fermentation also assists the formation of higher levels of alcohol than is produced in wines. Fortified wines are produced by the addition of distilled alcohol to wines. Distilled products are made from a variety of substrates with the addition of alcohol. Such products have no microbiological issues and are not considered further.

MICROORGANISMS IN FOODS 6

D Types of final products Ale types of beer are mainly produced in Great Britain and differ from the conventional European lager

beers on a number of parameters, e.g. malt type, yeast type, and fermentation temperature. The ale yeast (Sacc. cerevisiae) is always a top fermenting strain and fermentation is normally at temperatures of 20–23 ◦

C. The lager strains (Sacc. pasteurianus) are usually bottom fermenters and used at 12–17 ◦ C. Beer is classified according to alcohol content, typically ranging from 0% to 6% alcohol by volume, but there are no international standards for this classification. Generally speaking, wines are classified according to color (red, white, and ros´e) and alcohol content. Table wines have an alcohol content of 7–14% v/v. Fortified wines such as port, sherry, and madeira usually have an alcohol content of 14–21% v/v. Wines may also be classified according to the grape

variety, taste (dry, semidry, semisweet, and sweet), and content of CO 2 (still or sparkling).

II Initial microflora

A Grains The initial microflora of malt is dominated by microorganisms indigenous to the barley (Flannigan,

1969, 1996; see also Chapter 8). The initial microflora of adjuncts like maize and rice that are used in some breweries is also described in Chapter 8.

A variety of fungi (especially species of Fusarium and Aspergillus) may invade barley before malt- ing. Fusarium plays a role in cold, wet climates, invading the kernels during the growing season. Kernels infected with Fusarium species may be responsible for gushing (over-foaming) of the final beer (Flannigan, 1996).

During steeping, some microorganisms are capable of growth. Increases in bacterial numbers are vari- able, depending on temperature and a w , with Aureobacterium, Alcaligenes, Clavibacterium, Flavobac- terium, Erwinia, and Pseudomonas being the most common genera (Petters et al., 1988). Fungi that commonly increase in numbers include Fusarium, Mucor, Eurotium, Alternaria, and Rhizopus (Flan- nigan, 1996). Aspergillus clavatus is of particular concern, as this species can build to unacceptably high levels if malting temperatures are elevated or spontaneous heating occurs (Flannigan et al., 1984; Flannigan, 1986). In extreme cases, blue green mats of Asp. clavatus may form on grain during malting (Shlosberg et al., 1991). Under these conditions Asp. clavatus can be allergenic and is reported to be the cause of “malt workers’ lung” (Riddle et al., 1968; Flannigan, 1986).

Kilning reduces the microbial population substantially, but does not usually eliminate the flora (Flannigan, 1996).

6 The microflora of finished malt commonly consists of 10 8 –10 cfu/g, consisting largely of Erwinia, Pseudomonas, and Bacillus species. Lactobacilli can be of the order of 10 4 per gram (Flannigan,

1996). Fungal counts are commonly 10 3 –10 4 cfu/g, with great variability reported in the genera present (Flannigan, 1996). Malt may be stored or transported before use, during which time it may pick up moisture, often with resultant increases in storage fungi including Asp. candidus and Asp. versicolor (Flannigan, 1996).

B Grapes Many studies on the microflora of grapes have been published, but sound quantitative data are still

needed (Fleet and Heard, 1992). In general terms, the microflora on grapes is dominated by yeasts (Kloeckera, Hanseniaspora, Candida, Pichia, and Kluyveromyces spp.), LAB, acetic acid bacteria (Gluconobacter and Acetobacter spp.), and fungi (Botrytis, Penicillium, Aspergillus, Mucor, Rhizopus,

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III Primary processing

A Effects of processing on microorganisms Beer manufacture. The heat treatment of the malt and adjuncts (i.e. boiling to produce wort) eliminates

the initial microflora except for spore-forming bacteria and fungi. These microorganisms are usually of little relevance but may cause problems with low acid wines.

The yeast cultures used for the fermentation of beer are produced from pure cultures of selected strains of Sacc. pastorianus (lager beer) or Sacc. cerevisiae (ale beer). Growth of microorganisms in beer and wine is restricted by the alcohol content, the low pH, the presence of yeast metabolites (organic acids, fatty acids, and acetaldehyde), and the low redox potential created by the production of CO 2 . In beer, the content of oxygen is typically <0.5 mg/L. In beer, the microflora is also controlled by the presence of CO 2 (∼0.5% w/v). Further, the hops contain a number antimicrobial compounds, including α- and β-acids, iso-humulone, trans-isohumulone, trans- humulinic acid, and colupolone. Most lactic acid bacteria are inhibited by the levels of hop bitters used (15–55 mg/L of iso-alpha-acids). Leuconostoc and Lactococcus spp. are sensitive, as are most Lactobacillus and Pediococcus spp. However, some Lactobacillus and Pediococcus spp. isolated from spoiled beer can resist concentrations >65 mg/L of these acids (Simpson, 1993).

Winemaking. Aseptic crushing of sound, mature grapes will yield a total yeast population of

10 3 –10 5 cfu/mL. The apiculate yeast Kloeckera apiculata and Hanseniaspora species dominant, accounting for 50–70% of the population. Lesser populations of Candida, Cryptococcus, Rhodotorula, Pichia, and Kluyveromyces species will also be present. Fermentative species, i.e. Saccharomyces species, occur only at low levels (Martini and Martini, 1990). Numerous factors affect this population, including climate and weather at the time of picking, use of fungicides in the vineyard, and physical damage to the grapes (Fleet and Heard, 1992). The surfaces of winery equipment are also important sources of yeasts and other contaminants. Importantly, the winery equipment provides an important source of Sacc. cerevisiae, which also may or may not be added as a starter culture.

During fermentation, the viable populations of yeasts increase to 10 8 –10 9 cfu/mL, with growth following the typical growth curve for microorganisms in batch culture. In the absence of a starter culture,

a range of yeast species become established, including various species of Kloeckera, Hanseniaspora, Candida, and Pichia. These genera die off within 2–3 days of the start of fermentation, and are replaced by Saccharomyces species, which are able to withstand the increasing concentrations of alcohol produced (Fleet and Heard, 1992).

The fermentation process is influenced by the use of sulfur dioxide in the fermentation, traditionally considered to limit growth of yeasts other than Saccharomyces species. Recently, this view has been challenged (Heard and Fleet, 1988a). Fermentation temperature affects both the rate of fermentation and, probably more important, the ecology of the process (Heard and Fleet, 1988b; Fleet and Heard, 1992).

Lactic acid bacteria are also an integral component in the ecology of winemaking. The principal species associated with grape fermentation are Leuconostoc oenos, Pediococcus parvulus, Ped. pen- tosaceus, and various Lactobacillus species (Fleet and Heard, 1992).

B Spoilage Spoilage of beer. In the wort and during the first days of fermentation, oxygen is sufficient and the pH is

relatively high (5.0–5.5), consequently enterobacteria (e.g. Obesumbacterium proteus, Hafnia protea, Rahnella aquatilis, Enterobacter agglomerans, and Klebsiella terrigena) may proliferate, affecting

MICROORGANISMS IN FOODS 6

Table 17.1 Lactobacillus and Pediococcus species ranked according to importance in beer spoilage a

Lactobacillus

Group 1

Pediococcus Lb. brevis

Group 2

Group 3

Ped. damnosus Lb. lindneri

Lb. brevisimilis

Lb. delbrueckii

Ped. inopinatus Lb. curvatus

Lb. malefermentans

Lb. fermentum

Ped. dextrinicus b Lb. casei

Lb. parabuchneri

Lb. fructivorans

Ped. pentosaceus b Lb. buchneri Lb. coryneformis Lb. plantarum

a From Back (1987), Farrow et al. (1988), and Priest (1996). It should be noted that not all of the Lactobacillus species are now recognized as valid (Hammes and Vogel, 1995). b

Usually, no growth occurs in beer due to sensitivity to low pH, but may occur in pitching yeast and fermenting wort.

including Pichia, Brettanomyces, Dekkera, Debaromyces, Filobasidium, and Candida spp. may grow and produce acetic acid and esters, changing the flavor of the finished beer.

The lactic acid bacteria are considered the most hazardous of all beer spoilage microorganisms, especially Ped. damnosus. Whether introduced during fermentation or at bottling, LAB may grow in the beer, producing diacetyl (e.g. Ped. damnosus and Lb. brevis) or fruity (Micrococcus kristinae) off-flavors in the beer. The most important spoilage species in Lactobacillus are listed in Table 17.1.

Wild yeasts, defined as “yeasts not deliberately used and not under full control” (Priest, 1981) can grow in the fermenting beer, affecting the fermentation and producing off-flavors, such as phenolics. The group of wild yeasts able to spoil beer is very diverse and is normally divided into Saccharomyces and non-Saccharomyces wild yeasts. The Saccharomyces wild yeasts, dominated by Sacc. cerevisiae and Sacc. pastorianus (Jespersen et al., 2000), are often regarded as the most hazardous. In a study of yeast samples from 45 lager breweries, >50% of the wild yeast strains isolated belonged to Sacc. cerevisiae (van der K¨uhle and Jespersen, 1998). Wild yeasts other than Saccharomyces include Pichia, Candida, Kluyveromyces, Torulaspora, Brettanomyces, and Zygosaccharomyces species (Campell and Msongo, 1991; Campell, 1996; van der K¨uhle and Jespersen, 1998).

Over the years, brewers have had great success reducing the levels of oxygen in the beer to achieve better taste stability. The production of non-pasteurized or flash pasteurized beer is also increasing. As

a result of these developments, strictly anaerobic Gram-negative bacteria including Pectinatus cerevisi- iphilius and Megashaera spp. are becoming more and more important as beer spoilers, especially in beers with pH >4.1–4.3 and ethanol <5% (w/v). Growth of these bacteria may occur during maturation, but they are most often seen as contaminants during bottling. The anaerobic bacteria result in strong off-flavors of the beer due to their production of fatty acids (propionic, acetic, succinic, and butyric acids), mercaptans, dimethyl sulfide, and hydrogen sulfide (Seidel-R¨ufer, 1990).

Spoilage of wines. Similarly, growth of unwanted microorganisms on the grapes, in the must, during fermentation and maturation of wine can result in off-flavors and may affect the primary and secondary

fermentation. As with beer, wild yeasts including Zygosaccharomyces, Brettanomyces, and Schizosac- charomyces play an important role as spoilers causing estery, mousy, or phenolic off-flavors, haze or turbidity. Some yeasts can also produce low levels of a variety of sulfur compounds, adversely affecting aroma and flavor (Rauhut, 1992).

The oxygen levels of fermenting grapes and wines are higher than that of beer allowing acetic acid bacteria, including Gluconobacter and Acetobacter spp., to produce acetic acid or even ropiness. Lactic acid bacteria (Lactobacillus, Pediococcus, and Leuconostoc spp.) are to a large extent controlled by the alcohol but may cause acidity, diacetyl off-flavors, mousiness, etc., and ropiness. Spore-forming

721 Table 17.2 Occurrence of ochratoxin A in beer

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Positives (µg/kg) Origin of sample

Detection limit

Mean Range Germany

Incidence (%)

(µg/kg)

0.1–1.5 Canada (11 were imports)

0.1 NS

0.01 0.06 0.01–0.2 United Kingdom (14 were imports)

0.01 0.012 (median) 0.01–0.033 Total

Adapted from Scott (1996).

A major problem in wines is the growth of the spoilage yeast Zygosaccharomyces bailii. This yeast has a high tolerance to both ethanol and preservatives, as well as the ability to grow in high sugar concentrations (Sponholz, 1992). It can cause spoilage of white wines that contain residual sugars (glucose) by carbon dioxide production in the bottle, or by growth, which produces an undesirable haze. In warmer climates, white wines are often filter sterilized through membrane filters just before bottling to reduce the hazard from this yeast. Brettanomyces species can also cause wine spoilage by the production of haze or volatile acidic off-flavors (Sponholz, 1992). Certain other yeasts (Candida, Metschnikowia, and Pichia spp.) can form films on bottled wines and produce off-flavors due to acetaldehyde production (Sponholz, 1992).

Acetic acid and lactic acid bacteria occasionally cause wine spoilage also (Sponholz, 1992).

A variety of moulds may cause earthy and corky off-flavors mainly from growth on corks, wooden barrels, etc. (Lee and Simpson, 1993; Chattonet et al., 1994). The principal species are Penicillium glabrum and Pen. spinulosum (Lee and Simpson, 1992). Several possible mechanisms for cork taint exist, and these are revieweded by Lee and Simpson (1992).

C Pathogens There have been no reports of illness due to enteropathogenic microorganisms associated with beer or

wine. Mycotoxins in beer. Because beer is made from barley, it might be expected occasionally to contain

ochratoxin A. Data from 422 samples of beer are summarized in Table 17.2. Although 158 (37%) of the samples listed here were positive for ochratoxin A, the maximum level observed was only 1.5 µg/kg. Reported means ranged from 0.06 to 0.8 µg/kg. The latter figure was exceptional, and the true mean for European beers is probably near 0.1 µg/kg (Table 17.2). The beer fermentation process resulted in

a 2–13% reduction in ochratoxin A contamination (Scott et al., 1995; Scott, 1996). Mycotoxins in wines. Attention was drawn to the possibility of ochratoxin A contamination of wines

by Zimmerli and Dick (1995, 1996), who developed sensitive methodology for ochratoxin detection. Extensive data on ochratoxin A in wines from Europe and worldwide is now available and is summarized in Tables 17.3 and 17.4. Although the results indicate clearly that a high proportion of wines contain ochratoxin A, levels reported have almost always been low.

The highest ochratoxin A level observed in more than 200 samples reported in Table 17.3 was

7 µg/L in an Italian red wine. Methods of reporting make quantitative data difficult to extract, but it appears that figure was exceptional. Few of these or many other reported samples from Europe and elsewhere contained more than 1 µg/L ochratoxin A. H¨ohler (1998) reported that wines from Germany, France, and Spain had median concentrations of ochratoxin A <0.15 µg/L, samples from Italy, Portugal,

MICROORGANISMS IN FOODS 6

Table 17.3 Occurrence of ochratoxin A (ng/L) in major types of European wines

Number of

Number (%)

Type

Maximum White

samples

positive

90th percentile

1400 Ros´e

7000 From Majerus et al. (2000). Limit of detection 10 ng/L.

Table 17.4 Occurrence of ochratoxin A (ng/L) in wines worldwide Country

Maximum Germany

No. of samples

1850 South Africa

7000 From H¨ohler (1998).

samples from Tunisia was 1.6 µg/kg (Table 17.4). In a recent study of 600 wines from Australia, only one contained in excess of 0.5 µg/kg (Hocking et al., 2003).

However, because wine consumption is high in some populations, wine cannot be ignored as a source of ochratoxin A, especially in regions where this toxin is present in other foods as well (Pitt and Tomaska, 2002).