MICROBIOLOGY Textbook - 2012

CHAPTER 15. FOOD MICROBIOLOGY

15.9. BEER MICROBIOLOGY

15.9.2. Beer-Spoilage Microorganisms

Only certain types of microorganisms adapted to survive in the specific environment of a brewing facility can multiply in beer. The growth of many microorganisms is inhibited by hop antiseptic compounds, low nutrient content, acidic pH (5.4–4.6), low-Temperature beer maturation, and ethanol produced by Yeast. Nevertheless, contaminant microorganisms entering the production process gradually adapt to the environmental and technological conditions to such an extent that controlling them poses considerable challenges. Due to advanced microbiological analysis Methods, alongside well-known Bacteria contaminating wort and beer, new species of beer-spoilage agents have been identified in recent years, including strictly anaerobic bacterial strains of the genera Pectinatus and Megasphaera.

Sources of wort and beer contamination by undesirable microorganisms include raw Materials, propagation yeast, Water, air, equipment, utensils, pipelines, as well as the clothing and hands of operating personnel.

In brewing, both Gram-positive and Gram-negative bacteria can cause beer spoilage (Table 16).

Class="center">Table 16. Beer-spoilage microorganisms

Among Gram-positive microorganisms, lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Lactococcus, and Leuconostoc are the most common beer spoilers.

Lactobacilli are rods of varying length, non-motile, and do not form endospores or capsules. They cause both homo- and Heterofermentative lactic acid fermentation. L. brevis and L. casei are the species most frequently detected in beer.

Pediococci are homofermentative cocci arranged in pairs or tetrads. They have long been associated with beer microbiology and were originally referred to as beer sarcinae. Pediococci multiply at an optimum temperature of 21–25 °C, with a growth temperature range of 7–45 °C. They most commonly proliferate in bottom-fermented beer and rarely in top-fermented beer. Among the pediococci identified in beer, P. damnosus (synonym P. cerevisiae) is the most widespread and hazardous. This microbe is absent from raw materials and is found exclusively in beer, brewer's yeast, and wines.

Leuconostocs are heterofermentative, oval-shaped cocci occurring in pairs or short chains. These LAB are commonly found on vegetables, fruit, and fermenting plant matter. Leu. mesenteroides is the only species identified in breweries.

Lactococci. Among homofermentative cocci, Lactococcus lactis ssp. diacetylactis, which is capable of producing diacetyl from citrate, is occasionally encountered in brewing plants.

Micrococci, staphylococci, and kocuriae are widespread in breweries and can sometimes cause beer spoilage. However, they multiply poorly at pH 4.5 and below and are sensitive to hop components. Kocuria varians (formerly Micrococcus varians) is occasionally isolated from propagation yeast but is harmless. Kocuria kristinae is an atypical micrococcus (functioning as a facultative anaerobe). Strains of this species are relatively acid- and hop-tolerant and can impart an atypical taste and fruity aroma to beer.

Bacilli are Gram-positive, endospore-forming bacteria. Their spores are present in malt and survive wort boiling; however, these bacteria subsequently fail to multiply in fermenting wort due to low pH and the presence of hops. Thermophilic bacilli of the species B. coagulans and B. stearothermophilus have been isolated from malt, sugar wort, and post-boil equipment.

The presence of Gram-negative bacteria during the brewing process is highly undesirable. This group of microorganisms includes acetic acid bacteria, members of the family Enterobacteriaceae, and bacteria of the genera Zymomonas, Pectinatus, and Megasphaera.

Acetic acid bacteria are small Gram-negative rods that may be motile or non-motile and do not form endospores. They are strict aerobes, catalase-positive, and oxidase-negative. They multiply in long-stored wort, beer, and pitching yeast. In liquid media, acetobacters form surface rings or pellicles, and some strains produce dextrin. Their growth temperature ranges from 5 to 40 °C. They are characterized by resistance to hop resins, acids (capable of multiplying at pH 3.2–2.5), and alcohol (tolerating up to 8% ABV). Acetic acid bacteria are subdivided into the genera Acetobacter and Gluconobacter. Representatives of the genus Acetobacter can oxidize alcohol first to acetic acid and then to CO₂ and water. Bacteria of the genus Gluconobacter are morphologically similar to acetobacters, but unlike them, they do not oxidize acetate to CO₂ and water.

Enterobacteria. The family Enterobacteriaceae encompasses numerous genera, among which Citrobacter, Enterobacter, Hafnia, Klebsiella, Obesumbacterium, Proteus, Rahnella, and Serratia are encountered in breweries. As a rule, bacteria of the genus Escherichia and pathogenic genera Salmonella and Shigella have not been detected in brewing facilities.

It is now well established that enterobacteria inhibit or accelerate fermentation and significantly affect the taste and aroma of finished beer. It was previously believed that enterobacteria could not survive the adverse conditions of beer fermentation; however, they can persist in an "undetectable" state—meaning they are not revealed on standard diagnostic media. Nevertheless, these bacteria remain viable while in an inactive state.

Klebsiella terrigena is isolated at various stages of beer production. Unlike other coliform bacteria, this species is non-motile and forms a classical capsule. It is capable of multiplying at temperatures around 10 °C. Beer produced from wort contaminated with K. terrigena exhibits a characteristic phenolic off-flavor. This off-flavor is attributed to the deamination of Tyrosine by these bacteria.

Hafnia alvei (synonym Obesumbacterium proteus) also belongs to enterobacteria and appears as thick, short, non-motile, non-spore-forming rods measuring (0.6–1.5) × (1.0–6.0) µm, frequently joined in chains. It enters the brewing process via water and propagation yeast. In beer, it multiplies During the first day of fermentation, competing for nutrients with budding yeast. Hafnia alvei retards the fermentation process, resulting in elevated gravity and pH values in the beer. This species is capable of accumulating methyl sulfide, dimethyl sulfide, n-propanol, isobutanol, isopentanol, n-butanediol, and diacetyl in beer. Another feature of Hafnia alvei is its ability, under the anaerobic conditions of fermenting wort, to reduce nitrate to nitrite. The latter reacts with wort amines and amides to form N-nitrosamines.

Rahnella aquatilis (formerly Enterobacter agglomerans) consists of small Gram-negative rods measuring (2.0–3.0) × (0.5–0.7) µm. They are motile at 25 °C, but motility is lost at 37 °C. R. aquatilis multiplies readily in hopped and unhopped wort regardless of the presence of yeast. At standard wort gravity, this species survives the brewing process, accumulating in recycled pitching yeast. However, in high-gravity beer, R. aquatilis is inactivated at an alcohol concentration of 11–12% ABV. Beer produced from wort containing up to 10⁶ CFU/cm³ of R. aquatilis was found to contain abnormally high amounts of diacetyl (0.7 mg/dm³) and dimethyl sulfide (143 µg/dm³). The levels of these compounds in beer depend on the degree of contamination by this bacterial species.

The genus Zymomonas comprises two species: Z. mobilis and Z. anaerobia. Z. mobilis is more commonly encountered in brewing and appears as short, thick rods measuring (1.0–1.4) × (2.0–6.0) µm. The Cells typically occur in pairs, or in chains or rosettes in certain strains. Both motile and non-motile strains exist. Motile cells possess one to four polar flagella. Z. mobilis carries out Alcoholic Fermentation via a modified Entner-Doudoroff Pathway. Acetaldehyde, acetone, acetic acid, and lactic acid are byproducts of this fermentation. Strains of Z. mobilis are capable of multiplying at high ethanol concentrations (up to 10–12% ABV).

Anaerobic Gram-negative rods. For a long time, it was believed that only aerobic and facultatively anaerobic bacteria could multiply in beer; however, evidence emerged indicating that packaged beer can be spoiled by strictly anaerobic rods. The first of these was named Pectinatus cerevisifilus, and subsequently, new genera of anaerobic rods—Selenomonas and Zymophilus—were isolated from beer in Germany, Japan, and Scandinavian countries.

Pectinatus cereviphilus is a slightly curved, Gram-negative, non-spore-forming rod measuring (2.0–3.0) × (0.7–0.9) µm with rounded ends, occurring singly, in pairs, or sometimes in short chains. Older cells may exhibit spiral-shaped hyphae. These bacteria are motile by means of a polar flagellum. This species is an obligate anaerobe that ferments glucose with the Production of acetic and propionic acids.

Anaerobic Gram-negative cocci. Bacteria isolated from spoiled, foul-smelling beer samples have been identified as Megasphaera cerevisiae, belonging to the family Veillonellaceae.

Megasphaera cerevisiae consists of slightly elongated cocci with a diameter of 1.3–1.6 µm, found in pairs or occasionally in short chains. They are non-motile and non-spore-forming strict anaerobes. They reproduce within a temperature range of 15–37 °C, with an optimum of 28 °C. M. cerevisiae lacks catalase, does not produce indole, does not reduce nitrates to nitrites, but produces H₂S.

Wild yeasts contaminating wort and beer are conventionally divided into 4 main types: 1 — sugar-fermenting yeasts; 2 — killer yeasts; 3 — "wild" variants of brewing yeast strains; 4 — non-sugar-fermenting yeasts.

1. Yeasts of the genera Saccharomyces, Kluyveromyces, Torulaspora, and Zygosaccharomyces share common biochemical characteristics, allowing them to successfully compete with cultured S. cerevisiae strains while exerting virtually no inhibitory effect on them. However, if wild yeasts grow at a higher rate, their proportion in the pitching yeast will increase during subsequent fermentations. Many wild yeasts are non-flocculent, causing the beer to become turbid. In beer subjected to fining or other clarification treatments, the presence of contaminant yeasts can result in an opalescent haze. The yeast species S. diastaticus is present during both fermentation and secondary fermentation (lagering), meaning that even low levels of contamination can lead to turbidity, as well as phenolic and other off-flavors and off-odors.

2. Killer yeasts eliminate sensitive cultured yeasts, subsequently becoming dominant during fermentation. This leads to The Development of off-flavors and various sensory defects in the beer. In brewing practice, Saccharomyces spp. are the most probable strains of killer yeasts.

3. For most beer styles, a specific yeast strain or blend of strains is of critical importance. Substituting one strain for another can result in unsatisfactory product quality. The key Properties of Individual yeast strains include fermentation rate, flocculation, HEAD retention, and the profile and quantity of secondary aromatic fermentation by-products. Replacing one yeast culture with another will cause all these characteristics to deviate from the required specifications.

4. Within this group, yeasts of the genus Pichia are most frequently encountered, particularly the species P. membranifaciens. Yeasts of the genera Brettanomyces and 1Dekkera require oxygen and produce acetic acid as a metabolic product, whereas yeasts of the genera Pichia and Williopsis produce esters, which significantly alter the organoleptic Properties of the finished beer. Atmospheric oxygen significantly affects the COMPOSITION OF THE beer's contaminant microflora. Its access to bottled or canned beer creates favorable conditions for the proliferation of any aerobic yeasts that survive filtration and pasteurization. Yeasts of the genera Debaryomyces, Dekkera, Issatchenkia, Pichia, and Williopsis are capable of multiplying in beer only under aerobic conditions, as are many yeasts of the genus Candida.

To enhance the biological stability of beer, it is pasteurized or passed through fine-pore filters. The finished beer is tested for the following parameters:

✵ Total aerobic microbial count (TAMC) — not exceeding 500 CFU per 100 cm³;

✵ Coliform bacteria — absent in 10 cm³;

✵ Pathogenic bacteria, including Salmonella — absent in 25 cm³;

✵ Total yeast and mold count — not exceeding 40 CFU/cm³ combined.