GENERAL AND FOOD MICROBIOLOGY PART II - L. V. Krasnikova - 2016

TOPIC 12. MICROBIOLOGICAL CONTROL IN BREWING

12.1. Spoilage and contaminant microorganisms in brewing

Only certain types of microorganisms adapted to the specific conditions of the brewing process are capable of multiplying in beer wort and finished beer. The primary contaminants of pitching Yeast, wort, and beer include Gram-positive Bacteria of the genera Lactobacillus, Pediococcus, Leuconostoc, Micrococcus, Sarcina, Streptococcus, and Kocuria, as well as Gram-negative bacteria of the genera Acetobacter, Zymomonas, Citrobacter, Enterobacter, Hafnia, Klebsiella, and Obesumbacterium. In addition to bacteria, common brewing pests include wild Yeasts (a detailed description of which is provided in the textbook "General and Food Microbiology", 2016, Part I).

Bacteria of the genus Lactobacillus enter beer via wort, pitching yeast, or insufficiently treated Water. The species most frequently encountered in beer include L. brevis (hop-tolerant), L. diastaticus (possessing active amylases responsible for the Hydrolysis of dextrins in beer), as well as L. casei, L. plantarum, L. fermentum, L. coryneformis, and L. buchneri.

Beer spoilage caused by lactobacilli is characterized by a "silky turbidity", sometimes preceded by a buttery diacetyl odor. The lactic acid produced during Fermentation imparts a sour taste to the beer.

Lactic acid bacteria of the genus Pediococcus (formerly known as beer sarcinae due to the tetrad arrangement of their cocci) cause opalescent turbidity, fine-grained sediment, off-flavors, and a honey-like aroma in beer. Among pediococci, Pediococcus damnosus is the dominant species in beer, exhibiting the highest resistance to the antiseptic action of hops. Proliferation of pediococci can also increase beer viscosity; slime formation is attributed to P. acidilactici strains, which synthesize complex extracellular Heteropolysaccharides composed of glucose, mannose, and nucleic acid.

Heterofermentative lactic acid bacteria of the genus Leuconostoc are rarely found in breweries. The species Leuc. mesenteroides, isolated from beer, is capable of causing ropiness (slimes) through The Biosynthesis of the polysaccharide dextran.

Bacteria of the genera Micrococcus, Sarcina, Streptococcus, and Kocuria are widespread in brewing facilities and can occasionally lead to beer spoilage. Many species of these microorganisms are able to survive in beer for extended periods.

Certain micrococcal species detected in brewing belong to the genus Kocuria. Notably, Kocuria kristinae is an anaerobic Gram-positive micrococcus characterized by acid and hop resistance. Under anaerobic conditions, specific strains of this micrococcus can impart a fruity aroma and off-flavor to beer.

Acetic acid bacteria are traditionally divided into the genera Acetobacter and Gluconobacter. These represent an important group of Gram-negative rods capable of oxidizing alcohol to acetic acid under aerobic conditions. They enter the beer from wort alongside pitching yeast. Acetobacter aceti and Gluconobacter oxydans are notable for their resistance to the antiseptic properties of hops, as well as their tolerance to ethanol and acidic environments. Acetic acid bacteria spoil beer by imparting a sour taste, off-flavors, and abnormal aromas, while also causing turbidity and ropiness.

Genus Zymomonas. The genus Zymomonas comprises a single species, Zymomonas mobilis. The Cells of these Gram-negative bacteria are short, thick rods measuring 1.0–1.4 × 2.0–6.0 µm. They typically occur in pairs, though certain strains can form chains or rosettes, and many exhibit filamentous (hypha-like) Cell morphologies. Most strains are non-motile; motile strains possess one to four polar flagella. A distinctive feature of Zymomonas bacteria is the intensive fermentation of glucose and fructose via the Entner-Doudoroff Pathway, yielding alcohol and carbon dioxide. In addition, small amounts of acetic and lactic acids, acetaldehyde, acetone, and glycerol accumulate. Approximately half of all Zymomonas strains can proliferate at ethanol concentrations up to 10% ABV, while the strain Z. mobilis BS057 withstands ethanol concentrations of 12–13% ABV, which are achieved in high-gravity brewing. Beer contaminated with Zymomonas develops a rotten-apple or solvent-like fruity off-flavor.

Bacteria of the family Enterobacteriaceae. This family encompasses over 30 genera, of which the following are encountered in breweries: Citrobacter, Enterobacter, Hafnia, Klebsiella, Obesumbacterium, Rhanella, and Serratia.

Obesumbacterium proteus shares close similarities with Hafnia alvei and thus belongs to the family Enterobacteriaceae. O. proteus is frequently detected in pitching yeast as short, thick rods. This species thrives in both hopped and unhopped wort, exhibits ethanol tolerance, and can multiply across a wide pH range from 4.4 to 9.0. During fermentation, it proliferates alongside cultural yeast and inhibits the fermentation process. Its metabolic activity leads to increased levels of methyl sulfide, dimethyl sulfide, n-propanol, isobutanol, butanediol, and diacetyl in the beer. Beer produced using pitching yeast contaminated with O. proteus acquires a parsnip-like and fruity off-flavor and aroma.

Rhanella aquatilis was reclassified from the species Enterobacter agglomerans. Its cells appear as rods measuring 0.5–0.7 × 2.0–3.0 µm. They are motile at 25 °C, though motility decreases at 37 °C. Rhanella aquatilis ferments glucose with The production of acid and gas. It multiplies readily in both hopped and unhopped wort and accumulates in recycled yeast biomass.

Beer contaminated with Rhanella aquatilis contains abnormally high levels of diacetyl (up to 0.7 mg/dm³) and dimethyl sulfide (up to 143 µg/dm³). The beverage acquires an off-flavor and aroma characterized by fruity, milky, and sulfurous notes.

In addition to aerobic and facultatively anaerobic bacteria responsible for beer spoilage, brewery microbiologists have identified strictly anaerobic species: Pectinatus cerevisiiphilus, P. frisingensis, Selenomonas lacticifex, Zymophilus raffinovorans, and Megasphaera cerevisiae.

Pectinatus cerevisiiphilus. Cells appear as slightly Curved Rods measuring 0.7–0.9 × 3–30 µm with rounded ends. They are motile by means of flagella arranged in a crest-like tuft on the concave side of The Cell. Gram-negative, non-spore-forming, and strictly anaerobic. The optimal growth Temperature is approximately 30 °C. They ferment sugars to produce acetic and propionic acids, alongside minor quantities of lactic and succinic acids. Colonies on Agar media are circular, dense, white or light beige, shiny, and opaque.

Beer contaminated with bacteria of the genus Pectinatus contains high concentrations of hydrogen sulfide, imparting a rotten-egg odor to the beverage.

Selenomonas lacticifex. Cells of strains belonging to this species are curved rods measuring 0.9–1.1 × 3.0–6.0 µm, frequently with tapered ends, occurring in pairs or short chains. They exhibit a characteristic tumbling motility due to a bundle of up to 16 flagella located on the concave side of the cell. Gram-negative and non-spore-forming strict anaerobes. Glucose is fermented to yield acetic and propionic acids, carbon dioxide, and/or lactic acid. Colonies grown on modified MRS agar are circular, 2–3 mm in diameter, smooth, opaque, and yellowish.

Zymophilus raffinovorans. Cells of this microorganism are straight or slightly curved Gram-negative rods measuring 0.7–0.9 × 3.0–15 µm. They are motile, although motility may be lost during successive subculturing. Strictly anaerobic. On MRS medium, they form circular, smooth, opaque, yellowish colonies 1–2 mm in size. Glucose is fermented with the Production of acetic and propionic acids, occasionally with trace amounts of lactic acid.

Megasphaera cerevisiae. Cells of this species appear as elongated cocci with a diameter of 1.3–1.6 µm, occurring in pairs or occasionally in chains. Gram-negative, non-motile, and non-spore-forming. Strict anaerobes, catalase- and oxidase-negative. The optimal growth temperature is 28 °C. In beer, Megasphaera strains produce significant amounts of butyric acid, along with lesser quantities of acetic, isovaleric, valeric, caproic acids, and acetoin. Contamination of beer by Megasphaera bacteria results in unpleasant odors and turbidity. Because Megasphaera cerevisiae strains are sensitive to ethanol, they are primarily encountered in low-alcohol unpasteurized beers.

12.2. Quality control of pitching yeast

The principal quality indicators for pitching yeast are the morphological state of the cells, biological purity, and physiological properties.

Sampling. A sample of washed, thoroughly mixed pitching yeast is collected into a sterile flask using a clean Glass pipette lowered to the bottom of the vessel. The flask is immediately stoppered with cotton wool, and the yeast strain and generation are labeled on the container.

The morphological state of the cells is determined by Microscopy. The cells should be of uniform size, with thin walls, and homogeneous or finely granular Cytoplasm containing small vacuoles. The presence of a significant number of morphologically altered cells, especially when combined with reduced fermentative activity, indicates yeast culture degeneration. If budding cells are absent in the sample, and the yeast exhibits granular cytoplasm and large vacuoles, it indicates that the culture is old.

Determining the biological purity of yeast. A drop of a 10% KOH solution or 50% acetic acid is placed on a glass slide, and a drop of the test yeast suspension is added to it. This promotes the Breakdown of Proteins and fat particles, facilitating microscopic examination. The field of view should contain no more than 50 cells. Approximately 20 fields of view are examined, noting any wild yeast cells (distinguished from cultured yeast) and bacteria (rods, cocci, pediococci). Pitching yeast is considered suitable for production if it contains no more than 0.5% wild yeast and no more than 1% bacteria.

The bacterial count in pitching yeast can be determined by the pour-plate method through plating appropriate dilutions (10^-1; 10^-2; 10^-3, etc.) onto wort agar or yeast agar containing 1% glucose, 1% chalk, and an antibiotic (nystatin or actidione).

To detect wild yeast, nutrient media containing substances that inhibit the growth of cultured yeast are used. Wild yeast belonging to the genus Saccharomyces is plated on a medium containing crystal violet. Wild yeast not belonging to the genus Saccharomyces is identified by plating dilutions on a synthetic medium containing Lysine.

The physiological state of the yeast is determined by its Glycogen content. A drop of the yeast suspension is placed on a glass slide, mixed with an iodine solution, and covered with a coverslip. After 5–6 minutes, the specimen is examined under a Microscope. In well-nourished yeast, 70–75% of the cells contain glycogen. If a smaller number of glycogen-containing cells is found in the sample, it indicates Aging, prolonged storage under a layer of water in vats, or inadequate Nutrition.

Determining the final attenuation limit. The wort is poured into flasks, sterilized, cooled, and its initial dry matter (DM) content is determined. Then, depending on the flask volume, 10% of well-pressed yeast is added and thoroughly mixed. The flasks are placed in a thermostat at 25±1°C for 48 hours, after which the DM content is measured again.

The final degree of attenuation is determined using the formula

υ = [(Р-P₁) х 100] / Р,

where υ is the final degree of attenuation; Р is the initial DM content in the wort, %; P₁ is the final DM content after fermentation, %.

The final attenuation limit of pitching yeast is determined twice a month, especially when using new raw Materials.

12.3. Assessment of Finished Beer Quality

In finished beer, the total microbial count, coliform bacteria (coliforms), and biological shelf-life in commercial packaging are determined.

Detection of coliforms in beer is carried out using the fermentation method at least once a month by inoculation into a nutrient medium, or via the Membrane filtration method.

Fermentation method. Prior to testing, the beer is neutralized with a sterile 10% sodium bicarbonate solution to pH 7.2–7.4. Three tubes containing 9 cm³ of Kessler broth are each inoculated with 1 cm³ of the beer. The cultures are incubated at 37±0.5 °C for 24 hours. If there are no signs of growth, it is concluded that coliforms are absent in the tested beer. If gas is present in the Durham tubes of the Kessler medium, a streak inoculation must be performed from the fermented tubes onto The surface of Endo agar in a Petri dish. The dishes are incubated at 37±0.5 °C for 24 hours. From the typical colonies grown on Endo agar (dark red, with or without a metallic sheen), fixed smears are prepared and Gram-stained. The presence of small Gram-negative rods in the smear indicates the presence of coliforms in the beer.

Membrane filtration method. Filters with a pore size capable of retaining the majority of microorganisms (pore diameter of 0.45 nm) are selected for the analysis. For each sample package selected, 100 cm³ of beer is passed through a pre-sterilized membrane filter. Upon completion of filtration, the filter is rinsed with sterile water and then carefully placed, using flamed forceps, onto the surface of Endo agar in a Petri dish. Following incubation at 37±0.5 °C for 24 hours, the presence or absence of characteristic coliform colonies is recorded.

The biological stability of beer is determined by the time it takes for turbidity to develop. To do this, two bottles of beer from each packaged variety are selected and placed in an incubator at 20 °C. The bottles are inspected daily, and the time (in days) to beer turbidity, sediment formation, pellicle appearance, and other signs of spoilage is recorded. If turbidity appears earlier than the standard timeframe, the sediment or pellicle is examined under a microscope to identify the group of microorganisms responsible for the beer spoilage.

Topic Assignment:

1. Determine the morphological state of the pitching yeast cells.

2. Determine the biological purity of the pitching yeast.

3. Determine the final attenuation limit of the pitching yeast.

4. Familiarize yourself with the detection of coliforms in beer using the membrane filtration method.

Review Questions

1. What microorganisms act as contaminants in brewing? How do they affect beer quality?

2. How is the quality of pitching yeast evaluated based on the morphological state of the cells?

3. How is the final degree of wort attenuation by yeast determined?

4. How is the biological stability of beer determined?

5. What Methods are used to determine coliform bacteria (Enterobacteriaceae) in beer?