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

TOPIC 9. CHARACTERISTICS OF MICROORGANISMS USED IN DAIRY TECHNOLOGY

Various groups of microorganisms are used in the biotechnology of fermented dairy products, including lactic acid Bacteria, propionic acid bacteria, acetic acid bacteria, bifidobacteria, Yeasts, and Molds. Lactic acid bacteria play the most important role.

9.1. Lactic Acid Bacteria

Lactic acid bacteria (LAB) break down CARBOHYDRATES to produce primarily lactic acid, which is why they are grouped together. In addition to lactic acid, these bacteria accumulate other metabolites such as acetic acid, ethanol, carbon dioxide, and volatile compounds (acetaldehyde, diacetyl, and Fatty acids).

Morphology. In terms of Cell shape, LAB can be spherical or rod-shaped. They are Gram-positive, non-motile, and do not form spores or capsules (although some strains form a mucous capsule in young cultures). The only exception is the genus Sporolactobacillus, whose Cells are motile and produce endospores.

PHYSIOLOGICAL AND BIOCHEMICAL characteristics. Regarding oxygen requirements, LAB are facultative anaerobes. Lactic acid bacteria represent the only group of microorganisms that lack catalase yet are capable of growing in the presence of atmospheric oxygen. The enzyme lactoperoxidase performs the catalase function in these organisms. Because LAB lack heme-containing Enzymes, they derive energy exclusively through Lactic acid Fermentation, which is conventionally divided into homofermentative and heterofermentative pathways. In homofermentative fermentation, lactic acid is the primary metabolite; in heterofermentative fermentation, carbon dioxide, ethanol, and/or acetic acid are formed alongside lactic acid.

The Temperature range for LAB activity is quite broad: mesophilic species grow at an optimum temperature of 25–32 °C, with 10 °C being their minimum temperature. For thermophilic species, the optimal growth temperature ranges from 38 to 45 °C, while the minimum ranges from 20 to 22 °C. There is also evidence that certain lactic acid bacteria can grow at 3–5 °C.

Currently, the lactic acid bacteria group comprises 13 genera, including:

✵ Spherical: Lactococcus, Streptococcus, Leuconostoc, Enterococcus, Pediococcus, Lactosphaera, Oenococcus, Tetragenococcus, Vagococcus, Weissella.

✵ Rod-shaped: Carnobacterium, Lactobacillus, Paralactobacillus.

Spherical lactic acid bacteria. Among spherical LAB, the dairy industry utilizes lactococci of the subspecies Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. diacetylactis; streptococci of the subspecies Streptococcus salivarius subsp. thermophilus; and leuconostocs of the subspecies Leuconostoc mesenteroides subsp. cremoris and Leuconostoc mesenteroides subsp. dextranicum, The properties of which are described below.

Lactococcus lactis subsp. lactis (lactic streptococcus, abbreviated as Lac. lactis). Cells are spherical or oval, measuring 0.5–1.2 x 0.5–1.5 µm, occurring in pairs (diplococci) or short chains (Fig. 9.1, a). The optimal growth temperature is 28–32 °C.

Active strains of this species curdle milk within 4–6 hours, forming a smooth, firm coagulant. The ultimate acidity (determined after 5–7 days of incubation in milk at the optimal temperature) reaches 120–125 °T. Lac. lactis ssp. lactis reduces and curdles litmus milk, decomposes Arginine to produce ammonia, and does not grow in media containing 6.5% NaCl or in alkaline media at pH 9.5. Strains of this species are components of starter cultures for fermented milk beverages, cottage cheese, sour cream, cultured butter, and cheeses produced with a low second-scalding temperature.

Certain strains of Lac. lactis synthesize the bacteriocin nisin, which exhibits antagonistic activity against most Gram-positive bacteria (staphylococci, micrococci, bacilli, clostridia, lactobacilli, etc.). Nisin has no inhibitory effect on Gram-negative bacteria. It is used in the canning industry to suppress the growth of spore-forming bacteria.

Lactococcus lactis subsp. cremoris (creamy streptococcus — Lac. cremoris). Cells are spherical and arranged in short or long chains (Fig. 9.1, b). The optimal growth temperature is 25–30 °C, and growth ceases at 39–40 °C. Milk curdles within 6–8 hours, forming a firm coagulum with a slightly viscous or sour-cream-like consistency, which is due to the creamy streptococcus's ability to synthesize Polysaccharides. The ultimate acidity in milk does not exceed 110–115 °T.

Strains of this species do not produce ammonia from arginine and do not grow in media containing 4% NaCl or at pH 9.5. Creamy streptococcus is used in starter cultures for sour cream, cultured butter, and other fermented dairy products.

Some strains of this subspecies synthesize the bacteriocin lactococcin.

Lactococcus lactis subsp. diacetylactis (aroma-producing streptococcus — Lac. diacetylactis). Cells are arranged in diplococci and short chains (Fig. 9.1, c). The optimal growth temperature is 25–30 °C. It exhibits relatively weak acid-producing activity, curdling milk in 16–18 hours, with an ultimate acidity not exceeding 70–100 °T. The milk coagulum is firm, frequently containing gas bubbles (CO₂), and possesses a pleasant, specific aroma resulting from the accumulation of diacetyl. Strains of this species break down lactose and citrates to produce carbon dioxide, diacetyl, and acetoin. Two enzymes participate in citrate utilization: citrate permease, which transports citrate into The Cell across the cytoplasmic membrane, and citrate lyase, which splits citrate into acetate and oxaloacetate. The enzyme diacetyl reductase present in the cells reduces diacetyl to acetoin, leading to a decrease in aroma. This process can be slowed down by rapid cooling of fermented dairy products.

Aroma-producing streptococcus is a component of starter cultures for the majority of fermented dairy products.

Leuconostoc mesenteroides subsp. cremoris (Leuc. cremoris). Cells are spherical or lenticular, measuring 0.5–0.7 x 0.7–1.2 µm, connected in pairs or short chains (Fig. 9.1, d). The optimal growth temperature is 22–25 °C, with a minimum of about 5 °C. It does not curdle milk due to its low proteinase activity. Leuconostocs are capable of growing in milk when growth factors (Yeast or corn extract) are added. The ultimate acidity does not exceed 40–50 °T. After the medium pH drops to 5.0–4.5, it produces diacetyl; therefore, this species is used in multi-strain starter cultures for The production of cheese and cultured butter in combination with Lac. lactis and Lac. cremoris.

Class="center">Fig. 9.1. Spherical lactic acid bacteria: a — Lac. lactis; b — Lac. cremoris; c — Lac. diacetylactis; d — Leuc. cremoris; e — S. thermophilus

Streptococcus salivarius subsp. thermophilus (thermophilic streptococcus — S. thermophilus). Cells are oval or spherical with a diameter of 0.7–1.0 µm, frequently joined in long chains (Fig. 9.1, e). Active strains curdle milk within 3.5–4 hours at an optimal temperature of 40–42 °C. The growth temperature range is 20–50 °C. S. thermophilus is relatively heat-tolerant and can withstand a temperature of 75 °C for 15 minutes, which is why it forms part of the residual microflora in pasteurized milk.

The ultimate acidity in milk does not exceed 100–115 °T. Thermophilic streptococcus is sensitive to sodium chloride and antibiotic content in the environment: it ceases to grow in a medium containing 4% NaCl and 0.01 IU/cm³ of penicillin.

Some strains of thermophilic streptococci produce diacetyl and are capable of synthesizing exopolysaccharides, which leads to The formation of a viscous, mucilaginous curd.

Thermophilic streptococci are included in starter cultures for the production of ryazhenka, varenets, yogurt, and cheeses requiring a high second-scald temperature.

Leuconostoc mesenteroides subsp. dextranicum (Leuc. dextranicum) is morphologically similar to Leuc. cremoris. It coagulates milk at an optimum temperature of 22-25 °C within 3-4 days. The ultimate acidity reaches 70-80 °T.

The growth of leuconostocs is stimulated by The addition of manganese to the medium, which also leads to an increased synthesis of metabolites such as diacetyl, acetic acid, and carbon dioxide.

Leuconostocs are part of the natural microflora of kefir grains and play a major role in developing their characteristic flavor and aroma.

Many strains of Leuc. dextranicum synthesize the polysaccharide dextran from sucrose, which forms slime.

Rod-shaped lactic acid bacteria belong to the family Lactobacillaceae and the genus Lactobacillus, which is subdivided into three subgenera: Thermobacterium, Streptobacterium, and Betabacterium (according to Orla-Jensen).

According to their type of fermentation, lactobacilli are divided into three groups:

I - obligately homofermentative (thermobacteria);

II - facultatively heterofermentative (streptobacteria);

III - obligately heterofermentative (betabacteria).

The dairy industry utilizes the following species of lactobacilli (Fig. 9.2, a-i):

Lactobacillus delbrueckii subsp. bulgaricus (Bulgarian bacillus, abbreviated as L. bulgaricus). Cell morphology in milk consists of long and short rods measuring 5-20 x 0.8-1.0 µm (Fig. 9.2, a). When smears prepared from milk are stained with methylene blue, polyphosphate granules and sometimes unevenly stained cytoplasmic areas are frequently observed within the cells. The optimum growth temperature is 40-45 °C. Milk is coagulated within 4-6 hours. The ultimate acidity of milk reaches 200-300 °T. The milk curd may have a brittle or highly viscous, slimy texture. Strains of the Bulgarian bacillus produce acetaldehyde, which imparts a characteristic fruity aroma to the curd. The Bulgarian bacillus is sensitive to many Antibiotics present in raw milk, but resistant to bacteriophage infection.

Fig. 9.2. Rod-shaped lactic acid bacteria: a - L. bulgaricus; б - L. acidophilus; в - L. lactis; г- L. helveticus; д - L. rhamnosus; е - L. plantarum; ж - L. fermentum; з - L. brevis; и - L. casei

Strains of the Bulgarian bacillus are components of starter cultures used for the production of yogurt and southern, Bulgarian, and Mechnikov's sour milks.

Lactobacillus acidophilus (acidophilus bacillus). Cell morphology in milk: long and short rods measuring 3-40 µm in length and 1.0-1.5 µm in width (Fig. 9.2, b). In some strains, as in the Bulgarian bacillus, cellular granularity is observed. The optimum growth temperature is 37-38 °C. Milk is coagulated in 5-8 hours, and the ultimate acidity of milk is 260-280 °T. Some strains form a slimy curd.

The acidophilus bacillus is a normal resident of the intestinal microflora of humans and warm-blooded animals. Therefore, it is resistant to an alkaline environmental reaction (pH 8.3) and to the presence of phenol (0.3-0.4 %) and Bile (20 %) in the medium.

The acidophilus bacillus exhibits high antagonistic activity against putrefactive, opportunistic, and pathogenic microflora. It inhibits the growth of Salmonella, Shigella, Staphylococcus aureus, Escherichia, and others. This is because the acidophilus bacillus synthesizes several bacteriocins: acidophilin, lactocidin, acidocin B, and lactocins B and F. Consequently, L. acidophilus is classified among valuable probiotic cultures.

Lactic acid rods of this species are used for preparing acidophilin, acidophilus milk, and Fermented milk products for infants.

Lactobacillus delbrueckii subsp. lactis (lactic rod - L. lactis). Morphologically and biochemically, it is similar to the Bulgarian bacillus (Fig. 9.2, в). The optimum growth temperature is 40-42 °C. The ultimate acidity of milk fermented by L. lactis is 160-200 °T. It is used in the production of cheeses requiring a high second-scald temperature.

Lactobacillus helveticus (Swiss bacillus). Rods 2-6 µm long and 1.0 - 1.5 µm wide, occurring singly or in short chains (Fig. 9.2, г). The optimum growth temperature is 42-45 °C. Milk is coagulated in 5-6 hours, with an ultimate acidity of 300-350 °T. Strains of L. helveticus can be isolated from calf rennet. This species is used in starter cultures for cheeses with a high second-scald temperature.

Lactobacillus casei subsp. rhamnosus (L. rhamnosus) appears as short or long rods 1.5 µm in width, joined in chains (Fig. 9.2, д). Cellular granularity is frequently observed. The optimum growth temperature is about 30 °C. Unlike L. casei, it is capable of growing at a temperature of 45 °C and multiplying in a medium containing 6 % NaCl and 20 % bile. Compared to mesophilic lactococci, the proteolytic activity of L. rhamnosus is twice as high. The ultimate acidity in milk is 80-180 °T. It is used in starter cultures for the production of Emmental, Edam, Cheshire, and other cheeses. It is found in various types of cheese as a concomitant microflora.

Lactobacillus plantarum (L. plantarum). Rods of varying length, joined in long and short chains (Fig. 9.2, е). The optimum growth temperature is about 30 °C, and the ultimate acidity during growth in milk reaches 180 °T. In the dairy industry, this species plays a beneficial role during cheese ripening, as it can multiply after lactose fermentation by the starter microflora and at NaCl concentrations up to 6 %. In addition, Lactobacillus plantarum produces hydrogen peroxide and synthesizes the bacteriocins plantacin - substances that inhibit the growth of butyric acid bacteria and intestinal microflora, thereby preventing cheese defects such as early and late gas blowing. L. plantarum is also used as part of starter cultures for preparing silage and sauerkraut.

Lactobacillus fermentum (L. fermentum) consists of thick, short rods 0.5-0.9 µm in length (Fig. 9.2, ж).

Lactobacillus brevis (L. brevis). Small rods with rounded ends, measuring 0.7–1.0 × 2–4 µm (Fig. 9.2, c).

Lactobacillus casei subsp. casei (L. casei) — rods with square ends measuring 0.7–1.1 × 2–4 µm, most commonly arranged in chains (Fig. 9.2, d). The optimal growth temperature is 28–32 °C; growth ceases at 45 °C.

Lactobacillus buchneri (L. buchneri) — rods with rounded ends, measuring 0.7–1.0 × 2–4 µm.

Rods of the species L. brevis, L. fermentum, and L. buchneri belong to the group of obligately Heterofermentative lactic acid bacteria (betabacteria). They do not curdle milk by themselves, but upon the addition of yeast autolysate, they form a clot whose ultimate acidity can reach 150–160 °T. They ferment glucose to produce lactic acid, CO₂, ethanol, and small amounts of volatile acids. They produce DL-isomers of lactic acid.

Betabacteria participate in the ripening of cheeses with a low second-scalding temperature, contributing to the Formation of the cheese's eye pattern and flavor. They are also found in the stroma of kefir grains.

9.2. Bifidobacteria

Bifidobacteria belong to the family Actinomycetaceae, genus Bifidobacterium. They are part of the normal intestinal microflora of humans and animals. They exhibit high antagonistic activity against pathogenic and opportunistic microorganisms.

Morphology. Cell morphology is characterized by small, sometimes branching Y- or V-shaped rods, straight or curved, club-shaped or spatula-shaped (Fig. 9.3). Microbial cells occur singly, in pairs, in palisades, in rosettes, and rarely in chains. Cell dimensions are 0.5–1.3 × 1.5–8 µm. Bifidobacteria are Gram-positive, non-spore-forming, and non-motile. Some strains are capable of forming a microcapsule.

Fig. 9.3. Bifidobacteria

Bifidobacteria are strict anaerobes. Their optimal growth temperature is 36–40 °C, with temperature limits ranging from 20 to 50 °C. The optimal pH of the medium is 6–7; growth is inhibited at a pH below 4.5. During carbohydrate fermentation, bifidobacteria produce lactic and acetic acids in a 2:3 ratio. They multiply slowly in cow's milk because it is not their natural habitat. For laboratory cultivation, Blaurock's Liver-Cysteine medium or milk hydrolysate medium (MHM) is used. Following laboratory cultivation, they are capable of curdling milk within 10–12 hours. The ultimate acidity of the milk reaches 120–130 °T.

Strains of bifidobacteria are used to produce infant, therapeutic, and dietary fermented milk products (bio-yogurt, bifilact, bifidokefir, acidobifilin, etc.).

9.3. Propionic Acid Bacteria

Propionic acid bacteria belong to the family Propionibacteriaceae, genus Propionibacterium. The type species of this genus is Propionibacterium freudenreichii.

Propionibacterium freudenreichii are small, non-motile, non-spore-forming, Gram-positive pleomorphic rods measuring 0.5–0.8 × 1–5 µm. Cells can be coccoid, elongated, bifurcated, or branched (club-shaped forms occur); they are arranged singly, in pairs, or in V- or Y-shapes (Fig. 9.4).

Fig. 9.4. Propionibacterium freudenreichii

With respect to atmospheric oxygen, they are facultative anaerobes. Maximum growth is observed at an optimal temperature of 30–37 °C and a pH of about 7.0.

Propionic acid bacteria develop slowly in milk and curdle it within 5–7 days. The ultimate acidity of milk can reach 160–170 °T.

Propionic acid bacteria are used in cheesemaking for the production of cheeses with a high second-scalding temperature. After lactic acid fermentation concludes in the cheeses, the stage of propionic acid bacterial growth begins. During Propionic Acid Fermentation, these bacteria accumulate propionic and acetic acids as well as carbon dioxide. The volatile acids impart a specific flavor and aroma to the cheeses, while carbon dioxide forms the cheese's eye pattern.

9.4. Acetic Acid Bacteria

Acetic acid bacteria (acetobacters) belong to the genus Acetobacter, which includes seven species. The type species is Acetobacter aceti. Acetobacter aceti are small, straight or Curved Rods measuring 0.6–0.8 × 1.0–3.0 µm. Filamentous, ellipsoid, or swollen cell forms are also found. They occur singly or in chains (Fig. 9.5). They are Gram-negative, motile by means of peritrichous or laterally positioned flagella, and do not form endospores or capsules.

Fig. 9.5. Bacteria of the species Acetobacter aceti

With respect to atmospheric oxygen, these bacteria are obligate aerobes. Regarding their metabolic type, they are chemoorganotrophs. They oxidize ethanol to acetic acid, and lactate and acetate to CO₂ and H₂O. The best carbon sources for growth are ethanol, glycerol, and lactate; they do not hydrolyze lactose and starch.

Bacterial colonies grow only On the surface of solid nutrient media, whereas in liquid media they form a surface pellicle. They multiply poorly in milk and do not produce acid.

The optimal growth temperature is 30 °С, with an optimum pH of 5.4-6.3. Acetic acid bacteria are a component of kefir grains, playing a positive role in the Symbiosis of the microorganisms involved. However, their presence in sour cream, cottage cheese, and fermented milks can cause an undesirable odor, a distinct acetic acid flavor, and product ropiness.

Assignment for this topic:

1. Prepare fixed, stained smears of various species of lactic acid bacteria, examine them under a Microscope using a 90x oil immersion objective, and sketch the microscopic appearance.

2. Prepare a fixed, stained smear of a pure culture of bifidobacteria, examine it under a microscope using a 90x objective, and sketch the microscopic appearance.

3. Examine prepared slides of propionic acid and acetic acid bacteria and sketch their microscopic appearance.

Review Questions

1. Into which groups are lactic acid rods subdivided based on their carbohydrate fermentation pathway?

2. What is the relation of lactic acid bacteria to atmospheric oxygen?

3. By what mechanism do lactic acid bacteria generate energy?

4. What metabolic end products are formed during homo- and heterofermentative lactic acid fermentation?

5. What bacteriocins are synthesized by lactic acid bacteria?

6. Describe the morphology of bifidobacteria.

7. Provide a morphological characterization of bifidobacteria.

8. In the production of which dairy products are propionic acid bacteria utilized?

9. What is The Role of acetic acid bacteria in dairy products?