Exopolysaccharides (EPS)

Several species and strains of lactic acid bacteria are able to produce exopolysaccharides. The EP can be a capsule—either closely or loosely attached to cell wall, or even an excreted slime.

The strains that produce EP (e.g., Streptococcus thermophilus) may be used in industrial application, as stabilizers/texture improvers of yogurt.

The EP-producing strains (EPS) are often considered to be more resistant to bacteriophages than those nonencapsulated. The EP chemical composition can be different from strain to strain: some of them contain galactose and glucose, hexose-like-compo- nents, and rhamnose.

Inhibitory Substances

Lactic acid bacteria can produce inhibitory substances even if in small amounts. These compounds are hydrogen peroxide, diacetyl, bacteriocins, and some secondary compounds such as hypothiocyanate generated by the action of lactoperoxidase on hydrogen peroxide and thiocyanate. Hydrogen peroxide is the main inhibitory substance produced. It is generated by different mechanisms by some lactobacilli during their growth. The accumulation of hydrogen peroxide can occur because the lactobacilli do not have the catalase enzyme, but its production is active against some Gram-positive bacteria such as Staphylococcus aureus.

Diacetyl is known as the typical butter aroma but it also recognized as an antimicrobial substance. Its inhibitory action is 200 pg/mL for yeast and for Gram-negative bacteria is 300 pg/mL.

Bacteriocins are a heterogenous group of compounds that represent a widely antibacterial spectrum by a different mode of action and chemical properties, depending on genus and/or species of lactic acid bacteria. They are generally thermostable and part of the molecule is often a peptide. Among bacteriocins nisin and diplococcin, produced by lactococci, are well known. Nisin is effective against Gram-positive bacteria and also against Clostridium botulinum spores. Diplococcin, produced by Lactococcus subsp. cremoris, is active against only Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris.

Bacteriophage infection is the major cause of slow acid production in cheese plants today. Phages are viruses that can multiply only within a bacterial cell. Their multiplication occurs in one or two ways, named the lytic and lysogenic cycles. Lytic and lysogenic phages are also known as virulent and temperate, respectively. Lysis is caused by a lytic enzyme, called lysine, which is encoded in the phage DNA and hydrolyzes the cell wall of the host cell. The growth of a virulent phage in a sensitive host is characterized by both a latent period (from initial phage adsorption to the release of phage progeny after cell lysis, 10 to 140 minutes for Lactococcus spp.) and a burst size (the suddenly increased number of phage caused by the cell lysis, 10 to 300 minutes for Lactococcus spp.).

In the lysogenic cycle instead of phage multiplication, the phage DNA is inserted in the bacterial chromosome and multiplies with it. In this state, the host cell is immune to attack by its own phage and closely related strains of phage (superimmunity). In certain circumstances, temperate phages can be released from bacterial DNA, become lytic and multiply.

Most strains of LAB are lysogenic, but despite temperate phages are considered to be the source of phage for many bacteria; this has not yet shown for LAB starters, except for Lb.casei Shirota. To control phage it is important to identify their source: raw milk and cheese whey are considered to be an important source of phage and aerosols produced during fat separation from whey are probably a primary source of phage in cheese plants.

Important factors in controlling phage are (Fox et al., 2000):

  • • Use of a limited number of cultures
  • • Aseptic inoculation
  • • Use of phage inhibitory media heat treated at 85°C for 30 minutes
  • • Rotation of phage-unrelated strains
  • • Physical separation of starter and cheese-production areas
  • • Addition of starter and rennet together
  • • Chlorination of vats between fills
  • • Use of closed vats
  • • Physical separation of the cheese production area from the separator used to recoved fat from whey
  • • Heat treatment (>90° for 45 minutes) of starters containing phage before discarding it

Chlorination is a very effective phagicide. The final step in cleaning equipment, such as vats and filling lines, should include a chlorination component. Exposure to 100 ppm active chlorine for 10 minutes is sufficient to inactivate all phages on equipment surfaces.

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