Protective Cultures

Protective cultures are part of ancillary cultures. The term protective cultures has been applied to microbial food cultures (MFC), exhibiting a metabolic activity contributing to inhibit and/or control the growth of undesired microorganisms in food (EFFCA/ 2011/52; Medina and Nunez, 2011). Technologically harmful microorganisms (Enterococcus sp., E. coli, Clostridium sp., yeasts, and molds) and pathogenic bacteria (Listeria monocytogenes, S. aureus) metabolize food ingredients, compete for nutrients, and synthesize metabolites such as hydrogen, carbon dioxide, toxins, and bacetriocins, which lead to defects in cheese-like products and in the case of pathogens may affect human health (Aljewicz et al., 2015).

Protective cultures may reduce the number of technologically harmful bacteria. These cultures can be also a part of starters cultures and at the same time be used for technological purposes in fermented food preparation. High effectiveness of protective cultures is often related to high inoculation levels, which can affect food quality and increase costs compared with traditional chemical preservation.

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria as natural preservatives to control growth of spoilage and pathogenic bacteria in food.

LAB (lactic acid bacteria) are the main microorganisms involved in the fermentation and ripening of milk products, primarily producing lactic acid from lactose and rapidly lowering the pH, and secondarily contributing to biochemical changes in milk to develop texture, taste, and flavor of final products. But the biochemical changes may also be very important to inhibit and/or reduce the growth of undesired microorganisms, such as pathogen bacteria (e.g., Listeria monocytogenes, E. coli, etc.).

Natural antimicrobials produced by LAB include organic acids (lactic acid, acetic acid, formic acid, phenyllactic acid, caproic acid), carbon dioxide, hydrogen peroxide, diacetyl, ethanol, bacteriocins, reuterin, reutericyclin, and other bactericidal and/or bacteriostatic metabolites. These substances are released during the log phase and the stationary phase of bacterial growth (Aljewicz et al., 2015).

Thanks to implementation of the Hazard Analysis Critical Control Point (HACCP) plans, the cheese industries are now able to prevent or reduce the contamination of foodborne diseases related to cheese consumption. Nonetheless, a recent study on retail cheeses in the United Kingdom (Grattepanche et al., 2008) has shown 4% and 5% of cheeses made from unpasteurized milk and pasteurized milk, respectively, were unsatisfactory or borderline quality according to EC recommendations 2004/24 and 2005/175, due to the presence of Salmonella, Staphylococcus aureus, E. coli, and Listeria monocytogenes.

About the bacteriocins produced by LAB, several studies have demonstrated their capacity to inhibit or reduce foodborne pathogens (Dal Bello et al., 2012).

Bacteriocins are peptides with antimicrobial activity. They can be degraded by proteases in the gastrointestinal tract and do not interfere with human gut microbiota. The bacteriocins belong to four class ( Klaenhmmer, 1993; Ouwehand, 1998):

  • • Class I: lantibiotics (<5000 Da)
  • • Class II: small hydrophobic peptides, moderate (100°C) to high heat-stable (121°C)
  • (<10.000 Da), non-lanthionine-containing membrane-active peptides
  • - II a: Listeria active-peptides with Y-G-N-G-V-X-C near amino terminus
  • - II b : two-peptides bacteriocins
  • - II c: thiol-activated peptides
  • • Class III: large heat-stable proteins (>30 kDa)
  • • Class IV: complex bacteriocins proteins with lipid and/or carbohydrate

Some examples of class I are: Carnocin U149 (by Carnobacteriumpiscicola), Cytolisin L1, Cytolisin L2 (by Enterococcus faecalis), Lacticin 481 (Lactococcus lactis), Lactocin S (by Lactobacillus sake), Nisin A (by Lactococcus lactis subsp. lactis), Nisin Z (by Lactococcus lactis subsp. lactis NIZO 22186), Salivaricin (by Streptococcus salivarius 20P3) (Ouwehand, 1998).

Nisin, produced by lactococci, is the best-documented bacteriocin, and it is only approved by the Food and Drug Administration (USA) and by the European Union (listed as E234), so it can labeled as “a natural preservative" It has a broad spectrum of antagonist activity against enterococci, listeria, staphylococci, streptococci, clostridia, Campylobacter, Helycobacter pylori, Lactobacillus, Mycobacterium, Pediococcus, and Micrococcus (Ouwehand, A.C., 1998).

The stability of nisin depends on environmental condition, such as pH (acidity of food): in fact, it can be degraded by proteolytic enzymes in cheese, leading to a significant loss of activity during ripening.

Lactacin 3147, produced by Lactococcus lactis subsp. lactis DPC 3147, showed high stability over a wide range of pH. For instance, it did not show decreased activity during ripening (six months) in cheddar cheese This bacteriocin can be applied, as spray-dried powder, in cottage cheese production, reducing the number of L. monocytogens to < 10 ufc/g at 4°C within 5 days (Arques et al., 2015).

One example is bacteriocin belonging to class II is AcH, having a broad spectrum of activity against Listeria. It is produced by Pediococcus acidilactici. The production of this pediocin is reduced when final medium pH exceeds 5.0 (Grattepanche et al., 2008).

By contrast, bacteriocin WHE 92, produced by L. plantarum is not affected by pH values up to 6.0 and so it may usefully applied in cheese to inhibit Listeria monocytogenes (Grattepanche et al., 2008).

Streptococcus thermophilus and Enterococcus spp. are also able to produce antimicrobial substances with potential use in dairy products.

Thermophilin, produced by some strains of Streptococcus thermophilus, is included in class II: a bacteriocin with a spectrum activity against Streptococcus, Enterococcus, Lactococcus, Bacillus, and Listeria.

Streptococcus macedonicus produces macedocin ACA-DC 198, which is a lantibiotic bacteriocin (class I). Macedocin ACA-DC 198 inhibits several LAB and Clostridium tyrobutyricum. This last sporeformer affects semi-hard and hard cheeses such as Gouda and Swiss-type, Grana Padano, and Parmigiano Reggiano, leading to defects of flavor and late cheese blowing by production of high levels of CO2, butyric acid, and acetic acid. Macedocin ACA-DC 198 may be also useful against other Clostridia development.

Although some strains of Enterococci are pathogenic and are involved in the transfer of antibiotic resistance and/or virulence factors, others may be considered safe and used as starter or nonstarter lactic acid bacteria (NSLAB). NSLAB are present in many traditional Mediterranean cheeses where they may positively contribute to organoleptic characteristics during ripening through proteolysis, lipolysis and breakdown of citrate into aroma compounds.

Many enterococci produce bacteriocins belonging to heat-stable nonlantibiotics class II.

Indeed, cytolisin belongs to lantibiotic Class I, because it is responsible for hemolytic activity related to some Enterococcus faecalis strains.

Safety considerations, on the use of bacteriocins or their producer strains, is an important issue, since cross-resistances between different classes of bacteriocins and/ or antibiotics have already been reported. Applications of such bacteriocins and/or their strains must be carefully assessed, well considering the risk-to-benefit ratio.

 
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