Application of Bacteriocins and Protective Cultures in Dairy Food Preservation

1Bacteriocins are generally defined as peptides or proteins ribosomal synthesized by bacteria that inhibit or kill other related or unrelated microorganisms. Bacteriocins may have a narrow spectrum, by inhibiting bacteria taxonomically close, or a broad spectrum, by inhibiting a wide variety of bacteria.

2For the past years, bacteriocins have attracted considerable interest for their use as safe food preservatives, as they are easily digested by the human gastrointestinal tract. The use of bacteriocins as natural food preservatives fulfills consumer demands for high quality and safe foods without the use of chemical preservatives. However, the application of bacteriocins as food additives can be limited for various reasons, such as effectiveness of pathogen elimination or its high price. Nevertheless, research interest in bacteriocins has continued over the past years, as investigators continuing to search for new and more effective bacteriocins to address both biologic and economic concerns.

3The application of bacteriocins for biopreservation of foods usually includes the following approaches: inoculation of food with the bacteriocin-producer strain; addition of purified or semi-purified bacteriocin as food additive; and use of a product previously fermented with a bacteriocin-producing strain as an ingredient in food processing.

4An increasingly number of bacteriocins have been isolated and identified from Gram-positive and Gram-negative microorganisms. As a result, databases have been created to compile the information that can be used for the automated screening of bacteriocin gene clusters.

5For the past years, several classifications of bacteriocins have been proposed taking into consideration the first classification proposed by. Recently, in order to classify novel bacteriocins, proposed an adjusted classification scheme based on the biosynthesis mechanism and biological activity in accordance with other proposals. They propose three major classes: Class I – small post-translationally modified peptides; Class II – unmodified bacteriocins; and Class III – larger peptides (>10 kDa, thermo-labile), being each one subdivided into subclasses.

6Bacteriocins have distinct mechanisms of action and can be divided into those that promote a bactericidal effect, with or without cell lysis, or bacteriostatic, inhibiting cell growth. Most of the bacteriocins produced from LAB, in particular those inhibiting Gram-positive bacteria, exert their antibacterial effect by targeting the cell envelope-associated mechanisms. Several lantibiotics and some class II bacteriocins target Lipid II, an intermediate in the peptidoglycan biosynthesis machinery within the bacterial cell envelope and, by this way they inhibit peptidoglycan synthesis. Other bacteriocins use Lipid II as a docking molecule to facilitate pore formation resulting in variation of the cytoplasm membrane potential and ultimately, cell death. Nisin, the most studied lantibiotic, is capable of both mechanisms. Some bacteriocins damage or kill target cells by binding to the cell envelope-associated mannose phosphotransferase system (Man-PTS) and subsequent formation of pores in the cell membrane. Other bacteriocins can kill their target cells by inhibition of gene expression and protein production.

7Although there are several microorganisms that produce bacteriocins, those produced by the lactic acid bacteria (LAB) are of particular interest to the dairy industry. LAB have long been used in a variety of food fermentations by converting lactose to lactic acid, as well as producing additional antimicrobial molecules such as other organic acids, diacetyl, acetoin, hydrogen peroxide, antifungal peptides, and bacteriocins. As a result of their extensive use in traditional fermented products, most of the LAB are Generally Regarded as Safe (GRAS), granted by the American Food and Drug Agency (FDA). The European Food Safety Authority (EFSA) also granted the Qualified Presumption of Safety (QPS) status to most of the LAB genera, such as Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, and some Streptococcus. Nevertheless, species of the genus Enterococcus and some Streptococcus are pathogenic, thus, they do not have GRAS status and were not proposed for QPS status.

8Lactic acid bacteria bacteriocins are often active across a range of pH values, resistant to high temperatures and active against a range of food pathogenic and spoilage bacteria. In addition, LAB bacteriocins are sensitive to digestive proteases such as pancreatin complex, trypsin and chymotrypsin, and thus do not impact negatively on the gut microbiota.

9Bacteriocins have been used in the biopreservation of various foods, either alone or in combination with other methods of preservation, known as hurdle technology. Although results obtained from culture media may show that bacteriocins inhibit target organisms, the application of bacteriocins into foods must be tested to confirm their effectiveness. Many studies showed the potential of applying bacteriocins or bacteriocin-producing strains into foods, such as meat, dairy products, fish, alcoholic beverages, salads, and fermented vegetables. To date, only nisin (Nisaplin, Danisco) and pediocin PA1 (MicrogardTM, ALTA 2431, Quest) have been commercialized as food preservatives. Although other LAB bacteriocins offered promising perspectives to be used as biopreservatives, as for instance the enterocin AS-48 or lacticin 3147, no other bacteriocin has been proposed for industrial application. The screening of bacteriocins to be applied to foods requires the fulfillment of some important criteria. Producing strains should be food grade (GRAS or QPS), exhibit a broad spectrum of inhibition, present high specific activity, have no associated health risks, present beneficial effects (e.g., improve safety, quality, and flavor of foods), display heat and pH stability, and optimal solubility and stability for a particular food. Various authors have reported that inactivation of several foodborne pathogens by bacteriocins may differ greatly depending on the food matrix used. Therefore, the effectiveness of different bacteriocins to foodborne pathogens must be tested in all food systems.

10Recent applications of bacteriocins into dairy foods to control food-borne pathogens included the inoculation of food with LAB that produce bacteriocins (Table 1) or the addition of purified or semi-purified bacteriocins directly to food (Table 2). Applying bacteriocin-producing LAB strains as antibacterial starter cultures and protective cultures may confer an advantage over the use of semi-purified/purified bacteriocins. In most cases, bacteriocins are adsorbed into food matrices and are easily degraded, which results in a loss of antibacterial activity. Therefore, an alternative method is the incorporation of bacteriocins into food packaging films/coatings, which improve their activity and stability in complex food systems. Many researchers reported the potential of using immobilized bacteriocins in the development of antimicrobial packaging films to control food-borne pathogenic bacteria, such as L. monocytogenes. In fermented foods, the contamination with L. monocytogenes is a major concern. Several listeriosis outbreaks have been linked with the consumption of dairy products, in particular soft cheeses, causing a problem to dairy industry and public health authorities. Despite most dairy products, in particular cheeses, being made from pasteurized milk, contamination with Listeria still occurs. Cheeses are ready to use products and are usually conserved at refrigeration temperatures that allow the survival and growth of psychrotrophic bacteria, such as L. monocytogenes. Therefore, contamination can occur in later stages of dairy product processing. Consequently, Listeria active bacteriocins emerge as an ideal solution for preventing the growth of this pathogen after cooking or packaging. Bacteriocins can also be used to control adventitious non-starter flora, such as non-starter lactic acid bacteria (NSLAB) in cheese and wine, and in this way, contribute to the quality of the final product. In addition, bacteriocins can also be used to enhance food fermentation, accelerating cheese ripening, and even improve its flavor.

11Over 230 bacteriocins produced by LAB have been isolated and reported, but only half of them were identified at the protein or DNA levels. Moreover, a limited number of purified or semi-purified bacteriocins have been tested in food systems, especially in dairy foods.

12The bacteriocin nisin is classified as a class-Ia bacteriocin or lantibiotic and is the most characterized and commercially important bacteriocin. Nisin is licensed as a food preservative (E234) and is recognized to be safe by the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives. To date, eight types of nisin variants were discovered and characterized: nisins A, Z, F, and Q produced by Lactococcus lactis and nisins U, U2, P, and H produced by some Streptococcus strains. The commercially available form of nisin for use as a food preservative is NisaplinTM, with the active ingredient nisin A (2.5%) and other ingredients such as NaCl and non-fat dry milk.

13Nisin has antimicrobial activity against numerous Gram-positive bacteria, including LAB, pathogens such as Listeria and Staphylococcus, and the spore forming bacteria, Bacillus and Clostridium. One of the earliest applications of nisin was to prevent late blowing in cheese caused by gas-producing Clostridium spp..

14Nisin has been widely applied in cheese and pasteurized cheese spreads to replace nitrate for preventing the outgrowth of clostridia spores. Nisin has also been shown to be effective in the control of different pathogens such as L. monocytogenes and Staphylococcus aureus in dairy products. In a study of, nisin was shown to reduce L. monocytogenes and S. aureus in milk stored at refrigeration temperatures. Several studies also tested the addition of nisin to diverse cheese types (cottage cheese, cheddar, and ricotta-type cheeses) and show an effective reduction of L. monocytogenes growth, although limited to 1–3 log cycles. Several reports indicate that nisin A is not very active against L. monocytogenes, but its anti-listerial effect is enhanced by the reduction of pH and addition of NaCl.

15In Minas Frescal cheese, S. aureus counts were reduced by approximately 1.5 log cycles after addition of nisin. In processed cheese, Bacillus cereus and Bacillus subtilis were also inhibited by nisin. The use of nisin as an antimicrobial treatment extended the shelf-life of a Greek soft acid–curd cheese (Galotyri) by >21 days. Incorporation of nisin (at a level of 2.5 mg l-1) has also shown to increase shelf-life of Ricotta-type cheese by inhibiting the growth of L. monocytogenes for >8 weeks. Moreover, a high level of retention of nisin was observed over the 10-week storage period, with only 10–32% of nisin loss. also studied the effect of nisin on survival of the most resistant strains of L. monocytogenes in cottage cheese and showed a reduction to approximately 3 log cycles in 3 days. This bacteriocin was also tested in other pasteurized dairy products, such as chilled desserts, flavored milk, clotted cream, and canned evaporated milks, and was shown to reduce post process contaminating bacteria such as L. monocytogenes. The efficacy of a combination of nisin and bovicin HC5 against L. monocytogenes and S. aureus in fresh cheese was studied by. They observed a reduction of L. monocytogenes to undetected levels after 9 days of storage at 4°C, although the combination of bacteriocins did not prevent the growth of S. aureus.

16Several studies showed that the antimicrobial activity of nisin is affected by several factors including pH, temperature, composition, structure, and natural microbiota of food. Proteolysis in cheese-making process may also affect the activity of nisin and limit its antimicrobial efficacy. Nevertheless, some authors described a limited loss in nisin activity (10–32%) in Ricotta cheese after 10 weeks of storage. Moreover, nisin activity was not affected by proteases in a study on Emmental cheese.

17The application of nisin in dairy products is also limited to pH values lower than 7, as nisin greatly loses the activity at higher pH. Other studies also mention several limitations on the use of nisin in dairy products, due to its interaction with fat and other components in the food matrix. However, the role of fat in the activity of nisin is not entirely clear, as studies on heat-treated cream show the inhibition of B. cereus growth by low concentrations of nisin. Additionally, nisin was found to prevent spoilage bacteria and extend shelf-life in high-fat milk-based pudding. In contrast, homogenization of milk was show to reduce the anti-listerial effects of nisin, demonstrating that the treatment of foods may play an important role in the efficacy of bacteriocins such as nisin.

18The wide spectrum of inhibition associated with nisin includes LAB themselves. Therefore, in dairy foods which require LAB for fermentation processes, the application of nisin presents a great limitation. An alternative consists in employing other bacteriocins with a highly specific activity range. As a result, there has been a great interest in the search for new bacteriocins with a widespread range of antibacterial activity, stability in different food environments, tolerance to heat, and resistance to proteolytic enzymes.

19Pediocins are a class IIa bacteriocins produced by Pediococcus spp. and are commercially available under the name Alta 2341TM or MicrogardTM. This bacteriocin has been shown to be more effective than nisin against some food-borne pathogens such as L. monocytogenes and S. aureus and Gram-negative organisms such as Pseudomonas and Escherichia coli. The potential application of pediocins to dairy products is further enhanced by its stability in aqueous solutions, its wide pH range, and high resistance to heating or freezing. Despite this high potential, few studies have investigated the addition of pediocins to milk or dairy foods. Pediocin (PA-1) was found to reduce L. monocytogenes counts in cottage cheese, cream, and cheese sauce. The anti-listerial effect was noticeable over a wide temperature and pH ranges and was particularly effective at low initial L. monocytogenes contamination (102 cfu ml-1). Recently, described the production of food-grade pediocin from supplemented cheese whey medium. This semi-purified pediocin containing fermented cheese whey was shown to be effective in reducing S. aureus counts and enhancing shelf-life of raw buffalo milk.

20Lacticins are produced by certain strains of Lc. lactis and comprise lacticin 3147 and lacticin 481.

21Lacticin 3147 was isolated from an Irish kefir grain used for making buttermilk and is a two-component lantibiotic, requiring both structural proteins to give full biological activity. This bacteriocin exhibit antimicrobial activity against a wide range of food pathogenic and food spoilage bacteria in addition to other LAB. A lacticin 3147 powder preparation was shown to be effective for the control of Listeria and Bacillus in infant milk formulation, natural yogurt, and cottage cheese. However, the high concentrations of lacticin powder used, which represent 10% of product weight, were considered impracticable and non-economic by the authors.

22Lacticin 481 is a single-peptide lantibiotic that exhibits a medium spectrum of inhibition, mainly active against other LAB, Clostridium tyrobutyricum and L. monocytogenes. The use of non-purified lacticin 481 was show to have a mild bacteriostatic activity in milk stored at refrigeration temperatures. Yet, the application of semi-purified lacticin 481 to fresh cheeses stored at refrigeration temperatures reduced L. monocytogenes by 3 log cycles in 3–7 days. Nevertheless, the application of lacticins in food systems is not likely to ensure complete elimination of pathogens such as L. monocytogenes.

23Enterocins are produced by Enterococcus species and comprise a diverse group of bacteriocins, both in terms of their classification and inhibitory spectrum.

24Although most LAB have the GRAS status and can be used safely in food applications, bacteriocinogenic enterococci raise some safety concerns. Enterococci are among the pathogens that have been associated with a number of infections in humans. In addition, some enterococci may harbor virulence determinants and antibiotic resistance genes. While food enterococci have fewer virulence determinants than clinical strains, they are known for their capacity to exchange genetic information. Considering the safe concerns of using bacteriocinogenic enterococci, the use of purified enterocins may be considered more suitable for food consumption.

25Enterocin AS-48 is produced by Enterococcus faecalis and is a class IIc cyclic bacteriocin that is active against a number of Bacillus and Clostridium sp.. This is one of most studied bacteriocins, showing high stability to pH and heat, which makes a great candidate for application in food. tested the efficacy of the enterocin AS-48 for controlling staphylococci in skimmed milk. They observed a bactericidal effect proportional to the bacteriocin concentration (10–50 μg ml-1), but complete elimination of staphylococci was not achieved for any of the concentrations tested. This apparently lower effectiveness of AS-48 in milk when compared to culture medium could be attributed to a higher retention of the bacteriocin molecules by milk components and slower diffusion.

26investigated the antimicrobial effects of enterocin KP toward L. monocytogenes in skim, half fat, and full fat milks. Enterocin KP had a high anti-listerial effect, but this bactericidal effect decreased as both the fat content of milk and inoculation amount of L. monocytogenes increased. Also, reported an inhibitory effect of purified enterocin CCM 4231 (3200 AU ml-1) on the growth of S. aureus and L. monocytogenes in skimmed milk and yogurt. Still, the antagonistic effect of enterocin on S. aureus in yogurt was less influential after 24 h.

27Usually, enterocins are active against foodborne pathogens, such as Listeria spp. and Clostridium spp., but their application in food systems is not likely to prevent the re-growth of pathogens throughout the storage time. However, observed the reduction of L. monocytogenes counts in milk below the detection limit after 4 and 24 h by the combined effect of two bacteriocins, reuterin and enterocin AS-48.

28Recently, reported the efficacy of a semi-purified enterocin produced by an E. faecalis strain, in reducing the contamination of L. monocytogenes in fresh cheese in a dose-dependent manner. Moreover, the highest dose applied to cheeses (approximately 2000 AU g-1 of cheese) resulted in reduction of this pathogen below detection levels and this effect remained for all the storage time (72 h).

29Lactococcin BZ is produced by some strains of Lc. lactis spp. (lactis BZ) and has a wide antibacterial activity against Gram-positive and Gram-negative bacteria. This bacteriocin also displayed strong anti-listerial activity in milk. The partially purified lactococcin BZ (400–2500 AU ml-1) reduced L. monocytogenes counts to an undetectable level in both skim and full-fat milk during storage at 4 and 20°C. In addition, this anti-listerial activity was stable until the end of the storage period (25 days) and was not adversely affected by milk fat content.

30Aureocin A70 is a class II bacteriocin produced by a S. aureus strain isolated from pasteurized commercial milk. This bacteriocin exerted a bactericidal effect on a wide range of Gram-positive bacteria, including L. monocytogenes, and was tested in UHT-treated skimmed milk by. Aureocin A70 caused a time-dependent reduction in L. monocytogenes counts (5.51 log units) up to 7 days of incubation, but was insufficient to achieve the total elimination of the pathogen, resulting in <0.001% survivors.

31Genetically engineered bacteriocins have been proposed to overcome the narrow range of activity of most bacteriocins. reported the construction of a chimerical bacteriocin named Ent35-MccV. This hybrid bacteriocin combines in a single molecule the anti-listerial activity of enterocin CRL35 and the anti-E. coli activity of microcin V. This hybrid wide-spectrum bacteriocin was active against pathogenic strains of L. monocytogenes and E. coli O157:H7 and was effective in controlling the growth of both pathogens in skim milk.

32Despite the recent advances in bacteriocin research for food applications, the use of purified bacteriocins in the dairy industry remains limited. Frequently, the application of a bacteriocin alone does not provide sufficient protection against microbial contamination of dairy products. The high cost of bacteriocin isolation and purification also limits the commercial exploration of new bacteriocins. In addition, the restrictive food legislation of the health regulatory authorities (FDA and EFSA) limits the approval of new bacteriocins as food preservatives and, as a consequence, only two bacteriocins (nisin and pediocin) are currently commercially available.

33The use of bacteriocin-producing bacteria to control contamination microorganisms is an alternative for the use of purified bacteriocins as food additives. Many LAB genera and species have a long history of apparent safe use and they have been granted the GRAS and QPS status. In this regard, incorporation of such bacteria into foods offers a viable solution for controlling contamination microorganisms (Table 2). In addition, LAB are commonly used as starter cultures in food fermentations. Thus, researchers have explored the in situ production of bacteriocins by adding protective cultures that may grow and produce bacteriocins during the manufacture and storage of dairy foods. Many studies have also focused on the selection and development of bacteriocinogenic cultures as cell lysis-inducing agents to improve cheese maturation and flavor. In addition, the use of bacteriocin-producing LAB has been proposed to prevent late blowing occurring in cheeses. Late blowing defect is a major cause of spoilage in ripened cheeses, resulting in the appearance of texture and flavor defects, due to the ubiquitous presence of Clostridium spores. The most common strategies to reduce Clostridium spores are often not sufficient to prevent late blowing in cheeses and the use of bacteriocinogenic LAB emerges as an alternative strategy (Table 3).

34Among the various LAB species/strains producing bacteriocins, Lactococcus sp. has gained a particular interest in the biopreservation of dairy foods. tested the effect of in situ bacteriocin production of L. lactis ssp. lactis against L. monocytogenes, in a traditional fermented milk (lben). They found that L. monocytogenes decreased to below the detectable level within 24 h of storage at 7°C in lben fermented with the bacteriogenic starter culture. Moreover, the pathogen was efficiently inactivated from contaminated samples despite the high level of contamination (107 cfu ml-1) for up to 6 days of storage at 7°C.

35The application of nisin-producing Lactococcus sp. in dairy foods which requires LAB starters presents a problem, because the wide spectrum of inhibition associated with nisin includes LAB themselves. Still, produced a yogurt by incorporating a nisin-producing strain, L. lactis subsp. lactis, in raw milk. The bacteriocin-producing LAB were killed before the addition of the traditional yogurt cultures, and this resulted in increased storage life of the yogurts by preventing the growth of spoilage bacteria.

36Recently, reported a reduction in L. monocytogenes when three nisin A producing Lc. lactis strains were added to fresh cheese. However, the reduction on Listeria contamination was limited to 2 log units within 7 days of cheese storage. Likewise, the use of a nisin A-producing Lactococcus diacetylactis in a mixed starter culture was not effective in inhibiting L. innocua growth in Cheddar cheese. examined the inhibitory effect of another nisin-producing Lc. lactis on L. monocytogenes in Camembert cheese. In the presence of nisin-producing starter, the numbers of the pathogen decreased until the end of the second week, leading to a reduction of 3 log cfu g-1, but a regrowth of Listeria was observed in cheeses throughout ripening (6 weeks).

37A reduction of L. monocytogenes counts was observed in raw milk cheese by the use of two nisin-producing Lactobacillus lactis subsp. lactis as starter cultures. In contrast, the use of nisin-producing strains to control E. coli and L. monocytogenes in Feta and Camembert cheeses has shown limited results. E. coli O157:H7 survived the manufacturing process of both cheeses and was present in cheese made with nisin-producing strains at the end of 75 days of storage in greater numbers than the initial inoculum. The Feta cheese that contained nisin was the only cheese in which L. monocytogenes remained at the level of the initial inoculum after 75 days of storage. The use of bacteriocinogenic pediococci in dairy products also exhibited limited results due to their inability to ferment lactose.

38tested a plantaricin-producing Lactobacillus plantarum strain as an anti-listerial adjunct in the presence and absence of nisin-producing starters for the manufacture of cheeses. The combination of Lb. plantarum strain (at 108 cfu ml-1) with a nisin producer reduced Listeria to undetectable levels by day 28. Moreover, they found that Lb. plantarum was much more effective at inhibiting Listeria than the nisin producer alone.

39studied the anti-listerial potential of a food-grade strain Staphylococcus equorum, producing a bacteriocin named as micrococcin P1, in soft cheese. A remarkable reduction of L. monocytogenes growth was achieved, but this effect was dependent on the contamination level. In addition, a regrowth of the viable Listeria could be observed after 10–16 days of maturation. Likewise, detected a reduction of L. monocytogenes by 3 log units in cottage cheese, after incubation with a lacticin-481-producing Lc. lactis strain. However, after an initial period, L. monocytogenes counts increased to values comparable to the control.

40In addition to natural bacteriocinogenic strains, the heterologous production of bacteriocin by genetically engineered LAB was tested in dairy foods. A lacticin 3147 transconjugant generated via conjugation of bacteriocin-encoding plasmids was used successfully against L. monocytogenes in cottage cheese. Reductions of 99.9% were seen in cottage cheese held at 4°C after 5 days. In another study, a lacticin 3147 transconjugant presented also a protective effect when applied to the cheese surface, reducing L. monocytogenes by 3 log units. In contrast, this protective effect was not evident when a nisin-producing culture was used, possibly due to pH instability.

41The use of Enterococcus spp. in foods may represent a risk for consumers and requires a safety assessment by the European food authorities. However, enterococci are found naturally in some dairy foods, as they are used as starter cultures and are often part of the microbiota of artisanal cheeses. In addition, some strains of bacteriocinogenic enterococci were show to lack many of the virulence determinants. suggested that Enterococcus species could be safely used in food if virulence genes were absent. Consequently, several studies have also employed enterocin-producing enterococcus in food systems.

42The inhibitory effect of enterocin-producing enterococci against L. monocytogenes and S. aureus in dairy foods such as milk and cheeses was demonstrated by several authors (;;;,: ). Many enterococcal strains producing enterocins were isolated and found to be effective in controlling contamination in cheeses, without compromising the acid-producing activity of the starter and the organoleptic characteristics of the final product. Bacteriocinogenic E. faecalis strain was show to reduce L. monocytogenes counts in Manchego cheese by 1 and 2 log units after 7 and 60 days, respectively. Although inoculation of milk with E. faecalis strain reduced the rate of acid production in the curd, the flavor and bitterness of the final product were not influenced.

43Other bacteriocinogenic enterococci have also been reported to reduce L. monocytogenes in dairy foods. investigated two bacteriocinogenic enterococci strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch) isolated from cheeses. Growth of L. monocytogenes was inhibited in Minas cheeses containing E. mundtii up to 12 days of storage at 8°C. This bacterium displayed a bacteriostatic effect since Listeria counts remained similar to the initial inoculum. E. faecium strain was found to be less effective, as the bacteriostatic affect occurred only after 6 days at 8°C.

44Further studies also demonstrated the inhibitory effect of enterocin-producing enterococci strains against L. monocytogenes in raw milk. investigated the use of a multiple enterocin-producing bacterial strains possessing the structural entA, entB, and entP enterocin genes. Some strains exhibited a bacteriostatic effect on L. monocytogenes in raw milk incubated at 37°C for 6 h. When raw milk cultures were further incubated at 18°C, viable populations of the pathogen were reduced slightly (by 0.2–0.4 log cfu ml-1) after 24 h and up to 72 h.

45studied the effectiveness of an E. faecium strain in controlling L. monocytogenes in goat’s milk and goat’s traditional cheese (jben). Coculture experiments of E. faecium and L. monocytogenes in milk demonstrated that the pathogen was not eliminated, but when the bacteriocinogenic strain was previously inoculated in whole milk and left to grow for 12 h before contamination, Listeria was undetectable after 130 h of coculture. Moreover, addition of the bacteriocinogenic strain to jben cheese contaminated with L. monocytogenes prior to packaging, reduced the number of viable Listeria to undetectable levels, after 1 week of storage at 22°C.

46examined the inhibitory effect of enterocin-producing E. faecalis strains against L. monocytogenes in fresh cheese. Inoculation of milk with bacteriocinogenic E. faecalis strains was shown to reduce L. monocytogenes counts by 3–4 log units in fresh cheese compared to the control. The combination of two enterocin producers optimized the reduction of Listeria counts in fresh cheese, decreasing this pathogen by 4 log cfu g-1 in the first 3 days of storage and by 5 log cfu g-1 on day 7.

47Bacteriocin-producing LAB strains have also been assessed to improve cheese maturation and flavor. These LAB cultures may induce controlled lysis of starter and/or non-starter LAB (NSLAB) and subsequent intracellular release of proteinases and peptidases, resulting in rapid onset of proteolysis and cheese ripening. Lc. lactis producing lacticin 3147 was shown to accelerate cheese ripening and also prevent late blowing in cheese by the inhibition of clostridia growth.

48Various bacteriocin-producing strains were shown to possess a lytic effect on starter cultures. The use of bacteriocin-producing Lc. lactis ssp. cremoris as a starter adjunct in Cheddar cheese manufacture was show to increase the rate of starter lysis. Cheese manufactured with the bacteriocinogenic adjunct exhibited increased cell lysis and higher concentrations of free amino acids, with associated higher sensory evaluation scores. Another bacteriocin-producing starter Lc. lactis (a lacticin 3147 producer) was tested for controlling the proliferation of undesirable microorganisms during cheese manufacture. Cheeses made with lacticin 3147-producing starters exhibited significantly lower levels of NSLAB that remained constant over 6 months of ripening.

49Some strains of lacticin 481-producing Lc. lactis were also tested in cheese production and showed to cause partial lysis of starter lactococcal cultures, which continue to grow at a slower rate. As a direct result of starter lysis with concomitant enzyme release in the cheese matrix, these strains may be employed to increase cheese ripening. Additionally, a three-strain starter system was tested on cheese by. This system comprised a bacteriocin producer which causes the lysis of a second strain (sensitive to bacteriocin) and a third strain resistant to bacteriocin activity, for acid production during cheese manufacture. The experimental cheese made with this three-strain starter system showed an increase in lysis and decrease in bitterness compared to cheeses manufactured without the bacteriocin-producing adjunct.

50One of the approaches to improve the protective action of bacteriocins is the combination with other hurdles such as chemical additives (such as EDTA, sodium lactate, potassium diacetate, and others), heating, and high-pressure treatments.

51observed that the use of pediocin in combination with eugenol incorporated into polyhydroxybutyrate films worked in synergized form and provided an effective hurdle preventing food contamination. Other researchers used successfully the mixture of bacteriocins and EDTA in the sensitization of Gram-negative bacteria. Gram-negative bacteria become sensitive to bacteriocins if the permeability of their outer membrane is compromised with chelating agents, such as EDTA. demonstrated a synergistic effect of the combined use of nisin and the lactoperoxidase system (LPS) to control L. monocytogenes in skim milk. A listericidal effect (5.6 log units lower than the control milk) was observed in treatments containing nisin (10 or 100 IU ml-1) with LPS, after 24 h at 30°C. Moreover, when the two preservatives were added in two steps (LPS was added after 3 h and nisin after 5 h of growth), the difference in L. monocytogenes counts increased by 7.4 log units.

52Several authors also observed the synergistic effect of bacteriocins after temperature treatments. exposed bacteriocin-resistant bacteria to sub-lethal stresses, either at low or high temperatures, and treated with nisin and pediocin. They found that both bacteriocins were effective in reducing the cell viability. This synergistic effect was also observed in Gram-negative bacteria, which are normally insensitive to these bacteriocins.

53High pressure processing is a common technique for inactivating microorganisms at room temperature, but this treatment does not ensure the complete inactivation of microorganisms. Several studies have demonstrated the synergistic effect of bacteriocins such as nisin with high pressure processing on the inactivation of food microorganisms. It is well documented that the use of bacteriocins in combination with these processing techniques enhances bacterial inactivation. As an example, demonstrated the efficacy of the application of reduced pressures combined with bacteriocin-producing LAB to improve cheese safety.

54A common strategy for preservation of foods that are eaten raw or without further cooking is the application of edible films or coatings containing antimicrobial substances. The incorporation of antimicrobial compounds such as bacteriocins in edible coatings and films presents as an interesting alternative for ensuring the control of pathogenic microorganisms in food products.

55Edible coatings and films are composed of thin layers of biopolymers that modify the surrounding atmosphere of foods, forming a barrier between the food and the environment, improve the safety, quality, and functionality of food products without changing organoleptic and nutritional properties. The use of purified bacteriocins or bacteriocin-producing bacteria in the packaging system may be more effective in the inhibition of the growth of pathogenic and/or deterioration microorganisms throughout the extent of the latency phase. Hydrocolloids (proteins and polysaccharides) are the most extensively investigated biopolymers in edible coatings and films applied to cheese. They facilitate the incorporation of functional compounds such as bacteriocins and bacteriocin-producing bacteria and allow an increase in the stability, safety, and shelf-life of dairy foods.

56To date, few studies have investigated the effectiveness of incorporating bacteriocins and/or bacteriocin-producing LAB in coatings and films applied to dairy products. Some authors observed an inhibition of the growth of pathogenic microorganisms in foods packed with coatings and films containing antimicrobial metabolites synthesized by LAB or containing viable LAB in the film/coating matrix.

57Studies of the effectiveness of incorporating purified bacteriocins in edible coatings show a limited reduction of pathogens such as L. monocytogenes. Cheeses, particularly fresh cheeses, are highly perishable due to their high content in caseins, lipids, and water. The complexity of cheese composition and its manufacture support the development of pathogenic and deteriorating microorganisms that increase the risk of foodborne illness and reduce cheese quality and acceptability. By acting as additional hurdle, the application of edible coatings and films with incorporation of bacteriocins may overcome problems associated with post-process contamination, therefore enhancing the safety and extending the shelf-life of the cheese.

58incorporated nisin in films of sodium caseinate applied in semi-soft cheese and observed a small reduction in L. innocua counts (1.1 log cfu g-1) after a week of storage at 4°C. In another study, the incorporation of nisin and lacticin in cellulose coatings applied to Cheddar cheese reduced levels of L. innocua by 2 log cycles, and S. aureus by 1.5 log cycles. However, other studies have shown an effective reduction on pathogen growth. In Ricotta cheese coated with galactomannan and nisin, the growth of L. monocytogenes was prevented for 7 days at 4°C. The application of a coating in Port Salut cheese, consisting of tapioca starch combined with nisin and natamycin, reduced L. innocua counts above 10 cfu ml-1 during storage, acting as a barrier to post-process contamination.

59Recently, used a biodegradable film incorporated with cell-free supernatant (CFS) containing bacteriocin-like substances of Lactobacillus curvatus P99, to control the growth of L. monocytogenes in sliced “Prato” cheese. These films containing the bactericidal concentration of CFS were able to control L. monocytogenes for 10 days of storage at 4°C.

60This review highlights the most recent trends in the use of bacteriocins and bacteriocin-producer bacteria in dairy foods. Bacteriocins, either per se or produced by live bacteria, can be successfully incorporated into dairy products to assure safety, extending shelf-life and preserve quality. The use of purified and concentrated bacteriocins as food additives has been the preferred method due to more efficacy, compared to direct application of bacteriocinogenic cultures. However, the effectiveness of bacteriocins in food systems is often low due to several factors such as adsorption to food components, enzymatic degradation, poor solubility, or uneven distribution in the food matrix. On the other hand, the application of live bacteriocin-producing bacteria into dairy foods may overcome the limitations on the use of purified bacteriocins. The bacteriocinogenic LAB added to dairy products such as yogurts and cheeses will ensure continuous production of bacteriocins throughout maturation and storage, and may be included as starter/adjunct cultures in fermentation. The main difficulty associated with this application is the lack of compatibility between the bacteriocin-producing strain and other cultures required in the fermentation of dairy foods.

61Studies on application of bacteriocins and/or bacteriocin producers have been focused mainly on cheeses because most discovered LAB bacteriocins were effective against Listeria monocytogenes. This foodborne pathogen is often a great concern in traditional cheeses made from raw milk and as a post-processing contaminant in cheeses made from pasteurized milk. Cheeses are particularly vulnerable to the contamination of L. monocytogenes because they provide the appropriate growth conditions for this pathogen. Listeriosis out-breaks linked to the consumption of contaminated cheeses have been reported worldwide. Therefore, the application of bacteriocins as natural preservatives for improving cheese safety has attracted significant research interest in recent years. In contrast, very limited research has been focused on the application of bacteriocins to the preservation of milk, cream, yogurts, and other dairy products.

62Although most efforts have been devoted to discover novel bacteriocins with unique properties, more studies are necessary for effective application of bacteriocins in dairy foods in order to understand bacteriocin performance in the complex environment of food matrices. In addition, bacteriocins may be combined with other protection tools as part of hurdle technologies to ensure bio-preservation and shelf-life extension of dairy foods. Their use may lie in the combination with other preservation techniques, or in the incorporation in biofilms and active packaging. In the latter case, further studies are necessary to ensure the adaptation of edible coatings and films to bacteriocin activity and efficacy in dairy products.

63All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

64The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.