2. Application of Selected LAB or Bacteriocins in RTE Dry-Cured Meat Products
The meat industry has carried out extraordinary research efforts to minimize the appearance of outbreaks caused by foodborne L. monocytogenes. The application of selected LAB and/or their purified antimicrobial metabolites for the biopreservation of RTE dry-cured meat products has been increasing in the last years with promising results. Selected LAB strains or their metabolites have been directly incorporated into the meat products throughout the processing to reduce the hazard posed by the presence and growth of L. monocytogenes in these products.
With this aim,
Ll. sakei has been widely employed in several studies with different results. García-Diez and Patarata
[13] concluded that the addition of
Ll. sakei at a concentration of 6 log CFU/g did not provoke significant reduction in
L. monocytogenes counts in a Portuguese dry-fermented sausage. However, Ortiz et al.
[14] showed that
Ll. sakei, when added to meat batter in Iberian chorizo, showed an anti-listerial activity at either 7 or 20 °C, reducing by 2 log
10 units the pathogen counts. In addition, Vaz-Velho et al.
[15] demonstrated that
Ll. sakei was enough to minimise
L. monocytogenes counts (up to 2 log CFU/g) in a Portuguese salami-like product, Alheira. Selected
Lp. plantarum has also been used to inhibit and control
L. monocytogenes in RTE meat products. Thus, Kamiloglu et al.
[16] evaluated the effect of five
Lp. plantarum (initially inoculated at 7 log CFU/g) against
L. monocytogenes in sucuk, a traditional dry-fermented sausage from Turkey. They observed a decrease in
L. monocytogenes counts from 1 to 2.7 log CFU/g for the different
Lp. plantarum strains tested during ripening. In such work, they determined that acidification and production of bacteriocins and/or bacteriocin like peptides were the cause for the control of this pathogenic microorganism. Zanette et al.
[17] tested the anti-listerial activity of two
Lp. plantarum strains (one bacteriocin-producing strain and one bacteriocin non-producing strain) and found they were equally effective to limit
L. monocytogenes growth (≈1.7 log CFU/g reduction) from the initial levels of the pathogen (4 log CFU/g).
The combination of selected active LAB, such as
Ll. sakei (CRL1862), with bacteriocin combination and 2.5% lactic acid and acetic acid diminished the
L. monocytogenes counts at levels lower than 2 log CFU/g (from initial counts at 3–4 log CFU/g) in frankfurters from day 6 to day 36 at 10 °C
[18]. However, no significant additional reductions were observed when selected
Ll. sakei was evaluated in combination of packing under vacuum or modified atmosphere packaging. Nikodinoska et al.
[19] tested the antagonistic activity of
Lp. plantarum alone and combined with nitrite (at two concentrations) against the pathogenic bacterium in a chorizo sausage model. Counts of
L. monocytogenes were reduced with the addition of the LAB strain (ranging from 2.6 to 3.8 log CFU/g depending on the nitrite concentration used). In samples where nitrite was not added,
Lp. plantarum reduced
L. monocytogenes growth but not until the end of ripening. On the contrary, Macieira et al.
[20], who used bacteriocinogenic
Lp. plantarum cultures (at a concentration of 6 log CFU /g) in a traditional Portuguese fermented dry-cured sausage, did not have any antagonistic activity against
L. monocytogenes (initially inoculated at 5 log CFU/g).
In the study carried out by Sadaghiani et al.
[21], they checked the effect of one strain of
Lp. plantarum (initially inoculated at 7 log CFU/g) in ground raw beef alone and in combination with a garlic extract (1%). The LAB strain alone decreased the counts of the pathogen at 0.7 log CFU/g, but when combined with the garlic extract, this reduction was 1.5 log CFU/g.
Pediococcus (P) acidilactici has also been quite utilised as a biopreservative to control the development of
L. monocytogenes in RTE meat products. Cosansu et al.
[22] demonstrated that the bacteriocin-producing
P. acidilactici possessed a significant anti-listerial activity on sucuk but not on sliced turkey bread.
P. acidilactici produced a reduction of 3.3 log CFU/g
L. monocytogenes counts after 8 days of sucuk fermentation at mild temperatures (22–24 °C). On the other hand, Ortiz et al.
[14] showed that a starter culture containing
P. acidilactici in Iberian chorizo provoked an anti-listerial effect at 7 °C.
Other researchers have focused on looking for other LAB species as biopreservatives to counteract and minimize the growth of
L. monocytogenes in RTE meat products. Regarding
P. pentosaceus, it was added individually and in combination with
P. acidilacti in sliced fresh beef samples
[23]. This study concluded that the use of
P. pentosaceus alone or combined with
P. acidilacti is promising since they limited the
L. monocytogenes counts <2 log CFU/g on day 2.
Li. reuteri is another LAB species used as biopreservative in the meat industry. Sadaghiani et al.
[21] checked the anti-
L. monocytogenes activity of a
Li. reuteri strain in conjunction with garlic extract (1%) in beef, concluding that the combination of garlic extract with
Li. reuteri caused a 1.4 log count reduction, while
Li. reuteri alone only provoked a 0.5 log reduction. Orihuel et al.
[24] reported that a bacteriocinogenic
E. mundtii strain had limited anti-
L. monocytogenes activity in beef sausage when applied alone, but in combination with curing additives, reductions of 2 log CFU/g counts were achieved. Finally, Castellano et al.
[18] showed that the bacteriocin synthesized by
Ll. curvatus possessed some bacteriostatic effect in frankfurters but lower than that shown by the bacteriocin produced by
Ll. sakei.
Some metabolites synthesized by LAB have also been utilised as a biopreservative to control
L. monocytogenes in RTE meat products. Trinetta et al.
[25] studied the antagonistic effect of sakacin A, a bacteriocin produced by
Aureobasidium pullulans, when it was directly added to RTE turkey breasts and when incorporated in a pullulan film to package this product. Results showed that sakacin A directly applied to turkey decreased the
L. monocytogenes counts by more than 2 log CFU/g, while sakacin A-containing pullulan films diminished its counts 3 log CFU/g. Another bacteriocin that displayed anti
-L. monocytogenes activity was nisin when was added in RTE turkey ham
[26]. This bacteriocin was used in different concentrations (from 0.2 to 0.5%), and its antagonistic effect increased as the concentration did, keeping the
L. monocytogenes counts lower than the control in all treatments. Leucocin A is another bacteriocin used for
L. monocytogenes control purposes in RTE meat products. This bacteriocin produced by
Le. gelidum has been employed in wieners (sausages) to counteract
L. monocytogenes [27]. The antimicrobial activity of this bacteriocin was lower than the previous ones, obtaining only a reduction of 1 log CFU/g after 16 days of incubation at refrigeration temperatures.
3. Application of Selected LAB or Bacteriocins in RTE Dairy-Ripened Products
Most of the application of LAB species in dairy-ripened products have been reported in cheese throughout the ripening or storage. Thus, selected strains of
Ll. sakei and
Lp. plantarum used as protective cultures in soft cheese reduced the loads of
L. monocytogenes from 0.5 to almost 1 log CFU/g during 1375 h of storage at 15 °C
[28]. Higher reduction was found in semi-hard cheeses ripened with
L. brevis,
Lp. plantarum, and
E. faecalis, where
L. monocytogenes counts were reduced by 4 log CFU/g after 15 days of ripening in cheeses made with raw milk and after 21 days in cheese made with pasteurized milk
[29].
Selected
Lactococcus spp. has been widely used as protective cultures in cheese. Thus, Kondrotiene et al.
[30] found a significant reduction in
L. monocytogenes counts when three nisin A-producing
La. lactis strains were applied to fresh cheese. In addition, selected strains of
La. lactis subsp. lactis and
E. durans as individual or mixed cultures have also been reported to provoke a reduction of 2–3 log CFU/g of
L. monocytogenes during 35 days of storage at 4 °C of ultrafiltered cheese
[31]. These authors underlined the potential application of the above LAB strains in bio-control of this pathogen bacterium during storage of ultrafiltered cheese.
Ll. sakei, La. lactis, and
Carnobacterium strains selected from Gorgonzola cheese have been reported to provoke a notable inhibition at low level of contamination of
L. monocytogenes (2 log CFU/g) in this kind of cheese
[10]. This inhibition was found during the first stage of ripening (6 days), and
L. monocytogenes cells were maintained below the EC limit (<2 log CFU/g) for 60 days. However, these authors reported that when
L. monocytogenes was inoculated on the cheese surface at the end of ripening process (after 50 days; pH: 6.7), only one of the selected
La. lactis strains exerted a significant inhibition on the growth of this pathogen if the cheese was strictly maintained at 4 °C.
Morandi et al.
[10] underlined that the susceptibility of
L. monocytogenes biotypes to LAB antimicrobial activity is strain dependent. Thus, a blend of different LAB strains could represent a more effective tool to develop protective culture for ripened cheeses. In this sense, combinations of different LAB strains have been proposed to be used as protective cultures in cheese. The combination of
Lp. plantarum strain (initially inoculated at 8 log CFU/mL) with a nisin producer reduced
L. monocytogenes to undetectable levels in cheese by day 28 of ripening
[32]. Furthermore, these authors found that
Lp. plantarum was much more effective in inhibiting
L. monocytogenes when the nisin producer was attached than when it was alone.
Some studies have reported the use of bacteriocin produced by LAB for biopreservation of cheeses
[33][34]. Nisin is the most frequently used although it has been reported as efficient in control
L. monocytogenes only in fresh cheese
[35][36]. An increase in anti-
L. monocytogenes activity has been suggested when combining nisin with a second bacteriocin
[35]. Therefore, the use of nisin in combination with the IIa class bovicin HC5 in fresh cheese against
L. monocytogenes has been reported to provoke a 4 log reduction of this pathogen after 9 days at refrigeration storage
[37]. In ripened cheese, it has been proposed as most effective to use nisin-producing strain of
Lc. lactis subsp. lactis for the milk before cheese production, provoking an initial reduction higher than 2 log CFU/g
[38] since the use of nisin could have the problem of the regrowth during ripening of the surviving
L. monocytogenes [35]. Other bacteriocins, such as pediocins, enterocins, and lactacins, have also been used on the surface of cheese and mainly in fresh cheese
[39][40][41], but their utility in ripened cheese is limited. Thus, although it has been highlighted that the utilization of bacteriocins could contribute to the creation of low-salt and healthier formulations of cheeses and to the optimization of processing conditions without compromising the microbiological safety of these RTE foods
[42], the problem of the regrowth during ripening of surviving
L. monocytogenes should be considered, which it makes more effective the use of selected LAB than the direct addition of bacteriocins.
Furthermore, combinations of different preservation methods may act synergistically or provide higher protection than a single method alone
[43]. Thus, the combination of selected LAB with antimicrobial compounds has been proposed. In this sense, it has been proposed that selected
La. lactis be used in combination with acid/sodium lactate (LASL-L-lactic acid 61% (
w/w) and L-sodium lactate 21% (
w/w))
[44]. The former authors found a total inhibition of
L. monocytogenes strains in the first 50 days of ripening of Gorgonzola cheese when this combination was used, while LASL with selected
C. divergens was more effective in the second part of ripening when the pH was raised. These authors encouraged the use of LASL along with antimicrobial LAB rotation schemes during cheese ripening for the prevention and/or control of the
L. monocytogenes on the cheese surface of Gorgonzola cheese.
Finally, the use of active packaging with bacteriocins produced by selected LAB species is a promising strategy to control
L. monocytogenes in packaged cheeses. In fact, Contessa et al.
[45] described a film based on agar-agar incorporated with bacteriocin produced by a selected
Lc. casei to be used as active packaging in curd cheese. This active packaging provokes a reduction of 3 log
10 units of pathogen bacteria, such as
L. monocytogenes.
4. Conclusions and Future Remarks
L. monocytogenes is a serious concern in the RTE meat and dairy-ripened products industries. The use of LAB as protective cultures and/or their metabolites could be a promising tool to control L. monocytogenes in these kinds of products. Although LAB strains are present in most of the ripened foods as the natural microbial population, to find strains with anti-L. monocytogenes activity able to survive in conditions of ripened products, an appropriated selection methodology is necessary. This includes recovery of LAB isolates from different ripening/storage conditions and evaluation of the anti-listerial activity in food models simulating temperature, aw, and pH conditions of the processing. Then, final selection should be performed after evaluation of the most active strains in food matrices, following the challenge test methodology. As a result of the proposed isolation and selection methods for LAB strains with the ability to produce antimicrobial compounds, such as lactic acid and other organic acids, ethanol, diacetyl, carbon dioxide, hydrogen peroxide, bacteriocins, are available. In addition, the selected LAB strains can compete for nutrients and space with L. monocytogenes and some of them are able to eliminate this pathogen bacterium from biofilm and reduce its virulence and the ability of L. monocytogenes to survive. These strains have showed effectivity in meat and dairy-ripened products, achieving reductions form 2–5 log10 units of L. monocytogenes throughout the ripening process. This could be sufficient to guarantee the elimination of this pathogenic bacterium throughout the ripening/storage of RTE meat and dairy-ripened products when this pathogen contaminates these products at the usual levels (below 2 log CFU/g). This is of utmost importance since minimizing the risk of listeriosis caused by the consumption of these products improves food safety and meets the microbiological criteria of RTE foods throughout their shelf life. Bacteriocins could be also used to control L. monocytogenes, but their activity in these products could be limited by the regrowth during ripening or storage of the surviving strains of this pathogen. Thus, the combination of different active LAB strains and those bacteriocigenic ones could be the most appropriate strategies to control L. monocytogenes in ripened foods. Furthermore, the combination of selected LAB strains with antimicrobial compounds, such as acid/sodium lactate, and other strategies for active packaging could be the next step to eliminate the risk posed by L. monocytogenes in meat and dairy-ripened products.