Bacteriocinogenic LAB from cheeses – Application in biopreservation?

Trends in Food Science & Technology xx (2014) 1e12

Review

Bacteriocinogenic LAB from cheeses e Application in biopreservation?
cia Lorenzo Favaroa, Ana Lu b Barretto Penna and Svetoslav Dimitrov Todorovb,c,d,* a

Department of Agronomy Food Natural resources Animals and Environment (DAFNAE), University of Padova, Agripolis, Viale dell’Universit a 16, 35020 Legnaro, PD, Italy b Department of Food Engineering and Technology,
v~ Sao Paulo State University, Rua Cristo ao Colombo, 2265, 15054-000 S~ ao Jos
e do Rio Preto, SP, Brazil c Department of Food Science and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes 580, Bl. 14, 05508-000 S~ ao Paulo, SP, Brazil d
Departamento de Veterinaria, Universidade Federal de Vic¸osa, 36570 000 Vic¸osa, MG, Brazil (Departamento de Veterin
aria, Universidade Federal de Vic¸osa, 36570 000, Vic¸osa, MG, Brazil. Tel.: D55 31 3899 1463; fax: D55 31 3899 1457; e-mail: [email protected]) Over the last decade, there has been an explosion of basic and applied research on lactic acid bacteria bacteriocins, because of their potential as biopreservatives and inhibition of the growth of spoilage bacteria. Although bacteriocins can be produced during cheese production, their titers are much lower than those achieved in vitro fermentations under optimal physical and chemical conditions. Safety and technological traits of the bacteriocinogenic lactic acid bacteria (LAB) have to be * Corresponding author.

considered before their wide-spread applications. This review described the perspectives and hurdles to be solved in order to definitively disclose the potential of bacteriocins in the production of safe and healthy cheese commodities.

Introduction Traditionally, LAB have been applied as starter culture and competitive microbiota in dairy products for centuries. Based on their metabolic properties, LAB are generally employed because of their essential contribution to the flavor, texture and nutritional value in food products, besides their natural antimicrobial properties that extend the product shelf life. Frequently, the functional roles of LAB in milk fermentations are related to many beneficial effects. Firsty, LAB strains can serve to biopreserve the product due to the fermentation and reduction of pH, and consequently, the production of acids results in an extended shelf-life and enhanced safety. Moreover, LAB yields organic acids, carbonyl compounds and partial hydrolysis of the proteins and/or fats improving the sensory quality of the product. Furthermore, a LAB proliferation in cheese is often accompanied with the production of texturing compounds, such as exopolysaccharides, that improve the rheological properties of fermented milk products (i.e. viscosity and texture). In addition, LAB strains can produce antimicrobial compounds, such as bacteriocins with application in food preservativation and various health-promoting compounds, such as vitamins, antioxidants and bioactive peptides. Finally, they can act as probiotic micro-organisms contributing to therapeutic and organoleptic properties to fermented milks. Antibacterial compounds from LAB Natural antimicrobial compounds have been part of biopreservation practices since centuries. However, only after discover of antibiotics by Fleming and particularly after the detection of nisin, various antimicrobial peptides have been subject of research. Although antimicrobial peptides can be detected in all form of life (including animals, plant, microorganisms), the microbial antimicrobial peptides, commonly referred as bacteriocins, have become the focus of many biomedical and food-based research groups. Bacteriocins are produced by all bacterial species (Cotter, Hill, & Ross, 2005). Nevertheless, the main research efforts have been granted to LAB, their role in preservation of fermented foods and their bacteriocins (Heng, Wescombe,

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Please cite this article in press as: Favaro, L., et al., Bacteriocinogenic LAB from cheeses e Application in biopreservation?, Trends in Food Science & Technology (2014), http://dx.doi.org/10.1016/j.tifs.2014.09.001

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L. Favaro et al. / Trends in Food Science & Technology xx (2014) 1e12

Burton, Jack, & Tagg, 2007). Special attention has been given to inhibit foodborne pathogens and spoilage bacteria (De Vuyst & Vandamme, 1994; Garc
ıa, Rodr
ıguez, Rodr
ıguez, & Mart
ınez, 2010). The preservative effect of LAB is due to the production of one or more active metabolites, such as organic acids (lactic, acetic, formic, propionic acids), that intensify their action by reducing the pH of the media, and other substances, like fatty acids, acetoin, hydrogen peroxide, diacetyl, antifungal compounds (propionate, phenyl-lactate, hydroxyphenyl-lactate, cyclic dipeptides and 3-hydroxy fatty acids), bacteriocins (nisin, reuterin, reutericyclin, pediocin, lacticin, enterocin and others) and bacteriocin-like inhibitory substances e BLIS (Reis, Paula, Casarotti, & Penna, 2012). Thus, bacteriocins are just one specific share of a big range of bioinhibitor compounds produced by LAB. Antimicrobial entourage of LAB includes polymers, sugars, sweeteners, nutraceuticals, aromatic compounds and various enzymes, indicating that LAB have higher flexibility and wider application then just as starter cultures. Looking for antimicrobial compounds is crucial to provide an alternative to chemical additives, offering to the market new and appealing food products. The spectrum of activity of bacteriocins against certain foodborne pathogens and spoilage microorganisms, their resistance to high temperatures and low pH, and their sensitivity to human proteolytic enzymes are useful characteristics in the application of these compounds in food preservation (Masuda et al., 2011). Bacteriocins are ribosomally synthesized antimicrobial proteins, usually active against genetically related species (Cotter et al., 2005). However, based on the reports from the last decade, few bacteriocins may have application in controlling Gram-negative bacteria, some yeast, Mycobacterium spp. and even viruses (Schirru et al., 2012; Todorov et al., 2010), however, aminoacid sequences of only few of these unusual bacteriocins are provided (Todorov, Kruger, Martinez, LeBlanc, & Franco, 2012; Todorov et al., 2010). Many classifications have already been proposed (Cotter et al., 2005), but according to the most recent (Heng et al., 2007), bacteriocins of Gram-positive bacteria are grouped into four classes, based on their structure and function. Class I: lantibiotic peptides, class II: small non-modified peptides with <10 kDa, class III, large proteins with >10 kDa and class IV: cyclic proteins. Bacteriocins of class I are subdivided in three subgroups: type A corresponds to linear peptides, type B to globular peptides and type C are multicomponent bacteriocins. Type A bacteriocins are further divided in two subtypes: Subtype AI comprises nisin-like bacteriocins (nisin A, nisin U, streptin) and subtype AII comprises SA-FF22like bacteriocins (SA-FF22, lacticin 481, salvaricin A, sublancin 168). Mersacidin and cinnamycin, for instance, belong to the class I type B globular peptides. Class I type C examples are lacticin 3147 and cytolysin, being

both formed by more than one component, all necessary for biological activity. Bacteriocins of class II are also subdivided in three subgroups: type IIa corresponds to pediocin-like bacteriocins (pediocin Pa-1, carnobacteriocin B2, listerocin 743A and ubericin A), type IIb are multicomponent bacteriocins (lactococcin G, thermophilin 13, lactacin F and lactocin 705) and type IIc are miscellaneous bacteriocins, a diverse group that includes sakacins Q, T and X and aureocin A53. Bacteriocins of class III comprise the lysins (Class IIIa) and non-lytic bacteriocins (Class IIIb). Class IV bacteriocins include circulary inhibitory peptides and the prototype is enterocin AS-48 (Abriouel et al., 2005). One of the best studied bacteriocins is nisin (de Arauz, Jozala, Mazzola, & VessoniPenna, 2009). Wiedemann et al. (2001) described a model membrane study where lipid II (the principal transporter of peptidoglycan subunits from the cytoplasm to the cell wall) acts as a docking station for nisin. After interaction with the lipid II, nisin wedges itself into the cell membrane to form short-lived pores which disturb the integrity of the cytoplasmic membrane and causes the efflux of ions and other cell components. At high concentrations of nisin, pore formation may occur even in the absence of lipid II, resulting in the cell membrane containing at least 50% negatively charged phospholipids (Wiedemann et al., 2001). Under these conditions, the positively charged C-terminus of nisin is important for initial binding and antimicrobial activity. Mersacidine and the antibiotic vancomycin also bind to lipid II, but to a different part of the molecule (Cotter et al., 2005). Bacteriocins produced by LAB from cheese origin LAB, including various strains of Enterococcus spp. and Lactobacillus spp. are ubiquitous bacteria. Because of their ability to withstand heat stress and other adverse environmental conditions, they can occour in many fermented food products of both animal (milk, cheese, fermented sausage) and vegetable origin (Muller, Ulrich, Ott, & M€uller, 2001; Omar et al.,, 2004). For this reason, they are frequently present in many cheeses (Ross, Stanton, Hill, Fitzgerald, & Coffey, 2000) and they are often found in high numbers and are believed to contribute to cheese ripening and the development of their aroma due to their proteolytic and lipolytic activities, as well as the production of diacetyl (Foulqui
e-Moreno, Sarantinopoulos, Tsakalidou, & De Vuyst, 2006; Giraffa, 2003). Their additional ability to produce bacteriocins results in a better chance to compete with other microorganisms and facilitates the substrate colonization. The antimicrobial activity of LAB is related to factors such as decreased pH levels, competition for substrates and the production of substances with a bactericidal or bacteriostatic action, including bacteriocins. Countless papers have been published on bacteriocinproducing enterococci, primarily among strains of

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Enterococcus faecium associated with food ecosystems (Giraffa, 2003; Muriana, 1996). As reported in Table 1, several of the bacteriocin-producing strains have been isolated from dairy products (Abriouel et al., 2005; Ahmadova et al., 2013; Favaro et al., 2014; Ross et al., 2000; Schirru et al., 2012; Vera Pingitore, Todorov, Sesma, & Gombossy de Melo Franco, 2012). In the study of Mart
ın-Platero, Valdivia, Maqueda, and Mart
ınez-Bueno (2009), three different goat milk cheeses were selected as source of 95 enterococci, belonging to the species Enterococcus devriesei, Enterococcus faecalis and Enterococcus malodoratus. High numbers of these isolates (87%) were producers of antimicrobial compounds. In addition to the secretion of antimicrobial proteins, their safety traits were evaluated. The virulence factor genes gelE, esp, asa1, efaA and ace were not detected. However, the tyrosine decarboxylase (tdc) gene was found in all the E. faecalis and Enterococcus hirae isolates, but in none of the others studied Enterococcus spp. strains (Mart
ınPlatero et al., 2009). Other E. faecium strains have been isolated from Bulgarian homemade white brine cheese for their ability to produce bacteriocins with high inhibition activity against Listeria monocytogenes (Favaro et al., 2014). Their bacteriocin production and inhibitory spectrum were evaluated together with the occurrence of several bacteriocin genes (entA, entB, entP, entL50B). The E. faecium strains harbored at least one enterocin gene while the occurrence of virulence, antibiotic resistance and biogenic amines genes was found to be limited. Considering their strong antimicrobial activity against L. monocytogenes strains, the four E. faecium strains exhibited promising potential as bio-preservative cultures for fermented food productions. Moreover, due to their safety characteristics and

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technological properties, they could be used as adjunct NSLAB (non-starter lactic acid bacteria) rather than as starter culture (Favaro et al., 2014). Gonz
alez et al. (2007) investigated the antimicrobial activity of 395 LAB strains isolated from Genestoso, a traditional cheese produced in Asturias, in North-Western Spain. Genestoso, acid-curd cheese ripened over a short period, is produced on a purely craft basis and the output is very limited. Out of 395 screened microbes e Lactobacillus spp. (137 strains), Lactococcus spp. (125 strains), Leuconostoc spp. (58 strains) and Enterococcus spp. (75 strains) - bacteriocin production was recorded only in 24 strains with activity against E. faecalis, Lactobacillus plantarum and foodborne pathogens such as Staphylococcus aureus, Clostridium tyrobutyricum and L. monocytogenes. From Rigouta cheese, a Tunisian homemade starter-free fresh cheese from cow’s milk, it has been developed Lactococcin MMT24, as antimicrobial substance produced by Lactococcus lactis MMT24 (Ghrairi, Frere, Berjeaud, & Manai, 2005). The bacteriocin showed a narrow antimicrobial activity against closely related LAB with a bactericidal mode of action (Ghrairi et al., 2005). Non-starter LAB have been detected in high numbers in semi-hard cheeses during ripening, and may suppress harmful bacteria. Christiansen et al. (2005) isolated and identified more than 400 Lactobacillus spp. strains from Danish semi-hard cheeses. The majority of isolates belonged to the Lactobacillus paracasei complex and were classified into approximately 135 pulsotypes using pulsed field gel electrophoresis (PFGE). Almost half of the strains possessed anti-clostridial activity, and 10% disclosed a broad and promising activity as potential protective adjunct cultures (Christiansen et al., 2005).

Table 1. Bacteriocinogenic LAB strains isolated from cheese and their main target microbes. Cheese type

Strains

Main target microbes

References

Cheddar White-brined cheese

Leuconostoc mesenteroides E. faecium FAIR-E 198 E. faecium ST209GB, ST278GB, ST315GB, ST711GB Streptococcus thermophilus ACA-DC 0040 Streptococcus macedonicus ACA-DC 198 Lb. paracasei Lb. mesenteroides, Lb. paracasei subsp. paracasei, Lb. delbrueckii subsp. bulgaricus Lb. plantarum LCN 17, Lb. rhamnosus LCN 43 Lactococcus lactis

L. monocytogenes L. innocua L. monocytogenes, L. innocua, Listeria ivanovii subsp. ivanovii Clostridium sporogenes, Cl. tyrobutyricum Bacillus subtilis, Cl. tyrobutyricum

Daba et al. 1991 Sarantinopoulos et al., 2002 Favaro et al. 2014

Clostridium spp. Fungi, L. monocytogenes

Christiansen et al. 2005 Voulgari et al. 2010

L. monocytogenes

Nespolo & Brandelli, 2010

Staphylococcus aureus, L. monocytogenes Cl. tyrobutyricum, L. monocytogenes E. faecalis, L. innocua Salmonella enteritidis Brochothrix thermosphacta, L. monocytogenes, S. aureus E. faecalis and E. faecium

Dal Bello et al., 2010

Medium-hard cheese

Hard cheese

Semi-hard goat milk cheese Fresh cheese

E. faecium E. faecalis, E. hirae, E. avium Lb. paracasei subsp. paracasei Lc. lactis, E. faecalis, E. faecium Lc. lactis MMT24

Aktypis, Kalantzopoulos, Huis in’t Veld, & Ten Brink, 1998

Dal Bello et al., 2010 Mart
ın-Platero et al. 2009 Nikolic et al. 2008 Dal Bello et al., 2010 Ghrairi et al. 2005

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Gulahmadov et al. (2006) described the antibacterial activity of Lb. paracasei and Lactobacillus rhamnosus against Escherichia coli HB-101 and S. aureus CIP-9973 in traditional Azerbaijani cheeses. Further studies described the protective effect of bacteriocins and the control of flavor development during the cheese ripening, highlighting a novel approach to their potential usefulness (SobrinoL
opez & Mart
ın-Belloso, 2008). Streptococcus macedonicus ACA-DC 198, isolated from naturally fermented Greek Kasseri cheese (Tsakalidou et al., 1998) was described as producer of the lantibiotic macedocin. The expressed bacteriocin was characterized and the activity survived the stress conditions prevailing during Kasseri cheese processing. Macedonocin was produced during the initial stage of cheesemaking and it was detected until end of the ripening period. The bacteriocin showed a broad antimicrobial spectrum against, among others, Cl. tyrobutyricum (Georgalaki et al., 2002). Moreover, in other experiments, S. macedonicus ACA-DC 198 has been used as an adjunct culture in Kasseri cheese production without effecting the changes of the organoleptic properties of the final product (Anastasiou et al., 2007; De Vuyst & Tsakalidou, 2008). To study its ability to inhibit Cl. tyrobutyricum spore outgrowth under conditions prevailing during Kasseri cheese production and ripening, S. macedonicus ACA-DC 198 was used in three fermentations of skim milk (Anastasiou et al., 2009). The presence of macedocin was confirmed during the process and the results showed that macedocin effectively inhibited the germination of Clostridium spores (Anastasiou et al., 2009). The Bukuljac, a homemade cheese manufactured in Serbia from heat-treated goat’s milk without the addition of any starter culture, has been analyzed by Nikolic et al. (2008). Lb. paracasei subsp. paracasei revealed to be the predomimant strain (87%), and besides lactobacilli, 9% Lc. lactis subsp. lactis, 3% E. faecalis and 1% Le. mesenteroides were found. In addition, 50 out of 55 isolates exhibited also high antimicrobial activities (Nikolic et al., 2008). The use of bacteriocin producing strain to prevent late blowing in cheese, a serious microorganism-associated defect that can occur during cheese ripening, represents a promising alternative to the addition of lysozyme, which particularly gives the increasing concerns regarding its potential allergenicity. Mart
ınez-Cuesta et al. (2010) showed that late blowing can be controlled by adding lacticin 3147-producing Lc. lactis IFPL 3593 to the starter. Jamuna, Babusha, and Jeevaratnam (2005), Gao and Ju (2008), Al-Holy, Al-Nabulsi, Osaili, Ayyash, and Shaker (2012) focused to combine application of different hurdles with nisin to inhibit the spoilage or foodborne pathogens in cheeses. Al-Holy et al. (2012) demonstrated that mild heating in conjunction with nisin treatment (1000 and 1500 IUmL 1) resulted in a total elimination of Listeria innocua from brined white cheese stored under normal (4  C) and abused (10  C) refrigeration conditions. This finding is

significant since white cheese is a ready-to-eat food product that is vulnerable to colonization by L. monocytogenes and mostly will not be subjected to a subsequent biopreservation step that could reduce L. monocytogenes level in the product. Several species of enterococci (Enterococcus durans, E. hirae), lactobacilli (Lb. casei, Lactobacillus coryniformis, Lb. paracasei, Lb. rhamnosus, Lb. plantarum), pediococci (Pediococcus pentosaceus, Pediococcus acidilactici) and lactococci (Lc. lactis) have been described also to be antifungal as elegantly reviewed by Schn€ urer and Magnusson (2005) and Crowley, Mahony, and van Sinderen (2013). Their application in cheese sector could be very important as dairy products are particularly susceptible to fungal contaminations leading to food spoilage (off-flavor, deterioration of visual appearance) and important economic losses. However, only few studies (Schwenninger & Meile, 2004; Suomalainen & M€ayr€a-Makinen, 1999; Tawfik, Sharaf, Effat, & Mahanna, 2004) have tested in situ the capacity of these bacteria to prevent fungal spoilage in yogurts or fermented milks. A co-culture of Lb. paracasei subsp. paracasei and Propionibacterium jensenii was found to retard growth of various Candida species in an in situ yoghurt model as well as on cheese surface (Schwenninger & Meile, 2004). Cheeses are also susceptible to spoilage by psychrotolerant moulds able to withstand low oxygen environments such as Penicillium roqueforti. Three antifungal Lb. plantarum isolates demonstrated anti-mould capabilities when used as adjuncts during cheddar cheese production (Crowley et al., 2013). Furthermore, processed cheese slices and cheese shelf-life were improved after treatment with antifungal LAB against Aspergillus alternata (Garcha & Natt, 2012) or Aspergillus oryzae and Aspergillus niger (Muhialdini, Hassan, Sadon, Zulkifli, & Azfar, 2011). The use of the aforementioned isolates provides manufacturers with a natural option to the use of preservatives such as sodium benzoate, sorbic acids and natamycin in yoghurt and cheese production. Although very significant advances in the field of antifungal LAB have been achieved during the last decade, certain limitations and knowledge gaps still need to be addressed. Whilst there have been many publications on antifungal applications in recent years, just very few commercial cultures are available and not for cheese making, possibly due to the fact that the anti-fungal activity of any given strain is dependent on many physicoechemical parameters, the food production process and the ability of the strains to produce the compounds in situ in the food product (Crowley et al., 2013). Bacteriocins as food natural biopreservative In food industry it is essential to preserve the nutritive qualities of the raw material through the inhibition of spoilage and pathogenic bacteria. Based on the consumer’s demands, this needs to be realized through natural products

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or processes. The use of bacteriocins can help to reduce the addition of chemical preservatives as well as the intensity of heat treatment Alternatively, bacteriocins application, in combination with other conventional treatment as part of hurdle technology, could result in food which is more naturally preserved and better in sensorial and nutritional properties (Reis et al., 2012). Although several bacteriocins from LAB have been characterized to date, their usage as food preservatives is still very limited (Table 2) because of several technological or legislation barriers. Various bacteriocins may have potential applications in food, but they are not currently approved as antimicrobial food additives (Naghmouchi, Kheadr, Lacroix, & Fliss, 2007). The most extensively studied bacteriocins are nisin and pediocin PA-1 both having commercial applications in the food industry. Nisin was first marketed in England in 1953 and since then it has been approved for use in over 50 countries. Significantly, nisin is licensed as a food preservative (E234) and is recognized to be safe for food by the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives in 1969. There are numerous applications of nisin as a natural food preservative, including dairy products and processed cheese. In these cases, the bacteriocin is incorporated into the product as a dried concentrated powder, though not purified, preparation made with food-grade techniques. The FAO/WHO Codex Committee on milk and milk products allowed nisin as a food additive for processed cheese at a concentration of 12.5 mg (as pure nisin) per kilogram product (Reis et al., 2012). There are major differences in national legislations concerning the presence and levels of nisin in various food products. For instance, nisin can be added to cheese without limit in the United Kingdom, while a maximum concentration of 12.5 mg/kg in that food is allowed in Spain (Sobrino-L
opez & Martin-Belloso, 2008). In Brazil, Argentina, Italy and Mexico, nisin is permitted for application in cheese up to 12.5 mg/kg and up to 500 IU/g (Cleveland, Montville,

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Nes, & Chikindas, 2001). However, in USA much higher level of 10.000 IU/g is permitted (Cleveland et al., 2001). Nisin is innocuous, sensitive to digestive proteases and it does not influence sensory properties of the food products (Pongtharangkul & Demirci, 2004). For these reasons, it has been proved to be an effective natural food biopreservative. The chemical and physical properties of a food (raw or fermented) such as pH, proteins, fat and starch can influence the antimicrobial activity of bacteriocins. Limitation for application of nisin can occur at pH higher than 7, as nisin greatly loses the activity (de Arauz et al., 2009). The amphipatic nature of nisin limits widespread applications to various products because of its interaction with fat and other food components that reduces the antimicrobial potential of nisin in a food matrix. Sobrino-L
opez and Mart
ın-Belloso (2008) mentioned several limitations which curb the use of nisin in dairy products, such as its adsorption to fat and to surface of protein globules, a heterogeneous distribution in dairy product matrices, the inhibition of non-resistant starter cultures, or flavor alteration by the incorporation of nisin producing strains as starters. These are also the reasons for low application of nisin in the preservation of meat as it is in dairy products. The interference by cheese components such as phospholipids may occur, which limits the bacteriocin activity, especially when food presents high-fat content (de Arauz et al., 2009). Because of that, future research may also focus on the use of encapsulated nisin for more sustainable antimicrobial effect against pathogens and the combination of nisin with nonthermal treatment, such as ultrasound and microwave. Beside phospholipids, fat or proteins, some inorganic chemicals can have influence on bacteriocin mode of action. Chollet, Sebti, Martial-Gros, and Degraeve (2008) studied the effect of NaCl, fat and proteolytic enzymes on antimicrobial activity of nisin in a model system and Emmental cheese. Increasing anhydrous milk fat concentration caused a significant reduction of nisin bioactivity.

Table 2. Application of bacterioginocenic strains in cheese production. Bacteriocin/strains

Application

Main target microbes

References

Nisin

MicrogardÔ

semi-hard cheese cheddar-type cheese ricotta-type chees liquid cheese whey soft cheese cottage cheese

Sobrino-L
opez & Mart
ın-Belloso, 2008 Sobrino-L
opez & Mart
ın-Belloso, 2008 Sobrino-L
opez & Mart
ın-Belloso, 2008 Gallo et al. 2007 Kykkidou et al. 2007 von Staszewski and Jagus (2008)

Lc. lactis, Lc. cremoris E. casseliflavus Lacticin 3147 E. faecium, E. mundtii

low salt cheese fresh and soft cheese smear-ripened cheese fresh cheese

S. aureus Cl. sporogenes L. monocytogenes L. innocua L. monocytogenes Pseudomonas spp., Salmonella spp., L. innocua Micrococcus flavus NIZOB423, L. lactis subsp. cremoris L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes

Ayad, 2009 Iseppi et al. 2008 O’Sullivan et al. 2006 Vera Pingitore et al., 2012

MicrogardÔ a complex preparation (described into the text).

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However, higher NaCl content slightly enhanced nisin activity by influencing most probably the adsorption of the bacteriocin to the spoilage microorganisms. The study performed with Emmental cheese indicated that the nisin interacted with the cheese matrix, likely milk fat globules, but was not affected by proteases. Kykkidou, Pournis, Kostoula, and Savvaidis (2007) evaluated the use of nisin as an antimicrobial treatment for shelf-life extension of Galotyri, a Greek soft acid-curd cheese, stored aerobically under refrigeration for 42 days (Table 2). Among all microorganisms monitored and enumerated, lactobacilli, lactococci and yeast were the groups that prevailed in cheese samples, irrespective of antimicrobial treatment. Based primarily on sensory evaluation (appearance and taste) and a microbiological acceptability limit for yeast (5 log CFU/g), the use of nisin treatments extended the shelf-life of fresh Galotyri cheese stored at 4  C (Kykkidou et al., 2007). Gallo, Pilosof, and Jagus (2007) investigated the effect and interactions of pH (5.5, 6.0 and 6.5), temperature (7  C and 20  C) and nisin addition (100 IU/ml; 300 IU/ ml) on the growth and survival of L. innocua in liquid cheese whey. The growth kinetics of the spoilage microbe was dependent on the interaction of the three variables, particularly on the lag phase duration. The effectiveness of nisin was more pronounced at 7  C and as the pH decreased from 6.5 to 5.5. Considering that nisin is more active in acid or moderated neutral pH, this combined treatment may provide liquid cheese whey with an high degree of protection against L. innocua, particularly if employed in conjunction with low temperature (Gallo et al., 2007). Other antimicrobial compounds have been tested on cheese products in the last years. MicrogardÔ formulations are BLIS, such as diacetyl as well as lactic, propionic and acetic acid and other undefined low molecular-mass inhibitors around 700 Da (Al-Zoreky, Ayres, & Sandine, 1991), obtained by fermentation of grade A skim milk (MicrogardÔ 100) or dextrose (MicrogardÔ 200) with Propionibacterium shermanii or specific lactococci. MicrogardÔ products have been approved by the US Food and Drug Administration (FDA) for use in cottage cheese and they are estimated to be added to 30% of the cheese produced in the USA. Additionally, the FDA has approved propionbacterium metabolites as GRAS (Generally Recognized As Safe). Studies conducted with MicrogardÔ 100 demonstrated that it extends the shelf-life of cottage cheese by inhibiting spoilage and pathogenic bacteria, including Pseudomonas, Salmonella and Yersina and certain fungi (Al-Zoreky et al., 1991). MicrogardÔ 300 has been developed to target the Gram-positive bacteria (Lemay et al., 2002). Antimicrobial activity of MicrogardÔ individually or in combination with nisin against L. innocua in liquid cheese whey was investigated by von Staszewski and Jagus (2008). MicrogardÔ did not reduce the initial count of L. innocua during storage from 7 to 25  C, and showed a response

similar to the untreated whey. In comparison to MicrogardÔ, nisin exhibited an immediate bactericidal effect that was followed by recovery of L. innocua. Initially, a significant antagonistic action was detected when MicrogardÔ was combined with nisin in all the evaluated systems. However, during storage, different responses were observed by von Staszewski and Jagus (2008) and combining MicrogardÔ and nisin appears to be a feasible tool for extending the shelf-life of liquid cheese whey (von Staszewski & Jagus, 2008). Different bacteriocinogenic strains of Lc. lactis and Lactococcus cremoris were applied together with nisin by Ayad (2009) for the development of starter strain(s) to improve the safety and nutritional quality of Domiati cheese. All the tested strains can grow well together and appeared to produce typical Domiati cheese pleasant flavors, body and texture. Interestingly, the results reported by Ayad (2009) revealed that selected strains can be used for improving Domiati cheese quality with or without supplementing the system with nisin. The application of bacteriocinogenic cultures of Enterococcus casseliflavus and the bacteriocin enterocin 416K1 against L. monocytogenes in fresh and soft cheeses were reported by Iseppi et al. (2008) as bacteriocin entrapped in polymeric films. Izquierdo, Marchioni, Aoude-Werner, Hasselmann, and Ennahar (2009) described the use of E. faecium as adjunct culture in brine and smearing solution and observed the production of enterocin A and B. O’Sullivan, O’connor, Ross, and Hill (2006) investigated live-culture-producing lacticin 3147 as a treatment for the control of L. monocytogenes on the surface of smearripened cheese. Although application of the lacticin 3147 producer did not give complete elimination of the pathogen, the results demonstrated the potential of the bioprotectant for improving the safety of smear-ripened cheeses, particularly the cheeses containing low level of contamination. Reviriego, Fern
andez, and Rodr
ıguez (2007) proposed that recombinant Lc. lactis producers of nisin, pediocin PA-1 and enterocin A (respectively) may be used as starter cultures and have a potential benefit on the control of L. monocytogenes in cheese model system. Pinto et al. (2009) investigated the application of LAB as starter cultures in production of Minas Traditional Serro cheese to control L. innocua during cheese ripening and, unfortunately, only bacteriostatic effect against L. innocua was observed. Natural starter and non-starter LAB were not sufficient to guarantee the control of L. innocua even after 60 days of storage, as it is required by Brazilian legislation (Pinto et al., 2009). However the reported results seem to indicate that a combination of the selected high level bacteriocin producing LAB and the semi-purified bacteriocin or other permitted food preservative may be an answer for the more successful control of Listeria spp. in the cheeses production. Vera Pingitore et al. (2012) reported the bacteriocins production by two Enterococcus strains (Enterococcus

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mundtii CRL35 and E. faecium ST88Ch), isolated from cheeses. Both investigated strains were characterized for their capability to control growth of L. monocytogenes in experimentally contaminated fresh Minas cheese during refrigerated storage. Growth of L. monocytogenes was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8  C, evidencing a bacteriostatic effect, while E. faecium ST88Ch was less effective, inhibiting L. monocytogenes to grow only for 6 days at 8  C. Since in the reference cheeses containing nisin (12.5 mg/kg) and cheese inoculated with E. mundtii CRL35 showed similar reduction (less than 1 log difference), this research underlines the great potential of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese (Vera Pingitore et al., 2012). E. faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on L. monocytogenes in Munster cheese, a red smear soft cheese (Izquierdo et al., 2009). The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. After 7 days, L. monocytogenes was reduced to 50e100 CFU/mL on the cheese surface, while, adding E. faecium WHE 81 in the brine completely stopped L. monocytogenes to grow. On the contrary, in the control samples, L. monocytogenes counts exceeded 104 CFU/g. Interestingly, E. faecium WHE 81, which naturally occured in Munster cheese, did not adversely impact on the ripening process (Izquierdo et al., 2009). Enterocin AS-48 is one of most studied cyclic bacteriocin with a broad spectrum of activity showing remarkable stability to pH and heat, which makes it an ideal candidate for application as a food biopreservative. Efficacy of bacteriocin AS-48 for controlling staphylococci in a culture medium has been demonstrated (Grattepanche, Miescher-Schwenninger, Meile, & Lacroix, et al., 2008). Moreover, it was shown that chemical preservatives such as sodium nitrite, sodium lactate, sodium chloride, chelating agents, and moderate heat act synergistically with enterocin AS-48 against food-borne pathogens such as Bacillus cereus, S. aureus, Salmonella choleraesuis, and E. coli O157:H7 when applied in laboratory culture media (Grattepanche et al., 2008). In addition, the effectiveness of enterocin AS-48 to control S. aureus in skimmed milk and fresh cheese has been assayed by Mu~ noz et al. (2007) reporting the highest inhibition levels (4e2 log CFU/g below controls) within the first week of storage. Smearing operation approach was applied for pediocin AcH produced by Lb. plantarum WHE92 as a surface culture. The strain proved to be a viable strategy in reducing cheese contamination with Listeria spp. (Grattepanche et al., 2008), which are almost exclusively localized on the surface of smear cheeses. A semi-hard cheese produced from milk artificially contaminated with Cl. tyrobutyricum spores

7

(2.5  103 CFU/mL) was used as a model in the research of Bogovic Matijasi
c, Koman Rajsp, Perko, and Rogelj (2007) to study the ability of bacteriocin-producing Lb. gasseri K7 to inhibit clostridia. The added lactobacilli did not hinder the starter culture (St. thermophilus), but had an inhibitory effect on non-starter mesophilic lactobacilli. Late blowing, as a result of Cl. tyrobutyricum outgrowth and butyric acid fermentation, occurred in all cheeses, however it was reduced in cheeses processed with the addition of Lb. gasseri. The developments of cheese starter cultures with protective functionalities and the limits of their utilization (i.e., restricted in situ production, developed resistance of the target organisms) have been reviewed (Grattepanche, et al., 2008). However, very few protective cultures are marketed today, underlining the difficulty to develop such cultures. One of the main drawbacks of LAB bacteriocinogenic strains to be successfully applied as starter cultures is indeed the generally low occurrence of technological properties such as acidifying, lypolytic and proteolytic activities. Enterococcus and Lactobacillus spp. bacteriocinogenic strains have generally poor acidifying capacity in milk and only a limited number of strains was reported to be capable of hydrolyzing protein and lipids (Giraffa, 2003). To select for starter cultures having protective functionalities, we conducted an extensive survey of acidifying, lypolytic and proteolytic activities on more than 250 different LAB bacteriocinogenic strains (mainly Lactobacillus, Enterococcus, Streptococcus spp. isolates) obtained from several cheeses However, only 8% of the strains exhibited promising phenotypes in terms of lipase and protease production while very few showed interesting acidifying capacity (data not shown), indicating that the way to develop starter cultures containing bacteriocinogenic LAB is still a fascinating challenge. This is confirmed by the very limited availability of commercial products recommended for control of spoilage and pathogenic prevention in fermented dairy and meat product (for instance: Danisco DuPont, HoldbacÒ YM-B Plus, HoldbacÒ YM-C Plus, HoldbacÒ LC and HoldbacÒ Listeria, composed by Lb. rhamnosus and P. freudenreichii subsp. shermanii). What are the limitations? The application of bacteriocinogenic cultures in control of spoilage microorganisms cannot be presented as the panacea to solve the problems related to the preservation of fermented dairy products. From one side, starter or non-starter adjunct cultures can contribute to the safety of the final product, but it is necessary to consider that the antimicrobial compounds have their limitations. As a result, the best option is to look for the optimal combination of traditional and new ways of biopreservation. The use of bacteriocinogenic cultures have several limits related to the producer strain: such as i) no sufficient level of bacteriocin expression; ii) antagonism of other bacteria toward the producer strain; iii) inadequacy of producer

Please cite this article in press as: Favaro, L., et al., Bacteriocinogenic LAB from cheeses e Application in biopreservation?, Trends in Food Science & Technology (2014), http://dx.doi.org/10.1016/j.tifs.2014.09.001

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strain as a starter; iv) low capacity for bacteriocin production in food system; v) safety of the producer strain; vi) the interaction between produced bacteriocin and food matrix; vii) the effect of the physicochemical parameters on the bacteriocin activity. Antimicrobial substances and, particularly, bacteriocins are considered as primary metabolites. However, most of the time LAB expresses low levels of bacteriocins. In parallel with expression of the bacteriocin, immunity protein and induction factors are also expressed, since they are part of the same reading frame. In this way, the bacteriocin producer guarantees its own safety from the killing action of its own bacteriocin (Cotter et al., 2005). The presence of pathogenic bacteria can induce the production of bacteriocins, based on signals, mainly proteins produced by pathogenic bacteria, acting as bacteriocin production initiators. However, the biochemical machinery of LAB will need sufficient nutrients for the production of bacteriocins. Many studies have proved that bacteriocin production optimization highly dependent on the composition of medium, including type of sugars, organic nitrogen sources, minerals and vitamins (Audisio, Oliver, & Apella, 2001; Todorov & Dicks, 2005). Few research groups demonstrated that even the sort of organic nitrogen source has a critical role for bacteriocin production (Parente, Brienza, Ricciardi, & Addario, 1997; Schirru et al., 2014). Moreover, the type of carbohydrates is crucial for the growth and production of metabolites, including antimicrobial peptides. Preferable carbohydrates for most of LAB are glucose, sucrose, fructose and lactose. However, different growth rates and bacteriocin production levels have been recorded for many LAB (Parente et al., 1997; Schirru et al., 2014; Todorov & Dicks, 2005). Concerning that cheese is a peculiar environmental niche in which LAB are expected to grow and to secrete antimicrobial peptides, the first logical screening to be addressed should target the ability of LAB to grow and produce sufficient levels of bacteriocin(s) in cheese, where milk will be the major growth substrate. In addition, we have to consider that the production and ripening of many cheeses occur in the presence of NaCl and most of the time with ripening and storage in refrigeration temperature. In this case, “how LAB will produce bacteriocin(s) at this temperature?”, or “will we need to evaluate the bacteriocin produced and expressed on the previous stage of the cultivation or its production in the product (cheese itself)?”. Other key point is the storage temperature of most of cheeses (below 8  C). Such temperature is not detrimental for the survival of many LAB but does not allow them to grow abundantly, thus to produce sufficient amounts of bacteriocins. On the contrary, L. monocytogenes can survive and grow very well at cold temperatures. To solve this problematic issue, the use of semipurified bacteriocins could be very attractive. Furtado, Todorov, Landgraf, Destro, and Franco (2014) have demonstrated that L. lactis subsp. lactis DF04Mi, a bacteriocin producer isolated from goat milk, has the ability to produce

bacteriocin against L. monocytogenes. The same strain can grow and express bacteriocin(s) when cultured in milk reaching levels lower than those achieved in the commercial MRS broth. However, when the strain was applied in cheese, the effect on L. monocytogenes was similar to those detected with the non bacteriocin producing culture of L. lactis subsp. lactis (Furtado et al., 2014). In the same experiment, the better results, in terms of L. monocytogenes inhibition, have been obtained using commercial nisin. The authors argued that such differential effect could be linked to several environmental factors affecting mainly bacteriocins production and not nisin action. Specifically, the low temperature, together with different physical and chemical parameters in the cheese matrix may have inhibited the production and the following interaction of the bacteriocions with the target spoilage microbe. The high levels of nisin applied to the system, on the contrary, could be the reason for the effectiveness of the treatment. Most probably, in the studied conditions, L. lactis subsp. lactis DF04Mi is far away from the ability to produce quantity of antimicrobial peptides with effects similar to those achieved by nisin addition. Then, the reasonable point in the search for antimicrobial peptide producers will be the selection of LAB with natural high level of expression of antimicrobial peptides even in growth limiting conditions (i.e., lack of appropriate nutrients, sub-optimal temperature). This has been demonstrated by Vera Pingitore et al. (2012), who, towards the selection of potential biopreservation cultures, have firstly evaluated the effect of different environmental conditions on the efficacy of expressed bacteriocin(s) against L. monocytogenes. Factors such as level of bacteriocin production, adsorption of bacteriocin(s) to the target microorganism(s) were described to play an important role on the activity of antimicrobial peptides. In the same study, nisin was used as a control treatment and authors demonstrated that, in order to have a sufficient biopreservation effect, it was necessary to use a high level of bacteriocin producers. Moreover, it is important to guarantee that bacteriocin will be expressed in sufficient quantity in these growth/production limiting conditions and the expressed bacteriocin(s) would be sufficient to exhibit action against L. monocytogenes or other spoilage microorganisms. Another point that should be considered is that many LAB can express more then one bacteriocin. Poeta et al. (2007) demonstrated that some Enterococcus spp. can carry several genes. Todorov et al. (2012) recently overviewed information about several bacteriocin producing strains highlighting that many LAB express particular bacteriocin genes depending on the environmental conditions. The logic question is “what part of this gene(s) exactly can be expressed, how the environmental factors can influence the expression of this genes and on what level?”. Transferring the question of bacteriocinogenic cultures into cheese or other dairy products matrix, this point have even stronger significance. Since environmental conditions in dairy

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commodities are strictly limited, this will likely facilitate, reduce or even stop gene(s) expression. Further studies on this area will need to be performed using biomolecular tools and studying the expression of bacteriocins in these particular environments. The role of bacteriocins in different environments and control of the mechanisms of bacteriocin production and activity, however, has still not thoroughly explained, mainly because of the difficulties related to their detection in complex environments. A noteworthy and pioneering approach has been pursued by Trmci
c, Monnet, Rogelj, and BogovicMatijasi
c (2011) who quantified, in a cheese-like medium, the expression of nisin genes in Lc. lactis M78, a well-characterized nisin A producer isolated from raw milk. The expression of all 11 genes involved in nisin biosynthesis was evaluated during cheese production by real-time reverse transcription-PCR. Beside the contribution to the explanation of regulation of nisin biosynthesis, the cited study presented the first example of what could be done in the future to better understand the role of bacteriocins in different environments and the physiology of microorganisms growing in cheese. Considering that the most accepted mode of action of nisin is based on the interaction of nisin and lipid II (Cotter et al., 2005), it has been demonstrated that some bacteriocins, including nisin, when applied in high concentrations, can interact with the cell surface even if lipid II is not present. However, the presence of receptors with a lipid nature is a frequent for initial interaction between bacteriocin and target microorganism. Several authors already indicated that antimicrobial activity of nisin is very limited in meat products because of the high level of fat (de Arauz et al., 2009). Evaluating the wide variety of dairy products, we need to consider that fat can vary from 10 to 60% on dry basis in different cheese (Mistry, 2001). As a result, investigating cheese and LAB producing bacteriocins, special attention needs to be granted on fact that cheese has high protein, lipid and microelements concentrations, which may have a particular influence on the interaction with not only antimicrobial peptides, but with any bio- or chemical preservative. In dairy products, NaCl can be applied up to 6% (Schirru et al., 2012) as part of technological or sensorial characteristics of the product. Depending on the specific influence of NaCl on adsorption of each bacteriocin to test microorganism, the antimicrobial compound can be applied or not in the processing of the specific cheese. Similar influence of surfactants or food additives has to be studied for every potential LAB candidate for biopreservation in order to successfully fulfill the goal of guarantee the final achievements. Safety of LAB is another important point in bio-preservation. GRAS status for several Lactobacillus spp. has been granted. However, even if GRAS is not common for Enterococcus spp., they are part of the several fermented dairy products specially form Mediterranean area. Various

9

E. faecium have been reported to have an important role in the processing of dairy products (Foulqui
e Moreno et al., 2006; Franz, Stiles, Schleifer, & Holzapfel, 2003). On other side, Enterococcus spp., including E. faecium has been reported to be related with hospital infection (Vankerckhoven et al., 2004). The controversial position of Enterococcus spp. is part of long discussion “for” and “against”. In this perspective, it is fundamental to study the presence of virulence factors in potential candidates for biopreservations. For instance, Favaro et al. (2014) have demonstrated that E. faecium strains isolated from goat cheese can be considered as low level virulence factors carriers. Two out of the four studied strains gave positive results for asa1 (aggregation substance) and vancomicin B genes. However, all 4 strains have been positive for tdc (tyrosine decarboxylase). Nevertheless, none of other 10 virulence factors, antibiotic resistance or biogenic amines genes tested have been detected. Similar results regarding low virulence potential and safety of E. faecium have been observed in strains isolated from dairy products, and in opposite, a high spread of virulence determinants have been recorded in strains as Lb. plantarum (Todorov et al., 2012). Overall, we need to be aware that GRAS status of one species is not anymore sufficient to claim for safety of a particular strain. Horizontal gene transfer is a highly possible scenario that may occur in a real situation between different strains and species, and the spread of virulence genes can be realized. Any particular strain, candidate for a biopreservation role, need to be screened carefully for presence of virulence factors, antibiotic resistance or biogenic amines genes in order to guarantee both safety and potential beneficial properties. Conclusions Despite the great deal of basic and applied research on the identification, characterization and development of bacteriocinogenic LAB in cheese over the past four decades, very few formulations reached the market. This is largely explained by the hurdle of applying bacteriocinogenic strains in the context of industrial cheese production which needs robust and reproducible processes. The selection of competitive bacteriocinogenic strains in cheese environments and suitable starters with low sensitivity to the Table 3. Desirable traits of bacteriocinogenic LAB strains useful for cheese production. Trait

Research status

GRAS Producer of bacteriocins effectively working in the process Technological properties (acidifying activities, extracellular enzymes, texture activities) Capable of surviving and growing in the cheese production process

Research on going Few applications ready to use Research on going

Research on going

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bacteriocin(s) is essential for successful large-scale applications. Moreover, strict process control is crucial both to achieve proper growth of starter and adjunct cultures and to produce constant, high quality and safe cheese. The safety of such bacteriocinogenic cultures is an emerging priority for leading healthy lives and defining new cheese commodities for the market. This review focused on the desirable traits for a bacteriocinogenic LAB strain to be successfully used for cheese production. As reported in Table 3, before their commercial applications, few research perspectives have to be implemented. Overall, the perspective of using microbes as bio-preservative has huge potential and paves the way for a new concept of safe and healthier cheese production. Acknowledgments Dr. Favaro is recipient of “Assegno di ricerca Senior” grant from the University of Padova, Padova, Italy. Dr. Todorov has been supported by CNPq (Conselho Nacional de Desenvolvimento Cient
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