I. Dolenčić Špehar et al.: Antimicrobial activity of bacteriocins in cheese production, REVIEW | UDK: 637.352 | DoI: 10.15567/mljekarstvo.2020.0301 REcEIVED: 15.01.2020. | AccEptED: 20.04.2020. Antimicrobial activity of bacteriocins of Lactic Acid Bacteria on Listeria monocytogenes, Staphylococcus aureus and Clostridium tyrobutyricum in cheese production Iva Dolenčić Špehar, Darija Bendelja Ljoljić*, Zvjezdana Petanjek, Šimun Zamberlin, Milna Tudor Kalit, Dubravka Samaržija University of Zagreb, Faculty of Agriculture, Department of Dairy Science, Svetošimunska 25, 10000 Zagreb, Croatia *Corresponding author: [email protected] Mljekarstvo Abstract The generally accepted concept of the necessity of producing safe foods has indirectly influenced the 70 (3), 135-149 (2020) decision to replace chemical preservatives with natural ones. Bacteriocins, and in particular those syn- thesized by lactic acid bacteria (LAB) in the food industry, are considered to be their effective replace- ment. In controlling the growth of microbial pathogens and/or the occurrence of pathogenic bacteria in food, with the permitted nisin and pediocin, a significant antibacterial effect has been shown for most LAB bacteriocins. However, the use of purified bacteriocins as bio preservatives in cheese production is limited. To inhibit the growth of bacteria L. monocytogenes, S. aureus and C. tyrobutyricum in cheese, bacteriocinogenic LAB strains contained in primary, adjunct or protective culture are much more ac- ceptable in cheese production. Key words: bacteriocins and bacteriocinogenic LAB strains, inhibition, Listeria monocytogenes, Staphylococcus aureus, Clostridium tyrobutyricum, cheese 135 I.Dolenčić Špehar et al.: Antimicrobial activity of bacteriocins in cheese production, Introduction 2018; Venegas-Ortega et al., 2019). The use of LAB bacteriocin in the food industry is determined Primarily due to the high resistance of patho- by the type of food and the technology of its pro- genic bacteria to antibiotics, bacteriocins have be- cessing. In this regard, bacteriocins can be used to come equally important for the food industry (De improve food quality and safety: (i) by inoculation Vuyst and Leroy, 2007; Mokoena, 2017; Teix- of bacteriocin containing active LAB strains in the eira Barbosa et al., 2017), human (Mathur et form of primary, adjunct or protective culture, (ii) by al., 2015; Sivaraj et al., 2018; Dreye et al., 2019; adding a pre-fermented product containing bacte- Lopetuso et al., 2019) and veterinary medicine riocinogenic LAB strains, (iii) by direct addition of (Lagha et al., 2017; Abdelfatah et al., 2018; Vie- purified or partially purified bacteriocin, and (iv) -in co-Saiz et al., 2019). directly by incorporating bacteriocin into the pack- Consequently, compared to previous periods, aging protective film (De Vuyst and Leroy, 2007; the number of studies on the common and specific Borges and Teixeira, 2016). properties of bacteriocins, especially those synthe- In general, cheese is considered a safe food sized by lactic acid bacteria (LAB), has increased because of its physicochemical characteristics and significantly (Perez et al., 2014; Samaržija, 2015; the antimicrobial activity of its microbiome against Alvarez-Sieiro et al., 2016; Teixeira Barbosa et pathogenic bacteria (Fox et al., 2000; Farkye and al., 2017; Tumbarski et al., 2018; Venegas-Orte- Vedamuthu, 2002; Irlinger et al., 2015). Despite ga, 2019; Rahmeh et al., 2019). that, according to data available for 2015 published In the food industry, LAB bacteriocins are con- by the European Food Safety Authority (EFSA) and sidered as a real alternative to traditional food the European Centre for Disease Control (ECDC), ep- Mljekarstvo additives (Samaržija et al., 2009; Cotter et al., idemic poisoning caused by consumption of cheese 2013; Perez et al., 2014). However, due to tech- contaminated with pathogenic bacteria and/or nological constraints, and especially due to legal their toxins has been reported in many countries 70 (3), 135-149 (2020) restrictions, only nisin and pediocin PA-1/AcH are (EFSA and ECDC, 2016). This is especially true for allowed in the production of food (Favaro et al., the consumption of soft cheeses (≥50 % moisture) 2015). The criteria for authorizing the use of bac- contaminated with staphylococcal enterotoxins or teriocins in food production are numerous and re- Listeria monocytogenes (Choi et al., 2016; Babić et strictive: (i) the microbial strain that forms it must al., 2018). The potential use of bacteriocin as a bio have GRAS (Generally Regarded as Safe) status or preservative in these types of cheeses seems jus- QPS (Qualified Presumption of Safety) status, (ii) tified. On the contrary, for semi-hard (39 % - 50 % have a broad spectrum of inhibitory activity, (iii) moisture) and hard cheeses (<39 % moisture), which exhibit highly specific activity, (iv) show no adverse generally do not support the growth of pathogenic effect on health, (v) not have the ability to trans- bacteria after two months of ripening (Cogan and mit antibiotic resistance, (vi) demonstrate positive Beresford, 2002) the effect of bacteriocin may be effects on improving the safety, quality and taste significant for growth inhibition of Clostridium spp., of food, (vii) have good stability in a wide range of which cause late blowing defect. temperatures and pH values, and (viii) have opti- Compared to other types of fermented foods, the mal solubility and stability in a specific type of food use of bacteriocins in cheese production is limited, (Cotter et al., 2005; De Vuyst and Leroy, 2007; and the study of their actual effect in the cheese ma- Leroy and De Vuyst, 2010; Silva et al., 2018). In trix is extremely complex. This claim is equally true addition, once isolated bacteriocins become sus- regardless of whether they are used to prevent the ceptible to inactivation due to environmental and growth of pathogenic bacteria or microbial spoilage physio-chemical conditions, which limits their use. agents, or to improve the ripening and sensory prop- However, encapsulation of bacteriocins using na- erties of cheese. notechnology is considered as a good strategy for This review is a contribution to the analysis of sci- creating a protective barrier from environmental entific results on the effect of purified or semi-purified conditions and/or temporarily increasing its activity bacteriocins and bacteriocinogenic LAB strains on against target microbial species (Tumbarski et al., the growth inhibition of pathogenic bacteria S. aureus, 136 I. Dolenčić Špehar et al.: Antimicrobial activity of bacteriocins in cheese production, L. monocytogenes and C. tyrobutyricum in cheese. Also, which was not previously the case (Alvarez-Sie- the paper highlights research topics in the field that iro et al., 2016). Classification and mechanism of currently capture the interest of the professional and antibacterial action of bacteriocins of LAB for their scientific public. potentially wider application in the food industry, animal husbandry, aquaculture, medicine, veterinary medicine, the pharmaceutical industry or cosmetics Bacteriocins of lactic acid industry are described in detail in several excellent bacteria review articles (Cotter et al., 2013; Yang et al., 2014; Egan et al., 2016; López et al., 2016; Alva- rez-Sieiro et al., 2016; Teixeira Barbosa et al., In general, bacteriocins are a heterogeneous 2017; Silva et al., 2018; Tumbarski et al., 2018; group of ribosomally synthesized bioactive an- Lepetuso et al., 2019; Choyam et al., 2019). In timicrobial peptides or proteins of many types of this context it is also important to highlight the fact gram-positive and gram-negative bacteria, in- that certain strains of Enterococcus genus synthe- cluding certain types of archaea (O’Connor and size, different by chemical structure, bacteriocins of Shand, 2002; Nandane et al., 2007; Samaržija, the common name enterocins. More than 30 have 2015). Most bacterial species (~99 %) synthesize been isolated so far, the most studied of which is at least one bacteriocin. These can be post-trans- enterocin AS-48 synthesized by Enterococcus faeca- lationally modified by cellular enzymes or excreted lis (Hanchi et al., 2018). Notwithstanding the fact from the bacterial cell into the environment unmod- that enterococci are pathogenic in some cases and ified (Yang et al., 2014). For the bacterial cell which most of them (~88 %) are antibiotic resistant, mod- forms them, bacteriocins have a primarily protective ern genotyping techniques make it possible to iso- Mljekarstvo function against other microbial species competing late their safe bacteriocinogenic strains (Arbulu et for the same source of nutrients. The formation of al., 2016; Xi et al., 2018). Depending on the chem- bacteriocin is an evolutionarily inherited bacterial ical structure, some of them are classified into es- 70 (3), 135-149 (2020) ability of an effective mechanism of self-defence. tablished groups and some still cannot be classified. It is thought that a bacterial cell uses bacteriocin Many enterocins have been found to have a strong for survival in competition with closely related spe- inhibitory effect on foodborne pathogenic bacteria cies within a specific ecological habitat. Therefore, (Favaro et al., 2014). Their antibacterial activity is in most cases the inhibitory activity of bacteriocins preserved over a wide range of pH values (2-12), at is directed only at closely related species. However, higher heating temperatures (100 °C/1 hour)