Potential probiotic and safety characterisation of enterococcus bacteria isolated from indigenous fermented motal cheese

Abstract

Enterococcus spp. were isolated from Motal cheese (a traditional raw milk cheese in Iran) and some probiotic and safety properties were evaluated. Ten of the 52 isolated enterococci showed ideal survival rates under the simulated gastrointestinal tract and were therefore selected for further analysis. Based on the results, six out of ten tested isolates belonging to Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus avium and Enterococcus durans did not demonstrate the incidence of any major virulence factor, haemolytic activity or resistance to most of the antibiotics studied, especially vancomycin. Moreover, these six isolates inhibited the growth of Listeria monocytogenes, had adhesion ability to epithelial cells and showed high cholesterol assimilation and good auto-aggregation activities. The bottom line is that some naturally occurring Enterococcus strains isolated from Iranian Motal cheese have the potential to be used in functional food formulations due to their probiotic properties and safety.

Introduction

Enterococci form part of the environmental, food and clinical microbiology. Most enterococcal species comprise Gram-positive cocci that are catalase-negative, usually facultatively anaerobic and which grow in 6.5% NaCl and 40% bile salts, as well as pH 9.6. The optimal temperature for their growth is 10 °C and 45 °C and they can survive for 30 min at 60 °C (Ogier & Serror, 2008). This genus includes multiple species of bacteria such as Enterococcus faecium, Enterococcus faecalis, Enterococcus durans, Enterococcus hirae, Enterococcus cecorum, Enterococcus columbae, Enterococcus casseliflavus, Enterococcus pseudoavium, Enterococcus avium, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus malodoratus, Enterococcus sulfureus, Enterococcus dispar, Enterococcus raffinosus, Enterococcus flavescens, and Enterococcus saccharolyticus (Yerlikaya & Akbulut, 2020). These lactic acid bacteria (LAB) are often comprise a part of the common microflora of different kinds of traditional cheeses (Saavedra, Taranto, Sesma, & de Valdez, 2003) and other fermented foods (Nieto-Arribas et al., 2011) due to their tolerance to salt, heat, acid and temperature.

Enterococci are also involved in the human gastrointestinal tract; this is why they are known for their role as probiotics as well (Franz et al., 2001). Probiotics are defined as live microorganisms that confer a health benefit on the host when administered in adequate amounts (Verdenelli et al., 2009). Some researchers have comprehensively listed the characteristics of a successful probiotic (Ljungh & Wadstrom, 2006). The most important criteria for a potential probiotic strain are the ability to survive through the gastrointestinal (GI) tract and antimicrobial activity against intestinal pathogens (Verdenelli et al., 2009).

What is of great importance is that the enterococci are still not registered as GRAS (Generally Recognised as Safe) organisms (Ben Braïek & Smaoui, 2019; Hanchi, Mottawea, Sebei, & Hammami, 2018). One of the reasons might be their ability to develop resistance against a wide variety of antibiotics. In addition, several virulence factors such as aggregation substance (agg), sex pheromone (cpd and ccf), hyaluronidase (hyl), enterococcal surface protein (esp), cytolysin (cylA, cylB, and cylM), adhesion of collagen (ace), gelatinase (gelE), E. faecalis and E. faecium antigen A (efaAfs and efaAfm, respectively) have been identified in enterococci (Ahmadova et al., 2013; Cariolato, Andrighetto, & Lombardi, 2008). In the food sense, although enterococci are not highly virulent and there are no reports of enterococcal infections associated with cheese consumption (Dapkevicius, Sgardioli, Câmara, Poeta, & Malcata, 2021), it is feared that some of pathogenic enterococci strains have already entered into the food chain and genetically contaminated the culture strains, thereby behaving as the agents of food-borne illnesses (Hanchi et al., 2018; Joghataei, Yavarmanesh, & Dovom, 2017). The dualistic effects of enterococci as beneficial microorganisms or opportunistic pathogens have led researchers to evaluate their effects on consumer health, as well as the possibility of using them as a starter culture or probiotic in foods.

The effect of production technology, as well as geographical origin of traditional cheeses on the composition and diversity of their microbiota has been verified in several studies (Ahmadova et al., 2013; Hassanzadazar, Mardani, Yousefi, & Ehsani, 2017; Terzic-Vidojevic, Vukasinovic, Veljovic, Ostojic, & Topisirovic, 2007). Motal cheese is one of the old traditional indigenous fermented cheeses that is made from raw sheep milk or occasionally mixed raw sheep and cow milk in the northwest of Iran, Republic of Azerbaijan, Armenia and eastern Anatolia region of Turkey. This type of cheese contains high fat and salt with a crumbly texture and a strong taste. It is ripened in the skin of the sheep or goat and, therefore, the natural fermenting microbiota becomes predominant since the starter cultures are not used for cheese production (Azizi, Najafi, & Dovom, 2017). Since Enterococcus spp. are widespread in environment and due to the lack of heat treatment for this type of raw milk cheese, the cheese is usually infected with enterococci (Urbach, 1995). Although Motal cheese has been consumed by local people for many years without any harmful effects, identification of microbiota of this type of traditional cheeses is important for well-defining and introducing these products and thus improve its consumer acceptability outside the local region (Hassanzadazar et al., 2017; Kafili et al., 2009).

The few studies on the microbial diversity of the LAB population in Motal cheese have been limited to identifying different species of the Lactobacillus spp. (Azizi et al., 2017; Shanehb et al., 2013) and to the best of our knowledge, no study has so far investigated the natural enterococci of this type of Iranian traditional cheese. In addition, the numerous advantages of use of enterococci probiotics for humans such as treatment of antibiotic-associated diarrhoea (AAD) and irritable bowel syndrome, lowering of serum cholesterol, regulation of the immune system and anti-carcinogenic features (Ahl et al., 2016; Franz, Huch, Abriouel, Holzapfel, & Gálvez, 2011) and also vital role of Enterococcus spp. in the development of the technological and sensory properties of fermented foods (Graham, Stack, & Rea, 2020; Morandi, Brasca, Andrighetto, Lombardi, & Lodi, 2006; Nieto-Arribas et al., 2011), distinguish enterococci from other probiotic lactic acid bacteria. In the present study, enterococci in Iranian motal cheese were isolated and identified using phenotypic and genotypic methods. Then, the isolates were evaluated for major probiotic properties and safety aspects. The aim of this work was to answer the question whether natural enterococci from Motal cheese, which have been proven to be safe, could be used as probiotics for fermented food applications.