applied sciences Article Effectiveness of Bacteriocin-Producing Lactic Acid Bacteria and Bifidobacterium Isolated from Honeycombs against Spoilage Microorganisms and Pathogens Isolated from Fruits and Vegetables Chrysa Voidarou 1, Athanasios Alexopoulos 2, Anastasios Tsinas 1, Georgios Rozos 2, Athina Tzora 1, Ioannis Skoufos 1, Theodoros Varzakas 3,* and Eugenia Bezirtzoglou 4 1 School of Agriculture, University of Ioannina, 47100 Arta, Greece; [email protected] (C.V.); [email protected] (A.T.); [email protected] (A.T.); [email protected] (I.S.) 2 Department of Agricultural Development, Laboratory of Microbiology, Biotechnology and Hygiene, Democritus University of Thrace, 67100 Xanthi, Orestiada, Greece; [email protected] (A.A.); [email protected] (G.R.) 3 Department of Food Science and Technology, University of the Peloponnese, Antikalamos, 24100 Kalamata, Greece 4 Medical School, Laboratory of Hygiene and Environmental Protection, Democritus University of Thrace, 67100 Alexandroupolis, Greece; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +30-2721045279 Received: 29 August 2020; Accepted: 15 October 2020; Published: 19 October 2020 Abstract: Screening natural products for bacteriocin-producing bacteria may be the equilibrium point between the consumer demand for mild processing and the industry’s need for hazard control. Raw unprocessed honeycombs filled with oregano honey from the alpine mountainous territory of Epirus, Greece were screened for bacteriocinogenic lactic acid bacteria and Bifidobacterium spp., with inhibitory action towards some pathogens and spoilage microorganisms isolated from fresh fruits and vegetables (number and type of strains: three E. coli, two L. monocytogenes, two Salmonella spp., two B.cereus, two Erwinia spp., one Xanthomonas spp., L. innocua (ATCC 33090TM) and E. coli 0157:H7 (ATCC 69373)). Among the 101 collected isolates (73 Lactobacillus, 8 Lactococcus, 8 Leuconostoc and 12 Bifidobacterium species) from the oregano honeycombs (an original finding since there are no other reports on the microbial biodiversity of the flora of the oregano honey), 49 strains of lactic acid bacteria (LAB) and Bifidobacterium spp. were selected and tested for their bacteriocin-producing capacity (34 Lactobacillus, 6 Lactococcus, 5 Leuconostoc and 4 Bifidobacterium). The antibacterial activity exerted by the tested LAB and Bifidobacterium strains was not of the same potency. Our results suggest that the main molecules involved in the antimicrobial activity are probably bacteriocin-like substances (a conclusion based on reduced antibacterial activity after the proteolytic treatment of the cell-free supernatant of the cultures) and this antimicrobial activity is specific for the producing strains as well as for the target strains. The spoilage bacteria as well as the reference microorganisms showed increased resistance to the bacteriocin-like substances in comparison to the wild-type pathogens. Keywords: LAB; Bifidobacterium; BLS; fruits; vegetables; Oregano honey 1. Introduction According to EUROSTAT (ec.europa.eu) surveys, half of the EU population eats at least one portion of fruits and vegetables on a daily basis and this trend is increasing (the southern states are in the top three EU Member States in daily intake of fruit: Italy (85%), Portugal (81%) and Spain (77%)) [1]. Unsurprisingly, the WHO/FAO Joint Expert Consultation Report on Diet, Nutrition and the Prevention Appl. Sci. 2020, 10, 7309; doi:10.3390/app10207309 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 7309 2 of 18 of Chronic Diseases states that at least 400 g of fruits and vegetables per day are needed to prevent heart disease, certain types of cancer, diabetes and obesity [2]. Given these statistics, important food quality and food safety issues emerge. Fruits and vegetables are rich in humidity and carbohydrate content and hence characterized as ideal habitats for bacterial and fungal growth [3]. Among others, factors such as the presence of various parasites and the quality and origin of manure, harvest and postharvest treatment and skin lesions are of utmost importance for their contamination with pathogens and spoilage microorganisms. Various studies have shown the presence of Salmonella spp., E. coli O157:H7, Listeria monocytogenes, Bacillus cereus, Campylobacter spp., Yersinia enterocolitica, Clostridium botulinum and other pathogens, in addition to some viruses and parasites in fruits and vegetables [4,5]. Spoilage bacteria such as Erwinia carotovora and Xanthomonas campestris cause various types of lesions regarding texture and color thus reducing their quality and commercial value [6]. There are modern trends in food processing concerning food safety and prolonging shelf life. Consumers prefer minimally processed foods that are free of chemical preservatives. Given these demands, and the increasing resistance of pathogens and spoilage bacteria to antibiotics and other chemicals, the food industry is seeking alternative means of food preservation [7,8].Consequently, there is an increasing interest in so-called “green technologies,” including novel approaches to the minimal processing of food as well as the use of microbial metabolites such as bacteriocins on an industrial scale for “biopreservation” [9]. In order to control, or even prevent, the growth of such microorganisms, the application of bacteriocins-producing lactic acid bacteria (LAB) has been proposed [10]. LAB can be isolated from many raw wild fruits, vegetables and flowers [11,12]. Moreover, lactic and acetic acid bacteria can promote the spoilage of fruits, vegetables, fruit juices and beverages as residents at the outer layer of the skin of fruits and vegetables [6]. These bacteria are known for the fermentation of carbohydrates and the production of various organic acids such as lactic acid, which significantly lower the pH of fermented foods [12]. They also produce other compounds with antimicrobial action such as hydrogen peroxide, acetaldehyde and bacteriocins [13–15]. Bacteriocins are peptides with natural antibacterial activity, and there is a reason for their comparison to antibiotics. Many researchers propose the term “biological preservatives of foods” and stress the fact that bacteriocins are not used for clinical therapeutic purposes as antibiotics are [16]. They are synthesized in the ribosomes of the bacterial cell and secreted extracellularly. Bacteriocins are most effective against Gram-positive bacteria. The spectrum of their activity varies from narrow (against one species) to broad (against several species) [17,18]. The main advantage of bacteriocins is that their presence does not change the sensorial characteristics of foods. Their usage enables the reduction of the intensity of other means of preservation such as heat. These two characteristics make bacteriocins’ application compatible with modern consumer demands for the minimal and more natural treatment of foods [19]. Bacteriocins can be preferably added to foods as compounds rather than cultures of bacteriocinogenic LAB because, in the latter case, LAB can ferment the carbohydrates of foods [20]. Nisin, pediocin, enterocin AS-48, bovicin, enterocin 416K1 and bificin C6165 are some bacteriocins already tested against spoilage bacteria and pathogens but only the first two were granted approval as food additives. However, they are mostly used in other foods and their usage in the fruit and vegetable industry is still limited [19]. LAB have been established as “generally regarded as safe” (GRAS), a fact that makes them attractive candidates for industrial utilization [21,22]. There is ongoing research for new strains with potentially superior properties, such as being probiotic, and the production of active bacteriocins. Sources of such LAB are various natural products, which could be an ideal ecological niche for these microorganisms. Oregano honey is a very special and extremely rare natural product. It is produced by honeybees grazing on wild oregano plants in the alpine mountainous territory of Epirus, Greece. It is difficult to find it since there are very few producers. Unlike other types of honey, oregano honey has a bitter taste. It is consumed by rural populations of the area and data from local Appl. Sci. 2020, 10, x 3 of 18 by honeybees grazing on wild oregano plants in the alpine mountainous territory of Epirus, Greece. ItAppl. is difficult Sci. 2020, 10to, find 7309 it since there are very few producers. Unlike other types of honey, oregano honey3 of 18 has a bitter taste. It is consumed by rural populations of the area and data from local people suggest theypeople believe suggest oregano they believe honey oregano possesses honey therapeuti possesses therapeuticc properties properties against againstvarious various infections infections and gastrointestinaland gastrointestinal disorders. disorders. TheThe aim of this study is to screen the diversediverse autochthonous microbiota isolated from from the honeycombshoneycombs ofof oregano-grazing oregano-grazing bees bees for LABfor andLABBifidobacterium and Bifidobacteriumstrains and strains determine and thedetermine antibacterial the antibacterialactivity of these activity bacteriocin-producing of these bacteriocin-produc isolates againsting isolates some pathogens against some and spoilagepathogens microorganisms and spoilage microorganismsisolated from fresh isolated fruits from and vegetables. fresh fruits and vegetables. 2.2. Materials Materials and and Methods Methods 2.1.2.1. Sample Sample Collection