M: Food Microbiology & Safety JFS M: Food Microbiology and Safety Antistaphylococcal Effect of Enterocin AS-48 in Bakery Ingredients of Vegetable Origin, Alone and in Combination with Selected Antimicrobials PILAR MARTINEZ VIEDMA,HIKMATE ABRIOUEL,NABIL BEN OMAR,ROSARIO LUCAS L´ OPEZ, AND ANTONIO G´ ALVEZ ABSTRACT: The inhibitory effect of enterocin AS-48 against Staphylococcus aureus was investigated in various types of bakery ingredients. Antibacterial activity greatly depended on the food substrate, ranging from complete inactivation of S. aureus in liquid caramel (in which the bacterium survived poorly) to no significant inhibition (as in vanilla or chocolate creams). Significant reductions of viable counts in the range of 1.8 to 2.7 log units (P < 0.05) were achieved in substrates like pumpkin confiture or diluted almond cream stored at temperatures of 10 or 22 ◦ C. Given the very low activity detected in chocolate substrates, enterocin AS-48 was tested in combination with other antimicrobials. Bactericidal activity increased markedly for the combinations of AS-48 and 0.1% eugenol (v/v), 0.5% 2-nitropropanol (v/v), or 3% Nisaplin (w/v). Enterocin AS-48 could be applied in combination with other antimicro- bials for preservation of bakery ingredients against S. aureus. Keywords: additives, antibacterial, food preservation, foodborne pathogens Introduction S taphylococcus aureus is a regular commensal of human skin and respiratory tract mucosal surfaces. However, it may also cause foodborne intoxications due to ingestion of preformed en- terotoxin in the food (Halpin-Dohnalek and Marth 1989; Acco and others 2003; Le Loir and others 2003). S. aureus is the 3rd most common cause of confirmed food poisoning in the world (Bean and others 1997; Mead and others 1999; Tirado and Schmidt 2001; WHO 2002; EFSA 2006). Thus, S. aureus is of great concern in the food industry due to its metabolic versatility and the range of environmental conditions (temperature, pH, a w ) in which it can grow and produce enterotoxins (SEs), which indeed are of high sta- bility (ICMSF 1996; Ash 1997; USFDA 2000). According to Tranter (1996), 1 μg of staphylococcal enterotoxin A (SEA) is enough to in- duce symptoms and this amount can be produced when the popu- lation reaches 10 5 CFU/g. Several reports described the incidence of S. aureus in bakery ingredients and outbreaks of food poisoning related to the consumption of cream puffs, chocolate eclairs, and other cream-filled bakery products (Coughlin and Johnson 1941; Carrera Vara and others 1998; Rahman and others 1999), and also ice cream (Kanbakan and others 2004). Bakery ingredients may contain high levels of S. aureus as a result of contamination during or after processing due to the poor hygiene of personnel and equip- ments, which may also act as vehicles for transmission of staphylo- cocci to other foods. Therefore, the need to include additional hur- dles for the control of S. aureus in the food chain could be solved by using natural antibacterial substances like bacteriocins alone or in combination with other antimicrobial compounds. The safety and shelf life of bakery ingredients can be improved by application of different methodologies or hurdles to avoid or delay microbial growth like pasteurization treatments, packaging in con- MS 20090253 Submitted 3/20/2009, Accepted 6/23/2009. Authors are with ´ Area de Microbiolog´ ıa, Depto. de Ciencias de la Salud, Facultad de Cien- cias Experimentales, Univ. de Ja´ en, 23071-Ja´ en, Spain. Direct inquiries to author G´ alvez (E-mail: agalvez@ujaen.es). trolled atmosphere (Kotsianis and others 2002; Smith and others 2004), activated films (Silveira and others 2007), chemical preser- vatives, irradiation, modified atmosphere packaging, and so on. However, most of these procedures may cause loss of organoleptic properties of foods or compromise consumer acceptability. In this way, consumer demands are focusing increasingly on minimally processed food products, with less use of synthetic additives and at the same time without compromising food safety. The search of “naturally derived” antimicrobials such as organic acids, essential oils (Holley and Patel 2005), phenolic compounds (Serra and others 2008), and bacteriocins could be a good alternative to ensure food safety and to lower the intensity of preservation treatments when they act synergistically (Cleveland and others 2001; G´ alvez and oth- ers 2007). Since bakery ingredients are lightly preserved food prod- ucts (usually with a short shelf life) with a risk of food poisoning, they may be adequate candidates for testing bacteriocins as hurdles against growth of pathogenic bacteria. Nevertheless, application of bacteriocins in the preservation of bakery ingredients has seldom been reported (Turtell and Delves-Broughton 1998). One of the promising bacteriocins in food preservation is enterocin AS-48 produced by Enterococcus faecalis (G´ alvez and others 1986). This is a heat-stable cyclic peptide well character- ized at biochemical and genetic levels, that shows a bactericidal mode of action against a variety of Gram-positive bacteria, and also against some Gram-negatives especially in combination with outer-membrane damaging agents (reviewed by Maqueda and oth- ers 2004). Experimental work carried out against different food- borne pathogens in several food systems (including meat, dairy, and vegetable foods) indicates that the efficacy of this bacteriocin varies with the target bacterium and the type of food being tested (Mu ˜ noz and others 2004; Ananou and others 2005a,b; Grande and others 2006, 2007). AS-48 showed a strong inhibitory effect against S. aureus in vegetables sauces (Grande and others 2007) and in a meat model system (Ananou and others 2005c). Antistaphylo- coccal activity of AS-48 was potentiated by chemical preservatives (Ananou and others 2004, 2007; Grande and others 2007). M384 JOURNAL OF FOOD SCIENCE—Vol. 74, Nr. 7, 2009 C  2009 Institute of Food Technologists R  doi: 10.1111/j.1750-3841.2009.01288.x Further reproduction without permission is prohibited