Evaluation of the Antimicrobial Activity of Citrus Essences on Saccharomyces cerevisiae NICOLETTA BELLETTI,MAURICE NDAGIJIMANA,CLAUDIO SISTO, MARIA E. GUERZONI,ROSALBA LANCIOTTI, AND FAUSTO GARDINI* Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita ` degli Studi di Bologna, Via Fanin 46, 40127 Bologna, Italy The aim of this research was to assess the antimicrobial activity of nine different industrial essences used in a soft drink factory in relation to their composition, as well as to verify the role of vapor pressure on their bioactivity. The essences were tested against a Saccharomyces cerevisiae strain isolated from spoiled soft drinks. The tests were carried out by adding the essences directly to a liquid medium or into the headspace of closed systems inoculated with the yeast. The headspace composition was evaluated through a solid hase microextraction-gas chromatography technique. The use of a mass spectrometer allowed the identification of the peaks detected. The microbial growth was indirectly monitored by measuring the metabolic CO 2 released by the yeast. The results obtained indicated that the most effective essences were characterized by the highest concentration of some terpenes, such as citral, -pinene, and p-cymene. Moreover, all of the essences were more bioactive when added directly to the liquid medium. KEYWORDS: Citrus essences; terpenes; antimicrobial activity; Saccharomyces cerevisiae INTRODUCTION In recent years, the interest in the possible use of natural alternatives to food additives to prevent bacterial and fungal growth has notably increased. Plants and plant products can represent a source of natural alternatives to improve the shelf life and the safety of food. Also, they are characterized by a wide range of volatile compounds, some of which are important flavor quality factors (1). Recently, the interest in the application of essential oils to control plant and postharvest pathogens has increased and their potential role in food preservation has been exploited (2, 3). Plant essential oils have been studied for their antimicrobial activity against many microorganisms including several patho- gens (4, 5). In particular, the activity of oils from Labiatae (6- 9) and citrus fruits (10, 11) has been investigated. In addition, the action of single constituents of these oils has been exploited in order to better understand the cell targets of these molecules, to identify the most active molecules, and to balance the intrinsic variability of essential oils (2, 12). Commercial citrus essential oils are generally obtained from different parts of the plants by cold pressing (peels) or distillation (leaves) (13). Although fresh fruits and juices are the main commercial products of citrus fruits, their essential oils can also be used as ingredients in the pharmaceutical industry as well as in perfumery. In the food industry, essential oils and the essences derived from their concentrations are widely used as flavor enhancers in soft drinks, alcoholic beverages, and fruit- based products. In some cases, the composition of the flavoring essences can play an active role in the microbiological stability of the products. In fact, a recent industrial spoilage case, which involved more than 500000 orangeade bottles, has been at- tributed to the type of orange flavor added to the soft drinks (14). Citrus essential oils can have a very pronounced anti- microbial activity, even if their complexity and variability make it difficult to correlate their action to a specific component, as well as the possibility of antagonistic and synergistic effects. In general, the antimicrobial effects of essential oils have been mainly explained through the presence of C10 and C15 terpenes with aromatic rings and a phenolic hydroxylic group able to form hydrogen bonds with active sites of target enzymes (5, 7, 15, 16). Nevertheless, other active terpenes, as well as alcohols, aldehydes, and esters, can contribute to the overall antimicrobial effects of the essential oils (17). For this reason, Caccioni et al. (11) proposed a holistic approach to explain the antimicrobial capabilities of citrus essential oils, whose performances could be the result of a certain quantitative balance of various components. The aim of this research was to assess the inhibitory activity of nine different industrial essences used in a soft drink factory. These essences are used to flavor different kinds of soft drinks and can be added to the beverages at concentrations higher than 1000 ppm. Their antimicrobial activity was tested against the yeast strain Saccharomyces cereVisiae SPA, which was respon- sible for the spoilage episode described earlier (14). It is well- known that several procedures have been adopted to evaluate * To whom correspondence should be addressed. Tel: +390512096575. Fax: +390512096573. E-mail: fgardini@kaiser.alma.unibo.it. 6932 J. Agric. Food Chem. 2004, 52, 6932-6938 10.1021/jf049444v CCC: $27.50 © 2004 American Chemical Society Published on Web 10/16/2004