# AIP Bioluminescence for Rapid and Selective Detection of Bacteria and Yeasts in Wine
## 1 Introduction  
Microbial contamination may represent a consistent loss of money for wine producers, due to both yeasts and bacteria contaminating wines after primary fermentation. These would in fact lead to several defects, such as off-flavors or bottle explosions, causing the discard of products. Usually, wineries use culture-dependent methods to assess the microbial loads during the wine-making process. Although accurate, these are time-consuming, and the results are often obtained too late to be useful. Furthermore, certain strains of spoilage microorganisms exist in a viable but non-cultivable state giving the possibility of microbial contamination to arise quickly and without any warning [1]. Recognizing contaminations in time is fundamental to avoid economical losses but can be very tricky since a specific problem in wine can be provoked by different microorganisms such as lactic acid bacteria (LAB) or yeasts [2]. For these reasons it is important to develop a system that is faster than plating but also able to discriminate between different sources of microbial contamination. Several molecular-based testing methods have been developed to detect spoilage microorganisms in wine, such as bio- and nanosensors, fluorescence cells sorting, and real-time polymerase chain reaction (RT-PCR). These methods have their advantages, but they can be complex and require trained personnel. Commercial kits for microbial evaluation are also available for wineries, although they cannot simultaneously detect multiple spoilage microorganisms at one time [1]. In recent years an alternative was proposed to analyze food in a shorter time and with a higher level of accuracy with respect to culture-based methods: bioluminescence [3]. Since the beginning of life on Earth, cells have faced the problem of storing the energy produced by metabolism to sustain life processes, growth, and colonization of the environment. Energy in almost all theorganisms is stored in the bonds of adenosine triphosphate (ATP). The structure of ATP is a nucleoside triphosphate, consisting of a nitrogenous base (adenine), a ribose sugar, and three serially bonded phosphate groups. To release the energy stored in the bonds between each phosphorus, cells dephosphorylate the ATP, forming adenosine diphosphate (ADP) and adenosine monophosphate (AMP). ATP can be regenerated by reducing the chemical components of nutrients to synthesize new molecules of ATP. Cellular ATP can be measured thanks to bioluminescence systems based on the discovery of [PERSON] and [PERSON] [4]; they showed the involvement of fireflies' (_Photinus pyralis_) bioluminescence reaction in the production of a light signal that can be detected, measured, and expressed as relative light units (RLUs) and correlated with the number of cells in the analyzed media [5]. The reaction involves ATP, luciferin, luciferase, magnesium, and oxygen. Chemically, ATP transfers its energy to the luciferin and forms luciferyl adenylate. The luciferyl adenylate is then oxidized by the luciferase in the presence of magnesium ions to form oxyluciferin, carbon dioxide, adenosine monophosphate, and pyrophosphates, emitting light as a reaction result [6]. Luciferin reacts with ATP from microbial cells thanks also to specific reagents, added during the analysis, permitting in this way the formation of light from ATP produced by freshly lysed cells [7]. The produced luminous signal can be amplified and read by a bioluminometer. As ATP is rapidly degraded when the cells die, the measurement of bioluminescence is considered a reliable tool to detect microbial contamination. The improvement in the reagent quality and instrument sensitivity has led to the availability of several applications in many branches of the food industry, and many kits are now commercially available [7]. Carbonated beverages, beer, fruit juices, and wines have been analyzed by ATP assay [6; 7; 8]. However, the bioluminescence assay is a non-specific method, so it is not able to discriminate between different microbial sources of contamination.