**1. Introduction**

Olive oil production is a seasonal activity, and as such faces particular problems, which are more or less pronounced depending on the year-by-year situation, such as the global and micro climate conditions (rainfall and temperature), early or late ripening, decreased or surplus production (olive and oil yield) and even market positioning. Such factors affect harvesting decisions and may lead to inadequate/inefficient/insufficient oil production capacity in olive mills. Virgin olive oil (VOO) is obtained by several mechanical and physical processes, which begin with harvesting and post-harvest storage of olive fruits. The quality of VOO is closely related to the quality of olive fruits from which it is obtained [1]. In order to achieve high quality oil, it is recommended to process olives within 24 h after harvesting [2]. If processing is not done within that period, as for example when the capacity of available mills is insufficient, the fruits have to be stored for a certain period of time.

During a prolonged storage period, degenerative hydrolytic and oxidative processes in olive fruits start to develop. Under relatively high storage temperatures the proliferation of various microorganisms in olive fruit is accelerated, which often leads to many detrimental changes in olive physicochemical composition and the development of sensorial defects of fermentative origin, in the obtained olive oil [3,4].

Besides volatile compounds, principally associated with a decrease in olive oil sensory quality caused by prolonged olive fruit storage [4], other quality markers are affected as well. In olives

with ruptured drupe (unhealthy, overripe, fractured during picking or storage), the fatty acids are liberated by the action of enzymes. In the presence of ethanol, also a product of fermentation, fatty acid ethyl esters (FAEE) are formed by esterification of free fatty acids with ethanol [5,6]. Recent studies suggest that the FAEE concentration is considered an indicator of olive oil quality [7,8]. Higher storage temperatures, such as the ambient temperature of 25 ◦C, accelerate the processes of olive ripening [9]. As olive fruit ripens, its exocarp becomes thinner, and fruit tissues become softer [10]. Along with these changes, particular compounds are being extracted into oil, such as waxes from the waxy surface layer of the cuticle of olive fruit. The content of waxes in the oil can also be considered a quality indicator [6] although it is officially used as a marker for distinguishing olive from olive-pomace oil [8,11]. Phenolic compounds, in addition to their involvement in VOO taste characteristics, have antioxidant properties and, therefore, significantly contribute to the oxidative stability and health benefits associated with VOO [12]. Their presence in oil depends on the interaction between genetic (cultivar), environmental (cultivation, harvest and postharvest conditions) and physiological factors (fruit ripening degree and sanitary conditions), and processing conditions [13,14]. Previous studies revealed that phenolic compounds are strongly affected by olive fruit storage conditions, especially secoiridoids, whose concentrations decrease with longer storage times and higher storage temperatures [15,16]. Several authors have found that, due to genetically predetermined enzymatic activity, the polyphenol content of obtained VOO behaved differently among cultivars even if the fruits were stored under the same conditions prior to oil production [16,17]. Considering all the reasons mentioned above, optimal duration and temperature of fruit storage are obviously very important factors for obtaining high quality VOO in general [9] and are also crucial for preserving particular sensory profiles typical for VOO of certain cultivars, consisting of high levels of phenolic compounds and related high intensities of bitterness and pungency, as in the case of Croatian autochthonous Istarska bjelica and Rosinjola [18,19].

Currently, the main strategies for avoiding deterioration of olive fruit during storage and production of lower quality VOO include reducing the period between harvesting and processing by increasing the production capacity of olive mills, as well as applying conditions aimed to ensure lesser contact between fruits (larger storage spaces, perforated boxes, etc.) [20]. Storage of olive fruits at lower temperatures was also recognized as an alternative that could allow more flexibility during harvesting and oil production [17]. In general, low storage temperatures decrease water activity and inhibit microbial growth [21], slow down enzymatic and biochemical reactions [22], decrease fruit respiration and delay harvested fruit maturation [10,23]. On the other hand, low temperatures may cause physiological damage of the fruit [24] since ice crystals formed during frozen storage may break down the cell structure and at the same time allow the contact between various enzymes and substrates that could affect VOO quality [25,26]. Very low temperatures during olive fruits development and harvest, with related cycles of freezing and defrosting of fruits on the tree, are known to cause biochemical changes, which significantly modify its volatile compound and phenolic profiles [22] and could induce the development of the "frostbitten olives" sensory defect in olive oil [27].

Several studies have been conducted in order to investigate appropriate olive fruit storage conditions, including mostly ambient and refrigeration temperatures [9,16,21,23,28]. A few studies that examined the effects of frozen storage included either a very short storage period of one day [29], which might not be sufficient in practical conditions when delays in olive processing are larger, or a very long storage period of 6 months [26], which is not applicable from a practical point of view. These studies focused mostly on the basic quality parameters and phenolic and volatile profiles of the obtained oils and, to our knowledge, none of them investigated the influence of low storage temperatures on the contents of FAEE and waxes.

The aim of this study was to investigate the influence of low temperature during prolonged post-harvest storage of olive fruit on the production parameters (oil content in the fruits, oil yield and extractability index), composition (the content of pigments, FAEE, waxes and phenolic compounds) and sensory quality of olive oil obtained therefrom. The experiment was performed with olive fruits of two autochthonous Croatian cultivars, Istarska bjelica and Rosinjola, non-stored (control) and stored

for seven days at three different temperatures, including room temperature, refrigerating at +4 ◦C and freezing at −20 ◦C. The main intention was to evaluate the possibility of prolonging the storage period of olive fruits to a reasonable time still feasible in practice (one week), without compromising the chemical and sensory quality of VOO, which would possibly contribute to overcoming the problems with olive mill overloading during the harvesting period. In addition, particular attention was devoted to the influence of olive fruit storage temperature on both positive and negative sensory characteristics of the obtained VOO, in order to deepen the understanding of the origin of some VOO defects, such as "frostbitten olives", otherwise associated with the impact of low ambient temperatures during olive fruit maturation.

#### **2. Materials and Methods**
