# The Effect of Processing Methods on Phytochemical Composition in Bergamot Juice
## Materials and Methods  
### Study Design  
The effect of different processing methods on fresh bergamot juice (BJ) was evaluated by determination of proximate constituents (titratable acidity, total soluble solids, pectins, and free sugars) and by the occurrence of specific phytochemicals (flavanone glycosides, limonoid aglycones, and stachydrine) playing several biological functions in humans. In vitro assay, free radical scavenging activity (RSA) was also determined (Figure 1).  
Fresh bergamot fruits were harvested in January and February 2019 from the experimental field of SSEA agency (Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi, Reggio Calabria, Italy) according to fruits ripening. Once harvested, a random sampling (~10 kg of bergamots) was carried out in order to obtain the batch of fruits juice for laboratory (Extraction process 1: Ep-1). The other harvested fruits (~300 kg) were sent to a local industry that kindly processed the fruits to produce industrial juices using two different extraction systems (FMC in-line extractor (Ep-2) and screw press extractor (Ep-3)).  
Laboratory juice (\(n=3\)) was prepared by hand squeezing using a manual squeezer (Extraction process 1: Ep-1), filtered through a stainless-steel filter with 1.18 mm (mesh diameter) and placed in 100 mL aliquots in plastic bags, and stored at \(-20\)\({}^{\circ}\)C until used.  
Three samples of laboratory juice were debittered by Polystyrene-DVB Resins (Amberlite XAD-16) batch process according to [PERSON] et al [21]. The treatment was carried out in a 250 mL beaker containing 25 mL BJ and 5 g of exchange resin, with constant stirring for 20 min at 4 \({}^{\circ}\)C. Resin was removed by centrifugation for 10 min at 2000 rpm and at 4 \({}^{\circ}\)C.  
Industrial juices were obtained using two different extraction systems (Ep-2 and Ep-3):  
1. The Ep-2 system consists of separating the essential oil from the juice in a single operation by using an FMC in-line extractor (FMC Corporation, Fairway Avenue Lakeland, FL-USA.). The FMC squeezer is designed to separate the fruit into three different parts: peel and seeds; peel oil and washing water; and juice. In this extractor, a whole fruit is positioned in one of opposed inter-fitting cup halves which were provided with means for cutting the peel or rind at diametrically opposed areas. Concurrent movement of one or both cups toward each other compresses the fruit and produces the diametrically opposed cuts. Cutting and compressing ruptures the juice sacks and the juice flows through one of the cut areas of the peel, through a screen, and to a collecting trough.
2. In the Ep-3 system, oil extraction precedes juice extraction. In this case the essential oil is extracted from the whole fruit by rasping, using a machine called \"pelatrice.\" The rasped fruits are then moved into a juice extractor (POLYCITRUS ZX2, Fratelli Indelicato S.r.l., Catania, Italy). This machine consists of a screw press extractor extracting juice by crushing and pressing whole fruit with a rolling screw. The screw press type operates by pressing the screw horizontally while conveying the pomelo flesh along the perforated cylindrical container. Juices flow out via the perforated cylinder and the wastes are ejected at the end plate.  
Detailed descriptions of these processing were described by [PERSON] et al. [17]. In particular, the industrial juices were sampled during on-line processes, where 1 L of sample was firstly collected at regular intervals and then filtered through a stainless-steel filter (1.18 mm mesh diameter). The fruit juices were placed in 100 mL aliquots in plastic bags and stored at \(-20\)\({}^{\circ}\)C until used.  
Figure 1: Schematic diagram showing the experimental design applied in this study.