P U B L I C A T I O N S CODON Italian Journal of Food Science, 2023; 35 (2): 1–12 ISSN 1120-1770 online, DOI 10.15586/ijfs.v35i2.2304 1 P U B L I C A T I O N S CODON Effects of acidified apple juice before fermentation on ethyl carbamate and volatile components of apple distillate Zhicong Su, Yingying Han, Jinhua Du* College of Food Science and Engineering, Shandong Agricultural University, Tai’an, Shandong, China *Corresponding Author: Jinhua Du, College of Food Science and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, China. Email: djh@sdau.edu.cn Received: 18 November 2022; Accepted: 13 March 2023; Published: 11 April 2023 © 2023 Codon Publications OPEN ACCESS OPINION PAPER Abstract In order to eliminate ethyl carbamate (EC) content in apple distillate, Fuji apple juice was acidified to pH 3.0 by sulfuric acid (ST), malic acid (MT), lactic acid (LT), or citric acid (CT). The acidified juice was inoculated with yeast, fermented at room temperature, and distilled by double distillation. Acid treatment by ST (3.23 μg/L), MT (3.20 μg/L), LT (2.93 μg/L), and CT (3.57 μg/L) significantly eliminated EC from apple distillate. Combined with the EC content and sensory evaluation, it was suggested that the high-quality apple distillate could be obtained with lower EC if apple juice was treated with ST or MT before fermentation. Keywords: apple distillate, ethyl carbamate, sulfuric acid, malic acid, lactic acid, citric acid Introduction Apple is one of the main fruits in China. Its annual output is on the top in the world. However, some low- quality apples are not able to meet the market demand of fresh sales, resulting in a serious waste of resources. Consequently, the preparation of apple distillate not only reduces apple waste but also increases new apple prod- ucts and greatly improves the economic and social ben- efits. However, some harmful components, such as ethyl carbamate (EC) and cyanide, are formed during alcoholic fermentation and distillation. EC, a potentially geno- toxic and carcinogenic substance (Tu et al., 2018), has been recognized as a group 2A carcinogen by the World Health Organization’s (WHO) International Agency for Research on Cancer. In France, the maximum allowable level of EC is set as 150 μg/L for distilled spirits. The upper limit for EC in Canada is 400 μg/L in fruit spirits, 30 μg/L in wines, and 150 μg/L in wine spirits, brandies, and whiskies (Jia et al., 2022). Some studies have shown high levels of EC in distillates, for example, plum brandy contains up to 4750 μg/L of EC in tail during distillation (Balcerek et al., 2017), and the EC content in some sugar- cane spirits is between 42 μg/L and 5589 μg/L (Alcarde et al., 2011). It is essential to reduce the content of EC in apple distillate; however, no research has been conducted on the content of EC in apple distillate. Ethyl carbamate is found in many fermented food products and alcoholic beverages, such as wine, sake (rice beer), whisky, brandy, etc. (European Food Safety Authority, 2007). EC has the effects of oral toxicity, immunosuppression, and heart rate inhibition (Jiao et  al., 2014). Thus, the presence of EC in apple distillate is objectionable, and the EC content is expected to be as low as possible. In the process of making wine, five pathways of EC derived from urea, citrulline, cyanide, and 3a,6a- dimethylglycoluril react with ethanol (Wang et al., 2014b; mailto:djh%40sdau.edu.cn?subject= 2 Italian Journal of Food Science, 2023; 35 (2) Su Z et al. (Beijing, China). All the reagents used were of analytical grade. Apple distillate making Apple juice preparation Well-matured apple fruits were selected, washed, crushed, and squeezed to obtain apple juice. The result- ing juice was collected, divided into five samples (con- trol, ST, MT, LT, and CT). Each juice sample (35 L) was transferred into sterile fermenters (40 L) and treated as follows. Nothing was added to control. Other four juices were prepared by adding 10% ST, MT, LT, and CT to adjust their pH to 3.0. All the above-mentioned processes were carried out in triplicate (Figure 1). Fermentation Wine yeast CY 3079, 0.20 g/kg of juice, activated by apple juice was added into each of the above-prepared apple juices. Subsequently, the inoculated juice samples were  fermented at room temperature. The fermented apple juice was obtained after the completion of fer- mentation, that is, the residual sugar in the juice did not decrease over 3 consecutive days. The fermented juice was sampled daily for further analysis. Distillation The fermented apple juice was distilled by a double- distillation method. The first distillation was carried out in a 35-L Dibosk distiller comprising a stainless steel pot and condensing unit. The pot was heated on an induc- tion stove. The first distillate was obtained with 30% (v/v) alcohol. The second distillation was carried out in a 5-L glass conical flask heated on an electric furnace (Satora and Tuszynski, 2010). During the second distillation, the Zimmerli and Schlatter, 1991). Concerning distillation, urea is decomposed at a high temperature to cyanic acid, which reacts with ethanol to form EC (Schaber et al., 2004; Taki et al., 1992). Many studies have shown that EC production can be affected by controlling pH in wine (Uthurry et al., 2006). Lower pH affects citrulline metab- olism (Arena and Nadra, 2005) and thus reduces EC pro- duction during fermentation. Meanwhile, amygdalin in apple is hydrolyzed to hydrocyanic acid by β-glucosidase, which is then oxidized to isocyanic acid, and isocyanic acid reacts with ethanol to form EC, and low pH inhibits β-glucosidase activity. As far as we know, no study has focused on reducing EC content in fruit distillates by adjusting juice’s pH to 3.0 prior to fermentation. Sulfuric acid (ST), an inorganic acid is widely used in the preparation of ethanol (Sun et al., 2011). It is a listed food additive, as a flocculant agent, which can be used in fermentation processes in China (GB/T 2760) (National Health and Family Planning Committee of China, 2014). Malic acid (MT), lactic acid (LT), and citric acid (CT), as acidity regulators (GB/T 2760) (National Health and Family Planning Committee of China, 2014), are widely used in the food industry (Marques et al., 2020; Won et al., 2015). Hence, it is meaningful to apply ST, MT, LT, and CT to adjust the pH of apple juice before fermentation. The objective of this study was to investigate the effect of ST, MT, LT, and CT on cyanide and EC in apple dis- tillate. Apples were washed and juiced. Then ST, MT, LT, or CT was added to apple juice to adjust its pH to 3.0. The treated juice was fermented at room temperature to get fermented juice. The juice was distillated by a double- distillation method to obtain apple distillate. EC, cyanide, and volatile components present in the distillate were investigated. Materials and methods Materials Fuji apple (Malus domestica Borkh. cv. “Red Fuji”) fruits were purchased from a local fruit market (Tai’an, China). Commercial wine yeast Lalvin CY 3079 was purchased from Shanghai Jatou Industry and Commerce Co. Ltd. (Shanghai, China). ST was purchased from Laiyang Kant Chemical Co. Ltd. (Laiyang, China). DL-malic acid (Anhui Xuelang Biotechnology Co. Ltd, China) was ordered from a local food additives store. CT was pur- chased from the local food additive store, and LT was purchased from Henan Jindan Lactic Acid Technology Co. Ltd. (Henan China). Volatile standards (chromato- graphic grade) were obtained from China National Research Institute of Food and Fermentation Industries Apple Apples juice Control Treated juice Yeast CY3079 Fermented juice Distillation Apple distillates Fermentation Malic acid Lactic acid Citric acid Sulfuric acid Figure 1. Process of making apple distillates. Italian Journal of Food Science, 2023; 35 (2) 3 Acid treatment removes ethyl carbamate and affects volatile components from apple distillates occurring in the collision cell of triple quadrupoles, with an argon collision gas pressure of approximately 2.0 m Torr and an offset voltage of 20 eV. For quantitative analysis, the chosen fragments were monitored in mul- tiple reaction monitoring (MRM) modes: 74, 44, 62, and 89 m/z for EC, and 64 and 76 m/z for EC-D5. Then, selec- tive ion monitoring (SIM) of 62 m/z (EC) and 64 m/z (EC-D5) was used for the purpose of quantification. For quantification, peak area ratios of EC to EC-D5 were cal- culated as a function of the concentration of substances. Determination of cyanide The determination of cyanide is according to the Chinese national standard (National Health and Family Planning Committee of China. 2016; GB/T 5009.36). In a 50-mL beaker, 1 mL sample was taken; 5-mL of 2 g/L sodium hydroxide (NaOH) solution was added to the sample taken in beaker and and allowed to remain for 10 min. Then the beaker was heated on electric heating plate at 120°C till the solution was reduced to about 1 mL. It was transferred into 10-mL stopper colorimetric tube and the volume was adjusted to 5 mL by adding 2 g/L NaOH solution. Two drops of phenolphthalein indicator were added to the sample and the standard tube separately. Acetic acid was added to make the red color of the solution to fade; 2 g/L NaOH solution was used to adjust the color to near red. Phosphate buffer solution, 2 mL, and chloramine T solution, 0.2 mL, were added in turn with continuous shaking for 3 min. Then, 2 mL of isonicotinic-pyrazolone solution was added to the sample and diluted to 10 mL with water. Incubation was performed for 40 min in a water bath at 37°C constant temperature. Following incu- bation, the sample was taken out and the zero point was adjusted with a 1-cm colorimetric cup with a blank tube to measure absorbance at 638 nm. After the absorbance of 0-, 0.4-, 0.8-, 1.2-, 1.6-, and 2.0-mL cyanide standard intermediate solution into 10-mL stopper colorimetric tube, colorimetry was conducted according to sample determination to draw a standard curve. The cyanide content of the sample was measured by comparing to the standard curve of cyanide ion standard intermediate. Determination of methanol Methanol content was determined by gas chromatogra- phy with internal standard added according to the official reference method of Association of Official Analytical Chemists (AOAC, 1994; 940.06). The 100-mL sample was added to 50-mL deionized water and distilled to 100  mL. Internal standard (tert-amyl alcohol of 162 mg/L), 1 mL, was added to 10 mL of distillate; 1.0 μL of following three fractions were collected: the head (1% ethanol), the heart (83% ethanol), and the tail (16% eth- anol). A final alcoholic concentration of 68%–72% (v/v) was reached in the second distillate (SD). In order to avoid loss of volatiles, all samples were sealed and kept at 4°C until analysis. Before analysis, the heart was stan- dardized and the alcohol was diluted to about 40% (v/v). All analyses were performed on 40% (v/v) samples. Physicochemical analysis Physicochemical analysis was carried out according to the National Standards of the People’s Republic of China, GB/T 15038-2006. Alcohol content (% by vol- ume) and dry extract (g/L) were measured based on the Pycnometer method. Titrable acidity (g/L, tartaric acid equivalent) was determined by potentiometric titra- tion. pH values were directly measured using a labora- tory recording pH meter (FE20, Mettler Toledo, Zurich, Switzerland). Sugar content was titrated with Fehling’s reagent. Determination of ethyl carbamate Ethyl carbamate was analyzed by gas chromatography– mass spectrometry (GC-MS) method according to our previous study (Han et al., 2021) with some modifica- tions. Apple distillate samples, 2 mL, were mixed with 100 μL of EC-D5 (2 μg/mL in methanol solution), fol- lowed by addition of 0.30-g sodium chloride (NaCl). After ultrasonic dissolution for 10 min, the mixture was directly applied to solid phase extraction (SPE) car- tridge (SBEQ-CA3999, CNW technology, Germany), and allowed to remain for 10 min for adequate absorp- tion. The column was then washed with 10 mL n-hex- ane. Next, the analytes were extracted using 10-mL 5% ethyl acetate and diethyl ether solution. The eluate was mixed in a test tube and reduced to approximately 0.5 mL by a gentle stream of nitrogen. Subsequently, the resid- ual eluents were adjusted to 1 mL with methanol and directly injected into a GC-MS system (GCMS-TQ8030, Shimadzu, Tokyo, Japan). Substances were separated on a fused-silica capillary col- umn (VF-WAS, 60 m × 0.25 mm × 0.5 μm; Agilent, USA). Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The injector port was kept at 220°C in split- less mode. The starting temperature was held at 50°C for 1 min, then increased to 180°C at a rate of 8°C/min and held for 5 min. Finally, the temperature was increased to 240°C at a rate of 20°C/min and held for 5 min. The MS detector port and ion source temperature were set at 250°C and 230°C, respectively. GC-MS experiments were based on in-source collision-induced dissociation (CID) 4 Italian Journal of Food Science, 2023; 35 (2) Su Z et al. was performed according to the Chinese national stan- dard (General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. 2008; GB/T 11856) with some modifications. The evaluators were provided with 45-mL apple distillate in standard glass cups, coded with random numbers. Apple distillates were sniffed and tasted. Three aspects, includ- ing olfactory, gustatory, and typicality, were used to measure the quality of apple distillates. The descriptors of olfactory were fruity and vinous; for gustatory evalua- tions, considered descriptors were alcoholic and balance. During each session, expert judges first assessed the smell, and then they evaluated gustatory attributes after a short break. According to the characteristics of the sam- ples, the experts scored each descriptor with the highest score of 20 points. After descriptive analysis, the panel- ists assessed all samples for typicality according to their olfactory and gustatory tests and rated them with a max- imum score of 20 points. Finally, the experts wrote their comments on the samples after all the scores were com- pleted. The total score of the samples were the sum of all the scores. All samples were diluted with distilled water to an alcohol concentration of 40% (v/v). Mouthwash was used by tasters between analyses of two samples. Statistical analysis All the data were processed using SPSS Statistics 22. All pictures were drawn using Origin 2022. In Figure 3, red indicates positive correlation and blue shows negative correlation; the deeper the color, the greater the absolute value of correlation and stronger the correlation between them. Mean differences at p < 0.05 were considered as significant using Tukey’s test. All data were the average values of three replicates, analyzed in triplicate for each condition, and presented as mean values and standard deviations. Results and discussion Changes of titrable acidity and pH during fermentation All apple juice samples were fermented for 7 days, and the total sugar content was reduced to less than 1.4 g/L. In order to explore the influence of different acid treat- ments on fermented apple juice, titrable acidity and pH were tracked during fermentation. The pH of control was increased to 4.25 on the second day of fermentation and then decreased to 4.06 (Figure 2A). On the contrary, the titrable acidity content decreased on the second day of fermentation and then increased (Figure 2B). However, the pH of apple juice samples treated with different organic acids increased, but the corresponding titratable acidity content decreased during fermentation, which sample was directly injected into gas chromatography system equipped with a capillary column PEG-20 M (30 m × 0.5  mm × 0.25 μm; Dalian Zhonghuida Scientific Instrument, China) and a flame ionization detector. The temperature of injector and detector was 220oC. The oven temperature procedure was as follows: the initial temperature was maintained at 40oC for 4 min; it was then increased to 200oC at a rate of 3.5oC/min and held at 200oC for 10 min. The carrier gas was nitrogen with a flow rate of 1.0 mL/min. The split ratio was 50:1. Methanol quantification was determined by external standard method, but internal standard method was also used to improve the accuracy of results. Volatile compounds analysis The volatile compounds were analyzed by gas chroma- tography (López-Vázquez et al., 2010) with some modi- fications. Internal standard (10 μL), including tert-amyl alcohol and n-butyl acetate 162 mg/L, as well as 2-ethyl butyrate 186.6 mg/L, was added into 1 mL of sam- ple. Then, 1 μL of sample was directly injected into a Shimadzu 2010 chromatograph system with flame ioniza- tion detector. A capillary column CP-WAX 57CB (50 m × 0.25 mm × 0.2 μm; Agilent, USA) was used for this analy- sis. The temperatures of detector and injector were 260oC and 240oC, respectively. The oven temperature program was maintained at 35oC for 4 min, increased to 60oC at a rate of 2oC/min, continued to rise to 130oC at a rate of 10oC/min, and finally increased to and maintained at 205oC at a rate of 15oC/min. The carrier gas was nitro- gen with a flow rate of 1.35 mL/min, and the split ratio was 40:1. The qualitative analyses of volatile compounds were based on the comparison of retention time read from the chromatograms of both samples and standards. The quantitative analysis was performed according to the internal standard method. All detected carbonyl com- pounds, esters, higher alcohols, and acids were analyzed quantitatively. Sensory analysis The panel included three female and eight male analysts, with experience in the evaluation of fruit distillates and were trained to describe and recognize the evaluated odor qualities. The panelists were trained according to the ISO 8586 standard (International Organization for Standardization 2012). Prior to and during this study, monthly training was conducted to evaluate multiple flavor standards (acetoin, isoamyl acetate, ethyl acetate, ethyl lactate, acetaldehyde, 1-propanol, 1-hexanol, ace- tic acid, and head and tail distillation fractions) and dif- ferent spirits distilled from cider, hawthorn wine, and persimmon wine. The sensory analysis of the samples Italian Journal of Food Science, 2023; 35 (2) 5 Acid treatment removes ethyl carbamate and affects volatile components from apple distillates Control 4.4 (A) (B) 4.2 4.0 3.8 3.2 3.0 0 1 2 3 Fermentation time (d) pH 4 5 6 7 ST MT LT CT Control 10 8 6 4 2 0 0 1 2 3 Fermentation time (d) Ti tr ab le a ci di ty ( g/ L) 4 5 6 7 ST MT LT CT Figure 2. Changes in pH (A) and titrable acidity (B) during fermentation. Control: original apple juice; ST: sulfuric acid-treated juice; MT: malic acid-treated juice; LT: lactic acid-treated juice; CT: citric acid-treated juice. might be caused by the degradation of organic acids by yeast and LT bacteria (Lerena et al., 2016; Zhong et al., 2020). In all organic acid-treated samples, maximum change in titrable acidity was observed in MT sample, which decreased from 9.53 g/L to 7.92 g/L during the fermentation. The titrable acidity of LT- and CT-treated samples was reduced by 0.74 g/L and 1.16 g/L, respec- tively. Conversely, the pH of MT increased from 3.00 to 3.21. In summary, the titrable acidity of MT-, LT-, and CT-treated samples showed an overall decreasing trend during the fermentation process, while the titrable acid- ity of the control and ST-treated sample showed an insig- nificant decreasing trend. Physicochemical indices of fermented apple juices The basic physical and chemical indexes of fermented apple juices are shown in Table 1. All the fermented apple juice samples achieved complete fermentation, with the total sugar content ranging from 0.9 to 1.3 g/L and the alcohol content from 7.66 to 7.73% (v/v). This proposed that different treatments of apple juice samples before fermentation did not affect the fermentation capacity of yeast. The methanol content of all fermented apple juice samples was between 5.28 mg/L and 6.42 mg/L. The methanol content of ST, MT, LT, and CT was sig- nificantly lower than the control (p < 0.05), indicating that the methanol content could be significantly reduced by different acid treatments. In addition, EC was not detected in all the fermented apple juices. Volatile component of fermented apple juice samples In the fermented apple juice samples, 20 volatile com- pounds, such as carbonyl compounds, esters, higher alcohols, and acids, were detected by gas chromatogra- phy (Table 2). Three carbonyl compounds were identified in all samples. These could be produced by oxidation of alcohols or decarboxylation of acids (Xiao et al., 2015). Acetaldehyde contributed to the flavors of fermented apple juice samples with fruity, nut, and dried fruits aroma. However, acetaldehyde could impart pungent aroma if higher quantity is added. It was determined in all fermented apple juices; however, the maximum amount was determined in CT (123.26 mg/L). The lowest acet- aldehyde content was observed in ST, which was 26.69 mg/L. Acetoin was the only ketone detected and it pro- vided a buttery and cream aroma to the fermented apple juice samples (Welke et al., 2014). In this study, the con- tent of acetoin in ST, MT, LT, and CT were significantly reduced, compared to the control (p < 0.05). Acetoin reached the lowest level of 1.61 mg/L in ST. Although the amount of acetoin in fermented apple juices varies significantly, it is difficult for them to have a significant impact on the flavor of fermented apple juice samples because of its high sensory threshold. Esters are one of the most important volatile constituents of fermented apple juices, and were formed by the reac- tion of alcohol and free organic acids during fermentation (Villière et al., 2015). In this study, the contents of ethyl acetate and isoamyl acetate were above the threshold, 6 Italian Journal of Food Science, 2023; 35 (2) Su Z et al. Table 1. Physicochemical indices of fermented apple juice samples. Control ST MT LT CT Alcohol (%, v/v) 7.71 ± 0.04a 7.66 ± 0.06a 7.70 ± 0.03a 7.73 ± 0.04a 7.66 ± 0.01a Total sugar (g/L) 1.3 ± 0.1a 0.9 ± 0.0d 0.9 ± 0.0d 1.2 ± 0.0b 1.1 ± 0.0c Titrable acidity (g/L) 2.2 ± 0.0e 4.3 ± 0.0d 7.9 ± 0.0c 8.3 ± 0.0b 9.5 ± 0.0a pH 4.03 ± 0.01a 3.08 ± 0.00e 3.24 ± 0.00b 3.17 ± 0.00c 3.15 ± 0.00d Dry extract (g/L) 16.7 ± 0.0d 19.9 ± 0.2c 25.4 ± 0.0a 24.9 ± 0.0b 24.7 ± 0.0b Methanol (mg/L) 6.42 ± 0.08a 5.41 ± 0.00b 5.28 ± 0.14b 5.48 ± 0.69b 5.37 ± 0.18b Ethyl carbamate (μg/L) – – – – – All values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). Control: fermented apple juice from original apple juice; ST: fermented apple juice from sulfuric acid-treated juice; MT: fermented apple juice from malic acid-treated juice; LT: fermented apple juice from lactic acid-treated juice; CT: fermented apple juice from citric acid-treated juice. – indicates not detected. Table 2. Volatile component in fermented apple juice samples (mg/L). Compound Threshold Control ST MT LT CT Carbonyl compounds Acetaldehyde 0.5(1) 48.76 ± 5.72b 26.69 ± 0.25c 46.48 ± 7.08b 52.56 ± 4.87b 123.26 ± 6.23a Acetal 0.05(1) ND ND 0.85 ± 0.14b 0.89 ± 0.09b 1.49 ± 0.07a Acetoin 150(2) 31.85 ± 0.98a 1.61 ± 0.11c 12.72 ± 2.71b 12.21 ± 0.22b 14.08 ± 0.68b Esters Ethyl acetate 7.5 (1) 77.03 ± 4.08a 22.41 ± 2.32b 79.50 ± 3.49a 76.95 ± 0.73a 15.95 ± 0.05c Isoamyl acetate 0.03(3) 2.36 ± 0.17a 1.04 ± 0.04c 1.62 ± 0.07b 1.43 ± 0.02b 0.49 ± 0.13d Ethyl lactate 150(2) 7.18 ± 0.07c 4.13 ± 0.51c 31.77 ± 7.65b 73.39 ± 2.42a 13.19 ± 0.46c Ethyl decanoate 0.5(4) 0.07 ± 0.00a 0.04 ± 0.01b 0.07 ± 0.02a 0.05 ± 0.00a,b 0.04 ± 0.00b Higher alcohols 1-Propanol 50(3) 62.78 ± 0.97a 19.87 ± 0.16e 39.75 ± 0.34c 41.58 ± 0.52b 38.16 ± 0.21d 2-Methyl-1-propanol 40(1) 16.5 ± 0.06d 21.69 ± 0.20b 18.43 ± 0.11c 22.87 ± 0.09a 18.41 ± 0.40c 1-Butanol 150(3) 2.02 ± 0.04a 1.96 ± 0.09a 2.16 ± 0.17a 1.99 ± 0.06a 2.05 ± 0.02a 2-Methyl-1-butanol 0.32(4) 14.85 ± 0.12c 17.51 ± 0.19a 13.39 ± 0.42d 17.54 ± 0.02a 16.24 ± 0.04b 3-Methyl-1-butanol 30(4) 65.38 ± 0.39e 78.98 ± 1.07a 68.36 ± 0.88d 75.33 ± 0.32b 71.68 ± 0.79c 1-Hexanol 8(1) 2.39 ± 0.02a 2.16 ± 0.02a 2.1 ± 0.03a 2.55 ± 0.53a 1.95 ± 0.01a 2,3-Butanediol – 4.59 ± 0.00c 4.18 ± 0.02d 5.67 ± 0.08b 6.30 ± 0.05a 2.94 ± 0.12e 2-Phenylethanol 10(1) 0.53 ± 0.14b 0.8 ± 0.07b 0.82 ± 0.09b 0.76 ± 0.20b 1.99 ± 0.08a Acids Acetic acid 200(1) 244.87 ± 4.79a 108.3 ± 1.33c 254.22 ± 7.12a 230.22 ± 7.29b 46.68 ± 0.44d Propanoic acid 8.1(3) 2.26 ± 0.30a 1.33 ± 0.15b 2.57 ± 0.35a 2.81 ± 0.50a 1.99 ± 0.53a,b Butyric acid 10(1) 1.31 ± 0.23b 1.97 ± 0.02a 1.68 ± 0.06a 1.92 ± 0.08a 1.87 ± 0.11a Hexanoic acid 3(1) 5.73 ± 0.4a 4.47 ± 0.06c 4.56 ± 0.12c 4.82 ± 0.10b,c 5.36 ± 0.45a,b Octanoic acid 0.2(3) 14.92 ± 2.08a,b 10.89 ± 0.68c 13.34 ± 0.18a,b,c 12.06 ± 1.22b,c 15.18 ± 0.60a All values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). Control: fermented apple juice from original apple juice; ST: fermented apple juice from sulfuric acid-treated juice; MT: fermented apple juice from malic acid-treated juice; LT: fermented apple juice from lactic acid-treated juice; CT: fermented apple juice from citric acid-treated juice; ND: not detected (Guth, 1997;(1) Peinado et al., 2004;(2) Wang et al., 2017;(3) Wei et al., 2020(4)). Italian Journal of Food Science, 2023; 35 (2) 7 Acid treatment removes ethyl carbamate and affects volatile components from apple distillates fermented apple juices. They were described with cheese, rancid, and fatty notes, and were important for the bal- ance of complexity and fruity aromas of fermented apple juices (Sun et al., 2013). Physicochemical indices of apple distillates Cyanide and EC were not detected in the fermented apple juice or first distillates. As shown in Table 3, the highest concentration of cyanide was detected in the control (0.035 mg/L). The cyanide content of other treat- ment samples was significantly lower than that of the control. In this study, the content of EC in different treat- ments was in the range of 9.12–2.93 μg/L. Compared to the control, the content of EC in ST, MT, LT, and CT was significantly reduced (p < 0.05). The EC content of the control was 9.12 μg/L, which was 2.12–3.11 times that of other treatment samples. LT had the best effect on reducing EC by 67.87% compared to the control. The EC reduction effects of ST, MT, and CT were 64.58%, 64.91%, and 60.86%, respectively. Generally, the concen- tration of cyanide and EC in apple distillates could be effectively reduced by treating apple juice with different acids before fermentation. Volatile components of apple distillates As shown in Table 4, 24 volatile compounds were detected in the apple distillates by gas chromatography, including carbonyl compounds, esters, alcohols, and acids. Among carbonyl compounds, a total of four aldehydes and one ketone were detected. Only the contents of acetaldehyde and acetal exceeded the threshold in all apple distillates. Acetaldehyde was the most important aldehyde; its con- centration in different apple distillates ranged from 47.64 to 468.48 mg/L, and its content was significantly differ- ent (p < 0.05) in all distillates. The highest acetaldehyde content was detected in CT, and the lowest was observed in ST. In the control, the contents of acetaldehyde and acetal were 126.60 mg/L and 74.60 mg/L, respectively. It is worth mentioning that except for ST, the contents of and had a great influence on the aroma of apple juices. However, compared to the control, the content of ethyl acetate in ST and CT was significantly reduced to 54.62 mg/L and 61.08 mg/L, respectively, whereas the content of isoamyl acetate in other treatment samples was sig- nificantly decreased (p < 0.05). Significantly, although the content of ethyl lactate did not exceed the threshold, the MT- and LT-treated apple juice before fermentation could obviously increase the content of ethyl lactate in fermented apple juice samples. This result might be caused by the addition of MT and LT before fermenta- tion, which increased LT content during fermentation, thus increasing the content of ethyl lactate. Higher contents of alcohols in cider was synthesized by yeast through the glucose synthesis pathway or the cor- responding amino acid catabolism pathway, which was characterized by strong and pungent smell and had an important role in the aroma of fermented apple juices (Qin et al., 2018). Eight higher alcohols were detected in all treatment samples, of which 3-methyl-1-butanol was the most predominant alcohol in all fermented apple juices and its concentration was in the range of 65.38– 78.98 mg/L. Furthermore, both 3-methyl-1-butanol and 2-methyl-1-butanol were above the threshold in all fer- mented apple juices and provided the smell of alcohol, nail polish, and whiskey to fermented samples (Arcari et al., 2017). Compared to the control, the contents of both 3-methyl-1-butanol and 2-methyl-1-butanol in ST, LT, and CT were significantly increased (p < 0.05). As for 1-propanol, only the control exceeded the threshold of 1-propanol content, which was 62.78 mg/L, indicating that different acid treatments could significantly affect the production of 1-propanol. Five different volatile fatty acids were identified across fermented apple juices. Among these, acetic acid was the predominant volatile acid in all fermented apple juices. However, the content of acetic acid in ST and CT was significantly lower than that in the control (p < 0.05) and did not exceed the threshold, especially its content was only 46.68 mg/L in CT. Furthermore, the contents of hex- anoic acid and octanoic acid exceeded the threshold in all Table 3. Physicochemical indices of apple distillates. Control ST MT LT CT Alcohol (%, v/v) 40.2 ± 0.0a 40.2 ± 0.0a 40.2 ± 0.0a 40.1 ± 0.1a 40.2 ± 0.0a Cyanide (mg/L anhydrous ethanol) 0.035 ± 0.004a ND 0.015 ± 0.000c 0.010 ± 0.000c 0.028 ± 0.004b EC (μg/L) 9.12 ± 0.40a 3.23 ± 0.16b 3.20 ± 0.27b 2.93 ± 0.09b 3.57 ± 0.22b All values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). Control: apple distillate from original apple juice; ST: apple distillate from sulfuric acid-treated juice; MT: apple distillate from malic acid-treated juice; LT: apple distillate from lactic acid-treated juice; CT: apple distillate from citric acid-treated juice; ND: not detected. The concentrations of cyanide and EC were recalculated on the basis of 100% (v/v) ethanol. 8 Italian Journal of Food Science, 2023; 35 (2) Su Z et al. Table 4. Volatile components of apple distillates (mg/L). Threshold Control ST MT LT CT Carbonyl compounds Acetaldehyde 19.2(1) 126.60 ± 0.64d 47.64 ± 0.18e 191.39 ± 1.20c 207.92 ± 2.42b 468.48 ± 8.21a 2-Methylpropanal 1.3(4) 0.52 ± 0.03e 1.97 ± 0.04b 1.82 ± 0.03c 1.84 ± 0.03c 2.07 ± 0.05a Acetal 0.719(1) 74.60 ± 0.45d 59.54 ± 0.46e 92.55 ± 0.03c 111.95 ± 0.60b 148.33 ± 0.92a Acetoin – 6.70 ± 0.21b 0.45 ± 0.04e 4.98 ± 0.31c 16.03 ± 0.19a 3.77 ± 0.13d Furfural 44(4) 1.77 ± 0.12b,c 1.59 ± 0.06c 2.21 ± 0.02b 3.33 ± 0.39a 1.91 ± 0.04b,c Esters Ethyl formate – 1.46 ± 0.10d 3.01 ± 0.00c 3.78 ± 0.37b 2.84 ± 0.12c 5.31 ± 0.09a Ethyl acetate 32.6(3) 274.22 ± 3.08a 101.63 ± 0.64d 205.82 ± 1.17c 219.37 ± 0.60b 79.55 ± 1.07e Isoamyl acetate 0.245(3) 19.95 ± 2.29b 17.59 ± 0.74b,c 14.73 ± 0.39c,d 13.09 ± 0.10d 26.08 ± 1.56a Ethyl lactate 128(2) 8.21 ± 0.17d 5.59 ± 0.16e 21.65 ± 0.04b 74.74 ± 0.30a 18.76 ± 0.20c Ethyl octanoate 0.147(3) 1.45 ± 0.02a 0.91 ± 0.03c 0.80 ± 0.05d 1.00 ± 0.01b 0.88 ± 0.01c Ethyl decanoate 1.120(2) 0.02 ± 0.00b 0.02 ± 0.00a 0.01 ± 0.00c 0.02 ± 0.00b 0.01 ± 0.00c Alcohols 1-Propanol 54(3) 394.49 ± 3.29a 124.89 ± 0.22e 252.3 ± 0.95b 237.39 ± 0.76c 228.41 ± 0.41d 2-Methyl-1-propanol 28.3(3) 107.88 ± 0.76d 152.56 ± 2.72a 125.52 ± 0.07c 144.24 ± 0.40b 122.75 ± 0.32c 1-Butanol 2.73(2) 12.60 ± 0.46a 12.87 ± 0.29a 12.94 ± 0.04a 12.74 ± 0.08a 13.18 ± 0.17a 2-Methyl-1-butanol 45(1) 126.30 ± 4.58a,b 139.34 ± 1.78a 115.09 ± 4.00b 132.14 ± 7.11a 123.45 ± 10.48a,b 3-Methyl-1-butanol 179(3) 470.29 ± 1.84c 614.23 ± 8.07a 513.65 ± 4.54b 524.47 ± 4.88b 515.86 ± 0.81b 1-Hexanol 8(3) 21.04 ± 0.56a 20.77 ± 0.01a 19.43 ± 0.13b 20.73 ± 0.28a 19.37 ± 0.17b 2,3-Butanediol – 1.47 ± 0.03b 0.95 ± 0.18c 1.35 ± 0.09b 2.87 ± 0.00a 0.24 ± 0.02d 2-Phenylethanol 2.6(3) 1.74 ± 0.07b 1.76 ± 0.02b 1.45 ± 0.09c 1.95 ± 0.04a 1.14 ± 0.02d Methanol 25.54 ± 0.14a 22.58 ± 0.52c 23.86 ± 0.44b 23.47 ± 0.28b,c 22.45 ± 0.56c Acids Acetic acid 75.521(3) 4.60 ± 0.07a 2.27 ± 0.60c 1.11 ± 0.04d 3.66 ± 0.40b ND Butanoic acid 1.2(3) 1.85 ± 0.24a 0.92 ± 0.14b,c 1.21 ± 0.15b 0.37 ± 0.03d 0.68 ± 0.18c,d Hexanoic acid 2.52(3) 8.47 ± 0.35a 5.80 ± 0.31b 5.42 ± 0.25b 5.66 ± 0.29b 4.46 ± 0.33c Octanoic acid 2.7(3) 46.87 ± 0.77a 17.96 ± 3.48c,d 24.96 ± 0.28b 21.1 ± 0.30b,c 13.83 ± 2.53d All values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). Control: apple distillate from original apple juice; ST: apple distillate from sulfuric acid-treated juice; MT: apple distillate from malic acid-treated juice; LT: apple distillate from lactic acid-treated juice; CT: apple distillate from citric acid-treated juice; ND: not detected. – indicates not found. The concentrations of volatile compounds were recalculated on the basis of ethanol 40% (v/v) (Gao et al., 2014;(2) Wang et al., 2014a;(4) Willner et al., 2013;(1) Xiang et al., 2020(3)). acetaldehyde and acetal were significantly increased in other samples (p < 0.05). Compared to fermented juices, 2-methylpropanal and furfural were newly observed nat- ural products observed in apple distillates; these were formed due to the chemical reactions caused by high temperature during distillation (Awad et al., 2017). Six esters were also discovered in apple distillates (Table 4), among which the contents of ethyl acetate, iso- amyl acetate, and ethyl octanoate were above the thresh- old in all samples. Ethyl acetate was the most important ester, and its presence was consistent with the results in other apple distillates (Ledauphin et al., 2010; Versini et  al.,  2009). The lowest content of 79.55 mg/L was dis- covered in CT. The content of ethyl acetate in ST, MT, and LT was 101.63, 205.82, and 219.37 mg/L, respec- tively. Its concentration in apple distillates with different treatments was significantly different (p < 0.05). The con- tent of ethyl acetate in apple distillates was significantly reduced by acid treatments. Alcohols were the most important volatiles observed in distillates, and nine compounds were discovered in this study. The contents of 1-propanol, 2-methyl-1- propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 1-hexanol in all samples exceeded the threshold, Italian Journal of Food Science, 2023; 35 (2) 9 Acid treatment removes ethyl carbamate and affects volatile components from apple distillates Sensory evaluation of apple distillates According to the sensory descriptors mentioned in Table 5, we observed that MT had the highest fruity score, which was also consistent with the expert’s description of MT. ST had the lowest scores for fruity and typicality, but both vinous and alcoholic had high scores. The results indicated that although ST could affect the typicality of apple distillate, but ST would not have a seriously adverse effect on the quality of distillate. Concerning LT and CT, most of their scores of sensory descriptors were signifi- cantly lower than the control hence, LT and CT could have bad effects on the quality of distillate. In sensory evaluation, the control had the highest score of 92.27, but the CT score was lowest (85.73). The sensory evaluation scores from low to high were: LT (86.64), ST (88.64), and MT (91.09). The scores of control and MT were close to each other, which could be due to the close content val- ues of esters and alcohols. ST had the highest 3-methyl-1- butanol content of 614.23 mg/L (Table 4), which could be responsible for its strong alcoholic taste. Although CT had the highest acetaldehyde content (468.48 mg/L), it had the lowest content of ethyl acetate (79.55 mg/L), which could be the reason for its flavor defect and bitter taste. In summary, although the treatment of ST, MT, LT, and CT on apple juice before fermentation could reduce the EC but 3-methyl-1-butanol had the highest concentration as observed previously for apple distillates (Ledauphin et al., 2003). Compared to the control, content of 1-propanol in the acid-treated sample was decreased significantly (p < 0.05), and the lowest content found in ST was only 124.89 mg/L. However, the concentrations of 2- mthyl-1-propanol and 3-methyl-1-butanol in dif- ferent acid-treated samples were higher than that in the control (p < 0.05), and the highest contents of these two substances in ST-treated sample were 152.56 mg/L and 614.23 mg/L, respectively. However, the content of meth- anol in different treatment samples was decreased signifi- cantly (p < 0.05). Four different volatile acids were identified in all samples. The content of acetic acid in apple distillates was sig- nificantly reduced compared to that in fermented apple juice; this could be due to the removal of tail during dis- tillation. The contents of hexanoic acid and octanoic acid were above the threshold in all samples. In addition, the highest contents of hexanoic acid and octanoic acid in the control were 8.47 mg/L and 46.87 mg/L, respectively. Furthermore, the concentrations of these substances in other treatment samples were significantly decreased, and the lowest concentrations of hexanoic acid and octanoic acid were detected in CT sample, which were 4.46 mg/L and 13.83 mg/L, respectively. Table 5. Effects of different treatments on sensory scores of apple distillates. Sample Sensory descriptor Olfactory Gustatory Typicality Fruity Vinous Alcoholic Balance Control 17.91 ± 0.54b 17.55 ± 0.52a 18.09 ± 0.54b 19.27 ± 0.47a 19.45 ± 0.52a ST 17.09 ± 0.83c 17.55 ± 0.69a 19.18 ± 0.40a 18.27 ± 0.47b 16.55 ± 0.52c MT 18.82 ± 0.40a 17.82 ± 0.60a 16.91 ± 0.54c 18.27 ± 0.47b 19.27 ± 0.47a LT 17.73 ± 0.47b 16.64 ± 0.50b 16.27 ± 0.47d 17.82 ± 0.60c 18.18 ± 0.40b CT 16.91 ± 0.54c 17.45 ± 0.52a 16.73 ± 0.47c 16.82 ± 0.40d 17.82 ± 0.40b Sensory evaluation Score Control The distillate had a pleasant aroma, a full-bodied palate, and no miscellaneous flavors, and had the typical characteristics of apple distillate. 92.27 ± 0.90a ST The aroma of apple was weaker than that of the control and had a plastic odor. Strong alcoholic taste in the mouth. The overall coordination was good. 88.64 ± 1.12c MT It had the best apple aroma, no odor, the mouth was clean and refreshing, but the overall coordination was lower than the control. It also had the typical characteristics of apple distillates. 91.09 ± 0.94b LT It had a strong aroma of apple, but a weaker vinous. It also had a faint alcoholic taste in the mouth, which was unpleasant. 86.64 ± 1.21d CT The aroma of the wine was flawed compared to that of the control, with a lighter apple aroma, a bitter taste, and the heaviest wine. The overall coordination was not good. 85.73 ± 0.65e All values are expressed as mean values ± standard deviations (n = 11); different superscripted lowercase letters in the same column indicate significant difference (p < 0.05). Control: apple distillate from original apple juice; ST: apple distillate from sulfuric acid-treated juice; MT: apple distillate from malic acid-treated juice; LT: apple distillate from lactic acid-treated juice; CT: apple distillate from citric acid-treated juice. 10 Italian Journal of Food Science, 2023; 35 (2) Su Z et al. in distillates had a significantly positive correlation (0.72*). It has been reported that as a precursor substance of EC, cyanide content is significantly correlated with EC content (Wang et al., 2021). Therefore, the EC content of apple distillate can be decreased by reducing the cya- nide content. Furthermore, pH affected the production of ethyl acetate (0.73*), ethyl octanoate (0.93***), 1-pro- panol (0.91***), acetic acid (0.65*), butanoic acid (0.83**), hexanoic acid (0.91***), and octanoic acid (0.96***). In the meantime, pH was negatively correlated with 2-methyl- propanal (-0.99***), ethyl formate (-0.69*), 2-methpropa- nol (-0.77**), and 3-methyl-1-butanol (-0.70*). Overall, the pH of fermented apple juice plays an important role in the formation of volatiles in apple distillates. Conclusion Acid treatment by ST, MT, LT, and CT significantly removed EC from apple distillates produced by Fuji acid- ified apple juice. The lower the pH of fermented apple content in the final distillates, it also impacted the aroma, flavor, and taste. Therefore, the stored fermented apple juice would be treated with the acids to find their influence on EC and volatiles on the final distillate in of the following research. Correlation analysis of volatile constituents in apple distillates and basic physicochemical indexes of fermented apple juice In order to further analyze the effects of different treat- ments on cyanide, EC, and volatiles found in apple distil- lates, the correlation analysis chart was used to analyze the relationship of total sugar, titrable acidity, pH, dry extract, and alcohol in fermented apple juice, and that of cyanide, EC and volatiles in distillates. As shown in Figure 3, a sig- nificantly positive correlation is observed between the EC content in apple distillates and the pH of fermented apple juice (0.98***). It indicates that lowering the pH of fer- mented apple juice significantly reduces the EC content of apple distillates. In addition, the EC and cyanide contents Figure 3. Correlation analysis of volatile constituents in apple distillates and basic physicochemical indexes of fermented apple juice. Red indicates positive correlation and blue shows negative correlation. The deeper the color, the greater the abso- lute value of correlation and the stronger the correlation between them. AD: apple distillates. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0001. -0.29 0.74 -0.39 0.34 0.66 0.72 0.11 -0.73 0.15 0.59 0.27 -0.51 0.63 0.14 0.28 0.83 0.80 -0.55 -0.50 -0.73 0.44 0.39 0.31 0.65 0.26 0.64 0.63 0.019 -0.021 -0.71 0.97 -0.088 -0.13 -0.75 0.75 0.77 0.83 0.27 0.50 0.83 -0.42 0.053 0.53 -0.76 -0.69 0.19 0.53 -0.33 -0.73 -0.016 -0.49 -0.69 -0.74 -0.87 -0.74 -0.010 -0.36 -0.73 0.31 0.73 0.98 -0.25 -0.99 -0.32 0.082 -0.25 -0.69 0.73 0.13 -0.30 0.93 0.073 0.91 -0.49 -0.16 -0.70 0.091 0.20 0.65 0.83 0.91 0.96 0.44 -0.77 -0.0098 -0.26 -0.81 0.58 0.77 0.68 0.30 0.56 0.74 -0.31 0.17 0.56 -0.81 -0.66 -0.39 0.27 -0.36 0.035 -0.71 -0.38 -0.64 -0.70 -0.84 -0.71 0.11 0.45 0.13 0.20 -0.22 -0.35 -0.10 0.60 0.52 -0.41 0.66 -0.52 0.45 0.29 0.031 0.42 -0.10 -0.11 -0.36 0.41 0.39 0.58 0.095 0.32 0.44 0.62 -0.016 0.72 0.47 -0.64 0.36 0.034 -0.22 -0.045 0.34 0.58 -0.25 0.58 -0.49 0.84 -0.94 -0.52 -0.86 -0.10 -0.42 -0.087 0.53 0.39 0.57 -0.26 0.037 -0.23 -0.97 -0.33 -0.030 -0.36 -0.66 0.61 0.27 -0.40 0.94 0.14 0.85 -0.74 -0.080 -0.61 0.45 0.17 0.62 0.81 0.90 0.92 -0.032 -0.46 0.36 0.97 0.072 0.13 0.79 -0.41 0.63 0.16 -0.32 -0.78 0.043 -0.32 -0.41 -0.36 -0.69 -0.76 -0.64 -0.39 -0.58 -0.43 -0.38 0.54 0.42 -0.12 0.21 0.78 -0.77 -0.031 0.26 -0.94 -0.18 -0.87 0.68 0.092 0.63 -0.55 -0.33 -0.74 -0.82 -0.94 -0.98 -0.18 0.54 0.29 0.35 0.75 -0.34 0.45 0.39 -0.35 -0.75 0.011 -0.20 -0.34 -0.35 -0.62 -0.61 -0.55 -0.53 -0.62 -0.48 -0.16 0.47 0.92 -0.28 0.57 -0.50 0.92 0.20 -0.12 0.32 0.052 0.014 -0.41 0.27 0.52 0.49 -0.36 0.098 0.13 0.88 -0.33 -0.032 0.35 -0.57 0.97 -0.15 -0.19 0.023 0.28 0.040 -0.19 0.073 0.41 0.24 -0.57 -0.22 -0.17 0.82 -0.17 -0.77 0.45 -0.036 -0.78 -0.61 -0.49 0.11 -0.27 0.17 -0.83 -0.82 -0.94 -0.55 -0.90 -0.80 -0.55 0.72 -0.50 0.26 0.66 0.0059 0.79 -0.45 -0.23 -0.66 0.44 0.55 0.76 0.52 0.75 0.83 0.67 -0.62 -0.53 0.11 -0.29 0.11 -0.45 -0.12 -0.16 -0.35 -0.70 -0.43 0.089 -0.11 -0.078 -0.81 0.35 -0.16 -0.14 -0.045 0.35 0.074 -0.13 0.092 -0.42 0.23 -0.66 -0.25 -0.24 0.81 -0.13 0.33 0.78 -0.55 0.14 -0.53 0.67 0.41 0.79 0.64 0.91 0.88 0.22 -0.59 -0.29 0.54 0.80 0.60 0.83 0.71 0.58 0.085 0.42 0.15 0.21 -0.35 -0.88 -0.41 -0.93 0.19 0.051 0.51 0.66 0.72 0.86 0.19 -0.39 0.59 0.88 0.19 0.41 -0.071 -0.69 -0.46 -0.66 0.26 -0.35 0.21 -0.45 -0.66 0.65 -0.30 -0.66 -0.53 -0.52 0.59 -0.58 0.51 0.36 -0.25 0.11 -0.18 0.17 0.12 0.18 -0.23 -0.43 -0.41 -0.62 -0.17 0.81 0.88 0.25 0.66 0.50 0.80 0.67 -0.21 0.26 0.23 0.84 0.0036 0.53 0.35 0.36 0.83 0.74 0.76 0.82 0.94 0.50 0.10 To ta l s ug ar Ti tr ab le a ci di ty pH D ry e xt ra ct A lc ho l A D c ya ni de A D e th yl c ar ba m at e A D a ce ta ld eh yd e A D 2 -m et hy lp ro pa na l A D a ce ta l A D a ce tio n A D fu rf ur al A D e th yl fo rm at e A D e th yl a ce ta te A D is oa m yl a ce ta te A D e th yl la ct at e A D e th yl o ct on oa te A D e th yl d ec an oa te A D 1 -p ro pa no l A D 2 -m et hy l-1 -p ro pa no l A D 2 -m et hy l-1 -b ut an ol A D 3 -m et hy l-1 -b ut an ol A D 1 -h ex an ol A D 2 ,3 -b ut an ed io l A D 2 -p he ny le th an ol A D a ce tic a ci d A D b ut an oi c ac id A D h ex an oi c ac id A D o ct an oi ca ci d A D 1 -b ut an ol Total sugar 1 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 –1 Titrable acidity pH Dry extract Alchol AD cyanide AD ethyl carbamate AD acetaldehyde AD 2-methylpropanal AD acetal AD acetion AD furfural AD ethyl formate AD ethyl acetate AD isoamyl acetate AD ethyl lactate AD ethyl octonoate AD ethyl decanoate AD 1-propanol AD 2-methyl-1-propanol AD 2-methyl-1-butanol AD 3-methyl-1-butanol AD 1-hexanol AD 2,3-butanediol AD 2-phenylethanol AD acetic acid AD butanoic acid AD hexanoic acid AD octanoicacid AD 1-butanol Italian Journal of Food Science, 2023; 35 (2) 11 Acid treatment removes ethyl carbamate and affects volatile components from apple distillates and alcohol content in ‘heart’ fractions on the concentration of aroma volatiles and undesirable compoundsin plum brandies. 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