4.1. Volatile Aroma Composition
As to the authors’ knowledge, this is the first study to report the volatile profile from Honeycrisp juices. Substantiating our results, esters, aldehydes, and alcohols have been reported as major groups of volatile compounds in juices from different apple cultivars including McIntosh [
17], Jonagold [
13], Golden Delicious, Red Delicious [
18], Holsteiner Cox, Ingrid Marie, and Rajka [
19]. Our results indicated quantitative and qualitative differences in the level of volatile compounds between the two cultivars. Esters are known for their desirable aromatic notes in apple beverages, which are often described as “fresh apple”, “fruity”, and “sweet”, and it is reasonable to assume that the higher amount of total esters in Honeycrisp juices would increase its overall aroma and flavor [
20]. The higher content of 2-methyl-4-pentenal in McIntosh juice samples could offer desirable green grass and fruit aroma notes [
15]. Despite the greater content of total esters in Honeycrisp juices, the higher level of ethyl acetate in Honeycrisp juices may lead to the development of undesirable flavor as excessive amounts of fermentation volatiles, including ethyl acetate, which can generate off-flavors and aromas [
3]. The higher concentration of butanoate esters in McIntosh juices could provide juice with a favorable fruity, ripe, and sweet aroma [
2]. Due to the low odor threshold values (
Table 2) of acetate and butanoate esters, these groups of esters could generate a significant contribution to the final aroma of the juice samples. Accordingly, it has been demonstrated that acetate esters, including butyl acetate, 2-methyl butyl acetate, hexyl acetate, and ethyl butanoate, are major contributors to the typical apple-like aroma and flavor in many apple cultivars [
21,
22]. Similarly, hexyl acetate, butyl acetate, and ethyl butanoate were reported as the most important volatiles in Jonagold juice [
23].
In addition to straight-chain esters, a branched-chain ester, 2-methyl butyl acetate, was one of the most abundant volatile compounds detected in Honeycrisp and McIntosh juices. Owing to its lower threshold value, 2-methyl butyl acetate has been described as one of the most significant odor active volatile compounds in other apple cultivars, including Gala [
24].
It has been indicated that C-6 aldehydes, particularly (E)-2-hexenal and hexenal, are responsible for the fresh green aroma in apple juice [
25]. Excessive concentration of C-6 aldehydes (>2430 µg/L) in apple juice has been associated with negative odor impressions and thus led to lower sensory scores and is often denoted by sensory descriptors such as “artificial flavor, too green, and shampoo-like” [
2]. Thus, it is reasonable to assume the relatively high content of C-6 aldehydes in McIntosh juices might lead to the development of negative organoleptic properties.
The volatile compounds identified from McIntosh and Honeycrisp juices are a combination of primary (synthesized by the intact fruit) and secondary (synthesized in response to cellular disruption during juice processing) volatile compounds [
25]. The volatile compounds detected from intact fruit are mainly composed of esters and ethanol (
Figure 3C,D). These primary volatiles are synthesized by controlled enzymatic reactions mainly from fatty acid metabolism [
26]. It is well known that fatty acids are major precursors of aroma volatiles in several fruits, including apple, and the biosynthetic pathway includes beta-oxidation (primary volatiles) and lipoxygenase (LOX) action (secondary compounds) [
26]. The beta-oxidation pathway provides alcohols and acyl co-enzyme-A (CoA,), which are the main precursors for volatile ester production. Acyl CoAs are reduced by acyl CoA reductase to produce aldehydes, which in turn are reduced by alcohol dehydrogenase (ADH) to form alcohols that are converted to esters via the action of AAT [
25].
Secondary volatiles, which are mainly C-6 aldehydes and the associated alcohols, are formed by the LOX pathway from unsaturated fatty acids (linoleic and linolenic acids) when the fruit is crushed and exposed to oxygen [
26]. In our experiment, C-6 and C-5 aldehydes such as (E)-2-hexenal and 2-methyl-4-pentenal were detected only in juice samples but not in whole apple samples (
Figure 3). This is in agreement with other studies, which reported the higher content of C-6 aldehydes in apple juice compared to intact fruit [
27,
28]. As discussed earlier, the presence of aldehydes in juice samples but not in intact Honeycrisp and McIntosh apples is attributed to the oxidation of unsaturated fatty acids (linoleic and linolenic) during juice processing.
4.2. Effect of 1-MCP, Storage Atmosphere, and Juice Processing
According to the results presented, the content and composition of volatile compounds from clear and cloudy juice were strongly influenced by the different combinations of 1-MCP treatment, storage atmosphere, harvest maturity, and juice type. The subsequent impact of the levels of esters and aldehydes on the juice odor depends on its odor threshold value (i.e., the detection or recognition values, above which the compound can be detected by smell) and concentration [
21]. Based on the threshold values of volatile compounds summarized from the literature (
Table 2), esters and aldehydes have considerably lower threshold values as compared with alcohols. This means esters and aldehydes may have a key role in influencing the odor of the juice even at low concentrations. On the other hand, volatile compounds with higher threshold values (notably ethanol,
Table 2) might not have a large impact on the odor of apple juice. Hence, our discussion will focus on aldehydes and esters.
In McIntosh juices, whether it is clear or cloudy, our results indicated a remarkable reduction of all types of esters, aldehydes, most alcohols, and total volatile compounds when juices are extracted from 1-MCP-treated fruit stored in CA or RA. This is consistent with previous studies that found a substantial suppression of volatile aroma compounds in several apple cultivars that had been treated with 1-MCP before storage in RA or CA [
6,
7].
Unlike McIntosh, 1-MCP treatment alone (1-MCP + RA) in Honeycrisp apples did not alter the content of most volatile compounds except ethyl acetate and ethanol, which were substantially suppressed by the treatment. This effect might be attributed to the unusual response of this cultivar to 1-MCP treatment. In our previous study (unpublished) while 1-MCP + RA treatment in McIntosh apples inhibited ethylene production better than the control + CA/RA treatments, the same treatment in Honeycrisp produced the highest ethylene (49.03 µL kg
−1 h
−1) level, which was present at higher levels than in control fruit (17 µL kg
−1 h
−1). As reported in other cultivars [
6,
7] elevated ethylene production is usually accompanied by an increased level of volatile compounds and vice versa. Nevertheless, this trend did not occur in Honeycrisp apples. As there is no published information regarding the volatile profile of Honeycrisp fruit or juice, especially none focusing on 1-MCP treatment, it is difficult to account for the unusual response of this cultivar to 1-MCP treatment.
The observed inhibitory effect of CA storage on the content of volatile compounds is consistent with previous studies in different apple cultivars [
29,
30]. Reduced sensitivity to ethylene [
31] or suppressed ethylene production of CA-stored fruit [
31,
32] has been suggested as a mechanism by which volatile production could be inhibited in CA-stored apples. The biosynthesis of volatile compounds via beta-oxidation or the LOX pathway needs oxygen, and therefore their production could be slowed down by CA condition where the oxygen level is much lower than the RA atmosphere [
5].
Even though CA storage suppressed the content of most volatile compounds, it also enhanced some branched-chain esters detected from intact Honeycrisp apples (3-methyl-1-butyl acetate) as well as from Honeycrisp juices (2-methyl butyl acetate). In agreement with our observation, other studies also reported the increased level of branched-chain acetate esters in Delicious [
33], Gala [
30], and Fuji [
21] apples that were kept under low oxygen storage conditions. A study in pear fruit found an increased level of branched-chain esters associated with a higher level of amino acids, which are the main precursors of branched-chain esters [
34]. The higher concentration of (E)-2-hexenal in juices from 1-MCP and/or CA-treated Honeycrisp apples might be attributed to the suppressed ripening of the apples associated with lower ethylene production [
30]. Contrary to the results observed in late-harvested McIntosh juices, the suppressive effect of 1-MCP and/or CA storage was not clearly observed in juices extracted from fruit harvested at commercial maturity. These results are unexpected, and no explanation or corresponding results were found in the literature.
As compared to clear juices, cloudy juice samples from McIntosh and Honeycrisp apples had considerably higher levels of all the major esters, aldehydes, and total volatiles, which was most pronounced in Honeycrisp juices. Even though there is a lack of literature pertaining to the volatile composition of cloudy apple juices, one recent study reported higher levels of total esters in apple juice with pulp as compared to juice from concentrate [
18]. The reduction of esters in clear juice samples can be explained by the hydrolysis of esters by the action of esterase that is present in the commercial enzyme preparation [
26]. As mentioned in the methodology part, one of the major differences between the two juices is the absence (clear) or presence (cloudy) of ascorbic acid. The higher content of aldehydes in cloudy juices, which is processed with ascorbic acid addition, is consistent with a previous study [
13] that investigated the changes in the aroma value (the ratio of volatile concentration to odor threshold) of volatile compounds due to the addition of ascorbic acid (0.2% w/v) to the apple juice. Komthong et al. [
13] found considerably higher (4- to 5-fold) aroma value of (E)-2-hexenal and hexanal in juices treated with ascorbic acid than the control. Even though there is limited information about the exact mechanism of ascorbic acid reaction with volatile compounds, the reduced concentration of aldehydes in clear juice samples has been associated with the action of ADH during the clarification process. The lower content of aldehydes in clear juice samples was ascribed to the conversion of aldehydes to alcohols by the action of ADH during the enzymatic incubation [
26,
35]. The longer incubation period (about 3 h at 25 °C, in our case) would give additional time for different enzymatic reactions activated via endogenous enzymes, including ADH. In our experiment, the long incubation period was not part of cloudy apple juice preparation; instead, the juice was immediately cooled and then pasteurized. This immediate cooling, which was followed by pasteurization, could slow down and inactivate the action of the indigenous enzymes such as ADH. Hence, the preservation of aldehydes in cloudy apple juice could be attributed to the inhibition of ADH activity during processing [
26].
Generally, our study suggests that the content and composition of volatile aroma compounds in apple juice could be strongly influenced by the fruit quality and juice processing techniques. As each group of the volatile compound has a typical odor characteristic, the difference in their abundance associated with postharvest treatments and juice processing steps could affect the subsequent organoleptic quality of the juices. However, in future investigations, sensory evaluation is warranted to assess the consumers’ perception associated with the change of volatile aroma compounds observed in this study.