2.1. Major Chemical Compounds
summarizes the results of the determinations of dry matter and sugar contents in the analyzed products. The pressing of crushed and whole fruits affected the contents of both dry matter and sugars in juices. The content of dry matter and sugars in JUF was 16.87% and 87.31 g/100 g dm (dry matter), respectively, and was higher compared to JCF, i.e., 15.46% and 85.24 g/100 g dm, respectively. Kulling et al. [4
] reported a similar content of dry matter (15.5%) in commercially-produced chokeberry juice (100%, pasteurized, not from concentrated juice). The content of total sugars in the PDF was 46.15 g/100 g dm, which was 1.6- and 1.8-times higher compared to the PPUF and PPCF. This indicates that the dried powders made of pomace were significantly poorer in sugars than the PDF. This is beneficial, as they exhibit lower hygroscopicity and lower viscosity and are easier to disintegrate.
The contents of individual sugars were found to differ among the analyzed products. In PDF, the contents of sorbitol, glucose and fructose were 21.08, 14.64 and 10.43 g/100 g dm. In the case of PPUF, the contents of sorbitol and glucose were at the same level, i.e., 12.55 g/100 g dm, whereas fructose content was lower by ca. 50%. In PPCF, the content of sorbitol was 13.33 g/100 g dm, that of fructose 6.64 g/100 g dm and that of glucose 5.08 g/100 g dm [4
2.2. Identification of Phenolic Compounds in Black Chokeberry
presents the results of the identification of polyphenolic compounds in chokeberry products based on the comparison of their MS and MS/MS data, retention times, UV spectra of standards and published data. Twenty-seven phenolic compounds were detected in fruits, pomace and juice from crushed and uncrushed chokeberry fruits.
The first group of phenolic compounds was related to the anthocyanin family (Peaks 1, 3, 5, 7, 10, 11, 13). The anthocyanins identified in chokeberry for the first time were: cyanidin-3,5-hexoside-(epi)catechin with [M + H]+
737 and fragmentation at m
575, 423 and 287; cyanidin-3-pentoside-(epi)catechin with [M + H]+
707 and MS/MS fragmented m
557, 329 and 287; and cyanidin-3-hexoside-(epi)cat-(epi)cat with the [M + H]+
molecular ion at m
1025, showing typical fragments at m
575, 409 and 287. These compounds were identified by the molecular ion [M − H]+
, comparing their MS profiles with the fragmentation pathways observed, as well as the UV spectra and the retention time in pomegranate [30
]. Moreover, the following were detected: cyanidin-3-O
-galactoside and cyanidin-3-O
-glucoside with the molecular ion [M + H]+
449 and characteristic fragments at m
-arabinoside with [M + H]+
419 and MS/MS fragmented m
287; and cyanidin-3-O
-xyloside with [M + H]+
419 and fragmentation at m
287. Results regarding the presence of the last four anthocyanins are consistent with published data [5
Two types of flavonol derivatives (Peaks 17–23 and 25–28) with a fragment at m
301 and 315, characteristic for quercetin and isorhamnetin derivatives, respectively, were found in the chokeberry products (Table 2
). Quercetin derivatives are mainly flavonols found in chokeberry berries; seven quercetins as dihexoside (m
595), 3-robinobioside and 3-O
-galactoside and -3-O
463) and -O
593). These results agreed with recently-published data [5
]. Additionally, two isorhamnetin derivatives were found: -pentosylhexoside (m
609) and two -rhamnosyl-hexoside isomers (m
623). These results agreed with recently-published data [29
]. Another group that included five derivatives of phenolic acids was identified as caffeoylquinic acid derivatives. Three of them were characterized by the same [M − H]−
as 353, but assigned by different compounds as: neochlorogenic acid (Rt = 2.57 min), chlorogenic acid (Rt = 3.62) [5
] and cryptochlorogenic acid (Rt = 3.71), with λmax
= 325 nm for all. In addition, two phenolic acids were identified: 3-O
-p-coumaroylquinic acid with [M − H]−
337 and di-caffeic quinic acid with [M − H]−
515, MS/MS fragments at m
353 and 191. Chromatography standards of neochlorogenic, chlorogenic, cryptochlorogenic and 3,5-dicaffeoylquinic acid were used to confirm the identity of these compounds. These compounds were previously found by Slimestad et al. [34
] and Lee et al. [33
The next group of phenolic compounds identified in chokeberry products by the LC-PDA-ESI-MS/MS analysis belonged to the flavan-3-ol family and included monomers and dimers. (+)-Catechin and (−)-epicatechin had an [M − H]−
289. Chromatography with a standard was used to confirm their identity. Besides these compounds, one procyanidin dimer with m
] and one flavanone, i.e., eriodictyol-glucuronide with [M − H]−
463 and an MS/MS fragment at m
287, were found. The latter compound is largely responsible for the bitter taste of chokeberry. It was detected for the first time in chokeberry fruits by Slimestad et al. [34
In addition, the sum of polymeric proanthocyanidins was determined in the chokeberry products using the phloroglucinol method by UPLC-MS/MS. This method provides more detailed information on the proanthocyanidin fraction of these berries and products, especially when these compounds are difficult to detect in the UPLC-PDA analysis.
2.3. Comparison of Phenolic Compounds Detected in Black Chokeberry Products
The results of the determinations of the contents of polyphenolic compounds (PC) in the analyzed samples are presented in Table 3
and in Figure 1
. Their content in juices was significantly affected by the procedure of pulp preparation for pressing. In JCF, the content of polyphenols was higher by over 32% than in JUF. In the case of dried powders, the highest content of PC was obtained in the PPUF (24.45 g/100 g dm) and PPCF (15.61 g/100 g dm), and these values were significantly lower compared to the PDF (24.72 g/100 g dm). The main PC in the analyzed chokeberry products were: anthocyanins > procyanidin polymers >> phenolic acids ≥ flavonols > flavan-3-ols > flavanone. In an earlier study, Oszmiański et al. [5
] determined the total phenolic compounds in pomace from black chokeberry at 10.58 g/100 g dm, which was 2.3-times lower compared to the results obtained in this study for pomace and 1.5-times lower compared to whole dried fruits. These differences may be due to various chokeberry cultivation conditions and location and to various methods of material pretreatment for the preparation of products. In a study by Horszwald et al. [1
], who analyzed chokeberry powders made by different drying processes, the content of polyphenolics in freeze-dried chokeberry fruits was 27.63 g/100 g dm. This value differed insignificantly compared to the polyphenolic content determined in the powders made of pomace in our study, but was ca. two-times higher compared to PDF.
The main PC identified in chokeberry powders were anthocyanins, which constituted ~50% of total polyphenols (Table 3
). The total content of anthocyanins in the analyzed samples ranged from 6.68 in PDF to 12.16 g/100 g dm in PPUF. These results confirm that anthocyanins occur mainly in skin, wherein their concentration is the highest compared to the other morphological parts of fruits. Migration of anthocyanins from fruit skin to juice is determined, most of all, by skin damage. The appropriate crushing before pressing causes damage to cell walls and thus facilitates the migration of fruit components to the pressed juice. Pectolytic enzymes are often applied as they cause degradation of cell walls, which enables the degradation of the tissue structure, forming pectins. This process allows for an additional increase in pressing yield and, consequently, in the anthocyanin content of juice [35
]. These processes have a great effect on the concentration of anthocyanins in the end product [36
]. In the analyzed juices, anthocyanins were also the group of compounds with the highest concentrations, which ranged from 1.01 JUF to 2.12 g/100 g dm in JCF. The method of pulp preparation resulted in over two-fold differences in the anthocyanin content of juices. The content of anthocyanins in dried powders made of juice with the freeze-drying method determined by Horszwald et al. [1
] was 22.81 g/100 g dm, was 1.9-times higher compared to dry pomace and 3.4-times higher compared to whole dried fruits in our study. Differences in the contents of anthocyanins may result mostly from the juice production process. Procyanidin polymers (PP) were the second group of compounds in terms of the content in the analyzed products and represented ~40% of the total polyphenolic contents. They constitute an important group of health-promoting compounds and are responsible for the pungent taste of chokeberry. Procyanidin oligomers exhibit high affinity to proteins, thereby causing their denaturation. Such an effect is perceptible during the consumption of chokeberry fruits. However, PP are interesting due to their strong anti-inflammatory effects and beneficial impact on health, including antitumor and antiproliferative activities [17
The JUF contained over 1.6-times less PP than JCF (Table 3
), i.e., 1.47 and 2.37 g/100 g dm, respectively. The change in the mode of pulp preparation for pressing allowed the content of pungent and bitter PP to be reduced, which improving the organoleptic characteristics of the juice. Like anthocyanins, these compounds occur mainly in fruit skin: therefore, if whole fruits are crushed, they remain in the pomace.
In dry powders that may be applied as additives to nutraceuticals with health-promoting properties wherein a high content of PP is highly desirable, a considerably higher content of these compounds was obtained in dried pomace than in dried fruits. In our earlier investigations [5
], the content of PP in pomace was 8.19 g/100 g dm, which was 1.2-times lower compared to dried pomace and 1.3-times higher compared to dried fruits in the present study. An important compound identified in chokeberry products was eriodictyol-glucuronide, belonging to the group of flavanones, which represented ~0.01% of the total polyphenol contents. It is largely responsible for the bitter taste of chokeberry fruits. Its content in juices was significantly affected by the method of their preparation, i.e., JUF or JCF. The crushing of fruits before pressing caused a 1.5-fold increase in its content in the JUF. As in the case of PP, reduction of the content of eriodictyol-glucuronide improved the taste of chokeberry juice [6
Another group of compounds identified in chokeberry included phenolic acids (PA), which constituted ~7.2% of total phenolics (Table 2
and Table 3
). Chlorogenic acid was found to predominate, with its mean contents being 0.94 in powders and 0.56 g/100 g dm in juices. In turn, the lowest contents were determined for di-caffeic quinic acid, i.e., 0.001 in juices and 0.004 g/100 g dm in powders. PA occur in various morphological parts of fruits, in both soft pulp and hard skin. Therefore, their contents were insignificantly affected by the method of powder and juice preparation (Table 2
and Table 3
). The only difference, compared to the contents of anthocyanins and procyanidin polymers in the analyzed samples, was a higher content of PA in the PPUF than PPCF, i.e., 2.23 and 1.48 g/100 g dm, respectively. This may be due to a higher degree of PA oxidation during fruit crushing before pressing, as it is common knowledge that PA are good substrates of the enzyme phenoloxidase and that they are readily oxidized in enzymatic reactions.
Flavonols constituted another group of PC of chokeberry with the content representing ~1.5% of total polyphenols. Wang et al. [37
] reported in their study that, compared to anthocyanins, flavonols displayed higher antioxidative activity measured with the ORAC test. Like anthocyanins, flavonols occur mainly in fruit skin; therefore, their concentration in the JCF was over 2.4-times higher than in the juice made of whole fruits. In addition, their content was ca. two-fold higher in powder made of pomace than in PDF.
Another group of compounds occurring in black chokeberry and constituting ~1.3% of the total polyphenolics was flavan-3-ols (Table 2
and Table 3
). The JCF were characterized by a 1.2-times higher concentration of these compounds compared to the JUF. As in the case of PA, the PPUF contained more flavan-3-ols compared to PPCF, i.e., 0.44 and 0.31 g/100g·dm, respectively.
2.4. Antioxidant Activity of Powder and Juice with Black Chokeberry
The results of the ABTS test assaying the capability to reduce free radicals and of the FRAP test assaying the capability to reduce ferric ions are presented in Table 4
. The analysis of the antioxidative activity (AA) demonstrated differences in the ABTS and FRAP values between JCF and JUF and between PDF, PPUF and PPCF (Table 4
). The JCF were characterized by 1.6- and 2.1-times higher ABTS and FRAP values compared to JUF. This was due to the differences in the contents of polyphenolic compounds. The results of the ABTS test conducted for JCF (32.73 mmol Trolox/100 g dm) are comparable to the findings of previous studies carried out with chokeberry juices (31.41 mmol Trolox/100 g dm) [5
Comparison of the AA of powders revealed significantly higher values in the samples of dried chokeberry fruits than in those of dried pomace. The results of the ABTS method were 81.66 and 81.63 mmol Trolox 100 g·dm for PDF and PPUF, respectively, whereas the results of the FRAP method were 53.78 and 52.22 mmol Trolox/100 g dm, respectively. A considerably lower capability to reduce free ABTS radicals and of ferric ions was found for the PPCF, i.e., 59.94 and 32.61 mmol Trolox/100 g dm, respectively.
The AA determined with the ABTS test by Oszmiański et al. [5
] in pomace was 77.96 mmol Trolox/100 g dm and was slightly higher than that assayed in the reported study in pomace samples: 81.63 mmol Trolox/100 g dm. According to Horszwald et al. [1
], the capability to reduce free radicals determined with the ABTS method in powders made of freeze-dried chokeberry juice was 180.45 mmol Trolox/100 g dm and was two- and three-times higher compared to those assayed in dried pomace and fruits. In turn, the capability to reduce ferric ions analyzed with the FRAP method in chokeberry powders was 193.69 mmol Trolox/100 g dm and was ca. 3.5-times higher than in dried pomace and fruits. The difference was due to the use of various raw material, because in the work by Horszwald et al. [1
], the dried materials were made of juice, whereas in the present study, they derived from chokeberry fruits. In addition, the results could be affected by differences in the technology of juice production.
2.5. Effect of Chokeberry Pre-Treatment on the Content of Phenolic Compounds and Antioxidant Activity in Final Products
The results support the feasibility of modifying the production process of chokeberry juices and powders. The JUF enabled positive results to be achieved, as the juice was characterized by a lower content of compounds responsible for its pungent and bitter taste. In turn, pomace turned out to be a better raw material than fruits for the production of chokeberry powders, because it is easier to dry, requires less time and energy to evaporate water and contains more bioactive substances.
In order to depict differences in the contents of the analyzed components depending on the method of fruit pre-treatment for the production of chokeberry juices and powders, their percentage contents were calculated. The degree of fruit disintegration affected the juice pressing yield. The yield of JCF was 67%, which was significantly higher compared to the yield of JUF (47%). Large differences were found in the contents of sugars and polyphenols between juices obtained in particular variants. A greater amount of sugar (56%) migrated to JCF than during JUF (44%). The pressing of crushed fruits results in a higher content of polyphenols (70%) in juices. In the case of pressing the whole fruits, only 30% of these compounds were found in juice. This way of juice production contributed to a significantly higher content of bioactive compounds remaining in the pomace. Owing to this, the production of chokeberry PPUF material has two advantages. These juices have lower contents of PC and, therefore, are characterized by a milder taste. These compounds are present mainly in fruit skin and remain in pomace after pressing. This makes the pomace a valuable material for the production of powders with a high biological activity [34
]. In addition, during drying, the highest volume of water was evaporated from whole chokeberry fruits, i.e., 81%, whereas in pomace from whole fruits, it was 70%, and in pomace from crushed fruits, it was 56%. The lower the water content in the raw materials was, the higher was the yield of the drying process, i.e., 18.8%, 30.1% and 43.5%, respectively. The drying process for chokeberry pomace is easier and faster, and the pomace required the evaporation of a significantly smaller volume of water because ca. 50% of water was eliminated during drying.
The berry pre-treatment step significantly (p
< 0.05) affected the content of PC and AA in the final product obtained from PPUF and the contents of TPC and AA in the products from PPCF. A similar effect on PC was observed in blueberry products [38
]. The effects of berry pre-treatment on AA were previously described by other authors [39
]. Moreover, pre-treatment of berries before pressing significantly (p
< 0.05) affected the contents of PC and AA in the final juice obtained from crushed fruit compared to juice from uncrushed fruit.
The pre-treatment of berries significantly (p
< 0.05) affected final PC and AA in powders and juice. The powders obtained from uncrushed fruit showed significantly more (1.4-times) AA and 1.6-times more PC than crushed berries. Positive correlations were found for TPC content (total phenolics, anthocyanins, flavan-3-ols, flavanols and PA) and AA (the ABTS and FRAP methods) (Table 5
). These correlations showed that the AA of chokeberry products depends mainly on the content of PC.