3.2. Changes of Anthocyanins during Blueberry Juice Processing
Anthocyanin amounts were measured to determine the effects of processing on juice quality. Using the semi-quantitative LC–MS/MS peak area abundance data to compare anthocyanins, the most abundant major anthocyanin identified in the control rabbiteye sample was malvidin-3-pyranoside (
Table 3). Malvidin-3-galactoside was the most abundant anthocyanin found in NHB blueberries [
37]. Although peak resolution did not allow for the separation of the two 6 carbon sugars, herein, malvidin-3-galactoside was included in the recovery of malvidin-3-pyranoside. The least abundant major anthocyanin identified was peonidin-3-arabinoside. Comparing these results to a study by Srivastava et al. (2007) [
26], malvidin-3-pyranoside was also found to be the largest anthocyanin recovered; however, they concluded that cyanidin-3-pyranoside was the least abundant anthocyanin in “Tifblue” RAB juice.
After pressing, a large percentage of anthocyanins were not transferred to the PJ from the whole berry (control). With one exception, there was an approximate 70% decrease between control and PJ in all anthocyanins (
Table 3). However, malvidin-3-pyranoside (Mal-3-pyr) only had a 50% loss. Peonidin-3-arabinoside (Peo-3-ara) had the greatest loss (74%), decreasing from 5.1 ± 0.9 × 10
5 to 1.3 ± 0.2 × 10
5 counts. The anthocyanin decreases between the PJ and the pressed pasteurized unfiltered juice (PPJ) were minimal and not significantly different. This may be explained by the increased heat stability caused by possible polymerization and/or co-pigmentation of the anthocyanins. Co-pigmentation of anthocyanins with other anthocyanins and polyphenols increases the molecule’s heat stability [
45].
After the initial decrease from pressing the berries, the second greatest decrease in anthocyanins occurred when ultrafiltered juice was pasteurized (UFP). The major anthocyanins significantly decreased 40% to 53% after pasteurization in the UFP samples (
Table 3). Major anthocyanins containing delphinidin had the greatest degradation, with delphinidin-3-pyranoside (Del-3-pyr) decreasing from 8.4 ± 1.3 × 10
5 to 4.0 ± 0.6 × 10
5 counts. Delphinidin was less stable in elevated temperatures which resulted in greater decreases in relation to the other anthocyanins due to its greater heat lability [
26].
The minor anthocyanin trends after pressing were opposite compared to the major anthocyanins. Both cyanidin-3-(p-coumaroyl-glucoside) (Cya-3-cou) and petunidin-3-(p-coumaroyl-glucoside) (Pet-3-cou) increased (occasionally significantly) ~33% in PJ samples from their initial control levels. Delphinidin-3-(p-coumaroyl-glucoside) (Del-3-cou) significantly increased 50% from 0.5 ± 0.1 × 10
5 to 1.1 ± 0.3 × 10
5 counts after pressing (
Table 3). These increases may be due to the heat degradation process by which anthocyanins break down into chalcone structures which can then undergo transformation into a coumarin glucoside [
46]. One possible cause of the recoveries for the minor anthocyanins through processing involves the acylation of the anthocyanin molecule and its ability to increase stability by preventing hydration when exposed to pH changes and heat [
44,
47]. On the other hand, the greater anthocyanin degradation in the UFP juices as compared to the PPJ juices may be directly related to the role that co-pigmentation and anthocyanin polymers and/or polymerization play in the heat stability of the compounds themselves. Ultrafiltration removes many of the higher molecular weight co-pigments and polymers from the juice. It can be assumed that the larger the polymer or co-pigment, the greater the increase in heat stability of the compounds included in the polymer [
45].
There was a significant 61% decrease in total anthocyanins between the control and PJ samples in the juice processing steps. The control berries contained 185.2 ± 34.0 × 10
5 counts min
−1, while the PJ juice contained only 72.5 ± 13.4 × 10
5 counts (
Table 3). This was expected due to the large percentage of anthocyanins that remain in the press cake [
48]. There was an insignificant concentration increase in the amounts of anthocyanin content of the UF samples, as compared to the PJ (
Table 3). A concentrating effect regarding monomer anthocyanins has been observed in grape juice filtration using varying sizes of membranes [
49]. Utilization of filtration to increase the concentration of beneficial compounds in juices is a concept that is being explored in other fruits and can increase juice nutritional value [
50,
51,
52,
53]. Polyphenolics in the retentate (recirculated to the 100 L hopper) from the filtration process were not evaluated in this experiment. However, since sugars from controls (12.7 °Brix) increased in the residual retentate (16 °Brix), the residual retentate presumably accumulates other possible value-added by-products that the producer may capitalize upon, in addition to the press cake. Removal of the anthocyanin polymers could be utilized as more stable natural food colorings [
51]. Ultrafiltered juices (UFP) had significantly greater anthocyanin degradation due to pasteurization compared with the PPJ juice. A 40% decrease in total anthocyanins after pasteurization of UF juices, similar to microfiltered pomegranate juice [
54], may be attributed to anthocyanin degradation from pasteurization due to the removal of the co-pigmentation of the anthocyanins by ultrafiltration [
55].
Significant anthocyanin losses have also occurred in NHB blueberry juice and products [
19,
37,
48,
56,
57]. There was 58.8% loss of monomeric anthocyanins in enzyme-treated, non-clarified, batch-pasteurized NHB blueberry juice prepared from frozen blanched berries, but only 27.5% loss in centrifuge-clarified juice [
48]. A slightly different process using thawed, blanched NHB blueberries treated with pectinase, pressed, then clarified and followed by HTST (90 °C) pasteurization resulted in higher losses of 77.6% and 84.3% anthocyanin glycosides and anthocyanins, respectively [
19]. There was a 68.0% total anthocyanin loss in juice created with individually quick frozen (IQF) NHB blueberries that were partially thawed, heated to 43 °C, treated with pectinase, pressed, then HTST pasteurized (90 °C) [
37]. In puree/juice made with NHB berries that were manually crushed and machine-juiced, not filtered or clarified, then batch-pasteurized at 92 °C, there was a substantial 95.7% loss of total anthocyanins [
57]. In addition, there was a 76.2% and 79.1% anthocyanin loss in Powderblue and Tifblue RAB blueberry varieties, respectively, that were thawed 12 h at 5 °C, hot water blanched, homogenized in a blender, pectinase added, centrifuged, then batch-pasteurized (85 °C) [
26]. Overall, significant anthocyanin “losses” occur in processed blueberry juices and, herein, we did not prevent losses using a heated mash and UF with NFC juices.
The MS/MS PRC samples had unusually low anthocyanin peak area abundance amounts reported overall, compared to the remaining processing steps (
Table 3). However, the low reports of anthocyanins in the PRC were likely due to problems with the extraction method itself (discussed below in
Section 3.3.1) and did not reflect the actual amounts of anthocyanins found in the PRC. As discussed below, anthocyanidin recoveries from the PRC support the supposition that our extraction protocols for MS/MS PRC samples versus the liquid UPLC–UV samples were not comparatively effective, because the majority of anthocyanidins indeed remained in the PRC.