Many human studies reporting positive health outcomes have used freeze-dried wild blueberry powder, which is a natural source of concentrated polyphenolics. However, the freeze-dried WBB powder may be tart or astringent and not always palatable to consume. This can be problematic in feeding trials in children and adults. In our previous work, we developed five food products prepared with freeze-dried WBB powder that were evaluated for children’s acceptability and desire to eat. These results are useful in designing food products as well as menu items that could be used in clinical trials of WBB-rich diets. In addition to evaluating sensory properties, it is important to validate the storage stability of polyphenolics in these products, before use in clinical trials, to ensure that a consistent dose of polyphenolics can be maintained. Blueberry polyphenolics, especially anthocyanins, are unstable in various processed forms such as juices, jams, purees, and canned berries when stored at ambient temperature. Additionally, anthocyanins in freeze-dried WBB powder are susceptible to degradation when stored at ambient temperature with a reported half-life of 139 days at 25 ◦C. The mechanism responsible for loss of anthocyanins during storage is unknown, but anthocyanin losses are commonly accompanied by increased polymeric color values, suggesting that anthocyanins form polymers with proanthocyanidins. In addition to polymerization, many other factors can affect the stability of anthocyanins including exposure to elevated temperatures, light, oxygen, metals, sugars, and ascorbic acid. At present, refrigeration of blueberry products such as jam and juices is the best approach to mitigate polyphenolic losses during storage. This study was undertaken to determine the stability of anthocyanins, flavonols, plastic potting pots chlorogenic acid, and percent polymeric color in five blueberry products prepared with freeze-dried WBB powder.
Gummy, oatmeal bar, graham cracker cookie, and juice were stored at 21 ◦C and 4.4 ◦C and evaluated for anthocyanin, flavonol, and chlorogenic acid content and percent polymeric color over eight weeks of storage. An ice pop product stored at −20 ◦C was evaluated for its anthocyanin and chlorogenic acid content over eight weeks of storage. Samples of juice, ice pop, gummy, oatmeal bar, and graham cracker cookie, each containing 15 g of WBB powder per serving, were prepared and packaged as previously described. One serving of oatmeal bar, ice pop, and graham cracker cookie was equivalent to one piece each , a juice serving was 135 g, and a gummy serving was 7 pieces, or 113 g. The amount of 15 g of WBB powder used in product formulations was calculated and converted from previous animal studies to humans. This involved only the use of brief microwave heating to solubilize the ingredients in order to avoid thermal loss of phenolic compounds, but still obtain a ready-to-consume non-baked product. The blueberry juice and ice pop were prepared with an anthocyanin concentrate, previously extracted from the WBB powder. This procedure was used to produce juice and ice pop products with no particulates. The formulation was adjusted with water so the anthocyanin content ofthe products was equivalent to that found in 15 g of WBB powder per serving. The preparation and processing of the samples for the storage study were performed in two separate experiments, using the same sample of wild freeze-dried blueberries obtained from FutureCeuticals Inc. . The WBB powder was stored at 15.5 ◦C for four months between the two experiments. The samples from Experiment 1 were stored at 21 ◦C and the samples from Experiment 2 were stored at 4.4 ◦C. The ice pop products prepared in Experiment 1 were stored at −20 ◦C. Three samples of each packaged product were evaluated at time 0 and after 2, 4, 6, and 8 weeks of storage.
Polyphenolics were extracted by homogenizing 5 g of WBB-containing food product or 1 g of WBB powder in 25 mL of extraction solution containing methanol/water/formic acid , to the smallest particle size using a Euro Turrax T18 Tissuemizer for 1 min. Homogenates were centrifuged for 5 min at 10,864 × g. The pellet was re-extracted two additional times with 25 mL of extraction solution and centrifuged for 5 min at 10,864 × g. The filtrates were pooled and adjusted to 100 mL with extraction solvent in a volumetric flask. Prior to HPLC analysis, 5 mL of extract were dried in a Thermo Savant Speed Vac Plus SC210A and reconstituted in 1 mL 5% formic acid in water. All samples were passed through 0.45 µm nylon syringe filters into 1 mL HPLC vials prior to HPLC analysis. The ice pop and juice samples did not undergo extraction due to prior extraction of anthocyanins to make the concentrate used in the formulation but were filtered using the 0.45 µm nylon syringe filters prior to HPLC analysis. Anthocyanins and chlorogenic acid were analyzed by HPLC using the method of Cho and others. Samples were analyzed using a Waters HPLC system equipped with a model 600 pump, a model 717 Plus autosampler, and a model 996 photodiode array detector. Separation was carried out at room temperature using a 4.6 mm × 250 mm Symmetry C18 column preceded by a 3.9 mm × 20 mm Symmetry C18 guard column. The mobile phase was a linear gradient of 5% formic acid and methanol from 2% B to 60% B for 60 min at a flow rate of 1 mL/min. The system was equilibrated for 20 min at the initial gradient prior to each injection. Detection wavelengths of 320 nm and 510 nm were used to monitor chlorogenic acid and anthocyanin peaks, respectively. Individual anthocyanin monoglucosides and acylated anthocyanin derivatives were quantified as delphinidin, cyanidin, petunidin, peonidin, and malvidin glucoside equivalents using external calibration curves of a mixture of authentic standards .
Chlorogenic acid was quantified using external calibration curves of an authentic standard . Results are expressed as mg of anthocyanin or chlorogenic acid per g of WBB powder. Flavonols were analyzed by HPLC using the same HPLC system described above according to the method of Cho et al.. Separation was performed at room temperature on a 4.6 mm × 250 mm Aqua C18 column preceded by a 3.0 mm × 4.0 mm ODS C18 guard column. The mobile phase was a linear gradient of 2% acetic acid and 0.5% acetic acid in water and acetonitrile from 10% B to 55% B in 50 min and from 55% B to 100% B in 10 min at a flow rate of 1 mL/min. The system was equilibrated for 20 min at the initial gradient prior to each injection. A detection wavelength of 360 nm was used to monitor flavonol peaks. Flavonols were quantified as rutin equivalents using an external calibration curve of an authentic standard , with results expressed as mg of rutin equivalents per g of WBB powder.An analytical Hewlett Packard 1100 series HPLC instrument equipped with an autosampler, binary HPLC pump,raspberry container growing and UV/Vis detector was used. For HPLC/MS analysis, the HPLC apparatus was interfaced to a Bruker model Esquire-LC/MS ion trap mass spectrometer . Mass spectral data were collected with the Bruker software , which also controlled the instrument and collected the signal at 520 nm. Typical conditions for mass spectral analysis conducted in positive-ion electrospray mode for anthocyanins and negative-ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 mL/min, and a temperature of 300 ◦C. Data were collected in full scan mode over a mass range of m/z 50−1000 at 1.0 s per cycle. Characteristic ions were used for peak assignment. For compounds where chemical standards were commercially available, retention times were also used to confirm the identification of components. The effect of storage time on anthocyanins, flavonols, chlorogenic acid, and % polymeric color in each blueberry product was evaluated using the Fit Model platform of JMP, and the percent retention of each compound after 8 weeks of storage was calculated using the fit model equation. The effect of storage temperature on phenolic compounds stability was not evaluated in this study due to the length of time the WBB powder was stored between processing the products in Experiment 1 and Experiment 2 . During this four-month storage time, the powder stored at 15.5 ◦C presumably absorbed moisture evident by powder clumping, resulting in different amounts of polyphenolics in the products immediately after processing.
Principal component analysis was performed with the total and individual anthocyanins variables, using the Multivariate platform in JMP, on the mean value of each sample per time point and using the correlation method. Correlations among total anthocyanins and percent polymeric color were determined by pairwise correlations method in the multivariate platform of JMP.The WBB powder used to prepare the products contained at least 22 anthocyanins , which were identified by comparing their mass-to-charge values and elution orders with previous studies. Blueberries are unique in that three different sugars are commonly attached to the five anthocyanidins. This was confirmed in our study; however, we were unable to detect peonidin-3-arabinoside using our HPLC method. We were unable to obtain complete separation of all of the anthocyanins present in the extract due to the complexity of the anthocyanin profile. Peak 15 contained two co-eluting compounds, namely cyanidin-3- galactoside and cyanidin-3- galactoside, and peak 18 was composed of three co-eluting compounds, namely delphinidin-3-rutinoside, cyanidin-3- glucoside, and malvidin-3- galactoside. We were unable to identify peak 17, which appeared to be a delphinidin derivative based on its aglycone m/z of 303, but the molecular ion m/z value was ambiguous. Many of the anthocyanins were present in acylated form. Two of the cyanidin glycosides were acylated with malonic acid, whereas delphinidin, cyanidin, and malvidin galactosides as well as petunidin, peonidin, and malvidin glucosides were acylated with acetic acid moieties. The total anthocyanin content of the ice pop over eight weeks of storage at −20 ◦C is shown in Figure 1. The total amount of anthocyanins significantly decreased with storage time , but the percent retention remained high with 93% of total anthocyanins retained in the product after eight weeks. Consistent with our results, total anthocyanin content of frozen blueberries was stable over three months of storage at −20 ◦C . Changes in major individual anthocyanins in the ice pop over eight weeks of storage at −20 ◦C are shown in Figure S1. Most of the individual anthocyanins did not significantly decrease over storage. For the anthocyanins that decreased during storage, their percent retention after eight weeks remained over 87%: malvidin-3-glucoside , malvidin-3-galactoside , cyanidin-3-galactoside , malvidin-3- glucoside , petunidin-3-glucoside .The total anthocyanin content of the juice decreased with storage time for each storage temperature . The total anthocyanin content of juice stored at 4.4 ◦C and 21 ◦C is shown in Figure 2. After eight weeks of storage, the juice stored at 4.4 ◦C retained 90.7% of total anthocyanins compared with control samples , whereas the juice stored at 21 ◦C retained 69.1%. Concentrations of anthocyanins are known to readily decline during storage of blueberry juice at ambient temperature, but refrigeration is an effective treatment to ameliorate anthocyanin losses. Changes in the major individual anthocyanins in the juice stored at 4.4 ◦C and 21 ◦C over eight weeks of storage are shown in Figure S4. At 4.4 ◦C, peonidin-3-galactoside, cyanidin-3-arabinoside, malvidin-3-galactoside, malvidin-3-glucoside, and malvidin-3- galactoside remained stable over the eight weeks of storage. At 4.4 ◦C, all anthocyanins showed >50% retention, with the minimal percent retention being 57.7% for the unknown delphinidin derivative. This compound, however, did not significantly decrease over storage at 21 ◦C, along with the two co-eluting anthocyanins galactoside + cyanidin-3- galactoside. Besides these two compounds, the percent retention of anthocyanins at 21 ◦C ranged from 59% glucoside to 75.5% . The total anthocyanin content of the gummy product decreased with storage time for each storage temperature . The total anthocyanin content of the gummy product stored at 4.4 ◦C and 21 ◦C is shown in Figure 2. After eight weeks of storage, the gummy product stored at 4.4 ◦C and 21 ◦C retained 43.2% and 50.6%, respectively, of their original total anthocyanin content . Consistent with our findings, levels of total anthocyanins declined in gelatin gels prepared with grape pomace extract over 24 weeks of storage at 21 ◦C, with losses most pronounced in gels exposed to neon light.