**Changes in Polyphenolics during Storage of Products Prepared with Freeze-Dried Wild Blueberry Powder**

**Laura Lavefve <sup>1</sup> , Cindi Brownmiller <sup>1</sup> , Luke Howard 1,\*, Donovon Reeves <sup>1</sup> , Sean H. Adams 2,3 , Jin-Ran Chen 2,3, Eva C. Diaz 2,3,4 and Andy Mauromoustakos <sup>5</sup>**


Received: 16 March 2020; Accepted: 6 April 2020; Published: 9 April 2020

**Abstract:** Wild blueberry (WBB) powder can be added to the formulation of foods to encourage consumption of health-promoting polyphenolics, but the stability of polyphenolics throughout storage is important. We determined the stability of polyphenolics in five products (ice pop, oatmeal bar, graham cracker cookie, juice, and gummy product) prepared with WBB powder. Samples stored at 21 ◦C, 4.4 ◦C, or −20 ◦C (ice pops only) were analyzed at 0, 2, 4, 6, and 8 weeks for polyphenolic content and percent polymeric color. Total anthocyanins decreased over storage and storage temperatures in all products. However, the ice pop and the refrigerated juice both retained over 90% of their initial total anthocyanin content. The refrigerated oatmeal bar also showed good retention of anthocyanins (86%), but the gummy product retained only 43% and 51% when stored at 4.4 ◦C or 21 ◦C, respectively. The lower amount of polyphenolic compounds recovered in the gummies stored at 4.4 ◦C compared to 21 ◦C may be attributed to reduced extraction efficiency as a result of gel hardening at refrigerated temperature. Chlorogenic acid and flavonols were generally more stable than anthocyanins throughout storage.

**Keywords:** anthocyanin; chlorogenic acid; flavonol; polymeric color; storage; wild blueberry

#### **1. Introduction**

Lowbush "wild" blueberries (*Vaccinium angustifolium* Ait) are considered a nutrient-rich healthy food, due in large part to their exceptional phenolic content [1,2] and antioxidant activity [2]. Lowbush blueberries are particularly rich in anthocyanins and the anthocyanin profile is complex compared with other fruits [3,4]. They contain five of the six anthocyanidins commonly found in nature (delphinidin, cyanidin, petunidin, peonidin, and malvidin), which can have three different sugar moieties attached (galactose, glucose, and arabinose) as well as acyl groups such as acetyl-, malonyl-, or coumaryl- also attached to the sugar moieties [4,5]. Blueberries are also rich in proanthocyanidins [1,6], chlorogenic acid [7,8], and flavonols [7,9].

Diets rich in blueberries or their polyphenolic-rich extracts have been associated with lower cardiovascular risk, weight gain and metabolic syndrome, and neurological diseases (reviewed in [10]). In addition, studies involving blueberries have identified polyphenolic-derived phenolic acids that improve cell differentiation and proliferation of osteoblasts in vitro and promote bone growth and

limit bone loss in rodents [11–13]. These health-promoting effects are due to a myriad of mechanisms associated with blueberry polyphenolics, including prevention of oxidative stress and inflammation, and vaso- and lipid modulation [14–16]. Many human studies reporting positive health outcomes have used freeze-dried wild blueberry (WBB) powder [17–21], 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 (gummy, oatmeal bar, graham cracker cookie, juice, and ice pop) prepared with freeze-dried WBB powder that were evaluated for children's acceptability and desire to eat [22]. 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 [23–25]. 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 [26].

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 [27]. 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 [28]. At present, refrigeration of blueberry products such as jam [24] and juices [29,30] is the best approach to mitigate polyphenolic losses during storage.

This study was undertaken to determine the stability of anthocyanins, flavonols, 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.

#### **2. Materials and Methods**

#### *2.1. Standards and Solvents*

HiActive® North American wild blueberry powder was purchased from FutureCeuticals, Inc. (Momence, IL, USA). Rutin, chlorogenic acid, high performance liquid chromatography (HPLC) grade methanol, HPLC grade acetonitrile, potassium metabisulfite, formic acid, acetic acid, chlorogenic acid and rutin were purchased from Sigma-Aldrich (St. Louis, MO, USA). A standard mixture of delphinidin, cyanidin, petunidin, peonidin, pelargonidin, and malvidin glucosides was purchased from Polyphenols (Sandnes, Norway).

#### *2.2. Preparation of Blueberry Products*

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 [22]. One serving of oatmeal bar, ice pop, and graham cracker cookie was equivalent to one piece each (61 g, 121 g and 50 g, respectively), 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 [31]. The graham cracker cookies and oatmeal bars were prepared with minimal thermal treatment. 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 [22]. This procedure was used to produce juice and ice pop products with no particulates. The formulation was adjusted with water so the anthocyanin content of

the 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. (Momence, IL, USA). 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 (immediately after preparation) and after 2, 4, 6, and 8 weeks of storage.

### *2.3. Extraction of Polyphenolics from Freeze-dried Blueberries and Products*

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 (60:37:3 v/v/v), to the smallest particle size using a Euro Turrax T18 Tissuemizer (Tekmar-Dohrman Corp, Mason, OH, USA) 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 (Thermo Fisher Scientific, Waltham, MA, USA) and reconstituted in 1 mL 5% formic acid in water. All samples were passed through 0.45 µm nylon syringe filters (VWR, Radnor, PA, USA) 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.

### *2.4. HPLC Analysis of Anthocyanins and Chlorogenic Acid*

Anthocyanins and chlorogenic acid were analyzed by HPLC using the method of Cho and others (2004) [7]. Samples (50 µL) were analyzed using a Waters HPLC system (Waters Corp, Milford, MA, USA) 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 C<sup>18</sup> column (Waters Corp, Milford, MA, USA) preceded by a 3.9 mm × 20 mm Symmetry C<sup>18</sup> guard column. The mobile phase was a linear gradient of 5% formic acid (A) and methanol (B) 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 (3.75, 7.5, 15, 30, 60, 120, 240 mg/L; R <sup>2</sup> > 0.9977 for each anthocyanin glucoside) of a mixture of authentic standards (Polyphenols, Sandnes, Norway). Chlorogenic acid was quantified using external calibration curves (4, 8, 16, 32, 64, 128, 256 mg/L; R<sup>2</sup> = 0.9988) of an authentic standard (Sigma-Aldrich, St. Louis, MO, USA). Results are expressed as mg of anthocyanin or chlorogenic acid per g of WBB powder.
