Comparison of berry juice concentrates and pomaces and alternative plant proteins to produce spray dried protein–polyphenol food ingredients

Roberta Targino Hoskin , Jia Xiong and Mary Ann Lila *
Plants for Human Health Institute, Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, 600 Laureate Way, Kannapolis, NC 28081, USA. E-mail:

Received 19th July 2019 , Accepted 7th September 2019

First published on 9th September 2019

Spray dried functional food ingredients were prepared by complexing alternative plant protein sources – buckwheat flour alone or blended with pea and rice proteins, with polyphenol sources – blueberry, cranberry and purple muscadine grape extracts from juice concentrates and pomaces – to create colloidal aggregate powders. When fruit pomaces (rather than juice concentrates) were used as polyphenol resources, solid recovery was significantly enhanced, especially for matrices made with pea protein, buckwheat flour or pea–buckwheat blends (over two fold for pea protein–berry pomace aggregates). Polyphenol content and DPPH radical scavenging capacity were, in general, significantly greater for pomace-derived protein–polyphenol aggregates compared to those made with juice concentrates. In particular, the particles produced with muscadine grape pomace presented the highest (p < 0.05) phenolic content (147.3–174.3 mg g−1, 19.4–20.4 mg g−1 and 16.3–21.4 mg g−1 for total phenolic content, anthocyanins and proanthocyanidins respectively), and antioxidant activity (408.9–423.3 μmol TE per g) as well as good spray drying yield (38.6–63.4%). Buckwheat flour, despite its relatively low protein content (13.7%) relative to pea and rice protein isolates (84% and 89%, respectively) still demonstrated high capacity for sorption of flavonoid phytoactive compounds from the berry fruits. These results suggest an efficient plant-based approach to produce value-added protein–polyphenol aggregates with broad utility as healthy food ingredients.


There is a growing movement toward animal-free healthy ingredients and foods. This increasing trend comes at a time when there is an urgent need to address global health and environmental challenges. In fact, the food production sector has been regarded as one of the main causes of global climate change and plant-based diets are seen as a way to support agricultural practices that help promote sustainable energy, water and land use.1,2 Solid evidence supports a clear link between a dietary shift towards plant-based diets and better quality of life, longevity, lower risk of cardiovascular diseases and other health anomalies.3–5

In response to this marketplace demand, innovative sources of plant proteins have attracted the interest of health-conscious consumers, researchers and industry.6 Buckwheat, for example, is a gluten-free pseudo cereal with high nutritional value. It has been “rediscovered” due to its health benefits, sustainability and potential as a functional food source with a well-balanced amino acid profile high in lysine, polyunsaturated fatty acids, minerals and flavonoids, mainly rutin.7,8 It can grow in harsh soil and climatic conditions,9 and even though the USA is the sixth largest buckwheat world producer, it is still considered as a minor crop in the country.10 Historically, it has been a popular crop in Europe and Asia, and its world production has increased in the last decade.10 In addition, pulses like pea are considered “super foods” due to their relevant macro and micronutrients supply.11 These so-called “tiny powerhouses” are rich sources of both protein and fiber,12,13 and therefore, very effective in increasing satiety, reducing calorie intake, and managing body weight.14 In addition, the affordability, sustainability, and low environmental impact aspects of pulses in agricultural production15–17 justify their label as ‘nutritious seeds for a sustainable future’ by the Food and Agriculture Organization of the United Nations.18 Cereals like rice are short in lysine, but provide ample amounts of the amino acids missing in other plant proteins, so the combination of different plant sources can result in protein blends with a more complete amino acid composition and enhanced functionality.19

Berries have been a hot health research topic for decades due to their phytochemically-rich composition. Blueberries, with their diverse flavonoid profiles and high content, are one of the world's best recognized functional foods, still rising in consumer popularity.20–22 Similarly, cranberry, one of the top-ranked healthy fruits, is rich in anthocyanins and anti-infective A-type proanthocyanins.23,24 While muscadine grape, native to the Southeastern United States, is not as well-known as the other fruits, it has an inherently unique phytochemical profile rich in ellagic acid.25,26 The health-protective constituents found in berry fruits are also present in the pomaces, a waste product of the juicing industry consisting of residual berry skins, seeds and fibers.27–29 Berry-derived polyphenols from both fruits and pomaces are an attractive resource for development of novel ingredients for enrichment of packaged or processed foods.30

Our research group previously introduced an efficient strategy to capitalize on the natural affinity of medium polarity polyphenols to proteins, by formulating protein–polyphenol aggregates that deliver high quality edible protein as well as antioxidant and anti-inflammatory polyphenols.31–33 The green-chemistry complexation strategy can be adapted for specific food applications, such as plant-based foods and ingredients. Recently, we also demonstrated that spray drying, a popular industrially-friendly technique, is an efficient, rapid method to successfully produce stable phytochemical-rich protein–polyphenol aggregates.34,35 When spray drying protein–polyphenol complexes, no additional drying carriers are needed. The protein itself, as part of the pre-aggregated particles, acts as a smart drying aid, eliminating the need for addition of maltodextrin or other carbohydrate-based carrier commonly used in the food industry.36

In this work, we developed health-relevant protein–polyphenol aggregate particles based on berry (blueberry, cranberry and muscadine grape) flavonoid phytochemicals (from juice concentrates or pomaces) and alternative sustainable plant protein sources (buckwheat flour, pea protein isolate, rice protein isolate, or blends), characterized particle phytoactive content and bioactive potential, and identified particle formulations with the most desirable technological and phytochemical attributes to produce value-added plant-based ingredients for functional food innovations.

Materials and methods


Wild blueberry (Vaccinium angustifolium Aiton) juice concentrate (BJ, 65°Brix) and pomace (BP) were provided by Wyman's of Maine (Milbridge, ME, USA). Cranberry (Vaccinium macrocarpon Ait) juice concentrate (CJ, 50°Brix) and pomace (CP) were obtained from Ocean Spray Cranberries Inc. (Lakeville-Middleboro, MA, USA). Purple muscadine grape var. Noble (Vitis rotundifolia) juice concentrate (MJ, 70°Brix) and pomace (MP) were received from Muscadine Products Corporation (Wray, GA, USA). Upon receipt, all ground fruit pomaces (consisting of residual pulp, skin and seeds) were lyophilized. The juice concentrates and lyophilized fruit pomaces were kept frozen at −20 °C until use.

All protein sources were provided by Standard Process (Palmyra, WI, USA): buckwheat flour (B), 13.7% protein; pea protein concentrate (P), 83.6% protein and rice protein concentrate (R), 80% protein. In total, five experimental protein groups were tested: each protein source alone and two additional blended groups consisting of calculated amounts of buckwheat flour and pea protein (PB) or rice protein (RB) in order to reach a final protein concentration of 70%.

Preparation of protein–polyphenol aggregates

From juice concentrates. Initially, the blueberry, cranberry and muscadine grape juice concentrates were diluted 10, 5 and 10 times their volume in water, respectively. Juice concentrates were diluted because (a) their high viscosity would otherwise interfere with mixing and complexation and (b) to normalize total solids content (7.5–10 g per 100 mL) among juice concentrates and pomace extracts. After thorough homogenization, 10% (w/v) of each one of the five protein-rich groups was complexed with the diluted blueberry, cranberry or muscadine grape juices. After complexation, the solutions were homogenized using a magnetic stirrer and centrifuged at 8500 rpm for 30 min at 15 °C. The supernatant (containing most of the dissolved sugars from the juice) was discarded and the pellet was re-suspended in water to a volume corresponding to twice its weight. After vigorous homogenization for 30 min, the final solution was submitted to spray drying.
From fruit pomaces. Lyophilized pomaces were used to prepare concentrated fruit pomace extracts using a previously optimized protocol.35 Briefly, the lyophilized pomaces were blended with 50% ethanol solution (1[thin space (1/6-em)]:[thin space (1/6-em)]5 extraction ratio w/v). The mixture was transferred to a water bath at 80 °C for 2 h, and filtered under vacuum, followed by centrifugation at 4000 rpm for 20 min and ethanol evaporation in a rotary evaporator. Before spray drying, 10% (w/v) of protein-rich substrate was added to each of the concentrated liquid blueberry, cranberry or muscadine grape pomace extracts, then homogenized for 30 min using a magnetic stirrer to allow binding between proteins and polyphenols.
Spray drying. The protein–polyphenol aggregate particles were prepared by spray drying each one of the prepared solutions. Samples were coded according to the fruit (polyphenol) source (B (blueberry), C (cranberry) or M (muscadine grape) followed by J (juice concentrate) or P (pomace)) and the protein source (Pea (pea protein), Rice (rice protein), Buck (buckwheat flour), PB (blended pea + buckwheat) or RB (blended rice + buckwheat)). In total, 30 experimental protein–polyphenol groups using fruit juice concentrates or pomaces were prepared and analyzed.

The spray drying process (spray dryer B-290, Buchi Labortechnik AG, Switzerland) used air in co-current flow under the following previously optimized conditions: 1.5 mm nozzle, 30 mL min−1 of feed flow (controlled by a peristaltic pump) kept under constant magnetic stirring, drying air inlet temperature of 165 °C. The resulting spray dried protein–polyphenol aggregates were collected from the collection chamber, weighed, immediately sealed in Ziploc® bags, and stored at −20 °C until further analysis.

Percentage of solids recovery (spray drying yield). The production yield of spray dried (SD) protein–polyphenol matrices, expressed as percentage (%) of solids recovery, was determined as the ratio between the mass of powder obtained after drying and the weight of total solids in the feed solution according to a previously described protocol.37

Phytochemical content of protein–polyphenol aggregates

Elution of phytochemicals bound to aggregates. Briefly, aliquots (0.5 g) from each protein–polyphenol SD matrix were eluted with 8 mL 1% acetic acid in 80% methanol in water with sonication for 5 min at 55 °C followed by centrifugation for 10 min. The supernatant was collected and the procedure was repeated two additional times. The eluates were pooled together and brought to 25 mL in a volumetric flask with the extraction solvent.
Total polyphenol content (TPC). The TPC of the juice concentrates and pomace extracts (blueberry, cranberry and purple muscadine grape), protein sources (buckwheat flour, pea protein, and rice protein) and protein–polyphenol aggregates were quantified spectrophotometrically (Spectramax Plus 384, Molecular Devices, Sunnyvale, CA) by an adapted Folin–Ciocalteau method35 using microplates. Samples were read at 765 nm against a gallic acid (Sigma-Aldrich, St Louis, MO, USA) standard curve and results were expressed as gallic acid equivalent (mg GAE per mL or mg GAE per g sample).
Anthocyanin (ANC) and proanthocyanidin (PAC) determination by HPLC. Anthocyanins were analyzed by HPLC-DAD using an Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a photodiode array detector. Samples were filtered through a 0.2 μm PTFE syringe filter (Fisher Scientific, Fair Lawn, NJ, USA) and separation was performed using an RP Supelcosil-LC-18 column, 250 mm × 4.6 mm × 5 μm (Supelco, Bellefonte, PA, USA). The mobile phase consisted of 5% formic acid in H2O (A) and 100% methanol (B). The flow rate was 1 mL min−1 with a step gradient of 10, 15, 20, 25, 30, 60, 100, 100, 10 and 10% of solvent B at 0, 5, 15, 20, 25, 46, 50, 50, 57 and 60 min, respectively, at constant temperature (30 °C). Chemstation software was used to manage the HPLC data. The results were expressed in mg cyanidin-3-O-glycoside equivalence established from a standard curve built with concentrations between 2–175 μg μL−1. Proanthocyanidin separation was performed using an Agilent 1200 HPLC with fluorescence detector (HPLC-FLD), and a Develosil Diol normal phase column (250 mm × 4.6 mm × 5 μm, Phenomenex, Torrance, CA, USA). The binary mobile phase consisted of (A) acetonitrile/acetic acid (98[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v) and (B) methanol/water/acetic acid (95[thin space (1/6-em)]:[thin space (1/6-em)]3[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v/v). The flow rate was 0.8 mL min−1 with linear gradient at 35 °C, as follows: 0–35 min, 0–40% B; 35–40 min, 40–100% B; isocratic 100% B, 45 min; 100–0% B, 50 min; and 0% B to 55 min. Fluorescence detection was set at 230 nm excitation at and 321 nm emission.38 PAC components with different degrees of polymerization (DP1, DP2, DP3, DP4, DP5 and DP > 5) were identified and quantified with a calibration curve of procyanidin-B2 (0.05–0.50 mg mL−1).
Free radical scavenging activity with 2.2-diphenyl-1-pricrylhydrazil (DPPH) assay. Antioxidant capacity was determined based on a previous protocol.39 Briefly, 20 μL of eluted samples were added to 180 μL of DPPH solution (150 μmol L−1) in methanol–water (80[thin space (1/6-em)]:[thin space (1/6-em)]20, v/v) in a 96-well microplate. After 40 min in the dark at room temperature, the absorbance was measured at 515 nm. Solutions of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) at different concentrations (100–500 μM) were used to build a calibration curve and results were expressed as μmol Trolox equivalents (TE) per g.
Statistics. Results were expressed as means ± SEM. Two-way ANOVA, post hoc analyses of differences between experimental groups and Pearson's correlation analysis were conducted by Tukey's multiple comparison test using the software Prism 8.1.2 (GraphPad Software, San Diego, CA, USA). The principal component analysis (PCA) was carried out on the quantitative data obtained by the HPLC-DAD analysis for anthocyanins, and proanthocyanidins compounds. The dataset was organized in a matrix with 30 lines corresponding to protein–polyphenol aggregates and 31 columns corresponding to anthocyanin and proanthocyanidin data. The PCA calculation was performed using the software SPSS 24 (SPSS Inc., Chicago, IL, USA).

Results and discussion

Phytochemical content of polyphenol and protein substrates: total polyphenols, total anthocyanins and total proanthocyanidins

Prior to the complexation process to formulate the protein–polyphenol aggregates, all original substrates – polyphenol and protein sources – were analyzed with regard to their phytochemical content. Table 1 shows the concentration of phytochemicals inherent in all fruit substrates. The polyphenolic sources chosen for this study have some overlapping chemical characteristics, but important differences between them can be highlighted. Cranberry juice concentrate and pomace had inherently lower anthocyanin content relative to the other fruit tissues, but importantly had significant procyanidin content.40,41 Blueberry and muscadine grape (juice concentrates and pomaces) were characterized by high concentrations of anthocyanins (p < 0.05), while proanthocyanidins were relatively low in muscadine juice concentrate (Table 1). Similar to our results, previous reports showed that blueberry has a more concentrated anthocyanin content than cranberry.42 It is noteworthy that the juice-making process involves several sequential processing steps (thawing, milling, pressing, depectinization, centrifugation, evaporation and pasteurization) that can each affect the composition (e.g. as total solids and °Brix) of the final product. For example, cranberry juice concentrate undergoes a depectinization step involving heating and enzyme addition, that can potentially diminish the anthocyanin content of the final product.40,42 A significant content of the biologically active compounds, such as fibers, polyphenols and other phytoactives remain in the pomace generated as by-product of the industrial juicing process. For instance, blueberry pomace retains 25% to 50% of the proanthocyanidins present in fresh blueberries.43 In addition, skins and seeds of muscadine grape concentrate large amount of phenolics such as anthocyanins, but these materials (pomaces) are often discarded as juicing processing waste.44
Table 1 Total phenolics, anthocyanins and proanthocyanidins and level of proanthocyanidin polymerization in wild blueberry, cranberry and purple muscadine grape juice concentrates and pomacesa
a Results expressed as mean ± SEM. a–fMeans with different letters within the same row are significantly different at p < 0.05 by two-way ANOVA and Tukey's test. Sample identification: first letter B (blueberry), C (cranberry) or M (muscadine grape) followed by J (juice concentrate) or P (pomace); DP: degree of polymerization. Total phenolic content (TPC) quantified by Folin–Ciocalteau assay and expressed as gallic acid equivalent, total anthocyanin (ANC) quantified by HPLC-DAD as cyanidin-3-O-glucoside equivalents, proanthocyanidin (PAC) quantified by HPLC-FLD as proanthocyanidin B2 equivalents.
TPC, mg g−1 244.2 ± 3.3b 97.8 ± 1.8e 121.4 ± 5.2c 108.1 ± 2.9d 91.4 ± 4.1f 266.5 ± 5.0a
ANC, mg g−1 46.2 ± 5.5a 1.7 ± 0.1b 20.7 ± 2.9a 24.9 ± 0.2a 2.2 ± 0.1b 28.3 ± 4.6a
PAC, mg g−1 36.4 ± 4.7a 16.9 ± 0.5b 8.5 ± 0.7c 17.2 ± 0.4b 36.5 ± 1.8a 33.0 ± 1.4a
Proanthocyanidin composition (% of total PAC)
Monomers 27.1 11.3 20.7 9.3 10.7 52.2
Dimers 14.3 11.6 4.6 5.9 17.3 11.6
Trimers 7.6 6.3 19.6 4.0 7.4 13.3
Tetramers 7.0 3.9 14.2 3.6 6.2 6.4
Pentamers 5.1 4.1 3.1 3.1 4.7 3.4
DP > 5 39.0 62.9 37.8 74.0 53.6 13.1

As expected, the protein substrates had relatively low polyphenol content. Pea protein and rice protein had only 1.0 and 0.7 mg GAE per g respectively, but the TPC of buckwheat flour was 4 and 5-fold the TPC concentration of the former two (4.1 mg GAE per g). No anthocyanin was detected for any protein source. However, as opposed to pea and rice proteins that had no PAC detected, buckwheat flour showed 2.7 mg PAC g−1. Buckwheat flour is a gluten-free dietary source which has natural levels of health-relevant polyphenolic compounds which have been studied for various food applications.45 Although levels vary depending on the origin of buckwheat flour and method of processing, consistent phytochemical content, mainly rutin and PAC, has been reported in hulls, bran and endosperm of buckwheat seeds.46 For example, similar TPC (3.2 mg GAE per g) and PAC (1.6 mg g−1) values47 and total phenolic content in the range of 5.03 to 16.87 mg GAE per g[thin space (1/6-em)]48 have been previously reported for buckwheat flours.

Anthocyanin and proanthocyanidin profile of juice concentrates and pomaces

Wild lowbush blueberry, cranberry and muscadine grape happen to be the only commercial fruits native to North America and all of them are exceptionally rich flavonoid phytochemical resources. Twenty anthocyanin peaks were identified in blueberry juice concentrate and pomace (Table 2 and Fig. 1) which confirm previous reports on the highly diversified blueberry anthocyanin profile.22,49 As previously demonstrated, the more abundant anthocyanins found, both in blueberry pomace and blueberry juice concentrate, were malvidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-galactoside and petunidin-3-O-glucoside.22,35 The number of different anthocyanins might vary between blueberry varieties, but similar to previous reports, our study showed a large number of anthocyanin compounds, including acylated anthocyanins, in both lowbush (wild) blueberry juice and pomace.29
image file: c9fo01587f-f1.tif
Fig. 1 Typical chromatogram obtained by HPLC-DAD for anthocyanin profile detection in wild blueberry (A), cranberry (B) and purple muscadine grape (C) juice concentrates and pomaces.
Table 2 Concentration of anthocyanins (μg mL−1) identified by HPLC-DAD analysis in wild blueberry, cranberry and purple muscadine grape juice concentrates and pomacesa
a Results expressed as mean ± SEM. Sample identification: first letter B (blueberry), C (cranberry) or M (muscadine grape) followed by J (juice concentrate) or P (pomace). Anthocyanin identification: Del-gal: delphinidin-galactoside; Del-glu: delphinidin-glucoside; Del-diglu: delphinidin-3.5-diglucoside; Cya-gal: Cyanidin-galactoside; Del-ara: delphinidin-arabinoside; Cya-glu: Cyanidin-glucoside; Cya-diglu: Cyanidin-3.5-diglucoside; Pet-gal: Petunidin-galactoside; Cya-ara: cyanidin-arabinoside; Pet-glu: Petunidin-glucoside; Pet-diglu: Petunidin-3.5-diglucoside; Peo-gal: Peonidin-galactoside; Pet-ara: Petunidin-arabinoside; Peo-glu: Peonidin-glucoside; Peo-diglu: Peonidin-3.5-diglucoside; Peo-ara: Peonidin-arabinoside; Mal-gal: Malvidin-galactoside; Mal-glu: Malvidin-glucoside; Mal-diglu: Malvidin-3.5-diglucoside; Mal-ara: Malvidin-arabinoside; DAG: delphinidin-acyl-glucoside; PeoAG: Peonidin-acyl-galactoside CAG: Cyanidin-acyl-glucoside; MAGal: Malvidin-acyl-galactoside; PetAG: Petunidin-acyl-glucoside; MAGlu: Malvidin-acyl-glucoside. ND: Not detected.
Del-gal 200.9 ± 33.1 ND ND 220.5 ± 0.6 ND ND
Del-glu 386.1 ± 6.6 ND ND 291.1 ± 0.5 ND ND
Del-diglu ND ND 470.4 ± 90.2 ND ND 543.0 ± 50.5
Cya-gal 152.2 ± 24.0 28.4 ± 1.5 ND 190.4 ± 1.3 36.0 ± 0.3 ND
Del-ara 169.1 ± 29.5 ND ND 168.3 ± 4.1 ND ND
Cya-glu 268.1 ± 44.1 5.1 ± 0.3 ND 136.5 ± 0.6 3.1 ± 0.0 ND
Cya-diglu ND ND 668.5 ± 11.8 ND ND 242.4 ± 51.7
Pet-gal 145.8 ± 23.5 ND ND 148.3 ± 1.5 ND ND
Cya-ara 130.2 ± 22.1 48.8 ± 3.9 ND 76.4 ± 10.2 24.4 ± 0.1 ND
Pet-glu 334.6 ± 56.6 ND ND 253.4 ± 13.8 ND ND
Pet-diglu ND ND 545.1 ± 11.7 ND ND 621.1 ± 15.2
Peo-gal 60.0 ± 8.9 40.1 ± 2.5 ND 46.1 ± 1.0 55.0 ± 1.1 ND
Pet-ara 94.7 ± 18.2 ND ND 63.1 ± 4.8 ND ND
Peo-glu 165.4 ± 25.2 12.5 ± 0.9 ND 71.3 ± 3.4 9.2 ± 0.2 ND
Peo-diglu ND ND 670.5 ± 14.1 ND ND 300.6 ± 90.9
Peo-ara ND 37.6 ± 2.8 ND ND 26.8 ± 0.3 ND
Mal-gal 339.9 ± 56.7 ND ND 277.2 ± 58.5 ND ND
Mal-glu 590.7 ± 10.0 ND ND 328.3 ± 8.6 ND ND
Mal-diglu ND ND 258.4 ± 5.6 ND ND 332.7 ± 70.0
Mal-ara 191.8 ± 36.1 ND ND 146.2 ± 2.6 ND ND
DAG 130.7 ± 21.1 ND ND 100.9 ± 4.3 ND ND
PeoAG 24.9 ± 8.0 ND ND 12.5 ± 1.0 ND ND
CAG 66.1 ± 2.2 ND ND 42.1 ± 5.7 ND ND
MAGal 85.5 ± 16.4 ND ND 52.9 ± 0.8 ND ND
PetAG 95.4 ± 18.4 ND ND 59.8 ± 0.3 ND ND
MAGlu 261.9 ± 47.5 ND ND 134.5 ± 6.1 ND ND

Recognizing the phenolic composition of a food material is important in order to assess potential health-relevant value.50 The red color of cranberry fruit is due to the presence of four major anthocyanins: cyanidin-3-galactoside, peonidin-3-galactoside, cyanidin-3-arabinoside and peonidin-3-arabinoside.42 While cyanidin-arabinoside was the anthocyanin found at the highest concentration in the cranberry juice concentrate, peonidin-galactoside was the most prevalent in the cranberry pomace. Our results also confirmed the unique anthocyanin profile of purple muscadine grape previously reported in the literature, which consists of diglucosides.51,52 Very high anthocyanin concentrations were observed for all six peaks identified in muscadine grape samples and cyanidin-3.5-diglucoside and peonidin-3.5-diglucosides were predominant in muscadine grape juice. However, as noted above for cranberry, there were differences in the predominant phytochemical profiles between juice and pomace. Delphinidin-3.5-diglucoside and petunidin-3.5-diglucoside were predominant in muscadine grape pomace.

Spray drying yield (solids recovery)

Spray drying is a reliable physical method for preservation of phytoactive compounds in the pharmaceutical and food industries, since hydrophilic and hydrophobic phenolic compounds are easily entrapped, their bioactivity is preserved and their bioavailability can be enhanced.53 The spray drying yield, expressed as the percentage of solids recovery, is an important indicator for the industry, since higher recovery means more produced powder, less waste stream and therefore, more benefit. The most common reason for low product recovery is stickiness, commonly found in sugar and acid-rich food materials. It is especially critical when dealing with fruit-derived products such as juice concentrates, since low molecular weight sugars such as fructose, glucose, and sucrose and organic acids like citric, malic and tartaric acid, constitute more than 90% of the solids in fruit juices and purees. These molecules have low glass transition temperatures (Tg), which means that at temperatures normally used in spray dryers, they tend to stick to the walls of the dryer and decrease the spray drying yield.54

One way to avoid stickiness is to use temperatures substantially lower than the glass transition temperature of the material to be dried. However, this approach is not easily performed. Otherwise, one of the most common procedures is the use of drying carriers. While carbohydrate-based carriers (e.g., maltodextrin, gum Arabic) increase the glass transition temperature of the feed solution, proteins migrate to the droplet–air interface and form a high-protein-content film with high Tg when subjected to hot air inside the dryer. Protein-covered particles are able to resist to sticky interactions with the chamber wall, resulting in higher solids recovery.55,56 The migration to the surface and the formation of this glassy protective layer happens quickly, and in many cases, it is possible to successfully spray dry sugar-rich materials adding less protein when compared to their carbohydrate-based counterparts.36,57 Due to these advantages, proteins have been referred as “smart” drying aids for spray drying.55,56

Overall, the solids recovery was increased when fruit pomace extracts (rather than juices) were used to produce the protein–polyphenol aggregates (Table 3). Even though the juice concentrates were successfully diluted and the protein in the aggregate matrix acted as a drying aid/carrier to minimalize stickiness/hygroscopicity of the spray dried powders, the concentration of sugars and acids in the feed solution was still higher than for the pomace extracts, which were relatively sugar-free. We hypothesize that these sugars, although largely eliminated by separating the pelleted particles from the discarded supernatant, still contributed to a lower overall solids recovery compared to pomace-derived aggregates.

Table 3 Solids recovery (%), concentration of total phenolics, anthocyanins and proanthocyanidins and radical scavenging activity (DPPH) in spray dried protein–polyphenol aggregates produced with wild blueberry, cranberry and muscadine grape juice concentrates and pomaces and pea, rice protein and buckwheat flour or blended protein sourcea
  Protein–polyphenol aggregates Solids recovery, % TPC, mg g−1 ANC, mg g−1 PAC, mg g−1 DPPH, μmol g−1
a Results expressed as mean ± SEM. a–iMeans with different letters within the same row are significantly different at p < 0.05 by two-way ANOVA and Tukey's test. Sample identification: letters before dash identify the protein source: Pea (pea protein), Rice (rice protein), PB (blended pea + buckwheat), RB (blended rice + buckwheat), or buck (buckwheat flour); letters after dash identify the polyphenol source: B (blueberry), C (cranberry) or M (muscadine grape) followed by J (juice concentrate) or P (pomace). Total phenolic content (TPC) quantified by Folin-Ciocalteau assay and expressed as gallic acid equivalent, total anthocyanin (ANC) quantified by HPLC-DAD as cyanidin-3-O-glucoside equivalents, proanthocyanidin (PAC) quantified by HPLC-FLD as proanthocyanidin B2 equivalents; Radical scavenging activity quantified by DPPH assay as μ mol Trolox equivalents.
Blueberry juice concentrate Pea-BJ 33.4 37.5 ± 1.3i 2.2 ± 0.1b,c 1.0 ± 0.1f 23.5 ± 0.5i
Rice-BJ 39.8 37.7 ± 1.4i 1.2 ± 0.2c 1.0 ± 0.0f 23.3 ± 0.6i
PB-BJ 30.0 34.3 ± 0.5i 2.1 ± 0.2b,c 1.0 ± 0.1f 22.4 ± 0.2i
RB-BJ 34.2 38.8 ± 1.0i 1.5 ± 0.1c 1.2 ± 0.1f 24.3 ± 0.3i
Buck-BJ 26.9 39.9 ± 0.9i 2.4 ± 0.2b,c 1.5 ± 0.2f 27.7 ± 0.1i
Cranberry juice concentrate Pea-CJ 30.0 32.5 ± 1.5i 0.4 ± 0.0c 1.7 ± 0.3f 16.7 ± 0.4i
Rice-CJ 44.0 34.4 ± 1.2i 0.2 ± 00c 1.9 ± 0.1f 20.5 ± 0.4i
PB-CJ 33.0 34.2 ± 1.4i 0.4 ± 0.0c 2.0 ± 0.1f 20.1 ± 0.1i
RB-CJ 36.0 35.9 ± 0.8i 0.3 ± 0.0c 1.9 ± 0.1f 20.7 ± 0.4i
Buck-CJ 30.5 41.6 ± 0.9i 0.4 ± 0.0c 2.6 ± 0.1f 26.2 ± 0.5i
Muscadine grape juice concentrate Pea-MJ 30.0 33.0 ± 3.2i 1.5 ± 0.1c 0.4 ± 0.1f 19.2 ± 0.4i
Rice-MJ 44.0 30.5 ± 1.3i 0.9 ± 0.1c 0.3 ± 0.0f 24.5 ± 0.8i
PB-MJ 33.0 31.2 ± 1.2i 1.1 ± 0.1c 0.5 ± 0.0f 21.8 ± 0.5i
RB-MJ 36.0 32.7 ± 0.7i 0.8 ± 0.0c 0.4 ± 0.0f 24.8 ± 0.6i
Buck-MJ 30.5 31.0 ± 0.4i 0.7 ± 0.1c 0.6 ± 0.1f 28.3 ± 0.4h,i
Blueberry pomace Pea-BP 73.0 119.3 ± 1.2g 24.9 ± 2.1a 16.6 ± 2.1c 270.7 ± 10.4d
Rice-BP 25.7 67.8 ± 2.5f 16.7 ± 0.4a,b 4.7 ± 0.5e,f 95.4 ± 11.7n
PB-BP 37.7 107.7 ± 1.8e 24.1 ± 0.8a 13.6 ± 1.6c 206.4 ± 21.4d
RB-BP 33.6 82.5 ± 1.1d 21.3 ± 1.5a 6.3 ± 0.1e 149.2 ± 7.7e
Buck-BP 31.4 99.3 ± 2.5e 21.4 ± 1.5a 9.4 ± 0.7d 200.9 ± 3.3d
Cranberry pomace Pea-CP 61.3 83.6 ± 1.1d,f 2.0 ± 0.1c 16.2 ± 4.3c 96.2 ± 4.2f
Rice-CP 35.2 46.8 ± 0.5i 1.3 ± 0.1c 8.1 ± 0.6e 29.7 ± 0.1h,i
PB-CP 46.9 76.0 ± 1.5f 1.9 ± 0.2c 10.7 ± 1.4d,e 65.7 ± 6.9g
RB-CP 46.3 49.9 ± 0.7h,i 1.3 ± 0.1c 7.9 ± 0.2e 29.6 ± 0.2h,i
Buck-CP 40.4 77.1 ± 2.5f 1.8 ± 0.2c 13.5 ± 2.3c,d 79.9 ± 7.2g
Muscadine grape pomace Pea-MP 63.4 150.0 ± 1.6b 19.4 ± 1.4a 16.3 ± 1.2c 411.7 ± 7.1a,b
Rice-MP 52.0 131.4 ± 1.3c 18.6 ± 1.4a 16.5 ± 0.8c 384.3 ± 9.3c
PB-MP 55.9 147.3 ± 2.1b 20.4 ± 1.1a 17.7 ± 0.9a,b 408.9 ± 1.8b
RB-MP 38.6 133.9 ± 1.5c 18.8 ± 1.3a 15.6 ± 0.8b,c 382.3 ± 8.8c
Buck-MP 38.6 174.3 ± 2.3a 19.7 ± 2.2a 21.4 ± 2.3a 423.3 ± 5.1a

Interestingly, and contrary to our initial assumption, the protein content of the protein substrates (pea, rice, or buckwheat) was not directly correlated to solids recovery. Theoretically, higher protein content would lead to a more efficient formation of the protein-rich glassy film recovering the particles and promote higher solids recovery. Contrarily, although rice and pea protein concentrates have similar protein content (approx. 80%) they behaved differently in this study. While higher yields were observed for rice protein when producing juice concentrate-based particles, pea protein was more effective in terms of solids recovery when complexed with fruit pomaces (Table 3). Furthermore, despite the low protein content of buckwheat flour, it was possible to obtain polyphenol-buckwheat particles with 30–40% solids recovery. Similarly, no linear correlation between protein content and sorbing capacity was observed when producing cranberry juice-based protein–polyphenol particles using different types of protein.58 This indicates that other factors play a role in the complexation process and therefore, influence the capacity of each matrix to capture polyphenol components from the fruit source that in turn would affect the solids recovery.

Phytochemical content of protein–polyphenol aggregates

Overall, the phytochemical contents of the aggregates produced with fruit pomaces were higher than particles produced with fruit juice concentrates (Table 3). This finding is in agreement with visual observations of the spray dried protein–polyphenol aggregates. Independently of the type of protein or fruit used, the aggregates produced with fruit pomaces presented deeper purple (blueberry and muscadine grape pomace) or pink-red (cranberry) coloration, which is an indication of higher concentration of phenolic pigments, mainly anthocyanins. The pomace extract, a low sugar resource, was added directly to the protein source to accomplish complexation, which made the entire procedure, including the preparation of the feed solution and drying itself, easier and more productive. In fact, although already known for their high content of bioactive compounds, fruit and vegetable pomaces are still underexploited and have not yet been fully used in the food industry.59 This is partly justified by the challenges in recovering the bioactives from waste tissues in an efficient, economical and eco-friendly way.60 Here it is shown that the protein–polyphenol technology can be successfully used to concentrate and stabilize pomace-derived flavonoids into protein-rich matrices and expand their utility.

Altogether, the protein–polyphenol particles produced with pea protein, buckwheat flour and their blend (PB) presented the highest TPC, ANC, PAC and DPPH results among all samples produced with fruit pomaces (p < 0.05, Table 3). Moreover, muscadine grape pomace reached statistically higher results (p < 0.05) when complexed with these three protein sources, which suggests a high affinity between muscadine grape polyphenols, pea protein and buckwheat flour that leads to efficient complexation and drying.

Anthocyanin and proanthocyanidin profile of spray dried protein–polyphenol aggregates

In order to provide information regarding the compositional profile of anthocyanins and proanthocyanidins, as well as their relative abundance in the protein–polyphenol particles, further investigation was conducted. Table 4 shows the major anthocyanins identified in the protein–polyphenol particles. Overall, the anthocyanin profiles of the protein–polyphenol particles reflected well the anthocyanins present in each one of the berry sources. For example, malvidin-3-O-glucoside was the most abundant anthocyanin detected in both blueberry juice concentrate and pomace, and the same trend was found for protein–polyphenol particles made with either source of blueberry tissue. Similarly, peonidin-galactoside and petunidin-3,5-diglucoside were the major anthocyanins identified in cranberry and muscadine grape pomaces, respectively, as well as in the colloidal aggregate particles produced from these two substrates (Tables 2 and 4).
Table 4 Major anthocyanins identified by HPLC-DAD analysis in spray dried protein–polyphenol aggregates produced with wild blueberry, cranberry and purple muscadine grape juice concentrates and pomaces and pea, rice protein and buckwheat flour or blended protein sourcea
Protein–polyphenol aggregates Del-glu Del-diglu Cya-gal Cya-diglu Cya-ara Pet-glu Pet-diglu Peo-gal Peo-diglu Peo-ara Mal-gal Mal-glu Mal-diglu
a Results expressed as mean ± SD (mg cyanidin-3-O-glucoside per g). Sample identification: letters before dash identify the protein source: Pea (pea protein), Rice (rice protein), PB (blended pea + buckwheat), RB (blended rice + buckwheat), or buck (buckwheat flour); letters after dash identify the polyphenol source: B (blueberry), C (cranberry) or M (muscadine grape) followed by J (juice concentrate) or P (pomace). Anthocyanin identification: C3G: cyanidin-3-glucoside; Del-glu: delphinidin-glucoside; Del-diglu: delphinidin-3.5-diglucoside; Cya-gal: Cyanidin-galactoside; Cya-diglu: Cyanidin-3.5-diglucoside;Cya-ara: cyanidin-arabinoside; Pet-glu: Petunidin-glucoside; Pet-diglu: Petunidin-3.5-diglucoside; Peo-gal: Peonidin-galactoside; Peo-diglu: Peonidin-3.5-diglucoside; Peo-ara: Peonidin-arabinoside; Mal-gal: Malvidin-galactoside; Mal-glu: Malvidin-glucoside; Mal-diglu: Malvidin-3.5-diglucoside. ND: Not detected; —: values <0.2 mg C3G per g.
Aggregates produced with blueberry juice concentrate
Pea-BJ ND ND ND ND 0.2 ± 0.1 0.3 ± 0.1 ND
Rice-BJ ND ND ND ND 0.1 ± 0.1 0.2 ± 0.1 ND
PB-BJ ND ND ND ND 0.2 ± 0.1 0.3 ± 0.1 ND
RB-BJ ND ND ND ND 0.3 ± 0.1 0.2 ± 0.1 ND
Buck-BJ ND ND ND ND 0.2 ± 0.1 0.4 ± 0.1 ND
Aggregates produced with blueberry pomace
Pea-CJ 2.2 ± 0.2 ND 0.9 ± 0.1 ND 0.7 ± 0.1 2.1 ± 0.2 ND ND 2.4 ± 0.2 3.5 ± 0.2 ND
Rice-CJ 1.7 ± 0.1 ND 0.7 ± 0.1 ND 0.5 ± 0.1 1.4 ± 0.1 ND ND 1.5 ± 0.1 2.1 ± 0.1 ND
PB-CJ 2.2 ± 0.2 ND 0.9 ± 0.1 ND 0.7 ± 0.1 2.1 ± 0.1 ND ND 2.3 ± 0.1 3.2 ± 0.1 ND
RB-CJ 2.1 ± 0.1 ND 0.8 ± 0.1 ND 0.6 ± 0.1 1.8 ± 0.1 ND ND 1.9 ± 0.1 2.8 ± 0.1 ND
Buck-CJ 2.1 ± 0.2 ND 0.8 ± 0.1 ND 0.7 ± 0.1 1.8 ± 0.1 ND ND 1.9 ± 0.1 2.8 ± 0.1 ND
Aggregates produced with cranberry juice concentrate
Aggregates produced with cranberry pomace
Pea-BP ND ND 0.4 ± 0.1 ND 0.3 ± 0.1 ND ND 0.7 ± 0.1 ND 0.3 ± 0.1 ND ND ND
Rice-BP ND ND 0.3 ± 0.1 ND 0.2 ± 0.1 ND ND 0.5 ± 0.1 ND 0.2 ± 0.1 ND ND ND
PB-BP ND ND 0.4 ± 0.1 ND 0.3 ± 0.1 ND ND 0.7 ± 0.1 ND 0.3 ± 0.1 ND ND ND
RB-BP ND ND 0.3 ± 0.1 ND 0.2 ± 0.1 ND ND 0.5 ± 0.1 ND 0.2 ± 0.1 ND ND ND
Buck-BP ND ND 0.4 ± 0.1 ND 0.3 ± 0.1 ND ND 0.7 ± 0.1 ND 0.3 ± 0.1 ND ND ND
Aggregates produced with purple muscadine grape juice concentrate
Pea-CP ND 0.3 ± 0.1 ND 0.4 ± 0.1 ND ND 0.3 ± 0.1 ND 0.4 ± 0.1 ND ND ND
Rice-CP ND 0.2 ± 0.1 ND 0.2 ± 0.1 ND ND 0.2 ± 0.1 ND 0.2 ± 0.1 ND ND ND
PB-CP ND 0.2 ± 0.1 ND 0.3 ± 0.1 ND ND 0.2 ± 0.1 ND 0.3 ± 0.1 ND ND ND
RB-CP ND 0.1 ± 0.1 ND 0.2 ± 0.1 ND ND 0.2 ± 0.1 ND 0.2 ± 0.1 ND ND ND
Buck-CP ND 0.1 ± 0.1 ND 0.2 ± 0.1 ND ND 0.2 ± 0.1 ND 0.2 ± 0.1 ND ND ND
Aggregates produced with purple muscadine grape pomace
Pea-MP ND 4.9 ± 0.3 ND 2.6 ± 0.3 ND ND 5.6 ± 0.4 ND 3.3 ± 0.2 ND ND ND 2.9 ± 0.2
Rice-MP ND 4.8 ± 0.4 ND 2.4 ± 0.4 ND ND 5.5 ± 0.4 ND 3.1 ± 0.2 ND ND ND 2.8 ± 0.1
PB-MP ND 5.2 ± 0.4 ND 2.7 ± 0.4 ND ND 5.9 ± 0.3 ND 3.5 ± 0.2 ND ND ND 3.1 ± 0.1
RB-MP ND 4.9 ± 0.4 ND 2.4 ± 0.4 ND ND 5.5 ± 0.4 ND 3.1 ± 0.2 ND ND ND 2.8 ± 0.1
Buck-MP ND 5.1 ± 0.6 ND 2.5 ± 0.6 ND ND 5.8 ± 0.6 ND 3.3 ± 0.4 ND ND ND 2.9 ± 0.1

Normal phase HPLC was conducted in order to separate proanthocyanidin components in the protein–polyphenol particles according to their degree of polymerization (Fig. 2). Interestingly, all fruit juice concentrates showed a significant amount of highly polymerized PAC (Table 1), but aggregates produced with juice concentrates exhibited different profiles. For example, particles produced with blueberry and muscadine grape juice presented a high concentration of monomers (up to 50% of total PAC) instead of polymerized PAC. All aggregates derived from cranberry juice concentrate exhibited a high concentration of dimers, followed by monomers, similar to previous research when protein–polyphenol aggregates were produced with cranberry juice concentrates and several sources of protein.58 On the other hand, the pomace-derived particles captured the PAC components at levels similar to what was originally present in the pomaces. For instance, wild blueberry pomace particles exhibited a remarkable concentrations of highly polymerized (DP > 5) proanthocyanidins (50–79% of total PAC), while muscadine grape pomace particles presented a consistently high concentration of monomers (69–75% of total PAC), which is close to what was shown in Table 1 for blueberry and muscadine grape uncomplexed pomace extracts, respectively. Moreover, the particles produced with cranberry pomace presented a more diversified profile of PAC polymers, consisting mainly of dimers, monomers and DP > 5, in this order.

image file: c9fo01587f-f2.tif
Fig. 2 Proanthocyanidin (PAC) composition (% of total PAC) in spray dried protein–polyphenol aggregates produced with wild blueberry, cranberry and purple muscadine grape: juice concentrates (A) and pomaces (B). Sample identification: Letters before dash identify the protein source: Pea (pea protein), Rice (rice protein), PB (blended pea + buckwheat), RB (blended rice + buckwheat), or Buck (buckwheat flour); letters after dash identify the polyphenol source: B (blueberry), C (cranberry) or M (muscadine grape) followed by J (juice concentrate) or P (pomace).

Principal Component Analysis (PCA) was used to detect the most prominent phytochemical constituents (anthocyanins and proanthocyanidin components) of protein–polyphenol samples participating in the data variance and to verify whether any of the aggregates could be grouped according to their chemical composition (Fig. 3). It was carried out on two principal components that accounted together for 85.63% of the total variation among samples: the first (PC1; X-axis) and second principal component (PC2; Y-axis) explained 62.76% and 22.87% of the total variation in the data set, respectively. PCA results show the presence of three major groups that are mainly linked to the polyphenol source used to produce the particles: one cluster for aggregates from muscadine grape pomace, a second one for blueberry pomace aggregates and a third group where all the particles produced with juice concentrate clustered together. The protein–polyphenol particles produced with muscadine grape pomace (MP) were found close to the region of anthocyanin diglucosides, indicating that this characteristic is an important compositional attribute of muscadine pomace-based protein–polyphenol aggregates. The blueberry pomace particles were characterized by delphinidin and cyanidin-derived anthocyanins, while the aggregates produced with juice concentrate clustered together in the same region and were characterized by comparably low proanthocyanidin and anthocyanin contents.

image file: c9fo01587f-f3.tif
Fig. 3 Principal component analysis (PCA) loading plot (A) and score plot (B) applied to spray dried protein–polyphenol aggregates produced with wild blueberry, cranberry and purple muscadine grape: juice concentrates and pomaces. A 2D projection is presented using anthocyanin and proanthocyanidin compounds as variables.

Radical scavenging activity of spray dried protein–polyphenol aggregates

The radical scavenging capacities of the protein–polyphenol aggregates produced with juice concentrates and fruit pomaces are shown in Table 3. The term antioxidant refers to substances able to delay or mitigate oxidation reactions. Some of these reactions involve free radicals and lead to structural and functional damage of biomolecules and/or cell structures. Plant-based products have been extensively evaluated for their antioxidant activity and the DPPH method is one of the most popular assays due to its simplicity, low cost and efficacy, mainly in hydrophilic environments. The methodology is used to estimate the ability of antioxidants to scavenge free radicals and it is based on the reduction of a stable radical (DPPH˙) by the action of an antiradical compound present in the plant/food extract.61

Clearly, the antioxidant activity of protein–polyphenol aggregates produced with fruit pomaces was substantially higher than for the particles produced with juice concentrates. Pomace aggregates reached antioxidant activities 10-fold or higher than what was observed for fruit juice concentrates. Among all samples, the highest scavenging activity (p < 0.05) was observed for purple muscadine grape aggregates from pomace, reaching remarkable values around 400 μmol TE per g sample, independently of the type of protein used (Table 3).

A significant linear correlation was found between DPPH and TPC, ANC and PAC (r = 0.97, p < 0.0001; r = 0.72, p < 0.001 and r = 0.76, p < 0.001, respectively) in protein–polyphenol aggregates produced with fruit pomaces. This means that higher phenolic, anthocyanin and proanthocyanidin concentrations lead to higher antioxidant activity or in other words, these phytochemical components play an important role in the free radical scavenging activity observed. On the other hand, no significant correlation was found between TPC, ANC or PAC and the antioxidant activity (r = 0.31, r = 0.34 and r = −0.15, respectively; p > 0.05) in particles produced with fruit juice concentrates. Even though phenolic compounds are recognized as important antioxidants, other compounds such as protein, oligosaccharides and lipid molecules might also possess antioxidant activity. Another important consideration is that free radical scavenging is just one of the several possible mechanisms by which phytoactives exert their biological activity.62


Plant-based protein–polyphenol aggregates were efficiently produced by spray drying juice concentrates and pomaces from wild blueberry, cranberry and purple muscadine grape complexed to pea protein, rice protein, buckwheat flour and their blends. The spray drying process was especially favored when fruit pomaces were used as the polyphenol sources and high solids recovery (>40%) was observed for most of the experimental groups. Moreover, the edible particles produced with all three fruit pomaces presented higher phytochemical content, important health-related compounds and potent antioxidant activity. In particular, the protein–polyphenol particles produced with muscadine grape pomace and pea protein, buckwheat flour and their blend (PB) reached higher TPC, ANC and PAC, as well as higher antioxidant activity when compared to all other groups. The process shown here is based on a green chemistry approach that uses only food-grade reagents and suggests a rational alternative to transform the abundant fruit waste generated by the juicing industry into value-added food ingredients.

Conflicts of interest

There are no conflicts to declare.


We are grateful to Standard Process (Palmyra, WI, USA) for providing the pea protein, rice protein and buckwheat flour and for providing the funding to support this research. We also appreciate the support of Wyman's of Maine (Milbridge, ME, USA), Ocean Spray Cranberries (Lakeville-Middleboro, MA, USA) and Muscadine Products Inc. (Wray, GA, USA) for generously providing the fruit juice concentrates and pomaces utilized in this research.


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