The Influence of Temperature on the Antioxidants of Aronia mitschurinii Fruit Juice and Drinks

Abstract

Aronia mitschurinii is a cultivated species originating from crossbreeding the wild species Aronia melanocarpa, also known as the black chokeberry, and European mountain ash Sorbus aucuparia. The aronia fruit is dark purple, which can be attributed to the high content of anthocyanins, other flavonoids, and polyphenols. Aronia’s reputation as a superberry entices small farms to use it as a prospective specialty crop. The fruit’s high antioxidant content is a likely ingredient in several new products, such as jellies, jams, vitamin supplements, and beverages. Food applications of any fruit require high-temperature pasteurization as a major step in fruit processing. Many also require cooking. However, there are some major effects higher temperatures may have on antioxidants, such as isomerization and decomposition, both of which result in a loss of bioavailability of these key compounds. Here, we present a detailed investigation of the effect of temperature and time on the antioxidant content of Aronia mitschurinii juice and power aid drink. UV/vis methods were used to determine the total concentrations of anthocyanins, flavonoids, polyphenols, and tannins and to determine the optimal pasteurization and/or thermal processing conditions that would prevent significant loss in the antioxidant content in aronia products.

Introduction

Aronia mitschurinii, the intergeneric hybrid aronia plant, also referred to as the “black chokeberry”, has gained a popular reputation because of its extremely high content of phenolic compounds, which is significantly higher than the antioxidant capacity of famous açai berries.¹ Aronia fruits are characterized by their high content of bioactive substances, including valuable anthocyanins. Because of this, they are highly suitable base ingredients for the development of not only traditional jams, jellies, wine, and confitures but also health supplements and products, such as power aid drinks, which have been of substantial interest for polyphenol-rich berries in the past decade. Among these promising sources of natural antioxidants, aronia solidifies itself as one of the richest sources of polyphenols in the plant kingdom.²Aronia mitschurinii was originally bred in the 19th century by Russian scientist Ivan Mitchurin by crossbreeding Aronia melanocarpa with European mountain ash Sorbus aucuparia and then backcrossing the hybrid to Aronia melanocarpa.³ Several named cultivars are being cultivated for fruit processing throughout the world. It is an ideal plant for organic cultivation because it is somewhat resistant to pests and diseases.⁴ Aronia’s deep purple pigmentation arises from high contents of phenolic phytochemicals, especially anthocyanins,⁵ and was found to be one of the most powerful in vitro.(6) Additionally, it has been shown that the A. mitschurinii fruit contains over 40 times the antioxidant trapping ability of tomatoes.⁷ This Rosaceae family crop has a substantially higher antioxidant content compared to other Rosaceae relatives, such as peaches, apples, and pears.⁸ Aronia’s high content of phenolic antioxidants has stirred up the interest of scientists to study its cultural management, applications, and ability to capture free radicals in the body. From culinary applications, studies have also shown that wines made from the berry had the highest anthocyanin content compared to traditional red and white grape wines.⁹

The distinctive color of the aronia can be attributed to the fruit’s content of anthocyanins.¹⁰ Glycosylation of anthocyanins generally occurs on the 3-hydroxyl of the anthocyanin molecule; however, it can also occur in the 5 or 7 positions of flavylium ions.¹¹Figure 1 displays the common structure of anthocyanins at a pH of ∼3.6, the natural pH of aronia juice.¹²

Figure 1.

Basic structure of anthocyanins-3-glucosides at pH 3.6, the natural pH of aronia juice.

Depending on pH, anthocyanins can be neutral or ionic, and their color can change from deep red to blue or even light pink. Anthocyanins rank at the top among antioxidants due to their greater conjugation, allowing for greater stabilization upon capture of free radicals.

Until recently, the attention of researchers, nutritionists, and general health practitioners has been focused mainly on the role of aronia fruit products in the prevention and treatment of noncommunicable diseases. Antioxidants are important nutrients in the body that prevent oxidation between other molecules by reducing the amount of free radicals, which could be formed within our body by metabolic reactions or cell death.¹³ Antioxidants play a key role in the prevention of cancer, diabetes, and neurological and cardiovascular illnesses, among others.¹⁴ The results from previous studies have proven that crop treatment, the type of soil, the age of bush, and the breed of the berry can influence this fruit’s capacity for antioxidants.¹⁵

Aronia’s reputation of being a superberry leads to it being a likely candidate for small farms looking to increase their revenue. Such small farms often operate as both growers and producers of aronia’s edible products, including pasteurization and cooking. The process of pasteurization or cooking involves thermal exposure and, because of it, likely decomposition of part or all of the antioxidants. Thermal processing used for pasteurization involves heating aronia products to a variation of temperatures between 50 and 150 °C, with temperature depending on desired pH and product shelf life.¹⁵ There are two major influences of high temperatures on the antioxidant content of the fruit: isomerization of antioxidants and chemical decomposition.¹⁶ Anthocyanins, which contain a cinnamoyl derivative, have been proven to overcome this conversion.¹⁷ The reaction of polyphenols actually remained unknown as late as 2009.¹⁸ Theoretical calculations of possible decomposition reactions suggest that acylated and nonacylated anthocyanins cleaved the glucoside bond and then decomposed.^15,19 All calculated pathways start from the loss of the glycosidic linkage at position 3, followed by decomposition to flavonoids and then polyphenols, and finally end in several simple phenolics, including acids. While the first step of the loss of sugar seems to be the only process that is happening during that step, the rest of the stages involve several rearrangements and cleavages, and the scheme in Figure 2 presents a calculated summary of several plausible pathways that may happen all or in part and with several parallel reactions. While the exact kinetics and mechanism have not yet been investigated in detail, practical decomposition data showing the dependence of the antioxidant loss on the time and temperature of processing are valuable for growers and processors of aronia and other anthocyanin-rich superfruits to preserve as many antioxidants as possible in the processing products.

Figure 2.

Calculated pleasurable paths of thermal degradation of the anthocyanin type of antioxidants. Created based on calculations described by Wong et al. (1989) and Patras et al. (2010).

The viability of anthocyanins as a colorant is in part due to their stability. Previous studies have proven this ability to be influenced by pH, temperature, and the presence of light, oxygen, and metallic ions.²⁰ However, one study showed evidence that heat-labile factors can accelerate anthocyanin pigment destruction.²¹ Thermal analysis can provide more insight into the conversion of anthocyanins and the fate of antioxidants in pasteurization and cooking processes.

Here, we present our study of the dynamic change in the antioxidant concentrations of aronia juice, such as polyphenols, anthocyanins, and flavonoids, to withstand certain temperatures during different time periods. Total values of anthocyanins, flavonoids, and polyphenols have been analyzed for the aronia juice as a function of temperature and time to provide pasteurization and heating recommendations for preserving the antioxidant capacity of products. While measurements of the total antioxidant content by groups during the decomposition process provide accurate and useful data regarding the antioxidant loss, it cannot be interpreted into formal kinetics due to the complex character of the antioxidant content in the juice, where several substituted anthocyanins, flavonoids, and polyphenols decompose at the same time, and often, the product of decomposition of anthocyanins may be flavonoids and the product of decomposition of flavonoid may be the simple phenolic. So, the experimental data presented below are interpreted as an overall effect of increase, decrease, or no change in the concentration as a function of temperature and time without claiming particular mechanisms or reaction order.

Materials and Methods

General Procedures

Chemical reagents used included aluminum chloride (99% extra pure, anhydrous granules), ethyl alcohol (99% ACS spectroscopic grade), and quercetin hydrate (95%), which were purchased from Thermo Fisher Scientific. Potassium acetate (certified ACS Crystalline) and sodium carbonate anhydrous (HPLC grade powder) were purchased from Fisher Scientific. Folin and Ciocalteu’s Phenol reagent and gallic acid monohydrate (ACS reagent grade) were purchased from MP Biomedicals (Santa Ana, CA). Sodium acetate (Sigma Ultra minimum 99.0%) was purchased from Sigma-Aldrich. Double distilled water was used in all experiments. A Spectronic 10 Genesys spectrophotometer and Genesys 20 spectrophotometer were used for all UV/vis analyses (the project started with Spectronic 10 Genesys, but it was later replaced by Thermoscientiffic Genesys 20, the updated version of the same instrument with the name changed by the vendor). However, the specs of these two instrument versions and the principles of operation and output are almost the same. A Mettler Toledo pH meter was used to measure pH. Silicon oil for heating bath was purchased from Sigma-Aldrich. A Stuart Advanced Stirring Hot Plate & Free Temperature Controller, Ceramic, 120 V, by Cole-Parmer was used for heating, and time was controlled with a digital timer.

Sample Preparation

The aronia plant production was conducted at the University of Maryland’s Wye Research and Education Center (WyeREC) in Queenstown, MD. The soil was a Mattapex-Butlertown silt loam with a 0–2% slope and a pH of 6.1. Plants were treated with 3 g/bush/year of organic nitrogen. Fruit samples used in this study were taken from harvests in August 2020–2022 plants.

Fruit samples were maintained in a −25 °C freezer before juicing. The aronia fruit was partially defrosted right before juicing. The fruit was ground to a paste using a mortar, and subsequently, the juice was separated by vacuum filtration. For each heating series, fresh samples of the juice were prepared. Afterward, juice samples were stored in Eppendorf vials at −25 °C for a week before further analysis.

A basic power aid drink from aronia juice was prepared this way: a 16 oz bottle (473 mL), total volume consisting of 59 mL of aronia juice, 0.6 mL of stevia, 1 mL of mineral drops, 414 mL of distilled water, and 0.5 g of iodized table salt. Samples of the drink were stored like samples of the juice.

Controlled Heating

Controlled heating took place using a silicon oil bath equipped with a thermoregulator for temperature. The Aronia juice was divided into aliquots of 1500 μL and placed into individual Eppendorf vials to allow triplicate analysis at each time point and temperature series: 80, 90, 100, 110, and 120 °C. Initial measurements of anthocyanins, flavonoids, polyphenols, and tannins were made before the vials were placed in the silicon oil bath and are referred to as “0 min” heating time. Then, the vials were placed into the silicon oil with a magnetic stirrer for heat circulation and wire hooks along the side wall of the oil bath for easy removal.

After the samples’ initial immersion in the oil bath, phytochemical degradation was followed by removing three vials at 5, 10, 20, 30, 60, 90, and 120 min. Each sample vial was cleaned from oil and immediately frozen to stop the degradation process. Collected samples were then stored at −25 °C in the freezer until each type of antioxidant was analyzed, yet the storage time never exceeded 2 days. The collected triplet of vials for each time interval was used in triplicate for UV/vis analysis. The experimental setup is shown schematically in Figure 3.

Figure 3.

Thermal decomposition experimental setup.

Analysis of Total Anthocyanins

The anthocyanin pigment concentration was measured and calculated based on the procedure outlined by Lee et al.²² After they were defrosted in room temperature water, drink samples were vortexed (if needed). A portion of each sample was diluted 200 times into 0.025 M aqueous KCl and 0.4 M aqueous sodium acetate. Each dilution’s UV/vis absorbance was read at 520 and 700 nm using the Thermo Scientific Genesys 20 spectrophotometer. The instrument’s performance was validated by comparison measurements using the Thermo Scientific Genesys 20 spectrophotometer. The anthocyanin pigment concentration (APC) was calculated as cyanidin-3-glucoside equivalents in mg/L using Beer–Lambert’s law:

where A = absorbance = (A520 nm – A700 nm) pH 1.0 – (A520 nm – A700 nm) pH 4.5; MW (molecular weight) = 449.2 g/mol for cyanidin-3-glucoside (cyd-3-glu); DF = dilution factor established above (40×); l = path length in cm; ε = 26,900 molar extinction coefficient, in L/mol/cm, for cyd-3-glu; and 10³ = factor for conversion from g to mg.

Analysis of Polyphenols

Polyphenols were determined by following the standard procedures for the colorimetric analysis of plant materials outlined by Jakobek et al. (2012),²³ Jakobek et al. (2007),²⁴ Wojdyło et al. (2008),²⁵ Kobus et al. (2019),²⁶ Denev et al. (2018),²⁷ and Nowak et al. (2015).²⁸ The standard calibration was created using gallic acid solutions (50–500 mg/L, R²=0.9994) (Figure 4). To a small vial, 20 μL of the liquid sample was combined with 1580 μL of distilled water, then 100 μL of Folin–Ciocalteu’s reagent was added, and the mixture was left to incubate for 5 min at room temperature. Afterward, 300 μL of 20% w/v sodium carbonate solution was added, and the samples were placed in an oven to develop for 30 min at 40 °C. The absorbance was then measured at 765 nm, and the total polyphenol concentration (TPC) in mg of gallic acid equivalent (GAE)/g of juice (J) was then determined using the following equation:

where A = average absorbance and DF = dilution factor.

Figure 4.

Gallic acid linear regression. The standard deviations were calculated for all experiments using the three triplicate results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of the results within the 95% confidence limit.

Analysis of Flavonoids

Flavonoids were determined by following the standard procedures for the colorimetric analysis of plant materials outlined by Woisky and Salatino (1998)²⁹ and Chang et al. (2002).³⁰ The standard calibration was created using quercetin solutions (20–400 mg/L, R²=0.9988) (Figure 5). In a small vial, 200 μL of the liquid sample was combined with 600 μL of 95% ethanol in water, 40 μL of 1 M potassium acetate, and 40 μL of 10% w/v aluminum chloride. After a brief incubation period, 1120 μL of distilled water was added to the vial, and the sample was left to incubate at room temperature for 30 min. The absorbance was then measured at 415 nm, and the total flavonoid concentration (TFC) in milligrams of quercetin equivalent (QE)/g of juice (J) was then determined using the following equation:

where A = average absorbance and DF = dilution factor.

Figure 5.

Quercetin linear regression. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Analysis of Tannins

Tannins were determined by following the standard procedures for the colorimetric analysis of plant materials outlined by Amin (1997),³¹ Antoine et al. (2003),³² and Deshpande et al. (1986).³³ The standard calibration was created using tannic acid solutions (50–500 mg/L, R²=0.9987) (Figure 6). In a small vial, 300 μL of the liquid sample was combined with 100 μL of 0.01 M tetrazolium blue chloride and 100 μL of 0.2 M sodium hydroxide solution. Following a brief incubation period, the samples were then combined with 1500 μL of 80% ethanol in water, and the absorbance was measured at 527 nm. The total tannin concentration (TTC) in milligrams of tannic acid equivalent (TAE)/g of juice (J) was then determined using the following equation:

where A = average absorbance and DF = dilution factor.

Figure 6.

Tannic acid linear regression. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Limits of Detection and Statistics

The limit of detection (LOD) for anthocyanins was determined using the guidelines produced by the Clinical and Laboratory Standards Institute and the examples provided by Ervin and Kariuki.³⁴ To determine the limit of detection for anthocyanin, samples of pure cyanidine-3-glucoside were prepared at a concentration compatible with that in aronia juice. The sample was then serially diluted with its appropriate buffers, 0.025 M aqueous KCl or 0.4 M aqueous sodium acetate. For each standard, absorbance was measured at wavelengths of 520 and 700 nm using a spectrophotometer. When the value of absorbance did not change upon further dilution by a factor of 2, that concentration was defined as the limit of detection for anthocyanin, as shown in Figure 7.

Figure 7.

Cyanidin-3-glucoside LOD. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

The limit of detection for flavonoids was measured using the guideline produced by the Clinical and Laboratory Standards Institute and examples provided by Ervin and Kariuki.³⁴ The quercetin stock solution in 95% ethanol was prepared in triplicate with a concentration of 1500 mg/L measured against a blank of 95% ethanol. The sample was serially diluted by a factor of 2 until the absorbance measurements did not change. The final concentration was defined as the limit of detection for flavonoids. Absorbance was measured at 510 nm, as shown in Figure 8.

Figure 8.

Quercetin limit of detection. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

For the limit of polyphenol detection, the gallic acid stock solution in 20% ethanol was prepared in triplicate with a concentration of 1000 mg/L measured against a blank of distilled water. The sample was serially diluted by a factor of 2 until the absorbance measurements did not change. The final concentration was defined as the limit of detection for the polyphenols. Absorbance was measured at 765 nm, as shown in Figure 9.

Figure 9.

Gallic acid limit of detection. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

For the detection limit for tannins, the tannic acid stock solution in 20% ethanol was prepared in triplicate with a concentration of 1500 mg/L measured against a blank of distilled water. The sample was serially diluted by a factor of 2 until the absorbance measurements did not change. The final concentration was defined as the limit of detection for tannins. Absorbance was measured at 527 nm, as shown in Figure 10.

Figure 10.

Tannic acid limit of detection. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Each heating series was repeated three times, and samples for each heating time were measured by using a spectrophotometer three times. Then, the data were averaged, and all experiments’ standard deviations were calculated.

Results and Discussion

As mentioned by the theoretical calculations,²⁰ anthocyanins break their glucoside bonds, then gradually decompose into flavonoids and polyphenols first, and then decompose into simple phenolics. Aronia serves as a good model for the experimental study of the effect of extreme heating due to its unusually high antioxidant content, especially the content of anthocyanins, which is one of the highest known in fruits. With such a high content, any changes in concentrations, be it total anthocyanins, flavonoids, or polyphenols, can be detected accurately. In this project, harvested aronia fruit samples were immediately placed into −25 °C storage, and following acceptable processes of aronia juicing, the fruit was juiced while being nearly frozen. The portions of juice were heated in a silicon oil bath with thermoregulation, and the heating time was controlled with a timer. The totals of anthocyanins, flavonoids, and polyphenols in juice samples were determined spectrophotometrically using procedures published in the literature and were compared to the values before heating. Because the absolute concentration of antioxidants in aronia depends on the weather, irrigation, and fertilization, the absolute initial concentration of anthocyanins, flavonoids, and polyphenols may vary between harvest years. To normalize the decomposition effect observed for several years of harvesting, graphs representing the decomposition of various antioxidants were normalized as the percentage lost from the starting concentration, allowing for a future comparison between various harvests, plants, and products.

In this article, we report the loss of antioxidants during the decomposition process as a percentage of the original content of this antioxidant. Using this scale provides more accurate data. This is because the total content of antioxidants in a whole not yet decomposed sample may vary depending on the year of harvest, weather, treatment, and other factors. In contrast, the percentage loss of antioxidants depends only on the temperature and time of exposure to heat. For such purpose, the total content of the antioxidant was first determined in fresh, not heated juice/drink. This amount was counted as 100%. For samples after different exposure times to heat, the total content of antioxidants was determined using the same method and then compared to the total of unheated samples and expressed in % wt.

Decomposition of Anthocyanins

The time evolution of the overall concentration of anthocyanins measured at five different temperatures as a function of heating time is shown in Figure 11.

Figure 11.

Temperature dependence of the concentration of total anthocyanins (milligrams of C3GE/L) in aronia juice for the following temperatures: (A) 80 °C, (B) 90 °C, (C) 100 °C, (D) 110 °C, and (E) 120 °C.

As shown in Figure 11, the decomposition is rapid, and concentration decreases significantly after the first 5 min of heating, indicating very fast kinetics of all competing reactions of anthocyanins. The higher the temperature is, the faster is the decomposition. At 80 °C, about 17% of anthocyanins decomposed after 30 min of heat exposure, whereas the loss of half of anthocyanin content took 2 h. At 120 °C, after only the first 5 min of heating, the loss is a substantial 86%. Because anthocyanins are at the top of the decomposition chain, no other decomposition process would replenish the content of anthocyanins as the overall decomposition process happens.

The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Decomposition of Flavonoids

Flavonoids are secondary metabolites that can be found in the form of glycosides or aglycones in many vegetables and fruits.³⁵ Unlike anthocyanin decomposition, both the loss and resurgence of flavonoids were observed. The time evolution of the overall concentration of flavonoids measured at five different temperatures as a function of the heating time is shown in Figure 12. The concentration of total flavonoids was measured for the same samples used for the concentration of anthocyanins. Although the concentration of flavonoids never decreased beyond 50% of its initial concentration, with higher temperatures, the rate of flavonoid replenishment seemed to even increase. This becomes apparent in Figure 12e, as the final concentration of flavonoids at 120 °C after 120 min went on to exceed the initial concentration.

Figure 12.

Temperature dependence of the concentration of total flavonoids (quercetin equiv mg of QE/g of juice) in aronia juice for the following temperatures: (A) 80 °C, (B) 90 °C, (C) 100 °C, (D) 110 °C, and (E) 120 °C. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Flavonoids are intermediates in the degradation process of anthocyanins. At elevated temperatures, flavonoids decompose to polyphenolics, and at the same time, anthocyanins decompose to flavonoids. The nature of the change in the concentration of flavonoids depends on the ratio of the decomposition rates of anthocyanins and flavonoids. If anthocyanins decompose faster than flavonoids, flavonoid concentration is expected to increase with the heating time (Figure 13). If flavonoids (process 2) decompose faster than anthocyanins (process 1), we should expect to see a decrease in the concentration of total flavonoids. For temperatures below 120 °C, the rate of flavonoid decomposition outweighed that of anthocyanin decomposition, resulting in lower final concentrations. However, at 120 °C, the decomposition rate of anthocyanins increases and becomes moderately faster than the decomposition of flavonoids, leading to an increased flavonoid content.

Figure 13.

Possible decomposition effects (decreasing, increasing, or no change) on the concentrations of total flavonoids and total polyphenols. This graph does not claim a particular reaction order and just shows the overall effect.

It is important to point out that trends in Figure 13 only claim the overall effect of increase, decrease, or no changes in the total concentration of the group of antioxidants, whether it is anthocyanins, flavonoids, or polyphenols. Thus, anthocyanins are a group of several major compounds: cyananidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin. Moreover, in the third position, glucoside bonds bond most anthocyanins to sugars. Most are bonded to glucose, but some are bonded to galactose, rhamnose, and arabinose. Each such combination of anthocyanins and sugars will have its own mechanism and kinetics of decomposition. They may be similar, but accurate kinetics cannot be recorded in juice and may be investigated with individual compounds as a separate research project.

To better illustrate this phenomenon, we have performed thermal decomposition of a pure quercetin standard dissolved in water at a concentration similar to the initial concentration of total flavonoids in aronia juice. The results are presented in Figures 14 and 15, where Figure 14 shows the trend in the total content of flavonoids while decomposing our quercetin at the initial concentration, the same as its concentration in juice or extract. In such a case, we had quercetin before heating, and polyphenols were undetected. Also, C3G is not present in this solution, so nothing can produce additional amounts of quercetin during decomposition. As expected, in such a situation, the concentration of quercetin decreases as a function of the heating time. In contrast, in the case of aronia juice or drink, the concentration increased slightly after the initial loss. This increase was more significant at higher temperatures due to the fast decomposition of anthocyanins, resulting in the formation of flavonoids. Figure 15 illustrates the trend in total phenolics during the decomposition of the quercetin standard. Because quercetin decomposes to polyphenols and simple phenolics, the concentration of total phenolics increases slightly.

Figure 14.

Temperature dependence of the concentration of total flavonoids during the decomposition of quercetin standard dissolved in water at 100 °C: top, expressed as a percentage of loss; bottom, expressed in units of concentration (quercetin equiv mg of QE/mL). The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Figure 15.

Temperature dependence of the concentration of total polyphenols during the decomposition of quercetin standard dissolved in water at 100 °C: top, expressed as a percentage of loss; bottom, expressed in units of concentration (quercetin equiv mg of QE/mL). The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Decomposition of Polyphenols

As the temperature and time increase, anthocyanins and flavonoids decompose into polyphenols and then into simpler phenolics. The decomposition process of polyphenols is slower than that of flavonoids and much slower than that of anthocyanins. As a result, the decomposing polyphenols are being replenished by products of the decomposition of flavonoids, and their concentration does not change significantly over the 2 h of heating experiments, as shown in Figure 16.

Figure 16.

Temperature dependence of the concentration of total polyphenols (gallic acid equiv, mg of GAE/g of juice) in aronia juice for the following temperatures: (A) 80 °C, (B) 90 °C, (C) 100 °C, (D) 110 °C, and (E) 120 °C. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Figure 17 shows another model when we only have a solution of gallic acid (from the polyphenol group of antioxidants) but no anthocyanins or flavonoids are present. Pure gallic acid at an initial concentration equal to the average total polyphenol concentration in juice or extract decomposes slowly during the heating, decreasing the concentration of total phenolics. When decomposition was done for aronia juice of extract in the drink, the trend was to no change or a slight increase due to the additional formation of phenolics during the decomposition of flavonoids and anthocyanins in the juice.

Figure 17.

Temperature dependence of the concentration of polyphenols during the decomposition of pure gallic acid dissolved in water at 100 °C: top, expressed as a percentage of loss; bottom, expressed in units of concentration (mg of GAE/mL). The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Decomposition of Tannins

Tannins are characterized by a slight increase in concentration followed by a steady decrease at higher temperatures, as shown in Figure 18. This initial spike in concentration is not as prevalent in samples heated at low temperatures, making them ideal for steadily decreasing the tannin content of juice samples for short-term or long-term decomposition. At the highest temperatures, a trend similar to that of flavonoids was observed as tannins jumped to over 125% of their initial concentration. This observation has an important application to producers’ practice. While tannins are valuable antioxidants, their taste is bitter at high concentrations, and it is desirable to reduce the presence of tanning in edibles created from aronia to improve the taste. Processing at 110 °C helps to reduce the content of tannins more than twice over 1 h, while concentrations of flavonoids and polyphenols are not affected very significantly. The only antioxidants that will be affected are anthocyanins. In applications where the content of anthocyanins is less important, reducing the amount of tannins will help improve the taste of products.

Figure 18.

Temperature dependence of the concentration of total tannins (tannic acid equiv, mg of TAE/g of juice) in aronia juice for the following temperatures: (A) 80 °C, (B) 90 °C, (C) 100 °C, (D) 110 °C, and (E) 120 °C. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Thermal Decomposition of Aronia Power Drinks and Effect of Aronia Juice Concentration

The aronia power aid drinks, created with aronia juice, were also analyzed for their antioxidant content. What separates the drinks from the juice, in addition to some minor additives, is the dilution, as the drinks contain a large amount of water to control palatability, tannin prevalence, and sorbitol consumption. The decomposition was tested using only the base drink formulation, which consisted of water and aronia juice with no additional ingredients. This analysis allowed for the observation of whether or not the same trends presented in the pure juice sample will still apply to processed drinks that have been diluted. For comparison purposes, the basic drink formulation was selected for antioxidant analysis as it contained no additives that could potentially skew decomposition data.

Figure 19 illustrates how the polyphenol content is dependent on time and temperature variables. Compared to the results for pure juice, processed beverages created from the aronia have much sharper variations in concentration between any two given time periods; however, in the end, polyphenol content did not drop below 2% of the initial concentration at 0 min. In addition, concentrations rapidly exceeded 10% of their initial values when exposed to temperatures greater than 110 °C.

Figure 19.

Temperature dependence of polyphenol concentration on the following temperatures: (A) 90 °C, (B) 110 °C, and (C) 120 °C for a duration of 2 h. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

In Figure 20, flavonoid concentration has been observed for the same temperatures: 90 °C, 110 °C, and 120 °C over a 2 h heating period. In pure juice, it can be noted that there was a sharp decrease in flavonoid content of up to 40% within only the first 30 min of heating, a trend that did not reoccur in the beverage samples as time intervals following the initial concentration remained within a range of 20% of the original.

Figure 20.

Temperature dependence of flavonoid concentration on the following temperatures: (A) 90 °C, (B) 110 °C, and (C) 120 °C for a duration of 2 h. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

The lower overall concentration in beverage samples may have slowed the chemical kinetics, leading to a steady decline in all tannin temperatures. Compared with their concentrated counterparts, tannins never began to increase even at the most extreme temperatures. In Figure 21, the dependence of tannin concentration on time and temperature can be followed for the respective heating temperatures.

Figure 21.

Temperature dependence of tannin concentration on temperatures: (A) 90 °C, (B) 110 °C, and (C) 120 °C for a duration of 2 h. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Finally, in Figure 22, the anthocyanin concentration can be observed in the beverage samples during heating. We have observed effects similar to the decomposition of anthocyanins in pure juice. The major differences observed in the basic drink samples were that anthocyanin concentration degraded much more rapidly at 110 °C than at 120 °C and that, beyond 30 min, there was still a substantial amount of anthocyanins remaining in our samples from 120 °C. This unusual effect may have several possible explanations. First, the drink has several fundamental differences as compared to pure juice. The drink’s acidity is lower; the overall concentration of all antioxidants is about three times lower than in pure juice due to dilution with water and additives. It contains organic mineral drops that may significantly affect anthocyanins, which are present in their charged form at the pH of drinks. Organic stevia, a sugar alcohol obtained from plants, and added to drinks for sweetness, may also concurrent with anthocyanins in forming hydrogen bonds. As well, the presence of this sugar alcohol may interrupt the nature of the first decomposition step: hydrolysis of the glucoside bond. These factors may significantly influence two processes that often happen in anthocyanins: the already mentioned above formation of hydrogen bonds between molecules of anthocyanins and between anthocyanins and solvent/additives, and another phenomenon that is known as coagulation of aronia juice, in which anthocyanins become partially insoluble being in agglomerated form. While coagulation has not yet been described in the literature and its chemical and physical nature is a subject of further investigation, we often observe this phenomenon slowly happening even at freezing temperatures and becoming more significant as temperature increases. All these processes are concurrent to the decomposition and would be affected differently by temperature. In addition, some drink additives may act as inhibitors of the decomposition process and slow it down. However, due to the multiplicity of concurrent possible processes happening both in the juice and in drinks, it is difficult to study the kinetics of each process separately as an individual reaction.

Figure 22.

Temperature dependence of anthocyanin concentration on the following temperatures: (A) 90 °C, (B) 110 °C, and (C) 120 °C for a duration of 2 h. The standard deviations were calculated for all experiments using the three triplicates of results collected under each experimental condition. One-way ANOVA was performed to verify the statistical significance of results within the 95% confidence limit.

Assuming that 110 °C is not only the temperature at which we observe the slower decomposition of drinks but also the temperature at which a significant reduction in tannin content was observed, this might be the best option for processing both the juice and the drink.

Conclusions

Because pasteurization of aronia juice is required for limiting bacterial growth, this project aimed to examine the dynamics of decomposition of anthocyanins, flavonoids, polyphenols, and tannins in aronia as a function of temperature and exposure time to evaluate the best pasteurization and cooking techniques that would allow processing without affecting aronia’s antioxidant contents significantly.

We have shown that anthocyanins decompose rapidly in aronia juice and the power-aided drink produced from this juice. The decomposition rate increases with an increase in the temperature. Thus, at 80 °C, about 17% of anthocyanins decomposed after 30 min of heat exposure, whereas the loss of half of anthocyanin content took 2 h. At 120 °C, after only the first 5 min of heating, the loss is a substantial 86%. Because anthocyanins are at the top of the decomposition chain, no other decomposition process replenishes their content. Flavonoids and polyphenols decompose much more slowly due to overall slower kinetics and due to their partial replenishment, while anthocyanins decompose into flavonoids and when flavonoids decompose into polyphenols.

In both concentrated (juice) and diluted (drinks) samples, we have shown that applying temperatures of ∼80 °C will not result in the significant decomposition of any more than 17% of antioxidants, mostly anthocyanins, in the first 30 min of heating, and even with 2 h of heating, more than half of the total antioxidant content will remain. It was found that higher temperatures or exposure times substantially deterred anthocyanin concentration; however, this increased rate of anthocyanin decomposition promoted the generation of flavonoids, polyphenols, and tannins.

Glossary

Abbreviations Used

UV/vis

UV/visible spectrophotometry

HPLC

high-performance liquid chromatography

ACS

American Chemical Society

μL

microliter

V

volts

mL

milliliters

M

molar

LOD

limit of detection

C3GE

cyanidin-3-glucoside equivalents

PP

polyphenols

GAE

gallic acid equivalents

QE

quercetin equivalent

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c06369.

• Raw data of all measurements: totals of polyphenols, flavonoids, anthocyanins, and tannins, with calculations for juice and aronia drinks at each of the temperatures mentioned in the paper (PDF)

The AFRI-EWD-REEU program grant no. 2020-69018-30655, from the U.S. Department of Agriculture, National Institute of Food and Agriculture, and the USDA-NIFA Evans Allen formula grant at the University of Maryland Eastern Shore—assessing number 1016197, support his work.

The authors declare no competing financial interest.