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Article Improvement of Naturally Derived Food Colorant Performance with Efficient Formation from Sambucus nigra Using Caffeic Acid and Heat

Nicole Straathof and M. Monica Giusti * Department of Food Science & Technology, The Ohio State University, Columbus, OH 43210, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-614-247-8016

Academic Editor: Eulogio J. Llorent-Martínez   Received: 21 November 2020; Accepted: 15 December 2020; Published: 18 December 2020

Abstract: Consumers and regulations encourage the use of naturally derived food colorants. Anthocyanins (ACN), plant pigments, are unstable in foods. In aged red wines, ACN with a free hydroxyl group at C-5 condenses to form (PACN), which are more stable but form inefficiently. This study attempted to produce PACN efficiently using high cofactor concentration and heat. Elderberry anthocyanins were semi-purified and caffeic acid (CA) was dissolved in 15% ethanol and diluted with a buffer to achieve ACN:CA molar ratios of 1:50, 1:100, 1:150, and 1:200, then incubated at 65 ◦C for 5 days. The effect of temperature was tested using ACN samples incubated with or without CA at 25 ◦C, 50 ◦C, and 75 ◦C for 7 days. Compositional changes were monitored using uHPLC-PDA-MS/MS. Higher CA levels seemed to protect pigment integrity, with ACN:CA 1:150 ratio showing the highest tinctorial strength after 48 h. PACN content growth was fastest between 24 and 48 h for all ACN:CA ratios and after 120 h, all ACN had degraded or converted to PACN. PACN formed faster at higher temperatures, reaching ~90% PACN in 24 h and ~100% PACN in 48 h at 75 ◦C. These results suggest that PACN can form efficiently from elderberry ACN and CA if heated to produce more stable pigments.

Keywords: elderberry; natural food color; pigment; color stability; color intensity; CIELab; uHPLC; spectrophotometry

1. Introduction People experience colors daily that not only affect their feelings, but also their decisions. Food color is an important characteristic that has been used for over a century to help consumers identify and differentiate product safety, sensory, and quality characteristics [1]. However, many consumers do not trust artificial ingredients and look for natural alternatives. In one study, scientists determined that artificial colorants increase hyperactivity in children [2]. This study raised concerns and lead numerous countries to ban specific artificial food colorants. Naturally derived alternatives have become an important area of research, with anthocyanins (ACN) receiving a fair share of attention. These compounds are plant pigments found in many plants that act as powerful antioxidants and may provide health benefits. Unfortunately, they are unstable and lose their vibrancy over time. Therefore, scientists asked: What is a food that has color for a very long time? The answer was found in wine. Scientists studying wine found pyranoanthocyanins (PACN), which are -derived pigments. Pyranoanthocyanins were first discovered in red wine in 1996 as a crucial component of [3,4]. That same year, the structure was determined through NMR and showed the pyran

Molecules 2020, 25, 5998; doi:10.3390/molecules25245998 www.mdpi.com/journal/molecules Molecules 2020, 25, 5998 2 of 11 ring attached between the C-5 hydroxyl group and C-4 [5]. Because of the additional pyran ring, pyranoanthocyanins have a λmax that is hypsochromically shifted and expresses a more orange color compared to authentic anthocyanin pigments [4]. This additional ring makes PACN a much more stable pigment that is better suited for food processing environments [6]. Unfortunately, they are formed very inefficiently over time. As shown in wine, color development from PACN can take months to years [7]. In a food processing setting, storing the pigment for that amount of time is a cost not many are willing to spend. Increasing the formation rate of PACN is a relevant area of research. PACN require anthocyanins, a reaction partner, and storage time for formation [4]. PACN formation in food is dependent on the concentration of both the anthocyanins and the reaction partner. In wine, PACN form and cause color changes through aging, which can take months through to decades. Because of the increased stability of this pigment, studies look for ways to increase the rate of formation. Pinotin A, a hydroxyphenyl-pyranoanthocyanin, was shown to have the fastest formation in Pinotage wine. In this time period of 3 to 4 years, concentration of anthocyanins had significantly decreased, leading to an increase in the caffeic acid to anthocyanin ratio [8]. One study looked at using microwave-assisted extraction to increase PACN formation at differing grape ripeness levels [9]. Another study was able to reduce PACN formation to 42 days by increasing storage temperature to 25 ◦C[10]. However, with increased temperature, anthocyanin degradation also increases. The benefit of gaining a more stable pigment needs to be weighed with the loss of the initial pigment. Nonetheless, the efficient formation of PACN would be an asset to the food industry as they work towards eliminating artificial food colorants. It has been shown that increasing storage temperature from refrigeration to room temperature can increase PACN formation; however, temperatures beyond room temperature have not been evaluated [10,11]. These studies also found that caffeic acid is an effective cofactor in producing PACN. However, excessive caffeic acid used without obtaining significant increases in PACN formation would be a costly waste of product for producers. Additionally, too much caffeic acid may cause astringent flavor development [12]. Determining the amount of caffeic acid that is beneficial to the development of PACN without sacrificing other characteristics could save producers money as they switch to naturally derived colorants. This study consisted of two main objectives: to determine if using high incubation temperature would increase PACN formation significantly and to determine if using a higher concentration of cofactor and temperature would increase PACN formation enough for use as a naturally derived colorant. With the addition of significantly more heat, it is hypothesized that PACN formation and anthocyanin degradation will both increase. Furthermore, we expected that the increase in caffeic acid concentrations with a high incubation temperature would increase PACN formation, but that it may not be significant.

2. Results and Discussion There were three main anthocyanins eluted from the sample including -3-glucoside, cyanidin-3-sambubioside, and cyanidin-3-sambubioside-5-glucoside. The identification of cyanidin-3- glucoside and cyanidin-3-sambubioside confirmed that the elderberry sample was an ideal anthocyanin source for PACN formation. The free hydroxyl group at the C5 position of these main anthocyanins allowed for a cofactor to condense and form PACN. These results agreed with literature by Lee and Finn, which found that the first two small anthocyanin peaks are cyanidin-3-sambubioside-5-glucoside and cyanidin-3,5-diglucoside, respectively [13]. They determined that the next two larger anthocyanin peaks were cyanidin-3-sambubioside and cyanidin-3-glucoside, respectively. MS/MS data were used for pigment identification. A large portion of the sample was made up of peaks two and three (cyanidin-3-glucoside and cyanidin-3-sambubioside) (Figure1). Molecules 2020, 25, 5998 3 of 11 Molecules 2020, 25, x FOR PEER REVIEW 3 of 11 Molecules 2020, 25, x FOR PEER REVIEW 3 of 11

Figure 1. Elderberry anthocyanins before incubation with caffeic acid. uHPLC-PDA-MS/MS settings FigureFigurewere 1.as1. Elderberryfollows: 0.25 anthocyanins mL/min flow, before 50 °C incubationincubationoven temperature, withwith cacaffeicff 50eic μ acid.Lacid. injection uHPLC-PDA-MSuHPLC-PDA-MS/MS volume, and/ MSthe following settingssettings werewereacetonitrile asas follows:follows: gradient: 0.25 mLmL/min 0%/min at 0flow,flow, min, 5050 26% ◦°CC ovenat 19 min, temperature, 30% at 20 5050 to µμ L21L injection min, 0% volume, at 21 min. and the following acetonitrileacetonitrile gradient:gradient: 0% at 0 min, 26% atat 1919 min,min, 30%30% atat 2020 toto 2121 min,min, 0%0% atat 2121 min.min. With the main anthocyanins in this sample having a free hydroxyl group at the C5 position, this sampleWithWith was the main an ideal anthocyanins anthocyanins candidate in infor this this PACN sample sample formation. having having a freeWhen a free hydroxyl hydroxylthe sample group group was at the atanalyzed theC5 position, C5 position,using this the thissampleuHPLC-MS/MS-PDA sample was wasan ideal an ideal candidate methods candidate fordescribed PACN for PACN information. the formation. methods When Whensection, the thesample our sample resultswas wasanalyzed agreed analyzed usingwith using thosethe theuHPLC-MS/MS-PDAdescribed uHPLC-MS previously/MS-PDA methods by methodsLee and described describedFinn. The in inthetwo the methodspeaks methods that section, section,eluted ouraround our results results 18 minagreed agreed were with the thosethosePACN describeddescribedpeaks, but previouslypreviously when the by by sample Lee Lee and and was Finn. Finn. not The Theincubated, two two peaks peaks th thate amountthat eluted eluted aroundwas around very 18 minminima 18 wereminl. thewereIt was, PACN the however, PACN peaks, butpeaks,present when but thein when the sample sample the was sample containing not incubated, was notcaffeic incubated, the acid amount at lowth wase amountconcentrations very minimal. was very because It was,minima however, of l.the It timewas, present thathowever, inpassed the samplepresentbetween containingin samplethe sample preparation caff eiccontaining acid atand low caffeic analysis. concentrations acid at lowbecause concentrations of the time because that passedof the time between that samplepassed preparationbetweenAfter sample 24 and h analysis.incubationpreparation at and higher analysis. temperatures, the anthocyanin peak areas decreased faster, while the AfterAfterPACN 24 24 peak h h incubation incubation areas increased at athigher higher faster temperatures, temperatures, over time ththane theanthocyanin at anthocyaninlower temperatures peak peakareas areasdecreased (Figure decreased 2). faster, The faster,reversewhile whilethewas PACN thetrue PACN atpeak lower areas peak temperatures, increased areas increased faster with over fastergreater time over anthocyanin than time at than lower peak at temperatures lower area temperaturesand smaller (Figure PACN (Figure 2). The peak2 ).reverse The areas reversewasafter true 24 was hat of lower true incubation. at temperatures, lower temperatures, with greater with greater anthocyanin anthocyanin peak area peak and area smaller and smaller PACN PACN peak areas peak areasafter 24 after h of 24 incubation. h of incubation.

FigureFigure 2. 2.Retention Retention time time of of two two main main anthocyanins anthocyanins (ACN) (ACN) and and pyranoanthocyanins pyranoanthocyanins (PACN) (PACN) obtained obtained Figure 2. Retention time of two main anthocyanins (ACN) and pyranoanthocyanins (PACN) obtained fromfrom sample sample incubated incubated for for 24 24 h h at at 75 75◦C °C with with and and without without ca caffeicffeic acid acid (CA), (CA), 50 50◦ C°C with with ca caffeicffeic acid, acid, from sample incubated for 24 h at 75 °C with and without caffeic acid (CA), 50 °C with caffeic acid, andand 25 25◦C °C with with ca caffeicffeic acid. acid. Chromatograms Chromatograms viewed viewed between between 500 500 and and 520 520 nm. nm. uHPLC-PDA-MS uHPLC-PDA-MS/MS/MS settingsandsettings 25 °C were were with as ascaffeic described described acid. in Chromatogramsin Figure Figure1. 1. viewed between 500 and 520 nm. uHPLC-PDA-MS/MS settings were as described in Figure 1. ByBy comparing comparing PACN PACN formation formation and and anthocyanin anthocyanin degradation, degradation, it it can can be be seen seen that that at at lower lower temperaturestemperaturesBy comparing the the anthocyanin anthocyanin PACN formation degraded degraded and less lessanthocyanin butbut formedformed degradation, less PACN it (Figure can be 22).). seen On the thethat contrary, contrary,at lower at attemperatureshigher higher incubation incubation the anthocyanin temperatures, temperatures, degraded PACN less formationformation but form waswased greaterlessgreater PACN andand (Figure anthocyanin anthocyanin 2). On degradation degradationthe contrary, was was at greater.highergreater. incubation Therefore, Therefore, pasttemperatures, past a a specific specific time,PACN time, incubation incubation formation was waswas counterproductive counterproductive greater and anthocyanin because because it degradationit will will not not increase increase was greater.PACN Therefore,significantly past while a specific continuing time, incubation to degrad wase counterproductiveanthocyanin pigments because consistently. it will not increaseFigure 3 PACNshowed significantly a significant while increase continuing in PACN toformatio degradne at anthocyanin 75 °C compared pigments to 25 °C consistently. and 50 °C. Figure 3 showed a significant increase in PACN formation at 75 °C compared to 25 °C and 50 °C.

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PACN significantly while continuing to degrade anthocyanin pigments consistently. Figure3 showed a significant increase in PACN formation at 75 ◦C compared to 25 ◦C and 50 ◦C. Molecules 2020, 25, x FOR PEER REVIEW 4 of 11 Molecules 2020, 25, x FOR PEER REVIEW 4 of 11

Figure 3. Average percentage of pyranoanthocyanin (PACN) of total pigment of samples with caffeic Figure 3. Average percentage of pyranoanthocyanin (PACN) of total pigment of samples with caffeic caffeic acid incubated over time at 25 °C, 50 °C, and 75 °C. Calculated by Equation (4). Sample incubated at acid incubated over over time time at at 25 25 °C,◦C, 50 50 °C,◦C, and and 75 75 °C◦C.. Calculated Calculated by by Equation Equation (4). (4). Sample Sample incubated incubated at 25 °C for 72 h was not measured because of minimal PACN present and sample loss. Error bars at25 25°C◦ Cfor for 72 72h hwas was not not measured measured because because of of mini minimalmal PACN PACN present present and sample loss. Error bars represent the standard deviations. Significant difference from 25 °C incubation for the same represent thethe standard standard deviations. deviations. Significant Significant difference difference from 25from◦C incubation25 °C incubation for the same for incubationthe same incubation time with * p < 0.05; ** p < 0.01; *** p < 0.001. timeincubation with * timep < 0.05; with ** * p < 0.05;0.01; ** *** pp << 0.01;0.001. *** p < 0.001. Within 2424 h,h, there there was was 86% 86% PACN PACN and and within within 48 h 48 the h sample the sample was 100% was PACN.100% PACN. Incubating Incubating beyond Within 24 h, there was 86% PACN and within 48 h the sample was 100% PACN. Incubating beyond48 h decreased 48 h decreased total pigment. total pigment. Because ofBecause anthocyanin of anthocyanin sensitivity sensitivity to heat, degradation to heat, degradation of pigment of is beyond 48 h decreased total pigment. Because of anthocyanin sensitivity to heat, degradation of pigmentexpected is at expected high temperatures at high temperatures [14,15]. According [14,15]. toAcco Figurerding3, itto wouldFigure be3, nearlyit would pointless be nearly to pointless incubate pigment is expected at high temperatures [14,15]. According to Figure 3, it would be nearly pointless tobeyond incubate 48 h underbeyond these 48 h conditions. under these Even conditions though PACN. Even is morethough stable, PACN at 75 is Cmore incubation stable, thereat 75 may °C to incubate beyond 48 h under these conditions. Even though PACN is◦ more stable, at 75 °C incubationstill be minimal there pigment may still loss. be minimal At 48 h ofpigment incubation loss. underAt 48 h such of incubation a high heat, under the greatest such a high concentration heat, the incubation there may still be minimal pigment loss. At 48 h of incubation under such a high heat, the greatestof PACN concentration has already been of reachedPACN andhas continuedalready b incubationeen reached leads and to unnecessarycontinued incubation loss of pigment leads and to greatest concentration of PACN has already been reached and continued incubation leads to wastedunnecessary energy. loss of pigment and wasted energy. unnecessary loss of pigment and wasted energy. In the samples analyzed, at higher temperatures,temperatures, degradation of total pigment occurred more In the samples analyzed, at higher temperatures, degradation of total pigment occurred more rapidly than at lower temperatures (Figure4 4).). rapidly than at lower temperatures (Figure 4).

Figure 4. Average percentage of total pigment remaining in samples incubated with caffeic acid at 25 Figure 4. Average percentage of total pigment remaining in samples incubated with caffeic acid at 25 °C,Figure 50 °C, 4. Averageand 75 °C percentage over time; of 25 total °C at pigment 72 h wa remainings not measured in samples because incubated of minimal with pyranoanthocyanin caffeic acid at 25 ◦C, °C, 50 °C, and 75 °C over time; 25 °C at 72 h was not measured because of minimal pyranoanthocyanin present50 ◦C, and and 75 sample◦C over loss. time; Calculated 25 ◦C at 72with h wasEquation not measured (5). Error because bars represent of minimal the standard pyranoanthocyanin deviations. present and sample loss. Calculated with Equation (5). Error bars represent the standard deviations. presentSignificant and difference sample loss. from Calculated 25 °C incubation with Equation for the same (5). Error incubation bars represent time with the * p standard < 0.05; ** deviations. p < 0.01. Significant difference from 25 °C incubation for the same incubation time with * p < 0.05; ** p < 0.01. Significant difference from 25 ◦C incubation for the same incubation time with * p < 0.05; ** p < 0.01. Looking at the results shown in Figure 4, it could be argued that the production of PACN would Looking at the results shown in Figure 4, it could be argued that the production of PACN would be more efficient at 24 h versus 48 h because of the significant difference between 6 and 24 h versus be more efficient at 24 h versus 48 h because of the significant difference between 6 and 24 h versus the minimal difference between the former (Figure 3). The processing cost of storing and incubating the minimal difference between the former (Figure 3). The processing cost of storing and incubating the sample combined with the loss in pigment remaining may not be worth the minimal increased the sample combined with the loss in pigment remaining may not be worth the minimal increased PACN content. Although pigment was lost through the incubation of sample and formation of PACN content. Although pigment was lost through the incubation of sample and formation of PACN, the amount of PACN present in the sample was comparatively substantial. A similar study PACN, the amount of PACN present in the sample was comparatively substantial. A similar study

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Looking at the results shown in Figure4, it could be argued that the production of PACN would be more efficient at 24 h versus 48 h because of the significant difference between 6 and 24 h versus the minimal difference between the former (Figure3). The processing cost of storing and incubating the sample combined with the loss in pigment remaining may not be worth the minimal increased PACN content. Although pigment was lost through the incubation of sample and formation of Molecules 2020, 25, x FOR PEER REVIEW 5 of 11 PACN, the amount of PACN present in the sample was comparatively substantial. A similar study incubating samples at at 25 °C◦C required 42 days to obtain the PACN levels obtained within one day in this study at 75 °C◦C[ [10].10]. Being Being able able to to produce produce PACN PACN within within one day day versus versus several weeks or months increases the practicability of producing PACN inin thethe food industry as a naturally derived alternative to artificial artificial colorants. Because of the loss of anthocyanins and the form formationation of PACN, a color change occurred in the sample. According According to Figure5 5,, atat 2525 ◦°CC and 50 ◦°C,C, the Δ∆E*ab waswas greater greater at at 48 48 h h than than 24 24 h h with with caffeic caffeic acid. At At 75 75 °C,◦C, the the delta delta E Eincreased increased with with caffeic caffeic ac acidid between between both both 24 24and and 48 48h when h when compared compared to no to incubation.no incubation.

Figure 5. Average ∆ΔE*ab ofof samples samples incubated incubated at at 25, 25, 50, or 75 °C◦C between samples incubated for zero hours andand either either 24 24 or or 48 h48 with h with or without or without caffeic caffe acidic (CA). acid Error (CA). bars Error represent bars represent the standard the deviations. standard deviations. Figure5 showed that with the formation of PACN the ∆E*ab increased, indicating a change in the pigment.Figure Each 5 showed∆E*ab was that greaterwith the than formation five. This of pigmentPACN the could ΔE*ab therefore increased, not indicating replace anthocyanins, a change in thebut pigment. it may be Each used Δ inE* tandemab was greater with them. than five. This pigment could therefore not replace anthocyanins, but itThe may PACN be used content in tandem increased with consistently them. with an increase in caffeic acid concentration, but it did not increaseThe PACN significantly content increased (Figure6). consistently Therefore, under with the an conditionsincrease in tested caffeic it mayacid notconcentration, be worth adding but it didmore not than increase an ACN:CA significantly molar (Figure ratio of 6). 1:50. Therefore, under the conditions tested it may not be worth adding more than an ACN:CA molar ratio of 1:50. Because Figure 6 shows a consistent but not a significant increase in PACN present, the addition of more caffeic acid may have more of a negative effect than the benefits it provides. Too much caffeic acid may cause astringent flavor development and may become a costly investment for producers. Within 48 h, samples containing caffeic acid had all achieved PACN concentrations of at least 80%, while at 120 h all samples contained 100% PACN. With at least 80% PACN in the sample with ACN:CA molar ratios of 1:50, the sample may already contain enough pigment and have enough tinctorial strength for what is required. In Figure 7 we can see that through incubation, anthocyanin concentration decreased over time. We can also see that the anthocyanin concentration decreases more rapidly at higher concentrations of caffeic acid.

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FigureFigure 6. Average 6. Average total total pyranoanthocyanin pyranoanthocyanin (PACN)(PACN) as part part of of total total pigment pigment after after time time in incubation in incubation at 65 °C with samples containing anthocyanin to caffeic acid molar ratios of 1:0, 1:50, 1:100, 1:150, and at 65 ◦C with samples containing anthocyanin to caffeic acid molar ratios of 1:0, 1:50, 1:100, 1:150, 1:200. Error bars represent the standard deviations. Significant difference from anthocyanin to caffeic and 1:200. Error bars represent the standard deviations. Significant difference from anthocyanin to acid molar ratio of 1:50 for the same incubation time with * p < 0.05. caffeic acid molar ratio of 1:50 for the same incubation time with * p < 0.05.

Because Figure6 shows a consistent but not a significant increase in PACN present, the addition of more caffeic acid may have more of a negative effect than the benefits it provides. Too much caffeic acid may cause astringent flavor development and may become a costly investment for producers. Within 48 h, samples containing caffeic acid had all achieved PACN concentrations of at least 80%, while at 120 h all samples contained 100% PACN. With at least 80% PACN in the sample with ACN:CA molar ratios of 1:50, the sample may already contain enough pigment and have enough tinctorial strength for what is required. In FigureFigure7 6.we Average can see total that pyranoanthocyanin through incubation, (PACN) anthocyaninas part of total pigment concentration after time decreased in incubation over time. We can alsoat 65 see °C thatwith thesamples anthocyanin containing anthocyanin concentration to caffe decreasesic acid molar more ratios rapidly of 1:0, 1:50, at higher 1:100, 1:150, concentrations and of caffeic acid.1:200. Error bars represent the standard deviations. Significant difference from anthocyanin to caffeic acid molar ratio of 1:50 for the same incubation time with * p < 0.05.

Figure 7. Average percentage of anthocyanin of total pigment in sample containing anthocyanin to caffeic acid molar ratios of 1:0, 1:50, 1:100, 1:150, and 1:200 after hours of incubation at 65 °C. Error bars represent the standard deviations. Significant difference from ACN:CA molar ratio of 1:50 for the same incubation time with * p < 0.05, ** p < 0.01.

This would suggest that much of the loss of anthocyanins is attributed to the formation of PACN. As is also shown in Figure 6, we can see that between 24 and 48 h there was a significant (p-value < 0.05) shift from anthocyanins to PACN in samples containing caffeic acid. Some time after 48 h, anthocyanins were either completely degraded or converted to PACN, as a sample with no caffeic acid was the only sample with anthocyanins remaining at 120 h. Figure 8 showed a sample with ACN:CA molar ratios of 1:150 and 48 h of incubation had the greatest PACN conversion at 212% absorbance compared to the initial pigment.

Figure 7. Average percentage of anthocyanin of total pigment in sample containing anthocyanin to Figure 7. Average percentage of anthocyanin of total pigment in sample containing anthocyanin to caffeic acid molar ratios of 1:0, 1:50, 1:100, 1:150, and 1:200 after hours of incubation at 65 °C. Error caffeic acid molar ratios of 1:0, 1:50, 1:100, 1:150, and 1:200 after hours of incubation at 65 C. Error bars bars represent the standard deviations. Significant difference from ACN:CA molar ratio of◦ 1:50 for

representthe same the standardincubation deviations. time with * Significantp < 0.05, ** p di< ff0.01.erence from ACN:CA molar ratio of 1:50 for the same incubation time with * p < 0.05, ** p < 0.01. This would suggest that much of the loss of anthocyanins is attributed to the formation of PACN. As is also shown in Figure 6, we can see that between 24 and 48 h there was a significant (p-value < 0.05) shift from anthocyanins to PACN in samples containing caffeic acid. Some time after 48 h, anthocyanins were either completely degraded or converted to PACN, as a sample with no caffeic acid was the only sample with anthocyanins remaining at 120 h. Figure 8 showed a sample with ACN:CA molar ratios of 1:150 and 48 h of incubation had the greatest PACN conversion at 212% absorbance compared to the initial pigment.

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This would suggest that much of the loss of anthocyanins is attributed to the formation of PACN. As is also shown in Figure6, we can see that between 24 and 48 h there was a significant ( p-value < 0.05) shift from anthocyanins to PACN in samples containing caffeic acid. Some time after 48 h, anthocyanins were either completely degraded or converted to PACN, as a sample with no caffeic acid was the only sample with anthocyanins remaining at 120 h. Figure8 showed a sample with ACN:CA molar ratios of 1:150 and 48 h of incubation had the greatest PACN conversion at 212% absorbance compared to the initial pigment. Molecules 2020, 25, x FOR PEER REVIEW 7 of 11

Figure 8. AverageAverage pyranoanthocyanin (PACN) (PACN) concentrat concentrationion of of initial pigment concentration in in a samplesample containing containing anthocyanin anthocyanin to to caffeic caffeic acid acid mola molarr ratios of 1:0, 1:50, 1:100, 1:150, and 1:200 after

hours of incubation at 65 °C.◦C. The The sample sample without without caffeic caffeic acid was not observed because there was no PACN present. Error barsbars representrepresent thethe standard standard deviations. deviations. Significant Significant di differencefference from from anthocyanin anthocyanin to toca ffcaffeiceic acid acid molar molar ratio ratio of 1:50of 1:50 for fo ther the same same incubation incubation time time with with * p *< p0.05. < 0.05.

At this timetime ofof incubation,incubation, PACN PACN absorbance absorbance was wa greaters greater than than 125% 125% of the of initialthe initial sample sample for each for eachcaffeic caffeic acid containingacid containing the sample. the sample. This showedThis showed that withthat with the use the of us cae ffofeic caffeic acid, theacid, amount the amount of color of colorexpression expression in samples in samples incubated incubated for 48 hfor could 48 beh could greater be with greater PACN with formation PACN thanformation the pigment than the in pigmentthe initial in sample. the initial These sample. results These are solely results based are solely on absorbance, based on absorbance, specifically the specifically area under the the area curve under for theanthocyanins curve for anthocyanins and relative concentration. and relative concentratio Results couldn. Results not be compared could not tobe a compared standard curveto a standard because curveof the because lack of PACN of the standardlack of PACN samples. standard With sample greaters. color With expressiongreater color and expression stability, theand pigment stability, may the pigmentbe used tomay create be andused provide to create more and reliable provide colors more for reliable use in the colors food for industry. use in Sometimethe food afterindustry. 48 h, Sometimedegradation after overtook 48 h, degradation formation and overtook total pigment formation decreased, and total meaning pigment that decreased, under these meaning conditions that underincubating these past conditions 48 h was incubating not worth past the 48 loss h ofwas pigment, not worth unless the loss a pure of pigment, sample of unless PACN a is pure the goal.sample of PACN is the goal. 3. Materials and Methods 3. Materials and Methods 3.1. Materials 3.1. MaterialsStandardized organic elderberry (Sambucus nigra) was obtained from Artemis International, Inc. (FortStandardized Wayne, IN, USA). organic The elderberry following (Sambucus chemicals nigra) were was sourced obtained from from Fisher Artemis Scientific International, (FairLawn, Inc. NJ, (FortUSA): Wayne, ethyl acetate, IN, USA). methanol, The following HPLC-MS chemicals grade acetonitrile, were sourced HPLC-MS from Fisher grade Scientific water, and (FairLawn, hydrochloric NJ, USA):acid. Acetic ethyl acid acetate, and ca ffmethanol,eic acid were HPLC-MS sourced from grade Millipore acetonitrile, Sigma (Darmstadt, HPLC-MS Germany). grade water, Formic acidand hydrochloricwas purchased acid. from Acetic EMD Milliporeacid and Corporationcaffeic acid (Danvers,were sourced MA, from USA). Millipore Sigma (Darmstadt, Germany). Formic acid was purchased from EMD Millipore Corporation (Danvers, MA, USA).

3.2. Methods

3.2.1. Preparation of Elderberry Anthocyanin Extract Standardized elderberry powder was weighed (1.2 g) and solubilized in 1800 mL of distilled water. Sample was semi-purified using a C18 resin (Waters Corporation, Milford, MA, USA) through solid phase extraction. The column was activated with methanol and rinsed with three volumes of acidified water. The sample was added to the column until it was packed halfway, then two volumes of acidified water and three volumes of ethyl acetate were added. Anthocyanins were then eluted using three volumes of MeOH and collected. Methanol was evaporated out using a Buchi Rotovapor

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3.2. Methods

3.2.1. Preparation of Elderberry Anthocyanin Extract Standardized elderberry powder was weighed (1.2 g) and solubilized in 1800 mL of distilled water. Sample was semi-purified using a C18 resin (Waters Corporation, Milford, MA, USA) through solid phase extraction. The column was activated with methanol and rinsed with three volumes of acidified water. The sample was added to the column until it was packed halfway, then two volumes of acidified water and three volumes of ethyl acetate were added. Anthocyanins were then eluted using three volumes of MeOH and collected. Methanol was evaporated out using a Buchi Rotovapor (Buchi Corporation, New Castle, DE, USA) with a 35 ◦C water bath. The remaining semi-purified solution was resolubilized in 0.01% acidified water. The sample was stored at 18 C until use. This process − ◦ was repeated twice to produce a total of three replicates.

3.2.2. Determining Concentration of Monomeric Anthocyanins in Extract Concentration of monomeric anthocyanins in extract was determined according to the pH Differential method provided by Giusti and Wrolstad [16]. Sample was diluted separately with both potassium chloride buffer at pH 1.0 and sodium acetate buffer at pH 4.5 at a dilution factor where the λmax was below 1 for the sample diluted with potassium chloride buffer. The dilution factor was calculated by dividing the final volume of the sample with the final diluted volume of the sample and absorbance was measured on the SpectraMax M2 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Once both the pH 1.0 and pH 4.5 sample dilutions were equilibrated for 15 min, the spectrophotometer was used to measure absorbance at the λmax and 700 nm against distilled water. The following calculation was used to determine the absorbance (A), which was used in the latter equation to determine the monomeric anthocyanin pigment concentration.

A = (Aλ A ) (Aλ A ) (1) vis-max − 700 nm pH 1.0 − vis-max − 700 nm pH 4.5 Monomeric anthocyanin pigment (mg/liter) = (A MW DF 1000) (ε 1) (1) (2) × × × ÷ × A = Absorbance, MW = molecular weight of predominant anthocyanin, DF = Dilution factor, ε = Molar absorptivity of predominant anthocyanin.

3.2.3. Preparation of Caffeic Acid and Anthocyanin Sample To measure the effect of temperature, caffeic acid was added to 100% ethanol in Eppendorf tubes (Eppendorf, Hamburg, Germany), which were centrifuged for 5 min. The aliquots were removed, and these steps were repeated twice. This was used as a saturated caffeic acid in ethanol solution and 11 mL of EtOH with or without caffeic acid was added to 62.5 mL Phosphate Citrate buffer and 22.5 mL ACN in Phosphate Citrate buffer. To measure the effect of caffeic acid concentration, the weight of caffeic acid required in each sample was calculated using the molar mass of cyanidin-3-glucoside and the previously calculated monomeric anthocyanin pigment in milligrams per liter converted to moles in the sample. The ratios were then used to calculate the amount of moles of caffeic acid required to obtain the necessary anthocyanin–caffeic acid molar ratios. This was then converted to grams using caffeic acid’s molar mass and combined with 27 mL of EtOH. These EtOH samples with or without caffeic acid were added to 108 mL of Phosphate Citrate buffer and 42 mL of ACN in Phosphate Citrate buffer.

3.2.4. Incubation To measure the effect of temperature, samples were placed in Fisher Scientific Isotemp 637D incubators (Thermo Fisher Scientific, Waltham, MA, USA) at temperatures of 25 ◦C, 50 ◦C, and 75 ◦C. At hours 0, 3, 6, 24, 48, and 72, samples were removed from incubators, filtered using 0.3 micromolar Molecules 2020, 25, 5998 9 of 11

filters, and frozen in uHPLC vials until analyzed. Samples were removed from the freezer and left at room temperature for 15 min until thawed. To measure effect of caffeic acid, samples were placed in Fisher Scientific Isotemp 637D incubators (Thermo Fisher Scientific, Waltham, MA, USA) at 65 ◦C. At hours 0, 2, 4, 8, 24, 48, 72, 120, or 168 samples were filtered, frozen, and thawed following the same procedure as above.

3.2.5. Analysis of the Sample The uHPLC was coupled with a PDA detector and used a Restek reverse phase C-18 column with 50 mm, 2.1 mm, and 1.9 µm size, diameter and particle size respectively (restek Corporation, Bellefonte, PA, USA). The settings were as follows: 0.25 mL/min flow, 50 ◦C oven temperature, 50 µL injection volume, and the following acetonitrile gradient: 0% at 0 min, 26% at 19 min, 30% at 20 to 21 min, 0% at 21 min. The SpectraMax M2 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) was used with a standard 96-well microplate to obtain UV-Vis spectra and 35 µL of each sample was pipetted into separate wells and measured from 380 nm to 720 nm in 1 nm intervals with a 10-degree observer angle, regular transmission, and a D65 illuminant. The software ColorBySpectra was used to convert the UV-vis spectral data into color spaces data.

3.2.6. Statistical Analysis Data analysis to determine the statistical significance of research findings was performed. Microsoft Office 2019 Excel (Redmond, WA, USA) was used to calculate averages and standard deviations. It was also used to develop figures and tables. All statistical data were analyzed using RStudio (Boston, MA, USA). Linear relationships, parallel slope assumptions, and homogeneity of variance were tested between covariates and temperature and absorbance. Both one-way ANOVA and uncentered ANCOVA models were run between covariate, absorbance, and temperature (2-tailed, α = 0.01).

3.2.7. Treatment Performance Equations These equations quantified the performance efficiency of the pigments by determining the percent of PACN that formed, the percent of pigment that was PACN, and the amount of pigment which remained.

PACN yield percent = [(AUC500–520 nm C3S PACN + C3G PACN tn) (AUC500–520 nm ÷ (3) C3S + C3G at Day 0)] 100 ×

Percent PACN to total pigment percent = [(AUC500–520 nm C3S PACN + C3G (4) PACN tn) (AUC C3S +C3G + C3S PACN + C3G PACN at tn)] 100 ÷ 500–520 nm ×

Remaining pigment percent = [(AUC500–520 nm C3S + C3G + C3S PACN + C3G (5) PACN tn) (AUC C3S + C3G + C3S PACN + C3G PACN at t )] 100 ÷ 500–520 nm 0 × AUC = Area under the Curve, C3S = cyanidin-3-sambubioside, C3G = cyanidin-3-glucoside, PACN = Pyranoanthocyanins from C3G and C3R.

4. Conclusions The use of heat was effective for increasing PACN content. We found the use of heat and caffeic acid showed PACN increased at a faster rate over time than without these applications. We found significant evidence of this between 25 ◦C and 75 ◦C. Incubation of samples at 25 ◦C for 24 h retained the most pigment (98%) while incubation of samples at 75 ◦C for 72 h retained the least pigment (30%). However, at 75 ◦C incubation of samples, 90% converted to PACN within 24 h and 100% within 48 h. Because of pigment degradation over time, incubating for 24 h at 75 ◦C may be more beneficial under Molecules 2020, 25, 5998 10 of 11 these conditions to retain the pigment. At high incubation temperatures, the increase in caffeic acid concentration increased PACN content consistently, but not significantly. This brings into question the validity of the increasing caffeic acid due to the negative organoleptic effects it may pose to the finished product. The difference between no caffeic acid and the addition of caffeic acid was not significant in the amount of pigment remaining, although the samples containing caffeic acid had a slightly greater pigment remaining. Of the pigment remaining, the sample with caffeic acid would have a longer lasting and more stable pigment because of the PACN content. There was a correlation between the increase of PACN and the decrease of anthocyanins. The addition of heat, however, drastically decreased the amount of pigment that remained over time. Future work should consider how these results may be applied to the food industry.

5. Patents Patent pending. P2020-226-5636.

Author Contributions: Conceptualization, N.S. and M.M.G.; methodology, N.S.; software, M.M.G.; validation, N.S. and M.M.G.; formal analysis, N.S.; investigation, N.S.; resources, M.M.G.; data curation, N.S.; writing—original draft preparation, N.S.; writing—review and editing, N.S. and M.M.G.; visualization, N.S.; supervision, M.M.G.; project administration, N.S. and M.M.G.; funding acquisition, M.M.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded in part by the USDA National Institute of Food and Agriculture, Hatch Project OHO01423, Accession Number 1014136. Acknowledgments: Thank you to Gregory Sigurdson for technical assistance in the laboratory. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Clydesdale, F.M. Color as a factor in food choice. Crit. Rev. Food Sci. Nutr. 1993, 33, 83–101. [CrossRef] [PubMed] 2. McCann, D.; Barrett, A.; Cooper, A.; Crumpler, D.; Dalen, L.; Grimshaw, K.; Kitchin, E.; Sonuga-Barke, E. Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: A randomised, double-blinded, placebo-controlled trial. Lancet 2007, 370, 1560–1567. [CrossRef] 3. Sigurdson, G.T.; Tang, P.; Giusti, M.M. Natural Colorants: Food Colorants from Natural Sources. Annu. Rev. Food Sci. Technol. 2017, 8, 261–280. [CrossRef][PubMed] 4. Rentzsch, M.; Schwarz, M.; Winterhalter, P. Pyranoanthocyanins—An overview on structures, occurence, and pathways of formation. Trends Food Sci. Technol. 2007, 18, 526–534. [CrossRef] 5. Fulcrand, H.; Cameira dos Santos, P.J.; Sarni-Manchado, P.; Cheynier, V.; Favre-Bonvin, J. Structure of new anthocyanin-derived wine pigments. J. Chem. Soc. Perkin Trans. 1 1996, 7, 735–739. [CrossRef] 6. He, J.; Carvalho, A.R.F.; Mateus, N.; De Freitas, V. Spectral Features and Stability of Oligomeric Pyranoanthocyanin-flavanol Pigments Isolated from Red Wines. J. Agric. Food Chem. 2010, 58, 9249–9258. [CrossRef] 7. Brouillard, R.; Chassaing, S.; Fougerousse, A. Why are grape/fresh wine anthocyanins so simple and why is it that red wine color lasts so long? Phytochemistry 2003, 64, 1179–1186. [CrossRef] 8. Schwarz, M.; Hofmann, G.; Winterhalter, P. Investigations on Anthocyanins in Wines fromVitis viniferacv. Pinotage: Factors Influencing the Formation of Pinotin A and Its Correlation with Wine Age. J. Agric. Food Chem. 2004, 52, 498–504. [CrossRef] 9. Casassa, L.F.; Sari, S.E.; Bolcato, E.A.; Fanzone, M.L. Microwave-Assisted Extraction Applied to Merlot Grapes with Contrasting Maturity Levels: Effects on Phenolic Chemistry and Wine Color. Fermentation 2019, 5, 15. [CrossRef] 10. Hoehn, M. Altering pH, Temperature and Cofactors to Increase the Formation of the More Stable Anthocyanin Derived Pyranoanthocyanin. Master’s Thesis, The Ohio State University, Columbus, OH, USA, 2019. 11. Zhu, X.; Giusti, M.M. Comparing pyranoanthocyanin formation rates and yields using cyanidin-3-derivatives and pyruvic of caffeic acids. J. Food Chem. 2020, 341, 128776. 12. Gawel, R. Red wine astringency: A review. Aust. J. Grape Wine Res. 1998, 4, 74–95. [CrossRef] Molecules 2020, 25, 5998 11 of 11

13. Lee, J.M.; Finn, C.E. Anthocyanins and other polyphenolics in American elderberry (Sambucus canadensis) and European elderberry (S. nigra) cultivars. J. Sci. Food Agric. 2007, 87, 2665–2675. [CrossRef][PubMed] 14. Francis, F.; Markakis, P.C. Food colorants: Anthocyanins. Crit. Rev. Food Sci. Nutr. 1989, 28, 273–314. [CrossRef][PubMed] 15. Giusti, M.; Wrolstad, R.E. Acylated anthocyanins from edible sources and their applications in food systems. Biochem. Eng. J. 2003, 14, 217–225. [CrossRef] 16. Giusti, M.M.; Wrolstad, R.E. Characterization and measurement of anthocyanins by UV-Visible spectroscopy. In Handbook of Food Analytical Chemistry; Wrolstad, R.E., Acree, T.E., Decker, E.A., Penner, M.H., Reid, D.S., Schwartz, S.J., Shoemaker, C.F., Smith, D., Sporns, P., Eds.; John Wiley & Sons: Hoboken, NJ, USA, 2005; pp. F1.2.1–F1.2.13.

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