Gallic acid as a copigment enhance anthocyanin stabilities and color characteristics in blueberry juice

Abstract

The aim of this study was to evaluate the influence of adding copigment gallic acid (GA) on the stability of anthocyanin and color in blueberry juice, and assays were carried out with different anthocyanin:GA molar ratios (1:0, 1:1, 1:3, 1:5) in accelerated experiments (40 °C for 10 days). Results showed that the addition of GA made blueberry juice to appear more crimson color tonality, color saturation and anthocyanins stability. The most obvious hyperchromic effect appeared in juice with 1:5 of anthocyanin:GA molar ratios, and in this ratio, total anthocyanin content (137.67 mg/L) and main anthocyanin peonidin-3-glucoside content (51.68 mg/L) of the blueberry juice were higher than juice without copigment (total anthocyanin of 116.96 mg/L and peonidin-3-glucoside of 34.2 mg/L). Furthermore, anthocyanins in blueberry juice copigmented with molar ratios 1:5 of anthocyanin:GA were more stable at 4 °C than that at 25 °C and 40 °C. Thus, the addition of gallic acid at appropriate levels might be a promising juice process technology to obtain juices with high color quality and anthocyanin stability.

Electronic supplementary material

The online version of this article (10.1007/s13197-019-04175-w) contains supplementary material, which is available to authorized users.

Introduction

Blueberries (genus Vaccinium, family Ericaceae) are rich in polyphenols and appear dark purple on account of high amounts of anthocyanins (Prior et al. 1998). Due to an abundance of polyphenols, blueberry exhibits diverse biological properties such as antioxidant, cardioprotective, neuroprotective, antiinflammatory and anticarcinogenic properties (Benn et al. 2014; Signorelli et al. 2015). Blueberries are commonly processed into various forms including juice, wine, and jam. Blueberry juices are increasingly promoted and consumed due to nutritional and other health benefits (Michalska and Łysiak 2015; Diaconeasa et al. 2015).

Blueberry juice mainly contains 5 groups of anthocyanidins which include cyanidin (Cy), delphinidin (Dp), peonidin (Pn), malvidin (Mv), and petunidin (Pt) (You et al. 2011; Muller et al. 2012). Anthocyanins have relatively low stability and their degradation is influenced by factors such as pH, light, temperature, oxygen and non-covalent interactions with colorless compounds, coordination with metallic ions, and covalent reactions with other compounds present in the medium (Brenes et al. 2005; West and Mauer 2013). It is worth mentioning that the intermolecular copigmentation could better improve the stability of anthocyanins in berries (Markovic et al. 2000; Hernandez-Herrero and Frutos 2015; Fanzone et al. 2015; Chung et al. 2016).

Intermolecular copigmentation is a phenomenon in which pigments and copigments form complexes by weak hydrophobic and π–π interactions, and where the spectra of copigmented solutions usually display a shift of the visible λmax toward greater wavelengths (bathochromic effect) coupled with an increase of absorptivity (hyperchromic effect) (Castaneda-Ovando et al. 2009). A few copigments were successfully assayed in previous research, for example, the color and anthocyanin content of blueberry juice could be better retained through intermolecular copigmentation using the copigment vitexin, orientin and flavonoid C-glycoside (Pan et al. 2014). In addition, the intermolecular copigmentation of anthocyanins with sinapic acid, caffeic acid, ferulic acid, or rosemary polyphenolic extract could increase the stability of the anthocyanin (Sari et al. 2012; Kopjar and Pilizota 2009). Gallic acid (GA) is a low molecular weight polyphenol compound, which exhibits potent antioxidant properties (Abdelwahed et al. 2007). Previous research has stated that gallic acid facilitates the retention of anthocyanin in vitamin-C fortified cranberry juice (Roidoung et al. 2016).

There is some feasibility and benefits of using gallic acid in commercial juice production. The sources of food grade gallic acid are wide and it has a mildly sour taste (Srinivas et al. 2010). Moreover, in the human body, gallic acid could be absorbed better than other polyphenols (Daglia et al. 2014). In the present study, gallic acid as copigment was used in blueberry juice to enhance the stability of color and anthocyanins. The influence of different anthocyanin:GA molar ratios (1:0, 1:1, 1:3, 1:5) on the anthocyanins and chromatic parameters of juice was investigated, the stability of anthocyanins of blueberry juices was evaluated at different storage temperature and time, and copigmentation effect of GA on individual anthocyanins content in blueberry juice during storage has also been studied.

Materials and methods

Chemicals

The cyanidin-3-glucoside standard was purchased from Sigma-Aldrich (St. Louis, MO, USA). The food-grade gallic acid (99%) was obtained from Shanghai Kangting Bio-Technique Co. Ltd (Shanghai China). The Folin–Ciocalteu’s phenol reagent was purchased from Sinopharm Chemical Reagent (Shanghai, China). Other general chemicals with analytical grade were obtained from local suppliers.

Blueberry samples

Rabbiteye blueberry of cultivar ‘Baldwin’ was obtained from a commercial blueberry plantation located in Hefei (31° 52′ N, 117° 17′ E) in central-eastern China. The samples was frozen and stored at − 20 °C for subsequent chemical component analysis and making juice. Soluble solid content of the blueberry juice was 13.5°Brix, the pH was 3.3 and the concentration of titratable acid was 7.4 g/L.

Preparation of blueberry juice copigmented with gallic acid

The frozen blueberries were thawed at room temperature, blanched via steam for 3 min, and then cooled to room temperature. Steam blanching was performed in a food steamer at atmospheric pressure. Blanched berries were milled and treated with pectinase (Laffort, France) at a concentration of 0.05 g/kg fruit for 2 h at room temperature. To remove pomace, the blueberry puree by pectinase hydrolysis was strained through a piece of 200 mesh nylon mesh filter, and further centrifuged at 3100×g for 15 min by centrifugal machine (Xiangyi Laboratory Instrument Development Co., Ltd., Hunan, China). And at this moment, the blueberry juice containing 424.13 mg/L of total anthocyanin was prepared (the total anthocyanin was calculated with cyanidin-3-glucoside as standard).

In our previous experiment, the screening of the copigment, i.e., tea polyphenols, EGCG, P-coumaric acid and gallic acid (GA) were carried out, and gallic acid was confirmed as the copigment of blueberry juice to carry out the further research (Supplementary material, Fig. S1). Next, to obtain 1:0, 1:1, 1:3 and 1:5 ratio of anthocyanins to gallic acids in the copigmented juice samples, 0 mg, 16.06 mg, 48.19 mg and 80.31 mg GA was added to each 100 mL blueberry juice contained a total anthocyanin of 424.13 mg, respectively. Stir the juices until the gallic acid completely dissolved. The copigmented juices were pasteurized at 85 °C for 5 min for the next trials.

Storage stability of anthocyanins and color of blueberry juice

To evaluate the protection of anthocyanins and color by copigmentation of GA, blueberry juice with different anthocyanin/GA molar ratios (1:0, 1:1, 1:3, 1:5) were stored at 40 °C for 10 days to conduct an accelerated shelf life studies. The absorption spectra and individual anthocyanins content were determined on day 0 and day 10, respectively. The chromatic parameters [lightness (L*), chroma (C*) and hue angle (H*)] of blueberry juices were measured on day 0, 2, 5, 7 and 10. According to the degradation rates of anthocyanins in the blueberry juices, the changes of anthocyanins of juices at molar anthocyanins/GA ratios of 1:0 and 1:5 was further evaluated during storage at 4 °C for 50 days, 25 °C for 40 days and 40 °C for 10 days, respectively.

Measurements of hyperchromic effect and spectral shift

Anthocyanin copigmentation reactions can be detected by a hyperchromic effect or a bathochromic shift (Eiro and Heinonen 2002; Markovic et al. 2000). In this work, to evaluate the hyperchromic and bathochromic shift effect, the blueberry juice with different anthocyanin:GA molar ratios were diluted with deionized water according to their absorbance in advance, and then the absorption spectrum of juices were scanned in the visible range (between 400 and 700 nm) by a UV–visible light spectrophotometer with Lambda 35 UV–Vis systems (PerkinElmer, America).

Analysis of total anthocyanin content (TAC) and retention rate of TAC

The TAC and the retention rate of TAC of juices were estimated using the pH differential method as reported in our previous article (He et al. 2016). The samples of juice were diluted with pH 1.0 buffer and pH 4.5 buffer by the same multiple, respectively. The absorbance of diluted samples was measured at 510 nm and 700 nm. The TAC and the retention rate of TAC were calculated using the following formulas (1), (2) and (3):

$$A={\left(A_{510}-A_{700}\right)}_{\text{pH}1.0}-{\left(A_{510}-A_{700}\right)}_{\text{pH}4.5}$$ 1

$$TAC=\frac{\left(A\times MW\times DF\times V_{\text{e}}\times 1000\right)}{\epsilon \times 1\times M}$$ 2

$$RetentionrateofTAC=\frac{TA{C}_{\text{t}}}{TA{C}_0}\times 100\%$$ 3

where A₅₁₀ and A₇₀₀ is the absorbance detected at 510 nm and 700 nm, respectively; A is the difference in absorbance between pH 1.0 and 4.5, MW is the molecular weight of cyanidin-3-glucoside (449 g/mol); DF is the dilution factor; V_e is the extract volume, $\epsilon$ is the molar extinction coefficient of cyanidin-3-glucoside (29,600), and M is the mass of the blueberry extracted. Total anthocyanin content (TAC) was expressed as mg cyanidin-3-glucoside (C3G) equivalents per 1 L of juice (mg C3G/L). Further, TAC_t is the total anthocyanin content of storage for t day; TAC₀ is the total anthocyanin content of storage for 0 day. TAC (%, of initial) expresses the retention rate of total anthocyanin content.

Polymeric color analysis

Polymeric color was determined using the method modified by Zou et al. (2016). For analysis, 0.3 mL of distilled water or 0.90 M (mol/L) potassium metabisulfite was added to 4.2 mL of diluted sample. After equilibrating for 30 min, samples were evaluated at λ = 700, 510, and 420 nm. Color density (CD) was calculated by data from the experimental group with distilled water using the following formula:

$$CD=\left[\left(A_{420}-A_{700}\right)+\left(A_{510}-A_{700}\right)\right]\times K$$ 4

K was the dilution factor.

Polymeric color (PC) was determined from the experimental group with potassium metabisulfite using the following formula:

$$PC=\left[\left(A_{420}-A_{700}\right)+\left(A_{510}-A_{700}\right)\right]\times K$$ 5

Percent polymeric color (PPC) was calculated using the following formula:

$$PPC=\frac{\text{polymeric}\text{color}}{\text{color}\text{density}}\times 100\%$$ 6

Quantification of individual anthocyanins by Ultra Performance Liquid Chromatograph

Anthocyanin profiles in blueberry juice were analyzed by Ultra Performance Liquid Chromatograph (UPLC) using Waters Acquity Ultra-Performance™ LC system (Waters, Millford, USA). The chromatographic system consisted of a pump, an Agilent C18 column (2.1 × 150 mm, 2.6 μm), and a Waters Tunable UV detector. Quantification method of individual anthocyanins was determined according to the method described previously (He et al. 2016) with slight modifications. Briefly, the sample was filtered through 0.45 μm microfiltration membrane prior to UPLC analysis. The mobile phase consisted of 6% formic acid aqueous (A) and acetonitrile containing 6% formic acid (B). The gradient elution program was set as follows: 5–50% B (0–20 min), 50–5% B (20–22 min), and 5% B (22–25 min). The injection volume, the flow rate and column temperature were 5 μL, 0.3 mL/min and 30 °C, respectively. Anthocyanins were detected by their absorbance at 520 nm. The levels of individual anthocyanins were quantified as cyanidin-3-glucoside equivalents.

Individual anthocyanin identification by UPLC-DAD-ESI/MS

Anthocyanin analysis of blueberry juices were carried out using a Waters Acquity UPLC System. (Waters, Millford, US) equipped with a Diode Array Detector (DAD) and a Triple Quadrupole Mass Spectrometer. Identification of individual anthocyanins, including delphinidin-3-galactoside, delphinidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-glucoside, petunidin-3-galactoside, petunidin-3-galactoside, peonidin-3-galactoside, peonidin-3-glucoside, malvidin-3-galactoside, malvidin-3-glucoside and malvidin-3-arabinoside, was performed according to the method in the literature (He et al. 2016) with a slight modification. Samples were separated using an Agilent C18 column packed with Zorbax (2.1 × 150 mm, 2.6 μm). The column temperature was maintained at 30 °C. The sample was filtered through 0.45 μm microfiltration membrane prior to UPLC analysis. The injection volume was 5 μL. The flow rate was 0.3 mL/min. The mobile phase consisted of aqueous 0.2% formic acid (A) and acetonitrile containing 0.2% formic acid (B). The gradient program was set as follows: 5–50% B (0–10 min), 50–5% B (10–11 min), and 5% B (11–14 min). Equilibrium time between runs was 10 min. Anthocyanins were detected at 520 nm. The conditions of MS analysis were as follows: electrospray ionization (ESI) interface, nitrogen for drying, nebulizing gas and nebulizer pressure at 35 psi, dry gas flowing at 10 mL/min, 350 °C dry gas temperature, capillary voltage at 2500 V, and spectra recording in the positive ion mode with scans from m/z 10 to 1000.

Color measured

Color was determined with ColorQuest XE (Hunter Associates Laboratory Inc. Reston, VA, USA) (the 10° Standard Observer and Standard Illuminant D65). The chromatic parameters were assessed by using the method described previously (Zhang et al. 2016). The values a* and b* were used to calculate the hue angle $\left[\text{H}=arctan\left(\text{b}\ast /\text{a}\ast \right)\right]$ and chroma value $\left[{\text{C}=\left(\text{a}{\ast }^2+\text{b}{\ast }^2\right)}^{1/2}\right]$.

Statistical analysis

The experiments were conducted in triplicate and values were expressed as the means ± the standard deviation (SD). Data were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s range test Duncan using Prism™ v6.0 software. A P < 0.05 was considered to be statistically significant and the differences are denoted by different letters.

Results and discussion

Copigmentation effect

The copigmentation phenomenon is usually assessed by looking at the changes in λmax of the visible absorption spectrum. Figure 1 illustrated the visible spectra of blueberry juices with different anthocyanins/GA molar ratios. It was found that the addition of copigments induced the increase of absorbance over all the visible range (400–700 nm), where the hyperchromic effect was elicited by copigments. These copigmented juices also induced bathochromic shift in λmax (bathochromic effect). As shown in Fig. 1, it could be seen that the hyperchromic effect was dependent on the magnitude of the added GA, and the most obvious hyperchromic effect was observed in the juice at a molar anthocyanins/GA ratio of 1:5. Hyperchromic effect or a bathochromic shift indicated that anthocyanins and copigments formed an overlapping arrangement through intermolecular association (Eiro and Heinonen 2002; Markovic et al. 2000). Fanzone et al. (2015) presented the experimental results of positive correlation between malvidin-3-O-glucoside:dihydroquercetin-3-O-glucoside molar ratio and hyperchromic effect. Petrova et al. (2017) found that A_max increases with the increase in the concentrations of strawberry anthocyanin and quercetin at 40 °C and 50 °C. The hyperchromic effect could enhance with the increase of copigment content (Fanzone et al. 2015; Lorenzo et al. 2005; Gonzalez-Manzano et al. 2008).

Fig. 1.

Absorption spectra of blueberry juices with different anthocyanins/gallic acid molar ratios. GA: gallic acid; BJ: blueberry juice; BJ + GA (1:0), BJ + GA (1:1), BJ + GA (1:3) and BJ + GA (1:5): blueberry juice with 1:0, 1:1, 1:3 and 1:5 anthocyanin/gallic acid molar ratio

Effects of copigmentation on anthocyanin stability and percent polymeric color

The retention rate of total anthocyanin content (TAC) and percent polymeric color (PPC) from blueberry juices of different anthocyanin/GA (GA) molar ratios at 40 °C for 10 days were respectively shown in Fig. 2A, B. As demonstrated by Fig. 2A, the addition of GA with higher molar ratios enhanced the retention rate of TAC in blueberry juice (P < 0.05). The improvement of anthocyanin stability in blueberry juice is due to the effect of the added polyphenolics (e.g. GA). Phenolic compound act as antioxidants can protect anthocyanins from oxidation (Roidoung et al. 2016; Juurlink et al. 2014). And phenolic compound as copigments can interact with anthocyanins by binding covalent or noncovalent bond with anthocyanins to form anthocyanin-cofactor complexes to resist the non-oxidative degradation of anthocyanins, such as hydration and further molecular division (Giustim and Wrolstad 2003; Trouillas et al. 2016; Zhang et al. 2018). Previous studies indicated that the addition of caffeic acid significantly increased (P < 0.05) the stability of anthocyanins in model systems of Cabernet Sauvignon grape extracts (Gris et al. 2007). The addition of chlorogenic acid increased the stability of anthocyanin in blackberry juice during storage (Kopjar et al. 2012). And quercetagetin was effective to stabilize the anthocyanins of grape skin (Xu et al. 2015). Gallic acid can improve the stability of anthocyanin in anthocyanin-rich foods (Navruz et al. 2016; Liu et al.2016; Zhang et al. 2018; Kopjar and Pilizota 2009; Srinivas et al. 2010).

Fig. 2.

The total anthocyanin content (a) and percent polymeric color (b) in blueberry juices with different anthocyanins/gallic acid molar ratios at 40 °C for 10 days. GA: gallic acid; BJ: blueberry juice; BJ + GA(1:0), BJ + GA(1:1), BJ + GA(1:3) and BJ + GA(1:5): blueberry juice with 1:0, 1:1, 1:3 and 1:5 anthocyanin/gallic acid molar ratio; TAC: total anthocyanin content; PPC: percent polymeric color. Different lowercase letters represent significant differences between samples (P < 0.05)

Furthermore, Retention rate of TAC in blueberry juice increased with the increasing of the anthocyanins/GA molar ratio. It had the highest retention rate of TAC in the blueberry juice with the anthocyanins/GA molar ratio of 1:5. The result showed that the copigmentation of high content GA was more beneficial to improving the stability of anthocyanin in blueberry juice. A large number of studies have found that the copigmentation effect become stronger with the increase of the concentration of copigments (Brouillard et al. 1989; Ghasemifar and Saeidian 2014). At the temperature of 20°C and pH 2.7–5.7, the higher the molar ratio of chlorogenic acid was, the stronger the copigmentation effect of anthocyanins in the aqueous solutions was (Mazza and Brouillard 1990). Sari (2016) also found that the anthocyanin retention rate increased with the increase of the molar ratio of GA in copigmented solution.

Percent polymeric color (PPC) can represent a measure of the pigment resistance to bleaching, and it can also show the polymerization of anthocyanin (Cao et al. 2011). As shown in Fig. 2B, copigment GA had a positive effect on PPC in the blueberry juices with different anthocyanin/GA molar ratios. PPC in the blueberry juices declined as the increase of the anthocyanin/GA molar ratio. Lowest PPC was found in the blueberry juice with 1:5 anthocyanin/GA molar ratio. With the increase of copigment concentration, the decrease of PPC values may be attributed to delay the gradual degradation of anthocyanins or condensation reactions of anthocyanins with protein and other phenolics (Zou et al. 2016).

Characterisation of anthocyanins in blueberry juice and effect of copigmentation on stability of individual anthocyanins

The anthocyanin profiles of blueberry juice and the cyanidin-3-glucoside standard were present in Fig. 3. Eleven anthocyanins were identified by chromatographic (Fig. 3). Individual anthocyanins were further detected by UPLC-DAD-ESI/MS (Table S1). According to the mass spectrum (Table S1) and previous report (Prior et al. 2001; You et al. 2011), eleven anthocyanins were identified and they belong to 5 main groups of anthocyanidins which include cyanidin (Cyd), delphinidin (Dpd), peonidin (Pnd), malvidin (Mvd), and petunidin (Ptd). The chromatograph analysis (Fig. 3) showed that delphinidin-3-glucoside and peonidin-3-glucoside were the major anthocyanins in the blueberry juice, and the result was in accord with Pan et al. (2014).

Fig. 3.

UPLC chromatograms of blueberry juice (A) and cyanidin-3-glucoside standard (B) at 520 nm. The chromatographic peak, 1: Delphinidin-3-galactoside; 2: Delphinidin-3-glucoside; 3: Cyanidin-3-galactoside; 4: Cyanidin-3-glucoside; 5: Petunidin-3-galactoside; 6: Petunidin-3-galactoside; 7: Peonidin-3-galactoside; 8: Peonidin-3-glucoside; 9: Malvidin-3-galactoside; 10: Malvidin-3-glucoside; 11: Malvidin-3- arabinoside

Copigmentation effect of GA on individual anthocyanins in blueberry juice storage for 10 days at 40 °C was investigated by UPLC (Table 1). Table 1 indicates that, as a main anthocyanin in all blueberry juices, the content of peonidin-3-glucoside from GA (1:0), GA (1:1), GA (1:3) and GA (1:5) juices were 34.2, 38.36, 42.08 and 51.68 mg/L, respectively.

Table 1.

Contents of individual anthocyanins of blueberry juices with different anthocyanins/gallic acid molar ratios after storage for 10 days at 40 °C

Anthocyanin (mg/L)	RT (min)	Blueberry juices
		BJ + GA(1:0)	BJ + GA(1:1)	BJ + GA(1:3)	BJ + GA(1:5)
Delphinidin-3-galactoside	7.867	8.24 ± 0.67a	10.97 ± 0.79b	11.02 ± 0.93b	15.86 ± 0.50c
Delphinidin-3-glucoside	9.069	6.9 ± 0.45a	7.04 ± 0.62a	8.41 ± 0.91a	10.25 ± 1.02b
Cyanidin-3-galactoside	9.524	3.52 ± 0.12a	3.66 ± 0.37a	4.20 ± 0.34a	6.51 ± 0.40b
Cyanidin-3-glucoside	10.080	1.71 ± 0.06a	1.32 ± 0.13a	1.82 ± 0.40a	1.69 ± 0.33a
Petunidin-3-galactoside	10.667	10.59 ± 0.97a	12.60 ± 1.02ab	13.52 ± 1.88b	18.28 ± 1.56c
Petunidin-3-glucoside	11.835	3.99 ± 0.29a	4.18 ± 0.48ab	4.93 ± 0.56bc	5.65 ± 0.51c
Peonidin-3-galactoside	12.343	3.06 ± 0.18a	3.60 ± 0.26ab	3.88 ± 0.35b	5.17 ± 0.39c
Peonidin-3-glucoside	13.169	34.2 ± 2.88a	38.36 ± 2.39ab	42.08 ± 2.88b	51.68 ± 2.99c
Malvidin-3-galactoside	13.542	1.60 ± 0.11a	1.75 ± 0.14a	1.84 ± 0.23a	2.50 ± 0.20b
Malvidin-3-glucoside	14.083	3.04 ± 0.24a	3.14 ± 0.26a	3.51 ± 0.30a	4.37 ± 0.31b
Malvidin-3- arabinoside	14.879	9.22 ± 0.78a	10.67 ± 0.83ab	11.21 ± 0.91b	15.86 ± 0.89c

GA: gallic acid; BJ: blueberry juice; BJ + GA(1:0), BJ + GA(1:1), BJ + GA(1:3) and BJ + GA(1:5): blueberry juice with 1:0, 1:1, 1:3 and 1:5 anthocyanin/gallic acid molar ratio. Significance testing among the different samples was performed by one-way ANOVA followed by Duncan’s range test. Different lowercase letters represent significant differences between samples (P < 0.05)

Most of monomeric anthocyanin of juices (except Cyanidin-3-glucoside) at molar anthocyanins/ratio of 1:1, 1:3 and 1:5 increased than the juice without GA, while the addition of molar anthocyanins/GA ratio of 1:1 led to slight increase (Table 1). And with the increment of adding proportion on GA, the contents of monomeric anthocyanins of juice were gradually improved compared with the juice without GA (except Cyanidin-3-glucoside). GA exhibits potent antioxidant properties (Abdelwahed et al. 2007), a reason for higher antioxidant activities of juices protected anthocyanins from oxidative degradation with the increment of the addition proportion of anthocyanins/GA molar ratio. Gomez-Miguez et al. (2006) have observed a similar phenomenon that the presence of the caffeic acid at 1:1 copigment/pigment molar ratio does not impede the loss of the anthocyanin in model wine solutions. GA could improve the stabilities of cyanidin-3-rutinoside and cyanidin-3-glucosylrutinoside in sour cherry juice concentrates by protecting anthocyanins from oxidative degradation due to GA high antioxidant capacities (Navruz et al. 2016; Gil et al. 2000).

In the current study, with the increment of the addition proportion of anthocyanins/GA molar ratio, the content change of Cyanidin-3-glucoside showed the small fluctuations. Many factors have influenced copigmentation of monomeric anthocyanin, such as anthocyanin structure, concentration of anthocyanin relative to copigment and copigment structure (Eiro and Heinonen 2002). Besides, GA has a different protective effect on different monomeric anthocyanin, which may be related to the complexity of juice system during storage. Gallic acid, approximately from 300 to 928 mg/kg, has been explored in the production of sour cherry juice and Cabernet Sauvignon wine, and it has been found to retain more anthocyanins in juices and wines (Navruz et al. 2016; Liu et al.2016; Zhang et al. 2018). GA as a good copigment (Kopjar and Pilizota 2009; Srinivas et al. 2010), it played an important role in enhancing the stability of total individual anthocyanins in blueberry juice.

Stability of anthocyanin in blueberry juices storage at 4 °C, 25 °C and 40 °C

The retention rate of total anthocyanin in different blueberry juices during storage at 4 °C, 25 °C and 40 °C were given in Fig. 4A–C. The results showed that retention rate of total anthocyanin content (TAC) in blueberry juices presented decrease tendency with increasing of storage period at different storage temperature. Among them, the blueberry juice storaged at 4 °C had the highest retention rate of TAC (62.23–71.69%, 40 days), followed by 25 °C (12.83–23.04%, 40 days) and 40 °C (27.57–32.35%, 10 days). Anthocyanin, as a heat-sensitive substance, is highly susceptible to degradation under high temperature, and anthocyanins degradation could be accelerated with increase of storage temperatures, so low temperature is an effective method to delay anthocyanins degradation of blueberry juice (Syamaladevi et al. 2012; Kechinski et al. 2010; Buckow et al. 2010).

Fig. 4.

Changes in retention rate of total anthocyanin content of blueberry juices at a molar anthocyanins/gallic acid ratio of 1:5 during storage at 4 °C (A), 25 °C (B) and 40 °C (C). GA: gallic acid; BJ: blueberry juice; BJ + GA(1:0) and BJ + GA(1:5): blueberry juice with 1:0 and 1:5 anthocyanin/gallic acid molar ratio; TAC: total anthocyanin content. Different lowercase letters represent significant differences between samples (P < 0.05)

During storage period at different temperature, the anthocyanin degradation of blueberry juices with GA was significantly slower than the juice without GA (P < 0.05). The blueberry juice copigmented with GA had higher retention rate (4.23–13.65%) of TAC than untreated juice. In addition to higher antioxidant activities of GA protected anthocyanins from oxidative degradation (Abdelwahed et al. 2007; Navruz et al. 2016; Gil et al. 2000), GA and anthocyanin pigments in blueberry juice could form stable pigment-cofactor complexes through molecule association, which inhibits the hydration of anthocyanins and improves the retention rate of anthocyanins (Chen and Hrazdina 1981; Galli and Clemente 2013). Pan et al. (2014) also reported that anthocyanins in juices with copigment showed gentle downward trend, while control showed a faster downward trend during storage.

Effect of copigmentation on color stability

Color is considered the basis for assessing food quality. Moreover, color as a key sensory feature is thought to influence consumers’ preference, acceptability and ultimate choice of food (Zhang and Wang 2017; Bridle and Timberlake 1997). With the aim of evaluating the effect of copigmentation on the color expression of blueberry juice, chromatic analyses in the CIELab space were performed. The chromaticity parameters (L*, C*, and H*) of all juices were shown in Table 2. During storage, the addition of the copigment to the blueberry juice induced a decrease in lightness (L*) and a rise in chroma (C*) compared to juice without GA, indicating that the color of copigmented juice samples was more intense and saturated (Pan et al. 2014). After seventh days, juices with GA showed significantly (P < 0.05) lower value of hue angle (H*) and lightness (L*) compared with juice without GA, reflecting a more reddish color tonality of copigmented solutions. These changes were more marked with increasing copigment concentration. As reported by Shikov et al. (2008), the color stability was increased due to copigmented anthocyanins. The more color saturation are also connected with hyperchromic effect produced by copigmentation (Gomez-Miguez et al. 2006). The interaction between the anthocyanin and cofactors leads to hyperchromic and bathochromic effects, enhancing color intensity and stability (Marković et al. 2005; Zhang et al. 2015). Previous studies have shown that copigmentation can stabilize the color of natural anthocyanins (Gras et al. 2016; Chitgar et al. 2018; Fan et al. 2019).

Table 2.

Change of chromatic parameters lightness (L*), chroma (C*) and hue angle (H*) of blueberry juices with different anthocyanins/gallic acid molar ratios during storage at 40 °C

Chromatic parameters	Samples	Storage (days)
		0	2	5	7	10
L*	BJ + GA(1:0)	1.58 ± 0.02Aa	6.11 ± 0.01Bc	8.61 ± 0.02Cd	10.58 ± 0.01Dd	11.11 ± 0.01Ed
	BJ + GA(1:1)	1.56 ± 0.02Aa	6.07 ± 0.02Bb	8.18 ± 0.01Cc	9.88 ± 0.01Dc	10.49 ± 0.02Ec
	BJ + GA(1:3)	1.55 ± 0.02Aa	6.06 ± 0.01Bb	8.07 ± 0.03Cb	9.78 ± 0.02Db	10.41 ± 0.01Eb
	BJ + GA(1:5)	1.55 ± 0.02Aa	5.48 ± 0.01Ba	7.49 ± 0.06Ca	9.67 ± 0.02 Da	10.26 ± 0.01Ea
C*	BJ + GA(1:0)	11.54 ± 0.13Aa	33.92 ± 0.10Ba	39.12 ± 0.08Ca	42.56 ± 0.12 Da	43.99 ± 0.11Ea
	BJ + GA(1:1)	11.56 ± 0.09Aa	35.90 ± 0.13Bb	41.27 ± 0.10Cc	42.76 ± 0.09Db	44.51 ± 0.13Eb
	BJ + GA(1:3)	11.58 ± 0.08Aa	35.79 ± 0.07Bb	40.43 ± 0.09Cb	42.89 ± 0.06Db	44.47 ± 0.14Eb
	BJ + GA(1:5)	11.57 ± 0.09Aa	35.81 ± 0.08Bb	40.26 ± 0.14Cb	43.16 ± 0.10Dc	45.83 ± 0.11Ec
H*	BJ + GA(1:0)	13.58 ± 0.04Aa	17.06 ± 0.03Bb	21.09 ± 0.04Ca	23.58 ± 0.04Dd	24.72 ± 0.03Ed
	BJ + GA(1:1)	13.56 ± 0.05Aa	17.00 ± 0.06Bb	20.41 ± 0.03Ca	22.78 ± 0.05Dc	23.98 ± 0.04Ec
	BJ + GA(1:3)	13.58 ± 0.03Aa	17.00 ± 0.06Bb	20.22 ± 0.05Ca	22.58 ± 0.08Db	23.81 ± 0.03Eb
	BJ + GA(1:5)	13.55 ± 0.04Aa	16.17 ± 0.04Ba	19.29 ± 2.04Ca	21.58 ± 0.04 Da	23.72 ± 0.04Ea

GA: gallic acid; BJ: blueberry juice; BJ + GA(1:0), BJ + GA(1:1), BJ + GA(1:3) and BJ + GA(1:5): blueberry juice with 1:0, 1:1, 1:3 and 1:5 anthocyanin/gallic acid molar ratio. Significance testing among the different samples was performed by one-way ANOVA followed by Duncan’s range test. Different uppercase letters in the same row represent significant differences within sample and different lowercase letters represent significant differences between samples (P < 0.05)

Conclusion

According to the results acquired in this work, the high amount of gallic acid (GA) added into blueberry juice did indeed delay the degradation of main anthocyanins and prolong the color deterioration during the storage period. GA led to the hyperchromic effect and the increase in main anthocyanin content indicating the existence of copigmentation in blueberry juice. GA treatment do not only influence the color characteristics and anthocyanin compounds of juice, but also have a longer effect on the evolution of color and anthocyanins in juice during storage. In addition, the GA as a phenolic acid can bring juices appropriate taste and good functionality, improving the quality of blueberry juice. Therefore, the application of GA could be as a feasible and promising novel technology to produce a stabile blueberry juice.

Electronic supplementary material

Below is the link to the electronic supplementary material.