Influence of Steviol Glycosides on the Stability of Vitamin C and

Article pubs.acs.org/JAFC

Influence of Steviol Glycosides on the Stability of Vitamin C and Anthocyanins Łukasz Woźniak,* Krystian Marszałek, and Sylwia Skąpska Department of Fruit and Vegetable Product Technology, Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology, 36 Rakowiecka Street, 02-532 Warsaw, Poland S Supporting Information *

ABSTRACT: A high level of sweetness and health-promoting properties make steviol glycosides an interesting alternative to sugars or artificial sweeteners. The radical oxygen species scavenging activity of these compounds may influence the stability of labile particles present in food. Model buffer solutions containing steviol glycosides, a selected food antioxidant (vitamin C or anthocyanins), and preservative were analyzed during storage. The addition of steviol glycosides at concentrations of 50, 125, and 200 mg/L increased the stability of both ascorbic and dehydroascorbic acid (degradation rates decreased up to 3.4- and 4.5-fold, respectively); the effect was intensified by higher sweetener concentrations and higher acidity of the solutions. Glycosides used alone did not affect the stability of anthocyanins; however, they enhanced the protective effect of sugars; half-life times increased by ca. 33% in the presence of sucrose (100 g/L) and by ca. 52% when both sucrose (100 g/L) and glycosides (total 200 mg/L) were used. Steviol glycosides concentrations remained stable during experiments. KEYWORDS: anthocyanins, Stevia rebaudiana, steviol glycosides, vitamin C

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INTRODUCTION Stevia (Stevia rebaudiana Bertoni) is a perennial shrub native to Paraguay and Brazil. One of the major constituents of its leaves is a group of ent-kaurene diterpenoid glycosides commonly referred to as steviol glycosides. Their content varies depending on cultivation conditions, usually accounting for 4−20% of the leaf dry weight.1 Stevioside and rebaudioside A constitute about 90% of the total glycosides mass; apart from them, over 30 of these compounds were identified in S. rebaudiana leaves.2 From a practical point of view the most important property of steviol glycosides is their highly sweet taste (approximately 200−300 times sweeter than sucrose). The leaves of S. rebaudiana have traditionally been used for hundreds of years by Guarani Indians, but their application in Western diets is relatively new.3 Crude stevia extracts were first commercialized in Japan in the early 1970s, and since then they have gained popularity in East Asian and Latin American countries.4 The United States, Australia, New Zealand, and members of the European Union only very recently joined the list of countries authorizing the use of steviol glycosides; however, many consumers in those areas have already found stevia to be an interesting natural sweetener. Gastric juices and digestive enzymes from humans and animals are unable to degrade steviol glycosides. In addition, their high molecular masses along with hydrophilic character make their absorption in the intestines very unlikely; however, intestinal microbiota are able to convert glycosides into saccharides and an aglycone (steviol), which can penetrate into the bloodstream. Absorbed steviol is converted into steviol glucuronide in the liver and then excreted via the urinary and biliary tract.5 Besides their sweetening properties, steviol glycosides and related compounds offer therapeutic benefits including antiinflammatory, antiviral, antitumor, antihyperglycemic, antihy© XXXX American Chemical Society

pertensive, antidiarrheal, diuretic, and immunomodulatory effects.5,6 Some of the above-mentioned activities arise from the antioxidant properties of steviol glycosides and their metabolites. Studies conducted by Stoyanova et al.7 and Geuns et al.8 revealed that sweeteners from S. rebaudiana are very efficient reactive oxygen species scavengers: half-inhibitory concentrations on hydroxyl (HO•) and superoxide (O2•−) radicals were approximately 0.2 and 0.3 mM, respectively. The antioxidant properties of steviol glycosides suggest that these compounds might influence the quality of foods by delaying degradation of their labile ingredients such as ascorbic acid or phenolic compounds. Studies on the interactions between glycosides from S. rebaudiana and other food constituents are very scarce. Kroyer investigated the interactions in binary model solutions containing stevioside and water-soluble vitamins (ascorbic acid, thiamin, riboflavin, pyridoxine, and nicotinic acid) or low-calorie sweeteners (saccharin, cyclamate, aspartame, acesulfame, and neohesperidin dihydrochalcone).9,10 All samples were incubated in the dark, at a temperature of 80 °C for up to 4 h. The majority of the trials showed no significant change in the concentrations of the examined compounds; however, the protective effect of stevioside was observed in the case of the degradation of ascorbic acid. Its stability in the presence of glycoside (27% of ascorbic acid remained after 4 h) was significantly higher compared to that of the control sample (13% remained after 4 h). The stability of steviol glycosides in various foodstuffs was investigated by several research teams. Selected matrices were Received: August 26, 2014 Revised: October 28, 2014 Accepted: October 29, 2014

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dx.doi.org/10.1021/jf504001t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Table 1. Composition of Model Solutions To Study the Interactions between Steviol Glycosides and Anthocyaninsa symbol

C3G (mg L−1)

C3G/C C3G/Ste200 C3G/Ste125 C3G/Ste50 C3G/RebA200 C3G/RebA125 C3G/RebA50 C3G/Suc C3G/GF C3G/Mix P3G/C P3G/Ste200 P3G/Ste125 P3G/Ste50 P3G/RebA200 P3G/RebA125 P3G/RebA50 P3G/Suc P3G/GF P3G/Mix

100 100 100 100 100 100 100 100 100 100

P3G (mg L−1)

Ste (mg L−1)

RebA (mg L−1)

Suc (g L−1)

Glu (g L−1)

Fru (g L−1)

200 125 50 200 125 50 100 100 100 100 100 100 100 100 100 100 100 100

100

50

50

50

50

100

200 125 50 200 125 50 100 100

100

100

BA (mg L−1) 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150

a

The acidity of all solutions was pH 3.6 and was set using citric acid−sodium phosphate buffer prepared according to the method of Ruzin.20 Abbreviations: BA, benzoic acid; C3G, cyanidin-3-glucoside; Fru, fructose; Glu, glucose; P3G, pelargonidin-3-glucoside; RebA, rebaudioside A; Ste, stevioside; Suc, sucrose. Waters) was used. Separation of the 10 μL samples was performed within 12 min at a flow rate of 1 mL min−1 and a column temperature of 40 °C. Samples were eluted using a gradient of 1.8 mM hydrochloric acid (solvent A) and acetonitrile (solvent B), as follows (time, corresponding composition): 0 min, 65% A; 3 min, 65% A; 5 min, 60% A; 7 min, 60% A; 9 min, 65% A; 12 min, 65% A. Compounds were quantified using UV absorption at 210 nm. The L-ascorbic (AA) and L-dehydroascorbic acid (DHAA) contents were measured according to the a method proposed by OdriozolaSerrano et al.18 Before analysis, 1 mL of the sample was diluted with 1 mL of 0.01% phosphoric acid (determination of AA) or 1 mL of 1 g L−1 solution of dithiothreitol (DTT) in 0.01% phosphoric acid (determination of AA and DHAA sum). The samples with DTT were kept in the cold (4 °C) for at least 1 h before injection. Separation was done on a Sunfire C18, 5 μm, 4.6 mm × 250 mm analytical column with a Sunfire C18 Sentry guard cartridge, 5 μm, 4.6 mm × 20 mm (both Waters). Analysis of the 10 μL samples was performed within 10 min at a flow rate 1 mL min−1 and a column temperature of 25 °C. The samples were eluted isocratically using 0.01% phosphoric acid. The compounds were quantified using UV absorption at 245 nm. Anthocyanin concentrations were determined using a method proposed by Oszmiański19 using the same analytical column and guard column as described above. Separation of the 10 μL samples was performed within 26 min at a flow rate of 1 mL min−1 and a column temperature of 25 °C. The samples were eluted using a gradient of 80% solution of acetonitrile in 4.5% formic acid (solvent A) and 4.5% formic acid (solvent B), as follows (time, corresponding composition): 0 min, 0% A; 7 min, 15% A; 15 min, 15% A; 21 min, 100% A; 26 min, 0% A. Compounds were quantified using vis absorption at 520 nm. Glucose, fructose, and sucrose were determined according to EN 12630:1999 standard. For the analysis a Sugar-Pak I, 10 μm, 6.5 mm × 300 mm analytical column with a Sugar-Pak Guard-Pak insert, 10 μm (both Waters), was used. Separation of the 10 μL samples was performed within 18 min at a flow rate 0.5 mL min−1 and a column temperature of 90 °C. Samples were eluted isocratically using 0.1 mM calcium disodium EDTA. The compounds were quantified using a refractive index detector. Model Solutions. During research work, two series of experiments were conducted. The first investigated the interactions between steviol glycosides and two anthocyanins, cyanidin-3-glucoside and pelargoni-

carbonated drinks,11−14 dairy products and pastries,15,16 and hot beverages, model solutions, and solid sweetener.9,10 All of the cited publications show that the stability of glycosides decreases with increasing acidity (significant decomposition at room temperature occurred only in solutions with a pH 50% higher than those observed in the control samples. Pelargonidin-3-glucoside was more stable than cyanidin-3-glucoside. This fact is consistent with the principle that increased hydroxylation of the flavylium cation decreases the stability of anthocyanins.28 The effect of sweeteners was similar for both anthocyanins. The influence of sugar on the stability of anthocyanin in water solutions is dependent to a large extent on the temperature, concentration, and type of sugar. Low concentrations, below 20%, of most carbohydrates stabilize pigments during storage in cold and ambient temperatures; an exception is fructose, which shows a destabilizing effect.29−31 The results obtained in this study are consistent with these conclusions: anthocyanins were more stable in solutions containing sugars, and sucrose was also a better stabilizer than a mixture of glucose and fructose. Steviol glycosides did not affect the stability of anthocyanins, although they enhanced the protective properties of carbohydrates. The mechanism of this process is not yet known and should be examined in further research. Kinetics of Vitamin C Degradation. The mechanism of AA degradation is specific, as it depends on several factors, whether it follows an aerobic or anaerobic pathway. The first

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RESULTS AND DISCUSSION Stability of Steviol Glycosides. Concentrations of stevioside and rebaudioside in all of the model solutions investigated in the study were measured on the first and last days of each experiment (data not shown). The Tukey test did not reveal statistically significant differences between the initial and final concentrations of glycosides in any sample. These results are consistent with previous studies. Chang and Cook11 found that steviol glycosides in carbonated drinks are stable at room temperature. These results were later confirmed by many authors examining various food matrices.9,10,12−16 Kinetics of Anthocyanin Degradation. Anthocyanins are a group of flavylium cation based glycosides that act as pigments and antioxidants in plant tissues. The main route of their degradation leads through deglycosylation and cleavage to aldehyde and benzoic acid derivatives.22,23 The kinetics of anthocyanin (ACN) decrease is often described as a first-order reaction with results expressed as half-life time values (eq 1−3).24−27 k1

ACN → product

(1)

cACN = cACN,0 exp( −k1t )

(2) C



Article

step of both pathways leads to the formation of DHAA, through ascorbic radical as intermediate in the anaerobic pathway or the multistage aerobic pathway catalyzed by metal ions. The second step is the irreversible hydrolysis to 2,3diketogulonic acid (DKGA). DHAA retains part of the antioxidant and health-promoting activities of AA, although DKGA and further metabolites do not have such properites.32 Reduction of DHAA to AA occurs in the presence of reducing agents or enzymes such as dehydroascorbate reductase. Lack of such compounds in model solutions allows the above-mentioned reaction to be considered irreversible, so the degradation of ascorbic acid can be regarded as two consecutive first-order reactions (eq 4).33 k1

k2

AA → DHAA → DKGA

(4)

Changes in the concentration of compounds can be described with differential equations. Their solutions (eqs 5 and 6) can be used to determine the kinetic parameters of the reaction.

cAA = cAA,0 exp( −k1t ) c DHAA =

k1cAA,0 k 2 − k1

[exp(−k1t ) − exp(−k 2t )]

Figure 3. Influence of sweetener type, concentration, and acidity of solution on ascorbic acid degradation rate. Symbols and composition of model solutions are described in Table 2. Error bars represent standard deviation. Results were obtained from a duplicate analysis of two independent samples.

(5)

(6)

cAA = concentration of AA, cAA,0 = initial concentration of AA, cDHAA = concentration of DHAA, k1 = rate of first reaction, k2 = rate of second reaction, and t = time. Rates of reaction were found using linear (AA) and nonlinear regression (DHAA). The fit of the model kinetics to experimental data is shown in Figure 2. The experimental data are highly consistent with the assumed model of the kinetics; for each sample the coefficient of determination was >0.95 (significant at α = 0.05).

Figure 4. Influence of sweetener type, concentration, and acidity of solution on dehydroascorbic acid degradation rate. Symbols and composition of model solutions are described in Table 2. Error bars represent standard deviation. Results were obtained from a duplicate analysis of two independent samples.

effect was stronger: the rate of degradation was 26.6−41.9% lower than in the control sample. Higher concentrations of sweetener strengthened the protective effect; however, this relationship was not linearly proportional. The acidity of the solution influenced the stability of vitamin C and the stabilizing effect of glycosides from S. rebaudiana. Higher acidity of the solutions significantly improved the protective effect of steviol glycosides on AA stability and, to a lesser extent, on DHAA stability. In general, ascorbic acid degradation is fastest in a pH close to 4.0 due to the maximum occurrence of hydrogen ascorbate ions.32,34 Control samples containing sucrose (100 g L−1) were also examined. The protective effect of this carbohydrate was observed. Its strength did not depend on the acidity of the solution. The protective effect of steviol glycosides on the stability of ascorbic acid was observed by Kroyer; however, his experiments were very limited.9,10 Our research confirmed and expanded these results: the protective effect applied to both vitamin C forms, and it depended on the concentration of the sweetener

Figure 2. Fit between changes of ascorbic (AA) and dehydroascorbic (DHAA) acid concentration computed using kinetic equations (eqs 5 and 6; lines) and experimental data (dots) in sample pH4.0/Ste200 (composition of sample in Table 2).

A graphical representation of the influence of the type of sweetener and its concentration on the stability of vitamin C (AA and DHAA) at various pH values is shown in Figure 3 (AA) and Figure 4 (DHAA). Detailed data can be found in Table S2 in the Supporting Information. The addition of steviol glycosides significantly increased the stability of both ascorbic and dehydroascorbic acid. The rate of degradation of ascorbic acid decreased by 4.0−22.3%, depending on the conditions. In the case of DHAA the protective D



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(7) Stoyanova, S.; Geuns, J. M. C.; Hideg, É.; Van Den Ende, W. The food additives inulin and stevioside counteract oxidative stress. Int. J. Food Sci. Nutr. 2011, 62, 207−214. (8) Geuns, J. M. C.; Hajihashemi, S.; Claes, A. In Stevia: Six Months Beyond Authorisation. Proceedings of the 6th EUSTAS Stevia Symposium; Geuns, J. M. C., Ed.; Euprint: Heverlee, Belgium, 2012; pp 157−180. (9) Kroyer, G. T. The low calorie sweetener stevioside: stability and interaction with food ingredients. LWT−Food Sci. Technol. 1999, 32, 509−512. (10) Kroyer, G. T. Stevioside and stevia-sweetener in food: application, stability and interaction with food ingredients. J. Verbrauch. Lebensm. 2010, 5, 225−229. (11) Chang, S. S.; Cook, J. M. Stability studies of stevioside and rebaudioside-A in carbonated beverages. J. Agric. Food Chem. 1983, 31, 409−412. (12) Clos, J. F.; DuBois, G. E.; Prakash, I. Photostability of rebaudioside A and stevioside in beverages. J. Agric. Food Chem. 2008, 56, 8507−8513. (13) Wölwer-Rieck, U.; Tomberg, W.; Wawrzun, A. Investigations on the stability of stevioside and rebaudioside A in soft drinks. J. Agric. Food Chem. 2010, 58, 12216−12220. (14) Prakash, I.; Clos, J. F.; Chaturvedula, V. S. P. Stability of rebaudioside A under acidic conditions and its degradation products. Food Res. Int. 2012, 48, 65−75. (15) Prakash, I.; DuBois, G. E.; Clos, J. F.; Wilkens, K. L.; Fosdick, L. E. Development of rebiana, a natural, non-caloric sweetener. Food Chem. Toxicol. 2008, 46, S75−S82. (16) Jooken, E.; Amery, R.; Struyf, T.; Duquenne, B.; Geuns, J. M. C.; Meesschaert, B. Stability of steviol glycosides in several food matrices. J. Agric. Food Chem. 2012, 60, 10606−10612. (17) Bergs, G.; Burghoff, B.; Joehnck, M.; Martin, G.; Schembecker, G. Fast and isocratic HPLC-method for steviol glycosides analysis from Stevia rebaudiana leaves. J. Verbr. Lebensm. 2012, 7, 147−154. (18) Odriozola-Serrano, I.; Hernández-Jover, T.; Martín-Belloso, O. Comparative evaluation of UV-HPLC methods and reducing agents to determine vitamin C in fruits. Food Chem. 2007, 105, 1151−1158. (19) Oszmiański, J. Stabilization and application of anthocyanin chokeberry dye to colouring of beverages. ACTA Sci. Polym. Technol. Aliment. 2002, 1, 37−45. (20) Ruzin, S. E. Appendix II: Buffers. In Plant Microtechnique and Microscopy; Oxford University Press: New York, 1999; pp 223−234. (21) EU Comission. Commission Regulation (EU) No. 1131/2011. Off. J. Eur. Union 2011, L295, 205−211. (22) Sadilova, E.; Stintzing, F. C.; Carle, R. Thermal degradation of acylated and nonacylated anthocyanins. J. Food Sci. 2006, 71, C504− C512. (23) Seeram, N. P.; Bourquin, L. D.; Nair, M. G. Degradation products of cyanidin glycosides from tart cherries and their bioactivities. J. Agric. Food Chem. 2001, 49, 4924−4929. (24) West, M. E.; Mauer, L. J. Color and chemical stability of a variety of anthocyanins and ascorbic acid in solution and powder forms. J. Agric. Food Chem. 2013, 61, 4169−4179. (25) Patras, A.; Brunton, N. B.; O’Donnell, C.; Tiwari, B. K. Effect of thermal processing on anthocyanin stability in foods; mechanisms and kinetics of degradation. Trends Food Sci. Technol. 2010, 21, 3−11. (26) Song, B. J.; Sapper, T. N.; Burtch, C. E.; Brimmer, K.; Goldschmidt, M.; Feruzzi, M. G. Photo and thermodegradation of anthocyanins from grape and purple sweet potato in model beverage systems. J. Agric. Food Chem. 2013, 61, 1364−1372. (27) Hernández-Herrero, J. A.; Frutos, J. M. Degradation kinetics of pigments, colour and stability of the antioxidant capacity in juice model systems from six anthocyanin sources. Int. J. Food Sci. Technol. 2011, 46, 2550−2557. (28) Bąkowska-Barczak, A. Acylated anthocyanins as stable, natural food colorants − a review. Polym. J. Food Nutr. Sci. 2005, 14/55, 107− 116. (29) Rubinskiene, M.; Viskelis, P.; Jasutiene, I.; Viskeline, R.; Bobinas, C. Impact of various factors on the composition and stability of black currant anthocyanins. Food Res. Int. 2005, 38, 867−871.

and acidity of the sample. The protective effect of sucrose on ascorbic acid is consistent with earlier publications.35,36 To our best knowledge this is the first study concerning the influence of steviol glycosides on anthocyanin stability. It also considerably expanded earlier experiments regarding interactions between stevioside and vitamin C. The presented data demonstrate that the addition of glycosides from S. rebaudiana can increase the stability of vitamin C in water solutions of an acidic pH to a greater extent than sucrose used in sensory equivalent amounts. The addition of steviol glycosides also enhanced the protective effect of sucrose on anthocyanins. This fact may be of practical importance, indicating the additional benefits of using this natural noncaloric sweetener for fruit beverages. Our research was conducted on model solutions, which, on the one hand, simplifies following the changes occurring in the sample, but, on the other hand, eliminates the influence of other constituents of real matrices, for example, radicals and metal ions, which might significantly change the degradation pattern through Fenton or Haber−Weiss reactions. The observed phenomena should be further investigated in complex food samples.37

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ASSOCIATED CONTENT

S Supporting Information *

Anthocyanin half-lives and ascorbic acid and dehydroascorbic acid degradation rates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(L.W.) E-mail: [email protected]. Phone: (+48) 22 6063604. Fax: (+48) 22 8490426. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED

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REFERENCES

AA, ascorbic acid; ACN, anthocyanin (in general); BA, benzoic acid; C3G, cyanidin-3-glucoside; DHAA, dehydroascorbic acid; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography; P3G, pelargonidin-3-glucoside; RebA, rebaudioside A; Ste, stevioside

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Influence of Steviol Glycosides on the Stability of Vitamin C and

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