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Storage Stability of Aspartame in OrangeFlavored Soft DrinksTulin Yakici a & Muhammet Arici ba Firmenich Foreign Trade Co. Ltd. , Fulya , İstanbul , Turkeyb Departmant of Food Engineering , Faculty of Chemical andMetallurgical Engineering, Yildiz Technical University , Esenler,Istanbul , TurkeyAccepted author version posted online: 14 Jun 2012.Publishedonline: 31 Jan 2013.

To cite this article: Tulin Yakici & Muhammet Arici (2013) Storage Stability of Aspartame inOrange Flavored Soft Drinks, International Journal of Food Properties, 16:3, 698-705, DOI:10.1080/10942912.2011.565903

To link to this article: http://dx.doi.org/10.1080/10942912.2011.565903

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International Journal of Food Properties, 16:698–705, 2013Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print / 1532-2386 onlineDOI: 10.1080/10942912.2011.565903

STORAGE STABILITY OF ASPARTAME IN ORANGEFLAVORED SOFT DRINKS

Tulin Yakici1 and Muhammet Arici21Firmenich Foreign Trade Co. Ltd., Fulya, Istanbul, Turkey2Departmant of Food Engineering, Faculty of Chemical and MetallurgicalEngineering, Yildiz Technical University, Esenler, Istanbul, Turkey

In this study, “orange flavored soft drink” samples sweetened with 100% aspartame (0.5 g/L)were stored in three different pH values and temperatures. The samples with the pH valuesof 2.75, 3.25, and 4.57 were kept in 20, 30, and 40◦C temperatures over a period of 5 months.The remaining aspartame was determined at the 1st, 2nd, 3rd, 4th, and 5th months by HPLC.In the same pH groups, it was clearly observed that stability of aspartame decreased withincreasing storage temperature. It was also determined in each of the same pH groups thataspartame stability increased as the pH value was raised from 2.75 to 4.57. Aspartame wasthe least stable at pH 2.75 when stored in 40◦C, whereas it was the most stable at pH 4.57 and20◦C. At the end of 5th month in 40◦C, stability tests could not be performed and remainingaspartame could not be determined.

Keywords: Aspartame, Stability, pH, Temperature, Storage time.

INTRODUCTION

Records from the earliest civilizations show that man has always valued foods thattaste sweet. Historically, sugar has been used to sweeten most of the foods and drinks thatwe eat and drink in the last 100 years. Some health problems have been appearing dueto the high consumption of sugar and sugar-made foods.[1,2] It is determined that sugar isone of the main reasons of tooth decay, obesity, increasing blood sugar level and serumtriglycerides, and also it is dangerous for people who have diabetes.[3]

Aspartame is a dipeptide composed of two amino acids: L-aspartic acid and L-phenylalanine. Phenylalanine is an essential amino acid. These constituents exist in manyfoods and are metabolized exactly by the same way, as they would come from meat, cheese,fish, vegetables, fruit juice, or milk. Aspartame taste profile is the closest to sugar tasteamong all other artificial sweeteners and it is approximately 200 times sweeter than sugar.[4]

The stability of aspartame varies depending on time, temperature, pH, and wateractivity.[5–8] Aspartame is less stable in liquid systems and the stability is primarily of afunction of pH, temperature, and time. The aspartame molecule slowly hydrolyzes at lowpH to produce a tasteless molecule aspartyl-phenylalanine and methanol. An alternative

Received 10 November 2010; accepted 14 February 2011.Address correspondence to Muhammet Arici, Department of Food Engineering, Faculty of Chemical and

Metallurgical Engineering, Yildiz Technical University, Davutpasa Campus 34210, Esenler, Istanbul, Turkey.E-mail: muarici@yildiz.edu.tr

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route at pH 5 and above is that aspartame may cyclize to form its diketo-piperazine withthe elimination of methanol. These conversion products can be subsequently hydrolyzedto its individual amino acids—aspartic acid and phenylalanine. Stability during storage ofbeverages containing aspartame follows first-order kinetics and, to an extent, is predictableif parameters of pH and temperature are known. Expected results can, however, be con-founded by order interaction with ingredients that can have a positive or negative impacton stability and also perceived sweetness of beverages changes over time.[9]

Aspartame is progressively degraded to diketo-piperazine when stored at tempera-tures ranging from 30 to 80◦C in liquid solution. It is, therefore, not suggested to be used inhigh temperature treated foods (cooking, sterilization, etc.). Similarly, at elevated tempera-tures, aspartame has been shown to react with reducing sugars via the Maillard reaction.[10]

Aspartame stability is good at room temperature at pH 3.0–4.0 and maximum stabilityis observed at pH 4.2. The dipeptide structure is hydrolyzed below pH 3.4 and diketo-piperazine formation and crystallization occur above pH 5.0. In both cases, a transformationtakes place, which results in sweetness loss.[7,8] Buffer salts catalyze the degradation ofaspartame, with greater loss at higher buffer concentrations. In low to intermediate moisturesystems, aspartame degradation increases as water activity increases.[10]

While temperature is the major contributor to aspartame degradation, pH and reac-tive solutes also influence its stability. Aspartame is not recommended for use in liquid witha pH level higher than 7.0. It is important to choose appropriate pH levels and restrictedstorage conditions to obtain best aspartame stability in liquid preparations. Ultra high tem-perature (UHT), high temperature short time (HTST), and hot filling systems are usedeffectively. Shelf life is affected by acidity of medium. Products including phosphoric acidhave the shortest shelf life. Aspartame loss is less than 3% in the long time low temper-ature pasteurization method and even below 1% in the HTST pasteurization method. Theaspartame loss is at negligible levels in the UHT sterilization method.[6] Storage time, tem-perature, and pH do not only affect the sweetness potency of aspartame, but also makesthe shelf life of the product shorter. So, stability of aspartame and its degradation productsare significantly important in order to make a sufficient quality control of diet and diabeticfoodstuffs.[11]

The aim of this study is to determine the effects of different storage time and con-ditions on aspartame stability and sweetness of food products. The results of the presentstudy will help the technological problem faced during production and storage of foodscontaining aspartame and will contribute to the development of new products for peoplewho have a restriction for natural sugar intake and will lead to further scientific researchstudies.

MATERIALS AND METHODS

Materials

Orange flavored soft drinks with different pH values and sweetened with 100%aspartame (Ajinomoto, Switzerland) (0.5 g/L drinks) stored at different temperature condi-tions and the stability of samples were studied during 5 months. The samples were preparedin three different formulations, which belong to a private company. Table 1 shows thegeneral formulation of orange flavored soft drinks.

pH degrees of the samples were controlled with citric acid, which is the most effec-tive ingredient on pH. Citric acid was added to samples at the amounts of 4.05, 1.23, and

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Table 1 Orange flavored soft drink recipe used in the study.

Ingredients Amount (g)

Aspartame 0.500Citric acid anhydrous 4.050Sodium benzoate 0.100Potassium sorbate 0.200Ascorbic acid 0.050Trisodyum sitrat 0.100Orange flavor 0.500Clouding agent 1.000Sunset yellow 0.009Tartrazine 0.040Water 993.451

TOTAL 1,000

0.18 g/L for pH 2.75, 3.25, and 4.57, respectively. Trisodium citrate was used as an acidityregulator. Sodium benzoate (0.1 g/L) and potassium sorbate (0.2 g/L) were used to preventmicrobiological growth, and ascorbic acid (0.05 g/L) was used to prevent oxidation duringstorage.

Preparation of Samples

First, the acidity regulators, citric acid (4.05 g/L), ascorbic acid (0.05 g/L), andtrisodium citrate (0.1 g/L), were dissolved with aspartame (0.5 g/L). Sodium benzoate(0.1 g/L) and potassium sorbate (0.2 g/L) were dissolved in another beaker and addedinto the acidulant and aspartame mixture. Finally, a clouding agent (1 g/L), coloring agents(Sunset Yellow and Tartrazine, 0.09 and 0.04 g/L, respectively), and orange flavor (0.5 g/L)were added to the first mixture and stirred until all ingredients were dissolved and the finalmixture completed up to 1 liter.

Fifteen orange flavored soft drink samples with three different pH degrees (2.75, 3.26,and 4.57) were stored in incubators whose temperatures were fixed to 20, 30, and 40◦C for5 months of storage. Fifteen samples with their reference samples and totally 90 sampleswere stored in 30-mL bottles. The pH of the samples was measured with a Testo Model230 pH-meter (Testo, Sparta, USA).

Analysis of Aspartame

During the storage period, analysis was done on the 1st, 2nd, 3rd, 4th, and 5th monthsby the HPLC analyzing method on remaining aspartame % basis. Aspartame analyses wereconducted according to the method belonging to Ajinomoto Switzerland AG Company(Zug, Switzerland).[12] The manufacturer of the HPLC system was Waters Co. (Pump:Waters 515; Autosampler: Waters 717 Plus; Detectors: Waters 2487, Waters Co., Milford,USA).

Chromatographic Conditions

In this study, a Bondapak C18 column (Waters Co., Milford, USA) was used and a210-nm wavelength was preferred in UV detection. Orange flavored soft drink samples

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containing 500 mg/L aspartame, were filtered through a 0.45-µm filter. Aspartame-containing samples were dissolved in 10% acetonitril solution and 90% buffer solutionand aspartame was separated from the soft drink samples. Aspartame was eluted isocrati-cally with water–acetonitrile (90:10) including 0.00125 M Sodium-dihydrogen-phosphate,and its pH was fixed to 3.5 with phosphoric acid. The flow rate was fixed to 1.5 mL/minat room temperature. One mL orange flavored soft drink was diluted in 6.5 mL of solvents.Injection volume was used as 20 µL in this study. The mobile phase was degassed priorto analysis. The samples were identified and quantified through comparison of areas andretention times established with standards. The chromatograms presented limits of quan-tification and limits of detection of 2 and 5 µg/mL for aspartame, respectively. Recoveryof aspartame solution after sample preparation was >95%.

Statistical Analysis

All the experiments were done in triplicate. The data were analyzed by the SAS(Statistical Analysis Systems Institute, Inc., Cary, USA) statistical package program, anddifferences in the treatment effects on aspartame stability were compared using Duncan’sMultiple Range test.

RESULTS AND DISCUSSION

Results of the soft drinks with pH 2.75, which were stored at 40◦C during 148 days,could not be read. As shown in Fig. 1, the samples at the same pH (2.75) and stored at20◦C were analyzed at the end of the 1st, 2nd, 3rd, 4th, and 5th months. The remainingaspartame as percent of initial amounts were determined to be 90.3, 85.3, 80.9, 74.2, and64.5%, respectively. Concerning the samples at the same pH (2.75) and stored at the 30◦C,the remaining aspartame amount decreased significantly on the 34th day. Results at theend of the 1st, 2nd, 3rd, 4th, and 5th months showed the remaining aspartame as 74, 60.60,50.70, 38.30, and 28.84%, respectively (p < 0.01). The aspartame amount of samples storedat 40◦C after 1 month was 43.30% and decreased to 25.40, 14.80, and 7.40% at the 2nd, 3rd,

Figure 1 Stability of aspartame of orange flavored soft drink of pH 2.75 during storage at different temperatures.

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and 4th months, respectively. The remaining aspartame amount could not be determined asit was in a trace amount at the end of the total storage of 5 months.

The second group of the samples was sweetened with 100% aspartame and had a pH3.25 and was stored at 20, 30, and 40◦C during 5 months. The remaining aspartame amountswere determined to be 93.70, 92.60, 90.40, 86.60, and 82.10%, respectively. The remainingaspartame samples with a pH of 2.75 and stored at 30◦C, decreased significantly on the 1stand 2nd months. Analysis results at the end of the 1st, 2nd, 3rd, 4th, and 5th months showedthe remaining aspartame as 95.90, 79.20, 72, 63.10, and 51.50%, respectively (p < 0.01).When the temperature was 40◦C, the remaining aspartame values were 63.90, 50.10, 37.40,and 25.10% after the 1st, 2nd, 3rd, and 4th months, respectively. The remaining aspartameamount could not be determined after 5 months of storage as it was in a trace amount (p <

0.01) (Fig. 2).The third group of samples sweetened with 100% aspartame having a pH level of

4.57 have been stored at 20, 30, and 40◦C during the 5th month (Fig. 3). The remaining

Figure 2 Stability of aspartame of orange flavored soft drink of pH 3.25 during storage at different temperatures.

Figure 3 Stability of aspartame of orange flavored soft drink of pH 4.57 during storage at different temperatures.

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aspartame amounts were 97.96, 96.60, 95.00, 92.80, and 87.80% after the 1st, 2nd, 3rd,4th, and 5th months, respectively (p < 0.01). When the temperature was increased to 30◦C,the remaining aspartame amounts at the end of the first, 2nd, 3rd, 4th, and 5th months weredetermined to be 93.30, 89.70, 83.30, 74.60, and 60.30%, respectively (p < 0.01). Theanalytical results were not readable on the 14th day for orange flavored samples with a pHlevel of 4.57, which were stored at 40◦C (p < 0.01).

In this study, the lowest aspartame was observed for the samples with a pH 2.75 andstored at 40◦C; the highest stability was observed for the samples having a pH of 4.57 andstored at 20◦C. Woo and Chang[13] investigated the effects of temperature and pH on ther-mal stability of aspartame within the range of 60–100◦C temperature and pH 3.0–7.0 andthey reported that the lowest stability was observed at pH 7.0 and the highest aspartamestability at pH 4.0.

Bell and Labuza[14] evaluated the stability of aspartame in commercially sterilizedskim milk beverages that contained different buffer salts, buffer concentrations, and flavor.The effects of pH and temperature on aspartame stability in these dairy beverages were alsostudied. The half-lives were 1 to 4 days at 30◦C and 24 to 58 days at 4◦C, and decreasing thepH from 6.7 to 6.4 doubled the stability of aspartame. In the present study, results indicatedthat increasing the pH from 2.75 to 3.25 resulted in an increase in the remaining aspartamefrom 25.84 to 51.50% after 5 months of storage and enhanced the stability at 99.30% in thesamples stored at 30◦C. If the pH increases up to 4.57, the stability of aspartame increases17% more than pH 3.25 and 133% more than pH 2.75.

Fellows et al.[15] investigated the stability of aspartame in three different fruit prepa-rations at constant pH and stored at three different temperatures during 6 months. Theyreported the remaining aspartame to be around 60% at 21◦C after 6 months. According totheir results, estimated shelf life of aspartame was 2 months at 32.20◦C and 4–6 months at4.4◦C. In this study, at 20◦C after 5 months the remaining aspartame amounts were 65.4%at pH 2.75, 82.10% at pH 3.25, and 87.80% at pH 4.57. The remaining aspartame amountsat 30◦C after 2 months were 60.60% at pH 2.75, 79.20% at pH 3.25, and 89.70% at pH4.57. In another study, Fellows et al.[16] researched the stability of aspartame during thefermentation of milk and reported that the remaining aspartame in yoghurts was 95% afterincubation at 43◦C for 6 h and 90% for samples incubated at 32.2◦C for 13 h.

Rei et al.[17] investigated the stability of aspartame in cakes, which were packed inpolyethylene bags and stored at 25, 4, or −18◦C during 2 weeks. Subsequently, the recoveryof aspartame after baking and storage was determined, pH of the samples ranged from6.7 to 7.0. Recovery was found to be 24 and 41% for powder and encapsulated aspartame,respectively.

Wetzel and Bell[10] studied the chemical stability of encapsulated aspartame in sug-arless cakes and determined the remaining aspartame amount in various cake recipes.The stability of encapsulated aspartame in cakes varied between 22 and 58% dependingon the recipe. Between the range of pH 7 and pH 5, aspartame stability was increased8–16 times for each decreasing level of pH. In this study, the highest aspartame stabilitywas determined for the samples having a pH of 4.57.

Hence, the results of the study on the stability of aspartame in orange flavored softdrinks, the effects of storage conditions, storage time, and pH level, were found to be signif-icant on the stability of aspartame (p < 0.01). Increasing temperature decreased the stabilityof aspartame whereas increasing pH value between the ranges of pH 2.75–4.57 increasedits stability. Also, the interactions between storage temperature time, pH and time, andstorage temperature and pH on aspartame loss were found to be significant (p < 0.01).

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Increasing storage time and temperature together decreased the remaining aspartameamount. Remaining aspartame decreased quantitatively with the extended storage time andincreasing pH level between 2.75 and 4.57 increased the stability of aspartame. With theincreasing storage temperature and decreasing pH between 2.75–4.57 stability of aspartamedecreased as well. In the orange flavored soft drinks the triangular effect of storage temper-ature, pH value, and storage time on aspartame stability was found to be significant (p <

0.01).

CONCLUSION

In this research, orange flavored soft drinks that were prepared to be analyzed forstability of aspartame with a pH 2.75, 3.25, and 4.57 were stored at 20, 30, and 40◦C.In this study, the remaining aspartame amount decreased with increasing storage tempera-ture. Optimum aspartame and, thus, sweetness stability in fruit flavored soft drinks can bemaintained by controlling the storage conditions, temperature, and pH.

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