The Impact of Sweetener Type on Physicochemical Properties, Antioxidant Activity and Rheology of Guava Nectar during Storage Time

: This study aimed to evaluate the differences in physicochemical properties, antioxidant properties, and rheology between guava nectar with sucrose and guava nectar with neotame and stevia addition (0.01% and 0.05%). All parameters were investigated during refrigerated storage at 4 ◦ C for 15 days. The result showed that all sweetened guava juice samples led to the juice with the greatest presence of overall appearance and antioxidant property. The ﬂow behavior of sweetened guava juice was shear-thinning which was not different from guava juice without sweetener. However, the major volatile ﬂavor compounds identiﬁed in all guava juice samples were β -caryophyllene, α -caryophyllene, bisabolene, aromadendrene, α -humulene, and nerolidol, which is not different from guava juice without sweeteners during storage. It indicated that stevia and neotame are good alternative sweeteners to produce low caloric juice production. antioxidant activity, and rheology.


Introduction
Guava (Psidium guajava) is tropical fruit cultivated in tropical and subtropical regions. It contains high levels of antioxidants such as beta-carotene, lycopene which played an important role in combating free radicals. It is mostly consumed in fresh or processed form (puree, concentrated, and juice); guava juice is favored in tropical regions due to its sweet and refreshing flavor. Moreover, it is an excellent source of several important vitamins and minerals [1].
Nowadays, fruit juice consumption is increased due to its relief of thirst, nutrition, and easy digestibility [2]. Moreover, a large amount of refined sugar is used to sweeten fruit juice for preferable flavor, taste, and color [3]. They also play a crucial role in the appearance, physicochemical, and rheology of fruit juices [4]. However, it affected health problems, including obesity, diabetes, and hyperlipidemia [5]. Therefore, sweeteners are alternative compounds used to partly or completely replace sugar in fruit juice [6].
A sweetener is a substance used to replace sucrose which can provide the same sweet taste as sucrose. The type of sweetener used can influence the physicochemical properties which may limit its addition to a product [6] using each sweetener in situations for which it is best suited [7]. In addition, six sweeteners, including saccharin, aspartame, sucralose, neotame, acesulfame-K, and stevia have been classified under the generally recognized as safe (GRAS) category [8]. Several sweeteners are allowed for use in food and beverages [9].
Neotame is an artificial sweetener that has the same characteristics as aspartame. The sweetness is 5000 times that of sucrose, and is sweeter than aspartame. It also has a desirable flavor more than aspartame [10]. Derta and Bolini [11] reported that there was no significant difference in appearance and aroma of acerola nectar sweetened with sucrose, neotame, and stevia. In terms of vitamin C content and total titratable acidity, there was also no significant difference (p > 0.05).

Preparation Guava Nectar Formulation
The raw material was washed with running water and then cut into pieces. The pulp was mixed with distilled water in a ratio of 1:1 (w/v) by a blender. The mixture was transferred to a nylon bag which was applied pressure to separate the fruit juice from the pulp. Citric acid (0.15%, w/v) and NaCl (0.25%, w/v) were added to juice. The soluble solid was adjusted to 8 • Brix. After mixing, the beverage was homogenized in a rotor-stator type homogenizer (Walita ® , São Paulo, Brazil) for 5 min.

Preparation of Sweetened Guava Nectar
The guava nectar was mixed with neotame and stevia (0.01% and 0.05%) with 0.1% guar gum. Briefly, intense sweetener and guar gum were gradually mixed with guava nectar. For guava nectar without sweetener, sucrose was added to nectar for 4% (w/v), 0.15% (w/v) citric acid and 0.25% (w/v) NaCl as control. The mixture was homogenized and heated to 80 • C for 30 s, then cooled in the ice bath to 40 • C. Finally, guava nectar and sweetened guava nectar were filled in a pasteurized clear bottle (80 • C for 5 s), sealed, and stored refrigerator (4 • C) for 15 days. All juice samples were analyzed immediately after preparation. The shelf life of guava nectar and sweetened guava nectar were determined every 3 days for physicochemical properties, antioxidant activity, and rheology.

Analytical Methods for Shelf Life Evaluation
The pasteurized guava nectar shelf life was evaluated at 5 • C ± 2 • C to determine physicochemical properties and antioxidant activity which were performed on days 0, 3, 6, 9, 12, and 15.

Physico-Chemical Property
The cloud value was evaluated using a UV-1601 spectrophotometer (Shimadzu, Japan) that was calibrated with distilled water according to Abid et al. [19]. The samples were centrifuged at 618× g for 20 min, then absorbance at 660 nm of the supernatant was recorded. The transmittance rate was calculated as the following equation: where I is the light density that passes through a sample solution. I 0 is the light intensity.
The color of guava nectar samples was measured according to the method described by Wang et al. [20]. The colorimeter (ColorFlex EZ spectrophotometer, Horsham, PA, USA) was used to evaluate the color during storage.
The parameters including L*, a*, and b* of each sample were recorded. The results were expressed as total color difference (∆E) values which were calculated as follows where ∆L, ∆a* and ∆b* are a difference of lightness, redness, and yellowness, respectively, for 0, 3, 6, 9, 12 and 15 days of storage.
The pH of guava nectar and sweetened guava nectar was measured at 25 • C by using a digital pH meter (Denver Instrument, Denver, CO, USA) with a glass electrode.
The acidity of nectar was evaluated by using an auto titrator (Titriline 700, Columbus, OH, USA). Fruit juice (10 mL) mixed with 20 mL of distilled water that was titrated with 0.1 NaOH to a pH value of 8.3 as previously described by Xiang et al., (2018). The results were expressed as g citric acid/100 mL of guava nectar. Acidity (%) = mL of NaOH × 0.1 × 64 × 100 mL of juice × 1000

Total Carotenoid Content
Total carotenoid was determined by spectrophotometer (UV-1601 spectrophotometer, Shimadzu, Japan) as proposed by Kotíková et al. [21]. The nectar samples (2 mL) were added to 15 mL of acetone and incubated for 2 h. The excess acetone was removed by centrifuge at 4000× g for 5 min, then adjusted volume to 25  The ABTS •+ scavenging activity was performed according to Re et al. [22] as follows: The ABTS •+ solution was prepared by mixing two solutions of ABTS •+ (7 mM) and potassium persulfate (2.45 mM). The mixture was allowed to stand in the dark condition at room temperature for 16 h before use. The ABTS •+ solution was diluted to the absorbance of 0.700 ± 0.02 at 734 nm. Then, guava juice (1 mL) was added with 3 mL of ABTS •+ solution, mixed, and kept in the dark for 6 min. The absorbance was measured at 734 nm. The scavenging activity was calculated as: where A control is the absorbance of ABTS •+ solution without sample. A sample is the absorbance of ABTS •+ mixed with a sample.

DPPH • Radical Scavenging Activity Assay
Antioxidant activity was assessed as DPPH • radical scavenging activity according to Bhat and Goh. [23]. Briefly, 100 µL of the nectar was added to 2 mL of 0.1 mM DPPH • in 95% ethanol. The mixture was incubated at room temperature (dark condition) for 30 min. The absorbance was recorded at 517 nm with a spectrophotometer (UV-1601 spectrophotometer, Shimadzu, Japan). The antioxidant activity was expressed as the scavenging activity according to the equation where A control is the absorbance of DPPH • solution without sample. A sample is the absorbance of DPPH • mixed with a sample.

Flow Behavior and Apparent Viscosity of Guava Juice
The apparent viscosity of nectar samples was determined by using a rheometer (Haake Mars 40 model, Thermo Fisher Scientific, Waltham, MA, USA) equipped with cup and coaxial cylinder geometry. The shear rate was varied from 0.01 s −1 to 10 s −1 at 540 s −1 at 5 • C and 37 • C with a gap of 0.01 mm. The nectar samples (17 mL) were placed into a cup and allowed to rest at 5 • C and 37 • C for 120 s −1 [24]. A total of 30 data points were recorded at 15 s −1 during shearing.

Volatile Compound Gas Chromatography
The volatile compound of all guava nectar samples was performed by chromatographymass spectrometry (Agilent DB-WAX column, 60 m × 0.25 mm × 0.25 µm film thickness dimensions, Beijing, China) with flame ionization detector (FID). The sample (3 µL) was extracted by using headspace mode at 30 • C for 30 min. After that, volatile compounds were subjected to solid-phase microextraction (SPME fiber 50/30/µm Divinylbenzene/Carbon wide range/Polydimethylsiloxane stableflex 2 cm, Suppleco) and injection was performed in a splitless mode at 250 • C that helium was the carrier gas. The gas chromatography (GC) temperature was 50 • C for 2.5 min up to 200 • C in 4 min. The volatile compound was identified by comparing their GC retention time (RT) as proposed in NIST 2005 v 2.0 (Natl. Inst. of Standards and Technology, Gaithersburg, MD, USA) according to Guo et al. [25] with slight modification.

Statistical Analysis
The physicochemical and antioxidant properties were performed in triplicate in a Randomized Complete Block Design. The result was subjected to a one-way analysis of variance (ANOVA) with Statistical Analysis System (SAS, version 6.0) (SAS Institute, Cary, NC, USA). Duncan's Multiple Range Test was used to determine significant differences between means (p < 0.05). All data were presented as average values with the standard deviation.

Cloud Value
Turbidity is the cloudiness of nectar samples that occurred due to the colloid polysaccharides particles, including starch, pectin, and hemicellulose components [26]. Moreover, turbidity is an important quality criterion in fruit juice [27]. The cloud value was evaluated in guava nectar with sucrose as a reference control and guava nectar with neotame and stevia at different concentration levels (0.01% and 0.05%) as sweetened guava nectar. The result showed that guava nectar with 0.05% neotame had the highest cloud value. It was a significant difference with reference control, guava nectar with 0.01% neotame and guava nectar with stevia (0.01% and 0.05%). Moreover, turbidity was decreased with increasing storage time which was extremely significant in cold storage at 5 • C for 15 days (Table 1). Although in 3 days of storage reference control had the lowest cloudiness, guava nectar with 0.01% stevia had the highest cloud value. It implied that fruit particles in the nectar aggregated and degraded by oxidation-reduction reaction resulted in the decline of turbidity which was observed in reference control [28]. Therefore, the nectar had a high precipitate which allowed light more to pass through the nectar than nectar with less precipitate. Moreover, the appearance of reference control and sweetened guava nectar precipitate were recorded by observation. It was confirmed that storage time had a significant effect on turbidity. Pulp was separated from nectar at the start of storage (0 days) in reference control, while pulp-sweetened guava nectar precipitated at 6 days of storage and settled at bottom of the bottle ( Table 2). The cloudy appearance displayed the nectar homogenization. As a result, low cloudiness in all samples was observed during storage. It indicated an undesirable quality.

Color Attributes
Color attributes are an important standard to estimate the quality of fruit nectar that was related to consumers' satisfaction and requirements [29]. The color analyses were performed as color differences that were represented by ∆E at 0, 3, and 6, 9, 12 and 15 days of storage. Although the total difference was observed in the color of reference control and guava nectar with 0.01% and 0.05% stevia, there was no significant difference between guava nectar containing 0.01% and 0.05% neotame (Table 3).
Moreover, the color had a significant difference (p ≤ 0.05) during the storage period in reference control and sweetened guava nectar. It might be the destruction of the cell membrane which would lead to the loss of functional cell compartmentalization. Thus, it accelerates enzyme-substrate contact which promotes tissue browning [30]. However, the color variation was larger in reference control and guava nectar with neotame. The result demonstrated that guava nectar with 0.01% stevia showed the least color changes after 15 days. The addition of stevia might cause the inactivation of enzymes which prevent enzyme-substrate contact [30].  9.03 ± 0.05 A,a 6.88 ± 0.07 B,a 6.08 ± 0.08 D,a 6.82 ± 0.02 B,a 6.51 ± 0.02 C,a A-D Different capital letters in the same row show significant differences between the guava nectar with sucrose and sweetened guava nectar for the same day. a-f Different small letters in the same column show significant differences between storage days.

pH
The pH of reference control and sweetened guava nectar are presented in Table 4. The initial pH degrees of guava nectar were controlled with citric acid. The result showed that pH in reference control and sweetened guava nectar increased with extended storage time at 5 • C.  15 3.67 ± 0.02 A,a 3.57 ± 0.02 C,a 3.58 ± 0.03 B,a 3.57 ± 0.01 C,a 3.57 ± 0.01 C,a A-C Different capital letters in the same row show significant differences between the guava nectar with sucrose and sweetened guava nectar for the same day. a-e Different small letters in the same column show significant differences between storage days. It was indicated that poly-saccharides were hydrolyzed by acid which is responsible for the increase in pH [8]. However, reference control and guava nectar with different levels of sweetener (0.01%, 0.05% neotame and 0.01%, 0.05% stevia) had pH values ranged from 3.50 to 3.60 which were not significantly different (p > 0.05) as a function of storage time. It indicated that the substitution of sucrose with neotame and stevia did not affect the pH of the guava nectar. It was observed that the pH of guava nectar with stevia increased slightly with the increasing level of stevia at 0 days of storage due to the high pH of stevia [31]. In the beverage industry, the stability of sweeteners will be influenced by pH, temperature, and storage time. Stevia and neotame are also stable in acidic pH conditions at 2.9-4.5 [32]. Consequently, it is allowed for soft drinks and fruit nectar at acid pH [32].

Titratable Acidity (TA)
Titratable acidity in fruit nectar includes all the substances of an acid in the fruit nectar, of which the most acidic component is an organic acid. Titratable acidity was expressed as a percentage of the predominant acid which was citric acid (g/100 g). The decreasing trend of titratable acidity was presented from the initial to the 6th day of storage in both reference control and sweetened guava nectar (Table 5). Although pH was increased from 0 to 15 storage days, the acidity of all samples non-significantly decreased from 0 days to 15 days. Generally, the pH is always inversely proportional to acidity which acidity has decreased during the storage period so pH has increased [33]. This was also found in kinnow juice storage in the refrigerator [34] and mandarin juice storage at low temperatures [35]. The storage conditions of low temperatures and high humidity might be reduce the conversion of acids into sugar and salt by invertase enzymes in guava nectar [36]. Additionally, guava nectar with 0.01% neotame had the highest percent titratable acidity during storage. However, the different types and levels of sweeteners did not affect the pH and total acidity in guava juice.

Carotenoid Content
Carotenoids are natural pigments that contribute to both the appearance and attractiveness of beverages as well as additional nutritional value in the form of powerful dietary antioxidants. The result showed that the lowest carotenoid was found in the reference control during storage. However, total carotenoid content decreased significantly (p < 0.05) after storage in reference control and sweetened guava nectar (Figure 1). tion of the total carotenoid content was found in guava nectar during storage at 10 °C for 240 days. It indicated that oxidative degradation was the major cause of carotenoid loss. Additionally, a total carotenoid in guava nectar containing 0.01% and 0.05% neotame and stevia during storage days was no different. Thus, sweetener addition does not influence carotenoid content in guava nectar.

ABTS •+ Radical Scavenging
Total antioxidant capacity was measured by ABTS •+ and DPPH • method which were evaluated in vitro. As shown in Figure 2a,b, the trend of radical scavenging was decreased during the storage period in the reference control and sweetened guava nectar. The result showed that the ABTS •+ radical scavenging activity ranged from 54.55 to 29.43%. Guava nectar with 0.05% stevia had the highest ABTS •+ scavenging followed by 0.05% neotame, control, 0.01% stevia and 0.05% neotame exhibited the lowest radical scavenging by ABTS •+ assay. However, at day 0, the antioxidant of guava nectar with sucrose was not significant with sweetened guava nectar (0.01% neotame). All samples were observed with low antioxidant activity at the end of the storage. The ABTS •+ scavenging activity of sweetened guava nectar was higher compared to the control. It might be from carotenoid content and sweetener as well as the synergistic effect. Although total carotenoids in sweetened guava nectar decreased non-significantly in 3 days of storage, total carotenoids were significantly reduced after 3 days of storage. It suggested that sweeteners including, stevia and neotame as protective carotenoids in model systems [37]. Ordóñez-Santos and Vázquez-Riascos [38] reported that the reduction of the total carotenoid content was found in guava nectar during storage at 10 • C for 240 days. It indicated that oxidative degradation was the major cause of carotenoid loss. Additionally, a total carotenoid in guava nectar containing 0.01% and 0.05% neotame and stevia during storage days was no different. Thus, sweetener addition does not influence carotenoid content in guava nectar.

ABTS •+ Radical Scavenging
Total antioxidant capacity was measured by ABTS •+ and DPPH • method which were evaluated in vitro. As shown in Figure 2a,b, the trend of radical scavenging was decreased during the storage period in the reference control and sweetened guava nectar. The result showed that the ABTS •+ radical scavenging activity ranged from 54.55 to 29.43%. Guava nectar with 0.05% stevia had the highest ABTS •+ scavenging followed by 0.05% neotame, control, 0.01% stevia and 0.05% neotame exhibited the lowest radical scavenging by ABTS •+ assay. However, at day 0, the antioxidant of guava nectar with sucrose was not significant with sweetened guava nectar (0.01% neotame). All samples were observed with low antioxidant activity at the end of the storage. The ABTS •+ scavenging activity of sweetened guava nectar was higher compared to the control. It might be from carotenoid content and sweetener as well as the synergistic effect.

DPPH • Radical Scavenging
The antioxidant activity ranged from 3.01 to 32.26% which guava nectar with 0.05% stevia had the highest DPPH • scavenging value in 0 days of storage. However, guava nectar with different levels of neotame was no significant difference in DPPH • radical scavenging activity. It was expected that stevia powder contained a high amount of ascorbic acid which powerfully affects the antioxidant properties. Moreover, antioxidant capacity was related to carotenoid in guava nectar. In this study, it showed a decrease in DPPH • scavenging values in the presence of different carotenoid content. Although the antioxidant activity of reference control and sweetened guava nectar (0.1% stevia, 0.001% and 0.005% neotame) at the initial storage time decreased significantly different from 0, 3 and 6 days of storage, the sweetened guava nectar decreased not significantly DPPH • scavenging activity between 3 and after 6 days of storage. Moreover, the higher scavenging ABTS •+ activity than DPPH • might be related to the different structures of the radical and different molar ratios of the antioxidant to the radical molecules.

Volatile Compound
The volatile flavor of guava nectar was identified by gas chromatography at 0 and 15 days of storage which was reported as peak area percentages. As a result, the nectar contained aromatic volatile with low molecular weight, particularly alcohol and aldehyde. The major volatile in reference control and sweetened guava nectar was the sesquiterpenes, including β-caryophyllene, α-caryophyllene, bisabolene, aromadendrene, α-hu-

DPPH • Radical Scavenging
The antioxidant activity ranged from 3.01 to 32.26% which guava nectar with 0.05% stevia had the highest DPPH • scavenging value in 0 days of storage. However, guava nectar with different levels of neotame was no significant difference in DPPH • radical scavenging activity. It was expected that stevia powder contained a high amount of ascorbic acid which powerfully affects the antioxidant properties. Moreover, antioxidant capacity was related to carotenoid in guava nectar. In this study, it showed a decrease in DPPH • scavenging values in the presence of different carotenoid content. Although the antioxidant activity of reference control and sweetened guava nectar (0.1% stevia, 0.001% and 0.005% neotame) at the initial storage time decreased significantly different from 0, 3 and 6 days of storage, the sweetened guava nectar decreased not significantly DPPH • scavenging activity between 3 and after 6 days of storage. Moreover, the higher scavenging ABTS •+ activity than DPPH • might be related to the different structures of the radical and different molar ratios of the antioxidant to the radical molecules.

Volatile Compound
The volatile flavor of guava nectar was identified by gas chromatography at 0 and 15 days of storage which was reported as peak area percentages. As a result, the nectar contained aromatic volatile with low molecular weight, particularly alcohol and aldehyde. The major volatile in reference control and sweetened guava nectar was the sesquiterpenes, including β-caryophyllene, α-caryophyllene, bisabolene, aromadendrene, αhumulene and nerolidol (Table 6). These compounds were biogenetic, which was a response to the sweet and fruity aroma in guava [39]. Changes in the volatile flavor of the reference control and sweetened guava nectar during storage were observed. The result showed that all volatile flavors declined during the storage period due to chemical reactions and oxidation of free volatile compounds [40]. Among the volatiles present in all samples, guava nectar was observed to occur in various guava species [41]. β-caryophyllene, γ-caryophyllene and nerolidol are important in the characterization of tropical fruit flavor. Moreover, β-caryophyllene is often found together with α-humulene which is present in many aromatic plants [42]. In addition, the aroma of nerolidol is woody and reminiscent of fresh bark [43]. However, the major volatile flavor was detected during storage of bisabolene and aromadendrene. Although bisabolene was only detected as volatile at 0 days storage, and at 15 days, aromadendrene was only detected in 6 days.

Rheology Properties
Rheology is the deformation and flow behavior of fluid which was important for food quality. The apparent viscosity of reference control and sweetened guava nectar was obtained from the flow curve (shear rate versus viscosity). The results are expressed as the apparent viscosity of the reference control and sweetened guava nectar at 5 • C and 37 • C. It can be seen that viscosity decreased with increasing shear rate which presents the non-Newtonian behavior of a shear-thinning fluid [44]. As shown in Figure 3a-e, the viscosity was varied slightly different between reference control and guava nectar with 0.01% and 0.05% neotame. The guava juice with 0.01% and 0.05% stevia had a higher viscosity than the reference control and guava nectar with neotame, respectively. It might be caused by a decrease in the guava nectar water activities due to the replacement of sucrose with stevia [45]. Furthermore, this result agrees with the low apparent viscosity found in chocolate milk. In contrast, the storage time increased, and the viscosity decreased which was found in all guava nectar formulated [46]. However, all guava nectar formulated at 37 °C exhibited lower viscosity than guava nectar at 5 °C. This phenomenon might be caused by some The guava juice with 0.01% and 0.05% stevia had a higher viscosity than the reference control and guava nectar with neotame, respectively. It might be caused by a decrease in the guava nectar water activities due to the replacement of sucrose with stevia [45]. Furthermore, this result agrees with the low apparent viscosity found in chocolate milk. In contrast, the storage time increased, and the viscosity decreased which was found in all guava nectar formulated [46]. However, all guava nectar formulated at 37 • C exhibited lower viscosity than guava nectar at 5 • C. This phenomenon might be caused by some compound precipitates during storage at low temperatures.

Conclusions
The sweetened guava nectar with neotame and stevia at two levels could be effective to reduce the sugar content. Neotame and stevia improved the appearance of guava nectar during storage. Furthermore, carotenoid and antioxidant activity in sweetened guava nectar were higher than the reference control. Moreover, the physicochemical, including turbidity, color, pH and %TA of sweetened guava nectar provided resemble level compared to reference control. Therefore, stevia and neotame are good substitutes for sucrose which may keep nectar quality without affecting the physicochemical properties.