Introduction

Indonesia is a country with various types of tuber, including potatoes, sweet potatoes, and yam, which play an essential role in the local dishes. In addition, a widely used tuber in the food sector with unique characteristics is purple yam (Dioscorea alata L.), also known as “uwi ungu.” Several studies have shown that it has several similarities with sweet potatoes, but is more prominent in size. Purple yam has also been reported to have a predominant red-purple color, indicating high levels of anthocyanins, reaching 31-56.24 mg/100 g (db).^1,2

According to previous studies, anthocyanins are compounds prone to damage due to various factors, including temperature, pH, light, oxygen, and chemical structure.^3,4 Furthermore, the chemical stability of these compounds is primarily affected by the number and location of the hydroxyl and methoxy groups in the B-ring in their structure.⁵ A previous study reported that exposure to sunlight led to a 30% deterioration of anthocyanins, while only 29.1% remained after 30 minutes at 70 degrees Celsius.⁶ Elevated temperatures exceeding 40 °C can induce glycosylation, hydration reactions, and polymerization. In addition, the pigment of these compounds fades when exposed to light due to their ability to absorb visible light.⁷ In line with previous studies, anthocyanins typically exhibit a predominantly red color, with the dominant structure being flavylium cations at pH 1, gradually fading to yellow at alkaline pH. Degradation often occurs under alkaline conditions due to the transformation and hydration of flavylium cations.⁸ To avoid degradation, several studies have proposed the combination of different wall materials, which provides better protection for anthocyanins encapsulation compared to single wall materials. For instance, incorporating whey protein with maltodextrin (MD), β-cyclodextrin (CD), and/or gum Arabic has been shown to improve the thermal stability of inherent blueberry compounds.⁹ Anthocyanins from red raspberry encapsulation also showed stability at high temperatures using a combination of wall materials, comprising 2.5% soy protein isolate (SPI) and 2.5% gum arabic compared to soy protein alone.¹⁰ This indicates that effective strategies are needed to protect these compounds, thereby enhancing their application, stability, bioavailability, and color preservation through encapsulation.^6,8

In line with several studies, the successful nanoencapsulation of anthocyanins is largely dependent on 2 main factors. First, the choice of solvent used during the extraction process of the compounds from natural ingredients is essential. For instance, the extraction of anthocyanins from purple yam has been widely demonstrated using methanol solvent containing 1% (v/v) tartaric acid,² which is known to produce better extracts. The second main factor is the selection of coating materials as the encapsulants. These coating materials consist of a group of polysaccharides (gum arabic, modified starch, MD, alginate, pectin, chitosan, carrageenan, cellulose, and its derivatives, and cyclodextrin), proteins (gelatin, whey protein, caseinate, soy protein, gluten casein and zein) and lipids (lecithin, medium-chain triglycerides and glyceryl).⁸ Several studies have shown that MD is a cheap encapsulant with mild flavor and rapid dissolution properties but has low emulsification and protection capabilities. Meanwhile, whey protein, a functional protein with several capabilities, can cause discoloration when used in high quantities.¹¹ The combination of these two materials has been reported to be more effective in increasing polyphenol stability and antioxidant capability compared to single materials.^11–13

Various studies have succeeded in encapsulating anthocyanins extracts obtained from various sources, including red cabbage, brown rice, blueberries, black soybeans, and grapes. The biopolymers used during the process include a combination of whey protein isolate (WPI) and pectin, carboxymethyl chitosan, and lysozyme, leading to the production of encapsulation in sizes below 500 nm. The techniques commonly used in producing encapsulation include emulsification, coacervation, nanoprecipitation, supercritical fluid, ultrasonication, and spray drying.⁸ Among these techniques, spray drying typically produces solid powder from a solution using a drying “chamber”. This technique offers various advantages in terms of simplicity, speed, reproducibility, and efficiency. In addition, it has been widely used in various reports to encapsulate bioactive compounds due to its versatility and scalability.^12,13 Spray drying remains the most essential technique for bioactive component microencapsulation, enabling the production of easy-to-handle powdered microcapsules from liquid material in one simple and scalable operation.¹⁴

At present, there are no studies regarding the encapsulation of anthocyanins from purple yam tuber. Therefore, this study aimed to assess the encapsulation of anthocyanins from purple yam extract using different ratios of MD and WPI as encapsulants. The spray drier was used to produce the powder used during the process. The effect of MD:WPI ratio on the antioxidant activity and physical aspects of anthocyanins encapsulation from purple yam extract was also evaluated. The materials used are easily available, have relatively low production costs, and could be used by the public to improve the quality of life.

Materials and Methods

Materials

The materials used in this study included purple yam from Sleman, Yogyakarta, while whey protein isolate (WPI) and maltodextrin (MD) were obtained from the local market. Furthermore, the chemicals used were aquadest, citric acid, sodium citrate, ethanol, HCl, KCl, acetic acid, 2,2- Diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), sodium carbonate, Folin-Ciocalteu (Merck, Germany), sodium acetate, and gallic acid (Sigma Chemical Co., St Louis, United States).

Methods

Preparation of purple yam flour

The purple yam flour was prepared based on the method proposed in previous studies.¹⁵ In addition, the sample was first peeled, cut into slices, and crushed for 8 minutes, followed by drying for 10 hours at 50 °C in a cabinet drier until it had a moisture content of 10%. An 80-mesh sieve was then used to grind the dry materials into a powder. The acquired purple yam flour was prepared to continue the analysis.

Anthocyanin nanoencapsulation

Anthocyanin extract preparation

Anthocyanin extract was prepared by weighing 50 grams of the purple yam flour and then dissolving it with 500 ml of 70% ethanol containing 3% citric acid.¹⁶ During the procedures, ethanol was first prepared in water to achieve 70% ethanol, followed by the dissolution of 3% citric acid. The solution obtained was then stirred for up to 30 minutes and protected from the light. Subsequently, it was stored for 12-24 hours at 8-10 °C and filtered using Whatman filter paper no. 02. A rotary evaporator or rotavapor Buchi R II (BÜCHI Labortechnik AG, Flawil, Switzerland) was used to concentrate the anthocyanin extract at a temperature of 50 °C and a speed of 175–250 rpm.¹⁷

Nanoencapsulation of anthocyanin extract

The nanoencapsulation of anthocyanin extract was based on the method proposed by previous studies ¹⁸ with some modifications. The process started by weighing MD and WPI in various ratios of 1:3, 1:1, and 3:1 (w/w), as shown in Table 1. The mixtures obtained were dissolved with 250 ml of 0.1 M pH 3 citric buffer, placed in a 500 ml measuring flask, and stirred with a stirrer for 15      minutes at a speed of 400 rpm. Subsequently, the concentrated anthocyanin extract was added to the system up to 30% (w/w), followed by stirring for 15 minutes at 400 rpm. The citric buffer pH 3 was poured up to the mark in the 500 ml volumetric flask and stirred again for 15 minutes at 400 rpm. The samples were protected from light until the spray drying process was carried out. The spray drier was set up with an inlet temperature of 100 °C, an outlet temperature of 60 to 62 °C, and a drying airflow of 350 mL per hour.

Table 1: The maltodextrin/whey protein isolate ratio as encapsulants of purple yam flour anthocyanin extract.

Note: MD and WPI referred to maltodextrin and whey protein isolate, respectively.

Analysis Methods

Analysis of total anthocyanins

Total anthocyanins were measured using the technique suggested by a previous study.¹⁹

Analysis of Total Phenolic Content

The Folin-Ciocalteu technique was used to determine total phenolic levels with gallic acid as the standard.²⁰

Analysis of antioxidant activity

For the antioxidant activity test, the DPPH free radical scavenging capacity was determined.²¹ The scavenging capacity of free radicals was determined and reported as a percentage (%) RSA =% Radical Scavenging Activity, which was the percentage of DPPH bleaching. The equation was % RSA=1-(absorbance of sample)/(absorbance of control) X 100%.

Scanning Electron Microscopy (SEM) observation

The morphological and surface structures of anthocyanins nanoencapsulation were observed using SEM JEOL JSM-6510LA, auto Coater JEOL JEC-3000FC Auto Fine Coater.

Particle size

A particle size analyzer (Horiba scientific SZ-100) with dynamic light scattering was used to determine the particle size, polydispersity index, and zeta potential of anthocyanins nanoencapsulation.

Statistical analysis

This study used a completely randomized design with the encapsulants of MD: WPI (1:3; 1:1 and 3:1, (w/w)). Furthermore, the data were analyzed statistically by the Duncan New Multiple Range Test (DMRT) at a 95% degree of confidence using IBM SPSS Statistics 24.

Results

Antioxidant activity and total phenol content

Table 2 showed the effect of MD and WPI ratios on anthocyanins nanoencapsulation antioxidant activity. The antioxidant activity of anthocyanins encapsulants in this study varied from 35-65 %RSA. Furthermore, nanoencapsulation with MD:WPI (1:3) showed the highest antioxidant activity compared to other treatments. This result had a similar trend with the anthocyanins content, as shown in Table 2. Furthermore, the total phenol concentration was similarly affected by the encapsulant material ratios (p<0.05). The MD:WPI (1:1) treatment had a total phenol content of 868.44 mg GAE/100 g wb, which was the highest compared to treatment ratios of 1:3 and 3:1.

Table 2: Antioxidant activity, total phenol content and anthocyanin content at the various ratio of encapsulants

Note: the similar superscript in the identical column showed that there is no substantial difference (p>0.05).  

Anthocyanins content

Based on Table 2, the anthocyanins levels differed significantly (p<0.05) among different encapsulant ratios. Anthocyanins levels in the MD:WPI treatment (1:3) were the highest (60.83 mg/100 g) compared to others. Meanwhile, the results showed that the other nanoencapsulation contained 21-24 mg anthocyanin/100 g.

Color properties

Table 3: Color properties of anthocyanin nanoencapsulation at different ratio of encapsulants

Note: the similar superscript in the identical column showed that there is no substantial difference (p>0.05).

Morphology properties

In line with Figure 1, anthocyanins nanoencapsulation by using MD:WPI (1:3) had an irregularly round shape with several depressions that were not too deep. The MD:WPI (1:1) encapsulant treatment showed an irregular round shape with deeper depressions. Meanwhile, the MD:WPI (3:1) treatment had an irregular round shape with deeper, more numerous depressions, and some torn parts. The desired product morphology was tight, wrinkled, and without cracks because it allowed fewer compounds or extracts to diffuse and degrade.²² The encapsulant in this study with a tight shape and no cracks was the MD:WPI 1:3 treatment.

Figure 1: Anthocyanin nanoencapsulation (left) and the pictured that captured by the Scanning Electron Microscope of anthocyanin nanoencapsulation at the ratio of MD:WPI (1:3; 1:1 dan 3:1). Click here to view Figure

Particle size

Table 4 showed that the particle size of the samples was between 300.5 to 304.2 nm. The results of this study also revealed that the zeta potential value ranged from -30.4 to -31.9, which was categorized as a stable nanoparticle.

Table 4: The particle size of anthocyanin nanoencapsulation

Discussion

Table 2 showed the influence of maltodextrin (MD) and whey protein isolate (WPI) ratios on the antioxidant activity of anthocyanins nanoencapsulation. The antioxidant activity of anthocyanins nanoencapsulation in this study was significantly different (p<0.05) among the different ratios of encapsulants. Nanoncapsulation with MD:WPI (1:3) showed the highest antioxidant activity compared to other treatments. This result had a similar trend with the anthocyanins content, as shown in Table 2. The findings revealed that the total phenol concentration was similarly affected by the encapsulants ratios (p<0.05). The MD:WPI (1:3) treatment had the highest antioxidant activity at 65.16 + 2.87 (%RSA) with anthocyanins content of 60.83 + 1.56 mg/100 g, compared to the MD:WPI (1:1 and 3:1) treatments. Therefore, the higher the WPI, the higher the protection provided by these parameters. The total phenol content of encapsulants from MD:WPI (1:3) treatment was relatively high at 776.25+45.23 mg GAE/100g. A previous study showed that the encapsulation of grape seed extract phenolic with WPI and MD (4:1) protected antioxidant activity more effectively than using MD or WPI alone.²³ Encapsulation of phenolic compounds in grape seeds with whey protein-polysaccharide complex led to higher retention of phenolic compounds and antioxidant activity.²³ The use of WPI in the coating process was reported to be more effective in maintaining the bioactive content in the coated material.²⁴ Whey protein had good protection against oxidation compared to MD. However, its combination with MD as a coating became more effective in the encapsulation process because MD could produce an amorphous glass matrix acting as a barrier against oxidation.²⁵ Encapsulation treatment of purple grape extract with WPI and MD could maintain anthocyanins and antioxidant activity.¹² Protein had a good ability to package bioactive components.²⁶ Using MD and WPI in a 1:3 ratio in the nanoencapsulation process proved successful in encapsulating bioactive substances.

According to Table 2, the anthocyanins levels differed significantly (p<0.05) among different encapsulant ratios. Anthocyanins levels in the MD:WPI treatment (1:3) were the highest at 60.83 mg/100 g, compared to others. This finding was consistent with previous studies, that encapsulating purple corn anthocyanins with a combination of MD and gum arabic encapsulants gave better retention of anthocyanins compared to the use of MD encapsulation alone.¹⁴ Another study stated that the use of jaboticaba pomace with a combination of MD, pectin, and soy protein biopolymers increased the stability of anthocyanins due to light exposure.²⁷

In color parameters, the value for color L* showed changes in brightness or lightness. The results of statistical tests on the lightness parameter (L*) showed the presence of a significant difference. Furthermore, nanoencapsulation with MD:WPI (1:3) had the smallest value (78.13) (Table 3), which showed a darker color compared to other treatments. The dark color indicated the presence of a higher anthocyanins component compared to other treatments. This finding was in line with another study, stating that red onion ethanol extract with a low L* color test indicated high anthocyanins levels.²⁸ The chromatic color for red was shown by the a* value. In this investigation, the a* color value was statistically and significantly different with p<0.05. The highest a* value was obtained when using the MD:WPI ratio (1:3), indicating the presence of higher anthocyanins levels. The b* value represented the blue chromatic color, and it showed a statistical difference in this study (p<0.05). The MD:WPI (1:3) showed the highest b* color value. A decline in the b* color value indicated an increase in the blueness color value. This showed the presence of anthocyanins in the encapsulant of the results of this study. Therefore, the various ratios of encapsulants affected the color parameter of anthocyanins nanoencapsulation. The lower lightness and the higher parameter value indicated the presence of higher anthocyanins content in the sample.

Table 4 showed that the particle sizes of the samples were between 300.5 to 304.2 nm, indicating their monodisperse and nanoparticulate nature. Particle size was the most important characteristic of a nanoparticle system because it directly influenced the unique properties. Nanoparticles were included in the procedure when the particle size was less than 1000 nm.²⁹ Several studies have also successfully produced nanoencapsulation for various purposes. Encapsulation of black rice anthocyanins (Oryza sativa L.) with chitosan and alginate led to the production of nanoparticles measuring 358.5 – 467.9 nm.²⁹ Other reports found that the encapsulation of juwet seed extract (Syzygium cumini) showed a particle size of 395.9 – 820.67 nm.³⁰ Therefore, the encapsulation of anthocyanin extract from purple yam could be used to produce materials with nano-sizes. The zeta potential of nanoencapsulation was assessed along with particle size and polydispersity index. In addition, the zeta potential was an electric charge parameter between colloidal particles. The higher the value of this parameter, the better it prevented flocculation.³⁰ Zeta potential value of less than −30 mV or more than 30 mV indicated particle stability.³¹ The results of this study showed that the zeta potential value ranged from -30.4 to -31.9, which was a stable nanoparticle.

Conclusion

In conclusion, the best ratio of encapsulant for anthocyanins extract from purple yam flour nanoencapsulation was MDI:WPI (1:3). The results showed that the use of this ratio led to the retention of more bioactive components compared to the other. Therefore, nanoencapsulation had the potential to be applied in food and beverage systems as the carrier of anthocyanins extracted from purple yam flour.

Acknowledgement

The authors would like to thank our research team which has provided help during the work.

Conflict of Interest

The authors do not have any conflict of interest.

Funding Sources

The Ministry of Education, Culture, Research, and Technology through Hibah Penelitian Fundamental 2023 with contract number 0423.14/LL5-INT/AL.04/2023.

Authors’ Contribution

Siti Tamaroh : extracting anthocyanin, making encapsulation with a spray drier, anthocyanin test, color test, Scanning Electron Microscope test of anthocyanin nan encapsulation, Compiling the manuscript

Yuli Perwita Sari : evaporation of anthocyanin extract, total phenol test, antioxidant activity test, particle size test, data analysis

Data Availability Statement

Yes, the manuscript incorporates all datasets produced or examined throughout this research study.

Ethics Approval Statement

This research did not involve animals or humans for testing.

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