Encapsulation of aqueous Hibiscus sabdariffa L. extract in food-grade high internal phase pickering emulsions stabilized by soy protein isolate

Abstract

Hibiscus Sabdariffa L. is a highly versatile plant that finds applications in various industries due to its abundant nutrients, potent bioactive compounds such as phenolics and anthocyanins, and natural colour pigments. In addition to its use as a colouring agent, its positive health effects, such as antibacterial, antioxidant, anticholesterol and prevention of gastrointestinal problems, spread the consumption of the product for health purposes. However, the fact that bioactive ingredients are easily affected and damaged by many factors limits this product's use in many industries. Therefore, integrating Hibiscus Sabdariffa into food products and various formulations becomes more complex and limits the methods used. Encapsulation, a preferred method for much bioactive protection, appears at this point. Encapsulation comes to fields as a technique that allows us to apply in different areas, such as preserving these components and controlled release and integrating them into product formulations. Emulsification by encapsulation method has gained a solid place in industry and literature. High Internal Phase Emulsions are among the leading emulsions with high volume encapsulation and high stability against external factors. Similarly, double emulsion systems can enhance bioactive components' protection with their nested phase structure. The debate over the many adverse effects of preferred emulsifiers and stabilizers for emulsion stability, such as human health and environmental pollution, has accelerated scientific research into discovering alternative products. Plant proteins have gained tremendous interest in recent years as they have the potential to be an alternative to conventional emulsifiers and stabilizers. The various functional properties and amphiphilic characteristics indicate that these proteins are significantly effective in the stability of the emulsion system. Like many other types of plant proteins, soy protein is a preferred product due to its easy availability and processability. In this study, analyses were conducted to identify and evaluate the encapsulation of aqueous Hibiscus extract in the High Internal Phase Pickering Emulsions (HIPPE) and High Internal Phase Double Emulsions (HIPP-DE) systems stabilized by soy lecithin and soy protein isolate. The stability of the emulsions obtained, characteristics and the effects of in vitro gastrointestinal digestion on the phenolics and anthocyanins in the extract with the presence of soy protein and soy lecithin were investigated. The extraction of phenolics and anthocyanins from powdered Hibiscus calyces was realized by ultrasonic method, and water was used as a solvent. Emulsions with an internal volume of 80% have been created using soy protein isolate (SPI) gel and lecithin. A HIPPE resistant to phase separation could not be obtained using soya protein isolation gel alone, while emulsions (L4 and L6) containing 4% and 6% lecithin and phase separation resistance could be achieved. Combinations of different concentrations of lecithin and soy protein isolated gel stabilized other HIPPEs. After 24 hours of storage, HIPPEs stable against phase separation were obtained by homogenizing HIPP-DEs at a volume of 50% with a 6% SPI gel. When emulsions stored for 24 hours were observed, SPI gels and lecithin at varying concentrations acted as a synergistic mechanism that effectively prevented phase segregation and ensured stability in the emulsion system. All HIPP-DEs also showed superior resistance to phase separation. According to the CI results, an increase in the concentration of soy lecithin from 2% to 4% in HIPPE containing 6% SPI gel resulted, indicating increased stability, leading to a decrease in the CI value on day 1; however, no significant difference in CI was observed between HIPP-DEs. EAI and ESI values show a statistically significant increase when the SPI concentration increases from 2% to 4% in HIPPEs and HIPP-DEs with 4% and 6% lecithin concentrations. Still, surprisingly, no significant increase was observed with further increases in SPI. In contrast, the particle size of HIPPEs with 4% and 6% lecithin concentrations showed a significant decrease as the SPI gel concentration increased from 2% to 6%. In HIPPEs, the zeta potential was negative, and the absolute values increased by increasing the SPI gel concentration from 2% to 4%, while in L4S4, it was observed that the HIPPE had the highest absolute zeta potential (-41.21 ± 1.23 mV). However, an increase in SPI gel concentration from 4% to 6% decreased the absolute zeta potential value. The increase in the concentration of soy protein isolate (SPI) from 2% to 6% resulted in a significant decrease in the PDI values of HIPPE containing 4% and 6% lecithin. The lowest PDI values were obtained in L4S6 and L6S6 HIPPE with an SPI concentration of 6%. The same decline has been observed in HIPP-DEs. Different changes have been observed in the stability and characteristics of emulsions, as well as in the study of bioactive components and their properties. Encapsulation Efficiency (EE) determinations stated that HIPP-DEs produced higher EE than HIPPEs due to high SPI gel concentrations. The total anthocyanin content (TAC), total phenolic content (TPC), DPPH and ABTS of the resulting aqueous Hibiscus extract were analyzed. The TAC (mg Cy-3-GC equivalent/100 g), TPC (mg GAE/100 g), DPPH (mg TE/100g) and ABTS (mg TE/100g) values were found as 31.13±1.23, 2619.01±17.31, 335.12±1.21 and 223.21±2.56 respectively. Significant decreases in TPC in Hibiscus extract were observed with in vitro gastrointestinal digestion. In HIPPEs prepared with a concentration of 4 % lecithin (L4, L4S2, L4 S4, L4S6), the SPI gel concentration increased from 0 % to 4 %, while anthocyanins in the stomach environment showed more excellent stability compared to the intestinal environment. When the TPC of the digested phenolics of Hibiscus extract was compared to those of digestive HIPPEs and HIPP-DEs, higher TPC values were observed in all emulsions. Maximum TPC values in the stomach and intestinal digestion reached 709.82 ±2.06 mg/100g and 1160.71 ±21.01 mg/100g, respectively. In the stomach and intestinal phases of in vitro digestion, the rate of phenolic release has been significantly influenced by the pH of the environment. According to HPLC results, aqueous Hibiscus extract has detected anthocyanins of delphinidin-3-glucoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside. Four phenolic acids have also been detected in the extract and all emulsions, including gallic acid, syringic acid, ferulic acid and chlorogenic acid. After gastric digestion, a decrease in the content of chlorogenic acid, ferulic acid and gallic acid was observed, while syringic acid increased, showing a different tendency. The highest concentrations of each anthocyanins and phenolics concentrations were obtained in L6S6-D. As a result, changes in SPI gel and lecithin concentrations have been effective in many conditions, such as the stability of emulsions, their properties, and the effects of in vitro digestion on anthocyanins and phenolics. In addition, according to the results of encapsulation efficiency, it can be stated that HIPPEs and HIPP-DEs are effective emulsion systems in the encapsulation of Hibiscus extract.