“We used pectin extracted from residues of citrus fruit albedo [the layer of white spongy material inside the peel of oranges and lemons] and peel, with a degree of purity permitting human ingestion and excluding any kind of hazardous chemical,” said study author João Paulo Fabi, a professor at the University of São Paulo’s School of Pharmaceutical Sciences.
Methodology can be applied to other water-soluble compounds
“In addition to our review of the literature, we describe a novel technology for nanoencapsulation of bioactive compounds using pectin,” said Fabi. “This entails producing a pectin-lysozyme complex as a protective outer layer for a highly sensitive bioactive compound called anthocyanin.”
Lysozyme is “a safely edible substance obtained from egg white and used to enhance the stability of the end-product”. Anthocyanins are water-soluble pigments belonging to the flavonoid family. These phenolic compounds are found in all plants and are responsible for red, blue, and purple hues seen in flowers, fruit, leaves, stalks, and roots.
The authors say their methodology can be used to encapsulate other water-soluble bioactive compounds.
“We tested anthocyanin because of its challenging sensitivity to many factors, such as light, temperature, pH, and gut bacteria,” said co-author Thiécla Katiane Osvaldt Rosales, a postdoctoral researcher at the Nuclear and Energy Research Institute.
The study was published in the International Journal of Biological Macromolecules.
Nanotechnology with ingredients sourced from nature
A major advantage of the methodology is that no other compounds are added apart from pectin, lysozyme, and anthocyanin, the authors added.
“We used three compounds from sources in nature and mixed them in the laboratory to form a new product, without adding salts, ligands, or anything potentially toxic,” said Fabi. “Furthermore, the nanoparticles are not too small. Very tiny nanoparticles can penetrate barriers and cell membranes, entering the DNA and having toxic effects. The size we obtained is safe.”
Nanoencapsulation, a technique wherein a bioactive compound is encased within a matrix or inert material for preserving the coated substance, offers several advantages for nutraceuticals. It can be used to optimise stability of bioactive compounds and increase their bioavailability – the proportion that enters the bloodstream after absorption.
However, concerns have been raised about possible toxicity associated with the direct incorporation of engineered nanoparticles into foods.
Cost-effective method uses byproducts of citrus peel
Describing the process used to produce the nanoparticles, Rosales said: “Pectin and lysozyme are heated separately. The increase in temperature partly alters their structure, and they interact better when heated. They are then rapidly cooled to reach a temperature not harmful to anthocyanin, which is sensitive and fairly unstable.
“The three substances are blended in an aqueous suspension and agitated for an hour. The result is encapsulated anthocyanin. The suspension is then filtered to separate the non-encapsulated contents.”
Special care was taken with factors such as temperature and pH. “We tested the parameters for the purpose of optimisation, especially pH. If pH is too high, the anthocyanin breaks down. It can’t be too low, either. We found a pH of 5 to be optimal for interaction between the molecules,” Rosales said.
Meanwhile, the method does not require expensive equipment or procedures. “The material used for the nanocapsules, which comes from byproducts of citrus peel, would make the cost even lower for manufacturers. The pectin we used in our study is available commercially and is used by the food industry, mostly for gel formation in jam or as a thickener,” she added.
Pectin protects anthocyanin and its properties
Finally, the encapsulation was tested for efficacy in a digestion system simulated in the laboratory to mimic the gastric and intestinal phases.
“The result was that part of the anthocyanin was released during the digestive process, at the end of gastric digestion, and part remained in the nanostructure, with the possibility of release of this remainder in the gut or absorption together with the nanostructure. We believe this was a good outcome,” Rosales said. “Partial and gradual release suggests absorption of the compound starts before it enters the gut, with the nanoencapsulated remainder probably being released in the gut or fully absorbed with less structural alteration.”
The next step will be animal testing, she added.
“We have evidence that cells can absorb [the nanoparticles] in a non-toxic manner and that the pectin protects the anthocyanin and its properties,” she said. “We now have to test it in animals, observing the process of oral ingestion, absorption of the anthocyanin using specific markers for absorption, and the route followed in the organism. It’s important to verify the extent of absorption and the biological destination.”