**1. Introduction**

The microalga *Spirulina* has gained more attention in areas such as the pharmaceutical, food, poultry, and aquaculture industries for its nutritional and health benefits [1]. Certain therapeutic effects of *Spirulina* (reduced hyperlipidemia, obesity, and blood cholesterol; antioxidant and anticancer activity; immune system improvement; and increased beneficial intestinal bacteria) have been proven by pre-clinical and clinical studies, which are related to their bioactive constitution, e.g., phycocyanins, carotenoids, phenolic compounds, and polyunsaturated fatty acids. The green-blue microalgae are a rich source of proteins (60–70% of dry matter) and, due to the absence of cellulose in the cell wall, are very digestible. Thus, they have gained more attention in recent years as a food supplement, especially for athletes and vegetarians [2].

However, the undesirable taste, low digestibility, and high allergenicity of algae-based protein isolates for monogastric animals and humans have limited their application in the food industry. Protein hydrolysates and peptides can be excellent alternatives to overcome the problems associated with the direct consumption of protein isolates, as they have

**Citation:** Mohammadi, M.; Hamishehkar, H.; Ghorbani, M.; Shahvalizadeh, R.; Pateiro, M.; Lorenzo, J.M. Engineering of Liposome Structure to Enhance Physicochemical Properties of *Spirulina plantensis* Protein Hydrolysate: Stability during Spray-Drying. *Antioxidants* **2021**, *10*, 1953. https://doi.org/10.3390/ antiox10121953

Academic Editors: Li Liang and Hao Cheng

Received: 18 October 2021 Accepted: 2 December 2021 Published: 6 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

nutritional and health-promoting features and act as natural antioxidant agents in food preservation [3,4]. Although algae-derived peptides have various advantages, their instability during storage and under harsh conditions (e.g., in the gastrointestinal tract), low absorption efficiency, bitterness, reaction with the food matrix, and possible inactivation inhibit the application of hydrolysates in foods and beverages. Incorporating these bioactive compounds in lipid-based nanocarriers such as liposomes can be an appropriate solution to cover all of these problems and increase their efficacy under different conditions [5].

Liposomes have an enclosed vesicular structure and are able to accommodate both water-soluble and hydrophobic compounds in their internal aqueous core and bilayer space, respectively [6]. Moreover, the typical constituents of liposomes are completely natural, and their nontoxicity has led to the broad application of these vesicular systems in the encapsulation of various bioactive compounds [7,8]. However, the major drawbacks of this versatile carrier are the fluidity of the intravesicular space and the flexible bilayer structure, which can lead to the physical instability of vesicles (aggregation/flocculation and fusion/coalescence), resulting in changes in size and loss of liposome-incorporated bioactive materials over time. A possible solution to this problem is to engineer the liposomal structure [9]. Mostly, cholesterol has been applied as a stabilizing factor in vesicular systems because it can increase the packing of phospholipid molecules, reduce the fluidity of intravesicular space, and consequently create a more rigid and stable structure over time and under severe shear stress [10]. Moreover, the above-mentioned problems can be improved by depositing an oppositely charged biopolymer such as chitosan around the liposome surface through electrostatic interaction [11,12]. Several studies on improving hydrolysate stability during storage and processing using chitosan coating have been conducted [13,14].

To make a formulation that is more stable over time and more appropriate for industrial application, it can be transformed into powder form by spray-drying or freeze-drying. Freeze-drying is a more expensive technology and requires more time and energy compared with spray-drying. Thus, spray-drying technology is an economical strategy to make powdered liposomal dispersions, and it has a wide range of use in the food industry compared to other drying techniques. However, there have been no studies regarding the simultaneous use of different stabilizers and coating materials to increase the stability and bioactivity of liposome-containing *Spirulina* hydrolysates. Therefore, the aims of this research were as follows: (1) to explore the effect of cholesterol and γ-oryzanol as stabilizing agents and chitosan as a coating material on the mean diameter and encapsulation efficiency of *Spirulina* hydrolysate-loaded liposome, (2) to examine the antioxidant capacity of synthesized liposomes using the different methods, and (3) to estimate the stability of synthesized liposomes during storage. Finally, the optimum formulation for easy usage was converted into powder form and its physicochemical and structural properties and antioxidant activity were evaluated.
