**1. Introduction**

The spoilage of meat products, which is extremely harmful and destructive, significantly increases the risk to human health [1]. Therefore, it is necessary to detect meat freshness. In the past, total volatile basic nitrogen (TVB-N) was widely regarded as a useful method for meat freshness monitoring using the Kjeldahl method [2]. However, it is destructive to samples and time-consuming. In recent years, more studies have been interested in intelligent packaging systems for ''on-package" tracing in real-time. Intelligent food packaging is an effective tool for monitoring food conditions for consumers through intuitive changes. Meat corruption produces volatile amines, which results in an alkaline packaging environment. Therefore, pH indicator films, as a kind of intelligent sensor, have garnered wide attention because they can reflect freshness information through visual color changes. As a natural extract, anthocyanin presents visible color changes at different pH values and has been used in intelligent pH indicator films in recent years [3]. For instance, Zhang et al. successfully developed a novel film based on a mulberry anthocyanin extract for fish freshness monitoring [4]. However, most reported indicator films are based only on individual anthocyanins in the film-forming matrices, which makes them easily degraded in harsh environments (such as light and temperature). In addition, water-soluble

**Citation:** Zhang, J.; Yang, Y.; Zhang, J.; Shi, J.; Liu, L.; Huang, X.; Song, W.; Li, Z.; Zou, X.; Povey, M. High-Stability Bi-Layer Films Incorporated with Liposomes @Anthocyanin/Carrageenan/Agar for Shrimp Freshness Monitoring. *Foods* **2023**, *12*, 732. https://doi.org/ 10.3390/foods12040732

Academic Editor: M. Angela A. Meireles

Received: 7 December 2022 Revised: 9 January 2023 Accepted: 20 January 2023 Published: 8 February 2023

**Copyright:** © 2023 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/).

free anthocyanins easily leak out from the film matrix in a high-humidity environment, thereby affecting the indicator stability. This instability creates a barrier that limits the use of intelligent film in practical food packaging. Therefore, it is essential to use an effective method to improve the stability of anthocyanin indicators.

Nano/microencapsulation technology in particular has been verified as a useful method to enhance the stability of anthocyanins. In terms of encapsulation technology, liposomes are popularly prepared with phospholipids, oils, and different solvents [5]. The central aqueous cavity of liposomes can be used to improve the stability of hydrophilic active ingredients and increase bioavailability [6]. Liposomes are attractive because they can encapsulate anthocyanins without changing their structure. The prepared anthocyanin elderberry extract-loaded liposome has the highest encapsulation efficiency of 69% and storage stability [7]. The retention rate of anthocyanins in milk was effectively improved with liposomes prepared by Chi et al. [8]. Up to now, few researchers have reported the use of liposomes to encapsulate free anthocyanins in intelligent packaging films.

Compared with single-layer biopolymer films, bi-layer films have shown excellent mechanical properties and stability [9,10]. One layer serves as an indicator layer containing anthocyanins and the other as a protective layer. Agar, one of the most widely promising agents, is applied in food packaging films due to its good gelling properties and excellent film-forming materials [11]. Carrageenan, a natural biopolymer, is widely studied as a packaging film matrix due to its high gelling capacity [12]. Because of the stronger hydrogen bond interactions between their highly polar hydroxyl groups, agar and carrageenan are used together to improve their mechanical properties.

Therefore, in this study, a liposome was formed by encapsulating a butterfly bean flower anthocyanin extract (BA) in soybean lecithin, which was then added to carrageenan to develop as an indicator layer. In addition, agar was used as the protective layer of a bi-layer indicator to monitor shrimp freshness. The particle size, zeta potential, morphology, and encapsulation efficiency of the liposome were initially analyzed. The bi-layer indicator film was determined with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and its mechanical properties. Moreover, the temperature stability and the response sensitivity to pH solutions and ammonia of the bi-layer films were investigated before evaluating their application for shrimp freshness monitoring.
