Research progress of chilled meat freshness detection based on nanozyme sensing systems

It is important to develop rapid, accurate, and portable technologies for detecting the freshness of chilled meat to meet the current demands of meat industry. This report introduces freshness indicators for monitoring the freshness changes of chilled meat, and systematically analyzes the current status of existing detection technologies which focus on the feasibility of using nanozyme for meat freshness sensing detection. Furthermore, it examines the limitations and foresees the future development trends of utilizing current nanozyme sensing systems in evaluating chilled meat freshness. Harmful chemicals are produced by food spoilage degradation, including biogenic amines, volatile amines, hydrogen sulfide, and xanthine, which have become new freshness indicators to evaluate the freshness of chilled meat. The recognition mechanisms are clarified based on the special chemical reaction with nanozyme or directly inducting the enzyme-like catalytic activity of nanozyme.

Nanozyme-based detection method is listed as one of the top ten emerging technologies in chemistry in 2022 for "combining the power of natural and artificial catalysis" (Jiangjiexing, Wang, Zhangping, Sirong, & Zhu, & Qin., 2018;X. Zhang, Lin, Liu, Tan, & Xia, 2020).Nanozymes are nanomaterials with catalytic active sites and mimic the kinetic process of enzymatic reactions(H.Wei & Wang, 2013).They can obtain promising applications in rapid food safety testing with the advantages of high stability, low cost, easy production, and resistance to harsh experimental conditions (Liang & Xiyun., 2019;Lunjie Huang, Hongbin, & Wei, 2019).In the detection process, there are two main recognition mechanisms to realize the qualitative and quantitative detection of the target, including the direct reaction between targets and nanozymes, and the specific reaction between targets and the catalytic activity center of the nanozymes.Nanozyme sensing systems offer affordable, sensitive, specific, user-friendly, and rapid assessment of chilled meat freshness.
Hereby, this paper firstly summarized the main four types spoilage mechanisms of chilled meat, and their degradation products were used as indicators to evaluate the freshness of chilled meat.Secondly, the enzyme-like properties and recognition mechanisms of nanozyme sensing systems and their detection application for evaluating chilled meat freshness were analyzed in this paper.Thirdly, this paper was clearly elucidated two common recognition mechanisms between freshness indicators and nanozymes.At present, although nanozymesbased sensing systems had many superiorities in the biosensor detection field, we had to face some challenges in chilled meat freshness detection.This paper also provided some clues to overcome these problems, and to develop new intelligent, portable, rapid, and real-time efficient freshness detection methods in the future.

Enzymatic spoilage
Increasing evidence found that many endogenous enzymes in animals play an important role in ensuring the quality of chilled meat after slaughter (Fig. 1) (Bekhit et al., 2021).Calpains improve the tenderness of meat (Bhat, Morton, Mason, & Bekhit, 2018), and aminopeptidases regulate the flavor of meat (Nishimura, 1998).Meanwhile, exogenous proteases produced by bacteria also have similar roles (Jurado, García, Timón, & Carrapiso, 2007).Obviously, the process of enzymatic spoilage has a great influence on the freshness of chilled meat, which not only produce odor, but also produce harmful products.The packaging system, initial microbial composition and count, meat composition, pH value, temperature, and inhibitors can easily influence the process of enzymatic spoilage (Bekhit et al., 2021).

Lipid oxidation
The autooxidation, photo-oxidation, and enzymatic hydrolysis are the three primary oxidation pathways of lipid oxidation in chilled meat.Among them, the first two are of great significance in evaluating the freshness of chilled meat (Fig. 1) (Mariutti & Bragagnolo, 2017).Increasing evidences have proposed that the process of lipid oxidation may be mediated by lipases, lipoxygenases, cyclooxygenases, and several enzymatic systems involved in cholesterol oxidation (Papuc, Goran, Predescu, & Nicorescu, 2017).The progress of lipid oxidation can further induce the protein degradation process, thus leading to the decline of chilled meat quality.Furthermore, adjusting technological parameters in processing effectively inhibits lipoxygenase activity and further prevents the process of lipid oxidation (Jin, Zhang, Yu, Lei, & Wang, 2011).

Protein oxidation
The process of protein oxidation can lead to significant changes in the nutritional quality, physical properties, and sensory properties of chilled meat (Bekhit et al., 2021).Free amino acids (e.g., leucine, isoleucine, threonine, phenylalanine, and tryptophan) are produced by microbial proteases and endogenous proteases through the different degradation pathways of protein, which can be further utilized by decarboxylase to produce common biogenic amines and volatile amines.In addition, sulfur-containing amino acid can be utilized by specific sulfur-containing bacteria to produce hydrogen sulfide (Paczkowski & Schütz, 2011) (Fig. 1).Compared with the other three spoilage mechanisms, the process of protein oxidation is the major causes of spoilage, which can significantly reduce the freshness of chilled meat.

Volatile amines
During storage and transportation, microorganisms utilize glucose and amino acids to produce volatile amines that can generate obvious odors and reduce the freshness of chilled meat (Hazards, 2016) (Fig. 2).Nowadays, volatile amines serve as a crucial freshness indicator to evaluate the quality of chilled meat (Table 1) (Zhong et al., 2018).For example, a nanofilm was constructed by Zhai and their coworkers to efficiently detect the content of trimethylamine, and its LOD was 0.018 mM (Zhai et al., 2020).Thereafter an intelligent biopolymer film based on starch/carbon nanodots was created, and the visual color of the film changes from purple to green during the storage of pork (Koshy et al., 2021).It can be used as a low-cost portable visual indicator to monitor the freshness of pork.

Organic sulfides
Sulfur-containing amino acids (e.g., lysine and cysteine) can be decomposed by sulfur-containing bacteria into mercaptans (e.g., thiophenic acid, methanethiol), and further degraded to hydrogen sulfide (H 2 S) (Fig. 2) (Lin et al., 2022).Increasing evidences have proven that H 2 S can be used as an important indicator to evaluate the freshness of chilled meat (Table 1) (Yuan et al., 2020).For example, a H 2 S in situ and nondestructive detection sensor based on gellan gum-capped silver nanoparticles was developed to monitor the meat spoilage process in real time.The LOD of sensor was 1.09 μM, which showed a good selectivity towards H 2 S (Zhai et al., 2019).Then, a novel nanocomposite film label (AgNPs-BCNCs-MoO 3 NPs) was developed for the detection of H 2 S gas in a 1% w/v solution system (Sukhavattanakul & Manuspiya, 2021).The LOD and limit of quantitation (LOQ) of film label for H 2 S detection were 3.27 and 10.94 ppm, respectively.

Oxidase-like activity
Oxidases (OXD) use oxygen to produce reactive oxygen species and further oxidize substrate for a redox reaction (Yan et al., 2019).It has been found that a variety of metal-based and metal oxide-based inorganic nanomaterials (e.g., Ru, Au@Pt, CeO 2 , and N-CNMs) can mimic the OXD-like catalytic activity (Table 2) (Ding, Wang, Sun, & Lin, 2018;Wu & Wang, 2019).Many researches have proved that the formation of intermediates (e.g., singlet oxygen, oxygen, and superoxide anion) and the process of electron transfer greatly affect the OXD-like catalytic properties (C.Liu, Sang, & Yu, 2021).The OXD-like catalytic properties of nanozymes also exhibit through the value of K m and v max , and usually show stronger catalytic activity than natural enzymes.However, the specific catalytic mechanism of OXD-like is unclear.

Catalase-like activity
Catalases (CAT) are a kind of binding enzymes with iron porphyrin as a cofactor, which can decompose H 2 O 2 to produce O 2 and H 2 O (Yan et al., 2019).The CAT-like catalytic properties of nanozymes are closely related to the morphology, surface potency, and pH value (Table 2) (Wy et al., 2021).And then, the optimum pH value of CAT-like catalytic is alkaline environment, which is different from POD-like and OXD-like catalytic.Currently, CAT-like catalytic mechanisms mainly involve adsorption activation and redox reactions (Wy et al., 2021).Meanwhile, the intermediate products OH* (* referring to species adsorbed to the metal surface) and •OH play a crucial role in the catalytic process(J.Li, Liu, Wu, & Gao, 2015).

Superoxide dismutase-like activity
Superoxide dismutase (SOD) can catalyze the superoxide anion radical to produce O 2 and H 2 O 2 through the electron gain and loss (Yan et al., 2019).Nowadays, Pd, MnO 2 , PB, and fullerene nanomaterials have been reported to have excellent SOD-like catalytic properties (Table 2) (Wy et al., 2021).The chemical structure and surface ions of nanozymes jointly determine their SOD-like catalytic properties, and the optimum pH value is similar to CAT-like catalytic properties.The adsorption activation and electron transfer on the surface of the nanozymes are the two main SOD-like catalytic mechanisms.

Glutathione peroxidase-like activity
Glutathione peroxidase (GP x ) is an important peroxide-degrading enzyme with selenocysteine as its active center.Nicotinamide adenine dinucleotide phosphate (NADPH) can oxidize glutathione (GSSG) to generate glutathione (GSH) and promote the decomposition of H 2 O 2 into H 2 O.The GP x -like catalytic activity of nanozymes can be obtained ([E] is the enzyme or nanozyme concentration.K m is the Michaelis constant, ν max is the maximal reaction velocity and K cat is the catalytic constant, where K cat = ν max / [E] and the K cat / K m value indicates the catalytic efficiency of the enzyme or nanozymes.)by calculating the reduction content of NADPH (Yan et al., 2019).Currently, some nanomaterials (e.g., vanadium metal) have been shown that can mimic the catalytic activity of GP x -like (Table 2).The oxidation groups amount on the materials surface and the reaction process of H 2 O 2 to form peroxide bonds are two main reasons for inducing the catalytic activity of GP x -like.

Hydrolase-like activity
Increasing studies have proposed that the catalytic mechanism of hydrolase-like (Hyd-like) is closely related to the breaking of chemical bonds and the generation of free radicals (Wy et al., 2021).The CdTe nanoparticles modified by chiral cysteine that can mimic the catalytic properties of restriction endonuclease and specifically recognize and cleave restriction sites (Sun et al., 2018).Moreover, the chiral copper sulfide quantum dots (d/l-QDs) can mimic the catalytic activity of Hydlike and cause to the cleavage of peptide bonds between amino acids (Hao et al., 2019).There is little research on the field of food safety detection by using the catalytic activity of Hyd-like.

Mimicking the enzymatic microenvironment
Nanozymes have been reported to have multi-species enzyme catalytic properties by mimicking the microenvironment of natural enzyme catalysis (Fig. 3A)(Z.Wang et al., 2020).For example, the construct of natural horseradish peroxidase consists of a heme center and a polypeptide chain, which sever as the active site and active site microenvironment, respectively.Among them, the hydrophilic histidine (His) residues in the polypeptide are involved in the localization of H 2 O 2 .The specific catalytic process includes two main steps, the hydrogen bonding enters active site cavity, and further promotes the O -O bond cleavage to form Fe 4+ = O(C.P. Liu et al., 2016).It has been shown that a novel histidine-functionalized graphene quantum dot (His-GQD)/hemin complex can mimic the catalytic microenvironment of natural horseradish peroxidase for enzyme-like catalytic.Moreover, the AC@O group, O@CAOA group, and GQD group can mimic the heme active site, His ligand, and hydrophobic binding residues of natural horseradish peroxidase, respectively(C.P. Liu et al., 2016).

Mimicking the enzymatic active site architecture
The existence of metal atoms in the structure of nanozymes as the active site provides an important role in its enzyme-like activity (Fig. 3A)(Z.Wang et al., 2020).With the development of nanotechnology, the synthesis of nanozymes with high catalytic activity and strong selectivity will effectively promote the development of food safety detection methods(Z.Chen et al., 2018;J. C. Liu & Xiao, 2020).A type of M-N-C (M = Fe, Co, Mn, etc.) nanozyme is synthesis that can mimic the catalytic properties of natural enzyme.This design can reduce the phenomenon of easy aggregation and uneven distribution of metal atoms, and further improve the metal utilization rate and catalytic activity.Subsequently, a nanozyme (labeled as PMCS) with a remarkable POD-like catalytic activity was synthesized by using a metal-organic framework as a precursor, and the high catalytic property of PMCS is derived from the uniform distribution of single metal zinc atoms (Xu et al., 2019).

Application of nanozyme sensing systems
Currently, nanozyme sensing systems are gradually applied in the field of food safety detection (Table 3).The recognition mechanisms of systems mainly divided into two types (Song, Li, et al., 2022;Song, Zhang, et al., 2022): (1) Constructed a specific chemical reaction between the targets and nanozymes; (2) Regulated the enzyme-like activity of nanozymes in the targets presence (Fig. 3B).Chen et al., 2022;Song et al., 2022;Song et al., 2022).

Colorimetric detection methods
The colorimetric detection methods is widely used in rapid detection field because of its advantages of simplicity, portability, low cost, and so on (Duan et al., 2022).For example, an iron-doped polydopamine nanozyme (Fe-PDA) with POD-like catalytic activity was synthesized and used to detect the content of Hx(Y.Zhang et al., 2022).The xanthine oxidase (XOD) catalyzes the reaction of Hx and O 2 to generate H 2 O 2 , which is further catalyzed by the Fe-PDA nanozyme to produce •OH.The •OH can oxidize the colorless reduced 3,3′,5,5′ -tetramethylbenzidine ( re TMB) into blue oxidized TMB ( ox TMB), which can be quantified by measuring the absorbance at 652 nm.The LOD of the Fe-PDA nanozyme-base sensing system was 1.54 μM in the linear range of 5.13-200 μM (Fig. 4A).Similarly, a novel cerium oxide film (Ce NPs) with POD-like catalytic activity was prepared for Hx detection (Zheng et al., 2023).This detection mechanism is similar to the former, and it is Fig. 5.The fluorescent and electrochemical detection method is applied in detecting hypoxanthine(J.Chen, Lu, et al., 2020).A. TEM image, B. DLS analysis, C. N 2 adsorption-desorption isotherms, D. Principle of detecting aquatic freshness based on the fluorescence biosensor.E-H.The electrochemical detection method is applied in xanthine and hypoxanthine detection (Zhu et al., 2021).also achieved by multi-enzyme cascade catalytic reaction.The LOD of the Ce-NPs-based nanozyme sensing system was 35 μM in the linear range of 6.2-200 μM (Fig. 4B).Then, a nano-sensor (CuS-BSA-Cu 3 (PO 4 ) 2 ) with POD-like catalytic activity was constructed for dopamine detection (Swaidan et al., 2021).The presence of dopamine significantly inhibited the POD-like catalytic activity of the CuS-BSA-Cu 3 (PO 4 ) 2 nanozyme, and the LOD was 0.13 μM in the linear range of 0.05-100 μM.
The CuS-BSA-Cu 3 (PO 4 ) 2 nanozyme sensing system has excellent detection performance in the practical beef samples (Fig. 4C).Meanwhile, a gold-silver nano complexes nanoprobe (Ag-Au/AgCl) with high OXD-like and POD-like catalytic activity was investigated for spermine detection.The spermine can inhibit OXD-like and POD-like catalytic activity of Ag-Au/AgCl nanozyme (Kuo et al., 2018) (Fig. 4D).And then, a single-atom rhodium nanozyme (Rh SAzyme) with POD-like catalytic activity was prepared for XAN detection.XAN can be oxidized by XOD to produce H 2 O 2 , and the Rh SAzyme uses H 2 O 2 to exert its POD-like catalytic properties.The LOD of Rh SAzyme-based sensing systems was 0.73 μM in the linear range of 2-80 μM(Shuangfei Cai et al., 2018) (Fig. 4E).

Fluorescent detection methods
Nowadays, fluorescent detection methods play a vital role in the freshness indicators of chilled meat detection.For example, a platinum nanoparticles (Pt NPs) fluorescent biosensor with POD-like catalytic activity was prepared for Hx detection(J.Chen, Lu, et al., 2020).The fluorescence intensity of the Pt-NPs nanozyme sensing system is linearly proportional to the Hx concentration, and the LOD of system was 2.88 μM within the linear range of 8-2500 μM.The Pt NPs used in the sensing system can be reusable, and the recovery rate was 91% after three cycles (Fig. 5A-D).The methods for detecting chilled meat freshness based on the combination of nanozymes and fluorescence materials have the advantage of high sensitivity, velocity, low cost, and portability.However, the use of toxic and hazardous reagents hinders the application of fluorescent detection in food safety detection.The development of ecofriendly fluorescent materials has become one of the effective ways to solve the above problems.

Electrochemical detection methods
Electrochemical detection methods are crucial in the applications of chilled meat freshness detection due to their high sensitivity, rapidity, and portability.At present, voltammetry, amperometry, conductivity, and impedance methods have been reported to be used for detecting the freshness of chilled meat (Johnson, Atkin, Lee, Sell, & Chandra, 2019).For instance, a three-dimensional porous graphene flexible nanozyme electrode with enzyme-like kinetic characteristics was constructed for detecting the freshness indicators of XAN and Hx.The LOD of this system were 0.26 μM and 0.18 μM within the linear range of 0.3-179.9μM and 0.3-159.9μM, respectively (Zhu et al., 2021) (Fig. 5E-H).The results provide a better sensing platform for further constructing other electrochemical detection method to assess the freshness of chilled meat.However, although electrochemical detection methods have many excellent catalytic properties for detecting trace targets, the disadvantage of poor stability has limited their practical application.

Conclusions and perspectives
Chilled meat can be spoiled because of the process of lipid oxidation, enzyme degradation, protein oxidation, and microbial spoilage.Among them, the products of protein and ATP degradation have been as reliable freshness indicators for assessing chilled meat freshness.Such as biogenic amines, hydrogen sulfide, hypoxanthine, volatile amines and so on.These degradation products may directly or indirectly regulate the enzyme-like catalytic properties of nanozymes, which can be achieve specific detection by the means of colorimetric, fluorescent, and electrochemical signal transmission.However, these methods also have some detection drawbacks.Firstly, chilled meat is a complex food matrixes and the spoilage mechanism is also not easy to elucidate, which makes it difficult to ensure the accuracy of detection process.Secondly, The enzyme-like catalytic properties of nanozymes are unstable and their catalytic activity is still generally low.
Therefore, some measures should be taken to improve these problems existing in the current research.Firstly, the pre-treatment methods of practical samples need to be optimized and the specific freshness indicators need to be filtrated for different species of chilled meat.Secondly, the synthesis methods and conditions of nanozymes need to be improved and optimized, respectively.In this way, it can overcome the problems of catalytic properties.Thirdly, it is necessary to construct a novel cold-adapted nanozyme that can achieve better enzyme-like catalytic properties at cold temperature (0-4 • C).In conclusion, the high enzyme-like catalytic properties of nanozymes occupy a very important position in the field of freshness detection for chilled meat.The nanozyme sensing systems can be made as kinds of rapid detection labels, such as test strip, film, hydrogel, and so on in the future, which have significant advantages to meet current detection needs.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Table 1
Detection of indicators for assessing the freshness of chilled meat.
G.Song et al.

Table 2
Enzyme-like type and catalytic performance of nanozymes.

Table 3
Application of enzyme-like catalytic activity in freshness detection.